Rat Aortic Allografts Research Articles

TAK1 accelerates transplant arteriosclerosis in rat aortic allografts by inducing autophagy in vascular smooth muscle cells

Background and aimsThe proliferation and migration of vascular smooth muscle cells (VSMCs) are fundamental hallmarks of vasculopathy. Transforming growth factor β-activated kinase-1 (TAK1) plays a crucial role in mediating cellular functions, including autophagy, which has been recently linked to the regulation of VSMC functions and the development of vasculopathy. This study aims to better dissect how TAK1 controls VSMC proliferation and migration. MethodsA rat model of graft arteriosclerosis was employed to explore the influence of TAK1 signaling activation on VSMC proliferation, migration, autophagy, and neointima formation in vivo. Knockdown and pharmacological inhibition of TAK1 were utilized in cultured VSMCs to investigate the mechanisms underlying the progression of VSMC proliferation and migration. ResultsIncreased phosphorylation of TAK1 (Thr-184/Thr-187) was examined in SMα-actin positive cells in the medial and neointimal lesions of aortic allografts. Lentivirus-mediated Tak1 shRNA transfection of aortic allografts robustly suppressed neointimal formation and lumen stenosis, as well as autophagy and cell proliferative responses. In cultured PDGF-BB-incubated VSMCs, genetic and pharmacological inhibition of TAK1 markedly attenuated autophagy activation, and blocked the progression of cell cycle, proliferation, and migration responses. ConclusionsActivation of TAK1 in VSMCs in the setting of aortic transplantation is an early and critical event in VSMC proliferation and migration, as well as neointima formation, because it controls autophagy activation, constituting a potential molecular mechanism and target for preventing transplant vasculopathy.

Use of paclitaxel carried in lipid nanoparticles to treat aortic allograft transplantation in rats.

The aim of this study was to test whether lipid core nanoparticles loaded with paclitaxel (LDE-PTX) protect rat aortic allograft from immunological damage. Fisher and Lewis rats were used differing in minor histocompatibility loci. Sixteen Lewis rats were allocated to four-animal groups: SYNG (syngeneic), Lewis rats receiving aorta grafts from Lewis rats; ALLO (allogeneic), Lewis rats receiving aortas from Fisher rats; ALLO+LDE (allogeneic transplant treated with LDE), Lewis rats receiving aortas from Fisher rats, treated with LDE (weekly injection for 3 weeks); ALLO+LDE-PTX (allogeneic transplant treated with LDE-PTX), Lewis rats receiving aortas from Fisher rats treated with LDE-PTX (4 mg/kg weekly for 3 weeks). Treatments began on transplantation day. Thirty days post-transplantation, SYNG showed intact aortas. ALLO and ALLO+LDE presented intense neointimal formation. In ALLO+LDE-PTX, treatment inhibited neointimal formation; narrowing of aortic lumen was prevented in ALLO and ALLO+LDE. LDE-PTX strongly inhibited proliferation and intimal invasion by smooth muscle cells, diminished 4-fold presence of apoptotic/dead cells in the intima, reduced the invasion of aorta by macrophages and T-cells and gene expression of pro-inflammatory tumour necrosis factor-alpha (TNFα), interferon gamma (IFNγ) and interleukin-1 beta (IL-1β). LDE-PTX was effective in preventing the vasculopathy associated with rejection and may offer a potent therapeutic tool for post-transplantation.

Pharmacological Inhibition of Vanin Activity Attenuates Transplant Vasculopathy in Rat Aortic Allografts.

