J A C C : C A R D I O V A S C U L A R I M A G I N G V O L . 5 , N O . 9 , 2 0 1 2

© 2 0 1 2 B Y T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y F O U N D A T I O N I S S N 1 9 3 6 - 8 7 8 X / $ 3 6 . 0 0

P U B L I S H E D B Y E L S E V I E R I N C . h t t p : / / d x . d o i . o r g / 1 0 . 1 0 1 6 / j . j c m g . 2 0 1 1 . 1 2 . 0 2 7

C O N C E P T S O N T H E V E R G E O F T R A N S L A T I O N

IVUS Detection of Vasa Vasorum BloodFlow Distribution in Coronary ArteryVessel Wall

Regina Moritz, MD,* Diane R. Eaker, MSEE,* Jill L. Anderson, BA,*Timothy L. Kline, MSEE,* Steven M. Jorgensen, BSEE,* Amir Lerman, MD,†Erik L. Ritman, MD, PHD*

Rochester, Minnesota

There is an increased body of evidence to suggest that the vasa vasorum play a major role in the

progression and complications of vulnerable plaque leading to acute coronary syndrome. We propose

that detecting changes in the flow in the vascular wall by intravascular ultrasound signals can quantify

the presence of vasa vasorum. The results obtained in a porcine model of atherosclerosis suggest that

intravascular ultrasound-based estimates of blood flow in the arterial wall can be used in vivo in a

clinical research setting to establish the density of vasa vasorum as an indicator of plaque

vulnerability. (J Am Coll Cardiol Img 2012;5:935– 40) © 2012 by the American College of Cardiology

Foundation

ing,n ofart bto th

The role of vasa vasorum in the pathogenesisand complications of coronary artery diseasecontinues to emerge. Micro-computed tomog-raphy (MCT) and other methods have dem-onstrated that there is proliferation of vasavasorum early in the development of anatherosclerotic lesion and that this mightcontribute to plaque rupture (1). The in-creased density of vasa vasorum due to theangiogenesis and that these new vessels aremore fragile increases the chances of hemor-rhage from rupture of these new vessels.Thus, there is growing interest to detect thepresence of vasa vasorum in the vascular walland in the atherosclerotic lesion (mainly in

From the *Department of Physiology and Biomedical EngineerMinnesota; and the †Department of Internal Medicine, Divisioof Medicine, Rochester, Minnesota. This work was supported in pAll authors have reported that they have no relationships relevant

Manuscript received August 10, 2011; revised manuscript received De

the coronary circulation) at an early stage invivo. Motivated by this reasoning, we ex-plored the concept of the use of intramuralblood flow via intravascular ultrasound(IVUS) imaging to detect vasa vasorum den-sity in the arterial wall. Therefore, total flow(increased velocity � area of flow) within thearterial wall should be equivalent to thedensity of vasa vasorum in the imaged arterialwall. To evaluate this approach we performedboth in vivo IVUS with intramural flowcapability in coronary arteries of pigs and exvivo assessment of the vasa vasorum withMCT. Moreover, we enhanced heterogeneityof intramural blood flow distribution by em-

Mayo Clinic College of Medicine, Rochester,Cardiovascular Diseases, Mayo Clinic Collegey National Institutes of Health Grant HL065342.e contents of this paper to disclose.

cember 16, 2011, accepted December 22, 2011.

Wlpr

tTptAstsetCndCtaesdtfl

iTiNacMuaietTacsasptwr

gaRw

o

coronary artery

J A C C : C A R D I O V A S C U L A R I M A G I N G , V O L . 5 , N O . 9 , 2 0 1 2

S E P T E M B E R 2 0 1 2 : 9 3 5 – 4 0

Moritz et al.

IVUS Detection of Coronary Vasa Vasorum

936

bolizing some of the vasa vasorum, to provide arange of vasa vasorum densities over which wecould compare the IVUS intramural flow andMCT estimates.

