Testosterone protects cardiac myocytes from superoxide injury via NF-κB signalling pathways.

AIMS: Cellular and molecular mechanisms underlying the effects of androgenic hormone testosterone on the heart remain unclear. This study examined the impact of testosterone on viability of cardiac myocytes and the role of NF-κB signalling pathways. MATERIALS AND METHODS: Rat H9c2 myocytes were cultured in steroid-free media and incubated with hydrogen peroxide (H2O2, 200 µM, 6h). NF-κB expression was knocked down by RelA (p65) siRNA interference. Testosterone (5-100 nM, 24-48 h) was provided into the media and androgen receptor (AR) blocked by flutamide (100 nM). Cell apoptotic/necrotic death was determined by morphological examination and flow-cytometric analysis. Gene expression was examined by Western blotting analysis. KEY FINDINGS: Testosterone supplements reduced the superoxide-induced apoptotic/necrotic death, stimulated NF-κB (RelA) expression, activated Akt activity, and inhibited Caspase-3 expression in the cardiac myocytes. The hormonal effects were abolished by either AR blocker flutamide or NF-κB-knockdown. Testosterone also induced ERK1/2 activation, which was not affected by flutamide or NF-κB knockdown, and blocking the ERK activity did not affect the protective effect of the hormone on the cells. SIGNIFICANCE: This study demonstrates that exogenous testosterone supplementation protects cardiac myocytes from superoxide injury via AR mediation and dependent on normally functional canonical NF-κB (RelA/p50) signalling pathways. The NF-κB signalling may be an important key molecular basis for myocardial benefits of hormone (testosterone) therapy.

A phase I dose-escalation study to evaluate tolerability in a Western population to T89, a modern cardiovascular herbal medicine.

T89 (Dantonic) is a modern herbal medicine currently used in Chinese hospitals for the management of ischemic heart disease. This dose-escalation clinical trial aims to assess tolerability of Western people to T89. Healthy Australian adults of non-Asian background orally took a single dosage of 6, 8, 10, 12, 13, 14, 15, or 16 T89 capsules (6 people for each dose) and were assessed with respect to symptoms and physical signs, electrocardiogram, hematology, plasma biochemistry, and urinalysis. Secondary objectives were to determine the dose-limiting toxicity and maximum-tolerated dose. It found that a single dose of T89 up to 16 capsules was not associated with significant adverse events or abnormalities in clinical laboratory tests and electrocardiogram parameters, except minor symptoms reported included mild and transient dizziness, stomach discomfort, diarrhea, and involuntary muscular contraction. The incident rate of these symptoms was generally low (1/30, 3.3%) but increased (7/18, 38.9%) in higher dose (≥14 capsules) groups. No defined dose-limiting toxicity events occurred; so the study could not define the maximum-tolerated dose. In conclusion, a single dose of T89 up to 13 capsules, 4 times of a regular therapeutic dosage, is generally safe and tolerated by individuals of non-Asian background.

Polycystic ovary syndrome in patients with epilepsy: a study in 102 Chinese women.

PURPOSE: The incidence of polycystic ovary syndrome (PCOS) increases in women with epilepsy (WWE), which appears to vary with ethnicity. This study was conducted to determine the incidence and risk factors of PCOS in Chinese WWE. METHODS: The study was carried out in 102 of 139 Chinese WWE at reproductive ages, with 32 receiving valproic acid (VPA), 40 receiving other antiepileptic drugs (AEDs), and 30 without AEDs therapy. PCOS was defined as having 2 or more of the following components: polycystic ovaries, hyperandrogenism, and amenorrhoea or oligomenorrhoea (a/oligomenorrhoea). RESULTS: One or more isolated components of PCOS were found in 56 (54.9%) patients, with 29 (28.4%) having polycystic ovaries, 20 (19.6%) with a/oligomenorrhea, 7 (6.9%) with hyperandrogenism, and 13 (12.7%) with defined PCOS. Their average age at the start of seizure was 13.8±6.5 years, younger than that of patients without these disorders (16.9±8.6 years, p<0.05). VPA therapy increased the incidence of PCOS (11/32, 34.4%), in addition to increased blood levels of testosterone and luteinizing hormone (LH) as well as LH to FSH (follicle-stimulating hormone) ratio. No significant relationship was found between the incidence of PCOS and the type, duration, or frequency of seizures in these WWE. CONCLUSION: There is an increased incidence of PCOS in Chinese WWE at reproductive ages, by more than 2 times of that in the general population. Risk factors include seizures starting at a young age and VPA therapy.

