Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

II.EXPRESSION AND FUNCTION OF GROWTH FACTORS IN

OCULAR TISSUES

A.Cornea

The cornea is an avascular elastic tissue composed of three distinct cell layers including surface epithelial cells, stromal cells (keratinocytes) and endothelial cells. The surface of the cornea is composed of 5–6 non-keratinized stratified squamous epithelial cells. This cell layer is separated from the underlying keratinocytes by Bowman’s membrane. The keratinocytes and a single layer of corneal endothelial cells are separated by Descemet’s membrane. The surface of the cornea is bathed in the tear film, while the endothelial layer is in direct contact with aqueous humor. Growth factor mediated communication is important for corneal development, morphogenesis, homeogenesis and wound healing (Wilson et al., 2003). In fact communication between corneal epithelial cells and keratinocytes, and corneal endothelial cells and keratinocytes, appears to be regulated via soluble growth factors.

The presence and function of growth factors and growth factor receptors in the cornea has been examined through a number of reports. We will concentrate on five families of growth factors and their role in the cornea. These families include EGF, HGF/KGF, TGF-β, PDGF, and neurotrophins (NT).

1. Epidermal growth factor (EGF)

Epidermal growth factor (EGF) potentially affects the cornea via autocrine, paracrine and juxtacrine signaling mechanisms (Nakamura et al., 2001). The surface of the cornea is bathed in a tear film that contains EGF (Ohashi et al., 1989; van Setten et al., 1989) and thus can influence corneal epithelial cells that express both high and low affinity EGF receptors (Ohashi et al., 1989; Nakamura et al., 2001). Since topical EGF can penetrate into the anterior chamber

(Chan et al., 1991), EGF originating from lacrimal glands may find its way into the aqueous humor and influence corneal endothelial cells. The corneal endothelial cells also express high affinity EGFR while corneal stromal cells have been reported to express only low affinity receptors (Wilson et al., 1999a; Imanishi et al., 2000). Wilson et al. (1999a) reported the presence of mRNA for EGF in all three cell types that comprise the cornea while immunolocalization studies demonstrated higher levels of EGF protein in the superficial epithelial cells, the endothelial layer and a lower level in stromal cells.

Both in vitro and in vivo studies indicate that exogenous EGF can influence corneal cells. EGF was reported to inhibit epithelial cell differentiation and increase proliferation in a dose dependent fashion (Hongo et al., 1992; Wilson et al., 1999a; Imanishi et al., 2000). Interestingly, the mitogenic effect of EGF on corneal epithelial cells is dependent on the down-regulation of Pax6 (Li and Lu, 2005). Exogenous EGF can also stimulate corneal epithelial cell motility (Wilson et al., 1994). EGF has only a weak effect on stromal cell proliferation and this may be attributed to the expression of low affinityEGFRincornealstromalcells.Hongo et al. (1992) and Imanishi et al. (2000) have reported corneal endothelial cell proliferation in response to exogenous EGF.

2. Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF)

The relationship of HGF/KGF to corneal epithelial and keratinocyte communication has been studied extensively. Wilson et al. (1999b) have reviewed the role of these two growth factors in epithelial–stromal homeostasis. Both HGF and KGF mRNA and protein have been reported to be expressed by corneal keratinocytes but not in corneal epithelial cells while HGFR and KGFR are expressed in the corneal epithelium (Wilson et al., 1994, 1999a,b). It appears that both growth factors regulate corneal epithelial

cell differentiation, proliferation and motility (Wilson et al., 1994) upon release from keratinocytes, with subsequent activation on HGFR and KGFR expressed by corneal epithelial cells. Clinically this is important since it has been demonstrated that during corneal healing following wounding, the expression and secretion of HGF and KGF is significantly upregulated (Wilson et al., 1999a). In addition, the human tear film contains HGF and KGF thus bathing the outer surface of the corneal epithelial cell layer (Li et al., 1996; Tervo et al., 1997).

The binding of HGF to its receptor (c-Met) in corneal epithelial cells leads to activation of multiple signaling cascades. For example, upregulation of HGF following corneal injury increases epithelial cell migration, proliferation and enhances cell survival by activating anti-apoptosis signaling cascades. Corneal epithelial cell migration is activated via MAPK p38 whereas proliferation is dependent on ERK1/2 (Sharma et al., 2003) and the PI-3K/Akt-1/Bad pathway (Kakazu et al., 2004) regulates cell survival.

