Cadmium Exposure Enhances Ve-cadherin Expression In Endothelial Cells Through Suppression Of The ROCK Signaling

Objectives:The vascular endothelium is the target of cadmium(Cd),a global pollutant in the environment.Tobacco smoke,cadmium-contaminated food and industrial pollution are the main sources of human cadmium toxicity.Chronic exposure to Cd has great side effects on the human body,causing damage to various organs such as the kidneys,liver,lungs,pancreas and testicles.In addition,recent research reports that vascular endothelial cells(VECs)are another major target of Cd.Accumulated Cd can impair endothelial function at various molecular levels,including cell adhesion molecules,metal ion transporters,and protein kinase signaling pathways.Therefore,the research of the toxicity of Cd is important for the potential mechanism of VECs.Adhesion between cells is established by the interaction of the extracellular domain of endothelial cell cadherin(VE-cadherin).It has been reported that VE-cadherin m RNA expression is reduced in a chicken embryo model treated with 50 μM Cd.Therefore,the effect of Cd on VE-cadherin needs to be further studied.The Rho-associated coiled-coil kinase(ROCK)pathway regulates a variety of cellular functions including contraction,cytoskeletal organization,intercellular adhesion and permeability.However,whether Cd regulates VE-cadherin via the ROCK pathway is uncertain.The aim of this study was to investigate the role of the ROCK pathway in the regulation of Cd-induced VE-cadherin expression in human umbilical vein endothelial cells(HUVECs).Studying the mechanism by which cadmium increases cell permeability is important for the treatment of damage caused by cadmium poisoning.Method:1.HUVECs were treated with ROCK inhibitor Y27632 at a concentration gradient of5 μM,10 μM,20 μM for 12 hours,and the m RNA expression level of VE-cadherin affected by Y27632 was detected by q RT-PCR.HUVECs were treated with ROCK inhibitor Y27632 at a concentration gradient of 5 μM,10 μM,20 μM for 24 hours,and the protein level of VE-cadherin was detected by Western blot.2.Cd(10 μM)-induced HUVECs were treated with ROCK inhibitor Y27632(20 μM)for 24 hours,and the protein level of VE-cadherin was detected by Western blot.3.The HUVECs were exposed to 0.1 μM,1 μM,5 μM,10 μM,50 μM Cd,and the expression of VE-cadherin affected by Cd was detected by q RT-PCR and Western blot.4.HUVECs were exposed to 10 μM Cd,and the distribution of VE-cadherin in cells was detected by immunofluorescence.5.HUVECs were exposed to 10 μM Cd for 12 hours,and the phosphorylation level of myosin phosphatase myosin binding subunit(MYPT)was detected by Western blot.5.HUVECs induced by normal HUVECs and Cd(10 μM)were treated with Cd activator Narcislasine at a concentration of 10 μM for 24 hours.The expression of VE-cadherin was detected by Western blot.Result:1.VE-cadherin m RNA expression did not change at 0.1 μM,1 μM,5 μM Cd concentration.However,it was significantly upregulated at concentrations of 10 μM and50 μM.Western blot showed that Cd increased VE-cadherin protein expression after treatment with Cd at a concentration of 1 μM,5 μM,10 μM,and 50 μM.2.After treatment with 10 μM Cd,the fluorescence became stronger and higher levels of VE-cadherin were detected at the cell-cell junction between cells.3.HUVECs treated with 10 μM Cd showed a significant decrease in p-MYPT after 12 hours,indicating that Cd inhibits the Rho / ROCK pathway.4.10 μM Narciclasine reduced the m RNA and protein levels of VE-cadherin after Cd treatment.By pre-treating 10 μM Narciclasine,10 μM Cd did not increase VE-cadherin in HUVECs.5.Y27632 increased VE-cadherin m RNA expression at 10 μM and 20 μM.Y27632 also increases the protein level of VE-cadherin.Cd did not significantly alter the level of VE-cadherin in HUVECs by pretreatment with 10 μM Y27632.Conclusion:Our results indicate that high doses of Cd increase the expression of VE-cadherin and inhibit ROCK activity in HUVECs.Furthermore,Cd did not increase VE-cadherin expression in the presence of the ROCK inhibitor Y27632,suggesting that ROCK mediates Cd-induced VE-cadherin expression.It increases our understanding of Cd-induced vascular dysfunction.Taken together,our results indicate that Cd upregulates the expression of VE-cadherin by inhibiting ROCK activity.