THE RETINAL PIGMENT EPITHELIUM
Morten la Cour and Tongalp Tezel
I. II. III. IV. V. VI. VII.
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 RPE Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Photoreceptor Outer Segment Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Retinoid Metabolism and the Visual Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Production of Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 The RPE in Wound Healing and Proliferative Vitreoretinal Disease . . . . . . 264 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
ABSTRACT The retinal pigment epithelium (RPE) is a monolayer of cuboidal epithelial cells intercalated between the photoreceptors and the choriocapillaries. The human RPE incorporates some 3.5 million epithelial cells arranged in a regular hexagonal pattern. The density of RPE cells is relatively uniform throughout the retina, approximately 4000 cells/mm2. With age, the cell density decreases
Advances in Organ Biology Volume 10, pages 253–272. © 2006 Elsevier Inc. All rights reserved. ISBN: 0-444-50925-9 DOI: 10.1016/S1569-2590(05)10009-3
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particularly in the periphery, where it is reduced to approximately 2000 cells/ mm2 in individuals over 40 years. The peripheral RPE cells are larger and more pleomorphic than central cells (Harman et al., 1997; del Priore et al., 2002). In the primate retina, each RPE cell faces 30–40 photoreceptors, a number that is rather constant throughout the retina, although perhaps somewhat lower in the fovea (Robinson and Hendrickson, 1995). In fully developed primate retinas, no mitoses are seen in the RPE, and the epithelium is currently believed to consist of a stable, nondividing, pool of cells (Tso and Friedman, 1967). The retinal membrane of the RPE faces the subretinal space, which is the extracellular space surrounding the photoreceptor outer segments (Figure 1). Between the optic disc and the ora serrata, there are no anatomical contacts between the photoreceptors and the RPE. The RPE forms numerous long microvilli that interdigitate with the rod outer segments. In mammals, the cone outer segments are ensheathed by multilamellar specializations of the RPE, the so‐called cone sheaths. The epithelial cells are bound together by junctional complexes with tight junctions that separate the cells into an apical half that faces the retina and a basal half that faces the choroid. The nucleus and mitochondriae are located in the basal half of the cell. Numerous pigment granules, located predominantly in the apical cytoplasm, give the epithelium its macroscopic black appearance, from which it derives its name. The choroidal side of the RPE directly apposes Bruch’s membrane, a pentalaminar, approximately 2 mm thick, elastic membrane. The innermost part of Bruch’s membrane is the basement membrane of the RPE. The outer part of Bruch’s membrane is the basement membrane of the choriocapillaries. In between are two collagenous layers and a central elastic layer.
I. RPE FUNCTION The retinal pigment epithelial cells act as supportive cells for the photoreceptors. The best studied of these functions are the participation of the RPE in photoreceptor outer segment renewal, the storage and metabolism of vitamin A, the production of cytokines necessary for retinal development and survival, and the transport and barrier functions of the epithelium. These four aspects of RPE physiology will be discussed in separate sections later. Other, less well‐characterized functions of the RPE are the absorption of stray light by the melanin pigment in the epithelium, the scavenging of free radicals by the melanin pigment, and the drug detoxification by the smooth endoplasmic reticulum cytochrome p‐450 system (Shichi and Nebert, 1980).
The Retinal Pigment Epithelium
Figure 1. Diagram of the subretinal space, showing the relationship between the retinal pigment epithelium (RPE), the outer segment of the photoreceptors, the outer limiting membrane (OLM), and the Mu¨ller cells. The asterisk denotes the subretinal space. (From Steinberg R.H., Linsenmeier R.A. and Griff E.R. (1983). Vision Res. 23, 1315.)
