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Department of Clinical and Molecular Endocrinology (Y.Hiro., T.Y., N.S., T.S., M.Sa., M.Sh., Y.Hira.), Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan; Department of Pharmacology (S.T.), Osaka Medical College, Osaka 569-8686, Japan; and Department of Hypertension and Cardiorenal Medicine (N.K.), Dokkyo University School of Medicine, Tochigi 321-0293, Japan
Address all correspondence and requests for reprints to: Takanobu Yoshimoto, M.D., Ph.D., Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: tyoshimoto.cme{at}tmd.ac.jp.
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Introduction |
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In addition to its hypertensive effect, several recent studies have shown that aldosterone exerts its proinflammatory/profibrotic effect on cardiovascular tissues with the possible involvement of the local renin-angiotensin (Ang) system (RAS) (3). It has been reported that aldosterone up-regulates the expression of Ang II type 1 (AT1) receptor and potentiates Ang II-stimulated intracellular signaling and proliferation in rat vascular smooth muscle cells (VSMCs) (4, 5, 6). We have recently reported that aldosterone directly up-regulates Ang-converting enzyme (ACE) expression in rat endothelial cells through the MR-mediated and Janus kinase 2-dependent pathway (7). Because systemic RAS is completely suppressed under mineralocorticoid-excess conditions, the positive feedback loop of the local RAS is presumed to play some role in the development of aldosterone-induced cardiovascular injury. However, the pathophysiological role of aldosterone as a positive modulator of cardiovascular RAS and its relation to cardiovascular injury in vivo remain unknown.
On the other hand, recent studies have suggested that enhanced vascular oxidative stress resulting from increased superoxide anion (O2–) generation by reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase is partly involved in the development of blood pressure elevation and cardiovascular injury in mineralocorticoid-induced hypertension (8, 9, 10). Although it has been well recognized that Ang II exerts its cardiovascular effects mainly via AT1-mediated generation of reactive oxygen species (ROS) (11, 12, 13), the underlying mechanism(s) of mineralocorticoid-induced ROS generation and its relation to cardiovascular injury has not been well characterized.
Based on these observations, we decided to investigate whether vascular ACE gene expression is up-regulated in aldosterone-induced hypertensive rats (Aldo-rats) and, if so, whether local RAS and oxidative stress are involved in the development of hypertension and vascular injury, and finally to determine the possible molecular mechanism(s) underlying the aldosterone-induced vascular injury and oxidative stress.
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Materials and Methods |
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Experimental protocol
The Aldo-rats used in the present study were developed using the protocol described previously (3). In this model, animals were uninephrectomized and infused sc with 0.75 µg/h D-aldosterone by osmotic minipumps (Alzet 2004; Alza, Palo Alto, CA). Then, 1% NaCl and 0.3% KCl in tap water was provided to the rats ad libitum for 3 wk. The sham-operated Sprague Dawley rats with osmotic minipumps containing vehicle (9%, ethanol, 87%, propylene glycol, and 4%, dH2O) were provided with tap water ad libitum and used as controls. Aldo-rats were divided into four groups: 1) untreated, 2) treated with the selective MR antagonist eplerenone (EPL, 100 mg/kg·d, orally), 3) treated with the AT1 receptor blockade (ARB) (1 mg/kg·d in drinking water), and 4) treated with the superoxide dismutase mimetic tempol (TEM, 2 mM in drinking water). Systolic blood pressure (SBP) was measured in conscious semirestrained rats by a tail-cuff method (Manometer-Tachometer, model KN-21–1; Natsume Instruments, Tokyo, Japan) before and 1, 2, and 3 wk after treatments. Ten to 15 measurements were made in each rat per session, and the mean values were calculated. After 3 wk of treatments, the rats were anesthetized with pentobarbital (70 mg/kg, ip) and killed by decapitation.
For RNA extraction and ACE activity measurement, the aortic tissues were quickly excised, immediately frozen on dry ice, and stored at –80 C. For histopathological and immunohistochemical analyses, the equatorial regions from each left ventricle and thoracic aorta were fixed in 10% formalin, processed routinely, and paraffin embedded. For the histopathological study, 3-µm-thick sections from the left ventricle were stained with Masson’s trichrome.
