Roope Sihvola

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.

CYTOMEGALOVIRUS INFECTION AND CARDIAC ALLOGRAFT VASCULOPATHY

There is a wealth of clinical and experimental evidence indicating the interaction of cytomegalovirus (CMV) infection and rejection in cardiac and other solid organ allografts. A plausible explanation for this association comes from data showing that therapy with biologicals, sepsis, and rejection, all lead to the release of TNF‐α which, upon binding to its receptor, activates NF‐kB. TNF‐α is also able to stimulate the activity of the CMV‐IE enhancer/promoter region. CMV infection of several cell lines leads to NF‐kB activation. NF‐kB binding sites are present in regulatory regions of various cellular and viral genes, including the IE enhancer region of CMV. In a reciprocal situation, CMV infection, most likely via γ‐interferon, leads to upregulation of MHC antigens in the transplant and, thereby, to increased transplant immunogenicity. Thus, a vicious circle is induced. We have investigated in detail the pathobiology of CMV and allograft vasculopathy (chronic rejection) in experimental animals, using aortic and cardiac allografts as well as a trachea model. The results may be summarized as follows: Infection of the recipient with rat CMV results in an early inflammatory response in the aortic and cardiac allograft vascular adventitia and intima (endothelialitis) and in the airway wall of tracheal allografts. This early inflammatory response leads to enhanced intimal thickness in aortic and cardiac allografts and enhanced luminal occlusion of tracheal allografts. Timewise, this coincides with early activation of intragraft inflammatory leukocytes and increased mRNA of various growth factors and cytokines. When the recipients receive gancyclovir, the enhanced intimal response in aortic and cardiac allografts and luminal occlusion in tracheal allografts is entirely abolished. Gancyclovir treatment dramatically reduces the inflammatory response in the allograft, and thereby growth factor synthesis in response to injury. However, gancyclovir does not prevent the expression of IE antigen of CMV, suggested to inactivate tumor suppressor protein p53 predisposing smooth muscle cells to increased growth. Taken together, the effect of CMV infection on cardiac allograft dysfunction is bidirectional and biphasic. The bidirectional nature emerges from the observations that acute CMV infection may accelerate acute rejection, and, on the other hand, acute alloimmune response‐associated cytokine response may activate latent CMV infection. The biphasic effect of CMV on allograft dysfunction refers to its early and late detrimental effects, i.e. during the time of acute and chronic rejection. These two effects of CMV on allograft dysfunction emphasize the need for precise diagnosis of CMV infection in transplant recipients and pre‐emptive or prophylactic anti‐viral therapy. The benefits of this strategy may not be evident during the early post‐transplant period, but 5–10 years after transplantation they manifest as better graft survival Note .

Prevention of Cardiac Allograft Arteriosclerosis by Protein Tyrosine Kinase Inhibitor Selective for Platelet-Derived Growth Factor Receptor

BACKGROUND Increased immunoreactivity of platelet-derived growth factor (PDGF)-AA, -Ralpha, and -Rbeta in intimal cells correlates with the development of cardiac allograft arteriosclerosis, a condition for which there is little or no current therapy. Therefore, we hypothesized that PDGF may have a rate-limiting role in the development of this disease. METHODS AND RESULTS The hypothesis was tested in a rat model of heterotopic cardiac and aortic allografts using dark agouti (AG-B4, RT1(a)) donors and Wistar-Furth (AG-B2, RT1(u)) recipients. The recipients received CGP 53716, a selective PDGF-R protein tyrosine kinase inhibitor, 50 mg. kg-1. d-1, or vehicle for 60 days. Cardiac allograft recipients also received background cyclosporin A immunosuppression. Our results demonstrate that CGP 53716 significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac and aortic allograft recipients. When rat coronary smooth muscle cells were stimulated in vitro with PDGF-AA or -BB in the presence of interleukin-1beta or tumor necrosis factor-alpha, CGP 53716 significantly inhibited only AA-ligand-induced but not BB-ligand-induced replication. Concomitantly, in quantitative reverse transcriptase-polymerase chain reaction, interleukin-1beta or tumor necrosis factor-alpha stimulation specifically upregulated the expression of PDGF-Ralpha mRNA but not of other ligand or receptor genes in cultured smooth muscle cells. CONCLUSIONS We conclude that a PDGF-AA/Ralpha-dependent cycle is induced in the generation of allograft arteriosclerosis that may be inhibited by blocking of signaling downstream of PDGF-R.

Cytomegalovirus Infection–Enhanced Cardiac Allograft Vasculopathy Is Abolished by DHPG Prophylaxis in the Rat

BACKGROUND A wealth of clinical and experimental evidence exists for cytomegalovirus (CMV) infection as an accelerating factor in the development of cardiac allograft vasculopathy. In this study, the impact of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) on rat CMV infection-enhanced cardiac allograft vasculopathy is investigated. METHODS AND RESULTS Heterotopic rat cardiac allografts were performed from the DA to the WF rat strain, and the recipients were immunosuppressed with cyclosporine A 2 mg.kg-1.d-1 s.c. for a period of 90 days until the end of experiment. Two groups of recipients were infected intraperitoneally with 10(5) plaque-forming units of rat CMV, whereas one group was left noninfected and used as controls. One group of rat CMV-infected rats was treated with DHPG with an initial dose of 20 mg/kg i.p. and a maintenance dose of 10 mg/kg i.p. twice a day from 1 day before transplantation to 30 days after transplantation. Compared with noninfected rats, rat CMV infection was associated with a significant increase in intimal thickening, from 0.68 +/- 0.10 to 1.30 +/- 0.12 score units (P < .01), and double the number of vessels affected (P < .01). DHPG treatment significantly reduced intimal thickening in rat CMV-infected rats, from 1.30 +/- 0.12 to 0.68 +/- 0.13 score units (P < .01), and halved the number of vessels affected (P < .01). CONCLUSIONS The present results demonstrate that DHPG prophylaxis entirely abolishes the accelerating effect of rat CMV infection on cardiac allograft vasculopathy in immunosuppressed rat recipients, which is consistent with our earlier findings demonstrating a similar effect in nonimmunosuppressed rat aortic allografts. Taken together, these results suggest that DHPG might be useful in the prevention of CMV-accelerated cardiac allograft vasculopathy among heart transplant recipients.

