Heterotopic Rat Heart Transplantation Research Articles

Two good reasons for an angiotensin-II type 1 receptor blockade with losartan after cadiac transplantation: reduction of incidence and severity of transplant vasculopathy

Abstract Despite considerable progress in immunosupressive therapy, the incidence and severity of transplant vasculopathy (TVP) after cardiac transplantation have not declined. The renin-angiotensin system (RAS) plays a pivotal role in the proliferation of vascular smooth muscle cells (VSMCs) contributing to TVP. We compared the effects of an angiotensin-II blocker, losartan (AT1 blocker), and an angiotensin-converting enzyme (ACE) inhibitor, enalapril, on the incidence of diseased vessels and the severity of experimental TVP in the Lewis-to-Fischer rat heterotopic heart transplantation model. Recipients were randomly divided into six groups, group 1: no therapy, group 2: 3 mg/kg per day cyclosporine (CyA) s.c., group 3: CyA and 10 mg/kg per day losartan p.o., group 4: CyA and 40 mg/kg per day enalapril p.o., and groups 5 and 6: as groups 3 and 4, but additionally pre-treated with losartan or enalapril 7 days prior to transplantation. Eighty days after grafting, we assessed the incidence and severity of TVP, expressed as percentage of diseased vessels and mean vessel occlusion (MVO), by digitizing morphometry. CyA and CyA/enalapril post-treatmet significntly reduced MVO, compared with controls, but not the incidence. Additional reduction of MVO was achieved in CyA/enalapril pre-treatment and both CyA/losartan pre-and post-treatment groups when compared with CyA and untreated controls. However, only losaran post-treatment in combination with CyA reduced both incidence and MVO. Our reults validate the important role of the RAS in neointimal proliferation after cardiac transplantation. Losartan appears to be superior to enalapril in preventing TVP after experimental cardiac transplantation. Therefore, AT1 blockade with losartan might be a theraputic option for the prevention of TVP in human heart recipients.

Two good reasons for an angiotensin-II type 1 receptor blockade with losartan after cardiac transplantation: reduction of incidence and severity of transplant vasculopathy.

Despite considerable progress in immunosuppressive therapy, the incidence and severity of transplant vasculopathy (TVP) after cardiac transplantation have not declined. The renin-angiotensin system (RAS) plays a pivotal role in the proliferation of vascular smooth muscle cells (VSMCs) contributing to TVP. We compared the effects of an angiotensin-II blocker, losartan (AT(1) blocker), and an angiotensin-converting enzyme (ACE) inhibitor, enalapril, on the incidence of diseased vessels and the severity of experimental TVP in the Lewis-to-Fischer rat heterotopic heart transplantation model. Recipients were randomly divided into six groups, group 1: no therapy, group 2: 3 mg/kg per day cyclosporine (CyA) s.c., group 3: CyA and 10 mg/kg per day losartan p.o., group 4: CyA and 40 mg/kg per day enalapril p.o., and groups 5 and 6: as groups 3 and 4, but additionally pre-treated with losartan or enalapril 7 days prior to transplantation. Eighty days after grafting, we assessed the incidence and severity of TVP, expressed as percentage of diseased vessels and mean vessel occlusion (MVO), by digitizing morphometry. CyA and CyA/enalapril post-treatment significantly reduced MVO, compared with controls, but not the incidence. Additional reduction of MVO was achieved in CyA/enalapril pre-treatment and both CyA/losartan pre- and post-treatment groups when compared with CyA and untreated controls. However, only losartan post-treatment in combination with CyA reduced both incidence and MVO. Our results validate the important role of the RAS in neointimal proliferation after cardiac transplantation. Losartan appears to be superior to enalapril in preventing TVP after experimental cardiac transplantation. Therefore, AT(1) blockade with losartan might be a therapeutic option for the prevention of TVP in human heart recipients.