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture.

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be "beneficial" in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially "harmful". The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.

Oestradiol supplement minimises coronary occlusion-induced myocardial infarction and ventricular dysfunction in oophorectomised female rats.

BACKGROUND: Endogenous oestrogen deficiency after menopause is associated with high risk of acute cardiac events and the protection of exogenous oestrogen supplements remains uncertain. This study investigates whether oestrogen therapy protects the heart from ischemic injury in oophorectomised rats. METHODS: Sexually mature female Sprague-Dawley rats (6 for each group) with bilateral oophorectomy underwent selective ligation (occlusion) of left coronary artery for 4 weeks. 17ß-oestradiol (E2) supplements (10 µg, i.m., every other day) were started before (preventive-therapeutic supplement) or after coronary occlusion (therapeutic supplement). RESULTS: In oophorectomised rats plasma levels of E2 declined from 1301 ± 80 to 196 ± 48 pmol/L (p<0.01) and cardiac expression of oestrogen receptors (ER) decreased by ∼60%. E2 supplements recovered the ER expression. Selective ligation of left coronary led myocardial infarction in the left ventricle, with an increase in plasma cardiac troponin I (cTn-I), decrease in systolic blood pressure (SBP), and reduction of left ventricular pressures. Preventive-therapeutic but not therapeutic E2 supplement reduced cTn-I levels (from 21.9 ± 2.0 to 6.0 ± 0.3 ng/mL, p<0.01), minimised infarction (from 37.0 ± 1.2% to 18.1 ± 2.3%, p<0.05), increased SBP (from 82 ± 4.2 to 97 ± 4.4mm Hg, p<0.05), and improved left ventricular end pressures in the oophorectomised rats following coronary occlusion. CONCLUSION: Postmenopausal (ooporectomised) oestrogen supplement commenced before establishment of myocardial ischemia minimises myocardial infarction and ventricular dysfunction following the coronary artery occlusion. Cellular and molecular mechanisms underlying the cardiac protection of oestrogen therapy remain unclear, in which activation of cardiac ER expression and increasing in circulating CD90(+) stem cells may be involved.

A chinese herbal preparation containing radix salviae miltiorrhizae, radix notoginseng and borneolum syntheticum reduces circulating adhesion molecules.

Circulating adhesion molecules (CAMs), surface proteins expressed in the vascular endothelium, have emerged as risk factors for cardiovascular disease (CVD). CAMs are involved in intercellular communication that are believed to play a role in atherosclerosis. A Chinese medicine, the "Dantonic Pill" (DP) (also known as the "Cardiotonic Pill"), containing three Chinese herbal material medica, Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, has been used in China for the prevention and management of CVD. Previous laboratory and animal studies have suggested that this preparation reduces both atherogenesis and adhesion molecule expression. A parallel double blind randomized placebo-controlled study was conducted to assess the effects of the DP on three species of CAM (intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and endothelial cell selectin (E-selectin)) in participants with mild-moderate hypercholesterolemia. Secondary endpoints included biochemical and hematological variables and clinical effects. Forty participants were randomized to either treatment or control for 12 weeks. Treatment with DP was associated with a statistically significant decrease in ICAM-1 (9% decrease, P = .03) and E-Selectin (15% decrease, P = .004). There was no significant change in renal function tests, liver function tests, glucose, lipids or C-reactive protein levels and clinical adverse effects did not differ between the active and the control groups. There were no relevant changes in participants receiving placebo. These results suggest that this herbal medicine may contribute to the development of a novel approach to cardiovascular risk reduction.

Cardiovascular physiology of androgens and androgen testosterone therapy in postmenopausal women.

