Study On The Effect And Mechanism Of GDF11 On Re-endothelialization Of Injured Blood Vessels

Chapter 2 The effect of GDF11 on the remodeling of rat vascular structure after balloon injury of carotid arteryObjective:In this study we delivered adenovirus carrying GDF11 into the balloon-injured carotid artery area of rats by using intravascular injection to overexpress GDF11.We aimed to study the effects of GDF11 on the re-endothelialization and neointima formation in rats after carotid artery balloon injury.Methods:An adult Sprague-Dawley rat model of common carotid artery balloon dilatation injury was surgically established.A recombinant adenovirus carrying GDF11 was delivered into the common carotid artery and kept in situ for 30 min to overexpress GDF11.The animal models were randomly divided into four groups:normal group,without carotid artery balloon injury;carotid artery balloon injury group,no adenovirus injection after carotid balloon injury;vector control group,injection of adenovirus vector control virus after carotid artery balloon injury;GDF11 overexpression group,injection of adenovirus overexpressing GDF11 after carotid artery balloon injury(n=10 in each group).One week after the carotid artery balloon injury model was established,Evans blue staining and immunohistochemistry were used to detect the re-endothelialization of the balloon-injured blood vessels in rats.Two weeks after the establishment of the carotid artery balloon injury model,hematoxylin-eosin(HE)staining was used to detect neointima formation after balloon injury in rats.Results:After carotid artery balloon injury in rats,the expression level of GDF11 in the injured carotid artery did not change significantly.After adenovirus carrying GDF11 was transfected into rat carotid artery with balloon injury,the expression level of GDF11 in the GDF11 overexpression group increased,but there was no significant change between the vector control group and carotid artery balloon injury group.When the model was established 1 week,compared with the carotid artery balloon injury group,the results of Evans blue staining and immunohistochemical detection of CD31 showed that the re-endothelialization rate of the GDF11 overexpression group was significantly increased;when the model was established 2 weeks,the results of HE staining showed the neointima formation in the GDF11 overexpression group was significantly reduced.Conclusion:GDF11 promotes the re-endothelialization of balloon-injured blood vessels and improves the neointima formation of injured blood vessels.Chapter 3 The role and mechanism of GDF11 in protecting vascular endothelial cellsObjective:To verify the hypothesis that the protective effect of GDF11 on endothelial cells after carotid artery balloon injury and related mechanisms at the animal and cellular levels.Methods:The animal model construction and grouping are the same as the Chapter 2.Two weeks after carotid artery balloon injury,western blot was used to detect the expression of NLRP3 inflammasome-associated protein in the carotid area.At the cell level,lysophosphatidylcholine(lysoPC)induced human umbilical vein endothelial cells(HUVECs)damage model was established.The models were randomly divided into five groups:normal group,without any treatment;lysoPC group,HUVECs was cultured with lysoPC;GDF11 treatment group,GDF11(50ng/ml)pretreatment before lysoPC treatment cells;lysoPC+si-LOX-1 group,si-LOX-1 pretreatment before lysoPC treatment cells;lysoPC+4PBA group(4PBA(5mM)pretreatment before lysoPC treatment of cells)(n=5 in each group).HUVECs were treated with different concentrations of lysoPC to obtain the best concentration of lysoPC to induce cell damage in this experiment.CCK-8 was used to detect the effect of GDF11 pretreatment on lysoPC-induced cell damage.Western blot was used to detect the effect of GDF11 pretreatment on the expression of NLRP3 inflammasome-associated proteins in lysoPC-induced cells.When knocking down the expression of LOX-1 or applying the endoplasmic reticulum stress(ER stress)inhibitor 4PBA,western blot was used to detect the effect of lysoPC on the expression of NLRP3 inflammasome-related proteins in cells.Besides,the effect of knocking down the expression of LOX-1 on the expression of ER stress related proteins was tested by western blot.Western blot was also used to detect the effect of GDF11 pretreatment on the expression of LOX-1 and ER stress related proteins induced by lysoPC.Results:When the rat carotid artery balloon injury model was established for 2 weeks,compared with the carotid artery balloon injury group,western blot showed that the expression of NLRP3 inflammasome-related protein in the GDF11 overexpression group was significantly reduced.There was no significant change in the expression of NLRP3 inflammasome-related proteins in the vector control group.In the lysoPC-induced HUVECs injury model,compared with the lysoPC group,the activity of HUVECs in the GDF11 treatment group was significantly increased.Western blot showed that the expression of NLRP3 inflammasome-associated proteins in the GDF11 treatment group was significantly reduced.LysoPC can induce cell LOX-1 expression and ER stress related proteins expression.When the expression of LOX-1 was knocked down or the ER stress inhibitor 4PBA was applied,lysoPC induced a significant decrease in the expression of NLRP3 inflammasome-related proteins in cells.In addition,knockdown of LOX-1 can also inhibit the expression of ER stress-related proteins.The expression of LOX-1 and the expression of ER stress related proteins in the GDF11 treatment group were also significantly reduced.Conclusion:GDF11 can reduce endothelial cell damage;GDF11 can protect endothelial cells by inhibiting LOX-1 and ER stress related pathways to curb the expression of NLRP3 inflammasome-related proteins.Chapter 4 The effect of GDF11 on pyroptosis of vascular endothelial cellsObjective:At the cellular level,to study the protective effect of GDF11 on lysoPC-induced vascular endothelial cell damage by inhibiting pyroptosis of vascular endothelial cells.Methods:At the cell level,lysoPC induced HUVECs damage model was established as previous drescribed.The models were randomly divided into three groups:normal group,without any treatment;lysoPC group,HUVECs was cultured with lysoPC;GDF11 treatment group,GDF11(50ng/ml)pretreatment before lysoPC treatment cells(n=5 in each group).LDH release experiment and Hoechst/PI staining experiment were used to detect the effect of GDF11 pretreatment on lysoPC-induced cell plasma membrane damage.The caspase-1 enzyme activity experiment was used to detect the effect of GDF11 on lysoPC induced cell caspase-1 enzyme activity.Western blot was used to detect the effect of GDF11 on the expression of GSDMD-N,an active protein in HUVECs induced by lysoPC,which can promote the formation of plasma membrane holes.Besides,when knocking down the expression of LOX-1 or applying the ER stress inhibitor 4PBA,western blot was used to detect the effect of lysoPC on the expression of GSDMD-N.Results:In the lysoPC induced cell injury model,compared with the lysoPC group,HUVECs LDH release in the GDF 11 treatment group was significantly reduced,and the number of PI-positive cells was also significantly reduced in the GDF 11 treatment group.The caspase-1 enzyme activity in the GDF 11 treatment group was significantly reduced compared with the lysoPC group.LysoPC can induce the expression of GSDMD-N protein in cells.Inversely,in GDF11 treatment group GSDMD-N protein expression significantly decreased.In addition,western blot results further showed that knockdown of LOX-1 can inhibit the expression of GSDMD-N protein.Besides,ER stress inhibitor 4PBA also can inhibit the expression of GSDMD-N protein induced by lysoPC.Conclusion:GDF11 can reduce plasma membrane damage of vascular endothelial cells induced by lysoPC.GDF 11 can inhibit the expression of plasma membrane pore-forming protein GSDMD-N and inhibit pyroptosis of vascular endothelial cells.The inhibition of vascular endothelial cell pyroptosis by GDF11 is related to the inhibition of LOX-1 expression and inhibition of ER stress.