Elevated Platelet Galectin-3 and Rho-Associated Protein ...downloads.hindawi.com/journals/bmri/2019/8952414.pdf · Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity

Research ArticleElevated Platelet Galectin-3 and Rho-Associated ProteinKinase Activity Are Associated with Hemodialysis ArteriovenousShunt Dysfunction among Subjects with Diabetes Mellitus

Po-Wei Chen12 Ling-Wei Hsu3 Hsien-Yuan Chang12 Ting-ChunHuang12 Jia-Rong Yu2

Hsin-Yu Liao2 Cheng-Han Lee14 and Ping-Yen Liu 12

1Division of Cardiology Department of Internal Medicine National Cheng Kung University Hospital College of MedicineNational Cheng Kung University Tainan Taiwan

2Institute of Clinical Medicine College of Medicine National Cheng Kung University Tainan Taiwan3Institute of Basic Medical Sciences College of Medicine National Cheng Kung University Tainan Taiwan4Institute of Pharmacy and Pharmaceutical Sciences College of Medicine National Cheng Kung University Tainan Taiwan

Correspondence should be addressed to Ping-Yen Liu larrymailnckuedutw

Received 6 January 2019 Accepted 20 March 2019 Published 8 April 2019

Academic Editor Yu-Chang Tyan

Copyright copy 2019 Po-Wei Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Introduction Hyperglycemia is amajor factor in influencing the patency rate of arteriovenous shunts potentially associatedwith theRhoARho-associatedprotein kinase (ROCK) pathway Besides galectin-3mediates thromboticmechanisms in venous thrombosisand peripheral artery disease We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenousshunt dysfunction Methods We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction 29 patientsreceived a complete follow-up and each provided two blood samples which were collected at the first visit for occluded status ofarteriovenous shunts and 1 month later for patent status A Western blot assay for a myosin phosphatase target subunit (MYPT)was performed to examine Rho-kinase activity A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbentassay (ELISA) for circulating galectin-3 were completed Results Higher platelet MYPT ratios and galectin-3 levels were identifiedat occluded arteriovenous shunts (MYPT ratio 05 [03ndash14] vs 04 [03ndash06] p = 001 galectin-3 12 [04ndash16] vs 07 [01ndash12] p =00004) The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (84 [60ndash97] 120583gmL vs71 [45ndash91] 120583gmL p = 0009) Biomarker ratios (occludedpatent status) trended high in patients with poorly controlled diabetes(MYPT ratio 17 [10ndash30] vs 11 [07ndash13] p = 006 galectin-3 16 [13ndash34] vs 11 [08ndash19] p = 005) Conclusion High plateletROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction especially in patientswith poorly controlled diabetes

1 Introduction

Hemodialysis is the most common renal replacement ther-apy requiring permanent functioning vascular access Vas-cular access dysfunction is associated with substantial mor-bidity and mortality and presents a major economic burdento healthcare [1ndash4]

The arteriovenous (AV) shunt is the standard modeof repeated vascular access for hemodialysis in terms ofaccess longevity patient morbidity and long-term prognosisStudies have revealed that factors such as age diabetessmoking peripheral vascular disease hypotension and vessel

characteristics directly influence AV shunt patency rates [56]

In our daily practice catheter-based interventions aresuccessful in restoring flow inmore than 80 of hemodialysisaccess with thrombotic events Catheter-based interventionsmay have replaced surgical revision as the treatment ofchoice for thrombosed access [7] Despite this repeatedinterventions still occur in a short time for high-risk patientsNotably a recent meta-analysis has noted the low qualityof the evidence for medical adjuvant treatment to increasepatency of arteriovenous shunts [8]

HindawiBioMed Research InternationalVolume 2019 Article ID 8952414 9 pageshttpsdoiorg10115520198952414

2 BioMed Research International

A functional vascular access is critical for effectivehemodialysis AV fistula dysfunction largely reflects mat-urational failure whereas AV graft dysfunction is mainlydriven by recurrent stenosis and thrombosis in the venousanastomosis [9] The current understanding of the biologyof vascular access dysfunction remains inadequate and prob-lematic

Studies have shown that diabetes mellitus is a risk factorin the development of vascular access failure [10] Althoughthe mechanisms responsible for the higher rate of AV fistulafailure in subjects with diabetes are unclear peripheral arte-rial disease impaired vasodilatation secondary to endothelialdysfunction and increased thrombogenicity are believed tocontribute [11]

Platelet activation and inflammation may play essentialroles in the development of atherosclerotic disease Variousbiomarkers have been used to assess platelet activity andinflammation in different clinical settings Among themgalectin-3 levels and RhoARho-associated protein kinase(ROCK)-related protein expression in peripheral blood wereused in one study as therapeutic biomarkers to treat periph-eral artery disease [12]

ROCK is a serinethreonine kinase consisting of twoisoforms ROCK-I and ROCK-II which may mediate thedownstream signaling of the small guanosine triphosphate(GTP)-binding protein (BP) Rho [13 14] It is mainlyinvolved in regulating the shapes and movements of cells byacting on cytoskeletons Recent observations suggest that thebeneficial cardiovascular effects of statins may result at leastin part from the inhibition of ROCKs [13 14]

Hyperglycemia is a pertinent factor in the developmentof macrovascular complications in diabetes vascular smoothmuscle cell (VSMC) migration and proliferation also play acrucial role There is growing evidence that ROCK may beassociated with many cardiovascular conditions includinghypertension atherosclerosis coronary vasospasm myocar-dial hypertrophy and stroke through its action in VSMCcontraction endothelial function and inflammatory pro-cesses [14 15]

Furthermore elevated levels of plasminogen activatorinhibitor-1 (PAI-1) are associated with endothelial dys-function myocardial infarction and stroke especially inpatients with diabetes A study showed that the inductionof PAI-1 expression by hyperglycemia involves oxidativestress and protein kinase C (PKC) Hyperglycemia stimu-lates Rho-kinase activity via PKC- and reactive oxidativestressndashdependent pathways increasing PAI-1 gene transcrip-tion [15] In one study of murine lung endothelial cells(MLECs) Rho-kinase activity increased after exposure tohigh glucose whereas Rho-kinase activity was unchanged inROCK I+minusMLECs indicating that hyperglycemia stimulatedRho-kinase activity [15]

