Search

We reported previously that high concentrations of either estradiol-17beta (E(2)) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase-kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E(2) and DHT or protein bound hormones (E(2)-BSA or T-BSA), alone or in various combinations. High concentration of E(2) or E(2)-BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E(2) no longer inhibited (3)[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E(2), VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E(2) had only marginal effect on this interaction, and 30 nM E(2) reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E(2) and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E(2)-BSA was reversed in the presence of T-BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.

Background: 1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1alpha-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3., Methods and Results: We assessed the expression and activity of 25-hydroxyvitamin D3-1alpha-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17beta (30 nmol/L), raloxifene (3 micromol/L), and the phytoestrogens genistein (3 micromol/L), biochainin A (3 micromol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA increased by 43+/-13%, (P<0.05) 7+/-24% (P=NS), 176+/-28% (P<0.01), 65+/-11% (P<0.05), 152+/-24% (P<0.01), and 71+/-9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17beta and biochainin A also increased the generation of 1,25(OH)2D3 by 40+/-23% (P<0.05) and 55+/-13% (P<0.05), respectively., Conclusions: We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1alpha-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.

The present study was designed to determine whether some of the effects of estrogen on human vascular cell growth are exerted through membrane-binding sites, using native as well as novel protein-bound, membrane non-permeant estrogenic complexes. We measured changes in DNA synthesis and creatine kinase-specific activity (CK), after treatment with estradiol-17beta (E(2)), estradiol-17beta-6-(O)-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (E(2)-BSA), 6-carboxymethyl genistein (CG) or 6- carboxymethyl genistein bound to the high molecular protein keyhole limpet hemocyanin (CG-KLH), and 7-(O)-carboxymethyl daidzein (CD) or 7-(O)-carboxymethyl daidzein linked to keyhole limpet hemocyanin (CD-KLH). High concentrations of either E(2) or E(2)-BSA inhibited DNA synthesis in vascular smooth muscle cells (VSMC) (-39% +/- 28% v -32% +/- 15%). Estradiol as well as CG and CD increased DNA synthesis dose dependently in endothelial ECV-304 cells. The CG and CD, as well as CG-KLH and CD-KLH, stimulated DNA synthesis dose dependently in VSMC (66% +/- 2%, 100% +/- 12%, 66% +/- 6%, and 41% +/- 8% at 300 nmol/L, respectively). In contrast all forms of protein-bound hormones were unable to affect DNA synthesis in ECV-304 cells or CK in either cell type. In VSMC, both free and bound hormones increased mitogen-activated protein-kinase (MAPK)-kinase activity, which was blocked by UO126, an inhibitor of MAPK-kinase. Furthermore, the effects of E(2), E(2)-BSA, or CG-KLH on DNA synthesis were inhibited by UO126. Using the E(2)-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for E(2) in VSMC and ECV 304 cells. Hence, the effects of E(2) on DNA synthesis in human VSMC, but not in endothelial cells, are apparently exerted by membrane-binding sites for E(2) and do not require intracellular entry of E(2) through the classic nuclear receptor route.

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.

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) alpha 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 ERbeta 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 ERalpha 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.

Background: The aim of this study was to assess the effect of phytoestrogens on the human vascular wall in vitro., Methods: We compared the effects of E2 to those of genistein (G), daidzein (D), biochanin A (BA), equol (EQ), and quecertin (Qu) on 3[H] thymidine incorporation and creatine phosphokinase (CK) activity in human vascular smooth muscle cells (VSMC) and in a human endothelial cell line (E304)., Results: In VSMC, E2, the estrogen antagonist raloxifene (RAL), G, and D stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at higher concentrations. In contrast, BA and EQ had a monophasic stimulatory effect on 3[H] thymidine incorporation (87% +/- 9% and 54% +/- 17%, respectively) whereas Qu had only an inhibitory effect (-36 +/- 16% at 30 nmol/L). In E304 cells, all phytoestrogens stimulated DNA synthesis in a dose-related manner. In both cell types E2, RAL as well as all phytoestrogens increased CK-specific activity. The administration of phytoestrogens to immature female rats resulted in increased CK in the aorta (Ao) (60% to 220%) and in the left ventricle of the heart (Lv) (45% to 160%). Similar increases in Ao and Lv CK were also induced by E2 and all five phytoestrogens in ovariectomized (OVX) female rats. RAL antagonized phytoestrogen-induced CK activity in human vascular cells and in the rat Ao and Lv tissue but did not block phytoestrogen effects on DNA synthesis in human VSMC., Conclusions: Although phytoestrogens have estrogen-mimetic effects on cell growth and CK in cultured human vascular cells and on CK in rat vascular tissues in vivo, the effects on human VSMC replication are highly dependent on the concentration and the particular phytoestrogen under investigation.

