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Aldosterone mediates angiotensin II-induced interstitial cardiac fibrosis via a Nox2-containing NADPH oxidase

King’s College London, Cardiovascular Division, London, UK

¹Correspondence: King’s College London School of Medicine, New Medical School Bldg., Bessemer Rd., London SE5 9PJ, UK. E-mail: ajay.shah{at}kcl.ac.uk

SPECIFIC AIMS

Angiotensin II (angII) and aldosterone contribute to the development of interstitial cardiac fibrosis, at least in part through the activation of redox-sensitive signaling pathways. The source of reactive oxygen species (ROS) targeted by these hormones, its involvement in fibrosis, and the interrelationship between aldosterone and AngII in this regard is uncertain. We studied mice deficient in the Nox2 catalytic subunit of NADPH oxidase (Nox2^–/– mice) and wild-type littermate controls (WT) to probe the potential role of NADPH oxidase in aldosterone-induced cardiac fibrosis and the involvement of this mechanism in AngII-induced effects.

PRINCIPAL FINDINGS

1. AngII induces interstitial fibrosis through the activation of Nox2 oxidase and consequent NF-B activation, up-regulation of pro-fibrotic genes, and MMP activity
Nox2^–/– and WT mice treated with 2 wks’ subcutaneous (s.c.) AngII infusion (1.1 mg/kg/day) developed similar rises in systolic blood pressure (WT: 108.0±5.7 to 167.1±8.3 mmHg; Nox2^–/–: 113.1±5.5 to 151.4±5.9 mmHg) and left ventricular (LV)/body wt ratio. AngII infusion significantly increased myocardial NADPH oxidase activity in WT but had no significant effect in Nox2^–/– mice (Fig. 1 A). Interstitial cardiac fibrosis assessed in LV sections increased from 7.2 ± 0.7% to 11.5 ± 1.0% (P <0.05) in AngII-infused WT but was unaltered in Nox2^–/– mice (Fig. 1B ). AngII-induced fibrosis was inhibited by concurrent oral treatment with the antioxidant N-acetylcysteine, without any changes in blood pressure, consistent with an involvement of ROS.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of s.c. AngII infusion (1.1 mg/kg/day for 2 wk) on WT and Nox2–/– mice. A) Myocardial NADPH oxidase activity measured by 5 µM lucigenin-enhanced chemiluminescence. B) Interstitial fibrosis assessed by image analysis of Masson’s Trichrome stained sections. C–F) Messenger RNA transcripts assessed by real-time RT-PCR (data are normalized by GAPDH mRNA and expressed as arbitrary units [AU]). *P < 0.05.

AngII-infused WT hearts showed significant increases in collagen I and fibronectin mRNA expression by real-time RT-PCR (both 3-fold), whereas these were inhibited in Nox2^–/– mice (Fig. 1C, D ). No change in transforming growth factor ßbeta;1 (TGFßbeta;1) mRNA expression was found but connective tissue growth factor (CTGF) mRNA, which is implicated in AngII-induced vascular and cardiac fibrosis, was significantly increased in WT (Fig. 1E, F ). The increase in CTGF mRNA was inhibited in Nox2^–/– hearts. AngII infusion significantly increased MMP-2 activity in LV of WT mice as assessed by gelatin zymography (from 0.9±0.1 to 1.4±0.2 arbitrary units; P <0.05), whereas no significant change was found in Nox2^–/– hearts. Finally, AngII-infused WT hearts showed evidence of NF-B activation, as assessed by immunohistochemistry for p65 translocation, whereas NF-B was not activated in AngII-infused Nox2^–/– hearts.

2. AngII-induced activation of myocardial NADPH oxidase activity and interstitial fibrosis are inhibited by spironolactone
To investigate the possible involvement of mineralocorticoid receptor (MR) activation in the NADPH oxidase response to AngII, saline- or AngII-infused WT mice were treated concurrently with the MR antagonist spironolactone (200 mg/kg/day in chow). Spironolactone treatment significantly inhibited the AngII-induced increase in NADPH oxidase activity without affecting the AngII pressor response. Spironolactone also inhibited AngII-induced increases in interstitial fibrosis. Consistent with this, AngII-induced increases in the expression of collagen I and fibronectin mRNA expression were attenuated by spironolactone. These results suggest that MR activation plays an important role in AngII-induced myocardial NADPH oxidase activation and fibrosis.

3. Nox2 NADPH oxidase mediates the cardiac pro-fibrotic effects of MR-dependent hypertension in vivo
To directly investigate the role of Nox2 in MR-dependent fibrosis in vivo, we studied an established model of MR-dependent hypertension and fibrosis. WT and Nox2^–/– mice underwent unilateral nephrectomy, and received s.c. aldosterone (0.2 mg/kg/day for 4 wk) and 1% NaCl/0.3% KCl in drinking water ("ALDO" mice). Myocardial NAPDH oxidase activity increased significantly in WT ALDO mice but was unchanged in Nox2^–/– ALDO mice (Fig. 2 A). Interstitial cardiac fibrosis also increased significantly in WT ALDO mice (12.0±1.7% vs. 6.3±0.3%; P <0.05), but this was inhibited in Nox2^–/– ALDO mice (5.8±1.0% vs. 6.8±0.8%; P=NS; Fig. 2B ). Consistent with this, procollagen I and fibronectin mRNA expression were increased in WT ALDO mice but these increases were inhibited in Nox2^–/– ALDO mice (Fig. 2C, D ). ALDO-induced NF-B activation and MMP-2 activity were also inhibited in Nox2^–/– ALDO mice. These differences in NADPH oxidase activity and cardiac fibrosis between WT and Nox2^–/– mice were independent of blood pressure since systolic blood pressure in ALDO mice increased to a similar extent in both groups.

