Aortic Transplant Model Research Articles

Use of recombinase activation gene-2 deficient mice to ascertain the role of cellular and humoral immune responses in the development of chronic rejection

Introduction: Given its multifactorial etiology, the relative contribution of anti-donor cellular and humoral immune responses in the pathogenesis of chronic rejection is as yet ambiguous. We hypothesized that alloreactive T and B cells play a seminal role in the development of this lesion. Methods: To address this hypothesis, RAG-2 −/− mice were used as donors and recipients in a well-established murine model of aortic transplantation. Grafts were transplanted across the following groups: Group I: C3H → C3H; Group II: Wild-type [WT] 129Sv (H-2 b) → C3H (H-2 k); Group III: C3H → WT 129Sv; Group IV: 129SvEv RAG-2 −/− → C3H; and Group V: C3H → 129SvEv RAG-2 −/−. Grafts were harvested at d40 to 146 post-transplantation for morphologic and immunohistochemical analyses and semi-quantitative RT-PCR was employed to evaluate the intragraft mRNA expression of various immune mediators. Mixed lymphocyte reaction and complement-mediated alloantibody cytotoxicity assays were performed to determine anti-donor proliferative and humoral responses, respectively. Results: Unlike that across the syngeneic combination (Group I), marked intimal thickening with corresponding luminal narrowing was observed in the majority of the aortic allografts (Groups II–IV). On the contrary, the morphology of C3H aortic allografts harvested from the majority of the RAG-2 −/− was remarkably preserved. Correspondingly, anti-donor proliferative and humoral immune responses were undetectable in C3H → RAG-2 −/− recipients as was the intragraft mRNA expression of the Th 1 and the Th 2-type cytokines. Conclusions: Taken together, these data suggest that in this murine model of aortic allotransplantation, donor-specific cellular and humoral responses play a dominant role in the initiation and perpetuation of chronic rejection.

Effect of a novel anti-oxidant and anti-inflammatory compound on rodent allograft arteriosclerosis

Background: The long term success of cardiac transplantation continues to be limited by the development of graft coronary artery disease. AGI-1096 is a newly developed antioxidant and anti-inflammatory compound that inhibits the inducible expression of VCAM-1 and MCP-1 in endothelial cells (IC50=11μM and 5μM, respectively) as well as smooth muscle cell proliferation (IC50 =5.5μM), giving it the potential to prevent chronic allograft arteriosclerosis. In this study, we evaluated the efficacy of this compound using a rodent aortic transplantation model.

Elevating high-density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.

HDL cholesterol levels are inversely correlated with coronary heart disease risk in humans, and in animal studies, HDL elevation decreases formation and progression of foam-cell lesions. The potential for HDL to affect preexisting advanced atherosclerotic lesions is not known. To approach this issue, we used a novel mouse aortic transplantation model. ApoE-deficient (EKO) mice were fed a Western-type diet for 6 months, and thoracic aortic segments containing advanced lesions replaced segments of the abdominal aorta of 4-month-old EKO syngeneic mice not expressing (plasma HDL cholesterol approximately 26 mg/dL) or expressing (HDL approximately 64 mg/dL) a human apoAI (hAI) transgene. Both types of recipients had comparable non-HDL cholesterol levels. Five months after transplantation, mice were killed and grafts analyzed. Compared with lesion area in pretransplant mice (0.14+/-0.04 mm(2), mean+/-SEM), there was progression in the EKO recipients (0.39+/-0.06 mm(2), P<0.01). Compared with EKO recipients, hAI/EKO recipients had retarded progression (0.24+/-0.04 mm(2), P<0.05). Immunostaining for CD68 and other macrophage-associated proteins, monocyte chemoattractant protein-1, acyl coenzyme A:cholesterol acyltransferase, and tissue factor, in lesions of pretransplant and EKO recipient mice showed abundant macrophages. In contrast, compared with any other group, lesional macrophage area in hAI/EKO mice decreased >80% (P<0.003), and smooth muscle cell content (alpha-actin staining) increased >300% (P<0.006). The decrease in macrophages and increase in smooth muscle cells was primarily in the superficial subendothelial layer. Increasing HDL cholesterol levels in EKO mice retards progression of advanced atherosclerotic lesions and remodels them to a more stable-appearing phenotype.

