Expression Of MicroRNA-145 And Its Phenotypic Modulation Of Smooth Muscle Cell In Rat Carotid Allograft Vasculopathy

PART I Optimization and evaluation the model of rat carotid allograft vasculopathyObjective:To optimize the model of rat carotid transplantation and to evaluate whether the model can be used as an animal model to study cardiac allograft vasculopathy.Methods:Bilateral carotids from Wistar as donors were provided to the receptors of Wistar and BN respectively. High frequency ultrasound was used to detect the graft artery at four time points of 1 week,4 weeks,8 weeks and 16 weeks post-transplant, and neointima formation was observed. Six animals in each group were sacrificed at each time point and transplanted arteries were harvested. H&E staining, Masson staining, immunohistochemical staining of Ki-67 and a-SMA, double immunofluorescent labeling of Ki-67 and a-SMA and confocal imaging was used to evaluate neointimal formation and cell composition and whether they were in proliferative state. Stenosis rate was calculated by using both computer image processing system and data from high frequency ultrasound.Results:Forty eight out of 50 operations were successful (96%) and 2 were failure of thrombogenesis immediately post-transplant. There was neither postoperative infection nor death and weight of animals began to recover 5 days after operation. Obvious neointima was observed in allogeneic group at 4 weeks post-transplant. Neointima which was concentric became thicker over time and significant luminal stenosis was observed at 16 weeks post-transplant. However, only when 16 weeks post-transplant formed a few neointima in isogeneic group. High frequency ultrasound effectively detected the initial and progress of neointimal formation. There were a large number of a-SMA-positive cells in neointima and some of them were also ki-67 positive as confocal showed. a-SMA positive cells in media were significantly reduced and ki-67-positive cells in media and adventitia were significantly increased in allogeneic group(p<0.001). Stenosis rate calculated by computer image system and data from high-frequency ultrasound from 4 weeks to 16 weeks post-transplant in allogeneic group was 17.3%,37.6%,62.4% and 13.2%, 26.3%,42.0% at each time point respectively.Conclusions:Characteristics of allograft vasculopathy from carotid orthotopic transplantation of inbred rat consistent with that of CAV. The main cellular component of neointima was smooth muscle cell and some of them were in proliferative state. Smooth muscle cells in the media were dedifferentiated and then migrated to intima to form neointima. This model can be used as one animal model to study CAV.PART II Expression of microRNA-145 and its phenotypic modulation of smooth muscle cell in allograft vasculopathyObjective:To test the expression of microRNA-145 in the course of allograft vasculopathy (AV), and to explore the underling mechanism that microRNA-145 control the phenotypic switch of smooth muscle cell.Methods:Animals were divided into isograft group (n=24), allograft group (n=24) and normal control (n=16). Six and four animals were sacrificed in transplant group and normal control respectively at each time point of 1 week,4 weeks,8 weeks,16 weeks post surgery. Transplanted arteries and peripheral blood were harvested. Total RNA of artery and serum was extracted by using special RNA extraction kit. qRT-PCR technology was used to detect the expression of microRNA-145, SRF, Myocardin, KLF4 in arteries, and microRNA-145 in serum.Results:The expression of microRNA-145 was significantly decreased 1 week post-transplant in transplanted arteries. However, it was gradually increasing in syngeneic group from 4 weeks post-transplanted to 16 weeks and up to half of normal level in the end point, while that in allogeneic group was decreased to the minimum at 4 weeks post-transplanted and then increased slowly, and still significantly lower than normal level until 16 weeks post-transplanted (p<0.001). The expression of SRF and Myocardin was consistent with that of microRNA-145, with increasing of SRF, microRNA-145 expression was upregulated (r=0.887); the expression of Myocardin was increased with the increasing of microRNA-145 (r=0.996), suggesting that SRF can promote microRNA-145 expression, while microRNA-145 can promote Myocardin expression. Differently, KLF4 was negatively regulated by microRNA-145 (r=-0.911). With the decreasing of microRNA-145, expression of KLF4 was increased, when microRNA-145 expression was upregulated, KLF4 expression was downregulated. Expression of microRNA-145 in serum was significantly decreased 1 week post-transplanted, while begin to increase at 4 weeks post-transplanted and has been restored to the normal level in 16 weeks post-transplanted.Conclusions:MicroRNA-145 expression was significantly downregulated in the process of AV. MicroRNA-145 which was regulated by SRF, regulated its target genes such as Myocardin and KLF4 to control the phenotypic switch of smooth muscle cell in AV and promising as diagnostic and therapeutic targets.