Intraplaque Hemorrhage in Cardiac Allograft Vasculopathy

Brief Communication

Intraplaque Hemorrhage in CardiacAllograft Vasculopathy

C. Castellani1, A. Angelini1, O. J. de Boer2,C. M. van der Loos2, M. Fedrigo1, A. C. Frigo1,L. B. Meijer-Jorna3, X. Li2, H. J. P. Ploegmakers2,F. Tona1, G. Feltrin1, G. Gerosa1, M. Valente1,G. Thiene1 and A. C. van der Wal2,*

1Department of Cardiac, Thoracic and Vascular Sciences,University of Padua, Padua, Italy2Department of Pathology, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands3Symbiant Pathology Expert Center, Alkmaar, TheNetherlands�Corresponding author: Allard C. van der Wal,[email protected]

Plaque hemorrhage, inflammation and microvesseldensity are key determinants of plaque vulnerabilityin native coronary atherosclerosis (ATS). This studyinvestigates the role of intraplaque hemorrhage (IPH)and its relation with inflammation and microvesselsin cardiac allograft vasculopathy (CAV) in posttrans-planted patients. Seventy coronary plaques wereobtained from 12 patients who died because of CAV.For each patient we collected both native heart and theallograft, at the time of transplantation and autopsy,respectively. Intralesion inflammation, microvesselsand IPH were assessed semi-quantitatively. IPH wasobserved in 21/35 (60%) CAV lesions and in 8/35(22.9%) native ATS plaques, with a strong associationbetween fibrocellular lesions and IPH (p¼0.0142).Microvessels were detected in 26/35 (74.3%) of CAVlesionswith perivascular leakage as sign of endothelialdamage in 18/26 (69.2%). IPH was strongly associatedwith microvessels (p< 0.0001). Inflammation waspresent in 31/35 (88.6%) of CAV lesions. CAV IPHþlesions were characterized by presence of both freshand old hemorrhage in 12/21 (57.1%). IPH, associatedwith microvessel damage and inflammation, is animportant feature of CAV. Fresh and old intralesionhemorrhage suggests ongoing remodeling processespromoting the lesion progression and vulnerability.

Keywords: Atherosclerosis, cardiac allograft vascul-opathy, cardiac transplantation, inflammation, intra-plaque hemorrhage, microvessels

Abbreviations: AHA, American Heart Association; ATS,atherosclerosis; CAV, cardiac allograft vasculopathy;DCM, dilated cardiomyopathy; GFA, glycophorin A;ICM, ischemic cardiomyopathy; IPH, intraplaquehemorrhage; SMCs, smooth muscle cells; vWF, vonWillebrand factor

Received 05 July 2013, revised 11 September 2013 andaccepted for publication 23 September 2013

Introduction

Cardiac allograft vasculopathy (CAV) still represents the

Achilles’ heel of transplant procedures, accounting for

its increasing incidence in approximately 10% every year in

the long-term clinical follow-up (1). CAV is recognized as a

concentric thickening of the coronary artery wall due to

diffuse proliferation of smooth muscle cells (SMCs) in the

intima layer (neointima) (2). Its pathogenesis is complex

and can be related to different risk factors (3). A tight

correlation between acute rejection and the development

of CAV has been extensively reported (3). However, the

exact pathophysiological origin of CAV is not fully under-

stood (4,5). Previous studies have shown that inflammation

and oxidative stress in native coronary atherosclerosis

(ATS) contribute to rapidly progressive plaque destabiliza-

tion (6–8).

Recently several groups have also demonstrated that

intraplaque hemorrhage (IPH) is associated with the

development of ATS lesions and plaque instability (9–11).

Moreover, there is evidence in patients who died suddenly

from acute coronary syndromes that IPH may represent

a potent atherogenic stimulus both biochemically and

mechanically (12).

Red blood cells are rich in phospholipids and free

cholesterol, and their accumulation within the plaque plays

a key role in promoting lesion instability through necrotic

core expansion and inflammatory cell infiltration (12–14).