Women before menopause are at relatively lower risk of cardiovascular disease (CVD) compared with age-matched men and after menopause this gender advantage disappears. Androgen has been known to be an independent factor contributing to the higher male susceptibility to CVD, through adverse effects on lipids, blood pressure, and glucose metabolism. High androgen levels also contribute to CVD development in women with polycystic ovary syndrome as well as androgen abusing athletes and body builders. On the other hand, decline in androgen levels, as a result of ageing in men, is associated with hypertension, diabetes and atherosclerosis. Postmenopausal women, particularly those with oophorectomy are generally in low levels of sex hormones and androgen insufficiency is independently associated with the higher incidence of atherosclerosis in postmenopausal women. Androgen testosterone therapy (ATT) has been commonly used to improve well-being and libido in aging men with low androgen levels. The therapy has been demonstrated also to effectively reduce atherogenesis in these people. The use of ATT in postmenopausal women has increased in recent years and to date, however, the cardiovascular benefits of such therapy in these women remain uncertain. This review focuses on research regarding the impact of endogenous androgens and ATT on the cardiovascular physiology and CVD development in postmenopausal women.

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts.

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50-800 microg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H(2)O(2), 50-200 microM), or tumor necrosis factor alpha (TNFalpha, 40 ng/ml) for 24-48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [(3)H]-thymidine or [(3)H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 microg/ml) and double therapeutic (100 microg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200-800 microg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 microg/ml) significantly inhibited TNFalpha-induced apoptosis in myocytes, with 12.3+/-1.46% cells being apoptosis in CP treatment group and 37.0+/-7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by approximately 50% and approximately 90% reduction of AII-induced collagen synthesis at 50 and 100 microg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.

A preparation of herbal medicine Salvia miltiorrhiza reduces expression of intercellular adhesion molecule-1 and development of atherosclerosis in apolipoprotein E-deficient mice.

Cardiotonic pill (CP) is a pharmaceutical preparation of the herbal medicine Salvia miltiorrhiza. In vitro studies demonstrate that CP inhibits vascular endothelial expression of adhesion molecules and smooth-muscle proliferation, implying the possibility of antiatherosclerotic effects. This study employs an in vivo animal model to examine the potential therapeutic efficacy of CP on atherosclerotic development. Male apolipoprotein E-deficient (ApoE-/-) mice fed with an atherogenic (high fat) diet were administered with CP (90-120 mg/kg per day) via drinking water for 8 weeks. Hypercholesterolemia developed in the mice, with 22-fold increases in plasma levels of total cholesterol, 29-fold of LDL, and 7-fold of HDL. CP therapy did not significantly alter the lipid levels. Expression of intercellular adhesion molecule 1 significantly increased in circulating leukocytes and was abolished by CP therapy. Atherosclerosis significantly developed in the aorta and was attenuated by CP therapy, with an approximately 30% reduction in whole atherosclerotic lesions and an approximately 50% reduction in fibrous plaques in the artery. Thus, herbal medicine CP partly protects ApoE-/- mice from high-fat diet-induced atherogenesis. The protection is unlikely to be attributable to decreases in circulating cholesterol levels, but it might possibly relate to an inhibition of expression of adhesion molecules and other effects that remain unknown at this time.

Effects of four medicinal herbs on human vascular endothelial cells in culture.

BACKGROUND: Danshen (DS, Salvia miltiorrhiza), Shanchi (SQ, Panax notoginseng), Shanzai (SZ, Hawthorn) and Heshouwu (HSW, Polygonum multiflorum Thunb) are four medicinal herbs commonly used in traditional Chinese medicine and previously shown to have activity that may contribute to cardiovascular protection. This study aims to investigate effects of these herbs on vascular endothelial cells with respect to cell viability and expression of cellular adhesion molecules under inflammatory conditions. METHODS: Herbal extracts were prepared by an established industrial manufacturing process. Human umbilical vein endothelial cells (HUVEC) were incubated with the herbal extract under normal or serum-free culture and tumor necrosis factor (TNF) alpha stimulation. Cell apoptosis, apoptosis-associated gene expression, expression of cellular adhesion molecules, DNA synthesis, and growth were assessed via morphological examination, Annexin-V staining, Western blotting analysis, Flow-Cytometry, [(3)H]-thymidine incorporation assay, and cell number study. RESULTS: SZ and HSW significantly inhibited apoptosis in HUVEC undergoing serum deprivation and TNFalpha stimulation, accompanied by down-regulation of caspase-3 gene expression. DS and SQ significantly attenuated TNFalpha-induced expression of adhesion molecule VCAM-1 and also ICAM-1 by DS in the cells. All four herbs at therapeutic concentrations (100 microg/mL) inhibited DNA synthesis (10-42% decrease in [(3)H]-thymidine incorporation rates) and growth (5-10% decrease in cell numbers) in HUVEC under normal cultures. CONCLUSION: DS, SQ, SZ and HSW are physiologically active on human vascular endothelial cells. The actions by HSW and SZ to reduce apoptosis and DS and SQ to inhibit adhesion molecule expression may help protect endothelial function and inhibit atherogenesis, while their actions to inhibit DNA synthesis and cell growth may weaken the ability of endothelial repair. Further studies are needed to identify the chemical compounds responsible to these physiological effects by these herbs.

