Chapter 100 – Retinal Pigment Epithelium
MICHAEL F. MARMOR
• A melanin-containing epithelial layer that lies between the neural retina and choroid.
• Absorption of scattered light.
• Control of fluid and nutrients in the subretinal space (blood-retinal barrier function).
• Visual pigment regeneration and synthesis.
• Synthesis of growth factors to modulate adjacent structures.
• Maintenance of retinal adhesion.
• Phagocytosis and digestion of photoreceptor wastes.
• Electrical homeostasis.
• Regeneration and repair after injury or surgery.
The retinal pigment epithelium (RPE) is a vital tissue for the maintenance of photoreceptor function.  It is also affected by many diseases of the retina and choroid. Indeed, much of the pigmentary change that is visible clinically in retinal disorders takes place in the RPE (which is pigmented) rather than in the retina (which is transparent). Embryologically, the RPE is derived from the same neural tube tissue that forms the neural retina, but the cells differentiate into a transporting epithelium, the main functions of which are to metabolically insulate and support the overlying neural retina.
Cellular Architecture and Blood-Retinal Barrier
The RPE is a monolayer of cells that are cuboidal in cross section and hexagonal when viewed from above. The interlocking hexagonal cells are joined by tight junctions (zonulae occludens), which block the free passage of water and ions. This junctional barrier is the equivalent of the blood-retinal barrier formed by the capillary endothelium of the intrinsic retinal vasculature.
Cells of the RPE vary in size and shape across the retina. In the macular region, they are small (roughly 10–14?µm in diameter), whereas toward the periphery, they become flatter and broader (diameter up to 60?µm). The density of photoreceptors also varies across the retina, but the number of photoreceptors that overlie each RPE cell remains roughly constant (about 45 photoreceptors per RPE cell). This constancy has physiological relevance, in that each RPE cell is metabolically responsible for providing support functions to the overlying receptors.
In cross section, the RPE cell is differentiated into apical and basal configurations. On the apical side (facing the photoreceptors), long microvilli reach up between (and envelop) the outer segments of the photoreceptors ( Fig. 100-1 ). Melanin granules are concentrated in the apical end of the cell. The midportion of
Figure 100-1 Apical surface of human retinal pigment epithelium as seen through a scanning electron microscope. Fine microvilli cover the surface and reach up between the photoreceptor outer segments (which have been peeled away in this view).
the cell contains the nucleus and synthetic machinery (e.g., Golgi apparatus, endoplasmic reticulum) and digestive vesicles (lysosomes). The basal membrane lacks microvilli but has numerous convoluted infolds to increase the surface area for the absorption and secretion of material. The two membranes also have different ion channels and pumps.
The pigment that gives the RPE its name is melanin, which is present within cytoplasmic granules called melanosomes. Developmentally, the RPE is the first tissue in the body to become pigmented, and melanogenesis continues to some degree throughout life. However, in older age, melanin granules often fuse with lysosomes and break down, so the elderly fundus typically appears less pigmented. The role of melanin in the eye remains somewhat speculative. The pigment serves to absorb stray light and minimize scatter within the eye, which has theoretical optical benefits. However, visual acuity is not degraded in very blond fundi relative to heavily pigmented ones, so the magnitude of this effect is unclear. Further, the appearance of the fundus can be misleading with respect to the RPE, since the greatest racial differences are a result of choroidal pigmentation rather than RPE pigmentation. Melanin also serves as a free radical stabilizer, and it can bind toxins and retinotoxic drugs such as chloroquine and thioridazine, although it is unclear whether this effect is beneficial or harmful. Albino eyes lack melanin, but the poor visual acuity of most albinos is a result of foveal aplasia rather than optical scatter.
The other major RPE pigment is lipofuscin, which accumulates in RPE cells gradually with age. However, lipofuscin is found throughout the nervous system, and its significance within the eye remains to be determined. Some lipofuscin is present in childhood, but by old age, the cells can be severely clogged with the golden, autofluorescent pigment. Lipofuscin is thought to be derived from outer segment lipids that have been ingested and then digested by the RPE; it may represent membrane
fragments that have been damaged by light or oxidation. Because the substance accumulates in older eyes and these eyes may show RPE breakdown, as evidenced by drusen formation, RPE atrophy, and choroidal neovascularization, the question has been raised whether excess lipofuscin damages the RPE (or at least is a marker for cellular damage).  However, most elderly eyes have a considerable quantity of RPE lipofuscin, whereas only a few have clinically significant macular degeneration. A lipofuscin-like substance also accumulates in the RPE in Stargardt’s disease, fundus flavimaculatus, and Best’s vitelliform dystrophy, and may play a role in their pathogenesis.
