Ординатура / Офтальмология / Английские материалы / Essentials in Ophthalmology Pediatric Ophthalmology Neuro-Ophthalmology Genetics_Lorenz, Borruat_2008

for light induced apoptosis in several mouse models. Consequently, inhibiting the visual cycle can protect the retina against light damage. Application of 13-cis-retinoic acid reportedly slows down the visual cycle [52]. This effect is mediated by inhibition of RDH5, which catalyzes oxidation of 11-cis-retinal in the pigment epithelium before the chromophore is delivered back to the photoreceptor [52]. 13-cis-Retinoic acid has been shown to reduce the age-related accumulation of lipofuscin in the abcr–/– mouse model of AMD [43]. Accumulation of lipofuscin seems to contribute substantially to the etiology of Stargardt’s disease as well as of AMD, making application of 13-cis-retinoic acid a possible treatment strategy in these retinal diseases [43].

ever, in knockout mice with no expression of the Bcl-2 family members Bax and Bak the retina was protected against light damage. Despite these conflicting results, there is good evidence that Bcl-2 influences the cellular calcium homeostasis and modulates the anti-oxidative capacity of cells [17].

Anti-oxidants that showed a reportedly beneficial effect on retinal degeneration are DMTU and PBN, as it was revealed both in light induced apoptosis as well as in models of inherited retinal degeneration, although PBN was not sufficient in all inherited models examined. Taken together, these results indicate that anti-oxidative treatments are able to slow down certain forms of retinal degeneration.

11.3.1.3Strategies

for Neuroprotection Interfering with the Early Phase of Apoptosis

The early phase of apoptosis in acute bright light damage models is in agreement with, for example, with elevated intracellular calcium levels, the induction of oxidative stress, and aberrant mitochondrial function [18]. Several reagents and factors that can counteract these mechanisms had a protective effect in light-induced neurodegenerative animal models as well as in mouse models of inherited RP.

The calcium antagonist d-diltiazem is a blocker of calcium channels. It prevents light damage in mice, as revealed by the absence of TUNEL-posi- tive cells in the outer nuclear layer [19]. However, these data could not be reproduced in all studies, including those performed in the rd1-mouse and the P23H transgenic rat (for review see [59]).

Exposure to acute bright light is accompanied by changes in mitochondrial membrane integrity, and membrane leakage in these organelles might account for the induction of photoreceptor apoptosis in the respective animal models [18]. There are attempts to stabilize mitochondrial membranes by the over-expression of Bcl- 2 using a transgenic approach. While transgenic expression of Bcl-2 under the rhodopsin promoter in a study using constant white light had no protective effect, this was the case in a similar experiment performed by another group. How-

11.3.1.4Strategies Using Neuroprotective Cytokines that Showed Effects in Other Tissues

During application of cytokines in neurodegenerative retinal diseases, several promising candidates emerged, although the mechanism of cell rescue in the retina remains to be elucidated.

Lens epithelium derived growth factor (LEDGF) has a general anti-apoptotic effect that is mediated by a higher rate of expression of heat shock proteins and antioxidant proteins. In the eye, LEDGF protected retinal function during exposure to excessive light as well as after its application to the retinas of mice and rats carrying mutations responsible for retinal degeneration [1].

The expression of basic fibroblast growth factor (b-FGF) is endogenously upregulated when mouse retinas are exposed to excessive light, showing a neuroprotective effect if, for example, the retinas had been preconditioned with milder light before application of high doses [37]. Recombinant b-FGF was also injected intravitreally and showed a neuroprotective effect [8]. Successive attempts to preserve retinal morphology were undertaken by expressing b-FGF from virally delivered transgenes, for example in rats carrying a mutated rhodopsin gene (S334ter mutation), although retinal function could not be restored as effectively as retinal morphology in these experiments [35].

192 Retinal Research: Application to Clinical Practice

Pigment epithelium-derived factor (PEDF) originates in the eye and is neuroprotective after oxidative stress [55]. It had a robust neuroprotective effect when injected prior to light exposure and in two mouse models of inherited degeneration (rd1 and rd2) (for example see [8]).

