Intimal fibrosis in human cardiac allograft vasculopathy

doi:10.1016/j.trim.2011.07.001

Transplant Immunology

Volume 25, Issues 2–3, September 2011, Pages 124-132

https://doi.org/10.1016/j.trim.2011.07.001 Get rights and content

Abstract

Human Cardiac Allograft Vasculopathy (CAV) is one of the major complications for patients after heart transplantation. It is characterized by a concentric luminal narrowing due to (neo) intimal expansion in the coronary arteries of donor hearts after heart transplantation. In this process fibrosis plays an important role. Aim of this study is to analyze the factors and cells involved in this fibrotic process.

Coronary arteries from five heart transplantation patients and three controls were obtained at autopsy. Quantitative real-time PCR was performed on mRNA obtained from various arterial layers isolated by laser micro dissection. Positive gene expression was confirmed by immunohistochemistry and/or in situ hybridisation.

The strongest mRNA expression of fibrotic factors (predominantly pro-fibrotic) was found in the neo-intima. Especially, connective tissue growth factor expression was higher in the CAV vessels than in the controls. The lymphocyte activity of interferon gamma was only detected in CAV vessels. Furthermore as shown by in situ hybridisation, the lymphocytes producing interferon gamma also expressed transforming growth factor beta. Anti-fibrotic factors, such as bone morphogenic protein 4, were only expressed in CD3⁻/CD68⁻ stromal cells. Macrophages present in the CAV and control vessels showed to be of the M2 type and did not produce any fibrotic factor(s).

In conclusion, T-cells producing both interferon gamma and transforming growth factor beta, may play an important role in the fibrotic process in CAV vessels by upregulation of connective tissue growth factor production.

Highlights

► We study fibrotic process in Coronary artery vasculopathy (CAV) after HTx. ► Laser microdissection is used to obtain tissue of CAV vessels for Q-PCR analysis. ► To identify proteins/mRNA immunohistochemistry and in situ hybridisation are used. ► mRNA of fibrotic factors is predominantly found in the neo-intima of CAV. ► IFN-γ and TGF-β production by T-cells may be responsible for the fibrotic process.

Introduction

Cardiac Allograft Vasculopathy (CAV) is one of the major complications in long-term survival of heart transplant (HTx) recipients [1], [2]. CAV is characterized by a concentric intra-luminal obstructive thickening of the intima of the coronary arteries [3], [4]. A major difference between atherosclerosis and CAV is that the internal and external elastic laminae, normally destroyed in atherosclerosis, remain intact in CAV [5]. CAV clinically often presents as silent myocardial infarction, heart failure or sudden death [6], [7]. At present, there is no suitable preventive treatment or therapy for CAV available [6], [7].

The neo-intima (NI) seen in CAV vessels is composed of two distinct layers: 1) a luminal layer (NI-LL) consisting of loose connective tissue infiltrated by mononuclear cells, and 2) a layer (NI-SMC) composed of smooth muscle cells or myofibroblasts directly adjacent to the lamina elastica interna [8], [9], [10], [11].

Several factors are involved in the pathogenesis of CAV. The cellular immune response against the allograft is suggested to be one of major importance. Especially T-helper 1 cells seem to play a major role [11], [12], [13], [14]. Macrophages are obviously also involved, but their role has been studied in less detail [15]. Although the production of most cytokines is hampered in HTx patients (due to immunosuppressive treatment), interferon-γ (IFN-γ) can be detected and has been implicated as an important mediator of CAV [12], [13], [16], [17], [18].

As reported earlier the NI in CAV vessels sometimes consists almost completely of fibrotic tissue [3]. Our study focuses on the analysis of the fibrotic process in CAV and the role of T-cells and macrophages herein. Therefore we analyzed the expression of pro- and anti-fibrotic factors, extra-cellular matrix (ECM) components as well as the presence of macrophages in various layers of the arterial wall. Messenger ribonucleic acid (mRNA) expression was established by quantitative real-time PCR (Q-PCR) performed on distinct micro-dissected layers of the coronary arteries. Location of the expressed mRNAs was determined by in situ hybridization (ISH) and cells producing these factors were identified by double ISH and immunohistochemistry (IHC).

