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

d. Retinal neuroprotection by growth factors – A number of growth factors have been shown to protect the inner retina, outer retina, and retinal vasculature from a wide variety of pathogenic insults (Chaum, 2003). Fibroblast growth factors and ciliary neurotrophic factor (CNTF) are effective in protecting photoreceptors. FGF appears to provide ongoing trophic support for photoreceptors because photoreceptor expression of a dominant negative FGF receptor transgene causes retinal degeneration in mice (Campochiaro et al., 1996). Intraocular administration of FGF1 and FGF2 delays photoreceptor degeneration in genetic animal models of retinal degeneration and in light-induced photoreceptor degeneration. Retinal FGF2 expression is increased in response to a variety of retinal injuries including light, mechanical puncture, laser burn, and optic nerve crush. In fact, optic nerve crush protects photoreceptors from subsequent photic injury. FGF2 is expressed in RPE cells, the inner nuclear layer, and in photoreceptor inner segments. The expression of CNTF is increased when the retina is injured, and intraocular administration of CNTF inhibits photoreceptor degeneration in genetic models of retinal degeneration and in a model of retinal ischemia. CNTF receptors are found on neurons, RPE cells, Müller cells, and glia, but not on photoreceptors, suggesting that the neuroprotective action of CNTF is via these support cells. CNTF and FGF can act synergistically with other survival factors in the retina.

In addition to its anti-angiogenic activity, PEDF also is neuroprotective (Barnstable and Tombran-Tink, 2004). Intraocular viral delivery of PEDF protects photoreceptors from light damage (Imai et al., 2005) and protects the retina from ischemic damage (Takita et al., 2003). This therapeutic approach is also being evaluated clinically for CNV associated with AMD (Wei, 2005).

A significant amount of work has been done studying the neurotrophic effects of growth factors on retinal ganglion cells, because this cell type is progressively damaged in glaucoma. BDNF is a major

trophic factor for cultured RGCs and protects RGCs in several in vivo models of RGC damage. BDNF enhances and prolongs RGC survival after optic nerve injury and axotomy (Di Polo et al., 1998). However, Trk B expression in RGCs is down-regulated after axotomy, making the cells less sensitive to the neuroprotective action of BDNF. Trk B gene transfer to RGCs coupled with intravitreal BDNF administration significantly increased RGC survival after axotomy (Cheng et al., 2002). AAV. BDNF transduction of RGCs protected optic nerve axons in a rat model of ocular hypertensive glaucoma (Martin et al., 2003) and protected RGCs from excitotoxicity induced by intravitreal administration of NMDA (Schuettauf et al., 2004). Even target derived BDNF (injected into the superior colliculus) can protect RGCs from developmental RGC death (Ma et al., 1998). The neuroprotective effects of BDNF can be enhanced by concomitant administration of other growth factors, such as FGF2 and neurotrophin-3 (NT-3) (Logan et al., 2006). FGF2 administered to the optic nerve after optic nerve injury can increase BDNF and Trk B expression in RGCs (Soto et al., 2006). However, increased RGC survival does not necessarily result in protection of the optic nerve (Libby et al., 2005; Pernet and Di Polo, 2006), and useful therapies need to target both the RGC and optic nerve.

Several additional growth factors also have protective effects on RGCs. Ciliary neurotrophic factor (CNTF) can protect RGCs from axotomy-induced apoptosis, possibly by changing retinal glia cells to a more neuroprotective phenotype (van Adel et al., 2005). Glia derived neurotrophic factor (GDNF) is neuroprotective for cultured RGCs, and intravitreal administration protects RGCs in an optic nerve transection model (Yan et al., 1999). Intravitreal GDNF in biodegradable microspheres protected RGCs in DBA/2J mice, a naturally occurring mouse model of glaucoma (Ward et al., 2007). Repeated intraocular administration of NGF partially protects RGCs following optic nerve transection. AAV.FGF2

transduction of the retina protected RGCs in models of optic nerve crush and excitotoxicity (Schuettauf et al., 2004). EPO also protects RGCs in a rat model of ocular hypertensive glaucoma (Tsai et al., 2005), and weekly administration of EPO protects RGCs and optic nerve axons in the DBA/2J mouse model of glaucoma (Zhong et al., 2007). VEGF is best known for its vascular effects, but it is also neuroprotective. Transgenic mice that overexpress VEGF in neurons are more resistant to optic nerve transection-induced loss of RGCs (Kilic et al., 2006). BMPs, which are involved in ocular development, also promote RGC survival and neurite outgrowth (Kerrison et al., 2005).

