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Angiogenesis occurs in wound areas where the extracellular matrix consists of a fibrinous exudate, and in which growth factors (like VEGF) and inflammatory mediators (like TNFα) are present. Our aim is to analyse differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tube forming (angiogenic) endothelial cells and monolayer endothelial cells not involved in tube-formation. We applied a fibrin-rich three-dimensional matrix derived from human plasma to facilitate angiogenic sprouting of human foreskin microvascular endothelial cells (MVEC). These cells were characterized as blood vessel-derived MVEC without lymphatic cell content. After 7 days stimulation with VEGF and/or bFGF and TNF-α the culture consists of a monolayer and capillary sprouts that have grown into the fibrinous matrix. A method was developed to detach the distinct monolayer and tubule-forming populations of MVEC, keeping their cellular integrity intact to ensure subsequent mRNA extraction. Separately, the mRNA preparations were used as a template to produce cDNA samples that were radiolabeled and utilized in hybridization mixtures to probe Research Genetics gene arrays. Subsequent array analysis resulted in an inventory of differentally-expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real time PCR on the original RNA samples. In additions, we employed laser-capture microdissection on cross-sections of monolayers and capillary structures in a three dimensional plasma matrix. A selection of the differentially expressed genes was tested to demonstrate their direct functional involvement in angiogenesis. Our investigations demonstrate the feasibility of separation, analysis and verification of crucial to differential gene expression analysis by tube forming and non-tube forming endothelial cells.
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J. Vasc. Biol. 42, Sup:2 (2005) p16