Journal of Cardiovascular Pharmacology:
Aldosterone Impairs Vascular Endothelial Cell Function
Hashikabe, Yuko MD*; Suzuki, Kunihiro MD*; Jojima, Teruo MD*; Uchida, Kohsuke MD, PhD†; Hattori, Yoshiyuki MD*
Departments of *Endocrinology and Metabolism
†Pharmacology, Dokkyo University School of Medicine, Mibu, Tochigi, Japan
Reprints: Dr Y. Hattori, Dokkyo University School of Medicine, Dept of Endocrinology and Metabolism, Mibu, Tochigi 321-0293, Japan (e-mail: firstname.lastname@example.org).
Received for publication January 26, 2006; accepted February 18, 2006
Aldosterone is a mineralocorticoid hormone that plays an important role in regulating electrolyte balance and blood pressure and also participates in endothelial dysfunction. We evaluated the direct effect of aldosterone on human umbilical vein cells (HUVEC). Levels of eNOS phosphorylation by vascular endothelial growth factor were diminished, and the amount of NO produced in response to vascular endothelial growth factor measured as NO2−+NO3− was significantly decreased in cells previously incubated with aldosterone. Incubation with aldosterone for 24 h dose-dependently increased Nox4 mRNA expression in HUVEC. Although NF-κB was not apparently activated by aldosterone, mRNA levels of vascular cell adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVEC were significantly increased after incubation with aldosterone. Thus, aldosterone directly causes the dysregulation of endothelial cell function, which may be partly responsible for high blood pressure and atherosclerosis.
Aldosterone is a mineralocorticoid hormone that plays an important role in regulating electrolyte balance and blood pressure.1,2 Although the aldosterone escape mechanism is understood, aldosterone itself is often neglected in terms of the pathophysiological consequences of the activated rennin–angiotensin–aldosterone system (RAS) in arterial hypertension and chronic heart failure. The RAS is upregulated in the vasculature of atherosclerotic vessels,3 and recent data suggest that aldosterone mediates some of the effects of angiotensin II. Aldosterone participates in endothelial dysfunction, vascular fibrosis, and inflammation in the vasculature and is involved in the pathogenesis of hypertension.4–7
The Randomized Aldosterone Evaluation Study (RALES) uncovered a 30% survival advantage among patients with chronic heart failure administered the mineralocorticoid receptor blocker, spironolactone, in addition to standard therapy including diuretics and angiotensin-converting enzyme (ACE) inhibitors.8 In fact, ACE inhibitors only transiently suppress aldosterone production, and the RALES results suggest that aldosterone plays a significant role in cardiovascular diseases. Eplerenone, a selective mineralocorticoid receptor blocker, prevented relaxation in response to the endothelial-dependent agonist acetylcholine in a rabbit model of atherosclerosis.9 Moreover, eplerenone reduced the development of atherosclerosis and suppressed serum and macrophage oxidative stress in a mouse model of atherosclerosis.10 These findings indicate that the beneficial effect of aldosterone on cardiovascular abnormalities is achieved through a novel mechanism.
Because spironolactone improves endothelial dysfunction and increases NO bioactivity in patients with heart failure, aldosterone may impair endothelial function via direct action on endothelium, leading to the development of atherosclerosis. The present study evaluates the effect of aldosterone on endothelial cell function.
MATERIALS AND METHODS
Human umbilical vein endothelial cells (HUVEC) obtained from Sanko Junyaku (Tokyo, Japan) were cultured in EBM medium supplemented with 5% fetal calf serum under standard conditions. All cells in this experiment were used within 3 to 4 passages, and their specific immunohistochemical and morphological characteristics were confirmed.
To detect nitric oxide (NO) production within monolayers, bovine aortic endothelial cells were cultured in 24-well dishes and studied 1 day after reaching confluence. After incubation for 1 h, nitrite and nitrate levels (NO2− and NO3−) in the medium were measured using an automated NO detector coupled with a high-performance liquid chromatography system (ENO10, Eicom, Moraine, OH).
We dispersed HUVEC treated with vascular endothelial growth factor (VEGF) in the presence or absence of aldosterone using lysis buffer (Cell Signaling, Danvers, MA) containing 1 mmol/L phenylmethyl sulfonyl fluoride. Protein concentrations in lysates were measured using a Bio-Rad (Tokyo, Japan) detergent-compatible protein assay. Subsequently, β-mercaptoethanol was added at a final concentration of 1%, and the lysates were denatured by boiling for 3 min. Samples containing 10 μg of protein were resolved by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels, transferred to a polyvinyldienefluoride membrane (Bio-Rad), and incubated with anti-phospho-eNOS (ser1177p-eNOS) and anti-eNOS antibodies (1:1000, Cell Signaling). Antibody binding was detected using sheep anti-rabbit immunoglobulin G horseradish peroxidase (1:5000) and visualized using the ECL Plus system (Amersham, Buckinghamshire, UK).