Development of transplant vasculopathy is a major cause of graft loss and mortality in solid organ transplant recipients. Previous studies in mice have indicated that vanin-1, a member of the vanin protein family with pantetheinase activity, is possibly involved in neointima formation. Here, we investigated if RR6, a recently developed vanin inhibitor, could attenuate development of transplant vasculopathy. Abdominal allogeneic aorta transplantation from Dark Agouti to Brown Norway rats was performed. Surface neointima was quantified 2 and 4 weeks after transplantation. Systemic vanin activity was measured, and allograft leukocyte infiltration, glutathione-synthesizing capacity, matrix metalloproteinase 9 expression and neointimal smooth muscle cell (SMC) proliferation were assessed by immunohistochemistry. In vitro, the effects of RR6 on SMC proliferation (water-soluble tetrazolium-1 assay) and cytokine-induced apoptosis (flow cytometry) were investigated. RR6 treatment significantly reduced systemic pantetheinase activity during the 4-week follow-up period. RR6 attenuated neointima formation 4 weeks after transplantation. Neointimal SMC proliferation and medial SMC matrix metalloproteinase 9 expression were not altered by RR6. However, RR6 significantly reduced neointimal macrophage influx that was accompanied by increased GCLC messenger RNA expression. In vitro, RR6 inhibited platelet-derived growth factor-induced SMC proliferation and protected SMCs from TNF-α-induced apoptosis. Pharmacological inhibition of vanin activity attenuates development of transplant vasculopathy. This was accompanied by reduced macrophage infiltration and increased glutathione-synthesizing capacity. In vitro, RR6 reduced SMC proliferation and apoptosis that was not confirmed in vivo. Further in-depth studies are warranted to reveal the underlying mechanism(s) of RR6-induced attenuation of transplant vasculopathy in vivo.

Endothelial Cell Apoptosis Induces TGF-β Signaling-Dependent Host Endothelial-Mesenchymal Transition to Promote Transplant Arteriosclerosis.

Endothelial cells (ECs) apoptosis is an initial event in transplant arteriosclerosis (TA), resulting in allograft function loss. To elucidate the precise mechanisms of ECs apoptosis leading to neointimal smooth muscle cells (SMCs) accumulation during TA. We induced apoptosis in cultured ECs by overexpressing p53 through lentivirus-mediated transfection. ECs apoptosis induced the production of transforming growth factor (TGF)-β1 in both apoptotic and neighboring viable cells, leading to increased TGF-β1 in the culture media. Conditioned media from Ltv-p53-transfected ECs further promoted transition of cultured ECs to SM-like cells by activating TGF-β/Smad3, PI3K/Akt/mTOR, and MAPK/ERK signaling in a TGF-β-dependent manner. In transgenic rat aorta transplantation models, inhibition of ECs apoptosis in Bcl-xL(+/+) knock-in rat aortic allografts significantly reduced TGF-β1 production both in allograft endothelia and in blood plasma, which in turn decreased accumulation of SM22α+ cells from transgenic recipient ECs originally marked with EGFP knock-in in neointima and alleviated TA. Systemic treatment with SIS3, AP23573, or PD98059 also prevented recipient ECs-originated SM-like cells accumulation and intima hyperplasia in aortic allografts. These data suggest that allograft EC apoptosis induced recipient endothelial-mesenchymal (smooth muscle) transition via TGF-β signaling, resulting in recipient EC-derived SMC accumulation as a major mechanism of vascular remodeling during TA.

Adventitia and perivascular cells.

The past few years have seen significant advances in our understanding of the multiple and dynamic roles of the adventitia and its companion perivascular tissues for vessel wall homeostasis and disease. It is now becoming clear that signals originating from within the adventitia and from perivascular cells play essential roles in regulation of vascular development, physiology, artery wall remodeling, immune surveillance, and vascular disease.1–4 The adventitia is in contact with and completely surrounds the media and is the interface between the vessel wall and its neighboring tissues. It contains many different interacting cell types including fibroblasts, microvascular endothelium, nerves, resident macrophages, T cells, B cells, mast cells, and dendritic cells.5–9 The adventitia is also home to resident vascular progenitor cells whose formation and maintenance depend, in part, on sonic hedgehog signaling.1,10–12 Perivascular cells are in close contact with the adventitia, particularly for the aorta and coronary arteries. Perivascular tissue includes adipocytes, lymphatic vessels, perivascular nerves, and stromal cells exhibiting mesenchymal stem cell–like properties.3,13 The adventitia and periadventitial cells function in concert. They are linked by microvessels, nerves, and migratory cells to regulate vascular physiology, homeostasis, structural remodeling, and exert major influences on the progression or regression of vascular disease. Crosstalk between intima, media, and adventitia further links the adventitia-periadventitial unit to the rest of the vessel wall. Much of the work advancing our concepts of the adventitia and periadventitial tissues has been published in ATVB , and some of those key studies are discussed in this highlights article. In the following summary, areas that have attracted the most interest over the past 2 years in ATVB are reviewed including the multiple roles of perivascular adipose tissue (PVAT) on control of vascular physiology and remodeling, adventitial progenitor …