The cell signaling pathways associated with growth factor neuroprotection are complex and include the ERK, PI3K, and Jak/STAT pathways, although there is often overlapping pathways and cellular crosstalk involved (Chaum, 2003). Providing meaningful protection to RGCs needs to involve protection of the cell body, the optic nerve axon, and maintenance of the relevant afferent and efferent neuronal connections.

Inhibition of VEGF signaling remains a major therapeutic target for the treatment of retinal and choroidal neovascularization, as well as retinal edema. A number of different anti-VEGF approaches have been taken including: sequestering VEGF using aptamers, antibodies, and soluble receptors; inhibiting the VEGF receptor tyrosine kinase; blocking the expression of VEGF or VEGFR using RNAi; or inhibiting downstream signaling via protein kinase inhibitors. Therapeutically targeting VEGF has been successful in clinical studies of ranibizumab, a recombinant monoclonal antibody Fab fragment that binds and neutralizes VEGF. Monthly intravitreal injections of this agent prevent vision loss and improved visual acuity in AMD patients with CNV (Rosenfeld et al., 2006). In addition, ranibizumab therapy also reduced retinal thickness and improved visual acuity in patients with diabetic macular edema (Nguyen et al., 2006).

Although VEGF is currently an important therapeutic target for the treatment of retinal and choroidal neovascularization and retinal edema, it is important to remember that VEGF is not solely pathogenic. In addition to its role in angiogenesis, VEGF is also neurogenic and neuroprotective (Greenberg and Kunlin, 2005). VEGF receptors are present on neurons and astrocytes, supporting a direct effect of VEGF on these cells, and the expression of VEGFR2 is increased on neurons subjected to hypoxia and glucose deprivation (Gora-Kupilas and Josko, 2005). VEGF promotes axonal outgrowth in vivo and increases the length of neurites in cultured neurons. In addition, it protects neurons from a wide range of insults, including trophic factor withdrawal, ischemia, and excitotoxicity. Retinal ganglion cells in transgenic mice overexpressing VEGF in neurons were protected from axotomy-induced degeneration (Kilic et al., 2006).

e. Therapeutic use and delivery of growth factors to treat retinal diseases

BOX 5.1

Table 5.3 provides an overview of the current and potential use of specific GFs for the treatment of a variety of retinal diseases. Determination of which GF or set of GFs are the appropriate therapeutic targets for each disease state is based on our current understanding of disease pathogenesis, as well as effects of these agents in experimental preclinical models of the disease.

Another major therapeutic challenge is the delivery of the therapeutic agent to the target tissue (i.e. retinal vasculature, RGCs, photoreceptors, RPE, or choriocapillaris). Preconditioning, or exposure to subthreshold levels of stress, can upregulate endogenous protective growth factors

(Continued)

BOX 5.1 (Continued)

TABLE 5.3 Growth factors as therapeutic targets for the treatment of ocular diseases

that make the retina resistant to further insults that would normally induce disease.