The lipoxygenase pathway has been implicated in the growth, migration, and contraction of vascular smooth muscle cells (VSMCs). However, the precise type of lipoxygenase present in the vascular wall has not been characterized. In this study, we used a specific reverse-transcriptase polymerase chain reaction method with 2 sets of specific primers on total RNA and polyA (+)RNA of normal human VSMCs prepared from umbilical artery. Two forms of platelet-type 12-lipoxygenase mRNA were present in human VSMCs: the already published form cloned from human erythroleukemia cells and a variant form of platelet-type 12-lipoxygenase, which includes 2 additional sequences consistent with the 2 introns (D and E). This novel form of 12-lipoxygenase poly A (+)RNA was downregulated by lipopolysaccharide (10 ug/ml) and upregulated by epidermal growth factor (100 ng/ml) but was not affected by angiotensin II (10(-7) mol/l). We developed a rabbit anti-human platelet-type 12-lipoxygenase polyclonal antibody directed against a 24-amino acid peptide encoded within exon 4. Western immunoblotting of protein extracted from VSMCs and umbilical artery and platelet extract with this antibody showed a coordinate 110-kDa protein and the already-described 70-kDa band detected in platelets and cord homogenate. Another 120-kDa protein was consistently detected in cord extracts but not in platelet or VSMC homogenates. The immunohistochemistry study performed with the same antibody showed extensive cytoplasmic staining of VSMCs. The specific role of these different forms of platelet-type 12-lipoxygenase is subject to further investigation.

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.

Platelet-derived endothelial cell growth factor (PD-ECGF) is linked to angiogenesis in human cancer. Direct studies have demonstrated that PD-ECGF is a potent mitogen for endothelial cells in vivo. Because endothelial repair and smooth muscle cell proliferation are two processes that affect arterial wall structure and tone, we analyzed the effects of PD-ECGF on DNA synthesis and creatine kinase BB-specific activity (CK) in human umbilical artery smooth muscle cells (SMC) and in a human umbilical endothelial cell line (E304). In SMC, PD-ECGF (0.001 to 10 U/mL) inhibited DNA synthesis dose dependently (-24% + 6% to -63% + 15%) assessed by 3[H]thymidine incorporation into DNA, whereas in E304 it stimulated DNA synthesis dose dependently (30% + 4% to 100% + 4%). In both SMC and E304, however, PD-ECGF elicited an increase in CK-specific activity by 54% to 130% and 79% to 163%, respectively. These effects were reversed by a specific anti-PD-ECGF antibody. In E304 cells PD-ECGF enhanced 17beta-estradiol (E2) or dihydrotestosterone (DHT)-induced DNA synthesis from 56% to 122% and from 127% to 359%, and CK activity from 70% to 180% and from 90% to 190%, respectively. In SMC PD-ECGF, an inhibitor of 3[H]thymidine incorporation by itself, markedly enhanced the stimulatory effect of low concentrations of E2 and DHT on 3[H]thymidine incorporation. It also increased E2 and DHT CK induction from 40% to 140% and from 52% to 120%, respectively. In both E304 and SMC, PD-ECGF inhibited the proliferative and the CK-inducing effects of platelet-derived growth factor (PDGF) and immunoglobulin F1 (IGF1). Thus, PD-ECGF, an established growth promoter for endothelial cells, is a potent inhibitor of DNA synthesis in human arterial SMC. However, in both E304 endothelial cells and SMC, PD-ECGF enhances the stimulatory effect of low concentrations of gonadal steroids on 3[H]thymidine incorporation. PD-ECGF antagonizes PDGF- and IGF1-induced DNA synthesis in both E304 and SMC cells. By inhibiting arterial SMC proliferation and accelerating endothelial cell replication, PD-ECGF may buffer the effect of PDGF and favorably modulate arterial wall response to injury.

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 17beta-estradiol (E2), 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 E2 and DHT had a biphasic effect on [3H]thymidine incorporation into DNA: low concentrations (0.3 nmol/L for E2, 3 nmol/L for DHT) stimulated [3H]thymidine incorporation (+35% and +41%, respectively), whereas high concentrations (30 nmol/L for E2, 300 nmol/L for DHT) inhibited [3H]thymidine incorporation (-40%). In contrast, E2 (0.3 to 300 nmol/L) and DHT (3 to 3000 nmol/L) dose-dependently enhanced [3H]thymidine incorporation in E304 cells (peak, +85% for both). In VSMCs, high concentrations of E2 and DHT inhibited platelet-derived growth factor (PDGF)-or insulin-like growth factor (IGF-1)-induced DNA synthesis (-50% to 80%), whereas PDGF- or IGF-1-dependent DNA synthesis in E304 cells was further increased by E2. The antiestrogens tamoxifen and raloxifene mimicked the effects of E2 on DNA synthesis in both VSMCs and E304 cells. However, when coincubated with a stimulatory concentration of E2 (0.3 nmol/L), tamoxifen and raloxifene blocked E2-induced [3H]thymidine incorporation in E304 cells but not in VSMCs. Finally, the androgen antagonist flutamide inhibited the biphasic effects of DHT on VSMCs and blocked the increase in DNA elicited by DHT in E304 cells. The results suggest complex, dose-dependent, and cell-specific interactions of estrogens, androgens, and their respective antagonists in the control of cellular proliferation in the vascular wall. Gonadal steroid-dependent inhibition of VSMC proliferation and stimulation of endothelial replication may contribute to vascular protection and remodeling responses to vascular injury.