BACKGROUND & AIMS The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells. METHODS We analyzed hepatic stellate cells from C57BL/6 wild-type, Atg7F/F, and Atg7F/F-GFAP-Cre mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl4 or thioacetamide (TAA); liver tissues and stellate cells were analyzed. Autophagy was blocked in fibrogenic cells from liver and other tissues using small interfering RNAs against Atg5 or Atg7 and chemical antagonists. Human pulmonary fibroblasts were isolated from samples of lung tissue from patients with idiopathic pulmonary fibrosis or from healthy donors. RESULTS In mice, induction of liver injury with CCl4 or TAA increased levels of autophagy. We also observed features of autophagy in activated stellate cells within injured human liver tissue. Loss of autophagic function in cultured mouse stellate cells and in mice following injury reduced fibrogenesis and matrix accumulation; this effect was partially overcome by providing oleic acid as an energy substrate. Autophagy also regulated expression of fibrogenic genes in embryonic, lung, and renal fibroblasts. CONCLUSIONS Autophagy of activated stellate cells is required for hepatic fibrogenesis in mice. Selective reduction of autophagic activity in fibrogenic cells in liver and other tissues might be used to treat patients with fibrotic diseases.
Although hepatic fibrosis typically follows chronic inflammation, fibrosis will often regress after cessation of liver injury. Here we examined whether liver dendritic cells (DC) play a role in liver fibrosis regression using carbon tetrachloride (CCl4) to induce liver injury. We examined DC dynamics during fibrosis regression and their capacity to modulate liver fibrosis regression upon cessation of injury. We show that conditional DC depletion soon after discontinuation of the liver insult leads to delayed fibrosis regression and reduced clearance of activated hepatic stellate cells, the key fibrogenic cell in liver. Conversely, DC expansion induced either by Flt3L (Fms-like tyrosine kinase-3 ligand) or adoptive transfer of purified DC accelerates liver fibrosis regression. DC modulation of fibrosis was partially dependent on MMP-9, as MMP-9 inhibition abolished Flt3L-mediated effect and the ability of transferred DC to accelerate fibrosis regression. In contrast, transfer of DC from MMP-9 deficient mice failed to improve fibrosis regression. Conclusion Altogether, these results suggest that DC increase fibrosis regression, and that the effect is correlated with their production of MMP-9. These results also suggest that Flt3L treatment during fibrosis resolution merits evaluation to accelerate regression of advanced liver fibrosis.
SummarySustained progress in defining the molecular pathophysiology of hepatic fibrosis has led to a comprehensive framework for developing anti-fibrotic therapies. Indeed, the single greatest limitation in bringing new drugs to the clinical setting is lack of clarity about clinical trial and treatment endpoints, not the lack of promising agents. A range of treatments, including those developed for other indications, as well as those specifically developed for hepatic fibrosis, are nearing or in clinical trials. Most are focused on attacking features of either hepatic injury and/or activated stellate cells and myofibroblasts, which are the primary sources of extracellular matrix, or scar proteins. Thus, features of injury and stellate cell activation provides a useful template for classifying these emerging agents, and point to a new class of therapies for patients with fibrosing liver disease.
Kruppel like factor 6 (KLF6), a zinc finger transcription factor and tumor suppressor, is induced as an immediate-early gene during hepatic stellate cell (HSC) activation. The paradoxical induction of a tumor suppressor in HSCs during proliferation led us to explore the biology of wild type KLF6 (KLF6WT) and its antagonistic, alternatively spliced isoform KLF6SV1 in cultured HSCs and animal models. Methods The animal models generated include a global heterozygous KLF6 mouse (Klf6 +/−), and transgenic mice expressing either hKLF6WT or hKLF6SV1 under the control of the Collagen α2 (I) promoter to drive HSC-specific gene expression following injury. Results The rat Klf6 transcript has multiple splice forms that are homologous to those of the human KLF6 gene. Following a transient increase, all rat Klf6 isoforms decreased in response to acute CCl4 liver injury, and culture-induced activation. After acute CCl4, Klf6 +/− mice developed significantly increased fibrosis and enhanced fibrogenic mRNA and protein expression. In contrast, HSC-specific transgenic mice over-expressing KLF6WT or KLF6SV1 developed significantly diminished fibrosis with reduced expression of fibrogenic genes. Chromatin IP, and qRT-PCR in mouse HSCs over-expressing KLF6WT demonstrated KLF6WT binding to GC boxes in promoters of Colα1 (I), Colα2 (I), and β-Pdgfr with reduced gene expression, consistent with transcriptional repression by KLF6. Stellate cells over-expressing either KLF6WT or KLF6SV1 were more susceptible to apoptotic stress based on PARP cleavage. Conclusion KLF6 reduces fibrogenic activity of HSCs via two distinct mechanisms, direct transcriptional repression of target fibrogenic genes and increased apoptosis of activated HSCs. These results suggest that following its initial induction, sustained downregulation of KLF6 in liver injury may allow de-repression of fibrogenic genes and decreased stellate cell clearance by inhibiting apoptosis.
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