Cellular mechanisms underlying the cardiovascular actions of oestrogens.

Although pre-menopausal women enjoy relative cardiovascular protection, hormone (oestrogen+/-progestin)-replacement therapy has not shown cardiovascular benefits in post-menopausal women, suggesting that the effects of oestrogens on the cardiovascular system are much more complex than previously expected. Endothelial cells, smooth muscle cells, cardiac myocytes and fibroblasts, the cellular components of blood vessels and the heart, play important roles in cardiovascular health and disease. During the development and progression of cardiovascular disease, changes occur both in the structure and function of these cells, resulting in a wide range of abnormalities, which affect growth, death and physiological function. These cells contain functional oestrogen receptors and are targets for oestrogen action. This review focuses on recent studies on the effects of oestrogen on cardiovascular cell function. Oestrogens, particularly 17beta-oestradiol, exert multiple effects on cardiovascular cells, and these effects may contribute to the gender-associated protection against cardiovascular diseases.

Effects of 17beta-estradiol on growth and apoptosis in human vascular endothelial cells: influence of mechanical strain and tumor necrosis factor-alpha.

Vascular endothelial cell (EC) integrity is key to arterial health; endothelial dysfunction is linked to atherogenesis. Atherosclerosis shows a male preponderance, possibly related to the protective effect of estrogens in women. This study examined the effect of estrogens on growth, apoptosis and adhesion molecule expression in cultured human EC. The effects of 17beta-estradiol (E2) were studied in human umbilical vein endothelial cells (HUVEC) under normal culture conditions, and following exposure to cyclic mechanical strain or tumor necrosis factor alpha (TNFalpha). E2 enhanced HUVEC growth in serum-enriched media, in a concentration-dependent manner. This up-regulation of EC growth by E2 was associated with an increase in telomerase activity, assessed by PCR-based TRAP analysis. Cyclic strain enhanced [(3)H]-thymidine incorporation into DNA, and increased activation of mitogen-activated protein (MAP) kinase ERK1/2 and expression of early growth genes (Egr-1 and Sp-1); E2 attenuated the strain-induced ERK1/2 activation but not the early growth gene expression or DNA synthesis. TNFalpha (20 ng/mL) induced apoptosis in HUVEC, causing a decrease in DNA synthesis, increase in floating and Annexin-V-stained cell numbers, and morphological changes. TNFalpha also upregulated ERK1/2 activity and expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin). E2 significantly attenuated the effects of TNFalpha on ERK1/2 activity, apoptosis, and E-selectin expression in the cells. Thus, estradiol enhances growth and reduces TNFalpha-induced apoptosis in EC; enhanced EC growth may be mediated via upregulation of telomerase activity. These effects are possible cellular mechanisms underlying female gender-associated cardiovascular protection.

Effects of a Chinese herbal preparation on vascular cells in culture: mechanisms of cardiovascular protection.

