Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. METHODS: Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-ß1 with or without treatment with an ALK5/TGF-ß1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression. RESULTS: TGF-ß1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-ß1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-ß1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. CONCLUSIONS: TGF-ß1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.

Addition of trans fat and alcohol has divergent effects on atherogenic diet-induced liver injury in rodent models of steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are common causes of chronic liver disease. The overlap between ALD and NAFLD suggests the existence of metabolic steatohepatitis. Development of in vivo models that reflect various aspects of human steatohepatitis is essential for drug discovery. We aimed to characterize several models of steatohepatitis (SH) and to investigate whether the pathology could be modulated. Sprague-Dawley rats were fed a high-fat diet (HFD) for 9 wk, followed by either a high-fat, high-cholesterol and cholate diet (HFC) or a HFC diet containing 13% trans fat (HFC-TF). A subset received 15% ethanol-water twice a week for 12 wk. Serum triglycerides, cholesterol, LDL, HDL, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and rodent NH2-terminal propeptide of type III collagen (rPRO-C3) were assessed. The liver was weighed and evaluated using modified Nonalcoholic Steatohepatitis Clinical Research Network histological score system criteria. All diets induced hepatomegaly, but only HFC-TF increased the size of visceral adipose tissue. Trans fat augmented HFC-induced dyslipidemia, and cholesterol was higher and HDL was lower in the HFC-TF groups. Alcohol lowered triglycerides in both dietary groups. HFC elevated ALT and AST, which were lowered by trans fat. All diets induced histological SH, addition of trans fat induced more steatosis but less inflammation. Inclusion of alcohol augmented the HFC-induced inflammation. All diets induced mild fibrosis. Inclusion of trans fat and alcohol significantly increased rPRO-C3. The addition of trans fat reduced the HFC-induced inflammation but augmented steatosis and dyslipidemia. Inclusion of alcohol induced a more inflammatory and fibrogenic phenotype.NEW & NOTEWORTHY Alcoholic liver disease and nonalcoholic liver disease share significant overlap, which suggests the existence of metabolic steatohepatitis. Trans fat has been implicated in steatohepatitis development. Here, we show that the addition of trans fat to an atherogenic diet results in a more steatotic but less inflammatory phenotype, whereas the addition of alcohol to an atherogenic diet augments the inflammatory and fibrogenic properties of the diet.

Biochemical and histological characterisation of an experimental rodent model of non-alcoholic steatohepatitis - Effects of a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist and a glucagon-like peptide-1 analogue.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a prevalent disease that is highly associated with the metabolic syndrome and type II diabetes. The development of in vivo models that reflect all nuances of the human NASH pathology is essential for drug discovery and development. We aimed to further characterise a dietary induced model of NASH both biochemically and histologically. In addition, we also investigated whether pioglitazone and liraglutide, drugs that have both been investigated as potential NASH treatments, could modulate the pathological changes induced by the NASH diet. Furthermore, to aid the translation of data from pre-clinical in vivo models, we aimed to adapt the NASH Clinical Research Network (CRN) histological score system for use in rodent studies. METHODS: Sprague Dawley rats were fed a high-fat diet (HFD) for 9 weeks, after which they were switched to a high fat, high cholesterol and cholate diet (HFCC) for 12 weeks. The rats were divided into treatment groups, receiving either 30 mg/kg pioglitazone p.o. SID or liraglutide s.c. 200 µg/kg BID or the respective vehicles. Serum levels of triglycerides (TG), cholesterol (Chol), LDL, HDL, AST and ALT, as well as body weight were assessed in all subjects. Upon termination, the liver was weighed and evaluated histologically using modified NASH-CRN criteria. RESULTS: HFCC feeding induced severe hepatic injury and hepatomegaly as indicated by significant increases in AST, ALT and an increased liver weight. Additionally, HFCC feeding induced dyslipidaemia, significant increases in circulating cholesterol and LDL were observed. No obesogenic effect of the HFCC diet was observed, though the diet did induce insulin resistance. Histological analysis showed that the HFCC diet induced several NASH like features, though it did not induce the development of severe fibrosis. However, microgranulomas were often prevalent in addition to lobular inflammatory foci. Pioglitazone showed little efficacy upon both biochemical and histological features. However, liraglutide induced weight loss, improved glycaemic control, reduced ALT and AST and showed some beneficial effects upon steatosis and lobular inflammation. CONCLUSION: Similar to previous reports we have shown that the atherogenic diet, HFCC, induces a phenotype akin to that seen in human NASH patients. Despite inducing all histological features of NASH, HFCC feeding does not promote the development of significant fibrosis within rodents. Pioglitazone and liraglutide have been investigated as potential NASH treatments. Within this model of NASH we have shown that pioglitazone has little efficacy, whereas liraglutide reduced the levels of circulating aminotransferases and had some beneficial effects upon NASH histological parameters.