IPH correlates positively with both inflammation and the

presence of microvessels (15). Pathological examination of

unstable lesions has demonstrated that IPH is closely

associated with neoangiogenesis at site of microvessel

leakage (16).

For the first time we had the possibility to study and

compare in the same patients, CAV from explanted hearts

at autopsy with native coronary ATS from hearts removed

at transplantation.

In this setting, the aim of our study was to investigate

whether and to what extent IPH plays a role in the

American Journal of Transplantation 2014; 14: 184–192Wiley Periodicals Inc.

�C Copyright 2013 The American Society of Transplantationand the American Society of Transplant Surgeons

doi: 10.1111/ajt.12517

184

progression of CAV in patients transplanted for dilated

cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM).

Materials and Methods

Selection of patients, inclusion criteria

We selected 12 patients from the heart transplant registry at Padua

University who died because of CAV, onwhom autopsywas performed, and

from whom both the native heart and the graft removed at autopsy were

available for histopathology. Patients’ written consent for keeping the native

heart and further use for research purposes was obtained at transplantation.

The hearts retrieved at autopsywere kept for diagnostic and further research

purposes according to the permission of the family and by ethical committee

approval about research on archived materials (Padova Hospital, Ethical

Committee, June 2011).

Four of these patients had been transplanted because of DCM while

the other eight were transplanted for ICM. Overall we were able to evaluate

24 hearts. All major epicardial arteries were assessed macroscopically by

transverse sections every 0.5 cm and themost severe lesions from the three

main epicardial coronary arteries were identified. Among the most severe

lesions we included the culprit lesions responsible for the cardiac death. A

total of 70 lesions were included in the study, of which 35 were from native

hearts and 35 from posttransplant hearts.

Plaque histomorphological classification

Different types of lesion classification have been proposed in the literature

by Virmani et al (17). However at present, there is no internationally

acknowledged histological classification for the different types of CAV

lesions, but only a clinical and angiographic classification (18). Therefore, we

used the classification of native ATS as proposed by the American Heart

Association (AHA) (19,20) butwith somemodifications tomake it suitable for

CAV. Classification was carried out by two pathologists blindly.

Plaque morphologies are classified as follows:

(1) Early lesions including fatty streaks and intimal thickening (correspond-

ing to AHA classification I–III lesions).

(2) Fibrocellular lesions: concentric or eccentric fibromuscular intimal

hyperplasia with more than 50% of its area occupied by nucleated

cells (not included in AHA classification, and considered peculiar to

CAV).

(3) Fibrolipid lesions: concentric or eccentric lesions containing a necrotic

core with cholesterol clefts and extracellular lipid deposits covered by a

fibrous cap (AHA type IV).

(4) Fibrotic lesions: concentric or eccentric lesions composed of collagen

rich tissue, cellularity less than 50% and without significant lipid

deposition (AHA class Vc).

(5) Complicated lesions: lesions with intraplaque hematoma and/or super-

imposed mural thrombus (AHA class VI).

Four groups have been identified: (1) native hearts from DCM patients

(12 lesions); (2) transplanted hearts in DCM patients (12 CAV lesions); (3)

native hearts from ICM patients (23 lesions); (4) transplanted hearts in ICM

patients (23 CAV lesions).

Histological and immunohistochemical methods

Formalin-fixed coronary segments were embedded in paraffin and 4- to

5-mm sections were stained with hematoxylin and eosin, Azan-Mallory

staining for histomorphologic evaluation of the lesions.

Immunohistochemical single and double stains were performed on serial

sections as previously described (21), using mouse monoclonal anti

CD68, anti-mouse monoclonal SMA-1 (reactive with vascular SMCs),

CD31 (reactive with endothelial cells and platelets), anti-rabbit von

Willebrand factor (vWF; reactive with vWF), anti-LCA (reactive with

leucocytes) and anti-glycophorin-A (reactive with erythrocytes and erythro-

cyte membrane remnants). All antibodies were purchased from Dakopatts

(Glostrup, Denmark).

