Cardiovascular Research Day

November 3, 2017 Lexington Convention Center

ABSTRACT BOOK

Saha CardiovascularResearch CenterGILL HEART INSTITUTE

HealthCare®

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TABLE OF CONTENTS

Schedule for the Day ............................................................................................................................. 2

2017 Gill Heart Institute Young Investigator Award ................................................................. 6

2017 Distinguished Alumni Speaker .............................................................................................. 7

Featured Speakers ................................................................................................................................. 8

Event Supporters ................................................................................................................................. 11

Poster Judges ........................................................................................................................................ 13

Poster Participants ............................................................................................................................. 14

Abstracts ................................................................................................................................................ 16

Survey ................................................................................................................................................... 121

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SCHEDULE FOR THE DAY Friday, November 3 University of Kentucky Cardiovascular Research Day Lexington Convention Center | Bluegrass Ballrooms 8:15 am Guest Check –In Begins | Continental Breakfast | Bluegrass Prefunction 8:30 am Career Development Session for Trainees | Continental Breakfast | Thoroughbred Room 8 9:30 am Scientific Session I | Bluegrass Ballroom I Chair: Gregory Graf, Ph.D. and Gia Mudd-Martin, Ph.D., MPH, R.N.

9:30 am Alan Daugherty, Ph.D., D.Sc., Director, Saha Cardiovascular Research Center Welcoming Comments 9:35 am Trainee Presentations

Ahmed Al-Darraji | Ahmed Abdel-Latif Lab | University of Kentucky Azithromycin Therapy Reduces Cardiac Inflammation and Mitigates Adverse Cardiac Remodeling after Myocardial Infarction: Potential Therapeutic Targets Kelsey Conrad | Phil Owens Lab | University of Cincinnati Increased Circulating Trimethylamine N-oxide (TMAO) Augments the Incidence of Abdominal Aortic Aneurysm in Low Penetrant C57BL/6J Mice Ryan Allen, Ph.D. | Kasey Vickers Lab | Vanderbilt University Circulating Bacterial Small RNA Bound to LDL Induce Inflammatory Activation of Macrophages

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10:35 am Research Blitz I

David Henson | University of Kentucky Mohamed AboAly, MD | University of Kentucky Zheying “Jeff” Chen | University of Kentucky Dylan Colli | University of Kentucky Allison Cooke | University of Cincinnati Mihir Shah | University of Kentucky Jiansheng Huang, PhD | Vanderbilt University Aida Javidan, MS | University of Kentucky Michael Petriello, PhD | University of Kentucky Michael Kuefner | University of Tennessee Health Science Center Robin Shoemaker | University of Kentucky

10:45 am Featured Speaker

Calum MacRae, M.D., Ph.D. | Harvard Medical School Bridging the Gaps in Cardiovascular Translation

11:30 am Lunch | Scientific Session II | Bluegrass Prefunction

12:00 pm Welcoming Remarks

Mark Newman, M.D. | University of Kentucky Executive Vice President for Health Affairs

12:15 pm Distinguished Alumni Presentation

John Charles, Ph.D. | NASA Human Research Program at Johnson Space Center From the Bluegrass to Beyond the Blue

1:00 pm Poster Session | Bluegrass Ballroom II

1:00 pm Odd Numbered Posters

2:00 pm Even Numbered Posters

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SCHEDULE FOR THE DAY continued Friday, November 3 University of Kentucky Cardiovascular Research Day Lexington Convention Center | Bluegrass Ballrooms 3:00 pm Scientific Session III | Bluegrass Ballroom I Chair: Lisa Cassis, Ph.D. and Dan Rader, M.D.

3:00 pm Trainee Presentations

Brooke Ahern | Jon Satin Lab | University of Kentucky Cardiac Specific Rad Deletion Enhances Cardiac Function through Safe, Stable Positive Inotropic Support Ibra Fancher, Ph.D. | Irena Levitan Lab | University of Illinois at Chicago Hypercholesterolemia-induced endothelial dysfunction is rescued by overexpression of endothelial Kir2.1 in resistance arteries

3:30 pm Research Blitz II

Shayan Mohammadmoradi, MS | University of Kentucky Feiming Ye | University of Kentucky Chia-Hua Wu | University of Kentucky Hannah Russell | University of Cincinnati Katelyn Ahern | University of Virginia Hisashi Sawada, MD, PhD | University of Kentucky Courtney Turpin, MS | University of Kentucky Zhihong Yang, PhD | National Institutes of Health Miao Liu, PhD | University of Kentucky Bradley Wright | University of Kentucky

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3:40 pm Featured Speaker

Steven Houser, Ph.D. | Temple University Cardiac Repair and Injury

4:25 pm Gill Heart Institute Translational Early Career Award Recipient Kiran Musunuru, M.D., Ph.D., MPH | University of Pennsylvania

Discovery and Therapeutic Genome Editing of Cardiovascular Disease Genes

5:15 pm Networking Reception | Bluegrass Prefunction 6:00 pm Dinner and Awards Ceremony | Bluegrass Prefunction Special Presentation from Mark Stoops |University of Kentucky Head Football Coach

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2017 GILL HEART INSTITUTE YOUNG INVESTIGATOR AWARD

Kiran Musunuru, M.D., Ph.D., MPH Associate Professor Cardiovascular Medicine and Genetics Perelman School of Medicine University of Pennsylvania Dr. Musunuru received his medical degree from Weill Cornell Medical College, his Ph.D. from The Rockefeller University, and his Masters of Public Health from Johns Hopkins Bloomberg School of Public Health. He trained in Internal Medicine at Brigham and Women's Hospital and Cardiovascular Medicine at Johns Hopkins Hospital, followed by postdoctoral work at Massachusetts General Hospital and the Broad Institute of MIT and Harvard.

Dr. Musunuru's research focuses on the genetics of cardiovascular and metabolic diseases and seeks to identify naturally occurring genetic variants that predispose to or protect against disease and can be used to develop therapies to protect the entire population. In 2016, he received the Presidential Early Career Award for Scientists and Engineers from the White House, as well as the American Heart Association's Award for Meritorious Achievement.

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2017 DISTINGUISHED ALUMNI SPEAKER

John Charles, Ph.D. Chief Scientist, NASA Human Research Program at Johnson Space Center John B. Charles, Ph.D., is the Chief Scientist of NASA’s Human Research Program (HRP), responsible for the scientific direction of human research and technology development enabling astronauts to go beyond low Earth orbit and eventually to Mars. Previously he was HRP’s Associate Manager for International Science and led NASA’s space life sciences planning for the joint US/Russian one-year mission on ISS and the Twins Study.

Dr. Charles earned his B.S. in biophysics at The Ohio State University and his doctorate in physiology and biophysics at the University of Kentucky. He came to the Johnson Space Center in 1983 a postdoctoral fellow and became a civil servant in 1985. He is co-developer of the Shuttle-era fluid-loading countermeasure, and investigated the cardiovascular effects of space flight using ultrasound, re-entry data recording and in-flight lower body negative pressure on Space Shuttle astronauts and on crewmembers of the Russian space station Mir. He coordinated all of the NASA-sponsored biomedical, biological and microgravity science investigations as Mission Scientist for American astronaut missions on Mir, on STS-95, John Glenn’s Shuttle flight, and on STS-107, Columbia’s last mission in January 2003.

He is a Fellow of the Aerospace Medical Association and has been a member since 1983. He is also a Full Member of the International Academy of Astronautics (IAA) and co-chaired the 18th IAA “Humans in Space Symposium” in Houston in 2011.

He has published 75 scientific papers and space history articles and has received several professional awards, including National Space Club and Foundation Eagle Manned Mission Award (2017), the NASA Exceptional Achievement Medal (2014), the ASMA “Joe Kerwin Award” (2011), the “Hubertus Strughold Award” of the Space Medicine Association (2001) and the NASA Exceptional Service Medal (2000).

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FEATURED SPEAKER

Calum MacRae, M.D., Ph.D. Chief, Cardiovascular Medicine, Brigham and Women’s Hospital Associate Professor of Medicine, Harvard Medical School Calum MacRae is a cardiologist, geneticist and developmental biologist who trained in Edinburgh, London and Boston. He is Chief of Cardiovascular Medicine at Brigham and Women’s Hospital and Associate Professor of Medicine at Harvard Medical School. He is also an Associate Member at the Broad Institute and a Principal Faculty Member at the Harvard Stem Cell Institute. His research is focused on understanding the fundamental mechanisms of disease using human studies and

complementary efforts combining systems level modeling with empiric high-throughput biology in the zebrafish. His lab uses automated screens in fish to define the genetic architecture of disease and to explore gene-drug (or environment) interactions through the interrogation of large-scale chemical libraries. His interests include heart failure, arrhythmias and coronary disease, genomic medicine, innovation in phenotyping and the redesign of clinical care.

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FEATURED SPEAKER Steve Houser, Ph.D., FAHA Senior Associate Dean, Research Vera J. Goodfriend Endowed Chair, Cardiovascular Research Chair and Professor, Physiology Director, Cardiovascular Research Center Professor, Medicine For forty years, Steven R. Houser, Ph.D. , FAHA has been a constant presence at the Lewis Katz School of Medicine at Temple University. A committed alumnus, beloved instructor, scientific-leader and an internationally-recognized expert in cardiac biology and pathophysiology,

A widely respected and prolific cardiovascular researcher, Dr. Houser first came to Temple as a Ph.D. student in physiology before earning a research fellowship in Cardiology at the School of Medicine. In 1979 he joined the faculty full time, was named director of the Cardiovascular Research Center in 2003, chair of the Department of Physiology in 2006 and Senior Associate Dean of Research in 2013 (having previously held the position from 2003-2007). In 2015 he was appointed to the Vera J. Goodfriend Endowed Chair in Cardiovascular Research.

Outside of his Temple appointments, Dr. Houser has recently served as President of the American Heart Association. During Houser’s long association with the AHA, he has also served as a fellow, board member, chair of the research committee and president of the southeastern Pennsylvania affiliate. He has also worked on a number of national AHA committees.

His research group has helped define many fundamental features of the normal cardiac myocyte (a muscle cell) and identified defective molecular and cellular processes that produce abnormal cardiac myocyte function in cardiovascular disease. In 2012, the group was awarded a five-year $11.6 million grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) to develop new approaches to prevent, slow or reverse damage to the heart after a heart attack. He is a much sought-after expert in his field, having been an invited speaker at conferences and symposiums more than 200 times, a reviewer or editor for 13 peer-review journals, served as the chair or member of numerous NIH study sections and has published more than 210 manuscripts, papers or book chapters.

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SPECIAL PRESENTATION

Mark Stoops Head Coach, University of Kentucky Football Mark Stoops is in the middle of his fifth year as the University of Kentucky head football coach. In his first season, Coach Stoops inherited a program that came off a 2-10 season, averaging 22.1 points per loss. It took just four seasons to return the program to bowl eligibility and restore a winning culture. In addition to shaping the Wildcats’ performance on the field, Stoops has been instrumental in shaping the program off the field. Coach Stoops assisted in planning and fundraising for the $125 million

renovation of Kroger Field, as well as the new $45 million football complex. These renovations and the winning culture Coach Stoops brought with him to Lexington have led to multiple top-25 recruiting classes and optimism for the future of this program.

Coach Stoops grew up surrounded by a football family and is native of Youngstown, Ohio, one of the nation’s biggest football towns. After high school, he went on to play for Hall of Fame coach Hayden Fry at the University of Iowa, playing in four bowl games and winning 25 games in three years. After graduation, Coach Stoops spent two seasons as a graduate assistant coach under Coach Fry. After the ’91 season, he spent four years coaching high school football before entering the collegiate ranks. Coach Stoops made stops at several major programs including Iowa, USF, Wyoming, Houston, Arizona, Miami and Florida State. Fast forward to 2017, and Coach Mark Stoops is in his 28th season of coaching. He has compiled a 4-1 career BCS bowl record, won a BCS National Championship, and has developed 11 NFL 1st round draft picks.

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EVENT SUPPORTS

Gill Foundation of Texas

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The Saha Fund for Cardiovascular Research

and Education

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The Estate and Family of Mrs. Hager Koostra

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Mr. and Mrs. Bob Allen

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NOTES

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2017 POSTER JUDGES Douglas Andres Jason Brandon J. Mark Brown William Brown Lisa Cassis John Charles Cherry Croft Alan Daugherty Brian Delisle Sanda Despa Joyce Evans Ming Gong Gregory Graf Kathryn Gunn Zhenheng Guo Rebecca Haeusler Steven Houser Wendy Katz Kevin Pearson Joe Layne Sangderk Lee Norbert Leitinger Irena Levitan Zhenyu Li Xiangan Li MacRae Linton Robert Lodder Analia Loria Hong Lu Calum MacRae Andrew Morris

Kiran Musunuru Saskia Neher Fredrick Onono Phillip Owens Sabire Ozcan Romeo Papazyan Edwards Park Julie Pendergast Jonathan Satin Travis Sexton Mary Sheppard Sony Soman Venkateswaran Subramanian Lisa Tannock Elizabeth Tarling Ryan Temel Joel Thompson Jenelle Timmins-Persley Michael Tranter Vincent Venditto Kasey Vickers Nancy Webb Wally Whiteheart Jeremy Wood Changcheng Zhou Lin Zhu Zhihui Zhu Lin Zhu Zhihui Zhu

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2017 POSTER PARTICIPANTS 40 Mohamed AboAly 11 Brooke Ahern 85 Katelyn Ahern 8 Ahmed Al-Darraji 74 Ryan Allen 67 Yasir Alsiraj 86 Sarah Anthony 26 Kavya Balaji 49 Meenakshi Banerjee 89 Nicolas Barbera 32 Jazmyne Barney 45 Esias Bedingar 12 Dibyajyoti Biswal 88 Yedida Bogachkov 94 Lei Cai 17 Zheying Chen 13 Dylan Colli 72 Kelsey Conrad 73 Allison Cooke 55 Robert Correll 66 Marcielle de Beer 43 Mohamed El-Helw 56 Eman Elsawalhy 70 Ibra Fancher 44 Demetria Fischesser 63 Christy Gliniak 78 Aspen Gutgsell 35 Allison Hall 1 Dwight Harris 15 Peter Hecker 97 David Henson 10 Joseph Herald 31 Jessie Hoffman 38 Amanda Hoskins 41 Tianfei Hou 81 Jiansheng Huang 75 Aida Javidan 80 Ailing Ji 82 Shannon Jones 57 Smita Joshi

54 Tara Keenan 30 Peter Kekenes-Huskey 50 Morgan Kelly 96 Seonwook Kim 77 Alison Kohan 61 Michael Kuefner 92 Lindsey Lanzillotta 18 Bryana Levitan 53 Zhenyu Li 90 Yanzhang Li 93 Tong Li 7 Miao Liu 34 Shu Liu 33 Guogen Mao 51 Eric McGann 46 Gregory Milburn 84 Shayan Mohammadmoradi 9 Mohammad Javad Mollakazemi 60 Kathryn Moore 4 Chase Neumann 5 Laura Peterson 79 Michael Petriello 22 Joshua Preston 20 Seema Rao 62 Benjamin Roberts 71 Hannah Russell 21 Keegan Sammons 39 Kaitlyn Samuels 27 Hisashi Sawada 76 Leslie Sedgeman 48 Travis Sexton 14 Mihir Shah 68 Robin Shoemaker 6 Preetha Shridas 3 Tara Shrout 65 Daniel Silver 87 Loren Smith 91 Wenliang Song

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83 Stefanka Spassieva 47 Cody Sutphin 37 Joel Thompson 36 Himi Tripathi 64 Courtney Turpin 99 Clint Upchurch 24 Patrick Van Hoose 103 Sahar VarnoosfaderanI 59 Arielle Waller 25 Fang Wang 52 Robert Wendroth 98 Carrie Wiese

29 Patricia Wilson 58 Cassandra Woolley 28 Bradley Wright 19 Congqing Wu 23 Chia-Hua Wu 42 Liping Yang 69 Zhihong Yang 95 Lihua Yang 2 Feiming Ye 16 Xiang Ye 102 Jinchao Zhang 100 Lin Zhu 101 Wanying Zhu

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2017 ABSTRACTS

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Extracorporeal Membrane Oxygenation as a Rescue Measure in Patients Experiencing Respiratory Distress After Non-cardiac Thoracic Surgery

Dwight Harris 1 • Sibu Saha, MD 2 1College of Medicine, University of Kentucky • 2Cardiothoracic Surgery, University of Kentucky

Medical Student

Extracorporeal Membrane Oxygenation (ECMO) is a rescue measure used to treat patients in every age group from neonates to adults. ECMO is used to treat many different conditions including respiratory distress, cardiogenic shock, and trauma. In these patient groups ECMO has been extensively studied. Recently, physicians have begun using ECMO as a rescue measure in patients experiencing acute respiratory distress syndrome (ARDS) after non-cardiac thoracic surgery. Studies on the use of ECMO after non-cardiac thoracic surgery are lacking with only a hand full of reports published. The goal of this report is to present our experience with patients experiencing ARDS after non-cardiac thoracic surgery. We conducted a retrospective review of all patients that had received ECMO after thoracic surgery at the University of Kentucky from January 9, 2012 to January 9, 2017. We identified 8 patients. The average time on ECMO was 9.125 days, and the average hospital stay was 65.125 days. Of the eight patients placed on ECMO three of them survived to discharge. Of the 3 that survived to discharge one died within six months and two are less than four months out to follow up. The average charge per patient was calculated to be 1,053,551 dollars, and the average charge per day for ECMO was 16,177 dollars. The contribution margin per case was $109,200. ECMO is a tool that saves lives, but ECMO does not appear to have the same yield in patients experiencing ARDS after Thoracic surgery compared to other patient groups. ECMO after non-cardiac thoracic surgery is low yield and costly compared to other patient populations.

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2 Inhibition of Megalin Reduces Atherosclerosis and Improves Hypercholesterolemia-induced Lysosomal Dysfunction in Kidney

Feiming Ye 1 • Chia-Hua Wu 1 • Deborah Howatt 1 • Adam Mullick 2 • Mark Graham 2 • Anju Balakrishnan 1 • Craig Vander Kooi 3 • Jan Danser 4 • Jian-An Wang 5 • Alan Daugherty 1 • Hong Lu 1 1Saha Cardiovascular Research Center, University of Kentucky • 2Ionis Pharmaceuticals • 3Molecular and Cellular Biochemistry, University of Kentucky • 4Erasmus MC • 5Department of Cardiology, Zhejiang University

Graduate Student

Objective: Our previous study has demonstrated that hepatocyte-specific deficiency of angiotensinogen (hepAGT-/-) reduces atherosclerosis. Megalin interacts with AGT and determines its presence in proximal convoluted tubules of kidney. In this study, we investigated effects of megalin inhibition on hypercholesterolemia-induced atherosclerosis and kidney pathologies. Methods and Results: Male LDL receptor -/- mice at the age of 8 - 10 weeks were injected subcutaneously with PBS, control antisense oligonucleotides (ASO), or megalin ASO for 13 weeks. One week after the initial injection, mice were fed a Western diet enriched with saturated fat for 12 weeks. Inhibition of megalin profoundly reduced atherosclerotic lesion area, compared to the two control groups (Percent lesion area in PBS, control ASO and megalin ASO groups: 23 ± 2, 20 ± 2, 12 ± 1 %, respectively; P < 0.001). Histological analysis of kidney sections exhibited extensive vacuolar formation in proximal convoluted tubules of kidney in mice injected with PBS or control ASO, which were absent in mice injected with megalin ASO. Immunostaining of lysosomal-associated membrane protein 1 demonstrated that these vacuoles were derived from enlarged lysosomes. Same kidney pathology was also found in hepAGT+/+ mice in an LDL receptor -/- background fed the Western diet, but not observed in hepAGT-/- mice fed this diet. Conclusions: Megalin in proximal convoluted tubules of kidney and AGT derived from hepatocytes may interact to promote hypercholesterolemia-induced atherosclerosis and renal lysosomal dysfunction.

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3 Improving Patient Care on Left Ventricular Assist Device Support: Novel Outcome Predictors & Preventative Therapies

Tara Shrout 1 • Travis Sexton, PhD 2 • Susan Smyth, MD, PhD 3 1College of Medicine, University of Kentucky • 2Saha Cardiovascular Research Center, University of Kentucky • 3Chief, Division of Cardiovascular Medicine, University of Kentucky

Medical Student

Background: Left ventricular assist device (LVAD) implantation is an increasingly common and important intervention for heart failure. Despite advancements, implantation introduces a foreign body across which blood experiences aberrant sheer stress. Clinical complications of bleeding and stroke develop secondary to an acquired coagulopathy, and patients’ quality of life is an important outcome to consider. This study uniquely correlates early platelet function and biomarkers alterations with clinical outcomes, as well as patient quality of life measures to help predict, address, and explore complications with LVAD support. Objectives: 1. Evaluate correlations between predictive measures of platelet function, biomarkers, laboratory trends with clinical complications outcomes and LVAD types. 2. Characterize the role of a novel hemostatic agent as a preventative therapy for bleeding. 3. Understand patients’ quality of life on LVAD support. Design: March 2014-August 2016, 68 enrolled patients underwent implantation of Heartmate II (n= 48), Heartmate III (n=2), Heartware (n=18). Blood collections: 24-hours preoperatively and 1-, 24-, 72-, and 168-hours postoperatively. Follow-up collections: between 30-90-days postoperatively. Platelet function analyzed via impedance aggregometry, five agonists, and novel hemostatic agent. Biomarkers of coagulation, platelet activation, and inflammation analyzed. Daily clinical data collected. Participants: Consecutive-based sample. Sociodemographics: age, gender, ethnicity, co-existing conditions collected. Eligibility: First-time LVAD implant. Exclusion: Age<18, vulnerable populations. Withdraw n=1 (convenience). Measures and Outcomes: Platelets decreased 42.0% (p<0.001) in 72-hours with baseline recovery by day 7. Platelet ristocetin aggregation decreased 52% (p=0.0006) in 24-hours without baseline recovery by day 7. Of note, Angiopoietin-2/Angiopoietin-1 was increased 273% from healthy levels at baseline (p=0.05) and sustained after implantation. Outcomes: stroke 18.2%, gastrointestinal bleeding (GIB) 19.7%, mortality 15.2%. Significant associations exist between platelet function and GIB, and biomarkers (TNF-a, CD40L, ect) and stroke, GIB, and mortality. Further data will be presented. Conclusions and Relevance: LVADs can improve quality of life for patients with heart failure, however significant complications exist. Study results suggest that early platelet function and biomarker analysis may help predict complications such as stroke and bleeding, and thus serve as risk-stratification and targeted therapy tools for patients on LVAD support. Furthermore, novel therapies present an opportunity for aberrant coagulation rescue as well as possible prevention and treatment of adverse bleeding events.

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4 Identification of a Lipid Signature in Clear Cell Renal Carcinoma (ccRCC) Suggest a Role of Phosphatidylinositol Remodeling in Disease Progression

Chase Neumann 1 • Renliang Zhang, PhD 1 • Christopher Przybicin, MD 2 • Brian Rini, MD 3 • Mark Brown, PhD 1 1Cellular and Molecular Medicine, Cleveland Clinic • 2Robert J. Tomsich Pathology and Laboratory Medicine, Cleveland Clinic • 3Hematology and Oncology Taussig Cancer Institute, Cleveland Clinic

Graduate Student

The most lethal urological cancer and third most common urological cancer in the United States is clear cell renal carcinoma (ccRCC). Approximately 64,000 individuals in the United States are diagnosed with ccRCC annually, and more than 140,000 individuals die worldwide of ccRCC every year. The “clear cell” characteristic of this deadly kidney cancer is due to abnormal deposition of lipids in a large unilocular lipid droplet in cancer cells, and urinary biomarker studies have identified lipid droplet-associated proteins as predictive markers of disease progression. However, the mechanisms by which lipid metabolism impacts ccRCC progression are not well understood. We hypothesized that a group of mitogenic lipids exist within the ccRCC microenvironment that promote proliferation and metastatic potential. A high throughput Triple Time-of-Flight (TOF) LC-MS/MS lipidomics screen of metastatic ccRCC tumor samples and matched non-tumor adjacent tissue identifies a highly reproducible lipid signature of ccRCC. In agreement with previous reports, cholesteryl esters are increased in the tumor vs non-tumor. We also found a striking increase in phosphatidylinositol (PI) 36:4 and 34:1 in tumors. Membrane bound O-acyltransferase domain containing 7 (MBOAT7) is a lysophosphatidylinositol (LPI) acyltransferase that preferentially utilizes arachidonyl-CoA and 16:0 LPI as substrates. To further investigate the formation of 36:4 PI, an MBOAT7 product, we used TCGA datasets to analyze stage-specific expression of MBOAT7. MBOAT7 expression was found to increase with staging. This potential increase in phosphatidylinositol production, coupled with decreases in known phosphatase activity like PTEN, SACM1L, and MTM1 may lead to increased activation of the PI3K/Akt signaling activity. The impact of this work may suggest mechanisms why some patients are refractory to PI3K/mTOR inhibitors, while a small percentage (<10%) respond to rapalogs. Collectively, our studies have uncovered alterations in phosphatidylinositol metabolism in ccRCC that have allowed for refined identification of new potential therapeutic target.

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5 Potential Role of Muscle Specific Ribosomal Protein L3-like in Cardiomyocyte Transverse Tubule Formation

Laura Peterson 1 • Alexander Alimov, PhD 1 • Jon Satin, PhD 1 • Brian Delisle, PhD 1 • John McCarthy, PhD 1 1Physiology, University of Kentucky

Graduate Student

Ribosomes have long been thought as serving in a constitutive housekeeping function and possess no real regulative capacity. Recently however, ribosomal proteins have been shown to provide the ribosome with a regulatory capacity across phyla. Ribosomal protein paralogs arose from a duplication event millions of years ago and have contributed to the development of ribosome specialization. By allowing a ribosome to function in its original capacity and acquire new roles, some ribosomal protein paralogs have acquired specialized roles. Ribosomal protein L3-like (RPL3-like) is expressed only in skeletal muscle and the heart but its paralog, RPL3, is found ubiquitously throughout the body. Strikingly, the expression in the heart is limited to the ventricles and is not expressed in the atria. One of the major anatomical differences in the atria and ventricle is the patterning of transverse tubules (t-tubules) with the ventricles exhibiting a highly symmetrical, regular transverse pattern and the atria having an irregular pattern with more prominent axial projections. RPL3-like knock out in the adult mouse disrupts ventricular t-tubule organization. We hypothesize that RPL3-like containing ribosomes preferentially associate with transcripts that encode for proteins involved in t-tubule formation thereby controlling morphology.

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6 Serum Amyloid A Activates the NLRP3 Inflammasome in Macrophages

Preetha Shridas, PhD 1 • Maria de Beer, PhD 2 • Nancy Webb, PhD 3 1Internal Medicine, University of Kentucky • 2Physiology, University of Kentucky • 3Molecular and Biomedical Pharmacology, University of Kentucky

Faculty

Objectives: Interleukin-1beta (IL-1β) has been implicated in many chronic diseases including atherosclerosis and type 2 diabetes. Production of bioactive IL-1β is controlled by the inflammasome, a multi-protein complex that regulates caspase-1 activity. Serum Amyloid A (SAA) is an acute-phase protein whose levels in circulation can increase more than 1000-fold during severe infection and tissue damage. SAA is more modestly elevated in conditions associated with chronic inflammation, including obesity. SAA has been identified as the first known physiological mediator capable of activating the NRLP3 inflammasome. The objective of this study is to investigate the mechanisms involved in SAA-mediated inflammasome activation in macrophages. Methods/Results: J774 macrophage-like cells and mouse bone-marrow derived macrophages (BMM) were stimulated with 5-25 µg/ml purified lipid-free mouse SAA, concentrations corresponding to those typically observed in obese individuals. The analyses of culture media and cell lysates demonstrated that SAA dose-dependently induced both caspase-1 activation and IL-1β secretion. The ability of SAA to induce IL-1β secretion was significantly reduced in BMM deficient in NRLP3. There was significant suppression in IL-1β secretion by SAA when J774 cells were treated with SAA in the presence of caspase-1inhibitor, Z-YVAD-FMK. A P2X7-receptor antagonist, AA38079, did not have any effect on SAA-mediated IL-1β production. Inhibition of reactive oxygen species (ROS) and cathepsin-B activation by N-acetyl-L-cysteine and CA-074, respectively, inhibited inflammasome activation by SAA. Inhibiting cellular potassium efflux by glyburide also significantly reduced SAA-mediated IL-1β secretion. Pre-incubating SAA with HDL prior to cell treatments completely inhibited its ability to trigger inflammasome activation. HDL also abrogated SAA-mediated ROS generation in J774 cells. Conclusions: SAA-mediated NRLP3 inflammasome activation in macrophages is dependent on ROS generation, release of cathepsin-B, and potassium efflux, and is independent of the P2X7 receptor. Ongoing studies are investigating the cellular receptor(s) involved and the mechanism by which lipoproteins mask SAA’s effects. Blocking SAA-mediated inflammasome activation may ameliorate increased risk for type 2 diabetes and atherosclerosis in obese individuals.

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Diabetic Hfpef in a Rat Model of Systemic Amylin Dyshomeostasis

Miao Liu, PhD 1 • Nirmal Verma, PhD 1 • Analia Loria, PhD 1 • Sanda Despa, PhD 1 • Florin Despa, PhD 1 1Pharmacology and Nutritional Sciences, University of Kentucky

Postdoc

Diabetic heart failure with preserved ejection fraction (HFpEF) is a complex disease characterized by diastolic dysfunction, altered insulin sensitivity and multiple organ impairments. Amylin, a β-cell hormone co-secreted with insulin, participates in normal glucose regulation, but is also known to induce insulin resistance and to form pancreatic amyloid when oversecreted (hyperamylinemia). Accumulating data from several laboratories have confirmed that, in addition to pancreatic islets, the hearts, kidneys and brains of patients with type-2 diabetes contain also abnormally increased levels of aggregated amylin. Our overall hypothesis is that the interplay between insulin resistance and hyperamylinemia results in toxic accumulation of aggregated amylin in the microvasculature that adversely affects function of multiple organs, including, but not limited to, the heart. Here, we used rats overexpressing human amylin in the pancreas (HIP rats) to test whether a “human” hyperamylinemia predisposes to HFpEF. Wild-type (WT) littermates expressing non-amyloidogenic rat amylin served as control. Hearts and kidneys of HIP rats showed amylin deposition in capillaries, intravascular macrophage accumulation, microhemorrhages and loss of vascular endothelial cell coverage and tight junctions. These changes were associated with diastolic dysfunction, cardiac hypertrophy and mid-range cardiac ejection fraction (~50%). HIP rats also had increased blood pressure and renal dysfunction, including diuresis, natriuresis, creatinine clearance and microalbuminuria. Abundant amylin deposition was detected in HIP rat red blood cells (RBCs). Amylin-loaded RBCs have altered deformability and increased adherence to cultured endothelial cells. Intravenous infusion of RBCs from HIP rats in WT rats resulted in release of amylin in plasma and capillaries, attachment of RBCs to the vascular endothelium, intravascular macrophage accumulation and microhemorrhages. In conclusion, hyperamylinemia provokes systemic amylin dyshomeostasis which negatively affects cardiac and renal function via amylin-mediated microvascular injury. Circulating level of amylin may serve as a biomarker of diabetic HFpEF and therapeutic target to reduce the development/progression of HFpEF.