PHOTORECEPTOR OUTER SEGMENT TURNOVER
It has been known for more than 20 years that the outer segments of both rod and cone photoreceptors undergo continuous renewal. New membrane material is added at the base of the outer segments, and old membrane material is removed from the tip of the outer segments by RPE phagocytosis (Besharse and Defoe, 1998). In the Rhesus monkey, the renewal time for rod outer segments is 13 days in the parafoveal region and 9 days in the
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peripheral retina. These estimates are based on the classical experiments by Young, where he used pulse labeling of rod outer segment discs with radioactive amino acids and subsequent autoradiography (Young, 1971). In cones, a similar outer segment renewal process takes place. However, because of the continuity of the disc membranes with the outer segment cell membrane, the rate of cone outer segment membrane renewal cannot be assessed by pulse labeling. Count of phagosomes, containing outer segment material, within the RPE cells suggests that the rate of outer segment renewal is slower in cones than in rods (Anderson et al., 1980). The outer segment discs are shed in a circadian rhythm. In rods there is a burst of disc shedding and phagocytosis in the morning, immediately after light onset. In cones, there seem to be some species variability in the pattern of disc shedding (Besharse and Defoe, 1998). In rhesus monkeys there is a burst of disc shedding associated with light onset, although discs are also shed during the dark period in this species (Anderson et al., 1980). Retinal pigment epithelium phagocytosis is important for photoreceptor survival. This was first discovered in RCS rats, where a retinal dystrophy is caused by defective RPE phagocytosis of outer segment material (Chaitin and Hall, 1983). More recently, this dystrophy was linked to the MERKT gene (D’Cruz et al., 2000). Transfer of the MERKT gene has been shown to rescue photoreceptors in RCS rats (Vollrath et al., 2001). The MERKT gene product is a receptor tyrosinase kinase, necessary for normal RPE phagocytosis (Feng et al., 2002). MERKT gene defects have subsequently been shown to cause retinitis pigmentosa in humans, and a murine retinal dystrophy, similar to the one seen in RCS rats (Gal et al., 2000; Duncan et al., 2003). In Usher’s syndrome type 1b, a major deaf–blind disorder in humans, RPE phagocytosis is affected; with the causative defect in the myosine 7A gene (Gibbs et al., 2003). The amount of membrane material ingested and degraded by the RPE cells is impressive. Based on a quantitative study in the Rhesus monkey (Young, 1971), it can be calculated that each extrafoveal RPE cell, every day, must ingest and degrade a volume of rod outer segment material that corresponds to 7% of the volume of the RPE cell itself. Since the RPE cells do normally not divide, the burden of membrane material that these cells must ingest and degrade in their lifetime, far exceeds that of any other phagocytic cell. Probably as a consequence of this massive phagocytic load on the RPE cells, lipofuscin granules accumulate with age in these cells, and makes them autofluorescent (Nilsson et al., 2003). Exocytosis through the RPE choroidal cell membrane of lipofuscin and other waste products from phagocytosis may lead to the accumulation of hydrophobic material in Bruch’s membrane, and reduced water permeability of this membrane (Marshall et al., 1998). The major fluorophore in RPE lipofuscin is A2E, which is a product of retinal and ethanolamine (Sakai et al., 1996). Early
The Retinal Pigment Epithelium
accumulation of lipofuscin is seen in Stargardt’s disease, which is a macular dystrophy caused by a defective ABCR gene (Allikmets, 1997). The ABCR gene product seems to be involved in the transfer of all‐trans‐retinal from the disc lumen and into the cytosol of outer segments in rods (Weng et al., 1999). Accumulation of A2E and other indigestible retinoid metabolites in the rods might be cause of the excess RPE lipofuscin accumulation in Stargardt’s disease and possibly in age‐related macular degeneration as well (Mata et al., 2001).
RETINOID METABOLISM AND THE VISUAL CYCLE
The retinal pigment epithelium plays an important role in the uptake, storage and metabolism of vitamin A and related compounds. It has been known for more than 100 years that photoreception involves bleaching of the visual pigments, and that the RPE is required for the regeneration of these pigments, at least in rods (Marmor and Martin, 1978). The underlying mechanisms are now known in some detail (McBee et al., 2001). The retinoid 11‐cis‐retinaldehyde is the chromophore of the visual pigments in mammals. When light is absorbed by the visual pigment, the phototransduction cascade is initiated, and the visual pigment bleaches. During the bleaching process, the chromophore is released from the visual pigment and converted within the photoreceptor to all‐trans‐retinol, which is then released from the outer segment. Some of this all‐trans‐retinol finds its way to the retinal pigment epithelium, where it is reisomerized to 11‐cis‐retinol, oxidized to 11‐cis‐retinaldehyde, and subsequently transported back to the photoreceptor outer segments. The light induced movement of retinoids between the photoreceptors and the retinal pigment epithelium, and the involved transformations between the different retinoids, is denoted the visual cycle (Thompson and Gal, 2003). Retinoids enter the RPE cell via three different mechanisms: Firstly, all‐ trans‐retinol is taken up from the blood through the choroidal membrane of the RPE cells. All‐trans‐retinol circulates in the blood bound to a small, 21 kD, carrier protein, serum retinol binding protein, RBP, which is complexed to another and larger, protein, transthyretin. Autoradiographic studies have demonstrated specific membrane binding sites for RBP on the RPR choroidal membrane (Thompson and Gal, 2003, 1976). However, the receptor has not been identified with certainty, and the retinol‐uptake mechanism in the RPE choroidal membrane remains to be elucidated (Chader et al., 1998). Secondly, retinoid bound to the visual pigments enter the RPE cells via the phagocytosis of outer segment material (see previous section). Finally, retinoid is shuttled back and forth between the RPE and the outer segments as a part of the visual cycle. The all‐trans‐retinol that is liberated from the outer