(Immuno) histological assessment of plaque inflammation and

plaque hemorrhage

(1) IPH: IPHwas evaluated using glycophorin A (GFA) immunostain (22) and

Perls iron stain. IPH was classified as either recent-onset (extravascular

located clusters of GFAþ intact erythrocytes), old (deposits of GFAþerythrocyte membrane fragments or iron stain) or ongoing (lesions with

simultaneous presence of recent and old IPH). Plaque hemorrhage was

scored as 0 (absent) and 1 (present) in all cases.

(2) Inflammation: The topographic localization in lesions, of the CD68þ and

LCAþ areas, was scored as superficial (adjacent to the lumen) or deep

(inside the lesion). Inflammation was semi-quantitatively scored as 0:

absent, 1: present. The ratio between immunopositive staining macro-

phages and SMCswas scored as CD68/SMA< 10%, CD68/SMA¼ 10–

50% and CD68/SMA >50% in all plaques.

(3) Presence of microvessels: CD31 positive microvessels were scored as

follows: 0: absent, 1: <10% vessels and 2: >10% vessels.

(4) Microvascular dilatation and endothelial leakage: Dilatation of vessels

was evaluated as 0: absent, 1: present. Leakage of microvessels

was assessed by presence of a rim of perivascular staining with anti-

vWF as reported previously (12) and was scored as either 0: absent

(only staining of endothelial cells), 1: present (additional staining of

perivascular tissue).

Statistical analysis

Data are presented as counts and percentages. Association of morphologic

plaque characteristics was analyzed with chi-square or Fisher’s exact

test. The statistical significance was set at the 5% level. The analyses

were conducted with SAS 9.2 (SAS Institute, Inc., Cary, NC) for

Windows.

Results

Clinical profile of patientsClinical characteristics of all patients who were trans-

planted for either DCM or ICM are presented in Table 1.

Histomorphological classification of lesions in nativehearts and in transplanted heartsHistomorphological classification of the lesions in native

ATS and CAV is reported in Table 2.

In the DCM hearts, only rarely were ATS plaques detected

(9 lesions in 12 samples, of which 7 were early lesions).

In ICM hearts, 23 lesions were found, which were all

advanced plaques (including 1 lesion complicated by

thrombosis). By contrast, nearly all lesions studied from

the 12 autopsy hearts (CAV lesions) were advanced

plaques, of which 4 were complicated by thrombosis.

See Table 3 for details.

Intraplaque Hemorrhage in CAV

185American Journal of Transplantation 2014; 14: 184–192

IPH in CAV lesions versus native plaquesIn the allografts from DCM and ICM patients, altogether 21

out of 35 (60%) CAV lesions showed signs of either fresh

(presence of extracellular intact erythrocytes) or old (GFA

immunopositive erythrocyte remnants, membrane frag-

ments and/or iron deposits) IPH, while in the native ATS,

which were derived from the explant hearts of the same

patients, IPH was present in 8 of 35 (22.9%) lesions

(Figure 1A–C, p¼ 0.0016). Fresh IPH was present in 4/35

(11.4%) of CAV lesions and in 3/35 (8.6%) in ATS plaques

(Figure 1D). Old hemorrhageswere present in 5/35 (14.3%)

of CAV lesions versus 2/35 (5.7%) in ATS plaques

(Figure 1D–F). Fresh and old IPH, as ongoing IPH, were

present in 12/21 (57.1%) CAV lesions compared to 3/8

(37.5%, Figure 1D) ATS.

In CAV lesions IPH co-localized in the necrotic core with

extracellular lipid material, close to numerous cholesterol

clefts. Fresh and old IPH were localized in different sites in

12/21 (57.1%) plaques.

The incidence of IPH in CAV lesions did not differ

significantly from culprit (55.6%) and nonculprit (78.6%,

p¼ 0.2656) lesions.

IPH, microvessels, inflammation and endothelialleakage in CAV lesions versus native plaquesIntraplaque CD31-immunopositive microvessels were

present in 23/35 (65.7%) of the native ATS. Only in

8/23 (34.8%) of these cases the microvessels showed

a rim of perivascular immunostaining with anti-vWF anti-

body, indicating endothelial leakage as previously reported

(9,23).

