In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis.

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.

Galectin-3 regulates hepatic progenitor cell expansion during liver injury.

OBJECTIVE: Following chronic liver injury or when hepatocyte proliferation is impaired, ductular reactions containing hepatic progenitor cells (HPCs) appear in the periportal regions and can regenerate the liver parenchyma. HPCs exist in a niche composed of myofibroblasts, macrophages and laminin matrix. Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that binds to laminin and is expressed in injured liver in mice and humans. DESIGN: We examined the role of Gal-3 in HPC activation. HPC activation was studied following dietary induced hepatocellular (choline-deficient ethionine-supplemented diet) and biliary (3,5-diethoxycarbonyl-1,4-dihydrocollidine supplemented diet) injury in wild type and Gal-3(-/-) mice. RESULTS: HPC proliferation was significantly reduced in Gal-3(-/-) mice. Gal-3(-/-) mice failed to form a HPC niche, with reduced laminin formation. HPCs isolated from wild type mice secrete Gal-3 which enhanced adhesion and proliferation of HPCs on laminin in an undifferentiated form. These effects were attenuated in Gal3(-/-) HPCs and in wild type HPCs treated with the Gal-3 inhibitor lactose. Gal-3(-/-) HPCs in vitro showed increased hepatocyte function and prematurely upregulated both biliary and hepatocyte differentiation markers and regulated cell cycle genes leading to arrest in G0/G1. CONCLUSIONS: We conclude that Gal-3 is required for the undifferentiated expansion of HPCs in their niche in injured liver.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of ß3, ß5 or ß6 integrins or conditional loss of ß8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Metabolic pathways promoting intrahepatic fatty acid accumulation in methionine and choline deficiency: implications for the pathogenesis of steatohepatitis.

The pathological mechanisms that distinguish simple steatosis from steatohepatitis (or NASH, with consequent risk of cirrhosis and hepatocellular cancer) remain incompletely defined. Whereas both a methionine- and choline-deficient diet (MCDD) and a choline-deficient diet (CDD) lead to hepatic triglyceride accumulation, MCDD alone is associated with hepatic insulin resistance and inflammation (steatohepatitis). We used metabolic tracer techniques, including stable isotope ([¹³C4]palmitate) dilution and mass isotopomer distribution analysis (MIDA) of [¹³C2]acetate, to define differences in intrahepatic fatty acid metabolism that could explain the contrasting effect of MCDD and CDD on NASH in C57Bl6 mice. Compared with control-supplemented (CS) diet, liver triglyceride pool sizes were similarly elevated in CDD and MCDD groups (24.37 ± 2.4, 45.94 ± 3.9, and 43.30 ± 3.5 µmol/liver for CS, CDD, and MCDD, respectively), but intrahepatic neutrophil infiltration and plasma alanine aminotransferase (31 ± 3, 48 ± 4, 231 ± 79 U/l, P < 0.05) were elevated only in MCDD mice. However, despite loss of peripheral fat in MCDD mice, neither the rate of appearance of palmitate (27.2 ± 3.5, 26.3 ± 2.3, and 28.3 ± 3.5 µmol·kg⁻¹·min⁻¹) nor the contribution of circulating fatty acids to the liver triglyceride pool differed between groups. Unlike CDD, MCDD had a defect in hepatic triglyceride export that was confirmed using intravenous tyloxapol (142 ± 21, 122 ± 15, and 80 ± 7 mg·kg⁻¹·h⁻¹, P < 0.05). Moreover, hepatic de novo lipogenesis was significantly elevated in the MCDD group only (1.4 ± 0.3, 2.3 ± 0.4, and 3.4 ± 0.4 µmol/day, P < 0.01). These findings suggest that important alterations in hepatic fatty acid metabolism may promote the development of steatohepatitis. Similar mechanisms may predispose to hepatocyte damage in human NASH.

Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response.

BACKGROUND AND METHODS: In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. HPCs are located within a designated niche in close association with myofibroblasts and bone marrow (BM) derived macrophages. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Using the collagen Iα1((r/r)) mouse (r/r), which produces mutated collagen I resistant to matrix metalloproteinase degradation and has an exaggerated fibrotic response to liver injury, we test the relationship between collagen degradation, laminin deposition, and the HPC response. RESULTS: Chronic fibrotic carbon tetrachloride (CCl4) injury can induce a florid HPC response associated with dense laminin deposition. In the recovery phase after chronic CCl4 injury, r/r mice have a markedly attenuated HPC response compared to wild-types, together with persistence of collagen I and failure to deposit ECM laminin. Similar results were found in r/r mice given the choline-deficient ethionine supplemented diet, another model of the HPC response. In cross-over sex-mismatched BM transplantation (BMT) experiments between r/r mice and wild-types, the blunted HPC response of r/r mice was not rescued by wild-type BMT and likewise not conferred on to wild-type recipients by r/r BMT, demonstrating that the attenuated HPC response in r/r mice is a property intrinsic to the liver. CONCLUSION: Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.

Targeted treatments for cirrhosis.

The causes of hepatic scarring (fibrosis) are protean but, unchecked, all result in a common fate--the development of cirrhosis--with gross disruption of the normal liver architecture. Subsequent liver cell dysfunction and portal hypertension give rise to major systemic complications and premature death. Cirrhosis and its sequelae represent a huge, and global, healthcare burden. The success of liver transplantation and the development of efficacious antiviral regimens for hepatitis B and C should not be underestimated, but they also serve to highlight our current inability to manipulate the underlying fibrotic process in many patients with liver disease. Moreover, transplantation as a treatment is limited by organ availability, among other factors. The development of antifibrotic therapies is urgently needed and for this we require a mechanistic and evidence-based approach. Accumulating data from clinical and laboratory studies demonstrate that even advanced fibrosis and cirrhosis are potentially reversible. The hepatic stellate cells have been identified as the pivotal effector cells orchestrating the fibrotic process and, furthermore, reversibility appears to hinge upon their elimination. This review draws on recent scientific advances, and highlights emerging therapeutic interventions in liver fibrosis.