Effects Of Estrogen On Stress-induced Senescence Of Vascular Smooth Muscle Cells

Clinical and Epidemiological investigations have shown that women exhibit significantly higher incidence of cardiovascular disease after menopause or ovariectomy than before menopause, which indicates ovarian hormones, especially the estrogen, possess protective effects in the female’s cardiovascular system, however, the underlying mechanisms of the hormone’s cardiovascular-protective actions are still not fully clear.The unneglected phenomenon is that the senescent vascular smooth muscle cells (VSMCs) and endothelial cells exist in the atherosclerosis (AS) lesion site during the development of it. The senescent VSMCs abnormally secrete epithelial growth factor, pro-inflammatory factor, matrix metalloproteinase, collagenase to stimulate the proliferation of neighboring cells, to increase the inflammatory response, to destroy the local tissue microenvironment. The recent studies have shown that, the senescence or premature senescence of VSMCs caused by oxidative stress, oncogene activation, telomere shortening and so on, is an important reason to increase local inflammation, to promote sustainable development of AS lesion. One of the important physiological actions of estrogen is to delay aging and maintain the functions of female sexuality organs. Therefore, we speculate that estrogen maybe protect the cardiovascular system through delaying VSMC senescence.In addition, the recent analysis for clinical data by American Women’s Health initiative (WHI) showed that:estrogen replacement therapy reduced the incidence of cardiovascular disease among the women closing to menopause or early menopause. However, supplements of estrogen for women at late menopause or more than 60 years old increased the risk of cardiovascular disease. Therefore, we speculate the effects of estrogen on senescence of VSMCs are different because of the different aging degree of tissues. To test these above hypothesises, the researches have been performed from the following three aspects.1. Effects of estrogen on stress-induced premature senescence (SIPS) of vascular smooth muscle cells from young ratsObjectives:To observe the effects of estrogen on H2O2-induced-premature senescence of vascular smooth muscle cells from young rats. Methods:VSMCs of passage 2-3 were cultured from young (2 months) unpregnancy female SD rats’aortas, identified by immunocytochemical staining for a-SM actin, the cells with>90%α-SM actin positive cells were used in the examinations. The VSMCs were stressed by 150μM H2O2 in the presence or absence of the highest physiological concentration of 17-β-estradiol (10-8M E2), or 10-10～10-8M E2 with or without inhibitor of ERs (10-5M ICI182,780). The expression or activation of senescence-associated beta-galactosidase (SA-β-Gal) and senescence-associated marker, DcR2, were detected by cytochemical staining and flow cytometry.Results:(1) SA-β-Gal cell staining showed that:after 150μM H2O2-stress, there were a large number of SA-P-Gal positive cells (blue cytoplasm especially surrounding the nucleus). The typical senescent VSMCs showed an enlarged, flattened shape with granule or vacuolus in the cytoplasm. Pre-administration of the highest physiological concentration (10-8M) of E2 could significantly, but not completely, inhibited the H2O2-induced SIPS. However, this protective effect of E2 was completely blocked by the ER antagonist ICI 182,780 at a concentration of 10-5M.(2) Flow cytometric analysis for SA-β-Gal showed that there was no significant difference in percentages of SA-β-Gal positive cells between the VSMCs treated with 10-8M E2 alone and the control cultures. After 150μM H2O2-stress, the senescent VSMCs increased. Administration of 10-M E2 before stressing significantly inhibited the H2O2-induced SIPS, which was blocked by 10-5M ICI 182,780 completely.