There has been some debate

There has been some debate about which types of liver cells might be able to activate canonical Hh signalling during liver injury. These uncertainties reflect technical challenges imposed by imperfect and inconsistent reagent specificity and the nature of the signalling process itself, which is quite dynamic and regulated at multiple levels. These challenges are compounded by the fact that PC are thought be necessary for cells to activate canonical Hh signalling in response to Hh ligand exposure. Cells possess a single 0.25 µm diameter PC and this structure forms and regresses as cells exit and enter the cell cycle,[59], [60] making it quite difficult to visualise PC on any given cell in intact liver tissue, even with the best available approach (i.e. confocal microscopy with Lonafarnib to acetylated tubulin). This task is particularly daunting during the various phases of an active wound healing response. Indeed, it is conceivable that regeneration-related changes in PC contribute to the striking differences in Hh pathway activity in healthy and diseased livers. Current dogma posits that all healthy adult hepatocytes are devoid of PC, and hence cannot activate the canonical Hh pathway. This assumption merits renewed scrutiny in light of the aforementioned evidence suggesting that Hh signalling regulates hepatocyte metabolism during health.[42], [63] In addition, other reports in patients and animal models with liver disease have demonstrated Gli2 nuclear staining in periportal hepatocytes.[47], [62], [64], [65], [66], [67], [68] This suggests that some hepatocytes may be able to acquire PC and become responsive to Hh, since the activation of the transcription factor Gli2 is the result of the Hh pathway. On the other hand, Gli2 activation in such cells could be Hh- and Smo-independent, since Gli2 can be activated by other signalling pathways such as TGF-β (which increases after fibrogenic insults) and forkhead box C1 (which is upregulated in hepatocellular carcinoma). It is also possible that hepatocytes exhibit Hh-dependent, PC-independent, Smo-dependent or Smo-independent activation of Gli.[71], [72] In any case, several groups have demonstrated Smo activity in hepatocytes.[65], [72] Similar to healthy mature hepatocytes, liver sinusoidal endothelial cells are not thought to express PC in general. However, endothelial cells are known to become ciliated when exposed to increased hydrostatic pressure.[73], [74] The resultant Hh pathway activation induces a vasoconstrictive phenotype and loss of fenestration in liver sinusoidal endothelial cells, promoting angiogenesis, capillarisation and vascular remodelling that contributes to portal hypertension.[55], [56], [75], [76] In contrast to hepatocytes and liver sinusoidal endothelial cells, cholangiocytes and progenitor cells in healthy livers seem to express PC fairly consistently.[62], [77] Thus, heritable ciliopathies which alter Hedgehog signalling along PC (e.g. Bardet Biedl syndrome, adult polycystic kidney disease, Caroli’s disease) exhibit an aberrant biliary phenotype. Hh activation in bipotent liver epithelial progenitors induces proliferation, inhibits apoptosis and blocks differentiation along the mature biliary lineage.[38], [40], [62] Such dysregulated Hh signalling has been implicated in the pathogenesis of “acquired” cholangiopathies, including biliary atresia[79], [80], [81] and the ductular reaction that develops during many types of liver injury.[49], [51], [82] The ductular reaction is believed to reflect accumulation of immature liver epithelial cells. Hh pathway activation in liver progenitors expands the pool of cells available to replace epithelial cells that are dying after an acute or chronic insult. However, full recuperation of liver-specific functions requires precise modulation of Hh signalling, since complete differentiation of progenitors into mature liver epithelial cells seems to require repression of pathway activity.[38], [39] Like cholangiocytes and progenitors, some HSCs in healthy adult livers also appear to have PC. This conclusion is based on evidence that HSC in healthy livers are marked by both Patch- and Gli-reporter activity in transgenic mice,[84], [85], [86] similar to tissue-resident perivascular mesenchymal stem cell-like cells in multiple other tissues. Hh activation appears to promote differentiation of such cells into proliferative myofibroblasts.[84], [88], [89], [90] The mechanisms mediating this transdifferentiation have been delineated in HSC. They involve an epithelial-to-mesenchymal transition-like process whereby the cells transiently repress their expression of genes that favour a more quiescent, epithelial phenotype while inducing various factors that promote the transcription of mesenchymal genes and enhance the stability/translation of the respective mRNAs to promote proliferation, survival, migration, contractility, and angiogenesis.[91], [92], [93], [94] Interestingly, although ultrastructural studies of HSC and studies with confocal microscopy described PC in only 2.5% of HSC of healthy livers,[59], [68], [95] in vitro studies show that HSCs are highly responsive to both Hh ligand and antibodies that neutralise Hh.[84], [90] HSC also appear to be responsive to Hh ligands in vivo as indicated by evidence that the pool of hepatic myofibroblasts expands in parallel with Hh ligand accumulation following various liver insults, whereas during recovery, a decline in Hh associates with involution of that pool of cells. Further, in animal models, neutralising antibodies to Hh ligands promote myofibroblast inactivation, apoptosis and senescence. Treatments that reduced injury-related production of Hh ligands by hepatocytes also caused regression of myofibroblast populations in patients. HSCs are highly responsive to direct antagonists and agonists of Smo in vivo and in vitro,[84], [91] and direct manipulation of Gli factors also regulates HSC fate. Given this, it is conceivable that various canonical (i.e. PC-dependent) and non-canonical (i.e. PC-independent) pathways interact to modulate Hh signalling in HSC.