After approval from the Mayo Foundation Insti-tutional Animal Care and Use Committee, weemployed a porcine model of coronary atheroscle-rosis to evaluate the feasibility of the iConceptproposal. In 7 domestic female cross-bred swine aselective coronary artery catheterization and micro-embolization procedure was performed as describedpreviously (2). All pigs were anesthetized, heparin-ized, intubated, and ventilated. A guide catheterwas placed into the left anterior descending coro-nary artery (LAD), and then a 3-F catheter wasintroduced and advanced until its tip was positionedin the LAD. Then a suspension of 5,000 gold-coated, 100-�m-diameter microspheres (BioPal,

orchester, Massachusetts) was infused into theeft coronary artery. After the microembolizationrocedure, the pigs were allowed to recover andeceived a high-cholesterol diet, which consisted of

15% lard and 2% cholesterol (Harlan Lab-oratories, Madison, Wisconsin) for an ad-ditional 3 months. Next, the pigs wereagain anesthetized, and an IVUS imagingcatheter (Eagle Eye Gold catheter, Vol-cano Corporation, Rancho Cordova, Cal-ifornia) was introduced and advanced untilits tip was positioned in the distal LAD.The catheter tip contained a miniature,multi-element, solid state array ultrasound

ransducer operating at a frequency of 20 MHz.hen the catheter was connected to an automatedull-back device (Trak Back II, Volcano Corpora-ion) and pulled back at a constant speed of 1 mm/s.

patient interface module connected to the ultra-ound array excited the transducer elements toransmit ultrasonic energy to the surrounding tis-ue; it also amplified and processed the resultantcho signals from the transducer and sent these tohe system console (In-Vision System, Volcanoorporation). To visualize blood flow in the coro-ary artery wall due to vasa vasorum, the speciallyeveloped IVUS system (ChromaFlo, Volcanoorporation) was used. This program compared

emporally and spatially sequential images along thexis of the artery. Any differences in the position ofchogenic regions between images of the tissueurrounding the coronary artery are assumed to beue to blood flow in the arterial wall. The softwarehen colorized the de-correlation rate (i.e., blood

y

und

ing

ow speed) as a red overlay on the IVUS anatomic a

mage displayed in axial and longitudinal views (3).he resulting “AVI-movie” files were transformed

nto a stack of transaxial “tif” images (MATLAB,atick, Massachusetts). These were displayed with

n image analysis program (Analyze 9.0, Biomedi-al Imaging Resource, Mayo Clinic, Rochester,

innesota) as illustrated in Figure 1. The individ-al cross-sectional images along the arteries werenalyzed individually by creating a region-of-nterest (ROI) that encompassed the vessel wall. Tonsure that the entire arterial wall was included inhe ROI, the radius of the lumen (r) was measured.his measurement then defined the diameter of the

nnular ROI surrounding the arterial lumen. Byreating a binary image of the pixels with flowignal it was possible to sample just the red pixelsnd from them calculate the total flow value byumming the intensity of the red signal at eachixel. This summation within the ROI representedhe total blood flow within the vessel wall (i.e.,ithin the vasa vasorum). The total red signal of the

ed pixels (per mm2) was then plotted as a functionof axial distance along the coronary artery, therebycreating a vasa vasorum density profile along theaxial length of the coronary artery. The microem-bolization of vasa vasorum produced local regions ofreduced blood flow in the coronary artery vesselwall. After the IVUS procedure a midline sternot-omy was performed to allow access to the LAD.Then radiopaque contrast dye (Novaplus Om-nipaque, GE Healthcare, Princeton, New Jersey)was injected into the proximal LAD, and immedi-ately after the injection an approximately 5-cm-long segment of the LAD was harvested. Thisinvolved cutting free the segment with a marginwell outside the adventitia to protect and preserveall structures of the vessel wall. This isolated spec-imen was then snap-frozen. Once frozen the spec-imens were stored for subsequent scanning withcryostatic MCT. Subsequently, the frozen 5-cm-long specimen was cut into several 2-cm-longsegments. This cutting process caused some dam-age at the ends of each segment. Those individualspecimens were scanned as described previously (4).The stack of transaxial tomographic images (sidedimension of the cubic voxels was 18-�m, 16-bitray scale) was displayed and analyzed with imagenalysis software (Analyze 9.0, Biomedical Imagingesource, Mayo Clinic). The CT gray scale valuesere expressed in units of 1,000/cm.Within the CT images of the arteries, segments