N-Octanoyl-Dopamine Attenuates Transplant Vasculopathy in Rat Aortic Allografts by Smooth Muscle Cell Preservation.

N-Octanoyl-Dopamine Attenuates Transplant Vasculopathy in Rat Aortic Allografts by Smooth Muscle Cell Preservation.

Monocyte Chemoattractant Protein 1–Mediated Migration of Mesenchymal Stem Cells Is a Source of Intimal Hyperplasia

Intimal hyperplasia is considered to be a healing response and is a major cause of vessel narrowing after injury, where migration of vascular progenitor cells contributes to pathological events, including transplant arteriosclerosis. In this study, we used a rat aortic-allograft model to identify the predominant cell types associated with transplant arteriosclerosis and to identify factors important in their recruitment into the graft. Transplantation of labeled adventitial tissues allowed us to identify the adventitia as a major source of cells migrating to the intima. RNA microarrays revealed a potential role for monocyte chemoattractant protein 1 (MCP-1), stromal cell-derived factor 1, regulated on activation, normal T cell expressed and secreted, and interferon-inducible protein 10 in the induced vasculopathy. MCP-1 induced migration of adventitial fibroblast cells. CCR2, the receptor for MCP-1, was coexpressed with CD90, CD44, NG2, or sca-1 on mesenchymal stem cells. In vivo experiments using MCP-1-deficient and CCR2-deficient mice confirmed an important role of MCP-1 in the formation of intimal hyperplasia in a mouse model of vascular injury. The adventitia is a potentially important cellular source that contributes to intimal hyperplasia, and MCP-1 is a potent chemokine for the recruitment of adventitial vascular progenitor cells to intimal lesions.

Cordyceps sinensis Extracts Attenuate Aortic Transplant Arteriosclerosis in Rats

Transplant arteriosclerosis is a hallmark of chronic rejection and is still the major limiting factor affecting the success of long-term organ transplants. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs, and adequate therapy is not yet available. The aim of this study was to determine the role of Cordyceps sinensis extracts in reducing the formation of transplant arteriosclerosis in a rat aortic transplant model. Lewis rat aortic allografts were transplanted into Brown-Norway recipient rats. Recipients received 0.5, 1, 2, and 5 mg/kg of Cordyceps sinensis extracts (or control saline) daily via intragastric injection for 60 d. Grafts were harvested 60 d post-transplantation and intimal thickness determined microscopically following hematoxylin and eosin (H and E) staining and abdominal aorta protein profiles determined by Western blot analysis. Cellular localization was assessed by histology and immunohistochemistry and the serum analyzed for tumor necrosis factor alpha (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) by enzyme-linked immunosorbent assay (ELISA). C. sinensis administration resulted in a significant reduction in neointimal formation (neointimal thickness 8.27 ± 1.95 μm [0.5 mg/kg], 3.69 ± 1.43 μm [1 mg/kg], 3.69 ± 1.43 μm [1 mg/kg], 3.69 ± 1.43 μm [1 mg/kg] versus 11.42 ± 2.67 μm [control]) and in the proliferative activity of vascular smooth muscle cells. In addition, localized expression of TNF-α and ICAM-1 in transplant aortas was characterized by immunohistochemistry and immunoblot analyses demonstrating that C. sinensis treatment significantly reduced TNF-α and ICAM-1 levels compared with levels observed in controls (P < 0.05). Serum TNF-α and ICAM-1 levels were significantly reduced in C. sinensis-treated animals compared with controls (P < 0.05). C. sinensis treatment effectively reduced the formation of transplant arteriosclerosis in a rat aortic transplant model.