A technique of cervical aortic graft transplantation in mice

A new method of mouse aortic graft transplantation into carotid artery was developed with cuff technique. By harvesting the descending aorta of the donor using a small Teflon cuff (external diameter 0.6 mm, internal diameter 0.4 mm) and super fine-tip forceps, and modifying the method of mouse heterotopic heart transplantation with cuff technique, donor descending aortic allografts could be interposed in the common carotid artery of recipient mice. Histological analysis demonstrated neither evidence of tissue damage nor intimal thickening in isograft implanted over 100 days. We strongly recommend that this new model of aortic transplantation in mice is a simple and useful technique for vascular transplantation research.

Initial Inhibition of Tissue Factor Signalling Reduces Chronic Vascular Changes in Isogenic Rat Aortic Transplants

Vascular changes are considered the major histopathological indicator of chronic allograft dysfunction. These changes are characterized by intimal thickening caused by accumulation of primarily smooth muscle cells. Contributing factors may be of both immunological and nonimmunological origin. Cold ischemia has been shown to trigger intimal proliferation in the absence of alloantigen in an isogenic rat aortic transplant model. We have used this model to investigate the impact of inhibition of tissue factor (TF) signalling on the progression of intimal thickening. Group 1 was treated with recombinant FVIIa inhibited in its active site (rFVIIai), and group 2 served as untreated controls. At 8 weeks the intimal area was measured with image analysis. Medial areas and the proportion of medial necrosis were determined. Animals treated with rFVIIai showed significantly less intimal thickening compared with controls: median 0.147 vs. 0.256 mm2, respectively (p = 0.008). A positive correlation between intimal hyperplasia and medial necrosis (rs = 0.79, p = 0.01), as well as adventitial inflammation (rs = 0.83, p = 0.009), was found. TF mRNA was not detected in the neointima at 8 weeks, as determined by in situ hybridization.We conclude that active site inhibited FVIIa (rFVIIai) given prior to and directly after implantation of aortic transplants significantly reduces intimal hyperplasia caused by nonimmunological factors in this model.

Candesartan cilexetil reduces graft arteriosclerosis in aortic transplantation model in rat

To date established treatment of transplant arteriosclerosis is basically missing and there is a need for new therapeutic approaches. Angiotensin II (Ang II) and Ang II receptor type 1 (AT 1) are present in the vascular wall. Blocking of the AT 1 receptor by pharmacological agents may inhibit damaging effects of Ang II on endothelial and smooth muscle cells. The purpose of the study was to evaluate the effect of the AT 1 receptor blocker Candesartan cilexetil on the development of graft arteriosclerosis in a rat aortic transplant model. Two strain combinations were used for aortic transplantation: DA to PVG; and PVG to PVG. The animals received Candesartan cilexetil treatment (9.5±1.4 mg/kg/day) for 8 weeks. Candesartan cilexetil treatment reduced neointimal formation both in allografts (Qint 30.2±8.8% vs. 22.1±8.7%, P<0.05) and in isografts (Qint 15.5±4.4% vs. 6.7±3.3%, P=0.0001). Blocking of the AT 1 receptor signalling by Candesartan cilexetil was also associated with a reduced expression of TGF-β1. Macrophage infiltration was not affected by the treatment. Candesartan cilexetil treatment leads to reduced neointimal formation in aortic transplant. The positive effect of the drug might be partly explained by a reduction of TGF-β1 expression in the grafts. Candesartan treatment may provide another possibility for prevention of transplant arteriosclerosis and chronic rejection.

Development of a combined cardiac and aortic transplant model to investigate the development of transplant arteriosclerosis in the mouse

Background: The degree of transplant arteriosclerosis in murine cardiac allografts is difficult to assess. Aortic allografts represent an alternative model for evaluating the impact of novel transplant strategies on transplant arteriosclerosis in which the vascular changes can be quantified easily. However, it remains controversial as to whether vascular lesions seen in this model are equivalent to those that develop in solid-organ transplants. The aim of this study was to develop a model of combined cardiac and aortic transplantation to allow more precise quantification of transplant arteriosclerosis and to establish a correlation between the lesions that develop in the 2 types of graft. Methods CBA (H2 k) recipients received a C57BL/10 (H2 b) cervical cardiac allograft on Day 0 and a C57BL/10 (H2 b) abdominal aortic allograft on Day 1. Recipients were treated with anti-CD154 mAb (MR1) on Days 0, 2, and 4. We performed histology and morphometric measurements for both grafts 30 days after transplantation. Results We observed significant intimal proliferation in both the cervical cardiac and abdominal aortic allografts from recipients treated with anti-CD154 mAb (heart, 64% ± 9%; aorta, 67% ± 8%; n = 5). Abdominal aortic grafts transplanted alone into anti-CD154–treated recipients developed a degree of transplant arteriosclerosis equivalent to that seen in the aortic grafts of the combined group (aorta alone, 68% ± 9%, vs aorta + heart, 67% ± 8%; n = 5). Conclusions This combined cardiac and aortic transplant model permitted quantitative assessment of transplant arteriosclerosis while monitoring graft survival by cardiac palpation. Furthermore, development of transplant arteriosclerosis was equivalent in abdominal aortic allografts either in the presence or absence of an additional solid- organ transplant.

