TRPV4 Regulates The Migration And The Tube Formation Of Human Retinal Capillary Endothelial Cells

Background Transient receptor potential channel vanilloid（TRPV）4 is an important non-selective cationic protein channel.It is mainly distributed in vascular endothelial and smooth muscle cells,the function of TRPV4 in vascular endothelial cells has received much attention.TRPV4 plays a critical role in regulating the Ca²⁺ signaling pathway.It can participate many in vascular function,such as vascular tone and angiogenesis,by regulating the concentration of Ca²⁺ in vascular endothelial cells.Several studies have shown that activation of TRPV4 channels contributes to angiogenesis.4α-PDD promotes the growth of cerebral vascular collateral.In addition,some studies have shown that TRPV4 may be involved in tumor angiogenesis.There has a significant increase about TRPV4’s expression and activity in breast cancer vascular endothelial cells compared with normal skin microvascular endothelial cells.Arachidonic acid can promote tumor source through activate TRPV4.However,there has not been reported about the function of TRPV4 in human retinal capillary endothelial cells（HRCECs）.This study observed the expression and distribution of TRPV4 in HRCEC,then,we analyzed the effect of TRPV4 on Ca²⁺ influx in HRCECs.Secondly,we observed the effect of TRPV4 on HRCECs cell migration ability.Finally,we further verified the ability of TRPV4 on forming HRCECs.Method 1.In this study,we investigated the expression and distribution of TRPV4 in HRCECs by using Western blot analysis and immunofluorescence staining.Secondly,weused Calcium imaging to investigate the Ca²⁺ influx of TRPV4 in HRCECs.The effect of TRPV4 on Ca²⁺ influx in HRCECs cells was examined after treatment of HRCECs with TRPV4 specific agonist GSK and inhibitor HC067047.2.Secondly,we used the method of cell migration to detect the migration ability of HRCECs after treating HRCECs with GSK,HC067047 or sh RNA.3.Finally,we used Tube-formation assay experiments to determine the tube-forming ability of HRCECs.Results 1.Firstly,TRPV4 is expressed in HRCECs by Western blots and immunofluorescence assays,we also found a robust increase in intracellular Ca²⁺ levels after stimulating cells with GSK,while pretreatment with HC067047 significantly inhibited this effect.2.Secondly,we performed transwell assays with or without a TRPV4 inhibitor and found that TRPV4 can promote the migration of HRCECs.3.Finally,we found that TRPV4 can promote tube formation of HRCECs,GSK increased tube formation,while pretreatment with HC067047 and sh RNA blocked the effect.Conclusion 1.TRPV4 is expressed in HRCECs.2.TRPV4 is involved in the regulation of cellular function by mediating Ca²⁺ influx of HRCECs.3.TRPV4 promotes the migration ability of HRCECs by mediating Ca²⁺ influx,which can provide a basis evidence for the occurrence and development of angiogenesis.4.TRPV4 promotes the tube formation of HRCECs by mediating Ca²⁺ influx,which can provide a new target for the treatment of retinal neovascular diseases.