Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Mutations in LRAT result in early-onset retinal dystrophy in humans (Thompson et al., 2001). The product of LRAT activity is a substrate for RPE65, which produces 11-cis- retinol that is further oxidized to 11-cis-retinal in the RPE (Maeda et al., 2006a). Recently, functional recovery of photoreceptors was observed following 9-cis-retinal treatment in mice lacking the oxidative enzymes required for the final step in the reconversion of the bleached chromophore (Maeda et al., 2006b). These studies suggest that improvement in photoreceptor sensitivity is possible regardless of the primary genetic defect affecting the RPE biochemical synthetic pathway for 11-cis-retinal.

A major function of the RPE cells is to phagocytose photoreceptor outer segments, which are shed daily by the photoreceptors (Young and Bok, 1969). As a result, proteins that function in photoreceptors also affect neighboring RPE cells. ABCA4 is a member of the ATP-binding cassette transporter family and is localized in the outer segments of rod and cone photoreceptors (Cideciyan et al., 2004). Mutations in the ABCA4 gene are responsible for a number of photoreceptor degenerations in humans, such as Stargardt disease, atypical retinitis pigmentosa, and cone-rod dystrophy (Allikmets, 2000). Stargardt disease shares pathological features with AMD and is associated with accumulation of lipofuscin pigments in RPE cells and loss of central vision (Cideciyan et al., 2004). Mice lacking ABCA4, also known as rim protein (RmP), exhibit delayed dark adaptation with relatively normal retinal anatomy and photoreceptor sensitivity (Weng et al., 1999; Radu et al., 2003). Most important, these mice exhibit a dramatic accumulation of N-retinyli- dene-N-retinylethanolamine (A2E), the major fluorophore in lipofuscin that accumulates in RPE cells (Weng et al., 1999). Biophysical studies using recombinant protein and analysis of ABCA4 knockout mice suggest that ABCA4 functions as a flippase for N-retinylidene-phosphatidylethanolamine (N- ret-PE), the Schiff-base conjugate of all-trans-retinal and phosphatidylethanolamine. Hydrolysis of N-ret-PE in the photoreceptor cytoplasm releases all-trans-retinaldehyde, which is reduced to all-trans-retinol, which enters the RPE and is subsequently esterified by LRAT (Weng et al., 1999; McBee et al., 2001; Maeda et al., 2006b). In the absence of flippase activity, the N-ret-PE is thought to accumulate in the inner disc compartment. The models predict that following phagocytosis, outer segments containing high levels of the N-ret-PE adduct enter the phagolysosomal pathway in the RPE, which promotes the formation of A2E in lipofuscin upon light exposure. Accumulation of lipofuscin in the ABCA4 knockout is blocked when mice are reared in total darkness, confirming the light cycle dependency of this pathobiology (Mata et al., 2000).

Mutations in a different photoreceptor-specific protein known as elongation of very long chain fatty acids (ELOVL4) result in Stargardt disease in humans (Zhang et al., 2001).

Knockout of ELOVL4 in mice results in a lethal phenotype, while heterozygous mice exhibit very mild morphological defects in the retina (Raz-Prag et al., 2006). Mice genetically engineered to overexpress the human mutation in ELOVL4 exhibit lipofuscin accumulation in the RPE and progressive photoreceptor degeneration (Karan et al., 2005; Vasireddy et al., 2006). Although more work needs to be done to establish the biochemical activity of this gene, this evidence suggests that factors involved in long chain polyunsaturated fatty acid content in photoreceptors could play a significant role in the propensity for lipofuscin accumulation and retinal degeneration.

