Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

early phase ends, Na+ K+ ATPase (ATPase, adenosine triphosphatase) becomes concentrated at the apical surface, and the apical microvilli begin to elongate. In the second stage, the tight junctions become increasingly less permeable, presumably due to the alterations in the distribution of various tight-junction proteins. At this stage, the RPE now prevents the free diffusion of membrane proteins, and the basolateral membrane is remodeled. In the last stage of tight-junction formation, the composition of tight-junction protein isoforms stabilizes and the RPE begins to display barrier properties characteristic of a tight epithelium. Following completion of tight-junction formation, the RPE begins to express specialized proteins, such as the glucose transporter, that aid in the transport of essential nutrients from the RPE to the photoreceptors. As RPE maturation concludes, the RPE becomes fully functional and is able to interact with the photoreceptors.

Choroid Development

Blood vessels develop by vasculogenesis and angiogenesis. In vasculogenesis, primitive vascular cells assemble into a primitive capillary plexus, which is remodeled to define the pattern of the vascular architecture, whereas in angiogenesis, new vessels develop as sprouts from preexisting vessels. It is thought that the superficial vessels of the retina form by vasculogenesis at the optic nerve and expand along a gradient from the posterior to the anterior retina. However, new vessels then sprout via angiogenesis

and invade the retina to form intermediate and deep capillary beds. Nonetheless, the primitive vessels derived via vasculogenesis or angiogenesis must still be remodeled before they are considered mature. Remodeling involves the growth of new vessels and regression of others. In addition, alterations in lumen diameter and vessel wall thickness, which are dictated by the local needs of the tissue, must also occur.

The choroid develops from two principle embryonic tissues: the mesoderm and cranial neural crest cells. The mesoderm gives rise to the endothelial cells of the choroidal blood vessels, whereas the stromal cells, melanocytes, and pericytes are all derived from the neural crest cells. Choroid development begins early in eye development, which is not surprising considering that the differentiation of the ocular tissues relies upon the oxygen and nutrients supplied by the primitive vascular system. In early eye development, the oxygenation of the retina is supplied solely by the choroid and the transient hyaloid vascular system. The vascularization of the retina itself is actually a late event. Initially, tubes and spaces form in the surrounding periocular region of the optic vesicle. These tubes, which are lined by the mesodermal endothelium, expand to form a plexus as eye development proceeds. Concomitant with optic vesicle invagination, the primitive capillaries develop from this plexus adjacent to the RPE. In humans, choroidal capillaries completely encircle the optic cup by the 13-mm stage of development and remain separated from the retina by the basement

membrane of the RPE. At the anterior region of the optic cup, the choroidal plexus fuses to form a singular vessel known as the annular vessel, and during the second and third months of gestation, this primitive plexus is organized into a complex network. A well-defined choriocapillaris layer also appears at this stage. Pigmentation does not appear in choroidal melanocytes until late gestation – between 6 and 7 months; however, it is complete at birth.

BrM Development

In mice, the formation of the choroidal vessels precedes the deposition of BrM, and maturation of the BrM layers is not complete until 6 weeks following birth. However, in humans and monkeys, BrM layers are completely formed in utero. BrM formation commences near the RPE, and is initially comprised of a single basement membrane derived from the RPE. The RPE is able to synthesize many of the extracellular matrix (ECM) components that comprise BrM; therefore, it is not surprising that BrM development begins near the RPE. On the 17th to 18th day of gestation in rats, collagenous fibrils gradually accumulate in the space between the RPE and choriocapillaris endothelium and begin to form a three-dimensional meshwork within BrM. The meshwork consists of three sublayers: the basement membranes of the RPE and choriocapillaris endothelium, and a collagenous layer. The final component of BrM, the central elastic layer, appears on the fifth postnatal day. Initially, dense elastic deposits appear within the collagenous layer, and subsequently accumulate to form a single continuous layer, which separates the collagenous layer into an inner and outer layer. Finally, an immature, but five-layered BrM is formed by the ninth day after birth.

RPE–Choroid Complex: Structure and

Function

RPE Structure and Function

It is apparent that the RPE performs a variety of complex functions that are essential for proper visual function. If any aspect of one of the many roles that the RPE serves is disrupted or fails, retinal degeneration, loss of visual function, and ultimately blindness may result. Therefore, the RPE is an indispensable component of the RPE–choroid complex. Anatomically, the RPE is juxtaposed to the outer segments of the photoreceptors at its apical surface, extending long apical microvilli that surround the outer segments. These microvilli provide a means for a complex structural interaction between the RPE and photoreceptors. BrM, which is at the basolateral side of the RPE, separates the RPE from the fenestrated endothelium of the choriocapillaris.

