Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver.

Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in HSCs.

BACKGROUND AND AIMS: Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. APPROACH AND RESULTS: We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-ß2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. CONCLUSIONS: TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.

ASK1/ p38 axis inhibition blocks the release of mitochondrial "danger signals" from hepatocytes and suppresses progression to cirrhosis and liver cancer.

BACKGROUND AND AIMS: Apoptosis Signal-regulating Kinase 1 (ASK1) is activated by various pathological stimuli and induces cell apoptosis through downstream p38 activation. We studied the effect of pharmacological ASK1 inhibition on cirrhosis and its sequelae using comprehensive preclinical in vivo and in vitro systems. APPROACH AND RESULTS: Short-term (4-6 wk) and long-term (24-44 wk) ASK1 inhibition using small molecule GS-444217 was tested in thioacetamide-induced and BALB/c. Mdr2-/- murine models of cirrhosis and HCC, and in vitro using primary hepatocyte cell death assays. Short-term GS-444217 therapy in both models strongly reduced phosphorylated p38, hepatocyte death, and fibrosis by up to 50%. Profibrogenic release of mitochondrial DAMP mitochondrial deoxyribonucleic acid from dying hepatocytes was blocked by ASK1 or p38 inhibition. Long-term (24 wk) therapy in BALBc.Mdr2 - / - model resulted in a moderate 25% reduction in bridging fibrosis, but not in net collagen deposition. Despite this, the development of cirrhosis was effectively prevented, with strongly reduced p21 + hepatocyte staining (by 72%), serum ammonia levels (by 46%), and portal pressure (average 6.07 vs. 8.53 mm Hg in controls). Extended ASK1 inhibition for 44 wk in aged BALB/c. Mdr2-/- mice resulted in markedly reduced tumor number and size by ~50% compared to the control group. CONCLUSIONS: ASK1 inhibition suppresses the profibrogenic release of mitochondrial deoxyribonucleic acid from dying hepatocytes in a p38-dependent manner and protects from liver fibrosis. Long-term ASK1 targeting resulted in diminished net antifibrotic effect, but the progression to liver cirrhosis and cancer in BALBc/ Mdr2- / - mice was effectively inhibited. These data support the clinical evaluation of ASK1 inhibitors in fibrotic liver diseases.

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells.

Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM . We then generated Tilam -deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl 4 ) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM . Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo . In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl 4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

3D analysis of microvasculature in murine liver fibrosis models using synchrotron radiation-based microtomography.

Cirrhosis describes the development of excess fibrous tissue around regenerative nodules in response to chronic liver injury and usually leads to irreversible organ damage and end-stage liver disease. During the development of cirrhosis, the formation of collagenous scar tissue is paralleled by a reorganization and remodeling of the hepatic vascular system. To date, macrovascular remodeling in various cirrhosis models has been examined using three-dimensional (3D) imaging modalities, while microvascular changes have been studied mainly by two-dimensional (2D) light microscopic and electron microscopic imaging. Here, we report on the application of high-resolution 3D synchrotron radiation-based microtomography (SRµCT) for the study of the sinusoidal and capillary blood vessel system in three murine models of advanced parenchymal and biliary hepatic fibrosis. SRµCT facilitates the characterization of microvascular architecture and identifies features of intussusceptive angiogenesis in progressive liver fibrosis in a non-destructive 3D manner.

Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin+- and αSMA+ -HSC and CD68+- macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Hepatocyte mitochondria-derived danger signals directly activate hepatic stellate cells and drive progression of liver fibrosis.

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.

Lysyl Oxidase (LOX) Family Members: Rationale and Their Potential as Therapeutic Targets for Liver Fibrosis.

The cross-linking of structural extracellular matrix (ECM) components, especially fibrillar collagens and elastin, is strongly implicated in fibrosis progression and resistance to fibrosis reversal. Lysyl oxidase family members (LOX and LOXL1 [lysyl oxidase-like 1], LOXL2 [lysyl oxidase-like 2], LOXL3 [lysyl oxidase-like 3], and LOXL4 [lysyl oxidase like 4]) are extracellular copper-dependent enzymes that play a key role in ECM cross-linking, but have also other intracellular functions relevant to fibrosis and carcinogenesis. Although the expression of most LOX family members is elevated in experimental liver fibrosis of diverse etiologies, their individual contribution to fibrosis is incompletely understood. Inhibition of the LOX family as a whole and of LOX, LOXL1, and LOXL2 specifically has been shown to suppress fibrosis progression and accelerate its reversal in rodent models of cardiac, renal, pulmonary, and liver fibrosis. Recent disappointing clinical trials with a monoclonal antibody against LOXL2 (simtuzumab) in patients with pulmonary and liver fibrosis dampened enthusiasm for LOX family member inhibition. However, this unexpected negative outcome may be related to the inefficient antibody, rather than to LOXL2, not qualifying as a relevant antifibrotic target. Moreover, LOX family members other than LOXL2 may prove to be attractive therapeutic targets. In this review, we summarize the structural hallmarks, expression patterns, covalent cross-linking activities, and modes of regulation of LOX family members and discuss the clinical potential of their inhibition to treat fibrosis in general and liver fibrosis in particular.

Comparison of murine steatohepatitis models identifies a dietary intervention with robust fibrosis, ductular reaction, and rapid progression to cirrhosis and cancer.

Progressive fibrosis, functional liver failure, and cancer are the central liver-related outcomes of nonalcoholic steatohepatitis (NASH) but notoriously difficult to achieve in mouse models. We performed a direct, quantitative comparison of hepatic fibrosis progression in well-defined methionine- and choline-deficient (MCD) and choline-deficient, amino-acid defined (CDAA) diets with increasing fat content (10-60% by calories) in C57Bl/6J and BALB/cAnNCrl mice. In C57Bl/6J mice, MCD feeding resulted in moderate fibrosis at week 8 (up to twofold increase in total hepatic collagen content) and progressive weight loss irrespective of dietary fat. In contrast, CDAA-fed mice did not lose weight and developed progressive fibrosis starting from week 4. High dietary fat in the CDAA diet model induced the lipid metabolism genes for sterol regulatory element-binding protein and stearoyl-CoA desaturase-2 and increased ductular reaction and fibrosis in a dose-dependent manner. Longitudinal analysis of CDAA with 60% fat (HF-CDAA) feeding revealed pronounced ductular reaction and perisinusoidal bridging fibrosis, with a sevenfold increase of hepatic collagen at week 12, which showed limited spontaneous reversibility. At 24 wk, HF-CDAA mice developed signs of cirrhosis with pan-lobular "chicken wire" fibrosis, 10-fold hydroxyproline increase, regenerative nodules, portal hypertension and elevated serum bilirubin and ammonia levels; 80% of mice (8/10) developed multiple glypican-3- and/or glutamine synthetase-positive hepatocellular carcinomas (HCC). High-fat (60%) supplementation of MCD in C57Bl/6J or feeding the HF-CDAA diet fibrosis-prone BALB/cAnNCrl strain failed to result in increased fibrosis. In conclusion, HF-CDAA feeding in C57Bl/6J mice was identified as an optimal model of steatohepatitis with robust fibrosis and ductular proliferations that progress to cirrhosis and HCC within 24 wk. This robust model will aid the testing of interventions and drugs for severe NASH.NEW & NOTEWORTHY Via quantitative comparison of several dietary models, we report HF-CDAA feeding in C57Bl/6 mice as an excellent model recapitulating several key aspects of fibrotic NASH: 1) robust, poorly reversible liver fibrosis, 2) prominent ductular reaction, and 3) progression to cirrhosis, portal hypertension, and liver cancer within 24 wk. High fat dose-dependently activates SREBP2/SCD2 genes and drives liver fibrosis in e HF-CDAA model. These features qualify the model as a robust and practical tool to study mechanisms and novel treatments addressing severe human NASH.

Selective deletion of ENTPD1/CD39 in macrophages exacerbates biliary fibrosis in a mouse model of sclerosing cholangitis.

Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39-/-), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-ß1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39fl/fl and Cd39fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.

Distinct roles of ecto-nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl4) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient (Entpd2 null) mice. After chronic CCl4 intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deletion of NTPDase2 does not have a substantial effect on biliary-type fibrosis in the setting of DDC feeding. In injured livers, NTPDase2 expression extends from the portal areas to fibrotic septae in pan-lobular (CCl4-induced) liver fibrosis; the same pattern was observed, albeit to a lesser extent in biliary-type (DDC-induced) fibrosis. Liver regeneration after partial hepatectomy is not substantively impaired in global Entpd2 null mice. NTPDase2 protects from liver fibrosis resulting from hepatocellular injury induced by CCl4. In contrast, Entpd2 deletion does not significantly impact fibrosis secondary to DDC injury or liver regeneration after partial hepatectomy. Our observations highlight mechanisms relating to purinergic signaling in the liver and indicate possible therapeutic avenues and new cellular targets to test in the management of hepatic fibrosis.

Optimized Mouse Models for Liver Fibrosis.

Fibrosis is the excessive accumulation of extracellular matrix components due to chronic injury, with collagens as predominant structural components. Liver fibrosis can progress to cirrhosis, which is characterized by a severe distortion of the delicate hepatic vascular architecture, the shunting of the blood supply away from hepatocytes and the resultant functional liver failure. Cirrhosis is associated with a highly increased morbidity and mortality and represents the major hard endpoint in clinical studies of chronic liver diseases. Moreover, cirrhosis is a strong cofactor of primary liver cancer. In vivo models are indispensable tools to study the cellular and molecular mechanisms of liver fibrosis and to develop specific antifibrotic therapies towards clinical translation. Here, we provide a detailed description of select optimized mouse models of liver fibrosis and state-of-the-art fibrosis readouts.

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 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).

Macrophage recruitment by fibrocystin-defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis.

UNLABELLED: Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a ß-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvß6 integrin, an activator of latent local transforming growth factor-ß1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. CONCLUSION: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time.

Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

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.

Integrin αvß6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.

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.

A new Mdr2(-/-) mouse model of sclerosing cholangitis with rapid fibrosis progression, early-onset portal hypertension, and liver cancer.

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.

Extrahepatic platelet-derived growth factor-ß, delivered by platelets, promotes activation of hepatic stellate cells and biliary fibrosis in mice.

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.

Vascular endothelial growth factor promotes fibrosis resolution and repair in mice.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored. METHODS: We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays. RESULTS: VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9. CONCLUSIONS: In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function.