Abstract 267: Lentivirus Gene Transfer of the Endogenous Circulating RAGE Splice Form Blocks Mechanisms Leading to Atherosclerosis
The Receptor for Advanced Glycation End-products (RAGE) is a strong candidate gene for the development of atherosclerosis. We have previously shown that pharmacological blockade of RAGE (recombinant soluble RAGE) or genetic deletion of RAGE suppresses the development of vascular disease in animal models. Further, in human subjects the endogenous circulating levels of RAGE are lower in plasma of subjects with coronary artery disease, suggesting an innate protective role for circulating soluble (s)RAGE. Characterization of cardiovascular tissue and cells by RT-PCR revealed the presence of RAGE and the production of sRAGE by alternative splicing resulting from inclusion of intron 9 (RAGEint9) in normal human heart and in endothelial (EC), aortic smooth muscle (AoSMC) and monocyte/macrophage (MP) cells. Using an antibody generated against the unique C-terminus protein splice region of RAGEint9, we detected RAGEint9 in lysates and cultured media from EC, aortic (Ao) SMC and macrophages. Transfection of RAGEint9 suppressed RAGE-ligand S100B-stimulation of MMP-9 activity and levels by ~2–3 and ~3– 4-fold respectively, compared to wild-type RAGE expressing cells (p<0.0001). Upon S100B incubation, RAGEint9- vs. RAGE expressing cells generated significantly decreased levels of IL-6 in the supernatant (~1.6-fold; p<0.0001). We generated lentiviral constructs to enable gene transfer of RAGEint9 into primary human aortic SMCs and ECs. Lentivirus transduction of RAGEint9 into human AoSMCs, reduced S100B-stimulated levels of MCP-1 by ~2-fold compared to cells expressing full-length RAGE (p<0.05). Further, the migration of AoSMCs was blocked in RAGEint9 transduced cells in response to RAGE ligand stimulation. Finally, in ECs transduced with RAGE / RAGEint9 lentivirus, permeability assays were performed. The gene transfer of RAGEint9 into ECs blocked RAGE-ligand dependent endothelial permeability by 50%, p<0.001, the hallmark of atherosclerosis. We conclude that RAGEint9 gene therapy or endogenous stimulated expression may represent a novel therapeutic modality to enhance protection against atherosclerotic disease.