3. Transforming growth factor-beta (TGF-β)

Numerous reports have documented the presence of TGF-β and TGF-β receptors in corneal tissue (Pasquale et al., 1993; Wilson et al., 1994; Nishida et al., 1995; Joyce and Zieske, 1997; Wilson et al., 1999a; Imanishi et al., 2000). The major isoform of TGF-β present in the cornea appears to be TGF-β2 (Imanishi et al., 2000); however, TGF-β1 has been reported in small amounts in all three corneal layers (Imanishi et al., 2000). TGFreceptors have been reported to be located in corneal stromal cells and limbal and central corneal epithelial cells (Li and Tseng, 1995; Wilson et al., 1994). In corneal stroma cells, TGF-β has been shown to stimulate cell proliferation, migration, and alteration in the ECM synthesis including heparan sulfate (Brown et al., 1999). However, both TGF-β1 and TGF-β2 appear to inhibit corneal epithelial cell proliferation (Imanishi et al., 2000).

In order to avoid infection, a surface epithelial defect of the cornea must be repaired rapidly. A wound of the corneal epithelial layer is repaired via migration of epithelial cells followed by cell proliferation (Saika, 2006). Following cornea wounding, TGF-β appears to be upregulated and involved in the repair process (Saika, 2006). For example, corneal stroma cells express all three isoforms of TGF-β following injury (Saika, 2006). Also, endogenous TGF-β has been reported to enhance corneal epithelial cell migration, but inhibits cell proliferation (Saika, 2006). Interestingly, mice lacking Smad3 can repair wounded corneal epithelial cells. This may indicate that TGF-β may be acting via non-canonical signaling pathways such as p38, since it has been shown that TGF-β/p38MAPK signaling stimulates corneal epithelial cell migration (Saika, 2006).

4. Platelet-derived growth factor (PDGF)

Platelet derived growth factor (PDGF) consists of two disulfide-bonded polypeptide chains termed A and B. Therefore, three distinct isoforms of PDGF can be expressed by cells (e.g. PDGF-AA/AB/BB) and it is thought that each isoform has the potential to elicit different biological responses in cells. Platelet-derived growth factor is involved in a number of cell functions including proliferative and wound healing responses. High affinity PDGF receptors consist of either the α or β types. It is known that the α type PDGF receptor binds both the A and B chains while the β type PDGF receptor only binds the B chain. In the absence of PDGF, receptor subunits can exist individually or in reversible combinations. Stable dimers of PDGF receptors

exist in the presence of the PDGF ligand (e.g. αα, αβ or ββ).

Protein for PDGF-BB is present in corneal epithelial cells and concentrates in the basal lamina (Wilson et al., 2001). Both corneal stromal (Li and Tseng, 1995) and endothelial (Imanishi et al., 2000) cells

express receptors for PDGF. Exogenous PDGF treatment results in changes in the cytoskeleton in corneal epithelial cells and endothelial cells, as well as stimulation of migration of keratocytes and endothelial cells (Hoppenreijs et al., 1993; Knorr et al., 1992). Rabbit corneal endothelial cells proliferate in response to PDGF-BB, but not PDGF-AA (Imanishi et al., 2000). However, the role of PDGF in normal endothelial cells is questionable since PDGF has not been demonstrated in endothelial cells or in the anterior chamber under normal conditions (Hoppenreijs et al., 1996).

5. Neurotrophins (NT)

As previously indicated, the neurotrophin family of growth factors includes NGF, BDNF, NT-3 and NT-4. The majority of the work with respect to neurotrophins and the cornea revolved around the effects of neurotrophins on wound healing. Wound healing in the cornea is very complex and will not be discussed in detail in this chapter. The process is under the influence of several growth factors and proteases produced by the corneal epithelial cells, stromal keratinocytes and/or the corneal endothelial cells. The presence of Trk A, the high affinity NGF receptor, is expressed on the ocular surface (Lambiase et al., 1998; You et al., 2000).

