The first suggestion that the Hh pathway might

The first suggestion that the Hh pathway might be involved in the pathogenesis of chronic liver disease was reported at the beginning of this millennium by Shackel et al. who noted that Patch and Gli were upregulated in their microarray analysis of liver tissues from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Since that initial description, the Hh pathway has consistently been shown to be aberrantly activated in different human cholangiopathies and animal models of these diseases. For example, immunohistochemistry studies have demonstrated increased Hh pathway activity in human PBC and PSC relative to healthy age-matched controls,[68], [112], [113] with the main sources of Hh ligands being ductular g 15 in PBC and peribiliary gland progenitor cells in large bile ducts of patients with PSC.[112], [113] Progenitor cells and myofibroblasts are the main Hh-responsive cell types in both diseases.[112], [113] Importantly, the level of Hh activation strongly correlated with the degree of liver fibrosis in both PBC and PSC. Furthermore, genome wide association studies showed that genetic variants in the Hh pathway confer susceptibility to PBC. In paediatric cholestatic diseases such as biliary atresia, Alagille’s syndrome and progressive familial intrahepatic cholestasis, there is known activation of the Hh pathway. Further, high levels of Hh pathway activity correlate with dismal prognosis.[79], [115] In paediatric cholangiopathies, the Hh pathway not only promotes fibrogenesis, but also induces epithelial-to-mesenchymal transition in biliary progenitors, which arrests them in an immature phenotype and abrogates ductular morphogenesis.[51], [79] Studies in animal models of cholangiopathies not only replicated the evidence for excessive activation of the Hh pathway noted in human diseases, but demonstrated a causal association. For example, a zebrafish model of biliary atresia, caused by genetic deletion of glypican-1, could be reverted to a normal phenotype by administering an Hh inhibitor and replicated in wild-type zebrafish embryos simply by administering recombinant Shh. Rodent models of chronic bile duct ligation provide proof-of-concept that Hh signalling is also critical in the pathogenesis of cholangiopathies that are induced in adulthood. Bile duct ligation (BDL) in rats induces an exuberant fibroductular response that reproducibly culminates in biliary cirrhosis with accompanying high levels of Hh ligand production, profound downregulation of the Hh inhibitor Hhip, and striking nuclear accumulation of Gli, a Hh-regulated transcription factor. This liver phenotype can be reverted by subjecting BDL rats to Roux-en-Y hepaticojejunostomy, which decompresses the obstructed biliary tree. Such biliary diversion progressively silences the Hh pathway, causes regression of biliary fibrosis, and ultimately normalises the hepatic architecture.[48], [49], [51] Inhibiting the Hh pathway, either pharmacologically or through conditional disruption of the Smo gene abrogates liver injury and fibrosis in BDL mice.[53], [116] The reverse is also true, that is, genetically modified mice with an overactive Hh pathway demonstrate a more exuberant fibroductular response to BDL. Extensive data also demonstrate a role for the Hh pathway in the pathogenesis of alcoholic and non-alcoholic fatty liver disease. Studies of patients with alcoholic/non-alcoholic fatty liver disease consistently show that the level of Hh pathway activation correlates with the severity of liver cell injury/death, hepatic inflammation, liver fibrosis, and the risk of worse liver-related morbidity and mortality.[50], [64], [66], [68], [83], [117] Various animal models of non-alcoholic fatty liver disease (NAFLD) have confirmed that the level of Hh pathway activity increases in parallel with the severity of steatohepatitis and liver fibrosis.[47], [64], [118], [119], [120] In contrast, the Hh pathway seems to protect the liver from steatosis in several preclinical NAFLD models. For example, obese ob/ob mice develop massive steatosis with age but are relatively protected from steatohepatitis and progressive liver fibrosis. This phenotype results from monogenic deficiency of leptin; leptin has been proven to stimulate Hh signalling; and Hh signalling is severely reduced in ob/ob mice. Interestingly, a recent report indicates that the prevalence of NAFLD is higher in patients with germline mutations disrupting Smo than in the general population. This enrichment appears to be independent of obesity and seems to be driven predominately by the fact that reduced Smo function associates with hepatic steatosis. Indeed, expression of proinflammatory and profibrotic genes is generally low in such patients. Similarly, genetically modified mice with reduced Hh pathway activity caused by global haploinsufficiency of Gli-2 develop more steatosis but less inflammation than wild-type mice, when exposed to steatogenic diets. These findings in the livers of humans and mice with genetic inhibition of the Hh pathway are consistent with earlier publications proving that genetic activation of the Hh pathway directly inhibits lipogenesis in flies and mammals,[22], [44] as well as more recent papers showing that targeted genetic/pharmacological activation of Hh signalling reverses steatosis in fatty hepatocytes harvested from obesity-related mice models of NAFLD. Evidence linking reduced Hh activity with hepatic steatosis, and excessive Hh signalling with steatohepatitis and fibrosis, continues to accumulate as results of Hh pathway manipulation in additional animal models of NAFLD are published.[53], [57], [64], [118], [122], [123], [124]