Retinal Disease Models for Development of Drug and Gene

17 Retinal Disease Models for Development of Drug and Gene

Therapies

Leena Pitka¨nen University of Kuopio and Kuopio University Hospital, Kuopio, Finland

Lotta Salminen University of Tampere and Tampere University Hospital, Tampere, Finland

Arto Urtti University of Kuopio, Kuopio, Finland

I. INTRODUCTION In humans the retina is the innermost layer of the eye, which consists of

retinal pigment epithelium (RPE) and neural retina. The neural retina has several layers and various cell types, which are illustrated in Figure 1. RPE is

a single layer of hexagonal cells that maintains the homeostasis of neural retina. It has essential biochemical, physiological, physical, and optical func- tions in maintaining the visual system, including phagocytosis of rod outer segments, transport of substances between photoreceptors and choriocapil- laries, and uptake and conversion of the retinoids, which are needed in visual cycle. Together with endothelial cell linings of retinal capillaries, RPE forms the blood-retinal barrier. The neural retina is a complicated and delicate multilayer. The thickness of neural retina varies from 0.4 mm near the optic nerve to about 0.1 mm anteriorly at the ora serrata. The photoreceptors are the light-sensing part of retina. The electric impulses are amplified and integrated by bipolar, horizontal, amacrine, and ganglion cells. The principal glial cell of the retina is the Mu¨ller cell. The bipolar cells

516 Pitka¨nen et al.

Fig. 1 Light photomicrograph of a rat retinal section. The inner nuclear layer includes the nuclei of bipolar, amacrine, horizontal, and Mu¨ller cells. The nuclei of rods and cones are in the outer nuclear layer.

are the first and ganglion cells the second neuron of the visual pathway from photoreceptors to brain. Macula is the central part of retina located tempo- rally of optic nerve head between the upper and lower temporal vessels. Fovea is the central, approximately 1.5 mm wide sloping part of macula. Visual acuity is decreased quickly in the paramacular areas. Of the photo- receptors, the cones take care of photoptic and color vision and are located mainly in the macula. Rods are the main photoreceptor type in the periph- ery; they are specialized to scotopic vision.

The human retina may be affected by many vascular diseases such as occlusions, vasculitis, and anomalies. Retinopathy of prematurity (ROP) is

a retinal disease affecting premature children, where the growth of develop- ing retinal vasculature is interrupted. Diabetic retinopathy is a common cause of blindness, while arteriosclerosis, hypertension, and other cardio- vascular diseases may cause changes in the retinal vasculature. Neovascularization is also associated in macular degeneration.

In retinal detachment, fluid is collected in the potential space between the neural retina and RPE. In rhegmatogenous detachment, the fluid comes

Retinal Disease Models 517 from the vitreous cavity through a retinal hole or tear. Extravasation may

originate from choroid or retina and results in secondary retinal detach- ment. Retinal detachment caused by the traction of fibrous bands in vitreous is called traction retinal detachment. Traumas, intraocular inflammations, retinal or vitreal degeneration, or vitreal bleeding are etiological factors of retinal detachment. Proliferative vitreoretinopathy (PVR) is found in about 5% of retinal detachments. It is characterized by the formation of vitreal, epiretinal, or subretinal membranes after retinal reattachment surgery or ocular trauma. In some cases the membranes cause traction and distortion of retina. Severe postoperative PVR is the most common cause of failed retinal detachment surgery.

Retinoblastoma is a malignant retinal tumor with an incidence of about 1 : 20,000. The genetic abnormality of this disease located to 13q14. Both genes in this locus must be abnormal before this malignancy develops. In the nonhereditary form, mutation occurs only in the retinal cells. In the hereditary form the patient has inherited the first mutation from his or her parents, and 90% of these patients develop a clinical retinoblastoma.

In this chapter we present some recent development in the retinal disease models of animals. Models of retinal degeneration, proliferative diseases, and neovascularization are presented. These models are important tools in current research, since various growth factors, gene therapies, and transplantation strategies have demonstrated possibilities for treating severe retinal diseases.