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segments is shuttled to the RPE through the subretinal space. The interphotoreceptor matrix retinoid‐binding protein, IRBP, has been implicated as a carrier protein in retinoid transport through the subretinal space, since this protein is the most abundant protein in the subretinal space, and capable of binding both retinol and retinal in their all‐trans‐ as well as their 11‐cis configurations (Chader et al., 1998). However, the role of IRBP as the obligate carrier of retinoids in the subretinal space has been recently been challenged. In mice with a targeted disruption of the IRBP gene (IRBP‐/‐ gene knockout mice), it was found that the kinetics of 11‐cis‐retinal and rhodopsin recovery after a flash of light was only marginally slower in IRBP‐/‐ mice than in normal, wild‐type, mice (Palczewski et al., 1999). IRBP may play a role in retinoid transport between Mu¨ ller cells and cones, and it may play a role in retinal development (Gonzalez‐Fernandez, 2003). The fate of retinol inside the RPE cell has recently been reviewed (Thompson and Gal, 2003), and is illustrated in Figure 2. Once inside the RPE cell, the retinol is bound to a small, 16 kDa, carrier protein, cellular retinol‐binding protein (CRBP), which is a relative ubiquitous protein, not specific for the RPE. Retinol might then be esterified to a palmitate group derived from the membrane phospholipids lecithin. This reaction is catalyzed by lecithin‐retinol acyltransferase (LRAT). The retinyl ester is a stable, nontoxic form of the retinoid, and both the all‐trans‐ and the 11‐cis‐isomer of retinol is stored as retinyl esters within the RPE cell. The formation of retinyl esters is an important step in the synthesis of the 11‐cis‐isomer, since all‐trans‐retinyl ester is the substrate for the isomerohydrolase enzyme that catalyzes the combined hydrolysis of the ester bond and isomerization of the all‐trans‐retinol to 11‐cis‐retinol (Bernstein et al., 1987). Isomerization of all‐ trans‐retinol to the 11‐cis‐isomer is an endothermic reaction, and the energy required is probably derived from the energy‐rich ester bond in the retinylester (Dreigner et al., 1989). The isomerohydrolase enzyme has not been purified or cloned. Another RPE protein, RPE65, is necessary for the isomerization. RPE65 is distinct from the isomerohydrolase, although it is probably associated with it (Thompson and Gal, 2003). The 11‐cis‐retinol that is the product of the isomerization is bound to cellular retinaldehyde binding protein, CRALBP. This is a 36 kDa protein localized mainly in RPE cells and Mu¨ ller cells. It binds retinol and retinal, but is specific to the 11‐cis isomer. CRALBP might facilitate the isomerization reaction by trapping the 11‐cis product. The 11‐cis‐retinol can be reesterified by LRAT and stored in the RPE cell as 11‐cis‐retinylester, or it can be oxidized to 11‐cis‐retinal by 11‐cis‐retinol dehydrogenase (11‐cis‐RDH). This reaction uses NADþ as cofactor. While bound to CRALBP, 11‐cis‐retinal travels to the retinal membrane of the RPE cell where it is released. The mechanism for this is not clear.
The Retinal Pigment Epithelium
Figure 2. Diagram of retinoid trafficking in and around the retinal pigment epithelium (RPE) cell. ROS¼ rod outer segment; 11cis‐ROL ¼ 11–cis‐retinol; 11cis‐RAL ¼ 11‐cis‐retinal; at‐ROL ¼ all‐trans‐retinol; at‐RAL ¼ all‐trans‐retinal; RGR ¼ retinal G‐protein coupled receptor; RBP ¼ serum retinol binding protein, CRBP ¼ cellular retinol binding protein, CRALBP ¼ cellular retinal binding protein; RDH ¼ all‐trans‐ retinol dehydrogenase; 11‐cis‐RDH ¼ retinol dehydrogenase specific for 11‐cis‐ retinol; IRBP ¼ interphotoreceptor matrix retinoid binding protein.
An alternative pathway for the isomerization of all‐trans‐retinol to 11‐cis‐ retinol within the RPE cell is light dependent and resembles the mechanism used in eyes of cephalopods (e.g., octopus and squid). In this pathway the substrate for the isomerization reaction is all‐trans‐retinal. All‐trans‐retinol must therefore be oxidized to all‐trans‐retinal. This is accomplished by an all‐trans‐retinol dehydrogenase (all‐trans‐RDH). The isomerization is catalyzed by the enzyme retinal G‐protein coupled receptor (RGR), which binds all‐trans‐retinal covalently with a Schiff base linkage resembling the attachment in rhodopsin. Illumination of the RGR‐all‐trans‐retinal compound results in the formation of 11‐cis‐retinal. The RGR protein corresponds to
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retinochrome found in the myeloid bodies of cephalopod retinula cells (Thompson and Gal, 2003). Proteins involved in retinoid metabolism seem to be important for photoreceptor survival. Table 1 shows gene products, expressed in the RPE, in which genetic defects have been shown to cause retinal disease.