Real-Time Polymerase Chain Reaction (PCR)
Total RNA (2 μg) was incubated with DNase I for 15 min to synthesize the cDNA that was applied to PCR using the LineGene system (BioFlux, Tokyo, Japan) with the Nox411 (5′-TAACCAAGGGCCAGAGTATCACT-3′ and 5′-CCGGGAGGGTGGGTATCTAA-3′) and GAPDH (5′-AGTTCAAGAACGCCTTACCA-3′ and 5′-GCTCGCACACGGAAGTT-3′) primers under the following conditions: 95°C for 5 min, 40 cycles at 95°C for 15 s, 60°C for 15 s and 72°C for 30 s.
SV40-transformed mouse vascular endothelial cells (SVEC4) were stably transfected with a cis-reporter plasmid containing the luciferase reporter gene linked to 5 repeated NF-κB binding sites (pNFκB-Luc, Stratagene, La Jolla, CA) as described.12 NF-κB activation was then examined in several clones and luciferase activity was assayed using a kit (Stratagene). We also measured changes in the levels of NF-κB p50 and p65 in nuclear extracts from HUVEC using a transcription factor assay kit (Active Motif Japan, Tokyo). Nuclear extracts were prepared using the NE-PER nuclear extraction reagent (Pierce, Rockford, IL), and then p50 and p65 were quantified using Jurkat nuclear extract as the standard.
Data are presented as mean±SEM. Multiple comparisons were evaluated by ANOVA followed by Fisher protected least-significant difference test. A value of P<0.05 was considered statistically significant.
Effect of Aldosterone on VEGF-induced eNOS Phosphorylation and NO Production in HUVEC
Figure 1A shows that eNOS was phosphorylated (ser1177p-eNOS) 15 min after adding VEGF (100 ng/mL) to HUVEC and reached a maximum after 30 min. Levels of eNOS phosphorylation by VEGF were diminished in cells after incubation with aldosterone (1 μmol/L) for 16 h. Incubating HUVEC with VEGF (100 ng/mL) increased the concentration of bioactive NO in the cell supernatant (measured as NO2−+NO3−), whereas an incubation with aldosterone (1 μmol/L) for 16 h significantly decreased subsequent NO production (Fig. 1B).
Effect of Aldosterone on Induction of Nox4 mRNA in HUVEC
Incubating HUVEC with aldosterone for 24 h significantly increased the expression of Nox4 mRNA. Figure 2 shows that Nox4 mRNA levels were dose-dependently increased up to 1 κmol/L and then reached a plateau.
Effect of Aldosterone on NF-κB Activation
We examined whether aldosterone directly causes NF-κB activation by assessing the induction of NF-κB reporter gene expression in SVEC4 endothelial cells. Incubating SVEC4 cells with aldosterone for 2 h did not activate NF-κB-mediated gene transcription (Fig. 3A). In contrast, lipopolysaccharides (LPS) dose-dependently stimulated NF-κB-mediated gene transcription in endothelial cells (positive control; Fig. 3A). Likewise, levels of nuclear p65 and p50 in HUVEC were obviously increased after stimulation with LPS, but not aldosterone for 2 h (Fig. 3B).
Effect of Aldosterone on mRNA Expression of Vascular Cell Adhesion Molecule-1, E-Selectin, Monocyte Chemotactic Protein-1, and Intercellular Adhesion Molecule-1
The effect of aldosterone on the mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) in HUVEC was measured by real-time PCR. Figure 4 shows that incubating HUVEC with aldosterone for 24 h significantly increased the gene expression of VCAM-1, E-selectin, MCP-1, and ICAM-1. The expression of these genes induced by aldosterone was significantly inhibited by spironolactone or the NF-κB inhibitor, BAY11-7082 (data not shown).