f at least 10-mm length, with clearly distinguish-

A B B R E V I A T O N S

A N D A C R O N YM S

CMR � cardiac magnetic

resonance

CT � computed tomograph

IVUS � intravascular ultraso

LAD � left anterior descend

ble arterial wall, were identified for further analy-

ssRiWcosaraiiiastssdaam

a

cbHccbCrcivtf

dvTtaeapmrlspvst

J A C C : C A R D I O V A S C U L A R I M A G I N G , V O L . 5 , N O . 9 , 2 0 1 2

S E P T E M B E R 2 0 1 2 : 9 3 5 – 4 0

Moritz et al.

IVUS Detection of Coronary Vasa Vasorum

937

sis. The images of the 18-�m-thick cross-sectionallices within the segment (on average 950 slices/pecimen) were analyzed individually by creating aOI that encompassed the entire vessel wall, sim-

lar to the analysis of the IVUS flow datasets.ithin this ROI the average CT-number was

alculated, and this value was plotted as a functionf distance along the luminal axis of the arterialegment. This generated an “opacification profile”long the luminal axis of the segment that conveyedegions of varied perfusion within the arterial walls illustrated in Figure 1. The location of the MCTmage data was co-registered with the IVUS flowmage data by virtue of the branch points visual-zed in both images as illustrated in Figure 2. Theverage values for each of the CT slices in thepecimen were averaged and compared withhe average value of the IVUS slices in thoselices corresponding to the arterial segmentcanned with the MCT. Hence, the number ofata points is equal to the number of LADrterial segments scanned. The data are presenteds mean � SD for all arteries. The statisticalethod used was the regression coefficient (R2)

computed with Microsoft Excel 2003 (Microsoft,Redmond, Washington).

As illustrated in Figure 2, the variation of intra-mural blood flow and MCT contrast in the arterialvessel wall matched qualitatively quite well. Figure 3shows a linear relationship (R2 ranges between 0.90

Figure 1. IVUS and MCT Images of Coronary Arteries

Left panel is a single cross-sectional intravascular ultrasound (IVradial lines represent the tissue of the arterial wall, and the red(ChromaFlo, Volcano Corporation) data. The yellow concentric cfaces. The area between the circles is the region of interest thawall and was used as the index of vasa vasorum density withinphy (CT) image of approximately the same arterial cross-sectiontrast agent within the left anterior descending coronary artery luincrease in radiopacity above background radiopacity in this regthe vasa vasorum.

nd 0.96) between CT-number values obtained by i

ryo-MCT and the vasa vasorum density obtainedy IVUS image analysis in each of the 6 animals.owever, because there are different amounts of

ontrast within the arterial wall due to differentoronary flow rates and slightly different delaysetween injection of contrast and harvesting, theT gray-scale value to intramural flow intensity

atios varies between specimens. Figure 4 shows aomparison of a MCT image of a coronary arterynjected with Microfil (thereby showing individualasa vasorum) and an IVUS pull-back performed onhat same artery in vivo before harvesting that arteryor MCT scanning.

The iConcept study demonstrates the ability toetect and potentially quantify the degree of theasa vasorum in the coronary vascular wall in vivo.he current concept might emerge as a potential

ool to detect early plaque development and vulner-bility in vivo in human. The current study usesxisting intravascular technology and expends itspplication for the detection of vulnerable plaque. Arevious study (5) using a similar IVUS-based floweasurement to estimate the density of vasa vaso-

um in arterial walls has several differences thatikely explain the contradictory outcome of thattudy. It made the assumption that the method canrovide the actual visualization of the individualasa vasorum lumen cross-sections. The currenttudy extends this observation and demonstrateshat the measurements should not focus on actual

image of a left anterior descending coronary artery. The whitetures represent the IVUS-based vessel wall flow assessments represent the arterial lumen and abluminal adventitial sur-s sampled for quantitating the ChromaFlo signal in the arterialwall. The right panel is a cryostatic micro-computed tomogra-wn in the left panel. The white area is the intravascular con-n. The yellow region of interest outlines the arterial wall. Theof interest was used as an index of the blood volume within

US)feairclet watheshomeion

maging of the vasa vasorum, because the vessels of

J A C C : C A R D I O V A S C U L A R I M A G I N G , V O L . 5 , N O . 9 , 2 0 1 2

S E P T E M B E R 2 0 1 2 : 9 3 5 – 4 0

Moritz et al.

