Mechanism Analysis Of Endosulfan-induced Cytoskeleton Remodeling In HUVEC-C Cells

Endosulfan belongs to persistent organic pollutants,characterized by difficult degradation,bioaccumulation,long distance transfer and high toxicity.Our previous studies reported that endosulfan had endothelial toxicity,but the effect of endosulfan on endothelial barrier function is still unknown.In this study,human umbilical vein endothelial cells(HUVEC-C)were used to study the effect of endosulfan on endothelial cytoskeletion and signal pathways,and to reveal molecular mechanism in endothelial permeability due to cytoskeleton remodeling induced by endosulfan.In the present study,the effects of endosulfan on microfilament structure(F-actin)of cytoskeletion and focal adhesion kinase(FAK)distribution in HUVEC-C cells were analyzed by immunostaining.qRT-PCR was performed to examine mRNA expression levels of cytoskeletion-related genes ROCK2 and PXN.Western blot analysis was performed to examine expression of MMP-3,LAMC1,FAK and pFAK.RNAi experiments were utilized to investigate the influence of LAMC1 on cell variability,cytoskeleton and endothelial permeability.E-cadherin and β-catenin that function on cell-cell adheren junction was examined by western blot.Cx43 that is key gene in gap junctions was examined by qRT-PCR.Finally,coaster transwell system was utilized to study the time-effect and dose-effect of endosulfan on endothelial permeability in HUVEC-C cells.The results showed that endosulfan exposure caused the disruptions of actin cytoskeleton,downregulated ROCK and PXN,increased MMP-3 protein expression,and decreased expression of LAMC1,FAK and pFAK,indicating that endosulfan activated MMP3/LAMC1/FAK signaling pathway to disrupt endothelial cytoskeleton.LAMCI siRNAs significantly decreased in cell variability,disrupted actin cytoskeleton and elevated endothelial permeability,implying LAMC1 plays a critical role in maintaining the stucture and function of endothelial cells.Endosulfan affected adherens junctions via the downregulation of E-cadherin and β-catenin,and impaired gap junctions through downregulation of Cx43.Endothelial permeability was enhanced at 48 h after exposure to endosulfan in a dose-dependent manner.These findings suggested that endosulfan can disrupt endothelial cytoskeleton structure,subsequently weaken adherens junctions and gap junctions intercellular function,which increased endothelial permeability and thus reduced endothelial barrier function.At the first time,LAMC1 was found to be involved in the changes in cytoskeleton remodeling and endothelial permeability induced by endosulfan.This study provides the important theoretical basis that whether endosulfan acts as a risk factor,leads to imbalance of vascular homeostasis and thus causes cardiovascular diseases.