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.
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.
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
(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
. 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
, 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.
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:
) robust, poorly reversible liver fibrosis,
) prominent ductular reaction, and
) 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.
Telomere shortening is associated with stem cell decline, fibrotic disorders, and premature aging through mechanisms that are incompletely understood. Here, we show that telomere shortening in livers ...of telomerase knockout mice leads to a p53-dependent repression of all seven sirtuins. P53 regulates non-mitochondrial sirtuins (Sirt1, 2, 6, and 7) post-transcriptionally through microRNAs (miR-34a, 26a, and 145), while the mitochondrial sirtuins (Sirt3, 4, and 5) are regulated in a peroxisome proliferator-activated receptor gamma co-activator 1 alpha-/beta-dependent manner at the transcriptional level. Administration of the NAD(+) precursor nicotinamide mononucleotide maintains telomere length, dampens the DNA damage response and p53, improves mitochondrial function, and, functionally, rescues liver fibrosis in a partially Sirt1-dependent manner. These studies establish sirtuins as downstream targets of dysfunctional telomeres and suggest that increasing Sirt1 activity alone or in combination with other sirtuins stabilizes telomeres and mitigates telomere-dependent disorders.
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•Telomere dysfunction downregulates sirtuins in the liver in a p53-dependent manner•p53 represses sirtuins through transcriptional and post-transcriptional mechanisms•Telomere dysfunction is linked to a steep decline in NAD(+) upon additional damage•NAD(+) supplementation stabilizes telomeres and improves liver fibrosis
Telomere dysfunction is implicated in the promotion of tissue damage and fibrosis through mechanisms that are incompletely understood. Amano et al. show that telomere dysfunction in liver tissue downregulates sirtuins through p53-dependent mechanisms. Increasing NAD(+) stabilizes telomeres, dampens DNA damage response, and improves telomere-dependent fibrosis in a partially Sirt1-dependent manner.
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.
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.
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.
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.
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.
Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models.
define the function of complex resident microbes and their association relevant to PSC patients by ...studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (
) mice and microbial profiles in PSC patient cohorts.
We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF
mice and targeted metagenomic analysis in PSC patients.
GF
mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased
and
liver translocation. Colonisation of GF
mice with translocated
and
strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated
mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and
positively associated, with PSC patients' clinical severity by Mayo risk scores.
We identified novel functionally protective and detrimental resident bacterial species in
mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.
The controllable synthesis of carbon nanofibers (CNF) and composites based on CNF (Metals/CNF) is of particular interest. In the present work, the samples of CNF were produced via ethylene ...decomposition over Co-Pt (0–100 at.% Pt) microdispersed alloys prepared by a reductive thermolysis of multicomponent precursors. XRD analysis showed that the crystal structure of alloys in the composition range of 5–35 at.% Pt corresponds to a fcc lattice based on cobalt (Fm-3m), while the CoPt (50 at.% Pt) and CoPt3 (75 at.% Pt) samples are intermetallics with the structure P4/mmm and Pm-3m, respectively. The microstructure of the alloys is represented by agglomerates of polycrystalline particles (50–150 nm) interconnected by the filaments. The impact of Pt content in the Co1−xPtx samples on their activity in CNF production was revealed. The interaction of alloys with ethylene is accompanied by the generation of active particles on which the growth of nanofibers occurs. Plane Co showed low productivity (~5.5 g/gcat), while Pt itself exhibited no activity at all. The addition of 15–25 at.% Pt to cobalt catalyst leads to an increase in activity by 3–5 times. The maximum yield of CNF reached 40 g/gcat for Co0.75Pt0.25 sample. The local composition of the active alloyed particles and the structural features of CNF were explored.
Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values ...and reader-dependent variability. Here we used an automated image analysis software to measure fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl
-induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed using a fully automated, unsupervised software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl
) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and by standard histological staging systems. CPA showed a high correlation with HYP content for CCl
(r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl
and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models.
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