PRODUCTION OF CYTOKINES
Both photoreceptors and the choriocapillaries depend on the RPE for their survival. If the RPE is destroyed by chemical or mechanical means, the photoreceptors and the choriocapillaries atrophy (Peyman and Bok, 1972). As will be discussed further below, the RPE is necessary for the normal development of both the neuroretina and choroid (Raymond and Jackson, 1995; Zhao and Overbeek, 2001). The RPE cell has been shown to produce many growth factors, and the roles of some of these have begun to be elucidated. Vascular endothelial growth factor, VEGF or VEGF‐A, is a potent angiogenic factor, which is secreted by RPE cells in a polarized fashion, with 2–7 fold more being secreted towards the choroidal side of the epithelium (Blaauwgeers et al., 1999). The polarized secretion targets the vascular endothelium of the choriocapillaries, which expresses two different VEGF receptors, VEGFR‐1 and VEGFR‐2, preferentially towards its RPE facing side. During embryogenesis, a peak of VEGF production by the RPE is necessary for the development of the fetal choroidal vasculature (Witmer et al., 2003). VEGF also increases the permeability of vessel walls, and may contribute to the maintenance of the fenestrated phenotype of the choriocapillaries (Blaauwgeers et al., 1999). Ischemia, mechanical distortion, advanced glycation end products all upregulate VEGF production by RPE cells, and may result in
Table 1. Protein RPE65 LRAT RGR CRALBP 11‐cis‐RDH IRBP
RPE Gene Defects and Retinal Disease Disease Leber congenital amaurosis Leber congenital amaurosis Retinitis pigmentosa Recessive retinitis pigmentosa Fundus albipunctatus Retinal dystrophy in mice
Proteins involved in retinoid metabolism in the retinal pigment epithelium (RPE), where genetic defects cause retinal disease. LRAT ¼ lechitin retinol acyl transferase; RGR¼ retinal G‐protein coupled receptor; CRALBP ¼ cellular retinaldehyde binding protein; 11‐cis‐RDH ¼ retinol dehydrogenase, specific for 11‐cis‐retinol; IRBP ¼ Interphotoreceptor matrix retinoid binding protein. References in (Gonzalez‐ Fernandez, 2003; Thompson and Gal, 2003).
The Retinal Pigment Epithelium
choroidal and/or retinal neovascularization (Seko et al., 1999; Ohno‐Matsui et al., 2001; Uhlmann et al., 2002; Witmer et al., 2003). On the other hand, blockage of VEGF signalling inhibits choroidal neovascularization in a murine model (Kwak et al., 2000). The angiogenic stimulation by VEGF is balanced by an RPE‐ derived antiangiogenic factor, pigment epithelium derived factor, PEDF (Tombran and Barnstable, 2003). PEDF provides autocrine negative feedback of the angiogenic stimulation caused by VEGF, partly through upregulation of PEDF expression though VEGFR‐1 (Ohno‐Matsui et al., 2003). Apart from its antiangiogenic properties, PEDF has neurotropic and neuroprotective properties (Tombran and Barnstable, 2003). The balance between PEDF and VEGF has been proposed as a major mechanism keeping the ocular vasculature patent and avoiding pathological angiogenesis (Ohno‐Matsui et al., 2001). Several members of the FGF family are involved in autocrine and paracrine control of RPE growth, resistance to apoptotic cell death, and wound repair (Schweigerer et al., 1987; Bost et al., 1992; Yamamoto et al., 1996; Mascarelli et al., 2001). FGF‐2, previously named basic FGF, stimulates RPE growth, but also has neurotrophic and angiogenic properties. Failure of RPE to generate FGF signaling in transgenic mice results in disrupted photoreceptors and thinned choroid (Rousseau et al., 2000). The RPE cells have been shown to produce a number of other cytokines, with less well‐characterized roles (Campochiaro, 1998; Holtkamp et al., 2001).
The outer retina is avascular, and the choriocapillaries are the main source of oxygen and nutrients for the photoreceptors. The retinal pigment epithelium is interposed between the choriocapillaries and the photoreceptors, and the epithelial cells are bound together by tight junctions that effectively hinder transfer of water soluble compounds in‐between the cells (i.e., via the paracellular route). Hence, the RPE cells control the exchange of water‐soluble nutrients and metabolites between the choroid and the subretinal space. In vitro experiments have shown that the retinal pigment epithelium has a retina positive transepithelial potential between 2 and 15 mV. This potential is responsible for the cornea positive standing potential of the eye, which can be recorded by DC electroretinography (Gallemore et al., 1998). The transepithelial electrical resistance across isolated RPE preparations has been measured to be between 79 and 350 Ocm2 (Hughes et al., 1998). The isolated epithelium in vitro absorbs Naþ, Cl ‐, HCO3 , and Kþ. The transepithelial transport of these ions has been extensively studied in isolated preparations
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of frog and bovine RPE (Figure 3). In both preparations, a number of transport mechanisms have been identified in the RPE membranes, and models have been proposed that explain quantitatively the transepithelial transport of the major ions in these species (la Cour, 1993; Hughes et al., 1998). The retinal membrane of the RPE incorporates an electrogenic Naþ/ Kþ pump that pumps Naþ out of the cell and Kþ into the cell at the expenditure of metabolic energy. The Naþ/Kþ pump lowers the intracellular concentration of Naþ, which is below the electrochemical equilibrium for this ion. Secondary active transport systems use the energy invested in the inwardly directed Naþ gradient to drive transport of other ions. The retinal membrane incorporates three such secondary active transport systems: a Naþ:Kþ:2Cl cotransport system, a Naþ/Hþ exchange mechanism, and a Naþ:2HCO3 cotransport system. These transport systems accumulate Cl and HCO3 intracellularly above their electrochemical equilibrium.
