Angiotensin II and Aldosterone Regulate Gene Transcription Via Functional Mineralocortocoid Receptors in Human Coronary Artery Smooth Muscle
http://www.100md.com
循环研究杂志 2005年第4期
the Molecular Cardiology Research Institute (I.Z.J., M.E.M.), Department of Medicine
Division of Cardiology, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Mass
the Division of Cardiology (I.Z.J.), Brigham and Woman’s Hospital, Boston, Mass.
Abstract
Inhibition or blockade of the angiotensin-aldosterone system consistently decreases ischemic cardiovascular events in clinical trials. The steroid hormone aldosterone acts by binding to the mineralocorticoid receptor (MR), a ligand activated transcription factor that is a member of the nuclear hormone receptor superfamily. MR binds and is activated by aldosterone and cortisol with equal affinity, but MR activation by cortisol is diminished in tissues that express the cortisol-inactivating enzyme 11--hydroxysteroid-dehydrogenase-2 (11HSD2). Although previous studies support that the vasculature is a target tissue of aldosterone, MR-mediated gene expression in vascular cells has not been demonstrated or systematically explored. We investigated whether functional MR and 11HSD2 are expressed in human blood vessels. Human coronary and aortic vascular smooth muscle cells (VSMCs) express mRNA and protein for both MR and 11HSD2. The endogenous VSMC MR mediates aldosterone-dependent gene expression, which is blocked by the competitive MR antagonist spironolactone. Inhibition of 11HSD2 in coronary artery VSMCs enhances gene transactivation by cortisol, supporting that the VSMC 11HSD2 is functional. Angiotensin II also activates MR-mediated gene transcription in coronary artery VSMCs. Angiotensin II activation of MR-mediated gene expression is inhibited by both the AT1 receptor blocker losartan and by spironolactone, but not by aldosterone synthase inhibition. Microarray and quantitative RT-PCR experiments show that aldosterone activates expression of endogenous human coronary VSMC genes, including several involved in vascular fibrosis, inflammation, and calcification. These data support a new MR-dependent mechanism by which aldosterone and angiotensin II influence ischemic cardiovascular events, and suggest that ACE inhibitors and MR antagonists may decrease clinical ischemic events by inhibiting MR-dependent gene expression in vascular cells.
Key Words: hormones mineralocorticoid receptor nuclear receptors smooth muscle cells vascular biology
Introduction
Despite recent advances in our understanding of the biology of atherosclerosis, ischemic cardiovascular diseases remain the leading cause of morbidity and mortality in the developed world. Recent human clinical trials have shown that inhibition of the angiotensin-aldosterone system improves cardiovascular outcomes in patients with heart failure,1eC4 after myocardial infarction (MI),5 and at high risk for MI.6 In addition to predicted effects on blood pressure and end points such as ejection fraction and functional capacity, these studies revealed a surprising but consistent decrease in unstable angina, MI, and the need for coronary revascularization in those patients treated with angiotensin-converting enzyme-I or aldosterone antagonists.1eC6
The steroid hormone aldosterone is synthesized in the adrenal cortex in response to angiotensin II stimulation and functions to elevate systemic blood pressure through renal effects on electrolyte and volume balance.7 Until recently, the effects of mineralocorticoids on vascular disease have been attributed solely to these renal effects on systemic blood pressure. However, the reduction in ischemic events in clinical trials of angiotensin-converting enzyme inhibition or aldosterone antagonism is significantly greater than that expected from the modest decrease in systemic blood pressure in treated patients.3,4,6,8 In addition, in the RALES and EPHESUS studies of the aldosterone antagonists spironolactone and eplerenone, respectively, the doses of the antagonists used were below threshold for causing significant renal effects,2,5 suggesting that the mechanism of protection by these compounds may involve effects of aldosterone on extrarenal tissues.
In vitro and animal data support a direct role for mineralocorticoids in regulating vascular function and vascular smooth muscle cell (VSMC) proliferation. Spironolactone can directly relax isolated rat aortic rings.9 Stimulation of VSMC proliferation by the mitogen angiotensin II (Ang II) is inhibited by spironolactone10 and other aldosterone antagonists.11 In rats with aldosterone-induced hypertension, the aldosterone antagonist eplerenone decreases carotid artery cross-sectional thickness.12 These data are consistent with a direct effect of mineralocorticoids on VSMC function. However, the mechanism by which aldosterone may directly effect vascular pathophysiology is poorly understood.
Aldosterone is a steroid hormone that binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor.7 The MR was first cloned in 198713 and is a member of the steroid receptor family that includes the estrogen, progesterone, androgen, and glucocorticoid receptors.14 Steroid hormone receptors bind to specific DNA sequences (response elements [REs]) in the promoters of hormone-responsive genes and recruit cofactors in a ligand- dependent manner, thereby modulating gene expression.14 MR binds in vitro to glucocorticoid and mineralocorticoid response elements in a ligand-dependent manner.15 In the kidney, the focus of studies of MR to date, DNA binding by ligand-bound MR directly activates transcription of aldosterone-responsive genes such as the Na/K ATPase gene.16 Aldosterone and cortisol bind to human MR with equal affinity.13 Although plasma glucocorticoid concentrations are 100- to 1000-fold higher than those of aldosterone, aldosterone-responsive tissues express the cortisol-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (11HSD2), which converts cortisol to its 11-keto derivatives that have a low affinity for MR.17 11HSD2 is abundant in the kidney, which makes renal cells highly responsive to aldosterone.18,19 Deficiency or mutations in 11HSD2 results in the syndrome of apparent mineralocorticoid excess with hypertension and hypokalemia.20
Recent data suggest that the cardiovascular system and VSMCs have the capacity to respond directly to exogenous aldosterone and may produce aldosterone locally in response to angiotensin II.21 Previous studies have revealed aldosterone binding and 11HSD2 activity in rat and rabbit arteries,22eC24 and more recent studies demonstrate the presence of MR and 11HSD2 message in human aorta.25 However, the specific human vascular cell types expressing MR, the molecular mechanism of aldosterone activation of vascular MR, and effects of MR activation on the vasculature are not known. In this study, we sought to test the hypothesis that aldosterone and Ang II act directly on VSMCs to modulate MR-regulated gene expression in vascular cells and to explore whether aldosterone can directly alter expression of MR-responsive VSMC genes. Here, we show that human VSMCs express MR and 11HSD2, that vascular MR and 11HSD2 are functional, and that aldosterone and Ang II both can activate MR-mediated gene transcription in coronary artery VSMCs. We also demonstrate that aldosterone activates transcription of several endogenous VSMC genes of potential clinical relevance.