The RPE serves many functions that are vital to normal eye function and physiology. These functions have been categorized based on the characteristics of three classes of cells: epithelium, macrophage, and glia. The major function of an epithelium is to control the passage of substances from one extracellular space to another; therefore, the epithelial aspect of the RPE is its role in actively transporting ions, water, and metabolic end products from the subretinal space to the blood. The RPE is also involved in the uptake of nutrients, such as glucose, retinol, and fatty acids from the blood to nourish the photoreceptors. One of its most critical functions is the exchange of retinal with the photoreceptors. The photoreceptors are unable to reisomerize all-trans-retinal; therefore, it is transported to the RPE where it is reisomerized to 11-cis-retinal, and then transported back to the photoreceptors, a process known as the visual cycle of retinal.

The RPE also functions as a macrophage in that it phagocytoses the photoreceptor outer segments, which are constantly being renewed. As new membrane is added to the base of the outer segment, the older membrane is advanced toward the tip, and subsequently shed. The phagocytosis of the shed outer segments by the RPE is critical to normal photoreceptor function because it maintains the excitability of the photoreceptors. The vital nutrients, such as retinal, which are recycled and returned to the photoreceptors following outer segment digestion help rebuild the light-sensitive outer segments from the base of the photoreceptors.

The final role that the RPE plays is that of a glial cell. RPE resembles glial cells in the manner in which they respond electrically to changes in the concentration of extracellular K+, which is determined by the photoreceptors. The RPE possesses tight junctions at both its basolateral and apical membranes, which generate transepithelial resistance (TER); furthermore, the tight junctions at the basolateral surface form the RPE portion of the blood–retinal barrier.

Choroid Structure and Function

The choroid is a vascular bed that supplies nutrients and oxygen to the RPE and outer nuclear layer of the retina. It consists of larger vessels and a highly fenestrated choriocapillaris (Figure 2), whose endothelial basement membrane comprises one layer of BrM. Fenestrated endothelium is a characteristic of tissues that are involved in secretion and/or filtration. The photoreceptors are metabolically very active and the fenestrated choriocapillaris facilitates the transport of oxygen and nutrients to fulfill their metabolic needs. RPE tight junctions, in combination with sophisticated transport systems, determine which components of the choriocapillary secretions are transported to the photoreceptors.

RPE

BL col.

col.

BL

CC

Figure 3 Ultrastructure of the RPE–BrM–choriocapillaris. The RPE and choriocapillaris are separated by a thick (1–4 mm) elastic lamina, BrM, which consists of five layers: the RPE basement membrane (BL), an inner collagenous layer (col), a central elastic layer (el), an outer collagenous layer (col), and the choriocapillaris (cc) endothelium basement membrane (BL), respectively, from top to bottom. BrM acts as a barrier for macromolecules and regulates the diffusion of small molecules between the RPE and the choriocapillaris.

BrM Structure and Function

Located between the RPE and the choriocapillaris, BrM is a thick (1–4 mm) pentalaminar ECM, which consists of the RPE basement membrane, an inner collagenous layer, a central elastic layer, an outer collagenous layer, and the choriocapillaris endothelium basement membrane (Figure 3). BrM is comprised of several types of extracellular components, including many types of collagen, laminin, fibronectin, and proteoglycans. The collagenous layers of BrM are primarily composed of type I collagen and collagenous-associated proteins. It is believed that these components fortify the framework of BrM and aid in the resistance to the force that intraocular pressure exerts on the back of the eye. The basement membranes of the RPE and choriocapillaris endothelium are composed of type

IV collagen, which can indirectly interact with cells via laminin, and also binds to heparin and heparan sulfate proteoglycans. The central elastic layer consists of crosslinked elastin fibers that are associated with fibulin 5, an ECM protein that is thought to aid in elastin fiber assembly and serve as a link between the elastin fibers and cell surface receptors. BrM acts as a support and a barrier between the retina and the choroid, and is thought to support the many functions of the RPE. BrM is semipermeable, and therefore controls the transfer of molecules and cellular components between the RPE and the choroidal vasculature.

RPE–Choroid Interactions

Interactions During Development

The presence of the RPE is required for proper choroid development. Studies have shown that an intact, fully differentiated RPE is required for normal choroidal development; when the RPE is transdifferentiated into a neural retina by expressing fibroblast growth factor (FGF)-9 under the control of the tyrosine-related protein 2 (TRP-2) promoter, the choroid fails to develop. This is further illustrated in humans with colombas where RPE differentiation has failed and there are abnormalities in development of both the choroid and sclera. Basic fibroblast growth factor (bFGF) has also been implicated in choroidal development. Transgenic mice in which a dom- inant-negative bFGF receptor (FGFR1) was overexpressed in the RPE displayed choroidal abnormalities, such as incomplete and immature choroidal vessels.

