Modified culture protocol for testing anti-fibrotic drugs using precision-cut liver slices

Precision-cut liver slices (PCLS) have been widely utilized in various multicellular studies due to liver architecture and intercellular communication remain preserved. In studying anti-fibrotic drugs using PCLS, the culture method is currently based on a protocol for applications in drug metabolism and toxicity. This published protocol requires the pre-incubation step to incubate PCLS in a drug-free condition. However, this lag period may limit the effect of anti-fibrotic drugs. Therefore, we aimed to study whether a modification of culture protocol provided benefits in studying anti-fibrotic drugs. In this study, PCLS were cultured with LY2109761, the representative anti-fibrotic drug, by either published or modified protocol. Tissue viability and gene/protein expression of fibrosis-related markers of cultured PCLS were assessed. We found that, despite slightly depressed viability (ATP/protein of PCLS was 71-77% of those cultured by the published protocol), spontaneous fibrosis which is a distinctive feature of PCLS was greatly pronounced by the modified culture protocol. As a result, this novel culture protocol demonstrated apparent inhibitory effect of LY2109761 on the gene expression of fibrosis markers, particularly heat shock protein 47 and alpha smooth muscle actin (the inhibition was higher by 44% and 23% when compared to those cultured by published protocol respectively). This modified culture protocol could be a useful alternative for testing anti-fibrotic drugs using PCLS. Besides the differences in tissue viability and spontaneous fibrosis, the modified culture protocol might reduce time and cost of experiment

Exploring anti-fibrotic drugs:Focusing on an ex vivo model of fibrosis

Als een orgaan door ziekte beschadigd raakt, zet het lichaam een ingewikkeld proces in gang om de schade te herstellen. Soms wordt daarbij zoveel littekenweefsel (fibrose) gevormd, dat iemand van de regen in de drup belandt. In plaats van beter gaat het orgaan steeds slechter functioneren en in het ergste geval houdt er gewoon mee op. In de Westerse wereld is 45% van alle sterfgevallen het gevolg van ziekten die gepaard gaan met fibrose. Tot op heden zijn er geen effectieve medicijnen voorhanden. Met behulp van flinterdunne weefselplakjes, zg. precision-cut slices, heeft Luangmonkong het fenomeen leverfibrose buiten het lichaam (ex vivo) nagebootst en verschillende stoffen getest op hun anti-fibrotische werking. In deel 1 van dit proefschrift beschrijft Luangmonkong het pathofysiologisch mechanisme van leverfibrose en de anti-fibrotische werking van diverse kandidaat-medicijnen. Hij toont aan dat Galunisertib, een remmer van de pro-fibrotische factor TGF-beta, een veelbelovend middel is tegen leverfibrose. Om de klinische effectiviteit in kaart te brengen is aanvullend (humaan) onderzoek nodig. Ook laat Luangmonkong zien dat het gelijktijdig remmen van meerdere signaalroutes en groeifactoren een synergistisch effect heeft. Anders gezegd: 1 + 1 = 3. Dit impliceert dat een geschikt medicijn tegen fibrose op meerdere fronten actief moet zijn. In deel 2 beschrijft Luangmonkong de ontwikkeling van een nieuw experimenteel ziektemodel voor niet-alcoholische leververvetting. Dit model is een waardevolle toevoeging binnen het onderzoeksveld van orgaanfibrose en uitermate geschikt voor toekomstig geneesmiddelenonderzoek

Host microbiota dictates the proinflammatory impact of LPS in the murine liver

Gut microbiota can impact liver disease development via the gut-liver axis. Liver inflammation is a shared pathological event in various liver diseases and gut microbiota might influence this pathological process. In this study, we studied the influence of gut microbiota on the inflammatory response of the liver to lipopolysaccharide (LPS). The inflammatory response to LPS (1–10 μg/ml) of livers of specific-pathogen-free (SPF) or germ-free (GF) mice was evaluated ex vivo, using precision-cut liver slices (PCLS). LPS induced a more pronounced inflammatory response in GF PCLS than in SPF PCLS. Baseline TNF-α gene expression was significantly higher in GF slices as compared to SPF slices. LPS treatment induced TNF-α, IL-1β, IL-6 and iNOS expression in both SPF and GF PCLS, but the increase was more intense in GF slices. The anti-inflammatory markers SOCS3 and IRAK-M gene expression was significantly higher in GF PCLS than SPF PCLS at 24h with 1 µg/ml LPS treatment, and IL-10 was not differently expressed in GF PCLS than SPF PCLS. In addition, TLR-4 mRNA, but not protein, at basal level was higher in GF slices than in SPF slices. Taken together, this study shows that, in mice, the host microbiota attenuates the pro-inflammatory impact of LPS in the liver, indicating a positive role of the gut microbiota on the immune homeostasis of the liver

Murine Precision-cut Intestinal Slices as a Potential Screening Tool for Antifibrotic Drugs

BACKGROUND: Intestinal fibrosis is a hallmark of Crohn's disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. METHODS: Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1α1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor β (TGF-β) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. RESULTS: Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-β1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1α1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. CONCLUSIONS: Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis

A Pathophysiological Model of Non-Alcoholic Fatty Liver Disease Using Precision-Cut Liver Slices

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder closely related to metabolic syndrome. NAFLD can progress to an inflammatory state called non-alcoholic steatohepatitis (NASH), which may result in the development of fibrosis and hepatocellular carcinoma. To develop therapeutic strategies against NAFLD, a better understanding of the molecular mechanism is needed. Current in vitro NAFLD models fail to capture the essential interactions between liver cell types and often do not reflect the pathophysiological status of patients. To overcome limitations of commonly used in vitro and in vivo models, precision-cut liver slices (PCLSs) were used in this study. PCLSs, prepared from liver tissue obtained from male Wistar rats, were cultured in supraphysiological concentrations of glucose, fructose, insulin, and palmitic acid to mimic metabolic syndrome. Accumulation of lipid droplets was visible and measurable after 24 h in PCLSs incubated with glucose, fructose, and insulin, both in the presence and absence of palmitic acid. Upregulation of acetyl-CoA carboxylase 1 and 2, and of sterol responsive element binding protein 1c, suggests increased de novo lipogenesis in PCLSs cultured under these conditions. Additionally, carnitine palmitoyltransferase 1 expression was reduced, which indicates impaired fatty acid transport and disrupted mitochondrial β-oxidation. Thus, steatosis was successfully induced in PCLSs with modified culture medium. This novel ex vivo NAFLD model could be used to investigate the multicellular and molecular mechanisms that drive NAFLD development and progression, and to study potential anti-steatotic drugs

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor β Superfamily Type 1 Receptors.

The transforming growth factor β (TGFβ) superfamily includes TGFβ, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFβ superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor β receptor 1 (TGFBR1) kinase inhibitors designed to target TGFβ signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFβ-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFβ-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFβ- and BMP-regulated signaling pathways to disease states