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

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.