The RhoARho-kinase pathway is widely known in manycellular functions including contraction motility prolifera-tion and apoptosis and its excessive activity induces oxida-tive stress and promotes cardiovascular diseases Howeverlimited data is available on the stenosis and thrombosis of AVshunts [16 17]

Galectins are carbohydrate-BPs with high affinity togalactosides on cell surfaces and extra-cellular glycoproteinsGalectins are a family of 120573-galactoside-binding lectins withconserved carbohydrate-recognition domains (CRDs) Cur-rently 15 galectins have been identified in mammals whichare divided into three types based on domain organizationas follows prototype galectins with one single CRD tandem-repeat galectins with two CRDs and chimera-type galectinswith a single CRD connected to a long flexible N-terminaldomain [18]

The expression of galectin-3 has been detected in leuko-cytes mast cells and various organ tissues Various bio-logical functions are involved including cell apoptosis cellactivation and inflammation Galectin-3 BP and its recep-torligand galectin-3 are secreted proteins that can inter-act with each other to promote cell-to-cell adhesion Thispathway involves pathologic and proinflammatory signalingcascades [19 20]

In venous thrombosis galectin-3 BP and galectin-3 playcritical roles possibly through IL-6 and PMN-mediatedthrombotic mechanisms and are potential biomarkers inhuman venous thrombosis [20] Galectin-3 has been apromising prognostic marker It has a crucial role in inflam-mation and fibrosis Both experimental and clinical studieshave shown that galectin-3 is an independent predictor ofall-cause mortality cardiovascular death and occurrence ofheart failure following acute coronary syndrome [21]

We hypothesized that high ROCK activity and galectin-3 levels were associated with increased risk for arteriove-nous shunt dysfunction in patients with poorly controlleddiabetes

2 Methods

21 Study Population We prospectively enrolled 38 patientsdiagnosed with arteriovenous shunt dysfunction who werereferred to receive percutaneous intervention in our catheter-ization laboratory at National Cheng Kung University(NCKU)Medical Center andDou-Liu Branch fromFebruary2015 to November 2016

Details of the study protocol were explained to allparticipants and written informed consent was obtainedbefore the study The survey was conducted according to theprinciples in the Declaration of Helsinki and was approvedby the Medical Ethics Committee of NCKU Hospital [IRBA-ER-103-243 A-ER-104-201] All patients were confirmed ashaving AV shunt dysfunction by using invasive angiography

Among these patients 29 received a complete follow-upand each provided two blood samples which were collectedat the first visit (percutaneous intervention for AV shuntdysfunction) for occluded status of AV shunts and at least 1month later for patent status

Twenty milliliters of blood was drawn at the occludedsite of the AV shunt into an ethylenediaminetetraacetic acidtube After platelet extraction aWestern blot assay formyosinphosphatase target subunit (MYPT) and myosin light chain(MLC)were performed to predict ROCK activity Rho-kinase

BioMed Research International 3

activity was expressed as the ratio of phosphorylation levelsof MYPT divided by total MYPT A Western blot assay forplatelet galectin-3 was also completed

Among the enrolled subjects we divided our patients byHbA1C patients with poorly controlled diabetes (HbA1C gt8)patients with well controlled diabetes (HbA1C lt8) andpatients without diabetes (HbA1C lt65 without medication)We investigated the platelet ROCK activities and galectin-3levels of each group and analyzed their clinical values

22 Laboratory Section

221 Isolation of Human Platelets Blood was collected withan acid-citrate-dextrose solution (91 vv) in plus bloodcollection tubes (BD Vacutainer) After centrifugation at1000 rpm the supernatant platelet-rich plasma (PRP) wascollected and then a sample of PRP was centrifuged at 3000rpm After centrifugation platelet pellets were formed andseparated fromplatelet-poor plasmaThen the platelet pelletswere frozen and stored at minus80∘C until all samples had beencollected Besides platelet-poor plasma was also collectedfor further enzyme-linked immunosorbent assay (ELISA)analysis

222 Western Blot Analysis Platelets from patients werelysed by RIPA buffer (50 mM Tris-HCl (pH 74) 150 mMNaCl 1 mM EDTA 1 NP40 025 sodium deoxycholate 1mMdithiothreitol (DTT) 1120583gml aprotinin and 1 120583gml leu-peptin) with 25x proteinase inhibitors for 1 hour on ice Aftercentrifugation protein concentration was measured by BCAprotein assay and equal amounts of protein were preparedwithMQwater and 6X loading dye For protein denaturationmixed protein was heated at 100∘C for 5 minutes Protein wasloaded on sodium dodecyl sulfate (SDS)-containing 10 and15 polyacrylamide gel and electrophoresed at 70V for 30minutes and then at 100V for 2 hour Protein was transferredto polyvinylidene difluoride (PVDF) nylon membrane at300 mA for 3 hours After blocking transfer membranewas immersed into blocking buffer containing 5 skim milkat RT 1 hour After 3 times of washing by 1X TBST thetransfer membrane was immersed into primary antibod-ies at 4∘C overnight Specific antibodies for Western blotanalysis were galectin-3 (Abcam Cambridge UK) ROCK-II (BD Biosciences NJ USA) phosphor-(Thr853) MYPT1MYPT1 MLC2 (Cell Signaling Technology Inc DanversMA USA) phosphor-(Ser19) MLC and actin (MilliporeTemecula CA) On the second day the transfer membranewas washed 3 times by 1X TBST The second antibodieswere used to target primary antibodies at RT for 1 hourAfter 3 times of washing specific bands were detected by ahorseradish peroxidase-conjugated antibody and revealed byan enhanced chemiluminescence (ECL) Western blot system(PerkinElmer Waltham MA USA) We further analyzed thefilm and measured bandrsquos intensities by Image Pro software(Media Cybernetics Inc Rockville MD USA) and theprotein expression was normalized by actin Besides plateletsfrom healthy person were used for control in Western blotanalysis

223 Platelet Rho-Kinase Assay ROCK assays were per-formed on all platelet samples after extraction The sampleswere analyzed by Western blotting for the phosphorylationof MYPT with an antibody that specifically recognizedphosphorylated Thr853 and the phosphorylation of MLCspecifically on Ser19 We analyzed the phosphorylation ratiofor Rho-kinase activities

