ABSTRACT
OBJECTIVES: Vascularization is a major obstacle to clinical application of regenerative medicine. Engineered tissues must be able to generate an early vascular network that can quickly connect with the host vasculature. Recent research demonstrates that natural adipose tissues contain abundant stromal cells, which can give rise to pericytes. In this study, we aimed to investigate the application of human adipose stromal cells (ASCs) to vascularization, and the function of BMP4 protein during vascularization. MATERIALS AND METHODS: Immunofluorescence staining for α-SMA and PDGFR-ß were utilized to identify characteristics of ASCs/pericytes. They were then loaded into a collagen-fibronectin gel with endothelial cells to assess their vascularization ability, both in vitro and in vivo. RESULTS: We showed that the ASCs expressed some of the essential markers of pericytes and they were able to promote vascularization with endothelial cells in 3D culture, both in vitro and in vivo. BMP4 protein further promoted this vascularization. CONCLUSION: Adipose stromal cells promoted vascularization by endothelial cells and BMP4 protein further enhanced this effect.
Subject(s)
Adipocytes/physiology , Adipose Tissue/physiology , Bone Morphogenetic Protein 4/metabolism , Endothelial Cells/physiology , Neovascularization, Physiologic/physiology , Adipocytes/metabolism , Adipose Tissue/metabolism , Collagen/metabolism , Endothelial Cells/metabolism , Fibronectins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Stromal CellsABSTRACT
OBJECTIVE: To determine the inhibitory effect and mechanism of Notch signalling on adipogenesis of mouse adipose-derived stem cells (mASCs). MATERIALS AND METHODS: Varied concentrations of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT) were added to mASCs 3 days before adipogenic induction with insulin-containing differentiation medium. The process of adipogenesis and ability of lipid droplet accumulation were analysed using oil red-O staining. The Notch signalling pathway (Notch-1, -2, -3, -4, Hes-1 and Hey-1) and adipogenesis-related factors (PPAR-gamma, DLK-1/Pref-1 and Acrp) were tested using real-time PCR, Western blot analysis and immunofluorescence staining assays. RESULTS: We demonstrated that Notch-2-Hes-1 signalling pathway was inhibited dose-dependently by DAPT in mASCs. In addition, transcription of PPAR-gamma was promoted by DAPT before adipogenic induction, while inhibitor of adipogenesis DLK-1/Pref-1 was further depressed. At early stages of differentiation (2-4 days), adipogenesis in mASCs was advanced and significantly enhanced in 5 and 10 mum DAPT pre-treated cases. On day 4, in differentiated mASCs cases with DAPT pre-treatment, we also found promotion of activation of de-PPAR-gamma and depression of HES-1, DLK-1/Pref-1 mRNA and protein expression. CONCLUSIONS: We conclude that blocking Notch signalling with DAPT enhances adipogenesis of differentiated mASCs at an early stage. It may be due to depression of DLK-1/Pref-1 and promotion of de-PPAR-gamma activation, which work through inhibition of Notch-2-Hes-1 pathway by DAPT.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , PPAR gamma/physiology , Receptors, Notch/physiology , Stem Cells/cytology , Adipocytes/cytology , Adipocytes/drug effects , Adipogenesis/drug effects , Amyloid Precursor Protein Secretases/metabolism , Animals , Cell Differentiation/drug effects , Cells, Cultured , Dipeptides/pharmacology , Dose-Response Relationship, Drug , Mice , Mice, Inbred Strains , PPAR gamma/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Notch/metabolism , Signal Transduction , Stem Cells/metabolismABSTRACT
Macrophages play numerous roles in both physiologic and pathologic processes. Along with fibroblasts, they comprise the synovial tissue that forms the lining of musculoskeletal joint capsules and bursae, and they often envelop implants. During the process of phagocytosing prosthesis-related particles, macrophages in peri-implant tissue release inflammatory mediators. Little is known, however, about the response of these cells to mechanical perturbation, which often is a component of the physical environment of the cell. Mouse peritoneal macrophages were grown on a flexible membrane in vitro and a dynamic 1-Hz spatially uniform sinusoidal strain pattern imparted to the elastomeric substrate. The effect of mechanical strain on prostaglandin (PG) E(2) release was evaluated using cells that were activated by lipopolysaccharide (LPS) as well as by those that were not. The results are compared with the levels of PGE(2) stimulated by metallic particles. Strain magnitudes of 4 and 8% applied for 1 h resulted in almost a twofold increase in the release of PGE(2) from LPS-stimulated cells (p < 0.05) and nonstimulated macrophages (p < 0.07), compared with nonperturbated controls. No release was elicited by a challenge of metal particles. These findings demonstrate for the first time an effect of mechanical force on the release of an inflammatory mediator by macrophages. This response may help to explain the macrophage-mediated processes underlying the osteolysis associated with loose prostheses in bone and suggests a mechanism for the inflammation of synovial tissues by excessive mechanical strain.
