Role of mannose-binding lectin-2 polymorphism in the development of acute cellular rejection after transplantation for hepatitis C virus-induced liver disease.

UNLABELLED: The development of liver and graft disease is suspected to be affected by genetic diversity. Mannose-binding lectin-2 (MBL-2) is an important immunomodulatory factor that is involved in complement activation. The aim of our study was to elucidate the role of MBL-2 genotypes after liver transplantation (LT) for hepatitis C virus (HCV)-induced liver disease regarding the incidence of acute cellular rejection (ACR), graft inflammation, fibrosis development, and antiviral treatment response. METHODS: A group of 149 patients who underwent LT for HCV-induced liver disease were genotyped for MBL-2 (rs7096206; G/C) by TaqMan genotyping assay. We evaluated 518 post-LT protocol biopsies and at least 98 urgent liver biopsies regarding graft fibrosis stages, inflammation grades, and evidence for rejection within MBL-2 genotype groups. RESULT: No association of MBL-2 polymorphisms was observed regarding inflammation, fibrosis, and antiviral treatment outcome. However, the C allele of the MBL-2 gene (P = 0.001) and gender compatibility (P = 0.012) were factors significantly associated with the incidence of ACR. CONCLUSION: MBL-2 polymorphisms and gender are involved in the development of ACR after LT. CC genotype and gender match may be regarded as risk factors for ACR in HCV-positive graft recipients. Further studies are needed to confirm and verify this observation in non-HCV groups as well.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro.

Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

Modulation of collagen XVIII/endostatin expression in lobular and biliary rat liver fibrogenesis.

BACKGROUND/AIMS: The liver is the major source of collagen XVIII (C18), the precursor of the angiogenesis inhibitor endostatin. In human liver C18 is mainly expressed by hepatocytes. However, its quantitative and temporospatial expression patterns during liver fibrogenesis are unknown. METHODS: We used RNA quantification and in situ hybridization combined with cell-specific markers to study C18 compared to procollagen alpha1(I) and tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA expression in acute (single dose of CCl4) and chronic (biliary) rat liver fibrogenesis. RESULTS: C18 transcripts were only found in hepatocytes and bile duct epithelia of normal and fibrotic livers, and occasionally in arterial myocytes and hepatic stellate cells. 72 h after CCl4 injection, C18 mRNA levels remained unchanged, while procollagen alpha1(I) mRNA was increased at 72 h and TIMP-1 mRNA peaked at 12 h (P < 0.05). In biliary fibrosis C18 mRNA levels increased 1.8-fold, contrasting with 20- and 4-fold elevated procollagen alpha1(I) and TIMP-1 transcript levels, respectively. CONCLUSIONS: Hepatocytes and bile duct epithelia are the predominant sources of C18 in normal and fibrotic rat liver. Contrary to procollagen alpha1(I) and TIMP-1, C18 expression remains constant in acute fibrogenesis and is upregulated in biliary fibrosis. Modulation of epithelial C18 expression and its processing to endostatin could allow a liver-specific anticancer therapy.

Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha1(I) and TIMP-1.

BACKGROUND/AIMS: Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism. METHODS: Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen alpha1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) beta1 were determined by multi-probe ribonuclease protection assay. RESULTS: After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen alpha1(I), TIMP-1 and TGFbeta1 (P < 0.01). Silymarin downregulated elevated procollagen alpha1(I), TIMP-1 and TGFbeta1 mRNA levels by 40-60% (P < 0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III. CONCLUSIONS: Silymarin suppresses expression of profibrogenic procollagen alpha1(I) and TIMP-1 most likely via downregulation of TGFbeta1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

Matrix as a modulator of hepatic fibrogenesis.

