A cut above the rest? MMP-8 and liver fibrosis gene therapy

Liver fibrosis and cirrhosis represent the common pathological endpoint of chronic liver disease virtually regardless of the initiating etiology. I would guess that many readers of Gastroenterology have first hand experience of dealing with the clinical consequences of fibrosis and cirrhosis “at the sharp end” in the emergency room. We are all too familiar with the morbidity and mortality associated with cirrhosis. Whilst advances in the management of complications of cirrhosis such as transjugular intrahepatic portosystemic shunting have made some impact, currently our only definitive treatment for the underlying condition is liver transplantation. Therefore, we urgently require effective and targeted therapies for this condition. Past and current basic science research, in conjunction with corroborative studies in diseased human tissue, hold enormous promise for the development of such therapies. In particular, the results of detailed cell culture and animal model studies, which have taken place over the last 10 to 15 years, are now informing the design of mechanistic studies.1Bataller R. Brenner D. Hepatic stellate cells as a target for the treatment of liver fibrosis.Semin Liver Dis. 2001; 21: 437-451Crossref PubMed Scopus (444) Google Scholar Indeed, this data is now providing a platform from which clinical and nonclinical scientists are manipulating the fibrotic process in proof of concept experiments that are essential to identify therapeutic strategies; strategies which, given development, will be potentially applicable in the clinic. The article by Siller-López et al. in this edition of Gastroenterology is a report of just such a study.2Siller-López F. Sandoval A. Salgado S. Salazar A. Bueno M. Garcia J. Vera J. Galvez J. Hernández I. Ramos M. Aguilar-Cordova E. Armendariz-Borunda J. Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis.Gastroenterology. 2004; 126: 1122-1133Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Using an adenovirus to deliver one of the key mammalian collagenases (neutrophil collagenase, MMP-8) Siller-López et al. have shown that experimentally induced rat hepatic fibrosis and cirrhosis can be effectively manipulated to achieve regression of fibrosis. Liver fibrosis and cirrhosis are characterized by activation of hepatic stellate cells (HSC) to a myofibroblast-like phenotype.3Geert A.B. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.Semin Liver Dis. 2001; 21: 311-335Crossref PubMed Scopus (614) Google Scholar In this activated phenotype, activated HSC secrete collagens-1 and III which accumulate initially in the perisinusoidal space of Disse, but with progressive injury link vascular structures. Ultimately progression of this process leads to the architectural disruption which characterizes cirrhosis.2Siller-López F. Sandoval A. Salgado S. Salazar A. Bueno M. Garcia J. Vera J. Galvez J. Hernández I. Ramos M. Aguilar-Cordova E. Armendariz-Borunda J. Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis.Gastroenterology. 2004; 126: 1122-1133Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 4Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1895) Google Scholar Once secreted collagens-1 and III are and degraded by members of a family of neutral Zn and Ca dependent proteases, the metalloproteinases (MMPs).5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar Current evidence indicates that initiation of degradation of the fibrillar collagens-I and III is made by and limited to an MMP with interstitial collagenase activity. These MMPs (MMP-1, 8, and 13 in the human and MMP-13 in the rat and mouse) cleave collagen at a single site a quarter of the way along the molecule. This cleavage allows the collagen to partially unwind and renders it susceptible to degradation by more promiscuous MMPs and other proteases.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar More recently, evidence has been presented that MTI-MMP (MMP-14) and MMP-2 also have potential interstitial collagenase activity.6Aimes R.T. Quigley J.P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments.J Biol Chem. 1995; 270: 5872-5876Crossref PubMed Scopus (835) Google Scholar, 7Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover.Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar, 8Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. Membrane type 1 matrix metalloproteinase digests interstitital collagenas and other extracellular matrix macromolecules.J Biol Chem. 1997; 272: 2446-2451Crossref PubMed Scopus (834) Google Scholar One theme that emerged relatively early from the experimental studies of liver fibrosis was that progressive fibrosis was characterized not only by an exuberant secretion of collagens I and III and other matrix molecules, but a change in the pattern of their degradation. Early work demonstrated that with progression of experimental liver cirrhosis, collagenolytic activity was present but decreased when expressed relative to collagen content.9Perez-Tamayo R. Montfort I. Gonzalez E. Collagenolytic activity in experimental cirrhosis of the liver.Exp Mol Pathol. 1987; 47: 300-308Crossref PubMed Scopus (50) Google Scholar, 10Montfort I. Perez-Tamayo R. Collagenase in experimental carbon tetrachloride cirrhosis of the liver.Am J Pathol. 1978; 92: 411-420PubMed Google Scholar Subsequently workers in our group and others confirmed this observation and identified 2 potential mechanisms responsible for this failure of matrix degradation: regulation in the expression and activation of collagenases and expression of the potent metalloproteinase (and collagenase) inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) 1 and 2. Further work in several laboratories has suggested that this high level of TIMP expression profoundly inhibits MMP mediated matrix degradation in fibrotic liver.