FK409, a spontaneous nitric oxide releaser, attenuates allograft vasculopathy in a rat aortic transplant model.

Although systemic administration of NO donors has been shown to attenuate the development of neointimal hyperplasia in the balloon injury model, this strategy has not been tested in a model of allograft vasculopathy. In this study, we investigated the effect of FK409, a spontaneous NO releaser, on the development of allograft vasculopathy, using a rat aortic transplant model. Thoracic aortas from ACI rats were transplanted heterotopically into the abdominal aorta of Wistar-Furth rats. Postoperatively, recipients received FK409 orally every 8 hours from the day of transplantation to the time of euthanization. Morphometric and immunohistochemical analyses were performed on the aortic grafts 8 weeks after transplantation. Control allografts showed severe neointimal hyperplasia, which consists mainly of alpha-actin-containing vascular smooth muscle cells. The FK409-treated allografts showed a dose-dependent reduction (statistically significant compared with the control) in the neointimal thickness as the dose increased from 1 to 10 mg/kg (thrice per day). However, there was no significant difference in the neointimal thickness between groups treated with 10 and with 20 mg/kg. FK409 treatment (10 mg/kg) caused a significant decrease in DNA synthesis (5-bromo-2-deoxyuridine [BrdU] uptake), an increase in DNA fragmentation (terminal deoxynucleotidyltransferase-mediated uridine nick-end labeling [TUNEL]), and upregulation of Fas expression, in the neointimal vascular smooth muscle cells. These data suggest that FK409 attenuates the allograft vasculopathy in a rat aortic transplant model.

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.

Improved surgical technique for the establishment of a murine model of aortic transplantation.

Aortic allotransplantation is a reliable procedure to study the evolvement of chronic rejection in mice. The progressive nature of this process in mice is characterized by diffuse and concentric myointimal proliferation which is inevitably associated with variable degrees of luminal constriction. These vascular changes are comparable to those that are witnessed in organ allografts undergoing chronic rejection in humans, underscoring its utility as a model of choice for the study of the development of this lesion. Whilst improved surgical technique has resulted in markedly enhanced graft survival, the results are far from being acceptable. Realizing this limitation, we embarked on developing a modified technique for aortic transplantation which would allow for improved graft survival in mice. A bypass conduit was created by end-to-side anastomosis of a segment of the donor's thoracic aorta into the infrarenal portion of the recipient's abdominal aorta. Using this technique, the graft survival was >98% with evidence in allotransplanted aorta of morphological changes pathognomonic of chronic rejection. On the contrary, no histopathological anomalies were discerned in aortic grafts transplanted across syngeneic animals. This modified surgical approach ameliorates the unacceptably high graft loss associated with earlier techniques, further extending the utility of this model as a tool to study the molecular and cellular mechanisms rudiment to the evolvement of chronic rejection.

Progression of changes in arteries following cold storage preservation in UW and collins solution in a syngeneic aortic transplant model

Progression of changes in arteries following cold storage preservation in UW and collins solution in a syngeneic aortic transplant model

Influence of MHC class I and class II (mis)matching between donor and recipient on the cytomegalovirus induced accelerated graft arteriosclerosis in a rat aorta transplant model

Influence of MHC class I and class II (mis)matching between donor and recipient on the cytomegalovirus induced accelerated graft arteriosclerosis in a rat aorta transplant model

Influence of MHC class I and class II (mis)matching between donor and recipient on the cytomegalovirus induced accelerated graft arterioscierosis in a rat aorta transplant model

Influence of MHC class I and class II (mis)matching between donor and recipient on the cytomegalovirus induced accelerated graft arterioscierosis in a rat aorta transplant model