There are a variety of causes of corneal damage, including deliberate reshaping of the cornea in refractive surgery (PRK, LASIK, LASEK). Nerve growth factor (NGF) has been reported to be an important modulator of cornea wound healing (Micera et al., 2006) including healing both skin and corneal ulcers (Kawamoto and Matsuda, 2004). In vitro studies indicated that exogenous NGF caused both corneal epithelial cells (You et al., 2000) and stromal cells (Yamai et al., 2002) to undergo proliferation. In vivo studies by Lambiase et al. (1998) and Bonini et al. (2000) have reported that eye drops containing NGF stimulate corneal healing in patients suffering from

either neurotrophic or autoimmune corneal ulcers. Topical administration of NGF accelerated corneal reinnervation after LASIK surgery in rabbits (Joo et al., 2004). Aqueous humor levels of endogenous NGF affect corneal sensation and ocular surface dryness after refractive surgery in man (Lee et al., 2005).

B. Aqueous Humor

1. Growth factors in normal aqueous humor

The aqueous humor (AH) is a transparent fluid that supplies nutrients to the avascular cornea, lens and trabecular meshwork. The composition of AH is both quantitatively and qualitatively different than serum (Pavao et al., 1989). Growth factors present in normal human AH may be derived locally via cells of the trabecular meshwork, iris, ciliary body, lens and cornea. Growth factors reported to be present in normal human AH include epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), transforming growth factor-β2 (TGF-β2), platelet derived growth factor (PDGF) (Tripathi et al., 1996), vascular endothelium factor (VEGF) (Tripathi et al., 1998; Hu et al., 2002), hepatocyte growth factor (HGF) (ArakiSasaki et al., 1997; Shinoda et al., 1999) and interleukin-6 (IL-6) (Murray et al., 1990). The presence of growth factors in human AH is important because they can modulate normal cellular functions and may be involved in the pathophysiology of the cornea, lens and the trabecular meshwork (Tripathi et al., 1994; Wordinger et al., 1998). Table 5.2 summarizes growth factors that have been reported to be present in both normal and glaucomatous human aqueous humor samples.

2. Growth factors in glaucomatous aqueous humor

The concentration of specific growth factors in human AH has been reported to be elevated in various types of glaucoma.

Elevated growth factors in the AH may be related to the pathogenesis of glaucoma. For example, in primary open angle glaucoma (POAG), levels of TGF-β2 are higher than age-matched controls (Inatani et al., 2001; Ochiai and Ochiai, 2002; Picht et al., 2001; Tripathi et al., 1994). Since TGF-β2 has been demonstrated to influence the secretion and composition of the extracellular matrix (ECM), it has been proposed that elevated levels of TGF-β2 in glaucomatous AH increases ECM deposition and thus increases resistance to AH outflow, resulting in elevated intraocular pressure (IOP) (Welge-Lussen et al., 2001). The action of TGF-β2 may also be related to turnover of ECM in the trabecular meshwork via action on MMP/TIMP expression and activity.

Hu et al. (2002) demonstrated that vascular endothelial growth factor (VEGF) was present in human AH and was significantly elevated in glaucomatous eyes. While there were no significant differences between POAG, angle-closure and exfoliative glaucoma, significantly higher VEGF levels were seen in the AH from neovascular and uveitis glaucoma. Hu and Ritch (2001) reported that the concentration of HGF was significantly higher in AH from glaucomatous eyes when compared to cataract control eyes. In addition, eyes with exfoliative

glaucoma had significantly higher HGF concentrations than did eyes with POAG and angle-closure glaucoma. They further indicated that various ocular cells produce VEGF and HGF and the increased concentrations of VEGF and HGF in glaucoma was attributed primarily to increased local production of the growth factors.

C. Trabecular Meshwork (TM)

Numerous studies have reported the synthesis and secretion of growth factors and the expression of growth factor receptors by trabecular meshwork cells in a variety of species including porcine, bovine and human. Since the initial report of the isolation, characterization and culture of human TM cells by Polansky et al. (1979), studies have been reported on the effect of exogenous growth factors on TM cell function. This review will concentrate on the effect of exogenous growth factors on human trabecular meshwork cells.

The human trabecular meshwork cell expresses numerous growth factor receptors and can respond to endogenous growth factors in the aqueous humor, as well as to exogenous growth factors (Wordinger et al., 1998). In vitro treatment of human TM cells with HGF, EGF, IGF-I,

TNF-α, PDGF and FGF-2 stimulated cell proliferation while FGF-1, TGF-α, IL1α NGF and FGF-7 had no effect on cell proliferation. Significantly, treatment of human TM cells with TFG-β1 or TGF-β2 significantly inhibited EGF and FGF-2 stimulated TM cell proliferation (Wordinger et al., 1998). Although the functions are not known, mRNA for alternatively spliced growth receptors (TGF-βRII, HGFR and KGFR) have also been reported (Wordinger et al., 1999). The majority of studies involving the effect of exogenous growth factors on TM cells have centered on members of the TGF-β superfamily including BMP, TNF and interleukins and neurotrophins. They each will be discussed separately.