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8 Azithromycin Therapy Reduces Cardiac Inflammation and Mitigates Adverse Cardiac Remodeling after Myocardial Infarction: Potential Therapeutic Targets

Ahmed Al-Darraji, PharmD 1 • Dalia Haydar 2 • Lakshman Chelvarajan, PhD 3 • Himi Tripathi, PhD 3 • Bryana Levitan 4 • Shaojing Ye, PhD 3 • Vincent Venditto, PhD 5 • John Gensel, PhD 4 • David Feola, PhD 5 • Ahmed Abdel-Latif, MD, PhD 6 1Pharmacology and Nutritional Sciences, University of Kentucky • 2Pharmacy College, University of Kentucky • 3SAHA cardiovascular center, University of Kentucky • 4Physiology, University of Kentucky • 5College of Pharmacy, University of Kentucky • 6Cardiology, University of Kentucky

Graduate Student

Abstract Introduction and Hypothesis: Acute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality. Macrophages are fundamental components of post-AMI inflammation. Pro-inflammatory macrophages (M1-like) can lead to adverse cardiac remodeling and heart failure while regulatory/reparative macrophages (M2-like) enhance tissue healing. Shifting the balance between M1 and M2 macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly used macrolide antibiotic, polarizes macrophages towards an M2-like phenotype in animal and human studies. We hypothesized that using AZM can decrease adverse cardiac remodeling and improve heart function following MI. Methods and results: Male mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or vehicle control starting 3 days prior to MI and continued to day 7 post-MI. We observed significant reduction in mortality with AZM therapy. AZM-treated mice showed reduction in M1-like (CD45+/Ly6G-/F4-80+/CD86+) and increase in M2-like (CD45+/Ly6G-/F4-80+/CD206+) macrophages leading to significant reduction in the M1/M2 ratio in the heart and peripheral blood as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated with significant reduction in pro-inflammatory and increase in anti-inflammatory cytokine production as assessed by real-time PCR. AZM treatment was associated with increased neutrophil apoptosis, a known signal for shifting macrophages towards an M2-like phenotype. Finally, AZM treatment was associated with enhanced cardiac recovery, smaller scar size and enhanced angiogenesis. Conclusion: Azithromycin plays a cardioprotective role post-MI through attenuating inflammation and enhancing cardiac recovery. Long term and human translational studies are planned to examine the therapeutic applications of AZM.

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9 Cerebro-cardiac and Cerebro-respiratory Interactions while Listening to Songs

Mohammad Javad Mollakazemi 1 • Dibyajyoti Biswal 1 • Sridevi Thyagarajan 1 • Joyce Evans 1 • Abhijit Patwardhan, PhD 1 1Biomedical Engineering, University of Kentucky

Graduate Student

Introduction: Since ancient times listening to music has been thought to affect mental as well as physical wellbeing. Previous studies have shown that autonomic responses change while listening to songs, but the interactions between respiratory, cardiovascular and cerebral rhythms while listening to songs are not as well understood. In this study, we investigated relations between cerebral, cardiac and respiratory rhythms while subjects listened to a variety of songs. Materials and Methods: All study procedures were approved by the Institutional Review Board at the University of Kentucky. Data were collected from 8 adult subjects (4 males and 4 females). A pair of circumaural headphones was used to listen to songs. A short audio acuity test was administered to each subject to rule out overt hearing loss. The following signals were recorded during the study: 6 channels of EEG (F3, T3, P3, F4, T4, P4), ECG (lead II, Spacelabs), Respiration (abdominal and thoracic, AMI Inductotrace) and non-invasive continuous finger blood pressure (Finapres). Some results from cardio-respiratory variables are reported in a companion abstract. All signals were digitized online at a rate of 1000 samples/second. In order to determine the contribution of cognition and memory recall in the evoked responses, we scrambled local phase spectra for a song of the subject’s choosing while preserving the magnitude spectra. In addition, subjects listened to two songs, one with a slow and the other with a fast tempo. Data were collected during 10 minutes of control at the beginning of the study when the subjects sat quietly in the chair without listening to any music. To find interactions between cerebro-cardiac and cerebro-respiratory rhythms, frequency spectra, magnitude squared coherencies and transfer functions were computed for all possible combinations of EEGs and cardiovascular variable (RR intervals, computed from the ECG) and between EEGs and respiratory time series. Welch’s method with 100 sec segments, 50% overlap and a Hanning window were used for these computations. Results and Discussion: Coherence, integrated over dc-0.5 Hz (the Nyquist frequency used for cardiac and respiratory variables) between RR intervals and most channels of EEG increased during listening to songs relative to control. An increase was seen regardless of the type of song that was listened to. A similar increase in integrated coherence was also observed between respiratory oscillations and EEGs, again, regardless of type of the song. Comparing slow and fast tempo songs: the fast tempo song increased the coherence between RR intervals and EEGs and between respiratory oscillations and EEGs more than the slow tempo song. When subjects listened to the song of their choosing, the average coherence between cerebro-cardiac and cerebro-respiratory rhythms was higher than control, the phase randomized version of the same song had a somewhat lower coherence but was still higher than control. However, differences in integrated cerebro-cardiac and cerebro-respiratory coherencies between the song of choice and the phase randomized version of it were not consistent. The autospectra of the envelopes of all channels of the EEG showed an overall decrease in spectral power within the 0-5 Hz range while listening to songs. Conclusions: Although we had anticipated that the differences between the responses for the song of choice and its phase scrambled version would be small, our results do not show a consistent difference. Many subjects could identify the phase scrambled song as being the same or similar to the one of their choosing, which may explain this finding. The increase in integrated coherencies between respiration and EEGs is indicative of entrainment of respiration to the music. The increase in coherencies between EEGs and RR intervals, during listening to songs, also indicates entrainment of RR intervals with music; however, whether this entrainment happens in parallel with entrainment with respiration or it is sequential remains unknown. Collectively, these results show that listening to songs increases coherent entrainment between cerebro-cardio and cerebro -respiratory variables.

Acknowledgement: Supported by a grant from the National Science Foundation (EPSCoR RII Track-2).

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10 Evidence of Angiotensin II-dependent Obesity-induced Hypertension in Female Mice Exposed to Postnatal Neglect

Joseph Herald 1 • Jacqueline Leachman 1 • Analia Loria, PhD 1 1Pharmacology and Nutritional Sciences, University of Kentucky

Undergraduate

Previously, we have shown that female mice subjected to maternal separation with early weaning (MSEW), a model of postnatal neglect, display exacerbated diet-induced obesity and high blood pressure (BP) compared with control mice. Female MSEW mice show activated renin-angiotensin system components, including increased plasma renin activity and adipose tissue-derived angiotensinogen secretion. The goal of this study was to test whether augmented obesity-induced hypertension in female MSEW mice is AngII-dependent. Mouse MSEW was achieved by repeated, daily separations from the dam and 4-day early weaning. Normally reared controls (C) were weaned at postnatal day 21. Each experimental group of female weanlings was comprised of 6 mice each and derived from 3 different litters, that were placed on high fat diet (HFD, 60% kcal from fat). After 18 weeks, mice were implanted with radiotelemetry devices for BP measurement. At week 20, average 24-hr systolic blood pressure (SBP) was 134±2 mmHg in MSEW mice and 126±2 in C (P<0.05). No significant changes in mean arterial pressure, diastolic blood pressure or heart rate were observed between groups. We also determined the BP sensitivity to the acute administration of AngII (1, 10 and 50 ug/kg, s.c.). AngII-induced BP changes, assessed by BP area under the curve, were similar between MSEW and C mice at all doses (50 ug/kg dose:145±10 vs. 132±15 mmHgx30 min, respectively). Chronic enalapril treatment (2.5 mg/kg/day, drinking water, 7 days) was conducted to block endogenous AngII synthesis. Enalapril reduced SBP 15±2 mmHg in MSEW mice but only 6±1 mmHg in C mice (p<0.05). The BP response to acute AngII doses increased similarly in MSEW and C enalapril-treated mice, (50 ug/kg dose: 200±13 vs. 207±22 mmHgx30 min, respectively). In addition, BP and HR responses to acute injections (i.p) of mecamylamine (5 mg/kg), propranolol (5 mg/kg) or atropine (1 mg/kg) were similar between untreated MSEW and C mice, suggesting that exacerbated BP in female MSEW mice is independent of sympathetic or parasympathetic dysfunction. Taken together, these data provide evidence that increased BP in female MSEW mice results from elevated circulating AngII rather than enhanced AngII sensitivity or sympathetic nerve activity.

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11 Cardiac Specific Rad Deletion Enhances Cardiac Function through Safe, Stable Positive Inotropic Support

Brooke Ahern 1 • Bryana Levitan 2 • Mihir Shah 1 • Andrea Sebastian 1 • Zach Bessinger 3 • Radu Paul Mihail 3 • Landon Simpson 4 • Nathan Jacobs, PhD 3 • Douglas Andres, PhD 4 • Jonathan Satin, PhD 1 1Physiology, University of Kentucky • 2Gill Heart and Vascular Institute, University of Kentucky • 3Computer Science, University of Kentucky • 4Biochemistry, University of Kentucky

Graduate Student

Background: The cardiac L-type Calcium Channel (LTCC) is a heteromultimeric protein complex. Rad interacts with the main pore forming subunit (CaV1.2) and auxiliary subunit CaVβ2. Rad modifies LTCC function and Rad protein levels are reduced by pathological stimuli. Global, constitutive deletion of Rad results in an increase in calcium handling and improved ejection fraction without progression to heart failure. Goals: It is unknown if the effects of global Rad deletion are also exhibited in cardiac-specific Rad deletion, and if the effects are preserved long-term. Our hypothesis is that cardiac-specific Rad ablation causes positive inotropic effects that are maintained over time. Methods and Results: We crossed Radfl/fl onto αMHC-MerCreMer mice to create a cardiac-restricted inducible Rad knockout (cRadKO). Mice were induced for gene deletion at ~3 months of age. We assessed cardiac structure and function through echocardiography from 7 days through 9 months after cRadKO induction. Ejection fraction improved in females in comparison to wild type 7 days post knock out (762% vs. 594%; cRadKO n=16 vs. WT n=7, respectively; p=0.001). Improved function was stable for 9 months in females (774% vs. 587%;cRadKO vs. WT, respectively; p=0.01). Similar results were found in males. The key mechanism for improved function includes elevated trigger Ca2+ in cardiomyocytes measured as a Ca2+-transient departure velocity (48.72.4 units/s vs. 38.13.4 units/s; cRadKO n=8 vs. WT n=10, respectively; p=.014). Cellular Ca2+ synchrony was preserved in cRadKO despite chronic elevated Ca2+ homeostasis. Conclusion: Cardiomyocyte Rad deletion preserves increased ejection fraction over time through a mechanism including increased calcium handling resulting in safe, stable positive inotropic support for heart function.

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12 Auditory Entrainment of Respiration and RR Intervals

Dibyajyoti Biswal, PhD 1 • Javad Mohammad Mollakazemi, PhD 1 • Sridevi Thyagarajan, PhD 1 • Joyce Evans, PhD 1 • Abhijit Patwardhan, PhD 1 1Biomedical Engineering, University of Kentucky

Graduate Student

Introduction:- It is known that music has palliative effect in a number of patients. Listening to music is also known to evoke autonomically regulated cardiovascular and respiratory responses. In adults, these responses are a combination of sensory input and cognition. While it is known that listening to music evokes measurable responses in cardiovascular and respiratory variables, much is unknown in terms of causal links between these responses and the mechanisms by which they get entrained. Our overall objective is to determine the causal links between auditory stimuli, respiration and cardiovascular regulatory variables. Materials and Methods:- The study was approved by the Institutional Review Board at the University of Kentucky. Subjects gave informed consent. Subjects (n=8, equal numbers of males and females, age 18 to 35) sat in a comfortable chair and listened to songs through a pair of circumaural headphones. An internet based audio acuity test was administered to each subject to rule out overt hearing loss. The following measurements were made: non-invasive continuous finger blood pressure (Portapres, Finapres), ECG (lead II, Spacelabs), Respiration (abdominal and thoracic, AMI Inductotrace) and EEG. Results of analyses using EEG is reported elsewhere. All signals were digitized on-line at a rate of 1000 samples/second. To align the physiological data with the music, a square wave pulse was inserted at the beginning of the songs. For each study, the subject was asked to provide a song of their choice which in their opinion “moves” them. Data were collected during 5 trials in each study: control (10 minutes), songs of slow and fast tempo, the song of the subject’s choice, and a phase scrambled version of the song of the subjects’ choice. Each song was 3-4 minutes long. To minimize cognitive response, we scrambled the phase of the song of the subject’s chosen song by segmenting the songs and scrambling the phase spectrum of that segment while preserving the magnitude spectrum. From the recorded data, time series of RR intervals were generated. These time series and the respiratory signals were low pass filtered (cut off 5Hz) and subsampled at a rate of 10 samples/second. From the subsampled data auto-spectra of RR intervals and respiration, coherencies and transfer functions between these signals were computed using Welch’s method. Results and Discussion:- The most pronounced effect during listening to songs was in respiration. In all but one subject, the mean respiratory rate increased relative to control while listening to songs. In addition to an increase in mean rate, the power distribution in the autospectra of respiration markedly narrowed during auditory stimulation. Similar changes were seen in RR interval spectra, the so called HF power, i.e. respiratory region power, was more narrowly distributed while listening to songs. Coherence between RR intervals and respiration exhibited a broad band increase during listening to songs as compared with control. Although there was a slight tendency during phase scrambled song for respiratory spectra to move closer to those during control, these differences were not very robust. Conclusion:- Our results show that auditory stimulation does entrain respiration as well as RR interval. Taken together, these results suggest that RR intervals were entrained by respiration while listening to both slow and fast tempo songs. The increase in average coherencies suggests entrainment of respiration to auditory stimulation which in turn entrains RR intervals. Further analyses using phase synchronization indexes would be useful to better reveal the causal links in these entrainment processes. Supported by a grant from the National Science Foundation (EPSCoR RII Track-2).

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13 An Unbiased Classification Algorithm for Transverse Tubule Remodeling Within Murine Heart Failure Models.

Dylan Colli 1 • Bradley Stewart 2 • Peter Kekenes-Huskey, PhD 2 1Chemical and Materials Engineering, University of Kentucky • 2Chemistry, University of Kentucky

Undergraduate

Transverse tubules (TTs) are the main method of delivery of extracellular calcium into the myocyte, responsible for the initiation of calcium induced calcium release (CICR) necessary to carry out excitation contraction coupling (EC coupling) within ventricular myocytes. Disruptions in the regularly striated TT network are correlated with various degrees of calcium mishandling and are generally observed within heart failure models. Currently, TT remodeling is often judged qualitatively, subject to bias of the experimenter. To address the need to eliminate this bias, we propose a technique that utilizes: 1. Computer vision libraries. 2. Signal processing techniques such as matched filtering that provide signal-to-noise ratios, a quantitative measure of observed remodeling. Combining these methods, an unbiased classification algorithm is presented that is able to quantify both regions of TT loss and longitudinal remodeling as well as characterize whole myocytes by the magnitude of their deviation from the prototypical healthy myocyte. This allows for the expedient, and more importantly, unbiased and reliable classification of TT remodeling observed within murine ventricular myocytes.

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14 Cardiac specific Rad knockout Increases Calcium Release

Mihir Shah 1 • Andrea Sebastian 1 • Landon Simpson 2 • Doug Andres, PhD 2 • Jonathan Satin, PhD 1 • Brooke Ahern 1 1Physiology, University of Kentucky • 2Biochemistry, University of Kentucky

Undergraduate

Background: The L-type calcium channel complex (LTCC) associates with regulatory proteins including Rad that govern calcium channel function. Overexpression of Rad inhibits LTCC inward calcium current. In contrast, whole body constitutive Rad knockout in mice have higher calcium influx in cardiac myocytes. It is unknown whether the effect of constitutive whole body Rad knockout arise from development, from the influence of non-cardiac myocytes, or is a primary result of Rad reduction in cardiac myocytes. Our recently engineered inducible, cardiac myocyte restricted Rad-knockout mouse model allows us to address these possibilities. Hypothesis: Cardiac specific Rad knockout will recapitulate the same effects of global Rad knockout. Acute induction (1-4 weeks) of Rad knock out produces an immediate effect on cytosolic calcium entry. Methods: Cytosolic Ca2+ in single, live cells were analyzed using Fura-2. Cells were paced at 1 Hz. Twitch calcium, calcium reuptake, and release kinetics were measured before and after the addition of isoproterenol in both wild-type (Radfl/fl abbreviated as WT) and induced, Rad-/- cardiomyocytes (cRadKO). Results: Twitch calcium levels were significantly higher in cRadKO compared to WT (1.99±0.09 and 1.54±0.13 FU for WT and RadKO, respectively, p=0.008). cRadKO and WT twitch calcium significantly increased in response to acute isoproterenol (ISO; 2.63±0.13 and was 1.98±0.20 FU for WT and RadKO, respectively, p=0.0082). Calcium reuptake also increased with the knockout of Rad and in the presence of ISO. The onset velocity of calcium release increased in cRadKO (38.13±3.46 FU/ms and 48.71±2.38 FU/ms, for WT and cRadKO, respectively, p=0.01). The ISO response for onset velocity was retained with cRadKO, in parallel with amplitude effects. Conclusion: Reduction of Rad protein elevated baseline cytosolic calcium levels approaching that of ISO-stimulated WT cardiomyocytes. Thus, Rad knockout provides positive inotropic support to heart function by elevating baseline cytosolic Ca2+. cRadKO maintains modulated myocardial function to sympathetic activity.

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15 Atherosclerosis in the Cerebrovasculature of Nonhuman Primates Promotes Reactive Gliosis: A Potential Model for Vascular Dementia

Peter Hecker 1 • Lei Cai, PhD 1 • Elizabeth Head, PhD 2 • Donna M. Wilcock, PhD 3 • Ryan Temel, PhD 1 1Saha Cardiovascular Research Center and Department of Pharmacology and Nutritional Sciences, University of Kentucky • 2Sanders-Brown Center of Aging and Department of Pharmacology and Nutritional Sciences, University of Kentucky • 3Sanders-Brown Center of Aging and Department of Physiology, University of Kentucky

Graduate Student

Age- and lifestyle-dependent vascular insults drive and/or exacerbate cardio- and cerebro- vascular disease. While several animal models exist for studying vascular contributions to cardiovascular disease there are few models for studying the vascular contributions to cognitive impairment and dementia (VCID). Our lab is studying atherosclerosis progression and regression in male cynomolgus monkeys fed a high-fat/high-cholesterol diet. After 20 months on the atherogenic diet, a subset of monkeys are switched to standard nonhuman primate “chow” diet and are concomitantly treated with vehicle or a microRNA-33 antagonist (anti-miR-33). We hypothesize that anti-miR-33 treatment will create more stable atherosclerotic plaques by increasing cholesterol efflux from foam cells and dampening inflammation in the artery wall. While our initial interest was atherosclerosis in the coronary arteries, we have recently turned our attention to atherosclerosis in the main arterial network that supplies the brain. Evaluation of the circle of Willis (COW) revealed the presence of atherosclerotic plaques in major branch points of the large arteries. Immunohistochemistry (IHC) on arterial sections has begun to evaluate the size and molecular/cellular composition of these atherosclerotic plaques. Understanding that atherosclerosis could reduce cerebral blood flow (CBF) and/or promote endothelial dysfunction, we have started to examine brain sections from our monkeys for underlying neuropathology. Preliminary IHC data shows increased microglia and astrocyte reactivity around small cerebral vessels from a monkey with atherosclerosis versus a healthy, control animal. The marked increase in glial reactivity around small vessels may suggest extravasation or decreased CBF, both capable of driving neuroinflammation and downstream neurodegeneration. In the near future, we plan to pair MRI analysis of hypoperfusion and white matter lesions (WML) with histology and IHC assessments of WML, cerebral amyloid angiopathy, and astrocyte endfeet engagement with the vasculature. Although much work remains to be done, we are optimistic that we have created a unique and important translational model that mimics human cerebrovascular atherosclerosis and neuropathology associated with VCID.

32

16 Hypercholesterolemia Induces Acute Thymic Atrophy Through Blocking Thymocyte Differentiation and Expansion

Xiang Ye, PhD 1 • XiangAn Li, PhD 1 1Saha Cardiovascular Research Center, University of Kentucky

Postdoc

Background: Thymus is the primary site for T cell development, however its function is decreasing with age, defined as thymic involution. The age-related thymic atrophy reduces the ability to produce naïve T cells, which is one of the main factors contributes to lower immune-surveillance in the elderly. Other than aging, thymic atrophy also can be accelerated by various stressors, like chemotherapy, corticosteroids, disturbed lipid metabolism, infection, inflammation and psychological stress. However, our knowledge on thymic atrophy is very limited. Here we report that hypercholesterolemia induces acute thymic atrophy through inhibiting thymocyte expansion and differentiation. Methods and Results: To investigate the role of hypercholesterolemia, we use albumin driven cre to specifically knock out SRBI in the liver, causing defect in reverse cholesterol transport and thus hypercholesterolemia. One week of HCD induces acute thymic atrophy (thymus weight: SRBIflox/flox vs SRBIflox/flox-Alb-Cre, 28.22mg vs 9.28mg, p < 0.0001) in RCT defected mice, and a more dramatic decrease of thymocyte cellularity (5.9 X 10E7 vs 4.5 X 10E6). In order to know what happened with T cell development, we profiled development process using flowcytometry with CD4, CD8a, CD44 and CD25. We found that CD4+CD8a+ cell decreases to be only 18% of thymocyte from more than 80%, which indicates blocks in thymocyte differentiation. Besides, we also see an increase of DN1 (CD4-CD8a-CD25-CD44+) progenitor cells increased from 17% to 38% of double negatives (CD4-CD8a-). To exclude the possibility of accelerated cell apoptosis of certain cell type rather than ceased differentiation, we measured thymocyte apoptosis using annexin V staining. The results only show a very mild increase in cell apoptosis (annexin V+: 8% vs 11%) after 3 days of HCD treatment, which could not explain the dramatic change in thymocytes numbers alone. Then we performed gene expression profile on the thymus from mice after 3 days of HCD. Gene enrichment analysis using the differentially expressed genes reveals an enrichment of cell cycle genes, with many cell cycle-promoting genes decreased and cell cycle inhibiting genes increased. Consistently, BrdU incorporation combined with 7-AAD staining also indicates impaired thymocyte proliferation, the thymocytes reside in G2-M phase decrease from 2.8% to 1.7% of total thymocytes. The earlier developing cells are most impacted, with G2-M cells decrease from 5.3% to 1.4% of DN. Conclusions: Hypercholesterolemia induces acute thymic atrophy through inhibiting thymocyte expansion and differentiation. This study indicates high cholesterol diet could exert its impact on immune system through accelerating thymic atrophy.

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17 Cardiac Cycle Affects Ultrasound Measurements of Ascending Aortic Diameter in a Marfan Mouse Model

Zheying Chen 1 • Hisashi Sawada, MD, PhD 1 • Debra Rateri 1 • Alan Daugherty 1 • Mary Sheppard, MD 1 1Saha Cardiovascular Research Center, University of Kentucky

Graduate Student

Objective: Ultrasound measurements of aortic diameter are a common endpoint in preclinical studies. However, there is a lack of standardization in both image capture and analysis. For our study, we developed a standardized protocol for measuring ascending aortic diameter and examined effects of cardiac cycle in wild type and fibrillin-1 hypomorphic (FBN1mgR/mgR) mice. Methods and Results: Twelve week old male and female FBN1mgR/mgR mice were anesthetized and maintained at a heart rate of 450-550 beats per minute. Ultrasound images were captured using a Vevo 2100 system with a 40MHz tranducer. Images captured were standardized according to two anatomical landmarks: the innominate artery branchpoint and aortic valves. The largest luminal ascending aortic diameter between the sinotubular junction and the innominate artery were measured in mid-systole and end-diastole by two blinded, independent observers. Aortic diameters were significantly different (p<0.05) when comparing systole and diastole within gender and genotype. Interestingly, wild-type male (n=4) and female (n=3) mice exhibited a 19% and 15% expansion of the ascending aorta respectively during systole compared to diastole. This difference was not recapitulated in either male (n=6) or female (n=5) FBN1mgR/mgR mice (4% expansion in both; p<0.05 vs wild-type). Agreement between observers was excellent (R^2 = 0.99) but interobserver variability was a mean of .09 mm (%CV = 5%). Conclusion: As expected, there is a difference in aortic diameters between wild-type and FBN1mgR/mgR mice. Luminal aortic diameters in FBN1mgR/mgR vs wild-type mice of both genders are affected by cardiac cycle. Mid-systolic aortic expansion in wild-type vs FBNmgR/mgR mice were different. Error introduced by interobserver variability impacts ascending aortic measurements. Altogether, these phenomena may confound analyses of aortic dilation in FBN1mgR/mgR mice, especially when studying interventions with modest effect sizes.

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18 A Novel Approach for Modifying Intrinsic Heart Rate: RRad knock-down

Bryana Levitan 1 • Mihir Shah 2 • Joshua Rutland, MD 1 • Brooke Ahern 3 • Douglas Andres, PhD 4 • Jonathan Satin, PhD 3 1Cardiology, University of Kentucky • 2University of Kentucky • 3Physiology, University of Kentucky • 4Molecular and Cellular Biochemistry, University of Kentucky

Staff

Introduction: About 3 million people worldwide are dependent on pacemakers. Each year 600,000 pacemakers are implanted worldwide, for the primary indication of symptomatic bradycardia. Biological pacemakers represent an emerging technology for bradyarrhythmias that require a pacemaker such as sinoatrial nodal failure due to congenital disease or aging. Leading edge biological pacemaker approaches include implantation of induced, pluripotent stem cell-derived cardiomyocytes (iPSC-CMs); however, intrinsic rates of implanted cells might be insufficient to meet physiologic demands, and cell implantation carries a variety of obstacles. In the present report, we introduce a novel molecular mechanism for increasing heart rate. Rad is a monomeric G-protein and constituent of the L-type calcium channel macromolecular complex (LTCC). Rad inhibits LTCC inward calcium current. LTCC activity contributes to pacemaker depolarization. Therefore, we tested the hypothesis that reduction of cardiac Rad will result in a stable increased heart rate (HR). We suggest that knockdown of cardiac Rad could facilitate the development of rate support alone or in combination with cells as a biologic pacemaker. Methods & Experimental Design: Cardiac-restricted, inducible Rad knockout (cRadKO) female mice were implanted with radio-telemeters (model F10A, DSI, Minneapolis MN). Baseline recordings (continuous 72h) were followed by induction of Rad knockout (RadKO). Continuous recordings followed induction of RadKO at times specified in results. Intrinsic heart rate was measured after complete autonomic blockade with injection of propranolol (1 mg/kg) and atropine (1 mg/kg). Peak heart rate was achieved by injection of isoproterenol (ISO) at diurnal peak. Startle response was performed using a series of air jet challenges to provoke an increase in native adrenergic stimulation. Results: Baseline measures demonstrated normal diurnal rhythms of HR (daytime trough, night-time peak) without evidence of significant arrhythmias. Trough HR increased without evidence of significant arrhythmias 4 days post cRadKO, while peak HR remained unchanged. The increase in trough HR was sustained 1 month after cRadKO. During trough periods episodes of sinus bradycardia without SA node or AV node block were noted consistent with high vagal tone. Intrinsic HR was also significantly increased post-cRadKO over baseline. ISO challenge induced sinus tachycardia with a blunted response as compared to controls. During recovery 1-8 hours after ISO challenge both cRadKO and control mice exhibited transient episodes of bradyarrhythmias. All animals returned to pre-ISO rhythm without incident. The startle response did not elicit arrhythmias. Conclusion: Cardiac Rad deletion results in a stable increase in intrinsic heart rate with retained physiologic autonomic responsiveness. Rad depletion did not induce any significant arrhythmias under physiological stressors. We conclude that inhibition of Rad can be used to increase the intrinsic heart rate. This could represent a novel mechanism for tuning iPSC-CMs to more physiologic heart rates.

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19 Inflammasome Activation Triggers Blood Coagulation through Pyroptosis

Congqing Wu, PhD 1 1CVRC, College of Medicine

Postdoc

Disseminated intravascular coagulation (DIC) is a fatal complication of systemic bacterial infection. Mechanisms for blood coagulation induced by bacterial infection are often attributed to host inflammatory response to bacterial virulence factors, yet the molecular events linking bacteria sensing to initiation of the coagulation cascade remain unclear. Here we identify inflammasome activation as a trigger for coagulation induced by gram-negative bacterial products. Specifically, canonical inflammasome activation elicited by the conserved type III secretion system (T3SS) rod protein EprJ from E. coli induces systemic coagulation through caspase-1 activation, whereas noncanonical inflammasome activation by lipopolysaccharide (LPS), a major outer membrane component of gram-negative bacteria, produces similar effects involving caspase-11. Deficiency of gasdermin D (Gsdmd) or depletion of monocytes and macrophages blocks inflammasome-mediated coagulation. Together, our data unveil inflammasome-dependent pyroptosis leads to blood coagulation.

