Amadori-modified glycated albumin predominantly induces E-selectin expression on human umbilical vein endothelial cells through NADPH oxidase activation.
BACKGROUND
Protein glycation is closely linked to endothelial-cell dysfunction and vascular complications in diabetes. Glycated albumin is reported to induce cellular signaling similar to advanced glycation endoproducts (AGEs), however, cellular signaling remains obscure.


METHODS
We stimulated human umbilical vein endothelial cells (HUVECs) by glycated human serum albumin (Glc-HSA), determined E-selectin expression by real-time PCR and immunometric methods, and estimated cellular signaling by using various signaling molecule inhibitors and confocal microscopy.


RESULTS
Glc-HSA-induced E-selectin expression was 10 or 20 times more than that induced with 3 kinds of AGEs-HSAs, which was not suppressed by anti-receptor for AGEs (RAGE) antibody. Glc-HSA-induced E-selectin expression was completely suppressed by the NADPH oxidase inhibitor diphenylene iodonium chloride and the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine. Confocal microscopic analysis also revealed intracellular accumulation of ROS. Glc-HSA-induced E-selectin expression was suppressed by the phosphatidylinositol 3 kinase (PI3K) inhibitors wortmannin and LY294002, the protein kinase B (PKB) inhibitor ML-9, the IkappaB kinase (IKK) inhibitor BAY117082, and the Jun N-terminal kinase (JNK) inhibitor SP600125, On the other hand, the protein kinase C inhibitors calphostin C and H-7 did not suppress Glc-HSA-induced E-selectin expression.


CONCLUSIONS
Glc-HSA induces activation of NADPH oxidase, PKB-IKK and JNK, then E-selectin gene transcription is upregulated by nuclear-translocated NF-kappaB and AP-1.
Higai
Koji
K
Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan. koji@phar.toho-u.ac.jp
Shimamura
Ayano
A
Matsumoto
Kojiro
K
D002478:Cells, Cultured; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D017127:Glycation End Products, Advanced; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D014176:Protein Biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology*; D000093423:Glycated Serum Albumin
D019040:E-Selectin; D017127:Glycation End Products, Advanced; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012709:Serum Albumin; D019255:NADPH Oxidases; D051057:Proto-Oncogene Proteins c-akt; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D000093423:Glycated Serum Albumin