1. Transforming growth factor-β (TGF-β)

Tissue expression of TGF-β1, TGF-β2 and TGF-β3 and their receptors has been reported in the human TM. The majority of studies have examined the effect of TGF- β1 and TGF-β2 on cultured TM cells. Some studies have utilized the perfused human ocular anterior segment model. LütjenDrecoll (2005) has suggested that common factors may be involved in the pathogenesis of glaucoma in both the trabecular meshwork as well as the optic nerve head. Transforming growth factor-β may be one of these common factors. We have already indicated that TGF-β2 has been reported to be elevated in the aqueous humor of POAG patients. In addition human TM cells treated with exogenous TGF-β2 in concentrations comparable to those measured in the aqueous humor of glaucomatous eyes increase protein levels of fibronectin (FN) and tissue transglutaminase (WelgeLuessen et al., 2000), plasminogen activator inhibitor (PAI) (Fuchshofer et al., 2003), αβcrystalline (Welge-Luessen et al., 1999), myocilin (Tamm et al., 1999) and thrombospondin-1 (Flugel-Koch et al., 2004). Also, in anterior segment organ-cultured experiments, TGF-β2 treatment increased fibrillar deposition under the inner wall of

Schlemm’s canal and decreased outflow facility (Gottanka et al., 2004). In another experiment, Fleenor et al. (2006) utilized both cultured human TM cells and the perfused human ocular anterior segment model to demonstrate that TGF-β2 caused increased secretion of FN and PAI-1 and elevated IOP.

Liton et al. (2005a) demonstrated that mechanical stretch could elevate TGF-β1 in TM cells, as well as the transcription and secretion of IL-6 (Liton et al., 2005b). Also, Wordinger et al. (1998) demonstrated that exogenous TGF-β2 inhibited human TM cell proliferation stimulated by FGF-2 and EGF. Tamm et al. (1996) demonstrated TGF- β1 played a role in differentiating human TM cells towards a myofibroblast-like cell type by modulating the expression of α-smooth muscle actin.

2. Bone morphogenetic proteins (BMP)

The bone morphogenetic proteins (BMP) are members of the TGF-β superfamily of growth factors. Originally identified as osteoinductive cytokines that promote bone and cartilage formation (Wozney et al., 1988), BMPs are now known to control multiple functions in a variety of cells (Balemans and Van Hul, 2002). A combination of intracellular and extracellular antagonists tightly control the biological activity of BMPs. Recently, a group of unique, but structurally related, secreted BMP antagonists has been identified (von Bubnoff and Cho, 2001). Examples of secreted BMP antagonists include noggin, chordin, follistatin, and members of the DAN (Differential screening-selected gene and members Aberrative in Neuroblastoma) family including cerebus, caronte, and Drm/Gremlin (Down-regulated by mos) (CKTSF1B1). The mechanism of inhibition appears to be direct binding to BMP by these antagonists, thus preventing BMP from interacting with the receptor complex (Balemans and Van Hul, 2002). Drm/ Gremlin is a member of the DAN/Cerberus

family of BMP antagonists and is a highly conserved 20.7 kDa glycoprotein (Hsu and Economides, 1998). Gremlin heterodimerizes with BMP-2, BMP-4 and BMP-7 to prevent functional activity. Previous reports suggest that BMP antagonists are likely to play an important role in regulating multiple cell functions both during early development and in adult tissues.

We have previously reported the presence of BMPs, BMP receptors and selected BMP antagonists in both human TM tissues and isolated TM cells (Wordinger et al., 2002). We demonstrated that human TM cells and tissues express BMP-2, BMP- 4, BMP-5 and BMP-7, as well as high affinity BMP receptors, BMPR-IA, BMPR-IB and BMPRII. In addition BMP antagonists including DRM/gremlin cultured TM cells express follistatin, chordin and BAMBI.