METABOLISM AND MEMBRANE FUNCTION
Metabolism and Growth Factors
RPE cells are packed with mitochondria and engage actively in oxidative metabolism. Enzymes are synthesized for functions such as membrane transport, visual pigment metabolism, and digestion of wastes. The RPE contains the antioxidant enzymes superoxide dismutase and catalase, which minimize the formation of free radicals that can damage lipid membranes. The RPE contributes to the formation and maintenance of the interphotoreceptor matrix, which is critical for retinal adhesion, and to the elaboration of growth factors that modulate nearby tissues.
A number of growth factors are elaborated by RPE cells and serve to modulate not only the behavior of the RPE but also the behavior of surrounding tissues such as the choriocapillaris. Knowledge of these interactions is growing rapidly, and it is now recognized that the RPE is part of a complex system of cellular cross-talk that controls vascular supply, permeability, growth, repair, and other processes vital to retinal function. Factors produced by the RPE (though not necessarily exclusively) include platelet-derived growth factor (PDGF), which modulates cell growth and healing; pigment epithelium–derived factor (PEDF), which acts as a neuroprotectant and vascular inhibitor; vascular endothelial growth factor (VEGF), which can stimulate normal or neovascular growth; fibroblast growth factor (FGF), which can be neurotropic; and transforming growth factor (TGF), which moderates inflammation.
Membrane Properties and Fluid Transport
The RPE membrane contains a number of selective ion channels, as well as a number of active or facilitative transport systems for ions and for metabolites such as glucose and amino acids (e.g., taurine, which is essential to the photoreceptors). Different channels and transporters are present on the apical and basal membranes. For example, an electrogenic sodium-potassium pump occurs only on the apical membrane, whereas a chloride-bicarbonate exchange transporter occurs only on the basal membrane. The net effects of the asymmetrical transport systems are a movement of water across the RPE in the apical-to-basal direction and the generation of voltage across the RPE. It is important to recognize that both the movement of water and the transcellular potential are the sum of several transport systems that are moving ions and water in either direction. Thus, water transport can be diminished either by blocking a transporter that moves ions in the basal direction or by stimulating a transporter that moves ions in the apical direction.
The ability of the RPE to transport water is very powerful, and the RPE can pump fluid against a substantial gradient of hydrostatic or osmotic pressure. However, if the RPE barrier function is broken, fluid will leave the subretinal space more quickly ( Fig. 100-2 ) because of intraocular pressure and osmotic suction from the choroid. In other words, tight junctions are required to protect the neural environment of the retina, and because they are present, active transport by the RPE (and the expenditure of metabolic energy) is needed to keep the subretinal space dry.
These physiological observations have relevance for clinical disorders such as serous detachments. The unusual thing about a serous detachment is not that fluid gets in (given that a break
Figure 100-2 Fluid absorption from the subretinal space. These results are from rabbit experiments in which either serum or saline was injected beneath the retina to form fluid blebs. Over normal retinal pigment epithelium (RPE), serum was absorbed nearly as fast as saline, since the water was being absorbed actively. When the RPE barrier was broken (by the toxin sodium iodate), saline fluid was absorbed much more quickly than normal, and the absorption of serum hardly changed, since the absorption now was osmotic. (Adapted from Negi A, Marmor MF. The resorption of subretinal fluid after diffuse damage to the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1983;24:1475–9.)
Figure 100-3 Mechanism of serous detachment. When the retinal pigment epithelium (RPE) is normal, no serous detachment occurs beyond a focal site of leakage. When the RPE is compromised by choroidal or RPE disease that impairs outward fluid transport, a serous detachment forms until absorption across the exposed RPE balances the inward leak.
is present in the RPE barrier) but that fluid accumulates and persists (since the powerful RPE would be expected to pump it right back out). Disorders such as central serous chorioretinopathy probably involve diffuse pathological changes of the RPE-choroid complex that impair fluid absorption ( Fig. 100-3 ).
