RESUMO
Due to their bacterial ancestry, many components of mitochondria share structural similarities with bacteria. Release of molecular danger signals from injured cell mitochondria (mitochondria-derived damage-associated molecular patterns, mito-DAMPs) triggers a potent inflammatory response, but their role in fibrosis is unknown. Using liver fibrosis resistant/susceptible mouse strain system, we demonstrate that mito-DAMPs released from injured hepatocyte mitochondria (with mtDNA as major active component) directly activate hepatic stellate cells, the fibrogenic cell in the liver, and drive liver scarring. The release of mito-DAMPs is controlled by efferocytosis of dying hepatocytes by phagocytic resident liver macrophages and infiltrating Gr-1(+) myeloid cells. Circulating mito-DAMPs are markedly increased in human patients with non-alcoholic steatohepatitis (NASH) and significant liver fibrosis. Our study identifies specific pathway driving liver fibrosis, with important diagnostic and therapeutic implications. Targeting mito-DAMP release from hepatocytes and/or modulating the phagocytic function of macrophages represents a promising antifibrotic strategy.
Assuntos
Alarminas/imunologia , Células Estreladas do Fígado/imunologia , Hepatócitos/metabolismo , Cirrose Hepática/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alarminas/metabolismo , Animais , Apoptose/imunologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Hepatócitos/citologia , Hepatócitos/imunologia , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/imunologia , Fagocitose/imunologia , Tioacetamida/toxicidade , Adulto JovemRESUMO
BACKGROUND/AIMS: We studied the role of lysyl oxidase-like 2 (LOXL2) in collagen crosslinking and hepatic progenitor cell (HPC) differentiation, and the therapeutic efficacy of a LOXL2-blocking monoclonal antibody on liver fibrosis progression/reversal in mice. METHODS: Anti-LOXL2 antibody, control antilysyl oxidase antibody or placebo was administered during thioacetamide (TAA)-induced fibrosis progression or during recovery. Therapeutic efficacy in biliary fibrosis was tested in BALB/c.Mdr2-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice. Collagen crosslinking, fibrosis progression and reversal were assessed histologically and biochemically. HPC differentiation was studied in primary EpCAM(+) liver cells in vitro. RESULTS: LOXL2 was virtually absent from healthy but strongly induced in fibrotic liver, with predominant localisation within fibrotic septa. Delayed anti-LOXL2 treatment of active TAA fibrosis significantly reduced collagen crosslinking and histological signs of bridging fibrosis, with a 53% reduction in morphometric collagen deposition. In established TAA fibrosis, LOXL2 inhibition promoted fibrosis reversal, with enhanced splitting and thinning of fibrotic septa, and a 45% decrease in collagen area at 4â weeks of recovery. In the Mdr2-/- and DDC-induced models of biliary fibrosis, anti-LOXL2 antibody similarly achieved significant antifibrotic efficacy and suppressed the ductular reaction, while hepatocyte replication increased. Blocking LOXL2 had a profound direct effect on primary EpCAM(+) HPC behaviour in vitro, promoting their differentiation towards hepatocytes, while inhibiting ductal cell lineage commitment. CONCLUSIONS: LOXL2 mediates collagen crosslinking and fibrotic matrix stabilisation during liver fibrosis, and independently promotes fibrogenic HPC differentiation. By blocking these two convergent profibrotic pathways, therapeutic LOXL2 inhibition attenuates both parenchymal and biliary fibrosis and promotes fibrosis reversal.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Cirrose Hepática , Animais , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Hepatócitos/fisiologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular/métodos , Recuperação de Função Fisiológica/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
