Pathogenesis Of Cardiac Allograft Vasculopathy Research Articles

Proteomics early after heart transplantation and relation to coronary intimal changes and prognosis

BackgroundLong-term survival after heart transplantation (HTx) is limited by cardiac allograft vasculopathy (CAV). The pathogenesis of CAV is poorly understood, and treatment has not yet been well-established. In this exploratory study, we aimed to identify novel immune and non-immune biomarkers correlated to CAV development, and biomarkers predicting CAV related events. MethodsUsing a proteomic panel, 92 cardiovascular disease-related proteins were evaluated in 26 de novo HTx patients at 3- and 12-months post transplantation. Intima area changes were assessed using optical coherence tomography (OCT). Major adverse cardiac events (MACE) included significant CAV progression, heart failure, and cardiovascular death. ResultsMedian follow up was 6.8 years. We found changes in four inflammatory biomarkers: Matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase 2 (MMP-2) were significantly increased after 3 months in the group of patients with highest intima proliferation, and after 12 months in patients experiencing MACE, respectively. Monocyte chemotactic protein 1 (MCP-1) was increased after 12 months in patients experiencing MACE. Platelet derived growth factor subunit A (PDGF-A) was significantly increased after 3 months in the group of patients with highest intima proliferation. ConclusionsWe identified four biomarkers that may be associated with CAV development which differed significantly between groups of intima proliferation and MACE; MMP-2, MMP-3, MCP-1 and PDGF-A. Further studies are needed to examine whether our findings offer the basis to seek for new markers of graft vasculopathy or treatment options.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy

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.

Micro- and macrovascular cardiac allograft vasculopathy in relation to 91 cardiovascular biomarkers in heart transplant recipients-An exploratory study.

Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation (HTx), and the pathogenesis is not fully clarified. We aimed to investigate a wide range of biomarkers and their correlation with micro- and macrovascular CAV and major adverse cardiac events in HTx patients. We evaluated 91 cardiovascular disease-related proteins in 48 HTx patients using a novel proteomic panel. Patients were dichotomized according to micro- and macrovascular CAV burden determined by coronary angiography, optical coherence tomography, and 15 O-H2 O positron emission tomography imaging. Major adverse cardiac events included significant CAV progression, heart failure, treated rejection, and cardiovascular death. We found consistent differences in two proteins involved in cholesterol homeostasis: significantly increased proprotein convertase subtilisin/kexin type 9 (PCSK9) (p<.05) and significantly decreased paraoxonase 3 (PON3) (p<.05). N-terminal pro-brain natriuretic peptide (NT-proBNP) was significantly increased in patients with microvascular CAV (p<.05) and borderline significantly increased in patients experiencing major adverse cardiac events (p=.10) and patients with macrovascular CAV (p=.05). We identified consistent changes in two proteins involved in cholesterol homeostasis which may be important players in the pathogenesis of CAV: PON3 and PCSK9. NT-proBNP also showed consistent changes across all groups but only reached statistical significance in patients with microvascular CAV. Our results warrant further validation in future studies.

T cell repertoire analysis suggests a prominent bystander response in human cardiac allograft vasculopathy.

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 (2-6years 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.

Hepatitis B Seropositivity is Associated with Increased Cardiac Allograft Vasculopathy after Heart Transplantation

Viral triggers have been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), the Achilles heel of heart transplantation (HT). Cytomegalovirus is associated with impaired coronary endothelial function, and in pediatric HT patients a variety of viruses, particularly adenovirus, are associated with graft dysfunction or loss. Hepatitis B virus (HBV) is associated with vasculitis and vascular processes in non-HT patients. We thus investigated the association between HBV seropositivity and CAV after HT. The 173 HT patients assessed for HBV serology from 1997 to 2017 were divided into hepatitis B core antibody (HBcAb) positive (n=29) and HBcAb negative (n=144) groups. Primary outcomes were CAV and the combined end-point of CAV or cardiovascular mortality. Baseline patient and donor clinical and demographic data for the 2 groups were similar. Kaplan-Meier analysis showed that 20-year freedom from CAV was significantly higher in the HBcAb-negative vs HBcAb-positive group (80 vs 50%, log-rank p=0.003, Figure 1A). Similarly, 20-year freedom from CAV or cardiovascular mortality was higher for the HBcAb-negative group (75 vs 45%, log-rank p=0.018). Consistently, multivariable analysis showed HBcAb positivity to be independently associated with a significant ∼3-fold increased risk of CAV (95% CI 1.38-5.01, p=0.003, Figure 1B), and a 2-fold increased risk of CAV or cardiovascular mortality (95% CI 1.11-3.90, p=0.021). Hepatitis B seropositivity is independently associated with an increased risk of CAV in HT recipients. Future studies are needed to examine the effect of anti-viral therapies.