Within a given tissue, the actions of most growth factors are often counterbalanced by other growth factors, so that, normally, only small spatial and temporal changes occur in structure and function. Our most recent study demonstrated that TM cells are capable of secreting BMPs, and that BMP-4 selectively counteracted the action of TGF-β2 in TM cells with respect to ECM related proteins (Wordinger et al., 2007). It appears that BMP-4 plays a significant role in maintaining the normal function of the TM by modifying the action of TGF-β2. In addition, we demonstrated that the BMP antagonist DRM/Gremlin inhibits BMP-4 activity in cultured TM cells and increases outflow resistance in a perfusion cultured human eye anterior segment model. Significantly, we demonstrated that levels of both Gremlin mRNA and protein are elevated in glaucomatous human TM cell lines. We proposed that, in POAG, elevated DRM/Gremlin expression by TM cells inhibits BMP-4 regulation of TGF-β2 effects, leading to increased ECM deposition and elevated IOP (Wordinger et al., 2007). Fuchshofer et al. (2007) have confirmed that BMP proteins are antagonists for TGF- β2 in human TM cells. They reported that

treatment with TGF-β2 induced the expression of CTGF, TSP-1, and fibronectin collagen types IV and VI and PAI-1 in cultured human TM cells. All of these inductions were inhibited when TGF-β2 was added in combination with BMP-7.

3. Tumor necrosis factor and interleukins

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) have been reported to be involved in ECM metabolism within the TM. MMPs, including MMP-1 (collagenase-1), MMP-2 (gelatinase-A), MMP-3 (stromolysin-1) and MMP-9 (gelatinase-B), are produced by the human TM (Alexander et al., 1991; Samples et al., 1993; Pang et al., 2003b). In addition, Ando et al. (1993) reported the presence of MMPs in human AH.

IL-1α is a potent activator of MMP- 3 by cultured human TM cells (Samples et al., 1993; Alexander et al., 1998; Pang et al., 2003b). Functionally, perfusion of human organ culture eyes with IL-1α leads to a significant increase in AH outflow facility (Bradley et al., 1998). Fleenor et al. (2003) demonstrated the involvement of the AP-1 signaling pathway in IL-1α stimulated MMP-3 expression in human TM cells. They suggested that compounds that would activate the AP-1 pathway in the TM could upregulate the local production of MMP-3 and improve AH outflow. In fact Pang et al. (2003a) showed that tert-Butylhydroquinone, a compound that activates AP-1, did in fact upregulate MMP-3 expression and also increased aqueous outflow. Hosseini et al. (2006) reported that TNF, IL-1α and IL-1β increased MMP-3 and MMP-9 protein levels in TM cells and that the stimulation was regulated via the JNK signaling pathway. Zhang et al. (2006) reported that the MAPK, p38 and JNK signal transduction pathways were relatively unresponsive in glaucomatous TM cells as compared to normal TM cells following IL-1 treatment.

The clinical importance of TNF-1 and IL-1 in the human TM can be appreciated

from the fact that MMPs have been implicated as mediators of the IOP-lowering effect following laser trabeculoplasty and prostaglandin application in glaucoma patients. In 1996, Parshley et al. (1996) reported that laser trabeculoplasty induces MMP-3 by TM cells. Bradley reported that MMPs increased aqueous outflow facility (Bradley et al., 1998) and determined that laser trabeculoplasty induced the expression and secretion of IL-1β and TNF-α within the first 8 hours of treatment (Bradley et al., 2000). These growth factors mediated increased MMP-3 expression that subsequently initiated remodeling of the juxtacanalicular ECM to restore normal outflow facility, and have been demonstrated to increase aqueous outflow facility (Bradley et al., 1998). In addition prostaglandin receptor agonists increase the levels of MMP-1 and MMP-3 in cilary muscle (Lindsey et al., 1996; Weinreb et al., 1997), thus enhancing uveoscleral outflow of AH.

4. Neurotrophins and neurotrophic factors

Human TM cells have been reported to arise embryologically from the neural crest (Tripathi and Tripathi, 1989). A common feature of cells of neural crest origin is the ability to express and respond to neurotrophins and/or neurotrophic factors. Our laboratory was the first to report that human TM cells express both mRNA and protein for NGF, BDNF, NT-3 and NT-4 (Wordinger et al., 2000). In the same paper we reported that human TM cells also express high affinity Trk receptors (Trk A, Trk B and Trk C) as well as truncated Trk B and Trk C. Liu et al. (2001) reported that human TM cells express the ciliary neurotrophic factor (CNTF) tripartite receptor complex. This complex consists of CNTFRα, gp130 and LIRFβ. Both mRNA and protein were demonstrated via RT-PCR, western blot and immunohistochemistry but secretion of CNTF was not detected. Of additional interest was the report by Liton et al. (2005a), which showed

mechanical stretch of cultured human TM cells resulted in the secretion and transcription of IL-6. They also reported that IL-6 increased outflow facility and increased permeability through a monolayer of Schlemm’s canal cells (Liton et al., 2005b).