Figure 3. Model of mammalian RPE ion transport mechanisms. The retinal membrane incorporates an active Naþ/Kþ pump, an inward rectifying Kþ conductance of the Kir7.1 type, a rheogenic Naþ:2HCO3 cotransport system, a Naþ:Kþ:2Cl cotransport system, a Cl /HCO3 exchange mechanism, a Naþ/Hþ exchange mechanism, and a Hþ:water:lactate cotransport mechanism of the MCT1 type. The choroidal membrane incorporates a Kþ conductance, a Cl conductance, part of which is the CFTR channel, a Cl /HCO3 exchange mechanism, and a Hþ: lactate transport system of the MCT3 type. There are yet uncharacterized efflux mechanisms for Naþ, and water across the choroidal membrane. The figure also shows the aquaporin water channel AQP1 is located in the retinal membrane.
The Retinal Pigment Epithelium
The choroidal membrane incorporates a cAMP dependent Cl conductance that serves as exit mechanism for this ion (la Cour, 1992). Some of this cAMP dependent chloride conductance stems from the cystic fibrosis transmembrane conductance regulator (CFTR), which has recently been identified in human RPE choroidal membrane by reverse transcriptase PCR (Quinn et al., 2001; Blaug et al., 2003). The choroidal membrane also incorporates a cAMP independent Cl conductance, which is regulated by the intracellular Ca2þ concentration (Quinn et al., 2001). Bicarbonate exit is mediated by Cl / HCO3 exchangers in the RPE choroidal membrane. Most of the potassium that is pumped into RPE cells is recycled across the retinal membrane, through an unusual inward rectifying Kþ conductance, the major component of which is due to the Kir7.1 channel (la Cour et al., 1986; Segawa and Hughes, 1994; Hughes and Takahira, 1996; Yang et al., 2003). Some potassium exits the RPE cells via a smaller, yet uncharacterized Kþ conductance in the choroidal membrane (la Cour et al., 1986). The RPE cells incorporate transport systems for a number of organic nutrients and metabolites. The GLUT1 transport protein, which mediates facilitated transport of glucose across brain capillaries, is found in both the retinal and choroidal membrane of the RPE (Sugasawa et al., 1994). In photoreceptors, glycolysis is active even under aerobic conditions, and lactate has to be cleared from the subretinal space (Wang et al., 1997). The RPE incorporates the lactate transport systems MCT1 (MCT ¼ mono carboxylic acid transporter) in its retinal membrane and MCT3 in its choroidal membrane (la Cour et al., 1994; Philp et al., 2003). Other transport mechanisms, including transport systems for GABA, ascorbic acid, fluorescein, and amino acids, have also been described in the retinal pigment epithelium (Hughes et al., 1998). Ion transport across the RPE has been shown to be influenced by receptors in the retinal membrane of the epithelium. Both alpha‐ and beta receptors, as well as a purine receptor P2Y2 have been identified (Quinn et al., 2001; Yang et al., 2003). Stimulation of these receptors results in an increased chloride conductance in the choroidal membrane of the epithelium. The intracellular second messenger that conveys the signal from the retinal to the choroidal membrane is cAMP for the beta receptor, and Ca2þ for the P2Y2 purine receptor (Quinn et al., 2001; Yang et al., 2003). The retinal pigment epithelium from a number of species has been shown to absorb water (Hughes et al., 1998). This is consistent with the clinical observation that fluid under a rhegmatogenous retinal detachment absorbs quickly once the holes in the neurosensory retina are surgically closed. The rate of subretinal fluid resorption across the entire RPE in this setting has been estimated to be 2 ml per 24 hours. This corresponds to more than 50% of the aqueous secretion in that period (Marmor, 1998a). This high rate of water transport is probably not present under physiological conditions, since
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the retina with its tight extracellular spaces has a very low‐hydraulic conductivity (Tsuboi, 1987). Nevertheless, the retinal pigment epithelium has a large reserve capacity for removal of excess fluid from the subretinal space (Marmor, 1998a). The RPE water transport may have a role in the maintenance of normal retinal adhesion by exerting suction on the neurosensory retina. However, other mechanisms also seem to contribute to retinal adhesion (Marmor, 1998b). Under the pathologic condition of retinal detachment, the RPE capacity for fluid absorption is important for the surgical treatment of this condition. The RPE fluid absorption can be enhanced by stimulation of the purinoceptor P2Y2 as well as by beta adrenergic stimulation (Edelman and Miller, 1991; Peterson et al., 1997; Yang et al., 2003). Interestingly, the increased fluid absorption has been shown to increase the rate of resolution of experimental retinal detachments in rabbits (Yang et al., 2003). The mechanisms underlying RPE water transport is poorly understood. It is a clinical observation, confirmed in laboratory experiments, that subretinal fluid can be absorbed despite a high‐protein content (Marmor, 1998a). Proteinaceous retinal exudates can eventually be dehydrated to the extent that lipoprotein crystals precipitate in the retina as the so‐called hard exudates. Proposed mechanisms for RPE fluid transport must therefore be able to account for the apparent ability of the RPE to transport fluid against an osmotic gradient. It is known that the passive water permeability in the retinal membrane is larger than that in the choroidal membrane (la Cour and Zeuthen, 1993). This water permeability may be due to the water channel aquaporin‐1, AQP1, which recently was found to be expressed in the retinal membrane of human RPE (Stamer et al., 2003). Aquaporins are thought to be passive water channels. In order to explain vectorial water transport against an osmotic gradient, active transport systems are needed that can transport water against an electrochemical gradient (Stein and Zeuthen, 2002). The only such transport system so far known in the RPE is a Hþ: lactate: water cotransport system in the retinal membrane of both frog and porcine RPE (Zeuthen et al., 1996; Hamann et al., 2003). However, the physiological significance of this transport system is unknown at the present time.
