5-ethynyl-2--deoxyuridine and Inflammation

Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-βgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.

Topics: beta-Galactosidase; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cellular Senescence; Cytokines; Deoxyuridine; DNA Damage; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Inflammation; Mesenchymal Stem Cells; Multipotent Stem Cells; Phenotype; Signal Transduction; Telomere; Workflow

This study investigated whether mechano growth factor-E (MGF-E) peptide can regulate apoptosis and inflammation responses in fibroblast-like synoviocytes of osteoarthritis (OA).. A (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay was performed to evaluate cytotoxic effects of exogenous MGF-E peptide on OA fibroblast-like synoviocytes (OA-FLS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to check messenger RNA (mRNA) expression levels of lysyl oxidase (LOX) family members (LOX) after OA-FLS treatment using MGF-E peptide. A 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay was performed to identify the influence of MGF-E peptide on proliferation OA-FLS proliferation. Western blot was used to detect biomarkers of endoplasmic reticulum (ER) stress and inflammatory cytokines.. Exogenous MGF-E peptide has no obvious cytotoxic effects on OA-FLS and promotes LOX expression in OA-FLS, induce apoptosis and ER stress and down-regulate protein levels of tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β).. Our results suggest that MGF-E peptide possesses potential anti-inflammatory effects, induces cell apoptosis and facilitates repair of OA-FLS. Therefore, MGF-E peptide may have therapeutic potential in patients with OA.

Topics: Apoptosis; Blotting, Western; Cytokines; Deoxyuridine; Endoplasmic Reticulum Stress; Female; Fibroblasts; Humans; Inflammation; Insulin-Like Growth Factor I; Middle Aged; Osteoarthritis, Knee; Protein-Lysine 6-Oxidase; Real-Time Polymerase Chain Reaction; Synovial Membrane; Tetrazolium Salts; Thiazoles

The paucity of mammalian adult cardiac myocytes (CM) proliferation following myocardial infarction (MI) and the remodeling of the necrotic tissue that ensues, result in non-regenerative repair. In contrast, zebrafish (ZF) can regenerate after an apical resection or cryoinjury of the heart. There is considerable interest in models where regeneration proceeds in the presence of necrotic tissue. We have developed and characterized a cautery injury model in the giant danio (GD), a species closely related to ZF, where necrotic tissue remains part of the ventricle, yet regeneration occurs. By light and transmission electron microscopy (TEM), we have documented four temporally overlapping processes: (1) a robust inflammatory response analogous to that observed in MI, (2) concomitant proliferation of epicardial cells leading to wound closure, (3) resorption of necrotic tissue and its replacement by granulation tissue, and (4) regeneration of the myocardial tissue driven by 5-EDU and [(3) H]thymidine incorporating CMs. In conclusion, our data suggest that the GD possesses robust repair mechanisms in the ventricle and can serve as an important model of cardiac inflammation, remodeling and regeneration.

Topics: Animals; Cell Proliferation; Deoxyuridine; Disease Models, Animal; Granulation Tissue; Inflammation; Myocytes, Cardiac; Necrosis; Neovascularization, Pathologic; Pericardium; Regeneration; Thymidine; Ventricular Remodeling; Wound Healing; Zebrafish