Angiogenesis plays a central part not only in the development of retina but also in the visual impairment attributable to retinopathy in diabetes, retinal vascular occlusion, retinopathy of prematurity, sickle cell disease, and in age-related macular degeneration. The process of angiogenesis in the retina and other tissues is characterized by distinct phases or activities including an initial response to locally produced angiogenic factors and signals. This event is followed by a rapid upregulation of matrix-degrading enzymes or extracellular proteases (extracellular proteolytic mediators) that facilitate the breakdown of the capillary basal lamina and migration and subsequent invasion of activated endothelial cells into the surrounding extracellular tissues [2, 3]. Extracellular proteases help not only in the degradation of interstitial extracellular matrices (ECMs) and basement membranes but also in the recruitment of progenitor cells into the ECM during tissue remodeling. Proteases are expressed by normal cells in tissue remodeling events and also during pathological events such as tumor angiogenesis and metastasis. This chapter will review these extracellular proteases and discuss their potential roles in diabetic retinopathy and the development of therapeutic strategies targeting these molecules in preventing retinal neovascularization and diabetic macular edema.

Extracellular Proteases

The ECM is a complex assembly of proteins and polysaccharides which provides the physical support and organization to tissues. Cell-surface receptors on the plasma membrane bind to ECM and regulate intracellular signaling pathways that control cell migration and proliferation. Cell migration often involves the coordination of ECM proteolysis, adhesion, and signaling. The important enzymes that are primarily involved in the process of ECM proteolysis are the serine proteases that include (1) urokinase plasminogen activator (uPA) and (2) members of the family of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs).

Urokinase Plasminogen Activator System (uPA/uPAR System)

The proteolytically active urokinase (uPA) on the endothelial cell surface is critical for cell migration. The uPA is produced as an inactive single-chain protein known as pro-uPA, which binds to uPAR (uPA receptor) and is activated by plasmin [4]. Receptorbound pro-uPA is more rapidly cleaved by plasmin than the unbound form. The uPA is present in 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 (ATF) of the protein [5–7]. The ATF contains the growth factor and kringle domains of the protein that mediate binding to uPAR and play an important role in cell proliferation [8]. The main function of the uPA is to convert the inactive zymogen form of the enzyme plasminogen to plasmin, a broad spectrum of proteinase, which can cleave a variety of ECM components including collagen IV, fibronectin, and elastin including uPA (Fig. 1). The invasive and migratory potential of endothelial cells is largely determined upon the pool of active urokinase available on the cell surface. The uPA has also shown to directly activate the prohepatocyte growth factor/scatter factor (HGF/SF), and it also cleaves fibronectin and its own inhibitor, plasminogen activator inhibitor-1 (PAI-1), in

Fig. 1. uPA/uPAR in the degradation of ECM. Binding of inactive urokinase (pro-uPA) to urokinase receptor (uPAR) activates uPA. Active uPA proteolytically converts the inactive zymogen plasminogen to active plasmin, which then breaks down ECM components or activates latent growth factors such as transforming growth factor 1 (TGF-1). Plasmin can also degrade the ECM indirectly through activation of promatrix metalloproteinases (pro-MMPs).

a plasminogen-independent manner. The uPA/uPAR interaction represents a sensitive and flexible system to regulate proteolytic potential in endothelial cells. The uPAR is a cell-surface molecule that interacts with many potential ligands including uPA and vitronectin. The uPAR has also shown to be associated with several members of the integrin family which plays an important role in cell adhesion and migration [9]. This process is mediated through the low-density-lipoprotein-receptor-related protein (LRP), a multiligand receptor that can interact with both PAI-1 and uPAR. The uPA system also plays an important role in the activation of several MMPs and in the release and activation of growth factors stored in the ECM [10]. The contribution of the uPA/uPAR system to angiogenesis has been studied in several animal models of tumor angiogenesis, choroidal angiogenesis, and retinal angiogenesis. Many studies show that in addition to regulating proteolysis, uPAR is a signaling receptor that promotes cell motility, invasion, proliferation, and survival. Signaling through uPAR has been shown to activate many pathways involving kinases such as Ras–mitogen-activated protein kinase (MAPK) pathway [11]. These signaling events have been shown to involve the binding of its ligand such as uPA (independent of uPA proteolytic activity) and vitronectin.

The uPAR is a member of the lymphocyte antigen 6 (Ly-6) superfamily of proteins that are characterized by the Ly-6 and uPAR (LU) domain, also called the three-finger fold [12]. The LU domain folds into a globular structure with 5–6 antiparallel b-strands linked by 4–5 disulfide bonds [12, 13]. The uPAR contains three LU domains, designated D1–D3, connected by short linker regions, and these three domains pack together into a concave structure [14–16] in which the ligands such as uPA and vitronectin bind. Recent studies have indicated the importance of uPAR in human diseases, including many cancers. Hence, therapeutic targeting of uPAR is considered as an important concept to interrupt proteolytic cascades and block intracellular signaling in disease pathogenesis [17].