THE RPE IN WOUND HEALING AND PROLIFERATIVE VITREORETINAL DISEASE
The RPE is often considered a glial element in the retina (Bok, 1993). Despite being a resting epithelium in adult individuals, the epithelium has a high‐proliferative capacity, and after retinal trauma or rhegmatogenous retinal detachment, RPE cells can proliferate vigorously. The proliferating cells liberated from the epithelium can be seen clinically as free pigmented cells, so‐called tobacco dust, in the vitreous (Fisher and Anderson, 1998;
The Retinal Pigment Epithelium
Hiscott et al., 1999). The free RPE cells settle on all available surfaces, dedifferentiate, and acquire macrophage and fibroblast‐like characteristics. Eventually they may participate in the formation of contractile membranes, a condition called proliferative vitreoretinopathy (PVR), which is a feared complication of rhegmatogenous retinal detachment (Kirchhof and Sorgente, 1989). Several factors may induce this dedifferentiation, including vitreous, albumin, serum, activated macrophages, and several growth factors, including hepatocyte growth factor (HGF), and connective tissue growth factor (CTGF) (Kirchhof and Sorgente, 1989; Hinton et al., 2002). The dedifferentiation goes along with down‐regulation of proteins associated with the highly specialized functions and the cell shape of the RPE cells (Alge et al., 2003). Proliferative vitreoretinopathy might be characterized as an inappropriate and excessive wound‐healing response of RPE, which results in the distortion and tractional detachment of the neurosensory retina. Despite the high‐proliferative capacity of RPE cells, it is a clinical observation that the RPE monolayer has a limited capacity to heal acquired in situ defects resulting from aging, inflammation, surgery, or trauma (Grierson et al., 1994). Recent experimental studies of RPE wound healing have shown that the RPE cells have the capacity to repopulate experimentally induced defects in the RPE monolayer, but that concurrent damage to Bruch’s membrane may prevent healing of RPE defects in situ (Tezel and del Priore, 1999a; Wang et al., 2003). Healing of RPE defects requires attachment of RPE cells to Bruch’s membrane at the base of the defect. It has become apparent that this attachment is critically dependent on age and on the general status of this membrane. If the basal laminar layer of Bruch’s membrane is intact, RPE cells can attach to and repopulate both young and aged (>60 years) Bruch’s membrane, whereas they fail to attach and survive on the deeper layers of aged human Bruch’s membrane (Tezel and del Priore, 1999a). Age‐related changes such as cross‐linking and deposition of long‐spacing collagen, drusen formation, calcifications, cracks, or loss of inner layers can make these areas uninhabitable for RPE. Such RPE defects eventually lead to the development of progressive choriocapillaries and photoreceptor atrophy, and eventually turn into areas of geographic atrophy (del Priore et al., 1996). RPE wound healing does not necessarily involve cell proliferation. RPE cells are postmitotic and occasional cell loss does not stimulate cell proliferation, but is compensated for by the flattening and migration of the neighboring RPE cells (Grierson et al., 1994). This mechanism is an important role for the repopulation of small involutional RPE defects especially in the macula. Changes in the topographical density of aging human RPE suggest that there is a continuous proliferation and sliding of the peripheral RPE to cover midperipheral and macular RPE defects (del Priore et al., 2002). Mitosis, on the other hand, seems to be necessary to cover larger
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defects in the RPE monolayer (Hergott and Kalnins, 1991). Proliferating and migrating RPE cells become flat, display prominent stress fibers, and acquire the appearance of fibroblasts. This wound‐healing process requires upregulation of various adhesion molecules, cytokines, and signaling genes (Singh et al., 2001). At later stages of wound healing, cultured RPE cells produce extracellular matrix molecules and remodel their matrix (Kamei et al., 1998).
