Title
Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation
Author
Engelse, M.A.
Laurens, N.
Verloop, R.E.
Koolwijk, P.
van Hinsbergh, V.W.M.
TNO Kwaliteit van Leven
Publication year
2008
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.
Subject
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
To reference this document use:
http://resolver.tudelft.nl/uuid:3d79bff6-ce5e-4418-8a84-517302cc6cf8
DOI
https://doi.org/10.1007/s10456-007-9086-9
TNO identifier
240847
ISSN
0969-6970
Source
Angiogenesis, 11 (2), 153-167
Document type
article