DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells

Abstract

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes͞cm 2 for 6 or 24 h (1 dyne ‫؍‬ 10 N), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF͞GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine͞spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells. During the past 15 years, over 40 genes have been identified as being regulated by shear stress in endothelial cells (1-4). Shear stress responsive genes are involved in cell proliferation, differentiation, maintenance of vascular tone, thrombosis, cellmatrix and cell-cell adhesion, and modulation of the inflammatory͞immune system. The identification of such genes is important not only for developing a fundamental understanding of how endothelial cells work, but also for understanding and treating pathological conditions that are influenced by shear stress, such as thrombosis, restenosis, and atherosclerosis (5, 6).Most of the genes that have been shown to be regulated by shear stress were identified by using traditional techniques such as Northern blot analysis or reverse transcriptase PCR (7-9). The main limitation of these techniques is that only one gene or at best a handful of genes can be studied in one experiment. When multiple genes are studied by using traditional methods, the experiments usually require a reiteration of the detection procedure for each gene. Investigators must therefore be very selective in the genes they choose to study, necessitating a priori information linking the chosen genes to shear stress. Thus, these experiments generally tend to validate or disprove specific hypotheses and do not lead to the discovery of unexpected differentially expressed genes. However, DNA microarray technology allows researchers to study several thousands of genes at one time. In addition to identifying unexpected genes, this technology also has the power to lead to the development of new hypotheses concerning how cells respond to shear stress and identification of coregulated pathways responsive to the mechanical environment of the cell.We used DNA GeneFilter GF211 from Research Genetics (Huntsville, AL; ref. 10), which contains over 4,000 named human genes, to identify genes altered by shear stress in primary human umbilical vein en...

VEGF-C mediates cyclic pressure-induced endothelial cell proliferation

“…McCormick et al (17) used DNA microarray analyses to compare mRNA from HUVEC maintained under static (no-flow) conditions to those of cells exposed to 25 dyn/cm 2 steady fluid shear stress for 24 h. Of the reported 52 genes that were affected by shear stress (17), only the Rho B (a known signal transduction molecule) gene was also affected by exposure of HUVEC to cyclic pressure (Fig. 1).…”

Argininosuccinate Synthase Expression Is Required to Maintain Nitric Oxide Production and Cell Viability in Aortic Endothelial Cells

“…Gene expression studies, using DNA microarray analyses, demonstrated that a significant and coordinate up-regulation of AS gene expression occurred in response to fluid shear stress stimulation of NO production by human umbilical vein endothelial cells (21). Because extracellular arginine was again not limiting in these studies, the authors concluded that a prerequisite for shear stress-induced NO production, in the absence of synthesis of additional eNOS, was an increase in arginine regeneration via increased AS expression (21).…”

“…Recently, a DNA microarray analysis of shear stress-induced NO production demonstrated that up-regulation of AS was coordinated with an increase in NO production (21), supporting an important role for AS in endothelial NO production. If AS is essential for endothelial NO production then even in the presence of excess arginine, depletion of AS should decrease the capacity of endothelial cells to produce NO.…”

Transcriptional regulation of A33 antigen expression by gut-enriched Krüppel-like factor

“…Expression of GKLF is enriched in epithelial cells of the gastrointestinal tract (Ton-That et al, 1997;Behr and Kaestner, 2002) and the skin (Ton-That et al, 1997) and in vascular endothelial cells (Yet et al, 1998;McCormick et al, 2001;Gray et al, 2002). GKLF has been investigated with respect to its role in cellular differentiation, initially within the gastrointestinal tract and in the epidermis where GKLF is critical for late-stage differentiation of skin cells (Ohnishi et al, 2000;Behr and Kaestner, 2002).…”

“…The expression of GKLF is enriched in epithelial cells of the gastrointestinal tract (Ton-That et al, 1997;Behr and Kaestner, 2002) and the skin (Behr and Kaestner, 2002) and in vascular endothelial cells (Yet et al, 1998;McCormick et al, 2001;Gray et al, 2002;Nickenig et al, 2002). Phylogenetically and structurally, KLFs are closely related to Sp1 and Krox transcription factors and they recognize very similar 'GT-box' or 'CACCC element' sites on DNA (Mahatan et al, 1999;Black et al, 2001).…”