The Role Of Platelet Microparticles In Proliferation,Apoptosis And Autophagy Of Vascular Smooth Muscle Cells

After vascular injury,ECs are destroyed,and VSMCs are exposed and underwent functional changes such as abnormal proliferation,migration,autophagy,and apoptosis.However,with the process of the neointima formation,the molecular mechanism of VSMCs dysfunction during intimal injury is still unclear.Previous Studies have shown that after vascular injury,platelets can be activated and release PMPs,and participate in cell-to-cell communications.miRs play an important role in a variety of intracellular activities.This paper focuses on the function of PMPs-derived miRs in VSMCs proliferation,apoptosis and autophagy.In this paper,a model of rat carotid intima injury was constructed.The contralateral side was used as control.HE and immunofluorescence staining were used to detect the morphology of injured blood vessels and the protein expression of vWF,?-SMAand CD41.Based on miRs chip results,screening for highly expressed miRs.Fluorescence in situ hybridization was used for detection of platelets-derived miRs in VSMCs after vascular injury.RT-PCR was used to detect the expression levels of platelets-derived miRs in VSMCs after PMPs stimulation.Bioinformatics software combined with dual luciferase assay was used to detect miRs' s target gene.MiRs mimics or inhibitor was used to up-regulate or down-regulate miR-92a-3p or miR-30 in VSMCs and siRNA was used to inhibit target gene expression in VSMCs.Western blotting was used to detect protein expression levels of target genes and autophagy related proteins in VSMCs.Annexin V/PI combined with flow cytometry was used to detect apoptosis levels of VSMCs.BrdU ELISA was used to detect proliferation of VSMCs.Cyclic stretch with the magnitude of5% or 15% and frequency of 1.25 Hz was subjected to VSMCs in vitro by using FX-5000 T cyclic stretch loading system.Flow cytometry was used to detect the adhesion of PMPs in VSMCs.Cellular immunofluorescence was used to detect the expression of LC3 in VSMCs.The results showed that:(1)There were platelet aggregation and abnormal proliferation of VSMCs after intimal injury.After 4 weeks of injury,neointima occurred in the injury vascular and proliferating cells were mainly VSMCs.At the same time,platelets adhered to the exposed VSMCs.(2)PMPs regulate VSMCs proliferation through miR-92a-3p/ PTEN signalingpathway.Up-regulation of miR-92a-3p can significantly promote the proliferation of VSMCs.miRs chip results showed that miR-92a-3p was highly expressed in platelets.Fluorescence in situ hybridization confirmed that miR-92a-3p expression in neointimal VSMCs was higher than that of normal arteries.Up-regulation and down-regulation of miR-92a-3p combined with dual luciferase assay confirmed that the target gene of miR-92a-3p is PTEN.BrdU ELISA confirmed that miR-92a-3p up-regulation can promoted proliferation.(3)PMPs regulate VSMCs apoptosis through miR-30/Runx2 signaling pathway.Apoptosis of VSMCs was significantly increased after PMPs stimulation.miRs chip results showed that miR-30a-5p,miR-30b-5p,miR-30c-5p,miR-30d-5p,miR-30e-5p were highly expressed in platelets.RT-PCR detection the expression of miR-30a-5p,miR-30c-5p,miR-30d-5p and miR-30e-5p in VSMCs was significantly increased after stimulation with PMPs.Up-regulation of miR-30c-5p and miR-30e-5p was significantly promoted VSMCs apoptosis.Bioinformatics software predicts the target gene of miR-30 is Runx2.Both PMPs stimulated and up-regulated miR-30e-5p can inhibit the expression of Runx2.The dual luciferase assay confirmed that miR-30e-5p directly regulate Runx2.In addition,down-regulated Runx2 significantly promoted VSMCs apoptosis.(4)PMPs promote autophagy of VSMCs.Compared with 5% cyclic stretch,VSMCssignificantly increased the adhesion level of PMPs after 15% cyclic stretch.Immunofluorescence detected that the expression of LC3 was significantly increased in VSMCs after PMPs stimulation.At the same time,the protein expression levels of ATG5,ATG7 and ATG12 were significantly increased.Our study revealed that PMPs stimulation can up-regulation the expression of miR-92a-3p and miR-30e-5p in VSMCs,promoting proliferation of VSMCs via miR-92a-3p/ PTEN signaling pathway,and promote VSMC apoptosis via miR-30e-5p/ Runx2 signaling pathway.High cyclic stretch may enhance the autophagy of VSMCs by promoting the adhesion of PMPs to VSMCs and increasing the expression of ATG5,ATG7,ATG12 and LC3.This study suggests that PMPs and PMPs-derived miRs play an important role in the cell-cell communications between platelets and VSMCs.This study provides a new perspective for the search for potential targets for the diagnosis,clinical treatment,and efficacy evaluation of intimal injury vascular reconstruction diseases.