Materials and Methods
Reagents, Cell Lines, and Culture Techniques:
Aldosterone, spironolactone, cortisol (Research Plus, Inc), FAD286 (Novartis), Ang II (Sigma), Losartan (Merck), and glycerrhetinic acid (Masco Worldwide) were used as described with appropriate vehicle controls (see online Methods at http://circres.ahajournals.org). Primary VSMCs were harvested from human surgical and autopsy specimens as described,26 and immortalized cells were transformed by infection with retrovirus expressing human papilloma virus E6 or E7 (E6/E7) proteins.27 Specific VSMCs and cell culture conditions used are described in the online Methods supplement.
Reverse-Transcription Polymerase Chain Reaction and Quantitative Reverse-Transcription Polymerase Chain Reaction
Total cellular RNA was isolated from vascular cell lines and autopsy specimens as described in the online Methods supplement. RNA was reverse-transcribed with random hexamers and Superscript II (Invitrogen). Conventional and quantitative RT-PCR methods and primers (supplemental Table, available online at http://circres.ahajournals.org) are described in the online Methods supplement. Each quantitative RT-PCR sample was normalized to the expression of GAPDH and is expressed as fold change in aldosterone-treated samples compared with vehicle-treated samples.
Immunoblotting:
11HSD2 expression plasmid was a generous gift from Perrin C. White.28 Cell lysates were prepared as described in the online Methods supplement. Lysate from VSMCs (1 uL control, 30 uL for MR immunoblots, and 50 e蘈 for 11HSD2) was separated by 10% SDS-PAGE, transferred to nitrocellulose, immunoblotted with polyclonal antibodies raised against the unique MR N terminus (Santa Cruz) or the 11HSD2 C-terminal catalytic domain (Alpha Diagnostic International), and visualized by chemiluminescence techniques.
Immunoflourescence
Immortalized CO396 cells grown to 75% confluence were serum-starved 24 hours before overnight treatment with vehicle, aldosterone, or Ang II. The cells were then fixed and stained with polyclonal anti-MR antibody as described in the online Methods supplement.
Adenovirus Infection and Luciferase Assay
Adenovirus MMTV-Luc (luciferase gene regulated by the mineralocorticoid-inducible response element from the mouse mammary tumor virus [MMTV] LTR) was a generous gift from Phillip C. Hartig (Environment Protection Agency).29 Control adenovirus (luciferase gene regulated by an estrogen-inducible hormone response element [ERE-Luc]) was a generous gift from J.L. Jameson.30 Immortalized and primary human VSMCs were transduced with adenovirus MMTV-Luc or ERE-Luc as described in the online Methods supplement. Indicated concentrations of vehicle, ligand, and/or inhibitor were then added for 18 hours. Cells were lysed and luciferase activity was determined in duplicate as described.
Microarray Analysis
Human coronary VSMCs (CO396 P8-15) were treated with vehicle or aldosterone for 1 or 24 hours and total RNA was isolated from 4 independent experiments. RNA isolation, cDNA labeling, chip hybridization, slide-scanning, and image and data analysis software are described in the online Methods supplement. The cDNA was hybridized to slides containing the Human version 3.0 Operon probe set (Qiagen), which includes >34 000 70-mer probes printed by the Massachusetts General Hospital Microarray Core Facility. Genes with at least two quality replicates per time point were geometrically averaged and expressed as the fold ratio of aldosterone-treated cells compared with vehicle-treated cells for each time point.
Statistical Analysis
Adenoviral reporter assays were performed a minimum of three times on duplicate plates. Values are reported as mean-fold activation±standard error of the mean. Within-group differences were assessed with one-factor ANOVA. Post hoc comparisons were tested with the Student-Newman-Keuls test. P<0.05 was considered significant. Microarray and quantitative RT-PCR were compared by paired t tests (Table).
Results
Human Aorta and Coronary Artery VSMCs Express Mineralocorticoid Receptors
Total RNA was isolated from whole human aorta and coronary artery tissue as well as from cultured immortalized human aortic and coronary artery smooth muscle cells. RT-PCR with primers specific for the unique MR N-terminus resulted in amplification of MR mRNA from aortic and coronary tissue and VSMCs (Figure 1A). Direct sequencing of cDNA derived from the amplified mRNA confirmed that MR mRNA was amplified in all cases (data not shown). Controls lacking RT were consistently negative (data not shown).
MR protein expression was examined next in cell lysates from human aortic and coronary artery VSMCs. The characteristic 107-kDa MR protein13 was detected in both primary and immortalized coronary and aortic smooth muscle cells, and predominantly in supernatant fractions, as is characteristic for steroid hormone receptors solubilized in high salt14 (Figure 1B). Primary and immortalized cell lines shown were derived from the same source. In immortalized cells, MR protein expression levels did not change with cell passage (P8eC15, data not shown).
Expression and cellular localization of MR protein in human coronary artery VSMCs was studied by immunofluorescence microscopy (Figure 1C, top and middle). In the absence of ligand, MR protein expression was detectable mainly in the nucleus, but also in the cytoplasm of human coronary arterial VSMCs (Figure 1C, top). After exposure to aldosterone, MR was found solely in the nucleus of these coronary VSMCs (Figure 1C, middle), as is characteristic of liganded steroid hormone receptors.31,32 Pre-immune serum controls were consistently negative (data not shown). These data support that human aortic and coronary artery VSMCs express MR mRNA and protein and that aldosterone-activated MR is concentrated in the nucleus of human VSMC.