II. RETINAL DEGENERATION—GENETIC MODELS Retinal degeneration leads to impaired function of the photoreceptors and

consequently gradual loss of vision., Many types of degeneration are based on genetic factors, e.g., retinitis pigmentosa, a common term for various mutations causing retinal degeneration. In addition to genetic factors, envir- onmental factors (e.g., light exposure) may lead to retinal degeneration. Macular degeneration is the most common type of retinal degeneration, being the leading cause of vision loss in the industrial world. In the following sections we present some genetic and environmental animal models of ret- inal degeneration.

A. Natural Mutation Mouse Models The naturally occurring mouse rd (retinal degeneration) and rds (retinal

degeneration slow) photoreceptor dystrophies are recessively inherited. The mice have defects in the cGMP phosphodiesterase beta subunit gene

518 Pitka¨nen et al. (1,2) and in the peripherin gene (3,4). The rd mouse is a model of retinitis

pigmentosa in which a mutation of a rod-specific photophodiesterase leads to the rapid loss of photoreceptors during early postnatal life. Very little is known about the associated changes in the inner retinal neurons. Bipolar and horizontal cells of the rd mouse retina undergo dramatic morphological changes accompanying photoreceptor loss, demonstrating a dependence of second-order neurons on photoreceptors (5).

The rds phenotype is considered to be an appropriate model for per- ipherin 2/rds-mediated retinitis pigmentosa. Peripherin 2 glycoprotein is needed for the formation of photoreceptor outer discs. The photoreceptor cell is the primary site of the genetic defect that results in retinal dystrophy in the rds mouse model (6).

The protective effect of a number of survival factors on degenerating photoreceptors in mutant mice with naturally occurring inherited retinal degenerations, including retinal degeneration (rd/rd), retinal degeneration slow (rds/rds), nervous (nr/nr), and Purkinje cell degeneration (pcd/pcd), in three different forms of mutant rhodopsin transgenic mice and in light damage in albino mice were examined by La Vail et al. (7). The slowing of degeneration in the rd/rd and Q344ter (a naturally occurring stop codon mutation that removes the last five amino acids of rhodopsin) mutant mice demonstrated that intraocularly injected survival factors can protect photo- receptors from degenerating. Importantly, these animal models have the same or similar genetic defects as those in human inherited retinal degen- erations (7). Such models have also been used to improve the condition of photoreceptors by adeno-associated virus-mediated peripherin 2 gene ther- apy (8). The outcome of the gene therapy was dependent on the timing of the therapy (9).

B. Transgenic Mouse Models To generate transgenic animals, whole genes are injected into a fertilized egg

pronucleus. The genes associate randomly into the genome, and their expression is controlled by their own regulatory sequences. Due to the complexity of the photoreceptor biology, several genes can be used to gen- erate transgenic mouse models of retinal degeneration.

The VPP mouse carries three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals have slowly progressive degeneration of the rod photorecep- tors and subsequent changes in retinal function. These changes mimic auto- somal dominant retinitis pigmentosa of humans, which results from a point mutation (P23H) in opsin (10). The rate of photoreceptor degeneration in VPP mice seems to be adversely affected by the existence of the albino

Retinal Disease Models 519 phenotype (11). Light deprivation affects the rate of degeneration in pig-

mented transgenic VPP mice (12). To establish a transgenic mouse line with a mutated mouse opsin gene in addition to the endogenous opsin gene, a mutated mouse opsin gene was introduced into the germ line of a normal mouse. Simultaneous expression of mutated and normal opsin genes induces a slow degeneration of both rod and cone photoreceptors. The time course mimics the course of human autosomal dominant retinitis pigmentosa (13).

The biochemical, morphological, and physiological analyses of a transgenic mouse model for retinal degeneration slow (RDS) retinitis pig- mentosa have been carried out. RDS retinitis pigmentosa is caused by a substitution of proline 216 to leucine (P216L) in rds/peripherin. The phe- notype in P216L-transgenic mice probably caused by a combination of two genetic mechanisms: a dominant effect of the P216 substituted protein and a reduction in the concentration of normal rds/peripherin. The expression of the normal and mutant genes is similar to that predicted for humans with RDS-mediated autosomal-dominant retinitis pigmentosa. These mice may

be used as an animal model for this disease (14). The W70A transgenic mouse carries a point mutation (W70A) in the gene that encodes for the gamma-subunit of rod cGMP phosphodiesterase. This mouse represents a new model of stationary nyctalopia that can be recognized by its unusual ERG (electroretinogram) features (15).

