Background: Endothelial cells (EC) that make up the inner wall of blood vessels play an important role in angiogenesis and vascular recovery. Cardiovascular disease caused by dysfunction of ECs has been reported as a major cause of death worldwide. Despite significant research so far, the underlying mechanism of dysfunction of ECs in cardiovascular disease progression is not yet fully understood. Although therapeutic transplantation of autologous ECs is limited due to lack of cell availability, adiposederived stem cells (ADSCs), known for their ease of procurement and high potential for differentiation, will provide promising solutions to generate autologous ECs. Methods: This study investigated the optimal differentiation of ADSCs into ECs under EBM-2 culture conditions supplemented with VEGF and BMP-4 in hypoxia (2% O2). Results: During 14 days of in vitro differentiation, cells cultured in EBM-2 supplemented with VEGF showed the characteristics of early vascular ECs and some cells adopted polygonal forms. Conversely, cells cultured in EBM-2 and hypoxia supplemented with both VEGF and BMP-4 differentiated into the typical cobblestone morphology that appears in vascular ECs. As a result of immunostaining against the vascular ECs marker CD-31, CD-31 expression was increased under EBM-2 culture conditions with VEGF and VEGF + BMP-4 in hypoxia, but expression was insufficient in normal oxygenation (21% O2). In the flow cytometry analysis, high expression of CD-31 expression was observed under conditions including both VEGF and BMP-4 of hypoxia. Interestingly, in gene expression, the pluripotency marker OCT-3/4 was significantly reduced under hypoxic conditions, but SOX2 and NANOG expression were higher than under normal oxygen conditions. However, CD-31 expression was significantly higher under differentiation conditions in which VEGF and BMP-4 were added under hypoxia conditions. In a functional analysis, CD-31-positive ADSC-derived ECs differentiated under hypoxia had excellent tube formation and Dil-Ac-LDL uptake, which are important for vascular repair and function. Conclusions: These findings confirmed the therapeutic usefulness of ECs derived from ADSC for the treatment of cardiovascular disease due to the synergy effect of hypoxia and BMP-4.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Isolation and culture of swine umbilical vein endothelialcells (SUVEC)
    Isolatiuon and culture of porcine adipose-derivedstem cells (pADSCs)
    In vitro differentiation to endothelial cells (ECs)
    Immunocytochemistry
    Quantitative real-time PCR (qPCR)
    Flow cytometry (FACS) analysis
    Magnetic activated cell sorting (MACS)
    Tube formation assay
    Dil-ac-LDL uptake assay
    Statistical analysis
RESULTS
    Morphological changes during in vitro differentiationfrom hypoxic conditions to ECs
    Expression of CD-31 after in vitro differentiation ofpADSCs under hypoxic conditions
    Expression of pluripotency-related genes andendothelial gene of pADSCs differentiated in hypoxia
    Flow cytometry validation of CD-31 expression underhypoxic culture conditions
    Formation of capillary-like structures of pADSCderivedendothelial cells
    Assessment of function via Dil-Ac-LDL uptake inpADSCs-derived endothelial cells
DISCUSSION
CONCLUSION
REFERENCES

• Cheol Hwangbo(Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju 52828, Korea)
• Jung-Woo Choi(College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea)
• Sang-Hee Lee(College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea, School of ICT, University of Tasmania, Hobart 7005, Australia) Corresponding author
• Tae-Suk Kim(Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea)
• In-Won Lee(Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea)
• Dong-Ju Shin(Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea)
• Joon-Hee Lee(Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea, Institute of Agriculture & Life Science, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea) Corresponding author