For example, mild hypoxia can induce retinal HIF-1 and EPO, leading to neuroprotection of RGCs and photoreceptors from hypoxic retinopathy (Grimm et al., 2005). Transcorneal electrical stimulation rescues axotomized RGCs by increasing retinal levels of IGF-1 (Morimoto et al., 2005). Growth factors can also be delivered by introduction of exogenous cells into the eye. Intravitreal injection of lineage-negative bone marrow cells was able to rescue the retina and retinal vascular cells in mouse models of rd1 and rd10 retinal degeneration and in oxygen-induced retinopathy (Otani et al., 2004; Ritter et al., 2006). These cells appear to differentiate into microglia that provide trophic support to the retina (Ritter et al., 2006). Intravitreal injection of bone marrow stromal cells partially protected RGCs in a rat model of glaucoma,

and these cells expressed several different trophic factors (Yu et al., 2006). Specially engineered cells that express specific growth factors can be encapsulated and surgically transplanted into the vitreous cavity to slowly release controlled levels of these therapeutic agents (Thanos and Emerich, 2005). Encapsulated cell technology has been used to deliver therapeutic levels of CNTF that protected photoreceptors in a dog model of retinitis pigmentosa (Tao, 2006). This same technology is currently being tested clinically in RP patients (Sieving et al., 2006).

Several anti-VEGF therapies for wet AMD are being directly introduced into the eye by intravitreal injection. However, their ocular pharmacokinetics currently require intraocular injections every 4–6 weeks. A number of drug delivery platforms are being developed to provide longer-term delivery of the therapeutic agent. For example, PLGA nanospheres containing the bioactive PEDF peptide (82-121) provided longer-term retinal protection compared to PEDF alone in a model of retinal ischemia (Li et al., 2006). Biodegradable microspheres containing GDNF protected RGCs from glaucomatous damage in a mouse model of glaucoma (Ward et al., 2007). Recent studies have shown that bioactive proteins can be delivered through the sclera to the choroid and retina. Choroidal and retinal levels of full length PEDF were obtained after subconjunctival injection (Amaral et al., 2005). Transscleral delivery of anti-ICAM-1 antibodies significantly inhibited VEGFinduced leukostasis in rabbits (Ambati et al., 2000).

F. Optic Nerve Head

Two considerations will be discussed with respect to the role of growth factors in the pathogenesis of glaucoma and the ONH. The first consideration is the transport of neurotrophins in RGC axons located

in the LC region of the human ONH. The second consideration is synthesis, secretion and signaling of growth factors and/or neurotrophins within the human ONH by resident cell populations.

1. Transport of neurotrophins in RGC axons located within the optic nerve head

Since the initial report of Anderson and Hendrickson (1974) that elevated IOP affected rapid axoplasmic transport in the monkey optic nerve, numerous studies from both human eyes and experimental models of glaucoma suggest that elevated IOP obstructs both anterograde and retrograde axonal transport in RGC axons within the ONH. These reports have led to the hypothesis that obstructed transport might prevent the movement of critical molecules to the RGC body, resulting in RGC apoptosis and cell death (Pease et al., 2000; Quigley et al., 2000).

Neurotrophin/receptor complexes are retrogradely transported to the RGC body (DiStefano et al., 1992). Brain derived neurotrophic (BDNF) is of particular importance since it has been reported to positively influence RGC viability in vitro, during retinal development and following axotomy. Pease et al. (2000) demonstrated an interruption of BDNF retrograde transport and accumulation of Trk B at the ONH in both a rat acute IOP elevation model and a chronic monkey model of glaucoma. Quigley et al. (2000) also reported that retrograde axonal transport of BDNF in RGC was blocked by acute IOP elevation in rats. While it is known that RGC can synthesize BDNF themselves and that ONH astrocytes and LC cells can be additional sources of BDNF (Lambert et al., 2001), it is not known if these sources of BDNF have the same or different effect on RGC as BDNF transported to the RGC body in complex with the Trk B receptor (Quigley et al., 2000).

In an interesting article, Johnson et al. (2000) reported the chronology of ONH and retinal responses to elevated IOP in

the rat. While they reported depletion of endogenous neurotrophins (e.g. BDNF and NT-4) following elevated IOP, their study suggested that neurotrophin withdrawal was not the earliest alteration. They noted that the earliest alterations occurring in the rat ONH in response to elevated IOP involved astrocytes. In addition, they reported RGC apoptosis throughout the experiment and not necessarily associated with axonal transport obstruction. Thus, while blockage of neurotrophin transport may play a role in RGC death, early local changes in the glaucomatous ONH and within the RGC itself may be of equal importance.

