Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Abstract

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.

Figure 1. Immunocytochemical identification of cardiac fibroblasts

Isolated cardiac cells were incubated with antibodies to α-smooth muscle actin, fibronectin, vimentin or sarcomeric myosin and visualized with FITC-coupled secondary antibodies. The control was incubated without a primary antibody, as indicated.

Figure 2. Inhibition of DNA synthesis by Ang-(1-7) in cardiac fibroblasts

Cardiac fibroblasts were serum-deprived for 24 h and subsequently treated for 48 h with increasing concentrations of Ang-(1-7), in the presence of 1% FBS, 10 nM endothelin (ET-1), or 1 μM [D-Ala⁷]-Ang-(1-7), as indicated. DNA synthesis was detected by ³H-thymidine incorporation, which was added during the last 24 h of treatment. Data are presented as the percentage of mitogen-stimulated growth, in the absence of Ang-(1-7). n = 4-6, in triplicate. *denotes p < 0.05 and **denotes p < 0.01 compared to mitogen alone.

Figure 3. Inhibition of protein and collagen synthesis by Ang-(1-7) in cardiac fibroblasts

Cardiac fibroblasts were deprived of serum for 24 h and subsequently treated for 48 h with various concentrations of Ang-(1-7), in the presence of 1% FBS or 10 nM ET-1. Protein and collagen was detected by ³H-leucine and ³H-proline incorporation, respectively, which were added during the last 24 h of the treatment period. Data are presented as the percentage of mitogen-stimulated growth, in the absence of Ang-(1-7). n = 4 – 6, in triplicate. *denotes p < 0.05 and **denotes p < 0.01 compared to mitogen alone.

Figure 4. Ang-(1-7) reduces phospho-ERK1/ERK2

Cardiac fibroblasts pre-treated with 100 nM Ang-(1-7) [A7] were stimulated with 10 nM ET-1 (Panel A) or 100 nM Ang II (Panel B) and phospho-ERK1/ERK2 were determined using a phospho-specific antibody. Representative blots show ET-1 or Ang II-stimulated phospho-ERK1/ERK2 and actin immunoreactivity in the presence or absence of Ang-(1-7). Graphs show percent control of ET-1 or Ang II phospho-ERK1 and ERK2. n = cells isolated from 2 to 4 litters of neonatal pups, in duplicate or triplicate. * p <0.05 compared with Ang II alone; **denotes p < 0.01 compared with ET-1 treatment alone.

Figure 5. Ang-(1-7) up-regulates DUSP-1 in neonatal cardiac fibroblasts

Cardiac fibroblasts in serum-free media were treated with either 100 nM Ang-(1-7) or ET-1 (10 nM) and incubated for 6 h. A) DUSP1 mRNA isolated from cardiac fibroblasts was analyzed by RT real-time PCR. n= cells isolated from 4 litters of neonatal rat pups. * denotes p < 0.05, ** denotes p < 0.005, *** denotes p < 0.001 compared to treatment with ET-1. B) A 40-kDa immunoreactive protein was visualized by Western blot hybridization in cultured cardiac fibroblasts using a specific antibody to DUSP1 and quantified by densitometry, with β-actin as a loading control. n = cells isolated from 3 litters of neonatal rat pups. * denotes p < 0.05 compared to control (Con), in serum-free media alone.

Figure 6. Ang-(1-7) reduces COX-2 and PGES gene expression in cardiac fibroblasts

Cardiac fibroblasts were pre-treated with 100 nM Ang-(1-7) for 4 h and incubated with or without 10 nM ET-1 for increasing periods of time, as indicated. COX-2 mRNA (in Panel A) or PGES mRNA (in Panel B) were measured by RT real-time PCR. n = 5. For COX-2 mRNA in Panel A, ** denotes p < 0.005 compared to Ang-(1-7) or Ang-(1-7) and ET-1 treatment; for PGES mRNA in Panel B, * denotes p < 0.05 compared to Ang-(1-7) or Ang-(1-7) and ET-1 treatment.