Reversal of diabetes-induced rat graft transplant coronary artery disease by metformin

Background Induction of diabetes in rat heterotopic heart transplantation models leads to an accelerated form of severe transplant coronary artery disease (TxCAD). We undertook this study to determine whether treatment of diabetes with metformin would favorably affect TxCAD. Methods Heterotopic abdominal heart transplantation was performed in rat isograft and allograft models. After transplantation, diabetes was induced with streptozotocin. Fifty percent of the animals received metformin at 500 mg/kg twice daily. We quantitatively assessed TxCAD using histologic sections of harvested hearts at 30 and 60 days with computer-assisted morphometry. We compared vessels in the first tertile of the area distribution with vessels in the last tertile. Results Fasting glucose levels in metformin-treated animals were 161 ± 45 mg/dl compared with 400 ± 120 mg/dl ( p < 0.05) in untreated rats. Treatment with metformin led to decreased diabetes-induced TxCAD in the larger vessels. This effect was sustained during the study course in the isografts but not in the allografts. Treatment with metformin did not prevent progression of TxCAD in the smaller vessels at 60 days. Conclusions Metformin reduced luminal occlusion and severe TxCAD in the larger vessels but did not alter the course of TxCAD in the smaller vessels. These results may have therapeutic implications for patients.

Short-course methotrexate and long-term acceptance of fully allogeneic rat cardiac grafts: a possible mechanism of tolerance

Although a short course of methotrexate (MTX) has a potential immunoregulatory effect on clinical allograft rejection, little data are available about the drug, and the mechanism of hyporeactivity after withdrawal is still unknown. In previous studies, we achieved permanent graft acceptance through administration of a short course of high-dose MTX during heterotopic rat heart transplantation (HHT) in a combination of DA (MHC haplotype; RT1 a) to PVG/c (RT1 c) rats. A 3-week course of MTX (0.25 mg/kg/day) was administered intraperitoneally to the PVG/c recipients of a DA heart graft, and 11 of 16 rats survived longer than 300 days after HHT. The splenic lymphocytes obtained from one recipient showed high reactivity against donor type splenic lymphocytes, but others did not. All serum samples from recipients showed immunosuppressive activity. The serum had anti-donor antibodies. These results showed that tolerance induced by short-course MTX was maintained by a serologic factor believed to be anti-idiotypic antibodies.

A novel small animal model of left ventricular tissue engineering

Background: Complex congenital cardiac anomalies involving ventricular hypoplasia require either staged palliative reconstruction, converting the circulatory system to a single ventricle based pump, or allogeneic transplantation. Tissue engineering offers the potential for complete reconstruction of these defects, but is limited by the inability to model myocardial tissue engineering in a small animal. Our goal was to develop a small animal model for ventricular tissue engineering using rat heterotopic heart transplantation. Methods: Donor hearts were explanted after cardioplegic arrest and the left ventricular volume was augmented by the implantation of a biodegradable engineered construct. The heart was then transplanted heterotopically into syngeneic recipients creating either a volume loaded, functioning left ventricle, or a non-functioning left ventricle. Some of the engineered constructs were seeded with multipotent bone marrow-derived mesenchymal progenitor cells before implantation. Animals were evaluated by echocardiography, morphology, histology, and immunohistochemistry after 1 month. Results: A scaffolding constructed from polytetrafluoroethylene, polylactide mesh, and type I and IV collagen hydrogel resulted in minimal intracardiac inflammation without aneurysmal dilatation. Successful transplantation and differentiation of mesenchymal progenitor cells was accomplished using this scaffolding. No ventricular arrhythmias resulted from this surgical manipulation and echocardiography revealed both end systolic and diastolic volume augmentation with ventricular expansion. Conclusion: We have developed an in vivo model of ventricular tissue engineering using heterotopic heart transplantation. Future work will focus on construction of ventricular tissue around pre-fabricated vascular networks in order increase cellular engraftment for ventricular reconstruction.

Poly(ADP-Ribose) polymerase inhibition reduces reperfusion injury after heart transplantation.

The aim of the present study was to investigate the effects of the novel poly(ADP-ribose) polymerase (PARP) inhibitor PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) on myocardial and endothelial function after hypothermic ischemia and reperfusion in a heterotopic rat heart transplantation model. After a 1-hour ischemic preservation, reperfusion was started either after application of placebo or PJ34 (3 mg/kg). The assessment of left ventricular pressure-volume relations, total coronary blood flow, endothelial function, myocardial high energy phosphates, and histological analysis were performed at 1 and 24 hours of reperfusion. After 1 hour, myocardial contractility and relaxation, coronary blood flow, and endothelial function were significantly improved and myocardial high energy phosphate content was preserved in the PJ34-treated animals. Improved transplant function was also seen with treatment with another, structurally different PARP inhibitor, 5-aminoisoquinoline. The PARP inhibitors did not affect baseline cardiac function. Immunohistological staining confirmed that PJ34 prevented the activation of PARP in the transplanted hearts. The activation of P-selectin and ICAM-1 was significantly elevated in the vehicle-treated heart transplantation group. Thus, pharmacological PARP inhibition reduces reperfusion injury after heart transplantation due to prevention of energy depletion and downregulation of adhesion molecules and exerts a beneficial effect against reperfusion-induced graft coronary endothelial dysfunction.

