Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation
article
The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-α, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially 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 as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation. © 2007 Springer Science+Business Media B.V.
Topics
AngiogenesisCDC42GAPDifferential gene expressionFibrin matrixMicrodissectionbasic fibroblast growth factorcdc42gap proteincomplementary DNAmessenger RNAprotein derivativesmall interfering RNAtumor necrosis factor alphavasculotropinangiogenesisarticlecell populationcontrolled studyendothelium cellhumanhuman cellhuman cell culturemicrodissectionphenotypepriority journalprotein expressionreal time polymerase chain reactionRNA extractionBiological AssayBiological MarkersCell MovementCells, CulturedEndothelial CellsEndothelium, VascularGene Expression ProfilingGTPase-Activating ProteinsHumansLasersMicrodissectionNeovascularization, PhysiologicPhosphoproteins
TNO Identifier
240847
ISSN
09696970
Source
Angiogenesis, 11(2), pp. 153-167.
Pages
153-167
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