RESUMEN
Tissue engineering as an interdisciplinary field of biomedical sciences has raised many hopes in the treatment of cardiovascular diseases as well as development of in vitro three-dimensional (3D) cardiac models. This study aimed to engineer a cardiac microtissue using a natural hybrid hydrogel enriched by granulocyte colony-stimulating factor (G-CSF), a bone marrow-derived growth factor. Cardiac ECM hydrogel (Cardiogel: CG) was mixed with collagen type I (ColI) to form the hybrid hydrogel, which was tested for mechanical and biological properties. Three cell types (cardiac progenitor cells, endothelial cells and cardiac fibroblasts) were co-cultured in the G-CSF-enriched hybrid hydrogel to form a 3D microtissue. ColI markedly improved the mechanical properties of CG in the hybrid form with a ratio of 1:1. The hybrid hydrogel demonstrated acceptable biocompatibility and improved retention of encapsulated human foreskin fibroblasts. Co-culture of three cell types in G-CSF enriched hybrid hydrogel, resulted in a faster 3D structure shaping and a well-cellularized microtissue with higher angiogenesis compared to growth factor-free hybrid hydrogel (control). Immunostaining confirmed the presence of CD31+ tube-like structures as well as vimentin+ cardiac fibroblasts and cTNT+ human pluripotent stem cells-derived cardiomyocytes. Bioinformatics analysis of signaling pathways related to the G-CSF receptor in cardiovascular lineage cells, identified target molecules. The in silico-identified STAT3, as one of the major molecules involved in G-CSF signaling of cardiac tissue, was upregulated in G-CSF compared to control. The G-CSF-enriched hybrid hydrogel could be a promising candidate for cardiac tissue engineering, as it facilitates tissue formation and angiogenesis.
RESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a high-prevalence and progressive disorder. Due to lack of reliable in vitro models to recapitulate the consecutive phases, the exact pathogenesis mechanism of this disease and approved therapeutic medications have not been revealed yet. It has been proven that the interplay between multiple hepatic cell types and liver extracellular matrix (ECM) are critical in NAFLD initiation and progression. Herein, a liver microtissue (LMT) consisting of Huh-7, THP-1, and LX-2 cell lines and human umbilical vein endothelial cells (HUVEC), which could be substituted for the main hepatic cells (hepatocyte, Kupffer, stellate, and sinusoidal endothelium, respectively), encapsulated in liver derived ECM-Alginate composite, was bioengineered. When the microtissues were treated with free fatty acids (FFAs) including Oleic acid (6.6×10-4M) and Palmitic acid (3.3×10-4M), they displayed the key features of NAFLD, including similar pattern of transcripts for genes involved in lipid metabolism, inflammation, insulin-resistance, and fibrosis, as well as pro-inflammatory and pro-fibrotic cytokines' secretions and intracellular lipid accumulation. Continuing FFAs supplementation, we demonstrated that the NAFLD phenomenon was established on day 3 and progressed to the initial fibrosis stage by day 8. Furthermore, this model was stable until day 12 post FFAs withdrawal on day 3. Moreover, administration of an anti-steatotic drug candidate, Liraglutide (15 µM), on the NAFLD microtissues significantly ameliorated the NAFLD phenomenon. Overall, we bioengineered a drug-responsive, cost-benefit liver microtissues which can simulate the initiation and progression of NAFLD. It is expected that this platform could potentially be used for studying molecular pathogenesis of NAFLD and high-throughput drug screening. See also the graphical abstract(Fig. 1).
RESUMEN
Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.