stage (44, 45). In a separate study, no correlation was found between serum and vitreous levels of HGF in diabetic patients, and the high HGF level in vitreous was attributed to intraocular synthesis of HGF in these patients (46). These clinical observations raise the possibility of an important role for this growth factor in ocular angiogenesis. The function of HGF is dependent on the expression and activation of the Met receptor, a transmembrane tyrosine kinase encoded by the c-Met proto-oncogene (47). In a mouse model of retinal angiogenesis, we have shown that HGF and c-Met are upregulated in the retinas, and the HGF was active as evidenced by the increased presence of the phophorylated form of c-Met in the tissues (48). HGF stimulates the secretion of urokinase and expression of its receptor, uPAR in retinal endothelial cells. HGF also increases the migratory and invasive capacity of these cells which could be inhibited by the addition of the urokinase-derived peptide A6 (48). Inhibition of c-Met results in a 70% decrease in retinal angiogenesis and a 40% decrease in urokinase activity in the retina (Fig. 3). Interestingly, HGF has been shown to increase retinal vascular permeability at physiologically relevant concentrations with a magnitude similar to that of VEGF without altering retinal hemodynamics (49). Thus, HGF may play roles in both retinal angiogenesis and blood–retinal barrier alteration, and may be used as targets in diabetic retinopathy.

TUMOR NECROSIS FACTOR

Tumor necrosis factor, a 26 kDa transmembrane protein, has been shown to be expressed in retinas of human proliferative diabetic retinopathy (50, 51) and in animal models of retinal neovascularization (51, 52). It is expressed mainly by Muller cells and inner nuclear layer and in the outer nuclear layer during the angiogenic phase as well (52). TNFα is processed by a TNFα converting enzyme (TACE) to yield a 17 kDa soluble protein. TNFα functions through its binding to two receptors: p55, implicated in apoptosis and NFkB activation, and p75, involved with lymphocyte proliferation. TNFα increases the expression of several proteinases like MT1-MMP, MMP-3 and MMP-9 in cultured retinal microvascular endothelial cells. VEGF also plays a role in this process through its regulation of TNFα converting enzyme (TACE) and p55 mRNA in the vascular endothelial cells. The overlapping temporal expression of VEGF and TNFα suggests an interactive role of these two growth factors in the regulation of extracellular proteinases during the angiogenic process. Inhibition of TNFα significantly improves vascular recovery and reduces retinal neovascularization in the oxygen-induced retinopathy model (53). Also, in animals with TNF receptor p55 deficiency there is significantly reduced vascularization in the same model (54). TNFα has been shown to operate along with COX-2 in alteration of the blood–retinal barrier in early diabetic retinopathy, a newly recognized inflammatory disease (55).

EXTRACELLULAR PROTEINASES

Endothelial cell migration and invasion through the capillary basement membrane and extracellular matrix (ECM) are crucial steps in the angiogenic process. This process is tightly coupled to the induction and activation of certain extracellular proteinases. Historically, two families of proteinases have been studied in this process – the serine

Fig. 3. Expression of HGF and c-Met mRNA in control and experimental OIR mice using the realtime comparative Ct method of quantitation. HGF mRNA levels in retinas from experimental animals was significantly elevated on days 15 (P = 0.0093) and 17 (P = 0.0044) compared with controls when new blood vessels were forming (A). The pattern of c-Met mRNA expression in experimental animals was similar (B). The level of c-Met mRNA increased significantly on day 12 (P = 0.0043) and persisted through day 15 (P = 0.0126) and day 17 (P = 0.0121). Samples are from day 12, 15, and 17 control (C) and experimental (E) animals; n = 3. *Significantly greater than control. Experimental mice were treated with a single intraocular injection of c-met neutralizing antibody on day 13 and were analyzed for new vessel formation. Representative images of retinas from normal IgG (C) and anti-c-met-treated (D) animals. Quantitation of new vessels reveals a 70% reduction in the degree of angiogenesis in the eyes treated with the c-met antibody (e). *Significantly less than IgG-treated animals (P < 0.0001). Bar, 10 m. GF (reprinted from (48) with permission).

proteinase, urokinase plasminogen activator (uPA) and members of a family of zincdependent endopeptidases called matrix metalloproteinases (MMPs).

