The Effect Of Semaphorin3A On Transdifferentiation Of Human Glomerular Endothelial Cells Induced By Hypoxia

Objective:To investigate the effect of semaphorin 3A on the transdifferentiation of human glomerular endothelial cells induced by hypoxia.Methods:Part Ⅰ:In the first part,human glomerular endothelial cells（HGECs）were divided into normal oxygen group（21%O₂）,hypoxic group 1（1%O₂）and hypoxic group 2（5%O₂）.The morphological changes of cells in each group were observed under inverted phase contrast microscope after hypoxia stimulation for different times.The expression of Semaphorin3A in the supernatant of cells in each group was detected by ELISA,and the protein levels ofα-SMA,CD31 and VE-cadherin in cells in each group were detected by Western blotting（WB）.Part Ⅱ:Human glomerular endothelial cells（HGECs）were divided into normal oxygen group（21%O₂）,hypoxia group（1%O₂）and hypoxia intervention group（1%O₂+exogenous Semaphorin3A）.After 5 days of hypoxia stimulation,the expression ofα-SMA and CD31 protein in each group was detected by Western blotting（WB）.Results:（1）Compared with the normoxic group,the morphology of endothelial cells in hypoxic group 1 and hypoxic group changed from paving-stone to fusiform or polygonal fibroblasts,and the morphology of hypoxic group 1 was the most obvious on the 5th day.Compared with the hypoxic group 1,only a few cells in the hypoxic intervention group showed morphological changes,and most of the cells still had a paving-stone appearance.（2）Semaphorin3A was expressed in the supernatant of cell culture in the normoxic group,hypoxic group 1 and hypoxic group 2.Compared with the normoxic group,the content of Semaphorin3A in the hypoxic group 1 and hypoxic group 2 was decreased,and the difference of Semaphorin3A expression among the three groups on the third day was statistically significant（P<0.05）.（3）On the 3rd and 5th day of cell culture,the expression ofα-SMA protein in hypoxic group 1 and hypoxic group 2 was increased compared with that in normal oxygen group（P<0.05）;On the first and fifth day of cell culture,compared with the normal oxygen group,the expression of CD31 and VE-cadherin in hypoxia group 1 and hypoxia group 2 decreased,with statistical significance（P<0.05）.（4）Compared with the normal oxygen group,the expression of CD31 protein in the hypoxic group and the hypoxic intervention group was significantly decreased,but the decrease in the hypoxic intervention group was more obvious than that in the hypoxic group（P<0.05）;The expression ofα-SMA in hypoxic group was significantly increased（P<0.05）.After the intervention of exogenous Semaphorin3A,the expression ofα-SMA in hypoxic group was decreased compared with that in hypoxic group（P<0.05）,but was still higher than that in normoxic group,with statistical significance（P<0.05）.Conclusion:HGECs cells can secrete Semaphorin3A.Hypoxia promoted the occurrence of End MT in HGECs and inhibited the secretion of Semaphorin3A in HGECs.Exogenous Semaphorin3A can promote the transdifferentiation of human glomerular endothelial cells induced by hypoxia.Hypoxia inhibition of Semaphorin3A secretion may be a protective response of cells under hypoxia.