Human Vascular Cells Express Functional Mineralocorticoid Receptor
Steroid hormone receptors are ligand-activated transcription factors that regulate gene expression, thereby affecting effects on cellular physiology. A sensitive adenoviral reporter of MR-mediated gene expression29 was used to determine whether the VSMC MR is functionally capable of aldosterone-induced transcriptional transactivation in human coronary artery VSMCs. Gene expression was dose-dependently activated by aldosterone beginning at 1 nanomolar concentration of hormone (Figure 2A), consistent with the reported Kd for aldosteroneeCMR binding13 and physiologic aldosterone concentrations.33 Aldosterone was unable to activate an estrogen response element (ERE; control) reporter (Figure 2A). 30 Spironolactone, a competitive inhibitor of MR binding, blocked aldosterone-mediated MRE activation in VSMCs in a dose-dependent manner (Figure 2B), but alone it had no effect on basal MR reporter activity (Figure 2B), supporting that MRE activation by aldosterone in VSMCs requires hormone binding to endogenous MR.34 These data further support that the endogenous human coronary artery VSMCs MR is functional.
Human Vascular Cells Express Functional 11HSD2
Aldosterone-responsive tissues express the cortisol-inactivating enzyme 11HSD2. 11HSD2 mRNA was detected in immortalized human aortic and coronary artery VSMCs (Figure 3A), as confirmed by direct sequencing (data not shown). Controls lacking RT were negative (data not shown). Immunoblotting studies confirmed that primary and immortalized VSMCs express 11HSD2 protein (Figure 3B). 11HSD2 protein was detected predominantly in pellet fractions (Figure 3B), similar to nonvascular cells, in which 11HSD2 enzyme is localized to membranous endoplasmic reticulum.17,35
11HSD2 converts cortisol to cortisone, which does not bind or activate MR. 11HSD2 function in coronary artery VSMCs was tested using the 11HSD2 inhibitor, glycyrrhetinic acid (GA). DoseeCresponse curves for MRE-reporter activation by cortisol were generated in the absence or presence of GA (Figure 3C). At doses of GA exceeding its Ki for 11HSD2 (10 eol/L), a significant left-shift in cortisol-mediated MR activation was evident (Figure 3C; P=0.004). These data support that human VSMCs contain functional 11HSD2 that can inactivate cortisol.
Ang II Activates Gene Transcription of an MR Responsive Promoter in Coronary VSMCs.
Ang II-mediated VSMC proliferation can be inhibited by aldosterone antagonists.11 The effect of Ang II on MR cellular localization was studied first by immunoflourescence. Treatment of VSMC with Ang II, like aldosterone, results in accumulation of MR exclusively in the nucleus (Figure 1C, bottom). Ang II effects on MR-mediated gene transcription were next assayed in VSMCs (Figure 4). Ang II significantly activated the MRE-luciferase reporter without effect on ERE-luciferase (control) reporter in immortalized (Figure 4) and primary (Figure 5) human coronary VSMCs. Hormone-dependent activation of gene expression in primary VSMCs occurred to levels similar to those seen in immortalized cells (Figure 5; compare with Figures 2A and 4).
Ang II Activates the Mineralocorticoid Receptor Via the AT1 Receptor
The mechanism of Ang II activation of MR in VSMCs was studied. Activation of the MRE reporter by Ang II was inhibited by the AT1 receptor blocker losartan (Figure 6A), which alone had no effect on basal reporter transcription (Figure 6A). In adrenal cells, Ang II acts locally via the AT1 receptor to activate aldosterone synthase and aldosterone production.36 We tested whether human VSMCs synthesize aldosterone using 5 distinct approaches. Aldosterone synthase (AS) mRNA was undetectable by RT-PCR11,25 in immortalized human coronary VSMCs (data not shown). Similarly, AS RNA was undetectable in microarray studies of VSMC RNA (Table). Immunoblotting experiments also failed to detect AS protein expression in murine VSMCs (data not shown). Aldosterone release from human VSMCs in response to Ang II was undetectable by radioimmunoassay, in contrast to vascular endothelial cells.37 Finally, the aldosterone synthase inhibitor FAD286 (Novartis), at doses that completely inhibit AS, did not alter MRE-luciferase reporter activation in response to Ang II in human coronary VSMCs (data not shown). Taken together, these data strongly support that local production of aldosterone is not the mechanism of Ang II-mediated MRE activation in coronary VSMCs.
We next tested whether Ang II activation of the MRE-reporter requires MR. Transcriptional activation of the MRE reporter by Ang II in human coronary artery VSMCs was fully suppressible by spironolactone (Figure 6B; 10eC4 spironolactone). Spironolactone alone had no effect on basal reporter activation (Figure 6B).
Aldosterone Regulates Endogenous Gene Expression in Human Coronary VSMCs
To characterize endogenous VSMC gene expression regulated by aldosterone in human coronary artery VSMCs, we used a microarray approach (Table). Gene expression in immortalized human coronary artery VSMCs treated with aldosterone or vehicle was analyzed in 4 independent experiments. Two genes involved in renal sodium transport and known to be regulated in renal cells by aldosterone,16 the NaK-ATPase gene, and the epithelial sodium channel,38 were not regulated by aldosterone in VSMCs (Table). However, several VSMC genes were induced by aldosterone, including genes involved in vascular fibrosis, calcification, and inflammation (Table). Quantitative real-time RT-PCR studies of RNA from these cells were used as a second independent test of aldosterone-mediated gene expression and verified the microarray findings (Table).
