Catecholamines Block the Antimitogenic Effect of Estradiol on Human Coronary Artery Smooth Muscle Cells

0021-972X/04/$15.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 89(8):3922–3931 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2004-0115 Catecholamines Block the Antimitogenic Effect of Estradiol on Human Coronary Artery Smooth Muscle Cells RAGHVENDRA K. DUBEY, EDWIN K. JACKSON, DELBERT G. GILLESPIE, LEFTERIS C. ZACHARIA, AND BRUNO IMTHURN Department of Obstetrics and Gynecology, Clinic for Endocrinology, University Hospital Zurich (R.K.D., B.I.), 8091-CH Zurich, Switzerland; and Center for Clinical Pharmacology (R.K.D., E.K.J., D.G.G., L.C.Z., B.I.), Departments of Medicine (R.K.D., E.K.J., D.G.G., L.C.Z., B.I.) and Pharmacology (E.K.J., L.C.Z.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213 Sequential conversion of estradiol to catecholestradiols and methoxyestradiols by cytochrome-P450 (CYP450) and catechol-O-methyltransferase (COMT), respectively, contributes to the antimitogenic effects of estradiol on vascular smooth muscle cell (SMC) growth via estrogen receptor-independent mechanisms. Because catecholamines are also substrates for COMT, we hypothesize that catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic/inhibitory effects of estradiol on human coronary artery SMC growth (cell number, DNA synthesis, collagen synthesis, and SMC migration) and ERK1/2 phosphorylation in the presence and absence of catecholamines. Norepinephrine, epinephrine, isoproterenol, and OR486 (COMT inhibitor) abrogated the inhibitory effects of estradiol on SMC growth and ERK1/2 phosphorylation. The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, ␣- and ␤-adrenoceptor antagonists, respectively. The antimitogenic effects of 2-hydroxyestradiol, but not 2-methoxyestradiol, were abrogated by epinephrine, isoproterenol, and OR486. Catecholamines inhibited the conversion of both estradiol and 2-hydroxyestradiol to 2-methoxyestradiol, and SMCs expressed CYP1A1 and CYP1B1. Our findings suggest that catecholamines within the coronary arteries may abrogate the antivasoocclusive effects of estradiol by blocking the conversion of catecholestradiols to methoxyestradiols. The interaction between catecholamines and estradiol metabolism may importantly define the cardiovascular effects of estradiol therapy in postmenopausal women. (J Clin Endocrinol Metab 89: 3922–3931, 2004) E tion in lesions of mice lacking ER␣ (6), ER␤ (7), or both ER␣ and ER␤ (double knockout) (8) suggest that the antimitogenic effects of estradiol on SMC growth may be ER independent and involve yet another mechanism. Support for the participation of an ER-independent mechanism in mediating the antimitogenic effects of estradiol also comes from the observations that exogenous estradiol inhibits injury-induced neointima formation in gonadectomized, but not intact, male rats, even though vascular cells from both models express ERs (9). Pharmacological evidence for a role of ERs in mediating the antimitogenic effects of estradiol in SMCs is controversial. ICI182780, an ER antagonist, was effective in blocking the injury-induced neointima formation in one study (10), but not another (11). Due to similarity in its structure with estradiol, ICI182780 not only binds to the ER, but also blocks the metabolism of estradiol to 2-hydroxyestradiol (a potent inhibitor of SMC growth) by competing for cytochrome P450 (CYP450) enzymes (12). In in vitro studies with SMCs, we have demonstrated that ICI182780 blocks the antimitogenic effects of estradiol only at concentrations that inhibit the metabolism of estradiol to hydroxyestradiol (12, 13). Taken together, the above findings provide strong evidence that the antiproliferative actions of estradiol may be ER independent; however, the exact mechanism involved remains undiscovered. Endogenous estradiol is sequentially metabolized to cat- VIDENCE FROM MULTIPLE epidemiological and observational studies suggest that estradiol protects the cardiovascular system and is responsible for the lower incidence of coronary artery disease observed in premenopausal women compared with age-matched men (1). However, findings of randomized hormone replacement therapy trials for the primary and secondary prevention of cardiovascular disease do not support this idea (2, 3). The reasons for the negative findings remain unclear and emphasize the need for a greater understanding of the mechanisms by which estradiol influences the vasculature. Although estradiol is known to positively influence the vasculature via multiple mechanisms (4), its inhibitory effects on smooth muscle cell (SMC) growth play a key role in protecting blood vessels against vasoocclusive disorders. Based on the conventional mechanisms of steroid action, the growth inhibitory effects of estradiol are thought to be mediated via estrogen receptor (ER)␣ and/or ER␤ expressed by SMCs (5). However, recent findings that administration of exogenous estradiol inhibits injury-induced SMC proliferaAbbreviations: COMT, Catechol-O-methyltransferase; ER, estrogen receptor; FCS, fetal calf serum; PDGF-BB, platelet-derived growth factorBB; SMC, smooth muscle cell. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. 3922 Dubey et al. • Catecholamines Block Antimitogenesis by Estradiol echolestradiols (e.g. 2-hydroxyestradiol) by CYP450, and catecholestradiols are metabolized to methoxyestradiols (e.g. 2-methoxyestrdaiol) by catechol-O-methyltransferase (COMT). Catecholestradiols and methoxyestradiols have, respectively, little or no binding affinity for ERs, yet are potent inhibitors of cancer cell growth (14, 15). Because the antigrowth effects of estradiol are in part ER independent, we hypothesized that methoxyestradiols mediate the antiproliferative actions of estradiol on SMC growth. Subsequently, using pharmacological agents to block or induce the conversion of estradiol to catecholestradiols and methoxyestradiols and using COMT knockout mice, we provided strong evidence that methoxyestradiols mediate the antimitogenic effects of estradiol in SMCs (12, 13, 16). Moreover, we found similar effects in cardiac fibroblasts (17) and glomerular mesangial cells (18), cell types that are relevant for the cardiovascular system. Apart from metabolizing estradiol to methoxyestradiols, COMT is a key enzyme responsible for catabolizing catecholamines (19). Therefore, it is conceivable that increased levels of catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting the conversion of catecholestradiols to methoxyestradiols. This hypothesis is supported by the observations: 1) that postmenopausal compared with premenopausal women exhibit greater stress-induced increases in catech (...truncated)