The Effect And Mechanism Research Of Reticulocalbin-2 For The Arterial Intima Hyperplasia After Injury

Objective:Atherosclerosis is the main cause of coronary heart disease,ischemic stroke and peripheral artery disease.Atherosclerotic lesions may directly or indirectly cause arterial stenosis or occlusion,affect the blood supply of important organs,and cause corresponding ischemic symptoms.Percutaneous transluminal angioplasty and stent placement are the main methods for the treatment of occlusive arterial diseases.With the development of interventional devices and technology,endovascular treatment of occlusive arterial disease has made great progress.However,the problem of restenosis has not yet been completely solved.Neointimal hyperplasia is the main pathological process of restenosis after operation.Neointimal lesions are mainly derived from vascular smooth muscle cells,which proliferate and migrate from the medial area to the intima.Recent studies have suggested that vascular restenosis may be affected by dietary factors and genetic factors.In previous studies,we found significant differences in the susceptibility to arteriosclerosis between C3H/HeJ(C3H)and C57BL/6J mice after Apoe knockout.Through quantitative trait loci(QTL)analysis,it is found that Reticulocalbin 2(Rcn2)may be the key gene that causes this difference.In this study,we first identified the role of Rcn2 in the process of intimal hyperplasia after arterial injury by feeding high-fat diet and further explored that if knockout of Rcn2 could inhibit intimal hyperplasia through animal experiments.Secondly,it is clear that Rcn2 can inhibit the proliferation and migration of vascular smooth muscle cells and the inflammatory response induced by ox-LDL through cell test.Finally,the level of Rcn2 expression was detected in the plasma of the patients with arteriosclerosis obliterans of the lower extremities,and explore if Rcn2 could be a biological marker for predicting in-stent restenosis for femoropopliteal artery chronic total occlusion(CTO).Methods:1.In animal experiments,8-12 week old,20-25g weight C57BL/6 Apoe-/-mice(C57)and C57BL/6 Apoe-/-Rcn2-/-mice(Rcn2)were selected to establish carotid artery ligation injury model.One group of mice maintained general diet.The other group fed a high-fat diet 1 weeks before the carotid artery injury and maintained a high-fat diet until the end of the experiment.Carotid artery specimens were obtained at 1days,3 days after operation,and 1,2 and 4 weeks respectively.Pathological examination compared the area of the carotid artery,the area of the intima,the ratio of the intima and the area of the middle area.Immunohistochemical staining was used to identify the cell components of intimal lesion.The serum levels of pro-inflammatory factors(MCP-1and VCAM-1)and lipids were detected by El ISA.2,In vitro,primary cultured C57 Apoe-/-mouse aortic smooth muscle cells were selected and stimulated with different concentrations of ox-LDL(50ug/ml,100ug/ml,150ug/ml,200ug/ml)to stimulate vascular smooth muscle cells,and Rcn2 expression at mRNA level and protein level was detected.Vascular smooth muscle cells were transfected by siRNA,and the migration ability of vascular smooth muscle cells was detected by scratch test.MTT was used to detect the proliferation of vascular smooth muscle cells.The content of MCP-1 and VCAM-1 in cell supernatant was detected by ELISA.3,258 consecutive patients of femoralpopliteal artery CTO from January 2012 to December 2014 were selected.180 of the patients were included in the study,and the level of Rcn2 expression in the plasma of62 patients was detected and compared with 15 healthy controls.The correlation between the ratio of neutrophil to lymphocyte(NLR)and restenosis was also analyzed.Results:1.In the general diet group,C57 mice developed mild hyperlipidemia,the total cholesterol level was 308.5+60.3mg/dl,while the total cholesterol level in the high-fat diet group was 858.5+88.4mg/dl,which was severe hyperlipidemia(p<0.001).The levels of HDL cholesterol in the two groups were very low,and the levels of triglycerides were equal(159.3+37.1 mg/dl and 149.5+19.2mg/dl,p>0.05).The hyperlipidemia group appeared intimal hyperplasia 1 weeks after ligation,and the high fat diet group appeared intimal hyperplasia at the 2 week after ligation.With the increase of time,the two groups of intimal lesions were all increased.At the 4 week after ligation,the average intimal hyperplasia area of the carotid artery in the high-fat diet group was about 4 times higher than that in the general diet group[(112987±11395m~2 and 29539±7001 m~2,respectively);n=5,P<0.001].Histologically,1 weeks after the high fat diet and 2 weeks after the common diet,the intima was mainly composed of foam cells.At 2 weeks after the high fat diet,intimal hyperplasia is a typical fibrous plaque,which contains many foam cells covered by a fibrous cap.At 4 weeks,the intimal lesions of the two diet groups developed to a more advanced stage,including the narrowing of the neovascularization and the arterial lumen.The expression of Rcn2 is low in the intimal lesions of the geneal diet group,and is highly expressed in the intimal lesions of the high fat diet group.The plasma concentration of Rcn2 in the high fat diet group was significantly higher than that in the general diet group.