Lipofuscin appears in the RPE during senescence as a by-product of the retinoid visual cycle and phagocytosis of the outer segments (Dorey et al., 1989). Studies in the ABCA4 and ELOVL4 genetic models implicate phospholipid biosynthesis and appropriate processing of retinoid cycle by-products as key cellular events that affect accumulation of lipofuscin in the RPE. High levels of lipofuscin and A2E in RPE cells are toxic to cells and may contribute to degenerations observed in retinal disease patients (Sparrow et al., 2000; Suter et al., 2000). Recently, the photooxidation products of A2E have been shown to activate the complement cascade in vitro (Zhou et al., 2006). Strong genetic associations with AMD have been described recently for complement factor alleles in humans (Edwards et al., 2005; Haines et al., 2005; Klein et al., 2005). The light dependency of lipofuscin accumulation, along with other environmental factors, suggests that modulation of the visual cycle and complement cascade are potential therapeutic strategies for macular degeneration.

Recent studies have shown that small molecule antagonists of the visual cycle protect against light-induced degeneration and lipofuscin accumulation in disease models (Maiti et al., 2006). This pharmacological approach recapitulated the phenotype in the RPE65 knockout mice, which exhibited a reduction in lipofuscin during senescence (Katz and Redmond, 2001). Moreover, mice lacking RPE65 are resistant to the light-induced degeneration challenge assay (Grimm et al., 2000). Importantly, inhibiting the visual cycle protects against light-induced degeneration and attenuates lipofuscin accumulation in the ABCR knockout mouse as well (Sieving et al., 2001; Radu et al., 2003, 2005; Maiti et al., 2006).

Most knockouts are maintained on a mixed genetic background between 129/SvJ and C57BL/6J. These two inbred strains are homozygous for RPE65 alleles that render mice either susceptible (Leu450) or resistant (Met450) to lightinduced degeneration, most likely as a result of altered rhodopsin regeneration kinetics (Danciger et al., 2000; Kim et al., 2004). Indeed, the B6;129 F2 hybrid background shows different sensitivity to light damage when the RPE65 alleles are compared (figure 52.3). The ability to combine

different alleles of RPE65 in mice also harboring targeted mutations in other genes can reveal molecular modulators in the retinoid visual cycle. Collectively, these studies highlight the important interactions that occur between photoreceptors and RPE cells, and they identify new strategies to modulate human retinal diseases associated with lipofuscin accumulation and deficiencies in the visual cycle.

The intricate relationship that exists between the RPE and adjacent photoreceptors is critical to maintain normal visual function. The RPE is also important during retinal development (Strauss, 2005). Detailed studies of retinal development in hypopigmentation mutants have demonstrated that RGCs that normally project to ipsilateral central visual targets instead misroute at the optic chiasm and cross the midline, inappropriately reaching the contralateral central nuclei (LaVail et al., 1978; Dräger and Olsen, 1980; Jeffery, 1997). There is a delay in retinal development and rod number is decreased in albinos lacking functional tyrosinase, the key enzyme in the synthesis of melanin (Ilia and Jeffery, 1999). These studies implied that the enzymatic product of tyrosinase, which could be melanin, controls important aspects of retinal development.

Enzyme replacement therapy or product supplementation, as demonstrated in the retinoid cycle, is a viable

strategy and mouse models have provided examples of proof of concept–type experiments. For example, during melanin synthesis, tyrosinase oxidizes L-tyrosine to L-dopaquinone, which is further converted to melanin. One product of this enzymatic reaction is L-3,4-dihydroxyphenylalanine (L- dopa). Transgenic mice that express tyrosine hydroxylase in the RPE rescue the retinal abnormalities in albino mice (Lavado et al., 2006). This result was attributed to direct action of L-dopa during retinal development as the transgene was expressed in mice lacking functional tyrosinase and, therefore, could not convert L-dopa to melanin. This clever genetic experiment demonstrates that either L-dopa or a metabolite is critical for normal retinal development and that supplementation could rescue defects that arise due to a deficiency in tyrosinase (Ilia and Jeffery, 1999).

