R. Krebs

Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis.

Background—Cardiac allograft arteriosclerosis is a complex process of alloimmune response, chronic inflammation, and smooth muscle cell proliferation that includes cross talk between cytokines and growth factors. Methods and Results—Our results in rat cardiac allografts established alloimmune response as an alternative stimulus capable of inducing vascular endothelial growth factor (VEGF) mRNA and protein expression in cardiomyocytes and graft-infiltrating mononuclear inflammatory cells, which suggests that these cells may function as a source of VEGF to the cells of coronary arteries. Linear regression analysis of these allografts with different stages of arteriosclerotic lesions revealed a strong correlation between intragraft VEGF protein expression and the development of intimal thickening, whereas blockade of signaling downstream of VEGF receptor significantly reduced arteriosclerotic lesions. In addition, in cholesterol-fed rabbits, intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector that encoded mouse VEGF164 enhanced the formation of arteriosclerotic lesions, possibly secondary to increased intragraft influx of macrophages and neovascularization in the intimal lesions. Conclusions—Our findings suggest a positive regulatory role between VEGF and coronary arteriosclerotic lesion formation in the allograft cytokine microenvironment.

Angiopoietin-1 Protects Against the Development of Cardiac Allograft Arteriosclerosis

Background—Angiopoietin (Ang)–1 is an angiogenic growth factor that counteracts the permeability and proinflammatory effects of vascular endothelial growth factor and other proinflammatory cytokines. Recently, we demonstrated that vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Here, we studied the roles of Ang1, its natural antagonist Ang2, and their receptor Tie2 in rat cardiac allograft arteriosclerosis. Methods and Results—Heterotopic cardiac allografts and syngrafts were transplanted from Dark Agouti (DA) to Wistar-Furth rats and from DA to DA rats, respectively. Immunohistochemistry disclosed that only a few mesenchymal cells expressed Ang1 in normal hearts and syngrafts, whereas no immunoreactivity was found in cardiac allografts undergoing chronic rejection. Ang2 and Tie2 immunoreactivity was induced mainly in capillaries and postcapillary venules in chronic allografts when compared with syngeneic controls, but no immunoreactivity was found in arterial endothelium. Intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector encoding human Ang1 (Ad.Ang1) protected against the development of allograft arteriosclerosis. Ad.Ang1 perfusion reduced Ang2 expression in microcirculation, the numbers of graft-infiltrating leukocytes, and the level of immunoactivation and interstitial fibrosis, as well as both the incidence and intensity of intimal lesions. Ad.Ang1 perfusion also increased CD34+ stem cell counts in peripheral blood. Conclusions—Our findings suggest that the antiinflammatory properties of Ang1 may offer an entirely new therapeutic approach to prevent cardiac allograft arteriosclerosis.

Targeting Lymphatic Vessel Activation and CCL21 Production by Vascular Endothelial Growth Factor Receptor-3 Inhibition Has Novel Immunomodulatory and Antiarteriosclerotic Effects in Cardiac Allografts

Background— Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. Methods and Results— Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C+ inflammatory cell and hyaluronan receptor-1 (LYVE-1)+ lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3+, contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein–positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8+ effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62+ dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4+ T cells in chronically rejecting mouse cardiac allografts. Conclusions— These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel–targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

Donor Simvastatin Treatment Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury and Chronic Rejection Through Microvascular Protection

Background— Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Methods and Results— Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell–endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1&agr;, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-L-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-&bgr;1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-&bgr;1–induced microvascular endothelial-to-mesenchymal transition. Conclusions— Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

PDGF-A, -C, and -D but not PDGF-B Increase TGF-β1 and Chronic Rejection in Rat Cardiac Allografts

Objective—Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A–D)—potent mesenchymal cell mitogens—in rat cardiac allografts. Methods and Results—PDGFR-&agr; mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac¢hatn allografts. In acute rejection, PDGFR-&agr; immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors—except that of PDGF-B ligand—was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-&bgr;1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection. Conclusions—We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-&bgr;1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

Common Protective and Diverse Smooth Muscle Cell Effects of AAV-Mediated Angiopoietin-1 and -2 Expression in Rat Cardiac Allograft Vasculopathy

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.

Donor Simvastatin Treatment Prevents Ischemia‐Reperfusion and Acute Kidney Injury by Preserving Microvascular Barrier Function

