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

Abstract

Introduction: Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascula 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 seru deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors i uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEG stimulation on gene expression patterns of ASCs Methods: Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived mediu and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according t criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Microarray gen expressions were obtained using the Affymetrix HT HG-U133+ GeneChip®. Gene set enrichment analysis wa performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms. Transcription o 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 deprivatio and serum deprivation combined with VEGF, respectively. No significant differences in gene expression pattern between serum-deprived ASCs and serum-deprived ASCs combined with VEGF stimulation were found. Genes mos prominently and significantly upregulated by both conditions were growth factors (IGF1, BMP6, PDGFD, FGF9), adhesio molecule CLSTN2, extracellular matrix-related proteins such as matricellular proteins SMOC2, SPON1 and ADAMTS12, an 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 rathe than VEGF stimulation. Changes in expression of growth factors, matricellular proteins and matrix metalloproteinases i concert, diverge from direct pro-Angiogenic paracrine mechanisms as a primary consequence of the used protocol. I vitro serum starvation (with or without VEGF present) appears to favour cardioprotection, extracellular matrix remodellin and blood vessel maturation relevant for the late maturation phase in infarct healing.