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Baker Medical Research Institute, St. Kilda Central, Melbourne, Victoria 8008, Australia
Address all correspondence and requests for reprints to: Dr. Paul A. Komesaroff, Department of Medicine, Monash University, Alfred Hospital, Commercial Road, Prahran, Victoria 3181, Australia. E-mail: paul.komesaroff{at}med.monash.edu.au.
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Introduction |
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Interest in DHEA(S) stems from epidemiological studies showing an inverse relationship between cardiovascular mortality and plasma DHEA(S) levels in men (8, 9, 10). There is also limited evidence for a role in the pathophysiology of a number of other conditions, including immune disorders (11, 12), malignancies (13), and neurological dysfunction (14, 15, 16). Despite the apparent correlation between vascular disease and plasma levels of DHEA(S) and recent evidence that it inhibits human vascular smooth muscle cell proliferation independently of androgen receptors (AR) and estrogen receptors (ER) (17), the vascular effects of this hormone and its possible mechanisms of action remain unclear.
It is well recognized that cardiovascular disease is associated with dysfunction of the vascular endothelium (18, 19, 20), and a variety of actions of estrogens and androgens on the vascular endothelium have been described (21, 22, 23, 24, 25, 26, 27). In contrast, limited information is available about the effects of DHEA on endothelial function. We therefore sought to examine the actions of DHEA on the vascular endothelium in vitro by investigating the effects of DHEA on EC proliferation and subcellular pathways and in vivo by investigating its effects on endothelial function in healthy postmenopausal women.
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Materials and Methods |
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Bovine aortic ECs (BAECs) were isolated by enzymatic digestion from the aortae of young calves as previously described (28). Briefly, aortae were obtained aseptically from the slaughterhouse and transferred to the laboratory where they were transected longitudinally under sterile conditions. To obtain ECs, a sharp blade was passed gently over the luminal surface and rinsed into collagenase (3 mg/ml; Worthington Biochemical Corp., Freehold, NJ). The cell suspension so obtained was incubated for 5 min at 37 C, diluted with DMEM containing 20% fetal bovine serum (FBS) and centrifuged at 900 rpm (5 min). The cell pellet was resuspended in the same medium and incubated at 37 C in an atmosphere of 5% carbon dioxide in air. The medium was changed every 3 days, and over 2–3 wk the cells proliferated to form a monolayer that appeared microscopically as a cobblestone network. The cells were routinely passaged by digestion with 0.25% trypsin, and cells at passage 3–8 were used in the study; cells stained positively for von Willebrand factor and negative for smooth muscle 
-actin.
Human umbilical vein ECs (HUVEC; EA.hy926) were obtained from University of North Carolina (Chapel Hill, NC) (29). This cell line maintains a wide range of EC properties, including expression of von Willebrand factor and Weibel-Palade bodies. The cells (supplied by Dr. C.-J. Edgell) were cultured and maintained in DMEM with D-glucose at 4.5 g/liter; 10% FBS; 100 µmmol/liter hypoxanthine, 0.4 µmmol/liter aminopterin, and 16 µmmol/liter thymidine; 1000 U/liter penicillin; and 1 mg/liter streptomycin.
Cell proliferation
Cell proliferation was determined by direct counting of cell numbers. Cells were seeded in 24-well plates (10,000 cells/well) and grown to 60–70% confluence in FBS-enriched DMEM. After serum deprivation for 24 h, the cells were incubated with DHEA, 17ß-estradiol (17ß-E), or testosterone (T) at varying concentrations for 4 h and subsequently together with 2.5% FBS. After 48 h, cells were harvested and counted in an automatic cell counter (S.ST.II/ZM; Coulter Electronics Ltd., Luton, UK).