The extracellular matrix (ECM) provides cells with positional information and a mechanical scaffold for adhesion and migration. It consists of collagens, glycoproteins, proteoglycans, glycosaminoglycans and molecules that are bound specifically by the ECM, such as certain growth factors/cytokines, matrix metalloproteinases (MMPs) and processing enzymes such as tissue transglutaminase and procollagen propeptidases. This finely tuned ecosystem is dysbalanced in chronic fibrogenesis, which can be regarded as a continuous wound-healing process and which results in scar formation. Importantly, the ECM directs cellular differentiation, migration, proliferation, and fibrogenic activation or deactivation. Partially via defined oligopeptide sequences or structural domains, the ECM transfers specific signals to cells that act in concert with growth factors/cytokines. These signals either confer stress activation, with a resultant fibrogenic response, or stress relaxation, with a fibrolytic response. Alternatively, ECM-derived peptides can modulate angiogenesis, or growth factor and MMP availability and activity. Current ECM-related antifibrotic strategies are based on the identification and in vivo application of ECM-derived biomodulatory peptides, peptide sequences, or their nonpeptidic mimetics. The latter open the opportunity of oral application and an extended biological half-life. Examples are peptides derived from collagens VI (stress activation) and XIV (stress relaxation), or collagenous consensus peptides that remove ECM-bound MMPs and growth factors. Furthermore, certain peptides can be used as targeting structures to the fibrogenic lesion.

Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis.

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-beta1) and thus a potential target for antifibrotic treatment strategies. CTGF is up-regulated in disorders such as atherosclerosis, scleroderma, and fibrosis of kidneys and lungs. We investigated the temporospatial expression patterns of CTGF and TGF-beta1 mRNA in rat livers with acute fibrogenesis (after a single dose of CCl(4)) and with advanced fibrosis (6 weeks after complete bile duct occlusion). Multiprobe ribonuclease protection assay revealed increasing TGF-beta1 and CTGF mRNA levels 6 hours after injection of CCl(4), with peak levels after 72 hours. In biliary fibrosis TGF-beta1 and CTGF mRNA levels increased fourfold and sevenfold, respectively (P: < 0.001). In situ hybridization combined with cell-specific markers revealed CTGF transcripts in desmin-positive cells after a single dose of carbon tetrachloride, whereas no transcripts were found in normal livers. In biliary fibrosis, however, proliferating bile duct epithelial cells were the predominant source of CTGF mRNA. We conclude that in rat liver fibrogenesis CTGF is up-regulated in close association with TGF-beta1 and that, contrary to a previous report, not solely hepatic stellate cells but activated bile duct epithelial cells are the main source of this profibrogenic factor.

[Expression of collagen XVIII mRNA in rat liver fibrosis].

OBJECTIVE: To measure and study quantitatively the collagen XVIII mRNA in normal and fibrotic rat livers. METHODS: We used ribonuclease protection assay to investigate the collagen XVIII mRNA expression in rat liver fibrosis induced by complete bile duct occlusion (BDO). The expression level of procollagen 1 (XVIII) mRNA was compared with that of procollagen 1 (I) and tissue inhibitor of metalloproteinase 1 (TIMP1). RESULTS: mRNA levels of procollagen and TIMP 1 increased 20- and 4-fold in BDO rat livers, respectively. In contrast, hepatic procollagen 1 mRNA level increased only 1.8-fold in fibrotic rat livers. CONCLUSION: C XVIII mRNA is upregulated slightly in liver fibrosis, which is probably correlated with the fact that CXVIII is mainly expressed by hepatocytes.

Collagens serve as an extracellular store of bioactive interleukin 2.

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that interleukin 2 (IL-2), an important stimulator of T cell growth, preferentially binds to collagen types I, III, and VI and to a lesser degree to collagen types II, IV, and V, immobilized on polystyrene or nitrocellulose. These interactions are inhibited by denatured, single collagen chains or a subset of their cyanogen bromide peptides in a dose-dependent manner. Cross-inhibition experiments and ligand blotting of collagen-derived peptides point to a limited set of collagenous consensus sequences mediating the binding of IL-2. This interaction is saturable, with dissociation constants of approximately 10(-)(8) m, and estimated molar ratios of 4-6 molecules of IL-2 bound to one molecule of triple helical collagen. Furthermore, collagen-bound IL-2 stimulates proliferation of mouse lymphocytes. We conclude that its specific binding to the abundant interstitial collagens leads to a spatial pattern of bioavailable IL-2 which is dictated by the local organization of the collagenous extracellular matrix. This interaction may contribute to the particular phenotype of stromal lymphocytes and could be exploited for devising collagenous peptide analogues that modulate IL-2 bioactivity.

An oral endothelin-A receptor antagonist blocks collagen synthesis and deposition in advanced rat liver fibrosis.

