Abstract Background The interaction between intestinal myofibroblasts (iMFBs) and extracellular matrix (ECM) changes contributes to Crohn’s disease (CD) fibrosis, but the exact mechanisms are not yet clear. Our group recently discovered a selection of matrix-derived peptides (matrikines) that specifically appear in the intestinal tissue of patients with CD fibrostenosis.1 Here, we evaluate the in vitro effect of 19 matrikines (with various bioactivity likelihood) on primary human intestinal myofibroblasts (IMFBs), key cells in CD fibrosis. Methods Primary human iMFBS (500K/mL, Passage 4, Novabiosis) were cultured in serum-free medium (SFM) (DMEM high glucose, 1% sodium pyruvate, 1% non-essential aminoacids, 1% Antibiotics-Antimycotics and 3mg Gentamicin), complete medium (SFM with 10% fetal bovine serum), PDGF-bb 10ng/mL or treated with each of the matrikines at 10, 50, 100 μg/mL for 24hours. The effect of each matrikine on cell proliferation, gene expression and migration was assessed by BrdU, qPCR and Transwell assays, respectively. RNA sequencing data was analysed using the GSEA software. Results The effect on iMFBs proliferation and viability was tested with each of the 19 matrikines at 3 different doses (Table 1). Among 9 most likely-bioactive matrikines (M1-M9) (Figure 1), M1, M4, M7, M8 and M9 induced the highest effect in cell proliferation and were selected for further experiments. Gene expression assays showed upregulation of ACTA2 with M1 or M7 and upregulation of COL1A1 with M1, M7 or M8, indicating activation of the iMFBs (Figure 2). Transwell assays also showed that M1, M4 and M8 induce iMFB cell migration. A total of 1948, 1024, 460, 570, and 2749 differentially expressed genes (DEGs) were identified in iMBFs treated with M1, M4, M7, M8 and M9 vs no treatment, showing enrichment in pathways related to “smooth muscle contraction”, “fibroblast growth factor”, “cell migration”, “collagen formation”, “elastin formation” and “ECM organization”; most of which were also enriched by complete medium and/or PDGFbb that served as positive controls. However, pathways related to “proinflammatory and profibrotic mediators” or “vascular permeability” were only enriched in iMFBs treated with M1, M4, M8 and M9 or M1 and M9, respectively (Figure 3). Conclusion A selection of newly discovered CD-specific matrikines induce activation of iMFBs as well as inflammatory response and endothelial changes, suggesting an important role and therefore their potential value as biomarkers in the progression of intestinal fibrostenosis.

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture.

The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib reversed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvironment and could be employed for anti-fibrotic and anti-HCC drug screening.

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.

An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFβ signaling. This was also supported by the presence and release of higher concentration of endogenous TGFβ1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFβ1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFβ1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFβ-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.

BACKGROUND:The current methodologies for the identification of therapeutic targets for inflammatory bowel disease (IBD) are limited to conventional 2-dimensional (2D) cell cultures and animal models. The use of 3D decellularized human intestinal scaffolds obtained from surgically resected intestine and engineered with human intestinal cells may provide a major advancement in the development of innovative intestinal disease models. The aim of the present study was to design and validate a decellularization protocol for the production of acellular 3D extracellular matrix (ECM) scaffolds from the human duodenum.METHODS:Scaffolds were characterized by verifying the preservation of the ECM protein composition and 3D architecture of the native intestine and were employed for tissue engineering with primary human intestinal myofibroblasts for up to 14 days.RESULTS:Engrafted cells showed the ability to grow and remodel the surrounding ECM. mRNA expression of key genes involved in ECM turnover was significantly different when comparing primary human intestinal myofibroblasts cultured in 3D scaffolds with those cultured in standard 2D cultures on plastic dishes. Moreover, incubation with key profibrogenic growth factors such as TGFβ1 and PDGF-BB resulted in markedly different effects in standard 2D vs 3D cultures, further emphasizing the importance of using 3D cell cultures.CONCLUSIONS:These results confirm the feasibility of 3D culture of human intestinal myofibroblasts in intestinal ECM scaffolds as an innovative platform for disease modeling, biomarker discovery, and drug testing in intestinal fibrosis.

Hepatitis B virus (HBV) study is hampered by lacking of idea cell model which support effective HBV infection and meanwhile recapitulate hepatocyte biology function in vivo. In this study, we developed decellularized human liver scaffolds for cell culture and further applied for HBV infection. As a result, primary human hepatocytes (PHHs) engrafted into liver scaffolds and maintained differentiation with stable albumin secretion and liver-specific gene expression. Comparing to mono-layer cell culture, scaffold-based three-dimensional (3D) culture system significantly augment HBV DNA (including cccDNA), RNA level as well as HBsAg secretion. Moreover, HepG2-NTCP cells cultured on 3D system exhibited higher infection efficiency and longer infection period in vitro. In addition, HBV DNA level was suppressed when anti-HBV medicine Entecavir (ETV) introduced into HepG2-NTCP 3D system. Herein, we evaluated the potential of decellularized human liver scaffold-based in 3D cell culture and disclosed that scaffold-based 3D culture system can facilitate HBV infection in vitro. This 3D culture system could be further applied in HBV-related study. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1744-1753, 2019.

The neuropeptide adrenomedullin (ADM) shows anti-inflammatory activity but its role has not been investigated in liver diseases. We assessed the hepatic ADM expression in different inflammatory liver diseases (HCV, AIH, NASH) and the mechanism(s) by which ADM affects NF-κB activation in classical 2D and a new 3D model.