Ischemia‐reperfusion injury (IRI) after kidney transplantation may result in delayed graft function. We used rat renal artery clamping and transplantation models to investigate cholesterol‐independent effects of clinically relevant single‐dose peroral simvastatin treatment 2 h before renal ischemia on microvascular injury. The expression of HMG‐CoA reductase was abundant in glomerular and peritubular microvasculature of normal kidneys. In renal artery clamping model with 30‐min warm ischemia, simvastatin treatment prevented peritubular microvascular permeability and perfusion disturbances, glomerular barrier disruption, tubular dysfunction and acute kidney injury. In fully MHC‐mismatched kidney allografts with 16‐h cold and 1‐h warm ischemia, donor simvastatin treatment increased the expression of flow‐regulated transcription factor KLF2 and vasculoprotective eNOS and HO‐1, and preserved glomerular and peritubular capillary barrier integrity during preservation. In vitro EC Weibel–Palade body exocytosis assays showed that simvastatin inhibited ischemia‐induced release of vasoactive angiopoietin‐2 and endothelin‐1. After reperfusion, donor simvastatin treatment prevented microvascular permeability, danger‐associated ligand hyaluronan induction, tubulointerstitial injury marker Kim‐1 immunoreactivity and serum creatinine and NGAL levels, and activation of innate and adaptive immune responses. In conclusion, donor simvastatin treatment prevented renal microvascular dysfunction and IRI with beneficial effects on adaptive immune and early fibroproliferative responses. Further studies may determine potential benefits in clinical cadaveric kidney transplantation.

Platelet-derived growth factor receptor inhibition reduces allograft arteriosclerosis of heart and aorta in cholesterol-fed rabbits.

Background. Crosstalk between pro-inflammatory cytokines and platelet-derived growth factor (PDGF) regulates smooth-muscle-cell proliferation in cardiac-allograft arteriosclerosis. In this study, we tested the effect of STI 571, a novel orally active protein tyrosine kinase (PTK) inhibitor selective for PDGF receptor (PDGF-R) on transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Methods. Cardiac allografts were transplanted heterotopically from Dutch Belted to New Zealand White rabbits. A 0.5% cholesterol diet was begun 4 days before transplantation. Recipients received STI 571 5 mg/kg per day or vehicle intraperitoneally throughout the study period of 6 weeks. Cyclosporine A was given as background immunosuppression. Results. In cardiac allografts of vehicle-treated rabbits, 76.2±2.1% of medium-sized arteries were affected by intimal thickening, and the percentage of arterial occlusion was 45.0±5.0%. Treatment with STI 571 reduced the incidence of affected medium-sized arteries to 41.2±8.1% (P <0.05) and the arterial occlusion to 27.6±5.0% (P <0.05). In addition, we observed that STI 571 treatment reduced intimal lesion formation in proximal ascending aorta of transplanted hearts from 72.3±19.9 to 12.7±1.9 &mgr;m (P <0.05). Our results also show that STI 571 significantly inhibited accelerated arteriosclerosis in medium-sized arteries of recipients’ own hearts. Conclusions. The results of the present study suggest that PDGF-R activation may regulate the development of transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Thus, PTK inhibitors may provide new strategies for prevention of these fibroproliferative vascular disorders.

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

Increased Th17 rather than Th1 alloimmune response is associated with cardiac allograft vasculopathy after hypothermic preservation in the rat

BACKGROUND Preservation injury decreases patient survival and promotes the development of cardiac allograft vasculopathy. We investigated the sequential effects of hypothermic preservation on ischemia-reperfusion injury (IRI), subsequent innate immune activation, and adaptive immune response in rat cardiac allografts. METHODS Allografts were transplanted from fully major histocompatibility complex-mismatched Dark Agouti to Wistar Furth rats without pre-operative hypothermia or after 4 hours of hypothermic preservation. Recipients received cyclosporine A immunosuppression. The allografts were recovered at 6 hours (n = 6, 7), 24 hours (n = 6), 10 days (n = 5), and 8 weeks (n = 5). Immunohistochemical, histologic, and reverse-transcription polymerase chain reaction analysis was performed. RESULTS In IRI, significantly increased messenger RNA (mRNA) levels for Toll-like receptor 4, hyaluronan synthases (HAS)1-2 (p = 0.03), high-mobility group box 1 (p = 0.05), CD80/83 (p = 0.01, p = 0.048), and the cytokines tumor necrosis factor-alpha (p = 0.004), interferon-gamma (p = 0.012), and interleukin (IL)-6 (p = 0.019) were seen in allografts subjected to hypothermic preservation. During established alloimmune response, allografts subjected to hypothermic preservation expressed prominent infiltration of CD4+ T cells (p = 0.043) and dendritic cells (p = 0.029) and significantly up-regulated mRNA levels of CD80 (p = 0.036), chemokine (C-C motif) ligand 21 (p = 0.008), C-C chemokine receptor type 7 (p = 0.003), vascular endothelial growth factor-C (p = 0.016), and vascular endothelial growth factor receptor-3 (p = 0.02). These allografts also showed prominent mRNA upregulation of Foxp3 (p = 0.014), IL-17 (p = 0.038), and IL-23 (p = 0.043). Preservation significantly increased the incidence and intensity of allograft arteriosclerosis (p < 0.05) and cardiac fibrosis (p = 0.003) at 8 weeks. CONCLUSION Our results demonstrate that preservation injury induced a cascade leading to an innate immune response that modulated the adaptive immune response towards Th17 rather than Th1 T-cell response in rat cardiac allografts and ultimately enhanced cardiac fibrosis and arterial occlusion. Our results also suggest that this immune response was not regulated by the calcineurin inhibitor cyclosporine A.