The RPE is not a photoreceptive cell, and it generates no direct response to light. However, the asymmetrical transport properties of the apical and basal membranes generate a transepithelial voltage (called the standing potential), which can be modified
Figure 100-4 Electrical responses of the retinal pigment epithelium (RPE). Light induces the c wave, fast oscillation, and light peak (on electro-oculogram). Hypoxia and systemic drugs also modulate the voltage across the RPE. (Adapted from Marmor MF. Clinical electrophysiology of the retinal pigment epithelium. Doc Ophthalmol. 1991;76:301–13.)
secondarily by photoreceptor activity or by endogenously supplied substances ( Fig. 100-4 ).
When the photoreceptors respond to light, the potassium concentration falls for a few seconds in the subneural retinal space. The apical membranes of the RPE and the Müller cells respond by hyperpolarizing, which produces the c wave of the electroretinogram. This potassium change is transmitted slowly through the RPE cell, and roughly 1 minute later, a hyperpolarization appears at the basal membrane, which accounts for the fast oscillation of the electro-oculogram (EOG). This response involves basal chloride channels and is abnormal in some patients who have cystic fibrosis. Light activation of photoreceptors also causes the release of an unknown messenger substance that causes a basal RPE depolarization 5–10 minutes after the onset of light activation. This late basal depolarization is recorded clinically as the light response of the standing potential in the clinical EOG.
The basal membrane of the RPE can be activated independent of light, by chemical agents. For example, it hyperpolarizes several minutes after an intravenous injection of a hyperosmolar agent or acetazolamide, and it depolarizes after an oral dose of alcohol. The clinical significance of these nonphotic responses of the RPE remains to be determined.
PHOTORECEPTOR–RETINAL PIGMENT EPITHELIUM INTERACTIONS
Visual Pigment Regeneration
In 1877, Kuhne demonstrated that visual pigments regenerate to maintain the visual process. The primary rod pigment, rhodopsin, consists of a vitamin A aldehyde molecule bonded to a large protein (opsin); it is sensitive to light only when the vitamin A has the 11-cis conformation. Absorption of a photon converts the vitamin A to the all-trans form, which initiates the process of transduction and begins a series of regenerative chemical changes that are independent of vision. Vitamin A is split off from the opsin molecule and carried by a transport protein to the RPE. In the RPE, vitamin A may be stored in an ester form, but eventually it is isomerized back to the 11-cis form and recombined with opsin. The RPE is vital for this process and for the capture of vitamin A from the bloodstream to maintain its concentration within the eye.
The significance of this regenerative process is apparent in the time it takes to adjust to indoor lighting after a walk on a sunny beach or to the darkness inside a movie theater. In an unusual night-blinding disorder, fundus albipunctatus, this visual pigment regeneration process is severely prolonged, most often as
Figure 100-5 Retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments. The phagosome, containing the ingested material, enters the RPE cytoplasm, where it merges with lysosomes to facilitate digestion of the outdated membranes. (Adapted from Steinberg RH, Wood I, Hogan MJ. Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina. Philos Trans R Soc Lond. 1977;277:459–74.)
a result of defects in the retinal dehydrogenase (RDH) gene. Affected individuals may require 3–4 hours, instead of 30 minutes, to fully adapt to the dark. Defects in other regeneration cycle genes can cause retinitis pigmentosa, such as the gene for retinaldehyde binding protein (RLBP) and the RPE65 gene (involved in 11-cis retinol metabolism), which is responsible for severe early-onset disease.
Photoreceptor Renewal and Phagocytosis
Photoreceptors, like skin, are continually exposed to radiant energy (light) and oxygen (from the choroid), which facilitates the production of free radicals that can damage membranes over time. Thus, a process of cellular renewal is needed. Every day, upward of 100 discs at the distal end of the photoreceptors are phagocytosed by the RPE ( Fig. 100-5 ), while new discs are synthesized. The cellular renewal process has a circadian rhythm. The rods shed discs most vigorously in the morning at the onset of light, whereas cones shed more vigorously at the onset of darkness. The complete outer segments are renewed roughly every 2 weeks. Within the RPE, the phagocytosed discs become encapsulated in vesicles called phagosomes,  which merge with lysosomes so that the material can be digested. Necessary fatty acids are retained for recycling into outer segment synthesis, and waste products or damaged membrane material is digested across the basal RPE membrane. This is an impressive metabolic task for the RPE, since each cell must ingest and digest upward of 4000 discs daily. Some of this membranous material may persist within the RPE cell and contribute to the formation of lipofuscin.