Discussion
The data show that human VSMCs express functional MR, capable of sequence-specific, aldosterone-activated gene transcription at physiological (1 nM) hormone levels; that angiotensin II also activates MR-mediated gene transcription via the AT1 receptor; and that aldosterone activates endogenous human coronary VSMC genes involved in vascular fibrosis, inflammation, and calcification. Based on these data, we propose a model for MR activation of gene transcription in VSMCs by both aldosterone and angiotensin II (Figure 7). Previous studies have demonstrated aldosterone binding and 11HSD2 activity in rabbit and rat aorta and mesenteric arteries22eC24 and more recent studies reveal MR and 11HSD2 gene expression in human aorta25 and vascular cells.11,39eC40 However, the present studies show for the first time to our knowledge that the endogenous vascular MR is transcriptionally competent and regulates expression of endogenous genes. In nonvascular cells, MR has been shown to effect cell function by both genomic and nongenomic mechanisms.41 The studies shown here investigate and provide evidence for a genomic MR pathway in VSMCs, but do not examine whether nongenomic activation of MR occurs in VSMCs.
VSMCs also express a functional 11HSD2 that is capable of inactivating cortisol, as is characteristic of tissues that respond to aldosterone. Patients with mutations in 11HSD2 have hypertension (syndrome of apparent mineralocorticoid excess) and subjects consuming large quantities of GA-containing licorice also have elevations in blood pressure.20 Although this has been attributed to cortisol activation of renal MR, resulting in sodium and fluid retention,20 our data raise the possibility that vascular 11HSD2 mutations or inhibition may contribute to hypertension via direct effects of cortisol on local vascular MR activation.
The mechanism by which Ang II stimulates MR-mediated gene transcription is not yet entirely clear. Ang II stimulates nuclear localization of MR and spironolactone inhibits Ang II-mediated gene expression in VSMCs, supporting the role of the MR itself. Ang II-mediated MR activation is also inhibited by losartan, an inhibitor of the AT1 receptor, demonstrating a link between AT1 receptor activation and MR transcriptional activation. Even though the AT1 receptor has been implicated previously in Ang II activation of aldosterone synthase36 in the adrenal gland, there is controversy in the literature as to whether the cardiovascular system produces its own aldosterone. Some data support that the heart, VSMCs, and endothelial cells1121,25,37,42,43 express aldosterone synthase and produce aldosterone, whereas other investigators refute this.44,45 We were unable to detect aldosterone synthesis in VSMCs using several assays, including microarray and RT-PCR studies of aldosterone synthase mRNA expression, immunoblotting for aldosterone synthase protein expression, radioimmunoassay for aldosterone synthesis and release, and pharmacological aldosterone synthase inhibition, which had no effect on Ang II-mediated MRE-reporter activation. Although aldosterone synthase expression and function may depend on cell conditions, under the same conditions in which Ang II activates MR-dependent gene transcription, we found no evidence for aldosterone synthesis by VSMCs. Other mechanisms for the AT1-mediated activation of MR need to be considered next, including ligand-independent activation of MR by post-translational modifications such as phosphorylation, which occurs for several steroid hormone receptors, including MR, in other tissues.46,47
The aldosterone-stimulated expression of endogenous genes in VSMCs detected by microarray and quantitative RT-PCR is distinct from aldosterone-stimulated gene expression in nonvascular cells. For example, MR-responsive genes known to be involved in renal sodium regulation, including the NaKATPase16 and the epithelial sodium channel,38 were not regulated by aldosterone in our studies of human coronary artery VSMCs. Several new aldosterone-regulated genes were identified, including genes involved in vascular fibrosis, calcification, and inflammation. Type I and type III collagen were upregulated significantly by aldosterone in VSMCs and have been implicated previously in Ang II-mediated and aldosterone-mediated cardiac and vascular fibrosis.48eC50 Bone morphogenic protein 2, the parathyroid hormone receptor, bone-liver-kidney alkaline phosphatase, and type 1 collagen all were upregulated in these assays. These genes and their proteins are known to be involved in vascular calcification, a process associated clinically with an increased risk of cardiac ischemic events.51,52 Aldosterone antagonism can prevent aldosterone-induced and Ang II-induced vascular inflammation in animal models,53,54 and several inflammatory genes were induced in the microarray studies, including IL-16 (lymphocyte chemoattractant factor) and cytotoxic T-lymphocyteeCassociated protein 4. These and other aldosterone-regulated genes in VSMCs may play an important role in the pathophysiology of atherosclerosis and vascular dysfunction, and also underscore the importance of tissue specificity, a common theme in steroid hormone actions. The studies described here support a potential new mechanism for vascular disease and for the preventive effect of angiotensin-converting enzyme inhibitors and aldosterone antagonists for ischemic diseases. Further understanding of the molecular mechanisms by which aldosterone and angiotensin II regulate MR-dependent gene expression and which genes are most relevant to vascular pathophysiology may also lead to novel therapies for human vascular disease.
Acknowledgments
I.Z.J. thanks Perrin C. White, J.L. Jameson, Joshua A. Beckman, and Heather Gornik for their generosity with reagents and Mason Freeman, Glenn Short, Najib Elmessadi, and Danny Park for assistance with microarray processing and analysis. The microarray studies were supported by a core facility award from the National Institutes of Health (NIH; HL72358). This work was supported in part by NIH RO1 HL50569 and P50 HL63494 (to M.E.M.).
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Konishi A, Tazawa C, Miki Y, Darnel AD, Suzuki T, Ohta Y, Suzuki T, Tabayashi K, Sasano H. The possible roles of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 2 in cardiac fibrosis in the spontaneously hypertensive rat. J Steroid Biochem Mol Biol. 2003; 85 (2eC5): 439eC42.
Sun Y, Ramires FJ, Weber KT. Fibrosis of atria and great vessels in response to angiotensin II or aldosterone infusion. Cardiovasc Res. 1997; 35: 138eC147.
