Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy.
OBJECTIVE
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.
Leal
Ermelindo C
EC
Center of Ophthalmology of Coimbra, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
Manivannan
Ayyakkannu
A
Hosoya
Ken-Ichi
K
Terasaki
Tetsuya
T
Cunha-Vaz
José
J
Ambrósio
António Francisco
AF
Forrester
John V
JV
D000818:Animals; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002199:Capillary Permeability / Q000502:physiology*; D002448:Cell Adhesion; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D003930:Diabetic Retinopathy / Q000201:enzymology*; D004730:Endothelium, Vascular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007527:Isoenzymes / Q000502:physiology; D007962:Leukocytes / Q000378:metabolism; D018921:Leukostasis / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000502:physiology*; D062793:Occludin; D009887:Ophthalmoscopy; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D012171:Retinal Vessels / Q000502:physiology; D062826:Zonula Occludens-1 Protein
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; C568987:Ocln protein, rat; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019331:NG-Nitroarginine Methyl Ester