Vascular cell types

Cells in the inner retina are nourished by three vascular beds that develop during the first 2 postnatal weeks in mice. Cellular regulation of vascular retinal development in mice remains one of the most intensely studied biological questions, owing to the accessibility of the developing vasculature in the postnatal eye. All aspects of angiogenesis occur in this organ, and the increasing availability of mouse genetic

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Figure 52.3 Light-induced degeneration in 129;B6 mice that are homozygous for either the Leu450 allele or Met450 allele of the RPE65 gene. Each dot represents the mean thickness of the ONL/ INL from measurements obtained 400 μm on either side of the optic nerve head. A, Mice exposed to room light (40 lux) exhibit an ONL : INL ratio greater than 1.0 (n = 8). B, Mice homozygous

for the Leu450 allele in the RPE65 gene are sensitive to 5 hours of light damage (5,000 lux) as shown in this histological section (n = 6). Sections were generated 7 days post light exposure. C, Mice (n = 7) homozygous for the Met450 allele in the RPE65 gene are resistant to light damage.

models and the ability to apply agents directly to the postnatal eye have enabled detailed studies of cell types and mechanisms involved in physiological angiogenesis. Pathological angiogenesis contributes to diseases in humans such as cancer and is a serious threat to vision in “wet” AMD and diabetic retinopathy (Folkman, 1995). Therefore, studies of the mouse intraocular vasculature have provided some of the most exciting insights into cellular regulation of blood vessel development, maintenance, and pathobiology.

The process of vascularization in the mouse eye is well described, and several excellent reviews are available (Fruttiger, 2002; Dorrell and Friedlander, 2006). Before birth, the developing neural retina is avascular, although the hyloid vasculature nourishes the developing lens and surrounding tissues. As neuronal differentiation proceeds, increasing metabolic activity of the inner retina results in physiological hypoxia that is thought to initiate formation of the retinal vasculature (Zhang et al., 1999, 2003). The first vascular bed arises during postnatal days P1–P10 and is known as the superficial vascular plexus, which supports ganglion cells (Connolly et al., 1988). During the second postnatal week, the superficial vessels branch to descend toward the outer retina, where they elaborate into a second vascular plexus, which nourishes cells located in the distal INL. During the second and third postnatal weeks, a third vascular plexus forms through angiogenic sprouting at the level of the innermost region of the INL (figure 52.4).

The orderly appearance of retinal vasculature during the first 3 postnatal weeks is orchestrated by specific cell types and guidance cues. Evidence obtained in a number of species, including mice, indicates that the initial vascular plexus arises through the directed migration of endothelial cells along a preassembled grid of astrocytes (Dorrell and Friedlander, 2006). These astrocytes originate from the optic stalk and migrate radially prior to the arrival of endothelial cells at the optic nerve head. Genetic models have been instrumental in elucidating molecular details of these complex cellular interactions. For example, mice lacking platelet-derived growth factor-A (PDGF-A) exhibit a decrease in the complexity of the astrocytic grid, and consequently the coverage of the superficial vascular plexus is also decreased (Gerhardt et al., 2003). PDGF-A is expressed by RGCs and interacts with its receptor, PDGFR-α, present on astrocytes (Mudhar et al., 1993). Transgenic mice that overexpress PDGF under the control of the neuron-specific enolase promoter exhibit overgrowth of retinal vasculature due to increased density of astrocytes. Conversely, blocking PDGF-A function attenuates retinal vascular development by perturbing the complexity of the astrocyte network (Fruttiger et al., 1996). The cell adhesion molecule R- cadherin also appears to be important for the astrocytemediated guidance of the superficial vascular plexus (Dorrell

et al., 2002). Mice deficient in the nuclear hormone receptor Tlx exhibit impaired astrocyte development and loss of R- cadherin expression, with concomitant disruption in vascular development, further confirming the intimate association between astrocytes and the developing capillaries (Miyawaki et al., 2004). Astrocytes continue their important role in vascular biology in adult retina by enabling the formation of the blood-retina barrier (Janzer and Raff, 1987).

