Liver damage leads to an inflammatory response and to the activation and proliferation of mesenchymal cell populations within the liver which remodel the extracellular matrix as part of an orchestrated wound-healing response. Chronic damage results in a progressive accumulation of scarring proteins (fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Efforts to modulate the fibrogenesis process have focused on understanding the biology of the heterogeneous liver fibroblast populations. The fibroblasts are derived from sources within and outwith the liver. Fibroblasts expressing α-smooth muscle actin (myofibroblasts) may be derived from the transdifferentiation of quiescent hepatic stellate cells. Other fibroblasts emerge from the portal tracts within the liver. At least a proportion of these cells in diseased liver originate from the bone marrow. In addition, fibrogenic fibroblasts may also be generated through liver epithelial (hepatocyte and biliary epithelial cell)–mesenchymal transition. Whatever their origin, it is clear that fibrogenic fibroblast activity is sensitive to (and may be active in) the cytokine and chemokine profiles of liver-resident leucocytes such as macrophages. They may also be a component driving the regeneration of tissue. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.

Background & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of κB kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle α-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-κB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45β, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of κB kinase/nuclear factor-κB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of κB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of κB kinase/nuclear factor-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of κB kinase have potential as antifibrotics.

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-κB (NF-κB) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-κB DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor α (TNF-α), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-κB-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative IκB-α. Physiological significance of the lower persistent NF-κB activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-κB-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of IκB-α and IκB-β. Culture activation was accompanied by a long-term reduction in levels of IκB-α with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IκB-β expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility IκB-β antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC. Inhibition of NF-κB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-κB in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.