Recent studies have identified that the activation of protein kinase C (PKC) and increased diacylglycerol (DAG) levels initiated by hyperglycemia are associated with many vascular abnormalities in retinal, renal, and cardiovascular tissues. Among the various PKC isoforms, the beta- and delta-isoforms appear to be activated preferentially in the vasculatures of diabetic animals, although other PKC isoforms are also increased in the renal glomeruli and retina. The glucose-induced activation of PKC has been shown to increase the production of extracellular matrix and cytokines; to enhance contractility, permeability, and vascular cell proliferation; to induce the activation of cytosolic phospholipase A2; and to inhibit Na+-K+-ATPase. The synthesis and characterization of a specific inhibitor for PKC-beta isoforms have confirmed the role of PKC activation in mediating hyperglycemic effects on vascular cells, as described above, and provide in vivo evidence that PKC activation could be responsible for abnormal retinal and renal hemodynamics in diabetic animals. Transgenic mice overexpressing PKC-beta isoform in the myocardium developed cardiac hypertrophy and failure, further supporting the hypothesis that PKC-beta isoform activation can cause vascular dysfunctions. Interestingly, hyperglycemia-induced oxidative stress may also mediate the adverse effects of PKC-beta isoforms by the activation of the DAG-PKC pathway, since treatment with D-alpha-tocopherol was able to prevent many glucose-induced vascular dysfunctions and inhibit DAG-PKC activation. Clinical studies are now in progress to determine whether PKC-beta inhibition can prevent diabetic complications.

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The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGFneutralizing chimeric proteins were constructed byjoining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGFreceptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-,um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%,

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