results point to the pivotal role played by TSP-1 in mechanisms regulating both vasculogenesis and angiogenesis.

Tissue Inhibitor of Matrix Metalloproteinases

Tissue inhibitors of matrix metalloproteinases (TIMPs) control local MMP activity in tissues by a direct 1:1 binding stoichiometry. Four TIMPs (TIMP-1, -2, -3, and -4) have been identified in vertebrates (129), and their expression in relation to MMP is tightly regulated during tissue remodeling. ECM remodeling in response to a pathological stimulus, often involves unbalanced MMP activities and concomitant changes in TIMP levels. TIMP-1 primarily inhibits the activities of MMP-1, -3, and -9 and TIMP-2 inhibits MMP-2 (130, 131). TIMP-3 is exclusively localized to the ECM and is relatively insoluble, pointing to its potential in preventing matrix lysis and desequestration of growth factors from the matrix. In the retinas of normal mice, TIMP-2 mRNA and protein levels have been found to increase steadily between postnatal days 13–17. 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. A coincidental upregulation of MMP-2 and -9 and MT1-MMP mRNA was seen in experimental retinas as compared to ‘age-matched room air’ controls (132). The normal human retina constitutively expresses MMP-1 and TIMP-2, while retinas with PDR stained positive for TIMP-1, -2, and -3 (133). The role of TIMP-3 has been speculated in an age related macular degeneration. A point mutation of TIMP-3 gene has been implicated in patients with Sorsby’s fundus dystrophy, an autosomal dominant macular disease with earlier onset of symptoms similar to those of ARMD and characterized by choroidal neovascularization (134, 135).

CLINICAL IMPLICATIONS

Currently, the majority of the clinical trials in retinal diseases have targeted the VEGF molecule. So far two drugs, pegaptanib (Macugen), an aptamer against VEGF 165 and ranibizumab (Lucentis), a humanized monoclonal antibody fragment against all isoforms of VEGF have been approved by the FDA for use in exudative age-related macular degeneration. The Macugen DRS trial in PDR patients has shown regression of neovascularization in most of the patients by week 36 (136). Short-term results with intravitreal bevacizumab (Avastin) suggest a rapid regression of retinal and iris neovascularization secondary to PDR (137). A consistent biologic effect was noted, even with the lowest dose (6.2 g) tested, and the observation of a possible therapeutic effect in the fellow eye raises concern of systemic side effects from treatment with intravitreal bevacizumab. The orally administered protein kinase C (PKC) beta isoform-selective inhibitor ruboxistaurin (RBX) in subjects with moderately severe to very severe nonproliferative diabetic retinopathy (NPDR) did not prevent progression to PDR (138).

As the angiogenic cascade has been explored with more potential molecules involved, other therapeutic options using inhibitors other than VEGF inhibitors are being explored. At least in preclinical studies as pointed out in this chapter, many of these molecules have proved to be attractive targets for intervention in proliferative diabetic retinopathy. The rationale for searching other novel pathways is twofold. First, in macular

degeneration patients, clinical experience shows that the drugs, ranibizumab or bevacizumab have to be administered every 4 weeks as complete regression is not possible with just a few injections. Repeated intravitreal injections have potential side effects of infection, vitreous hemorrhage and retinal detachment, and also there may be unknown potential side effects from continuous VEGF inhibition as VEGF has been shown to be a neuroprotective agent. Secondly, a combination therapy using more than one antiangiogenic agent seems to be more attractive as one can attack different steps of the angiogenesis cascade rather than one. Such a combination therapy is common in glaucoma management where intraocular pressure-lowering agents are added one after another until the optimal therapeutic level is achieved. Also, the other scenario is possible where one can combine the current panretinal photocoagulation therapy with anti-VEGF or other anti-angiogenic agents.

What is the status of the antiangiogenic agents other than anti-VEGF agents that are promising in the treatment of proliferative diabetic retinopathy? The just completed Octreotide LAR Phase III study using the IGF-1 inhibitor, octreotide, has been shown to delay the progression of retinopathy, however the secondary endpoints of visual acuity and macular edema did not show any significant difference between placebo treated and octreotide treated patients (139). The urokinase inhibitor, A6 (Angstrom Pharmaceuticals, San Diego, CA), which is currently in a Phase II clinical trial in ovarian carcinoma patients, has been shown to be a promising agent in both retinal and choroidal angiogenesis in pre-clinical studies. Our group has recently shown the therapeutic effects of A6 on increased retinal vascular permeability in diabetic animals (140). Such a dual effect of A6 on both blood-retinal barrier and new vessels in diabetic retinopathy is an added advantage of this novel agent. Several MMP inhibitors, and integrin inhibitors are currently in clinical trial for different types of cancer (141), and many of these agents have been shown to be effective in retinal angiogenesis as well.

New therapies involving pharmacological intervention of retinal angiogenesis are currently being developed. These therapies, or a combination of them, would probably be more effective than the current destructive laser modalities. As we develop new drugs for anti-angiogenic effects, one needs to consider the fact that factors other than VEGF are also critical in the angiogenesis cascade, and one should target against these “other” molecules as well.

ACKNOWLEDGMENTS

Supported by National Institutes of Health RO1 12604-08 and Juvenile Diabetes Research Foundation International.

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