5 To clarify the relationship
between Hh pathway activation and OPN expression, day 4 culture-activated MF-HSCs were treated with cyclopamine to selectively inhibit Hh signaling. Cultures selleck kinase inhibitor were harvested 24 hours later, RNA was isolated, gene expression was assessed by way of QRT-PCR, and results were compared with parallel cultures that had been treated with tomatidine, an inactive cyclopamine analog. Inhibiting Hh signaling with cyclopamine attenuated induction of OPN gene expression (Fig. 4B; Supporting Information Fig. 4B). Conversely, addition of the Hh agonist SAG augmented OPN gene expression significantly (Fig. 4C). Therefore, endogenous OPN gene expression in MF-HSCs is regulated, at least in part, by the Hh pathway. To determine the relative importance of OPN as a downstream target of the Hh pathway in HSCs, day 4 primary MF-HSCs from Ptc+/− mice were incubated with OPN aptamers for 48 hours. At baseline, HSCs from Ptc+/− mice expressed
three-fold more gli2 mRNA than WT HSCs, confirming that Ptc deficiency enhances Hh signaling (Fig. 5A). Consistent with our CYC202 in vivo findings (Fig. 1), Ptc+/− HSCs also expressed more OPN mRNA than WT HSCs (Fig. 5B). Neutralizing OPN significantly reduced collagen and αSMA mRNA levels in Ptc-deficient HSCs (Fig. 5C,D) but had little effect on gli2 mRNA expression (Fig. 5E). These findings suggest that the Hh pathway mediates induction of certain fibrogenic genes indirectly through up-regulation of the Hh-responsive gene OPN. Because the mouse model of MCD diet–induced NASH 上海皓元医药股份有限公司 differs from human NASH in several aspects,30 it was important to determine whether OPN overexpression occurred in human patients with NAFLD. Coded liver sections from 11 patients with well-characterized NAFL (n = 3), NASH (n = 3), and NASH-related
cirrhosis (n = 5) were stained to demonstrate OPN and analyzed using computer-assisted morphometry. Expression of OPN was lowest in patients with NAFL and highest in patients with NASH-related cirrhosis (Fig. 6A,B). To further validate the association between NAFLD-related fibrosis stage and OPN expression, total liver RNA was isolated from a separate cohort of 36 patients with early (stage F0-F1) or advanced (stage F3-F4) NASH-related fibrosis (n = 18/group) and analyzed by way of QRT-PCR. In livers with advanced fibrosis, OPN gene expression was double that of livers with early fibrosis (Fig. 6C). Additional analysis was conducted using RNA and protein harvested from explanted livers with NASH-related cirrhosis and residual tissue from nondiseased donor livers (n = 6/disease). Livers with NASH-related cirrhosis contained over 10 times more OPN protein (Fig. 6D; Supporting Information Fig. 4) and 5 times more OPN mRNA compared with nondiseased control livers (Fig. 7A).