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1 United States Army Institute of Surgical Research and Clinical Investigation, Brook Army Medical Center, San Antonio 78234; and 2 Pharmaceutics Division, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712
To assess the feasibility of
using cDNA microarrays to understand the response of endothelial cells
to lipopolysaccharide (LPS) and to evaluate potentially beneficial
agents in treatment of septic shock, human umbilical vein endothelial
cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h. Total RNA was isolated and reverse-transcribed into
33P-labeled cDNA probes that were hybridized to human
GeneFilter microarrays containing ~4,000 genes. The mRNA levels of
several genes known to respond to LPS changed after
stimulation. In addition, a number of genes not previously
implicated in the response of endothelial cells to LPS also appeared to
be altered in expression. Nuclear factor-
B (NF-B) was shown to
play an important role in regulating genes identified from the
microarray studies. Pretreatment of endothelial cells with a specific
NF-B translocation inhibitor eliminated most of the alterations in
gene expression. Quantitative RT-PCR results independently confirmed
the microarray results for monocyte chemotactic protein-1 and
interleukin-8, and enzyme-linked immunosorbent assays demonstrated that
augmented transcription was followed by translation and secretion.
endothelium; endotoxin; gene expression profiling
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