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20 Clinical Use of the Amplatzer Septal Occluder in Percutaneous Closure: A Single-Center Experience

Seema Rao, MS 1 • Tyler Scaff 1 • John Gurley, MD 2 • Sibu Saha, MD 3 1College of Medicine, University of Kentucky • 2Cardiology, University of Kentucky • 3Cardiothoracic Surgery, University of Kentucky

Medical Student

Device therapy is a viable alternative to open heart surgery in the management of intracardiac defects. The Amplatzer Septal Occluder (ASO) is one such device that has been adapted to close a wide variety of cardiac defects with few complications and a high success rate. This is a retrospective review of all the patients who received the ASO between 2012-2016 at our institution. In total, 169 patients underwent percutaneous closure of a septal defect with Amplatzer during the timeframe studied, and of those, 91% received the device for an atrial septal defect or patent foramen ovale. All Amplatzer devices achieved successful closure without requiring a second procedure. Four patients sustained complications of the procedure, with two experiencing tamponade, one with hematoma, and one with cellulitis. Three fatalities occurred within thirty days and were attributed to comorbidities not related to the device. The ASO has performed very well at our institution and we expect it to serve additional functions as the field of transcatheter cardiology develops.

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21 Exposure to PCB126 during the Nursing Period Significantly Impairs Early-Life Glucose Tolerance

Keegan Sammons 1 • Sara Tenlep, MS 1 • Leryn Reynolds, PhD 1 • Hollie Swanson, PhD 1 • Kevin Pearson, PhD 1 1Pharmacology and Nutritional Sciences, University of Kentucky

Undergraduate

Polychlorinated Biphenyls (PCBs) are persistent environmental organic pollutants that are known to have detrimental health effects. In a mouse model in our lab, PCB126 exposure during pregnancy and nursing alters offspring body composition and glucose tolerance. The purpose of this study was to expose dams to PCB126 during the nursing period only. Female ICR mice were bred and half of the dams were exposed to either vehicle (safflower oil) or 1 µmole PCB126 per kg of body weight via oral gavage on postnatal days 3, 10, and 17 (n = 9/group). Offspring body weight, lean and fat mass, and glucose tolerance were measured. Both male and female offspring displayed normal body weights as well as body composition (p > 0.05). However, both male and female offspring that were exposed to PCBs during the nursing period had significantly impaired glucose tolerance at 3 weeks of age (p < 0.05). This persisted until 9 weeks of age in the female offspring (p < 0.05), but the difference disappeared as the male offspring aged (p > 0.05). Our earlier work suggests that in utero and postnatal PCB126 exposure predisposes offspring to having lower lean mass and impaired glucose tolerance later in life. However, our current study shows that exposure to PCB126 through the mother's milk impairs glucose tolerance in the short-term and is likely caused by impairments in insulin receptor signaling in the periphery as others have shown with direct PCB exposures in adult mice. Future experiments will investigate the mechanisms of dysfunction caused by in utero PCB126 exposure, which may be driving the increased risk of obesity and insulin resistance in adult offspring.

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22 Regulation of Akt Signaling by NQO1

Joshua Preston 1 • Andrea Di Francesco, PhD 2 • Krystle Kalafut 2 • Tyler Rhinesmith 2 • Clara Di Germanio, PhD 2 • Michel Bernier, PhD 2 • Rafael de Cabo, PhD 2 1Pharmacology and Nutritional Sciences, University of Kentucky • 2Translational Gerontology Branch, National Institute on Aging, National Institutes of Health

Undergraduate

Background: NQO1 is an inducible quinone reductase that participates in the cellular defense system in response to chemical and oxidative stress. By using NAD(P)H as electron donor, NQO1 enzymatic activity shifts the cellular redox state leading to a significant increase in the NAD+/NADH ratio. The propensity of NQO1 knockout mice to exhibit a diabetes-like phenotype and a null NQO1 polymorphism associated with metabolic syndrome phenotypes in humans suggests a role for this enzyme in cellular energetics and metabolism. Here, we explored a possible link between NQO1, an intracellular generator of NAD+, and nutrient-sensitive NAD+-dependent deacetylase activity in the regulation of Akt, which plays a central role in regulating cellular signaling and energy pathways critically involved in type 2 diabetes and associated metabolic disorders. Results: Pharmacological inhibition of NQO1 with the mechanism-based inhibitor Mac609 increased insulin-stimulated Akt phosphorylation in HepG2 and HeLa cell lines. Consistent with increased Akt activation, Mac609 promoted nuclear exclusion and inactivation of the downstream Akt targets, the gluconeogenic transcription factors forkhead box O1 (FOXO1) and O3 (FOXO3a) in liver-derived HepG2 cells treated with insulin. Conversely, siRNA-mediated NQO1 knockdown increased insulin- or serum-stimulated Akt phosphorylation. Similarly, MDA-MB-468 cells, which carry a polymorphic form of NQO1 that results in an absence of NQO1 protein and activity, showed markedly higher constitutive and insulin-stimulated Akt phosphorylation levels compared to an isogenic cell line stably expressing NQO1. We then explored the impact of pharmacological inhibition or genetic down-regulation of NQO1 on the global pattern of protein acetylation in HeLa cells. The reduction in NQO1 level and/or activity was associated with an increase in protein acetylation levels likely due to the inhibition of NAD+-dependent sirtuins. Lastly, cell treatment with the NAD+ precursor nicotinamide mononucleotide (NMN) inhibited Akt phosphorylation in a dose- and time-dependent manner, thus recapitulating the effects of NQO1 on Akt activity. Conclusions: We have identified a molecular mechanism whereby NQO1 functions as a modulator of insulin/Akt signaling pathway. These findings have implications for future basic and translational research on pathways that control energy homeostasis and diabetes.

39

23 Substitution of Leu11 and Tyr12 in Mouse Angiotensinogen Does Not Affect Angiotensin II-mediated Functions in Mice

Chia-Hua Wu 1 • Congqing Wu 1 • Feiming Ye 1 • Deborah Howatt 1 • Anju Balakrishnan 1 • Jessica Moorleghen 1 • Craig Vander Kooi 2 • Alan Daugherty 3 • Hong Lu 1 1Saha Cardiovascular Research Center , University of Kentucky • 2Molecular and Cellular Pharmacology, University of Kentucky • 3Saha Cardiovascular Research Center, University of Kentucky

Graduate Student

Background and Objective: Angiotensinogen (AGT) is the unique precursor of angiotensin II (AngII), which is a critical contributor to atherogenesis. Renin cleavage of AGT exhibits species specificity. It has been determined in in vitro studies that Leu11-Tyr12 in mouse AGT and Val11-Ile12 in human AGT are essential for species-specific renin cleavage. In this study, we compared adeno-associated viral (AAV) vectors encoding human AGT or mouse AGT with Leu11Val and Tyr12Ile mutations to determine whether substitution of these residues regulated AngII-mediated functions. Methods and Results: Male hepatocyte-specific AGT deficient (hepAGT-/-) mice were injected intraperitoneally with AAV vector containing a null insert or encoding human AGT, while their wild type littermates (hepAGT+/+) were injected with AAV containing the null insert. All mice were in an LDLR -/- background. Two weeks after AAV injections, mice were fed a saturated fat-enriched diet for 12 weeks. Administration of AAV encoding human AGT led to high plasma human AGT concentrations, but had no effect on plasma renin concentrations and hypercholesterolemia-induced atherosclerosis. In a subsequent study, AAV encoding mutated mouse AGT with Leu11Val and Tyr12Ile were injected into hepAGT-/- mice. Mutated mouse AGT resulted in a significant increase of plasma AGT concentrations. Plasma renin concentrations in hepAGT-/- mice repopulated with mutated mouse AGT were decreased to a level comparable to their concentrations in hepAGT+/+ mice injected with a null AAV. AAV-driven expression of mutated mouse AGT also augmented atherosclerosis in hepAGT-/- mice. Conclusion: Human AGT does not repopulate AngII-mediated functions, whereas mutations of Leu11Val and Tyr12Ile in mouse AGT, to mimic the two amino acids in human AGT, does not affect AngII-mediated effects.

40

24 Vascular Inflammatory Regulation of Lipid Phosphate Phosphatase 3 Expression

Patrick Van Hoose, PhD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1 1Cardiovascular Research Center, University of Kentucky

Postdoc

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3, is a cell surface enzyme that regulates lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) availability and signaling. Genome wide association studies in humans identified heritable single nucleotide polymorphisms (SNPs) in the final intron of PLPP3 that independently predicted coronary artery disease (odds ratio, 1.17; P=3.81×10–19). PLPP3 is dynamically regulated during vascular inflammation and the risk allele reduces gene expression by disrupting binding of CCAAT enhancer binding protein beta (CEBPβ). However, other mechanisms may control dynamic regulation of PLPP3. The USC Genome Browser identifies potential target sequences for NFκB responsive elements in the PLPP3 promoter, including three potential RelA (p65) binding sites. Previous work has established that smooth muscle expression of Plpp3 attenuates experimental atherosclerosis and development of intimal hyperplasia.These observations led to the hypothesis that dynamic regulation of PLPP3 is a crucial step in controlling vascular inflammation and disease progression. Coronary human smooth muscle cells (caHSMCs) were treated for 72hrs with 1µM angiotensin II (ATII) in the presence or absence of 1 µM parthenolide. Ldlr-/- mice were randomized to receive either vehicle saline or 1000ng/kg/min angiotensin II via osmotic mini-pump for 7 days. Following 72hr exposure to angiotensin II (ATII) PLPP3 expression increases in caHSMCs and is accompanied by increases in p65 but not CEBPβ expression. Parthenolide, an inhibitor of IκBα degradation, blocks ATII induced PLPP3 expression. To determine whether Plpp3 is upregulated in response to ATII in vivo, Ldlr-/- mice were treated with ATII for 7 days. Plpp3 and p65 expression remained unchanged in the aortic arch, thoracic aorta, abdominal aorta and mesenteric arteries.These observations suggest potential novel regulation of PLPP3 expression governed by an ATII-NFκB pathway that could be important in the context of vascular inflammation and disease.

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25 Myeloid β-catenin deficiency exacerbates atherosclerosis in low-density lipoprotein receptor-deficient mice

Fang Wang, PhD 1 • Zun Liu, PhD 1 • Se-Hyung Park, PhD 1 • Taesik Gwag, PhD 1 • Weiwei Lu, PhD 1 • Yipeng Sui, PhD 1 • Changcheng Zhou, PhD 1 1Pharmacology and Nutritional Science, University of Kentucky

Postdoc

Objective: The Wnt/β-catenin signaling pathway is an ancient and evolutionarily conserved pathway regulating essential aspects of cell fate determination, proliferation, migration and polarity. Canonical Wnt/β-catenin signaling has been implicated in atherosclerosis development but the cell-specific role of β-catenin in atherogenesis remains elusive. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, and this study aims to investigate the impact of β-catenin expression on macrophage functions and atherosclerosis development. Methods and Results: To investigate the role of macrophage Wnt/β-catenin signaling in atherogenesis, we generated myeloid-specific β-catenin-deficient low-density lipoprotein receptor-deficient mice (β-catenin∆myeLDLR-/-). As expected, deletion of β-catenin decreased macrophage adhesion and migration in vitro. However, β-catenin∆myeLDLR-/- mice had significantly increased atherosclerosis as compared with control littermates. Mechanistic studies revealed that β-catenin can directly regulate signal transducer and activator of transcription (STAT) pathway in macrophages, and ablation of β-catenin resulted in STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. Conclusions: This study demonstrates a critical role of myeloid β-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.

42

26 Vascular inflammation induced expression of lipid phosphate phosphatase

Kavya Balaji 1 • Patrick Van Hoose, PhD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1 1Cardiovascular Research Center, University of Kentucky

Undergraduate

Lipid phosphate phosphatase 3 (PLPP3) is a polymorphic gene that is a member of the phosphatidic acid phosphatase family. It encodes for the cell surface enzyme, lipid phosphate phosphatase 3 (LPP3) that regulates lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) availability and signaling. Genome wide association studies in humans identified heritable single nucleotide polymorphisms (SNPs) in PLPP3 that predicts coronary artery disease risk independently of traditional risk factors (odds ratio, 1.17; P=3.81×10–19). PLPP3 is dynamically regulated during vascular inflammation and the risk allele reduces gene expression by disrupting binding of CCAAT enhancer binding protein beta (CEBPβ). However, other mechanisms may control dynamic regulation of PLPP3. The USC Genome Browser identifies potential target sequences for NFκB responsive elements in the PLPP3 promoter, including three potential RelA (p65) binding sites. Hypothesis: These observations led to the hypothesis that angiotensin II, an inducer of vascular inflammation could regulate of PLPP3. Methods: Coronary human smooth muscle cells (caHSMCs) were treated for 12, 24, 48 and 72hrs hours with 1 M angiotensin II (ATII) in the presence or absence of parthenolide. Following treatment PLPP3 and p65 gene expression was examined. Results: The time course treatment of ATII revealed 72hr treatment upregulated PLPP3 gene expression in caHSMCs and this upregulation of PLPP3 was inhibited in the presence of parthenolide, an inhibitor of IκBα degradation. Conclusion: These observations suggest possible regulation of PLPP3 via an angiotensin II-NFkB dependent pathway, which may be important in the context of vascular disease.

43

27 LRP1 Deletion in Smooth Muscle Cells of the Outer Aortic Media Promotes Angiotensin II-induced Thoracic Aortic Aneurysm

Hisashi Sawada, MD, PhD 1 • Debra Rateri 1 • Mark Majesky, PhD 2 • Alan Daugherty, PhD 1 1Saha CVRC, University of Kentucky • 2Center for Cardiovascular Biology, University of Washington

Postdoc

Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional protein that is linked to several vascular pathologies. LRP1 deletion in smooth muscle cells (SMCs) accelerates angiotensin II (AngII)-induced thoracic aortic aneurysm (TAA). In association with TAA formation, there is medial thickening that is characterized by a transmural gradient in which pathology progressively increases from lumen to adventitial aspect. We hypothesized that deletion of LRP1 in the outer medial layers of the proximal thoracic aorta has a pivotal role in the pathogenesis of TAA. The aim of this study was to determine whether LRP1 deletion in the outer media accelerates AngII-induced TAA formation. Methods and Results: SMCs in the outer media of the ascending aorta are derived from the second heart field, as demonstrated by lineage tracing studies using Cre under the control of Mef2c. Therefore, we used Mef2c-driven Cre to delete LRP1 in SMCs of the outer medial layers. Female LRP1 flox/flox mice were bred to male Mef2c-Cre1/0 mice to generate study mice. We first confirmed LRP1 deletion in Cre1/0 mice by both immunostaining and Western blot. LRP1 was expressed ubiquitously across smooth muscle cells of all aortic medial layers in Cre 0/0 mice. In mice expressing Mef2c-Cre, aortic LRP1 protein was detected only in SMCs of the inner laminar medial layers. Western blotting demonstrated LRP1 protein abundance in Cre expressing mice was reduced by 43%. Saline or AngII (1,000 ng/kg/min) was infused by subcutaneous osmotic pumps for 28 days into 12 - 14 week-old male Cre0/0 and 1/0 mice. As expected, systolic blood pressure increased similarly in both AngII-infused Cre 0/0 and 1/0 mice compared to saline-infused mice. Aortic rupture occurred within 3 to 10 days after AngII infusion in 17% of AngII-infused Cre 0/0 mice, while LRP1 deletion in Cre 1/0 mice increased aortic rupture to 27%. Aortic diameter in the survivors was significantly increased in Cre1/0 mice compared to Cre0/0 mice. Histologically, elastin fragmentation was detected in the aorta of AngII-infused Cre 0/0 mice and greater in Cre1/0 mice. Conclusion: LRP1 in second heart field-derived SMCs of the outer media may play a critical role in the pathogenesis of TAA.

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28 Sexual Dimorphism of Angiotensin II-induced Thoracic Aortic Rupture in Mice with LRP1 Deficient Second Heart Field–derived Smooth Muscle Cells

Bradley Wright 1 • Hisashi Sawada, MD, PhD 1 • Jessica Moorleghen 1 • Richard Charnigo, PhD 2 • Debra Rateri 1 • Mark Majesky, PhD 3 • Alan Daugherty, PhD 1 1Saha CVRC, University of Kentucky • 2Biostatistics, University of Kentucky • 3Division of Cardiology, University of Washington

Undergraduate

Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis. Deletion of LRP1 in smooth muscle cells (SMCs) accelerates angiotensin II (AngII)-induced thoracic rupture and ascending aortic aneurysm. We demonstrated recently that SMCs in the outer medial layers of the ascending aorta were derived from second heart field (SHF). Deletion of LRP1 from SHF-SMCs significantly increased thoracic aortic rupture and aneurysms in male mice. The aim of this study was to determine if female sex influenced AngII-induced rupture and aneurysm in LRP1 deficient SHF-SMC mice. Methods and Results: Female LRP1 flox/flox mice were bred to male Mef2c-Cre 1/0 mice to generate study mice. Either saline or AngII (1,000 ng/kg/min) was infused by subcutaneous osmotic pumps for 28 days into 12-14 week old Cre 0/0 and Cre 1/0 mice of both sexes (N=12-31). As expected, AngII infusion increased systolic blood pressure in both sexes. During AngII infusion, aortic rupture occurred within 3 to10 days in 17% of Cre 0/0 mice, while LRP1 deletion in Cre 1/0 mice increased significantly to 27% in males. Aortic rupture rate in AngII-infused female mice was decreased significantly compared to male mice, as there were none in Cre 0/0 and within 6-14 days only 9% in Cre 1/0 female mice. Ultrasonography was used to measure ascending aortic dilation as an index of thoracic aneurysm. Ascending aortic diameter in the survivors was significantly increased in AngII-infused Cre 1/0 vs Cre 0/0 and saline-infused controls in both sexes. Conclusion: Although male and female Cre1/0 mice experienced similar dilation of the ascending aorta under AngII infusion, female Cre1/0 mice experienced a significantly increased rate of survival compared to male litter mates. Future studies will determine the mechanism of sexual dimorphism that reduces AngII-induced thoracic aortic rupture in females.

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29 SAA is not Just an HDL-Associated Lipoprotein

Patricia Wilson, MS 1 • Joel Thompson, PhD 1 • Frederick de Beer, MD, PhD 1 • Nancy Webb, PhD 1 • Lisa Tannock, MD, PhD 2 1Saha Cardiovascular Research Center, University of Kentucky • 2Endocrinology/Saha Cardiovascular Research Center, University of Kentucky

Staff

Cardiovascular disease (CVD) is the leading cause of death in developed nations despite widespread use of pharmacological interventions. Individuals with obesity and/or diabetes are at particularly high risk for CVD and research suggests this may be due to elevated levels of serum amyloid A (SAA). Brief and chronic elevations of SAA have been indeed found to be atherogenic in mouse models. The current dogma is that SAA is exclusively an HDL associated lipoprotein. However, we and others have reported SAA on apoB-containing lipoproteins in both humans and mice and several studies have recently suggested that SAA-LDL is a risk factor for CVD. The goal of this study was to determine if SAA can shift between HDL and apoB-containing particles, and if the presence of SAA on apoB particles affects their atherogenicity. To determine if SAA could be exchanged between lipoproteins in vitro, we first incubated SAA with each lipoprotein separately, then mixed the lipoprotein containing SAA with each of the other two lipoproteins. To determine is this exchange could also occur in vivo, we injected apoE-/- x SAA1.1/2.1-DKO mice with murine SAA complexed with individual murine lipoproteins. To elucidate the role of CETP in SAA shifting, some mice were preinjected with an adenovirus expressing CETP 72 hours prior to injection with SAA-HDL. We now have evidence that SAA does indeed shift between lipoproteins both in vitro and in vivo, facilitated by CETP, and that delipidated SAA will completely associate with any available lipoproteins although preferentially on HDL. The presence of SAA on apoB-containing lipoproteins has physiological relevance, as we have demonstrated that this augments the proteoglycan binding affinity of these particles thus increasing their atherogencity. Collectively our data shows that SAA can be exchanged from HDL to apoB-containing lipoproteins and that this exchange is pro-atherogenic.

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30 Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Peter Kekenes-Huskey 1 1Chemistry, University of Kentucky

Faculty

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4, 5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca 2+ . These findings motivate our core hypothesis that non-selective inward Ca 2+ conduction afforded by human amylin oligomers increase cytosolic and SR Ca 2+ load, which thereby magnifies intracellular Ca 2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca 2+ dysregulation, including whether enhanced SL Ca 2+ influx is sufficient to elevate cytosolic Ca 2+ load [6], and if so, how might amplified Ca 2+ transients perturb Ca 2+ -dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca 2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca 2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca 2+ loading of the sarcoplasmic reticulum (SR) and the greater fractional release may contribute to the Ca 2+ -dependent activation of calmodulin. Importantly, elevated Ca 2+ in the SR and dyadic space collectively drive greater fractional SR Ca 2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca 2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca 2+ dysregulation in pre-diabetes.

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31 PCB 126 Disrupts Gut Microbiota and Increases Intestinal Inflammation in a Mouse Model of Atherosclerosis

Jessie Hoffman, MS 1 • Michael Petriello, PhD 2 • Olga Vsevolozhskaya, PhD 3 • Richard Charningo, PhD 3 • Andrew Morris, PhD 2 • Bernhard Hennig, PhD 4 1Pharmacology and Nutritional Sciences, University of Kentucky • 2Division of Cardiovascular Medicine, University of Kentucky • 3Biostatistics, University of Kentucky • 4Animal and Food Sciences, University of Kentucky

Graduate Student

The gut microbiome is sensitive to diet and environmental factors and is involved in the regulation of many host metabolic responses. Dioxin-like pollutants contaminate many food sources, and thus human exposure begins within the gut, which may play a role in pollutant-induced systemic toxicity. Additionally, gut dysbiosis and inflammation pose risk factors for the development of cardiovascular diseases, specifically atherosclerosis. We hypothesize that the dioxin-like pollutant PCB 126 will perturb gut microbial populations and impair gut health, which may contribute to pollutant-induced systemic toxicity in an atherosclerotic mouse model. LDLr -/- mice were fed a low fat atherogenic diet (10% fat, 0.15% cholesterol) for 14 weeks and exposed to PCB 126 at week 2 and 4. Exposure to PCB 126 reduced gut microbial diversity and shifted populations at the phylum and genus levels in ways that mimic observations in chronic inflammatory diseases. Furthermore, PCB exposed mice exhibited increased markers of inflammation in intestinal and plasma samples. Interestingly, Cyp1a1 gene expression was increased in intestinal samples even 10 weeks after PCB exposure, indicating a slow continual passage of pollutants through the enterohepatic circulation. These data imply that PCB toxicity is already initiated in the gut through disruption of healthy microbiota, and increases in gut inflammation. These observations highlight a unique opportunity for dietary interventions that are beneficial for both gut and overall health. Further research should examine how nutritional components can combat pollutant induced toxicity initiated at the gut level.

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32 PCB 126 Exposure Increases Peripheral Vascular Disease Risk in Mice with Fatty Liver

Jazmyne Barney 1 • Banrida Wahlang, PhD 2 • Brendan Thompson 1 • Chunyan Wang, PhD 3 • Omer Hamad 3 • Jessie Hoffman, MS 4 • Michael Petriello, PhD 2 • Andrew Morris, PhD 2 • Bernhard Hennig, PhD 3 1Toxicology and Cancer Biology, University of Kentucky • 2UK Superfund Research Center, University of Kentucky • 3Animal and Food Sciences, University of Kentucky • 4Pharmacology and Nutritional Sciences, University of Kentucky

Graduate Student

Xenobiotic and energy metabolism is dependent on a functional liver. Exposure to environmental pollutants like polychlorinated biphenyls (PCBs) is associated with inflammatory diseases, including non-alcoholic fatty liver disease and atherosclerosis. A compromised liver may play a critical role in modifying the induction of PCB toxicity and inflammation of the peripheral vasculature. Over a 14 week study, male C57Bl/6 mice (n=10) were fed an amino acid control diet or a methionine-choline deficient diet (MCD) with or without oral gavage of PCB 126 (0.5mg/kg). Post euthanasia, tissue and blood were collected for histological, toxicological, and inflammatory evaluation. Regardless of diet, PCB 126 induced hepatic steatosis. The MCD+PCB126 group exhibited fibrosis and increased liver to body weight ratio, suggesting liver injury and toxicity. Mice fed MCD-diet and exposed to PCB 126 demonstrated altered expression of hepatic genes involved in carbohydrate and lipid metabolism, indicating metabolic dysfunction. With regard to effects of PCB 126 on extra-hepatic organs, all mice fed MCD diet had decreased expression of plasma leptin and resistin, and PCB 126 exposed groups appeared to have crown like structures in their epididymal adipose tissue, indicating the presence of inflammatory cells. In addition, MCD+PCB 126 mice displayed increased plasma inflammatory markers including Icam-1, Mcp-1, and Tnf-α. Interestingly, in the MCD+PCB 126 group, plasma ALT and AST levels were increased as well as pro-atherogenic trimethylamine-N-oxide (TMAO), implying simultaneous liver damage and increased peripheral vasculature disease risk. Together these results provide a novel linkage of a compromised liver to PCB-induced hepatic and vascular inflammation. These finding also a have translational component, suggesting environmental pollutants can cause inflammatory disease pathologies by stimulating cross-talk between individual organ systems. (Supported in part by NIEHS/NIH grant P42ES007380).

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33 Promoter Enhancer Interactions Regulating PLPP3 Gene Expression

Guogen Mao, PhD 1 • Shaojing Ye, PhD 1 • Susan S. Smyth, MD, PhD 2 • Andrew J. Morris, PhD 2 1Gill Heart and Vascular Institute, University of Kentucky • 2Gill Heart and Vascular Institute, Veterans Affairs Medical Center,, University of Kentucky

Staff

Common heritable intronic variants within the final fifth intron of the PLPP3 gene associate with significant inter individual differences in human coronary artery disease risk. PLPP3 is one of three closely related genes encoding lipid phosphate phosphatases 1-3 which are integral membrane enzymes that dephosphorylate lipid phosphate mono esters and some lipid polyphosphates. PLPP3 expression in vascular endothelial cells is required for mouse development and while individuals with heterozygosity for loss of function PLPP3 alleles have been identified homozygotes have not so PLPP3 is likely also essential for human development. Because the three PLPP genes have overlapping expression patterns and encode enzymes with very similar activities it is reasonable to hypothesize that differences in regulation of expression may underlie these unique functions of PLPP3 gene in atherosclerosis and development. We used informatic approaches to identify regulatory elements within the PLPP3 promoter and coronary artery disease risk haplotype block. We then cloned the relevant sequences into reporter constructs and used transfection assays and site directed mutagenesis to examine sequence and allele specific effects on promoter and enhancer activity and on how these interact to control gene expression. The ~1.5 KB PLPP3 promoter comprises a ~0.5 KB enhancer indicated by H3K4ms1/H3K27Ac markers and a ~1KB core promoter containing three NF-kB (p50/RelA and p42/RelB) binding sites. Chromatin immunoprecipitation and reporter assays indicate that all three of these are functional regulators of basal activity of the PLPP3 promoter with very strong upregulation of promoter activity observed in transfection assays with these transcription factors. Similar informatic approaches identified cFOS and C/EBP target sequences in accessible chromatin within the coronary artery disease risk associated haplotype block in the final intron of the PLPP3 gene. These sequences are conserved in mice and humans and interact with their cognate transcription factors in immunoprecipitation and gel shift assays and regulate expression of both generic promoters and the PLPP3 promoter in reporter gene/transfection assays. The cFOS and C/EBP target sequences exhibit allelic variation in humans with coronary artery disease risk variants decreasing transcription factor binding and transcriptional enhancer activity. Taken together these results identify an NF-kB driven pathway that likely explains the widely observed strong upregulation of PLPP3 expression during inflammation and atherosclerosis and suggest a mechanism by which coronary artery disease risk associated variants of intronic enhancer sequences could decrease PLPP3 expression and reduce the normally protective functions of this gene observed in experimental models of atherosclerosis.

50

34 Endothelial Mineralocorticoid Receptor and Neutrophils Mediate Aldo plus Salt-Induced Abdominal Aortic Aneurysm

Shu Liu 1 • Yu Zhong 1 • Zhenheng Guo 2 • Ming Gong 1 1Physiology, University of Kentucky • 2Pharmacology and Nutritional Sciences, University of Kentucky

Staff

Abstract Objective—We recently reported that administration of mice with aldosterone (Aldo) or deoxycorticosterone acetate (DOCA) plus salt induces AAA via mineralocorticoid receptor (MR). The current study defines the specific roles of endothelial and myeloid cell MR in DOCA-salt induced AAA. Approach and Results—A tamoxifen inducible endothelial cell (EC)-specific MR knockout mouse model (iECMRKO) and a myeloid cell-specific MR knockout mouse model (MyMRKO) were developed. The iECMRKO mice, but not the MyMRKO mice were protected from Aldo- or DOCA-salt-induced AAA. Mechanistically, EC-specific MR deletion had little effect on Aldo-salt-induced salt retention, hypertension, and renal fibrosis, but largely suppressed aortic elastin degradation, matrix metalloproteinase-2 (MMP-2) and MMP-9 upregulation, macrophage and neutrophil infiltration. Surprisingly, neutrophils, but not macrophages, were observed in the aorta 1 week after Aldo-salt administration in control mice, but not in iECMRKO mice. Treatment of C57BL/6 mice with an anti-PMN antibody selectively suppressed Aldo-salt-induced circulating Ly6G-postive neutrophils, but not CD4-postive leukocytes, and protected mice from Aldo-salt-induced AAA. In cell cultures, Aldo-induced endothelial adhesion molecules (E-selectin, P-selectin, and ICAM-1, but not VCAM-1) and proinflammatory cytokine (IL-6 and MCP-1) mRNA expressions were abolished in MR-deficient ECs. Importantly, Aldo-salt-induced ICAM-1 but not VCAM-1 protein upregulation was abolished in aortas from iECMRKO mice. Conclusions—Endothelial MR, but not myeloid cell MR, plays an important role in Aldo-salt-induced AAA. Moreover, ICAM-1, but not VCAM-1; and neutrophils, but not macrophages, mediate the early processes of Aldo-salt-induced and endothelial MR-mediated AAA development.

