Cytokines Released from Human Adipose-tissue–Derived Stem Cells By b-FGF Stimulation: Effects on Angiogenesis and Lymphatic Vessels Formation By IL-8 and CXCL1

Abstract

INTRODUCTION: Recently, adipose-derived stem cells (ADSCs) have been widely used in therapeutic such as wounds, ischemic limbs, and lymphedema, due to their ability to release various cytokines. In addition, ADSCs can improve the efficiency of fat transplantation. However, the molecular mechanisms in the paracrine effect, such as the types of cytokines released from ADSCs, are largely unclear. Previously, basic fibroblast growth factor (b-FGF) was reported to increase the proliferative capacity of ADSCs. In this study, we searched for cytokines released from ADSCs upon b-FGF stimulation and examined their effects on angiogenesis and lymphatic vessels formation. METHODS: In order to analyze the maximal effect of b-FGF stimulation, ADSCs obtained from a 45-year-old African-American man were cultured in either control or serum-free medium supplemented with 20, 25, and 50 μg/mL concentrations of b-FGF, respectively and 0.5 and 4 hours of stimulation time, respectively. qRT-PCR was performed on around 200 genes using TaqMan array in order to define the optimal condition for releasing cytokines with stimulation of b-FGF as stimulation condition (SC). Secondly, ADSCs were stimulated under either SC or SC without b-FGF (as a control) followed by qRT-PCR and ELISA analyses in order to select the gene and cytokines that show at least 1.5 times higher expression level than control, respectively. Based on the results from screening experiment, ADSCs derived from a 30-year-old woman (Asian), a 55-year-old man (White), and 47-year-old man (mix of African and White) were incubated under SC, and qRT-PCR was performed for the selected cytokines. Tube formation assay was performed with the 20 ng/ml concentration of selected cytokines for HUVEC (Human Umbilical Vein Endothelial Cells), TIME-GFP (Human Microvascular Endothelial Cells expressing green fluorescence protein), and HDLEC (Human Dermal Lymphatic Endothelial Cells). Finally, formed tube area (pixels)/total view area (pixels) of a still image was calculated with ImageJ software and we defined that as the tube formation rate (%). Unpaired t-test was used for statistically analysis, with significance set at P < 0.05. RESULTS: SC was determined as a combination of serum-free medium with 20 μg per mL of b-FGF for 4-hour incubation. By qRT-PCR analysis, the gene expression of CXCL1, HB-EGF, IL1B, and IL8 were upregulated and further confirmation by ELISA revealed that the secretion of CXCL1 and IL8 was promoted by b-FGF. Promotion of CXCL1 and IL8 induction by b-FGF was observed regardless of age, race, and gender by qRT-PCR analysis. Tube formation assay demonstrated that the rate of tube formation using HUVEC, TIME-GFP, and HDLEC with CXCL1 were 2.9-, 4.0-, and 1.5-fold increase compared with the control group (without CXCL1 and IL8), respectively, which were all statistically significant (P < 0.05). CONCLUSIONS: These findings suggest that b-FGF stimulation promotes the release of CXCL1 and IL8 from ADSCs, regardless of age, race, and gender in this study. These cytokines significantly promoted angiogenesis and lymphatic vessel formation and may promote various wound healing and engraftment of transplanted fat, as well as lymphatic vessel regeneration of lymphedema.