1. The use of traditional Chinese medicinal herbs or their pharmaceutical products for disease prevention and management is becoming increasingly popular in Western countries. Mixtures of various Chinese herbs have been used for the treatment of syndromes clinically overlapping Western cardiovascular syndromes. One modern preparation, known as the 'Cardiotonic Pill' (CP), is a pharmaceutical product derived mainly from a medicinal herb, Salvia miltiorrhiza bunge, and recently widely used in Chinese hospitals for the prevention and management of ischaemic cardiovascular diseases. Although the CP is believed to confer an extensive range of benefits, little is known about the physiological actions of this medicine, particularly at the cellular and molecular levels. Therefore, the aim of the present study was to explore possible cellular mechanisms of the CP on the cardiovascular system. 2. Cultured human vascular endothelial cells (EC) and vascular smooth muscle cells (VSMC) were exposed to the CP at various concentrations for periods ranging from hours to days. Cellular DNA synthesis was determined by a [(3)H]-thymidine incorporation assay, proliferation and death were assessed by investigations of cell numbers and apoptosis, whereas the expression of extracellular adhesion molecules was analysed by flow-cytometry and Western blotting. 3. The CP extract at concentrations of less than 200 microg/mL was not associated with cell damage. At doses beyond the therapeutic range (10-20 microg/mL), the CP appeared to exert a mild inhibitory effect on DNA synthesis and proliferation of EC in serum-enriched cultures. The CP significantly attenuated tumour necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in a dose-dependent manner, with 50 and 100 microg/mL CP producing decreases in the expression of ICAM-1 of 26-32% and 32-44%, respectively, and of VCAM-1 of approximately 23% and 27-42%, respectively. The CP did not affect apoptosis in EC under conditions of serum-deprivation. 4. In VSMC, the CP significantly inhibited platelet-derived growth factor BB-induced DNA synthesis and cell proliferation in a dose-dependent manner. The CP did not affect VSMC expression of adhesion molecules. 5. We conclude that the CP inhibits expression of ICAM-1 and VCAM-1 in EC and proliferation of VSMC in a manner that has potentially beneficial therapeutic effects.

Androgens stimulate human vascular smooth muscle cell proteoglycan biosynthesis and increase lipoprotein binding.

Vascular smooth muscle cell (VSMC) proliferation and proteoglycan biosynthesis are two critical contributors to the development of atherosclerosis. We investigated the effects of specific androgens, androstenedione, dihydrotestosterone, and testosterone, on proteoglycan biosynthesis in human VSMC derived from internal mammary arteries. Vascular SMCs were metabolically labeled with [(35)S]sulfate or [(35)S]methionine/cysteine to assess glycosaminoglycans (GAGs) or proteoglycan core protein, respectively. The electrophoretic migration of radiolabeled proteoglycans was assessed by SDS-PAGE. Proteoglycan-low density lipoprotein (LDL) interactions were assessed using LDL affinity columns. Treatment of VSMCs with androstenedione (100 nm), dihydrotestosterone (10 nm), or testosterone (100 nm) increased [(35)S]sulfate incorporation into GAGs by 24.8% (P < 0.05), 22% (P < 0.05), and 32.5% (P < 0.05), respectively. Treatment of VSMCs with testosterone did not alter [(35)S]methionine/cysteine incorporation into proteoglycan core protein, suggesting that the effect of testosterone was associated with an increase in GAG length. Dihydrotestosterone (10 nm) and testosterone (100 nm) treatment of VSMCs resulted in the synthesis of biglycan and decorin that showed reduced electrophoretic mobility by SDS-PAGE, indicating an increase in GAG length. The effect of testosterone treatment on [(35)S]sulfate incorporation and GAG length was reversed by pretreatment of VSMCs with flutamide (1 mum), an androgen receptor antagonist. Proteoglycans from VSMCs treated with testosterone showed 11% (P < 0.01) higher binding capacity to LDL compared with proteoglycans from untreated cells. These results suggest a possible proatherogenic action of androgens through an elongation of GAG chains on proteoglycans in an androgen receptor-dependent manner.

Dehydroepiandrosterone increases endothelial cell proliferation in vitro and improves endothelial function in vivo by mechanisms independent of androgen and estrogen receptors.