IVUS Detection of Coronary Vasa Vasorum

938

interest are �100 �m in lumen diameter. Thus, weused the sum of the blood flow within vasavasorum to quantify the total vasa vasorum flowrather than attempt to spatially resolve the vasavasorum, and we used MCT imaging—a power-ful method for in vitro detection and quantifica-tion of the 3D network of vasa vasorum—to compareour IVUS measurements. Interventional selectivecoronary angiography and CT as well as cardiacmagnetic resonance (CMR) angiography meth-ods are not capable of detecting very early lesionsthat do not have narrowing of the lumen. Cur-rently, multi-slice CT, CMR, IVUS, or opticalcoherence tomography are used to evaluate cor-onary artery wall pathology. However, the IVUSand optical coherence tomography methods, al-though providing important information aboutchanges in the material content in the arterialwall, have not been successful in quantifying thedensity of vasa vasorum in the arterial wall.

Figure 2. Comparison of Coronary Wall IVUS and MCT Perfusion

Upper-most panel is a longitudinal section along one left anteristack of intravascular ultrasound (IVUS) cross-sections obtained ddescending coronary artery lumen and the lumen of the major bare the longitudinal sections computed from the 3-dimensionaland its major branches are also outlined. The bright white spotimages are interrupted at the locations where the frozen arteryare the IVUS-based vessel wall flow assessment and MCT opacitythe artery.

However, before any noninvasive approach that

quantitates density of vasa vasorum can be im-plemented, it must be validated. An invasivemethod that can quantitate the density of vasavasorum in the coronary artery wall would beacceptable for this purpose. This method wouldthereby provide an objective method for evaluat-ing noninvasive imaging methods developed todetect early atherosclerotic changes in humans.Because the spatial distribution of vasa vasorumin these pigs was heterogeneous, the good corre-lation between the IVUS and MCT-based datacould be fortuitous, although unlikely to be so inall 6 specimens examined.

The rationale for using the density of vasa vaso-rum as an indicator of early atherosclerosis is 2-fold.First, it seems to be a direct indicator of the reactionof the arterial wall to early accumulation of fattymaterials. Second, the increased volume of bloodin the vasa vasorum as well as the increased

escending coronary artery coronary artery computed from theg the pull back of the IVUS catheter. The left anteriorches are outlined. The sequence of panels immediately below-micro-computed tomography (MCT) image data. The lumensthe arterial wall are the embolized microspheres. The MCTcut and some local damage occurred. The lower panelsrease at each cross-sectional location along the length of

or durinrancryos inwasinc

leakiness of the new vasa vasorum, provide a basis

tech

J A C C : C A R D I O V A S C U L A R I M A G I N G , V O L . 5 , N O . 9 , 2 0 1 2

S E P T E M B E R 2 0 1 2 : 9 3 5 – 4 0

Moritz et al.

IVUS Detection of Coronary Vasa Vasorum

939

for specific signals in CT and CMR images. Thedemonstration of the vasa vasorum in the vascularwall might potentially have implications for fu-ture therapeutic approach. In summary, the cur-

Y = 0.05x - 29.41R2 = 0.92

Ch

rom

aFlo

® A

rea

(mm

2 )

600 610 620 630 66403

3.5

0.7

1.6

1.4

1.2

1

0.91.11.31.51.71.92.1

4

4.5

5

5.5

6

Y = 0.07x - 40.72R2 = 0.96

Ch

rom

aFlo

® A

rea

(mm

2 )

600 625 630 635 640 60.5

2.3

Y = 0.04x - 26.7R2 = 0.90

Ch

rom

aFlo

® A

rea

(mm

2 )

CT# (1000/cm)