T-cadherin expression in cardiac allograft vasculopathy: Bench to bedside translational investigation

Coronary allograft vasculopathy (CAV) is the major limiting factor for long term survival after heart transplantation. The aim of this study was to identify gene candidates implicated in human CAV using a rat aortic allograft model in tandem with microarrays and quantitative real time PCR (Q-PCR). Rat abdominal aortas were isografted (5) or allografted (5) from Brown-Norway to Lewis rats and grafts were harvested after day 8, 25 and 60. Agilent microarrays were then used to highlight differentially expressed genes between isografted and allografted rat aortas. Further investigation of a selected candidate gene was performed on human coronary arteries. 1829, 2582 and 1925 genes (fold changes >2 or <2 and p values <0.05) were differentially expressed at day 8, 25 and 60 respectively between isografs and allografts. Seventeen candidate genes were selected according to significant differential expression at day 60. These rat candidate genes were then validated by quantitative real time polymerase chain reaction (Q-PCR). One of these candidate genes, T-Cadherin (T-Cad) was further investigated, using immunohistochemistry (IHC), in human coronary arteries showing CAV compared to classical atherosclerosis present in ischemic cardiomyopathy (ICM) and normal coronary arteries present in dilated cardiomyopathy (DCM). Results showed an over expression of T-Cad in CAV and classical atherosclerosis compared to normal coronary arteries. T-Cad was found to be over expressed in CAV. T-Cad could potentially act as a trigger for smooth muscle cells (SMCs) proliferation and vascular remodelling observed in CAV leading to a diffuse narrowing of the arterial lumen.

Antisense extracellular signal-regulated kinase-2 gene therapy inhibits platelet-derived growth factor-induced proliferation, migration and transforming growth factor-β1expression in vascular smooth muscle cells and attenuates transplant vasculopathy

Platelet-derived growth factor-BB (PDGF-BB) enables vascular smooth muscle cells (VSMCs) to proliferate, migrate and secrete connective tissue matrix, which are critical events in transplant vasculopathy. However, little is known about the intracellular pathways that mediate these biologic responses of VSMCs. Extracellular signal-regulated kinase (ERK) pathway plays a major role in cellular responses and vascular diseases. In this study, we observed that the inhibition of ERK2 activity by recombinant adenovirus encoding antisense ERK2 (Adanti-ERK2) significantly suppressed the proliferation, converting of cell cycle from G(1) phase to S phase and directed migration, and partially abrogated transforming growth factor-beta(1) (TGF-beta(1)) expression in VSMCs stimulated with PDGF-BB. Ex vivo gene transfer of Adanti-ERK2 into rat aortic allograft attenuated chronic transplant vasculopathy by the inhibition of VSMC proliferation and migration. In conclusion, ERK2 is involved in PDGF-BB-induced VSMCs proliferation, migration and TGF-beta(1) expression and may be a potential therapeutic target for transplant vasculopathy.

Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome

Acute coronary syndrome (ACS) represents a series of indications of sudden cardiac ischemia that involve a variety of causes, including acute myocardial infarction and unstable angina.1 ACS usually is associated with thrombus formation in the coronary artery or coronary artery vasospasm, resulting in myocardial ischemia. 2 Thrombus formation may involve platelet adhesion and degranulation on damaged or dysfunctional endothelium overlying the intact or ruptured atherosclerotic plaque, as well as microthrombi.3 Atherosclerotic plaque rupture represents a series of deleterious events linked to the breakdown of the fibrous cap. This process may occur as a result of the increased secretion of matrix metalloproteinases by lesion resident macrophages or enhanced apoptotic cell death within the fibrous cap. However, viable smooth muscle cells (SMCs) within the fibrous cap synthesize interstitial collagen fibers that enhance the structural integrity of the plaque and prevent its rupture.4

The origin of neointimal smooth muscle cells in transplant arteriosclerosis from recipient bone-marrow cells in rat aortic allograft

In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of alpha-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.