The role of alloantigen-independent factors in transplant arteriosclerosis

Chronic transplant dysfunction (CTD) is a major cause of late graft loss. Various investigations show that from 1-year post-transplantation there is a 3% to 9% annual loss of transplanted kidneys due to CTD. The major feature of CTD is transplant arteriosclerosis, characterized by concentric thickening of the intima, migration and proliferation of smooth muscle cells, medial necrosis, and adventitial inflammatory infiltrate. Although alloantigen-dependent factors are central in CTD, it is still a matter of debate whether alloantigen-independent factors, including ischemia and surgical manipulation, contribute to this process as well. Some clinical studies showed that prolonged cold ischemia is a risk factor, whereas others did not. Experimental studies of organ transplantation are also inconsistent with regard to the role of cold ischemia on CTD. The effects of surgical trauma per se are not uniform; Munger et al. demonstrated an effect of surgery, whereas others did not. The aim of this study was to elucidate the influence of two alloantigen-independent factors-cold ischemia and surgical trauma-on the development of transplant arteriosclerosis in a rat aorta transplantation model. To exclude organ specific factors on the development of CTD, the aorta graft was used, which is a proven chronic rejection model. To study these alloantigen-independent factors syngeneic grafts were compared with allogeneic ones.

Rationale for the use of antioxidant vitamins in clinical organ transplantation.

Rationale for the use of antioxidant vitamins in clinical organ transplantation.

Chronic rejection of concordant aortic xenografts in the hamster-to-rat model

Several groups have demonstrated that it is possible to obtain long-term graft survival of concordant xenografts. One of the important questions that remains is whether xenografts are susceptible to chronic rejection. To answer this question we used the aorta transplantation model. One centimetre of hamster aorta was interposed in the abdominal aorta of Lewis rat recipients. The recipients were either untreated (group 1), or treated with 10 mg/kg cyclosporine (CsA), given intramuscularly three times a week (group 2). Rats were sacrificed at day 7, 14, 21, 28, 56 and 84 and the thickness of the intima, the media and the adventitia was measured. Furthermore, the cellularity of the media and the adventitia was assessed by counting the number of nuclei per 0.05 mm 2 and immunohistochemistry of the aortic grafts was performed. Graft arteriosclerosis developed in aortic xenografts of both group 1 and group 2. In group 1, intimal lesions were already present from day 21 onwards in all rats, whereas in group 2 they were present only in 33% ( 2 6 ) of the rats. At day 84 all the grafts in group 1 were totally occluded, while those in group 2 were still open. The thickness of the media was slightly increased in both groups during the whole observation period, mainly due to edema. Although a few infiltrating macrophages could be seen, the number of nuclei per 0.05 mm 2 of the media remained constant during the first 21 days, but declined sharply from day 21 onwards, a consequence of disappearing myocytes. Thickness of the adventitia in both groups increased after transplantation due to infiltrating macrophages and T cells, reaching a peak at day 14. After day 14 the adventitial thickness in group 1 decreased rapidly to reach values comparable to group 2 from day 28 onwards. In conclusion, graft arteriosclerosis, as a sign of chronic rejection, occurs in concordant aortic xenografts. The lesions in the xenografts develop extremely rapidly, and, compared to data from the literature, faster than in aortic allografts. The process of chronic rejection in aortic xenografts can be reduced by CsA.

Aorta transplantation as a model to study hyperacute, acute, and chronic rejection of xenografts

Abstract: To explore the mechanism of rejection of vascularized xenografts, we performed discordant and concordant transplantation of aortic grafts. Forty‐one guinea pig‐to‐rat and 50 hamster‐to‐rat aortic transplantations were performed successfully. Recipient rats were either untreated or treated three times per week with 10 mg/kg of cyclosporine (CsA) starting the day before transplantation. Recipients were sacrificed at various timepoints after transplantation and the grafts were processed for histology and immunohistochemistry. Guinea pig aortic grafts were rejected hyperacutely in both the control and CsA treated group. Four hours after transplantation all endothelial cells and some of the medial smooth muscle cells had disappeared. Seven days after transplantation the media were totally acellular, whereas the adventitia was enlarged due to infiltrating cells, consisting mainly of macrophages. At day 56, only traces of the original graft could be identified.Hamster aortic grafts developed signs of chronic rejection. In control rats, the intimal lesions could already be demonstrated in 17% of the rats at day 14, and in all rats from day 28. CsA treatment inhibited the development of the intimal lesions. At days 56 and 84 the difference in intimal thickness between the control group and the CsA treated group was statistically significant. The cellularity of the media of the hamster grafts remained constant during the first 21 days in both groups but declined sharply thereafter as a consequence of migration or necrosis of myocytes. The thickness of the adventitia in both groups increased after transplantation, due to infiltrating macrophages and T cells, reaching a peak at day 14. In conclusion, the aorta transplantation model provides useful information with regard to the development of chronic rejection in concordant grafts. Discordant aortic grafts are rejected hyperacutely just like discordant heart grafts.

