DNA Micro-Array Gene Expression Profiling of Angiogenesis in Collagen Gel Culture

Abstract

Abstract:We examined angiogenesis-related gene expression profiles using collagen gel culture and a DNA chip. After isolation of total RNA from cultures before and after capillary tube formation, a mouse whole-genome array study was performed. Seventy-three out of over 35,000 transcripts were expressed after capillary tube formation. The majority of genes did not show any signifi cant differences between before and after capillary tube formation. However, there were 7 upregulated genes; tumor necrosis factor alpha-induced protein (Tnfαip) 2, vascular endothelial growth factor b (Vegfb), corticotropin releasing hormone receptor (Crhr) 2, vascular endothelial growth factor C (Vegfc), fi broblast growth factor (Fgf) 6, protein O-fucosyltransferase (Pofut) 1, hypoxia inducible factor 1 alpha subunit (Hif-1a). Eleven down-regulated genes were identifi ed; ras homolog gene family member B (RhoB), Fgf-2, Thrombospondin (Thbs) 1, Angiogenin (Ang) 3, guanine nucleotide binding protein alpha (Gna) 13, tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a), plasminogen (Plg), Elk3, upstream binding protein (Ubp) 1, integrin alpha V (Itgav), mitogen activated protein kinase (Mapk) 14. These up-and down-regulated genes identifi ed in this study offer the strategies which improve the myocardial substrate during and following a myocardial infarction, such as the regrowth of functional blood vessels to the ischemic myocardium.

Detection of the Hematopoietic Stem and Progenitor Cell Marker CD133 during Angiogenesis in Three-Dimensional Collagen Gel Culture

“…Using a three-dimensional (3D) collagen gel culture, we have conducted electron microscopic studies [14, 15] and immunohistochemical studies of fibroblast growth factor- (FGF-) 2 and FGF-9 [16]. Additionally, we have used 3D collagen gel cultures to test angiogenic and anti-angiogenic agents (TNF- α and thalidomide) [17, 18] to study vascular injury after laser microdissection [19] and for profiling of DNA microarray gene expression during angiogenesis [20]. The 3D collagen gel culture system provides a simple and rapid method to analyze angiogenesis.…”

Development of In Vitro Method for Assaying Anti-Angiogenic Effect of Drugs

“…The data were log transformed and the statistical significance of differences in intensity values was calculated by logfold-change (logFC) analysis (CodeLink Expression Analysis software, v 4.1). The majority of genes examined showed only small differences, with ratios ranging between 2.0 and -2.0 [120]. Negative logFC (< -2) indicate the logarithmic foldness of down-regulation.…”

“…We examined angiogenesis-related gene expression profiles using collagen gel culture and a DNA chip [120]. At day 5 of the culture, numerous cells migrated from the aortic explant into the collagen gel, but the capillary tubes had not yet formed.…”

“…This DNA microarray chip displays probe DNAs for about 35,000 mouse genes and expressed sequence tags [120]. For microarray analysis, RNA was purified using a RNeasy ® MinEluteTM Cleanup Kit (Qiagen).…”

“…The CodeLink ® Mouse Whole Genome array consists of probe sets representing over 35,000 transcripts [120]. Following normalization, p-value calculation for individual probes was done.…”