Abstract 693: Nox4 Mediates Cardiac Fibrosis while p47phox-based Nox2 is Required for the Inflammatory Response to Angiotensin II in Murine Cardiac Fibroblasts
Reactive oxygen species (ROS) mediate cardiac fibrosis induced by angiotensin II (Ang II) and NADPH oxidases (Nox) are a major source of ROS. In vitro, activation of Nox2 by Ang II requires the cytosolic subunit p47phox, while Nox4 does not. We recently demonstrated that deletion of p47phox leads to enhanced cardiac fibrosis in response to Ang II. In the current study, we found that Nox2 and Nox4, but not Nox1, are abundantly expressed in mouse cardiac fibroblasts (CFb), We hypothesized that Nox4 mediates the fibrotic response to Ang II, while p47phox-dependent Nox2 is required for the pro-inflammatory action of Ang II in CFb. Primary CFb isolated from C57BL6 (control) or p47phox knockout (p47KO) mice were treated with Ang II (100 nM) for 24 hours and RNA was extracted and processed for detection of Nox4, Nox2 and cardiac fibrosis markers (collagen I, fibronectin, smooth muscle α-actin (SM-α actin)). Nox4, collagen I, fibronectin and SM-α actin were upregulated in p47phox KO fibroblasts at baseline (2.75 ± 0.04, 3.02 ± 0.5, 1.84 ± 0.05 and 1.4 ± 0.2 fold respectively, versus control C57BL6; n = 3, p < 0.05) and in response to Ang II (6.31 ± 0.02, 6.18 ± 0.28, 3.55 ± 0.34 and 3.65 ± 0.5 fold respectively, versus control C57BL6; n = 3, p < 0.05). Deletion of Nox4 with small interference RNA against Nox4 (siNox4) prevented Ang II-induced Nox4, collagen I, fibronectin and SM-α actin upregulation in both C57Bl6 (by 81%, 64%, 92%, 88% respectively, n = 3, p < 0.05 versus control siRNA) and in p47phox KO CFb (by 94%, 65%, 89% and 79% respectively, n = 3, p < 0.05 versus control siRNA), but not the upregulation of inflammatory markers (plasminogen activator inhibitor-1 (PAI-1) or interleukin 6 (IL-6)). In contrast, deletion of p47 phox blocked Ang II-induced PAI-1 and IL-6 (by 60% and 73% respectively, n = 3, p < 0.05), but not fibrosis. Furthermore, deletion of Nox4 also inhibited the hydrogen peroxide released after stimulation for 3 hours with Ang II in both C57Bl6 (by 60%, n = 3, p < 0.05 versus control siRNA) and p47phox KO mice (by 87%, n = 3, p < 0.05 versus control siRNA). In conclusion, Ang II-induced cardiac fibrosis is mediated by Nox4, while Ang II-induced inflammation is mediated by p47phox-dependent Nox2. Thus, multiple NADPH oxidases are essential for pathological responses to Ang II.