Long-term type 1 diabetes enhances in-stent restenosis after aortic stenting in diabetes-prone BB rats.

Type 1 diabetic patients have increased risk of developing in-stent restenosis following endovascular stenting. Underlying pathogenetic mechanisms are not fully understood partly due to the lack of a relevant animal model to study the effect(s) of long-term autoimmune diabetes on development of in-stent restenosis. We here describe the development of in-stent restenosis in long-term (~7 months) spontaneously diabetic and age-matched, thymectomized, nondiabetic Diabetes Prone BioBreeding (BBDP) rats (n = 6-7 in each group). Diabetes was suboptimally treated with insulin and was characterized by significant hyperglycaemia, polyuria, proteinuria, and increased HbA(1c) levels. Stented abdominal aortas were harvested 28 days after stenting. Computerized morphometric analysis revealed significantly increased neointima formation in long-term diabetic rats compared with nondiabetic controls. In conclusion, long-term autoimmune diabetes in BBDP rats enhances in-stent restenosis. This model can be used to study the underlying pathogenetic mechanisms of diabetes-enhanced in-stent restenosis as well as to test new therapeutic modalities.

Dichotomous effects of rosiglitazone in transplantation-induced systemic vasodilator dysfunction in rats.

BACKGROUND: Transplantation-induced systemic endothelial dysfunction causes severe cardiovascular morbidity and mortality after transplantation. Interventions that improve systemic endothelial function after transplantation and furthermore reduce intragraft vascular dysfunction might improve graft and patient survival. Treatment with the PPARgamma agonist rosiglitazone is an intervention that potentially fulfills these criteria. In this study, we determined the effect of rosiglitazone treatment on transplantation-induced endothelial dysfunction and vasomotor activity in an experimental model for chronic transplant dysfunction in rats. METHODS: Lewis abdominal aortic allografts were orthotopically transplanted into Brown Norway recipients that received either regular chow or chow containing rosiglitazone (approximately 4.2 mg/day). Endothelium-dependent (response to metacholine) and total (response to sodium nitrite) vasodilatory responses were determined in autologous thoracic aortic rings using an ex vivo organ bath setup. Measurements were performed 8 weeks after transplantation. RESULTS: Aortic allografting induced systemic endothelial dysfunction as measured by reduced endothelium-dependent vasodilation in the recipient's vascular system. Rosiglitazone treatment restored endothelium-dependent vasodilatory responses to pretransplantation levels. However, rosiglitazone treatment reduced the total dilatory response despite normalized endothelial function, indicating impairment of vascular smooth muscle cell vasomotor activity. CONCLUSIONS: Rosiglitazone treatment after allogeneic transplantation restores endothelial function but impairs vascular smooth muscle cell vasomotor activity. This dichotomous effect of rosiglitazone might impede use of rosiglitazone after organ transplantation since this potentially increases cardiovascular risk despite improved endothelial cell function.

Non-bone marrow origin of neointimal smooth muscle cells in experimental in-stent restenosis in rats.

OBJECTIVE: To determine the contribution of bone marrow (BM)-derived cells in in-stent restenosis (ISR) and transplant arteriosclerosis (TA). METHODS: Non-transgenic rats WT F344(TG) (n = 3) received stent implantation 6 weeks after lethal total body irradiation and suppletion with bone marrow from a R26-hPAP transgenic rat. After 4 weeks the abdominal aortas were harvested, the stent was quickly removed, the abdominal aorta was snap-frozen in liquid nitrogen and 5 mum cryosections for stainings were cut. Additionally, DA aortic allografts were transplanted into WT F344(TG) (n = 3) and R26-hPAP(WT) (n = 3) BM-chimeric recipients. Immunohistochemistry (hPAP staining) and immunofluorescence (hPAP, alpha-SMA and OX1) was performed on all sections. RESULTS: Few hPAP-positive cells were observed in the neointima. Double stainings of hPAP-positive areas showed no alpha-SMA colocalization; OX-1 did show colocalization. CONCLUSIONS: Non-BM-derived cells are the predominant source of neointimal cells in ISR and TA. Vascular wall-derived progenitor cells may rather be the source of SMCs that contribute to ISR and TA, which may have implications for our quest for new therapeutic targets to treat these vasculopathies.

Rosiglitazone attenuates transplant arteriosclerosis after allogeneic aorta transplantation in rats.

BACKGROUND: Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts. METHODS: Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either approximately 5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro. RESULTS: Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P<0.01 vs. nontreated). Rosiglitazone reduced T cell alloreactivity which was not mediated through modulation of CD4+CD25+FoxP3+ regulatory T cells. Reduced development of transplant arteriosclerosis coincided with reduced intragraft expression of stromal-derived factor-1alpha and platelet-derived growth factor receptor-beta. Finally, rosiglitazone reduced growth-factor-driven proliferation of both medial and neointimal vascular smooth muscle cells in vitro, which was not mediated through PPARgamma. CONCLUSION: PPARgamma agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.

Role of progenitor cells in transplant arteriosclerosis.

To date, chronic transplant dysfunction (CTD) is recognized as the major cause of transplant loss long term after transplantation. CTD has the remarkable histologic feature that the luminal areas of the intragraft arteries become obliterated as a result of occlusive neointima formation. Neointimal lesions contain predominantly vascular smooth muscle cells (VSMCs) and extracellular matrix admixed with inflammatory cells. At the luminal side, neointimal lesions are covered with a monolayer of endothelial cells (ECs). The etiology of transplant arteriosclerosis (TA) is largely unknown, and adequate prevention and treatment protocols are not available. In contrast to the largely accepted "response-to-injury" hypothesis for the development of TA that attributes an important role to graft-derived ECs and VSMCs, recent data indicate that host-derived vascular progenitor cells play a major role in the development of TA. The process leading to TA appears to be heterogeneous, and neointimal ECs and VSMCs can be recruited from different sources, possibly depending on the severity and duration of vascular damage. These data suggest a significant role of host-derived circulating EC/VSMC progenitor cells, which may be partly bone marrow derived. Circulating vascular progenitor cells are potential targets for therapeutic intervention to ameliorate TA development. Therefore, identification of mediators and cellular mechanisms that promote recruitment of vascular progenitors to sites of injury is warranted to dissect their detrimental and possible beneficial effects in the development of TA.

