The Roles And Related Mechanisms Of Rho GTPases In Endothelial Dysfunction Induced By TNF-?

Objective In order to analyze the roles of Rho GTPases in regulating endothelial dysfunction triggered by tumor necrosis factor-?(TNF-?),we constructed endothelial dysfunction model in vitro by using human umbilical vein endothelial cells(HUVECs).Methods HUVECs were purchased for adherent culture.The silence of RhoA,Rac1 and Cdc42 in the vascular endothelial cells were conducted through siRNA technology,and then identified by RT-PCR analysis.Cells were divided into the control group and the model group(60 ng/ml TNF-? treatment for 4 h).And then the expression and activation of RhoA,Rac1,Cdc42 and Raf-1 were detected by Western Blot and pulldown assay.Cells were also divided into the control group,model group(60 ng/ml TNF-? treatment for 4 h),siRNA or inhibitor group,siRNA or inhibitor pretreatment + TNF-? group.The cell viability was measured by MTT assay,and cell apoptosis was detect by PI/Annexin V-FITC double staining and flow cytometry.The expression and activation of ROCK were detected by Western Blot.The expression,cleavage and phosphorylation of vimentin and also the activation of caspase3,caspase8 and caspase9 were detected by Western Blot.The cytoskeleton morphology and distribution of vimentin or phosphorylated vimentin were detected by immunofluorescence assay.Results 1.The RT-PCR analysis showed that the silence of RhoA,Rac1 and Cdc42 were successful.2.MTT assay showed that RhoA suppression could significantly increase the survival rate of endothelial cells induced by TNF-?.3.RhoA suppression exhibited a significant reduction of cell apoptosis induced by TNF-? through performing immunofluorescence assay and flow cytometry.4.Western Blot and pulldown assay showed that TNF-? could significantly induce the activation of RhoA,but had no significant effects on the expression and activation of Rac1 and Cdc42,indicating that the activation of RhoA mediates TNF-?-induced endothelial cell injury.5.Western Blot analysis showed that RhoA deficiency could significantly inhibit the expression of ROCK1 and the activation of ROCK induced by TNF-?,indicating that RhoA may mediate endothelial apoptosis by activating ROCK.6.MTT assay showed that Y-27632(ROCK inhibitor)pretreatment could significantly improve the survival rate of endothelial cells induced by TNF-?.7.Immunofluorescence assay and flow cytometry displayed that Y-27632 pretreatment could significantly decrease the apoptosis of endothelial cells induced by TNF-?,indicating that the activation of RhoA/ROCK signaling mediates TNF-?-induced endothelial cell injury.8.Western Blot showed that RhoA suppression or Y-27632 pretreatment could significantly inhibit the cleavage of vimentin and vimentin phosphorylation at serine 56,and improve the phosphorylation of vimentin at serine83.9.Western Blot showed that Y-27632 pretreatment could inhibit the cleavage of caspase3 and caspase8,but not that of caspase9,which indicates that RhoA suppression or ROCK inhibition inhibits the cleavage of vimentin induced by TNF-?through inhibiting the activation of caspase3 and caspase8.10.Immunofluorescence assay showed that RhoA deficiency or Y-27632 pretreatment significantly inhibited the structure and redistribution of vimentin or phospho-vimentin(Ser56 and Ser83)induced by TNF-?,indicating that RhoA/ROCK signaling pathway participates in the remodeling and redistribution of vimentin and phospho-vimentin(Ser56 and Ser83)induced by TNF-? through regulating vimentin phosphorylation,and therefore induces endothelial cell dysfunction.11.Western Blot showed that TNF-? could significantly induce the phosphorylation of Raf-1,indicating that Raf-1 might mediate TNF-?-induced endothelial dysfunction.12.Western Blot also showed that GW5074(Raf-1 inhibitor)pretreatment had no significant effects on the expression and activation of ROCK,indicating that the effects of Raf-1 on endothelial dysfunction may be independent of ROCK.13.Western blot analysis showed thatGW5074 or TBB(CK2 inhibitor)pretreatment had no significant effect on the cleavage of vimentin,but could significantly decrease the vimentin phosphorylation at Ser 56 and increase the phosphorylation at Ser 83.14.Immunofluorescence assay showed that GW5074 or TBB pretreatment could significantly inhibit the TNF-?-induced remodeling and redistribution of vimentin or phospho-vimentin(both at Ser56 and Ser83).15.MTT assay showed that GW5074 or TBB pretreatment could significantly increase the viability of endothelial cells induced by TNF-?.Immunofluorescence and flow cytometry showed that GW5074 or TBB pretreatment could significantly decrease the endothelial apoptosis induced by TNF-?.These results suggested that Raf-1/CK2 pathway participates in TNF-?-induced endothelial injury through regulating vimentin phosphorylation and the remodeling of vimentin and phospho-vimentin(Ser56 and Ser83).Conclusions Both RhoA/ROCK and Raf-1/CK2 signaling pathway participates in endothelial dysfunction induced by TNF-? through regulating the phosphorylation and remodeling of vimentin.In addition,RhoA/ROCK pathway activates caspase3 and caspase8,and then induces the cleavage of vimentin.