Aldosterone was purchased from Acros Organics (Geel, Belgium) and tempol from Sigma Chemical Co. (St. Louis, MO). ARB and EPL were generous gifts from Takeda Chemical Industries (Osaka, Japan) and Pfizer Inc. (New York, NY), respectively.
Quantification of mRNA
For measuring gene expression of ACE, adhesion molecules, growth factors, extracellular matrix-related factors, coagulation/fibrinolysis factors, and NAD(P)H oxidase components, the mRNA levels of rat ACE, osteopontin, matrix metalloproteinase-2 (MMP-2), platelet-derived growth factor-A (PDGF-A), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF), p22phox, gp91phox, and p47phox were quantified with real-time quantitative RT-PCR using fluorescent SYBR green technology (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany), as described previously (7, 12, 13). Rat acid ribosomal phosphoprotein P0 (ARPP P0) mRNA levels were quantified by TaqMan fluorescence methods as described previously (12, 13). Total RNA was extracted, first-strand cDNA synthesized, and amplification reaction performed as described previously (12, 13). PCR primers were synthesized by Greiner Bio-One (Tokyo, Japan), and the primers used for amplification are listed in Table 1
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Measurements of ACE activity and Ang II
Tissue extracts for measurement of ACE activity were prepared as described previously (14). The ACE activity was measured using a synthetic substrate hippuryl-His-Leu (HHL) (Peptide Institute, Inc., Osaka, Japan) that was specifically designed for ACE as described previously (14). Briefly, 50 µl tissue extract was incubated for 30 min at 37 C with 5 mmol/liter hippuryl-His-Leu in 250 µl of 10 mmol/liter phosphate buffer (pH 8.3) containing 300 mmol/liter NaCl. The reaction was terminated by adding 750 µl of 3% metaphosphoric acid, and then the mixture was centrifuged at 20,000 x g for 5 min at 4 C. The supernatant was analyzed using a reversed-phase column (RP-18, 4 mm id x 250 mm; IRICA Instrument, Kyoto, Japan). The results were normalized by determining the protein concentrations in the tissue extracts.
After extraction of plasma and aortic tissue samples (15), Ang II levels were measured by Ang II EIA kit (Bachem/Peninsula Laboratories, San Carlos, CA).
Immunohistochemical analysis
For evaluation of the lipid peroxidation by-product, immunohistochemical study using monoclonal anti-4-hydroxy-2-neonenal (4-HNE) (JaICA; Nikken SEIL Co. Ltd., Shizuoka, Japan) was performed as described previously (16). The aortic paraffin sections (3 µm thick) were deparaffinized, rehydrated, and boiled in 10 mmol/liter citric acid buffer for antigen retrieval. The sections were incubated with 5% normal horse serum for 30 min and then incubated overnight at 4 C with a primary antibody (0.5 µg/ml for anti-4-HNE in PBS). Negative controls were incubated with the same concentration of the corresponding IgG isotype (IgG1
; BioLegend, San Diego, CA). Antibody binding was visualized by the avidin-biotin-complex peroxidase method using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine tetrahydrochloride (Nichirei Bioscience, Tokyo, Japan).
Statistical analysis
Data are expressed as mean ± SEM from five different experiments. Differences between groups were examined for statistical significance using the unpaired t test or ANOVA with Dunn’s post hoc test, if they were appropriate. P values < 0.05 were considered to be statistically significant.
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Results |
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, a significant (P < 0.05) increase in SBP (186 ± 5 mm Hg) was induced by continuous aldosterone infusion compared with the control group (122 ± 4 mm Hg) at 3 wk after treatment. The increased SBP in Aldo-rats was significantly (P < 0.05) and equally decreased by cotreatment with EPL (160 ± 3 mm Hg), ARB (162 ± 3 mm Hg), and TEM (158 ± 4 mm Hg) after 3 wk.
