Background and AimsHepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix-degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP-1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N-cadherin at the cell surface.ResultsN-cadherin expression was upregulated in human HSC during activation in culture. Addition of function-blocking antibodies or a peptide targeting the extracellular domain of N-cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N-cadherin into 20-100 kDa fragments. MMP-2 became activated early during HSC apoptosis and directly cleaved N-cadherin in vitro. Addition of activated MMP-2 to HSCs in culture resulted in enhanced apoptosis and loss of N-cadherin.ConclusionsTogether, these studies identify a role for both N-cadherin and MMP-2 in mediating HSC apoptosis, where N-cadherin works to provide a cell survival stimulus and MMP-2 promotes HSC apoptosis concomitant with N-cadherin degradation.

Apoptosis of hepatic stellate cells (HSC) has previously been shown to occur during spontaneous resolution of experimental liver fibrosis. TIMP-1 has also been shown to have a key role because of its ability to inhibit apoptosis of HSC via matrix metalloproteinase (MMP) inhibition. This has led to further study of novel substrates for MMPs that might impact on HSC survival. N-Cadherin is known to mediate cell-cell contacts in fibroblasts. In this study we demonstrate that N-Cadherin is expressed by activated rat HSC. Furthermore, during apoptosis of HSC, the N-Cadherin is cleaved into smaller fragments. Apoptosis of HSC may be inhibited by TIMP-1. This is associated with reduced fragmentation of N-Cadherin. N-Cadherin may have an important role in supporting HSC survival while N-Cadherin cleavage may play a part in promoting HSC apoptosis in recovery from liver fibrosis.

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possi-bility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of (cid:1) v (cid:2) 3 integrin. (cid:1) v (cid:2) 3 integrin was expressed by activated, myo- fibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence (cid:1) v subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These (cid:1) v (cid:2) 3 antago- nists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/ BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by that stellate of (cid:1) v (cid:2) 3 ligands surrounding activated stellate cells 3 and (cid:1) v gene silencing using siRNA transfection) demonstrate that engagement of (cid:1) v (cid:2) 3 integrin is required to sustain both HSC proliferation and survival in culture. We suggest that this integrin might transduce proliferation and survival signals to HSCs during liver fibrogenesis when its matrix ligands accumulate, whereas loss of these ligands during resolution might prime HSCs for apoptosis. Such a mechanism might act generically to remove fibrogenic cells during wound healing in other tissues such as kidney, skin, intestine, and pancreas where myofibroblastic cells phe-notypically similar to activated HSCs are important effectors of fibrosis. Our studies show a novel integrin-dependent mechanism regulating HSC fate. To date there is little information about the integrins expressed by HSCs during activation. Cultured activated human HSCs express (cid:1) 1 , (cid:1) 2 , (cid:1) v , (cid:1) 6 , (cid:2) 1 and (cid:2) 4 integrin subunits (45), while a preliminary study shows that (cid:2) 1 integrin and several (cid:1) subunits became more highly expressed in situ in activated HSCs in human fibrotic liver (46). Using a well characterized tissue culture model of HSC activation, we show that both rat and human HSCs express (cid:1) v (cid:2) 3 integrin after activation in vitro . Expression of both subunits was regulated by ECM with expression being highest on type I collagen and vitronectin. As these proteins accumulate in fibrotic liver, this might allow a positive feedback loop to enhance HSC responsiveness to the accumulated ligands.

Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. Wiemann SU, Satyanarayana A, Tsahuridu M, Tillmann HL, Zender L, Klempnauer J, Flemming P, Franco S, Blasco MA, Manns MP, Rudolph KL. Department of Gastroenterology, Hepatology, and Endrocrinology, Medical School Hannover, GermanyTelomere shortening limits the number of cell divisions of primary human cells and might affect the regenerative capacity of organ systems during aging and chronic disease. To test whether the telomere hypothesis applies to human cirrhosis, the telomere length was monitored in cirrhosis induced by a broad variety of different etiologies. Telomeres were significantly shorter in cirrhosis compared with non-cirrhotic samples independent of the primary etiology and independent of the age of the patients. Quantitative fluorescence in situ hybridization showed that telomere shortening was restricted to hepatocytes whereas lymphocytes and stellate cells in areas of fibrosis had significantly longer telomere reserves. Hepatocyte-specific telomere shortening correlated with senescence-associated beta-galactosidase staining in 84% of the cirrhosis samples, specifically in hepatocytes, but not in stellate cells or lymphocytes. Hepatocyte telomere shortening and senescence correlated with progression of fibrosis in cirrhosis samples. This study demonstrates for the first time that cell type-specific telomere shortening and senescence are linked to progression of human cirrhosis. These findings give a novel explanation for the pathophysiology of cirrhosis, indicating that fibrotic scarring at the cirrhosis stage is a consequence of hepatocyte telomere shortening and senescence. The data imply that future therapies aiming to restore regenerative capacity during aging and chronic diseases will have to ensure efficient targeting of specific cell types within the affected organs.[FASEB J 2002;16:935–942]