Another transgenic mouse model with defective expression of the alpha subunit of the rod cGMP-gated channel was reported recently (16). Expression was reduced by antisense RNA. The low expression of the rod cGMP-gated channel causes a disease model that can be used to test thera- pies designed to slow down or cure retinal degenerations (16).

Mice (Pdegtm1/Pdegtm1) that are homozygous for a mutant allele of the gamma subunit of retinal cyclic guanosine monophosphate phospho- diesterase (PDE gamma) have a severe photoreceptor degeneration. Interestingly, the transgene that encodes the BCL2 protein was introduced by mating into the mutant background. Antiapoptotic transgene BCL2 delayed temporarily the degeneration of photoreceptors in this murine model of retinal degeneration (17).

C. Knockout Mouse Models Knockout mutation is created by transferring a gene that is inactivated by

mutation to pluripotent embryonal stem cells. They often find their copy in the genome and settle beside it and then change places by recombination. The cells with wanted recombination are transferred to blastocysts to pro-

520 Pitka¨nen et al. duce chimeric animals. Homozygous animals with the mutation can be

produced by mating.

A retinitis pigmentosa GTPase regulator-deficient mouse model for X- linked retinitis pigmentosa has been created by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins. Rod photoreceptors have a reduced level of rhodopsin, and subse- quently photoreceptors degenerate (18).

as an animal model of retinitis pigmentosa. In that case a gene encoding ciliary neurotrophic factor (CNTF) was delivered subretinally with adeno- associated virus-vector. CNTF gene therapy delayed the death of photore- ceptors (19).

exon 2 of the rhodopsin gene. They show a complete absence of functional serve during postnatal weeks 4–6 as a model for pure cone function (20).

These mice do not elaborate rod outer segments, and the photoreceptors are lost in 3 months. No rod ERG response is seen in 8-week-old animals. In

inner and outer segments of the cells display some structural disorganiza- tion. These animals may be a useful genetic background on which other mutant opsin transgenes can be expressed. (21).

Knockout mice with arrestin gene defect have been generated. Excessive light accelerated the cell death in pigmented arrestin knockout mice. Human patients with mutations leading to nonfunctional arrestin and rhodopsin kinase have Oguchi disease. This disease is a form of sta- tionary night blindness (22).

D. Rat Models The Royal College of Surgeons (RCS) rat is the first animal model with

inherited retinal degeneration. Although the genetic defect is actually not known, the RCS rat is widely used as a model of photoreceptor degenera- tion with relevance to retinitis pigmentosa and hereditary retinal dystrophies (23,24). Experiments with RCS rats have been used to demonstrate the beneficial effects of growth factors (like basic fibroblast growth factor, bFGF) on retinal degeneration (25).

Adenovirus-mediated gene transfer has been used to develop a rat model for photoreceptor degeneration. Recombinant adenovirus-mediated downregulation of cathepsin S (CatS) in the retinal pigment epithelium and/ or neural retina was achieved. These results demonstrate that the transient modulation of gene expression in RPE cells induced changes in the retina.

Retinal Disease Models 521 Despite the low expression of endogenous CatS in RPE cells, this enzyme

appears to play an important role in the maintenance of normal retinal function (26).

Transgenic rat P23H have been used as a model of autosomal domi- nant retinitis pigmentosa. Substitution of proline by histidine in position 23 in rhodopsin (P23H) is the most common human mutation in RP in the United States, with a prevalance of 15%. Several sublines of this strain have been developed. These lines have a similar genotype, but the rate of retinal degeneration varies. In line 1, almost complete degeneration is seen in 2 months, but in line 2 similar degeneration develops in one year. Similarly, there are many sublines of transgenic rats that carry a rhodopsin mutation S334ter with different rates of retinal degeneration. Ribozyme-directed clea- vage of mutant mRNAs slows the rate of photoreceptor degeneration in this rat model (27). d-cis-Diltiazem did not rescue photoreceptors of Pro23His rhodopsin mutation line 1 rats treated according to the protocol used in rd mouse (28). Extended photoreceptor viability by light stress has been detected in RCS rats but not in opsin P23H mutant rats (29). The photo- receptors of transgenic rats expressing either a P23H or an S334ter rhodop- sin mutation were protected from apoptosis by recombinant adeno- associated virus-mediated production of fibroblast growth factors fgf-2, fgf-5, and fgf-18 (30,31), while lens epithelium-derived growth factor pro- moted photoreceptor survival in light-damaged and RCS rats, but not in P23H rats (32).