The RAS is upregulated in the vasculature of atherosclerotic vessels.3 Local angiotensin II generated by the modulation of NADH/NADPH-dependent oxidases may result in NO inactivation, establishing the conditions for atherosclerotic progression. Indeed, a RAS blockade induced with an angiotensin II type 1 receptor blocker ameliorates endothelium-dependent vasomotion by decreasing the NADH oxidase activities in vessel walls and reduces experimental atherosclerosis.13 Recent data suggest that aldosterone mediates some of the effects of angiotensin II. The present study showed that aldosterone induces Nox4 mRNA in HUVEC, which leads to NADPH oxidase activation and O2− production, which may inactivate NO production by endothelial cells. Indeed, the present results showed that aldosterone decreased NO production from HUVEC stimulated with VEGF. This was at least partly caused by aldosterone decreasing the VEGF-induced phosphorylation of ser1177p-eNOS. Furthermore, the induction of superoxide and NO may lead to the production of peroxynitrite, which would subsequently oxidize tetrahydrobiopterin and cause “uncoupling” of the NOS reaction.14,15 Thus, if aldosterone facilitates eNOS uncoupling, then dysfunctional endothelium will produce more O2− and little NO. Indeed, mineralocorticoid receptor antagonism in patients with an upregulated RAS is associated with improved forearm blood flow, suggesting an effect on the NOS pathway.16
Incubating HUVEC with aldosterone significantly increased the gene expression of VCAM-1, E-selectin, MCP-1, and ICAM-1. The expression of all of these genes was inhibited by spironolactone, suggesting that this effect was mediated by mineralocorticoid receptor. Similarly, the expression of these genes induced by aldosterone was significantly inhibited by BAY11-7082, which selectively and irreversibly inhibits the cytokine-inducible phosphorylation of IκB.17 This finding suggested that the induction of these 4 genes is NF-κB dependent. However, aldosterone did not activate NF-κB in this study, although endothelial cells and inflammatory cells in the heart time-dependently sustain the activation of NADPH oxidase accompanied by 3-nitro-tyrosine generation and NF-κB activation in rats chronically treated with aldosterone and salt.18 Our observation may be explained as follows: Aldosterone induces Nox4 and then increases O2− production, which generates peroxynitrite that reacts with NO, resulting in delayed NF-κB activation.19 Thus, the mRNA expression of those adhesion molecules and chemokines in response to aldosterone may be responsible for the delayed NF-κB activation.
Pharmacological manipulation of the aldosterone pathway using a selective aldosterone receptor antagonist may improve NO availability and endothelial function. The results of the present study indicate that blocking the aldosterone receptor has potential antiatherosclerotic effects.
The authors are grateful to Masashi Ikeda, PhD (Institute for Medical Science, Dokkyo University School of Medicine), and Hiroko Satoh for technical assistance.
1. Arriza JL, Weinberger C, Cerelli G, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987;237:268–275.
2. Fejes-Toth G, Pearce D, Naray-Fejes-Toth A. Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc Natl Acad Sci USA. 1998;95:2973–2978.
3. Diet F, Pratt RE, Berry GJ, et al. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease. Circulation. 1996;94:2756–2767.
4. Duprez D, De Buyzere M, Rietzschel ER, et al. Aldosterone and vascular damage. Curr Hypertens Rep. 2000;2:327–334.
5. Stier CT Jr., Chander PN, Rocha R. Aldosterone as a mediator in cardiovascular injury. Cardiol Rev. 2002;10:97–107.
6. Schiffrin EL, Touyz RM. From bedside to bench to bedside: role of renin-angiotensin-aldosterone system in remodeling of resistance arteries in hypertension. Am J Physiol Heart Circ Physiol. 2004;287:H435–H446.
7. Keidar S, Kaplan M, Pavlotzky E, et al. Aldosterone administration to mice stimulates macrophage NADPH oxidase and increases atherosclerosis development: a possible role for angiotensin-converting enzyme and the receptors for angiotensin II and aldosterone, Circulation. 2004;109:2213–2220.
8. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999;341:709–717.
9. Rajagopalan S, Duquaine D, King S, et al. Mineralocorticoid receptor antagonism in experimental atherosclerosis. Circulation. 2002;105:2212–2216.
10. Keida Sr, Hayek T, Kaplan M, et al. Aviram, Effect of eplerenone, a selective aldosterone blocker, on blood pressure, serum and macrophage oxidative stress, and atherosclerosis in apolipoprotein E-deficient mice. J Cardiovasc Pharmacol. 2003;41:955–963.
11. Sorescu D, Weiss D, Lassegue B, et al. Griendling, Superoxide production and expression of Nox family proteins in human atherosclerosis. Circulation. 2002;105:1429–1435.
12. Hattori Y, Hattori S, Kasai K. Globular adiponectin activates NF-κB in vascular endothelial cells, which in turn induces expression of proinflammatory and adhesion molecule genes. Diabetes Care. 2006;29:139–141.
13. Warnholtz A, Nickenig G, Schulz E, et al. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999;99:2027–2033.
14. Laursen JB, Somers M, Kurz S. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001;103:1282–1288.
15. Milstien S, Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun. 1999;263:681–684.
16. Farquharson CA, Struthers AD. Spironolactone increases nitric oxide bioactivity, improves endothelial vasodilator dysfunction, and suppresses vascular angiotensin I/angiotensin II conversion in patients with chronic heart failure. Circulation. 2000;101:594–597.
17. Pierce JW, Schoenleber R, Jesmok G, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem. 1997;272:21096–21103.
18. Sun Y, Zhang J, Lu L, et al. Aldosterone-induced inflammation in the rat heart: role of oxidative stress. Am J Pathol. 2002;161:1773–1781.
19. Cooke CM, Davidge ST. Peroxynitrite increases iNOS through NF-κB and decreases prostacyclin synthase in endothelial cells. Am J Physiol Cell Physiol. 2002;282:C395–C402.
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