Low Molecular Weight Fucoidan Prevents Neointimal Hyperplasia After Aortic Allografting

Fucoidan, a new low molecular weight sulfated polysaccharide (LMWF), has previously been shown to mobilize bone marrow-derived progenitors cells via stimulation of stromal derived factor (SDF)-1 release. Mobilized progenitor cells have been suggested to repair intimal lesions after immune-mediated endothelial injury and thus prevent intimal proliferation. The aim of this study was to evaluate the effect of LMWF treatment in a rat aortic allograft model of transplant arteriosclerosis (TA). Aortic grafts were performed in Brown Norway (BN, donor) and Lewis (Lew, recipient) rats. The recipient rats were treated with LMWF (5 mg/kg/day) and sacrificed at 30 days. To determine the role of SDF-1 in mediating the effects of LMWF, a specific inhibitor of the SDF-1 receptor CXCR4, AMD 3100 (20 microg/kg/day), was used. The grafted segments were evaluated by morphometric (histochemical) analyses. Untreated aortic allografts exhibited severe intimal proliferation, indicative of TA. In contrast, LMWF treatment significantly prevented allograft intimal proliferation as compared with controls (5.7+/-3 vs. 66.2+/-6 microm, P<0.01) and permitted a normalization of the intima/media ratio (0.1+/-0.1 vs. 1.7+/-0.3, P<0.01). Further, LMWF treatment stimulated allograft reendothelialization, as evidenced by strong intimal endothelial nitric oxide synthase antibody and CD31 signals. Unexpectedly, AMD treatment failed to prevent the protective effect of LMWF on intimal thickening and AMD treatment alone was found to reduced intimal proliferation in allografts. We found that LMWF treatment reduced intimal thickness and induced the presence of an endothelial cell lining in the vascular graft at 30 days. Our findings may suggest a novel therapeutic strategy in the prevention of TA.

Trophic factor supplemented UW solution reduces intimal hyperplasia in the rat aortic transplant model

Background Chronic allograft nephropathy (CAN) is associated with delayed graft function, cold ischemic injury, and is an important cause of premature graft loss. A characteristic vascular lesion of CAN is intimal hyperplasia (IH). The goal of this study was to evaluate the effect of cold storage in University of Wisconsin solution supplemented with trophic factors (UW-TF) on IH in rat aortic isograft (IG) and allograft (AG) models. Methods F344 → F344 and Lewis → F344 orthotopic abdominal aortic transplants were performed after 48 h of cold storage in either UW or UW-TF solution with and without immunosuppression. Results Significant reduction in IH was observed when IG were stored in UW-TF solution compared to UW solution. A significant reduction in intimal inflammation was observed in UW-TF stored, nonimmunosuppressed AG. In immunosuppressed recipients, AG stored in UW-TF solution evidenced significantly less IH compared to those stored in UW alone. Conclusions UW-TF solution decreased IH in both alloindependent and dependent models.

