ABSTRACT
Tissue regeneration requires coordinated "teamwork" of growth factors, proteases, progenitor and immune cells producing inflammatory cytokines. Mesenchymal stem cells (MSC) might play a pivotal role by substituting cells or by secretion of growth factors or cytokines, and attraction of progenitor and inflammatory cells, which participate in initial stages of tissue repair. Due to obvious impact of inflammation on regeneration it seems promising to explore whether inflammatory factors could influence proangiogenic abilities of MSC. In this study we investigated effects of TNF-α on activity of adipose-derived stem cells (ADSC). We found that treatment with TNF-α enhances ADSC proliferation, F-actin microfilament assembly, increases cell motility and migration through extracellular matrix. Exposure of ADSC to TNF-α led to increased mRNA expression of proangiogenic factors (FGF-2, VEGF, IL-8, and MCP-1), inflammatory cytokines (IL-1ß, IL-6), proteases (MMPs, uPA) and adhesion molecule ICAM-1. At the protein level, VEGF, IL-8, MCP-1, and ICAM-1 production was also up-regulated. Pre-incubation of ADSC with TNF-α-enhanced adhesion of monocytes to ADSC but suppressed adherence of ADSC to endothelial cells (HUVEC). Stimulation with TNF-α triggers ROS generation and activates a number of key intracellular signaling mediators known to positively regulate angiogenesis (Akt, small GTPase Rac1, ERK1/2, and p38 MAP-kinases). Pre-treatment with TNF-α-enhanced ADSC ability to promote growth of microvessels in a fibrin gel assay and accelerate blood flow recovery, which was accompanied by increased arteriole density and reduction of necrosis in mouse hind limb ischemia model. These findings indicate that TNF-α plays a role in activation of ADSC angiogenic and regenerative potential.
Subject(s)
Adipose Tissue/cytology , Stem Cells/cytology , Stem Cells/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Actins/metabolism , Adipose Tissue/metabolism , Adult , Animals , Cell Cycle/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Flow Cytometry , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Male , Mice , Middle Aged , Stem Cells/drug effects , Young AdultABSTRACT
BACKGROUND: Multipotent mesenchymal stem/stromal cells (MSC) including adipose-derived stromal cells (ADSC) have been successfully applied for cardiovascular diseases treatment. Their regenerative potential is considered due to the multipotency, paracrine activity and immunologic privilege. However, therapeutic efficacy of autologous MSC for myocardial ischemia therapy is modest. We analyzed if ADSC properties are attenuated in patients with chronic diseases such as coronary artery disease (CAD) and diabetes mellitus type 2 (T2DM). METHODS AND RESULTS: ADSC were isolated from subcutaneous fat tissue of patients without established cardiovascular diseases and metabolic disorders (control group, n = 19), patients with CAD only (n = 32) and patients with CAD and T2DM (n = 28). ADSC phenotype (flow cytometry) was CD90(+)/CD73(+)/CD105(+)/CD45(-)/CD31(-) and they were capable of adipogenic and osteogenic differentiation. ADSC morphology and immunophenotype were similar for all patients, but ADSC from patients with CAD and T2DM had higher proliferation activity and shorter telomeres compared to control patients. ADSC conditioned media stimulated capillary-like tubes formation by endothelial cells (EA.hy926), but this effect significantly decreased for patients with CAD (p = 0.03) and with CAD + T2DM (p = 0.017) compared to the control group. Surprisingly we revealed significantly higher secretion of some pro-angiogenic factors (ELISA) by ADSC: vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) for patients with CAD and HGF and placental growth factor (PlGF) for patients with CAD + T2DM. Among angiogenesis inhibitors such as thrombospondin-1, endostatin and plasminogen activator inhibitor-1 (PAI-1) level of PAI-1 in ADSC conditioned media was significantly higher for patients with CAD and CAD + T2DM compared to the control group (p < 0.01). Inhibition of PAI-1 in ADSC conditioned media by neutralizing antibodies partially restored ADSC angiogenic activity (p = 0.017). CONCLUSIONS: ADSC angiogenic activity is significantly declined in patients with CAD and T2DM, which could restrict the effectiveness of autologous ADSC cell therapy in these cohorts of patients. This impairment might be due to the disturbance in coordinated network of pro- and anti-angiogenic growth factors secreted by ADSC. Changes in ADSC secretome differ between patients with CAD and T2DM and further investigation are necessary to reveal the MSC-involved mechanisms of cardiovascular and metabolic diseases and develop novel approaches to their correction using the methods of regenerative medicine.