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35 Identification of Candidate Long QT Syndrome Type 2 Patients Starting from Exome Sequences Identified in a Biobank Cohort

Allison Hall, MS 1 • Don Burgess, PhD 2 • Pierre Fwelo 1 • Jennifer Smith 3 • Corey Anderson, PhD 4 • Craig T. January, MD, PhD 4 • Ann Stepanchick, PhD 4 • Uyenlinh Mirshah, PhD 4 • Jonathan Luo, PhD 4 • Dustin Hartzel, PhD 4 • Michael Murray, MD 5 • Tooraj Mirshahi, PhD 5 • Brian Delisle, PhD 1 1Physiology, University of Kentucky • 2Physics, Asbury • 3University of Kentucky • 4University of Wisconsin • 5Geisinger Health System

Staff

Introduction: Every year congenital long QT syndrome (LQTS) is thought to cause sudden cardiac death in hundreds of individuals in the US. Genetic screening potentially could identify LQTS patients before it strikes. However, genetic analyses often find novel rare sequence variants of uncertain physiological significance, and little is known about genetic screening in unaffected populations. Hypothesis: LQTS type 2 (LQT2) is caused by loss-of-function mutations in the rapidly activating delayed rectifier K+ channel gene KCNH2 (Kv11.1). Screening for KCNH2 variants using an approach similar to the Comprehensive In Vitro Proarrhythmia Assay for drug testing will allow the identification of candidate LQT2 patients starting from exome sequences. Methods: Ten KCNH2 mutations from the NCBI ClinVar database listed as “pathogenic”, “suspected-pathogenic”, or “conflicting interpretations” were identified in 10,000 Whole Exome Sequences (WES) from the Geisinger MyCode® cohort. The KCNH2 mutations were screened using Western blot to quantify terminally glycosylated mature Kv11.1 protein (a proxy for Kv11.1 channel trafficking); patch-clamp to measure Kv11.1 channel current (IKv11.1); and computational simulations with a human ventricular action potential (AP) model to predict AP duration (a correlate for the QT interval). Results: Two of the KCNH2 mutations were trafficking-deficient to decrease mature Kv11.1 protein and peak IKv11.1, and five mutations altered normal Kv11.1 channel activation or deactivation. Simulating the decrease in IKv11.1 caused by the trafficking-deficient KCNH2 mutations increased AP duration by >30%, whereas the mutations that disrupted Kv11.1 channel gating did not predict significant changes in AP duration. De-identified Electronic Health Records (EHR) from the Geisinger MyCode® subjects showed that the corrected QT interval (QTc Bazette) for the patients that harbored the trafficking-deficient KCNH2 mutations was ≥480 ms, whereas the average QTc for all EHR database subjects was <440 ms. Conclusions: We conclude that comprehensive proarrhythmia screening for KCNH2 variants in an unselected population using exome sequencing facilitates the identification of candidate LQT2 patients. This strategy potentially can identify LQTS cohorts and initiate lifesaving preventive measures.

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36 Optimization of immunomagnetic separation for cryopreserved cord blood and apheresis mononuclear cell fractions derived endothelial progenitor cells

Himi Tripathi, PhD 1 • Lakshman Chelvarajan, PhD 1 • Brad J Berron, PhD 2 • Ahmed Abdel Latif, MD, PhD 1 1Gill Heart Institute and Division of Cardiovascular Medicine, University of Kentucky • 2Department of Chemical and Materials Engineering, University of Kentucky

Staff

Introduction: Cardiovascular tissue damage in acute myocardial infarction mainly arises from the loss of blood flow, leading to changes in the composition and function of the ischemic region and heat failure. Multiple studies have examined the utility of CD34+ endothelial progenitor cells (EPCs) for limiting tissue damage and promoting tissue repair after an ischemic event. However, isolation of EPCs using traditional fluorescent activated cell sorting is cumbersome, expensive and time consuming. In this study, we examined the efficacy of novel antigen based methods for EPC isolation from human cord blood. Methods and Results: Immunomagnetic cell sorting protocol to purify CD34+ cells from cryopreserved cord blood and apheresis samples was optimized using MACS ultrapure CD 34 microbead kit. Cell viability, CD 34 purity and endothelial progenitor cells phenotypic expression was assessed using flow cytometer and trypan blue assay. After thawing cryopreserved cord blood, we observed >80 ± 10% viable cells and 95±5% viable apheresis cells. CD34+ purity of 74 ± 16% and 94 ± 1.7% was achieved in cryopreserved cord blood and apheresis samples, respectively. Recovery of CD34+ cells was higher from apheresis (64.6%) in comparison to cord blood (31.3%). Post selection, the expression of EPCs markers was 65.6± 23.2 for CD31 and 90.3 ± 6.6 for CD133 in cord blood and 79.7± 5.5 and 69.6 ± 5.4 in apheresis samples. Additionally, we observed significantly higher recovery and system efficiency when using small input number of CD34+ cells (3.02 ± 2.7 X 106 vs. 31.3% ±54.1 X 106; p<0.01) cells. Conclusion: We have optimized antigen based selection protocol for EPCs from human cord blood and apheresis samples. This optimized protocol can enhance cell selection for clinical trials using a fast and time efficient method.

53

37

Elevated circulating TGF-beta is not the cause of increased atherosclerosis development in biglycan deficient mice

Joel Thompson, PhD 1 • Patricia Wilson, PhD 1 • Alex Wyllie 1 • Adrian Wyllie 1 • Lisa Tannock, MD 1 1Internal Medicine, University of Kentucky

Staff

Background: Vascular biglycan contributes to atherosclerosis development and increased biglycan expression correlates with increased atherosclerosis. However, mice deficient in biglycan have either no reduction in atherosclerosis or an unexpected increase in atherosclerosis. Biglycan deficient mice have systemically elevated TGF-beta, likely due to lack of sequestration of TGF-beta in extracellular matrix The purpose of this study was to determine if prevention of TGF-beta elevations in biglycan deficient mice affected atherosclerosis development. Methods: Biglycan deficient mice were crossed to Ldlr deficient mice. Diabetes was induced via streptozotocin and all mice were fed a high cholesterol diet. Diabetic biglycan wildtype and biglycan deficient Ldlr deficient mice were injected with the TGF-beta neutralizing antibody 1D11 or irrelevant control antibody 13C4. Results: Biglycan deficient mice had significantly elevated plasma TGF-beta levels, which was further increased by diabetes, and significantly increased atherosclerosis. There was a significant correlation between TGF-beta concentrations and atherosclerosis. However, despite nearly complete suppression of plasma TGF-beta levels in mice treated with the TGF-beta neutralizing antibody 1D11, there was no significant difference in atherosclerosis between mice with elevated TGF-beta levels and mice with suppressed TGF-beta levels. Conclusions: The increased atherosclerosis in biglycan deficient mice does not appear to be due to elevations in TGF-beta.

54

38 Gestational Diabetes Provokes Postpartum Cardiac Hypertrophy via Activation of Ca2+-Dependent Signaling

Amanda Hoskins 2 • Nirmal Verma, PhD 1 • Florin Despa, PhD 1 • Sanda Despa, PhD 1 1Pharmacology & Nutritional Sciences, University of Kentucky • 2University of Kentucky

Undergraduate

Introduction: Gestational diabetes mellitus (GDM) complicates 9% of pregnancies in the US and its incidence is growing. Women with prior GDM have higher risk of developing cardiac hypertrophy and dysfunction, but the underlying mechanisms are largely unknown. Hypothesis: GDM promotes pathological growth of the heart through Ca2+-dependent hypertrophy signaling. Methods and Results: Female rats that express the human isoform of amylin, an amyloidogenic peptide co-secreted with insulin, in the pancreatic β-cells (HIP rats) were used as a GDM model. WT littermates served as controls. In both groups, glucose tolerance decreased during pregnancy and recovered after giving birth, with HIP females remaining glucose intolerant compared to the WT throughout the study. Cardiac hypertrophy, assessed from heart weight-to-body weight ratio, heart weight-to-tibia length ratio and echocardiographic measurements of the left-ventricular wall, occurred in both HIP and WT females during pregnancy. By two months postpartum, heart size returned to the pre-pregnancy level in the WT but remained significantly larger in HIP females. To uncover the cause of hypertrophy in postpartum HIP females, we investigated the activation status of calcineurin/NFAT and CaMKII/HDAC hypertrophy pathways. Calcineurin/NFAT signaling, assessed from the nuclear-to-cytosolic localization of NFATc4, was reduced during late pregnancy in both groups. In WT females, this pathway returned to its baseline activation level within two months postpartum. However, in postpartum HIP females the ratio of nuclear-to-cytosolic NFATc4 was significantly larger than at baseline, indicating re-activation of this hypertrophy pathway. In contrast, the CaMKII/HDAC hypertrophy signaling was strongly activated in late pregnancy and returned to baseline postpartum in both HIP and WT females. Since calcineurin and CaMKII are activated by higher cytosolic Ca2+, we also analyzed myocyte Ca2+ cycling. Ca2+ transient decay was slower in myocytes from postpartum HIP females vs. baseline, while no differences occurred in the WT. Conclusion: Two months after a GDM-complicated pregnancy, female rats show cardiac hypertrophy that is likely caused by activation of calcineurin/NFAT hypertrophy pathway.

55

39 Effects of Molecular Clock Component Deletion in Mouse Cardiomyocytes on Long QT Syndrome Phenotype

Kaitlyn Samuels 1 • Elizabeth Schroder 1 • Tanya Seward1 • Brian Delisle, PhD 1 1Physiology, University of Kentucky

Undergraduate

Cardiac excitability is affected by various genetic and environmental perturbations, which is often the cause of a multitude of human cardiac diseases. Specifically, this study explored the effects of the genetic knockout of the Bmal1 circadian transcription factor in mouse heart cells superimposed upon a congenital Scn5a sodium ion channel genetic mutation representative of long QT syndrome type 3 (LQT3) in humans. Changes in electrocardiographic (ECG) properties were evaluated to investigate the changes in cardiac excitability in these transgenic mice. There were a significantly higher number of cardiac arrhythmias after the Bmal1 gene knockout than before the knockout, specifically an increased number of RR-interval pauses. There was also a consistently slower heart rate after the Bmal1 gene knockout. Finally, there was increased slope factor between the RR-interval and QT-interval after the Bmal1 knockout than before the knockout. These findings are consistent with previous similar investigations and serve to help provide a better understanding of the pathophysiology associated with cardiac genetic mutations.

56

40 The Prognostic Role of Elevated Myeloperoxidase in Patients with Acute Coronary Syndrome: A Systematic Review and Meta-Analysis

Mohamed Abo-Aly, MD 1 • Andrew kolodziej, MD 1 • Raphael Twerenbold, MD 1 • Christian Mueller, MD 1 • Ahmed Abdel-Latif, MD, PhD 1 1Division of cardiovascular medicine, Gill heart institution., University of Kentucky

Postdoc

Background: Many reports have shown the correlation between myeloperoxidase (MPO) and the pathogenesis of acute coronary syndrome (ACS). However, the prognostic role of MPO for major cardiac events or mortality in ACS patients has not been well studied. We sought to perform a systematic review and meta-analysis to examine the prognostic value of inpatient MPO level in patients presenting with ACS. Methods: PubMed and Cochrane databases were searched from 1975 to September 2017 for studies that investigated the prognostic value of serum MPO in patients with ACS. Studies should have dichotomized patients into a high MPO and a low MPO group, reported clinical outcomes according to the same cutoff value of MPO and followed up patients for at least 30 days to be eligible for enrollment. Data were analyzed using random-effects model to control heterogeneous reporting. Results: We retrieved 3896 studies for initial screening which yielded 14 studies including 9268 subjects with a median follow up of 8.7 ± 21.96 months for the final analysis. High serum MPO level significantly predicts the future risk of mortality (odds ratio [OR] 2.039; 95% confidence interval [CI]: 1.405-2.959; P=0.0176) and MACE (OR 1.42; 95% CI: 1.00-1.99; p=0.044). we also observed a string trend towards higher incidence of recurrent myocardial infarction (MI) in patients with high MPO level (OR 1.24; 95% CI: 0.99-1.54; p=0.054) in comparison to patients with low serum MPO. Conclusion: In this meta-analysis examining the long outcomes in ACS patients, high MPO levels were associated with worse clinical outcomes. These observations support the use of MPO as a novel clinical prognostic marker in patients with acute coronary syndrome.

57

41 Desynchrony of Tissue Oscillators and Compromised Blood Pressure Circadian Rhythm in a Novel Diabetic Db/Db-Mper2luc Mouse Model

Tianfei Hou, MS 1 • Wen Su, MD 1 • Ming Gong, MD, PhD 2 • Zhenheng Guo, PhD 1 1Department of Pharmacology and Nutritional Sciences, University of Kentucky • 2Department of Physiology, University of Kentucky

Graduate Student

Diabetic patients have a high prevalence of blood pressure (BP) circadian rhythm disruption mostly manifested as reduced BP dipping during the inactive phase (non-dipping profile). Accumulating evidence demonstrates a pathophysiological link between end-organ damage and non-dipping BP. Clock gene dysregulation may link diabetes with non-dipping BP but comprehensive study of clock gene oscillation dysregulation in diabetes has been hampered by the impractical requirement of intensive timed sampling. To address this issue, we generated a novel diabetic model, db/db-mPer2Luc mouse, by crossing the extensively used type 2 diabetic db/db mice with the clock gene Period 2 (mPer2) luciferase knock-in mPer2Luc mice. The db/db-mPer2Luc mice were obese, diabetic and had non-dipping BP. Real-time measurement of bioluminescence of the many tissues explanted from the db/db-mPer2Luc mice revealed that the phases of the mPer2 circadian oscillations shifted to different extents in aorta, mesenteric artery, kidney, liver, white adipose tissue and thymus, but no phase shift was detected in the central SCN tissue, or the adrenal gland and lung. The desynchrony of the tissue oscillators in the db/db-mPer2Luc mice was associated with compromised circadian rhythms in respiratory exchange ratio. Moreover, the time-of-day variations in vascular contractile responses and in baroreflex sensitivity were abolished. Taken together, we generated a novel diabetic db/db-mPer2Luc mouse model. Using this model, we revealed a desynchrony among tissue oscillators, a loss of the time-of-day variations in vascular contractile responses and baroreflex sensitivities, all of which likely contribute to the dampening of BP circadian rhythm in diabetes.

58

42 Lysophosphatidic Acid Receptor 4 Influence The development of Atherosclerotic Vascular Disease in Mouse Model

Liping Yang, MD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1 1Internal Medicine - Cardiology, University of Kentucky

Staff

Background: Lysophosphatidic acid (LPA) is one of the simplest phospholipid signaling molecules. LPA elicits diverse biological functions including cell proliferation, differentiation, migration, survival and apoptosis through binding and activating specific cell surface G-protein coupled receptors (LPA1-6). LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7 receptors are the endothelial differentiation gene (Edg) family. LPA4/p2y9/GPR23 of the purinergic receptor family and the related LPA5/GPR92 and LPA6/p2y5 have been identified as novel LPA receptors. LPA is generated by the secreted lysophospholipase D autotaxin (ATX) and inactivated by dephosphorylation by membrane lipid phosphate phosphatases (LPPs). Our previous work established that ATX and LPP3 contribute to atherosclerotic vascular disease, but little is known about the role of specific LPA4 receptors in atherosclerosis. We recently demonstrated upregulation of LPA4 in SMC undergoing a switch to assume a foam-cell like phenotype. The present work therefore aims to elucidate an association between LPA4 and experimental atherosclerosis in mice. Methods and Results: We generated mouse globally lacking LPA4 (LPA4-/-) on C57/BL background. Hyperlipidemia was elicited in the mice by PCSK9 virus injection at 5 weeks of age and then Western Diet feeding for 20 weeks. Although plasma cholesterol levels were similar, en face analysis of aortic atherosclerosis indicated a significant reduction in plaque area in LPA4-/- compared to LPA4-WT mice. Oil Red-O staining of aortic roots confirmed reduced neutral fat accumulation in the aortic roots of LPA4-/-mice. In sections taken at the level of the aortic root, CD68 and α-SMA immunoreactivity was lower in the absence of LPA4. SMCs isolated from mouse thoracic aorta were treated with ox-LDL to promote a foam cell phenotype, characterized by upregulation of CD68 expression. SMC isolated from LPA4-/- mice had lower CD68 mRNA after ox-LDL exposure than LPA4-WT cells. Conclusion: These findings revealed a role for LPA4 in vascular inflammation and the development of atherosclerotic vascular disease, potentially through effects of SMC phenotype and function.

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43

Intracoronary Versus Intravenous Adenosine-Induced Maximal Hyperemia for Fractional Flow Reserve Measurement: A Systematic Review and Meta-Analysis

Mohamed El-Helw, MD 1 • Mohamed Abo-Aly, MD 1 • Georges Lolay, MD 1 • Christopher Adams, MD 1 • Ahmed El-Sharaawy, MD 1 • Ahmed Abdel-Latif, MD, PhD 1 • Khaled Ziada, MD 1 1Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky

Postdoc

Background: Measurement of fractional flow reserve (FFR) is considered the gold standard technique for the invasive hemodynamic assessment of borderline coronary artery stenosis. Currently, intravenous (IV) adenosine is the recommended approach; however intracoronary (IC) administration is widely used due to its convenience and lower cost. The correlation between IV and IC administration to assess coronary blood flow is not well studied. Objective: This systematic review and meta-analysis is conducted to review the available literature that compared FFR measurements using bolus IC vs. standard continuous IV of adenosine infusion for detection of significant coronary artery stenosis. Methods: We systematically searched MEDLINE, EMBASE, Google scholar and the Cochrane Central Register of Controlled Trials databases. We reviewed data pertaining to the used adenosine doses, side effects of each method of administration and FFR values. We performed statistical analyses examining the sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and odds ratio in studies comparing bolus IC adenosine and continues IV adenosine infusion using random effects modelling. Results: We identified 8 studies addressing the primary review question. Compared to standard IV adenosine infusion, the overall sensitivity of IC adenosine is 0.843 (95% C.I. 0.701-0.925, P<0.01), overall specificity is 0.960 (95% C.I. 0.899-0.985, P<0.01), positive likelihood ratio is 19.133 (95% C.I. 7.993-45.798, P<0.01), negative likelihood ratio is 0.067 (95% C.I. 0.026-0.168, P<0.01) and diagnostic odds ratio is 280.206 (95% C.I. 94.810-828.141, P<0.01). Overall, hemodynamic side effects, symptoms and atrio-ventricular conduction delays were reported more frequently with IV adenosine. Conclusion: The current available literature suggests that IC adenosine is well tolerated and provides equivalent diagnostic accuracy compared to IV administration.

60

44 Recapitulating In Vivo Fibroblast Differentiation Using a Hydrogel Cell Culture System

Demetria Fischesser 1 • Onur Kanisicak, PhD 1 • Mario Perera 2 • Neil Ayres, PhD 2 • Jeff D Molkentin, PhD 1 1Molecular Cardiovascular Biology, Cincinnati Children's Hospital • 2Chemistry, University of Cincinnati

Graduate Student

Cardiac fibroblasts are resident interstitial cells known to maintain the extracellular matrix (ECM) under normal physiological conditions through the secretion of collagens and other adherent proteins. During injury, these fibroblasts take on an activated, contractile state and deposit additional ECM proteins to contribute to scar formation. While this response is initially beneficial to prevent rupture and further injury, continuous scar development leads to fibrosis, pathological ventricular remodeling and dysfunction, and eventual organ failure. Though much has already been discovered about the importance of fibroblasts during development and injury response, there are still many aspects of their functions that remain unclear. In order to decipher these fibroblast functions, many in vitro systems have been used to easily manipulate fibroblasts while attempting to mimic in vivo conditions. However, most of these systems have used plastic or gelatin-coated plastic as the surface medium. These have proven to be much stiffer than physiological tissue and provide a static system inhospitable to the dynamic changes of the activating fibroblast. In order to mimic an adaptable in vivo environment for fibroblast culture, we have developed a new hydrogel technology to understand fibroblast behavior and differentiation. This technology utilizes gelatin norbornene and a thiol conjugated to a Reversible Addition-Fragmentation Chain Transfer (RAFT) polymer which can dynamically mimic the stiffness of both healthy and fibrotic tissue, thus allowing for seamless modeling of injury and disease in various tissues. Through morphology and differentiation studies, we have found that there is a significant difference between fibroblasts plated on plastic versus softer hydrogels which mimic in vivo conditions. Fibroblasts plated on plastic and stiffer surfaces become very large and flat, and express markers of differentiation, such as alpha-Smooth Muscle Actin (αSMA), within days of plating. On softer hydrogels, however, the fibroblasts remain small and eventually develop long stellate appendages. They produce αSMA much more slowly and at a much less robust level than on stiffer surfaces. The morphology and differentiation pattern seen on softer hydrogels is highly comparable to fibroblasts observed in vivo. We will use these initial findings to better understand the activation and differentiation of fibroblasts and how, in injury, they first inhibit tissue damage and later enhance it. In the future, we plan to manipulate this hydrogel system to intentionally change the stiffness of the surface on which fibroblasts have already been plated to determine if we can mimic disease states as well as reverse the fibroblast activation phenotype seen previously.

61

45 Determination of The Effects Of High Fat Feeding On Autotaxin Activity

Esias Bedingar 1 • Frederick Onono, PhD 2 1University of Kentucky • 2Internal Medicine , College of Medicine

Undergraduate

Obesity is a major public health issue in the United States and is risk factor for several diseases such as coronary artery disease and certain types of cancers. Although the association between obesity and cardiovascular diseases is well known, the underlying molecular mechanisms are still unclear. One of the hypotheses proposed to explain the link between obesity and disease risk is that consumption of high fat diets or increased synthesis and storage of fats is associated with the production of bioactive lipids or lipid-derived molecules that promote disease. Lysophosphatidic acid (LPA) is a family of bioactive lysophospholipids well-recognized as an important signaling molecule acting primarily through actions at cell-surface G-protein coupled receptors. There are two major ways of LPA production. However, the enzyme responsible for making majority of the circulating LPA is autotaxin (ATX), a secreted phospholipase D. Autotaxin is strongly expressed in adipose tissue, thus synthesis of circulating LPA from dietary lipids could be further enhanced in obese individuals. In this study, we investigated the effects of fasting and high-fat feeding on the activity levels of ATX. Healthy volunteer subjects (n=11) were asked to fast overnight. In the morning, their blood was collected at t=0 for plasma preparation. Then, they were given a drink composed of boost protein shake and triglycerides and blood drawn at hourly intervals for up to 8 hours. Autotaxin activity was determined in the plasma using synthetic substrates. Preliminary results indicated that ATX activity is acutely sensitive to fasting and feeding, with the lowest activity observed following fasting and increasing with feeding of a high fat diet. To confirm the specificity of the ATX activity a novel inhibitor was used to inhibit ATX activity. Our findings indicate that ATX is dependent on the feeding status and is increased following feeding with a high fat diet. This study suggests the efficacy of ATX pharmacological inhibition could be enhanced if timed with the onset of feeding.

62

46 Sex-Specific Differences in Cardiac Fibrosis

Gregory Milburn 1 • Autumn Conger 1 • Cheavar Blair, PhD 1 • Maya Guglin, MD 2 • Gretchen Wells, MD, PhD 2 • Rebekah Waikel, PhD 3 • Kenneth Campbell, PhD 1 1Physiology, University of Kentucky • 2Cardiovascular Medicine, University of Kentucky • 3Biological Sciences, Eastern Kentucky University

Undergraduate

Cardiovascular disease is the leading causes of death in America, which prompted a concerted effort to better understand the causes and to develop innovative therapies. The etiology of heart disease is complex and depends on several factors such as age, race, and sex, but previous research conducted contains gaps, as it often did not look for difference between these groups. This study aims to fill one aspect of this research gap by examining sex-specific differences in cardiac fibrosis in failing and non-failing human hearts. Cardiac fibrosis is a result of cardiac remodeling and causes decreased heart function post cardiac damage. A previous study, conducted in our lab, used Nanostring analysis to examine sex-specific differences in the expression of genes related to fibrosis. The results of this study showed certain gene expressions were specific to sex or had an interaction between the sex and heart failure status. Several of these genes were regulators of collagen, a common type of cardiac fibrosis. We continued this study by quantifying collagen in human heart samples, collected from heart transplants and non-viable organ donors. These samples were stained using an established picrosirius red staining technique, turning the collagen tissue red and normal cardiac tissue yellow. These samples were then quantified using a k-means cluster, via a program written in lab, to eliminate any bias in determining red and yellow tissue. The results are pending as there are still samples left to be completed. We hope to continue pursuing this line of inquiry into sex-specific differences in heart disease by examining passive stiffness, of which collagen is a major component.

63

47 Dietary Effects on Lipoprotein-Associated Bioactive Mediators of Atherosclerosis

Cody Sutphin 1 • Maria Kraemer, PhD 2 • Pan Deng, PhD 2 • Courtney Hammill, PhD 2 • Susan Smyth, MD, PhD 2 • Andrew Morris, PhD 2 1Eastern Kentucky University • 2Cardiovascular Research Center, University of Kentucky

Undergraduate

Elevated circulating levels of atherogenic lipoproteins associate with increased risk of cardiovascular disease. Multiple lines of evidence implicate bioactive components of lipoprotein particles (such as oxidized phospholipids) as signaling molecules that provoke responses in blood and vascular cells that cause the development and progression of atherosclerosis. Studies from ourselves and others identify a role for the bioactive lipid mediator lysophosphatidic acid (LPA) as a proinflammatory atherogenic signal in preclinical models and clinical settings. LPA accumulates in atheromas, and mice lacking certain LPA receptors are protected from experimentally induced atherosclerosis while mice lacking the LPA degrading enzyme lipid phosphate phosphatase 3 (LPP3) exhibit accelerated atherosclerosis. These findings may explain why coronary artery disease risk is elevated in individuals with common genetic polymorphisms that decrease LPP3 expression. We hypothesized that LPA associated with low-density lipoprotein particles is sensitive to diet and enhanced by genetic hyperlipidemia. Plasma LDL-associated lipid species were analyzed in WT and LDLr-/- mice that were on either control or western diet. Mouse plasma was fractionated by size-exclusion chromatography using a calibrated Superose 6 column to separate lipoprotein species and then analyzed by targeted and untargeted HPLC coupled mass spectrometry to identify and quantitate LPA species, as well as other lipid-class species, that were altered in LDL-containing plasma fractions. We found that circulating levels of LPA are highly sensitive to high fat feeding in mice and that genetic manipulations that reduce lipoprotein clearance (Ldlr-/-) dramatically increase circulating levels of LDL-associated LPA while reducing albumin-associated LPA. Additionally, we identified unexpected increases in a broad range of atherogenic lipoprotein particle- associated lipid species when hyperlipidemic mice are put onto a western diet. These studies provide new insights into how diet could influence coronary artery disease risk by promoting increase in atherogenic lipoprotein associated LPA.

64

48 Ticagrelor Reduces Inflammation and Mortality in a Murine Model of Sepsis and Reduces Platelet-Leukocyte Aggregates and Inflammation In Pneumonia

Travis Sexton, PhD 1 • Guoying Zhang, MD 1 • Tracy Macaulay, PharmD 2 • Leigh Ann Callahan, MD 3 • Richard Charnigo, PhD 4 • Olga Vsevolozhskaya, PhD 4 • Zhenyu Li, PhD 1 • Susan Smyth, MD, PhD 2 1Cardiovascular Research Center, University of Kentucky • 2Gill Heart and Vascular Institute, University of Kentucky • 3Pulmonary, Critical Care & Sleep Medicine, University of Kentucky • 4Statistics, University of Kentucky

Staff

Background: Sepsis is a life-threatening and dysregulated response to infection that leads to numerous complications and carries substantial risk of mortality. Pneumonia is one of the most common precipitators of sepsis. Despite advances in treatment for sepsis and pneumonia, significant improvements have not been realized and high rates of cardiovascular events remain an issue. Retrospective analysis of clinical studies suggest anti-platelet therapy may improve outcomes in patients with pneumonia and sepsis. Methods: We conducted a human study with pneumonia patients (XANTHIPPE) and a murine study using a sepsis model to determine the effect of ticagrelor on inflammation, thrombosis, and lung function. Results: Among subjects with pneumonia not taking a P2Y12 antagonist at baseline, ticagrelor lowered the percent of leukocytes with attached platelets 11.75% at 24 hours compared to a 10.90% increase in placebo patients. Furthermore, ticagrelor lowered plasma IL-6 levels 83% at 24 hours compared to minimal change with placebo. Ticagrelor had a transient effect on markers for NETosis showing a significant 60% spike in MPO-NE complexes at 24 hours followed by a return toward baseline at 48 hours while placebo had no significant effect. Lung function tests also numerically improved with ticagrelor, although statistical significance was not achieved. In the murine sepsis model, disruption of the P2Y12 receptor protected against inflammatory response, lung permeability, and mortality. Conclusions: Our findings indicate a mechanistic link between platelets, leukocytes, and lung injury in settings of pneumonia and sepsis and suggest possible therapeutic approaches to reduce complications of pneumonia.

65

49 Endocytosis Mediates Platelets' Responses to Viruses In The Vasculature

Meenakshi Banerjee, PhD 1 • Sidney Whiteheart, PhD 1 1Molecular and Cellular Biochemistry, University of Kentucky

Postdoc

Platelet endocytosis is essential for Fibrinogen (Fg) uptake, receptor trafficking of integrins (αIIbβ3, αvβ3) and other surface receptors. However, the mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required and possible trafficking routes are understudied, in part due to a lack of viable experimental tools. Previously we showed the importance of ADP-ribosylation factor 6 (Arf6), which regulates αIIbβ3-mediated Fg uptake/storage and affects acute platelet functions e.g., clot retraction and spreading. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing Soluble N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called Cellubrevin/Vesicle Associated Membrane Protein-3 (VAMP-3) in platelet function. VAMP-3 KO platelets have defective uptake and accumulation of Fg, which led to enhanced platelet spreading on Fg and faster clot retraction. Using both Arf6 KO and VAMP-3 KO mutants as tools to probe the importance of endocytosis in platelets, we posited that platelet endocytosis could potentially be critical for actively sensing pathogenic damage in the vascular microenvironments and allowing platelets to act as immune cells. Previous reports show that platelets do endocytose viruses such as HIV-1 but the molecular machinery is ill-defined. In nucleated cells, responses to HIV-1 are mediated by virus phagocytosis/endocytosis, degradation to release Toll-like Receptor ligands, and subsequent receptor activation. Is this process recapitulated in platelets? Here we show that platelets indeed use VAMP-3 and Arf6-dependent pathways to endocytose HIV-1 virions, degrade retroviral particles to release TLR ligands, which initiate platelet activation and secretion. Consequently, HIV-1 uptake and subsequent activation is abolished in VAMP-3 and Arf6 KO mice. Collectively, our studies shed light on how platelets act at the early stage of pathogen recognition and are able to process them to initiate an immune response.

