Development of an ELISA for esRAGE and its application to type 1 diabetic patients.

We recently identified a naturally occurring soluble form of RAGE (the receptor for advanced glycation endproducts, receptor for AGE) in cultured human vascular cells, and named it endogenous secretory RAGE (esRAGE). esRAGE is generated by alternative RNA splicing and is able to capture AGE, and exerts protection against AGE-induced endothelial cell injury. In the present study, the presence of esRAGE in human circulation was demonstrated for the first time, and a highly sensitive and specific sandwich ELISA system for esRAGE was developed to see whether esRAGE could be related to an individual resistance to the development of diabetic vascular complications. Sera from 47 type 1 diabetic subjects without clinical nephropathy (urinary albumin excretion <300mg/g creatinine) and 55 healthy controls were analyzed by the ELISA. Circulating esRAGE concentrations in diabetic patients with simple and proliferative retinopathy (0.09+/-0.02ng/mL, n=16 and 0.08+/-0.02ng/mL, n=8, respectively) were significantly lower than in those without retinopathy (0.13+/-0.06ng/mL, n=23). The results indicate that esRAGE can be a useful biomarker to indicate individual variations in susceptibility to diabetic retinopathy.

Plasma level of endogenous secretory RAGE is associated with components of the metabolic syndrome and atherosclerosis.

OBJECTIVE: Advanced glycation endproducts, AGEs, and its specific receptor, RAGE, are involved in diabetic vascular complications. Endogenous secretory RAGE, esRAGE, has been identified as an alternatively spliced form of RAGE, and shown to act as a decoy receptor for AGE. Here, we measured plasma esRAGE level with a recently developed enzyme-linked immunosorbent assay (ELISA) and examined its association with atherosclerosis in age- and gender-matched 203 type 2 diabetic and 134 nondiabetic subjects. METHODS AND RESULTS: Plasma esRAGE was inversely associated with carotid or femoral atherosclerosis, as quantitatively measured as intimal-medial thickness (IMT) by arterial ultrasound. Stepwise regression analyses revealed that plasma esRAGE was the third strongest and independent factor associated with carotid IMT, following age and systolic blood pressure. Plasma esRAGE was significantly lower in diabetic patients (0.176+/-0.092 ng/mL) than nondiabetic controls (0.253+/-0.111). Of note, in all, diabetic or nondiabetic group, plasma esRAGE was significantly and inversely correlated with components of the metabolic syndrome including body mass index, blood pressure, triglyceride, HbA1c, or an insulin resistance index. Stepwise regression analyses showed that body mass index or insulin resistance index was the major factor determining plasma esRAGE in all, nondiabetic or diabetic population. CONCLUSIONS: esRAGE is a novel and potential protective factor for the metabolic syndrome and atherosclerosis.

Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N.

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457-Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218-Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.