2. Local synthesis, secretion and function of growth factors produced by cells within the human optic nerve head

a. Transforming growth factor-beta (TGF-β) –

Elevated levels of TGF-β1 and TGF-β2 have been suggested to mediate astrocyte activation and ECM remodeling in the glaucomatous ONH. For example, Pena et al. (1999) were the first to report an increased immunohistochemical localization of both TGF- β1 and TFG-β2 in the glaucomatous human ONH. They reported that intense staining for TGF-β2 was associated with ONH astrocytes, while TGF-β1 staining was associated with blood vessels. They failed to detect the presence of TGF-β3. Interestingly, they also noted that there was little or no expression of TGF-β isoforms in the normal human ONH. These reported results in the human glaucomatous ONH were substantiated in the glaucomatous monkey ONH by Fukuchi et al. (2001). They reported that glaucomatous eyes showed strong expression of TGF-β1 and TGF-β2 in the glial cells of the LC region. In an experimental rat model of glaucoma, Guo et al. (2005) demonstrated an increase in TGF-β2 in the ONH transition region. In addition there was a positive correlation between elevated IOP and TGF-β2 deposition.

In vitro studies have also been reported on the effect of TGF-β1 and TGF-β2 on cells

isolated from the human ONH. Fuchshofer et al. (2005) demonstrated that TGF-β2 modulated ECM component expression in cultured human ONH astrocytes. They reported that exogenous TGF-β2 was capable of inducing the expression of both ECM and basal lamina components including fibronectin, collagen type I, collagen type IV, CTGF, tissue transglutaminase (tTG) and TSP-1. Importantly they also demonstrated that the stimulatory effect of TGF- β2 was mediated via CTGF. When CTGF was inhibited via siRNA, the stimulatory effect of TGF-β2 on ECM components was significantly reduced.

Kirwan et al. (2005b) used gene array analysis to examine the effect of exogenous TGF-β1 on ONH lamina cribrosa (LC) cells. They concluded that exogenous TGF-β1 induced expression and release of ECM components by LC cells. Interestingly, they also reported that the BMP antagonist proteins Smurf-1 and Smurf-2 were upregulated following exogenous TGF- β1 treatment. This may be of significance since we have recently reported that in TM cells the BMP antagonist protein gremlin blocks the inhibition of BMP-4 on TGF-β2 stimulated fibronectin secretion. Thus it is possible in LC cells that Smurf-1 and/or Smurf-2 may also be upregulated leading to uncontrolled TGF-β stimulation of ECM expression.

Thus both in vivo and in vitro studies indicate that both TGF-β1 and TGF-β2 may be significant in understanding the pathophysiology of the glaucomatous ONH. In addition it appears that both ONH astrocytes and LC cells may play significant roles in maintaining the ECM components of the human ONH.

However, the central question concerning the increased expression of TGF-β in the glaucomatous ONH remains: what causes elevation of TGF-β in the glaucomatous ONH? One clue may come from the study of Kirwan et al. (2004). TGF-β has been implicated as a key molecule stimulated by mechanical stress (Sakata et al.,

2004). Kirwan et al. (2005a) demonstrated that cyclical stretch of LC cells induced significant increases in TGF-β1 mRNA synthesis after 12 hours and TGF-β1 protein secretion after 24 hours. They also reported that exogenous TGF-β1 induced a significant increase in cell media MMP-2 activity after 24 hours. They concluded that TGF- β1 and MMP-2 release from LC cells might facilitate ECM remodeling of the glaucomatous ONH.