665 667 669 671 673 675 677 679 60.8

1.8

CT# (1000/cm)

CT# (1000/cm)

Figure 3. Comparison of IVUS “Flow” Versus MCT Contrast Opa

Plots for each of the 6 pigs. Computed tomography number (CT#)IVUS-based vessel wall flow assessment area obtained by the IVUS

Figure 4. Comparison of MCT Anatomy and IVUS Perfusion

Left upper panel is a volume rendered display of an MCT image ofrounding vasa vasorum (VV). Left lower panel is a plot of the amouter distance for each CT slice along the axis of the artery. Right upwall flow signal in the arterial wall region. Right lower panel is a p

section along the length of the artery. Abbreviations as in Figure 2.

rent study demonstrated a high and significantcorrelation between the in vitro and the in vivomethods, such that this IVUS-based approach isan excellent candidate for assessing early athero-

Y = 0.02x - 15.09R2 = 0.91

570 590 610 630 650 6701

5

Y = 0.04x - 26.75R2 = 0.90

665 667 669 671 673 675 677 679 6810.8

1.8

Y = 0.10x - 56.78R2 = 0.94

CT# (1000/cm)

CT# (1000/cm)

CT# (1000/cm)

590 595 600 605 610 615 620 6253

6.5

1.5

0.91

1.11.21.31.41.51.61.7

22.5

33.5

44.5

6

5.5

5

4.5

4

3.5

Values

ined from the cryo-MCT contrast density data plotted versus thenique. Abbreviations as in Figure 2.

ontrast (Microfil)-injected epicardial coronary artery and its sur-of contrast around the epicardial lumen at up to 1 lumen diame-panel is an IVUS pull-back image with (red) IVUS-based vesself the IVUS-based vessel wall flow assessment signal in each cross-

50

45

81

city

obta

a cnt

perlot o

PPiS

2

J A C C : C A R D I O V A S C U L A R I M A G I N G , V O L . 5 , N O . 9 , 2 0 1 2

S E P T E M B E R 2 0 1 2 : 9 3 5 – 4 0

Moritz et al.

IVUS Detection of Coronary Vasa Vasorum

940

sclerosis changes during clinically indicated selec-tive coronary catheterization and as a means ofcalibrating noninvasive methods for detection ofearly atherosclerosis.

AcknowledgmentsThe authors thank Mrs. Jonella M. Tilford, Mrs.

on myocardial vascular blood volumeand perfusion in a pig model of coro-

3

4

phy imaging of arteScanning 2002;24:18

perform the animal studies and Ms. Delories C.Darling for editing and formatting the manuscript.

Reprint requests and correspondence: Dr. Erik L. Ritman,rofessor, Physiology and Medicine, Department ofhysiology and Biomedical Engineering, Alfred Build-

ng, 2-409, Mayo Clinic College of Medicine, 200 Firsttreet Southwest, Rochester, Minnesota 55902. E-mail:

Kay D. Parker, and Dr. Nitin Garg for helping to elran@mayo.edu.

R E F E R E N C E S

1. Ritman EL, Lerman A. Role of vasavasorum in arterial disease: a re-emerging factor. Curr Cardiol Reviews2007;3:43–55.

. Schmermund A, Lerman LO, Rum-berger JA, et al. Effects of acute andchronic angiotensin receptor blockade

nary microembolization. Am J Hyper-tens 2000;13:827–37.

. Crowe JR, O’Donnell M. Quantitativeblood speed imaging with intravascularultrasound. IEEE Trans Ultrason Fer-roelectr Freq Control 2001;48:477–87.

. Kantor B, Jorgensen SM, Lund PE,Chmelik MS, Reyes DA, Ritman EL.Cryostatic micro-computed tomogra-

rial wall perfusion.6–90.

5. Redwood A, Holmes DR III, RobbRA. Using ChromaFlo intra-vascular ul-trasound (IVUS) to analyze adventitial vasavasorum distribution: considerations andrecommendations. Proceedings SPIE2006;6143:614309-01–614309-06.

Key Words: arterial wall yatherosclerosis y diffusion y

embolization y perfusion.