new vascular tubes [51]. Remodeling of the ECM in angiogenesis is exerted by MMPs, which are induced by angiogenic stimuli such as VEGF and Ang-2 [52].

Neovascularization and the switch to subsequent fibrosis in PDR can be considered as a wound healing-like response [53]. Fibrosis is the deposition and cross-linking of collagen in the terminal phase of the normal wound healing response [54, 55], which has mainly been studied in the skin. Wound healing in the skin is initiated by tissue injury [56–58], which involves vascular damage, hemorrhage, and activation of the clotting system. The subsequent response can be divided into three phases: an inflammatory phase, a proliferative phase, and a maturation phase [56].

During the inflammatory phase, angiogenic and profibrotic cytokines and growth factors are released from activated cells, such as platelets and macrophages. In the proliferation phase, fibroblasts contribute to the synthesis of the ECM [59], and endothelial cells form “sprouts” and new capillaries. Sprouting angiogenesis is initiated by the presence of a fibrin matrix and growth factors at the wound healing edge [60]. Besides ECM components, fibroblasts also produce growth factors and various enzymes such as proteases which are of importance for reepithelialization and angiogenesis. During the wound healing response, the ECM itself serves as a reservoir for growth factors, thereby regulating their activity and presentation to receptors. In the proliferation phase, formation of ECM, angiogenesis, and reepithelialization take place [56].

In the maturation phase, angiogenesis ceases whereas the production of ECM continues [56]. Under normal conditions, after this switch from angiogenesis to fibrosis, ECM production ceases when sufficient quantities of collagen have been synthesized [56, 61–63]. Then, remodeling of the newly formed ECM reduces the wound thickness and increases the strength of the regenerating tissue. This breakdown of collagen is tightly regulated by a balance between proteases such as MMPs and their endogenous inhibitors such as TIMPs [64, 65].

Most features of the wound healing response in human skin can also be recognized in pathological wound healing responses characterizing various disease states in other organs. These pathological conditions have in common that tissue-specific wound healing responses are initiated, but that the wound healing process is not properly terminated, leading to pathological fibrosis [54, 66]. This is a situation in which normal scarring progresses to excessive production, limited degradation, altered deposition, and/or contraction of the ECM, probably due to an imbalance between proand antifibrotic factors causing a profibrotic state.

Several eye conditions lead to blindness by the involvement of wound healing-like responses culminating in scarring or excessive fibrosis (see Section on “CTGF in the Eye”). Although the initial wound healing response may have a functional meaning in restoring ocular integrity, it also results in loss of visual function and is therefore deemed to be pathological [67, 68].

CTGF STRUCTURE AND FUNCTION

CTGF is a member of the CCN family of growth factors, named after the first three members identified, Cyr61 (CCN1), CTGF (CCN2), and Nov (CCN3), but also includes CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) as well [69–71]. CTGF exhibits a unique domain structure, made up of five modules including a signal peptide,

Fig. 2. Modular structure of the CTGF protein. CTGF consists of an N-terminal secretory signaling peptide (SP) and four distinct domains, through which CTGF binds extracellular ligands like VEGF, TGF-b, and fibronectin, and cell surface proteins like integrins and heparin-sulfate proteoglycans. (Asterisks) Hinge region. CTGF can be cleaved by proteases, such as MMPs, in between the domains. Cleavage products can accumulate in biological fluids and may serve as clinical markers.

encoded by five exons (Fig. 2) [71]. CTGF exerts its biological activities by interactions with ECM components, such as fibronectin, extracellular signaling molecules, and cell surface proteins, such as integrins, through its various interaction domains [70, 72–76]. Most likely, CTGF also indirectly regulates signaling by modulating the activity of other growth factors [77, 78]. For instance, binding of CTGF and VEGF suppresses VEGFinduced angiogenesis, and cleavage of CTGF by MMPs recovers the angiogenic activity of VEGF [79].

