Abstract 127: Nox5 Expression In A Vascular Smooth Muscle Cell-specific Manner Induces Fibroblast To Myofibroblast Differentiation

Abstract

Mice expressing human Nox5 (hNox5) in VSMC exhibit cardfiovascular fibrosis, where mechanisms are unknown. We postulated that VSMC-Nox5 promotes fibroblast to myofibroblast differentiation that contributes to fibrosis. Fibroblasts were cultured from wildtype (WT) and hNOX5 mice. Mice (20 weeks old) were infused with Ang II (600 ng/Kg/day) for 28 days and renal fibrosis/inflammation studied. Markers of myofibroblasts (αSMA), and pro-fibrotic and inflammatory signaling molecules were assessed by qPCR and immunoblotting. Inflammatory infiltrate was assessed by FACS. Fibroblasts from Nox5 mice exhibited increased mRNA of markers of myofibroblast αSMA (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), as well as, pro-fibrotic markers, 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. mRNA expression of CD36 (2 -ddC :1.37±0.07 vs. WT 86±0.24), TNFα (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 mice (p<0.05). In Nox5 mice fibroblasts, ROS production and TGFβ protein expression (AU:1.8±0.05 vs. WT 1.4±0.06), as well as TGFβR2 gene expression (2 -ddC :2.04±0.17 vs. WT 0.57±0.12), were increased (p<0.05). mRNA of DNMT3a and TET2, DNA methylation regulatory enzymes, were also increased in fibroblasts from Nox5 mice, p<0.05. Kidneys from Ang II-infused Nox5 mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT, as well as increased protein expression of TGFβ (AU: 3.59±0.8 vs. WT 1.54±0.2) and IL-11 (AU: 0.64±0.08 vs. WT 0.39±0.04), p<0.05; where levels of macrophage F4/80+ cells (%:24±2 vs WT 18±1, p<0.05) and levels of cytotoxic CD8+ T cells were increased, p<0.05. Kidney expression of vimentin (AU:1.01±0.05 vs WT 0.85±0.03) and αSMA (AU:0.44±0.03 vs WT 0.33±0.01), were increased in Nox5 mice (p<0.05). Nox5 regulates fibrosis by inducing fibroblast-to-myofibroblast differentiation, possibly through increased ROS. These processes may be important in Nox5-assocated cardiovascular-renal fibrosis.