The relative hypoxia of the newborn retina is believed to stimulate expression of various pro-angiogenic factors that drive endothelial cell proliferation and migration (Stone et al., 1995; Gerhardt and Betsholtz, 2005; Dorrell and Friedlander, 2006). VEGF-A is among one of the best characterized of these pro-angiogenic factors, and alternative splicing of Vegf mRNA generates multiple isoforms that have different diffusion capabilities within the extracellular matrix (Ferrara et al., 2003). VEGF is produced by astrocytes prior to the arrival of endothelial cells (Stone et al., 1995; Gariano, 2003; Gerhardt et al., 2003). At the leading edge of the developing capillaries, specialized endothelial cells termed tip cells extend and retract actin-rich filopodia along the glial cell network and the surrounding matrix (Dorrell et al., 2002; Gerhardt et al., 2003). These endothelial tip cell pioneers are followed closely by endothelial cells in the stalk, which are proliferating to form a patent capillary network superimposed on the astrocytic grid (Gariano, 2003; Gerhardt and Betsholtz, 2005). Studies in mice designed to express individual isoforms of VEGF-A demonstrate that tip cell migration and normal vascular patterning in the developing retina and brain are dependent on the establishment of tightly regulated VEGF-A gradients (Ruhrberg et al., 2002; Stalmans et al., 2002; Gerhardt et al., 2003). Moreover, VEGF-A signaling through VEGF receptor 2 (VEGFR2) affects simultaneously tip cell migration and stalk cell proliferation, and the cellular mechanism for this effect remains an area of intense research (Gerhardt et al., 2003; Gerhardt and Betsholtz, 2005). VEGFR2 protein is highly expressed in tip cells, and antagonism of VEGFR2 signaling retracts tip cell filopodia, providing further support that VEGF-A is involved in the guided migration of tip cells.

Genetic knockout studies targeting a single allele of VEGF-A in mice result in lethality between E11 and E12. The heterozygous embryos displayed defective vascularization in several organs, confirming the essential role of VEGF in blood vessel formation (Carmeliet et al., 1996; Ferrara et al., 1996). Moreover, knockout of the VEGFR2 results in embryonic lethality around day 9 and is characterized by failed angiogenesis and hematopoiesis, along with disorganized blood vessels (Shalaby et al., 1995).

These studies established a clear role for VEGF-A signaling in physiological angiogenesis. The clinical utility of VEGF in pathological angiogenesis is evidenced as VEGF-

Figure 52.4 The retinal vasculature in mice contains three vessel beds. A, Whole-mount retinal preparation from a wild-type mouse stained with isolectin B4 conjugated to fluorescein. The retinal vasculature covers the entire retinal surface. B–D, Images taken at different depths through the retinal vasculature, starting at the most superficial layer, the nerve fiber layer (NFL [B]), which provides capillaries to the inner plexiform layer (IPL [C]), which are continuous with those capillaries in the outer plexiform layer (OPL [D]). E, Retinal whole mount obtained from a P6 mouse and stained

blocking antibodies are now approved for wet AMD and colorectal cancer. Of note, the phenotypes observed in the heterozygous knockout of VEGF predicted the clinical efficacy of VEGF inhibition and provide a benchmark of the embryonic lethal phenotypes in mouse knockouts as a reliable indicator for potentially very potent modulators of vascular development and homeostasis.

The endothelial network, initially sculpted by astrocytes, is further remodeled into the mature vasculature that exhibits a stereotypical pattern. This process involves vascular

with isolectin B4. The optic nerve head is at the lower left of the image, and the retinal periphery is in the upper right. The vasculature is developing toward the retinal periphery through the movement of the migration front (arrows). F, A higher magnification image of the migration front shows pioneer endothelial cells, termed tip cells, that direct the migration of the vasculature by extending actinrich filipodia. Endothelial cells in the stalk form the nascent capillary bed.

pruning to establish a mature vessel density concurrent with the establishment of hemodynamic flow and increased oxygen levels in perfused tissue (Alon et al., 1995; Benjamin et al., 1998). Stabilization into a functional vessel requires intimate association with mural cells, such as pericytes and smooth muscle cells (Fischer et al., 2006). The dynamic interactions that occur between pericytes and endothelial cells are little understood at the molecular level, and studies using mouse genetic models are making inroads into this complex interaction among these cell types ( Jain, 2003;

Armulik et al., 2005). The clinical significance of this question is great, as loss of pericytes and the appearance of microaneurysms are early hallmarks of retinopathy in diabetic patients (Speiser et al., 1968). Continued vessel occlusion results in increased vascular permeability, edema, and the formation of new vessels that proliferate into the vitreous.