Tintu Y, Demer LL. Recent advances in multifactorial regulation of vascular calcification. Curr Opin Lipidol. 2001; 12: 555eC560.
Bostrom K, Watson KE, Stanford WP, Demer LL. Atherosclerotic calcification: relation to developmental osteogenesis. Am J Cardiol. 1995; 75: 88BeC91B.
Rocha R, Martin-Berger CL, Yang P, Scherrer R, Delyani J, McMahon E. Selective aldosterone blockade prevents angiotensin II/salt-induced vascular inflammation in the rat heart. Endocrinology. 2002; 143: 4828eC4836.
Rocha R, Rudolph AE, Frierdich GE, Nachowiak DA, Kekec BK, Blomme EA, McMahon EG, Delyani JA. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol Heart Circ Physiol. 2002; 283: H1802eCH1810.(Iris Z. Jaffe, Michael E.)
Division of Cardiology, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Mass
the Division of Cardiology (I.Z.J.), Brigham and Woman’s Hospital, Boston, Mass.
Abstract
Inhibition or blockade of the angiotensin-aldosterone system consistently decreases ischemic cardiovascular events in clinical trials. The steroid hormone aldosterone acts by binding to the mineralocorticoid receptor (MR), a ligand activated transcription factor that is a member of the nuclear hormone receptor superfamily. MR binds and is activated by aldosterone and cortisol with equal affinity, but MR activation by cortisol is diminished in tissues that express the cortisol-inactivating enzyme 11--hydroxysteroid-dehydrogenase-2 (11HSD2). Although previous studies support that the vasculature is a target tissue of aldosterone, MR-mediated gene expression in vascular cells has not been demonstrated or systematically explored. We investigated whether functional MR and 11HSD2 are expressed in human blood vessels. Human coronary and aortic vascular smooth muscle cells (VSMCs) express mRNA and protein for both MR and 11HSD2. The endogenous VSMC MR mediates aldosterone-dependent gene expression, which is blocked by the competitive MR antagonist spironolactone. Inhibition of 11HSD2 in coronary artery VSMCs enhances gene transactivation by cortisol, supporting that the VSMC 11HSD2 is functional. Angiotensin II also activates MR-mediated gene transcription in coronary artery VSMCs. Angiotensin II activation of MR-mediated gene expression is inhibited by both the AT1 receptor blocker losartan and by spironolactone, but not by aldosterone synthase inhibition. Microarray and quantitative RT-PCR experiments show that aldosterone activates expression of endogenous human coronary VSMC genes, including several involved in vascular fibrosis, inflammation, and calcification. These data support a new MR-dependent mechanism by which aldosterone and angiotensin II influence ischemic cardiovascular events, and suggest that ACE inhibitors and MR antagonists may decrease clinical ischemic events by inhibiting MR-dependent gene expression in vascular cells.
Key Words: hormones mineralocorticoid receptor nuclear receptors smooth muscle cells vascular biology
Introduction
Despite recent advances in our understanding of the biology of atherosclerosis, ischemic cardiovascular diseases remain the leading cause of morbidity and mortality in the developed world. Recent human clinical trials have shown that inhibition of the angiotensin-aldosterone system improves cardiovascular outcomes in patients with heart failure,1eC4 after myocardial infarction (MI),5 and at high risk for MI.6 In addition to predicted effects on blood pressure and end points such as ejection fraction and functional capacity, these studies revealed a surprising but consistent decrease in unstable angina, MI, and the need for coronary revascularization in those patients treated with angiotensin-converting enzyme-I or aldosterone antagonists.1eC6
The steroid hormone aldosterone is synthesized in the adrenal cortex in response to angiotensin II stimulation and functions to elevate systemic blood pressure through renal effects on electrolyte and volume balance.7 Until recently, the effects of mineralocorticoids on vascular disease have been attributed solely to these renal effects on systemic blood pressure. However, the reduction in ischemic events in clinical trials of angiotensin-converting enzyme inhibition or aldosterone antagonism is significantly greater than that expected from the modest decrease in systemic blood pressure in treated patients.3,4,6,8 In addition, in the RALES and EPHESUS studies of the aldosterone antagonists spironolactone and eplerenone, respectively, the doses of the antagonists used were below threshold for causing significant renal effects,2,5 suggesting that the mechanism of protection by these compounds may involve effects of aldosterone on extrarenal tissues.
In vitro and animal data support a direct role for mineralocorticoids in regulating vascular function and vascular smooth muscle cell (VSMC) proliferation. Spironolactone can directly relax isolated rat aortic rings.9 Stimulation of VSMC proliferation by the mitogen angiotensin II (Ang II) is inhibited by spironolactone10 and other aldosterone antagonists.11 In rats with aldosterone-induced hypertension, the aldosterone antagonist eplerenone decreases carotid artery cross-sectional thickness.12 These data are consistent with a direct effect of mineralocorticoids on VSMC function. However, the mechanism by which aldosterone may directly effect vascular pathophysiology is poorly understood.