(18.93+1.76ng/ml,33.03+15.01ng/ml,n=5,p=0.029).The high-fat diet Rcn2 mice had mild hyperlipidemia,the total cholesterol level was significantly lower than that of C57 mice(264±56mg/dl and 858.5±88.4mg/dl,n=5,p<0.001),HDL cholesterol and triglyceride were not different in the two groups.Rcn2 knockout obviously inhibited the intimal hyperplasia of the carotid artery in C57 Apoe-/-mice under the high fat diet.It mainly inhibits the migration of vascular smooth muscle cells from the middle layer to the intima,and inhibits the expression of MCP-1 in the vascular wall cells.At 4 weeks after operation,the concentrations of MCP-1(202.3±39.6ng/ml,349.3±67.1ng/ml,n=5,p=0.009)and VCAM-1(110.6±20.7ng/ml,240.3±50.1ng/ml,n=5,p=0.002)in plasma of Rcn2 mice were significantly lower than those of the mice.2,we choose different concentrations of ox-LDL(50ug/ml,100ug/ml,150ug/ml,200ug/ml)to stimulate vascular smooth muscle cells for 4 hours,the mRNA expression and protein level of Rcn2 are highest at150ug/ml.After transfection of Si Rcn2 into vascular smooth muscle cells,ox-LDL was stimulated for 24 hours.MTT test showed that Rcn2 could inhibit ox-LDL induced smooth muscle cell proliferation(p<0.05).The number of vascular smooth muscle cells passing through scratches was significantly reduced(p<0.05)after silenting Rcn2 by a flat scratch test.The concentration of MCP-1 and VCAM-1 in the supernatant of ELISA showed that silent Rcn2 inhibited the secretion of MCP-1 and VCAM-1(p<0.05)by ox-LDL induced smooth muscle cells.3,a total of 180 patients who underwent stent implantation of the femoralpopliteal artery CTO(male 141 cases,female 39 cases)were included in this study.According to the occurrence of ISR and the time of ISR,the patients were divided into 3 groups:non ISR group,early ISR group(1 years)and late ISR group.There were significant differences in baseline NLR,diabetes,hyperlipidemia,mean lesion length,and the number of outflow channels in the non ISR group and the early ISR group.In the early ISR group,baseline NLR was significantly higher than that in the late ISR group(P=0.03),and there was a difference in the rate of balloon dilation(P<0.01)and the incidence of distal embolization(P<0.01)between the two groups during the treatment of ISR.Univariate analysis showed that baseline NLR is more than3.62 and the early ISR,NLR<3.62 and NLR=3.62 of baseline group of early ISR rates were 16%and 32.72%(P<0.01).Multivariate logistic analysis showed that the baseline NLR=3.62 is a independent predictor of early ISR in patients with femoralpopliteal artery CTO.The plasma concentrations of Rcn2 were measured in 62 patients and 15healthy controls.Restenosis was divided into no restenosis group and restenosis group based on the 1 years of follow-up.It was found that the concentration of Rcn2 in the three groups was statistically different(F=29.59,P<0.001).The concentration of Rcn2was positively correlated with NLR,and the concentration of Rcn2 was associated with low density lipoprotein cholesterol and the degree of ischemia.The operator's operation characteristic curve analysis(ROC)uses Rcn2 and early ISR to define the Rcn2 cut-off value.The area under the ROC curve was 0.901,the concentration of Rcn2 was 80.43ng/ml,and the sensitivity and specificity were 79.92%and 83.72%respectively(p<0.001).Conclusion:1.High fat diet obviously aggravate the intimal hyperplasia of C57 mice after carotid artery injury.Intimal lesions mainly include vascular smooth muscle cells,foam cells and neovascularization.Rcn2 is highly expressed in the intima lesions of C57mice induced by high fat diet.Rcn2 knockout can obviously inhibit the hyperplasia of intima.It mainly inhibits the proliferation,migration and inflammation of vascular smooth muscle cells.2,ox-LDL can induce the increase of Rcn2 expression in vascular smooth muscle cells.Silencing Rcn2 can effectively inhibit the proliferation and migration of vascular smooth muscle cells induced by ox-LDL.Silencing Rcn2 can effectively inhibit the secretion of proinflammatory cytokines in vascular smooth muscle cells induced by ox-LDL.3,Baseline NLR more than 3.62 is an independent predictors of early ISR femoralpopliteal artery stenting.Baseline NLR is associated with the degree of difficulty of reintervention of and occurrence of distal embolism.The expression of Rcn2 in femoralpopliteal artery CTO increased significantly.Rcn2 has a positive correlation with NLR and is related to the degree of lower limb ischemia and low density lipoprotein cholesterol levels in the patients.Rcn2 could be a biological marker for restenosis after interventional therapy in arteriosclerosis obliterans of the lower extremities.