[Show abstract][Hide abstract] ABSTRACT: Background & Aims: Platelet-derived growth factor beta (PDGF-B) is the major mitogen for hepatic stellate cells (HSC). We studied the cellular sources of PDGF-B and tested the antifibrotic efficacy efficacy of anti-platelet therapy and a novel high affinity anti-PDGF-B monoclonal antibody (mAB), MOR8457, in mouse models of biliary fibrosis. Methods: Cellular sources of PDGF-B were studied using QRT-PCR, ELISA and immunohistological methods. Platelet depletion was achieved with anti-CD41 mAb. MOR8457 was administered i.p. weekly in Mdr2-/- and in mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Liver fibrosis was evaluated by histology, biochemical determination of collagen content and QRT-PCR. Results: While protein levels of PDGF-B in liver and serum were elevated in fibrotic Mdr2-/- mice, PDGF-B mRNA was not increased. Platelet clusters were detected in sinusoids and identified as extrahepatic source of PDGF-B protein in Mdr2-/- mice. Platelet depletion with anti-CD41 antibody normalized hepatic PDGF-B protein within 48h, reduced the HSC activation marker αSMA and shifted gene expression towards a fibrolytic profile. Anti-platelet therapy with low-dose dietary aspirin significantly improved long-term, but not short-term, fibrosis outcomes in the Mdr2-/- model. Treatment of Mdr2-/- mice with MOR8457 for 6 weeks resulted in a dose-dependent decrease in fibrosis, with up to 45% reduction of collagen deposition and >50% inhibition of profibrogenic transcripts. Antifibrotic activity of MOR8457 was confirmed in the mechanistically different model of DDC-induced biliary fibrosis. In human disease, serum PDGF-BB levels were elevated in patients with liver fibrosis of various etiologies in early histological fibrosis stages (F1-2, n=16, p<0.05), as compared with patients with no significant fibrosis (F0, n=12). There was a correlation between serum PDGF-BB level, histological stages (F0-2, r=0.5762) and serum levels of liver fibrosis-specific 7S domain of type IV collagen (r=0.8947). Conclusions: Platelet activation and homing to the liver is the major source of PDGF-B dependent fibrogenic activation of HSC in biliary fibrosis. Pharmacological targeting of platelets with anti-platelet therapy or selective antibody inhibition of PDGF-B potently attenuate biliary fibrosis progression.
65th Annual Meeting of the American Association for the Study of Liver Diseases, Boston; 11/2014
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND & AIMS:
Platelet-derived growth factor-β (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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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 ± 355I U/L compared to 93 ± 65 IU/L in nonfibrotic), 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.
American Journal Of Pathology 05/2013; 183(1). DOI:10.1016/j.ajpath.2013.03.018 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND/AIM: Apoptosis Signal-Regulating Kinase 1 (ASK1) plays a critical role in mediating oxidative stress-induced apoptosis and inflammation. We hypothesized that hepatocyte apoptosis drives fibrosis progression and can be targeted therapeutically using a novel small molecule ASK1 inhibitor. Serum markers of cell death and apoptosis were studied in relation to hepatic fibrosis progression, and the antifibrotic efficacy of ASK1 inhibition was evaluated in a mouse model of TAA-induced liver fibrosis and in primary human hepatic stellate cells.
METHODS: Progressive liver fibrosis was induced in mice according to optimized, escalated oral gavage dose of carbon tetrachloride (CCL4) or intraperitoneal injection thioacetamide (TAA) for 1-12 weeks. Caspase-cleaved cytokeratin 18 (M30) levels, in serum were determined by ELISA and correlation analysis was performed on classical parameters of liver injury and fibrosis. Antifibrotic efficacy of an ASK1 inhibitor at 0.05, 0.15, and 0.3% in diet was tested in pre-established progressive TAA-induced liver fibrosis from 3 to 6 weeks of fibrosis induction.
RESULTS: During progression of CCL4-induced murine liver fibrosis, serum M30 levels, as a marker of apoptosis, increased in parallel with fibrosis progression. Interestingly, serum M30 levels correlated well with extent of fibrosis (hepatic collagen content as hydroxyproline, r=0.77), while unspecific markers of hepatic injury ALT and AST did not (r=0.06, r=0.40, respectively).
Targeting ASK1 with the small molecule inhibitor at 0.15% and 0.3% in diet significantly reduced hepatic collagen levels following chronic administration of TAA in BALB/c mice (34 and 37% of reduction by hydroxyproline, p<0.05), while a lower dose of 0.05% was ineffective. Both 0.15% and 0.3% doses strongly and dose-dependently reduced TGFβ1 mRNA levels. Sirius red staining showed histological improvement of liver fibrosis in both 0.15% and 0.3% group and dose-dependent decrease in TUNEL positive hepatocytes. Serum M30 levels were reduced by 40%, and ALT decreased up to 32% in ASK1 inhibitor-treated fibrotic animals as compared with the control group. No adverse events or toxicity in fibrotic or healthy mice with ASK1 inhibition were observed during the study. In primary human hepatic stellate, ASK1 inhibitor downregulated TGF-β-induced α-SMA expression and collagen deposition
1) Serum apoptosis marker M30 demonstrates good correlation with the degree of fibrosis in progressive liver fibrosis models.
2) Selective targeting of ASK1 using a small molecule inhibitor is safe and an effective antifibrotic treatment in pre-clinical settings.
63th Annual Meeting of the American Association for the Study of Liver Diseases, Boston; 11/2012
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND/AIMS: There are a total of 11 HDAC isoforms in humans and pan-HDAC inhibition has been reported to have an antifibrotic effect on hepatic stellate cells in vitro. However, antifibrotic therapy will likely require more selective HDAC isoforms-specific drug due to potential serious side effects expected with long-term pan-HDAC therapy. We tested two novel, orally available, small molecule inhibitors with selectivity for HDAC1&2 (GS-443023-6) and HDAC1,2&3 (GS-432780-5) in the pre-established thioacetamide (TAA)-induced progressive liver fibrosis model in BALB/c mice.
METHODS: Progressive liver fibrosis was induced by TAA according to optimized escalating dosing protocol (100-400mg/kg, 2x week i/p) in seventy-five male BALB/c mice for six weeks. HDAC inhibitors (GS-443023-6, GS-432780-5) in two doses (1.5 and 15mg/kg) or vehicle control (placebo) were administered via daily oral gavage starting from week 3 through week 6 of fibrosis induction (n=12-15/group). Antifibrotic efficacy was evaluated by histology, hepatic collagen levels via hydroxyproline, profibrogenic gene expression and serum transaminases levels.
RESULTS: Both HDAC inhibitors were effective in inhibiting progression of ongoing liver fibrosis even when the start of treatment was delayed (3-6 weeks). Collagen levels were reduced by 12% and 28% with GS-443023-6, and by 13% and 45% with GS-432780-5 for low and high dose groups, respectively (p<0.05). Connective tissue staining revealed dose-dependent inhibition of bridging fibrosis formation. Both drugs significantly and dose-dependently suppressed profibrogenic gene expression: procollagen-α1(I) (18-50%), TGFβ1 (18-55%), α-SMA (63- 100%), TIMP-1 (46-77%) and MMP-2 (16-36%). Serum ALT levels were reduced by up to 50% and 67% with GS-443023-6 and GS-432780-5 treatments, respectively (p<0.05). For all parameters, therapeutic effect of GS-432780-5 appeared to be more potent than GS-443023-6. No adverse events, weight loss, or hepatotoxicity were observed in fibrotic or healthy mice receiving either compounds during the study. Class I HDACs 1 and 3 (but not 2) mRNA was upregulated in liver tissue from cirrhotic patients vs. normal controls. In vitro, both HDAC inhibitors attenuated PDGF-BB and TGFβ-induced collagen deposition and α-SMA expression in human hepatic stellate cell cultures.
CONCLUSIONS: 1) Two selective HDAC inhibitors strongly and dose-dependently attenuated progression of pre-established liver fibrosis in mice.
2) Chronic selective HDAC inhibition appears to be safe in healthy mice and mice with chronic liver injury.
3) Antifibrotic activity of novel selective HDAC inhibitors should be evaluated in future clinical trials.
63th Annual Meeting of the American Association for the Study of Liver Diseases, Boston; 11/2012
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND/AIMS: Platelet-derived growth factor beta (PDGF-BB) is the major mitogen for hepatic stellate cells and myofibroblasts via its receptor (PDGFRβ). A paracrine PDGF-BB/PDGFRbeta loop between activated cholangiocytes and myofibroblasts was proposed as a central driving force of biliary fibrosis. We studied the cellular sources of PDGF-BB and tested the therapeutic efficacy of a novel anti-PDGF-B monoclonal antibody in two mouse models of biliary fibrosis.
METHODS: Three doses of anti-PDGF-B mAB (1, 10 and 100mg/kg), or isotype control (KLH, 100mg/kg) were administered i.p. once a week in 6 weeks old Mdr2-/- mice for 6 weeks and in mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-diet for 3 weeks (n=10-16 per group). One group received the non-selective tyrosine kinase inhibitor Gleevec (50mg/kg/day p.o.) as a comparator. Liver fibrosis was evaluated by histology, biochemical determination of collagen and analysis of profibrogenic gene expression by QRT-PCR.
RESULTS: PDGFRβ transcripts strongly correlated with fibrosis markers. Interestingly, while protein levels of PDGF-BB in liver homogenates were elevated 3-fold in fibrotic Mdr2-/- livers compared to WT controls, PDGF-B mRNA was not increased, indicating an extrahepatic source of PDGF-BB in biliary fibrosis. Western blot of Mdr2-/- liver homogenates showed strong positivity for the platelet-marker CD41 compared to CD41-negative WT livers. In Mdr2-/- mice, CD41+ platelet clusters were localized to periductular areas, and serum PDGF-BB (ELISA) was elevated >3-fold, suggesting significant intrahepatic platelet activation and degranulation. Weekly treatment of Mdr2-/- mice with anti-PDGF-B antibody resulted in an up to 99.3% depletion of PDGF-BB serum levels, a dose-dependent decrease of onion-skin fibrosis, up to 45% reduction of collagen deposition and a >50% inhibition of TGFβ1, TGFβ2, COL1A1 and α-SMA mRNA expression. Importantly, by all studied parameters anti-PDGF-B antibody outperformed the non-selective tyrosine kinase inhibitor Gleevec. Finally, a potent antifibrotic activity of the anti-PDGF-B antibody was validated in a second, mechanistically different model of DDC-induced biliary fibrosis (28 and 40% reduction of collagen content at 10 and 100mg/kg dosing regimen, respectively).
CONCLUSIONS: 1) In biliary fibrosis PDGF-BB is supplied from extrahepatic sources via platelet activation and degranulation rather than being produced locally.
2) A therapeutic anti-PDGF-B antibody significantly and dose-dependently inhibited progression of liver fibrosis in two mouse models of biliary fibrosis, outperforming non-selective tyrosine kinase inhibition.
63th Annual Meeting of the American Association for the Study of Liver Diseases, Boston; 11/2012
[Show abstract][Hide abstract] ABSTRACT: 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.
Hepatology Research 01/2012; 42(6):601-10. DOI:10.1111/j.1872-034X.2011.00960.x · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
PLoS ONE 06/2010; 5(6):e11256. DOI:10.1371/journal.pone.0011256 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.