Pharmacological antagonism of estrogen receptors (ER) and androgen receptors (AR)
To determine whether the effects of DHEA on cell proliferation are mediated via AR or ER, measurements of cell proliferation were performed in the presence and absence of the AR antagonist flutamide or the ER antagonist ICI 182,780 (Tocris Cookson, Inc., Bristol, UK), added 2 h before the addition of the hormones. Both antagonists were specifically chosen not only as selective for AR or ER, respectively, but importantly as being without partial agonist effects on either receptor.
Western blot analysis
Cells (10,000 cells/ml) were seeded in 60-mm culture dishes and grown to near confluence in FBS-enriched DMEM. After serum deprivation for 24 h, the cells were treated with DHEA at varying concentrations for 4 h and then with FBS (2.5%) for 15 min for analysis of MAPK activity. For analysis of endothelial nitric oxide synthase (eNOS) expression, the cells were treated with DHEA for 8, 16, and 24 h after serum deprivation. Cells were harvested at various time points, and total cell protein was determined after addition of lysis buffer (20 mmol/liter Tris-HCl, pH 7.7; 250 mmol/liter NaCl; 2 mmol/liter EDTA; 2 mmol/liter EGTA; 0.5% Nonidet P-40; 10% glycerol; 20 mmol/liter ß-glycerophosphate; 1 mmol/liter Na3VO4; 10 µg/ml leupeptin; 5 µg/ml aprotinin; 1 µmmol/liter pepstatin; 1 mmol/liter phenylmethylsulfonylfluoride; and 1 mmol/liter dithiothreitol). Protein (20 µg) was separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellular filters (Millipore Corp., Bedford, MA). After blocking in 5% nonfat milk overnight, the filters were hybridized with the first antibody, monoclonal p44/42 (phospho/dephospho) MAPK (Thr202/Tyr204; New England Biolabs, Beverly, MA), or eNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibody for 1 h. After addition of horseradish peroxidase-conjugated second antibody (DakoCytomation, Carpinteria, CA) for an additional hour, the filters were submersed in enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ) for 1 min and exposed to x-ray film for 1–10 min to visualize the protein bands. For sample load control, nonspecific protein bands on the gel were visualized by staining with Coomassie Blue (0.5 g in 500 ml methanol, 200 ml acetic acid, and 300 ml water). For relative quantification bands were scanned in a PowerLook Scanner (Seiko Epson Corp., Nagano, Japan).
Subjects
Thirty-six healthy, postmenopausal, normotensive, nondiabetic, nonsmoking women, aged 42–70 yr, not taking any form of hormonal therapy or other medication likely to affect vascular function were recruited from the general community. All subjects gave written informed consent for their participation in the study, which was approved by the Alfred Hospital ethics review committee. Before participation in the study, subjects underwent a screening process to ensure their suitability.
The study followed a randomized, double-blind, placebo-controlled design. Subjects were assigned the active treatment of DHEA (two 50-mg capsules of pharmaceutical grade DHEA, taken orally) or matching placebo. Subjects were studied at baseline and after 12 wk of treatment. At each visit, blood samples were collected to measure blood levels of the hormones DHEA, E, and T; fasting lipids; and endothelial function.
Flow-mediated dilation (FMD) of the brachial artery
FMD was assessed by established techniques (22). A high resolution ultrasound transducer was placed over the brachial artery to measure its diameter before, during, and after reactive hyperemia. Briefly, after a 15- to 20-min rest period, the right brachial artery was scanned with a 7.5-Hz linear array transducer over a longitudinal section 5–7 cm above the right elbow. A blood pressure cuff around the forearm distal to the target area was inflated to a pressure of 250 mm Hg (4.5 min) and then abruptly deflated, after which a second scan was performed continuously for 90 sec. After an additional 10 min of rest, a final scan was performed over the same area. The ultrasound images were recorded on videocassette. The diameter of the brachial artery was measured from the tunica intima at a fixed distance from the chosen marker. The mean diameters of the brachial artery before, during, and after reactive hyperemia were calculated from three cardiac cycles synchronized with the R-wave peaks on the electrocardiogram. To assess endothelium-independent vasodilation of the brachial artery the same ultrasound scanning protocol was used before and after the administration of 300 µg sublingual glyceryl trinitrate (GTN). The coefficient of variation for the brachial artery measurements was 0.34 ± 0.03, and that for the percent change after reactive hyperemia was 0.45 ± 0.05%.