Chemokines and allograft rejection: narrowing the list of suspects.

Chemokines and allograft rejection: narrowing the list of suspects.

Photodynamic therapy with motexafin lutetium (Lu-Tex) reduces experimental graft coronary artery disease.

Motexafin lutetium (Lu-Tex) is a photodynamic therapy (PDT) agent that localizes in atheromatous plaque in which it can be activated by far-red light. Lu-Tex biolocalization was examined in graft coronary artery disease (GCAD) with a rodent allograft model. After photoactivation, the effect on intimal proliferation was assessed. A PVG to ACI rat heterotopic heart transplantation model was used. Lu-Tex (10 mg/kg) was intravenously administered 90 days after transplantation. Photoactivation was performed 24 hr after Lu-Tex administration. A light-emitting diode, central wavelength of 742 nm, was used to illuminate the intraperitoneally placed allografts via a laparotomy (light fluence of 75 J/cm2 at a power density of 75 mW/cm2). Animals were divided into four groups according to postoperative treatments: PDT with Lu-Tex injection and light illumination (n=21), Lu-Tex injection and laparotomy (n=14), laparotomy with light only (n=14), and laparotomy only (n=16). GCAD was quantitatively assessed 14 days after treatments. Lu-Tex localized in atherosclerotic plaque in vessels with GCAD. PDT significantly reduced both the percent of affected vessels and intimal proliferation compared to all other control study groups. alpha-Smooth muscle cell actin and anti-rat macrophage antibody-positive areas were significantly reduced within the neointima in allografts treated with PDT compared to all other study groups. PDT significantly reduced atherosclerotic lesions of GCAD. Lu-Tex-mediated PDT may, therefore, be a potential method for treating accelerated atherosclerosis associated with transplantation.

A murine model of left ventricular tissue engineering

Purpose: Donor organ shortage has severely limited widespread availability of heart transplantation. In vivo efforts to engineer myocardium have been limited by the high morbidity associated with manipulation of the native heart. Our purpose is to create a model of ventricular tissue engineering through a modification of the rat heterotopic heart transplant.

A Non-peptide Functional Antagonist of the CCR1 Chemokine Receptor Is Effective in Rat Heart Transplant Rejection

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Monitoring of intragraft pressure of rejecting organs: increased tissue pressure can be reduced by hyaluronidase therapy.

The present study was undertaken in order to: (a) develop a new technique for measurement of interstitial pressure, (b) study the intragraft pressure of rejecting and non-rejecting organs, and (c) study the effect of treatment with the hyaluronan-degrading enzyme hyaluronidase on intragraft pressure. Treatment with hyaluronidase has previously been demonstrated to result not only in reduction of tissue hyaluronan but also in ameliorated interstitial edema, and we suggested that the diminished edema would lead to a reduced interstitial pressure as well. At day 5 after syngeneic or allogeneic rat heterotopic heart transplantation, the interstitial pressure of the cardiac grafts was measured using a microtip pressure sensor. Subsequently, the allogeneically grafted animals received a continuous intravenous infusion of either hyaluronidase (total dose: 60,000 U/kg) or vehicle during 2 hr; meanwhile, the interstitial pressure was monitored. The intragraft pressure measurement technique was found to give reproducible results. The interstitial pressure of the rejecting (allogeneic) grafts was considerably higher than that of the non-rejecting (syngeneic), i.e., 12.3+/-1.6 mmHg vs. 1.1+/-0.6 mmHg (P<0.001). Hyaluronidase infusion effectively reduced the interstitial pressure as compared with vehicle treatment. By 20 min, the pressure had been reduced by 28% (P<0.01 compared with vehicle treatment); after 1 hr, by 49% (P<0.001); and after 2 hr, by 68% (P<0.01). By using modern technology for tissue pressure measurements, we found that the strongly increased interstitial pressure of rejecting organs can be instantly reduced by intravenous administration of the hyaluronan-degrading enzyme hyaluronidase.

The relationship between acute rejection and chronic rejection is highly dependent on specific MHC matching: a multi-strain rat heterotopic heart transplant study.