224 Galectin-3 and Galectin-3 BP ELISA Galectin-3 andgalectin-3 BP levels were measured by using an ELISA kit(RampD Systems Inc Minneapolis MN USA catalogue num-bers DGAL30 and DGBP30B) According to the instructionsthe detectable concentrations of galectin-3 and galectin-3 BPwere 25 ngmL and 100 ngmL The scanning procedure wasperformed by using a microscope (BX51 Olympus) at 20timesmagnification and pictures were collected

225 Immunohistochemical Assessment Thrombus samplesfrom the occluded shunts of patients were fixed in formalinand then paraffin-embedded tissue blocks were cut into 5-120583m-thick slides Samples were deparaffinized rehydratedand then covered with 3 H2O2 for 10 min After blockingwith 5 bovine serum albumin the slides were incubatedwith primary antibodies (galectin-3) overnight at 4∘C Sec-ondary antibodies were incubated for 1 h the next day Forstaining 331015840-Diaminobenzidine was used as the chromogenwhich was counterstained with hematoxylin

23 Statistical Analysis Statistical analyses were performedby using SPSS Version 220 (IBM Corp Armonk NYUSA) Continuous data were presented as mean plusmn standarddeviation or median (interquartile range) depending on thedistribution Dichotomous data were presented as numbersand percentages Comparisons were conducted with non-parametric statistics by using the Wilcoxon rank sum testor the MannndashWhitney U test for continuous variables Amultivariate linear regression analysis was used to analyzecorrelations between potential associated factors and ourbiomarkers Variables with p lt 02 in a univariate linearregression analysis and other associated factors of specialconcern were also included

3 Results

31 Baseline Characteristics Twenty-nine enrolled patientsreceived a complete follow-up during our study The meanage of our patients was 662 years and 448 of the patientswere male Among the enrolled patients 655 had a historyof diabetes and 344 were classified as having poorly con-trolled diabetes based on HbA1C gt8 Detailed informationabout the baseline characteristics of the patients is presentedin Table 1

32 Biomarkers in Occluded versus Patent ArteriovenousShunts (Plasma) We first compared the occluded and patentAV shunt conditions in each patient Circulating galectin-3and galectin-3 BP were measured by using a galectin-3- andgalectin-3-BP ELISA


P= 017

Shunt condition

Circ

ulat

ing

Gal

ectin

-3

Occluded056

8

10

12

14

Patent

(a)

P= 0009

Shunt condition

Circ

ulat

ing

Gal

ectin

-3 B

P

Occluded033

5

7

9

11

13

Patent

(b)

Figure 1 Biomarkers in occluded versus patent arteriovenous shunts (plasma)

Table 1 Baseline characteristics of patients with end-stage renaldisease and shunt occlusion

Characteristic End-stage renal disease patients withshunt occlusion (N =29)

Personal factorsAge 662 plusmn 112Gender (Male) 13 (448)BMI 234 plusmn 45Smoking 2 (68)

Medical historyHypertension 20 (689)Hyperlipidemia 7 (241)Coronary artery disease 3 (103)Stroke 5 (172)Diabetes mellitus 19 (655)HbA1C gt8 10 (344)Insulin use 10 (344)Statin use 5 (172)Antiplatelet use 8 (275)

Shunt conditionProsthetic graft 16 (551)Total occlusion 15 (517)Urokinase usesect 5 (172)Repeated interventionsectsect 23 (793)

Gender male BMI kgm2 HbA1C data n () or mean plusmn standarddeviationsect urokinase use during catheter intervention sectsect within a year afterenrollment

The plasma galectin-3 ELISA was similar between theoccluded status and the patent status of AV shunts (131[117ndash141] ngmL vs 129 [122ndash134] ngmL p = 017) (Fig-ure 1(a)) The plasma galectin-3 BP ELISA was significantlyhigher at the occluded status of AV shunts (84 [60ndash97]120583gmL vs 71 [45ndash91] 120583gmL p = 0009) (Figure 1(b))

33 Biomarkers in Occluded versus Patent ArteriovenousShunts (Platelets) After platelet extraction a Western blotassay for ROCK activity was completed by using the previ-ously published protocol [22] ROCK activity was expressedas the ratio of phosphorylation levels of MYPT divided bytotal MYPT (Figure 2)

Platelet MYPT ratios were significantly higher foroccluded AV shunts 05 (03ndash14) vs 04 (03ndash06) p =001 Platelet galectin-3 was significantly higher for occludedAV shunts 12 (04ndash16) vs 07 (01ndash12) p = 00004(Figure 2)

The platelet MYPT ratio was significantly higher at theoccluded status of AV shunts 05 (03ndash14) vs 04 (03ndash06)p = 001 (Figure 2(a)) Platelet galectin-3 was significantlyhigher at the occluded status of AV shunts 12 (04ndash16) vs07 (01ndash12) p = 00004 (Figure 2(b)) Actin was used as aloading control in Western blot analysis Besides plateletsfrom healthy person were used for control in Western blotanalysis

34 Characterizing the Possible Interaction between ShuntOcclusion and Biomarkers in Diabetes We found higherplatelet MYPT ratios and galectin-3 values in occluded AVshunts than in patent shunts We then considered the valuesof biomarkers as occluded status divided by patent statusin each patient We used the ratios of biomarker values torepresent the degrees of elevated biomarkers in occluded AVshunts

A high platelet MYPT ratio difference was also noted inpatients with poorly controlled diabetes 17 (10ndash30) vs 11(07ndash13) p = 006 Also a high platelet galectin-3 differencewas noted in the patients with poorly controlled diabetes 16(13ndash34) vs 11 (08ndash19) p = 005 (Figure 3)

A higher platelet MYPT ratio difference was also notedin patients with poorly controlled diabetes 17 (10ndash30) vs 11(07ndash13) p = 006 (Figure 3(a)) A higher platelet galectin-3 difference was noted in patients with poorly controlleddiabetes 16 (13ndash34) vs 11 (08ndash19) p = 005 (Figure 3(b))


ROCK2

p-MYPT

p-MLC

t-MYPT

t-MLC

actin

Occluded PatentOccluded Patent

P= 001

Shunt condition

MYP

T ra

tio

00

05

10

15

252565

20

(a)

Shunt condition

(Fol

ds o

f hea

lthy

cont

rol)

Gal

ectin

-3

0

1

2

3

448 P= 00004

Galectin-3

actin


(b)

Figure 2 Biomarkers in occluded versus patent arteriovenous shunts (platelets)