RPE develops from a layer of ciliated and pseudostratified neuroectodermal precursor cells that form the outer layer of the optic cup. At the 4th week of gestation, these cells start melanogenesis by the activation of the tyrosinase promoter and form simple cuboidal epithelia (Oguni et al., 1991). They are initially hexagonal in shape, and have short microvilli. A smooth RPE basal lamina can be identified in as early as the 5th week of gestation (Fu and Li, 1989). RPE basal lamina, elastic layer, and choriocapillaries basal lamina form the primordial Bruch’s membrane (Marmorstein et al., 1998). At around the 11th week of gestation, the inner and outer collagen layers appear flanking the central elastic layer, thus completing the pentalaminar structure of the adult Bruch’s membrane (Fu and Li, 1989). Further development of the embryonic RPE waits for the maturation of fetal photoreceptors. As photoreceptor’s outer segments start to develop into the subretinal space, RPE extends long‐apical microvilli to ensheath them. In order to adapt to photoreceptors’ high need of nutrients, RPE cells develop basal infoldings that increase their surface area for uptake and transport purposes (Marmorstein et al., 1998). Maturation of photoreceptors also induce polarization of certain proteins, such as apically polarized Naþ, Kþ‐ATPase, N‐CAM, EMMPRIN, aVb5 integrin, and basally polarized a6b1 integrin (Marmorstein et al., 1998). Junctional complexes are observed as early as the 6th week of gestation (Oguni et al., 1991). Survival and development of primordial RPE is controlled by a number of transcription and growth factors, such as brain derived neurotrophic factor, BDNF (Liu et al., 1997; Hackett et al., 1998). Interestingly, RPE precursors can be induced to transdifferentiate into neural retinal tissue by FGF stimulation (Galy et al., 2002). Growth of RPE is required for the development of the choroid, neural retina, and vitreous. Neural retina and vitreous fail to develop in transgenic mice lacking RPE due to expression of diphtheria toxin‐A within the developing epithelial cells (Raymond and Jackson, 1995). Similarly, choroid fails to develop in transgenic mice where RPE is transdifferentiated into neural retina, due to expression of FGF9 in the developing RPE (Zhao and Overbeek, 2001).
The Retinal Pigment Epithelium
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Sakai, N., Decantur, J., Nakanishi, K. and Eldred, G.E. (1996). Ocular age pigment ‘A2E’: An unprecedented pyridinium bisretinoid. J. Chem. Soc. 118, 1559–1560. Schweigerer, L., Malerstein, B., Neufeld, G. and Gospodarowicz, D. (1987). Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells. Biochem. Biophys. Res. Commun. 143, 934–940. Seko, Y., Fujikura, H., Pang, J., Tokoro, T. and Shimokawa, H. (1999). Induction of vascular endothelial growth factor after application of mechanical stress to retinal pigment epithelium of the rat in vitro. Invest. Ophthalmol. Vis. Sci. 40, 3287–3291. Singh, S., Zheng, J.J., Peiper, S.C. and McLaughlin, B.J. (2001). Gene expression profile of ARPE‐19 during repair of the monolayer. Graefes Arch. Clin. Exp. Ophthalmol. 239, 946–951. Stamer, W.D., Bok, D., Hu, J., Jaffe, G.J. and McKay, B.S. (2003). Aquaporin‐1 channels in human retinal pigment epithelium: Role in transepithelial water movement. Invest. Ophthalmol. Vis. Sci. 44, 2803–2808. Steinberg, R.H., Linsenmeier, R.A. and Griff, E.R. (1983). Three light‐evoked responses of the retinal pigment epithelium. Vision Res. 23:1315, 1983. Sugasawa, K., Deguchi, J., Okami, T., Yamamoto, A., Omori, K., Uyama, M. and Tashiro, Y. (1994). Immunocytochemical analyses of distributions of Na, K‐ATPase and GLUT1, insulin and transferrin receptors in the developing retinal pigment epithelial cells. Cell Struct. Funct. 19, 21–28. Tezel, T.H., Kaplan, H.J. and del Priore, L.V. (1999b). Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch’s membrane. Invest. Ophthalmol. Vis. Sci. 40, 467–476. Tsuboi, S. (1987). Measurement of the volume flow and hydraulic conductivity across the isolated dog retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 28, 1776–1792. Uhlmann, S., Rezzoug, K., Friedrichs, U., Hoffmann, S. and Wiedemann, P. (2002). Advanced glycation end products quench nitric oxide in vitro. Graefes Arch. Clin. Exp. Ophthalmol. 240, 860–866. Vollrath, D., Feng, W., Duncan, J.L., Yasumura, D., D’Cruz, P.M., Chappelow, A., Matthes, M. T., Kay, M.A. and La Vail, M.M. (2001). Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc. Natl. Acad. Sci. USA 98, 12584–12589. Wang, H., Ninomiya, Y., Sugino, I.K. and Zarbin, M.A. (2003). Retinal pigment epithelium wound healing in human Bruch’s membrane explants. Invest. Ophthalmol. Vis. Sci. 44, 2199–2210. Wang, L., Tornquist, P. and Bill, A. (1997). Glucose metabolism in pig outer retina in light and darkness. Acta Physiol. Scand. 160, 75–81. Weng, J., Mata, N.L., Azarian, S.M., Tzekov, R.T., Birch, D.G. and Travis, G.H. (1999). Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice. Cell 98, 13–23. Witmer, A.N., Vrensen, G.F., Van Noorden, C.J. and Schlingemann, R.O. (2003). Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 22, 1–29. Yamamoto, C., Ogata, N., Yi, X., Takahashi, K., Miyashiro, M., Yamada, H., Uyama, M. and Matsuzaki, K. (1996). Immunolocalization of basic fibroblast growth factor during wound repair in rat retina after laser photocoagulation. Graefes Arch. Clin. Exp. Ophthalmol. 234, 695–702. Yang, D., Pan, A., Swaminathan, A., Kumar, G. and Hughes, B.A. (2003). Expression and localization of the inwardly rectifying potassium channel Kir7.1 in native bovine retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 44, 3178–3185. Young, R.W. (1971). The renewal of rod and cone outer segments in the rhesus monkey. J. Cell Biol. 49, 303–318. Zeuthen, T., Hamann, S. and la Cour, M. (1996). Cotransport of Hþ, lactate, and H2O by membrane proteins in retinal pigment epithelium of bullfrog. J. Physiol. (Lond.) 497, 3–17.