The pathogenesis of primary sclerosing cholangitis (PSC) and the mechanistic link to inflammatory bowel disease remain ill-defined. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1)/clusters of differentiation (CD) 39, the dominant purinergic ecto-enzyme, modulates intestinal inflammation. Here, we have explored the role of CD39 in biliary injury and fibrosis. The impact of CD39 deletion on disease severity was studied in multidrug resistance protein 2 (Mdr2)-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse models of sclerosing cholangitis and biliary fibrosis. Antibody-mediated CD8+ T-cell depletion, selective gut decontamination, experimental colitis, and administration of stable adenosine triphosphate (ATP) agonist were performed. Retinoic acid-induced gut imprinting on T cells was studied in vitro. Over half of Mdr2-/-;CD39-/- double mutants, expected by Mendelian genetics, died in utero. Compared to Mdr2-/-;CD39+/+, surviving Mdr2-/-;CD39-/- mice demonstrated exacerbated liver injury, fibrosis, and ductular reaction. CD39 deficiency led to a selective increase in hepatic CD8+ T cells and integrin α4ß7, a T-cell gut-tropism receptor. CD8+ cell depletion in Mdr2-/-;CD39-/- mice diminished hepatobiliary injury and fibrosis. Treatment with antibiotics attenuated, whereas dextran sulfate sodium-induced colitis exacerbated, liver fibrosis in Mdr2-/- mice. Colonic administration of αß-ATP into CD39-sufficient Mdr2-/- mice triggered hepatic CD8+ cell influx and recapitulated the severe phenotype observed in Mdr2-/-;CD39-/- mice. In vitro, addition of ATP promoted the retinoic acid-induced imprinting of gut-homing integrin α4ß7 on naive CD8+ cells. CD39 expression was relatively low in human normal or PSC livers but abundantly present on immune cells of the colon and further up-regulated in samples of patients with inflammatory bowel disease. Conclusion: CD39 deletion promotes biliary injury and fibrosis through gut-imprinted CD8+ T cells. Pharmacological modulation of purinergic signaling may represent a promising approach for the treatment of PSC. (Hepatology Communications 2017;1:957-972).
RESUMO
UNLABELLED: Integrin αvß6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFß1). In human liver cirrhosis, αvß6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvß6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvß6-dependent TGFß1 activation. Freshly isolated αvß6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvß6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvß6 antibody is recapitulated by TGFß1 neutralization and rescued by addition of bioactive TGFß1. Genetic disruption or selective targeting of αvß6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. CONCLUSION: These results suggest that αvß6 is a promising target for chronic fibrotic liver diseases and associated cancers.
Assuntos
Antígenos de Neoplasias/fisiologia , Carcinogênese , Integrinas/fisiologia , Cirrose Hepática/etiologia , Células-Tronco/fisiologia , Animais , Colangite Esclerosante/etiologia , Fibrose/etiologia , Hepatócitos , Humanos , Fígado/patologia , Neoplasias Hepáticas/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversalin vivousing chronic administration of irreversible LOX inhibitor ß-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl4)-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl4withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its resolution.-Liu, S. B., Ikenaga, N., Peng, Z.-W., Sverdlov, D. Y., Greenstein, A., Smith, V., Schuppan, D., Popov, Y. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.
Assuntos
Colágeno/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Aminopropionitrilo/administração & dosagem , Aminopropionitrilo/farmacologia , Animais , Tetracloreto de Carbono , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Progressão da Doença , Fibrose , Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We previously characterized the Mdr2(Abcb4)(-/-) mouse as a reproducible model of chronic biliary liver disease. However, it demonstrates relatively slow fibrosis progression, possibly due to its fibrosis-resistant genetic background. We aimed to improve the model by moving it onto a fibrosis-susceptible background. We generated novel BALB/c.Mdr2(-/-) mouse via genetic backcross onto highly fibrosis-susceptible BALB/c substrain, identified in inbred mouse strain screening. Liver fibrosis, portal pressure, and hepatic tumor burden in BALB/c.Mdr2(-/-) mice were studied up to 1 year of age in direct comparison to parental strain FVB.Mdr2(-/-). BALB/c.Mdr2(-/-) mice developed periductular onion-skin type fibrotic lesions and pronounced