Aldosterone-induced inflammation in the rat heart : role of oxidative stress

Abstract

Heart failure and hypertension have each been linked to an induction of oxidative stress transduced by neurohormones, such as angiotensin II and catecholamines. Herein, we hypothesized that aldosterone (ALDO) likewise induces oxidative stress and accounts for a proinflammatory/fibrogenic phenotype that appears at vascular and nonvascular sites of injury found in both right and left ventricles in response to ALDO/salt treatment and that would be sustained with chronic treatment. Uninephrectomized rats received ALDO (0.75 micro g/hour) together with 1% dietary NaCl, for 3, 4, or 5 weeks. Other groups received this regimen in combination with an ALDO receptor antagonist, spironolactone (200 mg/kg p.o. daily), or an antioxidant, either pyrrolidine dithiocarbamate (PDTC) (200 mg/kg s.c. daily) or N-acetylcysteine (NAC) (200 mg/kg i.p. daily). Unoperated and untreated age- and gender-matched rats served as controls. We monitored spatial and temporal responses in molecular and cellular events using serial, coronal sections of right and left ventricles. Our studies included: assessment of systolic blood pressure; immunohistochemical detection of NADPH oxidase expression and activity; analysis of redox-sensitive nuclear factor-kappaB activation; in situ localization of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha mRNA expression; monitoring cell growth and infiltration of macrophages and T cells; and analysis of the appearance and quantity of fibrous tissue accumulation. At week 3 of ALDO/salt treatment and comparable to controls, there was no evidence of oxidative stress or pathological findings in the heart. However, at weeks 4 and 5 of treatment, increased gp91(phox) and 3-nitrotyrosine expression and persistent activation of RelA were found in endothelial cells and inflammatory cells that appeared in the perivascular space of intramural coronary arteries and at sites of lost cardiomyocytes in both ventricles. Coincident in time and space with these events was increased mRNA expression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha. Macrophages, lymphocytes, and proliferating endothelial and vascular smooth muscle cells and fibroblast-like cells were seen at each of these sites, together with an accumulation of fibrillar collagen, or fibrosis, as evidenced by a significant increase in ventricular collagen volume fraction. Co-treatment with spironolactone, PDTC, or NAC attenuated these molecular and cellular responses as well as the appearance of fibrosis at vascular and nonvascular sites of injury. Furthermore, elevated systolic blood pressure in ALDO-treated rats was partially suppressed by spironolactone or either antioxidant. Thus, chronic ALDO/salt treatment is accompanied by a time-dependent sustained activation of NADPH oxidase with 3-nitrotyrosine generation and nuclear factor-kappaB activation expressed by endothelial cells and inflammatory cells. This leads to a proinflammatory/fibrogenic phenotype involving vascular and nonvascular sites of injury found, respectively, in both normotensive and hypertensive right and left ventricles. Spionolactone, PDTC, and NAC each attenuated these responses suggesting ALDO/salt induction of oxidative/nitrosative stress is responsible for the appearance of this proinflammatory phenotype.

Figure 1.

Expression of gp91^phox and 3-nitrotyrosine as detected by immunolabeling, NF-κB activation by RelA labeling, macrophage infiltration by ED-1 labeling, and cell proliferation by BrdU labeling are presented at sites of cardiac repair induced by ALDO treatment. Positive staining appears as brown. A: In rats receiving 4 weeks of ALDO treatment, NADPH oxidase was expressed by inflammatory cells within perivascular space (not shown) and sites of microscopic injury. B: 3-Nitrotyrosine staining was present in inflammatory cells at these sites. C: Activated NF-κB was observed in vascular endothelial cells (not shown) and inflammatory cells found in the injured myocardium of both ventricles. D: Numerous ED-1-positive macrophages appeared within the perivascular space and sites of scarring. E: Abundant BrdU-positive cells were likewise seen at these sites. F: Negative control for gp91^phox and 3-nitrotyrosine. Original magnifications: ×360 (A–C, F); ×280 (D, E).

Figure 2.

Transcription of ICAM-1, MCP-1, and TNF-α in the rat heart by in situ hybridization. In the normal rat heart, low levels of ICAM-1 (A), MCP-1 (E), and TNF-α (I) gene expression were present in both right (RV) and left (LV) ventricles. At 4 weeks of ALDO/salt treatment, gene expression of ICAM-1 (B), MCP-1 (F), and TNF-α (J) were increased within the perivascular space of intramural coronary arteries (arrowheads) and sites of myocardial scarring (S) in both ventricles. These responses were markedly attenuated by co-treatment with PDTC (C, G, and K, respectively) or with Spi (D, H, and L). NAC was similarly protective (not shown). High (H) and low (L) optical densities are color coded and presented at the left side of A.

Figure 3.

Quantitative optical density of ICAM-1 mRNA in the control rat hearts and those hearts obtained from ALDO-, ALDO + Spi-, ALDO + PDTC-, and ALDO + NAC-treated rats. Compared to controls, ICAM-1 mRNA was markedly increased in both ventricles in ALDO-treated rats for 4 weeks, which was prevented by co-treatment with either Spi, PDTC, or NAC. *, P < 0.01 compared to controls; ^†, P < 0.01 compared to ALDO-treated rats.

Figure 4.

Quantitative optical density of MCP-1 mRNA in control hearts and those hearts obtained from ALDO-, ALDO + Spi-, ALDO + PDTC-, and ALDO + NAC-treated rats for 4 weeks. Compared to controls, MCP-1 mRNA was markedly increased in ALDO-treated rats, which was prevented by co-treatment with either Spi, PDTC, or NAC. *, P < 0.01 compared to controls; ^†, P < 0.01 compared to ALDO-treated rats.

Figure 5.

Quantitative optical density of TNF-α mRNA in control rat hearts and those hearts obtained ALDO-, ALDO + Spi-, ALDO + PDTC-, and ALDO + acetylcysteine-treated rats. Compared to controls, TNF-α mRNA was markedly increased in ALDO-treated rats, which was prevented by co-treatment with either Spi, PDTC, or NAC. *, P < 0.01 compared to controls; ^†, P < 0.01 compared to ALDO-treated rats.

Figure 6.

Collagen volume fraction (%), a marker of fibrosis, in control rat hearts and those hearts obtained from ALDO-, ALDO + Spi-, ALDO + PDTC-, and ALDO + NAC-treated rats. Compared to controls, collagen volume fraction was markedly increased in the right and left ventricles of ALDO-treated rats, which was prevented by co-treatment with either Spi, PDTC, or NAC. *, P < 0.01 compared to controls; ^†, P < 0.01 compared to ALDO-treated rats.