P= 006

DM control

MYP

T ra

tio d

iffer

ence

0

1

2

3

4

（＜1gt8 （＜1lt8

(a)

P= 005

DM control

Gal

ectin

-3 d

iffer

ence

0

1

2

3

4

8

（＜1gt8 （＜1lt8

(b)

Figure 3 Characteristics of the possible interaction between shunt occlusion and biomarkers in diabetes


Figure 4 Immunohistochemical stain of galectin-3 in thrombotictissue from an occluded AV shunt

35 Galectin-3 Is Increased Locally at Totally OccludedAV Shunts Thrombus samples from totally occluded AVshunts were obtained during scheduled invasive proceduresGalectin-3 expression was identified by using an immunos-taining technique Similar to previous data on venous throm-bosis [20] an abundant amount of galectin-3 was stained inthe thrombus samples from occluded AV shunts (Figure 4)

DAB was used as the chromogen for staining whichwas counterstainedwith hematoxylin immunohistochemicalstain of galectin-3 in thrombotic tissue (white arrow) Abun-dant quantities of galectin-3 were stained in our thrombussamples from occluded AV shunts

36 Clarifying the Correlation between Thrombus Burden andBiomarkers Different vessel condition and thrombus burdenare possible variant factors to AV shunt dysfunction Tofurther analyze the mechanisms underlying thrombosis wedivided them into subgroups based on the shunt charactersand severity of occlusion

361 Prosthetic Graft vs Autologous Fistula Compared withautologous fistulas prosthetic grafts showed a significantlyhigher platelet galectin-3 level 27 (15ndash45) vs 12 (08ndash14)p = 0002 (Figure S1a) The platelet MYPT ratio was similarbetween prosthetic grafts and autologous fistulas 14 (10ndash32)vs 11 (08ndash17) p = 016 (Figure S1b)

362 Total Occlusion vs Subtotal Occlusion Thedifference inplatelet galectin-3 was similar between totally and subtotallyoccluded AV shunts 21 (11ndash46) vs 13 (10ndash21) p = 024(Figure S2a) A similar difference in the platelet MYPT ratiowas also noted between totally and subtotally occluded AVshunts 15 (08ndash33) vs 11 (10ndash18) p = 080 (Figure S2b)

37 Multivariate Linear Regression Analysis We found that ahigher MYPT ratio for occluded status was associated withHbA1C (Beta coefficient 0658 p lt0001) Furthermore ahigher galectin-3 difference was associated with HbA1C (Betacoefficient 0377 p = 0044) The shunt characteristics andseverity of occlusion were not associated with our biomarkersor the ratio of biomarker values in the multivariate linearanalysis (Tables S1 and S2)

4 Discussion

We analyzed 29 patients with AV shunt dysfunction whoreceived a catheter-based intervention We found that plateletMYPT ratios and galectin-3 levels were relatively high atthe time of AV shunt occlusion In patients with poorlycontrolled diabetes higher plateletMYPT ratios and galectin-3 levels were more likely than in the patients with wellcontrolled diabetes In patients with prosthetic grafts thedegree of elevated platelet galectin-3 was higher than inpatients with autologous fistulas In summary high ROCKactivity and galectin-3 levels were associated with increasedrisk for AV shunt dysfunction especially in patients withpoorly controlled diabetes

41 Hyperglycemia Is Associated with AV Shunt Dysfunc-tion through the RhoAROCK Pathway Inducing PlateletActivation MYPT ratios in occluded status appeared to beassociated withHbA1C Furthermore a higher plateletMYPTratio difference was noted in patients with poorly controlleddiabetes We supposed that hyperglycemia is associated withAV shunt thrombosis through the RhoAROCK pathwayinducing platelet activation

The Rho family of GTP binding proteins also commonlyreferred to as the RhoGTPases are pertinent regulators of theplatelet cytoskeleton and platelet function These low molec-ular weight GTPases act as signaling switches in the spatialand temporal transduction and amplification of signals fromplatelet cell surface receptors to the intracellular signalingpathways that drive platelet function The Rho GTPasefamily members RhoA Cdc42 and Rac1 have emerged askey regulators in the dynamics of the actin cytoskeletonin platelets and play roles in platelet aggregation secretionspreading and thrombus formation Rho GTPase regulatorsincluding guanine nucleotide exchange factors and GTPase-activating proteins and other downstream effectors may alsoregulate platelet activation and function [23]

In a previous study hyperglycemia stimulated Rho-kinase activity via PKC- and oxidative stressndashdependentpathways leading to increased PAI-1 gene transcription [15]These potential mechanisms can be applied to our clinicalresults and associated with AV shunt thrombosis through theRhoAROCK pathway inducing platelet activation

In previous review article galectin-3 was one of theadvanced glycosylation end product (AGE)-binding proteinsand participated in some diabetic complications involvingthose in the nervous tissue kidney and vessels Availabledata showed that hyperglycemiawas associatedwith galectin-3 upregulation Then advanced AGE-binding protein andadhesive lectin might influence development of diabeticcomplications [24]

Besides we also observed a potential role for galectin-3 in hepatocyte adipocyte and myocyte insulin resistancesuggesting that galectin-3 can link inflammation to decreasedinsulin sensitivity Inhibition of galectin-3 could be a newapproach to treat insulin resistance [25] Galectin-3 inhibitorwas investigated to be applied to the fields of heart failurecancer and nonalcoholic steatohepatitis [26]


In our study a trend of higher platelet galectin-3 differ-ence was noted in patients with poorly controlled diabetesand higher galectin-3 difference was associated with HbA1Cbymultivariate analysis Potential relationship between sugarcontrol and galectin-3 was also noted this time

Based on previous literature and our study resultsgalectin-3 inhibitor play a role in mediating inflammatorypathways and platelet activation andmight benefit the groupsof insulin resistance

42 Galectin-3 Participates in Platelet Activation Signifi-cantly higher platelet galectin-3 and circulating galectin-3 BPlevels were noted for occluded AV shunts than for patentstatus in this study

Platelets can be activated by soluble molecules includingthrombin thromboxane A2 adenosine diphosphate andserotonin or by adhesive extracellular matrix proteins suchas Von Willebrand factor We described a recent advancedpathway in the activation of platelets by noncanonical plateletagonists such as galectins [27]