Dehydroepiandrosterone (DHEA) may be beneficial in cardiovascular health, but mechanisms of DHEA action in the cardiovascular system are unclear. We have therefore 1) determined DHEA effects on the proliferation of cultured endothelial cells (EC), 2) compared effects of DHEA with estradiol (E) and testosterone (T), and 3) examined DHEA effects on subcellular messengers. We have in addition examined effects of DHEA (100 mg/d, 3 months) in 36 healthy postmenopausal women on blood pressure, lipids, and endothelial function, assessed noninvasively in large vessels by flow-mediated dilation of the brachial artery during reactive hyperemia, and in small vessels by laser Doppler velocimetry with iontophoresis of acetylcholine. DHEA, E, and T all increased EC proliferation; the effect of E was abolished by the estrogen receptor antagonist ICI 182,780, and that of T was abolished by the androgen receptor antagonist flutamide; neither blocked the effect of DHEA. In vitro, DHEA increased EC expression of endothelial nitric oxide synthase and activity of extracellular signal-regulated kinase 1/2. In vivo, DHEA increased flow-mediated dilation and laser Doppler velocimetry and reduced total plasma cholesterol. Thus, DHEA increases EC proliferation in vitro by mechanism(s) independently of either androgen receptor or estrogen receptor and in vivo enhances large and small vessel EC function in postmenopausal women.

The isoflavone metabolite cis-tetrahydrodaidzein inhibits ERK-1 activation and proliferation in human vascular smooth muscle cells.

Phytoestrogens have recently been proposed as alternatives to estrogens for cardiovascular protection; however, the effect of their metabolites on vascular biology is unclear. We studied the effect of a red clover-derived isoflavone metabolite cis-tetrahydrodaidzein (cis-THD) on human vascular smooth muscle cell (VSMC) proliferation. Cis-THD significantly inhibited platelet-derived growth factor (PDGF) BB-induced DNA synthesis (10% at 1 nmol/L, 17% at 10, 100 nmol/L; 17beta-estradiol: 27% inhibition at 1, 10 nmol/L, 33% at 100 nmol/L). Cis-THD reduced PDGF BB-induced increase in cell numbers. Cis-THD showed high binding affinity to estrogen receptors (ER) by ER competitor assays; its inhibitory effect on DNA synthesis was abolished by the ER antagonist ICI 182780 (100 nmol/L), indicating ER-mediation. Immunoprecipitation assays revealed that cis-THD inhibited PDGF BB-stimulated activation of mitogen-activated protein (MAP) kinase ERK-1 by 34% at 1 nmol/L, 58% at 10 nmol/L, and 81% at 100 nmol/L, while MAP kinase JNK and p38 activities were unaltered. Thus, the isoflavone metabolite cis-THD inhibits PDGF-induced ERK-1 activation and cell proliferation in human VSMC, suggesting a potential beneficial effect in cardiovascular protection.

Endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells: insights from the aromatase-knockout mouse.

BACKGROUND: Altered proliferation and death of vascular smooth muscle cells (VSMCs) are important mechanisms in vascular growth and remodeling. This study examined the effect of endogenous estrogens on VSMC proliferation. METHODS AND RESULTS: An estrogen-deficient animal model, the aromatase-knockout (ArKO) mouse, was used. Primary cultures of VSMCs were established from aortas of 11-week-old male and female ArKO and wild-type (WT) mice. In ArKO cells, the absence of aromatase cytochrome P450 mRNA expression was demonstrated by reverse transcription-polymerase chain reaction; Western blotting showed normal expression of estrogen receptor protein. Proliferative responses to serum or platelet-derived growth factor-BB were lower in ArKO than WT cells; 17beta-estradiol (E2, 10 nmol/L) enhanced the response to platelet-derived growth factor-BB in ArKO cells but inhibited the response in WT cells. E2 inhibited activity of mitogen-activated protein kinase ERK1/2 in WT but not ArKO cells. Apoptosis-related death caused by tumor necrosis factor-alpha was greater in ArKO than in WT cells; this effect in ArKO was attenuated by E2. Differences in VSMC proliferation between ArKO and WT occurred in both sexes. Morphological studies in aortas derived from male mice at 1 year of age demonstrated that medial smooth muscle area was approximately 10% less in ArKO than WT mice at this age. CONCLUSIONS: Deficiency of endogenous estrogens reduces proliferation and enhances apoptosis-related death in VSMCs; exogenous E2 corrects these abnormalities.

High glucose abolishes the antiproliferative effect of 17beta-estradiol in human vascular smooth muscle cells.

