Published online before print December 2, 2002, doi: 10.1161/01.RES.0000049100.22673.F6
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Submitted on October 19, 2001
Revised on October 9, 2002
Accepted on November 13, 2002
DNA Microarray Profiling to Identify Angiotensin-Responsive Genes in Vascular Smooth Muscle Cells. Potential Mediators of Vascular Disease
Alexandre H. Campos ;
From the Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Ga.
* To whom correspondence should be addressed. E-mail: ggibbons{at}msm.edu.
Angiotensin II (Ang II) induces changes in vessel structure by its capacity to activate genes that are coupled to signaling pathways such as extracellular signal-regulated kinase (ERK), p38, and phosphatidylinositol 3-kinase (PI3K). Using a DNA microarray containing 5088 genes and expressed sequence tags, we initially established a database of replicated experiments (n=4) to define the variances in mRNA expression in response to Ang II versus vehicle treatment. We observed a wide range of values for the coefficients of variation in a gene-specific manner. Guided by power calculations, we used statistical inference on a sufficient number of experimental replicates to minimize the number of false-negatives and define a subset of Ang II-responsive genes (P<0.05). To further characterize the molecular circuitry that couples Ang II stimulation with mRNA expression, we assessed expression profiles in the presence and absence of inhibitors of ERK, p38, and PI3K. Using two different methods of computational cluster analysis, we identified a subset of six matricellular proteins (eg, osteopontin and plasminogen activator inhibitor-1) that are coordinately upregulated by Ang II via an ERK/p38-dependent pathway. In addition, these cluster analyses identified calpactins I and II as novel Ang II-responsive genes. Given that Ang II promotes vascular lesion formation, we examined whether this matricellular gene cluster was also coordinately regulated in vivo. Indeed, we demonstrate that both calpactin I and osteopontin are upregulated in response to vascular injury. Taken together, the combined use of DNA microarrays, statistical inference, and cluster analysis identified novel, coordinately regulated Ang II-responsive genes that may mediate vascular lesion formation.
Key words: vascular smooth muscle cells • angiotensin II • DNA microarray • matricellular genes
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