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FURTHER READING Besharse, J.C. and Defoe, D.M. (1998). Role of the retinal pigment epithelium in the photoreceptor membrane turnover. In: The Retinal Pigment Epithelium. (Marmor, M.F. and Wolfensberger, T.J., Eds.), 1st edn., pp. 152–172. Oxford University Press, New York. Bok, D. and Heller, J. (1976). Transport of retinal from the blood to the retina: An autoradiographic study of the pigment epithelial cell surface receptor for plasma retinol‐binding protein. Exp. Eye Res. 22, 395–402. Chaitin, M.H. and Hall, M.O. (1983). Defective ingestion of rod outer segments by culutred dystrophic rat pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 24, 812–820. Edelman, J.L. and Miller, S.S. (1991). Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 32, 3033–3040. Fisher, S.K. and Anderson, D.H. (1998). Cellular responses of the retinal pigment epithelium to retinal detachment and reattachment. In: The Retinal Pigment Epitelium. (Wolfensberger, T.J. and Marmor, M.F., Eds.), pp. 406–419. Oxford University Press, New York. Fu, J. and Li, F.M. (1989). Embryonic development and structure of human Bruch’s membrane. Zhonghua Yan Ke Za Zhi 25, 18–19. Harman, A.M., Fleming, P.A., Hoskins, R.V. and Moore, S.R. (1997). Development and aging of cell topography in the human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 38, 2016–2026. Hergott, G.J. and Kalnins, V.I. (1991). Expression of proliferating cell nuclear antigen in migrating retinal pigment epithelial cells during wound healing in organ culture. Exp. Cell Res. 195, 307–314. Hughes, B.A. and Takahira, M. (1996). Inwardly rectifying Kþ currents in isolated human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 37, 1125–1139. Kirchhof, B. and Sorgente, N. (1989). Pathogenesis of proliferative vitreoretinopathy. Modulation of retinal pigment epithelial cell functions by vitreous and macrophages. Dev. Ophthalmol. 16, 1–53. la Cour, M. and Zeuthen, T. (1993). Osmotic properties of the frog retinal pigment epithelium. Exp. Eye Res. 56, 521–530. Marmor, M.F. and Martin, L.J. (1978). 100 years of the visual cycle. Surv. Ophthalmol. 22, 279–285. Peyman, G.A. and Bok, D. (1972). Peroxidase diffusion in the normal and laser‐coagulated primate retina. Invest. Ophthalmol. 11, 35–45. Raymond, S.M. and Jackson, I.J. (1995). The retinal pigmented epithelium is required for development and maintenance of the mouse neural retina. Curr. Biol. 5, 1286–1295. Robinson, S.R. and Hendrickson, A. (1995). Shifting relationships between photoreceptors and pigment epithelial cells in monkey retina: Implications for the development of retinal topography. Vis. Neurosci. 12, 767–778. Segawa, Y. and Hughes, B.A. (1994). Properties of the inwardly rectifying Kþ conductance in the toad retinal pigment epithelium. J. Physiol. (Lond.) 476, 41–53. Shichi, H. and Nebert, D.W. (1980). Drug metabolism in ocular tissues. In: Extrahepatic Metabolism of Drugs and Other Foreign Compounds. (Gram, T.E., Ed.), 1st edn., pp. 333–363. MTP press limited. Snodderly, D.M., Sandstrom, M.M., Leung, I.Y., Zucker, C.L. and Neuringer, M. (2002). Retinal pigment epithelial cell distribution in central retina of rhesus monkeys. Invest. Ophthalmol. Vis. Sci. 43, 2815–2818. Stein, W. and Zeuthen, T. (2002). In Molecular Mechanisms of Water Transport, 1st edn., pp. 1–475. Academic Press, London.
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Tezel, T.H. and del Priore, L.V. (1999a). Repopulation of different layers of host human Bruch’s membrane by retinal pigment epithelial cell grafts. Invest. Ophthalmol. Vis. Sci. 40, 767–774. Thompson, D.A. and Gal, A. (2003). Genetic defects in vitamin A metabolism of the retinal pigment epithelium. Dev. Ophthalmol. 37, 141–154. Tombran‐Tink, J. and Barnstable, C.J. (2003). PEDF: A multifaceted neurotrophic factor. Nat. Rev. Neurosci. 4, 628–636. Tso, M.O.M. and Friedman, E. (1967). The retinal pigment epithelium. I. Comparative histology. Arch. Ophthalmol. 78, 641–649. Zhao, S. and Overbeek, P.A. (2001). Regulation of choroid development by the retinal pigment epithelium. Mol. Vis. 7, 277–282.