The RPE also secretes a variety of growth factors and an increasing body of evidence indicates that RPEderived vascular endothelial growth factor (VEGF) is essential to choroidal development. Studies in humans and rodents have illustrated that both VEGF and its

receptor, VEGFR2, are highly expressed by the RPE and the underlying mesenchyme, respectively, at the time of choriocapillaris formation. Furthermore, mice with an RPE-specific deletion of VEGF presented a variety of defects, including microphthalmia, loss of visual function, and the complete absence of the choriocapillaris. In addition, the RPE itself was discontinuous, suggesting that RPE-derived VEGF is not only important for choroidal development, but also for RPE survival. Whether the RPE abnormality is secondary to the defects in choroidal development or due to a direct effect of VEGF on RPE has not been elucidated.

Interactions in the Adult

Normal RPE–choroid interactions are not only important during development, but also in the adult. Several studies have revealed the impact of RPE loss on choroidal structure and function. The presence of an intact RPE is critical to proper choroidal function, as surgical RPE removal causes several changes throughout the choroid. Both large choroidal vessels and the choriocapillaris display a reduction in circulation, and depending upon the extent of RPE removal, choroidal nonperfusion can be permanent due to fibroblast infiltration. A landmark study in which the RPE was selectively destroyed by sodium iodate treatment illustrated that within 1 week following iodate injection, the choriocapillaris had reduced fenestrations and displayed signs of atrophy, such as degenerating endothelial cells, and pericapillary basal laminae that had begun to separate from the apparently shrunken endothelium. Together, these data illustrate the critical role the RPE plays in both survival and in the maintenance of the choriocapillaris.

Growth factor secretion

RPE secretes a variety of growth factors, including VEGF, FGFs, transforming growth factor-b (TGF-b), and ciliary neutrophic factor (CNTF). The RPE also secretes pig- ment-epithelium-derived factor (PEDF), which functions not only to maintain the retina by acting as a neuroprotective factor, but also to provide antiangiogenic activity to inhibit endothelial cell proliferation, thereby stabilizing the choriocapillaris endothelium. VEGF, a well-characterized angiogenic factor, also plays a role modulating blood vessel permeability. Differential splicing of VEGF premessenger RNA (mRNA) gives rise to multiple isoforms, with the most notable being VEGF120, VEGF164, and VEGF188 in mice, and VEGF121, VEGF165, and VEGF189 in humans. VEGF120 does not bind heparin sulfate proteoglycans (HSPGs) and is readily diffusible, whereas VEGF164 is partially sequestered on the cell surface and in the ECM. VEGF188, with high affinity for heparan sulfate, is therefore primarily cell-surface- and matrix-associated. The RPE primarily expresses the

diffusible isoforms, VEGF164 and VEGF120, while VEGF188 is virtually undetectable. RPE-derived VEGF is essential for both survival and maintenance of the underlying choriocapillaris endothelium. Interestingly, PEDF and VEGF are secreted by the RPE in a polarized fashion in opposing directions. PEDF is secreted to the apical surface of the RPE to support the neurons and photoreceptors of the retina, whereas VEGF is primarily secreted to the basolateral surface where it acts on the choroidal endothelium (Figure 4). Despite the fact that a small fraction of total VEGF, a potent angiogenic factor, is secreted to the apical surface by the RPE, the outer nuclear layer of retina remains completely avascular, presumably due to the balance between antiangiogenic (PEDF) and angiogenic (VEGF) factors.

Receptor expression

In parallel with the many factors secreted by the RPE, the choroid expresses a number of corresponding receptors, including FGF receptors, type I and II TGF-b receptors, and VEGFR2. Of particular interest is the fact that VEGFR2 is observed primarily in the choriocapillaris adjacent to the RPE, with substantially less VEGFR2 expression observed in the major vessels of the choroid. Furthermore, VEGFR2 is localized to the apical surface of the choriocapillaris endothelium and to the photoreceptors where it is constitutively activated, which is surprising given that adult choroidal vasculature is mature and quiescent.

Isoform-specific VEGF mouse model

In support of a critical role for RPE-derived VEGF in the maintenance of the adult choriocapillaris, we have shown that the absence of soluble VEGF isoforms in the RPE leads to changes that recapitulate the classical features of dry age-related macular degeneration (AMD). Mice expressing only VEGF188 (i.e., lacking the diffusible isoforms that they normally express) display signs of RPE dysfunction, such as increased autofluorescence, loss of barrier proprieties, and accumulation of basal deposits that are similar to drusen, extracellular deposits that build up beneath the basement membrane of the RPE within BrM. These changes occur prior to the formation of both focal choroidal atrophy and RPE attenuation, which progress to large areas of RPE loss. The abnormalities are age dependent and increase in severity over time.