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar, 11Iredale J.P. Murphy G. Hembry R.M. Friedman S.L. Arthur M.J.P. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1 (TIMP-1) implications for regulation of matrix degradation in liver.J Clin Invest. 1992; 90: 282-287Crossref PubMed Scopus (184) Google Scholar, 12Iredale J.P. Benyon R.C. Pickering J. McCullen M. Northrop M. Pawley S. Hovell C. Arthur M.J.P. 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 (940) Google Scholar, 13Dudas J. Kovalszky I. Gallai M. Nagy J.O. Schaff Z. Knittel T. Mehde M. Neubauer K. Szalay F. Ramadori G. Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.Am J Clin Pathol. 2001; 115: 725-735Crossref PubMed Scopus (66) Google Scholar, 14Knittel T. Mehde M. Grundmann A. Saile B. Scharf J.G. Ramadori G. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat.Histochem Cell Biol. 2000; 113: 443-453Crossref PubMed Scopus (207) Google Scholar, 15Yoshiji H. Kuriyama S. Miyamoto Y. Thorgeirsson U.P. Gomez D.E. Kawata M. Yoshii J. Ikenaka Y. Noguchi R. Tsujinoue H. Nakatani T. Thorgeirsson S.S. Fukui H. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model.Hepatology. 2000; 32: 1248-1254Crossref PubMed Scopus (237) Google Scholar Thus, a logical therapeutic approach to fibrosis might be to enhance matrix degradation via up-regulating or inducing expression of a collagenase or inhibiting the effect of the TIMPs.1Bataller R. Brenner D. Hepatic stellate cells as a target for the treatment of liver fibrosis.Semin Liver Dis. 2001; 21: 437-451Crossref PubMed Scopus (444) Google Scholar, 16Iimuro Y. Nishio T. Morimoto T. Nitt T. Stefaanovic B. Choi S.K. Brenner D.A. Yamaoka Y. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology. 2003; 124: 445-458Abstract Full Text PDF PubMed Scopus (208) Google Scholar Siller-López et al. chose the former approach. Using an adenovirus, Siller-López et al. designed a construct, which would express pro-MMP-8 in mammalian cells. Initially using HeLa cells they demonstrated that cell transduction would result in expression of pro-MMP-8. All metalloproteinases are expressed in a pro-form and require activation to become functional. Interestingly, the transduced HeLa cells did not activate the pro-metalloproteinase-8 and effective collagenase activity in the media of these cells was only seen after an organomerucurial was used to achieve protease activation. The workers then went on to use the adenovirus in vivo to express MMP-8 in models of liver fibrosis induced by CCl4 and bile duct ligation. One powerful aspect of this study is that the authors here demonstrated the efficacy of their adenoviral approach in 2 mechanistically distinct models of liver fibrosis. In addition, by studying the effects of MMP-8 expression achieved through adenoviral transduction of liver during spontaneous recovery from bile duct ligation induced fibrosis (following biliodigestive anastomosis), the authors offer strong evidence that this therapeutic approach will enhance recovery from liver fibrosis. Although the adenovirus expressing MMP-8 was identical to that used with the HeLa cells, activation of pro-MMP-8 was demonstrable in the fibrotic/cirrhotic liver in vivo. Whilst the authors did not identify the specific activating factor, this result is perhaps not surprising as many potential candidates to activate MMPs, may be present in the fibrotic liver. Foremost amongst these candidates are probably uPA and MMP-14 which are expressed by activated stellate cells in a focused manner.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar In vivo uPA may well be a critical regulator of collagenase activation. Indeed, studies previously published by the same group showed that by inducing expression of uPA matrix proteolysis could be induced in the fibrotic liver.17Salgado S. Garcia J. Vera J. Siller F. Bueno M. Miranda A. Segura A. Grijalva G. Segura J. Orozco H. Hernandez-Pando R. Fafutis M. Aguilar-Cordova L.K. Armendariz-Borunda J. Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy.Mol Ther. 2000; 2: 545-551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar By placing undue emphasis on biochemical and histological changes in liver, it is easy to forget the importance of demonstrating that manipulations of experimental animal models have functional consequences. These consequences are profoundly important when we consider the clinical relevance of a particular therapeutic approach. Another strength of the study by Siller-López et al., and one for which the authors are to be congratulated, is the assessment of clinical parameters after experimental adenoviral manipulation. The effective decrease in fibrosis that the authors observed, following their adenoviral collagenase treatment, was associated with a reduction in ascites score and a reduction in the presence of varices and presumably, therefore, portal hypertension. At its core then, the study by Siller-López et al. demonstrates proof of concept that enhancing collagenase activity, achieved via adenoviral delivery, is an effective means of reducing liver fibrosis in progressive fibrotic/cirrhotic injury and during spontaneous recovery from fibrosis. The study by Siller-López et al. complements a similar investigation already published in Gastroenterology, which demonstrated the effect of antifibrotic action of adenoviral delivered MMP-1 (collagenase-1 or interstitial collagenase).16Iimuro Y. Nishio T. Morimoto T. Nitt T. Stefaanovic B. Choi S.K. Brenner D.A. Yamaoka Y. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology. 2003; 124: 445-458Abstract Full Text PDF PubMed Scopus (208) Google Scholar Taken together, these studies represent significant steps toward identifying an effective antifibrotic approach to chronic liver disease. Like most cutting-edge research, however, this study has left several key questions unanswered and has thrown up some intriguing and sometimes counterintuitive results which will undoubtedly be the subject of further work. One intriguing observation is that the animals treated with the control adenovirus (GFP-expressing) have reduced expression of TIMPs compared with normal untreated animals. This is perhaps rather unexpected as there is now a wealth of evidence to suggest that TIMPs are increased in fibrotic animals relative to normal untreated animals and indeed, this pattern is observed also in human liver disease.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar, 13Dudas J. Kovalszky I. Gallai M. Nagy J.O. Schaff Z. Knittel T. Mehde M. Neubauer K. Szalay F. Ramadori G. Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.Am J Clin Pathol. 2001; 115: 725-735Crossref PubMed Scopus (66) Google Scholar, 14Knittel T. Mehde M. Grundmann A. Saile B. Scharf J.G. Ramadori G. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat.Histochem Cell Biol. 2000; 113: 443-453Crossref PubMed Scopus (207) Google Scholar, 18Benyon R.C. Iredale J.P. Goddard S. Winwood P.J. Arthur M.J.P. Expression of tissue inhibitor of metalloproteinases-1 and -2 is increased in fibrotic human liver.Gastroenterology. 1996; 110: 821-831Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar This result suggests that the GFP-expressing adenovirus may not be quite as neutral as it at first appears. Indeed, this intriguing observation may only be understood by building and studying the comparative effects of an adenovirus expressing pro-MMP-8 and an adenovirus expressing a mutated pro-MMP-8 that was functionally inactive. Transduction with an MMP-8 expressing adenovirus also resulted in up-regulation of MMP-2 in one model (CCl4 intoxication) but not bile duct ligation, whilst TIMP expression demonstrated the reverse of that pattern. The mechanisms mediating these regulations may be very critical to understanding recovery from fibrosis as the data suggests that cleavage of collagen may initiate a response characterized by expression of other MMPs. MMP-8 has a focused substrate specificity.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar Therefore, any secreted MMP-2, which would be expected to degrade those collagens initially cleaved by MMP-8, may function as a downstream effector. In addition, liver fibrosis is characterised by the presence of other matrix molecules and their degradation may result from the presence of MMPs with a wide substrate specificity such as MMP-2.5Benyon R.C. Arthur M.J.P. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (447) Google Scholar A further observation made by the authors was that by inducing collagen degradation, not only was there an improvement in histology, but an increase in hepatocellular proliferation. The link between hepatocellular proliferation and matrix degradation is an intriguing one. When studying fibrosis, it is easy to become fixated on changes in the matrix and the cells, which regulate those changes. The regulation of hepatocyte proliferation is central to the restitution of normal tissue architecture and function. The data presented in this paper builds on an increasing body of evidence suggesting that matrix turnover profoundly regulates hepatocytes proliferation during recovery from liver fibrosis.16Iimuro Y. Nishio T. Morimoto T. Nitt T. Stefaanovic B. Choi S.K. Brenner D.A. Yamaoka Y. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology. 2003; 124: 445-458Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 19Issa R. Zhou X. Trim N. Millward-Sadler H. Krane S. Benyon C. Iredale J.P. 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.FASEB J. 2003; 17: 47-49Crossref PubMed Scopus (178) Google Scholar The work presented by Siller-López et al. therefore provides key experimental evidence that in hepatic fibrosis, inducing expression of MMP-8 results in a loss of the fibrotic tissue and a return to more normal architecture. Together with these changes, hepatocellular proliferation occurs and liver function improves. So what of the future? We live in exciting times. As a result of the strides forward that have been made in understanding the pathogenesis of liver fibrosis, it is now realistic to anticipate that over the next decade and as a result of studies such as that by Siller-López et al., targeted treatments for liver fibrosis will become available. Following the tragic death of Jesse Gelsinger it seems likely that drug-regulating authorities will be extremely reticent about this form of adenoviral approach; an approach which theoretically could move rapidly from bench to clinical trials. Alternative delivery systems and a firm and detailed understanding of the effects of both the delivery system and the delivered drug to the liver and more distant organs are essential prerequisites for this endeavour to move toward clinical application. Nevertheless, the study reported here and similar studies recently reported in Gastroenterology16Iimuro Y. Nishio T. Morimoto T. Nitt T. Stefaanovic B. Choi S.K. Brenner D.A. Yamaoka Y. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology. 2003; 124: 445-458Abstract Full Text PDF PubMed Scopus (208) Google Scholar have ensured that manipulating matrix degradation is now firmly on the map as a potential therapy for liver fibrosis.