In addition to biochemical measures, these disease state models can be monitored on the basis of retinal morphology (number of outer nuclear layers) and ERG (a and b waves).

E. Cat Models Abyssinian cats with recessively inherited rod-cone degeneration have been

introduced (33). Photoreceptor allografts were examined to determine the viability and influence of such transplants on the host retina of the cats. Also, clinical and pathological features, light and electron microscopy, and the electrophysiology of an autosomal dominant, early-onset feline model of rod/cone dysplasia (Rdy cats) have been documented (34–36). The immu- nohistochemical changes in the retina and photoreceptor cell death of this model have also been studied (37).

F. Transgenic Pig Model Transgenic pigs that express a mutated rhodopsin gene (Pro347Leu) were

generated (38). These transgenic pigs provide a large animal model to study

522 Pitka¨nen et al. the protracted phase of cone degeneration in retinitis pigmentosa and for

preclinical treatment trials.

G. Dog Models Canine rcd1 model of retinitis pigmentosa is caused by a null mutation in

the PDE6B gene. Treatment of rcd1-affected dogs with d-cis-diltiazem did not modify the photoreceptor disease (39).

Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (red2; collie) in dogs are early-onset forms of progressive retinal atrophy, which serve as models of retinitis pigmentosa in humans (40).

Swedish Briard dogs have a very slowly progressive retinal dystrophy that is inherited in an autosomal recessive manner. The lipid and fatty acid compositions of plasma, retina, and retinal pigment epithelium were ana- lyzed in this model (41). These studies provide evidence for yet another animal model of inherited retinal degeneration with a defect in retinal poly- unsaturated fatty acid metabolism. The fatty acid pattern in affected dogs resembles that in the retina in n-3 fatty acid deficiency.

III. RETINAL DEGENERATION—LIGHT-INDUCED MODELS Retinal damage by light has two distinct action spectra. One peaks in the

ultraviolet A (UVA) and the other in the midvisible wavelength. It was shown in the Long Evans rat that UVA and green light can produce histo- logically dissimilar types of damage. UVA light in particular produces severe retinal damage at low irradiation levels (42).

Albino rats were continuously exposed to blue light for 1–7 days. Continuous exposure of albino rats to moderate blue light for 2–5 days selectively eliminated most of the photoreceptors while leaving the RPE intact (43).

Monocularly aphakic gray squirrels were exposed for 10 minutes to monochromatic near-ultraviolet radiation to determine if their yellow pig- mented lens protected retinal tissue from photochemical damage. In aphakic eyes the retinas revealed irreversible lesions to the photoreceptors. Eyes exposed to ultraviolet radiation with their lenses intact were devoid of sig- nificant retinal lesions. This study represents a model system for studying the potential damaging effects of near-UV radiation to the aphakic eyes of humans (44).

Constant fluorescent light can also be used to generate light-induced degeneration model. Albino rats of the F344 strain were exposed to 1 or 2 weeks of constant light, either with or without intravitreal or subretinal