The impact of acute rejection, immunosuppression, and infection, specifically cytomegalovirus infection, on the development of chronic rejection in the cardiac allograft, has been the subject of a large number of investigations. One of the difficulties in finding associations has been the marked immunologic heterogeneity of the patient population coupled with the lack of the ability to HLA match. Furthermore, the ideal animal model, which duplicates as well as controls for this immunologic heterogeneity, is lacking. To try to simulate differences in HLA matching, immunosuppression regiments and cytomegalovirus infection, heterotopic heart transplantation was performed in two separate, complete MHC mismatch, rat strain combinations (WF-LEW, BN-LEW) requiring chronic immunosuppression and employing four separate immunosuppression/infection protocols. Animals were followed for 6 months, killed, and rejection and vascular changes were scored blinded to the group. The mean vascular and acute rejection scores were not significantly different between treatment regiments for either specific strain combination. There was a trend for the subtherapeutic groups to have higher vascular scores. Overall, there were no significant differences in vascular scores between the WF-LEW and BN-LEW groups (1.25+/-0.18 vs. 1.13+/-0.20, P=NS). Similar numbers of WF-LEW and BN-LEW exhibited cellular infiltration and necrosis of the allograft, but the intensity of the response (rejection score) was more severe in the WF-LEW combination (4.54+/-0.22 vs. 3.92+/-0.21, P=0.052) when limiting the analysis to those with myocyte necrosis. There was no significant correlation between acute rejection and vascular lesion severity in the WF-LEW combination (r=0.22, P=NS) but a high correlation between these parameters in the BN-LEW combination (r=0.74, P<0.0001). These data suggest that, although acute rejection and chronic rejection are related, MHC matching may influence their interdependence. These data also may explain why the clinical association between acute and chronic rejection is difficult to demonstrate.

Modified Technique for Heterotopic Rat Heart Transplantation Under Cardioplegic Arrest

The development of microsurgical techniques offers a valuable opportunity to use small animals for experimental studies of vascularized organ transplants. Availability of inbred strains, natural resistance to infection, and economy make the rat an ideal animal model to investigate the effects of heart transplantation. The recent high interest and substantial laboratory activity with regard to posttransplantory immunological tissue reactions and apoptotic tissue processes led us to optimize transplantation technique by improving myocardial protection during ischemia and thereby minimizing adverse effects of the transplantation procedure itself. Thus the present report details the technique of heterotopic heart transplantation in rats using cardioplegic arrest.

Coronary artery disease after heart transplantation and its relation to cytomegalovirus

The most common cause of death and retransplantation after heart transplantation is a rapidly progressive, obliterative vascular disease involving the coronary arteries, termed cardiac allograft vasculopathy (CAV). Most believe that this is a form of chronic rejection. Several clinical series have suggested an association between cytomegalovirus and CAV. Rat cytomegalovirus enhances the development of CAV in rat heterotopic heart or aortic transplantation models. The mechanism(s) by which cytomegalovirus might have an impact on the severity of chronic rejection include the augmentation of vascular growth factors, the alteration in the alloimmune response directly or the alteration of cytokines and cell adhesion molecules, enhancing cellular and humoral interactions. We previously reported that the infection of smooth muscle cells by cytomegalovirus resulted in the alteration of major histocompatibility complex class I molecules on the smooth muscle cell surface. In a subsequent report we demonstrated that a sublethal inoculum of cytomegalovirus produced no cytopathology in smooth muscle cells yet had the same viral burden as fibroblasts, which demonstrated cytopathology. The identical effects on major histocompatibility complex class I were observed in smooth muscle cells, and cytokine gene transcription was altered, favoring a proinflammatory milieu. These and most in vitro studies are carried out with the use of traditional laboratory strains of cytomegalovirus. We have subsequently demonstrated major genotypic differences between laboratory and clinical strains of cytomegalovirus that are associated in differences in biological activity in vitro. These include differences in tropism for vascular cells, differences in cell surface antigen expression, and differences in mesenchymal growth factor gene expression. All of these may have important implications with regard to associating cytomegalovirus with CAV. (Am Heart J 1999;138:S469-S472.)

