Intimal fibrosis in human cardiac allograft vasculopathy
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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2014, AtherosclerosisCitation Excerpt :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].