(3) Flow cytometric analysis for DcR2 showed that the expression of DcR2 significantly increased after 150μM H2O2-stress. The high level expression of it induced by H2O2 was decreased by pre-administration of 10-10～10-8 M E2 in a dose-dependent manner, which was blocked completely by 10-5M ICI182,780.Conclusions:(1) Physiological concentrations of E2 inhibited, but not completely, H2O2-induced premature senescence of VSMCs from young rats in a dose-dependent manner, which indicates E2 possesses a certain effect on anti-SIPS of VSMCs.(2) The effect of E2 on anti-SIPS of VSMCs could be blocked by ICI 182,780 completely which indicates this effect of E2 is mediated by ERs. 2. Mechanisms of estrogen on anti-SIPS of VSMCs from young ratsObjectives:In the first part examination, we have found that E2 possesses the effect on anti-SIPS of VSMCs from young rats. The following experiments were performed to explore the mechanisms of E2’s anti-SIPS.Methods:VSMCs of passage 2-3 cultured from young (2 months) female SD rats’ aortas were stressed by 150μM H2O2 in the presence or absence of 10-8M E2, with or without 10-5M ICI 182,780. The activation or expression of oncogene Ras, p38, PRAK, p53, p21, p16 and Rb were analyzed through Pull-Down assay or Western Blot.Results:(1) Pull-Down assay revealed that 150μM H2O2-stress caused a sharp increase in GTP-Ras, while not affecting total Ras expression, thus leading to a significant rise in GTP-Ras/Total-Ras ratio. Pre-administration of the highest physiological concentration (10-8M) of E2 could significantly, but not completely, reverse the H2O2-induced Ras activation and this effect of E2 was completely blocked by 10-5M ICI 182,780.(2) Western Blot analysis showed that H2O2-stress led to increases in the level of p-p38, PRAK, p53, p21, p16 and Rb, as well as decrease in the level of p-Rb, and those were significantly inhibited by administration of 10-8M E2 before stressing.Conclusion:(1) Estrogen inhibits SIPS of VSMC from young female rats through suppressing H2O2-induced activation of oncogene Ras to down-regulate both Ras-p38-PRAK-p53-p21-Rb and Ras-p16-Rb pathways.(2) The effect of estrogen on suppressing oncogene Ras’s activation is mediated by ERs.3. Effects of estrogen on SIPS of vascular smooth muscle cells from old ratsObjectives:The experiment results of the first and second parts show that E2 possesses the action on anti-H2O2-induced premature senescence of VSMCs from young rats. The following experiments were performed to investigate whether the action of E2’s anti-SIPS is changed in VSMCs from old female body.Methods:The VSMCs of passage 2 from old (18 months) female SD rats were induced by 150μM H2O2 containing different concentrations of E2 (10～10～10～8M), or 10-8 M E2 plus 10-5M ICI 182,780 or 10-5M ABT (CYP450 inhibitor which blocks transformation of E2 into catecholestradiol. The detections of SA-P-Gal positive cells by flow cytometry were performed with the same methodology for the VSMCs from young rats.Results:(1) Flow cytometric analysis showed that H2O2-stress led to a significantly increased population of SA-β-Gal positive cells (1.85-fold higher than control cultures). But unexpectedly, pre-administration of E2 at 10-10 and 10-9M had no inhibitive effect on H2O2-induced VSMCs senescence; and even more, at its highest physiological concentration (10-8M) of E2 showed an opposite action, increasing H2O2-induced senescence.(2) The senescent-promoting action of E2 at 10-8M was enhanced by 10-5M ICI 182,780.(3) The senescent-promoting action of E2 at 10-8M was eliminated by 10-5M ABT.Conclusion:(1) The effect of E2 on anti-SIPS of VSMCs almost disappears in old rat, and even more, the highest physiological concentration (10-8M) of E2 promotes SIPS of the VSMCs from old rat.(2) The senescent-promoting action of E2 at 10-8M was enhanced by 10-5M ICI 182,780, which indicates this effect is not mediated by ERs and completely covers the effect of E2 on anti-SIPS.(3) The senescent-promoting action of E2 at 10-8M was completely eliminated by 10-5M ABT, which indicates the effect of higher concentration of E2 on promoting SIPS of VSMCs from old rat is mediated by its metabolisms.