Tratwal Josefine, Mathiasen Anders Bruun, Juhl Morten, Brorsen Sonja Kim, Kastrup Jens, Ekblond Annette
Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet, University Hospital Copenhagen, Juliane Maries Vej 20, dept. 9302, Copenhagen, 2100, Denmark.
Stem Cell Res Ther. 2015 Apr 13;6(1):62. doi: 10.1186/s13287-015-0062-9.
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.
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.
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).
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.
在多项动物研究和临床试验中，已使用血管内皮生长因子（VEGF）刺激间充质基质细胞和脂肪组织来源的基质细胞（ASC）以达到再生目的。VEGF刺激被认为可促进血管生成，且VEGF刺激通常在血清剥夺条件下进行。潜在的再生分子机制众多，而相关因素的作用尚不确定。本研究的目的是探讨体外血清剥夺和VEGF刺激对ASC基因表达模式的影响。
比较了在完全培养基中培养的ASC、在血清剥夺培养基中培养的ASC以及在血清剥夺培养基中用VEGF刺激的ASC的基因表达。通过流式细胞术根据国际细胞治疗协会设定的标准确认ASC的特征。使用Affymetrix HT HG-U133+ GeneChip®获得基因芯片基因表达。使用京都基因与基因组百科全书和基因本体术语进行基因集富集分析。通过定量PCR确认所选感兴趣基因的转录。
与完全培养基中的ASC相比，血清剥夺和血清剥夺联合VEGF分别使190个和108个基因发生显著改变。在血清剥夺的ASC和血清剥夺联合VEGF刺激的ASC之间未发现基因表达模式的显著差异。在这两种条件下最显著上调的基因是生长因子（IGF1、BMP6、PDGFD、FGF9）、粘附分子CLSTN2、细胞外基质相关蛋白，如基质细胞蛋白SMOC2、SPON1和ADAMTS12，以及增殖抑制剂（JAG1）。下调最显著的基因包括基质金属蛋白酶（MMP3、MMP1）、增殖标志物（CDKN3）和GREM2（一种BMP6拮抗剂）。
观察到的ASC基因表达变化的决定性因素被证明是血清饥饿而非VEGF刺激。生长因子、基质细胞蛋白和基质金属蛋白酶表达的变化共同作用，与直接的促血管生成旁分泌机制不同，这是所用方案的主要结果。体外血清饥饿（无论是否存在VEGF）似乎有利于心脏保护、细胞外基质重塑以及与梗死愈合后期成熟阶段相关的血管成熟。