Galectin-3 BPwas formerly known asMac-2 binding pro-tein and was initially described as a tumor-secreted proteinGalectin-3 BP was recently reported as a large oligomericprotein composed of approximately 90 kDa subunits witheach one containing numerous cysteines andN-glycosylationsites [27] Galectin-3 BP has the ability to bind to differentgroups of proteins via these subunits Furthermore galectin-3 BP is heavily glycosylated which increases its affinity tolarger groups of proteins Galectin-3 BP binds to galectin-3as a binding protein modulating galectin-3 activities such asthe promotion of cell-cell adhesion Galectin-3 BP also bindsto galectin-1 and then modulates the inflammatory activityof galectin-1 Galectin-3 BP as a binding galectin protein iscrucial for galectin mediated biological processes [27]

43 Potential Connection between ROCK and Galectin-3 Based on previous data in our laboratory room theexpression of ROCK2 and galectin-3 increased in activemacrophage After ROCK inhibitor the expression ofgalectin-3 decreased in the active macrophage [28]

In summary hyperglycemia stimulated ROCK activityleading to increased PAI-1 gene transcription These potentialmechanisms can be applied to our clinical results and asso-ciated with AV shunt thrombosis through the RhoAROCKpathway inducing platelet activation Galectin-3 was oneof the downstream factors in RhoAROCK pathway andgalectin-3 can be mediated by ROCK inhibitor

44 Galectin-3 and Fibrosis We also found that approxi-mately 73 of our patients had the problems of drainingvein stenosis In previous data more than 60 of stenosiswas located in the venous anastomosis and about 20 inthe venous outlet for a total of 80 Correcting stenosismay decrease the risk of thrombosis and improve graftpatency [29] However some of the draining veins weredifficult to treat by using balloon angioplasty due to fibroticchanges which are different from atherosclerotic changes ofthe peripheral artery

Galectin-3 plays a vital role in the promotion of fibrosis[30] Fibrosis is a consequence of inflammation Galectin-3 activates fibroblasts which are responsible for collagendeposition leading to fibrosis [30 31] Galectin-3 is involvedin the synthesis of new matrix components such as typeI collagen Furthermore it also modulates the degradationof extracellular matrix components through tissue inhibitormetalloproteinases and matrix metalloproteinases [27]

Galectin-3 has been evaluated as an important biomarkerof heart failure and cardiac fibrosis andmay also be associatedwith renal fibrosis There was growing evidence of highgalectin-3 associated with elevated risk of renal deterioration[24 32] and galectin-3 seemed to have a potential role intreatment of kidney disease [32]

Based on this hypothesis galectin-3 was also found tobe independently associated with progressive renal disease intype 2 diabetes [33] However limited studies were designedto focus on the role of galectin-3 in hemodialytic patientsVascular access dysfunction was critical point for hemodia-lytic patients and diabetes affected the risk of shunt failure[34] In addition to traditional medications for potential riskfactors current literature reported that drug-eluting balloonfor recurrent AV shunt stenosis seemed to be a safe andbeneficial therapy [35] Based on the results of our studygalectin-3 inhibition might be a potential target for drug-eluting balloon to treat draining vein fibrosis in venousanastomosis

45 Limitations The main limitation of the present studyis its small sample size Although the clinical presentationprovided a potential correlation between our biomarkers andHbA1C our sample size was too small to fulfill the thresholdof the multivariate linear regression analysis

Moreover no angiographic score was established todifferentiate thrombus with the burden of AV shunt occlu-sion We only divided our patients into two groups totallyoccluded shunts and non-totally occluded shunts Thrombusburden was not demonstrated in our methods therefore wecould not explore the potential correlation between galectin-3 and thrombus burden even if a higher galectin-3 differencewas identified in prosthetic grafts which tend to impose arelatively large thrombus burden on our daily practice

Unlike other cardiovascular research selecting appro-priate study endpoints in clinical trials of AV shunt dys-function is challenging Overall only 20 of our patientsdid not receive repeated catheter intervention for AV shuntdysfunctionwithin a year after enrollmentUnder this clinicalsituation it was difficult to identify potential biomarkersfor understanding AV shunt patency which is evident inother clinical trials For example the Dialysis Access Con-sortium Fistula Thrombosis Trial examined whether dailyclopidogrel (versus placebo) prevented early AVfistula failure[36] Clopidogrel significantly reduced AV fistula thrombosiswithin 6 weeks but it did not significantly increase AVfistula suitability for dialysis within 6 months Thus theantithrombotic effects of clopidogrel did not culminate inimproved AV fistula functionality

Unlike the results in isolated platelets plasma galectin-3 levels did not increase at occluded status of AV shunt


in our study Potential reason might be our limitation oflaboratory methods We aimed to investigate the potentialrole of platelet activity so our blood samples were collectedwith platelet-poor plasma and platelet pellets Western blotanalysis was done for platelet galectin-3 but we only cando the ELISA analysis for galectin-3 in platelet-poor plasmaFurther studies might be warranted for the roles of galectin-3in the microenvironment of vessel thrombosis

46 Future Work This is the first study to demonstrate thepotential role of galectin-3 and ROCK activity in AV shuntdysfunction Platelet MYPT ratios and galectin-3 levels werehigher at the time of AV shunt occlusion The degree ofelevated platelet MYPT ratios and galectin-3 levels appearedto be higher in patients with poorly controlled diabetesFurther animal study is recommended to clarify the causalrelationship between the ROCK pathway and galectin-3

Impressive advances in the biology of galectins and theirrole in cell homeostasis have been made in recent yearsCurrently available information indicates that galectins areexpressed and secreted by several cell types in normal andpathological conditions In summary regarding galectin-3 asa soluble mediator capable of triggering platelet activationoffers new opportunities that will provide further insightinto the mechanisms bridging inflammatory responses to theformation of thrombus

5 Conclusion

High platelet ROCK activity and galectin-3 levels are associ-ated with increased risk of arteriovenous shunt dysfunctionespecially in patients with poorly controlled diabetes

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Ethical Approval

The hospitalrsquos research and ethics committee approved thestudy design

Consent

Informed consent was obtained from all participants prior toenrollment

Disclosure

Theauthors take responsibility for all aspects of the reliabilityand freedom from bias of the data presented and theirdiscussed interpretation The preliminary abstract of thismanuscript was presented before at the poster section of ESCCongress and the link of European Heart Journal was listedas follows httpsacademicoupcomeurheartjarticle38suppl 1ehx493P53734086734