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). The aldosterone-induced vascular inflammatory changes and myocardial fibrosis were ameliorated by cotreatment with EPL, ARB, or TEM (Fig. 1
). These results suggest that Aldo-induced hypertension and vascular injury involves MR- and AT1-mediated oxidative stress.
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). The Aldo-induced ACE gene expression and enzyme activity was completely blocked by cotreatment with EPL, whereas ARB or TEM had no effect (Fig. 3). These results suggest that aldosterone increases vascular ACE expression via the MR-dependent mechanism that is independent of Ang II-mediated oxidative stress.
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). Plasma Ang II concentrations were significantly (P < 0.05) decreased in the Aldo-rats compared with those in the control, whereas aortic Ang II contents in Aldo-rats were significantly (P < 0.05) increased compared with those in the control, suggesting that local aortic RAS is increased in the Aldo-rats under the suppressed systemic RAS.
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). Expression of all of these proinflammatory genes induced by aldosterone was completely inhibited by cotreatment with EPL (Fig. 5). By contrast, the inhibitory effects of ARB and TEM varied depending on each given gene (Fig. 5). ARB and TEM equally inhibited osteopontin, ICAM-1, and PAI-1 mRNA expressions that were induced by aldosterone (group 1 gene); TEM, but not ARB, inhibited Aldo-induced up-regulation of VCAM-1 and VEGF mRNA (group 2 gene); neither ARB nor TEM cotreatment affected Aldo-induced up-regulation of MMP-2 and PDGF-A mRNA (group 3 gene). As summarized in Table 2, each given gene is categorized into three groups according to the differential inhibitory responses to EPL, ARB, and TEM. These results suggest that Aldo-induced vascular proinflammatory gene expression differentially involves the AT1 receptor-mediated redox-sensitive mechanism.
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).
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). By contrast, cotreatment with either ARB or TEM significantly decreased Aldo-induced p47phox mRNA levels, whereas either treatment did not affect the Aldo-induced up-regulation of p22phox or gp91phox mRNA. Taken together, the present findings suggest that Aldo-induced vascular oxidative stress is mediated via induction of several components of NAD(P)H oxidase among which p47phox is preferentially up-regulated by AT1-mediated oxidative stress.
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Discussion |
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Even if systemic RAS is completely suppressed in mineralocorticoid-treated hypertensive rats, the present result, which shows that ARB exerts its strong antihypertensive and vasculo-protective effects in aldosterone-treated rats suggests that local RAS is partly involved in the development of blood pressure elevation and vascular injury in these animal models. Recently, it has been shown that aldosterone up-regulates ACE gene expression in neonatal rat cardiomyocytes (19) and in rat endothelial cells (7). The present in vivo result, that vascular ACE is up-regulated by excess aldosterone, agrees with the results of the previous in vitro studies (7, 19). Our data are in agreement with a recent in vivo study showing that ACE protein expression is increased in the heart of aldosterone-induced hypertensive rats (3, 20). Furthermore, it is noteworthy that plasma Ang II levels are decreased, but aortic Ang II contents are increased in the Aldo-rats, suggesting that local aortic Ang II generation in the vasculature is increased under the mineralocorticoid excess state. Because ACE is a key, but not a rate-limiting, enzyme for Ang II generation in local RAS (21, 22), it is most likely that the increased local Ang II generation by the up-regulated vascular ACE in Aldo-rats may partly contribute to the development of blood pressure elevation and vascular injury even under the suppressed systemic RAS. Furthermore, because it has been shown that intracellular signaling of Ang II in VSMCs is amplified by aldosterone (4, 5), the cardiovascular effect of Ang II may be further augmented by excess aldosterone. In agreement with the present study, it has very recently been reported that the ARB losartan ameliorated blood pressure elevation and cardiac fibrosis in Aldo-rats (9). When considered together with the previous in vitro findings (3, 4, 5, 6, 7), our data strongly suggest that local RAS activation is partly involved in the pathophysiology of cardiovascular injury in aldosterone-induced hypertension.
