Xiaoying Zhou

Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration

Collagen‐I, which predominates in the neomatrix of fibrotic liver, regulates hepatocyte and hepatic stellate cell (HSC) phenotypes. Recovery from liver fibrosis is accompanied by hepatocyte regeneration, matrix degradation, and HSC apoptosis. Using mice bearing a mutated collagen‐I gene (r/r mice), which confers resistance to collagenase degradation, we have investigated the hypothesis that collagen‐I degradation is critical to HSC apoptosis and hepatocyte regeneration during recovery from liver fibrosis. During a 28‐day recovery period after 8 wk of CCl4 treatment, wild‐type (WT) livers had significantly (43%) decreased hydroxyproline (OHP) content. In r/r livers, however, OHP content remained elevated at peak fibrosis levels. Expressed markers of activated HSC (α‐smooth muscle actin, collagen‐I), elevated at peak fibrosis, dropped to control levels in WT livers after 28 days but remained raised in the r/r livers. Moreover, relative to WT livers, r/r livers had significantly reduced stellate cell apoptosis and hepatocyte regeneration during the recovery period. Using extracted collagen‐I from each genotype as culture substrata, relative to r/r, we show that WT collagen‐I promotes hepatocyte proliferation via stimulation of integrin αvβ3. Failure to degrade collagen‐I critically impairs HSC apoptosis and may prevent the effective restoration of hepatocyte mass in liver fibrosis.

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors

BACKGROUND/AIMS Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation. METHODS Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. RESULTS Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells. CONCLUSIONS This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.

Expression of matrix metalloproteinase‐2 and ‐14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis

Abstract: Background/Aims: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP‐2 and MMP‐14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis.

Impaired Proteolysis of Collagen I Inhibits Proliferation of Hepatic Stellate Cells IMPLICATIONS FOR REGULATION OF LIVER FIBROSIS

Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, α-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin αvβ3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via αvβ3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.