66

50 Association between Plasma Cholesterol Levels and the Development of Xanthomas, Malaise, and Inappetance in Cynomlgus Monkeys

Morgan Kelly 1 • Courtney Burkett 1 • Sierra Paxton 1 • Lei Cai 1 • Ryan Temel, PhD 1 1Cardiovascular Research Center, University of Kentucky

Undergraduate

Cardiovascular disease is the leading cause of death in the United States. There is a positive correlation between coronary heart disease (CHD) and LDL cholesterol (LDL-C) levels. Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events caused by atherosclerotic lesion rupture. Therefore, finding a therapy capable of regressing or stabilizing atherosclerotic lesions is a priority for academic and pharmaceutical researchers. By stimulating macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in mice causes aortic atherosclerotic lesions to acquire a more stable composition. However, the preclinical mouse studies have limited translational value since mice do not develop coronary artery atherosclerosis and express only one of the two miR-33 family members found in humans. In contrast, nonhuman primates (NHPs) have miR-33a and miR-33b and develop coronary artery atherosclerosis and thus are a good model to determine the therapeutic potential of miR-33 antagonism in humans. In order to determine whether miR-33a/b antagonism can regress or stabilize atherosclerotic lesions, 36 male cynomolgus monkeys were fed for 20 months a diet high in fat and cholesterol, which increased LDL-C and drove atherosclerosis formation. During this period, the animals developed xanthomas and callosities due to cholesterol deposition in the skin. In addition, some of the monkeys went through prolonged periods of malaise and inappetance. The objectives of this project were to review the veterinary records and determine 1) the number of days on atherogenic diet needed to first observe xanthomas and callosities and 2) the number of incidences of malaise and prolonged inappetance per monkey. This data was then correlated to the average total plasma cholesterol (TPC) concentration during the 20 months on atherogenic diet. A statistically significant, negative correlation was observed between the number of days to initial callosity formation and TPC (p= 0.049 and = 0.19). There were no statistically significant correlations between TPC and the other clinical observations. We are in the process of data collection but hypothesize that coronary artery atherosclerotic lesion size will be greater in animals that went through periods of malaise and inappetance. In addition, we hypothesize that the days for xanthoma/callosity formation will be inversely associated with plaque size. This information may allow our lab to predict atherosclerosis severity in monkeys based upon clinical observations.

67

51 Circadian disruption and atherosclerosis in ApolipoproteinE-deficient mice

Eric McGann 1 • Jeffery Chalfant, MS 1 • Deborah Howatt 2 • Julie Pendergast, PhD 1 1Biology, University of Kentucky • 2Saha Cardiovascular Research Center, University of Kentucky

Staff

The circadian system is a network of molecular clocks located throughout the body. These clocks coordinate daily rhythms of behavior and physiology with environmental cycles. Shift work, which chronically disrupts circadian rhythms, increases the risk of developing cardiovascular diseases. The mechanisms by which this happens, however, are largely unknown. Our long-term goal is to determine how disruption of circadian rhythms causes cardiovascular disease. Atherosclerosis is the build-up of plaques in arteries and can lead to myocardial infarction and stroke. In this study, we sought to investigate whether circadian disruption accelerates atherosclerosis in mice. Wild-type mice do not develop atherosclerotic lesions. Therefore, we studied C57BL/6J ApolipoproteinE-deficient (ApoE-/-) mice because they spontaneously develop atherosclerotic lesions. We first characterized circadian rhythms in ApoE-/- mice. We found that circadian behavioral rhythms, including free-running periods of activity in constant darkness and constant light, phase angles of entrainment, and phase shifts to light pulses, were similar between wild-type and ApoE-/- mice. These data show that ApoE-/- mice had no deficits in their circadian behavior rhythms or light responsiveness. Next, we determined if molecular circadian rhythms in tissues were altered in ApoE-/- mice. We analyzed the expression of the circadian timekeeping protein, PERIOD2, in central and peripheral tissues using a luciferase reporter. We found that PERIOD2::LUCIFERASE rhythms in tissues were similar in wild-type and ApoE-/- mice. We next determined the effect of constant light on the development of atherosclerosis in ApoE-/- mice. After 3 months in constant light, locomotor activity was arrhythmic or the rhythm was severely disrupted. In addition, atherosclerotic lesion area was increased in ApoE-/- mice in constant light compared to those in control 12h light-12h dark condition. Together, these data demonstrate that ApoE-/- mice have normal circadian rhythms and chronic circadian disruption accelerates atherosclerosis in ApoE-/- mice. This study was funded by National Institutes of Health grant P20GM103527, the Gertude F. Ribble Trust, and the University of Kentucky.

68

52 Does Light Pollution Affect the Development of Atherosclerosis?

Robert Wendroth 1 • Eric McGann 1 • Deborah Howatt 2 • Julie Pendergast, PhD 1 1Department of Biology, University of Kentucky • 2Saha Cardiovascular Research Center, University of Kentucky

Undergraduate

Exposure to light at night has been steadily increasing since industrialization. Light at night comes from light bulbs, light pollution produced by cities, and more recently from the widespread use of personal electronics (smart phones, tablets, laptops) at night. Previous studies in outbred mice have shown that dim light at night (DLAN) disrupts circadian rhythms and increases body weight, but it is unknown whether DLAN affects the development of cardiovascular disease. In this project, we are testing the hypothesis that DLAN accelerates the development of atherosclerotic lesions in C57BL/6J Apolipoprotein E-deficient (ApoE-/-) mice. We single-housed male ApoE-/- mice in light-tight boxes at 7 weeks of age for 1 week in 12h light:12h dark (12L:12D) and then in DLAN (12h light: 12h dim 5 lux light) for 12 weeks. Control mice were maintained in 12L:12D for 13 weeks. Body weights did not differ between ApoE-/- mice in 12L:12D and DLAN. In 12L:12D, ApoE-/- mice had a robust rhythm of eating behavior that peaked during the night. However, after a few days in DLAN, ApoE-/- mice had a low-amplitude eating behavior rhythm. Preliminary data also suggest that ApoE-/- mice in DLAN have increased atherosclerotic lesion area compared to ApoE-/- mice in 12L:12D. These data suggest that DLAN disrupts eating behavior rhythms and may also increase atherosclerosis.

69

53 TRAF3 negatively regulates platelet activation and thrombosis

Rui Zhang, PhD 1 • Guoying Zhang, MD 1 • Binggang Xiang, MD, PhD 1 • Xiaofeng Cheng, MD, PhD 2 • Lijang Tang, MD 3 • Shaojun Shi, MD 4 • Yani Liu, MD 4 • Xun Ai, PhD 5 • Ping Xie, PhD 6 • Zhenyu Li, MD, PhD 1 1Division of Cardiovascular Medicine, University of Kentucky • 2Taizhou Hospital, Wenzhou Medical University • 3Department of Cardiology, Zhejiang Hospital • 4Department of Pharmacy, Huazhong University of Science and Technology • 5Department of Physiology and Biophysics, Rush University • 66Department of Cell Biology and Neuroscience, Rutgers University

Faculty

CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelets also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin αIIbβ3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.

70

54 Impact of miR-33 antagonism on metabolic parameters in nonhuman primates

Tara E. Keenan 1 • Tong Li, MD 1 • Lei Cai, PhD 1 • Sierra M. Paxton 1 • Courtney R. Burkett 1 • Peter I. Hecker 1 • Ryan E. Temel, PhD 1 1Saha Cardiovascular Research Center and Department of Pharmacology and Nutritional Sciences, University of Kentucky

Undergraduate

Introduction: MicroRNA-33a (miR-33a) and miR-33b regulate lipid homeostasis by inducing degradation or blocking translation of mRNAs encoding proteins that control cholesterol efflux and fatty acid oxidation. By stimulating macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization, antagonism of miR-33a in mice reduces atherosclerotic. However, some mouse studies have shown that disruption of miR-33 function has negative metabolic effects such as increased plasma and hepatic triglycerides. Mouse studies have limited translational value since mice express only one of the two miR-33 family members found in humans. Since nonhuman primates (NHPs) have miR-33a and miR-33b, NHPs are the best preclinical model for determining the therapeutic potential of miR-33 antagonism. The objective of this project was to determine whether miR-33 antagonism has adverse metabolic effects on NHPs. Methods: Liver was collected via laparotomy from 12 chow fed male cynomolgus monkeys. Male cynomolgus monkeys (n=36) were fed for 20 months a high fat/high cholesterol diet, which caused hypercholesterolemia and atherosclerosis development. At the end of the 20-month progression phase, a subset of monkeys (n=12) were euthanized to collect tissues. The remaining monkeys were switched to a cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-33 (n=12) for 6 months. Blood was collected at 20 months of progression and 2, 4, and 6 months of regression and the serum was sent to ANTECH Diagnostics for chemical analysis. Liver lipid content was biochemically determined. Results: Feeding the monkeys a chow diet for 6 months caused serum triglycerides (TG) to significantly increase and total cholesterol (TC) to significantly decrease. However, serum TG and TC were similar for the vehicle and anti-miR-33 treated monkeys. The progression group had significantly greater hepatic TC and TG compared to the regression groups. Hepatic TC and TG levels of the vehicle and anti-miR-33 groups were similar and had returned to the baseline levels observed in monkeys fed only chow. In line with the liver lipids changes, serum ALT and AST levels were reduced to a similar extent in the regression NHPs. In addition, body weights and serum glucose concentrations were similar for the vehicle and anti-miR-33 treated NHPs during the 6-month regression phase. Conclusion: Metabolic parameters are normal in chow-fed NHPs treated with miR-33 antagonist.

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55 ATF6 proteins regulate cardiac hypertrophy during pressure overload in the mouse heart

Robert Correll, PhD 1 • Jeffrey Lynch, PhD 1 • Michelle Sargent 1 • Allen York 1 • Jeffery Molkentin, PhD 1 1Molecular Cardiovascular Biology, Cincinnati Children's Hospital

Postdoc

Objective: The activating transcription factor 6 (ATF6) branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) plays a critical protective role in the heart’s response to acute injury or long-term hemodynamic stress that results in cardiac hypertrophy. Here we seek to elucidate the molecular mechanisms by which engagement of ATF6 proteins results in protective signaling in the context of pressure overload in the mouse heart. Methods and Results: Overexpression of thrombospondin-4 (Thbs4) results in a protective ER stress response and mice gene-deleted for Thbs4 demonstrated compromised ER stress signaling, inhibition of ATF6α processing and nuclear localization, and decreased survival after transverse aortic constriction (TAC) or myocardial infarction (MI) surgery. Using gene-deleted mice lacking ATF6α we demonstrate that Thbs4-mediated upregulation of ER protein chaperone expression and expansion of the ER compartment requires ATF6α expression. Furthermore, mice lacking ATF6α or the related protein ATF6β show defects in the hypertrophic response after pressure overload, leading to accelerated decompensation and failure after long-term TAC surgery, accompanied by reduced expression of ER protein chaperones. Conclusion: We find that both ATF6α and ATF6β proteins are required for compensatory hypertrophy and ER chaperone mobilization following pressure overload in the mouse heart. We hypothesize that the inability to upregulate chaperone expression during pressure overload results in diminished ER protein folding capacity that impairs the hypertrophic response. These results position ATF6 proteins as essential regulators of compensatory cardiac hypertrophy following chronic hemodynamic stress.

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56 Isolation and characterization of primary bone marrow mesenchymal stem cells.

Eman El-sawalhy, MD 1 • Lakshman Chelvarajan, PhD 2 • Ahmed Abdel-Latif, MD, PhD 1 1Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky • 2Saha cardiovascular research center, University of Kentucky

Postdoc

•Introduction: Myocardial infarction remains a major clinical problem and the leading cause of mortality in the world. Bone marrow derived stem cells have the capacity to participate in cardiac repair and regeneration of compromised heart muscle. The aim of this study is to generate highly enriched mesenchymal stem cells to inject 3x106 into the hearts of mice recovering from a myocardial infarction. •Methods: We isolated bone marrow (BM) from crushed long bones and hip bones of 6-8 weeks old GFP positive mice (C57BL/6). The stem cells were cultured in MesenCult media supplemented with MesenPure. Cells were cultured under either normoxic or hypoxic conditions (4% O2) for about two weeks. The cultures were then passaged when they reached 80% confluence. The cells were analyzed by flow cytometry using mesenchymal stem cell (CD90.2 and SCA-1) and hematopoietic (CD45) markers. •Results: As expected, the baseline bone marrow cells were negative for both stem cell markers, CD90 and Sca-1, while expressing the hematopoietic marker, CD45 (80%). During early expansion all the CD45– cells were Sca1+CD90–. Following subsequent passaging of the enriched cells, they began to express CD90. By the end of the second passage, the proportion of Sca1+CD90+ mesenchymal stem cells was 65%, while cells expressing Sca-1 alone was down to 32%. Most of the live healthy cells remained GFP+ throughout the expansion and passaging. Conclusion: Mesenchymal stem cells can be isolated and enriched from murine bone marrow in sufficient amounts to be used in our in vivo study to enhance regeneration of cardiomyocytes and restore cardiac function after myocardial infarction.

73

57 Fine Tuning Platelet Secretion to Modulate Hemostasis

Smita Joshi, MS 1 • Irina Pokrovskaya, MS 2 • Brian Storrie, PhD 2 • Sidney W. Whiteheart, PhD 1 1Molecular and Cellular Biochemistry, University of Kentucky • 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences

Graduate Student

Globally, occlusive thrombotic events: e.g., heart attacks and cerebral strokes, cause > 50% of total deaths attributed to the noninfectious disease. However, aggressive attempts to limit thrombosis cause bleeding, which can be equally catastrophic. What is needed is a strategy to limit clot formation, but not prevent it. Platelets play a critical role in controlling bleeding by sensing vascular damage and releasing a host of components to seal breaches. This secretion process is mediated by Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptors (SNAREs) and their regulators. To drive secretion, vesicle (v)-SNARE on granules and target (t)-SNARE on the plasma membrane (PM) form a trans-bilayer complex that mediates membrane fusion. Syntaxin 11 and SNAP-23 form the functionally relevant t-SNARE heterodimer. For v-SNAREs, platelets contain Vesicle-Associated Membrane Protein (VAMP)-2, -3, -4, -5, -7, and -8. We focused on how the platelet VAMPs influence secretion and whether modulating secretion can modulate clot formation. To address this goal, we genetically titrated the different VAMPs to define their roles in exocytosis and hemostasis. We gathered global VAMP-3-/- and VAMP-8-/- animals. To overcome embryonic lethality of global VAMP-2 deletion, we generated platelet-specific VAMP-2/3-/- mice by using a tissue-specific promoter that facilitates expression of tetanus toxin that cleaves VAMP-2 and 3. We crossed these with VAMP-8-/- mice to create platelet-specific VAMP-2/3/8-/- mice. Structural analysis of wild-type and VAMP-deficient platelets showed that the α granule cargo solubilization/decondensation follows granule fusion. To define the structure of secretion, activation intermediates were fixed at various time points, post stimulation, and electron microscopy was performed. The data indicate that granule decondensation is time- and agonist concentration-dependent. Moreover, decondensation of granule cargo was VAMP dependent. Three dimensional EM analyses indicate that VAMP-8 plays a major role in compound, intra-granule fusion and also contributes to single, granule-PM fusion. Our structural data elucidate how platelet secretion occurs at the cellular level and explains the complex secretion kinetics previously reported in activated platelets. To further measure the functional importance of the VAMPs, ex vivo secretion assays were used to monitor the kinetics and the extent of release from all three platelet granules (dense, α, and lysosomes). Only VAMP-2/3/8-/- platelets showed a robust defect in secretion (~70% decrease), more than observed for VAMP-8-/- platelets (~50%). When we studied the effects of secretion on hemostasis, only VAMP-2/3/8-/- mice showed significantly increased tail-bleeding times and delayed arterial thrombosis. VAMP-8-/- animals did show a delay in thrombus formation but no overt bleeding diathesis. Our data show that small differences in secretion kinetics alter hemostasis, thus by modulating platelet secretion, we can control thrombus formation without inducing pathological bleeding. These data identify the secretory machinery as a viable target to control occlusive cardiovascular diseases. Our work is the first comprehensive study showing how by targeting secretion we can achieve the long-sought balance between occlusive thrombosis and spurious hemostasis. Additionally, by titrating amounts and types of VAMPs in platelets we have created a valuable set of animals to precisely analyze the role of platelet secretion in other vascular processes.

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58 The XY sex chromosome complement augments Ang-II induced aortic arch aneurysms in female LDLr-/- mice

Cassandra Woolley 1 • Yasir Al-Siraj 1 • Sean Thatcher, PhD 1 • Lisa Cassis, PhD 1 1Department of Pharmacology and Nutritional Sciences, University of Kentucky

Undergraduate

Objective: Sex difference in cardiovascular disease has been a subject of research efforts aiming to improve the efficacy and approach of therapeutics available. In humans, abdominal aortic aneurysms (AAAs) display sexual dimorphism, with higher risk for development in males but with females exhibiting more rapid AAA growth rates and AAA ruptures at smaller aneurysm sizes. Previous studies performed in our laboratory have demonstrated that testosterone increases incidence and rupture rate of angiotensin II (AngII)-induced AAA in hyperlipidemic mice. After establishing this relationship, the four-core mouse model was used to analyze the role of sex chromosomes separate from sex hormones in aneurysm pathology. Female XY LDLr-/- mice are more susceptible to AngII-induced AAA than female XX LDLr-/- mice. The XY chromosome complement demonstrated more severe pathology and higher rupture rate. However, studies examining the role of sex chromosomes in the aortic arch between XX and XY female mice have not been performed. The purpose of this preliminary study was to observe whether differences were present between female XX and XY mice in formation of the aortic arch aneurysm. Methods and Results: Female XX and XY LDLr-/- mice were placed on a Western diet (TD.88137) one week prior to implantation of a 28-day AngII osmotic pump. The pumps administered AngII (1,000 ng/kg/min) for 28 days to induce aneurysm formation. The body weight of the mice was recorded weekly. Baseline measurements of the aortic arch diameter were made at D0 using ultrasound and found to be significantly different between XX and XY females (XX, 1.4 ± 0.03; XY, 1.5 ± 0.05 mm; p<0.05). Ultrasound was also used to measure the aortic arch diameter at D7 with a significant difference observed (XX, 1.5 ± 0.06; XY, 1.7 ± 0.06 mm; p<0.05). A subsequent measurement was made at D27, though no significant difference was found. We believe this lack of significance was due to the increased rupture rate and lower survival of female XY mice compared to the female XX mice (XX, 71% survival; XY 33% survival; p=0.2). After conclusion of the study, ex-vivo measurements of the arch aortic diameter and abdominal aortic diameter were performed after perfusion-fixing the aortas of the mice (XX, 1.4 ± 0.08; XY, 1.7 ± 0.17 mm; p=0.1). Measurements of abdominal aortic diameter were consistent with findings in previous studies. Conclusions: Not many studies have been performed analyzing sex chromosome effect in aneurysm pathology. The results of this preliminary study suggest positive correlation of XY chromosome complement with aortic arch aneurysm formation. Combined with results of previous studies, this suggests the XY complement is influencing overall aortic aneurysm susceptibility. In the future, it would be necessary to comprehensively analyze potential causes of this observed sex-chromosome difference.

75

59 Multilevel Mechanical Testing of Cardiac Valves

Arielle Waller 1 • Daniel Perry 1 • Luke Knudson 1 • RS Baker 2 • David Morales, MD 3 • Farhan Zafar, MD 2 • Daria Narmoneva, PhD 1 1Biomedical Engineering, University of Cincinnati • 2Division of Pediatric Cardiothoracic Surgery, The Heart Institute, Cincinnati Children's Hospital • 3Division of Pediatric Cardiothoracic Surgery, The Heart Institute, Cincinnati Children's Hospital

Graduate Student

The lack of adequate heart valve replacement options represents a major problem in pediatric care, where multiple operations are required to accommodate patient growth. Bioengineered valves may provide a viable alternative to existing strategies. Knowledge of the relationship between macro- and micro-biomechanical properties and functional behavior of native or engineered valve tissue is critical for success of bioengineered valves, yet it is still poorly understood. Here, a tubular tricuspid valve bioprosthesis made of small intestinal submucosa-derived extracellular matrix (SIS-ECM) was used as a valve replacement in the in vivo sheep model. Current study developed distinct multilevel testing approaches to determine valve tissue biomechanical properties and quantify the structure-function and behaviors. The following analyses were performed: (i) tissue/organ level uniaxial testing of valve samples; (ii) microanalyses (cellular/subcellular level) of adjacent full-thickness valve sections using atomic force microscopy (AFM) to determine valve mechanical properties. The AFM approach was used in combination with MATLAB-based quantitative 2D histological mapping of valve matrix content (collagen & proteoglycans), for functional analyses of biomechanical behavior. Results demonstrated that valve tensile elastic modulus was higher for the valve annulus vs. the cusp, consistent with proteoglycan rich regions on the distal end of the cusp, and a collagen rich region on the proximal end of the cusp and fibrosa. Compression stiffness from AFM testing demonstrated an increased Young’s modulus for the fibrosa vs. spongiosa, with intermediate values for atrialis (mitral and tricuspid) and ventricularis (aortic and pulmonary), consistent with a collagen-rich fibrosa, proteoglycan-rich spongiosa, and elastin-rich atrialis/ventricularis. In summary, this demonstrates feasibility of this multilevel testing approach in functional assessment of bioengineered valve and its ability to provide necessary mechanical function immediately upon implantation, and to attain structural and biomechanical properties of the native tissue during growth and remodeling.

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60

CHROME: a Long Non-coding RNA that Regulates Cholesterol Homeostasis

Kathryn Moore, PhD 1 1Medicine, New York University

Faculty

Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, many of which are not conserved in lower mammals. The majority of these lncRNAs remain functionally uncharacterized and may have important implications in human physiology and disease. We identified a primate-specific lncRNA, CHROME, which is increased in the plasma and atherosclerotic plaques of individuals with coronary artery disease compared to healthy controls. Using gain- and loss-of-function approaches, we show that CHROME functions as a competing endogenous RNA of microRNAs (miRNAs) that repress cellular cholesterol efflux and plasma high density lipoproteins (HDL) levels, and regulates the concentration and biological functions of these miRNAs. CHROME knockdown in primary hepatocytes increases levels of its miRNA binding partners, thereby reducing expression of their target gene networks, hepatic cholesterol and phospholipid efflux, and the formation of nascent HDL particles. Consistent with these findings, hepatic levels of CHROME are positively correlated with plasma levels of HDL cholesterol in healthy individuals. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

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61 Secretory phospholipase A2 group IIA (PLA2G2A) enhances metabolism and insulin sensitivity

Michael Kuefner 1 • Kevin Pham 1 • JeAnna Redd 2 • Erin Stephenson, PhD 3 • Innocence Harvey 2 • Xiong Deng, PhD 1 • Dave Bridges, PhD 2 • Eric Boilard, PhD 4 • Marshal Elam, MD, PhD 1 • Edwards Park, PhD 1 1Pharmacology, University of Tennessee Health Science Center • 2Nutritional Sciences, University of Michigan School of Public Health • 3Physiology, University of Tennessee Health Science Center • 4Infectious Diseases and Immunity, CHUQ Research Center and Division of Rheumatology

Graduate Student

Secretory phospholipase A2 group IIA (PLA2G2A) is a member of a family of secretory phospholipases that have been implicated in inflammation, atherogenesis, and antibacterial actions. Here, we evaluated the role of PLA2G2A in the metabolic response to a high fat diet. C57BL/6 (BL/6) mice do not express PLA2g2a due to a frameshift mutation. We fed BL/6 mice expressing the human PLA2G2A gene (IIA+ mice) a fat diet and assessed the physiologic response. After 10 weeks on the high fat diet, the BL/6 mice were obese, but the IIA+ mice did not gain weight or accumulate lipid. The lean mass in chow- and high fat-fed IIA+ mice was constant and similar to the BL/6 mice on a chow diet. Surprisingly, the IIA+ mice had an elevated metabolic rate, which was not due to differences in physical activity. The IIA+ mice were more insulin sensitive and glucose tolerant than the BL/6 mice, even when the IIA+ mice were provided the high fat diet. The IIA+ mice had increased expression of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), and PPARγ coactivator 1α (PGC-1α) in brown adipose tissue (BAT), suggesting that PLA2G2A activates mitochondrial uncoupling in BAT. Our data indicate that PLA2G2A has a previously undiscovered impact on insulin sensitivity and metabolism.

78

62 Making Good: Secretory Quality Control in Lipoprotein Lipase Trafficking

Benjamin Roberts 1 • Saskia Neher, PhD 1 1Biochemistry and Biophysics, UNC Chapel Hill

Graduate Studnet

Lipoprotein lipase (LPL) is an essential vascular regulator of serum triglycerides. Patients without LPL activity suffer from hypertriglyceridemia. Chronic elevated blood triglycerides increase the risks of acute pancreatitis, atherosclerosis, and cardiac disease. There is no cure for LPL deficiency available in the United States. Of the over 70 clinical LPL clinical mutations, some affect intracellular LPL trafficking. It is clear that misfolded LPL is subject to post-Endoplasmic Reticulum (ER) quality control, which results in its degradation. The regulation of this process and the LPL partners that promote LPL degradation are not well understood. To study atypical LPL trafficking we fluorescently tagged two clinically-identified LPL variants to follow intracellular LPL transport. We also characterized the trafficking of WT LPL produced in cells lacking its folding chaperone Lipase Maturation Factor 1. Finally, we used mass spectrometry to search for intracellular binding partners unique to misfolded LPL. Using these techniques, we quantified post-ER quality control of folded and misfolded LPL. These studies enhance our understanding of the regulation in LPL trafficking and help to explain the consequences of some LPL mutations resulting in LPL deficiency.

79

63 Gut Microbial Trimethylamine (TMA) Lyase Activity Coordinates Circadian Rhythms in Host Hepatic Lipid and Bile Acid Metabolism

Christy Gliniak, PhD 1 • Rebecca Schugar, PhD 1 • Robert Helsley, PhD 1 • Anthony Gromovsky 1 • Chase Neumann 1 • Zeneng Wang, PhD 1 • Stanley Hazen, MD, PhD 2 • J. Mark Brown, PhD 2 1Department of Cellular and Molecular Medicine, Cleveland Clinic • 2Department of Cellular and Molecular Medicine, Center for Microbiome & Human Health, Cleveland Clinic

Faculty

From cyanobacteria to humans, circadian rhythms evolved to allow an organism to adapt and anticipate environmental cues, particularly events that regulate energy metabolism. Misalignment of circadian rhythms are associated with increased incidence of obesity, diabetes, cardiovascular disease, cancer, and other inflammatory disorders. Intestinal microbial composition and structure displays circadian rhythmicity, and the gut microbiome itself can regulate host endogenous circadian rhythms. However, it is not well understood how gut microbes regulate host metabolism and circadian rhythms. Gut microbes contribute to the production of the circulating metabolite trimethlyamine-N-oxide (TMAO), which is associated with cardiovascular disease in humans, and has been shown to enhance atherosclerosis and thrombosis potential in mice. Microbes produce trimethlyamine (TMA) from dietary choline or carnitine, which is later converted by the host to TMAO, primarily by host liver flavin monooxygenase 3 (FMO3). Previous studies have shown that pharmacologic inhibition of microbial TMA lyase activity or inhibition of FMO3 results in decreased atherosclerosis in mice, and the expression of the TMAO producing enzyme FMO3 exhibits circadian rhythmic patterns in the liver. Here we hypothesized that the co-metabolites TMA and TMAO may serve as gut microbe-derived signals that entrain host metabolic circadian rhythms, thereby impacting cardiometabolic disease. To test this we treated C57BL/6 mice with a highly potent and selective second-generation TMA lyase inhibitor CC08, and examined effects on circadian rhythms in host metabolism. As expected, mice treated with CC08 displayed reduced plasma TMA/TMAO levels across an entire 24-hour period. Unexpectedly, TMA lyase inhibition resulted in increased expression of the key nuclear receptors that regulate the circadian clock including Bmal1 (Arntl1) and Rev-erbα (Nr1d1) during the light cycle in the liver. The well known circadian cycling of transcription factors orchestrating hepatic fatty acid metabolism such as Srebp1c and Pparα were markedly altered in CC08-treated mice. TMA lyase inhibition resulted in elevated hepatic expression of Srebp1c during the light cycle, yet suppressed hepatic Pparα expression during the dark cycle. Furthermore, the expression of the bile acid-sensing nuclear receptor Fxr was lower in TMA lyase-inhibited animals during the dark cycle, which is associated with alterations in hepatic Fxr target gene expression and circulating bile acid levels. Collectively, these data suggest that pharmacologic inhibition of gut microbial TMA lyase activity can alter host transcriptional programs that dictate hepatic circadian rhythms in lipid and bile acid metabolism. These data provide the first clues into mechanisms by which TMA lyase inhibitors may protect mice against cardiometabolic disease.

