Endogenous Inhibitors of Proteases

Tissue Inhibitors of Metalloproteinases (TIMPs)

Tissue inhibitors of metalloproteinases (TIMPs) are specific endogenous inhibitors that bind MMPs in 1:1 stoichiometry. Four types of TIMPs have been identified, which have overlapping activities with respect to their inhibition of most soluble MMPs [3, 57]. TIMP molecules block the activity of MMPs by binding to the active-site cleft, in a manner similar to their substrates. TIMP-2 and TIMP-3 are good inhibitors of MT1MMP, MMP-19, and ADAM-17, while TIMP-1 is poor in this respect. TIMPs also have biological effects unrelated to metalloproteinase inhibition. TIMP-4 has been shown to be localized mostly in the vascular tissues. TIMP-3, but neither TIMP-1 nor TIMP-2, is involved in binding to the VEGF receptor-2 (KDR) and competes for debinding of VEGF to this receptor. Overexpression of TIMP-3 can induce apoptosis. TIMP-1 and TIMP-2 display antiapoptotic properties and indirectly induce cell signaling. A point mutation of the TIMP-3 gene has been implicated in Sorsby’s fundus dystrophy, an autosomal dominant macular disease similar to wet macular degeneration, but with earlier onset of symptoms [58, 59]. Studies have also characterized protein molecules such as RECK, a2-macroglobulin (a2M), and tissue factor pathway inhibitor-2 to inhibit the MMP activity [60–62].

Plasminogen Activator Inhibitors (PAI)

Plasminogen activator inhibitors (PAIs), which are members of the serine proteinase inhibitor (SERPIN) family, regulate the proteolytic activity of uPA through the inhibition of uPA and plasmin formation. PAI-1 and PAI-2 have been found to interact with urokinase in 1:1 ratio to inhibit enzyme activity and cause enzyme/inhibitor internalization and turnover [63]. These serine protease inhibitors (SERPINS) bind covalently to their targets, inhibiting proteolytic activity [64]. PAI-1 has been shown to be a strong prognostic marker for several cancer types [65].

PROTEASES IN RETINAL NEOVASCULARIZATION

Significant upregulation of uPA (both the 54and 32-kDa isoforms) along with increases in secretion and activation of MMP-2 and -9 was observed in the retinas of animals with neovascularization [45]. These results suggest that proteolytic activity and its regulatory mechanisms might play an important role in the angiogenic process. Various studies have shown that the plasma levels and the fibrovascular epiretinal membranes MMP-2 and -9 levels were significantly elevated in patients with PDR [66–71]. Examination of proteases in epiretinal neovascular membranes removed surgically from humans with PDR showed a similar increase in the levels of uPA and proform and active form of MMP-2 and -9 as compared to normal retinas [72]. It has been also shown that the pro-MMP-2 is efficiently activated in the fibrovascular tissues of PDR through interaction with MT1-MMP and TIMP-2 [73], indicating its increased role in PDR. Further, MMP-2 deficiency in mice has been shown to reduce the retinal angiogenesis [74]. Type 1 diabetes subjects with retinopathy have displayed elevated systemic levels of