ductular reaction starting from 4 weeks of age. Compared to parental strain, BALB/c.Mdr2(-/-) mice demonstrated dramatically accelerated liver fibrosis, with threefold increase in collagen deposition and bridging fibrosis/early signs of cirrhosis at 12 weeks. This was accompanied by early-onset severe portal hypertension and twofold to fourfold increase in profibrogenic transcripts Col1a1 [procollagen α1(I)], Tgfb1, and Timp1. Primary liver cancers in BALB/c.Mdr2(-/-) developed earlier, with greater tumor burden compared to FVB.Mdr2(-/-). BALB/c.Mdr2(-/-) mice have unprecedented degree and rapidity of hepatic fibrosis progression and clinically relevant cirrhosis complications, such as early-onset portal hypertension and primary liver cancers. This new model will facilitate development of antifibrotic drugs and studies into mechanisms of biliary fibrosis progression.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Colangite Esclerosante , Hipertensão Portal , Cirrose Hepática , Neoplasias Hepáticas , Animais , Colangite Esclerosante/genética , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Hipertensão Portal/genética , Hipertensão Portal/metabolismo , Hipertensão Portal/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND & AIMS: Platelet-derived growth factor-ß (PDGFB) is a mitogen for hepatic stellate cells (HSCs). We studied the cellular sources of PDGFB and the effects of a high-affinity monoclonal antibody against PDGFB (MOR8457) in mouse models of biliary fibrosis. METHODS: Cellular sources of PDGFB were identified using quantitative reverse-transcription polymerase chain reaction, biochemical, and immunohistologic methods. Mice with advanced biliary fibrosis, MDR2(Abcb4)-null mice, and C57Bl/6 (control) mice were placed on 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diets and were given weekly intraperitoneal injections of MOR8457. Platelets were depleted from MDR2-null mice by injection of an antibody against CD41, or inhibited with diets containing low-dose aspirin. Liver tissues were collected and analyzed by quantitative reverse-transcription PCR and histologic and biochemical analyses. RESULTS: Levels of PDGFB protein, but not messenger RNA, were increased in fibrotic livers of MDR2-null mice, compared with control mice. Platelet clusters were detected in the hepatic endothelium, in close proximity to HSCs, and were identified as a source of PDGFB protein in MDR2-null mice. Levels of the PDGFB were increased in serum samples from patients with early stages of liver fibrosis of various etiologies (F1-2, n = 16; P < .05), compared with nonfibrotic liver tissue (F0, n = 12). Depletion of platelets from MDR2-null mice normalized hepatic levels of PDGFB within 48 hours, reducing levels of a marker of HSC activation (α-smooth muscle actin) and expression of genes that promote fibrosis. Diets supplemented with low-dose aspirin reduced circulating serum and hepatic levels of PDGFB and significantly reduced progression of fibrosis in MDR2-null mice over 1 year. MOR8457 produced a dose-dependent decrease in liver fibrosis in MDR2-null mice, reducing collagen deposition by 45% and expression of fibrosis-associated genes by 50%, compared with mice given a control antibody. In vitro, platelets activated freshly isolated HSCs (induction of α-smooth muscle actin and fibrosis-associated genes) via a PDGFB-dependent mechanism. MOR8457 also reduced liver fibrosis in mice placed on DDC-supplemented diets. CONCLUSIONS: Platelets produce PDGFB to activate HSC and promote fibrosis in MDR2-null mice and mice on DDC-supplemented diets. Antiplatelet therapy or selective inhibition of PDGFB might reduce biliary fibrosis in patients with liver disease.
Assuntos
Ductos Biliares Extra-Hepáticos/metabolismo , Plaquetas/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/imunologia , RNA Mensageiro/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.
Assuntos
Hepatectomia , Cirrose Hepática/fisiopatologia , Regeneração Hepática , Alanina Transaminase/sangue , Animais , Biomarcadores/metabolismo , Morte Celular , Colágeno/metabolismo , Imunofluorescência , Estimativa de Kaplan-Meier , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Biomarkers are becoming increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.
