A Preliminary Research On The Molecular Mechanism Of Transmembrane Protein ESDN Regulating VEGF Receptor Recycling In Vascular Endothelial Cells

Objective:Modern medical studies have shown that endothelial dysfunction is usually associated with the pathophysiological processes of several clinically common diseases and critical diseases,including hypertension,atherosclerosis?As?and coronary artery disease and other cardiovascular diseases,as well as diseases such as tumors and diabetes mellitus.Among many factors regulating endothelial cell function,vascular endothelial growth factor?VEGF?is one of the strongest growth factors that stimulate angiogenesis,and directly acts on endothelial cells and plays a role in promoting cell proliferation and neovascularization.VEGF plays its biological role by binding with three tyrosine kinase receptors,namely,vascular endothelial growth factor receptor?VEGFR-1,VEGFR-2,and VEGFR-3?,mediating a series of cell behavior including cell migration,survival and proliferation etc.Among these receptors,VEGFR-2 is the main receptor mediating the function of endothelial cells,which promotes the cascade phosphorylation of downstream signaling molecules by activating the activity of VEGFR-2 after their combination.The biological and behavioral changes of endothelial cells induced by abnormal VEGFR-2 activity are the cellular and molecular biological basis of many diseases.Among the many regulatory steps affecting the activity of VEGFR-2,the dynamic balance of endocytosis and receptor recycling is a key step in the regulation of receptor activity,which affects the process of angiogenesis and arterogenesis,and is closely related to the pathophysiological process of many cardiovascular diseases and tumor angiogenesisEndothelial and smooth muscle cell-derived neuropilin-like molecule?ESDN?is a type-I transmembrane protein originally cloned from human coronary artery and highly metastatic lung cancer cells.It contains a CUB domain and a coagulation factor V/VIII homologous structure,which is similar to the structure of Neuropilins.Previous research by the our research group has shown that ESDN promotes VEGF-mediated endothelial cell growth,migration,proliferation,permeability increase and other biological functions through regulation of VEGFR-2 activity,while down-regulation or deletion of ESDN expression has the opposite effect.However,ESDN as a receptor-regulated protein,the molecular mechanism of which regulating the dynamic balance of endocytosis and recycling of VEGFR-2 remains unclear.In this study,we used siRNA of cultured human umbilical vein endothelial cell?HUVEC?to knock down the expression of ESDN.By detecting the changes of its affinity to the markers of VEGFR-2 and receptor recycling,we explored the primary molecular mechanism of ESDN regulating endocytosis and receptor recycling.The primary culture technology of vascular endothelial cells was improved in vitro,and the cultured mouse vascular endothelial cells were used to further verify the regulation of ESDN on VEGFR-2 function.Methods:1 Methods for the culture of mouse thoracic and abdominal aortic vascular endothelial cells.Three 8-week-old WT and Esdn^-/-mice were selected.After the skin was disinfected with 75%alcohol,the thoracic and abdominal aortas of the mice were removed under sterile conditions and placed separately according to the genotype.The adipose tissue around the blood vessels was removed under the microscope and cut into vascular rings with a width of 1-2 mm,which were inoculated into matrix gel?Corning?in the ultra-clean workbench according to the genotype,and then added to the endothelial cell media?ECM,Science?containing 100 U/mL heparin?TCI?and cultured in 37?and 5%CO₂ cell incubator.2 Identification of vascular endothelial cells of thoracoabdominal aorta in mice.Because arterial vessels contain not only endothelial cells,but also smooth muscle cells and fibroblasts,it is necessary to test the specificity and purity of cultured cells.When the growth of cells reached logarithmic phase,and some cells slides were cultured on the slides.Immunofluorescence staining was performed by the specific antigen CD31 of endothelial cell surface.Some cells were lysed into protein lysate.The expression of cell surface specific receptor VEGFR-2 was detected by electrophoresis and cell specificity was detected.In addition,10⁸ cells were collected for flow cytometry analysis of CD31 monochrome fluorescence staining to detect the purity of cells.3 Culture of HUVEC and knockdown of ESDN expression by siRNA in HUVEC.HUVEC was inoculated in a 60 mm culture dish,cultured in antibiotic-free medium containing 5%fetal bovine serum?FBS?,incubated in37?and 5%CO₂ cell incubator.When the cells reached 40%-50%fusion,the cells were transfected with 20 nmol/L ESDN siRNA or general negative control?RiboBio?siRNA.The transfection was carried out according to the instructions of Lipofectamine RNA iMax kit.After 8 hours of transfection,when about 80%of the cells were fused,the medium was replaced by a low-sugar medium without FBS and antibiotics.After 16 hours of starvation,the cells were stimulated with vascular endothelial growth factor-AA?10ng/mL?at different times?0,5,15,30 minutes?.Proteins were collected and the changes of vascular endothelial growth factor signaling pathway were detected by Western