Inducible nitric oxide synthase and vascular endothelial growth factor are colocalized in the retinas of human subjects with diabetes.

PURPOSE: Nitric oxide (NO) mediates vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. This study was undertaken to study the cellular distribution of inducible nitric oxide synthase (iNOS) and VEGF in the retinas from human subjects with diabetes mellitus. In addition, glial reactivity and peroxynitrite generation were detected by immunolocalization of glial fibrillary acidic protein (GFAP) and nitrotyrosine, respectively. METHODS: Eight post-mortem eyes from four consecutive subjects with diabetes mellitus and eight eyes from four subjects without diabetes and without known ocular disease were prospectively collected and examined. We used immunohistochemical techniques and antibodies directed against iNOS, VEGF, GFAP, and nitrotyrosine. RESULTS: In retinas from all subjects without diabetes, weak GFAP immunoreactivity was confined to nerve fibre and ganglion cell layers. There was no immunoreactivity for iNOS, nitrotyrosine, and VEGF. All diabetic retinas showed GFAP induction in Müller cells and GFAP upregulation in nerve fibre and ganglion cell layers. All diabetic retinas showed cytoplasmic immunoreactivity for iNOS, and VEGF in ganglion cells, cells in the inner nuclear layer, and glial cells. In serial sections, ganglion cells and cells in the inner nuclear layer expressing VEGF were localized in the same area of iNOS-expressing ganglion cells and cells in the inner nuclear layer. Six retinas from three subjects with diabetes showed immunoreactivity for nitrotyrosine in vascular endothelial cells in inner retinal layer. CONCLUSIONS: iNOS and VEGF are colocalized in diabetic retinas. Increased GFAP immunoreactivity is a pathological event in the retina during diabetes.

Immuno-histochemical evaluation of conjunctival remodelling in vernal keratoconjunctivitis.

PURPOSE: To study the expression of the extracellular matrix (ECM) proteins, tenascin, laminin, and fibronectin in the conjunctiva of patients with active vernal keratoconjunctivitis (VKC). METHODS: Conjunctival biopsy specimens were obtained from nine patients with active VKC and 6 normal control subjects. The presence and distribution of tenascin, laminin, and fibronectin were assessed microscopically with immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against tenascin, laminin, and fibronectin. RESULTS: In normal conjunctiva, weak immunoreactivity for tenascin was localized to the walls of blood vessels in the upper substantia propria. Weak immunoreactivity for laminin was located at the epithelial-stromal junction and in the walls of blood vessels. Staining for fibronectin was absent. In VKC specimens, intense immunoreactivity for tenascin was noted in the substantia propria associated with the inflammatory infiltrate and in the perivascular stroma. Intense immunoreactivity for laminin around all stromal vessels and fibrillar immunoreactivity among basal epithelial cells were noted. There was no immunoreactivity for fibronectin. CONCLUSION: Our data indicate increased deposition of tenascin and laminin in the conjunctiva from patients with active VKC. Our findings suggest that tenascin and laminin might play distinct roles in chronic inflammation seen in VKC.

Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus.

PURPOSE: Inducible nitric oxide synthase has been implicated in the pathogenesis of cerebral ischemic damage, in the angiogenic process and in diabetic vascular damage. This study was undertaken to determine whether inducible nitric oxide synthase is present in the retinas from human subjects with diabetes mellitus. METHODS: This was an experimental immunohistochemical prospective study. Ten postmortem eyes from five subjects with diabetes mellitus, 10 eyes from five subjects without diabetes and without known ocular disease, and two eyes from one subject with unilateral ocular ischemic syndrome secondary to severe carotid artery obstruction were examined. We used immunohistochemical techniques and antibodies directed against inducible nitric oxide synthase, glial fibrillary acidic protein, and vimentin. The main outcome measure was immunoreactivity for these antibodies. RESULTS: Immunoreactivity for inducible nitric oxide synthase was not observed in retinas from all subjects without diabetes and without ocular disease. Six retinas from three subjects with diabetes and nonproliferative retinopathy, and the retina from the eye with ocular ischemic syndrome showed immunoreactivity for inducible nitric oxide synthase in cells with elongated processes. Based on morphology and on glial fibrillary acidic protein and vimentin immunoreactivity, this inducible nitric oxide synthase immunoreactivity appeared to localize to retinal Müller glial cells. CONCLUSIONS: These observations suggest that Müller cells may be involved in the microvascular remodeling of the diseased retina and that high concentrations of nitric oxide produced by inducible nitric oxide synthase could contribute to neurotoxicity and angiogenesis that occur in diabetic retinopathy.