PS-B01-7: VASCULAR SMOOTH MUSCLE CELL NOX5 REGULATES FIBROBLAST PHENOTYPIC SWITCH AND CARDIOVASCULAR FIBROSIS

Abstract

Objective: We previously observed that mice expressing human Nox5 (hNox5) in VSMC exhibit vascular dysfunction and cardio-renal fibrosis, where mechanisms are unknown. Here we postulated that VSMC-Nox5 promotes fibroblast phenotypic changes leading to myofibroblast differentiation and pro-fibrotic responses. Design and Methods: Fibroblasts were cultured from wildtype (WT) and hNOX5 mice (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. Results: Fibroblasts from Nox5 + SM22 + mice exhibited increased mRNA of pro-fibrotic markers, such as Col1A1 (2-ddC:1.74 ± 0.16 vs. WT 0.67 ± 0.11), Col3A1 (2-ddC:1.74 ± 0.18 vs. WT 0.96 ± 0.24) and TIMP3 (2-ddC:2.65 ± 0.25 vs. WT 0.38 ± 0.07), p < 0.05. Myofibroblast phenotype (SMC) markers mRNA, aSMA (2-ddC:1.54 ± 0.05 vs. WT 0.78 ± 0.17) and Myocd (2-ddC:1.36 ± 0.17 vs. WT 0.39 ± 0.22) were also increased, p < 0.05. mRNA expression of CD36 (2-ddC:1.37 ± 0.07 vs. WT 86 ± 0.24), TNFa (2-ddC:1.32 ± 0.2 vs. WT 0.71 ± 0.17) and TNFR1 (2-ddC:1.26 ± 0.04 vs. WT 1.02 ± 0.10) were increased, while CD68 expression was decreased (2-ddC:0.82 ± 0.11 vs. WT 1.36 ± 0.18) in fibroblasts from Nox5+SM22+ mice (p < 0.05). In Nox5 mice fibroblasts, ROS production and TGFb protein expression (AU:1.8 ± 0.05 vs. WT 1.4 ± 0.06), as well as TGFbR2 gene expression (2-ddC: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). Gene expression of DNMT3a and TET2, DNA methylation regulatory enzymes, were also increased in fibroblasts from Nox5+SM22+ mice, p < 0.05. Conclusion: VSMC-Nox5 regulates fibroblast phenotypic switch leading to fibrosis; a process that may involve increased production of ROS and TGFb, and activation of DNA methylation pathways.