We examined effects of 17beta-estradiol (E(2)) on human vascular smooth muscle cell (VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l) glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were greater in high than normal glucose concentrations; the difference in DNA synthesis was abolished by a protein kinase C (PKC)-beta inhibitor, LY-379196 (30 nmol/l). Western blotting showed that PKC-beta(1) protein increased in cells exposed to high glucose, whereas PKC-alpha protein and total PKC activity remained unchanged, compared with normal glucose cultures. In normal glucose, E(2) (1-100 nmol/l) inhibited PDGF-induced DNA synthesis by 18-37% and cell proliferation by 16-22% in a concentration-dependent manner. The effects of E(2) were blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating ER dependence. In high glucose, the inhibitory effect of E(2) on VSMC proliferation was abolished but was restored in the presence of the PKC-beta inhibitor LY-379196. Thus high glucose enhances human VSMC proliferation and attenuates the antiproliferative effect of E(2) in VSMC via activation of PKC-beta.

Testosterone (T) enhances apoptosis-related damage in human vascular endothelial cells.

Androgens may contribute to higher cardiovascular risk in men via deleterious effects on vascular endothelial cells (EC). We examined the effects of androgens on male human umbilical vein EC (EA.hy926) in culture. [(3)H]Thymidine incorporation assays showed that after 24-h serum deprivation, testosterone (T) (but not dehydroepiandrosterone nor 17beta-E2) induced significant dose-dependent decreases in DNA synthesis (10-16% at 1-100 nmol/liter); the AR antagonist flutamide (100 nmol/liter) abolished this effect of T. After 48-h serum deprivation, typical apoptotic DNA patterns were detected in agarose gels, and the number of floating cells indicative of severe damage was significantly greater after T treatment for 48 and 72 h (13.7 +/- 0.5% and 30.2 +/- 2.5%, respectively) than the control values (9.7 +/- 1.05% and 23.7 +/- 3.0%). Analysis of attached cells by annexin V-fluorescein isothiocyanate/propidium iodide staining showed that after 48-h serum deprivation, T significantly increased the number of cells in the early (16.0 +/- 1.1%) and late (8.3 +/- 0.3%) stages of apoptosis compared with control (6.8 +/- 1.0% and 4.0 +/- 0.2%, respectively); such increases in apoptosis-related damage were also observed, to a lesser degree, in serum-enriched culture. Western blotting showed that B cell leukemia/lymphoma-2 protein (Bcl-2) expression decreased significantly in serum-deprived EC treated with T. Thus, T reduces DNA synthesis and enhances apoptosis after serum deprivation in EC, possibly related to reduced Bcl-2 expression.

Dehydroepiandrosterone inhibits human vascular smooth muscle cell proliferation independent of ARs and ERs.

Dehyroepiandrosterone (DHEA), an adrenal-derived steroid, has been clinically implicated in protection against coronary artery disease and experimentally in inhibition of atherosclerosis and plaque progression. Because DHEA is enzymatically metabolized to androgens or estrogens, it is not clear whether DHEA exerts effects directly or after conversion to these hormones, both of which are associated with well-characterized pathways of action. We therefore examined the effects of DHEA on proliferation of human vascular smooth muscle cells (VSMCs) in culture in the presence or absence of the ER antagonist ICI 182,780 and the AR antagonist flutamide and compared them with the effects of 17beta-estradiol, androstenedione, and T. We also determined the affinity of DHEA for ERs and ARs in VSMC and its specific binding in intact cells. To explore a possible mechanism for DHEA action in these cells, we measured the phosphorylation of ERK-1, c-jun N-terminal protein kinase, and p38 (three members of the MAPK superfamily). Both DHEA and 17beta-estradiol significantly inhibited platelet derived growth factor (PDGF)-BB-induced increases in VSMC proliferation, whereas androstenedione and T increased proliferation. Although E2-induced inhibition of the PDGF effect was abolished by ICI 182,780 and T-induced stimulation was abolished by flutamide, neither receptor antagonist altered the inhibitory effect of DHEA. Binding studies confirmed the presence of both ERs and ARs; DHEA showed minimal affinity for either receptor but bound specifically and with high affinity to putative receptors in intact cells. Following 4-h incubation with DHEA (1-100 nM), ERK1 phosphorylation was significantly reduced in a dose-dependent manner, whereas neither c-jun N-terminal protein kinase nor p38 kinase activity was altered by either PDGF-BB or DHEA. DHEA inhibits human VSMC proliferation by a mechanism independent of either ARs or ERs, presumably via a DHEA-specific receptor that involves ERK1 signaling pathways.