Chlamydia Pneumoniae Infection Is Not Associated With Chronic Transplant Dysfunction in a Rat Aortic Allograft Model

Long-term survival of solid-organ transplants is limited as a result of chronic transplant dysfunction (CTD), which is characterized by occlusion of intragraft vascular tissue due to myointimal hyperplasia. Recent studies have shown a role for infections in vascular pathologies. For example, Chlamydia pneumoniae ( Cpn) has been shown to aggravate atherosclerosis, and Cpn immunoglobulin (Ig)G titers correlate with severity of allograft atherosclerosis after cardiac transplantation. In this study, we evaluated the effect of Cpn infection on CTD using a rat aortic allograft model. Orthotopic abdominal aorta transplantations (Tx) were performed with Brown Norway rats as donors and Lewis rats as recipients. Rats were humanely killed at 1 or 8 weeks after surgery. The graft was processed for DNA isolation and histological examination. Influx of macrophages and T cells was assessed using immunohistochemistry. At 1 week after Tx, the perivascular influx of inflammatory cells in the graft was not affected by Cpn infection. Furthermore, only limited numbers of Cpn DNA copies were found in the graft at 1 week after Tx. In addition, Cpn did not alter the severity of myointimal hyperplasia in the rat aortic allograft model at 8 weeks after surgery. Our data suggested that, in the rat aortic allograft model, Cpn infection did not influence the influx of inflammatory cells or the severity of CTD.

Effect of a Novel Inducible Nitric Oxide Synthase Inhibitor in Prevention of Rat Chronic Aortic Rejections

Cytotoxic nitric oxide (NO) is produced during ischemia-reperfusion injury and acute and chronic rejection in allografts by expression of inducible (i) NO synthase (NOS). Therefore, continuous inhibition of iNOS may prevent early graft dysfunction and immune injury (rejection) and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity that works by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n > or = 6) were involved in this study. FR260330, tacrolimus, or both were administered orally for 14 or 90 days, according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. Both low and high doses of FR260330- or tacrolimus-treated grafts showed significantly decreased intima/(intima+media) ratios at day 90 compared with placebo controls. Combination therapy of low-dose FR260330 with low-dose tacrolimus produced a significant decrease of intima/(intima+media) ratios with intact endothelium compared with placebo controls. Anti-alpha-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. A selective inhibitor of NOS, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose FR260330 with tacrolimus produces significant protection of immune injury and may serve to improve long-term graft survival and function.

Seeding of Recipient Bone Marrow Cells Reduces Neointimal Hyperplasia of De-endothelialized Rat Aortic Allograft

Transplant vasculopathy is a leading cause of graft failure and a major contributor to the lack of success with small caliber vascular allografts. In this study we evaluate techniques of bone marrow cell seeding on small caliber vessels and assess the impact of this tactic on neointimal hyperplasia in de-endothelialized rat aortic allografts. In a preliminary study, bone marrow cells from Lewis rats were seeded onto the chemically de-endothelialized luminal surface of the abdominal aorta of WKY rats - with or without fibrin glue. In the allograft transplantation model, de-endothelialized fresh aortic allografts of WKY rats were orthotopically transplanted into Lewis recipients either directly (n = 6) or after recipient bone marrow cell seeding (n = 6). Histological evaluation was performed at 28 days. Bone marrow cells were able to adhere to the de-endothelialized aortic wall owing to the use of fibrin glue, but were unable to do so without fibrin glue. In the de-endothelialized allograft transplantation model, recipient bone marrow seeding led to a significant reduction of the ratio of intimal to medial area (0.40 +/- 0.08 versus 0.79 +/- 0.08, P = 0.0077). Some of the seeded cells remained in the intima for 4 weeks and some infiltrated the media, expressing CD31 or alpha-SMA. The results suggest that recipient bone marrow cell seeding on de-endothelialized aortic allograft is feasible with the use of fibrin glue and that this technique reduces neointimal hyperplasia of the graft.