Cardiac allograft vasculopathy: Insights on pathogenesis and therapy

Cardiac allograft vasculopathy (CAV) is a unique accelerated form of coronary vascular disease affecting heart transplant recipients. This complication is a significant contributor to medium- to long-term post-transplant morbidity and mortality. There is a high prevalence of CAV with approximately one in three patients developing CAV by 5years post-transplant. Morphologically, CAV is characterized by concentric coronary intimal hyperplasia in both the epicardial arteries and intramural microvasculature. Although several immune and non-immune factors have been identified, their precise pathogenic mechanisms, interactions, and relative importance in the development of CAV are not well defined. The advent of improved imaging surveillance modalities has resulted in earlier detection during the disease process. However, overall management of CAV remains challenging due to paucity of treatment. This review aims to discuss key concepts on the pathogenesis of CAV and current management strategies, focusing on the use of mammalian target of rapamycin inhibitors.

Platelet aggregation and response to aspirin therapy in cardiac allograft vasculopathy

Long-term survival after heart transplantation (HTx) is compromised by cardiac allograft vasculopathy (CAV) characterized by coronary macro- and microvascular disease. The pathogenesis of CAV is unclear and may involve coronary thrombosis. We investigated whether HTx patients with CAV had higher platelet aggregation and turnover than HTx patients without CAV and healthy controls. Furthermore, we investigated the anti-platelet effect of low-dose aspirin in HTx patients. We included 57 patients who had undergone HTx (median 8.3 years from HTx) and 57 healthy controls. Platelet aggregation was measured on-aspirin and off-aspirin using impedance aggregometry with adenosine diphosphate (ADP) and arachidonic acid (AA). We evaluated platelet turnover by flow cytometry, CAV burden by coronary angiography and echocardiography, and microvascular function by echocardiographic coronary flow velocity reserve (CFVR). Off-aspirin, HTx patients with CAV (n = 21) had higher ADP-induced platelet aggregation than healthy controls (p < 0.01) and HTx patients without CAV (n = 36) (p < 0.05). Aspirin treatment reduced AA-induced platelet aggregation in both HTx groups, but HTx patients with CAV had higher platelet aggregation on-aspirin than HTx patients without CAV (p < 0.05). Platelet turnover did not differ between HTx patients with CAV and HTx patients without CAV (p > 0.34). HTx patients with lower CFVR values had higher platelet aggregation than HTx patients with higher CFVR values (p < 0.05). Off-aspirin, platelet aggregation was higher in HTx patients with CAV than in HTx patients without CAV and healthy controls. On-aspirin, platelet aggregation was higher in HTx patients with CAV than in HTx patients without CAV. Aspirin monotherapy may not provide sufficient platelet inhibition in HTx patients with CAV.

Clonal Composition and Single-Cell Characterization of T-Cell Infiltrates in Cardiac Allograft Vasculopathy

