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

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-alpha, 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.

Molecular Mediators of Angiogenesis

“…Consistent with this, recent evidence demonstrated that overexpression of Cdc42 GTPase activating protein (Cdc42 GAP), a protein which catalyzes GTP hydrolyzation and consequently decreases the active conformation of Cdc42, attenuates EC lumen formation while siRNA knockdown of Cdc42 GAP increased lumenogenesis 160 . In vitro , ECs with lower levels of Cdc42 GAP expression have been shown to be more likely to form tubes and lumens in comparison to ECs with a higher level of expression 160. Although the exact mechanistic role of Cdc42 in EC lumen formation is still unclear, this and other downstream GTPase pathway proteins demonstrate potentially promising therapeutic targets for pro or anti-angiogenesis therapies 161…”

Identification of Vascular and Hematopoietic Genes Downstream of etsrp by Deep Sequencing in Zebrafish

“…The few survivors experience premature aging associated with heightened genomic instability [36]. In vitro evidence suggests cdc42GAP1 negatively modulates angiogenesis [37]. Although it is expressed ubiquitously in mammals, it is predominantly expressed in the developing vasculature and heart in zebrafish at the stage examined here (Figure 3B).…”

Staphylococcus aureus recruits Cdc42GAP through recycling endosomes and the exocyst to invade human endothelial cells

“…The vesicles deliver Cdc42GAP, which then deactivates Cdc42 and thereby terminates further delivery of the vesicles and Cdc42GAP. Preliminary results from our laboratory suggest that this regulatory circuit might also control endothelial cell functions known to be instrumental in angiogenesis-like migration of endothelial cells and the formation of anastomoses between nascent endothelial cell tubules (Engelse et al, 2008;Koh et al, 2008Koh et al, , 2009.…”