Nox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products

Alejandra San Martin, Rocio Foncea, Francisco R. Laurindo, Roberto Ebensperger, Kathy K. Griendling, Federico Leighton

Research output: Contribution to journalArticlepeer-review

84 Scopus citations

Abstract

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/106 cells, p < 0.001) or coelenterazine (277,907 ± 71,295 vs 120,456 ± 4140 LU/106 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-κB 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-κB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-κB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

Original languageEnglish (US)
Pages (from-to)1671-1679
Number of pages9
JournalFree Radical Biology and Medicine
Volume42
Issue number11
DOIs
StatePublished - Jun 1 2007

Bibliographical note

Funding Information:
This work was supported by Projects PUC-PBMEC 2001–2003, Fondecyt 1020486, HL075209 and FAPESP 00/12154-2. The authors thank Drs. Ohara Agusto, Sergei Dikalov, and Marcelo Pedro for their technical help with EPR measurements and Dr. Victoria Velarde for her valuable discussions during the performance of these studies.

Keywords

  • Advanced glycated end products
  • Diabetes
  • Free radicals
  • Inflammation
  • NADPH oxidase
  • Nox1
  • ROS
  • Superoxide
  • iNOS

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