Purpose T-cells are implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), yet their clonal composition and function are incompletely defined. We examined the T-cell receptor β (TCRβ) repertoire from explanted grafts with CAV and characterized their phenotype at the single-cell level. Methods TCRβ repertoire analysis was carried out in 4 cardiac explants with CAV by next generation sequencing using DNA extracted from 1) coronary artery (CA), 2) endomyocardium (endo) 3) peripheral blood and 4) cardiac biopsies from the same patients. Blood and intragraft T cells were also phenotyped at the single cell level using a strategy combining index sorting, 13-colour flow cytometry and single-cell RT/PCR for key cytokines, transcription markers and TCRβ chain sequencing. Unique TCRβ chain gene rearrangements were used to link single-cell and TCR repertoires. Results Overlap between intragraft and blood T cell repertoires was observed in all 4 cases with most high frequency rearrangements present in both compartments (Fig. 1a). The clonality index was higher in the blood compared to the graft. In contrast, there was minimal overlap in the B cell repertoire from the same samples (example in 1b). Strikingly, in each case, a common high frequency TCRβ in the blood was also amongst the top rearrangements in the CA, endo, and a previous biopsy (Quilty lesion or cellular rejection) obtained 2 to 6 years earlier (1c). Using their unique TCRβ sequence as a link to the single-cell phenotype data, we identified these clones as CD8+CD45RA+Tbet+ T-cells evocative of effector memory RA T cells (Temra). Conclusion Our studies demonstrated considerable overlap between the graft and blood TCR repertoire in CAV suggestive of a bystander T cell response in situ. However, these findings do not rule out the existence of individual clones being expanded in the graft. Moreover, the Temra phenotype of persistent high frequency clones offers a first indication of the type of T cells infiltrating cardiac allografts.

Leukotriene B4 Contributes to Development of Cardiac Allograft Vasculopathy

Purpose Cardiac allograft vasculopathy (CAV) is a highly prevalent vaso-occlusive disease that is a leading cause of graft failure and mortality after heart transplantation. While the pathogenesis of CAV remains incompletely understood, histologic evidence suggests that macrophages, which constitute an important part of the innate immune response, may play an important role. Prior studies in pulmonary hypertension have shown that macrophage-derived leukotriene B4 (LTB4) induces proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We hypothesized that LTB4 plays a similar role in the development of CAV. Methods Human studies: We performed immunofluorescence staining of sections from human CAV+ and CAV- coronary arteries with CD68 (macrophage marker) and 5-LO (5-lipoxygenase, LTB4-producing enzyme), and measured serial LTB4 plasma levels in 14 patients with angiographic CAV and 14 matched CAV- controls. Rat model: We performed aortic transplants in a rat CAV model (Fischer into Lewis orthotopic aortic transplants, n=11) with allogeneic transplants as controls (n=6), and performed CD68 and 5-LO staining of aortic cross-sections of mice with and without aortic intimal hyperplasia. We then treated rat allogeneic transplants with bestatin (protease inhibitor that blocks LTB4 production) to determine whether LTB4 inhibition can prevent the intimal proliferation seen in this rat CAV model. Results Immunofluorescence staining showed that 5-LO co-localized with CD68+ macrophages in the intimal layer of CAV+ human arterioles and in allogeneic rat aortic transplant models. Plasma LTB4 levels were higher in CAV+ cases compared to CAV- controls (n=14 per group, p Conclusion Macrophage-derived leukotriene B4 may contribute to development of CAV. Blockade of LTB4 synthesis has the potential to prevent CAV development after heart transplantation.

Novel Cytokine Score and Cardiac Allograft Vasculopathy

To date, there are no established noninvasive biomarkers available for prediction of cardiac allograft vasculopathy (CAV) after orthotopic heart transplantation (OHT). Inflammatory processes are supposed to play a central role in the pathogenesis of CAV. Recent studies have suggested that immune mediators could serve as biomarkers for cardiovascular diseases. We hypothesized particular cytokines or a combination thereof may serve as noninvasive biomarkers for CAV. Plasma cytokines were screened from 27 patients with CAV and 27 patients without CAV after OHT. The concentrations of interleukins-4, -6, -10, -21, -23, -31, -33, interferon gamma, tumor necrosis factor alpha, and the soluble activation marker CD40 ligand were determined using Luminex-based multiplex analyses. Although concentrations of all cytokines except interferon gamma were on average higher in the CAV group, there were no significant differences between the groups for any 1 cytokine. Using a binary logistic regression model, we were able to develop a probability score for detecting patients at elevated risk for advanced CAV with a sensitivity of 92.31% and a specificity of 60.71% (receiver-operating characteristic area under the curve 0.799 ± 0.06; p<0.0001). In conclusion, analyzing the concentration of specific inflammatory cytokines could be meaningfully included in evaluation of CAV after OHT.

Elevated serum vascular endothelial growth factor and development of cardiac allograft vasculopathy in children.