Morphological and Functional Alterations in Glycerol Preserved Rat Aortic Allografts

Glycerol preservation is an effective method for long-term preservation of skin allografts and has a potential use in preserving arterial allografts. We evaluated the effect of glycerol concentration and incubation period on vessel-wall integrity of rat aortic allografts. No significant differences were measured in breaking strength (2.3 +/- 0.3 N) and bursting pressure (223 +/- 32 kPa) between standard glycerolized and control segments (1.7 +/- 0.3 N, 226 +/- 17 kPa). Isometric tension measurements showed complete lack of functional contraction and relaxation capacity in allograft segments prepared according to all preservation protocols. Morphologically, thickness of the vessel-wall media diminished after preservation using low (30/50/75%) or high (70/85/98%) concentrations of glycerol, as compared to control segments (i.e. 81 +/- 2.4 microm, 95 +/- 5.6 microm and 125 +/- 3.5 microm, respectively). Confocal microscopy and Fourier analysis demonstrated that vascular collagen and elastin bundle orientation had remained unaltered. Electron microscopy showed defragmentation of luminal endothelial cells. In conclusion, glycerol preservation of rat aorta resulted in an acellular tissue matrix, which maintained biomechanical integrity and extracellular matrix characteristics. The next step in the investigation will be to test the concept of glycerol preservation of arterial allografts in a vascular transplantation model.

Conversion from cyclosporine A to mycophenolate mofetil protects recipient kidney and prevents intimal hyperplasia in rat aortic allografts

Background Recent studies have demonstrated that complete conversion from cyclosporine A (CsA) to mycophenolate mofetil (MMF) prolongs graft survival in patients undergoing clinical organ transplantation. We investigated the effects of conversion from CsA to MMF on recipient kidneys and transplant arteriosclerosis in a rat aortic allograft model as a high responder combination. Methods DA (MHC haplotype, RT1 a) rat abdominal aortic grafts were orthotopically transplanted into Lewis (RT1 l) rats. The recipients were divided into four oral treatment groups: (1) vehicle group, (2) CsA group (15 mg/kg/day), (3) CsA/MMF40 group (conversion from CsA 15 mg/kg/day to MMF 40 mg/kg/day on day 14), and (4) CsA/MMF20 group (conversion from CsA 15 mg/kg/day to MMF 20 mg/kg/day on day 14). On day 28 after transplantation, the rats were sacrificed and the hematoserological parameters were analyzed. The grafted aortas and recipient kidneys also were evaluated histologically and immunohistochemically. Results The CsA group developed serological renal dysfunction, arteriolar hyalinosis, and apoptosis in the recipient kidneys, whereas the CsA/MMF40 and CsA/MMF20 groups did not. In the vehicle group, we observed remarkable intimal hyperplasia and marked inflammatory cell infiltration including macrophages and T cells. In the CsA group, intimal hyperplasia was evident without infiltration of macrophages or T cells. In the CsA/MMF40 and CsA/MMF20 groups, intimal hyperplasia was abrogated, while adventitial infiltration of and adhesion to the endothelium by macrophages and T cells occurred. Conclusions Conversion from CsA to MMF protected recipient kidneys and prevented transplant arteriosclerosis. However, insufficient immunosuppression by MMF might reactivate immune cells. This conversion therapy has preventive potential in transplant patients with CsA-associated nephrotoxicity and transplant arteriosclerosis.

THE EFFECT OF A NOVEL INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITOR IN PREVENTION OF CHRONIC AORTIC REJECTION IN RATS

P1219 Background: Cytotoxic nitric oxide (NO) is produced during ischemia/reperfusion (I/R) injury, acute and chronic rejection in the allograft by expression of inducible NO synthase (iNOS). Therefore, continuous inhibition of iNOS may prevent early graft disfunction and immune injury (rejection), and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. Methods: Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n ≥ 6) were involved in this study. FR260330 and/or tacrolimus were administered orally for 14 or 90 days according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. Results: Both low and high doses of FR260330 or tacrolimus treated grafts showed significantly decreased intimal/(intimal + media) ratios at days 90 compared to placebo controls. Combination therapy of low-dose of FR260330 with low-dose of tacrolimus produced significant decrease of intimal/(intimal + media) ratios with intact endothelium to compare with placebo controls. Anti-α-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. Conclusion: A selective inhibitor of nitric oxide synthase, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose of FR260330 with tacrolimus produces significant protection of immnue injury and may serve to improve long-term graft survival and function.