Pericyte recruitment lags behind vessel formation (Benjamin et al., 1998). Pericyte coverage progresses from arterioles to venules, and the process does not reach completion before 3 weeks from birth. Remodeling of retinal vessels is complete before the establishment of pericyte coverage, indicating that mural cell investment marks the end of vascular remodeling capacity. Several gene knockout studies have shown that mural cells affect blood vessel branching, remodeling, and stabilization. For example, mice with a targeted deletion in either angiopoietin-1 or its receptor Tie2 exhibit defects in angiogenesis and vessel remodeling and maturation (Sato et al., 1995; Suri et al., 1996). Loss of pericytes and smooth muscle cells is evident in these animals. Most of the evidence suggests that these defects arise via an intrinsic block in signaling within endothelial cells, which specifically express Tie2, although alternative models have been suggested (Armulik et al., 2005). Endothelial tip cells secrete platelet-derived growth factor-B (PDGF-B), presumably in response to VEGF signaling (Lindahl et al., 1997; Dorrell and Friedlander, 2006). Mice deficient in PDGF-B are embryonic lethal and exhibit attenuation in pericyte proliferation and decreased vessel coverage. Vascular hemorrhaging, presumably as a result of vessels that are not fully invested by pericytes, is a common pathology observed in these mice. Pericyte loss, microvascular aneurysms, and lethality occur in mice lacking the PDGF-B receptor, which is expressed on mural cells (Benjamin et al., 1998; Hellstrom et al., 2001). In the absence of appropriate pericyte coverage, endothelial cells proliferate and become permeable, leading to impaired perfusion and hypoxia. These highlighted examples, and many others identified through mouse genetic studies, demonstrate a reciprocal cell-cell communication in which endothelial cells promote mural cell proliferation and migration and mural cells help support and stabilize endothelial cells (Hellstrom et al., 1999; Armulik et al., 2005).

Retinopathy of prematurity in premature infants is a pathogenesis of retinal blood vessels that arises as a result of high oxygen exposure to compensate for the underdeveloped lungs. Exposure of developing retinal vasculature to high oxygen decreases VEGF expression, leading to dropout of newly formed capillaries (Alon et al., 1995; Benjamin et al., 1998). Upon return to room air (normoxic conditions), VEGF is upregulated and neovascularization occurs. Pericyte investment is thought to be important for the ability of endothelial cells to respond to increasing oxygen tension.

Fully invested capillaries, as is the case in mature vasculature, are largely refractory to the oxygen-induced vasobliteration (Alon et al., 1995; Benjamin et al., 1998). Novel strategies aimed at modulating cellular interactions between pericytes and endothelial cells are likely to emerge, given their important role in establishing and maintaining vascular integrity.

Additional guidance cues have been discovered through knockout mice that act to repel and keep tip cell endothelial pioneers on track toward formation of the complex vascular network. Using a knock-in reporter gene to monitor Unc5B expression, Lu and colleagues demonstrated high levels of Unc5B in endothelial tip cells in the retina and arteriole endothelial cells in more mature vessels (Lu et al., 2004). Unc5 proteins are membrane-bound receptors for axon guidance molecules known as netrins. Depending on the genetic background, homozygous Unc5B deficient mice were lethal by E12.5, exhibiting severe vascular phenotypes. Exuberant branching of vessels was observed in several organs, and endothelial tip cells demonstrated increased filopodial extensions in mice lacking Unc5B.

Vascular patterning arises through cellular interactions with both attractive and repulsive cues, which is reminiscent of guidance mechanisms that control the trajectories of neuronal projections during CNS development. Additional molecules involved in axon guidance have been shown to be involved in vascular patterning through gene knockout studies in mice. This list includes plexins, semaphorins, neuropilins, ephrins, and their receptors (Carmeliet and Tessier-Lavigne, 2005; Eichmann et al., 2005). The discovery of tip cells in endothelium that closely resemble growth cones of developing axons suggests that mechanisms acting during axonal guidance may directly apply to those controlling blood vessel patterning. Conversely, manipulations using cellular or molecular strategies in the relatively simple architecture of mouse retinal vasculature may yield insights into mechanisms that could be applied to promote reestablishment of neuronal connections in neurodegenerative conditions affecting the eye, such as glaucoma.