Cardiac allograft vasculopathy (CAV) is a leading cause of retransplantation and death in pediatric heart transplant recipients. Our aim was to evaluate the association between serum vascular endothelial growth factor-A (VEGF) and CAV development in the pediatric heart transplant population. In this retrospective study performed at a university hospital, VEGF concentrations were measured by enzyme-linked immunosorbent assay in banked serum from pediatric heart transplant recipients undergoing routine cardiac catheterization. In subjects with CAV (n = 29), samples were obtained at 2 time-points: before CAV diagnosis (pre-CAV) and at the time of initial CAV diagnosis (CAV). In subjects without CAV (no-CAV, n = 16), only 1 time-point was used. VEGF concentrations (n = 74) were assayed in duplicate. Serum VEGF is elevated in pediatric heart transplant recipients before catheter-based diagnosis of CAV (no-CAV mean: 144.0 ± 89.05 pg/ml; pre-CAV mean: 316.2 ± 118.3 pg/ml; p = 0.0002). Receiver-operating characteristic curve analysis of pre-CAV VEGF levels demonstrated an area under the curve of 87.7% (p = 0.0002), with a VEGF level of 226.3 pg/ml predicting CAV development with 77.8% sensitivity and 91.7% specificity. VEGF is similarly elevated in subjects with angiographically diagnosed CAV and in those with normal angiography but intravascular ultrasound (IVUS) evidence of CAV. The increase in serum VEGF before onset of detectable CAV is fundamental to its utility as a predictive biomarker and suggests further investigations of VEGF in the pathogenesis of CAV are warranted in the pediatric heart transplant population.

Biomarker Discovery in Cardiac Allograft Vasculopathy: Novel Aptamer Proteomic and MicroRNA Profiling

Cardiac allograft vasculopathy (CAV) limits long-term outcomes after heart transplantation. Biomarkers may aid noninvasive diagnosis and prognostication of CAV, and enable personalized treatment. The novel Slow Off-rate Modified Aptamer (SOMAscan) proteomic and NanoString nCounter microRNA (miRNA) assays provide rapid high precision evaluation of a large number of analytes, capturing a broad range of potentially important biological functions in CAV pathogenesis. The purpose of this study was to identify candidate biomarkers for CAV utilizing these novel methods.

Cardiac Allograft Vasculopathy: A Review of Risk Factors and Pathogenesis

Heart transplant remains the gold standard therapy for patients with end stage heart disease and offers improved survival and quality of life. Significant progress has been achieved in improving one year mortality after heart transplantation. Nonetheless, long-term graft survival has not changed significantly over the past few decades. Long term survival of heart transplant recipients is limited by chronic rejection, cardiac allograft vasculopathy (CAV), and malignancy. CAV is a major contributor for graft failure and mortality after the first year of in heart transplant recipients. CAV is a proliferative vasculopathy characterized by diffuse myointimal hyperplasia and progressive narrowing of the graft vessels. Both immune dependent and independent factors have been shown to contribute to the pathogenesis of CAV. Understanding these risk factors is essential in developing preventative and therapeutic strategies. Angiography with Intravascular ultrasound (IVUS) has become the key diagnostic tool in the early detection of CAV as well as prognostication. Echocardiographic assessment of allograft function in conjunction with coronary angiographic findings are used in assessing the severity of CAV. Adjustment of immunosuppression and statins remain the initial steps in the management of CAV. Retransplantation is the definitive treatment for severe CAV, however, the paucity of organs along with increased mortality associated with retransplantation makes it a less desirable option. Although significant progress has been achieved in the understanding of pathogenesis, risk factors for CAV and plaque morphology, knowledge gaps still persists and further research is warranted. Progress is still needed in developing diagnostic modalities that could detect early CAV, as well as preventative and therapeutic strategies that will facilitate graft tolerance and immune modulation. This review focuses on summarizing the pathogenesis and risk factors for CAV.

Immunological and Fibrotic Mechanisms in Cardiac Allograft Vasculopathy.