Conflicts of Interest

There are no real or potential conflicts of interest involved inthis publication

Acknowledgments

This study was funded by research grants from the Nation-al Cheng Kung University Hospital in Tainan Taiwan(NCKUH-10405041 NCKUH-10504024) This manuscriptwas edited by Wallace Academic Editing

Supplementary Materials

Supplementary Figure Legends Figure S1 Interplay betweendifferent shunt types and the biomarkers Compared withautologous fistula prosthetic grafts showed a significantlyhigher platelet galectin-3 level 27 (15ndash45) vs 12 (08ndash14)p = 0002 (Figure S1a) The platelet MYPT ratio was similarbetween different shunt types 14 (10ndash32) vs 11 (08ndash17)p = 016 (Figure S1b) Figure S2 Interplay between thrombusburden and biomarkers The platelet galectin-3 difference wassimilar between totally and subtotally occluded AV shunts21 (11ndash46) vs 13 (10ndash21) p = 024 (Figure S2a) A similarplatelet MYPT ratio difference was noted between totally andsubtotally occluded status 15 (08ndash33) vs 11 (10ndash18) p =080 (Figure S2b) Supplementary tables Table S1 Factorsassociated with the MYPT ratio at occluded status of AVshunt Table S2 Factors associated with galectin-3 differences(occludedpatent status) (Supplementary Materials)

References

[1] C J Hill andD G Fogarty ldquoChanging trends in end-stage renaldisease due to diabetes in theUnitedKingdomrdquo Journal of RenalCare vol 38 no 1 pp 12ndash22 2012

[2] B S Grace P Clayton and S P McDonald ldquoIncreases inrenal replacement therapy in Australia and New ZealandUnderstanding trends in diabetic nephropathyrdquo Nephrologyvol 17 no 1 pp 76ndash84 2012

[3] BMannsM Tonelli S Yilmaz et al ldquoEstablishment andmain-tenance of vascular access in incident hemodialysis patientsa prospective cost analysisrdquo Journal of the American Society ofNephrology vol 16 no 1 pp 201ndash209 2005

[4] K R Polkinghorne S P Mcdonald R C Atkins and P G KerrldquoVascular access and all-cause mortality a propensity scoreanalysisrdquo Journal of the American Society of Nephrology vol 15no 2 pp 477ndash486 2004

[5] G E Smith R Gohil and I C Chetter ldquoFactors affecting thepatency of arteriovenous fistulas for dialysis accessrdquo Journal ofVascular Surgery vol 55 no 3 pp 849ndash855 2012

[6] T Hod R N Desilva B K Patibandla Y Vin R S Brown andA S Goldfarb-Rumyantzev ldquoFactors predicting failure of AVldquofistula firstrdquo policy in the elderlyrdquo Hemodialysis Internationalvol 18 no 2 pp 507ndash515 2014

[7] J A Bittl ldquoCatheter interventions for hemodialysis fistulas andgraftsrdquo JACC Cardiovascular Interventions vol 3 no 1 pp 1ndash112010


[8] N C Tanner and A Da Silva ldquoMedical adjuvant treatment toincrease patency of arteriovenous fistulae and graftsrdquo CochraneDatabase Systematic Reviews Article ID CD002786 2015

[9] K A Nath and M Allon ldquoChallenges in developing newtherapies for vascular access dysfunctionrdquoClinical Journal of theAmerican Society of Nephrology vol 12 no 12 pp 2053ndash20552017

[10] H J T Huijbregts M L Bots C H A Wittens Y C SchramaF L Moll and P J Blankestijn ldquoHemodialysis arteriovenousfistula patency revisited Results of a prospective multicenterinitiativerdquo Clinical Journal of the American Society of Nephrol-ogy vol 3 no 3 pp 714ndash719 2008

[11] K Konner ldquoPrimary vascular access in diabetic patients anauditrdquo Nephrology Dialysis Transplantation vol 15 no 9 pp1317ndash1325 2000

[12] J Sheu P Lin P Sung et al ldquoLevels and values of lipoprotein-associated phospholipase A2 galectin-3 RhoAROCKand endothelial progenitor cells in critical limb ischemiapharmaco-therapeutic role of cilostazol and clopidogrelcombination therapyrdquo Journal of Translational Medicine vol12 no 1 p 101 2014

[13] M Dong B P Yan and C M Yu ldquoCurrent status of rho-associated kinases (ROCKs) in coronary atherosclerosis andvasospasmrdquo Cardiovascular amp Hematological Agents in Medic-inal Chemistry vol 7 pp 322ndash330 2009

[14] P-Y Liu Y-W Liu L-J Lin J-H Chen and J K LiaoldquoEvidence for statin pleiotropy in humans differential effects ofstatins and ezetimibe on Rho-associated coiled-coil containingprotein kinase activity endothelial function and inflamma-tionrdquo Circulation vol 119 no 1 pp 131ndash138 2009

[15] Y Rikitake and J K Liao ldquoRho-kinasemediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascu-lar endothelial cellsrdquo Circulation vol 111 no 24 pp 3261ndash32682005

[16] H Shimokawa S Sunamura and K Satoh ldquoRhoARho-Kinasein the cardiovascular systemrdquo Circulation Research vol 118 no2 pp 352ndash366 2016

[17] J-N Roan S-Y Fang S-W Chang et al ldquoRosuvastatinimproves vascular function of arteriovenous fistula in a diabeticratmodelrdquo Journal of Vascular Surgery vol 56 no 5 pp 1381e1ndash1389e1 2012

[18] P Argueso and N Panjwani ldquoFocus on molecules galectin-3rdquoExperimental Eye Research vol 92 no 1 pp 2-3 2011

[19] R Dong M Zhang Q Hu et al ldquoGalectin-3 as a novelbiomarker for disease diagnosis and a target for therapy(Review)rdquo International Journal of Molecular Medicine vol 41pp 599ndash614 2018

[20] E P DeRoo S K Wrobleski E M Shea et al ldquoThe role ofgalectin-3 and galectin-3-binding protein in venous thrombo-sisrdquo Blood vol 125 no 11 pp 1813ndash1821 2015

[21] L Agnello G Bivona B Lo Sasso et al ldquoGalectin-3 in acutecoronary syndromerdquoClinical Biochemistry vol 50 no 13-14 pp797ndash803 2017

