NOX5 EXPRESSION IN A VASCULAR SMOOTH MUSCLE CELL-SPECIFIC MANNER INDUCES FIBROBLAST PHENOTYPIC SWITCH AND PRO-FIBROTIC EFFECTS

Abstract

Objective: Mice expressing human Nox5 (hNox5) in vascular smooth muscle cells (VSMC) exhibit redox-sensitive vascular dysfunction and cardio-renal fibrosis. Mechanisms underlying pro-fibrotic effects are unclear. Here we assessed whether hNox5 in VSMCs promotes fibroblast phenotypic changes leading to myofibroblast differentiation and pro-fibrotic/inflammatory responses. Design and method: Fibroblasts were cultured from wildtype (WT) and mice expressing hNOX5 in VSMC (Nox5+SM22+). Mice (20 weeks old) were infused with Ang II (600 ng/Kg/day) for 28 days and renal fibrosis/inflammation studied. Markers of myofibroblasts, pro-fibrotic and inflammatory phenotypes were assessed by qPCR and immunoblotting. Inflammatory infiltrate was assessed by FACS. Results: Fibroblasts isolated from Nox5+SM22+ mice exhibited increased mRNA expression of pro-fibrotic markers, such as Col1A1 (2-ddCt: 1.74 ± 0.16 vs. WT 0.67 ± 0.11), Col3A1 (2-ddCt: 1.74 ± 0.18 vs. WT 0.96 ± 0.24) and TIMP3 (2-ddCt: 2.65 ± 0.25 vs. WT 0.38 ± 0.07), p < 0.05. Myofibroblast phenotype (SMC) markers mRNA, aSMA (2-ddCt: 1.54 ± 0.05 vs. WT 0.78 ± 0.17) and Myocd (2-ddCt: 1.36 ± 0.17 vs. WT 0.39 ± 0.22) were also increased in fibroblasts from Nox5 mice, p < 0.05. In terms of inflammatory phenotype, we observed that mRNA expression of CD36 (2-ddCt: 1.37 ± 0.07 vs. WT 86 ± 0.24), TNFa (2-ddCt: 1.32 ± 0.2 vs. WT 0.71 ± 0.17) and TNFR1 (2-ddCt: 1.26 ± 0.04 vs. WT 1.02 ± 0.10) were increased, while CD68 expression was decreased (2-ddCt: 0.82 ± 0.11 vs. WT 1.36 ± 0.18) in fibroblasts from Nox5+SM22+ mice (p < 0.05). In Nox5 mice fibroblasts, key regulators of fibrosis such as ROS production and TGFb protein expression (AU: 1.8 ± 0.05 vs. WT 1.4 ± 0.06), as well as TGFbR2 gene expression (2-ddCt: 2.04 ± 0.17 vs. WT 0.57 ± 0.12), were increased (p < 0.05). Kidneys from Ang II-infused Nox5+SM22+ mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT. Kidney expression of vimentin (AU: 1.01 ± 0.05 vs WT 0.85 ± 0.03) and aSMA (AU: 0.44 ± 0.03 vs WT 0.33 ± 0.01), markers of myofibroblast differentiation, were increased in Nox5 mice (p < 0.05). In kidneys from Nox5+SM22+ mice, increased frequencies of macrophage F4/80 + (%: 24 ± 2 vs WT 18 ± 1, p < 0.05). Conclusions: Our findings suggest that Nox5 in VSMCs influences fibroblast phenotypic switching to myofibroblasts that likely influences cardiovascular and renal fibrosis in hypertension.