Laser Doppler velocimetry (LDV) with direct current iontophoresis
Blood flow responses to the endothelial vasodilator acetylcholine (ACh) and the smooth muscle-mediated, nonendothelial dilator sodium nitroprusside (SNP) were measured by LDV with direct current iontophoresis. LDV employs the Doppler shift of the reflection of a low energy laser beam from moving erythrocytes to quantify microvascular perfusion (30), and iontophoresis uses a direct electrical current to introduce charged molecules into the skin.
LDV was performed as previously described (30). Blood flow was measured with a laser Doppler imager (Moor Instruments, Ltd., Devon, UK) via a 633-nm (helium-neon) infrared light from a low power laser. Changes in blood flow were recorded on a laptop computer using Moor Instruments laser Doppler perfusion measurement package version 3.01, and the total response was calculated as the area under the curve with purpose-written software. Solutions of either ACh (BDH Chemicals, Poole, UK) or SNP (David Bull Laboratories, Mulgrave, Australia) previously mixed in a methyl cellulose gel (10%, wt/vol), each at a concentration of 10 mg/ml, were placed in the chambers. To iontophorese solutions, a current of 0.1 mA was administered for 30 sec after two baseline scans. SNP was administered with a cathodal charge, and ACh with an anodal charge. Subjects rested for 20 min before testing to establish a stable baseline. The coefficient of variation for blood flux assessed using this method is 0.15 ± 0.05.
Biochemical analysis
Fasting venous blood samples were drawn on the day of testing for analysis of lipids and hormone levels. Total, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, and total triglycerides were determined enzymatically with Roche/Hitachi 912 analyzer (Roche Diagnostics, Indianapolis, IN) by the Alfred Hospital Pathology Service (Melbourne, Australia). Venous blood samples were obtained from subjects for the quantitative determination of DHEA, E, estrone, T, and SHBG. Plasma hormone levels were measured by Mayne Health, Dorevitch Pathology (Heidelberg, Melbourne, Australia), using commercial RIA kits to measure plasma levels of DHEA (ICN Pharmaceuticals, Costa Mesa, CA), E (Orion Diagnostica, Espoo, Finland), estrone (Diagnostics Biochem Canada, Inc., London, Ontario, Canada), SHBG (Bayer Delfia, Helsinki, Finland) and T (BioSource Europe, Nivelles, Belgium).
Statistical analysis
All data are presented as the mean ± SEM. Statistical analysis was performed by t test and for multiple comparisons by ANOVA. The null hypothesis was rejected at P > 0.05.
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Results |
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DHEA, 17ß-E, and T at 1–100 nmol/liter increase FBS-induced cell proliferation of BAEC in a dose-dependent manner, with a maximum effect of DHEA to 138 ± 3% of the control value compared with 17ß-E (119 ± 4%) and T (127 ± 4%; Fig. 1
). These results show that DHEA increases FBS-induced BAEC proliferation in a fashion similar to, and to an extent at least as great as, that of estrogen or androgen. Similar results were found in cultured HUVEC.
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The stimulatory effect of DHEA (10 nmol/liter) on FBS-induced BAEC proliferation was not blocked by either flutamide or ICI 182,780 (100 nmol/liter). However, flutamide completely abolished the stimulatory effects of T, and ICI 182,780 blocked the effects of 17ß-E (Fig. 2). This study thus shows that the actions of DHEA are not mediated by either AR or ER; flutamide and ICI 182,780, alone or combined, had no effect on cell proliferation.
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DHEA (0.1–100 nM) significantly enhanced FBS-induced activation of MAPK extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVEC, assessed by the increased expression of p44/42 protein, to a plateau at 1 nM (Fig. 3).