Recently, it has been reported that the prorenin/renin receptor could play a role in the local RAS activation (23, 24). Thus, it is possible to speculate that prorenin may be increased and/or activated in the vessel wall via renin/prorenin receptor under the mineralocorticoid excess conditions. Although we examined expression of the renin/prorenin receptor in the membrane fraction of various rat tissues by receptor binding assay using radiolabeled handle-protein decapeptide as a ligand, its specific bindings were barely detectable in the aorta, heart, kidney, and liver but abundantly detectable only in the brain (Yoshimoto, T., and M. Shichiri, unpublished observations). Although it has been reported that renin/prorenin receptor mRNA and protein are expressed in various rat tissues evaluated by Northern blot analysis and immunohistochemical method (24), it remains to be determined whether they are functionally expressed on the cell surface.
Because all the proinflammatory gene expressions examined were completely inhibited by cotreatment with eplerenone, aldosterone was suggested to induce up-regulation of these gene expressions via MR. However, it should be noted that the inhibitory effects of TEM and ARB on the aldosterone-induced expression of these proinflammatory genes in the aortic tissue varied (Table 2); ARB and TEM equally inhibited osteopontin, ICAM-1, and PAI-1 expression (group 1), whereas TEM, but not ARB, inhibited VCAM-1 and VEGF expression (group 2), and neither ARB nor TEM affected ACE, MMP-2, or PDGF-A expression (group 3).
ARB- and TEM-sensitive expression of group 1 genes (osteopontin, ICAM-1, PAI-1, and p47phox) in the aortic tissue of aldosterone-treated rats suggests that local RAS activation and oxidative stress are mainly involved in the expression of such vascular proinflammatory/fibrinolytic genes. In accordance with this, it has recently been shown that ACE inhibitor (imidapril) decreases cardiac mRNA levels of proinflammatory/profibrotic factors (TGF-ß, monocyte chemoattractant protein-1, and type I and III collagen) in Aldo-rats (20). By contrast, TEM-sensitive but ARB-insensitive expression of group 2 genes (VCAM-1, VEGF) in the aortic tissue of Aldo-rats suggest that oxidative stress triggered by factors other than Ang II is involved in the induction of group 2 genes under conditions of mineralocorticoid-excess. Although expression of VCAM-1 and VEGF mRNA in vascular cells has been shown to be induced by Ang II via a redox-sensitive pathway (13, 25), Ang II is not a sole determinant of redox state under conditions of mineralocorticoid excess. Indeed, it has been shown that aldosterone directly stimulates O2– production through NAD(P)H oxidase in cardiovascular cells in vitro (26, 27). Callera et al. (26) have recently revealed that aldosterone activates NAD(P)H oxidase via a c-Src-dependent pathway in VSMCs, whose effect was abrogated by EPL. Therefore, it is inferred that O2– generation triggered by factors other than Ang II is sufficient for induction of group 2 genes under the conditions of mineralocorticoid excess. ARB- and TEM-insensitive expression of group 3 genes (ACE, MMP2, PDGF-A, gp91phox, and p22phox) in the aortic tissue of aldosterone-induced rats suggests that mechanisms other than local RAS and redox state are responsible for group 3 gene expression. In fact, aldosterone has been shown to induce a diversity of intracellular signaling and cellular responses, such as activation of various protein kinases via genomic and nongenomic pathways (28, 29). Taken together, diverse pathways downstream of MR activation, including local RAS activation, redox state, and/or other yet uncharacterized signals may differentially regulate vascular proinflammatory gene expression.
The present immunohistochemical study demonstrated that the increased accumulation of 4-HNE, a lipid-peroxidation by-product and a marker for oxidative stress (30), in endothelium of aldosterone-treated rats was blocked not only by EPL or TEM but also by ARB to a similar degree. The inhibitory effect of ARB on oxidative stress damage in the aortic tissue suggests that local RAS activation is partly involved in the development of oxidative stress damage as well as the redox-sensitive proinflammatory gene expressions. Our results are in agreement with those of a recent study that showed that vascular superoxide generation was decreased by losartan or TEM in aldosterone-induced hypertension even under conditions of normal salt intake (9). Thus, regardless of the salt status, oxidative stress induced by aldosterone per se is partly responsible for the vascular injury.
