Egr-1 target genes in human endothelial cells identified by microarray analysis
Introduction
Vascular endothelium is a non-thrombogenic surface of normally quiescent cells that lies at the inner surface of blood vessels. The integrity of this endothelium is fundamental for maintaining the homeostasis of vasculature. Injury of this lining results in dramatic changes in the functional characteristics of the endothelium, rendering it adhesive and prothrombotic. These initial events are well correlated with subsequent inflammation and proliferation in vasculature, which is associated with the development of vascular diseases such as atherosclerosis and restenosis (Ross, 1999).
Early growth response factor-1 (Egr-1) was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli, including growth factors, cytokines, hypoxia, physical forces, and injury, all of which are implicated in the progress of vascular diseases (Silverman and Collins, 1999). As a transcription factor, Egr-1 can induce expression of a set of vasculature genes, such as PDGF-A and B chain, bFGF, TGFβ, TNFα, and intracellular adhesion molecule-1 (Silverman and Collins, 1999). Recent studies demonstrated that Egr-1 expression is elevated in both atherosclerotic lesions and neointima after vascular injury McCaffrey et al., 2000, Santiago et al., 1999a. Interestingly, reduction of Egr-1 expression by the Egr-1 antisense technology decreased intimal hyperplasia in balloon-injured carotid through inhibiting smooth muscle cell migration and proliferation Santiago et al., 1999b, Fahmy and Khachigian, 2002. Taken together, these findings suggest that Egr-1 is the key mediator in orchestrating the functional characteristics of the vessel wall after injury.
To further understand the role of Egr-1 in vasculature, we used commercially available oligonucleotide-based microarrays from Affymetrix (U95Av2 GeneChips, Santa Clara, California) to quantitate the Egr-1-mediated changes in expression of more than 12,000 mRNAs in human vascular endothelial cells.
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Cell culture and adenovirus infection
Human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) were purchased from Bio-Whittaker (San Diego, California). HUVECs were cultured in endothelial cell growth medium-2 (EGM-2, Bio-Whittaker) containing 5% FBS, human basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), human epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), 50 μg/ml gentamicin, 50 ng/ml amphotericin-B, hydrocortisone, and ascorbic acid. HASMCs
Overexpression of Egr-1 in HUVEC
To overexpress Egr-1 in HUVEC, we utilized a recombinant adenovirus (AdEgr-1*) that co-expresses the green fluorescent protein (GFP) and the constitutively active Egr-1*, which is resistant to repression by endogenous NAB transcriptional corepressors. After infection with AdEgr-1* or the control virus AdGFP at a titer of ∼5 pfu/per cell for 24 h, almost all of cells (>99%) were GFP-positive (Fig. 1A). No apoptosis was observed in HUVEC infected with adenovirus at up to 10 pfu/cell in this
Discussion
To further explore the role of Egr-1 in vasculature, we have employed adenovirus-mediated Egr-1 overexpression in human vascular endothelial cells (HUVEC) to identify novel Egr-1 target genes using Affymetrix microarray analyses. In the present study, 229 up-regulated and 75 down-regulated genes were identified with ≥3-fold changes in HUVEC. Among them, several genes have been previously identified as Egr-1 target genes. However, most of them are novel Egr-1 target genes. These results will
Acknowledgements
This work was partially supported by the NIH (grants HL068878, HL03676, and S06GM08248) and the Swiss National Science Foundation (grant 31-57125.99 to M.U.E.). M.F. and Y.L. are supported by the fellowships from the American Heart Association Southeast Affiliate.
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