Unraveling the Spider Web of Hepatic Stellate Cell Apoptosis

Abstract

See “Angiotensin II activates IκB kinase phosphorylation of RelA at Ser536 to promote myofibroblast survival and liver fibrosis,” by Oakley F, Teoh V, Ching-A-Sue G, et al, on page 2334. See “Angiotensin II activates IκB kinase phosphorylation of RelA at Ser536 to promote myofibroblast survival and liver fibrosis,” by Oakley F, Teoh V, Ching-A-Sue G, et al, on page 2334. The process of stellate cell trans-differentiation into myofibroblast-like cells, a key event in hepatic and pancreatic fibrogenesis, is associated with the activation of pathways promoting cell survival.1Elsharkawy A.M. Oakley F. Mann D.A. The role and regulation of hepatic stellate cell apoptosis in reversal of fibrosis.Apoptosis. 2005; 10: 927-939Crossref PubMed Scopus (368) Google Scholar This phenomenon has been described also in other cell types that participate in the wound healing response in different organs and, together with the remarkable increase in cell proliferation, leads to hyperplasia of extracellular matrix (ECM)-producing cells, which represent a key factor in chronic wound healing and tissue fibrogenesis. Considering the natural history of chronic liver diseases (CLD), and particularly chronic hepatitis C,2Poynard T. Bedossa P. Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups.Lancet. 1997; 349: 825-832Abstract Full Text Full Text PDF PubMed Scopus (2786) Google Scholar it is possible that the acceleration in the rate of fibrosis progression observed in the late phases of the disease occurs when a critical mass of profibrogenic cells is reached and is accompanied by a progressive exhaustion of the molecular mechanisms regulating ECM degradation and remodelling. According to current knowledge, it has been speculated that any feasible treatment able to reduce ECM-producing cell hyperplasia would lead to a reduced rate of fibrosis progression or even to fibrosis regression when associated with a treatment favoring the cessation of tissue damage. In this context, it is likely that hepatic stellate cell (HSC) apoptosis represents a major mechanism. Accordingly, data from animal models indicate that recovery from acute or chronic injury is characterized by apoptosis of HSC and, as a consequence, reduction of tissue inhibitor of metalloproteinases levels and progressive degradation of the fibrotic matrix.3Iredale J.P. Hepatic stellate cell behaviour during resolution of liver injury.Semin Liver Dis. 2001; 21: 427-436Crossref PubMed Scopus (296) Google Scholar, 4Issa R. Williams E. Trim N. et al.Apoptosis of hepatic stellate cells: involvement in the resolution of biliary fibrosis.Gut. 2001; 48: 548-557Crossref PubMed Scopus (280) Google Scholar, 5Iredale J.P. Benyon R.C. Pickering J. et al.Mechanisms of spontaneous resolution of rat liver fibrosis Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (937) Google Scholar, 6Murphy F.R. Issa R. Zhou X. et al.Inhibition of apoptosis of activated hepatic stellate cels by TIMP-1 is mediated via effects on MMP-inhibition: implications for reversibility of liver fibrosis.J Biol Chem. 2002; 277: 11069-11076Crossref PubMed Scopus (386) Google Scholar Along these lines, regression of liver fibrosis and, possibly, cirrhosis has been reported in patients with CLD,7Dufour J.F. DeLellis R. Kaplan M.M. Reversibility of hepatic fibrosis in autoimmune hepatitis.Ann Intern Med. 1997; 127: 981-985Crossref PubMed Scopus (275) Google Scholar, 8Wanless I.R. Nakashima E. Sherman M. Regression of human cirrhosis: morphologic features and the genesis of incomplete septal cirrhosis.Arch Pathol Lab Med. 2000; 124: 1599-1607PubMed Google Scholar, 9Hammel P. Couvelard A. O'Toole D. et al.Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of the common bile duct.N Engl J Med. 2001; 344: 418-423Crossref PubMed Scopus (367) Google Scholar once achieved with cessation of the causative agent. Although the possibility for regression of complete cirrhosis is unlikely,10Desmet V.J. Roskams T. Cirrhosis reversal: a duel between dogma and myth.J Hepatol. 2004; 40: 860-867Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar specific induction of apoptosis in HSC may represent a realistic objective for cell-targeted therapy of liver fibrosis at almost any stage of the disease. From this perspective, it is important to stress that human HSC are characterized by a much higher resistance to apoptosis when compared to rodent HSC and that anti-apoptotic proteins such as Bcl-2 are highly expressed in fibrogenic cells within human liver tissue undergoing active fibrogenesis.11Novo E. Marra F. Zamara E. et al.Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.Gut. 2006; 55: 1174-1182Crossref PubMed Scopus (150) Google Scholar This indicates that meaningful indications for therapy of human CLD will be attained only when addressing this issue in human cell models and possibly, in therapeutic clinical trials. Based on this background, the identification of factors and pathways promoting HSC survival seems to be central, because it may provide the basis for pharmacologic treatments able to induce HSC apoptosis. Candidate survival factors for HSC include transforming growth factor β1, insulin-like growth factor-1, tissue inhibitor of metalloproteinase-1, and type I collagen3Iredale J.P. Hepatic stellate cell behaviour during resolution of liver injury.Semin Liver Dis. 2001; 21: 427-436Crossref PubMed Scopus (296) Google Scholar, 12Friedman S.L. Liver fibrosis: from bench to bedside.J Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar, 13Gentilini A. Lottini B. Brogi M. et al.Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cells.Fibrogenesis Tissue Repair. 2009; 2: 1Crossref PubMed Scopus (20) Google Scholar together with persistent activation of nuclear factor-κB (NF-κB). Activation of NF-κB is a key event in the activation of HSC in cell culture models14Elsharkawy A.M. Wright M.C. Hay R.T. et al.Persistent activation of nuclear factor-κB in cultured hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkB family proteins.Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar, 15Lang A. Schoonhoven R. Tuvia S. et al.Nuclear factor kappaB in proliferation, activation, and apoptosis of rat hepatic stellate cells.J Hepatol. 2000; 33;: 49-58Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar and is accompanied by a sustained transcriptional repression of IκBα, the inhibitor of NF-κB.16Oakley F. Mann J. Ruddel R. et al.Basal expression of IκBα is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and its activator Notch 1.J Biol Chem. 2003; 279: 24359-24370Crossref Scopus (79) Google Scholar NF-κB regulates a number of genes acting as potent inhibitors of the cellular apoptotic machinery and its activation promotes survival of HSC in the presence of apoptotic stimuli.15Lang A. Schoonhoven R. Tuvia S. et al.Nuclear factor kappaB in proliferation, activation, and apoptosis of rat hepatic stellate cells.J Hepatol. 2000; 33;: 49-58Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Wright M.C. Issa R. Smart D.E. et al.Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhance the resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar IκBα is further inactivated by several agents, including tumor necrosis factor-α and lipopolysaccharide through the phosphorylation of serine residues operated by IKKβ, a component of the IKK complex which acts downstream to the tumor necrosis factor-α and lipopolysaccharide receptors.18Hayden M.S. Ghosh S. Shared principles in NF-κB signalling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar However, it is likely that additional IKK-dependent regulatory checkpoints are active because IKK inhibitors such as sulphasalazine inhibit myofibroblast NF-κB activity and induce apoptosis despite the low level of IκBα expression.19Oakley F. Meso M. Iredale J.P. et al.Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerates recovery from rat liver fibrosis.Gastroenterology. 2005; 128: 108-120Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar The complex web of regulatory checkpoints leading to the constitutive activation of NF-κB in activated HSC, discovered largely by the authors of the work published in this issue of Gastroenterology,20Oakley F. Teoh V. Ching-A-Sue G. et al.Angiotensin II activates IκB kinase phosphorylation of RelA at Ser536 to promote myofibroblast survival and liver fibrosis.Gastroenterology. 2009; 136: 2334-2344Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar has raised the possibility of targeting several regulatory steps with molecules ultimately acting as proapoptotic and antifibrotic agents. In the present study, Oakley et al20Oakley F. Teoh V. Ching-A-Sue G. et al.Angiotensin II activates IκB kinase phosphorylation of RelA at Ser536 to promote myofibroblast survival and liver fibrosis.Gastroenterology. 2009; 136: 2334-2344Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar demonstrate the existence of a constitutively active positive feedback fibrogenic signaling loop in activated HSC leading to active NF-κB (Figure 1). This loop has 2 major points of relevance and originality: (A) the phosphorylation of the Ser536 residue of RelA, which is required for efficient nuclear transport of RelA-containing NF-κB, progressively increases during the cell culture transdifferentiation of HSC, thus representing an additional molecular feature of the activation process, and (B) angiotensin II, acting in an autocrine fashion, induces phosphorylation of RelA via IKK and the stimulation of NF-κB–dependent transcription of cell survival genes. The existence of this pathway expands further the web of regulation of NF-κB activation and explains the effect of sulphasalazine mentioned. The positive feedback loop is completed by a direct link between NF-κB activation and the expression of the angiotensin II precursor angiotensinogen. These findings, in addition to their intrinsic value in the understanding the complexity of the mechanisms regulating HSC survival, have relevant clinical implications. Angiotensin II, in addition to its vasoconstrictor action, has been demonstrated to act as a pleiotropic cytokine with important pro-fibrogenic effects in different organs including the liver.21Moreno M. Bataller R. Cytokines and renin-angiotensin system signaling in hepatic fibrosis.Clin Liver Dis. 2008; 12: 825-852Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Angiotensin II is locally synthesized in the injured liver and has been shown to induce profibrogenic actions in HSC including stimulation of cell proliferation and expression of profibrogenic genes.21Moreno M. Bataller R. Cytokines and renin-angiotensin system signaling in hepatic fibrosis.Clin Liver Dis. 2008; 12: 825-852Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar In addition, upon activation, HSC express all the components of the renin–angiotensin system and synthesize angiotensin II22Bataller R. Sancho-Bru P. Ginès P. et al.Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.Gastroenterology. 2003; 125: 117-125Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar that may act in an autocrine fashion. Importantly, a genetic polymorphism associated with increased synthesis of angiotensin II has been revealed to be associated with faster fibrosis progression in patients with chronic HCV infection.23Powell E.E. Edwards-Smith C.J. Hay J.L. et al.Host genetic factors influence disease progression in chronic hepatitis C.Hepatology. 2000; 31: 828-833Crossref PubMed Scopus (376) Google Scholar Accordingly, drugs blocking the renin–angiotensin system have been proposed as antifibrotic agents. Indeed, inhibition of the renin–angiotensin system at different levels attenuates fibrosis progression in animal models of liver fibrosis.24Bataller R. Sancho-Bru P. Gines P. et al.Liver fibrogenesis: a new role for the renin-angiotensin system.Antioxid Redox Signal. 2007; 7: 1346-1355Crossref Scopus (137) Google Scholar In this connection, data provided in the paper by Oakley et al20Oakley F. Teoh V. Ching-A-Sue G. et al.Angiotensin II activates IκB kinase phosphorylation of RelA at Ser536 to promote myofibroblast survival and liver fibrosis.Gastroenterology. 2009; 136: 2334-2344Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar show that administration of the angiotensin-converting enzyme inhibitor captopril to rats with bile duct ligation leads to fibrosis regression despite continuous injury and, remarkably, is associated with the loss of P-Ser536-RelA in cells associated with fibrotic tissue. It is therefore likely that the expression of P-Ser536-RelA in liver tissue undergoing progressive fibrogenesis represents a biomarker of the predominantly angiotensin II-driven survival mechanism in activated HSC leading to active NF-κB and, at the same time, an indicator of the effective response to the treatment with anti-angiotensin II agents. The study provides also elements for translation into large scale clinical applications in humans: a more marked pretreatment expression of P-Ser536-RelA in liver tissue obtained from patients with chronic hepatitis C undergoing treatment with losartan for 18 months was associated with a higher response to the treatment. Although these findings need to be confirmed and validated in larger series of patients, they represent a clear innovation both in theoretical and practical terms. The possibility of employing drugs able to block angiotensin II as antifibrogenic is indeed real. Many effective compounds able to block the renin–angiotensin system at different levels are available, their side effects are limited and well known, and, in addition, it is possible to tailor the use of these agents according to the patient characteristics. Of relevance, positive therapeutic effects of the angiotensin receptor 1 (AT1) blocker candesartan have been reported in patients with compensated cirrhosis with a significant reduction of the hepatic venous pressure gradient associated with decreased serum levels of direct markers of fibrosis.25Debernardi-Venon W. Martini S. Biasi F. et al.AT1 receptor antagonist Candesartan in selected cirrhotic patients: effect on portal pressure and liver fibrosis markers.J Hepatol. 2007; 46: 1026-1033Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Importantly, these effects occurred at a dosage of candesartan not affecting mean arterial pressure and therefore possibly related to reduced fibrosis. In addition, the drug was very well tolerated, even when administered chronically. In conclusion, the work by Oakley et al highlights the central role of angiotensin II in promoting liver fibrosis with different molecular mechanisms including the regulation of key survival pathways and the more and more likely possibility of employing anti-angiotensin II drugs as antifibrotic agents in everyday practice. But more than anything this work is a very good example of translational research with possible immediate applications in clinical practice. Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser536 to Promote Myofibroblast Survival and Liver FibrosisGastroenterologyVol. 136Issue 7PreviewThe transcription factor nuclear factor-κB (NF)-κB promotes survival of hepatic myofibroblasts and fibrogenesis through poorly defined mechanisms. We investigated the activities of angiotensin II and IκB kinase (IKK) in regulation of NF-κB activity and the role of these proteins in liver fibrosis in rodents and humans. Full-Text PDF