That RPE phagocytosis is important is shown by a peculiar strain of rat (RCS) that lacks the ability to phagocytose outer segments. Within weeks after the birth of these animals, outer segment debris begins to accumulate in the subretinal space, and the photoreceptors degenerate. This is not the problem in most cases of retinitis pigmentosa, however, because eyes examined histologically have had shortened rather than lengthened outer segments. The general process of RPE phagocytosis and its relationship to lipofuscin formation may be more relevant to the process of aging within the RPE cell and the development of age-related macular degeneration.
Interphotoreceptor Matrix and Retinal Adhesion
The interphotoreceptor matrix (IPM) is not simply a sticky glue. It contains complex molecules, such as glycosaminoglycans, and has an elaborate structure in which domains of distinct chemical
characteristics surround the rods and cones. These can be demonstrated by staining the matrix material with fluorescent binding molecules ( Fig. 100-6 , A). The matrix serves several functions, including physical support of the photoreceptors, transfer of nutrients and visual pigments, and formation of an adhesive bond between the retina and RPE. The quality of these functions is largely controlled by the RPE, not only through the synthesis of matrix materials and transport proteins but also acutely through the transport of ions and water. The degree to which the IPM is hydrated or dehydrated alters its bonding properties and viscosity.
Retinal adhesion is a complex process involving several complementary and interactive mechanisms. The neural retina is pressed in place by the vitreous gel, intraocular fluid pressure, and RPE water transport, which drive or pull water through the semipermeable tissue. Also, some physical resistance prevents separation of the outer segments from the enveloping RPE microvilli. The strongest mechanism for bonding the retina to the RPE space appears to be the IPM. When neural retina is freshly peeled from the RPE, the IPM material stretches dramatically before it breaks, which shows that it is firmly attached to both neural retinal and RPE surfaces ( Fig. 100-6 , B). It is also important to recognize that, despite these physical forces of adhesion, the strength of neural retinal adhesion is constantly and acutely dependent on metabolism.  For example, the retinal adhesive force drops to near zero within minutes after death, and adhesive strength can be reversibly restored or enhanced by tissue oxygenation. The likely basis of these metabolic effects is RPE
Figure 100-6 Cone sheaths of the interphotoreceptor matrix, shown by fluorescent staining with peanut agglutinin. Cone tips indent the sheaths from above; the retinal pigment epithelium (RPE) is on the bottom. A, The matrix sheaths are short in a normal eye. B, They stretch dramatically before breaking as the retina is peeled from the RPE. This shows that matrix material bonds across the subretinal space. (Reproduced with permission from Hageman GS, Marmor MF, Yao X-Y, Johnson LV. The interphotoreceptor matrix mediates primate retinal adhesion. Arch Ophthalmol. 1995;113:655–60.)
control of the hydration and local ionic environment of the subretinal space and the bonding properties of the IPM material. This may explain why rhegmatogenous detachment is more frequent in older eyes (which may be metabolically less competent) and why serous neural retinal detachment can be associated with local ischemic conditions such as eclampsia and severe hypertension.
Neural retinas do not detach easily, which is a reflection of these multiple mechanisms for keeping the retina in place. However, after a retinal detachment is repaired and the fluid is absorbed, time is required to restore all these mechanisms and regain full adhesive strength. Resynthesis of matrix domains after enzymatic destruction requires about 2 weeks, and additional time may be needed for the RPE and photoreceptors to regain full microvillous intercalation.
REPAIR AND REGENERATION
Although of neural origin, the RPE can be a pluripotential tissue. In amphibians, RPE cells can regenerate lens, neural retina, and other components of the eye; mercifully, this does not take place in humans. Nevertheless, the RPE is capable of local repair (unlike the neural retina), and cells may migrate and take on altered characteristics. After a laser burn, for example, the RPE cells that surround the burn begin to divide, and small cells fill the defect to form a new blood-retinal barrier within 1–2 weeks. In degenerative disease, such as retinitis pigmentosa, RPE cells migrate into the injured neural retina and sometimes come to rest around vessels to contribute to the characteristic bone spicule appearance. An overly vigorous RPE response can lead to duplicated layers of RPE cells and RPE scarring, which may be part of a macular degenerative process. In the extreme, RPE cells contribute to proliferative vitreoretinopathy. Growth factors from the RPE may, at times, help contain unwanted proliferation, and at other times they may stimulate vascular or fibrous growth. Functionally, the most useful RPE repair characteristic is the ability to heal defects. The value of photocoagulation for macular edema and proliferative diabetic retinopathy may, in part, depend on the ability of RPE cells to seal laser scars, reestablish a degree of normal transport, and avoid unnecessary leakage of proteins into the subretinal space.
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