ESDN Modulates Vascular Remodeling Through Accelerating Membrane Receptor Endocytosis In Endothelial Cells

Objective:Abnormal angiogenesis contributes to a series of pathological diseases,including cardiovascular diseases,tumors,etc.Therefore,the study of molecules mechanisms on angiogenesis-related diseases is crucial for clinical treatment.Neuropilin（NRP）has been shown to promote the binding of vascular endothelial growth factor（VEGF）to VEGF receptor 2（VEGFR-2）,which plays an important role in tumor growth and angiogenesis effect.Previous studies have shown that endothelial and smooth muscle cells derived neuropilin-like factor（ESDN）,a new I type of transmembrane protein derived from endothelial and smooth muscle cells,can promote the proliferation and migration of vascular endothelial cells by promoting the VEGF signaling pathway.Since the endocytosis of membrane receptor is an efficient way to activate signaling pathways,the mechanism of ESDN regulation on membrane receptor endocytosis and transport is still unknown,as well as the effect on other receptors.In this study,two different cell membrane receptors,VEGFR-2 and low density lipoprotein receptor（LDLR）were chosen to evaluate the role of ESDN in receptor endocytosis and recycling,clarifing the mechanism of ESDN modulating signal via regulating receptor internalization and transportation,and its possible mechanisms for regulating angiogenesis.Methods:Part I:The transmembrane protein ESDN modulates angiogenesis by promoting VEGFR-2 recycling in ECsPulmonary microvascular endothelial cells（PMVEC）of wild type（WT）and Esdn gene knockout(Esdn^-/-)mice were cultured in vitro,stimulated with VEGF₁₆₄,and then isolated membrane cytoplasmic proteins,detecting the differential distribution of VEGFR-2 on surface and cytoplasm.Co-immunoprecipitation（Co-IP）and immunofluorescence（IF）methods were used to detect whether the interaction between VEGFR-2 and its related vesicle transporters Rab5 or Rab11 was changed in the situation of Esdn gene deletion.Rab5 was knocked down or Rab11 overexpressed in PMVEC of WT and Esdn^-/-mice,subsequently stimulated ECs with VEGF₁₆₄,exploring the effect of ESDN on VEGF signaling by affecting VEGFR-2 recycling process.We used IF to detect the variety on binding of VEGFR-2 to lysosome（Lysosome Tracker）in PMVEC of WT and Esdn^-/-mice.Finally,we determined the function of ESDN on the degradation of VEGFR-2.Additionally,we conducted animal models mimic ischemia,in which left hindlimb femoral artery ligation was performed to create hind limb ischemia in endothelial cell-specific Esdn knockout mice(Tek Cre^+/-Esdn^flox/flox)and control mice(Tek Cre^-/-Esdn^flox/flox).The right side is a sham group,the blood flow recovery of the left hind limb was detected by the Doppler blood flow detection system at day 3,7,14 and 21after the operation,separately.The gastrocnemius muscle on both sides were acquired on the 14th day after the operation.IF detected the difference in the expression of angiogenesis marker,NG-2 and CD31,between different groups,eventually determined the absence of Esdn gene in endothelial cells and its effect on angiogenesis.Part II:The transmembrane protein ESDN promotes LDLR endocytosis in endothelial cellsThe primary WT and Esdn^-/-mice PMVECs were isolated,cultured and purified in vitro,the total protein was extracted,labeled with TMT peptides in protease hydrolyzed solution,after thatthe peptides were separated,followed by analyzed with chromatography-mass spectrometry.Quantitative proteomic bioinformatics analysis of protein identified the association of ESDN with cholesterol metabolism related molecules.Validate the result in mouse tissues and endothelial cells.High-fat Diet of WT and Esdn^-/-mice,Tek Cre^-/-Esdn^flox/floxand endothelial cell-specific Esdn knockout mice Tek Cre^+/-Esdn^flox/flox,plasm biochemical indicators and lipid deposition in arotic roots were detected after 3months.Human umbilical vein endothelial