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, Jose Cunha-Vaz, Antonio Francisco Ambrosio, John V. Forrester

Research output: Contribution to journalArticle

215 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, N-G- 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 & Visual Science
Issue number11
Publication statusPublished - Nov 2007


  • endothelial growth-factor
  • intercellular adhesion molecule-1
  • glycation end-products
  • protein-kinase-C
  • in-vivo
  • oxidative stress
  • leukocyte entrapment
  • tyrosine nitration
  • cells
  • rats


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