In CAV lesions, 26 out of 35 (74.3%, p¼ 0.4339 vs. ATS

microvessels) showed presence of intimal microvessels,

and 18/26 (69.2%) of thempresented perivascular anti-vWF

immmunostaining, indicating vessel leakage (Table 4).

Inflammation was identified as areas with CD68þ and

LCAþ cells, indicating presence of macrophages and

leukocytes. Inflammation was present in 31/35 (88.6%)

of the CAV lesions and in 25/35 (71.4%) of the ATS lesions

(Table 4, p¼0.1102 vs. ATS lesions). In the ATS plaques, it

was not possible to define the presence of inflammation in

one early lesion. A superficially located inflammatory infil-

trate was more commonly found in CAV lesions (26/35,

74.3%) than in ATS lesions (14/35, 40%, p¼ 0.0047).

Inflammatory infiltrates that were located deep inside the

lesions were found both in CAV (25/35, 71.4%) and in ATS

lesions (19/35, 54.3%) with no statistically significant

differences between them (Table 4). Superficial and deep

inflammation together were found in 20/35 (57.1%)

CAV lesions compared to 6/35 (17.1%) ATS plaques

(p¼0.0121). Moreover, in CAV lesions, the IPH events

were strongly associated with presence of microvessels,

20/21 (95.2%) IPH positive plaques (p¼0.0009) as in ATS

group (p¼ 0.0316).

In CAV lesions areas with IPH frequently co-localized with

microvessels and perivascular anti-vWF immunostaining

and with inflammatory cells at the same sites within the

plaques (Figure 2).

Table 1: Baseline clinical characteristics

pt

Age at

HT (yrs)

Indication

to HT

Survival

(yrs) Hypertension Diabetes Smoking

Donor

age (yrs)

PRA

(%)

Mismatch

(AB and DR)

Ischemic

time (min)

Total

rejection

score

Severe

rejection

score

1 21 DCM 13 y n n 11 na 5/12 (42%) 120 1 0.18

2 55 DCM 18 y y n 20 na 2/12 (17%) 120 1.3 0.09

3 45 DCM 17 n n n 28 0 4/12 (33%) 180 0.41 0.06

4 27 DCM 17 n n n 16 na 4/12 (33%) 120 0.46 0.04

5 59 ICM 1.3 n n n 41 na 3/12 (25%) 60 1.4 0.13

6 63 ICM 6 y y n 19 0 4/12 (33%) 180 0.6 0.27

7 40 ICM 3.3 n n n 27 0 4/12 (33%) 120 1.57 0.19

8 47 ICM 1.5 n n n 58 0 6/12 (50%) 60 1.19 0.19

9 51 ICM 1.1 y n n 27 0 2/12 (17%) 180 1.65 0.3

10 53 ICM 10 y y n 16 0 3/12 (25%) 120 2.6 0.36

11 52 ICM 1.2 y n n 40 0 na 180 1.38 0.62

12 59 ICM 3.6 n n n 30 0 4/12 (33%) 120 1 0.73

DCM, dilated cardiomyopathy; HT, heart transplantation; ICM, ischemic cardiomyopathy; min, minutes; n, no; na, not available; PRA, panel

reactive antibodies; pt, patient; y, yes; yrs, years.

Table 2: Plaque classification scheme and morphology of ATS

and CAV plaques

Plaque classification

Native ATS

(n¼35),

n (%)

CAV

(n¼35),

n (%)

Total

(n¼70),

n (%)

1. No lesions 3 (8.6) 0 (0) 3 (4.3)

2. Early lesions 7 (20) 3 (8.6) 10 (14.3)

3. Fibrocellular lesions 3 (8.6) 17 (48.6) 20 (28.6)

4. Fibrotic lesions 8 (22.9) 3 (8.6) 11 (15.7)

5. Fibrolipid lesions 13 (37.1) 8 (22.9) 21 (30)

6. Complicated lesions 1 (2.9) 4 (11.4) 5 (7.1)

ATS, native atherosclerotic plaques in explant hearts; CAV, cardiac

allograft vasculopathy plaques in graft hearts at autopsy.