b. Neurotrophins and neurotrophic factors –

Lambert et al. (2001) reported that both mRNA and protein for each of the neurotrophins (e.g. NGF, BDNF, NT-3 and NT-4), three full-length Trk receptors (e.g. Trk A, Trk B and Trk C) and two truncated Trk receptors (e.g. Trk B-T and Trk C-T) were detected in human ONH tissue, and cells isolated from the human ONH, including LC cells and ONH astrocytes. In addition, secretion of neurotrophins was observed from cells isolated from the human ONH. The effect of exogenous neurotrophins on Trk receptor phosphorylation, cell proliferation and neurotrophin secretion by cells isolated from the human ONH has also been reported (Lambert et al., 2004a). Exogenous neurotrophins caused phosphorylation of specific Trk receptors indicating that cells within the human ONH express functional Trk receptors. In addition exogenous neurotrophins stimulated cell proliferation and neurotrophin secretion. Using in vitro conditions that mimic ischemia increased the expression and secretion of neurotrophins by cells isolated from the human ONH (Lambert et al., 2004b). These studies indicate that paracrine/autocrine signaling via neurotrophins may occur within the human ONH, and may be involved in the pathogenesis of glaucoma.

In addition to the neurotrophins, cells of the human optic nerve head express glial cell line derived neurotrophic factor (GDNF) and the GDNF receptor complex (Wordinger et al., 2003). Lamina cribrosa

cells, ONH astrocytes and ONH tissues express mRNA and protein for GDNF, Ret and GRFa-1. Secretion of GDNF by cells isolated from the human ONH was not detected. However, exogenous GDNF caused a significant increase in cell proliferation of LC cells but not ONH astrocytes.

c. Bone morphogenetic proteins – Wordinger et al. (2002) first reported the presence of bone morphogenetic proteins (BMPs) and their high affinity receptors (BMPRs) in human ONH tissues and cells isolated from the human ONH. ONH tissues and cells expressed BMP-2, BMP-4, BMP-5 and BMP-7. In addition they also noted the presence of the high affinity BMP receptors BMP-RIA, BMP-RIB and BMP-RII. In the same study, mRNA for BMP antagonist proteins gremlin, follistatin, chordin and BAMBI were reported to be expressed by ONH astrocytes and LC cells. The authors concluded that the BMP signaling pathway may be involved in the normal formation and function of the human ONH.

More recently, Zode et al. (2007) have reported BMP-4 and Smad signaling proteins are present in human ONH tissues and isolated ONH astrocytes and LC cells. In addition exogenous BMP-4 treatment of ONH astrocytes and LC cells resulted in downstream signaling via the canonical Smad pathway. Thus cells within the human ONH may respond to locally released BMP via paracrine and/or autocrine mechanisms.

G. Roles of Growth Factors in Dry Eye

Dry eye affects approximately 10–15% of the population over the age of 30, and its prevalence increases with age. This condition can be very irritating and in some cases vision threatening. Dry eye has a number of etiologies all of which lead to dysfunction of the ocular surface and secretory glands. Unstable tear film causes ocular surface inflammation and epithelial disease. The lacrimal gland makes and secretes the

water, electrolytes, and proteins composing the aqueous layer of the tear film, which is under neural control. Defective lacrimal gland function causes aqueous tear deficient dry eye syndrome. The lacrimal gland makes a wide variety of growth factors and neurotrophic factors and their receptors (e.g. TGFβ, FGF2, NGF, PDGF) (Nguyen et al., 1997). EGF stimulates lacrimal gland acinar cells in culture (Schonthal et al., 2000) and stimulates lacrimal gland secretion (Dartt, 2004). Tears obtained from dry eye patients have lower levels of EGF compared to controls (Ohashi et al., 2003).

Neurterin is a neurotrophic factor for some neurons and a member of the TGFβfamily of growth factors. Neurterindeficient mice have defects in their autonomic and sensory nervous system and they also develop dry eye. Mice lacking the neurturin receptor develop dry eye (Rossi et al., 1999) and neurturin-deficient mice have a number of ocular surface changes that mimic the dry eye phenotype (Yeh et al., 2003). These studies indicate that neurturin functions in regulating the ocular surface–lacrimal gland–neural network.

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