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Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of  in vitro  serum deprivation and VEGF stimulation on gene expression patterns of ASCs.    Methods  Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Microarray gene expressions were obtained using the Affymetrix HT HG-U133+ GeneChip®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms. Transcription of selected genes of interest was confirmed by quantitative PCR.    Results  Compared to ASCs in complete medium, 190 and 108 genes were significantly altered by serum deprivation and serum deprivation combined with VEGF, respectively. No significant differences in gene expression patterns between serum-deprived ASCs and serum-deprived ASCs combined with VEGF stimulation were found. Genes most prominently and significantly upregulated by both conditions were growth factors ( IGF1, BMP6, PDGFD, FGF9 ), adhesion molecule  CLSTN2 , extracellular matrix-related proteins such as matricellular proteins  SMOC2 ,  SPON1  and  ADAMTS12 , and inhibitors of proliferation (JAG1). The most significantly downregulated genes included matrix metalloproteinases ( MMP3 ,  MMP1 ), and proliferation markers ( CDKN3 ) and  GREM2  (a  BMP6  antagonist).    Conclusion  The decisive factor for the observed change in ASC gene expression proves to be serum starvation rather than VEGF stimulation. Changes in expression of growth factors, matricellular proteins and matrix metalloproteinases in concert, diverge from direct pro-angiogenic paracrine mechanisms as a primary consequence of the used protocol.  In vitro  serum starvation (with or without VEGF present) appears to favour cardioprotection, extracellular matrix remodelling and blood vessel maturation relevant for the late maturation phase in infarct healing.

stem cell research and therapy

Influence of vascular endothelial growth factor stimulation and serum deprivation on gene activation patterns of human adipose tissue-derived stromal cells