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Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

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Author: Engelse, M.A. · Laurens, N. · Verloop, R.E. · Koolwijk, P. · Hinsbergh, V.W.M. van
Type:article
Date:2008
Institution: TNO Kwaliteit van Leven
Source:Angiogenesis, 2, 11, 153-167
Identifier: 240847
doi: doi:10.1007/s10456-007-9086-9
Keywords: Angiogenesis · CDC42GAP · Differential gene expression · Fibrin matrix · Microdissection · basic fibroblast growth factor · cdc42gap protein · complementary DNA · messenger RNA · protein derivative · small interfering RNA · tumor necrosis factor alpha · vasculotropin · angiogenesis · article · cell population · controlled study · endothelium cell · human · human cell · human cell culture · microdissection · phenotype · priority journal · protein expression · real time polymerase chain reaction · RNA extraction · Biological Assay · Biological Markers · Cell Movement · Cells, Cultured · Endothelial Cells · Endothelium, Vascular · Gene Expression Profiling · GTPase-Activating Proteins · Humans · Lasers · Microdissection · Neovascularization, Physiologic · Phosphoproteins

Abstract

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.