blot.4 Immunofluorescence Identification of ESDN expression regulates co-localization of endocytosis marker proteins such as VEGFR-2 and EEA1,Rab5,Rab7 or Rab11 in HUVEC.To study the co-localization of endocytosis marker proteins such as VEGFR-2,EEA1,Rab5,Rab7 and Rab11 in HUVEC and its possible molecular mechanism,HUVEC were cultured to cell slides.When ESDN expression was knocked down by siRNA,the culture medium was removed at different time?0,5,15,30 minutes?when stimulated by VEGF-AA?10ng/mL?.Using 4%polyformaldehyde fixed cell slides,the VEGFR-2?anti-mouse antibody,Santa Cruz?and EEA1?anti-rabbit antibody,Cell Signaling?were labeled on the same cell slide,and the co-localization of the two proteins was detected by fluorescence confocal microscopy after the second antibody?sheep anti-mice red fluorescent antibody and sheep anti-rabbit green fluorescent antibody,Lifetechnologies?.The co-localization of the two proteins was identified by the same method as that of Rab5,Rab7and Rab11.5 Immunco-precipitation identified the regulation of ESDN expression on the interaction between VEGFR-2 and EEA1,Rab5,Rab7 or Rab11 in HUVEC.Immunocoprecipitation technique was used to further verify the interaction of ESDN expression with EEA1,Rab5,Rab7 and Rab11 in HUVEC.After siRNA knocked down the expression of ESDN in cultured HUVEC,Protein lysates were collected by stimulation with VEGF-AA?10ng/mL?at different times?0,5,15,30 minutes?,and then incubated with VEGFR-2 antibody magnetic beads?Invitrogen?for 16 hours,western blotting was used to detect the expression of EEA1,Rab5,Rab7 or Rab11.It was determined that the regulation of ESDN expression mainly affected the interaction between VEGFR-2 and what kind of endocytic marker molecule.Results:1 Mouse aortic endothelial cells were successfully cultured in vitro.Specificity and purity of endothelial cells from thoracic and abdominal aortas of WT and Esdn^-/-mice primarily cultured were detected in vitro.Western Blot showed that the cell surface receptor VEGFR-2 was positive,immunofluorescence results showed that the specific protein CD31 of endothelial cell surface was also positive,and flow cytometry showed that more than 90%of the cells were positive for CD31 staining.Finally,the results showed that the primary culture method of murine thoracic and abdominal aortic vascular endothelial cells in vitro was successful and could be used for subsequent experiments.2 Down-regulation of ESDN expression inhibited the signal pathway of VEGF.To investigate the relationship between ESDN deletion and angiogenesis,HUVEC was cultured in vitro,and siRNA knocked down the expression of ESDN.After serum starvation,the protein was collected at different time after treatment with VEGF-AA?10 ng/mL?.Western blot was used to detect the phosphorylation level of VEGFR-2 and the changes of its downstream signaling pathway.The results showed that the down-regulation of ESDN expression inhibited the activation of phosphorylation of VEGFR-2 induced by VEGF-AA,which could reach its peak at 5 minutes of stimulation of VEGF.At the same time,downstream signaling molecules related to endothelial cell proliferation and migration were detected.The results showed that the down-regulation of ESDN expression inhibited the phosphorylation of MAPK-ERK1/2 and p38 induced by VEGF-AA,both of which reached their peak at 5 minutes of stimulation by VEGF-AA.These results suggest that the down-regulation of ESDN expression may inhibit the phosphorylation of the downstream signal molecules MAPK-ERK and p38 by inhibiting the phosphorylation of the VEGF-AA-induced by VEGFR-2.3 Down-regulation of ESDN promoted the interaction of VEGFR-2 and Rab5 in the cytosol,and inhibited the interaction of VEGFR-2 and Rab11 in the cytosol.In order to further study the mechanism of down-regulation of ESDN expression on the phosphorylation of VEGFR-2 and its downstream signaling molecules MAPK-ERK1/2 and p38,siRNA knocked down the expression of ESDN in HUVEC.After serum starvation and being treated with VEGF-AA?10 ng/mL?at different time points,immunofluorescence and immunoprecipitation techniques were used to research the co-localization between VEGFR2 and vesicle transport-related proteins such as EEA1,Rab5,Rab7 or Rab11 in cytoplasmic matrix,respectively.The results showed that co-localization changes between EEA1,Rab7 and VEGFR-2 in the cytosol were not detected at the time of VEGF stimulation,regardless of whether ESDN expression was down-regulated or not.After down-regulation of ESDN expression,VEGF stimulated for 5 minutes,promoted the binding of VEGFR-2 and Rab5 in the cytoplasm,and inhibited the binding of VEGFR-2to Rab11 in the cytoplasm,and the dephosphorylation of VEGFR-2 in the cytoplasm.Finally,the process of inhibiting the return of VEGFR-2 to the cell membrane is inhibited.Conclusion:1.The primary culture method of vascular endothelial cells of thoracic and abdominal aorta in mice was successfully improved.2.The down-regulation of ESDN expression inhibits the signal pathway of VEGF in the vascular endothelial cells.3.Down-regulation of ESDN promotes the binding of VEGFR-2 to Rab5in the cytosol,and inhibits the binding of VEGFR-2 to Rab11 in the cytoplasm,dephosphorylation of VEGFR-2 in the cytosol,and finally the process of inhibiting the return of VEGFR-2 to the cell membrane is inhibited.