The availability of large numbers of genetic knockouts, combined with reliable markers to identify individual cell types, will continue to illustrate the power of mouse models to reveal additional cell types and molecules that affect both physiological and pathological angiogenesis (Jain, 2003). Many gene deletions in mice result in prenatal lethality or reduced viability as a consequence of blood vessel defects. In viable animals, genes affecting blood vessel formation and vascular integrity can be identified using noninvasive imaging techniques, such as angiography. For example, disruption of the endothelial cell–specific protein EGFL7 results in abnormal ocular blood vessel anatomy in some adult mice (figure 52.5A). EGFL7 is a secreted protein

Figure 52.5 EGFL7 affects vascular anatomy. A, Angiography of wild-type mice (control) and mice lacking the secreted protein EGFL7 (−/−). In control eyes, blood vessels project directly toward the retinal periphery, while branches dive down toward the deeper levels of the retina to form the capillary beds. In the EGFL7−/− retina, vessels often meander on their way toward the retinal periphery. B–E, In situ hybridization of Egfl7 in the postnatal mouse eye. B, At P3, Egfl7 expression in endothelial cells is present in the migrating front of the developing capillaries (arrow). Hyloid

that is highly expressed during embryonic and neonatal development (figure 52.5B–E). Expression levels decrease in mature vasculature, but Egfl7 expression is upregulated in vascular injury and in tumor models (Parker et al., 2004; Campagnolo et al., 2005). The abnormal vasculature ob-

vessels and the choriocapillaris surround the eye, and iris vessels also express Egfl7. C, No signal in the P3 eye is observed when an Egfl7 sense probe is used in the hybridization. D, At P6, the developing vasculature approaches the retinal periphery and Eglf7 is present in these developing endothelial cells (arrows). E, Higher magnification of the P6 retina shown in D. Egfl7 expression is present on the nerve fiber layer and the choriocapillaris (arrows), consistent with a developing retinal vasculature in the mouse eye.

served in the eye of adult animals is due to a developmental defect in endothelial cell migration. Therefore, genetic screens in mice using embryonic lethality or noninvasive imaging in viable mice will lead to the identification of new targets for pathological angiogenesis.

Cell types in the anterior chamber

Glaucoma is a disease that affects RGCs, amacrine cells, and the optic nerve. This sight-threatening condition is often associated with increased ocular pressure, a major risk factor for glaucomatous disease. Regulation of intraocular pressure (IOP) is a balance between the production of aqueous humor at the ciliary body and drainage of aqueous humor at the iridocorneal angle (Civan and Macknight, 2004). Aqueous humor is produced by cells in the ciliary body that are nourished by a rich vascular supply. The humor travels through the pupil into the anterior chamber, where it encounters a series of cell types derived from the neural crest and mesoderm (Gould et al., 2004). These cells collectively form the drainage structures known as the conventional and uveoscleral (sometimes referred to as the nonconventional) outflow pathways. Traditionally, the conventional outflow pathway is thought to be dependent on pressure and involves cell types present in the trabecular meshwork, Schlemm’s canal, and episcleral veins (Crowston and Weinreb, 2005). Aqueous humor is also drained through the uveoscleral pathway, which is a route through extracellular spaces in the iris, ciliary muscle, choroid, and sclera, where it ultimately collects in the lymphatics. The facility of the uveoscleral outflow remains an area of intense investigation (Crowston and Weinreb, 2005).

