Abstract 3193: A New Functional Genomic Approach to Identify Soluble Mediators of Atherosclerotic Plaque Stabilization
One of the major drawbacks and potential pitfalls of the use of large scale gene expression studies is the lack of functional assays early in the selection of potential candidates. To circumvent the selection of non-functional candidates we developed a novel functional genomic approach to identify soluble mediators of atherosclerotic plaque stabilization. In this approach, conditioned medium of transfected human cells, containing soluble mediators, is used to stimulate human macrophages which are subjected to functional assays. A SSH (suppression subtractive hybridization) cDNA library, containing over 5000 clones of genes differentially expressed between human stable, advanced atherosclerotic plaques and plaques containing thrombus, was recloned in mammalian expression vectors representing all possible reading frames. The resulting libraries were of good diversity and insert sizes ranged from 150bp to over 1500bp. To identify soluble mediators of plaque stability, pools containing 8–12 individual cDNAs were chemically transfected into HEK293 cells and conditioned media of these cells were used to stimulate/suppress IL-6 cytokine production by THP-1 derived macrophages. Construction and subsequent transfection of β-amyloid and TGF-β1 expression plasmids, which served as positive assay controls, in HEK293 cells led to quantifiable β-amyloid and TGF-β1 protein production. Addition of HEK293 cells conditioned medium to THP-1 derived macrophages led to IL-6 production of 73.3±11.7pg/ml for the β-amyloid construct and 63.4±8.2pg/ml for the TGF-β1 construct. All negative controls, an anti-sense β-amyloid construct and an empty vector, resulted in substantially lower IL-6 levels, 5.8±2.5pg/ml and 9.7±3.6pg/ml respectively. Moreover, this approach revealed the candidate gene construct 70G7 with an IL-6 production of 51.7±6.6pg/ml. Sequence analysis of clone 70G7 revealed a 10 AA ORF which was out of frame, but on the nucleotide level 100% homologous to fibronectin-1 cDNA. In conclusion, we have shown the possibility to identify known and unknown soluble mediators of atherosclerotic plaque stabilization by a functional genomics approach, which facilitates high-throughput screening of large expression libraries.