The biological functions of CTGF are diverse and cell and context dependent. CTGF was first discovered in conditioned media of endothelial cells as a molecule affecting the activity of fibroblasts [80]. CTGF is induced during wound healing [81], is overexpressed in fibrosis [82, 83], and acts as an essential downstream mediator for most of the profibrotic activity of TGF-b, in particular in stimulation of ECM production [66], and fibroblast proliferation [84–86]. The synergy between CTGF and TGF-b1 may be explained by binding of the unique TGF-b response element of CTGF, which enhances receptor binding and signaling activity of TGF-b (Fig. 2). For example, skin fibrosis in newborn mice was persistent only after coinjection of both TGF-b1 and CTGF, and not after injection of TGF-b1 or CTGF alone [87, 88]. In humans, CTGF is upregulated in diseases that are characterized by pathological fibrosis including renal diseases of various etiology, liver, lung, cardiovascular diseases, and in the eye.

Biological functions of CTGF include induction of angiogenesis, chondrogenesis, osteogenesis, and control of cell proliferation and differentiation, migration, adhesion, apoptosis, and survival of fibroblasts [10, 89], but the exact function of CTGF in normal tissues is not known yet; CTGF is expressed in the placenta during embryo implantation [90] and during the development of ovarian follicles [91]. Recently, a role CTGF was suggested in (nonfibrotic) tissue repair in the eye, as it was required for reepithelialization in human cornea [92].

CTGF IN THE EYE

CTGF in Ocular Fibrosis

It has been suggested that CTGF functions in the eye primarily as a profibrotic growth factor. In the human eye, CTGF has been identified in various diseases complicated by fibrosis, both in the anterior and posterior segments [93–100]. Several major eye conditions lead to blindness due to scarring or pathological fibrosis [101] as a consequence of tissue-specific wound healing responses. In subretinal neovascularization as well as in PDR and other ischemic retinopathies, these responses are driven by neovascularization, like in skin wound healing. In other conditions such as proliferative vitreoretinopathy (PVR), these responses are mainly avascular. There is accumulating evidence that CTGF is an important pathogenic factor in these conditions. For instance, in the vitreous of patients with PVR, CTGF is present in higher levels as compared to nonproliferative retinal diseases [101, 102], in correlation with TGF-b [103]. In human PVR membranes, CTGF has been identified as well [104–106].

CTGF in Ocular Angiogenesis

CTGF has been suggested to play a role in ocular angiogenesis. In the rat eye, corneal micropocket implants containing murine CTGF induced neovascularization [7]. Moreover, CTGF and VEGF colocalized in vascular cells in human choroidal neovascular membranes, and levels of CTGF were increased in the vitreous of patients with active PDR [107]. However, VEGF-induced angiogenesis was inhibited by combined exogenous administration of CTGF and VEGF in the back of mice, as well as in a mouse model of hindlimb ischemia, as a result of binding of VEGF by CTGF [108, 109]. When CTGF is upregulated by VEGF [11], it can reduce the bioavailability of VEGF through direct binding. The involvement of CTGF in angiogenesis in ocular disease in general and in DR in particular is also questionable because of findings in human PDR and in distinct angiogenesis models applied to CTGF transgenic mice [101, 110, 111]. In human PDR, CTGF levels consistently correlated with degree of fibrosis and not with angiogenesis activity [101, 111]. In studies in transgenic mice lacking the CTGF gene, vascular outgrowth from metatarsals of 17-day-old CTGF−/− embryos, cultured in the presence or absence of VEGF, did not differ significantly from outgrowth of wild-type or heterozygous CTGF+/− metatarsals [110]. These data indicate that CTGF is not required for (VEGF-induced) angiogenesis in this model. Secondly, the effect of CTGF gene deletion was investigated in two ocular angiogenesis models. In the oxygeninduced retinopathy model [112], in which retinal hypoxia-induced VEGF overexpression causes preretinal angiogenesis, differences between CTGF+/+ and CTGF+/− mice were not observed. In another ocular angiogenesis model, choroidal neovascularization was induced in CTGF+/+ and CTGF+/− mice by laser burns [113, 114], but statistical differences between CTGF+/+ and CTGF+/− mice were not found [110]. Taken together, these data suggest that CTGF is a dispensable factor in the complex interplay of hypoxic signaling and VEGFor wound healing-driven ocular angiogenesis.