Although cell types involved in fluid dynamics have been well described, the molecular mechanisms involved in this process are only now being discovered. This exciting area of research is made possible by the development of several methods to measure IOP, determine the aqueous humor flow and episcleral venous pressure, and calculate total outflow facility in mice (John et al., 1998; Aihara et al., 2003a, 2003b; Crowston et al., 2004a). Mice have IOPs that are consistent with those in normotensive humans (Savinova et al., 2001). The dynamics of humor production and turnover are also comparable to those in humans (Aihara et al., 2003a), and mice respond to pharmacological agents that affect human aqueous humor production and outflow facility (Avila et al., 2001a, 2002; Aihara et al., 2002; Husain et al., 2006). These characteristics are leading to great interest in using mouse models to study processes associated with regulation of IOP and glaucoma.

Prostaglandin analogues are among the most widely prescribed drugs to manage ocular hypertension and glaucoma in the clinic. The mechanism by which these analogues lower IOP is still under investigation, but evidence suggests that IOP reductions occur by increasing outflow facility (Weinreb et al., 2002; Crowston and Weinreb, 2005). Prostaglandin analogues have been tested in a number of species, and a single topical application in mice lowers IOP within 6 hours (Aihara et al., 2002; Ota, Murata, et al., 2005; Husain et al., 2006). The intraocular metabolites of these

analogues bind with high affinity to the FP receptor expressed primarily in cell types associated with aqueous production and flow (Anthony et al., 2001; Weinreb et al., 2002). Mice deficient in several of the prostanoid receptors have been studied to clarify the role of individual receptors involved in IOP effects of these hypotensive agents (Crowston et al., 2004b, 2005; Ota, Aihara, et al., 2005; Ota et al., 2006a, 2006b). A single application of latanoprost, travaprost, bimatoprost, or unoprostone to wild-type mice or mice deficient in the EP1 or EP2 receptors was effective at lowering IOP, indicating that EP receptors are not involved in the pharmacological actions of these analogues. In contrast, when these agents were applied to mice lacking the FP receptor, their ocular hypotensive action was no longer observed. These results clearly demonstrate that the FP receptor is required during the initial IOP-lowering effects of these clinical agents.

In the previous example, genetic knockouts provided the ability to determine the molecular target of clinically important compounds. Application of pharmacological agents to mice that lack specific genes also enables the direct test of onversus off-target side effects (Zambrowicz et al., 2003). However, the phenotype of the knockout is also capable of predicting the potential utility of modulators for clinical applications. For example, several secreted and membrane proteins, such as type 1 collagen, aquaporin 1 and aquaporin 4 (AQP1 and AQP4, respectively), and the adenosine receptor 3 (AR3) have been shown to affect IOP in knockout mice. Mice deficient in type 1 collagen exhibited elevated IOP, while knockout of AQP1, AQP4, or AR3 resulted in lower IOP (Avila et al., 2001b; Zhang et al., 2002; Aihara et al., 2003c). The few studies that have focused on cellular mechanisms that control aqueous humor facility have concluded that mice appear to be excellent models in which to study the dynamics of pressure regulation (Lindsey and Weinreb, 2005). Combined with the increasing number of mouse genetic knockouts and new techniques amenable to the small mouse eye, the future looks very bright for new therapies aimed at specific cell types in the outflow pathway.

Ocular cell types are derived from the neural ectoderm, surface ectoderm, and mesenchymal and neural crest. Mouse genetics have played a leading role in elucidating cell migrations and communications that form and maintain the complexity and function of this organ. Therapeutic strategies under investigation or in clinical practice have established proof of concept in genetic models, and the predictive efficacy of the therapeutic was revealed through studies of gene knockouts. Mouse knockouts in challenge assays for pathological stresses, such as ischemia, ocular inflammation, and dry eye, can reveal individual genes with important functions in disease mechanisms (Da and Verkman, 2004; Pflugfelder et al., 2005; Liao et al., 2006). Mouse genetics

will continue to be the core technology to study cell-cell interactions and physiological responses in mammalian biology and contribute to the next generation of therapeutics aimed at treating ocular disease.

acknowledgments The author thanks Kim Paes (Lexicon), Claire Gelfman (Lexicon), Weilan Ye (Genentech), and Maike Schmidt (Genentech) for scientific contributions to this chapter and Alex Turner for critical review of the manuscript.

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