title: The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth.
abstract: Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGE(Sf9))and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGE(CHO)) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGE(Sf9). In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGE(CHO) exhibited a significantly higher bioactivity relative to sRAGE(Sf9) to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGE(CHO) is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGE(CHO) significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGE(CHO) reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGE(Sf9) showed no effect. The administered sRAGE(CHO) is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGE(CHO) may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications.
  
  
  CONCLUSIONS
  The specific N-glycoform modification is the key underlying sRAGE bioactivity Markedly reduced sRAGE dose to attenuate neointimal hyperplasia and inflammation Provide a molecular target for glycobioengineering of sRAGE as a therapeutic protein Blocking RAGE alarmin ligands during acute injury phase offsets neointimal growth.
authors.affiliation: Laboratory of Cardiovascular Science, National Institute on Aging, 251 Bayview Boulevard, Baltimore, MD, 21224, USA.
authors.forename: Hyun-Jin
authors.initials: HJ
authors.lastname: Tae
authors.forename: Ji Min
authors.initials: JM
authors.lastname: Kim
authors.forename: Sungha
authors.initials: S
authors.lastname: Park
authors.forename: Noboru
authors.initials: N
authors.lastname: Tomiya
authors.forename: Geng
authors.initials: G
authors.lastname: Li
authors.forename: Wen
authors.initials: W
authors.lastname: Wei
authors.forename: Natalia
authors.initials: N
authors.lastname: Petrashevskaya
authors.forename: Ismayil
authors.initials: I
authors.lastname: Ahmet
authors.forename: John
authors.initials: J
authors.lastname: Pang
authors.forename: Stefanie
authors.initials: S
authors.lastname: Cruschwitz
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Riebe
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Morrell
authors.forename: David
authors.initials: D
authors.lastname: Browe
authors.forename: Yuan Chuan
authors.initials: YC
authors.lastname: Lee
authors.forename: Rui-ping
authors.initials: RP
authors.lastname: Xiao
authors.forename: Mark I
authors.initials: MI
authors.lastname: Talan
authors.forename: Edward G
authors.initials: EG
authors.lastname: Lakatta
authors.forename: Li
authors.initials: L
authors.lastname: Lin
mesh_terms: D000818:Animals; D001168:Arthritis / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D015415:Biomarkers / Q000378:metabolism; D016466:CHO Cells; D002339:Carotid Arteries / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D058426:Neointima / Q000188:drug therapy / Q000378:metabolism*; D051381:Rats; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000008:administration & dosage / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D015415:Biomarkers; D008024:Ligands; D016328:NF-kappa B; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011994:Recombinant Proteins