Xiao-Ming Zhao

Association of Thrombospondin-1 and Cardiac Allograft Vasculopathy in Human Cardiac Allografts

Background —Despite the expression of angiogenic growth factors in transplanted hearts, neovessel formation appears scant. We therefore hypothesized that cardiac allografts contain endogenous inhibitors of angiogenesis. In particular, we tested the involvement in cardiac allografts of thrombospondin-1 (TSP-1), a matrix glycoprotein that inhibits angiogenesis and facilitates smooth muscle cell (SMC) proliferation. Methods and Results —Levels of TSP-1 mRNA in endomyocardial biopsy samples of human cardiac allografts substantially exceeded those in normal hearts. The ratio of TSP to GAPDH mRNA determined with quantitative RT-PCR was 6.54±1.6 in cardiac allografts versus 0.26±0.02 (P =0.001) in normal hearts. Analysis in sequential biopsies revealed a strong association between persistent elevation of TSP-1 in allografts and the development of cardiac allograft vasculopathy (CAV). The CAV score was 2.4±0.8 in patients with persistent TSP-1 elevation compared with 0.2±0.2 in patients without elevation (P =0.001). Immunohistochemistry demonstrated intense expression of TSP-1 in cardiac allografts, predominantly in cardiac myocytes and neointimal SMCs. In vitro experiments demonstrated that T cells expressed TSP-1, acidic fibroblast growth factor, and vascular endothelial cell growth factor on allogeneic stimulation. Cytokines known to be elevated in cardiac allografts (interleukin-1&bgr;, interferon-&ggr;, and tumor necrosis factor-&agr;) induced TSP-1 in SMCs but inhibited TSP-1 in endothelial cells. Conclusions —Persistent elevation of TSP-1 in cardiac allografts correlates with the development of CAV. Allogeneic stimulation induces angiogenic growth factors and TSP-1 in T cells. Cytokines differentially regulate TSP-1 expression in SMCs versus endothelial cells. Increased levels of TSP-1 in human cardiac allografts may alter vascular responses to angiogenic growth factors by inhibiting angiogenesis and promoting SMC proliferation characteristic of CAV.

Expression of cytokine genes in human cardiac allografts: correlation of IL‐6 and transforming growth factor‐beta (TGF‐β) with histological rejection

Cytokines may play critical roles in allograft rejection. Currently, a clear pattern of cytokine production that correlates with rejection has not emerged. Our preliminary studies suggested a trend toward increased IL‐6 and TGF‐β gene expression in cardiac allografts during rejection. We have extended these studies using reverse transcriptase/polymerase chain reaction (RT/PCR) to detect the expression of IL‐6. TGF‐β, and T cell receptor β chain constant region (TCR‐β genes in 21 additional consecutive myocardial biopsies obtained from six heart transplant patients and from five pre‐transplant donor hearts. Cytokine gene expression was compared with histological diagnosis of rejection. There was strong correlation between IL‐6 as well as TGF‐β gene expression, and histological rejection (6/8 biopsies with versus 0/7 without rejection (P = 0.006) and 7/9 biopsies with versus 0/7 without rejection (P=0.003) respectively). Neither IL‐6 nor TGF‐β transcripts were detected in any pre‐transplant donor heart. TCR‐β chain mRNA was found in all allograft biopsies regardless of the presence of rejection, but was absent in pre‐transplant donor hearts. Our results indicate that expression of IL‐6 and TGF‐β is highly correlated with allograft rejection and thus may play an important role in regulation of cardiac ailograft rejection. T cell infiltration of allografted myocardium is invariably detected by PCR regardless of histological rejection. The long‐term functional significance of these cells in transplanted hearts needs further investigation.

The expression of acidic fibroblast growth factor (heparin-binding growth factor-1) and cytokine genes in human cardiac allografts and T cells.

The purpose of this study was to investigate the role of cytokines and growth factors in cardiac allograft rejection and vasculopathy (CAV). The polymerase chain reaction (PCR) was used to detect the expression of IL-1, IL-2, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, TGF-beta, TCR-beta chain and aFGF genes in 21 myocardial biopsies obtained from 9 heart transplant patients. There was no statistically significant correlation between cytokine gene expression and rejection, although a trend toward increased IL-6 and TGF-beta expression was noted with rejection (6 of 10 biopsies with vs. 1 of 7 without rejection, and 4 of 9 biopsies with vs. none of 7 without rejection, respectively). IL-2 gene expression was detected in only 2 of 21 biopsies, both positive for rejection. IL-1, IL-4, IL-5, CD8, IFN-gamma, and TNF-alpha were not detected in any of the biopsies. TCR-beta chain mRNA was found in all biopsies, indicating the invariable presence of T cells regardless of histologic diagnosis of rejection. The aFGF gene was expressed in the majority (18 of 21) of biopsies, and its presence was not correlated with rejection. In addition to mRNA for the complete coding sequence of aFGF, two alternatively spliced mRNAs for aFGF were present in myocardial biopsies. Because aFGF and TCR beta genes were expressed in most biopsies, we determined whether aFGF mRNA was expressed in T cells; aFGF transcripts were found in 2 of 5 T-cell clones examined. Thus, aFGF mRNA in cardiac allografts may have been induced within the myocardium or elaborated by infiltrating T cells. The presence of mRNA for aFGF, a potent endothelial and smooth muscle cell mitogen, in allograft myocardium suggests that aFGF may play a role in the pathogenesis of CAV.

Modification of alternative messenger RNA splicing of fibroblast growth factor receptors in human cardiac allografts during rejection.