Castellani et al

186 American Journal of Transplantation 2014; 14: 184–192

IPH, microvessels, inflammation and vessel leakagein relation to plaque morphologyPresence of IPH, neoangiogenesis, inflammation and

vessel leakage in CAV lesions were also evaluated in

relation to the specific type of plaque morphology (Table 4).

IPH was found in 11/17 (64.7%) of fibrocellular lesions, the

lesion type specific for CAV. Three out 11 of these lesions

(27.3%) had fresh IPH, 2/11 (18.2%) had old IPHwhile 6/11

(54.5%) had features of both fresh and old IPH in the

same lesion. Moreover, 13/17 (76.5%) fibrocellular lesions

also showed presence of microvessels, 15/17 (88.2%) had

inflammatory infiltrates, and 11/17 (64.7%) had perivascular

anti-vWF immunostaining (Figure 3).

Moreover there was a strong association between

fibrocellular lesions and IPH (p¼0.0142).

Relation of CAV lesions phenotype and the nativepathology leading to transplantationIPH was detected in more than half of CAV lesions (52.2%)

from ICM patients. CAV lesions from ICM and DCM

Figure 1: Analysis of intraplaque hemorrhage (IPH). (A) The graph shows the percentage (%) of IPH in native atherosclerotic (ATS)

lesions and in cardiac allograft vasculopathy (CAV). IPH is present in 21/35 (60%) of CAV lesions compared to ATS 8/35 (22.9%, p¼0.0016);

(B) graph shows the percentage of IPH in CAV and ATS lesions separated in fresh, old and for both fresh and old staining. Fresh IPH was

present in 4/35 (11.4%) of CAV lesions and in 3/5 (8.6%) in ATS plaques. Old hemorrhagewas present in 5/35 (14.3%) of CAV lesions versus

2/35 (5.7%) in ATS plaque lesions. Fresh and old IPH, as ongoing IPH phenomenon,were present in 12/21 (57.1%) CAV lesions compared to

ATS with 3/8 (37.5%); (C) immunostaining for glycophorin A antibody that identifies fresh staining for IPH. Original magnification 40�; (D)

immunostaining for glycophorin A, antibody that identify staining for IPH. Original magnification 40�.

Table 3: Plaque classification scheme and morphology according to the native pathology leading to transplantation


DCM (n¼12) ICM (n¼23)

Native ATS, n (%) CAV, n (%) Native ATS, n (%) CAV, n (%)

1. No lesions 3 (25) 0 (0) 0 (0) 0

2. Early lesions 7 (58.3) 0 (0) 0 (0) 3 (13)

3. Fibrocellular lesions 0 (0) 8 (66.7) 3 (13) 9 (39.1)

4. Fibrotic lesions 0 (0) 1 (8.3) 8 (34.8) 2 (8.7)

5. Fibrolipid lesions 2 (16.7) 2 (16.7) 11 (47.8) 6 (26.1)

6. Complicated lesions 0 (0) 1 (8.3) 1 (4.3) 3 (13)

CAV, cardiac allograft vasculopathy lesions in allograft at autopsy; DCM, lesions from patients transplanted because of dilated

cardiomyopathy; ICM, lesions from patients transplanted because of ischemic cardiomyopathy; native ATS, native atherosclerotic plaques

in explanted hearts.