D. Role of Growth Factors in Myopia

Over 1 billion people worldwide have myopia. In most cases, the inability to focus on distant objects is due to an elongated globe involving remodeling of the sclera. Both genetic and environmental factors are involved in the development of myopia. Both FGF2 and TGFβ1 have been implicated in the development of form deprivation myopia in chicks. In mammalian models of myopia, down regulation of TGFβ in the sclera is associated with decreased collagen synthesis (Jobling et al., 2004). Although scleral FGF2 levels are not altered in a mammalian myopia model, levels of FGFR1 change during myopia and may play a role in scleral remodeling (Gentle and McBrien, 2002). Genetic association studies looking at growth factor gene polymorphisms have suggested that TGFβ1 (Lin et al., 2006), TGFβ-induced factor (Lam et al., 2003), and HGF (Han et al., 2006) genes are involved in predisposition to myopia.

E. Retina

A wide variety of growth factors (GFs) play many important roles in the retina. During retinal development GFs are involved in differentiation and programmed cell death. The normal functions and homeostasis of retinal neurons, photoreceptors, astroglial, and vascular endothelial cells are regulated by GFs. Numerous retinal diseases are caused by altering this balance of growth factors, with increased or decreased expression causing and/or propagating the disease. Lastly, therapies directed at providing neurotrophic support or antagonizing

pathogenic GFs are being explored and used clinically to treat retinal diseases.

1. Growth factors and retinal development

Several well-characterized signaling pathways involved in embryogenesis and normal development also play important roles in the development of the retina. Wnt and Fzd signaling components are expressed in the developing retina, and activation of canonical Wnt β-catenin signaling is stageand location-dependent during development (Liu et al., 2006b). Wnt signaling is involved in the development of the ciliary epithelium, differentiation and proliferation of retinal cells, as well as regulation of axonal guidance. Recent evidence also supports the role of Wnt signaling in angiogenesis (Goodwin et al., 2006). Fibroblast growth factor (FGF), hedgehog (Hg), and bone morphogenetic protein (BMP) signaling pathways interact and have pleiotrophic effects in retinal development. Fibroblast growth factors participate in retinal cell proliferation and retinal ganglion cell (RGC) guidance and target recognition, while hedgehog proteins regulate retinal precursor cell proliferation and differentiation, photoreceptor (PR) differentiation, and axonal guidance (Dakubo and Wallace, 2004). In contrast, TGFβ and BMP4 mediate physiological programmed cell death, or developmental pruning, of retinal neuronal cells including retinal ganglion cells (RGCs) (Beier et al., 2006; Franke et al., 2006).

2. Involvement of growth factors in retinal diseases

a. Retinal degenerations – Over 180 different genetic loci and 130 genes have been identified that cause various forms of heritable retinal degeneration (RetNet at http://www.sph.uth.tmc.edu/RetNet/). Mutations in these genes can cause abnormal PR architecture and disc morphogenesis, defects in phototransduction, metabolic overload, as well as RPE abnormalities. Although Wnt signaling plays a prominent

role in the development of the retina, Wnt signaling components are also expressed in the adult retina. Mutation in membrane type FRP (mFRP), an inhibitor of Wnt signaling, is responsible for retinal degeneration (rd6) mice (Kameya et al., 2002), and altered expression of other FRPs has been reported in retinitis pigmentosa retinas (Jones et al., 2000).