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DHEA (1–100 nM) significantly increased the expression of eNOS protein in BAEC in a dose-dependent manner, with a maximum stimulation of 174 ± 15% of the control value (Fig. 4A). The time dependence of the increase in eNOS expression was also explored, with a maximum response of 177 ± 9% to 100 nM DHEA after 16 h (Fig. 4B).
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As shown in Table 1, subjects in both treatment groups were well matched for baseline characteristics. DHEA administration significantly increased serum levels of DHEA (from 16 ± 2 to 35 ± 5 nmol/ml; P < 0.05), T (0.6 ± 0.005 to 2.4 ± 0.8 nmol/liter; P < 0.05), and estrone (from 10.7 ± 1.6 to 28.4 ± 4.6 pmol/liter; P < 0.05), with no effect on E (59 ± 20 and 60 ± 11 pmol/liter) or SHBG (38 ± 3, 30 ± 3 nmol/liter; P = 0.09; see Table 2). Although total plasma cholesterol concentrations decreased after 12 wk of DHEA administration (from 6.2 ± 0.2 to 5.5 ± 0.2 U; P < 0.05), there were no significant changes in the concentrations of LDL (3.4 ± 0.3 and 3.2 ± 0.2 U; P = 0.14) or HDL (1.9 ± 0.1 and 1.6 ± 0.1 U; P = 0.1) or in LDL/HDL ratios (1.72 ± 0.2 and 1.85 ± 0.2 U; P = 0.10). No differences were observed in total plasma HDL or LDL cholesterol levels or in LDL/HDL ratio after treatment with placebo.
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There were no differences in baseline measures of FMD between the two treatment groups (DHEA and placebo, 8.4 ± 0.7% and 10.7 ± 1.1%). After 12 wk of DHEA administration, brachial artery FMD increased significantly (14.5 ± 1.1%; P < 0.05), with no change after placebo (as shown in Fig. 5A).
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]. In all subjects, resting brachial artery diameter did not change over the duration of the study. Effects of DHEA on cutaneous microvascular reactivity
Endothelium-dependent cutaneous blood flow in response to iontophoresed ACh was significantly increased after 12 wk of DHEA administration compared with baseline values [3192 ± 331 arbitrary flux units (AU) vs. 1583 ± 168 AU; P < 0.05]. No differences were observed in ACh-induced dilation over 12 wk in the placebo group (1809 ± 226 and 1971 ± 219 AU; Fig. 6A).
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Discussion |
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Although DHEA and DHEAS are quantitatively the most abundant circulating adrenal steroids in humans, their extensive metabolism to estrogens and androgens (and, until recently, the lack of evidence for a functioning receptor) has led to the assumption that they exert their effects predominantly or uniquely via conversion to steroid metabolites (5, 6, 31, 32). In addition, despite the recognized importance of the endothelium for vascular function and repeated suggestions that DHEA may act to influence endothelial function, the possible clinical relevance of its actions has remained controversial. The present study appears to be the first to provide evidence that DHEA(S) at physiological levels affects endothelial function in vivo and in vitro in a potentially beneficial manner by mechanisms at least in part independent of both androgen and estrogen receptors.
Our finding that DHEA increases the proliferation of bovine ECs is consistent with the known effects of this hormone in influencing proliferation in other tissues, such as vascular smooth muscle cells (33), fibroblasts (34), T lymphocytes (35), and preadipocytes (36). The actions of DHEA on ECs to stimulate MAPK mirrors the responses seen with estrogens and androgens (37, 38) and is consistent with our previous finding that DHEA inhibits platelet-derived growth factor-BB-induced MAPK phosphorylation in vascular smooth muscle cells (39). The significant increase in eNOS expression in ECs of human origin extends the recent report of such a report in bovine ECs (7) and similarly mirrors the increases recently reported after DHEA administration in vivo (40). Of potential significance, the marked differences in time course and dose-response characteristics of the effects of DHEA on MAPK and eNOS suggest actions via more than one receptor and/or effector pathway.
