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

173 Citations (Scopus)

Abstract

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
Volume48
Issue number11
DOIs
Publication statusPublished - Nov 2007

Keywords

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

Cite this

Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy. / Leal, Ermelindo C.; Manivannan, Ayyakkannu; Hosoya, Ken-Ichi; Terasaki, Tetsuya; Cunha-Vaz, Jose; Ambrosio, Antonio Francisco; Forrester, John V.

In: Investigative Ophthalmology & Visual Science, Vol. 48, No. 11, 11.2007, p. 5257-5265.

Research output: Contribution to journalArticle

Leal, Ermelindo C. ; Manivannan, Ayyakkannu ; Hosoya, Ken-Ichi ; Terasaki, Tetsuya ; Cunha-Vaz, Jose ; Ambrosio, Antonio Francisco ; Forrester, John V. / Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy. In: Investigative Ophthalmology & Visual Science. 2007 ; Vol. 48, No. 11. pp. 5257-5265.
@article{24d58689fe204470b27d85d99e27c9ad,
title = "Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy",
abstract = "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.",
keywords = "endothelial growth-factor, intercellular adhesion molecule-1, glycation end-products, protein-kinase-C, in-vivo, oxidative stress, leukocyte entrapment, tyrosine nitration, cells, rats",
author = "Leal, {Ermelindo C.} and Ayyakkannu Manivannan and Ken-Ichi Hosoya and Tetsuya Terasaki and Jose Cunha-Vaz and Ambrosio, {Antonio Francisco} and Forrester, {John V.}",
year = "2007",
month = "11",
doi = "10.1167/iovs.07-0112",
language = "English",
volume = "48",
pages = "5257--5265",
journal = "Investigative Ophthalmology & Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "11",

}

TY - JOUR

T1 - Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy

AU - Leal, Ermelindo C.

AU - Manivannan, Ayyakkannu

AU - Hosoya, Ken-Ichi

AU - Terasaki, Tetsuya

AU - Cunha-Vaz, Jose

AU - Ambrosio, Antonio Francisco

AU - Forrester, John V.

PY - 2007/11

Y1 - 2007/11

N2 - 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.

AB - 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.

KW - endothelial growth-factor

KW - intercellular adhesion molecule-1

KW - glycation end-products

KW - protein-kinase-C

KW - in-vivo

KW - oxidative stress

KW - leukocyte entrapment

KW - tyrosine nitration

KW - cells

KW - rats

U2 - 10.1167/iovs.07-0112

DO - 10.1167/iovs.07-0112

M3 - Article

VL - 48

SP - 5257

EP - 5265

JO - Investigative Ophthalmology & Visual Science

JF - Investigative Ophthalmology & Visual Science

SN - 0146-0404

IS - 11

ER -