Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-β has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-β would have undesired systemic side effects due to the multiple physiological functions of TGF-β. Further characterization of the downstream signaling pathway(s) involved in TGF-β-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-β-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-β/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders.

Both reactive oxygen species (ROS) and Forkhead box O (FOXO) family transcription factors are involved in the regulation of adipogenic differentiation of preadipocytes and stem cells. While FOXO has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXO during adipogenesis are not clear. Here we examined how ROS and FOXO regulate adipogenesis in human adipose-derived stem cells (hASC). The identity of isolated cells was confirmed by their surface marker expression pattern typical for human mesenchymal stem cells (positive for CD29, CD44, CD73, CD90, and CD105, negative for CD45 and CD31). Using a standard adipogenic cocktail consisting of insulin, dexamethasone, indomethacin, and 3-Isobutyl-1-methylanxthine (IDII), adipogenesis was induced in hASC, which was accompanied by ROS generation. Scavenging ROS production with N-acetyl-L-cysteine or EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase, inhibited IDII-induced adipogenesis. We then mimicked IDII-induced oxidative stress through a lentiviral overexpression of Nox4 and an exogenous application of hydrogen peroxide in hASC and both manipulations significantly enhanced adipogenesis without changing the adipogenic differentiation rate. These data suggest that ROS promoted lipid accumulation in hASC undergoing adipogenesis. Antioxidant enzymes, including SOD2, catalase, and glutathione peroxidase were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing, which also suppressed significantly IDII-induced adipogenesis. Our findings demonstrated a balance of ROS generation and endogenous antioxidants in cells undergoing adipogenesis. Approaches targeting ROS and/or FOXO1 in adipocytes may bring new strategies to prevent and treat obesity and metabolic syndrome.

The mechanism of the relaxant action and the structure-activity relation of flavonols (fisetin, quercetin, and 3,3',4'-trihydroxyflavone) and flavones (apigenin, chrysin, and luteolin) were examined in rat isolated thoracic aorta. The control responses to flavonols and flavones were compared with responses observed after the removal of the endothelium or in the presence of the L-type Ca2+ channel blocker, nifedipine (10(-7) M). The effects of flavonoids on contraction caused by the influx of extracellular Ca2+ and agonist-induced release of intracellular Ca2+ also were investigated. The flavones exhibited endothelium-independent vasorelaxation, whereas the removal of the endothelium significantly decreased the sensitivity of the relaxant responses to the flavonols without affecting the maximal relaxation. In the presence of nifedipine, the responses to apigenin, luteolin, and quercetin were significantly inhibited, but relaxation to chrysin, fisetin, and 3,3',4'-trihydroxyflavone was unaffected. All flavonols and flavones caused concentration-dependent inhibition of the contractile responses to exogenous application of Ca2+ and the release of intracellular Ca2+ stimulated by phenylephrine. Of the six flavonoids examined, 3,3',4'-trihydroxyflavone was the most potent when causing vasorelaxation or inhibition of contraction caused by the influx or release of Ca2+. In conclusion, these studies provide evidence that the hydroxyl substitution in the carbon 3 position that characterizes the flavonols is important in stimulating endothelium-dependent vasorelaxation, and the absence of hydroxyl substitution on the A phenolic ring enhances the relaxant action.