Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway

Abstract

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase-H2O2-chloride system of activated phagocytes is highly up-regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxidase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochemical studies performed in the present study revealed marked colocalization of HOCl-modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myeloperoxidase-H2O2-chloride system, is a high affinity ligand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE-like structures, induced a rapid, RAGE-dependent activation of extracellular signal-regulated kinase 1/2 and up-regulation of the proinflammatory mediator monocyte chemoattractant protein-1. Cellular activation could be blocked either by a specific polyclonal anti-RAGE IgG and/or a specific mitogen-activated protein-kinase kinase inhibitor. The present study demonstrates that HOCl-modified albumin acts as a ligand for RAGE and promotes RAGE-mediated inflammatory complications.

Figure 1

Immunofluorescence and double immunofluorescence for RAGE staining. 5 µm frozen sections of abdominal aorta (type II) were incubated with polyclonal anti-human albumin antiserum (A), polyclonal anti-human-sRAGE antiserum (prepared as described in the methods section) (B–D), mAb clone 2D10G9 (recognizing HOCl-modified epitopes) (A, C), and mAb anti-CD141 (an endothelial marker; B) as primary antibodies. Preabsorbtion of anti-sRAGE IgG was performed with a 5-fold molar excess of purified sRAGE for 30 min (D). Cy-2/-3 labeled secondary antibodies were used.

Figure 2

sRAGE binding to HOCl-modified albumin. A) Albumin was immobilized onto wells of plastic dishes and modified by indicated HOCl-concentrations. After washing and blocking of excess binding sites with native albumin, ¹²⁵I-sRAGE (3 µg/ml) was added to coated wells. Subsequently, wells were washed and radioactivity was counted. B) Native albumin (Alb) and freshly prepared HOCl-albumin (oxidant:protein molar ratio of 25:1, 50:1, and 100:1) was immobilized onto wells of plastic dishes. After washing and blocking of excess binding sites with native albumin, ¹²⁵I-sRAGE (3 µg/ml) was added to coated wells. Subsequently, wells were washed, and radioactivity was counted. C) Albumin (Alb), AGE-Alb, HOCl-Alb (oxidant:protein molar ratio of 25:1, 50:1, and 100:1) and MPO-Alb (modified by the MPO-H₂O₂-chloride system; oxidant:protein molar ratio of 50:1) were immobilized onto wells of plastic dishes. After washing and blocking of unspecific binding sites, indicated concentrations of ¹²⁵I-sRAGE (1, 3, 10, and 30 µg/ml) were added to coated wells. Subsequently, wells were washed and radioactivity was counted. Binding of ¹²⁵I-sRAGE to control albumin-coated wells (nonspecific binding) was subtracted from binding values to modified-albumin to calculate specific binding. Only specific data are shown. D) Dose-response curves for ¹²⁵I-sRAGE (5 µg/ml) binding inhibition to AGE-BSA-coated microtiter wells by albumin (Alb), AGE-Alb, HOCl-Alb (oxidant:protein molar ratio of 25:1, 50:1, and 100:1) and MPO-Alb (modified by the MPO-H₂O₂-chloride system; H₂O₂:Alb molar ratio of 50:1). Results represent mean ± sd (n=3) of one experiment out of four.

Figure 3

RAGE-dependent Erk1/2 MAP-kinase activation. A) RAGE-HEK cells were incubated with indicated concentrations of albumin (Alb) or HOCl-Alb (oxidant:protein molar ratio of 25:1, 50:1, and 100:1) for 20 min. B) RAGE-HEK cells were incubated at indicated time periods with 200 µg/ml Alb or HOCl-Alb (oxidant:protein molar ratio of 100:1). C) RAGE-HEK cells were incubated with 1 mg/ml Alb, 1 mg/ml AGE-Alb, or 200 µg/ml HOCl-Alb (oxidant:protein molar ratio of 100:1) for 20 min in the absence or presence of 200 µg/ml polyclonal anti-sRAGE IgG. Anti-sRAGE IgG was added to the cells 2 h before addition of AGE-Alb or HOCl-Alb. After cell lysis, SDS-PAGE, and Western blot analysis, phosphorylation of Erk1/2 (pErk1/2) was determined by use of phospho-specific Erk1/2-kinase specific antibodies. Blots were reprobed with anti-Erk antibody to ensure equivalence of loading. A typical experiment out of three is shown.

Figure 4

RAGE-dependent MCP-1 transcript expression by HOCl-albumin. Control HEK and RAGE-HEK cells were incubated for 3 h with indicated concentrations of albumin (Alb) and HOCl-Alb (oxidant:protein molar ratio of 100:1) in the absence or presence of polyclonal anti-sRAGE IgG (200 µg/ml) or the specific MAP-kinase kinase inhibitor PD 98059 (25 µM). MCP-1 transcripts were determined by RT-PCR as described in the Methods section. Results are the average of duplicates of one representative experiment out of three, and error bars represent the ranges of the measurements. MCP-1 expression of nontreated RAGE-HEK cells were used as controls.