The biology of estrogen in endothelial cells

The rapid development of atherosclerosis following menopause and its prevention by estrogen replacement therapy suggests a potent anti-atherogenic effect of estrogen on the arterial wali Since estrogens exert their effect through an intranuclear estrogen receptor (ER) we have investigated whether human microvascular endothelial cells (HMVEC) isolated by collagenase digestion from liposuction specimens express ERs All 15 lots of HMVEC (PM) investigated by immunofluorescence demonstrated the presence of an intranuclear ER. To mimic the postmenopausal state in humans, oopherectomized New Zealand white female rabbits (OVX) were used as models. Using this model we have investigated a) whether rabbit aortic cndothelial cells (RAEC) express ERs, b) if estrogen deprivation leads to any alteration in ER levels and c) since estrogens have recently been shown to facilitate endothelium dependent vasodilatation and such an action may be mediated by nitric oxide (NO) and/or endothelins (ET), we also investigated whether estrogen regulates the levels of endothelial cell nitric oxide synthase (ECNOS) and ET in the rabbit model. RAEC grown in culture (P3) were stained immunohistochemicaily for ER. All 6 RAEC cultures from OVX rabbits stained for ER with markedly greater intensity than those from controls. Western Blot analysis for ECNOS demonstrated that all 6 RAEC homogenates from OVX animals showed a marked decrease in ECNOS when compared to those of controls. On the other hand, EC homogenates from OVX animals had significantly (p<0.05) higher levels of endothelins (mean=13 0 ±2 4 vs. 2.1 ±0 4 frnol/mg protein) lhan those from controls, as measured by EL1SA. Since NO inhibits the attachment of leukocytes to ihe endothelium and exerts antimitogenic effects and since these phenomena may be cardinal in atherogenesis, the possibility that oopherectomy can affect the levels of intercellular adhesion molecule-1 (ICAM-I) and proliferating ceil nuclear antigen (PCNA) (a cell-cycle regulated protein whose levels correlate directly with rates of cellular proliferation and DNA synthesis) was investigated. 1CAM-1 and PCNA levels, assayed for by western blotting and immunocytochemistry, respectively, were markedly elevated in RAEC from OVX animals. It has previously been shown that stromal cells from human subcutaneous fat express aromatase activity and since stromal cells include microvascular endothelial cells too, the possibility that HMVEC could express aromaiase was also investigated Using RT-PCR and in-siiu hybridization, the presence of aromatase P450 mRNA was demonstrated in HMVfcC Early passage cultures al confluence, were assayed for aromatase activity by [³H] water release assay. RAEC cultures from OVX rabbits had more aromatase activity than those from control rabbits (13 ± 6.27 vs. 6.88 + 3.68 pmol/mm/mg protein). These novel observations of oopherectomy-induced changes in the levels ol proteins that affect vascular function indicate possible mechanisms which may be implicated m the increased incidence of atherosclerosis in postmenopausal women.