cells（HUVEC）were cultured in vitro,Esdn was knocked down followed by LDL stimultion.The uptake of LDL in endothelial cells and intracellular cholesterol deposition were detected.Result of IF showed that the connection between LDLR and its endocytic protein Clathrin was changed in the case of Esdn knockdown,clearing the possible mechanism of ESDN regulation on LDLR endocytosis.Results:Part I:The transmembrane protein ESDN promotes angiogenesis by promoting VEGFR-2 recycling in ECsPMVECs were stimulated with VEGF₁₆₄ for 30 min in vitro,the VEGFR-2 expression on the surface was significantly decreased,however,the expression in the cytoplasm was obviously increased.Co-IP and IF results showed that the binding of VEGFR to the vesicle transporter Rab5 was enhanced after Esdn deletion,while the binding with Rab11 was weakened,indicating that ESDN inhibits the vesicle transport process of activated VEGFR-2 in the cytoplasm.After simultaneously intervening the expression of Rab5 or Rab11 in cells,the downstream VEGF signaling was also significantly affected,indicating that ESDN attenuates the VEGF signaling by inhibiting the receptor recycling of VEGFR-2.In the absence of Esdn,the interaction time of VEGFR-2 with lysosome was prolonged,indicating that ESDN deletion accelerated the degradation of VEGFR-2 accumulated in the cytoplasm.Finally,the conclusion was confirmed in mice that the hindlimb blood flow recovery of endothelial cell-specific Esdn knockout mice Tek Cre^+/-Esdn^flox/flox was slower.These results indicated that ESDN could retain VEGFR-2 in the cytoplasm by inhibiting the recycling process of VEGFR-2,increasing its degradation,resulting in the weaken of the downstream VEGF signaling and finally inhibiting angiogenesis.Part II:The transmembrane protein ESDN promotes LDLR endocytosis in endothelial cellsThe proteomic analysis of total proteins in PMVEC showed there were 65up-regulated and 84 down-regulated protein related to lipid metabolisim in Esdn^-/-mice compared with WT mice.The 149 proteins were subjected to GO secondary classification,GO enrichment analysis,and cluster analysis based on the enrichment results.It was found that Esdn deletion affected the activity of cell membrane receptors and the binding of lipoprotein particles.The LDLR expression in Esdn^-/-mice was significantly higher（about 2.1 times）than that in WT mice.This result was verified in mouse tissues and endothelial cells,as well.Animal models showed that after 3 months of high-fat diet,Esdn^-/-mice and Tek Cre^+/-Esdn^flox/flox mice,LDL expression in the plasma in Esdn^-/-mice and Tek Cre^+/-Esdn^flox/flox mice was significantly higher than that of the control group,as well as the lipid deposition in aortic roots.After knockdown of Esdn in HUVEC and LDL stimulation,it was found that the uptake of LDL was significantly reduced,and the lipid accumulation in the cytoplasm was also significantly reduced,indicating that knockdown of Esdn inhibited the endocytosis of LDLR.Since LDLR is endocytosed into the cytoplasm by Clathrin,the interaction between LDLR and Clathrin is weaken after Esdn knockdown,indicating that Esdn knockdown inhibited the endocytosis process of LDLR,resulting in decreased LDL uptake and increased LDL accumulation in plasma,ultimately,promoted lipid deposition in the body.Conclusion:1.Esdn deletion inhibits the recycling of VEGFR-2 to the cell membrane in endothelial cells,and its retention in the cytoplasm increases the degradation of VEGFR-2,resulting in the inhibited VEGF signaling,and finally inhibits angiogenesis.2.Esdn deletion inhibits the endocytosis of LDLR by inhibiting the combination of LDLR and Clathrin in endothelial cells,which increases the accumulation of LDL in plasma and accelerates lipid deposition in mice.3.ESDN affects the biological pathways mediated by VEGFR-2 and LDLR by regulating their endocytosis and recycling,suggesting that ESDN may regulate other membrane receptors through similar pathways.