Ciliary neurotrophic factor (CNTF) is reportedly upregulated after pre-conditioning with milder light in a similar way as b-FGF, and also after injury of ganglion cells [9]. While after light-induced damage the injection of CNTF alone protected the retina, in models of inherited diseases the delivery of a transgene was necessary to provide the long-term elevated levels of CNTF necessary for neuroprotection in these genetic models.

Brain-derived neurotrophic factor (BDNF), either applied directly or indirectly through release from transgenic cell transplants, protected the retina from light-induced degeneration [29]. Viral delivery of a BDNF transgene, but not in-

11 jection of recombinant BDNF, slowed down cell death in several inherited mouse and rat models of retinal diseases [12].

It has been suggested that VEGF induces pathologic symptoms in AMD especially neovascularization of the retina in later stages of the disease. Therefore, several VEGF antagonists have been developed and tested in animals, but also in patients with neovascular AMD. Among those showing modest benefits in clinical trials is pegaptanib, an RNA molecule binding VEGF165 but not other isoforms of VEGF-A. Another VEGF antagonist tested in patients is ranibizumab, a Fab fragment of an antibody that binds all isoforms of VEGF-A. Repeated intraocular injections of ranibizumab resulted in stabilization of vision in the majority of patients, with substantial improvement in vision in about a third of the patients [40]. VEGF Trap is another VEGF antagonist that has been administered in clinical studies intravenously to patients suffering from AMD resulting in significantly reduced retinal thickness [40].

Despite some promising results in cell preservation following the different treatments, several further aspects need to be taken into account. The rescue of neurons by application of neuroprotective factors does not necessarily correlate

with functional rescue of the respective cells in their environment, as researchers often had to admit after taking a closer look at their results. Functional tests as well as effective neuronal signaling are necessary in order to evaluate whether there is satisfactory protection and restoration of retinal function. In addition, the mode of applying a factor seems to influence its effect on neuroprotection: some cytokines were ineffective when injected intravitreally, whereas their transgenic expression achieved a significant cell rescue effect. This is especially true in inherited models of retinal degeneration, where often long-term expression of a factor is essential for its beneficial effect. Moreover, the long-term expression of factors may be important in another respect: considering the relatively rapid turnover of vitreous liquid, a single intra-vitreal injection might not be enough to sufficiently protect photoreceptors and adjacent tissue in the presence of persistent pro-apoptotic stimuli. The majority of data indicate that factors need to be present in the diseased tissue over extended periods of time in order to be protective. Even so, there is no proof that long-term application of factors can be managed in small laboratory animals let alone in the human retina, which is larger and has a substantially longer lifetime.

Summary for the Clinician

■Neuroprotective strategies are promising at the experimental level, but mostly lack long-term therapeutic effects.

11.3.2Cell Therapy

for the Diseased Retina

The replacement of retinal cells lost during the course of a retinal degenerative disease is a strategy that is currently being investigated heavily. In general, one may think of several different approaches. The ex vivo approach uses cultured cells that are expanded and sometimes induced in culture before being transplanted back into the diseased tissue. A further possibility within

this approach is to enhance the therapeutic potential of these cells by genetic engineering (ex vivo gene therapy). The in vivo approach, in contrast, tries to stimulate endogenous stem cells within the diseased tissue. Here we review and discuss progress in the retinal transplantation approaches and also in approaches targeted toward endogenous cell replacement.

11.3.2.1Cell Transplantation in the Retina

11.3.2.1.1 General Considerations

Prerequisites to success in the transplantation approach are: (1) establishment of appropriate cellular connections between transplanted cells and the local circuitry inside the visual system, and (2) a significant restoration of eyesight as assessed by behavioral tests. Transplanting retinal layers from healthy individuals to diseased retina aims to replace the injured or degenerated cells with new functional tissue.