Section snippets

Patient population

Coronary arteries of five non-transplanted and five HTx patients were obtained at autopsy. Informed consent of all patients was obtained prior to HTx. Anonymous use of redundant tissue for research purposes is part of the standard treatment agreement with patients in our hospital [19]. All HTx patients were treated with a triple immunosuppressive therapy of cyclosporine A, azathioprine and steroids. Characteristics of the patients and of the arteries used in the different experimental

mRNA expression of fibrotic factors, cytokines and ECM components

The Q-PCR data of mRNA expression in the various layers of the coronary arteries are presented in Fig. 1, Fig. 2. In control arteries expression of the tested genes was higher in the tunica intima compared to the media. Bone morphogenic protein (BMP) 7, Interferon gamma (IFN-γ) and Matrix-metalloproteinases (MMP) 9 were not expressed in control arteries. In general, the mRNA expression measured in CAV NI-SMC and media was low.

Q-PCR data of the anti- and pro-fibrotic factors and cytokines are

Discussion

CAV has long been considered as a result of solely SMC migration from the tunica media to the tunica intima [1], [3]. However, a SMC layer is also present in the intima of coronary arteries from healthy individuals and CAV is actually characterized by a fibrotic response in the NI-LL [11]. This study focussed on the characterization of the fibrotic process in human CAV vessels.

Our results showed that the mRNA expression of the fibrotic factors TGF-β, CTGF and PAI1/serpine was high in the NI-LL

Study limitations

The small number of patients and the small numbers of controls are a main limitation of this study. Coronary arteries were only included if they contained a clear NI layer with loose connective tissue and an infiltrate of mononuclear cells and when sufficient vessel tissue could be obtained. Besides, they (both CAV and controls) should not have any form of atherosclerosis which could bias our results. These criteria meant that of our population only five CAV patients remained. However, this was

Disclosures

The authors of this manuscript have no conflicts of interest to disclose as described by transplant immunology.