Table 4: Plaque classification scheme and morphology


CAV (n¼35), n (%)

Total

(n¼35)

No

lesions

Early

lesions

Fibro

cellular

lesions

Fibrotic

lesions

Fibrolipid

lesions

Complicated

lesions

0 (0) 3 (8.6) 17 (48.6) 3 (8.6) 8 (22.9) 4 (11.4)

IPH, n (%) Positive 0 (0) 0 (0) 11 (64.7) 1 (33.3) 6 (75) 3 (75) 21 (60)

Negative 0 (0) 3 (0) 6 (35.3) 2 (66.6) 2 (25) 1 (25) 14 (40)

Inflammation, n (%) Positive 0 (0) 1 (33.3) 15 (88.2) 3 (100) 8 (100) 4 (100) 31 (88.6)

Negative 0 (0) 2 (66.6) 2 (11.8) 0 (0) 0 (0) 0 (0) 4 (11.4)

Microvessels, n (%) Positive 0 (0) 0 (0) 13 (76.5) 3 (100) 6 (75) 4 (100) 26 (74.3)

Negative 0 (0) 3 (100) 4 (23.5) 0 (0) 2 (25) 0 (0) 9 (25.7)

Vessel leakage, n (%) Positive 0 (0) 0 (0) 11 (64.7) 3 (100) 3 (37.5) 3 (75) 20 (57.1)

Negative 0 (0) 3 (100) 6 (35.3) 0 (0) 5 (62.5) 1 (25) 15 (42.9)

CAV, cardiac allograft vasculopathy; IPH, intraplaque hemorrhage.

Figure 2: Overviewof IPH,microvessels andendothelial leakage inCAV lesions. (A) Hematoxylin and eosin (H&E) of a cross-section of

a cardiac allograft vasculopathy (CAV) lesion. The bar indicates 500mm. (B) Detail of the black insert in (A). Immunostaining for glycophorinA,

showing extravasation of intact erythrocytes, indicating fresh hemorrhage. The bar indicates 100mm. (C) Detail of the black insert in (A).

Immunostaining for CD31 indicates presence ofmicrovessels in the plaque. Note as areaswith intraplaque hemorrhage (IPH) in CAV lesions

frequently co-localized with presence of microvessels. Bar indicates 100mm. (D) Detail of the black insert in (A). Immunostaining for von

Willebrand factor (vWF): leakage of microvessels is identified by the presence of diffuse perivascular vWF deposits. Note as areas with IPH

in CAV lesions co-localized also with microvessel and vessel leakage. Bar indicates 100mm.

Castellani et al


presented both fresh and old hemorrhage with a mean

percentage of 40%and 16%, respectively. Hemorrhage co-

localized with lipid and necrotic core in these lesions.

Inflammation was a common pattern in most of the CAV

lesions (78.3% and 50% from ICM and DCM). No

differences were identified between superficial and deep

inflammation (Table 5).

Discussion

The main result of our study is that IPH, which is regularly

observed in native ATS and considered responsible for

episodes of sudden plaque growth (9–11), is also an

important feature in CAV. Such IPH could be observed in

our study both in CAV lesions (60%) and in native ATS

Figure 3: Intraplaque hemorrhage localization. (A) Hematoxylin and eosin (H&E) stain of a cross-section of a cardiac allograft

vasculopathy lesion (2�); insert shows intraplaque hemorrhage. (B) Details of black insert in (A), immunostained for glycophorin A, showing

erythrocytes fragments, which indicates old hemorrhage. Bar indicates 100mm. (C) Perls staining for detection of iron (black deposits),

indicating old hemorrhage that co-localizedwith glycophorin A, in erythrocytemembrane. Bar indicates 100mm. (D) Adjacent tissue section

immunostained for von Willebrand factor showing leakage of vessels. Bar indicates 100mm. (E) Immunostaining for SMA/CD68, showing

the presence of inflammation around necrotic core. Bar indicates 100mm.



(22.9%) in a high percentage. In both conditions, and with

the use of specific staining for erythrocyte membrane

remnants (9,15), these IPH could be identified as being

either of recent (fresh) or old onset, supporting the concept

of repetitive ongoing IPH as an important mechanism of

plaque progression (15).

The contribution of IPH to the progression of coronary

atheroma was already reported in native ATS by Kolodgie

et al (9), but, as far as we know, no previous articles

have reported contribution of IPH to CAV lesions re-

modeling. Several other aspects like angiogenesis, micro-

vessel leakage and inflammation, which are known to

be related to IPH in native ATS, were also frequently

present in CAV (15,16). Similar to the situation described

in detail in native coronary ATS (9,15), ongoing hemor-

rhages can be involved in the progressive growth of CAV

lesions.

