Abstract-The cardiovascular effect of estrogen is currently under intense investigation, but the role of androgens in vascular biology has attracted little attention. Because endothelial repair and vascular smooth muscle cell (VSMC) proliferation affect atherogenesis, we analyzed the effects of 17␤-estradiol (E 2 ), dihydrotestosterone (DHT), and sex hormone antagonists on DNA synthesis in human umbilical VSMCs and in E304 cells (a human umbilical endothelial cell line). In VSMCs, both E 2 and DHT had a biphasic effect on [ 3 H]thymidine incorporation into DNA: low concentrations (0.3 nmol/L for E 2 , 3 nmol/L for DHT) stimulated [ 3 H]thymidine incorporation (ϩ35% and ϩ41%, respectively), whereas high concentrations (30 nmol/L for E 2 , 300 nmol/L for DHT) inhibited [ 3 H]thymidine incorporation (Ϫ40%). In contrast, E 2 (0.3 to 300 nmol/L) and DHT (3 to 3000 nmol/L) dose-dependently enhanced [ 3 H]thymidine incorporation in E304 cells (peak, ϩ85% for both). In VSMCs, high concentrations of E 2 and DHT inhibited platelet-derived growth factor (PDGF)-or insulin-like growth factor (IGF-1)-induced DNA synthesis (Ϫ50% to 80%), whereas PDGF-or IGF-1-dependent DNA synthesis in E304 cells was further increased by E 2 . The antiestrogens tamoxifen and raloxifene mimicked the effects of E 2 on DNA synthesis in both VSMCs and E304 cells. However, when coincubated with a stimulatory concentration of E 2 (0.3 nmol/L), tamoxifen and raloxifene blocked E 2 -induced

These findings suggest that estrogen modulates the monocyte numbers and its effects may be mediated through the ER in the monocytes.

We have previously reported that estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in an endothelial cell line (E304). In SMC both gonadal steroids stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at high concentrations, whereas in E304 cells E2 and DHT dose dependently enhanced DNA synthesis. In both cell types gonadal steroids also induced the specific activity of creatine kinase BB (CK). Previous evidence suggets that the in vitro and in vivo CK responses to gonadal steroids in bone cells are upregulated by pretreatment with vitamin D analogs due to increased level of cellular estrogen receptors (ER). Here we analyzed the interaction of the vitamin D analogs hexafluorovitamin D (FL), JK-1624 F2-2 (JKF), and CB 1093 (CB) with gonadal steroids in regulating DNA synthesis and CK activity in human vascular cells in vitro. In E304 cells, daily treatment with FL, JKF, or CB (1 nmol/L for 3 days) increased DNA synthesis by 110 +/- 11%, 65 +/- 16%, and 88 +/- 23% respectively. In contrast, the same analogs inhibited 3[H] thymidine incorporation by 52 +/- 21%, 46 +/- 19%, and 50 +/- 10%, respectively, in SMC. In both cell types all three analogs increased CK by 25% to 75% and amplified the CK response to E2 and to DHT by twofold to threefold. In E304 cells the vitamin D analogs also increased DNA response to gonadal steroids from 50% to 60% to 200% to 280%. In SMC these analogs did not modify the DNA synthetic response to a low E2 concentration, but prevented the suppression of DNA synthesis exerted by high concentrations of E2 and DHT. Vitamin D inhibitors known to block cellular calcium mobilization, had no effect on the proliferative activity induced by vitamin D analogs. However, the inhibitor of the nuclear effects of vitamin D, ZK 159222, blocked the stimulatory effects of CB on DNA synthesis in E304 cells. Finally, both 1,25(OH)2 D3, and JKF decreased the expression of ERbeta proteins in SMC and increased the ERalpha isoform in E304 cells by 40% to 75%. The results indicate that vascular cells are targets for both vitamin D and gonadal steroid action and suggest a possible interaction between these hormones in the regulation of cell proliferation via modulation of vascular ER or interaction with proteins associated with ER.

The novel genistein (G) derivative, 6-carboxymethyl genistein (CG) was evaluated for its biological properties in comparison with G. Both compounds showed oestrogenic activity in vitro and in vivo. On the other hand G and CG differed in the following parameters: (i) only CG displayed mixed agonist-antagonist activity for oestrogen receptor (ER) in transactivation assays and (ii) only CG was capable of attenuating oestrogen (E 2 )-induced proliferation in vascular smooth muscle cells and of inhibiting oestrogen-induced creatine kinase (CK) specific activity in rat tissues. On the other hand only G enhanced the stimulatory effect on CK specific activity in the uterus. In comparison to the selective oestrogen receptor modulator (SERM) raloxifene (RAL), CG showed the same selectivity profile as RAL in blocking the CK response to E 2 in tissues derived from both immature and ovariectomized female rats. Molecular modelling of CG bound to the ligand binding domain (LBD) of ER predicts that the 6-carboxymethyl group of CG almost fits the binding cavity. On the other hand, molecular modelling of CG bound to the LBD of ER suggests that the carboxyl group of CG may perturb the end of Helix 11, eliciting a severe backbone change for Leu 525, and consequently induces a conformational change which could position Helix 12 in an antagonist conformation. This model supports the experimental findings that CG can act as a mixed agonist-antagonist when E 2 is bound to its receptors. Collectively, our findings suggest that CG can be considered a novel SERM with unique effects on the vasculature, bone and uterus.