To assess whether the effects of DHEA on endothelial function in vitro are also seen in vivo, we administered DHEA to healthy menopausal women, a group with consistently low circulating estrogen and androgen levels. In this study DHEA, total T, and estrone, but not E, levels increased. T concentrations remained within the physiological range and at substantially lower levels than those reported to produce direct vascular effects (27), effects that have been variously disputed (41, 42, 43, 44, 45); this makes it unlikely that the changes seen were due to androgen action. Furthermore, although the vasodilatory and endothelial effects of estradiol are well documented, there is no evidence that the weak estrogen estrone is capable of influencing endothelial function in the absence of changes in E levels. Accordingly, it appears likely that the changes observed represent a DHEA-specific effect on the endothelium.
The vascular endothelium contributes to cardiovascular homeostasis by regulating the caliber of blood vessels in response to a changing hemodynamic and hormonal environment, primarily by producing and secreting vasoactive substances that alter vasomotor tone and maintain blood flow (46). Flow-induced endothelium-dependent vasorelaxation of large conduit vessels, experimentally demonstrated by FMD, is augmented by increased synthesis and release of nitric oxide from the endothelium and correspondingly reduced by inhibition of nitric oxide synthase (46). ACh-induced vasorelaxation, as shown here in the cutaneous microvasculature, also reflects the release of nitric oxide from the endothelium (47, 48). There is substantial evidence that reduced endothelial function in both large and small blood vessels is associated with both increased risk of cardiovascular disease and established cardiovascular disease, and that improvements in endothelial function reduce other risk factors and improve clinical end points. The present study therefore suggests that the changes in large vessel and microvascular endothelial function observed after oral administration of DHEA are potentially beneficial, although further studies with long-term clinical end points are necessary to confirm this.
The demonstration that DHEA influences endothelial function in a potentially beneficial manner both in vivo and in vitro, that its actions are linked to specific subcellular mechanisms, and that these actions are independent of classical sex hormone receptors raises the question of the receptor mechanism that subserves these effects. It has recently been shown (7) that in bovine ECs DHEA binds to a functional G protein-coupled receptor located on the plasma membrane; the extent to which the effects described in the present studies reflect DHEA action via this receptor awaits exploration. In summary, our findings implicate the peripheral vasculature as a potential site for DHEA action and extend our current understanding of the mechanisms by which DHEA may contribute to the amelioration of cardiovascular disease. These results suggest that DHEA and its receptor may offer possibilities for novel therapies for the management of cardiovascular risk and established cardiovascular disease.
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Footnotes |
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Current address for S.L., A.D., and P.A.K.: Department of Medicine, Monash University, Alfred Hospital, Commercial Road, Prahran, Victoria 3181, Australia. E-mail: shanhong.ling{at}med.monash.edu.au.
Current address for J.W.F.: Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia. E-mail: john.funder{at}med.monash.edu.au.
Current address for K.S.: Stanford University, Palo Alto, California 94305. E-mail: ksudhir{at}cvmed.stanford.edu
Abbreviations: ACh, Acetylcholine; AR, androgen receptor; AU, arbitrary flux unit; BAEC, bovine aortic EC; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; E, estradiol; 17ß-E, 17ß-estradiol; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; FMD, flow-mediated dilation; GTN, glyceryl trinitrate; HDL, high-density lipoprotein; HUVEC, human umbilical vein EC; LDL, low-density lipoprotein; LDV, laser Doppler velocimetry; SNP, sodium nitroprusside; T, testosterone.
Received September 5, 2003.
Accepted June 4, 2004.
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, Cultured in FBS-free DMEM; 
, cultured in 2.5% FBS DMEM. *, Significant difference from FBS-induced EC proliferation.
, significant difference from the hormone alone treatment.
