MMP-9 and MMP-9/TIMP-1 ratio. This increased level of MMP-9 has been suggested to be surrogate biomarkers of retinopathy in type 1 diabetic patients free of other vascular complications [75].

Further characterization of roles of MMPs in diabetic retinopathy revealed various molecular aspects of its activity and regulation. Hyperglycemic condition induces the increased activation of retinal capillary MMP-2 and MT1-MMP and decreases in TIMP-2. This activation was inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2 [76]. The hyperglycemia-induced activation of MMP-9 accelerates apoptosis of retinal capillary cells, a phenomenon that predicts the development of diabetic retinopathy, and the activation of MMP-9 is downstream of H-Ras. Also, inhibition of high glucose–activated MMP-9 by pharmacologic inhibitor or siRNA ameliorated accelerated apoptosis in the retinal endothelial cells [77]. Interestingly, the human retinal pericytes treated with high glucose levels have been shown to have increased MMP-2 activity leading to increased ECM turnover while there was no MMP-9 activity observed in those cells. Thiamine and benfotiamine correct the increase in MMP-2 activity due to high glucose in HRP, while increasing TIMP-1 levels in the pericytes [78]. MMP inhibitor such as a2M has been shown to play a key role in the control and regulation of the retinal neovascularization involved in the pathogenesis of PDR [79]. The transcription factor such as AP-1 and JUN has been shown to regulate retinal MMP synthesis during neovascularization. The importance of transcriptional factor as a therapeutic target that regulates the expression of MMPs such as MMP-2 in microvascular endothelial growth and retinal neovascularization is also considered [80, 81].

Angiogenesis and matrix degradation are an important step in endothelial cell migration and proliferation. Evidence has indicated the role of serine proteases, such as tissue plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and PAI, in the retinal neovascularization. In PDR, the vitreous levels of these proteases are increased [82]. At cellular level, hyperglycemic condition has been shown to alter the levels of t-PA and PAI in the retinal microvascular endothelial cells [83]. In an animal model of hypoxia-induced retinal neovascularization, it was found that the expression of the urokinase receptor (uPAR) was required to mediate an angiogenic response. uPAR−/− mice demonstrated normal retinal vascularity but showed a significant reduction (by 73%) in the extent of pathological neovascularization as compared to wild-type controls (Fig. 4). The expression of uPAR mRNA was upregulated in experimental animals during the active phase of angiogenesis, and uPAR protein was localized to endothelial cells in the superficial layers of the retina [84–86].

TISSUE INHIBITOR OF MATRIX METALLOPROTEINASES IN RETINAL NEOVASCULARIZATION

In the retinas of normal mice, TIMP-2 mRNA and protein levels have been found to increase steadily between postnatal days 13 and 17. This was in contrast to retinas of mice with hypoxia-induced retinal angiogenesis, in which TIMP-2 mRNA and protein remained low and significantly less than in retinas of “room air” controls [84]. Thus, a temporal correlation between proteases (MMP-2 and -9 and MT1-MMP) and TIMP-2 was seen in retinas with neovascularization as compared to controls [85].

Fig. 4. AbsenceoftheurokinasereceptoruPARreducedtheextentofretinalneovascularization in the mouse. (A) Representative section of the retina from an experimental oxygen-treated P17 C57BL6 mouse demonstrating numerous neovascular tufts on the surface of the retina (arrows). (B) A similar section from an experimental oxygen-treated P17 uPAR−/− mouse with many fewer vascular tufts (arrows). (C) Quantitation of neovascularization in C57BL6 and uPAR−/− mice. The uPAR−/− mice demonstrated 73% less neovascularization compared with the normal C57BL6 mice. Values are the mean ± SEM for n = 4 mice in each group (eight eyes, 15–20 sections/eye). *Significantly less than in C57BL6 mice, P < 0.01 (reproduced with permission from McGuire et al. [84]).

Inhibition of Retinal Angiogenesis by MMP Inhibitors

Inhibition of MMP activity is considered an important therapeutic option in the prevention of diabetic retinopathy. Preclinical studies have shown the importance of these inhibitors in the prevention of retinal neovascularization. Systemic injection of a broadspectrum MMP inhibitor, BB-94 (1 mg/kg), in the murine model has been shown to suppress retinal neovascularization by 72% [45] (Fig. 5). The retinas of BB-94-treated animals demonstrated a significant decrease in the levels of active forms of MMP-2 and MMP-9 compared to controls. In a mouse model of OIR, the extent of preretinal neovascularization was drastically reduced in MMP-2−/− (75%) and MMP-9−/− mice (44%) at postnatal day 19, compared to wild-type control mice [45]. The functional association of MMP-2 and avb3 on the cell surface of angiogenic blood vessels points to the ability of MMPs to regulate cell adhesion and integrin-mediated behavior.