Sulfasalazine inhibits reperfusion injury and prolongs allograft survival in rat cardiac transplants

Introduction: Reperfusion injury is an inflammatory cell-mediated response that causes tissue damage immediately following transplantation, and has been implicated in the development of acute and chronic rejection. NF-κB is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1 following reperfusion. We hypothesized that treatment with sulfasalazine, a potent inhibitor of NF-κB, would decrease adhesion molecule expression, decrease reperfusion injury, and prolong allograft survival in rat cardiac transplants. Methods Heterotopic rat heart transplants were performed. Donor allografts were treated with saline, sulfasalazine (SSA), or lipopolysaccharide (LPS), a potent inducer of NF-κB activity. Reperfusion injury was assessed with cardiac edema (percent wet weight), neutrophil infiltration (MPO activity), and histologic damage (contraction band necrosis). Immunohistochemistry was performed to analyze protein expression. Acute rejection was determined by daily palpation. Results Treatment with a single 100 mg/kg intraperitoneal injection of sulfasalazine decreased reperfusion injury compared to saline controls (MPO activity, saline: 2.1 ± 0.3, SSA: 1.2 ± 0.31, P < 0.005; % wet weight, saline 77.6 ± 1.1%; SSA 75.8 ± 1.0%, P < 0.005; contraction band necrosis, saline: 13.1 ± 2.5%, SSA: 6.1 ± 3.4%, P < 0.001). LPS administration increased all parameters of reperfusion injury. Treatment with sulfasalazine prior to LPS also decreased reperfusion injury compared to LPS and saline groups. Sulfasalazine treatment decreased ICAM-1 and VCAM-1 protein expression. Administration of 500 mg/kg sulfasalazine increased graft survival to 15.4 ± 1.8 days compared to saline (6.8 ± 1.4 days, P < 0.005). Conclusion Treatment with sulfasalazine is an effective method to decrease reperfusion injury and prolong allograft survival in a rat cardiac transplantation model.

A simple new model of physiologically working heterotopic rat heart transplantation provides hemodynamic performance equivalent to that of an orthotopic heart

Background T he widely used non–volume-loaded abdominal heterotopic heart transplant (NL) in rats undergoes atrophy after transplantation. Various techniques have been designed to load the transplanted heart because of its potential immunological impact. Our aim was to create a volume-loaded heterotopic heart transplantation model (VL) capable of ejection and practical for routine studies. Using this model, we tested the hypothesis that VL isografts would retain myocardial performance comparable to native hearts (NH). Methods Heterotopic hearts were transplanted using and end-to-side anastomosis between the donor’s superior vena cava and the recipient’s abdominal inferior vena cava. The right ventricle loads the left ventricle (LV) via a direct anastomosis of the pulmonary artery to the left atrium. The LV ejects volume through an end-to-side anastomosis of the donor’s aorta to the recipient’s abdominal aorta. Hemodynamic data (systolic and diastolic LV pressures, dP/dt max and min, tau) were studied in-situ (at baseline and after adding volume) and in a Langendorff perfusion system (at baseline and after stimulation with isoproterenol) 2 weeks after transplantation. Results In situ systolic pressure and diastolic function of VL was superior to NL, and β-adrenergic stimulated performance in the Langendorff perfusion of VL showed hemodynamic performance equivalent to NH, unlike NL which had a diminished response. Conclusion This technique results in a volume-loaded ejecting heart transplant model that preserves anatomical structures. The VL can be evaluated in situ and after explantation in Langendorff perfusion system and may offer advantages if workload of the graft is of significance to the study performed.

Atrophy of Myocardium and Its Myocytes by Left Ventricular Assist Device

To the Editor: Dipla and coworkers1 believe that their data support the notion that a temporary left ventricular assist device (LVAD) obviates the need of cardiac transplantation of the failing heart. Barry2 supports this notion. The LVAD is a double-edged sword. The beneficial effects of this device are well known. Its action ensures sufficient nutrition to the peripheral tissues so that they can recover from starvation and also eliminates or decreases the harmful effects of the increased catabolic products produced by the starved tissues. Harmful effects of the LVAD are due to the rapid, profound decrease of cardiac work (contraction) that leads to atrophy of the myocardium and its myocytes. An excellent description of the morphological changes that occur in myocardial atrophy is present in Bargmann and Doerr’s textbook.3 Modern textbooks of medicine and cardiology ignore this subject, with the exception of Braunwald’s,4 which devotes a single sentence published in fine print to it. This is as …

Adenovirus-mediated gene transfer of the beta2-adrenergic receptor to donor hearts enhances cardiac function.