Retinal Disease Models 523 bFGF solution injected 2 days before the start of light exposure. Constant

light exposure causes a decrease in the thickness of the outer nuclear layer and blocks ERG responses. The results indicated that the photoreceptor rescue activity of bFGF is not restricted to inherited retinal dystrophy in the rat. The light damage is an excellent model for studying the normal function of bFGF and its survival-promoting activity (45). It has been shown that, in the retina, basic fibroblast growth factor delays photorecep- tor degeneration in Royal College of Surgeons rats with inherited retinal dystrophy. bFGF also reduces or prevents the rapid photoreceptor degen- eration produced by constant light in the rat. This light-damage model was used to assess the survival-promoting activity in vivo of a number of growth factors and other molecules. Photoreceptors can be significantly protected from the damaging effects of light by intravitreal injection of eight different growth factors, cytokines, and neurotrophins. They act through several distinct receptor families. In addition to basic fibroblast growth factor, effective photoreceptor rescue was obtained with brain-derived neurotrophic factor, ciliary neurotrophic factor, interleukin 1 beta, and acidic fibroblast growth factor. Less activity was seen with neurotrophin 3, insulin-like growth factor II, and tumor necrosis factor alpha, while nerve growth fac- tor, epidermal growth factor, platelet-derived growth factor, insulin, insulin- like growth factor I, heparin, and laminin did not show any protection (25).

IV. PROLIFERATIVE VITREORETINOPATHY Proliferative vitreoretinopathy (PVR) is found in about 5% of retinal

detachments. The cellular evens of PVR include migration of glial cells, pigment epithelial cells, and fibrocytes into the vitreous cavity, where they proliferate and transform and dedifferentiate. The cells may interact with endogenous membranous components of the vitreous. This leads to the formation of vitreal, epiretinal, and subretinal membranes and traction ret- inal detachment (47). Severe postoperative PVR is the most common cause of failed retinal detachment surgery. Animal models of PVR are based on environmental injuries.

A. Cell Injection Injury can be caused by intraocular injection of fibroblasts into rabbit eye.

This was used as a model to test treatment of PVR by gene therapy. A classification of severity of PVR in this model has been published (48–50).

The extent of PVR by cells that do or do not express the receptors for platelet-derived growth factor (PDGF) was investigated. Mouse embryo

524 Pitka¨nen et al. fibroblasts was derived from PDGF receptor knock-out embryos. They do

not express PDGF receptors and induced PVR poorly when injected into the eyes of rabbits. PDGF made an important contribution to the develop- ment of PVR in this animal model. Furthermore, there was a marked dif-

capable of inducing PVR (51).

B. Dispase PVR can be induced by injecting dispase intravitreally to rabbits (Dutch

belted, New Zealand white). Proliferative vitreoretinopathy developed in response to subretinal or intravitreal dispase, with or without retinal break. Severity of PVR was correlated with increasing doses of dispase. The dispase model of PVR is easy to perform, and it permits a clear view of the retina. This model showed a high success rate in development of PVR (52), and intravitreally administered prinomastat decreased development of PVR in this experimental model (53).

C. Combined Models

A proliferative vitreoretinopathy model was generated in albino rabbits by combing some factors that probably cause the disease. The eyes were injected with platelet-rich plasma, and in addition they underwent cryother- apy or vitrectomy or both procedures. Total retinal detachment and giant holes were obtained more often in experimental eyes than in controls. Microscopic investigation showed intravitreal or preretinal proliferation of fibroblast-like cells (54).

Another combination model involves retinotomy with removal of vitr- eous, cryotherapy, and platelet-rich plasm injection. This is an efficient model of PVR: retinal detachments were produced in 100% of rabbit eyes (55).

In a similar model (56) combined therapy of systemic methylpredni- solone, sodium diclofenac, and colchicine was combined with topical atro- pine, adrenaline, and dexamethasone phosphate. The therapies were useful in treating experimental PVR.

D. Laser Laser-induced retinal injury can be used to provoke PVR formation. For

example, pigmented rabbits underwent argon laser panretinal photocoagu- lation in one eye. Then cultured fibroblasts were implanted into the intact

Retinal Disease Models 525 vitreous of both eyes. More severe PVR developed in the eye with prior

panretinal photocoagulation than in the controls (57).

E. Other Models Platelet-rich plasma causes PVR after injection into the vitreous in rabbit

eyes (59). It contributed more effectively to the development of an experi- mental porcine PVR than PDGF. The efficacy depends on the platelet con- centration of the plasma. It seems that other growth factors and plasma components may interact synergistically with PDGF in the pathogenesis of PVR (58).

