Inventory of 'atherosclerotic genes' induced or repressed in endothelial cells and smooth muscle cells by differential display rt-pcr

Abstract

Initiation and progression of atherosclerosis requires differential expression of a distinct set of known and novel genes in various cell types, e.g vascular endothelial cells (ECs) and smooth muscle cells (SMCs). We set out to identify genes with a reproducibly altered, time-dependent expression in either cultured human ECs or SMCs, activated by TNF-a or by conditioned medium of oxLDL activated monocytes (CM-MC). An unbiased method (differential display RT-PCR) was employed to make an inventory of differential gene expression. Theoretically, the combinations of 12 anchored primers (5'T,NN) and 12 arbitrary 10-mers displays approximately 80% of a cell's mRNA repertoire. Depending on the abundance of differential transcription, confirmation was done by Northern blotting, RNase protection or by semi-quantitative RT-PCR. Accordingly, we identified 106 TNF-a/CM-MC responsive genes in cultured ECs and 46 genes in SMCs. Strikingly, only 3 known genes (GM-CSF, IL-8, IAP-C) were found to be induced both in ECs and SMCs, illustrating that ECs and SMCs express different genes in response to the same atherogenic stimulus. Probes corresponding to these genes are then employed for in situ hybridization with human specimen, obtained either from major vascular surgery or from donors, representing different stages of the disease. At present, in situ hybridization demonstrated SMC-specific expression in specimen for three novel unknown genes. The in vim mRNA expression pattern markedly differs, suggesting a differential function in early or advanced lesions. Full-length cDNA cloning and, subsequent, functional characterization of the corresponding gene products is in progress