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.

VEGFR-1 and -2 Regulate Inflammation, Myocardial Angiogenesis, and Arteriosclerosis in Chronically Rejecting Cardiac Allografts

Objective—Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. Methods and Results—Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow– or non–bone marrow–derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. Conclusions—These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

Crosstalk of endothelin-1 and platelet-derived growth factor in cardiac allograft arteriosclerosis

OBJECTIVES In this study, we investigated the crosstalk of endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) in coronary artery smooth muscle cell (SMC) proliferation in the rat cardiac allograft model. BACKGROUND Previous studies have suggested an independent role of ET-1 and PDGF in the development of cardiac allograft arteriosclerosis (i.e., chronic rejection). METHODS Heterotopic heart transplantations were performed from Dark Agouti to Wistar Furth rats. Grafts were harvested after five days in an acute rejection model and after 60 days in a chronic rejection model. In the in vitro part of the study, SMC proliferation and migration were quantitated, as well as messenger ribonucleic acid (mRNA) levels of ET-1 and PDGF ligands and receptors after growth factor stimulation. RESULTS Acute rejection induced both ET-1 receptors in the arterial wall. On linear regression analysis of chronically rejecting cardiac allografts, a strong correlation between intimal thickening and immunoreactivity of ET-1 and ET receptors A and B (ET(A) and ET(B)) in the arterial walls was observed. Treatment with Bosentan, a mixed ET-1 receptor antagonist, significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac allografts, as well as total intragraft ET(A) and ET(B) mRNA expression and intimal cell ET-1 and receptor immunoreactivity. This was associated with significantly reduced intragraft PDGF beta-receptor (PDGF-Rbeta) mRNA expression. In contrast, CGP 53716, a protein tyrosine kinase inhibitor selective for the PDGF receptor, did not reduce intragraft ET-1, ET(A) or ET(B) mRNA expression. In rat coronary artery SMC cultures, ET-1 stimulation significantly upregulated PDGF-Ralpha and -Rbeta mRNA expression and augmented PDGF-BB-mediated SMC proliferation as well as PDGF-AB- and PDGF-BB-mediated SMC migration. CONCLUSIONS Our results suggest that the ET-1/PDGF-Rbeta/PDGF-BB axis may operate in SMC migration and proliferation in cardiac allograft arteriosclerosis, thus explaining the marked beneficial effects of blocking the signaling downstream of ET-1 receptors.

Prevention of cardiac allograft arteriosclerosis by protein-tyrosine kinase inhibitor selective for platelet-derived growth factor receptor

Background—Increased immunoreactivity of platelet-derived growth factor (PDGF)-AA, -R a and -Rb in intimal cells correlates with the development of cardiac allograft arteriosclerosis, a condition for which there is little or no current therapy. Therefore, we hypothesized that PDGF may have a rate-limiting role in the development of this disease. Methods and Results—The hypothesis was tested in a rat model of heterotopic cardiac and aortic allografts using dark agouti (AG-B4, RT1) donors and Wistar-Furth (AG-B2, RT1 ) recipients. The recipients received CGP 53716, a selective PDGF-R protein tyrosine kinase inhibitor, 50 mg z k 21 z d, or vehicle for 60 days. Cardiac allograft recipients also received background cyclosporin A immunosuppression. Our results demonstrate that CGP 53716 significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac and aortic allograft recipients. When rat coronary smooth muscle cells were stimulated in vitro with PDGF-AA or -BB in the presence of interleukin-1 b or tumor necrosis factora, CGP 53716 significantly inhibited only AA-ligand–induced but not BB-ligand–induced replication. Concomitantly, in quantitative reverse transcriptase–polymerase chain reaction, interleukin-1 b or tumor necrosis factor-a stimulation specifically upregulated the expression of PDGF-R a mRNA but not of other ligand or receptor genes in cultured smooth muscle cells. Conclusions—We conclude that a PDGF-AA/R a–dependent cycle is induced in the generation of allograft arteriosclerosis that may be inhibited by blocking of signaling downstream of PDGF-R. (Circulation. 1999;99:2295-2301.)

Angiogenic growth factors in cardiac allograft rejection.

Normal adult vasculature is in a quiescent state. In transplanted hearts, peri- and postoperative ischemic and alloimmune stimuli may be interpreted as inadequate tissue perfusion leading to activation of angiogenic signaling. Although this may have protective functions, improper activation of cardiac allograft endothelial cells and smooth muscle cells may actually result in impaired survival of cardiac allografts. In this paper, we review the current knowledge on angiogenic growth factors, vascular endothelial growth factor, angiopoietins, and platelet-derived growth factor in cardiac allografts. We also discuss the potential for therapies aimed at angiogenic growth factors in preventing and treating cardiac allograft rejection and transplant coronary artery disease.

Immunobiology of Chronic Cardiac Allograft Rejection

Cardiac transplantation is currently the only method available to return patients with end-stage heart disease to normal life. The success of intrathoracic organ transplantation has increased constantly over the past decade as a result of new surgical techniques, immunosuppressive protocols, and innovations in managing acute rejection and infection, particularly cytomegalovirus (CMV) infection. Despite the substantial improvement in early survival, the long-term survival rate has not increased during the past decade.