An Ex Vivo Aorta Culture Model to Study Vascular Cellular Senescence.

Animal studies on vascular aging pose a few limitations. One of the most important reasons for this is the absence of a fast and efficient model of vascular tissue aging. In this study, ex vivo aortic culture and Matrigel subcutaneous implantation are combined to develop a new model for studying vascular cellular senescence. Eight-week-old C57BL/6J mice are used to obtain aortas. Bleomycin is used to induce aortas senescence in vitro. Then, aortas are transplanted to the acceptor mice with Matrigel. Senescence is evaluated using western blotting, quantitative polymerase chain reaction, and senescence-associated beta-galactosidase activity. Inflammatory cytokines are detected using Luminex Liquid Suspension Chip. RNA levels are analyzed by transcriptome sequencing. The results revealed that vessels in the bleomycin group exhibited significant senescence than those in the control group that can be enhanced by stripping vessel adventitia. The levels of cytokines such as interleukin (IL-2, IL-1ß, and IL-6 increased significantly in the ex vivo model. Furthermore, transcriptome sequencing revealed 56 significantly differentially expressed genes (DEGs) in ex vivo model vessels compared with those in naturally aging aortas. In conclusion, this study introduces a cost-effective and time-saving vessel senescence model for vascular cellular senescence.

Lin28b delays vasculature aging by reducing platelet-derived growth factor-beta resistance in senescent vascular smooth muscle cells.

BACKGROUND AND AIMS: Platelet-derived growth factor-ß (PDGFB) is an important mediator of vascular smooth muscle cell (VSMC) proliferation, and PDGFB resistance is observed in senescent VSMCs. Lin28b is a stemness regulator in the embryo; however, its role in vasculature aging and VSMC senescence is unknown. We aimed to investigate whether Lin28b could restore the VSMC response to PDGFB and delay vasculature aging. METHODS: ApoE-/- mice were fed a high-fat diet for different weeks to establish an aging model. PDGFB resistance was observed using EdU staining in vessel culture in vitro. Quantitative polymerase chain reaction and in situ hybridization were used to detect let-7 expression. Senescence was identified by Western blotting, senescence-associated beta-galactosidase activity or Sudan Black B staining, and VSMC function was determined using CCK-8, migration, and enzyme-linked immunosorbent assays. RESULTS: Vessels from aged mice showed poor responses to PDGFB stimulation compared with those from young mice; similar results were found in senescent VSMCs. The expression levels of Lin28b and PDGF receptor-ß were downregulated in aging vasculature and senescent VSMCs, whereas let-7 family levels increased with aging and VSMC passage growth. Transfection of VSMCs with let-7c induced PDGFB resistance and accelerated VSMC senescence, whereas blocking let-7c restored PDGFB reactions in VSMCs. Overexpression of Lin28b protein by lentivirus resulted in the restoration of PDGFB reactions and delayed VSMC senescence, which was blocked by a let-7c mimic. CONCLUSIONS: This study reveals the role of Lin28b in delaying vasculature aging by decreasing senescent VSMC PDGFB resistance mediated by let-7.

MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells.

LIN28A aberrant expression contributes to the development of human malignancies. However, the LIN28A expression profile remains to be clarified. Herein, we report that LIN28A expression is directly associated with the methylation status of its two CpG island sites in pancreatic cancer cells. First, Bisulfite sequencing reveals that PANC1 cells possess the higher methylation rate at LIN28A CpG islands compared with SW1990 and PaTu8988 cells. Subsequently, LIN28A expression is increased at both mRNA and protein levels in pancreatic cancer cells treated with 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor. Further Chromatin immunoprecipitation (ChIP) assays indicate that methyl-CpG-binding protein 2 (MeCP2) binds preferentially to the two hypermethylated CpG islands sites at LIN28A promoter compare to MBD3. Expectedly, MeCP2 knockdown transcriptionally activates LIN28A expression in above cells, rather than MBD3 knockdown. Moreover, LIN28A overexpression remarkably improves OCT4, NANOG and SOX2 expression, and the ability of sphere and colony formation, and enhances the capacities of invasion in PaTu8988 and SW1990 cells, whereas LIN28A knockdown significantly inhibits the above malignant behaviors in PANC1 cells. These findings suggest that LIN28A is epigenetically regulated via MeCP2 binding to methylated-CpG islands, and may play a crucial role in pancreatic cancer progression.

Discovery of Metal Ions Chelator Quercetin Derivatives with Potent Anti-HCV Activities.

Analogues or isosteres of α,γ-diketoacid (DKA) 1a show potent inhibition of hepatitis C virus (HCV) NS5B polymerase through chelation of the two magnesium ions at the active site. The anti-HCV activity of the flavonoid quercetin (2) could partly be attributed to it being a structural mimic of DKAs. In order to delineate the structural features required for the inhibitory effect and improve the anti-HCV potency, two novel types of quercetin analogues, 7-O-arylmethylquercetins and quercetin-3-O-benzoic acid esters, were designed, synthesized and evaluated for their anti-HCV properties in cell-based assays. Among the 38 newly synthesized compounds, 7-O-substituted derivative 3i and 3-O-substituted derivative 4f were found to be the most active in the corresponding series (EC50 = 3.8 µM and 9.0 µΜ, respectively). Docking studies suggested that the quercetin analogues are capable of establishing key coordination with the two magnesium ions as well as interactions with residues at the active site of HCV NS5B.

Design, synthesis and biological evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3ß (GSK-3ß).

Glycogen synthase kinase-3ß (GSK-3ß) plays a key role in type II diabetes and Alzheimer's diseases, to which non-ATP competitive inhibitors represent an effectively therapeutical approach due to their good specificity. Herein, a series of small molecules benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of GSK-3ß have been designed and synthesized. The in vitro evaluation performed by luminescent assay showed most BTZ derivatives have inhibitory effects in micromolar scale. Among them compounds 6l, 6t and 6v have the IC50 values of 25.0 µM, 27.8 µM and 23.0 µM, respectively. Moreover 6v is devoid of any inhibitory activity in the assays to other thirteen protein kinases. Besides, SAR is analyzed and a hypothetical enzymatic binding mode is proposed by molecular docking study, which would be useful for new candidates design.