The Effect And Mechanism Of Epsin In Carotid Intimal Hyperplasia

Objective: Carotid artery stenosis is the main causes of ischemic stroke.Due to the advantages of low risk of injury and fewer surgical complications,intravascular intervention has gradually become one of the main treatment methods for carotid artery stenosis.Studies have found that the formation of carotid restenosis is closely related to neointimal hyperplasia,which limits the long-term efficacy and safety of carotid angioplasty.Therefore,exploring the progression mechanism of carotid neointimal formation has important clinical significance for preventing the development of intimal hyperplasia and the formation of restenosis.Epsin is a protein family involved in reticuloprotein dependent endocytosis,and its members include Epsin1 and Epsin2.Studies have found that Epsin1 and Epsin2 play a very important role in maintaining normal cardiovascular development and nervous system homeostasis.Therefore,this study intends to use endothelial cells as a test hypothesis to explore the role and possible mechanism of Epsin in carotid intimal hyperplasia.Methods: Twelve adult clean male C57BL/6J mice,each weighing about 24 g,were randomly divided into two groups,six in each group,which were divided into sham operation group and carotid artery ligation model group.Immunohistochemical staining was used to observe the expression of Epsin1,Epsin2,and CD31 labeled endothelial cells in the carotid vascular tissues of mice in the carotid artery ligation group and sham group7 days after the operation.After being treated with small interfering RNA from endothelial cells Epsin1 and Epsin2 for 24 hours,they were stimulated with VEGF for 24 hours.Further,Western blotting was used to detect the expression changes of Epsin1 and Epsin2 on endothelial cell proliferation indicators PCNA,mitochondrial dynamic proteins Drp1 and OPA1,and endothelial matrix metalloproteinase MMP2.The effects of Epsin 1 and Epsin 2 on the migration function of endothelial cells were observed using migration and scratch experiments.Mito Traker staining was used to detect the effects of Epsin1 and Epsin2 on the mitochondrial morphology of endothelial cells,and JC-1 staining was used to detect the effects of knocking down Epsin1 and Epsin2 on the mitochondrial potential of endothelial cells.Finally,a co culture model of endothelial cells and smooth muscle cells was established.Upper endothelial cells were knocked down for 24 hours and stimulated with VEGF for 24 hours.Western blotting was used to detect the proliferative index PCNA of smooth muscle cells.Migration and scratch experiments were used to detect the migration ability of smooth muscle cells.Results: In the carotid artery ligation group,Epsin1 and Epsin2 were significantly higher in endothelial cells than in the sham group.Knocking down Epsin promoted endothelial cell proliferation and migration,protected endothelial cell function by inhibiting the expression of Drp1 in endothelial cells,while knocking down Epsin1 and Epsin2 reduced the secretion of MMP2 in endothelial cells,indirectly affecting the proliferation and migration of co cultured smooth muscle.Finally,a co culture model of endothelial cells and smooth muscle cells was established.Upper endothelial cells were knocked down for24 hours and stimulated with VEGF for 24 hours.Western blotting was used to detect the proliferative index PCNA of smooth muscle cells.Migration and scratch experiments were used to detect the migration ability of smooth muscle cells.Conclusion:This experimental data can conclude that the down-regulation of Epsin expression in blood vessels may be an important strategy for the treatment of carotid intimal hyperplasia.