151. Decoding the Paracrine Effect of bFGF-Stimulated Adipose Tissue-derived Stem Cells on Angiogenesis and Lymphangiogenesis

PURPOSE: Recently, adipose-derived stem cells (ADSCs) have been widely used in therapy such as chronic wounds, ischemic limbs, and lymphedema, due to their ability to release various cytokines. The molecular mechanisms of their paracrine effect remain largely unclear. Previously, basic fibroblast growth factor (bFGF) was reported to increase the proliferative capacity of ADSCs. In this study, we searched for cytokines released from ADSCs upon bFGF stimulation and examined their effects on angiogenesis and lymphatic vessel formation. METHODS: After preliminary experiment, stimulation condition was determined as stimulation with b-FGF concentrations of 20μg/mL for 4 hours as it provided the highest transcription for IL-8 and CXCL-1 in ADSCs. ADSCs were incubated under stimulation conditions and under control serum free medium. Expression of cytokines was measured with qRT-PCR. Tube formation assay was conducted evaluating Human umbilical vein endothelial cells (HUVECs), Telomerase-immortalized human microvascular endothelium cells expressing green fluorescence protein (TIME-GFP), and Human dermal lymphatic endothelial cells (HDLECs) co-cultured with recombinant IL-8 or CXCL-1, and with conditioned media of bFGF-stimulated ADSCs. Neutralizing antibodies (NAb) for IL-8 and CXCL-1 were used to assess interference of these factors. RESULTS: After stimulation with bFGF, qRT-PCR analysis showed gene expression of CXCL1, HB-EGF, IL1B, and IL8 was upregulated. Further confirmation by ELISA revealed that the secretion of CXCL1 and IL8 was promoted by bFGF. Promotion of CXCL1 and IL8 induction by bFGF was observed regardless of age, race, and gender by qRT-PCR analysis. Tube formation assay demonstrated an increased rate of tube formation for HUVEC, TIME-GFP, and HDLEC when independently co-cultured with CXCL-1 or IL-8 or with conditioned media of bFGF-stimulated ADSCs in comparison to their controls (p <.05). IL-8 NAb in conjunction with CXCL-1 NAb significantly reduced the rate of formation for HUVEC and TIME-GFP (p <.05). CONCLUSION: Our findings suggest that bFGF 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 wound healing and engraftment of transplanted fat, as well as lymphatic vessel regeneration in lymphedema.