Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy

Ermelindo C. Leal, Ayyakkannu Manivannan, Ken Ichi Hosoya, Tetsuya Terasaki, José Cunha-Vaz, António Francisco Ambrósio, John V. Forrester

Research output: Contribution to journalArticlepeer-review

206 Citations (Scopus)


PURPOSE. Nitric oxide (NO) is involved in leukostasis and blood-retinal barrier (BRB) breakdown in the early stages of diabetic retinopathy (DR), but it is unclear which NO synthase (NOS) isoforms are primarily involved. In this study, the authors aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO-mediated leukostasis and BRB breakdown. METHODS. Diabetes was induced with streptozotocin for 2 weeks. Mice were treated with a NOS inhibitor, NG-nitro-L- arginine methyl ester (L-NAME), which shows a preference for constitutive isoforms over iNOS. Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat-mounted retinas with confocal microscopy, in vivo with a scanning laser ophthalmoscope, and in vitro in a retinal endothelial cell line. ICAM-1, occludin, and ZO-1 levels were assessed by Western blot, flow cytometry, or immunohistochemistry. Nitrotyrosine content was assessed by immunohistochemistry. RESULTS. Diabetes increased leukostasis within retinal vessels and BRB permeability, which were reduced by L-NAME. Similar effects were observed in diabetic iNOS knockout mice. In diabetic mouse retinas, ICAM-1 protein levels increased, whereas the immunoreactivity of tight junction proteins, occludin and ZO-1 decreased, in correlation with increased protein levels of all NOS isoforms. Those effects were prevented by L-NAME and also in diabetic iNOS knockout mice. High glucose and nitrosative/oxidative stress also increased leukostasis caused by ICAM-1 upregulation. CONCLUSIONS. These results indicate that the iNOS isoform plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction protein downregulation.

Original languageEnglish
Pages (from-to)5257-5265
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Issue number11
Publication statusPublished - 2007 Nov
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience


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