Development of transplant vasculopathy in aortic allografts correlates with neointimal smooth muscle cell proliferative capacity and fibrocyte frequency.

OBJECTIVE: Transplant vasculopathy consists of neointima formation in graft vasculature resulting from vascular smooth muscle cell recruitment and proliferation. Variation in the severity of vasculopathy has been demonstrated. Genetic predisposition is suggested as a putative cause of this variation, although cellular mechanisms are still unknown. Using a rat aorta transplant model we tested the hypothesis that kinetics of development of transplant vasculopathy are related to neointimal smooth muscle cell proliferative capacity and fibrocyte frequency, the latter being putative neointimal smooth muscle ancestral cells. METHODS: Aortic allografts were transplanted in Lewis and Brown Norway, as well as MHC-congenic Lewis.1N and Brown Norway.1L recipients. Severity of transplant vasculopathy was quantified 4, 8, 12 and 24 weeks after transplantation. Host-endothelial chimerism, as a reflection of vascular injury, was determined by specific immunofluorescence. Neointimal smooth muscle cell proliferative capacity was determined in vitro and in situ. Fibrocyte frequency and phenotype were determined after in vitro culture by cell counting, immunofluorescence and in situ zymography. RESULTS: Compared to Lewis, Brown Norway recipients developed accelerated transplant vasculopathy which is dependent on the presence of Brown Norway non-MHC-encoded determinants. Accelerated transplant vasculopathy was associated with increased levels of host-endothelial chimerism and increased neointimal smooth muscle cell proliferation, the latter being accompanied by increased endothelial and smooth muscle cell-derived neuropilin-like protein mRNA expression. Moreover, accelerated transplant vasculopathy was associated with increased frequency of circulating gelatinase-expressing CD45(+)vimentin(+) fibrocytes. CONCLUSION: Susceptibility for transplant vasculopathy appears to be genetically controlled and correlates with neointimal smooth muscle cell proliferative capacity and circulating fibrocyte frequency.

Angiogenic sprouting from the aortic vascular wall is impaired in the BB rat model of autoimmune diabetes.

BACKGROUND: Diabetes is associated with impaired neovascularization leading to reduced revascularization of ischemic tissue and impaired wound healing. Endothelial progenitor cells in diabetes were previously shown to be numerically reduced and functionally impaired. We hypothesize that diabetes also has a long-term effect on angiogenic cells residing in the vessel wall. To test this hypothesis, angiogenic sprout formation from ex vitro cultured aortic rings isolated from diabetic and non-diabetic BioBreeding (BB) rats was assessed. METHODS: Diabetes prone BB (BBDP) rats spontaneously develop autoimmune diabetes were suboptimally treated with insulin by subcutaneous implantation of slow-release insulin-pellets. Neonatally thymectomized BBDP rats, pre-diabetic BBDP rats and diabetes resistant BBDR rats served as non-diabetic controls. After follow-up thoracic aortas were harvested and cultured in vitro in Matrigel to induce sprout formation. Sprout length was quantified after 4, 7, 10 and 14 days of culture. The total number of sprout-derived cells was measured and in vitro proliferative capacity of sprout cells was quantified. Finally, expression of Flk-1, CD31 and smooth muscle alpha-actin on sprout cells was determined. RESULTS: Mean blood glucose levels in diabetics were significantly elevated compared with non-diabetics. Both long-term and short-term diabetes significantly reduced sprout formation (p<0.05 vs. non-diabetics). Reduced sprout length in diabetics was reflected by significantly reduced numbers of sprout cells that could be isolated (p<0.05 vs. non-diabetics). Isolated sprout cells from diabetics revealed significantly reduced proliferative capacity after in vitro culture (p<0.05 vs. non-diabetics). Immunofluorescent staining indicated an endothelial phenotype of both freshly isolated and in vitro cultured sprout cells as indicated by CD31 and Flk-1 expression and absence of smooth muscle alpha-actin expression. CONCLUSIONS: Diabetes in BB rats impairs angiogenic sprouting from cells residing in the vascular wall, independent of effects on circulating cells or circulating angiogenic/anti-angiogenic factors. The angiogenic impairment of diabetic sprout cells is, to some extent, imprinted upon the cells.

Cytomegalovirus-enhanced development of transplant arteriosclerosis in the rat; effect of timing of infection and recipient responsiveness.

Cytomegalovirus (CMV) is put forward as a risk factor for transplant arteriosclerosis (TA). In this article, we studied CMV-enhanced development of TA in rats in different donor/recipient combinations in relation to the timing of infection. Recipient rats transplanted with an aortic allograft (BN to Lew) were infected with rat CMV (RCMV) at different time-points relative to transplantation. The virus-induced effects on TA development were also determined in other strain combinations (PVG to AO and DA to WF). Finally, transmission of RCMV from aortic grafts and its effect on TA was studied. RCMV infection enhanced TA development only in Lew recipients and only after infection early post-transplantation (days 1-5). Virus transmission to the recipient only occurred from 5 and 10 days infected aortic donor-grafts, however without affecting TA development. These data indicate that the acute alloresponse and acute CMV infection need to occur simultaneously to enhance TA. This effect, however, appears to be strain combination dependent and therefore cannot be generalized.