Aldosterone is a steroid hormone that binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor.7 The MR was first cloned in 198713 and is a member of the steroid receptor family that includes the estrogen, progesterone, androgen, and glucocorticoid receptors.14 Steroid hormone receptors bind to specific DNA sequences (response elements [REs]) in the promoters of hormone-responsive genes and recruit cofactors in a ligand- dependent manner, thereby modulating gene expression.14 MR binds in vitro to glucocorticoid and mineralocorticoid response elements in a ligand-dependent manner.15 In the kidney, the focus of studies of MR to date, DNA binding by ligand-bound MR directly activates transcription of aldosterone-responsive genes such as the Na/K ATPase gene.16 Aldosterone and cortisol bind to human MR with equal affinity.13 Although plasma glucocorticoid concentrations are 100- to 1000-fold higher than those of aldosterone, aldosterone-responsive tissues express the cortisol-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (11HSD2), which converts cortisol to its 11-keto derivatives that have a low affinity for MR.17 11HSD2 is abundant in the kidney, which makes renal cells highly responsive to aldosterone.18,19 Deficiency or mutations in 11HSD2 results in the syndrome of apparent mineralocorticoid excess with hypertension and hypokalemia.20
Recent data suggest that the cardiovascular system and VSMCs have the capacity to respond directly to exogenous aldosterone and may produce aldosterone locally in response to angiotensin II.21 Previous studies have revealed aldosterone binding and 11HSD2 activity in rat and rabbit arteries,22eC24 and more recent studies demonstrate the presence of MR and 11HSD2 message in human aorta.25 However, the specific human vascular cell types expressing MR, the molecular mechanism of aldosterone activation of vascular MR, and effects of MR activation on the vasculature are not known. In this study, we sought to test the hypothesis that aldosterone and Ang II act directly on VSMCs to modulate MR-regulated gene expression in vascular cells and to explore whether aldosterone can directly alter expression of MR-responsive VSMC genes. Here, we show that human VSMCs express MR and 11HSD2, that vascular MR and 11HSD2 are functional, and that aldosterone and Ang II both can activate MR-mediated gene transcription in coronary artery VSMCs. We also demonstrate that aldosterone activates transcription of several endogenous VSMC genes of potential clinical relevance.
Materials and Methods
Reagents, Cell Lines, and Culture Techniques:
Aldosterone, spironolactone, cortisol (Research Plus, Inc), FAD286 (Novartis), Ang II (Sigma), Losartan (Merck), and glycerrhetinic acid (Masco Worldwide) were used as described with appropriate vehicle controls (see online Methods at http://circres.ahajournals.org). Primary VSMCs were harvested from human surgical and autopsy specimens as described,26 and immortalized cells were transformed by infection with retrovirus expressing human papilloma virus E6 or E7 (E6/E7) proteins.27 Specific VSMCs and cell culture conditions used are described in the online Methods supplement.
Reverse-Transcription Polymerase Chain Reaction and Quantitative Reverse-Transcription Polymerase Chain Reaction
Total cellular RNA was isolated from vascular cell lines and autopsy specimens as described in the online Methods supplement. RNA was reverse-transcribed with random hexamers and Superscript II (Invitrogen). Conventional and quantitative RT-PCR methods and primers (supplemental Table, available online at http://circres.ahajournals.org) are described in the online Methods supplement. Each quantitative RT-PCR sample was normalized to the expression of GAPDH and is expressed as fold change in aldosterone-treated samples compared with vehicle-treated samples.
Immunoblotting:
11HSD2 expression plasmid was a generous gift from Perrin C. White.28 Cell lysates were prepared as described in the online Methods supplement. Lysate from VSMCs (1 uL control, 30 uL for MR immunoblots, and 50 e蘈 for 11HSD2) was separated by 10% SDS-PAGE, transferred to nitrocellulose, immunoblotted with polyclonal antibodies raised against the unique MR N terminus (Santa Cruz) or the 11HSD2 C-terminal catalytic domain (Alpha Diagnostic International), and visualized by chemiluminescence techniques.
Immunoflourescence
Immortalized CO396 cells grown to 75% confluence were serum-starved 24 hours before overnight treatment with vehicle, aldosterone, or Ang II. The cells were then fixed and stained with polyclonal anti-MR antibody as described in the online Methods supplement.
Adenovirus Infection and Luciferase Assay
Adenovirus MMTV-Luc (luciferase gene regulated by the mineralocorticoid-inducible response element from the mouse mammary tumor virus [MMTV] LTR) was a generous gift from Phillip C. Hartig (Environment Protection Agency).29 Control adenovirus (luciferase gene regulated by an estrogen-inducible hormone response element [ERE-Luc]) was a generous gift from J.L. Jameson.30 Immortalized and primary human VSMCs were transduced with adenovirus MMTV-Luc or ERE-Luc as described in the online Methods supplement. Indicated concentrations of vehicle, ligand, and/or inhibitor were then added for 18 hours. Cells were lysed and luciferase activity was determined in duplicate as described.
Microarray Analysis
Human coronary VSMCs (CO396 P8-15) were treated with vehicle or aldosterone for 1 or 24 hours and total RNA was isolated from 4 independent experiments. RNA isolation, cDNA labeling, chip hybridization, slide-scanning, and image and data analysis software are described in the online Methods supplement. The cDNA was hybridized to slides containing the Human version 3.0 Operon probe set (Qiagen), which includes >34 000 70-mer probes printed by the Massachusetts General Hospital Microarray Core Facility. Genes with at least two quality replicates per time point were geometrically averaged and expressed as the fold ratio of aldosterone-treated cells compared with vehicle-treated cells for each time point.
Statistical Analysis
Adenoviral reporter assays were performed a minimum of three times on duplicate plates. Values are reported as mean-fold activation±standard error of the mean. Within-group differences were assessed with one-factor ANOVA. Post hoc comparisons were tested with the Student-Newman-Keuls test. P<0.05 was considered significant. Microarray and quantitative RT-PCR were compared by paired t tests (Table).
Results
Human Aorta and Coronary Artery VSMCs Express Mineralocorticoid Receptors
Total RNA was isolated from whole human aorta and coronary artery tissue as well as from cultured immortalized human aortic and coronary artery smooth muscle cells. RT-PCR with primers specific for the unique MR N-terminus resulted in amplification of MR mRNA from aortic and coronary tissue and VSMCs (Figure 1A). Direct sequencing of cDNA derived from the amplified mRNA confirmed that MR mRNA was amplified in all cases (data not shown). Controls lacking RT were consistently negative (data not shown).
MR protein expression was examined next in cell lysates from human aortic and coronary artery VSMCs. The characteristic 107-kDa MR protein13 was detected in both primary and immortalized coronary and aortic smooth muscle cells, and predominantly in supernatant fractions, as is characteristic for steroid hormone receptors solubilized in high salt14 (Figure 1B). Primary and immortalized cell lines shown were derived from the same source. In immortalized cells, MR protein expression levels did not change with cell passage (P8eC15, data not shown).