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of beta2-adrenergic receptors (beta2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human beta2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the beta2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the beta2-AR agonist, zinterol. Treatment with the beta2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean beta-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 +/- 286 versus beta2-AR, 6250.6* +/- 432.5 mmHg/s; n = 10, *P < 0.02). beta2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of beta2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

ORGAN PRESERVATION BY CURCUMIN+EURO-COLLINS IS EQUIVALENT TO UNIV. OF WISCONSIN SOLUTION IN BOTH IN-VIVO AND EX-VIVO MODELS

440 Curcumin (CUR), a component of the spice turmeric, enhances hypothermic organ preservation. Here we demonstrate both this enhancement for Euro-Collins (EC) solution in both an ex-vivo model of liver preservation and in-vivo model of heterotopic heart transplantation, making EC equivalent to University of Wisconsin (UW) solution in both models. Ex-Vivo: Sprague-Dawley rat livers underwent oxygenated perfusion after being flushed with different preservation solutions: UW or EC±100 μM CUR solution and stored at 4°C for 24 hours (10 in each group). The livers in the normal (NL) group (10) were immediately perfused. The livers were then perfused for 120′ via the portal vein with 37°C oxygenated Krebs-Henseleit solution at 18 cm H2O; flow rates, measured serially and shown here as the maximal rates (cc/gm-min). Bile production rates (μl/gm-min) and liver enzyme (AST, ALT-U/gm) release into the perfusate were also measured over the 120 minute perfusion. (Table 1)Table 1In-Vivo: To study the in-vivo effects of CUR in a vascularized transplant, we used a well-established heterotopic rat heart transplant model. Following procurement, rat hearts were flushed with either EC or UW solution alone or EC or UW solution containing 80 mg/l CUR. Each group was stored at 4°C for 12 (6 per group) or 18 (10 per group) hours, then transplanted into another rat using standard microsurgical techniques. Graft function was evaluated daily by palpation. Because the transplants were performed between animals of the same strain, no immunosuppression was used. Results: long-term (>7 days) graft survival; §p<0.05 vs. EC, T-test (Student) (Table 2)Table 2In both the ex-vivo perfusion model and the in-vivo transplant model, the addition of CUR to EC enhanced its organ preserving properties, making it the equivalent of UW.

A NEW SYNTHETIC VLA-4 BLOCKER PREVENTS DEVELOPMENT OF CHRONIC REJECTION IN A RAT CARDIAC TRANSPLANT MODEL

197 Introduction: Chronic rejection after cardiac transplantation leads to neointimal proliferation of small and large arteries in the allograft and limits long-term survival. The pathology of this disease is still poorly understood and different factors contribute to the development of neointimal inflammation and the proliferation of vascular smooth muscle cells. VLA-4, a β1-integrin, interacts with VCAM-1, a member of the immunoglobuline superfamily, and with the extracellular matrix molecule fibronectin. VCAM-1 and fibronectin are upregulated during the development of chronic rejection. By the application of monoclonal antibodies to VCAM-1 chronic rejection was diminished. The objective of this study was to evaluate the antiproliferative effect of a new, synthetic, soluble VLA-4 blocker on the development of chronic rejection after heterotopic rat heart transplantation. Methods: 42 rat hearts were transplanted from Lewis to Fisher rats and randomly divided into 3 groups. Group 1, the control group, received no treatment, group 2 received 3 mg/kg/d/s.c. Cyclosporine A (CsA) and group 3 was treated with a combination of 3 mg/kg/d/s.c. CsA and 10 mg/kg/d/s.c. VLA-4 blocker. 20 and 80 days after grafting the extent of neointimal proliferation in arteries was assessed by digitizing morphometry in paraffin embedded specimens. Results: 20 days after transplantation neointimal proliferation in the control group reached 73.5% ± 18.8%, in the CsA group 41% ± 16.7% (p<.05) In the CsA + VLA-4 blocker group the neointimal proliferation was reduced to 17.9% ± 11.3% (p<.05 compared with control and CsA). 80 days after transplantation neointimal proliferation in the control group was determined at 87.5% ± 1.2%, compared with 46.7% ± 4.7% in the CsA group (p<.05). In the CsA + VLA-4 blocker group the neointimal proliferation was reduced to 24.4% ± 9.9% (p<.05 compared with control and CsA). Conclusion: Interactions between adhesion molecules are essential steps in allograft recognition and rejection. ICAM-1 and VCAM-1 mediate the binding of T-lymphocytes to activated endothelia via the lymphocyte function associated antigen (LFA-1) and the very late antigen 4 (VLA-4). Our results suggest that interactions of VLA-4 with VCAM-1 and fibronectin play an important role in the pathology of chronic rejection after cardiac transplantation and that neointimal proliferation may successfully be reduced by the additional treatment with the new VLA-4 blocker.