Reversibility of allograft arteriosclerosis after retransplantation to donor strain.

The rat aortic transplant model was modified to investigate whether the vascular wall changes on chronic rejection are reversible. DA (RT1a) aortas were first transplanted to WF (RT1a) recipients. The first transplantation was accompanied, as described earlier, by an increase in intimal cellularity and thickness and typical arteriosclerotic changes of chronic rejection in the allograft intima. A second transplantation was made to DA, WF or to (DAxWF)F1 recipients 10 days-2 months after the first transplantation. In all retransplantations performed at any one of the indicated timepoints, the thickness and number of nuclei of the intima continued to increase. These observations demonstrate that, after an initial trigger, allograft arteriosclerosis proceeds and is irreversible despite elimination of histoincompatibility.

JE Gene Expression in an Animal Model of Acute Arterial Graft Rejection

Transplant arteriosclerosis (TA) is an immune mediated vascular injury that results in rapid intimal thickening and parenchymal ischemia. The monocytes that infiltrate the transplanted organ's vessels may mediate this effect. One of the cell signals that may initiate monocyte infiltration in rat models of acute and chronic vessel wall rejection is the product of the JE gene, a potent and selective chemoattractant for monocytes. We used combinedin situhybridization and immunocytochemistry to localize JE gene expression in specific cell types using a rat aortic transplant model of acute arterial graft rejection. Within 3 days of transplantation, there was JE mRNA expression in adventitial mesenchymal cells, probably fibroblasts, and this signal increased until 10 days post-transplantation. During this period, the number of adventitial monocytes, identified by immunocytochemistry, increased around the mesenchymal cells expressing JE. Intimal JE mRNA expression between 7 and 20 days localized to undefined mesenchymal intimal cells and occasionally blood-borne monocytes. There was no JE gene expression detected in the intima after 20 days. JE mRNA expression at 20 days was limited to α-actin-positive vascular smooth muscle cells in the outer aspect of the media. At 40 and 60 days, there was no JE hybridization signal in the media or adventitia despite increasing medial monocyte infiltration. The temporal sequence of JE mRNA expression, starting in the adventitia, intima, and finally the media, preceded or coincided with monocyte/macrophage infiltration. These results support our hypothesis that early JE gene expression may lead to the initial monocyte recruitment in acute vessel wall rejection. Because JE gene expression is downregulated by glucocorticoids, their immunosuppressive effect may be partly due to decreased JE gene expression by transplanted vessels.

INHIBITION OF INTIMAL HYPERPLASIA IN RAT AORTIC ALLOGRAFTS WITH CYCLOSPORINE

The rat aortic transplant model of chronic rejection was used to study the effect of cyclosporine (CsA) on the development of intimal hyperplasia. ACI and Lewis rat strains were used to create isograft and allograft CsA nontreated and treated groups. After orthotopic abdominal aortic transplantation, recipients received either no treatment, CsA 2.5 mg/kg/day, CsA 5 mg/kg/day, or CsA 10 mg/kg/day by gavage. Treated grafts were harvested at 3 and 6 months after transplantation, and computer image digital analysis was used to measure intimal and medial areas of graft cross-sections. At 3 months, the reduction in percent intima was 62% (P = 0.005), 74% (P = 0.002), and 97% (P < 0.0001) for the 2.5-, 5-, and 10-mg/kg allograft groups, respectively. There was a 93% (P < 0.0001) reduction in percent intima at 6 months in the 10-mg/kg allograft group. CsA treatment also preserved the aortic media. In comparison to nontreated isografts, medial area in nontreated allografts was decreased by 37% at 3 months after transplantation. In contrast, medial area was not significantly changed in CsA-treated recipients (10 mg/kg/day) in comparison to nontreated isografts. More importantly, medial nuclear density was preserved in the CsA-treated recipients in comparison to nontreated allografts and was similar to treated or nontreated isografts. In conclusion, daily high dose CsA treatment was found to markedly inhibit intimal hyperplasia in rat aortic allografts up to 6 months after transplantation, which suggests that CsA at an adequate dosage can suppress chronic rejection.