Expression and cellular localization of MR protein in human coronary artery VSMCs was studied by immunofluorescence microscopy (Figure 1C, top and middle). In the absence of ligand, MR protein expression was detectable mainly in the nucleus, but also in the cytoplasm of human coronary arterial VSMCs (Figure 1C, top). After exposure to aldosterone, MR was found solely in the nucleus of these coronary VSMCs (Figure 1C, middle), as is characteristic of liganded steroid hormone receptors.31,32 Pre-immune serum controls were consistently negative (data not shown). These data support that human aortic and coronary artery VSMCs express MR mRNA and protein and that aldosterone-activated MR is concentrated in the nucleus of human VSMC.
Human Vascular Cells Express Functional Mineralocorticoid Receptor
Steroid hormone receptors are ligand-activated transcription factors that regulate gene expression, thereby affecting effects on cellular physiology. A sensitive adenoviral reporter of MR-mediated gene expression29 was used to determine whether the VSMC MR is functionally capable of aldosterone-induced transcriptional transactivation in human coronary artery VSMCs. Gene expression was dose-dependently activated by aldosterone beginning at 1 nanomolar concentration of hormone (Figure 2A), consistent with the reported Kd for aldosteroneeCMR binding13 and physiologic aldosterone concentrations.33 Aldosterone was unable to activate an estrogen response element (ERE; control) reporter (Figure 2A). 30 Spironolactone, a competitive inhibitor of MR binding, blocked aldosterone-mediated MRE activation in VSMCs in a dose-dependent manner (Figure 2B), but alone it had no effect on basal MR reporter activity (Figure 2B), supporting that MRE activation by aldosterone in VSMCs requires hormone binding to endogenous MR.34 These data further support that the endogenous human coronary artery VSMCs MR is functional.
Human Vascular Cells Express Functional 11HSD2
Aldosterone-responsive tissues express the cortisol-inactivating enzyme 11HSD2. 11HSD2 mRNA was detected in immortalized human aortic and coronary artery VSMCs (Figure 3A), as confirmed by direct sequencing (data not shown). Controls lacking RT were negative (data not shown). Immunoblotting studies confirmed that primary and immortalized VSMCs express 11HSD2 protein (Figure 3B). 11HSD2 protein was detected predominantly in pellet fractions (Figure 3B), similar to nonvascular cells, in which 11HSD2 enzyme is localized to membranous endoplasmic reticulum.17,35
11HSD2 converts cortisol to cortisone, which does not bind or activate MR. 11HSD2 function in coronary artery VSMCs was tested using the 11HSD2 inhibitor, glycyrrhetinic acid (GA). DoseeCresponse curves for MRE-reporter activation by cortisol were generated in the absence or presence of GA (Figure 3C). At doses of GA exceeding its Ki for 11HSD2 (10 eol/L), a significant left-shift in cortisol-mediated MR activation was evident (Figure 3C; P=0.004). These data support that human VSMCs contain functional 11HSD2 that can inactivate cortisol.
Ang II Activates Gene Transcription of an MR Responsive Promoter in Coronary VSMCs.
Ang II-mediated VSMC proliferation can be inhibited by aldosterone antagonists.11 The effect of Ang II on MR cellular localization was studied first by immunoflourescence. Treatment of VSMC with Ang II, like aldosterone, results in accumulation of MR exclusively in the nucleus (Figure 1C, bottom). Ang II effects on MR-mediated gene transcription were next assayed in VSMCs (Figure 4). Ang II significantly activated the MRE-luciferase reporter without effect on ERE-luciferase (control) reporter in immortalized (Figure 4) and primary (Figure 5) human coronary VSMCs. Hormone-dependent activation of gene expression in primary VSMCs occurred to levels similar to those seen in immortalized cells (Figure 5; compare with Figures 2A and 4).
Ang II Activates the Mineralocorticoid Receptor Via the AT1 Receptor
The mechanism of Ang II activation of MR in VSMCs was studied. Activation of the MRE reporter by Ang II was inhibited by the AT1 receptor blocker losartan (Figure 6A), which alone had no effect on basal reporter transcription (Figure 6A). In adrenal cells, Ang II acts locally via the AT1 receptor to activate aldosterone synthase and aldosterone production.36 We tested whether human VSMCs synthesize aldosterone using 5 distinct approaches. Aldosterone synthase (AS) mRNA was undetectable by RT-PCR11,25 in immortalized human coronary VSMCs (data not shown). Similarly, AS RNA was undetectable in microarray studies of VSMC RNA (Table). Immunoblotting experiments also failed to detect AS protein expression in murine VSMCs (data not shown). Aldosterone release from human VSMCs in response to Ang II was undetectable by radioimmunoassay, in contrast to vascular endothelial cells.37 Finally, the aldosterone synthase inhibitor FAD286 (Novartis), at doses that completely inhibit AS, did not alter MRE-luciferase reporter activation in response to Ang II in human coronary VSMCs (data not shown). Taken together, these data strongly support that local production of aldosterone is not the mechanism of Ang II-mediated MRE activation in coronary VSMCs.
We next tested whether Ang II activation of the MRE-reporter requires MR. Transcriptional activation of the MRE reporter by Ang II in human coronary artery VSMCs was fully suppressible by spironolactone (Figure 6B; 10eC4 spironolactone). Spironolactone alone had no effect on basal reporter activation (Figure 6B).