Cardiac allograft vasculopathy (CAV) has a high prevalence among patients that have undergone heart transplantation. Cardiac allograft vasculopathy is a multifactorial process in which the immune system is the driving force. In this review, the data on the immunological and fibrotic processes that are involved in the development of CAV are summarized. Areas where a lack of knowledge exists and possible additional research can be completed are pinpointed. During the pathogenesis of CAV, cells from the innate and the adaptive immune system cooperate to reject the foreign heart. This inflammatory response results in dysfunction of the endothelium and migration and proliferation of smooth muscle cells (SMCs). Apoptosis and factors secreted by both the endothelium as well as the SMCs lead to fibrosis. The migration of SMCs together with fibrosis provoke concentric intimal thickening of the coronary arteries, which is the main characteristic of CAV.

THERAPEUTIC EFFECT OF VEGF INHIBITION IN CARDIAC ALLOGRAFT VASCULOPATHY

Cardiac allograft vasculopathy (CAV) affects approximately 70% of heart transplants and represents the greatest limitation for long term survival. This expression of chronic rejection is characterized by a widespread and concentric thickening of the donor heart macrovessels which increases over time, eventually resulting in tissue ischemia and ultimately graft failure. Vascular endothelial growth factor (VEGF) has been shown to be endogenously over expressed within both human and animal model heart allografts. Significant host bone marrow-derived stem cell migration to the allograft has been previously demonstrated, contributing new endothelial cells to micro vessels. It is thought that VEGF induced graft neo-angiogenesis and inflammation may be central mechanisms involved in the pathogenesis of CAV. To investigate the therapeutic effect of the inhibition of VEGF expression in CAV. Female 129J donor hearts were heterotopically transplanted into C57/B16 males and maintained on Western diet in the presence or absence soluble VEGF receptor 1 (sVEGFR1). At 21 days post transplantation, significant reductions in both the average grade of luminal narrowing (P<0.05) and the percentage of vessels affected (P<0.005) were observed in sVEGFR-1treated transplants. Administration of sVEGR1 also significantly (P<0.05) reduced average wet heart weight as compared to vehicle controls while mean ventricular cross-sectional area remained similar. Bone marrow (BM) was subsequently harvested from male C57/B16 mice. The effect of VEGF inhibition on bone marrow mediated micro-vascular outgrowth and endothelial cell migration and proliferation were assessed using in vitro assays of aortic ring angiogenesis, wound healing and proliferation, respectively. Compared to non-treated controls, treatment of aortic rings with sVEGFR1 significantly (P<0.05) reduced bone-marrow mediated microvascular outgrowth length and area. Treatment of Human Coronary Artery Endothelial Cells (HCAEC) with sVEGR1 significantly (p<0.05) reduced bone marrow mediated endothelial cell migration and proliferation. These results indicate that VEGF inhibition via administration of sVEGR1 may reduce the severity and incidence of CAV through reduced myocardial edema and neo-angiogenesis. We provide in vitro evidence for the role of VEGF signaling in BM mediated microvascular outgrowth, and endothelial cell migration and proliferation, supporting the function of the VEGF inhibition strategy. Strong evidence that manipulation of the VEGF signaling axis within the allograft ameliorates CAV prognosis could pave the road for a novel approach to CAV treatment and prevention.

Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.

Anomalous expression of heat shock protein 27 and glucose regulated protein 78 contributed to cardiac allograft vasculopathy

Objective To observe the protein expression of heat shock protein 27 (HSP27) and glucose regulated protein 78 (GRP78) in rats transplanted hearts,and provide some clues to elucidate the pathogenesis of cardiac allograft vasculopathy (CAV).Methods The hearts were transplanted from Lewis to Sprague-Dawely rats as allograft (n =8) and from Lewis to Lewis rats as isograft (n =8) based on Ono' s model.The protein expression of HSP27 and GRP78 was detected by using immunohistochemistry [streptavid-inperoxidase (SP) method].Results The expression of HSP27 and GRP78 was stained uniformly in cardiac isografts,but that was anomalous in cardiac allografts,which showed higher expression on cardiac muscle (2.24 ± 0.36 vs.1.04 ± 0.10,P ＜ 0.01 ; 1.51 ± 0.12 vs.0.85 ± 0.30,P ＜ 0.05) and lower expression on blood vessels in biopsies with CAV (2.24 ±0.36 vs.0.25 ±0.08,P ＜0.01 ; 1.51 ±0.12 vs.0.16 ± 0.10,P ＜ 0.01).Conclusion The less expression of HSP27 and GRP78 on blood vessels decreased the defense to immunologic injury,which stimulated the over-proliferation of vascular smooth muscle cells to repair the vessels damage in heart allografts.Meanwhile,these protective reactions could incur the development of CAV in heart allografts. Key words: Cardiac allograft; Vasculopathy; Heat shock protein 27; Glucose regulated protein 78

