Racl accelerates endothelial cell senescence induced by hypoxia {sl in vitro}
Han Yaling, Yu Haibo, Yan Chenghui, KANG Jian, Meng Zimin, Zhang Xiaolin, Li Shaohua, Wang Shiwen
Department of Cardiology, General Hospital of Shenyang,The Institute of Cardiovascular Research, PLA.Shenyang 110016,Liaoning;USA;Institute of Geriatric Cardiology, General Hospital of PLA.Beijing 100853
Abstract
To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cells (HUVECs) senescence,we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-#beta#-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-#beta#-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G_(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96h. Accompanied with these changes, activated Rac1 expression increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVECs senescence, HUVECs were transiently infected with the constitutively active forms of Rac1 (V12Rac1) or dominant negative forms of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs,V12Rac1-HUVECs) after hypoxia for 48h and 96h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-#beta#-Gal activation, PAI-1 expression increase, G_(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96h hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96h. All the results identified that the activation of Rac1 might accelerate HUVECs senescence induced by hypoxia and that inactivation of Rac1 could block partly the cells senescence. To further investigate the mechanism of HUVECs senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggested that activation of Rac1 accelerated endothelial cells senescence and inhibition of Rac1 activity prevented HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.
Key words: Rac 1;Hypoxia;Endothelial cell;senescence
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Citing This Article:
Han Yaling, Yu Haibo, Yan Chenghui, KANG Jian, Meng Zimin, Zhang Xiaolin, Li Shaohua, Wang Shiwen. Racl accelerates endothelial cell senescence induced by hypoxia {sl in vitro}. Acta Physiol Sin 2006; 58 (3): (in Chinese with English abstract).