Such IPH could result from either incorporation of mural

thrombus by means of fibrocellular organization of the

thrombus mass (24), or leakage or rupture of intralesional

microvessels (9).

Independently from the diseases leading to heart trans-

plantation, all CAV lesions seem to have similar features

undergoing similar remodeling process. In this setting

genetic predisposition does not seem to play a role in the

progression of lesion hemorrhage.

Based on meticulous quantitative studies on native ATS,

Kolodgie et al (9) postulated that erythrocyte membranes,

which have a very high lipid content, derived from

hemorrhages, could lead to an abrupt increase in the level

of free cholesterol in plaques, resulting in expansion of the

necrotic core and the potential for plaque destabilization.

Our finding that deposits of erythrocytemembrane–derived

material were detected in necrotic core close to the

cholesterol cleft and at the level of superficial fibrous layer is

in keeping with Kolodgie et al’s findings (9). IPH may

stimulate CAV by being a source of free cholesterol and

Table 5: Plaque characteristics in CAV, according to the native pathology leading to transplantation


CAV from ICM (n¼23), n (%)

Total,

n¼23

No

lesions

Early

lesions

Fibrocellular

lesions

Fibrotic

lesions

Fibrolipid

lesions

Complicated

lesions

0 (0) 3 (13) 9 (39.1) 2 (8.7) 6 (26) 3 (13)

IPH, n (%) Positive 0 (0) 0 (0) 5 (55.6) 1 (50) 4 (66.7) 2 (66.7) 12 (52.2)

Negative 0 (0) 0 (0) 4 (44.4) 1 (50) 2 (33.3) 1 (33.3) 8 (34.8)

Inflammation, n (%) Positive 0 (0) 1 (33.3) 7 (77.8) 2 (100) 6 (100) 3 (100) 19 (82.6)

Negative 0 (0) 2 (66.7) 2 (22.2) 0 (0) 0 (0) 0 (0) 4 (17.4)

Microvessels, n (%) Positive 0 (0) 0 (0) 5 (55.6) 2 (100) 4 (66.7) 3 (100) 14 (60.9)

Negative 0 (0) 3 (100) 4 (44.4) 0 (0) 2 (33.3) 0 (0) 9 (39.1)

Vessel leakage, n (%)) Positive 0 (0) 0 (0) 5 (55.6) 2 (100) 1 (16.7) 2 (66.7) 10 (43.5)

Negative 0 (0) 3 (100) 4 (44.4) 0 (0) 5 (83.3) 1 (33.3) 13 (56.5)

CD68/SMC <10% 0 (0) 1 (33.3) 7 (77.8) 0 (0) 0 (0) 0 (0) 8 (34.8)

10–50% 0 (0) 2 (66.7) 2 (22.2) 1 (50) 2 (33.3) 1 (33.3) 8 (34.8)

>50% 0 (0) 0 (0) 0 (0) 1 (50) 4 (66.7) 2 (66.7) 7 (30.4)


CAV from DCM (n¼12), n (%}

Total, n¼12

No

lesions

Early

lesions

Fibrocellular

lesions

Fibrotic

lesions

Fibrolipid

lesions

Complicated

lesions

0 (0) 0 (0) 8 (66.7) 1 (8.3) 2 (16.7) 1 (8.3)

IPH, n (%) Positive 0 (0) 0 (0) 6 (75) 0 (0) 2 (100) 1 (100) 9 (75)

Negative 0 (0) 0 (0) 2 (25) 1 (100) 0 (0) 0 (0) 3 (25)

Inflammation, n (%) Positive 0 (0) 0 (0) 8 (100) 1 (100) 2 (100) 1 (100) 12 (100)

Negative 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Microvessels, n (%) Positive 0 (0) 0 (0) 8 (100) 1 (100) 2 (100) 1 (100) 12 (100)

Negative 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Vessel leakage, n (%) Positive 0 (0) 0 (0) 6 (75) 1 (100) 2 (100) 1 (100) 10 (83.3)