BACKGROUND & AIMS: Endothelin 1 induces contraction, proliferation, and collagen synthesis of hepatic stellate cells in vitro, which may be mediated via the endothelin A receptor. It is unknown if specific blockade of the endothelin A receptor inhibits hepatic fibrosis in vivo. METHODS: Groups of 10-20 rats with bile duct occlusion were treated with the nonpeptide endothelin-A receptor antagonist LU 135252 at 80 mg. kg(-1). day(-1) from week 1-6 or from week 4-6, or with LU at 10 mg. kg(-1). day(-1) from week 1-6. Animals with bile duct occlusion alone and sham-operated rats without or with LU at 80 mg. kg(-1). day(-1) over 6 weeks served as controls. After 6 weeks, parameters of fibrogenesis were determined. RESULTS: LU treatment led to improved histology, paralleled by a dose-dependence up to 60% reduction of liver collagen, even when administered at an advanced fibrosis stage. This was accompanied by a decreased messenger RNA of hepatic procollagen alpha1(I) and tissue inhibitor of metalloproteinase 1, 2 major effectors of fibrosis, and of serum procollagen type III, a surrogate marker of liver fibrogenesis. CONCLUSIONS: Selective endothelin-A receptor blockade can dramatically reduce collagen accumulation in rat secondary biliary fibrosis, a model refractory to most potential antifibrotic agents. Endothelin-A receptor antagonists are promising antifibrotic agents in chronic liver disease.

Collagens in the liver extracellular matrix bind hepatocyte growth factor.

BACKGROUND & AIMS: Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, binds to heparan sulfate. Because immunoreactive HGF can be detected in the interstitial extracellular matrix (ECM), where little heparan sulfate is found, the aim of this study was to investigate binding of HGF to several collagens and noncollagenous ECM proteins in vitro. METHODS: 125I-labeled HGF was incubated with collagens I-VI, single collagen chains and their cyanogen bromide peptides, with fibronectin, fibrinogen, and laminin that were either immobilized on polystyrene or blotted to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biological activity of collagen-bound HGF was investigated in cell culture. RESULTS: HGF displayed binding of moderate affinity (Kd approximately 10(-9) mol/L) to immobilized collagen types I, III, IV, V, and VI. Binding of HGF to all collagens could be inhibited by single chains of either collagens I, III, or VI. Fragmentation with cyanogen bromide indicated unique collagenous peptides mediating the interaction. Collagen-bound HGF induced primary hepatocyte proliferation and MDCK cell scattering in a dose-dependent manner. CONCLUSIONS: Interstitial collagens I, III, V, and VI serve as abundant, low-affinity binding sites for HGF in the ECM. This interaction is mediated by unique collagenous peptides, opening the potential to modulate HGF availability and activity by collagen-derived peptide analogues.

Alcohol and liver fibrosis--pathobiochemistry and treatment.

In Western societies roughly 50% of all cases of liver cirrhosis are related to alcohol abuse. The oxidative metabolite of ethanol, acetaldehyde, often in conjunction with viral or metabolic liver disease, is implicated as the major cause for liver fibrogenesis. Acetaldehyde damages cell membranes, initiates lipid peroxidation and forms noxious protein adducts, resulting in the activation of Kupffer cells and perisinusoidal lipocytes/portal fibroblasts. The activation of lipocytes and fibroblasts to a proliferative and collagen-producing myofibroblast-like phenotype is triggered by the release of fibrogenic factors such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) from the activated Kupffer cells. Due to the socioeconomic burden inflicted by cirrhosis, antifibrotic treatment is urgently needed. Strategies to prevent or reverse cirrhosis must interrupt the continuous process of pathological wound healing in the liver. An antifibrotic effect has been demonstrated for the interferons, prostaglandins E and relaxin. Polyunsaturated lecithin, silymarin and ursodeoxycholic acid, agents with a high hepatotropism and a good safety-profile, appear to have antifibrotic properties. Targeted approaches include the specific removal of matrix-bound fibrogenic growth factors and the induction of stress-relaxation of the activated mesenchymal cells by biologically active matrix-peptides and their stable analogues. Since serum tests for the non-invasive assessment of collagen synthesis and degradation in the liver are now available, rapid progress in the development and clinical application of antifibrotic drugs can be anticipated.