Aldosterone Regulates Endogenous Gene Expression in Human Coronary VSMCs
To characterize endogenous VSMC gene expression regulated by aldosterone in human coronary artery VSMCs, we used a microarray approach (Table). Gene expression in immortalized human coronary artery VSMCs treated with aldosterone or vehicle was analyzed in 4 independent experiments. Two genes involved in renal sodium transport and known to be regulated in renal cells by aldosterone,16 the NaK-ATPase gene, and the epithelial sodium channel,38 were not regulated by aldosterone in VSMCs (Table). However, several VSMC genes were induced by aldosterone, including genes involved in vascular fibrosis, calcification, and inflammation (Table). Quantitative real-time RT-PCR studies of RNA from these cells were used as a second independent test of aldosterone-mediated gene expression and verified the microarray findings (Table).
Discussion
The data show that human VSMCs express functional MR, capable of sequence-specific, aldosterone-activated gene transcription at physiological (1 nM) hormone levels; that angiotensin II also activates MR-mediated gene transcription via the AT1 receptor; and that aldosterone activates endogenous human coronary VSMC genes involved in vascular fibrosis, inflammation, and calcification. Based on these data, we propose a model for MR activation of gene transcription in VSMCs by both aldosterone and angiotensin II (Figure 7). Previous studies have demonstrated aldosterone binding and 11HSD2 activity in rabbit and rat aorta and mesenteric arteries22eC24 and more recent studies reveal MR and 11HSD2 gene expression in human aorta25 and vascular cells.11,39eC40 However, the present studies show for the first time to our knowledge that the endogenous vascular MR is transcriptionally competent and regulates expression of endogenous genes. In nonvascular cells, MR has been shown to effect cell function by both genomic and nongenomic mechanisms.41 The studies shown here investigate and provide evidence for a genomic MR pathway in VSMCs, but do not examine whether nongenomic activation of MR occurs in VSMCs.
VSMCs also express a functional 11HSD2 that is capable of inactivating cortisol, as is characteristic of tissues that respond to aldosterone. Patients with mutations in 11HSD2 have hypertension (syndrome of apparent mineralocorticoid excess) and subjects consuming large quantities of GA-containing licorice also have elevations in blood pressure.20 Although this has been attributed to cortisol activation of renal MR, resulting in sodium and fluid retention,20 our data raise the possibility that vascular 11HSD2 mutations or inhibition may contribute to hypertension via direct effects of cortisol on local vascular MR activation.
The mechanism by which Ang II stimulates MR-mediated gene transcription is not yet entirely clear. Ang II stimulates nuclear localization of MR and spironolactone inhibits Ang II-mediated gene expression in VSMCs, supporting the role of the MR itself. Ang II-mediated MR activation is also inhibited by losartan, an inhibitor of the AT1 receptor, demonstrating a link between AT1 receptor activation and MR transcriptional activation. Even though the AT1 receptor has been implicated previously in Ang II activation of aldosterone synthase36 in the adrenal gland, there is controversy in the literature as to whether the cardiovascular system produces its own aldosterone. Some data support that the heart, VSMCs, and endothelial cells1121,25,37,42,43 express aldosterone synthase and produce aldosterone, whereas other investigators refute this.44,45 We were unable to detect aldosterone synthesis in VSMCs using several assays, including microarray and RT-PCR studies of aldosterone synthase mRNA expression, immunoblotting for aldosterone synthase protein expression, radioimmunoassay for aldosterone synthesis and release, and pharmacological aldosterone synthase inhibition, which had no effect on Ang II-mediated MRE-reporter activation. Although aldosterone synthase expression and function may depend on cell conditions, under the same conditions in which Ang II activates MR-dependent gene transcription, we found no evidence for aldosterone synthesis by VSMCs. Other mechanisms for the AT1-mediated activation of MR need to be considered next, including ligand-independent activation of MR by post-translational modifications such as phosphorylation, which occurs for several steroid hormone receptors, including MR, in other tissues.46,47
The aldosterone-stimulated expression of endogenous genes in VSMCs detected by microarray and quantitative RT-PCR is distinct from aldosterone-stimulated gene expression in nonvascular cells. For example, MR-responsive genes known to be involved in renal sodium regulation, including the NaKATPase16 and the epithelial sodium channel,38 were not regulated by aldosterone in our studies of human coronary artery VSMCs. Several new aldosterone-regulated genes were identified, including genes involved in vascular fibrosis, calcification, and inflammation. Type I and type III collagen were upregulated significantly by aldosterone in VSMCs and have been implicated previously in Ang II-mediated and aldosterone-mediated cardiac and vascular fibrosis.48eC50 Bone morphogenic protein 2, the parathyroid hormone receptor, bone-liver-kidney alkaline phosphatase, and type 1 collagen all were upregulated in these assays. These genes and their proteins are known to be involved in vascular calcification, a process associated clinically with an increased risk of cardiac ischemic events.51,52 Aldosterone antagonism can prevent aldosterone-induced and Ang II-induced vascular inflammation in animal models,53,54 and several inflammatory genes were induced in the microarray studies, including IL-16 (lymphocyte chemoattractant factor) and cytotoxic T-lymphocyteeCassociated protein 4. These and other aldosterone-regulated genes in VSMCs may play an important role in the pathophysiology of atherosclerosis and vascular dysfunction, and also underscore the importance of tissue specificity, a common theme in steroid hormone actions. The studies described here support a potential new mechanism for vascular disease and for the preventive effect of angiotensin-converting enzyme inhibitors and aldosterone antagonists for ischemic diseases. Further understanding of the molecular mechanisms by which aldosterone and angiotensin II regulate MR-dependent gene expression and which genes are most relevant to vascular pathophysiology may also lead to novel therapies for human vascular disease.
Acknowledgments
I.Z.J. thanks Perrin C. White, J.L. Jameson, Joshua A. Beckman, and Heather Gornik for their generosity with reagents and Mason Freeman, Glenn Short, Najib Elmessadi, and Danny Park for assistance with microarray processing and analysis. The microarray studies were supported by a core facility award from the National Institutes of Health (NIH; HL72358). This work was supported in part by NIH RO1 HL50569 and P50 HL63494 (to M.E.M.).
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