S. van Soest, R.D. Fontijn, H. Pannekoek and A.J.G. Horrevoets.
Department of Biochemistry, AMC, Amsterdam.
Endothelial cells play an important role in the onset and progression of atherosclerosis. Activation of these cells by an atherogenic stimulus induces the expression of cell adhesion molecules like E-selectin and VCAM-1, which results in the recruitment and invasion of monocytes into the vessel wall. The altered properties of activated endothelial cells in a pathogenic condition reflect altered patterns of gene expression. The aim of this study is to identify new genes involved in the response of endothelial cells to atherogenic stimuli.
Stimulation of human umbilical vein endothelial cells (HUVEC) with TNFa is used as a model system for activated endothelium. Differential display analysis of stimulated vs. non-stimulated HUVEC yielded 106 differentially expressed gene-fragments (DD-fragments). A number of the DD-fragments represent known genes (22/106) or genes present in the EST databases (38/106). The remaining DD-fragments (48/106) represent currently unknown genes. Considering the size of the obtained set of candidate genes we chose to implement the microarray technology in order to analyze the expression profile of all DD-fragments simultaneously.
A microarray was constructed that contains the DD-fragments and ESTs, as well as a number of genes with an established role in vascular pathology, and housekeeping genes to be used for standardization. The gene fragments were PCR amplified and printed onto glass slides, which were subsequently hybridized with fluorescently labeled cDNA probes generated by reverse transcription of total mRNA. Direct comparison of the expression profiles of stimulated and non-stimulated cells was accomplished by simultaneous hybridization with two probes, labeled with different fluorophores. Quantification and analysis of the first results confirmed the differential expression of a number of control genes, e.g. MnSOD and ICAM, demonstrating the feasibility of this approach, as well as several new genes.
Similar results were obtained with probes generated from T7-amplified RNA.
With the chosen set-up, combining Differential Display with microarray analysis, we obtained a flexible and efficient screening method for the identification of differentially expressed genes. The synthesis of amplified RNA from minute amounts of mRNA, will enable us to analyze the expression of the whole set of “atherosclerotic” genes simultaneously in minute amounts of human vascular tissue material, providing a link to the in vivo situation.