C5a-induced gene expression in human umbilical vein endothelial cells

Abstract

The endothelium plays a critical role in the inflammatory process. The complement activation product, C5a, is known to have proinflammatory effects on the endothelium, but the molecular mechanisms remain unclear. We have used cDNA microarray analysis to assess gene expression in human umbilical vein endothelial cells (HUVECs) that were stimulated with human C5a in vitro. Chip analyses were confirmed by reverse transcriptase-polymerase chain reaction and by Western blot analysis. Gene activation responses were remarkably similar to gene expression patterns of HUVECs stimulated with human tumor necrosis factor-alpha or bacterial lipopolysaccharide. HUVECs stimulated with C5a showed progressive increases in gene expression for cell adhesion molecules (eg, E-selectin, ICAM-1, VCAM-1), cytokines/chemokines, and related receptors (eg, VEGFC, IL-6, IL-18R). Surprisingly, HUVECs showed little evidence for up-regulation of complement-related genes. There were transient increases in gene expression associated with broad functional activities. The three agonists used also caused down-regulation of genes that regulate angiogenesis and drug metabolism. With a single exception, C5a caused little evidence of activation of complement-related genes. These studies indicate that endothelial cells respond robustly to C5a by activation of genes related to progressive expression of cell adherence molecules, and cytokines and chemokines in a manner similar to responses induced by tumor necrosis factor-alpha and lipopolysaccharide.

Figure 1

Microarray cluster of 1260 genes. Gene activation values of <2.6-fold increases above control values were not considered to be of significance (see text). HUVECs were stimulated with LPS (1 μg/ml) for 4 hours; TNF-α (1 ng/ml) for 4 hours; and C5a (50 ng/ml) for 30 minutes, 2 hours, and 4 hours. The control (cntrl) represents a labeling control between two identically stimulated samples. A: The black vertical bar 1 is a cluster of genes up-regulated in response to stimulation by C5a, LPS, or TNF-α. The black vertical bar 2 represents a cluster of genes down-regulated in response to 4 hours of stimulation with C5a, LPS, or TNF-α. The black vertical bar 3 represents genes that are transiently down-regulated in response to C5a at 30 minutes and 2 hours. B: Expanded sections are from black vertical bars 1 and 2. Induced genes selected have cytokine/chemokine and transcription-related functions. The selected repressed genes are associated with cytoskeletal functions. C: An expanded section is from black vertical bar 3. Transiently repressed genes in response to 50 ng/ml of C5a were associated with metabolic functions.

Figure 2

Semiquantitative gene and protein expression based on microarray, RT-PCR, and Western blotting techniques. A: Lines represent groups of clustered genes induced after 30 minutes (early), 2 hours (mid), or 4 hours (late) stimulation with C5a (50 ng/ml). B: RT-PCR confirmation of selected gene expression in response to C5a (50 ng/ml) stimulation is shown. Data are representative of n = 3. C: Western blots for protein expression of selected genes are displayed. Cell lysates were collected after 2, 8, 18 hours after stimulation with C5a. Control lysates were from cells without C5a stimulation. Loading conditions were confirmed by β-tubulin content.