Accelerated coronary atherosclerosis in cardiac transplants (cardiac allograft vasculopathy, CAV) is characterized by coronary intimal hyperplasia. Acidic fibroblast growth factor (aFGF) is a potent mitogen for vascular smooth muscle cells and endothelial cells, and its expression is increased in cardiac allografts, suggesting it may play a role in the pathogenesis of CAV. The activity of aFGF is dependent on binding to transmembrane receptors. To investigate whether receptors for aFGF are also induced after transplantation, polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to analyze expression of four receptors for aFGF (FGFR1-FGFR4). Expression of mRNA encoding extracellular immunoglobulin-like domains of FGFR1 was increased 35-fold in cardiac allografts compared with normal hearts and was predominantly present in cardiac myocytes and vascular structures. Alternatively spliced mRNA that encodes transmembrane forms of FGFR1, which contain the signal-transducing tyrosine kinase domains, was induced in allografts during rejection, in infiltrating cells, vascular structures, and myocytes. In vitro experiments showed that differential expression of FGF receptor isoforms was induced by aFGF, and also by IL-6 and TGF-beta, which are expressed in cardiac allografts during rejection. The results show that expression of both aFGF and its receptors is altered in cardiac allografts and suggest that these events are important in the pathogenesis of CAV.

Confirmation of alternatively spliced platelet-derived growth factor-A chain and correlation with expression of PDGF receptor-alpha in human cardiac allografts.

PDGF is a potent mitogen for vascular smooth muscle cells (SMC) and may play an important role in the pathogenesis of cardiac allograft vasculopathy (CAV). Two isoforms of PDGF-A chain exist as a result of alternative mRNA splicing that either includes (long-form) or excludes (short-form) exon 6. Short-form PDGF-A is expressed in both resting and activated cells, while the long-form is present predominantly in activated cells. Using RT/PCR, we have found previously that long-form PDGF-A chain was expressed in human cardiac allografts but not in normal human hearts. In the experiments reported here, we studied the cellular distribution of PDGF-A chain isoforms and expression of PDGF receptor-alpha in cardiac allografts. In situ hybridization and immunohistochemistry confirmed the PCR data and demonstrated that expression of long-form PDGF-A chain was diffusely increased in cardiac allografts, predominantly in myocytes and vascular structures. Expression of PDGF receptor alpha also was induced in cardiac allografts and was not detected in any of the normal hearts. Induction of PDGF receptor alpha in cardiac allografts was associated with the presence of long-form PDGF-A chain. In vitro experiments with human endothelial cells demonstrated that aFGF, IL-6, and TGF-beta, which are produced in cardiac allografts in vivo, induced expression of long-form PDGF-A chain. Expression of long-form PDGF-A chain and its receptor was markedly increased in cardiac allografts, predominantly in vascular structures and myocytes. Alterative splicing of PDGF-A chain variants may be mediated by growth factors and cytokines produced in vivo.

Association of acidic fibroblast growth factor and untreated low grade rejection with cardiac allograft vasculopathy

Acidic fibroblast growth factor (aFGF) is a potent growth factor for vascular smooth muscle cells and may mediate vasculopathy in cardiac allografts subjected to chronic immunological injury. Therefore, we examined cardiac expression of aFGF, the number of rejection episodes, and other potential risk factors in 32 heart transplant patients who underwent intravascular ultrasound (IVUS) for detection of cardiac allograft vasculopathy (CAV). As defined by IVUS, CAV was present in 21 patients and absent in 11 patients (follow-up time: 52 +/- 21 vs. 51 +/- 12 months, respectively, P = NS). The level of aFGF in myocardial biopsies obtained at the time of IVUS was measured by semiquantitative reverse transcriptase polymerase chain reaction and expressed as the aFGF:GAPDH ratio. Higher level of aFGF were associated with CAV (mean aFGF:GAPDH ratio was 1.45 +/- 0.99 in patients with vs. 0.18 +/- 0.12 in patients without CAV [P < 0.001]). A strong association was found between high levels of cardiac aFGF and CAV, as 18 of 19 patients (95%) with high levels of aFGF (aFGF:GAPDH > 1) but only 3 of 13 patients with low levels of aFGF had CAV (P < 0.001). The relative risk of high level of aFGF for CAV was 4.1. Untreated low grade rejection (ISHLT I), but not treated high grade rejection (ISHLT > 2), was also associated with CAV (average number of untreated low grade rejection episodes was 3.5 +/- 1.8 in patients with vs. 2.1 +/- 1.0 in patients without CAV [P = 0.04]). Among other risk factors examined (age, sex, serum cholesterol, blood pressure, CMV infection, dose of immunosuppressants, and ischemic time), only triglycerides were higher in patients with than those without CAV (P = 0.003). We conclude that increased cardiac production of aFGF is significantly associated with CAV, which suggests that aFGF may serve as an important mediator in CAV. Untreated low grade rejection also poses an increased risk for CAV.

Longitudinal Analysis of Fibroblast Growth Factor Expression After Transplantation and Association With Severity of Cardiac Allograft Vasculopathy

BACKGROUND Vascular smooth muscle cell growth factors are postulated to contribute to cardiac allograft vasculopathy (CAV). Few data quantitatively address the timing, location, or stimuli for growth factor expression and relationship to CAV. METHODS AND RESULTS Acidic fibroblast growth factor (aFGF) mRNA expression was determined in serial endomyocardial biopsies during the first year after transplantation. Patients with high levels of aFGF mRNA and elevations after the early posttransplant period had significantly more severe CAV than patients with low aFGF and no late elevations. CONCLUSIONS Parenchymal aFGF expression varies between patients and in the same patient over time and correlates with development of CAV.