Effects Of SOCC-related Molecules On Differential Regulation Of VSMC And VEC Proliferation In Rats

Objective:Coronary heart disease is a major disease that threatens human health.At present,one of the main treatment methods for coronary heart disease is percutaneous coronary intervention(PCI).Remove the bracket.While inhibiting vascular smooth muscle cells to prevent postoperative restenosis,drug stents also delay arterial re-endothelialization,and the possible complications are mainly thrombosis.In order to prevent complications,patients after PCI often need to take dual antiplatelet therapy over 1 year.Although it can prevent thrombosis,it also increases the risk of bleeding complications.Most of the current research focuses on how to fully degrade the adhesive material between the stent and the drug,or the fully degradable stent itself(biodegradable stent),but there is less research on the drug coating of the stent.As an important second messenger in cells,calcium ions play an important role in the process of cell proliferation,among which store-operated calcium channels(SOCC)are widely expressed in various mammalian cells,including smooth muscle cells,endothelial cells,neural Domestic and foreign studies have shown that SOCC participates in the proliferation of various cells by regulating the concentration of calcium ions in cells.In this study,starting from the calcium store-operated calcium channel that regulates proliferation,we hope to find a target molecule from SOCC-related molecules that inhibits the proliferation of smooth muscle cells(VSMC)without affecting the proliferation of endothelial cells(VEC).The main flaws provide new research directions.Methods:Part I(SOCC-related molecule expression level):we select 12 SOCC-related molecules expressed in rat vascular smooth muscle cells and vascular endothelial cells:STIM1,STIM2,Orai1,Orai2,Orai3,NCX1,NCX2,TRPC1,TRPC3,TRPC4,TRPC5 and TRPC6 were cultured in primary rat coronary artery smooth muscle cells and endothelial cells.Western Blot and RT-PCR were used to detect the protein and mRNA expression levels of SOCC-related molecules in primary rat coronary artery VSMC and VEC,respectively.Preliminary screening target molecule.Part II(gene transfection and proliferation detection):the sh-TRPC3 adenovirus interference vector and the sh-TRPC5 adenovirus interference vector were constructed,and the interference efficiency was detected by RT-PCR.The proliferation of vascular smooth muscle cells and vascular endothelial cells was detected by CCK-8 and PCNA,respectively,and the distribution of cell cycle was detected by flow cytometry.The intracellular calcium pool was depleted by thapsigargin,and the dynamic change of intracellular calcium ion concentration was measured by calcium ion imaging technology to further determine the role of target molecules in the proliferation of VSMC and VEC.Part III(in vivo verification):the SD rat carotid artery balloon pull-off model was established,and after treatment with sh-TRPC3 and sh-TRPC5,HE staining,immunohistochemistry,immunofluorescence and other methods were used to analyze the proliferation of angiogenesis intima and vascular media.Paamycin was used as a control to compare the repair of endothelial cell layers and to verify the effect of target molecules in in vivo experiments.Results:Part I:The primary rat coronary artery smooth muscle cells and endothelial cells that can be stably passaged were successfully established.VSMC were triangular or short-fusiform in the early stage,which can be stably transmitted to the 10th generation;VEC cells are polygonal in the early stage when the density is low,and there may be slender pseudopodia between cells,showing a typical "paving stone-like" distribution.,which can be stably transmitted to the 9th generation.After detecting the protein expression level and mRNA expression level,it was found that the trend of the protein expression level and mRNA expression level of SOCC-related molecules was consistent.The expressions of STIM1,TRPC1,TRPC3 and TRPC5 in VSMC were significantly higher than those in VEC(P<0.05).The expression of NCX1,STIM2,Orai1,Orai2,TRPC4,TRPC6 was lower than that of VEC(P<0.05),and there was no significant difference between NCX2 and Orai3 in the two cells(P>0.05).Combined with the literature,STIM1 and TRPC1 can inhibit the proliferation of endothelial cells,which is inconsistent with the research purpose,so we chose TRPC3 and TRPC5 as candidate factors for subsequent experiments.Part Ⅱ:it is successfully that transfected sh-TRPC3 and sh-TRPC5 adenovirus interference vectors to VSMC and VEC,and after transfecting cells to detect the interference efficiency,select the best adenovirus interference vector for the experiment.CCK-8 proliferation assay found that TRPC3 or TRPC5 could inhibit the proliferation of smooth muscle cells,but had no significant effect on endothelial cell proliferation.The distribution of cell cycle showed that in VSMC,interference with TRPC3,compared with NSC group,decreased G0/G1 phase,decreased S phase,and increased G2/M phase(P<0.05);and after interference with TRPC5,compared with NSC group,G0/G1 phase decreased,G2/M phase increased(P<0.05),S phase remained basically unchanged,indicating that interfering with TRPC3 or TRPC5 blocked the cell cycle of VSMC in G2/M phase.In VEC,interference with TRPC3 or TRPC5 had no significant changes in G0/G1 phase,S phase,and G2/M phase,indicating that interference with TRPC3 or TRPC5 had no significant effect on the cell cycle of VEC.The detection of cell proliferating nuclear antigen(PCNA)showed that the expression of PCNA decreased significantly when TRPC3 was interfered with,compared with the control group(P<0.05).The percentage of cells in S phase after TRPC5 was not significantly affected.Interference with TRPC3 or TRPC5 had no significant effect on VEC proliferation.The dynamic changes of calcium ion concentration detected by calcium imaging technology showed that in VSMC,interfering with TRPC3 or TRPC5 could inhibit thapsigargin(TG)-mediated calcium influx(P<0.05),but the effect was not obvious in VEC(P>0.05).Part III:the successful establishment of the SD rat carotid artery balloon pull-out model.Interfering with TRPC3 or TRPC5 could inhibit the formation of neointima(P<0.05).Interfering with TRPC3 or TRPC5 had no effect on the vascular media(P>0.05).Interfering with TRPC3 or TRPC5 had no significant effect on the repair of the endothelial cell layer.Compared with rapamycin,the repair effect of the endothelial cell layer was better than that of rapamycin(P<0.05).Conclusions:1.The expression levels of SOCC-related molecules in rat coronary VSMC and VEC were compared for the first time,and differences were found:STIM1,TRPC1,TRPC3,TRPC5 were expressed at higher levels in rat coronary VSMC,while NCX1,STIM2,Orai1,Orai2,TRPC4,and TRPC6 were more expressed in VEC,suggesting that SOCC-related molecules may have differences in regulating the proliferation of VSMC and VEC.2.Silencing TRPC3 or TRPC5 can change the distribution of VSMC cell cycle,arrest the cell cycle of VSMC in G2/M phase,and then inhibit the proliferation of VSMC,but silencing TRPC3 or TRPC5 does not affect the proliferation of VEC.3.In VSMC,silencing TRPC3 or TRPC5 inhibited thapsigargin-mediated calcium influx,but had no significant effect in VEC.4.Silencing TRPC3 or TRPC5 in SD rats can effectively inhibit the formation of neointima,but does not hinder the repair of endothelial cell layer.TRPC3 and TRPC5 are expected to provide new research directions for the study of drug-eluting stent coating drugs.