Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells.

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue. Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL. Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels. In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase.

Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.