Negative 0 (0) 0 (0) 2 (25) 0 (0) 0 (0) 0 (0) 2 (16.7)

CD68/SMC 10% 0 (0) 0 (0) 4 (50) 1 (100) 0 (0) 0 (0) 5 (41.7)

10–50% 0 (0) 0 (0) 1 (12.5) 0 (0) 0 (0) 1 (100) 2 (16.7)

>50% 0 (0) 0 (0) 3 (37.5) 0 (0) 2 (100) 0 (0) 5 (41.7)

CAV, cardiac allograft vasculopathy; DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy, IPH, intraplaque hemorrhage.

Castellani et al


macrophage activation. Our lesions are representative of

the heterogeneity of CAV lesions. Lipid could result from

lipid insudation from blood or from disrupted erythrocytes

membranes.

IPH frequently occurs in ATS, and pathological studies have

demonstrated that IPH and plaque rupture are associated

with increased microvessel density (9). In our study, IPH

was strongly associated with presence of microvessels.

IPH derived from microvascular disruption or leakage is

considered to contribute to necrotic core expansion, which

precedes plaque instability and rupture (9,25). Repeated

hemorrhage events, in our study, occurred in at least

one-third of patients with previous IPH.

Iron accumulation due to hemoglobin breakdown can act as

a catalyst in the formation of free radicals that modify LDL

cholesterol and foam cells (26). Thus, iron accumulation

deposits are not only a histological marker of previous

hemorrhage but also a possible source of reactive oxygen

species, and could activate the redox state inducing

inflammatory reaction and activating matrix metallopro-

teases, leading to coronary plaque instability via reactive

oxygen species generation (26).

CAV is a process that negatively affects survival after heart

transplantation. The concentric growth is generally believed

to be caused by the influx and growth of SMCs originating in

the tunica media of the coronary arteries (6).

In this work, we also identified infiltrating inflammatory

cells both superficial and deep in the plaque. There was no

difference in plaque inflammation phenotype between ATS

and CAV. Moreover in CAV lesions, both superficial and

deep inflammation were found in 57.1% compared to ATS

lesions with only 19.4%.

Limitation of the Study

For the first time we had the possibility to study and

compare within the same group of patients CAV from

explanted hearts at autopsy with native coronary ATS from

the heart removed at transplantation. The inclusion criteria

adopted in this study led to a relatively constrained data set,

with an overall number of lesions analyzed around 70. Even

though the limited amount of data available suggests

caution in extrapolating general patterns from this study,

our results provide new insight on the mechanisms

underlying the formation of CAV lesions. Further work

will be needed to expand the current database and better

assess the statistical significance of these results.

Conclusions

Findings from this study suggest the importance of

repeated hemorrhage into the plaque as an additional

mechanism of CAV progression.

Neoangiogenesis combined with endothelial leakage

was a constant feature in CAV lesions. Incorporation of

luminal thrombi cannot be entirely excluded. However,

microvessels were constantly present in association with

hemorrhage while plaque fissure was absent.

Repeated IPH and the resulting consequences related

to trapped erythrocytes and activated macrophages may

be important factors in the rapid progression of CAV.

Understanding the mechanism by which plaque angiogen-

esis, vessel growth and IPHoccur in CAVmay help to define

a better therapeutic and preventive strategy.

Imaging modalities such as contrast-enhanced ultrasound

and dynamicMRI have the potential to evaluate intraplaque

neovascularization and IPH noninvasively, improving our

stratification of ‘‘high-risk’’ patients (10,27,28). IPH may

serve as surrogate for events that are stronger than intima-

media thickness (29). Using microbubbles as vehicles

for targeted drug therapy could be foreseen as future

therapeutic intervention (30).

Acknowledgments

This work was supported by a research grant from University of Padua

(nos. CPDR099073, CPDA108809, 60A07-5074 and 60A07-8587).

Disclosure

The authors of this manuscript have no conflicts of interest

to disclose as described by the American Journal of

Transplantation.

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