Dual Effects Of Estrogen On Vascular Smooth Muscle Cells: An Estrogen Receptor-mediated Proliferative Vs. An Estrogen Metabolite-induced Pro-senescent Actions

It was well documented that E2 had opposite actions on the same cells in different conditions. Why does one hormone have so complex effects on one cell type? To address this question, the present study dissected the estrogen receptor (ER)-and estrogen metabolite-mediated effects of E2 on rat vascular smooth muscle cells (VSMCs) using a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) and an ER antagonist ICI 182,780, respectively.Part 1. Dual effects of estrogen on vascular smooth muscle cellsObjective:To investigate the effects of estrogen on vascular smooth muscle cells (VSMC), When the activity of CYP450s or ERs was blocked.Methods:The VSMCs isolated from the aortas of female SD rat and sub-cultured in passages of 3-4 were treated with different concentrations of 17β-estradiol (E2) (0M-10-5M). To dissect the ER- and estrogen metabolite-mediated effects of E2 on VSMCs, the VSMC were divided into three groups:①E2 group:the VSMCs was cultured with different concentrations of E2 (0M-10-5M);②ABT+E2 group:in which the VSMC were pre-treated with ABT for 30min before administration of 0M-10-5M E2;③ICI+E2 group: in which the VSMC were pre-treated with ICI 182,780 for 30min before administration of 0M-10-5 E2. The growth, cell cycle progression, premature senescence (SA-(3-Gal positive) and senescence-associated protein p21 were analyzed with cell counting assay, flow cytometry and Western blot.Results:1. The effects of E2 on the growth of the VSMCsCell counting assay showed that in the concentrations from 10-9 M to 10-8 M, E2 had concentration-dependent promoting effect on the growth of the VSMCs; but at a concentration higher than 10 M, the growth-promoting effect of E2 gradually decreased and switched to a growth-inhibiting action at 10-5M. Under the same conditions, when the activity of CYP450s was blocked by pre-administration of ABT, the growth-promoting effect of E2 was significantly enhanced and did not reverse at the concentrations higher than 10-8M, but maintained at an elevated plateau; whereas when the ERs were blocked by pre-administration of ICI 182,780, E2 showed a pure concentration-dependent growth-inhibiting effect on the cells.2. The effcets of E2 on the cell cycle of the VSMCsFlow cytometric analysis for cell cycle progression showed that administration of E2 in concentrations from 10-9 M to 10"8 M caused a slight (but statistically significant) increase in replication index (RI) of VSMCs and this proliferative effect of the hormone was due to an acceleration of cell cycle progression from G1 to S phases (the population of the VSMCs at G0/G1 phase decreased, and those at S and G2/M phases increased); but when the concentrations of E2 were higher than 10-8M, a self-inhibition of the hormone's proliferative action (the raised RI returned back with the further increase in E2 concentration) due to an inhibited G1 to S transition was observed. When CYP450s was blocked by ABT, the proliferative effect of E2 was significantly enhanced and the self-inhibition in high concentrations disappeared. Whereas when ERs were blocked by ICI 182,780, E2 significantly reduced the RI of the VSMCs in a concentration-dependent manner in concentrations higher than 10"8 M; and this anti-proliferative action was related to a significantly increased G0/G1 population. Although RI of VSMCs in 10-9 M+ICI group was not significantly changed in comparison to control (0 M E2) cells, it was sharply decreased than that of the cells treated with E2 alone at the same concentration (10-9ME2 group).3. The effects of E2 on the VSMC premature senescenceFlow cytometric analysis for VSMC premature senescence showed that administration of E2 in concentrations from 10-9 M to 10-8 M slightly reduced the populations of senescent (SA-β-Gal positive) cells, but when its concentrations were higher than 10-8 M, the hormone showed a concentration-dependent opposite (pro-senescent) effect. When the activity of CYP450s was blocked by pre-administration of ABT, E2 displayed an enhanced anti-senescence effect at all concentrations tested. Whereas when the ERs were blocked by ICI 182,780, E2 had a pure and