The first experiments in this field were performed in 1959 using material from fetal eyes injected into the anterior chamber of rat eyes [47]. In the 1980s, similar experiments with pieces of RPE followed [25], and then between 1986 and 1992 the first data on embryonic and neonatal retinal cell aggregates transplanted into lesioned retina were published (reviewed by [4]). Research on improving transplantation techniques focused on the composition of transplants (cell aggregates or pieces of tissue of different size), the cell types transplanted, and the way of delivering them to the graft site.

To prove the adequate integration of donor tissue into the host photoreceptor layer one needs to distinguish between the two, which can be achieved by labeling the cells prior to transplantation. In preclinical studies, this was achieved mostly by genetically labeling the cells with cytoplasmic reporters such as green fluorescent protein (GFP) or beta-galactosidase, or by nuclear markers (e.g., 3H-thymidine, bromodeoxyuridine or by detecting Y chromosomes in male tissue transplanted to female recipients). Only with cytoplasmic stains can the cell pro-

cesses of transplanted cells be followed, because cell-to-cell contacts become visible. The GFPmouse – all of its cells show green fluorescence – has been widely used for experiments on retinal transplantation. Another promising attempt is to use transgenic rats expressing human placental alkaline phosphatase (hPAP) in the cytoplasm of all cells. Grafts from these animals can later be detected by histochemistry or immunohistochemistry in the host eye.

Immunological rejection of grafted cells or tissue needs to be considered seriously when thinking of future therapeutic concepts for human retinal degenerative diseases and their possible cure by allogeneic transplantation. In principle, the subretinal space has been shown to be rarely accessible to immunogenic elements, similar to the CNS. This “immune privilege” was deduced from data showing that allografts of neonatal retina and also other foreign antigens do not elicit a classic immune response in the subretinal space. A prerequisite for these results was an intact blood–brain barrier [58]. Nevertheless, upregulation of microglia expressing major histocompatibility complex (MHC) class I and II antigen was detected after allogeneic subretinal transplantations in mice and rats. These microglia could be found in the transplant and surrounding host tissue [34]. It is not yet known why this activation of microglia does not elicit rejection of the foreign tissue. Fetal tissue has yet to develop inner retinal vessels and therefore it elicits less of an immune response than postnatal tissue. As a consequence, the number of microglia is less in fetal versus postnatal tissue [4].

11.3.2.1.2Transplantation of Retinal Sheets

Earliest attempts to restore retinal function by transplantation were undertaken using retinal pieces of different size or dissociated cells (reviewed by [4]). However, in nearly 100% of treated rodents the grafted cells formed spherical structures, so-called rosettes, due to mechanical disruption of transplanted material. These roundshaped artifacts have the inner retinal layers on the outside, clasping photoreceptors that point

194Retinal Research: Application to Clinical Practice

with their outer segments towards the lumen of the rosettes [51]. Gouras and Tanabe established a micro-aggregate procedure, in which neonatal retina was cut into pieces small enough to pass through an injection needle without mechanical disruption, e.g. by shearing forces. Sheets integrated randomly at the proper orientation to the host RPE and survived well for at least 9 months [26]. In addition it has been shown that grafting material to the subretinal space was more advantageous to the laminar organization of transplants than grafting it to the epiretinal space [2]. In a different approach, Silverman used vibratome sectioning of postnatal-day-8 rat retinal wholemounts and transplanted the resulting retinal sheets into the subretinal space. In order to avoid rosette formation by the transplanted photoreceptors during these experiments, it was necessary to include the inner retina within sheets, indicating the importance of Müller cells for correct retinal

lamination [53]. A similar method using vibra- 11 tome sectioning was later applied to isolate photoreceptors from human post-mortem eyes [31].

Aramant and Seiler have developed a method to transplant sheets of fetal retinal neuroblastic progenitor cells into the subretinal space of rat eyes (reviewed in [4]). Healthy RPE provided by host or donor tissue was a prerequisite for the successful establishment of lamination resembling a normal retina. Authors saw the repair of degenerated retina after the transplantation of fetal tissue, also proven by visually evoked responses detected in areas of the superior colliculus corresponding to the transplant. These results seem to be especially promising, as not only protection but also repair of damaged tissue could be seen in these experiments. However, it became clear that transplantation cannot reverse all stages of disease to the healthy state: when photoreceptor degeneration in the host has advanced too far, including neovascularization and tight adherence of the retina to its RPE, no restoration of lamination can be achieved, because the force necessary to detach the host retina upon transplantation of donor tissue causes major tissue disruption in the recipient [3].

Clinical trials of retinal transplantation have been performed using adult (allogeneic and autologous) as well as fetal material in order to restore or prevent loss of vision in retinal degen-

erative diseases. When using RPE allografts in AMD patients, long-term beneficial effects were inhibited by inflammatory events and rejection of transplanted cells inside the recipient eye, although this effect could be inhibited using immunosuppressive treatment. Similarly, rejection was not observed in autologous transplantation of adult RPE cells in patients with wet AMD, who reported subjective improvements in vision after the treatment (reviewed in [3]). Another group established the transplantation of fetal retinal sheets together with its RPE in patients with RP or AMD [42]. Vision was not significantly improved by this treatment, although no apparent rejection was observed.

11.3.2.1.3Transplantation

of Stem and Progenitor Cell Populations

Stem-cell-based therapies are being introduced to the clinic in a wide range of human illnesses. Regarding neurodegenerative diseases of the eye, the use of stem and progenitor cells has been expected to be a promising tool for the replacement of injured or irreversibly declining tissue. The main focus of present cell therapy development is the replacement of lost photoreceptors by transplantation of suitable cells into the subretinal space between the outer retinal layers and the RPE. The subretinal space was established as preferred location for grafts in retinal damage. Several cell types with stem and progenitor characteristics have been investigated for their potential in retinal transplantation, including transplantation of embryonic stem cells, iris pigment epithelium, Schwann cells, retinal progenitor cells, fetal and adult neural stem cells, and bone marrow mesenchymal cells. The application of a retinal prosthesis was considered as an alternative. More recently, ex vivo genetic modification of transplanted cells has become an interesting modality. This chapter concentrates on stemand progenitor-cell-based therapy of retinal degenerative diseases.

Stem and progenitor cells are defined by unique properties: they proliferate, they self-re- new and they give rise to a multitude of differentiated cell types. While embryonic stem cells

derived from the inner cell mass of the blastocyst can develop into virtually any type of tissue and are therefore considered pluripotent, fetal or adult stem cells are generally more restricted with regard to their differentiation potential and are considered multipotent. In this context, it is widely accepted that neural stem cells of the developing or adult brain can develop into neurons, astrocytes, and oligodendrocytes (Fig. 11.1), and that retinal progenitors generate all the different retinal cell types, while hematopoietic stem cells give rise to all types of hematopoietic cells. However, some data suggest that in rare events transdifferentiation of hematopoietic or bone-marrow-derived mesenchymal stem cells into cells of neuro-ectodermal phenotype can occur, although these findings are strongly debated.

11.3.2.1.3.2Fetal Stem

and Progenitor Cell Populations

from the Retina

Fetal stem and progenitor cell populations that might be relevant for cell transplantation strategies for the retina are those derived from the fetal brain or retina. These cells have the potential to differentiate into neurons and, in the case of fetal retinal progenitors, to differentiate into retinal-specific neurons such as photoreceptors. Therefore, we focus here on progenitors from the developing retina and recapitulate retinal development.

The mammalian eye is generated during development from bilateral evaginations of the di-

11.3.2.1.3.1 Embryonic Stem Cells

Embryonic stem cells are derived from the inner cell mass of very early embryos (blastocysts). Their massive impact on biological and medical sciences derives from two unique characteristics that distinguish them from all other cell types. First, they can indefinitely be maintained as undifferentiated cell populations (self-renewal) and therefore represent an unlimited supply of material for cellular-based replacement therapies. Second, embryonic stem cells are pluripotent, possessing the capacity to create all cell types that constitute an adult organism including the reproductive cells of the germ line. The field of embryonic stem cell research is trying to develop from a basic science discipline to a highly relevant clinical issue for replacement therapy approaches. Very recent data demonstrated that embryonic stem cells can be triggered to differentiate efficiently into retinal neurons indicating their therapeutic potential for retinal diseases [32]. Despite their broad capacity to generate a great multitude of differentiated cells, the use of embryonic stem cells is significantly limited due to ethical issues in humans. Since substantial evidence has emerged that stem cells are present in the adult human eye, more effort has been focused on the development of feasible treatments using these cells.

Fig. 11.1. Neural stem cell tree. Neural stem cells are characterized by their potential to proliferate, self-re- new and to generate the three main cell types of the CNS: neurons, astrocytes and oligodendrocytes

196Retinal Research: Application to Clinical Practice

encephalic neuroepithelium forming the optic vesicles. Coordinated invagination of ectodermal tissue results in the lens placode, while the optic vesicles form a bilayered structure, the so-called eyecup. The retinal pigment epithelium (RPE) develops from the outer layer, while the neural retina is derived from the inner layer of this optic cup. During later developmental steps, multipotential retinal stem cells develop from the inner layer, giving rise to the basic cell types of the adult retina. The mammalian retina is populated through proliferation of these stem cells and differentiation of daughter cells. This process happens along a conserved pattern, although there is considerable overlap between the generation of different cell types: the first daughter cells in the retina are the retinal ganglion cells (RGCs), followed by cones and amacrine cells, horizontal cells, rods, bipolar cells and Müller glia. In the mature tissue, RGCs can be found at the inner

surface of the retina, and photoreceptors com- 11 prising rods and cones form the outer margin next to the RPE. Between these two outermost layers of the retina lie the cells of the inner nuclear layer including bipolar, amacrine, and horizontal cells. The Müller cells span the entire retina and descend from retinal stem cells, while the two other types of glial cells in the eye, the astrocytes of the inner retinal surface and the oligodendrocytes that clasp the optic nerve, migrate

to the retina during development.

Retinal progenitors can be isolated from the developing retina and expanded in culture [23]. They are restricted to a bipotent fate and give rise to neurons and glia, but not oligodendroglia, suggesting certain molecular differences between retinal progenitors and neural stem cells.

in the adult mammalian retina, a number of different cell types might function as sources for somatic neural stem cells. These cells can be derived from the margin of the ciliary body (CB), the pigment epithelium layer (RPE) and the sensory retina (SR) (Fig. 11.2).

In vitro experiments suggest the presence of multipotent neural progenitor cells in the CB or the ciliary marginal zone of the adult mammalian eye [56]. Under the culture conditions used in these studies, pigmented cells from the CB, but not the SR, RPE or other retinal structures, formed neurospheres consisting of pigmented and non-pigmented cells. Several cells in these neurospheres expressed Nestin and Chx10, both markers for somatic neural precursor cells and retinal progenitors. Some CB-derived cells differentiated into retinal neurons and glia. Despite the lack of convincing clonal analysis, pigmented cells in the CB were proposed in these studies to be multipotent neural stem cells [56].

As Müller glia cells are among the last cells in the retina to develop, their ability to generate retinal progenitor cells during development was excluded. However, after injury, Müller glia cells undergo reactive gliosis, a process associated with cell proliferation and the upregulation of glial fibrillary acidic protein (GFAP) [21]. After an N-methyl-d-aspartate lesion in postnatal

11.3.2.1.3.3Stem Cells

from the Adult Retina

It was proposed that the adult mammalian retina

– unlike its poikilothermic vertebrate analogs in fish, amphibians or reptiles – is devoid of proliferative or regenerative capacity. However, recent data suggest that there is indeed such proliferative capacity, as has been proven during the identification of stem or progenitor cells in the adult mammalian eye. Several studies have shown that

Fig. 11.2. Putative stem and progenitor cells in the adult retina. The adult mammalian retina has putative stem and progenitor cell populations: Müller glial cells, RPE cells, cells from the ciliary body and from the ciliary marginal zone

chicken retina, cell proliferation is induced and triggers the expression of the retinal progenitor markers CASH-1, Pax6 and Chx10 in Müller glia. Newly born cells differentiate into retinal neurons, into Müller glia or remain undifferentiated [24], suggesting that Müller glia might be a potential source for de-differentiating cells that acquire a somatic neural stem cell phenotype.

The RPE is of neuroectodermal origin as it derives from the neural plate and descends from precursors that later generate neural retina. The mature RPE consists of a mosaic of fully differentiated, polygonal cells between the choroid and the neural retina. This single cell layer is strongly important in processes essential to vision such as the metabolism of intermediates of the visual cycle and the phagocytosis of photoreceptor outer segments (reviewed in [7]). While in birds and amphibians RPE cells are able to either transdifferentiate into retinal neurons and glia or dedifferentiate into multipotential retinal stem or progenitor cells, homeothermic vertebrates have apparently lost this capacity. In mammals, RPE cell proliferation is described as a consequence of retinal detachment surgery. The resulting cells partially trans-differentiate and acquire neural progenitor and neuronal features [22], including expression of β III tubulin and voltage-gated Na+ channels. However, they do not de-differentiate into a multipotent somatic neural stem cell and neither do they trans-differentiate completely to acquire the full phenotypic pattern of a nerve cell or regenerate a retina (reviewed in [14]).

11.3.2.1.3.4Transplantation of Stem and Progenitor Cells

to the Degenerated Retina

In the field of retinal degenerative diseases, much hope has been placed on the potential use of stem and progenitor cells to restore vision. Transplantation of tissue or single cells/cell aggregates may be especially useful at stages of disease where the majority of photoreceptors have disappeared and neuroprotective approaches are doomed to failure. However, cell replacement is a challenging task, and several obstacles need to be overcome

in order to develop efficient strategies. First of all, cell delivery techniques need to be improved. Nowadays, retinal sheets can be transplanted to the subretinal space, although single cell transplantations are much more difficult with regard to the formation of a three-dimensional network [61]. The capacity of grafted cells to survive in the host retina is probably limited and needs to be increased in order to establish long-term improvement of vision in retinal diseases. Another hurdle in efficiently applying or inducing stem cells in the eye is the lack of sufficient protocols regarding purposeful differentiation of stem and progenitor cells.

Some of the only cell transplants with an established clinical application in diseases of the eye are corneal limbal epithelial stem cells (LESCs) used in corneal defects. These LESCs show characteristics of stem or progenitor cells in having a high capacity for self-renewal and being poorly differentiated. LESCs can be found in the basal layer of the limbus between the cornea and the conjunctiva. Although descending from mesodermal tissue, these cells are being explored with regard to their capacity for the repair of retinal structures (for review see [36]. Additionally, autologous transplantation of RPE cells in AMD has been established [6].

Embryonic stem (ES) cells have been considered a powerful source for ocular regeneration due to their high proliferative capacity and differentiation potential. However, ethical considerations inhibit the widespread use of these cells in most parts of the world. While differentiation protocols for ES cells have recently been improved and established towards retinal cell type differentiation [32], the success of ES cell transplantations in the past was low due to immunological rejection [38].

Neural stem cells, which can be derived from the adult brain and propagated in vitro in the presence of FGF and EGF, have also been transplanted into retinal degeneration models. The grafted cells integrated into the laminar structures of the retina and extended processes into the optic nerve head [50, 54], but no expression of retina-specific markers was observed. When transplanted into the immature retina, neural stem cells adopted expression profiles similar to those of retinal neurons [48].

198

11

Retinal Research: Application to Clinical Practice

Retinal progenitor cells from the fetal retina [60], the postnatal retina [23] and the adult mammalian CB [56] have been characterized in detail. Transplantation studies with these cells indicate that the degree of integration and migration into the host retina depends on the age or stage of the diseased or injured recipient retina. Grafted retinal progenitor cells express the retina-specific marker opsin [11], but their differentiation potential seems to be limited to the glial lineage after transplantation to an adult host with retinal degeneration [60]. Some of the material transplanted into the retina is summarized in Table 11.3.

Summary for the Clinician

■Cell transplantation seems to be a promising approach, at least in the preclinical setting.

■Grafted embryonic and fetal stem and progenitor cells have the potential to replace degenerated retinal tissue. However, major ethical concerns and limited availability seem to make them an unlikely candidate for standard therapy.

■Adult stem cell transplantation might have some clinical relevance in future. However, the growth and differentiation potential of these cells is yet not sufficient.

11.3.2.2Application of Transgenes or Genetically Engineered Stem and Progenitor Cells

One of the major limitations in successful transplantation strategies is probably the fact that cells are transplanted into a pathological, hostile environment. This environment is unlikely to provide the necessary stimuli for differentiation and integration of grafted cells. Ex vivo gene transfer has been shown to harbor the potential to overcome this barrier. The advantage of these strategies is the inclusion of survival-promoting factors into the grafts. Factors applied in this way could also act in an autocrine manner to simulate a physi-

ological environment for differentiation after transplantation and integration into the retina. Ex vivo gene transfer could facilitate neuroprotection and thereby prevent retinal cell loss in RP, AMD or glaucoma.

Several studies have been conducted that address optic nerve degeneration and gene transfer via viral vectors, most of them using recombinant adeno-associated viruses. Gene therapy focuses on: (1) providing growth factors to protect resident neurons or improve graft integration, (2) delivering anti-angiogenic proteins that may help to overcome secondary adverse effects of retinal diseases, and (3) gene replacement strategies for autosomal recessive retinal diseases (reviewed in [46]).

Results from the application of survival factors by gene therapy indicated that the delivery of neurotrophins such as nerve growth factor (NGF) [33], ciliary neurotrophic factor (CNTF) [10] or brain-derived neurotrophic factor (BDNF) [16] by viral vectors can rescue photoreceptors and RGC within the optic nerve in degeneration models. Genetically modified human-derived RPE cells, which over-express BDNF, have been shown to promote cell survival [30] and to inhibit aberrant retinal neovascularization [39]. An FGF transgene has also been implemented to endogenously stimulate regeneration in degenerative retinal models, where it provoked axonal outgrowth of adult RGC after optic nerve injury [49]. An approach using small interfering RNA (siRNA), which targeted VEGF, effectively inhibited ocular neovascularization in a mouse model for AMD [45]. This suggests that, besides the viral vectors, siRNA techniques also harbor the potential to address retinal degeneration and neural protection by targeting factors that drive disease mechanisms.

Summary for the Clinician

■Progress in the development of safer vectors and new technologies such as siRNA make gene therapy a highly promising therapeutic approach.

Cell transplants in clinical trials

One of the most promising ideas for replacement strategies in the retina is the stimulation of endogenously persisting stem or progenitor cell populations. Even though it has been reported that the mammalian retina is devoid of regenerative capacities, numerous studies have indicated that, with appropriate stimuli, regeneration can be induced, especially in chicken and neonatal mammalians (reviewed in [44]). Many attempts have been made to overcome the quiescence that stops endogenous stem and progenitor cell proliferation and differentiation in the diseased retina.

With the observation that glial cells of the CNS provide a source of neural regeneration [28], focus has been placed on the glial cell type of the retina, the Müller glia. Fischer and Reh showed that Müller glia cells respond to injury or exogenous growth factors by de-differentiation, proliferation and expression of neuronal and glial markers [24]. First indications for successful endogenous stimulation of Müller glia regeneration

that Müller glia cells were stimulated to proliferate in response to the toxic injury. Furthermore, the cells produced bipolar cells and rod photoreceptors and their numbers could be promoted by the application of retinoic acid. The authors convincingly showed that they could partially control the fate of the newly generated neurons with extrinsic factors and intrinsic factors. The analysis of the integration of newly generated neurons and their functionality remains to be elucidated. Müller glia cells might be an endogenous source of retinal progenitor cells and may become a target for both drug delivery and gene therapies to effectively treat retinal degenerative diseases.

The reasons for the limited or nonexistent proliferation of retinal stem and progenitor cells in the adult retina might be different; for example, the lack of a sufficient amount of mitogens might be a limiting factor. Alternatively, anti–stem-cell proliferative activities might be present in the adult retina. This hypothesis was recently introduced by the work of Close et al. [13], which sug-

gests that TGF-beta1 might be a paracrine-inhib- iting factor derived from mature retinal neurons that limits retinal progenitor cell proliferation [13]. In a similar context, TGF-beta1 has recently been described to be an inhibitor of neurogenesis in the adult brain [57]. Future experimental approaches might be targeted towards the elimination of such activities to restore retinal stem and progenitor proliferation and functional regeneration.

Summary for the Clinician

■The presence of stem and progenitor cells in the adult retina makes these cells a very promising drug target, which might be stimulated to regenerate the retina. However, the preclinical development is still at a very early step.

11

References

1.Ahuja P, Caffe AR, Holmqvist I, Soderpalm AK, Singh DP, Shinohara T, van Veen T (2001) Lens epithelium-derived growth factor (LEDGF) delays photoreceptor degeneration in explants of rd/rd mouse retina. Neuroreport 12:2951–2955

2.Aramant R, Seiler M (1991) Cryopreservation and transplantation of immature rat retina into adult rat retina. Brain Res Dev Brain Res 61:151–159

7.Bok D (1993) The retinal pigment epithelium: a versatile partner in vision. J Cell Sci Suppl 17:189–195

8.Cao W, Tombran-Tink J, Elias R, Sezate S, Mrazek D, McGinnis JF (2001) In vivo protection of photoreceptors from light damage by pigment epithe- lium-derived factor. Invest Ophthalmol Vis Sci 42:1646–1652

9.Casson RJ, Chidlow G, Wood JP, Vidal-Sanz M, Osborne NN (2004) The effect of retinal ganglion cell injury on light-induced photoreceptor degeneration. Invest Ophthalmol Vis Sci 45:685–693

10.Cayouette M, Behn D, Sendtner M, Lachapelle P, Gravel C (1998) Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse. J Neurosci 18:9282–9293

11.Chacko DM, Rogers JA, Turner JE, Ahmad I (2000) Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat. Biochem Biophys Res Commun 268:842–846

12.Chong NH, Alexander RA, Waters L, Barnett KC, Bird AC, Luthert PJ (1999) Repeated injections of a ciliary neurotrophic factor analogue leading to long-term photoreceptor survival in hereditary retinal degeneration. Invest Ophthalmol Vis Sci 40:1298–1305

13.Close JL, Gumuscu B, Reh TA (2005) Retinal neurons regulate proliferation of postnatal progenitors and Muller glia in the rat retina via TGFbeta signaling. Development 132:3015–3026

nal sheet transplantation. Prog Retin Eye Res 23:475–494

4.Aramant RB, Seiler MJ (2002) Retinal transplantation – advantages of intact fetal sheets. Prog Retin Eye Res 21:57–73

5.Aramant RB, Seiler MJ, Turner JE (1988) Donor age influences on the success of retinal grafts to adult rat retina. Invest Ophthalmol Vis Sci 29(3):498–503

6.Binder S, Stolba U, Krebs I, Kellner L, Jahn C, Feichtinger H, Povelka M, Frohner U, Kruger A, Hilgers RD, Krugluger W (2002) Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: a pilot study. Am J Ophthalmol 133:215–225

therapy for retinal degeneration: retinal neurons from heterologous sources. Semin Ophthalmol 20:3–10

15.del Cerro M, Notter MF, del Cerro C, Wiegand SJ, Grover DA, Lazar E (1989) Intraretinal transplantation for rod-cell replacement in light-damaged retinas. J Neural Transplant 1(1):1–10

16.Di Polo A, Aigner L, Bray GM, Aguayo AJ (1998) BDNF gene transfer to the retina reduces lightinduced damage to photoreceptor cells. Soc Neurosci

17.Distelhorst CW, Shore GC (2004) Bcl-2 and calcium: controversy beneath the surface. Oncogene 23:2875–2880