Several lines of evidence have indicated that NAD(P)H oxidase-derived superoxide plays a key role in the oxidative stress and inflammatory response by aldosterone in the cardiovascular tissue (8, 9, 10, 17). The present results showing that aortic mRNA levels of p22phox, gp91phox, and p47phox were increased in aldosterone-induced hypertension are consistent with those of previous studies showing that Ang II and aldosterone increase mRNA levels of NAD(P)H oxidase components (p22phox, gp91phox, and p47phox) in cardiovascular cells in vivo and in vitro (20, 31). Unlike p22phox and p47phox expressed in all three layers of the vasculature, gp91phox is mainly expressed in the intimal and the adventitial layers, but not in the medial smooth muscle cell layer, as well as in leukocytes (31). Because gene expression analysis was performed using RNA extracted from the whole aortic tissue in the present study, the increased gp91phox mRNA expression in the aorta could represent the changes in the intimal and the adventitial layers. Alternatively, infiltration of leukocytes to the aortic tissue may be responsible for the increased gp91phox mRNA expression. Moreover, the present study has demonstrated that TEM and ARB preferentially decreased p47phox mRNA level but not that of p22phox or gp91phox. Recently, Iglarz et al. (9) have reported that increased vascular NAD(P)H oxidase activity in aldosterone-induced hypertension is inhibited by cotreatment with losartan. When considered together, the results indicate that local RAS activation in aldosterone-induced hypertension partly contributes the to increased vascular oxidative stress resulting from preferential induction of p47phox, an essential component of vascular NAD(P)H oxidase (32, 33).
The proposed mechanisms of aldosterone-induced vascular injury deduced from the present study is illustrated in Fig. 8. Vascular inflammation, oxidative stress, and hypertension are thereby involved in the development of aldosterone-induced vascular injury, all of which are mediated by MR activation and subsequent vascular NAD(P)H oxidase activation. Both Ang II-dependent and Ang II-independent pathways may contribute to the underlying mechanisms of the increased vascular NAD(P)H oxidase expression. Local RAS activation by vascular ACE expression preferentially up-regulates vascular p47phox expression, whereas vascular p22phox and gp91phox are up-regulated via an Ang II-independent pathway. The resultant increased NAD(P)H oxidase activity may partly contribute to the development of vascular oxidative stress and proinflammatory/profibrotic gene expression in addition to blood pressure elevation, thereby leading to vascular injury, whereas redox-independent MR signaling may also play some role in the development of vascular inflammation.
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Acknowledgments |
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Footnotes |
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Author Disclosure Information: Y.Hiro., T.Y., N.S., T.S., M.Sa., S.T., N.K., M.Sh., and Y.Hira. have nothing to declare.
First Published Online January 11, 2007
Abbreviations: ACE, Angiotensin converting enzyme; Ang, angiotensin; Aldo-rats, aldosterone-induced hypertensive rats; ARB, AT1 receptor blockade; ARPP P0, acid ribosomal phosphoprotein P0; AT1, Ang II type 1; EPL, eplerenone; 4-HNE, 4-hydroxy-2-neonenal; ICAM-1, intercellular adhesion molecule-1; MMP-2, matrix metalloproteinase-2; MR, mineralocorticoid receptor; NAD(P)H, reduced nicotinamide adenine dinucleotide phosphate; PAI-1, plasminogen activator inhibitor-1; PDGF-A, platelet-derived growth factor-A; RAS, renin-angiotensin system; ROS, reactive oxygen species; SBP, systolic blood pressure; TEM, tempol; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cell.
Received August 22, 2006.
Accepted for publication January 2, 2007.
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, Nontreated control rats; 
, Aldo-rats; 
, Aldo-rats cotreated with EPL; 
, Aldo-rats cotreated with ARB; x, Aldo-rats cotreated with TEM. *, P < 0.05 vs. control; 
, P < 0.05 vs. Aldo-rats.
