Testosterone Promotes Tube Formation Of Human Umbilical Endothelial Cells Via Activation Of Smadl Protein

BackgroundCardiovascular disease(CVD)is the leading cause of death.Sex steroid hormone androgen has the impact on the development and progress of CVD.However,the cardiovascular outcomes of androgen therapy has been debated.In this aspect,several epidemiological studies have demonstrated an inverse relationship between serum level of testosterone(T)and the incidence rate of CVD,cardiovascular mortality or extent/severity of coronary atherosclerosis both in men and in women.Moreover,the latest largest cohort showed that normalization of T level was associated with reduced incidence of myocardial infarction and mortality in men.Likewise,in a double-blind,randomized,placebo-controlled study in women with heart failure,T therapy was associated with significant improvements on surrogate cardiovascular outcomes.These studies manifested the potential cardiovascular benefits of T,while its detailed actions in cardiovascular system and the underlying molecular mechanisms remain to be further investigated.Angiogenesis is the formation of new blood vessels from the pre-existing endothelium and it is crucial for wound healing and organ regeneration in multiple disease processes.T has a positive effect on angiogenesis in the repair of cardiovascular damage.Tube formation capacity is critical for vascular regeneration/repair.Smadl is enriched in endothelium and plays a key crucial role in angiogenesis.Mice defective in Smad1 died in utero due to defective vasculature formation and the impaired endothelial Smadl resulted in defective angiogenesis.There had reported that T can promote angiogenesis.However,currently there is no evidence showing the relevance of T on endothelial Smadl activation.ObjectivesTo investigate the effects of testosterone promotes tube formation and the tole of Smadl in this process.Moreover,the molecular mechanism under the testosterone effects was investigated.Methods and ResultsIn this study,we investigated the effects of T on Smadl activation and tube formation of cultured human umbilical endothelial cells(HUVECs).Our results showed that T rapidly increased endothelial Smad1 phosphorylation.This effect was mimicked by cell-impermeable T-BSA conjugates and was not altered by transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide.T-induced Smadl phosphorylation was blocked by ERK1/2 and c-Src inhibitors or their specific siRNAs,while it was reinforced by ERK1/2 or c-Src overexpression.Indeed,T rapidly activated ERK1/2 and c-Src signalings and c-Src was confirmed as the upstream of ERK1/2.Moreover,caveolae disruptor methy1-β-cyclodextrin(β-MCD)blocked Smadl activation induced by T.The association of caveolin-1 with androgen receptor(AR)or c-Src was detected by immunoprecipitation and it was significantly increased by rapid T stimulation.Furthermore,fractional analysis showed that AR and c-Src were expressed in caveolae-enriched membrane fractions.T promoted tube formation of HUVECs,which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1.ConclusionsIn conclusion,T increased tube formation of endothelial cells isolated from veins by stimulating Smadl phosphorylation in a nongenomic manner,which was mediated by signals from AR/c-Src located in caveolae to ERK1/2 cascade.These findings may shed new light on the relevance of T to its vascular functions.