concentration-dependent pro-senescent effect on the VSMCs.The dual effects of E2 on VSMC senescence upon different concentrations were further confirmed at the Western blot analysis for another cellular senescent marker p21. It was shown that E2 reduced expression of p21 at the concentration of 10-8 M, but enhanced its expression at 10-5M. At both concentrations of E2, pre-administration of ABT reduced and pre-administration of ICI 182,780 enhanced the expression of the senescent marker, respectively.Conclusions:1. E2 has Dual effects on vascular smooth muscle cells in different concentration: under the highest plasma level (10-8 M), a mild and concentration-dependent promoting effect on growth of VSMCs; over 10-8 M, the growth-promoting effect of E2 gradually reversed to a growth-inhibiting action.2. When the activity of CYP450s was blocked, the growth-promoting effect of E2 increased and did not reverse at high concentrations, but was maintained at a plateau, accompanied by decreased levels of cellular senescence, indicating a pure growth-promoting effect of E2 mediated by ERs; and also suggested the growth-inhibiting effects of E2 on VSMC derived from the metabolite-induced pro-senescent actions.3. When the ERs were blocked by pre-administration of ICI 182,780, E2 showed a pure growth-inhibiting effect on the same cells, accompanied by increased levels of cellular senescence. This also suggested that pro-senescent actions of E2 was mediated by its metabolite.4. The complex effect of E2 is due to the balance of two opposite actions:one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.Part2. The mechanism for metabolite-induced pro-senescent actions of estrogen on vascular smooth muscle cells.Objective:In this part, we investigated the mechanism of estrogen metabolite-mediated effects of E2 on VSMCs (to investigate the mechanism of metabolite-induced pro-senescent actions, when the ERs were blocked by pre-administration of ICI 182,780,.).Methods:The VSMC were divided into three groups as above. Estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with liquid chromatography-mass spectrometry,DCF-DA and comet assay, respectively.Results:1. The change of the estrogen metabolitesLC-MS showed that the major estrogen metabolites,2-and 4-hydroxyestradiols, were only slightly increased in VSMCs grown in 10-8 M E2 in comparison to control cells (grown in 0 M E2) in which the two metabolites were undetectable. However, a sharp increase in these metabolites, particularly in 2-hydroxyestradiol, were found in the VSMCs grown in 10-5M E2. Block of ER by ICI 182,780 did not affect, but deactivation of CYP450s by ABT eliminated the production of the two E2 metabolites.2. The effcets of E2 on ROS in VSMCsROS in VSMCs grown in 10"8M E2 slightly decreased in comparison to control cells (grown in 0 M E2). However, an obvious increase in ROS was found in the VSMCs grown in 10-5M E2. Block of ER by ICI 182,780 reversed the effect of 10-8M E2 on ROS from inhibitive to promotive and enhanced the 10-5M E2-induced ROS generation. Whereas block of CYP450s by ABT reduced ROS to a level slightly lower than control level.3. The effcets of E2 on DNA damage in VSMCsComet assay showed that in comparison to control (0 M E2) cells, the level of DNA damage represented by OTM slightly decreased in VSMCs grown in 10-8M E2, but significantly increased in the cells grown in 10-5M E2. Block of ER by ICI 182,780 reversed the effect of 10-8 M E2 on DNA damage from inhibitive to promotive and enhanced the 10-5M E2-induced DNA damage. Whereas block of CYP450s by ABT reduced OTM to a level slightly lower than the control.Conclusions:1. The E2 major metabolites (2- and 4-hydroxyestradiols) accumulated with increase of E2, which was accompanied by increased levels of intracellular ROS, DNA damage (OTM value), and cellular senescence. It suggested that DNA damage, leading to VSMC premature senescence, induced by E2 metabolites not by ERs.2. When the VSMC was cultured in physiological concentrations, decreased levels of intracellular ROS, DNA damage (OTM value) was induced by E2. All these changes were reversed by block of ER by ICI 182,780, it suggested that administration of physiological concentrations of E2 slightly inhibited senescence of VSMCs mediated by ERs.