Matrix Metalloproteinases

The MMPs are a family of zinc-containing endopeptidases that are capable of degrading various components of ECM. These proteases are produced as latent proenzymes that are activated proteolytically. At least 21 different types of MMPs have been identified to date. Based on their structure/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), stromelysins (MMP-3, MMP-10, and MMP-11), and the nontraditional MMPs (matrilysin or MMP-7 and metalloelastase or MMP-12) and the membrane-type MMPs (MT-MMPs) [3, 18]. There are at least five distinct types of MT-MMPs (MMP-12, -15, -16, -17 and -21), and these MMPs are bound to cell surface through C-terminal transmembrane domain or glycosylphosphatidylinositol anchor. The MT-MMPs can degrade gelatin, fibronectin, and other ECM substrates [19, 20].

The basic structure of the MMPs contains the following domains that include (a) preor signal-peptide domain that directs MMPs to the secretory or plasma membrane insertion pathway; (b) prodomain that confers latency to the enzymes by occupying the active-site zinc, making the catalytic enzyme inaccessible to substrates; (c) zinc-con- taining catalytic domain; and (d) hemopexin domain or the C-terminal domain which mediates interactions with substrates and confers specificity of the enzymes, and also, it is connected to the catalytic domain by a flexible hinge region or linker region [21] (Fig. 2).

Various members of the MMPs have been implicated in a wide range of physiological and pathological processes, including wound healing, angiogenesis, inflammation, and tumor metastases [22–24]. During the physiological and pathological processes, the MMP functions included the proteolytic cleavage of ECM structures and destruction of cell-surface proteins and proteinase inhibitors. In addition to their capacity to degrade a large variety of ECM molecules, MMPs are known to process a number of bioactive molecules, and in many cases, MMP action leads to the proteolytic activation or release of latent signaling molecules and proteases including cytokines [25]. MMPs regulate a variety of cell behaviors such as cell proliferation, migration, differentiation, apoptosis, and host defense (Fig. 3).

Studies have shown that MMPs are one of the important molecules in the cascade of angiogenesis process and can be considered as proangiogenic agents. Specific MMPs have been shown to induce angiogenesis by detaching the pericytes from vessel wall and thereby releasing ECM-bound angiogenic growth factors. Also, this process has been implicated in the exposure of cryptic proangiogenic integrins binding sites in the ECM through the cleavage of endothelial cell–cell adhesion [26, 27]. Degradation of ECM releases ECM/basement membrane–sequestered angiogenic factors such as VEGF, bFGF, and TGF-b [28]. MMPs have been shown to have multiple effects on endothelial cells themselves. As mentioned earlier, MMPs facilitate endothelial cell migration and tube formation [29, 30]. Exogenous MMP-9 has been shown to enhance endothelial cell growth in vitro [31]. The cleavage of the ectodomain of VE-cadherin by MMPs is considered as an important event in the breaking of cell–cell adhesions [32]. MMPs involved in angiogenesis have been shown to originate from the infiltrating inflammatory cells or from endothelial cells. MMPs are synthesized in response

to diverse stimuli including cytokines, growth factors, hormones, and oxidative stress [33, 34]. Basic fibroblast growth factor (bFGF) induces endothelial MMP-9 expression via AP-1 [35]. Stimulation of endothelial cells by bFGF also upregulates the expression of uPA and integrin avb3 which then leads to the activation MMPs [36]. VEGF has also been indicated in the expression of MMP-1 [37], and also, the inflammatory cytokine TNF-a has been shown to upregulate the MMP-2 and -9 expressions [38]. Factor such as thrombin has been shown to activate the pro-MMP-2 directly in the endothelial cells [29]. Release of NO by inflammatory cells has been shown to transcriptionally upregulate MMP-13 and its activation by endothelial cells [34]. A connective tissue growth factor (CTGF) forms an inactive complex with VEGF165, and cleavage of CTGF by MMPs has been shown to release active VEGF165 [39]. MMP-2 has been indicated in the release of latent TGF-1, while MMP-2 and MMP-9 cleave the latency-associated peptide to activate TGF-b1 [40, 41].

The presence of MMPs in the eye has been demonstrated as early as 1968 in the cornea through its proteolytic activity on collagen substrate [42]. MMPs have been indicated in many eye disorders such as age-related macular degeneration [43], proliferative diabetic retinopathy (PDR) [44, 45], glaucomatous optic nerve head damage [46], vitreal liquification [47], and vitreoretinopathy [48, 49]. The cellular origin of the MMPs in these studies is still not clear, but it is likely that the expression would come from the resident cells, invading vasculature, and the inflammatory cells [50]. The importance of MMPs in the retinal pathology is currently well known, and many recent studies have demonstrated the presence of various MMPs such as MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and MMP-14 that are expressed at different retinal tissues (Table 1). Regardless of the sources in the retina, MMPs are considered as an attractive therapeutic target to treat proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME).