Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

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

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Biomarkers are 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: 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 they can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and can substantially improve early detection of cancer compared with clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and to point-of-care diagnostics

The ectonucleotidase ENTPD1/CD39 limits biliary injury and fibrosis in mouse models of sclerosing cholangitis

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 α4β7, 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 αβ‐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 α4β7 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

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

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.National Institutes of Health (U.S.) (Bioengineering Research Partnership R01 CA124427)Kathy and Curt Marble Cancer Research FundNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32CA159496-01

Broad-Spectrum Matrix Metalloproteinase Inhibition Curbs Inflammation and Liver Injury but Aggravates Experimental Liver Fibrosis in Mice

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)-α-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 CCl4was administered to investigate acute liver injury in mice pretreated with Marimastat, mice deficient in Mmp9, or mice deficient in both TNF-α 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-α 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 CCl4administration 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

Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis

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

Selective targeting of lysyl oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression and accelerates its reversal

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 4 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

Marimastat treatment ameliorated hepatic injury and the inflammatory response following repeated carbon tetrachloride (CCl4) administration.

<p>Marimastat treatment significantly reduced serum alkaline phosphatase levels (A), and resulted in a 14-fold decrease of serum ALT (B), indicating decreased hepatic injury. Serum TNF-α receptor II (p75) levels as measured by ELISA decreased following Marimastat treatment, suggesting successful inhibition of TNF-α converting enzyme (TACE) and an ameliorated inflammatory response. IL-6 serum levels as measured by ELISA increased following Marimastat treatment, suggesting hepatoprotection and stimulated liver regeneration (D). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; ALT, alanine aminotransferase; TNF, tumor necrosis factor; IL, interleukin; *, P<0.05; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error.</p

Marimastat decreases hepatic stellate cell (HSC) activation, but increases recruitment of inflammatory cells.

<p>Chronic carbon tetrachloride (CCl4) administration in animals treated with Marimastat resulted in a decreased activation of HSCs, as identified by alpha-smooth muscle actin (α-SMA) staining (A). Quantification revealed that following chronic CCl4 administration, Marimastat treated animals had a 74% decrease of activated HSCs, compared to controls (B). Liver sections from animals that were chronically challenged with CCl4 showed that resident T cells (CD3, C,D) and macrophages (F4/80, E,F) counts increased up to 2-fold upon Marimastat treatment. Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; α-SMA, alpha-smooth muscle actin; *, P<0.05; **, P<0.01; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error. Original magnification: 200×.</p