Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways

Abstract

Connective tissue growth factor (CTGF) is a polypeptide implicated in the extracellular matrix synthesis. Previous studies have provided evidence that angiotensin II (Ang II) promotes collagen synthesis and regulates collagen degradation. We investigated whether or not CTGF mediates the profibrotic effects of Ang II in the heart and kidneys and the role of calcineurin-dependent pathways in CTGF gene regulation. In transgenic rats harboring human renin and angiotensinogen genes, Ang II induced an age-dependent increase in myocardial CTGF expression, which was 3.5-fold greater compared to normotensive Sprague Dawley (SD) rats. CTGF overexpression correlated closely with the Ang II-induced rise in blood pressure. CTGF mRNA and protein were located predominantly in areas with leukocyte infiltration, myocardial, and vascular lesions and co-localized with TGFbeta(1), collagen I, and collagen III mRNA expressions. Ang II induced CTGF mRNA and protein to a lesser extent in the kidneys, predominantly in glomeruli, arterioles, and in the interstitium with ample inflammation. However, no expression was found in the right ventricle or pulmonary arteries. Blockade of calcineurin activity by cyclosporine A completely normalized Ang II-induced CTGF overexpression in heart and kidney, suppressed the inflammatory response, and mitigated Ang II-induced cell proliferation and apoptosis. In contrast, blockade of mTOR (target of rapamycin) pathway by everolimus, further increased the expression of CTGF even though everolimus ameliorated cell proliferation and T-cell-mediated inflammation. Our findings provide evidence that CTGF mediates Ang II-induced fibrosis in the heart and kidneys via blood pressure and calcineurin-dependent pathways.

Figure 1.

Systolic blood pressure (SBP, A), cardiac hypertrophy (CW/BW, B), 24-hour albuminuria (dU-Alb, C), and myocardial CTGF mRNA expression (D) in SD and dTGR in postnatal week 4. dTGR had higher blood pressure, cardiac weight-to-body weight ratio, and urinary albumin excretion rate compared to SD rats. Myocardial CTGF mRNA expression was similar in SD and dTGR. CTGF mRNA expression was measured by real-time RT-PCR and calculated in relation to GAPDH mRNA expression of the corresponding sample. Values are mean ± SEM. *, P < 0.05 vs. SD, n = 6 to 10.

Figure 2.

Systolic blood pressure (SBP, A), 24-hour albuminuria (dU-Alb, B), and CTGF gene expressions in the heart and kidney (C) in dTGR during postnatal weeks 4 to 7. CTGF mRNA expression was measured by Northern blot and calculated in relation to GAPDH mRNA expression of the corresponding sample. There was a age-dependent increase in blood pressure, albuminuria, and CTGF mRNA expression in dTGR. Values are mean ± SEM, n = 4 to 10 in each group.

Figure 3.

Representative photomicrographs of the CTGF mRNA and protein expressions in the kidney and heart of dTGR and SD rats. Faint CTGF mRNA expression was found in the heart (A) and kidney (C) of SD rat, whereas in dTGR, myocardial (B), and renal (D) CTGF mRNA expressions were intense. In situ hybridization with hematoxyline counterstaining. CTGF mRNA-positive label can be seen especially in the vascular media and in areas with inflammatory response (magnification, ×200). E and F show the CTGF protein expression in the kidney and heart of dTGR, respectively. Immunopositive label locates mainly in the vasculature. Hematoxyline counterstaining (magnification, ×400).

Figure 4.

Effects of CsA and SDZ RAD treatments on CTGF gene expression in the kidney (A) and heart (B), on cell proliferation (C) and apoptosis (D) in dTGR. Cell proliferation in the kidney was assessed by anti-PCNA immunohistochemistry. Values represent the percentage of positive signal per total surface area. The number of apoptotic cells (excluding leukocytes) was counted in 10 randomly selected high-power fields per sample (n = 4 to 6). CsA prevented Ang II-induced increases in renal and myocardial CTGF mRNA expressions, cell proliferation, and apoptosis. Values are mean ± SEM. *, P < 0.05 compared to SD controls; ^†, P < 0.05 compared to dTGR; ^‡, P < 0.05 compared to CsA-treated dTGR.

Figure 5.

CTGF mRNA expression in the pulmonary artery and right ventricle of dTGR and SD rats. There was only a faint CTGF mRNA expression in the pulmonary arteries of SD rats (A) and dTGR (B). CTGF mRNA in situ hybridization (magnification, ×400). No significant difference in CTGF mRNA expression was found in the right ventricle of SD and dTGR rats (C).