In addition to these genes, retinal degeneration can occur via loss of trophic support or increased expression of pathogenic levels of growth factors. Both cone and rod photoreceptors require continued trophic support for survival. Genetic defects in rod photoreceptors often lead to the eventual degeneration of cone photoreceptors. Rods make a cone trophic factor, rod derived cone survival factor (RdCSV), that promotes the survival of cultured rod photoreceptors (Leveillard et al., 2004). Trophic factors supporting RPE cells include FGF2, IGF-1, and PEDF (Chaum, 2003). Retinal detachment separates photoreceptors from RPE cells, which often leads to photoreceptor degeneration. In addition disruption of important PR–RPE interactions, surrounding retinal glia cells (Müller cells and astrocytes), elaborate several cytokines and growth factors, including TNFα, IL- 1β, and MCP-1, which may be responsible for the photoreceptor degeneration associated with this condition (Nakazawa et al., 2006a).

b. Damage to retinal ganglion cells and optic nerve – Glaucoma is a group of optic neuropathies that are characterized by cupping and excavation of the optic disc, optic nerve axonopathy, and progressive death of retinal ganglion cells. Retinal ganglion cells require continued trophic support for survival. Removal of retrograde trophic support by damaging the optic nerve, or removal of trophic factors from RGC cultures, leads to apoptosis. Brain derived neurotrophic factor (BDNF) is a major RGC survival factor. Both BDGF and its receptor Trk B are expressed by RGCs as well as in

optic nerve head cells. BDNF and Trk B are retrograde transported to RGC soma from the target tissue (LGN) in the brain. This retrograde transport is blocked at the optic nerve head in glaucoma (Pease et al., 2000), suggesting that loss of trophic support is at least one pathogenic mechanism involved in glaucomatous retinopathy.

Tumor necrosis factor alpha (TNFα) appears to play a pathogenic role in glaucomatous damage to the retina and optic nerve. Retina and optic nerve TNFα and TNFα receptor expression is elevated in glaucomatous eyes (Yuan and Neufeld, 2000; Tezel et al., 2001). TNFα is generated by retinal glia in response to hypoxia and elevated pressure. RGCs have TNFα receptors, and stimulation by TNFα leads to cell death (Tezel and Wax, 2000). Retinal TNFα is increased in a mouse model of laser-induced ocular hypertension, and glaucomatous damage to RGCs and the optic nerve is blocked by inhibiting TNFα or the TNFα receptor (Nakazawa et al., 2006b). In addition, intravitreal administration of TNFα can cause glaucoma-like microglia activation, RGC loss, and optic nerve degeneration in normal mice (Kitaoka et al., 2006; Nakazawa et al., 2006b). Activation of the epidermal growth factor receptor (EGFR) also appears to play a pathogenic role in glaucomatous damage to the retina and optic nerve head. EGFR is expressed on RGCs and on optic nerve head astrocytes. Injury to the optic nerve activates the EGFR, which generates reactive optic nerve head astrocytes. Administration of EGFR inhibitors protects RGCs in a rodent model of glaucoma (Liu et al., 2006a) and promotes regeneration of injured optic nerve fibers (Koprivica et al., 2005), demonstrating the potential importance of this signaling system in glaucoma.

c. Retinal and choroidal neovascularization –

Retinal and choroidal neovascularization are major causes of visual impairment and blindness in the world. The formation and maintenance of vasculature in a tissue is regulated by pro-angiogenic and

anti-angiogenic factors. In some ocular disease states, this delicate balance is disturbed, leading to pathological neovascularization. Several different angiogenic growth factors are responsible for these damaging effects to the eye (Kvanta, 2006). Retinal ischemia or hypoxia, which occurs in diabetic retinopathy (DR), retinopathy of prematurity, and retinal vein occlusions (RVO), can lead to the formation of abnormal blood vessels. Vascular endothelial growth factor (VEGF) is a survival factor for developing immature vessels, but it is also a potent angiogenic factor and vascular permeability factor. VEGF has several different alternatively spliced isoforms that bind and activate two receptor tyrosine kinases, VEGFR1 and VEGFR2. It appears that VEGFR2 is the main mediator of neovascularization and enhanced vascular permeability (Ferrara et al., 2003). Hypoxia induces VEGF expression in retinal vascular endothelial cells, pericytes, Müller cells, and RPE cells. Increased intraocular VEGF levels have been found in patients with active proliferative retinopathies (PDR, RVO) and in animal models of ischemiainduced neovascularization. VEGF is also responsible for the macular edema associated with diabetic retinopathy and exudative AMD. Although the role of hypoxia in AMD-associated choroidal neovascularization (CNV) is less clear, VEGF and VEGF receptors have been found in CNV membranes (Rakic et al., 2003). Aqueous humor VEGF levels are also increased in AMD patients with CNV (Tong et al., 2006). In addition, certain anti-VEGF therapies do appear to improve vision or slow vision loss in patients with wet AMD (Rosenfeld et al., 2006). PEDF is made by RPE and Müller cells and is a natural inhibitor of angiogenesis (Barnstable and TombranTink, 2004). Retinal and choroidal neovascularization may be determined by the balance of pro-angiogenic VEGF and antiangiogenic PEDF (Tong and Yao, 2006).

Although VEGF has justifiably received considerable attention for its role in retinal

neovascularization, other growth factors also appear to be involved. Placental growth factor (PlGF) is a member of the VEGF family, which is expressed in retinal vascular endothelial cells and pericytes. VEGFR1 is the PlGF receptor and is found on pericytes and vascular smooth muscle cells. Unlike VEGF, PlGF is not induced by hypoxia, but its expression is regulated by VEGF. PlGF is found in the vitreous and neovascular membranes of patients with PDR and CNV. Mice deficient in PlGF have reduced levels of neovascularization in models of oxygen-induced retinopathy (Carmeliet et al., 2001) and CNV (Rakic et al., 2003), further supporting its involvement in retinal neovascularization. Hypoxia also regulates the expression of erythropoietin (EPO), which is best known for its ability to stimulate red blood cell formation, but it can also stimulate vascular endothelial cell proliferation and angiogenesis. Levels of EPO are elevated in ischemic retinas, and intravitreal EPO levels correlate with PVR disease status. Retinal angiogenesis can be reduced by inhibition of EPO (Watanabe et al., 2005a).

Insulin-like growth factor-1 (IGF-1) is involved in normal retinal vascular development. IGF-1 and IGF-1 binding proteins are expressed in retinal vascular, neuronal, and glial cells, and their expression is altered by hypoxia and hyperglycemia (Wilkinson-Berka et al., 2006). IGF-1 deficient mice have abnormal retinal vascular development, leading to retinal hypoxia and increased VEGF expression (Hellstrom et al., 2001). Intravitreal IGF-1 levels are increased in PDR, and IGF-1 is found in CNV membranes for AMD patients. Intravitreal administration of IGF-1 stimulates retinal neovascularization (Danis and Bingaman, 1997), and decreasing IGF-1 levels inhibits oxygen-induced retinopathy in mice (Smith et al., 1997). Hypoxia-induced retinal neovascularization is reduced in mice deficient in endothelial IGF-1 receptor (Kondo et al., 2003), providing further support for the

role IGF-1 plays in pathologic angiogenesis. However, IGF-1 works in concert with other growth factors such as VEGF, PDGF, and FGF (Wilkinson-Berka et al., 2006), and IGF- 1 is required for maximum VEGF activation, vascular endothelial cell proliferation and survival (Smith, 2005).

Angiotensins (Ang1 and Ang2) and their receptor Tie2 regulate vascular remodeling and maturation (Eklund and Olsen, 2006). Ang1 is an agonist and Ang2 an antagonist of the Tie2 receptor tyrosine kinase. All three components have been found in PDR epiretinal membranes and in CNV membranes from AMD patients. Retinal Ang2 expression is increased in an animal model of oxygen-induced retinopathy (Hackett et al., 2000). Inhibition of the Tie2 receptor decreases retinal neovascularization in a mouse model of oxygen-induced retinopathy and laser-induced CNV in mice (Hangai et al., 2001). These studies demonstrate that the Ang/Tie2 signaling pathway plays a major role in pathologic retinal angiogenesis.

Platelet derived growth factor (PDGF) is involved in recruiting pericytes to the vascular endothelium and in stabilizing the vascular bed. There is a high ratio of pericytes to vascular endothelial cells in the retina, and loss of pericytes is a hallmark of diabetic retinopathy. Inhibition of PDGF decreases retinal pericyte number, making the retina more susceptible to oxygeninduced retinopathy (Wilkinson-Berka et al., 2004). Tissue specific PDGF and PDGF receptor deficient mice develop retinal microvascular abnormalities that resemble many features of diabetic retinopathy (Betsholtz et al., 2004).

In addition to vascular endothelial cells, CNV membranes contain trans-differenti- ated RPE cells that contribute to the fibrous component of the membrane. The presence of TGF-β and CTGF in these membranes may stimulate these myofibro-blasts to produce extracellular matrix and further promote angiogenesis (Watanabe et al., 2005b).