Expression Of RCAN1 And CnA In Tissues Of In-stent Restenosis After Intervention Of Lower Extremity Atherosclerotic Obliterans And Significance

Objective:To investigate the expression of the regulator of calcineurin 1(RCAN1)and calcineurinA(CnA)in tissues of stent restenosis after intervention of atherosclerotic obliterans(ASO),and to explore the relationship with restenosis.Methods:Superficial femoral arterial tissue from 15 arteriosclerosis obliterans patients undergoing lower extremity amputation for in-stent restenosis in our department from September 2013 to June 2016 were collected.Accord to the position of the stent,the arterial tissue is divided into three groups: the stenosis groups(Restenosis),the proximal end of the stent groups(Proximal),and the distal end of the stent groups(Distal).The morphological changes of target vascular were observed by optical microscope after HE stain and Masson stain.Western blotting was used to detect the protein levels of RCAN1,CnA,Proliferating Cell Nuclear Antigen(PCNA)in three groups,respectively.mRNA expression of RCAN1,CnA were observed by qPCR.The characteristics of RCAN1 and CnA distribution was observed using the methods of immunohistochemistry and immunofluorescence.In addition,co-immunoprecipitation was used to validate protein interactions between RCAN1 and CnA in vascular tissue of ASO.Result:Histological examination showed that the three groups with varying degrees of intimal hyperplasia.The vascular tissue morphological obvious changes in restenosis group after stent implicated within 3-6 months.In restenosis group,stented vascular was covered with new tissues and vascular lumen occlusion and VSMCs was observed in the tissue of stented intracavity hyperplasia.There were intimal hyperplasia and derangement of smooth muscle in proximal group.In distal group,the intima was slightly thickened,with a slight intimal hyperplasia and the endothelial cells were arranged.The expression of RCAN1 protein and mRNA in Distal groups was significantly elevated compared with Proximal groups(P<0.05).The expression of RCAN1 protein and mRNA in Proximal groups was higher than Restenosis group,with statistically significant differences(P <0.05).The CnA mRNA and protein were up-regulated in Restenosis group compared with Proximal group and Distal group(p<0.05).The expression PCNA in Restenosis groups was significantly elevated compared with Proximal groups and Distal groups(P<0.05).Immunohistochemistry showed that the numerous intimal cells were positive for smooth muscle a-actin(SMA)in Restenosis group.Immunohistochemistry and immunofluorescence analyses showed that RCAN1 and CN were mainly expressed in the cytoplasm of vascular smooth muscle cells(VSMC).Co-immunoprecipitation analyses showed that protein-protein interactions between RCAN1 and CnA in arterial tissue.Conclusion:The low expression of RCAN1 and the high expression of CnA are probably related to the occurrence of stent restenosis,and the signaling pathway of RCAN1-CN may be involved in the regulation of stent restenosis.