Fig. 5. Use of a matrix metalloproteinase inhibitor suppresses the development of retinal neovascularization. (A) Hematoxylin–eosin-stained cross section from the retina of a mouse exposed to 75% oxygen for 5 days followed by room air for an additional 5 days. Capillary tufts are present on the vitreal side of the inner limiting membrane, characteristic of the angiogenic response in this tissue (arrow). (B) Representative hematoxylin-and-eosin-stained section from the retina of an experimental mouse treated with BB-94 1 mg/kg, on postnatal days 12, 14, and 16. (C) Similar section from an experimental animal stained with diamidinophenylindole showing individual endothelial cell nuclei that belong to new vessels (arrow). (D) Similar section from the retina of a BB-94-treated mouse stained with diamidinophenylindole showing a significant reduction in the number of neovascular nuclei. Only a single endothelial cell nucleus is present on the vitreal side of the inner limiting membrane. Scale bars: (A, B) 166 mm; (C, D) 113 mm (reproduced with permission from Das et al. [45]).

Inhibition of Retinal Angiogenesis by Inhibitors of the uPA/uPAR System

A peptide inhibitor of the urokinase system, A6 (an octapeptide that inhibits the interaction of uPA with uPAR), was able to reduce the extent of retinal neovascularization and uPAR expression in the experimental animals. Intravitreal injection of an adenoviral vector carrying the murine ATF has been shown to inhibit retinal neovascularization by 78% in the oxygen-induced retinopathy level [84]. These results suggest that inhibition of the urokinase receptor might be a promising target for antiangiogenic therapy in the retina.

PROTEASES IN DIABETIC MACULAR EDEMA

The vitreous level of MMP-9 has been shown to be higher in diabetic subjects with DR than with the diabetic subjects without DR. This study indicates a potential role of MMPs in the pathogenesis of DR [87]. Furthermore, in an animal model of diabetes,

both MMP-2 and MMP-9 were elevated in the retinas [88]. The MT1-MMP was also increased along with MMP-2 in the diabetic animals, and concomitant to this, there was an increased apoptosis of pericytes in the diabetic retina when compared to the normal retina. This may further accelerate the BRB alteration in the diabetic state. We have shown that retinal vascular permeability was significantly increased in rats following 2 weeks of diabetes coincident with a decrease of VE-cadherin expression. This increased vascular permeability could be inhibited with an MMP inhibitor [89]. Treatment of endothelial cells with AGE-BSA led to a reduction of VE-cadherin staining on the cell surface and increased permeability, which was MMP-mediated. This suggests that MMPs have a direct role in the alteration of endothelial permeability [89]. Treatment of cells with specific MMPs or AGEs resulted in cleavage of VE-cadherin from the cell surface. These observations suggest a possible mechanism by which diabetes contributes to BRB breakdown through proteolytic degradation of VE-cadherin. The ability of a broad-spectrum MMP inhibitor in the breakdown of BRB suggests a potential alternative therapeutic strategy to the treatment of diabetic macular edema. High glucose can activate many soluble mediators such as AGE, ROS, and inflammatory cytokines, which can increase MMP expression and activity in the diabetic state.

The role of MMP-9 is implicated in the alteration of barrier function which is shown to be mediated by TGF-b [90]. Studies have hinted that diabetes causes retinal inflammation which unleashes a sequelae of events resulting in the vascular leakage. Retinal inflammation attracts increased leukocytes to the retina which then bind to the vascular endothelium. The binding of leukocyte to the endothelial cells can also activate cellular proteases that may clip off VE-cadherin and its associated protein from the cell surface resulting in endothelial monolayer alteration.

Inhibition of Proteases in the Prevention of Blood–Retinal

Barrier in Diabetes

MMPs have emerged as regulators of endothelial barrier function in several tissues. Studies have demonstrated an increased expression of MMPs in the retinas of diabetic animals. The proteolytic degradation of vascular endothelial (VE)-cadherin from the surface of cultured endothelial cells by MMP-9 has been shown to increase the vascular monolayer permeability. An inhibitor of MMP-9 (Batimastat (BB-94)) was able to block diabetes-induced vascular permeability and prevented the loss of VE-cadherin in the retinal vasculature. These study result indicates a role for extracellular proteases in the alteration of the BRB seen in diabetic retinopathy and can be a potential therapeutic target for treating DME [89].

We have shown that the increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE)-cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin [91]. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was also significantly inhibited with A6. Treatment of endothelial cells with specific MMPs or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play