Nox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products.
Vascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cell (VSMC) ROS production are unclear. We studied the source and downstream targets of AGE-mediated ROS and reactive nitrogen species production in these cells. Significant increases in superoxide production in AGE-treated VSMC were measured using lucigenin (7650+/-433 vs 4485+/-424 LU/10(6) cells, p<0.001) or coelenterazine (277,907+/-71,295 vs 120,456+/-4140 LU/10(6) cells, p<0.05) and confirmed by ESR spectroscopy. These signals were blocked by the flavin-containing oxidase inhibitor diphenylene iodonium (DPI). AGE-stimulated NF-kappaB activity was abolished by DPI and the superoxide scavenger MnTBAP. AGE differentially regulated VSMC NADPH oxidase catalytic subunits, stimulating the transcription of Nox1 (201+/-12.7%, p<0.0001), while having no effect on Nox4. AGE also increased 3-nitrotyrosine formation, which was inhibited by MnTBAP, DPI, or the NOS inhibitor L-NAME. Regarding the source of NO, AGE stimulated inducible nitric oxide synthase mRNA (1 vs 9.7+/-3.0, p=0.046), which was abolished by a NF-kappaB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-kappaB-dependent mechanism involving ROS generation by a Nox1-based oxidase.
San Martin
Alejandra
A
Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322, USA. msanmar@emory.edu
Foncea
Rocio
R
Laurindo
Francisco R
FR
Ebensperger
Roberto
R
Griendling
Kathy K
KK
Leighton
Federico
F
D000818:Animals; D002478:Cells, Cultured; D017127:Glycation End Products, Advanced / Q000633:toxicity*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014652:Vascular Diseases / Q000209:etiology*
D017127:Glycation End Products, Advanced; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013481:Superoxides; C002744:3-nitrotyrosine; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614712:NOX1 protein, rat