[22] P-Y Liu and J K Liao ldquoA method for measuring rho kinaseactivity in tissues and cellsrdquo Methods in Enzymology vol 439pp 181ndash189 2008

[23] J E Aslan andO JMccarty ldquoRhoGTPases in platelet functionrdquoJournal of Thrombosis and Haemostasis vol 11 no 1 pp 35ndash462013

[24] F Pricci G Leto L Amadio et al ldquoRole of galectin-3 asa receptor for advanced glycosylation end productsrdquo KidneyInternational Supplements vol 58 no 77 pp S31ndashS39 2000

[25] P Li S Liu M Lu et al ldquoHematopoietic-derived galectin-3causes cellular and systemic insulin resistancerdquoCell vol 167 no4 pp 973ndash984e12 2016

[26] K Kingwell ldquoDiabetes turning down galectin 3 to combatinsulin resistancerdquo Nature Reviews Drug Discovery vol 16 no1 p 18 2016

[27] J A Diaz E Ramacciotti and T W Wakefield ldquoDo galectinsplay a role in venous thrombosis a reviewrdquo ThrombosisResearch vol 125 no 5 pp 373ndash376 2010

[28] Y W Wang and P Y Liu Roles of ROCK and its associatedpathways during acute vascular thrombosis events [Masterrsquosthesis] Institute of Clinical Medicine College of MedicineNational Cheng Kung University Taiwan 2015

[29] I D Maya R Oser S Saddekni J Barker and M AllonldquoVascular access stenosis comparison of arteriovenous graftsand fistulasrdquo American Journal of Kidney Diseases vol 44 no5 pp 859ndash865 2004

[30] N C Henderson A C Mackinnon S L Farnworth et alldquoGalectin-3 expression and secretion links macrophages to thepromotion of renal fibrosisrdquoTheAmerican Journal of Pathologyvol 172 no 2 pp 288ndash298 2008

[31] N C Henderson A C Mackinnon S L Farnworth etal ldquoGalectin-3 regulates myofibroblast activation and hepaticfibrosisrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 103 no 13 pp 5060ndash5065 2006

[32] S Chen and P Kuo ldquoThe role of galectin-3 in the kidneysrdquoInternational Journal of Molecular Sciences vol 17 no 4 p 5652016

[33] K C B Tan C-L Cheung A C H Lee J K Y Lam Y Wongand S W M Shiu ldquoGalectin-3 is independently associatedwith progression of nephropathy in type 2 diabetes mellitusrdquoDiabetologia vol 61 no 5 pp 1212ndash1219 2018

[34] C Yen C Tsai Y Luo et al ldquoFactors affecting fistula fail-ure in patients on chronic hemodialysis a populationndashbasedcasendashcontrol studyrdquo BMC Nephrology vol 19 no 1 p 213 2018

[35] A Z Khawaja D B Cassidy J Al Shakarchi D G McGroganN G Inston and R G Jones ldquoSystematic review of drugeluting balloon angioplasty for arteriovenous haemodialysisaccess stenosisrdquo Journal of VascularAccess vol 17 no 2 pp 103ndash110 2016

[36] L M Dember G J Beck M Allon et al ldquoEffect of clopidogrelon early failure of arteriovenous fistulas for hemodialysisa randomized controlled trialrdquo The Journal of the AmericanMedical Association vol 299 no 18 pp 2164ndash2171 2008

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


A functional vascular access is critical for effectivehemodialysis AV fistula dysfunction largely reflects mat-urational failure whereas AV graft dysfunction is mainlydriven by recurrent stenosis and thrombosis in the venousanastomosis [9] The current understanding of the biologyof vascular access dysfunction remains inadequate and prob-lematic

Studies have shown that diabetes mellitus is a risk factorin the development of vascular access failure [10] Althoughthe mechanisms responsible for the higher rate of AV fistulafailure in subjects with diabetes are unclear peripheral arte-rial disease impaired vasodilatation secondary to endothelialdysfunction and increased thrombogenicity are believed tocontribute [11]

Platelet activation and inflammation may play essentialroles in the development of atherosclerotic disease Variousbiomarkers have been used to assess platelet activity andinflammation in different clinical settings Among themgalectin-3 levels and RhoARho-associated protein kinase(ROCK)-related protein expression in peripheral blood wereused in one study as therapeutic biomarkers to treat periph-eral artery disease [12]

ROCK is a serinethreonine kinase consisting of twoisoforms ROCK-I and ROCK-II which may mediate thedownstream signaling of the small guanosine triphosphate(GTP)-binding protein (BP) Rho [13 14] It is mainlyinvolved in regulating the shapes and movements of cells byacting on cytoskeletons Recent observations suggest that thebeneficial cardiovascular effects of statins may result at leastin part from the inhibition of ROCKs [13 14]

Hyperglycemia is a pertinent factor in the developmentof macrovascular complications in diabetes vascular smoothmuscle cell (VSMC) migration and proliferation also play acrucial role There is growing evidence that ROCK may beassociated with many cardiovascular conditions includinghypertension atherosclerosis coronary vasospasm myocar-dial hypertrophy and stroke through its action in VSMCcontraction endothelial function and inflammatory pro-cesses [14 15]

Furthermore elevated levels of plasminogen activatorinhibitor-1 (PAI-1) are associated with endothelial dys-function myocardial infarction and stroke especially inpatients with diabetes A study showed that the inductionof PAI-1 expression by hyperglycemia involves oxidativestress and protein kinase C (PKC) Hyperglycemia stimu-lates Rho-kinase activity via PKC- and reactive oxidativestressndashdependent pathways increasing PAI-1 gene transcrip-tion [15] In one study of murine lung endothelial cells(MLECs) Rho-kinase activity increased after exposure tohigh glucose whereas Rho-kinase activity was unchanged inROCK I+minusMLECs indicating that hyperglycemia stimulatedRho-kinase activity [15]

The RhoARho-kinase pathway is widely known in manycellular functions including contraction motility prolifera-tion and apoptosis and its excessive activity induces oxida-tive stress and promotes cardiovascular diseases Howeverlimited data is available on the stenosis and thrombosis of AVshunts [16 17]