Assuntos
Biomarcadores/urina , Monitorização Fisiológica , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Humanos , Cirrose Hepática/urina , Espectrometria de Massas , Camundongos , Nanopartículas , Neoplasias/urina , Peptídeo Hidrolases/urinaRESUMO
AIM: While low grade inflammation persists in both visceral fat and hepatic tissue in obesity, these changes often result in progressive disease and fibrosis only in the liver and not in adipose tissue. We hypothesized that a tissue-specific difference in obesity-induced inflammatory cell infiltrate may be responsible for such organ difference in susceptibility to fibrosis. METHODS: Mice were fed either standard chow or a high fat diet over 19 weeks. Hepatic steatosis was assessed by histology and quantified via magnetic resonance spectroscopy. Immunohistochemistry staining for macrophage subsets and quantitative reverse transcription-polymerase chain reaction for matrix metalloproteinase (MMP)- and fibrosis-related gene expression was performed in paired livers and visceral (epididymal) fat pads at early (9 weeks) and advanced (19 weeks) stages of progressive diet-induced obesity. RESULTS: Up to 19 weeks of high fat feeding led to the development of obesity and hepatic steatosis, as well as increased gene expression of Mmp12, Mmp13 and Timp1 in predominantly adipose tissue, and to a lesser extent of liver tissue. In contrast to visceral fat, cell counts for macrophages as well as profibrogenic gene signaling in liver tissue during development of diet-induced obesity remained largely unchanged. CONCLUSIONS: Development of diet-induced obesity in the mouse increased inflammatory macrophages counts in adipose tissue rather than the liver. This was associated with greater increases in MMP expression in adipose tissue compared with liver. We propose that attenuated hepatic MMP expression in livers and adipose tissue of obese mice shifts the balance of fibrogenesis/fibrolysis and predispose the liver to development of fibrosis.
RESUMO
BACKGROUND & AIMS: The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice. METHODS: Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction. RESULTS: Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated. CONCLUSIONS: TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.
Assuntos
Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Cirrose Hepática Experimental/enzimologia , Fígado/patologia , RNA/genética , Transglutaminases/genética , Animais , Apoptose/genética , Progressão da Doença , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas de Ligação ao GTP/biossíntese , Fígado/enzimologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transglutaminases/biossínteseRESUMO
BACKGROUND: Liver fibrosis is characterized by excessive synthesis of extracellular matrix proteins, which prevails over their enzymatic degradation, primarily by matrix metalloproteinases (MMPs). The effect of pharmacological MMP inhibition on fibrogenesis, however, is largely unexplored. Inflammation is considered a prerequisite and important co-contributor to fibrosis and is, in part, mediated by tumor necrosis factor (TNF)-alpha-converting enzyme (TACE). We hypothesized that treatment with a broad-spectrum MMP and TACE-inhibitor (Marimastat) would ameliorate injury and inflammation, leading to decreased fibrogenesis during repeated hepatotoxin-induced liver injury. METHODOLOGY/PRINCIPAL FINDINGS: Liver fibrosis was induced in mice by repeated carbon tetrachloride (CCl4) administration, during which the mice received either Marimastat or vehicle twice daily. A single dose of CCl4 was administered to investigate acute liver injury in mice pretreated with Marimastat, mice deficient in Mmp9, or mice deficient in both TNF-alpha receptors. Liver injury was quantified by alanine aminotransferase (ALT) levels and confirmed by histology. Hepatic collagen was determined as hydroxyproline, and expression of fibrogenesis and fibrolysis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. Marimastat-treated animals demonstrated significantly attenuated liver injury and inflammation but a 25% increase in collagen deposition. Transcripts related to fibrogenesis were significantly less upregulated compared to vehicle-treated animals, while MMP expression and activity analysis revealed efficient pharmacologic MMP-inhibition and decreased fibrolysis following Marimastat treatment. Marimastat pre-treatment significantly attenuated liver injury following acute CCl4-administration, whereas Mmp9 deficient animals demonstrated no protection. Mice deficient in both TNF-alpha receptors exhibited an 80% reduction of serum ALT, confirming the hepatoprotective effects of Marimastat via the TNF-signaling pathway. CONCLUSIONS/SIGNIFICANCE: Inhibition of MMP and TACE activity with Marimastat during chronic CCl4 administration counterbalanced any beneficial anti-inflammatory effect, resulting in a positive balance of collagen deposition. Since effective inhibition of MMPs accelerates fibrosis progression, MMP inhibitors should be used with caution in patients with chronic liver diseases.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Ácidos Hidroxâmicos/farmacologia , Cirrose Hepática/prevenção & controle , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Alanina Transaminase/sangue , Animais , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/sangue , Regulação para Baixo/efeitos dos fármacos , Cirrose Hepática/sangue , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.