Repeated episodes of thrombosis as a potential mechanism of plaque progression in cardiac allograft vasculopathy

The pathogenesis of cardiac allograft vasculopathy (CAV) remains complex and may involve multiple mechanisms. We tested the hypothesis that the multilayer (ML) appearance, an intravascular ultrasound (IVUS) finding suggestive of repetitive thrombosis, is associated with plaque progression in heart transplant (HTx) recipients. Our study population consisted of 132 HTx recipients undergoing at least two grayscale and virtual histology (VH)-IVUS examinations. A retrospective serial analysis was performed between the first (baseline) and the last (follow-up) IVUS data during a median follow-up of 3.0 years. The subjects were divided into two groups based on the presence of the ML appearance on the baseline IVUS. At baseline, subjects with ML appearance (n = 38) had a longer time elapsed since transplant, larger vessel volume, and larger plaque volume than those without (n = 94) (all P < 0.01). Intraluminal thrombi and plaque ruptures were identified only in subjects with ML appearance (P < 0.01 vs. those without). More subjects with ML appearance at baseline developed subsequent ML formation compared with those without [21 (55%) vs. 22 (23%), P < 0.01] during follow-up. There was an increase in plaque volume, necrotic core volume, and dense calcium volume in subjects with ML appearance (all P < 0.01 vs. those without). Multivariable linear regression analysis showed that ML appearance was a potential predictor of plaque progression (regression coefficient 0.28, 95% CI 0.10-0.45, P < 0.01). The current study demonstrates that a finding of ML appearance, indicative of repeated episodes of mural thrombosis, is not infrequent in asymptomatic HTx recipients and possibly contributes to progression of CAV.

Implication for transglutaminase 2-mediated activation of β-catenin signaling in neointimal vascular smooth muscle cells in chronic cardiac allograft rejection

Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical β-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of β-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection. Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of β-catenin protein and expression of several components of β-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures. Activation of β-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of β-catenin target genes, as well as the accumulation and nuclear localization of the β-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of β-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for β-catenin activation by platelet-derived growth factor in cardiac tissue. β-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection.

478 TNF -α and IL-8 Predict Cardiac Vasculopathy after Cardiac Transplantation

Purpose: Cardiac allograft vasculopathy (CAV) is a major cause of mortality after heart transplantation. Yearly coronary angiograms are performed post transplantation to screen for CAV. Accurate non-invasive screening tests for CAV might decrease the need for coronary angiograms. Cellular and humoral immune responses have been implicated in the development of CAV; we hypothesized that immune function and cytokine expression could potentially provide information regarding the development of CAV. Methods and Materials: We measured Immuknow, concentration of 12 cytokines and sCD30 in blood collected at the time of routine endomyocardial biopsy and when clinically indicated. CAV was defined as new coronary artery disease (CAD) detected by angiography or a significant worsening (increase in coronary stenosis 50% or more) of previously diagnosed CAD. Results: We collected 162 samples from 56 cardiac transplant recipients. CAV was identified in 4 patients. Significant correlation was observed between CAV and TNFand IL8 (P 0.001). ROC curve was constructed to test the performance of TNFand IL8 in predicting CAV and showed a maximum efficiency for TNFat 38ng/ml (sensitivity 66.67%, specificity 97.74%), and a maximum efficiency for IL8 at 2.2ng/ml (sensitivity 66.67%, specificity 82.71%). No significant correlation between CAV and other immune function parameters was identified. Conclusions: Our data suggest strong correlation between CAV and TNFwhich initiates the proinflammatory response. A significant correlation is also seen with IL-8 which is a potent proinflammatory and chemotactic cytokine. These findings may indicate a major role for these cytokines in the pathogenesis of CAV and should be validated in a larger patient cohort.