Methoxyestradiols Mediate the Antimitogenic Effects of 17β-Estradiol

Abstract

Background-Studies using pharmacological agents suggest but do not prove that the antimitogenic effects of estradiol are caused by conversion of estradiol to hydroxyestradiols (mediated by CYP450s) followed by methylation of hydroxyestradiols to methoxyestradiols (mediated by catechol-O-methyltransferase, COMT). Methods and Results-To test this hypothesis more rigorously, we used aortic smooth muscle cells (SMCs) from mice lacking COMT (COMT-KO). Wild-type (WT) but not COMT-KO SMCs efficiently converted 2-hydroxyestradiol to 2-methoxyestradiol. Both WT and COMT-KO SMCs expressed estrogen receptors. Estradiol and 2-hydroxyestradiol concentration-dependently inhibited serum-induced DNA synthesis, cell numbers, and collagen synthesis in WT but not COMT-KO SMCs. 2-Methoxyestradiol inhibited DNA synthesis, cell numbers, and collagen synthesis in both WT and COMT-KO SMCs. Conclusions-These data provide strong evidence that the vascular antimitogenic effects of estradiol are estrogen receptor-independent and involve the sequential conversion of estradiol to hydroxyestradiols and then to methoxyestradiols. (Circulation. 2003;108:2974-2978.)Key Words: coronary disease Ⅲ hormones Ⅲ metabolism Ⅲ muscle, smooth Ⅲ receptors S eventeen ␤-estradiol (estradiol) inhibits the growth of vascular smooth muscle cells (SMCs), even in mice lacking estrogen receptors (ERs). 1-3 Because methoxyestradiols (major endogenous metabolites of estradiol with no affinity for ERs) inhibit growth of cancer cells, 4 it is feasible that the vascular antimitogenic effects of estradiol are mediated by methoxyestradiols. Using pharmacological agents, previous studies demonstrate that metabolism of estradiol to hydroxyestradiols by CYP450 followed by methylation of hydroxyestradiols by catechol-O-methyltransferase (COMT) to form methoxyestradiols is critical for estradiol-induced inhibition of SMC growth. 5 In the present study, we use aortic SMCs from COMT-knockout (COMT-KO) mice 6 that express ERs but lack methylation capability to test our hypothesis that the vascular antimitogenic effects of estradiol are mediated via an ER-independent mechanism involving formation of methoxyestradiols. MethodsCOMT-KO mice were developed by Dr Karayiorgou. 6 Aortic SMCs were cultured from male mice by the explant technique. SMC purity was tested by immunostaining with SMC-specific antibodies as described previously. 7 Cells in passage 2 were used.3 H]proline incorporation, and cell proliferation were conducted as previously. 7 The absence of COMT was confirmed by incubating SMCs from wild-type (WT) and COMT-KO mice for 2 hours with 2-hydroxyestradiol (2 mol/L) and analyzing 2-hydroxyestradiol/2-methoxyestradiol by high-performance liquid chromatography, as previously. 8 ER␣ and ER␤ expression were analyzed by Western blots. 9 Experiments were conducted in triplicate (repeated 3 or 4 times). Results are expressed as meanϮSEM. Statistical analyses were performed with ANOVA and Fisher's least significant difference test. A value of PϽ0.05 was considered statistically ...

Early endothelial dysfunction as a marker of vasculogenic erectile dysfunction in young habitual cannabis users

“…This has suggested that the effects of estradiol would be independent from the activity of its own receptors. 29 In our patients, adrenal and pituitary axes were not evaluated; we have not found any significant steroid hormone or prolactin alterations as well as any alteration in testosterone/estradiol ratios, but we cannot rule out an ER activation to explain vascular damage.…”

Correlation of 2-Methoxyestradiol Levels in Cord Blood and Complications of Prematurity

“…The lack of correlation of 2ME2 to complications of prematurity may be because of ineffective 2ME2 levels, even among the premature infants born with the highest 2ME2 levels (0.200 -1.35 ng/mL). For in vitro studies, induction of apoptosis and inhibition of HIF-1␣ occur at micromolar 2ME2 concentrations (Ͼ300 ng/mL) (4,5,40) and inhibition of MAP kinase activity and of DNA synthesis was observed at 100 nM (30 ng/mL; 41,42).…”

Estrogens and atherosclerosis: insights from animal models and cell systems

“…Finally, 2-ME, an estrogen metabolite with no affinity to classical ERs, was shown to prevent injury-induced neointima formation in rats (Barchiesi et al 2006). Several studies in vitro documented that 2-ME inhibits VSMC proliferation at both G 0 /G 1 and G 2 /M cell cycle phase and downregulates cyclones D and B, while upregulating p27 -a negative regulator of VSMC growth, and that these effects are missing in VSMC lacking COMT that do not effectively convert E 2 to 2-ME (Zacharia et al 2003, Barchiesi et al 2006, Dubey & Jackson 2009). Collectively, these results suggest that at least part of the atheroprotective effects related to the effects of estrogens on VSMC are mediated in a fashion independent of classical ERs.…”