80

64 Thrombospondin 1 (TSP1) Deficiency Protects Against Diet-Induced Fatty Liver Disease

Courtney Turpin, MS 1 • Heather Norman-Burgdolf, PhD 2 • Ling Yao, MS 1 • Yanzhang Li, PhD 3 • Shuxia Wang, MD, PhD 1 1Department of Pharmacology and Nutritional Sciences, University of Kentucky • 2Department of Dietetics and Human Nutrition, University of Kentucky • 3Department of Internal Medicine, University of Kentucky

Graduate Student

Background: Non-alcoholic fatty liver disease (NAFLD) is a widespread disease, affecting around 20-30% of American adults with prevalence on the rise in many age groups. Obesity is thought to play a contributing role in the occurrence of this disease. NAFLD progression includes an initial state lipid accumulation, which is worsened by additional stresses. Thrombospondin 1 (TSP1) is a secreted matricellular protein that has been implicated in playing a role in causing obesity-associated inflammation, lipid accumulation, and fibrosis in various tissues, but TSP1 expression in the liver and its possible role in pathogenesis are not known. Methods: Eight-week-old control and TSP1 deficient mice were fed at either a low-fat (10% kcal from fat) or high-fat (60%kcal from fat) for 16 weeks. Liver histology, gene expression, and immunohistochemistry were performed to observe the effect of TSP1 deficiency on the liver physiology. Results: TSP1 expression was found to be significantly increased in control high-fat diet fed mice liver tissue. Liver histology showed less lipid accumulation in the TSP1 deficient mice fed a high-fat diet, which was further confirmed by reduced triglyceride levels. Although there was no significant change fatty acid uptake in the whole liver, there was a significant decrease in CD36 expression in the hepatocytes of the deficient mice, which also had reduced fatty acid uptake. There was also a significant decrease in de novo lipogenesis genes PPARγ and SREBP1c, but with normal feeding, there was no significant change in triglyceride production or secretion. In addition to the changes in lipid content, there was a reduction in inflammatory markers and the fibrosis marker Collagen1α1, but there were no observed changes in fibrosis histology staining. Discussion and implications: The findings of this study suggest that TSP1 expression could play a role in the development of the hallmark signs of NAFLD. Further studies using a NAFLD animal model and cell culture could help elucidate the mechanism of its involvement. In summary, the data suggests that TSP1 has involvement in lipid accumulation and the resulting inflammation and fibrosis that are seen in progressed disease states of NAFLD.

81

65

An obesity-generating diet drives the cancer stem cell phenotype and glioblastoma progression.

Daniel J Silver, PhD 1 • Gustavo A Roversi 1 • Anthony Gromovsky 1 • J Mark Brown, PhD 1 • Justin D Lathia, PhD 1 1Cellular and Molecular Medicine, Cleveland Clinic

Staff

Glioblastoma (GBM) is the most prevalent and lethal brain cancer. The disease occurs in two to three per 100,000 adults annually and accounts for approximately half of all brain cancers. While there are no known causes for GBM, obesity is an established risk factor for cancer in general. Recent work confirmed that overweight and obese women are at greater risk of developing glioma compared to women of healthy body weight. This finding represents a major change in the consideration of body mass for brain cancer patients, a factor that has been largely ignored in the clinic. Furthermore, it is well-established that consumption of a Western-pattern diet leads to increased rates of obesity. For these reasons, we hypothesized that obesity accelerates GBM progression by driving tumor cell proliferation and/or cancer stem cell enrichment. We have tested this hypothesis in vivo using three syngeneic glioma models transplanted into the brains of immune-competent C57BL/6 mice. Mice were maintained on either an obesity-generating, high-fat diet (HFD) or a standard rodent chow diet. Tumor-bearing mice fed the HFD succumbed to disease nearly two-fold faster than those fed the chow diet. For example, mice transplanted with the GL261 syngeneic glioma model and maintained on HFD were culled, on average, 16 days earlier than those maintained on chow. Furthermore, we noted a nearly three-fold increase in the frequency of tumor generation in obese animals compared to lean, suggesting that systemic obesity enriches for a tumor-initiating cancer stem cell population in the brain. Tumor cells treated directly with oleic acid or linoleic acid in vitro demonstrated increased viability and enhanced sphere formation compared to vehicle controls. These data indicated that certain lipid components of the obesity-generating diet were sufficient to drive tumor cell proliferation and the emergence of the cancer stem cell state. In order to identify the set of lipid species contributing to accelerated disease progression, we profiled the major lipids present in tumors of obese mice compared to tumors of lean mice using untargeted lipidomics. This analysis revealed four putative oncogenic lipids. These species were robustly expressed in tumors resected from obese mice compared to tumors resected from lean mice. We have additionally identified two potentially tumor-suppressive lipids that were expressed strongly in the contralateral hemisphere of lean mice compared to tumors resected from either lean or obese mice. This work confirms a shift in the lipid profile of tumors developing in the brains of the obese mice and suggests that select lipid species may directly drive the increased tumor cell proliferation and enhancement in the cancer stem cell compartment that we observe in vivo.

82

66

Serum Amyloid A3 is a High Density Lipoprotein-associated Acute Phase Protein

Maria De Beer, PhD 1 • Ailing Ji, PhD 2 • Victoria Noffsinger 2 • Preetha Shridas, PhD 2 • Frederick De Beer, MD 2 • Lisa Tannock, MD 2 • Nancy Webb, PhD 2 1Physiology, University of Kentucky • 2Internal Medicine, University of Kentucky

Faculty

Acute phase serum amyloid (SAA) is a family of evolutionarily conserved, secreted proteins that exerts innate functions relevant to obesity and diabetes. In humans, two SAA isoforms (SAA1 and SAA2) are highly induced in the liver and extrahepatic tissues under the regulation of inflammatory cytokines. During severe inflammation, SAA1/2 levels can increase >1000-fold in plasma, where it is found associated with HDL. Mice produce an additional acute phase SAA, SAA3, which is thought to be produced mainly by adipocytes and macrophages and has not previously been found circulating on HDL. Objectives: The goal of this study was to investigate whether SAA3 serves as a third liver-derived, HDL-associated acute phase SAA in mice. Methods: Using isoform-specific oligonucleotide primers for qRT-PCR, we determined that SAA3 is transcriptionally induced to a similar extent (~2500-fold) compared to SAA1.1/2.1 (~6000-fold) in livers of C57BL/6 mice 19 hr after lipopolysaccharide (LPS) injection (100 µg/mouse). SAAs were also robustly induced in fat tissue (SAA1/2~100-fold; SAA3~400-fold). The analysis of primary mouse hepatocytes and in situ hybridization of mouse liver sections indicated that liver-derived SAAs are produced by hepatocytes and not other stromal cells, including Kupffer cells. All 3 SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ~20% of SAA1/2. After separation by FPLC, virtually all of plasma SAA1/2 eluted with the HDL fraction, whereas ~15% of plasma SAA3 appeared to be lipid poor/free. HDL isolated from acute phase mouse plasma by density gradient ultracentrifugation was subjected to isoelectric focusing to determine the relative recovery of the various SAA isoforms. Whereas the bulk of plasma SAA1.1 was found in the d=1.063-1.21 fraction, only ~50% of SAA2.1 and ~10% of SAA3 was recovered after ultracentrifugation. These findings suggest that SAA3 may be more loosely associated with HDL compared to SAA1.1/2.1, which may give rise to lipid poor/free SAA3 that is susceptible to more rapid clearance in vivo. Conclusions: We conclude that SAA3 is a major hepatic acute phase SAA in mice that may produce systemic effects during inflammation. Future studies investigating SAA pathobiology in mice must take into account the previously under-studied SAA3.

83

67 An XX Sex Chromosome Complement Promotes Hypercholesterolemia and Atherosclerosis in Ldlr-/- Mice Fed Western Diet

Yasir Alsiraj, MS 1 • Sean Thatcher, PhD 1 • Lei Cai, PhD 2 • Ryan Temel, PhD 2 • Patrick Tso, PhD 3 • Lisa Cassis, PhD 1 1Pharmacology and Nutritional Sciences, University of Kentucky • 2Saha Cardiovascular Research Center, University of Kentucky • 3Department of Pathology and Laboratory Medicine, University of Cincinnati

Graduate Student

Objectives: Sex hormones are primary contributors to sexual dimorphism of cardiovascular diseases, but little is known about the influence of genes on sex chromosomes as mediators of cardiovascular sex differences. In this study, we hypothesized that genes on sex chromosomes influence the development of hypercholesterolemia and atherosclerosis. Methods and Results: Transgenic male mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were bred to female Ldlr-/- mice to generate female and male mice with an XX or an XY sex chromosome complement. Male (M) and female (F) mice were fed a Western diet (Teklad TD88137) for 3 months. XX mice exhibited increased body weights compared to XY mice, regardless of gonadal sex (FXX, 38 ± 1.8; FXY, 30.6 ± 1.3 g; P<0.05; MXX, 51.5 ± 1.2; MXY, 41.7 ± 1.8 g; P<0.05), and this difference was associated with increased energy intake in XX (M or F) mice. XX mice had increased serum cholesterol concentrations regardless of gonadal sex (FXX, 2175 ± 174; MXX, 3150 ± 370; MXY, 1149 ± 227; FXY, 776 ± 128 mg/dl; P<0.05). Fat absorption was increased significantly in XX compared to XY Ldlr-/- mice challenged acutely with a 5% sucrose polybehenate diet (FXX, 93.9% ± 3.6; MXX, 92.6% ± 3.3 vs. FXY, 89.4% ± 3.2; MXY, 89.4% ± 4.3; p<0.05). However, neutral fecal sterol was not different between groups. VLDL production was influenced by gonadal sex (higher in M than F), but not by genotype in Ldlr-/- mice fed standard murine diet. Rather, intestinal mRNA abundance of diacylglycerol O-acyltransferase 2 (DGAT2) and microsomal triglyceride transfer protein (MTTP) were increased significantly in gonadectomized XX compared to XY mice (M or F). The extent of atherosclerosis in aortic arch was significantly increased in XX compared to XY mice, regardless of gonadal sex (FXX, 37 ± 2.1; MXX, 38 ± 3.6; MXY, 24 ± 3.6; FXY, 20 ± 3.2;; % lesion surface area; P<0.05). Similar effects were observed in aortic sinus. Conclusion: Results suggest that an XX sex chromosome complement promotes Western diet-induced hypercholesterolemia and hypertriglyceridemia, contributing to increased atherosclerotic lesion formation. Increased expression of intestinal genes mediating fat absorption and triglyceride-rich lipoprotein assembly may contribute to increased atherosclerosis of XX mice. Funding: These studies were supported by the National Institutes of Health Heart Lung and Blood Institute (R01 HL107326; LC), NIDDK (5U24DK059630), and from the American Heart Association (YA; predoctoral fellowship 14PRE20030018).

84

68 Adipocyte deficiency of ACE2 increases systolic blood pressures of obese female C57BL/6 mice

Robin Shoemaker, PhD 1 • Wen Su, MD 2 • Ming Gong, PhD 2 • Lisa Cassis, PhD 1 1Pharmacology and Nutritional Sciences, University of Kentucky • 2Physiology, University of Kentucky

Postdoc

Background: We demonstrated that sexual dimorphism of obesity-hypertension was associated with differential activity of adipocyte angiotensin-converting enzyme 2 (ACE2) in male versus (vs) female mice. These data suggest that adipocyte ACE2 regulates blood pressure by influencing the balance of angiotensin II (AngII, a substrate of ACE2) vs angiotensin-(1-7) (Ang-(1-7)), which differs in male and female mice. We hypothesized that deficiency of ACE2 in adipocytes increases blood pressure of HF-fed female mice. Methods/Results: Mice with adipocyte ACE2 deficiency were developed from breeding Ace2fl/fl mice to transgenic C57BL/6 mice with heterozygous transgenic expression of Cre recombinase driven by the adiponectin promoter (Ace2Adipo). Female or male Ace2fl/fl and Ace2Adipo mice (8 weeks old) were fed a HF (60% kcal as fat) or low fat (LF; 10% kcal from fat) diet for 16 weeks, after which blood pressure was quantified by radiotelemetry. Systolic blood pressures (SBP) were not different in LF-fed female mice of either genotype (24 hr average SBP [mmHg]: Ace2fl./fl: 121+/-1; Ace2Adipo: 121+/-1; p>0.05). However, SBP was significantly increased in HF-fed female Ace2Adipo vs Ace2fl/fl mice (Ace2fl/fl: 127+/-2; Ace2Adipo: 133+/-3; p<0.05). As AngII is an ACE2 substrate, we quantified blood pressure responses of female mice to an acute challenge of AngII (20µg/kg body weight, sc). AngII administration increased SBPs of LF and HF-fed female mice of both genotypes. However, SBP responses to AngII were augmented in HF-fed Ace2Adipo female mice compared to HF-fed Ace2fl/fl controls (baseline vs AngII SBP [mmHg]: LF Ace2fl/fl: 122+/-8 vs 180+/-4; LF Ace2Adipo: 127+/-5 vs 173+/-7; HF Ace2fl/fl: 121+/-3 vs 183+/-3; HF Ace2Adipo: 138+/-7 vs 197+/-43; p<0.05). In contrast to females, HF-fed Ace2Adipo male mice had similar SBPs compared to HF-fed Ace2fl/fl males (24 hour SBP [mmHg]: Ace2fl/fl: 127+/-2; Ace2Adipo: 133+/-3; p>0.05). Conclusions: Deficiency of ACE2 in adipocytes promoted the development of obesity-induced hypertension in female mice. Moreover, female adipocyte ACE2 deficient mice exhibited augmented blood pressure responses to AngII. These data support the hypothesis that adipocyte ACE2 contributes to sex differences in obesity-induced hypertension.

85

69 Fish oil-derived long-chain monounsaturated fatty acid (LCMUFA) for cardiovascular diseases : a novel approach for cardioprotection

Zhihong Yang, PhD 1 • Scott Gordon, PhD 1 • Milton Pryor 1 • Shuibang Wang, PhD 2 • Robert Danner, MD, PhD 2 • Alan Remaley, MD, PhD 1 1Lipoprotein Metabolism Section, CPB/NHLBI, National Institutes of Health • 2Critical Care Medicine Department, Clinical Center, National Institutes of Health

Postdoc

Cardiovascular disease (CVD) remains the leading cause of death, disability, and healthcare expense in the United States and is also a major healthcare problem worldwide. Numerous studies have shown cardiovascular benefits of fish oil, and most of these favorable effects have been attributed to omega-3 fatty acids. Fish oils, however, also contain varying amounts of other unusual types of fatty acids not commonly found in other food sources. For example, oils derived from saury, pollock and herring are all enriched in long-chain monounsaturated fatty acids (LCMUFA) with aliphatic tails longer than 18 C atoms (i.e., C20:1 and C22:1 isomers combined). Compared with well-studied omega-3, limited information is available on the role of LCMUFA in cardiovascular health. In the current study, we first examined the effect of saury fish oil-derived LCMUFA concentrate on the pathogenesis of atherosclerosis in atherosclerosis animal model. In LDLR-deficient female mice, we observed that 12-week supplementation of 2% LCMUFA on a western diet significantly decreased atherosclerosis lesion areas and accumulation of macrophages, compared with western diet (control) or western diet supplemented with 2% olive oil enriched in shorter-chain MUFA oleic acid (C18:1 n-9), although there were no differences in plasma lipoprotein profiles between the three groups. LCMUFA diet also decreased plasma inflammatory cytokine levels, and improved cholesterol efflux capacity to apoB-depleted plasma in BHK cells overexpressing ABCA1. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated hepatic PPAR signaling pathway. To clarify the individual effect of LCMUFA isomers, we further produced concentrated C20:1 and C22:1 fractions, and fed LDLR-deficient mice a western diet supplemented with 5% C20:1, C22:1, or not (control) for 12 weeks. In good agreement with the first study, both LCMUFA isomers showed beneficial effects on atherosclerosis development, systematic inflammation, and cholesterol efflux capacity, without lowering plasma lipids. It is suggested that the atheroprotective effect of LCMUFA is likely due to both isomer fractions. Alterations in lipoprotein proteome revealed by LC-MS/MS proteomic analysis were favorably correlated with reduction in atherosclerotic plaque areas. For mechanistic study, we hypothesized that some of the beneficial effect of LCMUFA were derived from their metabolite, and we synthesized LCMUFA-derived ethanolamides to estimate their effect on PPAR activation. In vitro PPAR transactivation assay revealed a beneficial role of LCMUFA-derived ethanolamides in PPAR transcriptional activity. Based on these positive findings, we are now conducting a double-blind crossover clinical trial of LCMUFA-rich saury oil to estimate the effect of LCMUFA-rich diet on lipoprotein metabolism in adults (ClinicalTrials.gov Identifier: NCT03043365). In conclusion, our research showed for the first time that fish oil-derived LCMUFA-rich diet attenuates atherosclerosis, possibly by regulating PPAR signaling pathway and modulating lipoprotein proteome, other than lipid-lowering effects. Although omega-3 fatty acids are generally considered the major active components in fish lipids, our findings provide novel insights into potential cardioprotective effect of LCMUFA-rich fish oil, and build on past efforts to understand the impacts of MUFA on health outcomes.

86

70

Hypercholesterolemia-induced endothelial dysfunction is rescued by overexpression of endothelial Kir2.1 in resistance arteries

Ibra Fancher, PhD 1 • Sang Joon Ahn, PhD 2 • Crystal Adamos 3 • Catherine Osborn 3 • Catherine Reardon, PhD 4 • Godfrey Getz, MD, PhD 4 • Shane Phillips, PhD 3 • Irena Levitan, PhD 3 1Medicine, University of Illinois at Chicago • 2Yale University • 3University of Illinois at Chicago • 4University of Chicago

Postdoc

Rationale: Hypercholesterolemia induces endothelial dysfunction, a key stage in the development of cardiovascular disease. Impairment of nitric oxide (NO) dependent vasomotor function is established as an important factor. We recently identified endothelial Kir2.1 as upstream mediators of flow-induced NO production. Our earlier studies established that endothelial Kir is suppressed by cholesterol. Objective: We tested the hypothesis that hypercholesterolemia induces microvascular endothelial dysfunction through suppression of Kir2.1, which we propose to be responsible for the inhibition of flow-induced NO production and the loss of NO-dependent component of flow-induced vasodilation (FIV). Methods and Results: Kir2.1 currents and their sensitivity to flow are significantly suppressed in microvascular endothelial cells exposed to acetylated-LDL without any change in Kir2.1 expression. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe-/- mice, Kir2.1+/-/Apoe-/- exhibit the same blunted FIV and flow-induced NO response as Apoe-/- and Kir2.1+/- alone, as we showed earlier Kir2.1+/- arteries show significant impairment of (FIV) and NO production. Endothelial-specific overexpression of Kir2.1 via adenoviral transduction of arteries from Apoe-/- and Kir2.1+/-/Apoe-/- mice results in full rescue of FIV and NO production in both hypercholesterolemic models. Conversely, endothelial-specific expression of dominant-negative Kir2.1 results in impairment of FIV in WT arteries but has no further effect on the blunted FIV in Apoe-/- arteries. No differences were detected in Kir2.1 or eNOS expression between WT and Apoe-/- mice. Notably, a full rescue of the FIV response by endothelial-specific Kir2.1 was observed in Apoe-/- mice with and without the addition of high fat diet. Conclusions: We conclude that hypercholesterolemia-induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow-induced NO production. The stages downstream of flow-induced Kir2.1 activation appear to be intact because overexpression of endothelial Kir2.1 rescues FIV and flow-induced NO production in arteries from hypercholesterolemic mice.

87

71 Fibrinogen Depletion Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm

Hannah Russell 1 • Keith Saum 1 • Alexandra Sundermann 2 • Shannon Jones 1 • Anders Wanhainen, MD, PhD 3 • Todd Edwards, PhD 2 • Lori Holle 4 • Alisa Wolberg, PhD 4 • A. Phillip Owens III, PhD 1 1Internal Medicine, University of Cincinnati • 2Medicine and Public Health, Vanderbilt University Medical Center • 3Surgery, Uppsala University • 4Pathology, UNC Chapel Hill

Graduate Student

Background: Fibrinogen and fibrin [collectively fibrin(ogen)] provides physical and biochemical support to a developing clot and is one of the most crucial independent risk factors for cardiovascular diseases (CVDs). In addition to clot formation, fibrin(ogen) also promotes wound healing and powerful inflammatory and immune responses via engagement of leukocytes. Increased levels of circulating fibrin(ogen) degradation products are correlated with increased diameter and progression of the inflammatory disease abdominal aortic aneurysm (AAA). However, a causal link between fibrinogen and AAA has not yet been established. The objective of this study was to determine the role of fibrinogen depletion in a mouse model of AAA. Methods and results: We first determine whether AAA resulted in elevated procoagulant activity by quantifying plasma levels of thrombin anti-thrombin (TAT) and thrombin generation via calibrated automated thrombography (CAT). Compared to controls, both mice and humans with AAA had significantly elevated TAT as well as CAT parameters velocity index, peak height, and endogenous thrombin potential. To identify a role of fibrinogen in AAA, low density lipoprotein receptor deficient (Ldlr-/-) mice were injected intraperitoneally with scrambled anti-sense oligonucleotide (ASO) or β-fibrinogen ASO (30 mg/kg) 3 weeks prior to experimentation and throughout the study. Administration of fibrinogen ASO successfully achieved > 90% depletion of fibrinogen. After 3 weeks of ASO, mice were fed a high fat/cholesterol ‘Western’ diet for 1 week prior to and throughout infusion with angiotensin II (AngII; 1,000 ng/kg/day) for 28 days. Compared to controls, fibrinogen depletion decreased abdominal diameter (33% decrease; P = 0.001), atherosclerotic lesion area (~63% decrease; P = 0.001), and inflammatory cytokines (>75% decreased IL-1 and IL-6; P = 0.001). Fibrinogen depletion also decreased aneurysm incidence (P < 0.05) and rupture-induced death (P = 0.078). Conclusions: Our results show that AAAs are associated with elevated TAT and thrombin generation in both patients and mice, and that fibrinogen depletion attenuates AAA incidence, diameter, rupture-induced death, atherosclerosis, and inflammation. We propose that increased procoagulant activity accelerates the rate of fibrinogen to fibrin conversion, which promotes inflammation and increases cardiovascular disease. Further studies will define whether reducing plasma fibrinogen may be a novel treatment strategy.

88

72 Increased Circulating Trimethylamine N-oxide (TMAO) Augments the Incidence of Abdominal Aortic Aneurysm in Low Penetrant C57BL/6J Mice

Kelsey Conrad, MS 1 • Shannon Jones 1 • Robert Helsley, PhD 2 • Rebecca Schugar, PhD 2 • Zeneng Wang, PhD 2 • Stanley Hazen, MD, PhD 2 • J. Mark Brown, PhD 2 • A. Phillip Owens III, PhD 1 1Internal Medicine, University of Cincinnati • 2Cellular and Molecular Medicine, Cleveland Clinic

Graduate Student

Background: The gut microbiota is a metabolically active endocrine organ critical to the maintenance of cardiovascular health. Nutrients common in high fat foods (phosphatidylcholine, choline, L-carnitine) are metabolized by microbial enzymes to form the gut microbial metabolite trimethylamine (TMA). Metabolism by the host enzyme flavin-containing monooxygenase 3 (FMO3) converts TMA to the pro-inflammatory molecule trimethylamine N-oxide (TMAO). Human clinical trials have correlated high levels of circulating TMAO to an increased risk of cardiovascular diseases. However, this meta-organismal pathway has not been evaluated in the context of abdominal aortic aneurysm (AAA). The objective of this study was to determine the effects of a high choline diet on the development of AAA. Methods: C57BL/6J male (n=20) and female (n=20) mice were fed either a standard chow control diet (n = 10 each sex) or a choline-rich diet (1%; n = 10 each sex) for 5 weeks. After 1 week of diet, basal abdominal ultrasounds were performed and angiotensin II (AngII; 1,000 ng/kg/min) was infused for 28 days via implantation of osmotic minipumps. Termination ultrasounds were performed on day 27 and mice were sacrificed on day 28. Aortas were harvested for evaluation of aneurysm progression and plasma was analyzed for the metabolites TMA, TMAO, and choline. To determine whether TMAO was elevated in human patients with AAA, plasma samples from participants with fast growing AAAs (n = 85), slow growing AAAs (n = 84), and normal (non-aneurysmal) aortas (n = 115) were analyzed for plasma TMAO levels via liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Administration of a choline-rich diet augmented the incidence (P < 0.02) and aortic diameter (P < 0.001) of AAAs in both male and female mice versus placebo-fed mice. Plasma levels of TMA, TMAO, and choline were significantly elevated in choline-fed mice versus normal chow (P < 0.05). Importantly, circulating levels of plasma TMAO were significantly elevated in a step-wise fashion with the rate of aneurysm growth versus non-aneurysmal control patients (fast growing > slow growing > normal patients; P < 0.001). Conclusions: Our results indicate increases in circulating TMAO augments the growth status of aneurysms in human patients and the incidence of AAA in a low penetrant C57BL/6J mouse model.

89

73 A specific rotamer of apolipoprotein A-I enables Lecithin-cholesterol acyl transferase activation by discoidal HDL

Allison L. Cooke 1 • Jamie C. Morris 1 • John T. Melchior, PhD 1 • Rong Huang, PhD 1 • W. Gray Jerome, PhD 2 • Scott E Street 1 • W. Sean Davidson, PhD 1 1Pathology, University of Cincinnati • 2Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center

Graduate Student

Investigating the structure of apolipoprotein (apo)A-I, the primary protein component of high density lipoprotein (HDL), is important for understanding how HDL interacts with lipid-modifying proteins to mediate its cardioprotective functions in plasma. HDL lipid discs contain two molecules of apoA-I arranged in an antiparallel, stacked ring-structure. ApoA-I is composed of 10 tandem, amphipathic, alpha-helical repeats that encapsulate lipid for plasma transport on the hydrophobic face of the ring-structure, and allows HDL to interact with modifying proteins on the hydrophilic face. For example, lecithin-cholesterol acyl transferase (LCAT) is a critical lipoprotein-associated enzyme that converts cholesterol to cholesterol ester for transport out of peripheral cells. It was hypothesized that changing the orientation of apoA-I on HDL discs can modulate LCAT reactivity. To test this, site-directed cysteine-point mutagenesis was used to generate different orientations (rotamers) of apoA-I on HDL discs of similar size and composition: K133C- helix 5 of one apoA-I molecule adjacent to helix 5 of its antiparallel partner (5/5 rotamer), K206C- helix 5 adjacent to helix 2 (5/2 rotamer), and K195C-helix 5 adjacent to helix 1 (5/1 rotamer). Initially, an LCAT activation assay revealed LCAT had significantly increased reactivity to discs with the 5/5 rotamer relative to wild-type or the other rotamers (p<0.001). Surface plasmon resonance showed that despite increased activation by the 5/5 rotamer, LCAT binding affinity to HDL discs was not dependent on apoA-I rotamer orientation. Finally, chemical cross-linking and mass-spectrometry indicated LCAT interacts with a hybrid epitope composed of adjacent helices 5-7 and 3-5 on apoA-I molecules in the 5/5 rotamer discs. This study demonstrates how the rotamer orientation of apoA-I can modulate an HDL interaction, and may provide a basis for the association and reactivity of HDL with other plasma proteins.

90

74 Circulating Bacterial Small RNA Bound to LDL Induce Inflammatory Activation of Macrophages

Ryan Allen, PhD 1 • Shilin Zhao, PhD 2 • Marisol Ramirez, MS 1 • Bradley Richmond, MD, PhD 3 • Timothy Blackwell, MD 3 • Quanhu Sheng, PhD 2 • Kasey Vickers, PhD 1 1Cardiovascular Medicine, Vanderbilt University Medical Center • 2Center for Quantitative Sciences, Vanderbilt University Medical Center • 3Allergy and Inflammation, Vanderbilt University Medical Center

Postdoc

Chronic, sub-acute inflammation is an active component of many human diseases of diverse etiology, characterized by sustained recruitment and activation of macrophages. Macrophages are phagocytic, myeloid-derived cells that are resident in nearly every tissue to provide innate defense against invasive pathogens. Upon injury, macrophages respond to remove damaged cells and cell debris and facilitate tissue repair. However, in disease, prolonged activation of macrophages promotes tissue remodeling, often to the detriment of tissue function. This is classically observed in the development of atherosclerosis, in which monocytes infiltrate the artery wall, differentiate to macrophages, and phagocytize LDL to become cholesterol-engorged “foam cells”, which rapidly develop a pro-inflammatory phenotype that contributes to further cardiovascular disease. Our lab previously reported that high-density lipoproteins (HDL) traffic miRNAs between cells as part of intercellular gene-regulation networks that we proposed could mediate anti-inflammatory properties of HDL. Although low-density lipoproteins (LDL) have also been reported to traffic miRNA, more recent data from small-RNA sequencing (sRNA-SEQ) indicates that LDL-associated sRNA are primarily exogenous, with most sRNA fragments originating from bacteria. Most interestingly, taxonomic analysis of bacteria contributing sRNA to the LDL pool revealed a profile distinct from any characterized profile of the comprehensive human microbiome. Additionally, antibiotic mediated ablation of the gut microbiome failed to disrupt lipoprotein-sRNA pools of mice, suggesting that bacterial sRNA fragments are not linked to the gut microbiome. However, manipulation of housing conditions was capable of inducing stark changes in the landscape of bacterial sRNA bound to lipoproteins of mice, indicative of a potential role for the environment in shaping the lipoprotein-sRNA fingerprint. In support of this, we characterized the lipoprotein-sRNA profiles of wild-type and polymeric IgA receptor deficient (Pigr-/-) mice, a model of compromised airway mucosal immunity, and found reduced abundance of bacterial sRNA fragments, most notably of Proteobacteria and Firmicutes. Although many potential functions remain to be explored, we hypothesized that exogenous sRNA associated with LDL may influence the inflammatory potential of LDL upon uptake by artery-wall macrophages by activation of endosomal, nucleic acid-sensing toll-like receptors (TLR), which are highly expressed in macrophages and facilitate innate immunity. To test this hypothesis, we treated primary mouse macrophages and PMA-stimulated THP1 cells with physiologically relevant concentrations of fresh, native LDL (nLDL) and observed induction of pro-inflammatory cytokine expression. Strikingly, partial silencing of nucleic acid-sensing TLRs blunted this nLDL induced activation of pro-inflammatory gene expression. Conversely, treatment of human hepatoma cells, which express low levels of exogenous RNA-sensing TLRs, with nLDL failed to induce pro-inflammatory cytokine expression. Taken together, our data identify a novel, non-lipid cargo of LDL that is capable of modulating inflammatory gene expression in macrophages and may contribute to the pathogenesis of atherosclerosis, and many other metabolic diseases.

