大鼠移植动脉硬化加快模型的建立

目的　建立一种简捷 ,有代表性且稳定的移植物动脉硬化模型。方法　将SD大鼠的腹主动脉分别冷缺血 1、2 4、48h行SD→SD及SD→Wistar的原位腹主动脉移植 ,观察术后植入段血管病理改变、TGF β1表达及手术前后过氧化脂质的变化。结果　SD→SD及SD→Wistar缺血1h组分别于术后 10周及 6周见内膜明显增厚 ,而缺血 2 4h组只需 2周 ;各组移植后 2h过氧化脂质均明显高于术前 ,术后 4、2 4h与术前比差异无显著性 (P >0 .0 5 ) ;强化缺血组TGF β1不论是SD→SD还是SD→Wistar均于术后 1周即出现高表达。结论　以SD/Wistar作为供 /受体行腹主动脉移植 ,强化冷缺血损伤 ,可加快移植物动脉硬化 ,可望成为新型慢排模型。     

Development of a mouse aortic transplant model of chronic rejection

Chronic rejection is the most common cause of late graft failure after solid organ transplantation. A model of chronic rejection, the rat aortic allograft, has histologic features that parallel those in the vessels of human transplanted organs. However, the molecular tools required to dissect the immunology of chronic rejection are unavailable in the rat. We developed aortic transplantation in the mouse as a new model of chronic rejection. This will allow the use of the diversity of recombinant cytokines and monoclonal antibodies available for the mouse and its well-defined genetics to investigate chronic rejection in greater detail. We describe the perioperative care and surgical technique for the model in which a 1 cm segment of donor thoracic aorta was used to replace a section of recipient abdominal aorta below the renal arteries and above the aortic bifurcation. Mortality rates were initially high (70%) due to thrombosis and shock. Changes in technique and operator facility resulted in a high rate of success (75%). After 192 operations, the current success rate is >80%. Mice free from complications at 12 hrs postop had indefinite survival, and after 2 months the typical vascular lesion of chronic rejection was present. This new model of chronic rejection will be a valuable tool to study the molecular immunology and genetics of chronic rejection. © 1995 Wiley-Liss, Inc.     

Inhibition of TNF-α reduces transplant arteriosclerosis in a murine aortic transplant model

Experimental and clinical data provide evidence that TNF-α contributes to acute and chronic allograft rejection. In this study, we explored the effect of TNF-α blockade using the chimeric monoclonal antibody infliximab on the development of transplant arterisoclerosis in a fully mismatched aortic allograft model. Post-transplant treatment of CBA (H2^k) recipients with 250 μg infliximab (cumulative dose 1.25 mg) reduced luminal occlusion of C57Bl/6 (H2^b) aortic grafts on day 30 from 77 ± 5% in untreated controls to 52 ± 6%. Increasing the dose of anti-TNF-α antibody had no further beneficial effect. Treatment with human control immunoglobulin had no effect on intima proliferation. Under TNF-α blockade, ICAM-1 and PDGF mRNA expression within the grafts was strongly reduced, whereas iNOS expression was enhanced. The data show that TNF-α blockade using infliximab can reduce the development of transplant arteriosclerosis in fully mismatched murine aortic grafts.     

Investigation into the onset and progression of transplant arteriosclerosis in a mice aortic retransplantation model

Long-term function of vascularized human organ grafts is often limited by transplant arteriosclerosis and can lead to graft failure. Here, we have analyzed the impact of an initial rejection episode on the later development of transplant arteriosclerosis. Following transplantation of allogeneic abdominal aortic segments in mice, aortic grafts were retransplanted into either immunodeficient or syngeneic recipients. Retransplantation of grafts from immunocompetent into immunodeficient mice as early as 2 days after the primary transplant resulted in intimal proliferation and obstruction of the graft lumen 30 days after the primary transplant. In contrast, retransplantation of the grafts into donor syngeneic B10 recipients within 7 days did not result in the development of transplant arteriosclerosis. These data suggest that the adaptive immune system can induce intimal proliferation by an initial lethal hit that is sustained by the innate response. However our data demonstrate that development of chronic rejection can be inhibited, in this case by retransplantation into a syngeneic host.     

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis

BACKGROUND: Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.     

Combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis in murine aortic allografts

Our group has shown that platelet inhibition with clopidogrel, an antagonist of the P2Y12 adenosine diphosphate receptor on platelets, reduced the formation of transplant arteriosclerosis. The aim of this study was to investigate whether a combination of cyclosporin or everolimus with clopidogrel has a beneficial effect on the development of transplant arteriosclerosis. Fully MHC mismatched C57Bl/6 (H2^b) donor aortas were transplanted into CBA.J (H2^k) recipients and mice received either clopidogrel alone (1 mg/kg/day) or in combination with cyclosporin (2 mg/kg/day) or everolimus (0.05 mg/kg/day). Grafts were analysed by histology and morphometry on day 30 after transplantation. In mice treated with clopidogrel alone, transplant arteriosclerosis was significantly reduced [intima proliferation 56 ± 11% vs. 81 ± 7% (control)/n = 7]. Daily application of everolimus reduced the development of transplant arteriosclerosis compared with untreated controls [intima proliferation of 29 ± 9% vs. 81 ± 7% (control)/n = 7]. Strikingly, combination of clopidogrel and everolimus almost abolished the formation of transplant arteriosclerosis [intima proliferation: 11 ± 8% vs. 81 ± 7% (control)/n = 7]. By contrast, combination of cyclosporin and clopidogrel compared with clopidogrel alone showed no additive effect. These results demonstrate that combination of platelet‐ and mammalian target of Rapamycin‐inhibition can dramatically reduce the development of transplant arteriosclerosis.     

Antisense ERK1/2 oligodeoxynucleotide gene therapy attenuates graft arteriosclerosis of aortic transplant in a rat model

Chronic rejection is a major cause of transplant loss that is effected by the extracellular signal-regulated kinases (ERK) pathway. This study investigated the effects of antisense ERK1/2 oligodeoxynucleotide(ODN) gene therapy on chronic rejection. METHODS: Lewis (RT1(1)) rats served as recipients of Brown-Norway (BN, RT1n) grafts. The BN rat abdominal aortas were harvested and orthotopically grafted into Lewis rats. The recipients were divided into three groups: (1) control group (n = 9), (2) random ODN transfer group (n = 10), and (3) antisense ODN transfer group (n = 10). At day 60 after transplantation, the recipients were sacrificed; the grafted aortas were evaluated histologically and immunohistochemically. ERK1/2 protein expression in the grafts was determined using Western Blot assays. Serum levels of slCAM-1 were detected by ELISA. RESULTS: In the control group and random ODN transfer group, we observed a remarkable degree of intimal hyperplasia and inflammatory cell infiltration, including macrophages and T cells. Compared with the control group, antisense ERK1/2 ODN gene therapy resulted in a significant reduction in neointimal proliferation (P < .01), inhibition of ERK1/2 protein expression (P < .01), decreased graft infiltration with CD4+ T lymphocytes (P < .01), CD8+ T lymphocytes(P < .05), and ED-1 macrophages (P < .01) with decreased serum levels of sICAM-1 (P < .05). We obtained a negative correlation between ERK1/2 expression and immune cell infiltration or ICAM-1 level. CONCLUSIONS: Antisense ERK1/2 gene therapy can attenuate graft arteriosclerosis so as to protect aortic allografts. The protection seemed to correlate with inhibition of inflammatory infiltration, implying that the ERK1/2 signal transduction pathway plays an important role in the process of chronic vascular rejection.     

Aging and transplant arteriosclerosis in absence of alloreactivity and immunosuppressive drugs in a rat aortic model: recipient age's contribution

BACKGROUND: Almost half of all transplanted vascularized organ grafts will be lost to transplant arteriosclerosis sometime posttransplantation. Organ shortage for primary transplants and retransplants has led to donor-pool expansion to include elderly donors, knowing that aging per se promotes arteriosclerosis. The current understanding that donor age negatively affects organ and/or patient survival outcome is undermined by variables such as the use of immunosuppressive drugs, their toxicity to the graft, degree of donor-recipient histocompatibility, and the resulting chronic rejection. The purpose of this study was to determine whether the donor's age or recipient's age matters the most in transplant arteriosclerosis in the absence of such variables. METHODS: A syngeneic combination was used where young (2-month-old) and old (22-month-old) donor aortas were injured to initiate neointimal thickening, then transplanted into age-mismatched recipients for 14, 60, and 90 days and then assessed for neointimal thickening. Base level injury response due ischemia and surgery was evaluated in age-matched and noninjured aortic grafts, respectively. RESULTS: Young aortas invariably developed thicker neointima when transplanted into old recipients than when transplanted into young ones. Correspondingly, old aortas transplanted in young recipients consistently developed less neointimal thickening than when transplanted into old recipients. CONCLUSIONS: Our findings strongly suggest that the severity of age-related neointima formation is primarily determined by the recipient's age rather than the donor's age. Therefore, in addition to focusing on donor-specific tolerance induction, strategies aiming at increasing the lifespan of vascularized organ grafts also have to take into consideration the recipient's aging milieu.     

Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.

BACKGROUND: Graft arteriosclerosis is a major cause of death after allotransplantation of organs such as the heart or the kidney. Aortic allotransplantation in mice is a useful experimental model to study the mechanisms of this pathology. However, the conventional heterotopic aortic model is limited by a high morbidity and is technically difficult to perform. We developed a new simple method for aortic transplantation in mice. METHODS: The infrarenal aorta from the donor mouse was anastomosed to the recipient's aorta at the same position using a sleeve technique. Orthotopic aortic transplantation was performed in 45 mice, 5 isografts and 40 allografts. No immunosuppression was given, and the mice were killed at day 15 or 30. The graft was examined macroscopically, and several histologic sections were made. RESULTS: The overall survival rate was 78%. The incidence of thrombosis was low (4 cases) compared with previously published series. Histology of aortas revealed typical aspects of rejection in the allografts with a chronic picture at day 30. No significant lesion was observed in isografts. CONCLUSIONS: We have developed a model of orthotopic aortic transplantation in mice. This new model is easy to carry out and has a low incidence of thrombosis, probably because there is no size discrepancy between donor and recipient aortic segment.     

Abdominal aortic aneurysmectomy in long-term cardiac transplant survivors.

Two thirds of the 114 patients undergoing heart transplantation at Stanford (Calif) Medical Center have had atherosclerotic cardiac disease. Two of these patients have been found in the late posttransplant period to have symptomatic abdominal aortic aneurysms requiring resection. We present the clinical course and successful operative treatment of these two patients, with emphasis on the prophylaxis of infection and maintenance of hemodynamic, metabolic, and immune stability. Close surveillance of the long-term cardiac transplant survivor should be directed towards other manifestations of atheroscleortic disease.     

Abdominal aortic aneurysm repair in a patient with a cardiac transplant

We describe successful elective abdominal aneurysm repair in a patient with a cardiac transplant. In light of the unique physiology and pharmacology of the denervated heart, this presented an unusual combination of complex problems. Whereas the normally innervated heart increases cardiac output via neural stimuli, the denervated heart relies primarily on the Frank Starling mechanism which is dependent on preload and myocardial contractility. Thus, rapidly changing haemodynamic variables associated with aortic cross-clamping require scrupulous attention to the maintenance of adequate preload as well as myocardial function which can only be manipulated by direct-acting agents. We conclude that the denervated heart will readily compensate for the haemodynamic changes brought about by infrarenal aortic crossclamping if a high-normal preload is maintained and if the transplanted donor heart is free of pathology with good inherent myocardial contractility.     

Circulating smooth muscle cell plasticity in the development of transplant arteriosclerosis

To date, chronic transplant dysfunction (CTD) is recognized as the major cause of long-term transplant loss (>1 year) after transplantation. CTD presents histologically with obliterated intragraft arteries as a result of intimal hyperplasia referred to as transplant arteriosclerosis (TA). Neointimal lesions predominantly consist of vascular smooth muscle cells (VSMCs) intermingled with some inflammatory cells. The pathogenesis of TA is believed to be multifactorial, and many risk factors have been identified. Because the precise pathogenetic mechanisms underlying TA are still largely unknown, adequate prevention and treatment protocols are not available. In this review, we discus the origin (donor vs recipient, bone marrow vs non-bone marrow) of neointimal endothelial cells (ECs) and VSMCs in TA lesions, which were formerly believed to be solely graft-derived. On the basis of the data obtained in both clinical and experimental transplantation, it appears that the process leading to TA is heterogeneous and that neointimal ECs and VSMCs can be recruited from different sources, possibly depending on the severity of vascular damage. These data suggest a significant role of host-derived circulating EC-VSMC progenitor cells, which may be partly bone marrow-derived. These circulating progenitor cells are potential targets for therapeutic intervention to ameliorate TA development or occlusive vascular disease in general.