The future R&D landscape in non-alcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is emerging as a major public health issue for the 21st century and is associated with significant liver-related morbidity and mortality. At present, there are no approved drug therapies for NASH. Consequently, NASH has become the focus of significant public and private research and development. In this review, we highlight the research and development (R&D) challenges and opportunities in this emerging therapeutic area. In particular, we consider the impact of the development of new biomarker strategies on clinical trial execution and design, and the positioning of single and combination therapies in future approaches to the treatment of NASH.

Corrigendum: The RA-MAP Consortium: a working model for academia-industry collaboration.

This corrects the article DOI: 10.1038/nrrheum.2017.200.

The RA-MAP Consortium: a working model for academia-industry collaboration.

Collaboration can be challenging; nevertheless, the emerging successes of large, multi-partner, multi-national cooperatives and research networks in the biomedical sector have sustained the appetite of academics and industry partners for developing and fostering new research consortia. This model has percolated down to national funding agencies across the globe, leading to funding for projects that aim to realise the true potential of genomic medicine in the 21st century and to reap the rewards of 'big data'. In this Perspectives article, the experiences of the RA-MAP consortium, a group of more than 140 individuals affiliated with 21 academic and industry organizations that are focused on making genomic medicine in rheumatoid arthritis a reality are described. The challenges of multi-partner collaboration in the UK are highlighted and wide-ranging solutions are offered that might benefit large research consortia around the world.

Tissue turnover of collagen type I, III and elastin is elevated in the PCLS model of IPF and can be restored back to vehicle levels using a phosphodiesterase inhibitor.

BACKGROUND: The aim of this study was to develop and validate a model for pulmonary fibrosis, using ex vivo tissue cultures of lungs from bleomycin treated animals, enabling the investigation of fibrosis remodeling using novel biomarkers for the detection of ECM protein fragments. The combination of in vivo and ex vivo models together with ECM remodeling markers may provide a translational tool for screening of potential treatments for IPF. METHODS: Twenty female Sprague-Dawley rats, twelve weeks of age, were administrated either two doses of bleomycin (BLM) (n = 14) or saline (n = 6) I.T., two days apart. Ten rats were euthanized at day seven and the remaining ten rats at day fourteen, after the last dose. Precision-cut lung slices (PCLS) were made and cultured for 48 h. Ten female Sprague-Dawley rats, twelve weeks of age, were administrated either two doses of BLM (n = 7) or saline (n = 3) I.T., two days apart. The rats were euthanized fourteen days after the last dose. PCLS were made and cultured for 48 h in: medium, medium + 100 µM IBMX (PDE inhibitor), or medium + 10 µM GM6001 (MMP inhibitor). Turnover of type I collagen (P1NP, C1M), type III collagen (iP3NP, C3M) and elastin degradation (ELM7) was measured in the supernatant of the cultured PCLS. RESULTS: P1NP, C1M, iP3NP, C3M and ELM7 were significantly increased in supernatants from BLM animals (P ≤ 0.05 - P ≤ 0.0001) when compared to controls. P1NP, C1M, iP3NP, C3M and ELM7 were significantly increased in supernatants from day seven BLM animals compared to day fourteen BLM animals (P ≤ 0.05 - P ≤ 0.0001). P1NP, C1M, iP3NP, C3M and ELM7 were significantly decreased when adding IBMX to the culture medium of fibrotic lung tissue (P ≤ 0.05 - P ≤ 0.0001). C1M, C3M and ELM7 were significantly decreased when adding GM6001 to the culture medium (P ≤ 0.05 - P ≤ 0.0001). Sirius Red and Orcein staining confirmed the presence of collagen and elastin deposition in the lungs of the animals receiving BLM. CONCLUSIONS: The protein fingerprint technology allows the assessment of ECM remodeling markers in the BLM PCLS model. By combining in vivo, ex vivo models and the protein fingerprint technology in the fibrotic phase of the model, we believe the chance of translation from animal model to human is markedly increased.

Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment.

OBJECTIVE: Aggrecan is a critical component of cartilage extracellular matrix. Several members of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1ß (IL-1ß) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated. METHODS: MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1ß in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1ß or meniscotibial ligament transaction was carried out in MMP17 null mice. RESULTS: The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1ß mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1ß into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17. CONCLUSION: These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: prominent role of Wnt-induced signaling protein 1.

OBJECTIVE: Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS: Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS: Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION: This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

Characterisation of cell-penetrating peptide-mediated peptide delivery.

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia

Expression profiling of metalloproteinases and their inhibitors in cartilage.

OBJECTIVE: To profile the expression of all known members of the matrix metalloproteinase (MMP), ADAMTS, and tissue inhibitor of metalloproteinases (TIMP) gene families in normal cartilage and cartilage from patients with osteoarthritis (OA). METHODS: Human cartilage was obtained from femoral heads at joint replacement for OA or following fracture to the femoral neck. Total RNA was purified, and gene expression was assayed using quantitative real-time polymerase chain reaction. RESULTS: Several members of the above gene families were regulated in OA. Genes that showed increased expression in OA were MMP13, MMP28, and ADAMTS16 (all at P < 0.001), MMP9, MMP16, ADAMTS2, and ADAMTS14 (all at P < 0.01), and MMP2, TIMP3, and ADAMTS12 (all at P < 0.05). Genes with decreased expression in OA were MMP1, MMP3, and ADAMTS1 (all at P < 0.001), MMP10, TIMP1, and ADAMTS9 (all at P < 0.01), and TIMP4, ADAMTS5, and ADAMTS15 (all at P < 0.05). Correlation analysis revealed that groups of genes across the gene families were coexpressed in cartilage. CONCLUSION: This is the first comprehensive expression profile of all known MMP, ADAMTS, and TIMP genes in cartilage. Elucidation of patterns of expression provides a foundation with which to understand mechanisms of gene regulation in OA and potentially to refine the specificity of antiproteolytic therapies.

Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor.

Matrix metalloproteinases (MMPs) and their inhibitors are important in connective tissue re-modelling in diseases of the cardiovascular system, such as atherosclerosis. Various members of the MMP family have been shown to be expressed in atherosclerotic lesions, but MMP9 is consistently seen in inflammatory atherosclerotic lesions. MMP9 over-expression is implicated in the vascular re-modelling events preceding plaque rupture (the most common cause of acute myocardial infarction). Reduced MMP9 activity, either by genetic manipulation or through pharmacological intervention, has an impact on ventricular re-modelling following infarction. MMP9 activity may therefore represent a key mechanism in the pathogenesis of heart failure. We have determined the crystal structure, at 2.3 A resolution, of the catalytic domain of human MMP9 bound to a peptidic reverse hydroxamate inhibitor as well as the complex of the same inhibitor bound to an active-site mutant (E402Q) at 2.1 A resolution. MMP9 adopts the typical MMP fold. The catalytic centre is composed of the active-site zinc ion, co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402). The main differences between the catalytic domains of various MMPs occur in the S1' subsite or selectivity pocket. The S1' specificity site in MMP9 is perhaps best described as a tunnel leading toward solvent, as in MMP2 and MMP13, as opposed to the smaller pocket found in fibroblast collagenase and matrilysin. The present structure enables us to aid the design of potent and specific inhibitors for this important cardiovascular disease target.