Estrogen therapy is associated with reduced risk of coronary artery disease in postmenopausal women. The atheroprotective effects of estrogen may in part be due to favorable changes in lipids, but other biological effects may also be important. Cytomegalovirus, a potential atherogenic agent, causes rapid increases in intracellular reactive oxygen species which are essential for transcription factor (NFkappaB) activation. NFkappaB in turn activates the major immediate early promotor (MIEP), with subsequent expression of the major immediate early protein product IE-72 which transactivates the MIEP, all critical events for viral replication. Because estrogen and the anti-estrogen tamoxifen have antioxidant properties, we examined the effect of estrogen and tamoxifen on generation of reactive oxygen species using an intracellular fluorescent marker (dichlorofluorescein), and on the early intracellular events following cytomegalovirus infection of human coronary smooth muscle cells. We found that 17beta-estradiol at physiological concentrations (0.1- 0.5 nM) dose-dependently inhibited cytomegalovirus-induced reactive oxygen species accumulation, as did tamoxifen at therapeutic concentrations (5-500 nM); progesterone (2.5-25 nM) was ineffective. To determine the relevance of this finding, we next co-transfected smooth muscle cells with a reporter gene under the control of the MIEP, and with an IE-72 expression vector. IE-72 stimulated MIEP transcriptional activity 2-3 fold, an effect inhibited by estrogen and by tamoxifen. Estrogen or tamoxifen, but not progesterone, lowered viral titers and reduced cytopathic effects (plaque formation) by 50% at 8 days after cytomegalovirus infection. We conclude that estrogen and tamoxifen have anti-cytomegalovirus effects by inhibiting reactive oxygen species generation following infection of coronary smooth muscle cells. To the extent that cytomegalovirus is a risk factor for atherosclerosis, this antioxidant effect may contribute to the anti-atherogenic effect of hormone therapy. We further demonstrated that pretreatment with estradiol inhibits expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and protein (both are induced by viral infection); 3-methoxyestrone treatment was ineffective. We also found that infected smooth muscle cells release TNFalpha into the supernatant, and pretreatment with estradiol attenuated TNFalpha release. These anti-inflammatory properties of 17beta-estradiol are shared by the stereoisoner 17alpha-estradiol, which in contrast to 17beta-estradiol has only weak affinity for the estrogen receptor, suggesting non-receptor dependent mechanisms.