The Urokinase Plasminogen Activator System (uPA/uPAR System)

The urokinase plasminogen activator is present in endothelial cells in two molecular forms, a 54 kDa high molecular weight form and a 32 kDa low molecular weight form which lacks the amino terminal fragment of the protein (56, 57). The amino terminal fragment contains the growth factor and kringle domains of the protein that mediate binding to uPAR and is postulated to play a role in cell proliferation (58, 59). uPA is

Matrix Metalloproteinases

The matrix metalloproteinases are a family of zinc-dependent proteases, originally identified for their ECM degrading capabilities. At least 21 different types of MMPs have been identified to date. Based on their substrate specificity and cellular localization MMPs are grouped into the collagenases (MMP-1, MMP-8, and MMP-13), the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10, MMP-11), and the non traditional MMPs (matrilysin/MMP-7, metalloelastase or MMP-12) and the membrane type MMPs,(MT-MMPs) (66). There are at least five distinct types of MT-MMPs: MMP-12, -15, -16, -17 and -21 and MT1-MMP has been studied in relation to its ability to regulate focused matrix lysis and the activation of MMP-2 (66). In addition to their capacity to degrade a large variety of ECM molecules, MMPs are known to process a number of bioactive molecules. MMPs regulate a variety of cell behaviors such as cell proliferation, migration, differentiation, apoptosis and host defense. In relation to retinal neovascularization, MMPs are implicated in the angiogenic process for their involvements in mainly endothelial cell migration and invasion and posttranslational modification of anti-angiogenic growth factor receptors.

Proteinases in Retinal Neovascularization

Significant upregulation of uPA (both the 54 and 32 kDa isoforms) along with increases in secretion and activation of MMP-2 and -9 were observed in the retinas of animals with neovascularization (67). These results suggest that proteolytic activity and its regulatory mechanisms might play an important role in the angiogenic process. There has been increasing evidence that the plasminogen activators play an important role in the progression of this disease. Increased levels of VEGF and TPA have been accounted for in the vitreous fluid of patients with PDR (68). Several growth factors such as HGF, TGF-B, VEGF and certain angiostatic drugs have been shown to induce angiogenesis by increasing proteolytic activity in endothelial cells (48, 69). Examination of proteinases in epiretinal neovascular membranes removed surgically from humans with PDR showed a similar increase in the levels of uPA, pro and active forms of MMP-2 and -9 as compared to normal retinas (70) (Fig. 5). The pro-MMP2 and TIMP-2 have been reported in the normal and diabetic vitreous (71, 72), and pro-MMP-9 in diabetic vitreous (71). A recent study has shown that activity of both MMP-2 and its physiological activator MT1-MMP are significantly increased in retinas of diabetic animals, and increased MMP-2 activity compromises retinal pericyte survival possibly through MMP-2 action on ECM proteins (73).

In an animal model of hypoxia-induced retinal neovascularization, it was found that expression of the urokinase receptor was required to mediate an angiogenic response. uPAR−/− mice demonstrated normal retinal vascularity but showed a significant reduction in the extent of pathological neovascularization as compared to wild type controls (74). 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. A peptide inhibitor of the urokinase system, A6, was able to reduce the extent of retinal neovascularization and uPAR expression in the experimental animals (74) (Fig. 6). In another study, the adenovirus-mediated delivery of a uPA/uPAR antagonist inhibited endothelial cell

Fig. 6. Absence of the urokinase receptor uPAR reduced the extent of retinal neovascularization 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 (arrow). (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 (reprinted from (74) with permission).

suppress retinal neovascularization by 72% (67) (Fig. 7). 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. In the same study, the efficacy of three matrix metalloproteinase inhibitors with different selectivities was tested in a rat model of retinopathy of prematurity. Intravitreal injections of the MMP inhibitors resulted in a significant reduction in the angiogenic response in experimental animals in comparison with uninjected controls (77). Elevated levels of ADAM-15/ MMP-15, a membrane anchored glycoprotein and disintegrin, were reported in endothelial cells and Adam 15−/− mice demonstrated a major reduction in neovascularization in a model of retinopathy of prematurity (78). The functional association of MMP-2 and αvβ3 on the cell surface of angiogenic blood vessels points to the ability of MMPs to