View larger version (7K): [in this window] [in a new window]   Figure 2. Effects of aldosterone infusion on (A) Myocardial NADPH oxidase activity; (B) Interstitial fibrosis; (C, D) Real-time RT-PCR analysis of mRNA transcripts. Data are normalized by GAPDH mRNA and expressed as arbitrary units (AU). *P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

The mechanisms underlying the development of interstitial cardiac fibrosis in hypertensive heart disease are incompletely understood. Activation of the renin-angiotensin-aldosterone system (RAAS) is implicated but the underlying pathways through which this signals increased fibrosis remain unclear. The current study provides significant new information regarding this question. First, we show that critical events in the cardiac fibrosis induced by AngII are the Nox2-NADPH oxidase-dependent activation of the transcription factor NF-B, up-regulation of profibrotic genes, and MMP-2 activation. Secondly, we demonstrate that MR activation contributes significantly to AngII-induced myocardial NADPH oxidase activation and interstitial fibrosis in vivo, since these effects are inhibited by the spironolactone. Thirdly, we show that Nox2 is also pivotally involved in the development of cardiac fibrosis in a model of MR-dependent hypertension. Taken together, these results suggest a central role for the Nox2 oxidase in the cardiac fibrosis that accompanies RAAS activation (Fig. 3 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. Role of Nox2-containing NADPH oxidase in interstitial cardiac fibrosis induced by AngII or aldosterone (MR-mineralocorticoid receptor).

It is increasingly appreciated that NADPH oxidases are major sources of ROS in the cardiovascular system. At least 5 oxidase isoforms are described, of which the main ones expressed in heart are Nox2 and Nox4. AngII can activate the Nox2 oxidase via the AT₁ receptor, and we have previously reported that cardiac hypertrophy induced by short-term subpressor AngII infusion is inhibited in Nox2^–/– mice. The present study indicates that Nox2 also plays a critical role in AngII-induced cardiac fibrosis in that both NADPH oxidase activation and interstitial fibrosis were completely inhibited in Nox2^–/– mice. Furthermore, a number of redox-sensitive pathways potentially involved in the Nox2-dependent induction of cardiac fibrosis were defined, namely the activation of the transcription factor NF-B and of MMP2, both of which are implicated in the regulation of extracellular matrix remodeling.

Complex interactions between aldosterone and AngII are recognized in many pathological settings and it has been shown that AngII-induced cardiac injury can be attenuated by MR antagonists. However, the potential role of MR activation in AngII-induced up-regulation of NADPH oxidase and subsequent downstream effects is unknown. In the current study, we provide the first evidence that MR activation is involved in AngII-induced myocardial NADPH oxidase activation and subsequent interstitial cardiac fibrosis in vivo. Thus, the MR antagonist spironolactone inhibited both the increase in myocardial NADPH oxidase activity and the profibrotic effects of AngII. MR activation per se has been suggested to cause cardiac oxidative stress in previous studies, and recently it was found that aldosterone could activate NADPH oxidase in macrophages. A key novel observation in the current study is the finding that Nox2 is centrally involved in the development of cardiac fibrosis in ALDO mice, in which the up-regulation of both myocardial NADPH oxidase activity and pro-fibrotic genes are inhibited in Nox2^–/– animals. These results therefore support a central role for the Nox2 oxidase in the interstitial cardiac fibrosis induced not only by AngII but also aldosterone.

Previous work has shown that cardiomyocyte hypertrophy and interstitial fibrosis may be independently regulated processes that do not necessarily parallel each other in conditions such as hypertension. Although increased cardiac load may lead to interstitial cardiac fibrosis, this is not a simple linear relationship. An important finding in the current study was the significant dissociation between interstitial fibrosis, blood pressure and cardiac hypertrophy both in response to AngII and ALDO. Thus, fibrosis was inhibited in AngII-infused Nox2^–/– mice whereas these mice developed similar rises in blood pressure and cardiac mass to WT animals. The increase in cardiac mass is in line with previous work showing that Nox2 is critical for the cardiac hypertrophic response to subpressor AngII but not that to pressure overload. Similarly, the inhibition of AngII-induced fibrosis by spironolactone in the current study was independent of blood pressure. Nox2^–/– mice subjected to ALDO also developed similar rises in blood pressure and heart/body wt ratio to WT whereas fibrosis was inhibited. Taken together, these results support a Nox2-mediated regulation of interstitial cardiac fibrosis in response to either AngII or ALDO, which is independent of effects on blood pressure or cardiac hypertrophy.

The present findings could potentially be of relevance to clinical heart disease and its treatment. Recent clinical trials demonstrated that MR antagonists significantly improved cardiac remodeling and reduced mortality in patients with impaired cardiac function post-MI (the EPHESUS study) or those with chronic heart failure (the RALES study). Spironolactone also improves forearm vascular endothelial function in patients with heart failure, a setting in which increased oxidative stress is implicated. However, the effects of MR antagonists in clinical hypertensive heart disease have not been addressed in detail. The current data indicate a key role for MR-dependent activation of a Nox2 oxidase in interstitial cardiac fibrosis induced by either AngII or aldosterone. The Nox2 oxidase could potentially represent a useful therapeutic target for modulating interstitial fibrosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4642fje

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