Galectins are carbohydrate-BPs with high affinity togalactosides on cell surfaces and extra-cellular glycoproteinsGalectins are a family of 120573-galactoside-binding lectins withconserved carbohydrate-recognition domains (CRDs) Cur-rently 15 galectins have been identified in mammals whichare divided into three types based on domain organizationas follows prototype galectins with one single CRD tandem-repeat galectins with two CRDs and chimera-type galectinswith a single CRD connected to a long flexible N-terminaldomain [18]

The expression of galectin-3 has been detected in leuko-cytes mast cells and various organ tissues Various bio-logical functions are involved including cell apoptosis cellactivation and inflammation Galectin-3 BP and its recep-torligand galectin-3 are secreted proteins that can inter-act with each other to promote cell-to-cell adhesion Thispathway involves pathologic and proinflammatory signalingcascades [19 20]

In venous thrombosis galectin-3 BP and galectin-3 playcritical roles possibly through IL-6 and PMN-mediatedthrombotic mechanisms and are potential biomarkers inhuman venous thrombosis [20] Galectin-3 has been apromising prognostic marker It has a crucial role in inflam-mation and fibrosis Both experimental and clinical studieshave shown that galectin-3 is an independent predictor ofall-cause mortality cardiovascular death and occurrence ofheart failure following acute coronary syndrome [21]

We hypothesized that high ROCK activity and galectin-3 levels were associated with increased risk for arteriove-nous shunt dysfunction in patients with poorly controlleddiabetes

2 Methods

21 Study Population We prospectively enrolled 38 patientsdiagnosed with arteriovenous shunt dysfunction who werereferred to receive percutaneous intervention in our catheter-ization laboratory at National Cheng Kung University(NCKU)Medical Center andDou-Liu Branch fromFebruary2015 to November 2016

Details of the study protocol were explained to allparticipants and written informed consent was obtainedbefore the study The survey was conducted according to theprinciples in the Declaration of Helsinki and was approvedby the Medical Ethics Committee of NCKU Hospital [IRBA-ER-103-243 A-ER-104-201] All patients were confirmed ashaving AV shunt dysfunction by using invasive angiography

Among these patients 29 received a complete follow-upand each provided two blood samples which were collectedat the first visit (percutaneous intervention for AV shuntdysfunction) for occluded status of AV shunts and at least 1month later for patent status

Twenty milliliters of blood was drawn at the occludedsite of the AV shunt into an ethylenediaminetetraacetic acidtube After platelet extraction aWestern blot assay formyosinphosphatase target subunit (MYPT) and myosin light chain(MLC)were performed to predict ROCK activity Rho-kinase











3 Results




P= 017

Shunt condition

Circ

ulat

ing

Gal

ectin

-3

Occluded056

8

10

12

14

Patent

(a)

P= 0009

Shunt condition

Circ

ulat

ing

Gal

ectin

-3 B

P

Occluded033

5

7

9

11

13

Patent

(b)
















ROCK2

p-MYPT

p-MLC

t-MYPT

t-MLC

actin


P= 001

Shunt condition

MYP

T ra

tio

00

05

10

15

252565

20

(a)

Shunt condition

(Fol

ds o

f hea

lthy

cont

rol)

Gal

ectin

-3

0

1

2

3

448 P= 00004

Galectin-3

actin


(b)


P= 006

DM control

MYP

T ra

tio d

iffer

ence

0

1

2

3

4

（＜1gt8 （＜1lt8

(a)

P= 005

DM control

Gal

ectin

-3 d

iffer

ence

0

1

2

3

4

8

（＜1gt8 （＜1lt8

(b)










4 Discussion



























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























3 Results




P= 017

Shunt condition

Circ

ulat

ing

Gal

ectin

-3

Occluded056

8

10

12

14

Patent

(a)

P= 0009

Shunt condition

Circ

ulat

ing

Gal

ectin

-3 B

P

Occluded033

5

7

9

11

13

Patent

(b)
















ROCK2

p-MYPT

p-MLC

t-MYPT

t-MLC

actin


P= 001

Shunt condition

MYP

T ra

tio

00

05

10

15

252565

20

(a)

Shunt condition

(Fol

ds o

f hea

lthy

cont

rol)

Gal

ectin

-3

0

1

2

3

448 P= 00004

Galectin-3

actin


(b)


P= 006

DM control

MYP

T ra

tio d

iffer

ence

0

1

2

3

4

（＜1gt8 （＜1lt8

(a)

P= 005

DM control

Gal

ectin

-3 d

iffer

ence

0

1

2

3

4

8

（＜1gt8 （＜1lt8

(b)










4 Discussion



























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















P= 017

Shunt condition

Circ

ulat

ing

Gal

ectin

-3

Occluded056

8

10

12

14

Patent

(a)

P= 0009

Shunt condition

Circ

ulat

ing

Gal

ectin

-3 B

P

Occluded033

5

7

9

11

13

Patent

(b)
















ROCK2

p-MYPT

p-MLC

t-MYPT

t-MLC

actin


P= 001

Shunt condition

MYP

T ra

tio

00

05

10

15

252565

20

(a)

Shunt condition

(Fol

ds o

f hea

lthy

cont

rol)

Gal

ectin

-3

0

1

2

3

448 P= 00004

Galectin-3

actin


(b)


P= 006

DM control

MYP

T ra

tio d

iffer

ence

0

1

2

3

4

（＜1gt8 （＜1lt8

(a)

P= 005

DM control

Gal

ectin

-3 d

iffer

ence

0

1

2

3

4

8

（＜1gt8 （＜1lt8

(b)










4 Discussion



























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















ROCK2

p-MYPT

p-MLC

t-MYPT

t-MLC

actin


P= 001

Shunt condition

MYP

T ra

tio

00

05

10

15

252565

20

(a)

Shunt condition

(Fol

ds o

f hea

lthy

cont

rol)

Gal

ectin

-3

0

1

2

3

448 P= 00004

Galectin-3

actin


(b)


P= 006

DM control

MYP

T ra

tio d

iffer

ence

0

1

2

3

4

（＜1gt8 （＜1lt8

(a)

P= 005

DM control

Gal

ectin

-3 d

iffer

ence

0

1

2

3

4

8

（＜1gt8 （＜1lt8

(b)










4 Discussion



























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























4 Discussion



























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of







































5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























5 Conclusion


Data Availability


Ethical Approval


Consent


Disclosure




Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Elevated Platelet Galectin-3 and Rho-Associated Protein ...downloads.hindawi.com/journals/bmri/2019/8952414.pdf · Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity