Intermittent hypoxia induces fibroblast activation: An in vitro approach in NIH/3T3 cells

Obstructive sleep apnea (OSA) is recognized as an independent cardiovascular (CV) risk factor. Intermittent hypoxia (IH), the landmark feature of OSA, represents the major detrimental factor triggering CV complications. In rodent models, IH contributes to cardiac remodeling, mainly characterized by early interstitial fibrosis. However, the direct effect of IH on cardiac fibrosis, as well as involved mechanisms are unclear. The aim of this study is to investigate in vitro whether IH induces fibroblast activation. NIH/3T3 murine fibroblasts were exposed to normoxic condition (N) or fast IH cycles, including 5-min normoxia (16% O2) followed by 5-min hypoxia (2% O2), in the presence or not of 10 ng/ml transforming growth factor (Tgf-∝). Cell proliferation was assessed after 48 h N or IH exposure by MTT assay; cell migration was evaluated during 12 h epxosure by scratch assay; mRNA expression of several markers of fibrosis was evaluated after 6 h exposure. Fibroblast differentiation was evaluated through α Smooth Muscle Actin (Acta2) mRNA expression and fibrosis through expression of Collagen 1 (Col1a1), lysyl oxidase (Lox), Tgf∝ and Tgf∝ receptor (Tgf-∝R). Data were analyzed by 2-ways Anova (Impact of IH and Tgf-β), followed by a Tukey post-hoc test. Our results confirm the well-known role of Tgf-∝ on fibroblast activation, characterized by increased proliferation (P = 0.004) and mRNA expression of Acta2 (P = 0.002), Col1a1 (P = 0.008) and Lox (P = 0.04). Independently of Tgf-β, IH does not increase cell proliferation, tends to increase fibroblasts migration (P = 0.1) and significantly increases mRNA expression of Acta2 (P = 0.002) and Col1a1 (P = 0.05), whereas increases in Lox and Tgf-β expression do not reach significance (P = 0.06 and P = 0.08). Interestingly, concomitant stimulation with Tgf-β and IH does not potentiate cell proliferation and migration, but synergistically increases Acta2, Col1a1 and Lox mRNA expression. Our in vitro results shows that IH induces fibroblast activation, promoting migration, differentiation into myofibroblast and expression of fibrotic markers. Furthermore, IH and Tgf-∝ seem to exert synergistic effects on fibrosis. Further studies are needed to understand specific mechanisms involved in IH-induced fibrosis.