Cellular signaling via receptor for advanced glycation end products (RAGE) results in pro-inflammatory responses. RAGE-mediated inflammation has been implicated in inflammatory diseases including diabetes, atherosclerosis, and Alzheimers disease. The spliced or proteolytically cleaved form of RAGE is referred as soluble RAGE (sRAGE), which functions as a natural decoy counter-effecting RAGE signaling. It has been demonstrated in animal models that administration of sRAGE blocks atherogenesis, and stabilizes existing plaques on the vessel wall. In addition, sRAGE also prevents the formation of neointima prompted by vascular injuries and hence inhibits restenosis. We have developed Chinese Hamster Ovary (CHO) cell lines that stably express sRAGE, and the accompanied affinity purification strategies that produce homogenous sRAGE. We also generated recombinant Adenovirus that expresses sRAGE. Systemic studies of sRAGE application in restenosis animal models have been completed, and data have been analyzed. Our results showed that sRAGE produced in our laboratory exhibits 500-1000 x higher efficacy than that of sRAGE produced in insect sf9 cells. In addition to blocking restenosis, we also tested sRAGE blockage on infarct animal models and obtained promising preliminary results. We also performed studies to explore the molecular basis of the observed high potency of sRAGE and found that N-glycan structure in sRAGE contributes to its bioactivity. To further develop sRAGE as an effective therapeutic product, we used GeneOptimizer algorithm from Invitrogen to optimize T7-sRAGE----this tool removes sequence repeat, killer motifs, splice sites and RNA secondary structures in the cDNA sequence and optimize codon usage (for CHO cell) and GC content without changing protein sequence. We also tested and found that the new sRAGE cDNA has a higher level of expression than that of native cDNA . This step should enhance future sRAGE scale-up production. To study the role of circulating sRAGE in aging-associated diseases and life-span, we have generated a transgenic mouse model, RAGE-null/sRAGEhigh. Currently, we have completed congenic biomarker-assisted backcrossing, and will breed the new mice to a population for phenotype characterization and longevity studies. The mice will also be crossed to different genetic background/disease models for studying additional aging-associated diseases.