91

75 Inducible Depletion of Calpain-2 Attenuates Obesity-accelerated Abdominal Aortic Aneurysms in mice

Aida Javidan, MS 1 • Weihua Jiang, MS 1 • Jessica Moorleghen, MS 1 • Venkateswaran Subramanian, PhD 2 1Saha Cardiovascular Research Center, University of Kentucky • 2Physiology, University of Kentucky

Graduate Student

Background and Objective: Recent clinical studies demonstrated that abdominal adiposity is associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are non-lysosomal calcium dependent cysteine proteases that are highly expressed in human and experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we identified that calpain-2 (a major ubiquitous isoform) plays a critical role in Angiotensin II (AngII)-induced AAA formation in mice. In addition, calpain inhibition strongly suppressed adipose tissue inflammation in obese mice. The purpose of this study was to determine the functional contribution of calpain-2 in obesity-accelerated AAA. Methods and Results: Calpain-2 floxed mice that were hemizygous for β-actin Cre-ERT2 were produced by breeding male Cre-ERT2 to female calpain-2 floxed mice. At 8 weeks of age, male non-Cre littermates (Cre-) and Calp-2 x Cre-ERT2 (Cre+) mice were injected with tamoxifen (25 mg/kg, i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion of calpain-2 protein in the aorta and periaortic adipose tissue from Cre+ mice compared to non-Cre littermates. Mice were fed a high fat diet (60% Kcal) for 20 weeks. After 16 weeks of diet feeding, mice were infused with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. Depletion of calpain-2 had no effect on high fat diet-induced body weight gain, fat mass, glucose and insulin tolerance. Interestingly, calpain-2 depletion significantly attenuated AngII-induced expansion of ex-vivo maximal diameter of abdominal aortas in obese mice (Cre-: 1.4 ± 0.14; Cre+: 0.9 ± 0.04 mm; P<0.001). In addition, calpain-2 depletion significantly reduced the incidence of AngII-induced AAAs in mice (Cre-: 75%, Cre+: 7%; P< 0.001). Conclusion: These findings suggest that calpain-2 plays a critical role in AngII-induced AAA development in diet-induced obese mice.

92

76 Pancreatic Beta Cell Export of miR-375 to High-Density Lipoproteins is Regulated by Cellular Ion Fluxes.

Leslie Sedgeman 1 • Carine Beysen, PhD 2 • Marisol Ramirez-Solano 3 • Quanhu Sheng, PhD 4 • Yan Guo, PhD 4 • Scott Turner, PhD 2 • Kasey Vickers, PhD 3 1Molecular Physiology and Biophysics, Vanderbilt University • 2Kinemed, Inc. • 3Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center • 4Department of Cancer Biology, Vanderbilt University Medical Center

Graduate Student

microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL) transport and deliver functional miRNAs to recipient cells, including endothelial cells and hepatocytes. However, the mechanism of export is not understood. Since miR-375 expression in the islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to export miR-375 to HDL through in vitro export assays, incubating HDL with INS1 beta cells or primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and primary islets in vitro. To determine if cholesterol transporters contribute to HDL-miR-375 export from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-375 to HDL. On the other hand, inhibition of the KATP channel with tolbutamide resulted in the suppression of HDL-miR-375 export. Similarly, export of miR-375 to HDL was blunted from islets of two mouse models lacking functional KATP channels (Kir6.2 and SUR1 KO mice). Our work suggests that miR-375 export to HDL is regulated by cellular dynamics, including ion fluzes in the beta cell. We are currently investigating the relationship between HDL-miR-375 export, insulin secretion, and miRNA processing in pancreatic beta cells to further elucidate the mechanism(s) controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol transporters.

93

77 The Role of ApoC-III in the immune system

Alison Kohan, PhD 1 • Cayla Rodia 1 1Nutritional Sciences, University of Connecticut

Faculty

While we know that CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a powerful tool in the resolution of gut inflammation, and that their secretion of IL-10 is critical to inflammatory bowel disease (IBD) remission, there is a major gap in identifying mechanisms for increasing Tregs in the intestine. Recently, we have found that intestinal Tregs and plasma IL-10 are dramatically increased in mice overexpressing human apoC-III. These intestinal Tregs significantly protect apoC-III transgenic mice mice from experimental colitis, including weight loss, diarrhea, and TNF-α secretion. While apoC-III overexpression is protective, loss of apoC-III in apoC-III-/- mice severely impacts colitis sensitivity; more than 50% of apoC-III-/- mice die in response to dextran sodium sulfate (DSS)-colitis induction, compared to wild-type controls that all survive. Supporting our studies in these mouse models are human studies which show that both plasma and ileal apoC-III levels are reduced in patients during active IBD flare-ups. We have now determined that apoC-III acts specifically on Tregs and CD103+dendritic cells in the intestine and mesenteric lymph nodes, and that treatment of Tregs ex vivo with apoC-III-containing lipoproteins inhibits their ability to take up extracellular fatty acids. This inhibitory role of apoC-III on lipid uptake is well known, but this is the first time this role has been described in Tregs, thus defining a new and biologically important function of this apolipoprotein in the immune system. We hypothesize that a critical function of apoC-III is to regulate lipid uptake and metabolism in intestinal Tregs, which results in increased tolerogenicity in the gut.

94

78 Understanding inhibition of lipoprotein lipase by angiopoietin-like protein 4

Aspen Gutgsell 1 • Saskia Neher, PhD 1 1Biochemistry and Biophysics, UNC Chapel Hill

Graduate Student

More than three million cases of hypertriglyceridemia are diagnosed every year in the United States. Hypertriglyceridemia, or elevated levels of plasma triglycerides, is a major risk factor cardiovascular disease and the leading cause of death worldwide. Two major sources of plasma triglycerides are dietary fat and stored fat in adipose tissue. Triglycerides are packaged into lipoprotein particles and reahernmoved from circulation by lipoprotein lipase (LPL) in the capillaries of muscle, heart, and adipose tissue. In the absence of LPL, patients have severe hypertriglyceridemia, life-threatening pancreatitis, and fatty skin lesions. A family of proteins known as angiopoietin-like proteins (ANGPTL3, 4 and 8) temporally regulate LPL to control the delivery of fatty acids to certain tissues. More specifically, ANGPTL4 inhibits LPL in adipose tissue during periods of fasting. Recently, genome wide association and exome sequence studies identified individuals with loss of function mutations in ANGPTL4 as having a unique lipid profile that significantly decreases their risk for developing cardiovascular disease. Their profile consists of low plasma triglycerides and high levels of “good” cholesterol, commonly known as HDL. The direct relationship between LPL activity and plasma triglyceride levels lends LPL as an ideal target for triglyceride lowering therapeutics. More specifically, preserving LPL activity by blocking ANGPTL4 inhibition is hypothesized to generate a “cardio protective” lipid profile by lowering plasma triglycerides and increasing HDL cholesterol in patients with high triglycerides. However, the interaction between LPL and ANGPTL4 is poorly understood as no structural information is available. It was originally believed ANGPTL4 irreversibly inhibits LPL by dissociating it into inactive monomers. However, our group has shown that ANGPTL4 noncompetitively inhibits LPL, while leaving the active dimeric structure intact. Since this discovery, little information has surfaced regarding exactly how or where ANGPTL4 and LPL interact. Therefore, our aim is to characterize the ANGPTL4/LPL interaction and generate a structural model for this complex.

95

79 Serum Levels of Dioxin-Like Pollutants Are Positively Associated With the Cardiometabolic Disease Risk Biomarker TMAO in Leaner Individuals

Michael Petriello, PhD 1 • Richard Charnigo • Manjula Sunkara • Sony Soman • Marian Pavuk 2 • Linda Birnbaum 3 • Andrew Morris, PhD 4 • Bernhard Hennig 5 1Cardiovascular Research Center, University of Kentucky • 2CDC Agency for Toxic Substances and Disease Registry • 3NCI at NIEHS • 4Cardiovascular Medicine, University of Kentucky • 5Superfund Research Center, University of Kentucky

Postdoc

Trimethylamine N-oxide (TMAO) is a diet and gut microbiota-derived metabolite that has been linked to cardiovascular disease risk in human studies and animal models. TMAO levels show wide inter and intra individual variability in humans that can likely be accounted for by multiple factors including diet, the gut microbiota, levels of the TMAO generating liver enzyme Flavin-containing monooxygenase 3 (FMO3) and kidney function. We recently found that dioxin-like (DL) environmental pollutants increased FMO3 expression to elevate circulating diet-derived TMAO in mice, suggesting that exposure to this class of pollutants might also contribute to inter-individual variability in circulating TMAO levels in humans. To begin to explore this possibility we examined the relationship between body burden of DL pollutants (reported by serum lipid concentrations) and serum TMAO levels (n=340) in the Anniston, AL cohort, which was highly exposed to polychlorinated biphenyls (PCBs). TMAO concentrations in archived serum samples from the Anniston Community Health Survey (ACHS-II) were measured, and associations of TMAO with 28 indices of pollutant body burden, including total dioxins toxic equivalent (TEQ), were quantified. Twenty-three (22 after adjustment for multiple comparisons) of the 28 indices were significantly positively associated with TMAO. Multivariate modeling revealed that total dioxins TEQ was significantly associated with TMAO among females (except at high BMIs) but not among males. Our results from this cross-sectional study indicate that exposure to DL pollutants may contribute to elevated serum TMAO levels. With the observation that dioxins were only associated with TMAO in leaner individuals, we have begun to mechanistically study the relationship between dioxin exposure, FMO3/TMAO, and cardiometabolic disease by characterizing a mouse model that develops atherosclerosis but not adipose tissue expansion. We examined the effects of PCB 126 on markers of systemic inflammation and atherosclerotic lesion size. Exposed mice exhibited significantly increased plasma cytokine levels and accelerated atherosclerotic lesion formation. More work needs to be completed to determine the role of TMAO and FMO3 in these processes (Supported in part by NIEHS/NIH grant P42ES007380).

96

80 Insulin signaling regulates apolipoprotein (Apo) AI secretion from hepatocytes

Ailing Ji, PhD 1 • Xuebing Wang 1 • Lisa Bennett 2 • David Graf 2 • Gregory Graf, PhD 2 • Deneys van der Westhuyzen, PhD 1 1Cardiovascular Research Center, University of Kentucky • 2Pharmaceutical Sciences, University of Kentucky

Staff

Insulin resistance is associated with increased risk for cholesterol gallstones as well as the development of diabetic dyslipidemia in which plasma levels of HDL cholesterol are reduced. HDL is the primary cholesterol carrier in the reverse cholesterol transport (RCT) pathway, the process by cholesterol is delivered from peripheral organs to the liver for elimination in the bile. Therefore, we hypothesized that insulin signaling is a regulator of hepatobiliary cholesterol clearance from the plasma and secretion into bile. To directly test the role of insulin signaling, we utilized mice harboring insulin receptor flanked by loxP sites (IRfl/fl) in combination with adenoassociated viral vectors containing no transgene (empty) or Cre recombinase to generate control and liver insulin receptor knock out mice (LIRKO), respectively. As with previous LIRKO strains (albumin-Cre, adenoviral-Cre), LIRKO mice showed markedly reduced insulin receptor mRNA and protein in liver, but not skeletal muscle or adipose tissue as well as impaired glucose tolerance when compared to controls. LIRKO mice had increased biliary cholesterol secretion as well as increased expression of the ABCG5 ABCG8 sterol transporter, the primary mediator of biliary cholesterol secretion. Levels of SR-BI, the primary HDL receptor were unchanged as were rates of HDL clearance from plasma and selective delivery of HDL cholesterol to the liver. None-the-less, plasma HDL cholesterol and ApoAI were reduced. Despite these reductions, ApoAI protein, but not mRNA, was increased in the liver of LIRKO mice. Immunofluorescence microscopy revealed that ApoAI accumulated in membrane-bound inclusions that stained positively for markers of early, late and recycling endosomes and of lysosomes. These structures persisted in primary hepatocyte cultures where rates of ApoAI secretion into the culture medium were reduced. To determine the intracellular itinerary of ApoAI, we developed a chimera of mCherry fluorescent protein and human ApoAI. mCherry ApoAI was capable of forming nascent HDL, associated with HDL in plasma, and partially restored HDL cholesterol in ApoAI-deficient mice. However, this mCherry-ApaAI failed to co-localize with a probe for acidic compartments in live cells, suggesting that the compartment is unique or that the accumulation of ApoAI neutralizes the endosomal/lysosomal vesicles in which it accumulates. Additional studies will be required to investigate the route by which ApoAI arrives and accumulates in cells with impaired insulin signaling.

97

81 Scavenging reactive aldehydes with 5'-O-pentyl-pyridoxamine (PPM) improves HDL function and reduces atherosclerosis in Ldlr deficient mice

Jiansheng Huang, PhD 1 • Linda Zhang, PhD 2 • Patricia Yancey, PhD 1 • Huan Tao, PhD 1 • Lei Ding 3 • YouMin Zhang 1 • John Oates, MD 4 • Venkataraman Amarnath, PhD 5 • Jackson Roberts , PhD 2 • Sean Davies, PhD 2 • MacRae Linton, MD 4 1Medicine, Vanderbilt University Medical Center • 2Pharmacology, Vanderbilt University • 3Vanderbilt University Medical Center • 4Medicine and Pharmacology, Vanderbilt University Medical Center • 5Pathobiology, Vanderbilt University Medical Center

Postdoc

Background: Lipid peroxidation products impair the cholesterol efflux capacity of high-density lipoprotein (HDL) and contribute to the development of atherosclerosis. The effect of inhibition of HDL dysfunction by scavengers on HDL function and whether scavenging reactive aldehydes with PPM protects against the development of atherosclerosis was examined. Methods and Results: HDL of familial hypercholesterolemia (FH) subjects has impaired ability to promote cholesterol efflux and FH-HDL contains 5-fold more malondialdehyde crosslinks (MDA-Lys) than control HDL. In vitro studies found that the reactive aldehyde malondialdehyde (MDA) crosslinks apolipoprotein AI (apoAI) and impairs the ability of HDL to promote cholesterol efflux from Apoe-/- macrophages in a MDA dose dependent manner. Western blot analysis of apoAI revealed that the reactive aldehyde scavenger 5'-O-pentyl-pyridoxamine (PPM) abolished MDA-mediated crosslinking of apoA-I in HDL (at a molar ratio of MDA to HDL of 1:5) by 80% (P<0.05). PPM maintained the cholesterol efflux capacity of MDA treated HDL in Apoe-/- macrophages. Furthermore, PPM significantly improved the ability of HDL to promote cholesterol efflux in Ldlr-/- mice fed a Western diet (WD) for 16 weeks (P<0.05), indicating that PPM protects HDL from modifications by the reactive aldehyde MDA and improves the cholesterol efflux capacity of MDA-HDL in Ldlr-/- mice. Importantly, administration of 1 mg/mL of the reactive aldehyde scavenger PPM, versus 1 mg/mL of the nonreactive analogue PPO, to Ldlr-/- mice consuming a WD for 16 weeks reduced the extent of proximal aortic atherosclerosis by 45% (P<0.05). Immunohistochemistry studies revealed that PPM reduced the macrophage content and decreased the number of TUNEL positive cells by 55% (P<0.05) and by 60% (P<0.01) in advanced atherosclerotic lesions of Ldlr-/- mice, respectively. In addition, the necrotic core area was dramatically reduced by 52% (P<0.05) in advanced atherosclerotic lesions in Ldlr-/- mice treated with PPM compared to control group. Conclusions: Treatment with PPM, a reactive aldehyde scavenger: 1) inhibits MDA-ApoA1 adduct formation thereby preserving HDL cholesterol efflux capacity; 2) improves the ability of HDL to promote cholesterol efflux in Ldlr-/- mice fed on a WD for 16 weeks; 3) reduces the macrophage content and atherosclerotic lesion area of Ldlr-/- mice; 4) decreases the number of apoptotic cells and the necrotic core area in lesions of Ldlr-/- mice. These results support the therapeutic potential of PPM in the treatment of atherosclerotic cardiovascular disease.

98

82 Protease-activated Receptor 2 Deficiency Attenuates the Formation of Atherosclerosis in Mice

Shannon Jones 1 • Adrien Mann 1 • Kelsey Conrad, MS 1 • Keith Saum 1 • David Hall, MS 2 • Lisa McKinney 1 • Nathan Robbins, MS 1 • Joel Thompson, PhD 3 • Abigail Peairs, PhD 2 • Michael Tranter, PhD 1 • Nigel Mackman, PhD 4 • A. Phillip Owens III, PhD 1 1Internal Medicine, University of Cincinnati • 2Nutritional Sciences, University of Cincinnati • 3Endocrinology and Molecular Medicine, University of Kentucky • 4Medicine, UNC Chapel Hill

Staff

Objective – Protease-activated receptor 2 (PAR2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. While PAR2 is present in coronary atherosclerotic lesions, the relevance of this finding has not been investigated in experimental models. The objective of this study was to determine the effect of PAR2 deficiency on the development of atherosclerosis. Approach and Results – PAR2 mRNA and protein expression is increased in human carotid artery and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To determine the effect of PAR2 deficiency on atherosclerosis, male low density lipoprotein receptor deficient (Ldlr-/-) mice (8-12 weeks old) that were Par2+/+ or Par2-/- were fed a fat and cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that PAR2 on non-hematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly attenuated levels of the chemokine monocyte chemoattractant protein 1 (MCP-1) in the circulation and macrophage content in atherosclerotic lesions. Mechanistic studies using ex vivo vascular smooth muscle cells showed that PAR2 deficiency is associated with reduced production of MCP-1 mRNA and protein release into the supernatant resulting in less monocyte migration and infiltration. Conclusions – Our results indicate PAR2 deficiency is associated with attenuation of atherosclerotic inititation and reduces lesion progression by blunting MCP-1 induced monocyte infiltration.

99

83 Deoxysphingolipids - mediators in taxane-induced peripheral neuropathy

Katrin Anne Becker, PhD 1 • Anne-Kathrin Uerschels, MD 2 • Jacek Bielawski, PhD 3 • Joan Colglazier 4 • Erhard Bieberich, PhD 5 • Erich Gulbins, PhD 1 • Stefka Spassieva, PhD 5 1Molecular Biology , University of Duisburg-Essen • 2Neurosurgery, University of Duisburg-Essen • 3Biochemistry and Molecular Biology , Medical University of South Carolina • 4Medicine, Medical University of South Carolina • 5Physiology, University of Kentucky

Faculty

Taxanes are chemotherapy drugs used in wide variety of cancers. However, there efficacy can be hindered by a major dose limiting side effect - peripheral neuropathy. Although, the clinical symptoms of taxane-induced peripheral neuropathy are well documented, the molecular mechanism is currently not well understood and there are no treatment options available. In our previous work, we have shown that in the plasma of breast cancer patients treated with taxane, the levels of minor class of lipids, the deoxysphingolipids, were associated with incidence and severity of neuropathy (Kramer et al, FASEB J, 2015). In our current study, we used a mouse model to test whether taxane treatment results in increased levels of deoxysphingolipids in the dorsal root ganglia and spinal cord. Mice were three times intraperitoneally injected with taxane. Lipids were extracted from the isolated ganglia and spinal cord and subjected to quantitative mass spectrometry analyses. We observed significant elevation of deoxysphingolipid levels only in the dorsal root ganglia. In addition, we compared in vitro in neuronal cultures, the toxic effect of deoxysphingosine, a deoxysphingolipid, and sphingosine, a structurally similar sphingolipid. Importantly, our in vitro data showed that only deoxysphingosine treatment resulted in morphological changes and toxicity in the neurons. Taken together our data suggest that in the dorsal root ganglia, the neurotoxic effect of the systemic taxane treatment is likely mediated by deoxysphingolipids.

100

84 Reduced Low-Density Lipoprotein Receptor-Related Protein-1 in Mature Mice Modestly Effects Hypercholesterolemia and Atherosclerosis

Shayan Mohammadmoradi, MS 1 • Deborah A. Howatt 1 • Anju Balakrishnan 1 • Jessica J. Moorleghen 1 • Mark Graham 2 • Adam Mullick 2 • Hong Lu, PhD 1 • Alan Daugherty, PhD, DSc 1 1Saha Cardiovascular Research Center, University of Kentucky • 2Ionis Pharmaceuticals

Graduate Student

Background and Objectives: Low-density lipoprotein receptor-related protein-1 (LRP1) has been hypothesized to serve as a receptor for the removal of selected lipoprotein particles from plasma. However, mice with somatically engineered mutation of LRP1 have not provided a clear understanding of the specific lipoprotiens that are regulated by LRP1. Therefore, the aim of this study was to reduce LRP1 using antisense oligomers in mature mice to determine effects on lipoprotein metabolism and atherosclerosis. Methods and Results: LRP1 antisense oligonucleotide (ASO; 50 mg/kg, injected once per week) or its control ASO was administered subcutaneously to 8-week old male low-density lipoprotein (LDL) receptor deficient mice (n=10/each group). All groups were fed a saturated fat-enriched diet for 8 weeks started 1 week after the first injection of ASOs. There was no difference in body weight between the two groups. Profound reduction of LRP1 was confirmed by qPCR and Western blotting. Whole body inhibition of LRP1 elicited a modestly increased total plasma cholesterol compared to its control group. Size exclusive chromatography analyses demonstrated the increased cholesterol was due to increased VLDL and LDL-cholesterol. Despite augmented hypercholesterolemia, whole body inhibition of LRP1 did not increase atherosclerotic lesion size. LRP1 was also reduced in a hepatocyte-specific manner by administration of an LRP1 ASO coupled with GalNac to target the asialofeutin receptor. LDL receptor deficient mice were injected with GalNAc LRP1 ASO or its control ASO (5 mg/kg/week). Hepatic-specific inhibition of LRP1 did not change plasma cholesterol concentration, lipoprotein distribution and atherosclerosis. Conclusion: Profound reductions of LRP1 in mature mice modestly increased hypercholesterolemia and had no effect on atherosclerosis in LDL receptor deficient mice.

101

85 Oxidized neutral lipid lipolysis as a novel regulator of insulin signaling during acute stress

Katelyn Ahern 1 • Garrett Mullins, PhD 1 • Vidisha Raje, PhD 1 • Vlad Serbulea 1 • Norbert Leitinger, PhD 1 • Thurl Harris, PhD 1 1Pharmacology, University of Virginia

Graduate Student

The acute stress response following an injury or surgery often results in systemic insulin resistance (IR) and hyperglycemia which can lead to increased morbidity and mortality. Post-operative IR is downstream of stress hormones such as catecholamines, but the underlying mechanisms driving IR are unknown, thus limiting therapeutic development. We have previously demonstrated that β-adrenergic stimulation of adipocytes causes inhibition of the mTOR complexes and leads to the development of IR in a lipolysis-dependent manner. Therefore, we hypothesize that a product of lipolysis is responsible. We find that lipid extracts isolated from forskolin-stimulated 3T3-L1 adipocytes inhibit the activity of the mTOR complexes in vitro. However, when tested, “traditional” products of lipolysis had no effect on mTOR complexes. Interestingly, stimulating oxidation of fatty acids using tert-butyl hydroperoxide further exacerbates mTOR inhibition, while antioxidant treatment reverses this effect, suggesting that the active species is an oxidized fatty acid. In fact, incubation of mTOR complex with in vitro auto-oxidized fatty acids is sufficient to inhibit kinase activity. These findings reveal a previously unrecognized mechanism of oxidized fatty acid signaling and mTOR complex regulation in the development of peripheral IR during acute stress. Our future work will focus on identifying these species in vivo using our novel targeted liquid chromatography-mass spectrometry method and characterizing their physiological role in response to stress-inducing events known to stimulate reactive oxygen species production.

102

86 Pharmacological inhibition of HuR improves survival and reduces adverse cardiac remodeling following left-ventricular pressure overload

Sarah Anthony 1 • Xiaoqing Wu, PhD 2 • Lisa Green 1 • Michelle Nieman 3 • John Lorenz, PhD 3 • Jack Rubinstein, MD 1 • Liang Xu, PhD 2 • Michael Tranter, PhD 1 • Burns Blaxall, PhD 4 1Division of Cardiovascular Health and Disease, University of Cincinnati • 2Molecular Biosciences, University of Kansas • 3Pharmacology and Systems Physiology, University of Cincinnati • 4Department of Pediatrics, Division of Molecular Cardiovascular Biology, Heart Institute, Cincinnati Children's Hospital

Staff

Human antigen R (HuR) is a widely expressed RNA binding protein that has been implicated in numerous human diseases including cancer, neurological disorders, and cardiovascular disease. We have previously shown that HuR is both necessary and sufficient for induction of hypertrophic signaling pathways in isolated primary myocytes. In addition, data from our lab is the first to suggest that HuR expression and activation is increased in failing human hearts. To determine the role of HuR in hypertrophic signaling in vivo, we created an inducible cardiomyocyte-specific HuR deletion mouse, and showed that genetic deletion of HuR reduces pathology using a transverse aortic constriction (TAC) model of pressure-overload-induced hypertrophy, adverse cardiac remodeling, and heart failure. In this work, we sought to recapitulate this reduction in pathology using KH-3, a novel pharmacological inhibitor of HuR, to determine the translational potential of HuR inhibition as a viable therapeutic target. Twenty wild-type mice were randomized to either vehicle or KH-3 at four weeks post-TAC, a time-point consistent with substantial development of cardiac hypertrophy, and monitored via serial echocardiography for a further seven weeks. Our results show that treatment with KH-3 increased survival relative to vehicle controls. In addition, as compared with vehicle, KH-3 treatment significantly abated the continued progression of left ventricular (LV) hypertrophy. This was accompanied by a significant preservation of LV ejection fraction and reduction in LV chamber dilation in KH-3 treated mice. Importantly, chronic KH-3 treatment had no effects on systemic blood pressure and no observable adverse reactions. In conclusion, these results suggest that inhibition of HuR is a promising therapeutic approach to treat pathological LV hypertrophy.

103

87 Perioperative high density lipoprotein particle changes and the risk of acute kidney injury after cardiac surgery

Loren Smith, MD, PhD 1 • Derek Smith, PhD 2 • Alan Remaley, MD, PhD 3 • MacRae Linton, MD 4 • Frederic Billings IV, MD 5 1Anesthesiology, Vanderbilt University Medical Center • 2Biostatistics, Vanderbilt University Medical Center • 3National Heart, Lung, and Blood Institute, National Institutes of Health • 4Medicine and Pharmacology, Vanderbilt University Medical Center • 5Anesthesiology and Medicine, Vanderbilt University Medical Center

Faculty

Acute kidney injury (AKI) after cardiac surgery occurs in up to 30% of patients and increases the risk of postoperative myocardial infarction and death. No effective treatments exist. Increased intraoperative oxidative stress independently predicts AKI after cardiac surgery. High density lipoproteins (HDL) have anti-oxidant capacity. We hypothesize that perioperative HDL cholesterol concentration (HDL-C), HDL particle concentration (HDL-P) and HDL composition are associated with the risk of postoperative AKI. We analyzed data from a prospective, 393-subject trial of perioperative atorvastatin to prevent AKI after cardiac surgery. Statin-using patients were randomized to placebo or 80mg atorvastatin the morning of surgery and 40mg on postoperative day 1. Stain-naïve patients were randomized to placebo or 80mg the day prior to surgery and 40mg daily thereafter during hospitalization. The associations between 1) HDL-C, 2) small HDL particle concentration, and 3) the change in HDL-P from anesthetic induction to postoperative day two with maximum serum creatinine change from baseline in the first 48 postoperative hours were assessed using two-component latent variable mixture models adjusted for known AKI risk factors. Regression analyses assessed interactions of chronic statin use, perioperative atorvastatin treatment, and HDL-C on AKI risk. We quantified HDL particle size by NMR Lipoprofile test in 90 subjects. HDL-C and HDL-P change over time were assessed with mixed effects models adjusted for AKI risk factors. We quantified myeloperoxidase (MPO) activity of apolipoprotein B-depleted serum in 30 subjects using a bioluminescence assay. A higher preoperative HDL-C was independently associated with a decreased postoperative serum creatinine change (p=0.02). The association between a high HDL-C and an attenuated increase in serum creatinine was strongest in chronic statin-using patients (p=0.008) and was enhanced with perioperative atorvastatin treatment (p=0.004) and increasing chronic statin dose (p=0.003). HDL-C did not change during the perioperative period (p=0.60), however, HDL-P decreased (p<0.001). A larger decrease in HDL-P from induction to postoperative day two was independently associated with a greater postoperative rise in serum creatinine (p=0.02). A higher preoperative small HDL particle concentration was independently associated with a decreased postoperative rise in serum creatinine (p=0.02). Further, preoperative apoB depleted serum associated MPO activity showed a trend toward being lower in patients with smaller postoperative serum creatinine change than in patients with higher postoperative serum creatinine change (p=0.07). Future work will focus on assessing perioperative HDL anti-oxidant capacity by quantifying MPO and paraoxonase-1 activity in our trial patients with the goal of identifying the biological mechanism underlying the association between HDL and AKI after cardiac surgery.

104

88 The Effects of Hypercholesterolemia on Wound Healing and Endothelial Angiogenic Properties

Yedida Bogachkov 1 • Lin Chen, MD, PhD 2 • Myung-Jin Oh, PhD 3 • Luisa A. DiPietro, PhD 2 • Irena Levitan, PhD 4 1Pharmacology, University of Illinois at Chicago • 2Periodontics, University of Illinois at Chicago • 3Medicine, University of Chicago • 4Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago

Graduate Student

Nearly 31 million adults in the US have elevated total cholesterol levels over 240 mg/dL, and almost 32% of US adults have LDL cholesterol levels of 200mg/dL or above. Our study focuses on the impact of dyslipidemia on wound healing and wound angiogenesis. Using ApoE-/- mice, a known dyslipidemic model and a skin punch biopsy wound healing assay, we show that ApoE -/- mice show significantly delayed wound closure on days 2-7 post-wounding compared to control mice. These same ApoE-/- also display significantly decreased angiogenesis on days 10 and 14 post wounding, based on PECAM staining for endothelial cells, indicative of endothelial cell infiltration of the wound bed and new blood vessel growth. Furthermore, macrophage infiltration is also reduced. This significant suppression of wound angiogenesis is also seen when LDL is directly applied to the wounds via subcutaneous injections on C57BL6 mice. Additionally, using a matrigel plug assay, we show significantly suppressed angiogenesis in C57BL6 mice fed a Western Diet for 40 weeks. These results utilize an Isolectin B4 probe, which is indicative of endothelial cell infiltration into the matrigel plug and new blood vessel growth. Finally, in vitro studies on human aortic endothelial cells were pursued showing LDL significantly decreases endothelial cell proliferation, whereas its oxidized form, oxLDL, shows an increase in proliferation. In sum, we have shown in vivo that hypercholesterolemic mice have a decreased angiogenic potential, and in vitro, excess LDL leads to a decrease in angiogenic potential of aortic endothelial cells.