Conference Abstract| July 01 2002 Inhibition of Apoptosis of Activated Hepatic Stellate Cells by Timp-1 is Mediated via Effects on Mmp Inhibition: Implications for Reversibility of Liver Fibrosis F. Murphy; F. Murphy 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar R. Issa; R. Issa 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar X. Zhou; X. Zhou 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar H. Hussain; H. Hussain 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar S. Ratnarajah; S. Ratnarajah 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar P. Soloway; P. Soloway 1Rosewell Park Cancer Institute, New York, USA Search for other works by this author on: This Site PubMed Google Scholar H. Nagase; H. Nagase 2 The Kennedy Institute, Imperial College, London, UK Search for other works by this author on: This Site PubMed Google Scholar Arthur Mjp; Arthur Mjp 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar RC Benyon; RC Benyon 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar JP Iredale JP Iredale 1Liver Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, SO16 6YD Search for other works by this author on: This Site PubMed Google Scholar Clin Sci (Lond) (2002) 103 (s47): 40P. https://doi.org/10.1042/cs103040P Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation F. Murphy, R. Issa, X. Zhou, H. Hussain, S. Ratnarajah, P. Soloway, H. Nagase, Arthur Mjp, RC Benyon, JP Iredale; Inhibition of Apoptosis of Activated Hepatic Stellate Cells by Timp-1 is Mediated via Effects on Mmp Inhibition: Implications for Reversibility of Liver Fibrosis. Clin Sci (Lond) 1 July 2002; 103 (s47): 40P. doi: https://doi.org/10.1042/cs103040P Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsClinical Science Search Advanced Search This content is only available as a PDF. © 2002 The Biochemical Society and the Medical Research Society2002 Article PDF first page preview Close Modal You do not currently have access to this content.

Liver fibrosis represents a major worldwide healthcare burden. Current therapy is limited to removing the causal agent. This approach is successful in some diseases; particularly haemochromatosis and chronic viral hepatitis. However, for many patients treatment is not possible, while other patients present to medical attention at an advanced stage of fibrosis. There is therefore a great need for novel therapies for liver fibrosis. The hepatic stellate cell has been recognised to be responsible for most of the excess extracellular matrix observed in chronic liver fibrosis. The detailed understanding of hepatic stellate cell biology has allowed the rational design of novel antifibrotic therapies. This review describes for the general reader the novel emerging therapies for liver fibrosis.

Liver fibrosis represents the final common pathway for the majority of chronic liver insults (alcohol, autoimmune or viral injury).Current evidence indicates that the central mediator of fibrosis is the hepatic stellate cell (HSC).During fibrotic injury, these retinoid rich cells proliferate and undergo a phenotypic transformation to myofibroblast-like cells, a process termed activation.Previous work by the our group has demonstrated that activated HSC also express the powerful tissue inhibitors of metalloproteinases (TIMPs) 1 and 2, suggesting that matrix degradation is inhibited during fibrogenesis.This hypothesis is supported by findings that spontaneous recovery from liver fibrosis is associated with a diminution of TIMP expression and an increase in collagenase activity with consequent matrix degradation (1).A further finding in this study is that sustained apoptosis of HSC accompanies recovery from fibrosis.This has highlighted the role of TIMP-1 and -2 in HSC proliferation, survival and apoptosis. AIMS.To determine the effect of TIMP-1 on human and rat hepatic stellate cell proliferation and apoptosis.