V. NEOVASCULARIZATION Neovascularization is involved in various diseases (e.g., cancer, psoriasis),

and the mechanism of angiogenesis has intensely been studied. Neovascularization complicates the treatment of many retinal diseases, and, therefore, appropriate animal models are needed.

A. Laser-Induced Neovascularization Subretinal neovascularization (NV) can be induced by intense laser photo-

coagulation in monkey eyes (60). In pigs, the laser-induced branch of retinal venous obstruction with rose bengal develops neovascularization of the optic nerve head and retina (612). This process was assisted by photody- namic thrombosis. A model of retinal ischemia and associated NV estab- lished by venous thrombosis was produced. After anesthesia, eyes of pigmented rats received an intraperitoneal injection of sodium fluorescein prior to laser treatment. With a blue-green argon laser, selected venous sites next to the optic nerve head were photocoagulated (64).

B. Angiogenic Factor Several ocular NV models are based on exposing the retina to excess of

angiogenic compounds. The effect of increased vascular endothelial growth factor (VEGF) expression in the retina was investigated using transgenic mice in which bovine rhodopsin promoter is coupled with the gene for human VEGF. This study demonstrated that overexpression of VEGF in the retina was sufficient to cause intraretinal and subretinal NV and pro- vided a valuable new animal model (62).

526 Pitka¨nen et al. Controlled-release systems have been developed in order to provide a

long-term supply of angiogenic factors to the retina at defined levels. Ethylene–vinyl acetate copolymer pellets release VEGF slowly into the vitr- eous cavity of rabbits and primates. This induces neovascularization. Sustained intravitreal release of VEGF caused widespread retinal vascular dilation and breakdown of the blood-retinal barrier. Retinal NV seems to require persistent high levels of VEGF at the retinal surface. This can be achieved in rabbits more easily than in primates (63).

In alternative controlled-release models, subretinal implantation of bFGF-impregnated gelatin microspheres is used to induce subretinal neo- vascularization in the rabbit (65).

C. Ischemia Relative hypoxia triggers formation of neovessels in the retina. When one-

week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then to room air, the retinal neovascularization occurs between postnatal days 17 and 21. This model can then be used to study the therapeutic strategies (66). In a similar ischemia-induced ocular neovascularization model, the expres- sion of Flk-1 and neuropilin-1 was restricted on neovascularized vessels, suggesting that these molecules may play important roles in retinal NV (67).

NV studies with ischemic models suggest that PaO 2 fluctuation is more important than extended hyperoxia for retinal neovascular response in rats (68). Indeed, a cycled hypoxia/hyperoxia (10–50% O 2 ) protocol followed by normoxia (20% O 2 ) has been used as a retinal model of retinopathy of prematurity to induce neovascularization in rat pups (69,70). The time course and degree of proliferative vascular response after hyperoxic insult were examined in dogs after oxygen-induced retinopathy. In the neonatal dog, revascularization after hyperoxic insult involves a per- iod of marked vasoproliferation peaking 3–10 days after a return to room air. Oxygen-induced changes in the extravascular milieu probably affect the pattern of reforming vasculature and possibly restrict the growth anteriorly (71).

D. Genetic Models NV of the RPE occurs earlier in a line of P23H mutant rhodopsin transgenic

mice than in most other mice and rats. The temporal course of RPE NV in P23H mice was compared with that of two other retinal degeneration mutants with a similar time course of photoreceptor cell loss. The findings suggest that the P23H mutant rhodopsin transgenic mouse may be a useful model for studying the regulation of NV in the outer retina (72).

Retinal Disease Models 527

E. Other Models NH4Cl gavage in the neonatal rat produced a metabolic acidosis–induced

retinopathy that may be a model for retinopathy of prematurity. Acidosis is induced by high-dose acetazolamide. Independently of hyperoxemia or hypoxemia, the treatment is associated with preretinal neovascularization in the neonatal rat (73).

A consistent model of preretinal NV in the rabbit was developed by partially digesting the posterior virtreous with repeated injection of hyalur- onidase. Then 250,000 homologous dermal fibroblasts were injected intravi- treally (74). Neovascular events observed in this model agree with those previously described for diabetic retinopathy and retinopathy of prematur- ity in humans (75).

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