105

89 Coarse-Grained Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of Cholesterol Molecules

Nicolas Barbera, MS 1 • Manuela Ayee, PhD 1 • Belinda Akpa, PhD 2 • Irena Levitan, PhD 3 1Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago • 2Department of Molecular Biomedical Sciences, North Carolina State University • 3Department of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago

Graduate Student

Hypercholesterolemia, elevated plasma levels of cholesterol, is a major risk factor in the development of atherosclerosis. Our previous studies have shown that inwardly rectifying K+ (Kir) channels, which play an important role in endothelial function, are suppressed by enriching cells with cholesterol. Additionally, earlier work done by our group and others has shown that cholesterol regulates Kir2 channels, a sub-family of Kir channels expressed in endothelial cells, through direct interactions at non-annular interaction sites. However, while a putative binding site has been identified, the dynamics of the binding process and cholesterol’s structural effect on the protein remain poorly understood. To address these questions, we used coarse grained molecular dynamics simulations of Kir2.2, a subfamily of Kir2, in model membranes containing cholesterol and POPC to interrogate their molecular interactions at the microsecond timescale. Our simulations show that rather than single ligand-channel binding, interactions between cholesterol and the channel are both complex and numerous, with an average of 15-20 cholesterol molecules interacting with the protein at any given time. These interactions occur at a range of timescales and at distinct annular and non-annular sites on the surface of the protein. Additionally, we observed spontaneous diffusion between these sites and between the protein surface and the surrounding membrane. At present, we are exploring the functional significance of these various interactions and the impact of this collective action on the structure-function relationships governing Kir2 channel activity.

106

90 Reduced HDL in mice overexpressing SR-BI is associated with alterations in the acute inflammatory response to endotoxemia

Yanzhang Li, PhD 1 • Ailing Ji, PhD 2 • Xuebing Wang 2 • Andrea Trumbauer 2 • Maria C. de Beer, PhD 3 • Frederick C. de Beer, MD 4 • Nancy R. Webb, PhD 5 1IM-Endocrinology, University of Kentucky • 2Cardiovascular Research Center, University of Kentucky • 3Physiology, University of Kentucky • 4Internal Medicine, University of Kentucky • 5Pharmacology and Nutritional Sciences, University of Kentucky

Faculty

Objectives: Serum amyloid A (SAA) is an acute phase protein (APP) produced mainly by the liver during an acute phase response (APR). During the APR, virtually all of SAA circulating in the blood is associated with HDL. SR-BI is an HDL receptor and also can bind SAA. Previous reports showed that in SR-BI transgenic mice, hepatic uptake of SAA is increased, and this uptake is involved in the pro-inflammatory effects of SAA. Whether SR-BI regulates SAA expression in response to the inflammatory stimulation is not yet investigated. Methods/Results: Ten week-old male C57BL/6 mice were administered 1x1011 particles of a replication-defective adenoviral vector expressing mouse SR-BI (AdSR-BI) or an adenoviral vector containing no transgene (Adnull) as control. At 72h after adenovirus infusion, the mice were injected i.p. with 1mg /kg body weight LPS. Plasma and tissues were collected at selected time points after LPS injection. As expected, overexpression of SR-BI produced a dramatic reduction in HDL-C (56.52 ± 2.338 mg/dL vs 2.08 ± 1.555 mg/dL for Adnull versus AdSR-BI). Plasma levels of inflammatory cytokine IL-1b, TNFa and IL-6 were robustly higher in AdSR-BI-injected mice compared to control mice. Lympohcyte numbers were also signicantly higher in AdSR-BI-injected mice compared to control mice 12h after LPS injection. In contrast, SAA in plasma was much lower in AdSR-BI-injected mice compared to control mice. Before LPS injection, SAA could only be detected at very low levels in the plasma by ELISA (20.7±8.3 ug/ml). LPS induced a robust increase in plasma SAA levels as early as 2h post-LPS treatment in Adnull-treated mice (1100.0±108.7 ug/ml). Notably, increases in plasma SAA were significantly lower in AdSR-BI-treated mice (122.7±9.5 ug/ml) 2h after LPS injection. At 12h after LPS injection, plasma levels of SAA were 10-fold lower in AdSR-BI-treated mice compared to control mice (12.9±0.24 mg/ml vs 1.2±0.21 mg/ml). The results from western blot and real-time RT-PCR showed that hepatic SAA expression was significantly decreased in the liver of SR-BI overexpressed mice but significantly increased in hepatocytes of SR-BI knock out mice compared to that of control mice. Conclusions: Our results demonstrate that decreased levels of HDL in SR-BI-overexpressing mice are associated with alterations in acute inflammatory responses. Future studies will investigate the relationship between reduced SAA and increased inflammatory cytokines in mice with increased hepatic SR-BI overexpression.

107

91 A patient with very high plasma level of high-density lipoprotein cholesterol (HDLc) and sudden cardiac arrest

Wenliang Song, MD 1 • Ginger Milne, PhD 2 • John Fahrenholz, MD 3 • John McPherson, MD 1 • John Oates, MD 4 • Dan Roden, MD 1 • MacRae Linton, MD 1 1Cardiology, Vanderbilt University Medical Center • 2Clinical Pharmacology, Vanderbilt University • 3Medicine, Vanderbilt University Medical Center • 4Medicine, Vanderbilt University

Postdoc

Synopsis: A 58-year-old female with plasma HDLc of 110-150 mg/dL suffers from a very debilitating facial flushing for the past several years. She also had two episodes of sudden cardiac arrests. Purpose: To find a pathological condition that can explain the constellation of symptoms this patient has, which may provide important insights of HDL metabolism Methods: Extensive clinical work-ups to evaluate her constellation of symptoms and basic laboratory experiments including HPLC/MS/MS to evaluate specific biomarkers for rare orphan diseases Results: This patient's coronary angiogram was normal. However, acetylcholine challenge produced significant coronary spasm. Bone marrow biopsy excluded systemic mastocytosis. Urinary prostaglandin D2 metabolites increased after flushing attacks, which suggests the patient has a condition of mast cell activation syndrome. Her clinical constellation of symptoms mimic the niacin's pharmacological effect. Genetic investigation may shed lights on the mechanism of HDL metabolism and niacin's therapeutic effect. Conclusions: Isolated mast cell activation syndrome may cause coronary spasm. Rare gene mutation may be responsible for her condition. Future genetics investigation may shed lights on the mechanism of HDL metabolism.

108

92 Human Antigen R (HuR) Regulates Structure and Function of Brown Adipose Tissue

Lindsey Lanzillotta 1 • Zach Taylor 1 • Kaila Yamamoto 1 • Sarah Anthony 1 • Shannon Jones 1 • George Yoshida 1 • A. Phillip Owens III, PhD 1 • Michael Tranter, PhD 1 1Internal Medicine, University of Cincinnati

Graduate Student

Human antigen R (HuR) is an RNA binding protein widely expressed throughout the body, including in both white (WAT) and brown (BAT) adipose tissue. Recent work from our lab has shown that adipocyte-specific HuR knockout (adipo-HuR-/-) mice gain less weight following a high fat diet (HFD) compared to wild type (WT) controls. In this study, we focused on the role of HuR deletion on the function of BAT, which mediates non-shivering thermogenesis. Histological analysis revealed that, while the BAT of the chow fed adipo-HuR-/- mice appeared normal, the BAT of the HFD fed adipo-HuR-/- mice appears less dense with a cell size that more closely resembles WAT. Upon further examination of BAT function, our results show that, when subjected to cold stress (4C), adipo-HuR-/- mice are less cold tolerant compared to their WT counterparts . We also show that, compared to the WT mice, adipo-HuR-/- mice have decreased mitochondrial density and expression of uncoupling protein-1 (UCP-1) in their BAT. This may be caused by a HuR-dependent decrease in expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1a), which plays a key role in mitochondrial biogenesis and is upregulated during exposure to cold (4C). In conclusion, our results indicate that HuR regulates both the structure and function of BAT, at least in part through modulation of PGC-1a mRNA expression. Support or Funding Information: This work was supported by a University of Cincinnati Heart, Lung, and Vascular Institute Near Horizons Grant (MT, APO).

109

93 Impact of miR-33 antagonism on atherosclerotic plaque size and cholesterol content in nonhuman primates

Tong Li, MD 1 • Lei Cai, PhD 1 • Sierra Paxton 1 • Courtney Burkett 1 • Peter Hecker, MS 1 • Ryan Temel, PhD 1 1CVRC, University of Kentucky

Graduate Student

Introduction: Elevated LDL cholesterol (LDL-C) is a major risk factor for coronary heart disease (CHD). Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events caused by atherosclerotic lesion rupture. By stimulating macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in mice reduces the cholesterol content and size of atherosclerotic lesions. Mouse studies have limited translational value since mice do not develop coronary artery atherosclerosis and express only one of the two miR-33 family members found in humans. Since nonhuman primates (NHPs) have miR-33a and miR-33b and develop coronary artery atherosclerosis, NHPs are the best preclinical model for determining the therapeutic potential of miR-33 antagonism. We hypothesize that NHPs treated with miR-33 antagonist will have decreased plaque size and cholesterol content. Methods: Male cynomolgus monkeys (n=36) were fed for 20 months a high fat/high cholesterol diet, which caused severe hypercholesterolemia. At the end of the 20-month progression phase, a subset of monkeys (n=12) were euthanized to collect tissues. The remaining monkeys were switched to a cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-33 (n=12) for 6 months. Formalin-fixed right coronary arteries (RCA) were split into five ~3 mm sections, paraffin embedded, and cut into 5 µm slices. Lesion area, defined as the region between the lumen and internal elastic lamina, was measured for each RCA section and then averaged for each animal. Cholesterol was extracted from a section of the fixed thoracic aorta and the free and total cholesterol levels were determined by GC-FID. Esterified cholesterol (EC) = total cholesterol – free cholesterol (FC). Results: No significant difference among the progression, vehicle, and anti-miR-33 groups were observed for RCA lesion area (P=0.60) and thoracic aorta FC (P=0.24). Thoracic aorta EC was significantly increased in the progression versus the regression groups (P<0.0001) but was similar for the vehicle and anti-miR-33 groups (P=0.94). Conclusions: Antagonism of miR-33 for 6 months does not decease RCA plaque size. Regardless of anti-miR-33 treatment, consumption of a cholesterol-lowering diet for 6 months significantly reduces thoracic aorta EC, a readout of foam cell content. It is possible that our NHPs treated with anti-miR-33 versus vehicle for a 6-week regression period will have significantly less plaque EC.

110

94 Impact of miR-33 antagonism on coronary artery atherosclerotic plaque composition in nonhuman primates

Lei Cai, PhD 1 • Tong Li, MD 1 • Sierra Paxton 1 • Courtney Burkett 1 • Peter Hecker, MS 1 • Ryan Temel , PhD 1 1CVRC, University of Kentucky

Staff

Introduction: Elevated LDL cholesterol (LDL-C) is a major risk factor for coronary heart disease (CHD). Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events caused by atherosclerotic lesion rupture. By stimulating macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in mice causes atherosclerotic lesions to acquire a more stable composition. Mouse studies have limited translational value since mice do not develop coronary artery atherosclerosis and express only one of the two miR-33 family members found in humans. Since nonhuman primates (NHPs) have miR-33a and miR-33b and develop coronary artery atherosclerosis, NHPs are the best preclinical model for determining the therapeutic potential of miR-33 antagonism. We hypothesize that miR-33a/b antagonism will stabilize coronary artery atherosclerotic plaques of NHPs. Methods and Results: Male cynomolgus monkeys (n=36) were fed for 20 months a high fat/high cholesterol diet, which caused severe hypercholesterolemia. At the end of the 20-month progression phase, the hearts from 12 animals were perfused with formalin under physiological to preserve the structure of the coronary arteries. The remaining monkeys were switched to a cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-33 (n=12) for 6 months. LDL-C for both treatment groups was rapidly reduced by the chow diet and returned to baseline levels by the 3rd month of the regression phase. The anti-miR-33 versus the vehicle group had a sustained and significant elevation in HDL-C that was linked to a significant increase in hepatic ABCA1, a miR-33 target. Following dissection and paraffin embedding of the right coronary arteries (RCA), 5 µm slices were analyzed by histology and immunohistochemistry (IHC). The RCA atherosclerotic lesions from both the progression and regression groups were characterized by intimal smooth muscle cell proliferation, collagen deposition, elastin degradation, and medial layer destruction. While necrotic cores were a common feature, there was a minimal amount of plaque calcification. Macrophages were found at high levels in the progression group lesions but were almost absent from the plaques of both the anti-miR-33 and vehicle groups. Conclusions and Future Directions: The qualitative histology and IHC results show that the monkeys developed RCA atherosclerotic plaques similar to early fibroatheromas observed in humans. By quantifying the area occupied by the various plaque components, we will determine whether antagonism of miR-33a/b allowed the lesions to attain a more stable composition. In addition, to determine whether anti-miR-33 changes the amount or type of macrophages in lesions, we will analyze coronary arteries from monkeys subjected to 6 weeks, as opposed to 6 months, of regression.

111

95 Targeting of HB-EGF against hypertension and renal damage

Lihua Yang, MS 1 • Seonwook Kim 1 • Debra Rateri 1 • Richard Lee, PhD 2 • Mark Graham, PhD 2 • Sangderk Lee, PhD 1 1Saha Cardiovascular Research Center, University of Kentucky • 2Cardiovascular Antisense Drug Discovery Group, Ionis Pharmaceuticals

Staff

Study goal: Previous reports suggested that the heparin binding EGF-like growth factor (HB-EGF), which is an EGFR ligand, is positively involved in the development of renal disease. In this study, we tested the effects of the HB-EGF targeting using antisense oligonucleotide (ASO) administration on the blood pressure and renal damage induced by AngII infusion in mouse model systems. Experimental design and results: To test the effects of the HB-EGF ASO administration on the AngII-induced hypertension, C57BL/6 mice (male, 10 weeks of age) were pretreated with control and HB-EGF ASO for 2 weeks and cotreated with AngII infusion (1,000ng/min/kg) for additional 4 weeks. We monitored blood pressure changes during 6 weeks using standard tail-cuff procedure. We observed that the HB-EGF ASO administration significantly downregulated basal and AngII-induced blood pressure in the system. The HB-EGF targeting also reduced the kidney size. To test the effects of the HB-EGF targeting on the renal damage, the LDLR KO mice (male, 16 weeks of age) were pretreated with control and HB-EGF ASO and cotreated with high fat diet (HFD; 42% calorie from fat, 0.2% cholesterol) and AngII-infusion (1,000ng/min/kg) for additional 4 weeks. At the termination step, we compared plasma creatinine and BUN levels as markers of the status of the renal damage. The result showed that the HB-EGF ASO administration effectively protected against HFD and AngII-induced renal damage in the mouse model system. Conclusion: These results suggested that the HB-EGF signaling positively involved in regulating blood pressure and renal damage. The targeting of the HB-EGF signaling might be an approach to delay the development of the hypertension associated with renal damage.

112

96 HB-EGF is a Key Positive Regulator of Hepatic VLDL Secretion

Seonwook Kim 1 • Lihua Yang, MS 1 • Debra Rateri 1 • Ryan Temel, PhD 1 • Richard Lee, PhD 2 • Mark Graham, PhD 2 • Sangderk Lee, PhD 1 1Saha Cardiovascular Research Center, University of Kentucky • 2Cardiovascular Antisense Drug Discovery Group, Ionis Pharmaceuticals

Staff

Study goal: Unbalanced hepatic VLDL production (assembly/secretion) is a risk factor for the development of hypertriglyceridemia or non-alcoholic fatty liver disease (NAFLD) under metabolic stress conditions like metabolic syndrome. Previous reports showed that the circulatory heparin-binding EGF-like growth factor (HB-EGF), which is a ligand of EGFR, is correlated with cholesterol level and risk of coronary artery disease in human. From these previous data, we tested the hypothesis that the HB-EGF signaling is a positive regulator of VLDL production in the liver. Experimental procedure and results: In Hep-G2 cells, the recombinant HB-EGF induced increase of the apoB secretion, which is an essential component of VLDL particle. In C57BL/6 mice, the administration of recombinant HB-EGF increased hepatic VLDL secretion; in contrast, the HB-EGF antisense oligonucleotide (ASO) administration or the tail-vein injection of EGFR blocker BIBX1382 induced significant suppressions of the hepatic VLDL secretion. The HB-EGF ASO administration effectively downregulated circulatory lipid levels (TG and cholesterol) associated with VLDL particles in multiple mouse strains (C57BL/6, LDLR KO, and apoE KO). At the same time, the targeting induced accumulation of neutral lipids in the liver, specifically, TG and cholesterol ester. Conclusion: These results indicate that the HB-EGF signaling positively regulates VLDL secretion in the liver under normal and metabolic stress condition. Future study of the interaction with insulin signaling for the regulation would be interesting in understand the pathology of metabolic symptom.

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97 Anti-ApoA-I antibodies induced using an epitope-specific immunostimulatory liposomal formulation exacerbate atherosclerosis in dyslipidemic mice

David Henson 1 • Robert Kline IV, MS 1 • Vincent Venditto, PhD 1 1Pharmaceutical Sciences, University of Kentucky

Graduate Student

Autoantibodies targeting apolipoprotein A-I (ApoA-I) have been identified in patients with chronic inflammatory diseases including rheumatoid arthritis, lupus and obesity and correlate with cardiovascular disease progression. Although correlated, the exact role of these antibodies has not been fully elucidated. Induction of antibodies in mice through immunization, rather than passive administration of antibodies derived from other species, offers a unique opportunity to explore the impact of anti-ApoA-I antibodies in the context of atherosclerosis. Furthermore, the ability to modulate epitope-specific immune responses offers a strategy to study the impact of antibody responses toward specific domains within the full protein. To achieve these objectives, we prepared an immunostimulatory liposomal formulation containing peptides derived from the LCAT domain of ApoA-I. We first determined that mice immunized with the complete formulation induce robust antibody responses toward the epitope, as well as the full-length protein. These data suggest that antibody responses toward ApoA-I are either not controlled by immunological tolerance mechanisms or our formulation was capable of breaking tolerance. Although successful at inducing a robust immune response, the antibodies alone failed to induce atherosclerosis in wild-type mice. We then studied the role of antibodies in the context of dyslipidemia utilizing the AAV-PCSK9 gain-of-function mutant and western diet. After establishment of dyslipidemia, mice were immunized with the immunostimulatory liposomal formulations. Once again, mice immunized with the complete formulation achieved robust antibody responses, which persisted for 150 days. Furthermore, mice immunized with the complete formulation had a statistically significant elevation in atherosclerosis over control mice. These data highlight our ability to modulate the anti-ApoA-I antibody response using epitope-specific liposomal formulations and provides a platform to study the role of these antibodies in an immunologically intact system. This work was supported by a pilot grant through the Center for Research in Obesity & Cardiovascular Disease COBRE, NIH (P20GM103527) and a Scientist Development Grant from the American Heart Association (17SDG32670001). DH is supported by a UK Center for Clinical and Translational Science training grant from the National Center for Advancing Translational Sciences, NIH (TL1TR001996).

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98 HDL-miR-223 Communication Pathway in vivo.

Carrie Wiese 1 • Leslie Roteta 1 • Ryan Allen, PhD 2 • Wanying Zhu 2 • Kasey Vickers, PhD 2 1Molecular Physiology & Biophysics, Vanderbilt University • 2Cardiovascular Medicine, Vanderbilt University Medical Center

Graduate Student

High-density lipoproteins (HDL) stably transport microRNAs (miRNAs) through the blood and facilitate a HDL-miRNA communication pathway. Previously, we demonstrated that macrophages and other myeloid cells export miR-223 to HDL in vitro. Furthermore, HDL-miRNAs, including miR-223, are delivered to recipient human coronary artery endothelial cells (HCAECs) where miR-223 is not transcribed. Upon transfer of miR-223 to endothelial cells, the validated miR-223 target intracellular adhesion molecule 1 (Icam-1) expression is significantly suppressed. While HDL-miRNA transfer and cellular gene regulation have been demonstrated in vitro, intercellular communication has not been established in vivo. We utilized bone marrow transplantation between C57/B6J (WT) and Mir223-/- mice to define the physiological role of HDL-miRNA pathway in regulating cellular gene expression. To restore the HDL-miR-223 communication pathway within Mir223-/- mice, we transplanted WT bone marrow into lethally irradiated Mir223-/- mice resulting in a significant increase in HDL-miR-223 levels. Subsequently, a significant increase in miR-223 was identified in recipient tissues including whole liver, hepatocyte isolates, and aortic endothelium. Conversely, HDL-miR-223 pathway was depleted by transplanting Mir223-/- bone marrow into lethally irradiated WT mice, which resulted in a significant reduction of miR-223 on HDL. The reduction in HDL-miR-223 levels correlated with reduced miR-223 levels in recipient cells including endothelial and hepatocyte. Furthermore, altering miR-223 levels in endothelial cells and hepatocytes resulted in altered mRNA levels of putative targets, which we have validated as miR-223 targets. In conclusion, myeloid cells export miR-223 to extracellular carriers such as HDL, which results in delivery of miR-223 to recipient cells where it regulates gene expression.

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99 Dynamic hepatic oxidized phospholipid composition promotes non-alcoholic fatty liver disease progression

Clint Upchurch 1 • Vlad Serbulea 1 • Norbert Leitinger, PhD 1 1Pharmacology, University of Virginia

Graduate Student

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, affects 25% of individuals within the United States. NAFLD is a progressive pathology initiating with accumulation of fat within the liver (hepatic steatosis) followed by inflammation (steatohepatitis) and liver fibrosis. Currently, drivers of NAFLD progression are poorly understood. We propose that oxidized phospholipids (OxPLs) within the liver promote NAFLD pathophysiology. We developed a liquid chromatography-mass spectrometry method to quantify oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) in liver tissue and observed an increase in full-length PAPC oxidation products (OxPAPC) in mice fed a high fat diet (HFD). HFD-fed mice exhibit extensive hepatic steatosis, the first stage of NAFLD. Separately, using immunohistochemistry we observed an increase in liver OxPLs in mice treated with carbon tetrachloride, a model of liver damage. Furthermore, hepatic stellate cells, the major contributor to fibrosis, show an increase in fibrotic gene expression in response to treatment with OxPAPC. Taken together, OxPLs play a role in stellate cell over-activation in response to liver damage and subsequent fibrosis.

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100 Short-Term Exercise Training Differently Modifies Aspects of HDL Function in Lean, Obese and Diabetic Subjects

Lin Zhu, PhD 1 • Nicolas Musi, MD 2 • John Stafford, MD, PhD 1 1Diabetes, Vanderbilt University Medical Center • 2Diabetes, University of Texas Health Science Center

Faculty

The triglyceride (TG)-lowering effects of exercise have been well documented. In contrast, mechanisms for the cardiovascular protective effects of exercise are not well established. We previously showed that short-term exercise training improves insulin sensitivity but does not inhibit inflammatory pathways in immune cells from insulin-resistant subject. The purpose of this study is to evaluate metabolic changes of lipoproteins after short-term exercise training in lean, obese and type 2 diabetic (T2D) subjects. Lean, obese nondiabetic, and obese T2D individuals (n=6 per group) underwent a high intensity aerobic exercise program for 15 days. Blood lipid parameters and HDL functions were analyzed before and after exercise. Blood cholesterol levels were higher in obese and T2D than in lean subjects. Exercise training primarily reduced LDL- and HDL-cholesterol (17%-21%, P<0.05) in obese subjects. However, exercise mainly reduced VLDL- and LDL-cholesterol in T2D subjects (30%-20%, P<0.05). Baseline blood TG levels were higher in obese and T2D subjects by 50% and 100%, respectively, compared with lean subjects (P<0.01). The exercise training program did not affect blood TG in lean subjects but lowered blood TG in both obese and diabetic subjects. In obese subjects the reductions in TG were in LDL- and HDL-TG, whereas in T2D subjects TG reductions were in VLDL-, LDL and HDL-TG. In line with this change, the anti-oxidant activity of HDL was improved by exercise training in obese subjects. In addition, the ratio of total TG to HDL-cholesterol, an index that positively correlates with CHD risk, was slightly increased in obese subjects and was not affected by exercise training in lean and obese subjects. The total TG:HDL ratio was increased 3.4-fold in T2D in comparison to lean subjects (4.7 vs 1.4, T2D vs Lean, P<0.001) , which was dramatically decreased by exercise training (4.7 vs 2.7, Pre vs Post, P<0.05). Our results show the efficacy of short-term exercise training to improve HDL function, which might be related to the changes of TG and/or cholesterol by exercise in different lipoprotein particles.

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101

Identification of a novel lipoprotein microRNA carrier in plasma

Wanying Zhu 1• Danielle L. Michell 1 • Ryan M. Allen 1 • Kasey C. Vickers 1

Vanderbilt University Medical

Staff

Recently, multiple groups have detected of microRNAs (miRNA) in plasma by high-throughput sequencing. Circulating sRNAs are likely protected from RNases in plasma through their association with lipid carriers. Our lab previously reported that lipoproteins (HDL and LDL) transport miRNAs in plasma within potential intercellular communication networks. Based on these previous studies, we sought to define the distribution of miRNAs across lipid carriers in plasma.

Methods: To specifically precipitate carriers, whole plasma and pre-purified HDL (density-gradient ultracentrifugation) treated with lipid removal agents (LRA and Cleanascite) for the separation of lipid-associated miRNAs. Plasma samples from humans and mice were fractionated using size-exclusion chromatography (FPLC, Superdex 200 Increase in triplicate). Each fraction was assessed for total cholesterol, triglycerides, protein, phospholipids, and markers for lipoproteins and extracellular vesicles by western blotting. Carrier sizes in each fraction were quantified using dynamic light scattering. Total RNA was isolated from whole fractions and separated contents, lipid pellets and supernatants, after centrifugation. Real-time PCR and high-throughput sequencing were used to quantify miRNAs.

Results: Results demonstrated that Cleanascite efficiently removed lipids from plasma without damaging lipoproteins, as determined by the lack of phospholipids and apolipoproteins in the supernatants. Candidate miRNAs (miR-24-3p, miR-26a-5p, miR-146a-5p, miR-92a-3p, and miR-223-3p) were detected in fractions corresponding to HDL and LDL based on the distribution of cholesterol and triglycerides. Strikingly, candidate miRNAs also entirely separated with lipids in fractions that contained low cholesterol and high phospholipid content with a mass corresponding to very small HDL. These fractions were found to contain apoA-I and spherical particles <10nm in diameter.

Conclusions: miRNAs in plasma are predominantly associated with cholesterol-poor, phospholipid-rich spherical particles containing apoA-I.

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102

The Ral-Exocyst Pathway Regulates Platelet Secretion

Jinchao Zhang1 and Sidney W. Whiteheart1

1 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky

Postdoc

In Platelets, Exocyst complex has been identified as an octamer consisting of Sec3, Sec5, Sec6, Sec8, Sec10, Sec 15, Exo70 and Exo84 in platelets. The complex plays an essential role in tethering vesicles to the plasma membrane and regulating dense core granule secretion in platelets. RalA/B (family members of GTPases) are regarded as the upstream regulator of the Exocyst complex, and RalA/B binds to Sec5 and Exo84 in a GTP-dependent manner.

RalA/B, sharing over 88% identity in sequences, directly interact with Exo84 and Sec5 in the Exocyst complex. The role of RalA/B are interchangeable, but RalA has the higher ability to bind to the Exocyst components in GTP binding manner in cells. Several groups suggest that interrupting the association Sec3 and Exo70 with plasma membrane could disrupt exocytosis in other cells. However, it is little known how the role of the Exocyst complex in platelet secretion.

Here we ask the question whether inhibitors of RalA/B could affect granule secretion or not in platelets. And what is the exact role of the Exocyst complex in granule secretion? How is the Exocyst complex regulating vesicles tethering in secretion process? Firstly, specific RalA/B inhibitors, RBC8 and BQU57, were used to investigate the role of RalA/B in granule secretions. From the biochemical perspective, these inhibitors affect RalA/B bound to their downstream effector (RLP76) due to the Ral-GDP bound forms. We also found the inhibitors of RalA/B affects platelet spreading (data are not shown here). At the same time, we also answer the questions of the localization of Rals and Exo70 (one of the Exocyst component) as well as their association with F-actin.

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103 Alternating heights of the R wave in ECG: possible link with depolarization alternans Sahar Varnoosfaderani1, David Wasemiller1, Siqi Wang1, Paul Anaya2, and Abhijit Patwardhan1. 1Department of Biomedical Engineering and 2Division of Cardiovascular Medicine. University of Kentucky.

Alternans of the T wave in the ECG (TWA) is widely investigated as a potential predictor of ventricular arrhythmia, however, clinical trials show that TWA has high negative predictive value but poor positive predictive value. A possible reason that TWA has a large number of false positives is that a pattern of alternans, concordant alternans, may not be as arrhythmogenic as discordant alternans. Currently, it is not possible to discern the pattern of alternans using clinical ECGs. However, our research using tissue and cellular level electrophysiological measurements suggest that depolarization alternans which affects the pattern of alternans may manifest itself in the amplitude of the R wave of the ECGs. In order to investigate the link between depolarization alternans and changes in ECGs, we used a mathematical model which simulated ECGs from the cellular level changes observed in our experimental studies. These results suggest that the changes in ECGs should appear as alternating pattern of the heights of the R wave. However, there are a variety of factors which may also cause the R wave heights to change. We use signal analysis and statistical modeling to determine the link between the observed changes in R wave heights and depolarization alternans. Results from ECGs recorded from patients show that heights of the R wave can change as predicted by our experimental results and mathematical models. These results support further exploration of the link between depolarization and repolarization alternans as it has the potential to improve the positive predictive value of TWA.

Supported by grants from the Kentucky Science and Engineering Foundation (KSEF RDE18) and NSF EPSCoR RII Track-2.

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