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HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab’)₂) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.
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While they found a strong relation between DSA presence and that of ISHLT CAV grade ≥II, no difference was reported in terms of OCT-derived mean intimal area increase between DSA positive and negative patients. CAV is characterized by accelerated concentric fibrous intimal hyperplasia along the length of coronary vessels, and its early diagnosis is of crucial importance, as identification of such patients may favourably affect CAV-related allograft failure rates [24]. Optical coherence tomography (OCT) enables the thin intimal layer to be clearly differentiated from the tunica media, a crucial factor in determining early progression of CAV, as early as during the first year after HTx [16,5].
Recent studies suggested potential positive correlations between HLA-specific antibodies and development of cardiac allograft vasculopathy (CAV).
This prospective two-center study investigated early progression of CAV by coronary optical coherence tomography in 1 month and 12 months after heart transplantation (HTx) in 104 patients. Detection and characterization of donor specific (DSA) and MHC class-I polypeptide-related sequence A (MICA) antibodies were performed before, 1, 6 and 12 months after transplantation.
During the first post-HTx year, we observed a significant reduction in the mean coronary luminal area (P < .001), and progression in mean intimal thickness (IT) (P < .001). DSA and anti-MICA occurred in 17% of all patients, but no significant relationship was observed between presence of DSA/anti-MICA and IT progression within 12 months after HTx. In contrast, we observed significant association between presence of DSA (p=0.031), de-novo DSA (p=0.031), HLA Class II DSA (p=0.017) and media thickness (MT) progression.
Results of our study did not identify a direct association between presence of DSA/anti-MICA and intimal thickness progression in an early period after HTx. However, we found significant relationships between DSA and media thickness progression that may identify a newly recognized immune-pathological aspect of CAV.
T cell repertoire analysis suggests a prominent bystander response in human cardiac allograft vasculopathy
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T cells are implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), yet their clonality, specificity, and function are incompletely defined. Here we used T cell receptor β chain (TCRB) sequencing to study the T cell repertoire in the coronary artery, endomyocardium, and peripheral blood at the time of retransplant in four cases of CAV and compared it to the immunoglobulin heavy chain variable region (IGHV) repertoire from the same samples. High-dimensional flow cytometry coupled with single-cell PCR was also used to define the T cell phenotype. Extensive overlap was observed between intragraft and blood TCRBs in all cases, a finding supported by robust quantitative diversity metrics. In contrast, blood and graft IGHV repertoires from the same samples showed minimal overlap. Coronary infiltrates included CD4⁺ and CD8⁺ memory T cells expressing inflammatory (IFNγ, TNFα) and profibrotic (TGFβ) cytokines. These were distinguishable from the peripheral blood based on memory, activation, and tissue residency markers (CD45RO, CTLA-4, and CD69). Importantly, high-frequency rearrangements were traced back to endomyocardial biopsies (2–6 years prior). Comparison with four HLA-mismatched blood donors revealed a repertoire of shared TCRBs, including a subset of recently described cross-reactive sequences. These findings provide supportive evidence for an active local intragraft bystander T cell response in late-stage CAV.
Prognostic Biomarkers for Precision Medicine in Heart Transplant: Is Galectin-3 the One?
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Ischemia-Reperfusion Injury Reduces Long Term Renal Graft Survival: Mechanism and Beyond
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Blood flow resistance increases 16-fold, with a 50% reduction in the luminal radius (Mitchell and Libby, 2007). Therefore, arterial remodelling could severely compromise graft perfusion, whilst occlusion of arterioles could result in stellate infarcts, which may develop into fibrotic lesions (Huibers et al., 2011). Studies have shown that ischemia-reperfusion injury accelerates the development of graft vascular disease and the early ischemia-associated recruitment of neutrophils.
Ischemia-reperfusion injury (IRI) during renal transplantation often initiates non-specific inflammatory responses that can result in the loss of kidney graft viability. However, the long-term consequence of IRI on renal grafts survival is uncertain. Here we review clinical evidence and laboratory studies, and elucidate the association between early IRI and later graft loss. Our critical analysis of previous publications indicates that early IRI does contribute to later graft loss through reduction of renal functional mass, graft vascular injury, and chronic hypoxia, as well as subsequent fibrosis. IRI is also known to induce kidney allograft dysfunction and acute rejection, reducing graft survival. Therefore, attempts have been made to substitute traditional preserving solutions with novel agents, yielding promising results.
Distinct phenotypes of cardiac allograft vasculopathy after heart transplantation: A histopathological study
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The MNCs are captured in diffusely organized fibrous tissue [14]. The intimal fibrotic process is likely caused by T-cells producing interferon gamma (IFNγ) and transforming growth factor beta (TGFβ) which induces the growth of the connective tissue [15–18]. For clinical purposes the ISHLT proposed a standardized way to define CAV using angiography [19].
Long-term survival after heart transplantation (HTx) is hampered by cardiac allograft vasculopathy (CAV). Better understanding of the pathophysiological mechanisms of CAV might have considerable consequences for therapeutic approaches in the future. The aim of the present study was to investigate the histological phenotypes of CAV in relation with clinical patient characteristics.
Coronary cross-sections from 51 HTx patients were obtained at autopsy. CAV was observed in 42 patients (82%). Three histological CAV phenotypes were identified (H-CAV 1–3). No CAV (H-CAV 0) is as seen in normal coronary arteries; intimal thickening consisting of a layer of longitudinal oriented smooth muscle cells. In H-CAV 1 to 3 a second intimal layer is formed, on top of the longitudinal oriented smooth muscle cell layer, with predominantly mononuclear inflammatory infiltrate in loose connective tissue (H-CAV 1), smooth muscle cells in different orientation (H-CAV 2), or a fibrotic intimal lesion (H-CAV 3). H-CAV type was significantly related with time after transplantation, age at transplantation, the amount of atherosclerotic disease and the occurrence of infection. In addition, morphometric analysis revealed that higher H-CAV types have a relatively larger intimal area, that is compensated for by expansive arterial remodeling of the artery.
CAV in an ongoing process that can be classified into three different phenotypes; inflammatory lesions, lesions rich of smooth muscle cells and fibrotic lesions. Our results suggest that these phenotypes are related to time after transplantation, age at transplantation, the amount of atherosclerotic disease and the occurrence of infection.

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Intimal fibrosis in human cardiac allograft vasculopathy

Abstract

Highlights

Introduction

Section snippets

Patient population

mRNA expression of fibrotic factors, cytokines and ECM components

Discussion

Study limitations

Disclosures

J Heart Lung Transplant

Am J Cardiol

J Heart Lung Transplant

Am J Transplant

J Heart Lung Transplant

Am J Transplant

Am J Pathol

Lab Invest

Trends Immunol

Cardiac allograft vasculopathy: a review

Circulation

Histopathology of graft coronary disease

J Heart Lung Transplant

Vascular remodeling in transplant vasculopathy

Circ Res

Allograft vasculopathy versus atherosclerosis

Circ Res