Marco A. Briones-Orta

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

INTRODUCTION Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Iron enhances hepatic fibrogenesis and activates TGF-β signaling in murine hepatic stellate cells

Background: Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). Materials and Methods: We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF‐&bgr;) signaling in murine HSC. Cells were treated with holotransferrin (0.005‐5 g/L) for 24 hours, with or without the iron chelator deferoxamine (10 &mgr;M). Gene expressions (&agr;‐SMA, Col1‐&agr;1, Serpine‐1, TGF‐&bgr;, Hif1‐&agr;, Tfrc and Slc40a1) were analyzed by quantitative real time‐polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF‐&bgr; RII and phospho‐Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme‐linked immunosorbent assay. Results: HSC expressed the iron‐uptake protein transferrin receptor 1 (TfR1) and the iron‐export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8‐fold (P < 0.03) and ferritin accumulation (iron storage) by 2‐fold (P < 0.01), and activated HSC with 2‐fold elevations (P < 0.03) in &agr;‐SMA messenger RNA and collagen secretion, and a 1.6‐fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF‐&bgr; pathway with TGF‐&bgr; messenger RNA elevated 1.6‐fold (P = 0.05), and protein levels of TGF‐&bgr; RII and phospho‐Smad2 increased by 1.8‐fold (P < 0.01) and 1.6‐fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes &agr;‐SMA (0.2‐fold; P < 0.05) and TGF‐&bgr; (0.4‐fold; P < 0.01) and reduced levels of TGF‐&bgr; RII and phospho‐Smad2 proteins. Conclusions: HSC express iron‐transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF‐&bgr; pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.