Suppression Subtractive Hybridization Identifies Distinctive Expression Markers for Coronary and Internal Mammary Arteries

Abstract

Objective-We sought to identify differentially expressed genes in the athero-prone coronary artery and athero-resistant internal mammary arteries. Methods and Results-Using suppressive subtraction hybridization, we generated reciprocal cDNA collections of representative mRNAs specific to porcine coronary arteries versus porcine mammary arteries. We screened 1000 suppressive subtraction hybridization cDNA clones by dot blot array and sequenced 600 of those showing the most marked expression differences. Northern blot, in situ hybridization, and immunostaining confirmed the differential gene expression patterns identified by the dot blot arrays. Genes associated with mammary arteries included claudin-10 and h-cadherin, which are genes associated with tight junctions and intermediate junctions. In contrast, genes associated with proatherosclerotic processes, such as lipid retention and metabolism, inflammation, and cell growth, were preferentially expressed in coronary arteries. Conclusions-Normal coronary arteries have gene expression program that is significantly different than internal mammary arteries. These differences may partly explain the resistance of coronary arteries and internal mammary arteries to atherosclerosis.

Genome-Wide Expression Studies of Atherosclerosis

“…A drawback of combining samples from different sites is that differences in shear stress, embryonic origin, or elastic versus muscular phenotype between arteries may have an effect on expression profiles and thus obscure the data. 7 Therefore, the use of samples from a single site may reduce the variability in gene expression and fewer samples may be needed for the same statistical power. Four studies used "selected arrays" to evaluate vascular expression levels of a few hundred genes, 10,16,17,27 selected because of their involvement in apoptosis for example.…”

“…Our analysis reveals the methodological limitations in the published studies and highlights challenges for future genomic profiling. animals (7 studies; Table 2), but larger animals such as rats 4 -6 and pigs 7 have also been used.…”

“…In 3 studies, 7,26,27 macrodissection was used to separate the smooth muscle cell (SMC)-rich fibrous cap, 26,27 media, 7,26 and nonatherosclerotic intima. 26 To this end, the adventitia was trimmed, the endothelium was scraped off, and the fibrous cap was dissected from the necrotic core and shoulder regions.…”

Arterial Smooth Muscle Cell Heterogeneity

“…A very recent study with suppressive subtractive hybridization that was performed in the pig has compared the coronary artery, predisposed to atherosclerosis, with the mammary artery, which is resistant to atherosclerosis. 106 Genes that are preferentially associated with the mammary artery are involved in cell-cell junction formation, whereas genes that are preferentially expressed in the coronary artery are implicated in lipid metabolism, inflammation, and cell proliferation. However, the presence of distinct SMC populations within the same vessel has not been taken into account in these studies.…”

Thematic review series: The Pathogenesis of Atherosclerosis. Toward a biological network for atherosclerosis

“…Nevertheless, such studies will importantly complement in vivo studies (7). In particular, expression array analyses of cells in response to genetic, nutritional, or pharmacologic perturbations should help in the formulation or validation of network models.…”