Choroidal change impact on RPE

The RPE–choroid interactions do not merely function to serve the choroid; changes in the choroid have also been shown to impact the RPE. Choroidal ischemia has been reported to lead to opaque RPE lesions and subsequent serous retinal detachment. Furthermore, very early studies on the effects of choroidal congestion on RPE–retina function revealed that increased choroidal pressure may

cause RPE–retina dysfunction by altering normal fluid movement across the RPE, modifying the integrity of the subretinal space.

RPE–Choroid Changes with Age

Various changes occur in the RPE–choroid complex with aging. In light of the close association among the RPE, BrM, and choroid, it is not surprising that an alteration in a single component of this complex compromises the normal RPE–choroid interaction and ultimately leads to disease. Studies have shown that the density and diameter of the choriocapillaris and medium-sized choroidal vessels substantially decline with age, resulting in decreased choroidal blood volume and blood flow. The aging RPE exhibit changes such as a reduction in cell density and a loss of RPE melanin. Melanin pigmentation is believed to play a protective role, acting to protect cells against oxidative stress. Oxidative changes in RPE melanin may be attributed to complexing of melanin with lipofuscin, pigment granules composed of lipid-containing residues of lysosomal digestion, which generate reactive oxygen species upon excitation with blue light, thereby making the aged RPE more susceptible to oxidative damage.

BrM Changes

Changes in BrM include increased thickness, accumulation of lipids, and subsequent alterations of the BrM

permeability. Collectively, these changes can limit the diffusion of water-soluble proteins, leading to a range of problems. Furthermore, drusen are known to accumulate in the aging eye (Figure 5). There is a strong correlation between the presence of drusen and ocular pathology, such as AMD. It has been speculated that drusen lead to a gradual reduction in the diffusion of RPE-derived factors, such as VEGF, which may be causal in the atrophy of segments of the RPE and underlying choriocapillaris, known as geographic AMD. Drusen accumulation and coalescence may also lead to breaks in BrM that are believed to be the initiating event in the formation of choroidal neovascularization (CNV), in which new blood vessels sprout from preexisting choroidal vessels and invade the overlying RPE and retina. The development of CNV associated with wet AMD leads to visual loss due to the fact that the neovessels are leaky and cause damage to the surrounding tissues.

Gene Expression

Gene expression of the RPE–choroid also changes with age. There is considerable upregulation of the expression of genes and proteins involved in leukocyte extravasation, and the accumulation of leukocytes at the RPE–BrM interface suggests that leukocytes are possibly recruited to aid in the removal of cellular waste. Furthermore, there have been reports of increased macrophages in the aged RPE–choroid; mice harboring mutations that render them

deficient in macrophage recruitment display hallmarks of AMD. It is also thought that the aged RPE–choroid synthesizes proteins that not only attract leukocytes, but that also activate the complement pathway, which is a part of the immune response and can lead to inflammation. Recent associations between polymorphisms in a member of the complement pathway reinforce the role of inflammation in the development of AMD.

Conclusions

Despite the fact that the RPE and choroid are separated by BrM, there is a great deal of interaction between the tissues. The presence of an intact, fully differentiated RPE is not only required for proper choroidal development, but is also essential for survival and maintenance of adult choriocapillaris endothelium specializations

(fenestrations) and integrity. The RPE secretes a variety of factors, one of the most notable being VEGF. The secretion of VEGF by the RPE is somewhat of a double-edged sword. Although VEGF is vital to choroidal homeostasis during both development and in adult, breakdown of the RPE barrier upon direct contact with choroidal endothelial cells is thought to involve a VEGF-mediated mechanism, for VEGF has been shown to mediate the vessel growth and permeability associated with wet AMD. Thus, maintenance of proper RPE–choroid interaction is vital to normal function, and any perturbation to this system can ultimately lead to disease.

See also: Breakdown of the RPE Blood–Retinal Barrier; Choroidal Neovascularization; Developmental Anatomy of the Retinal and Choroidal Vasculature; Immunobiology of Age-Related Macular Degeneration; RPE Barrier.

Further Reading

Gogat, K., Le Gat, L., Van Den Berghe, L., et al. (2004). VEGF and KDR gene expression during human embryonic and fetal eye development.

Investigative Ophthalmology and Visual Science 45(1): 7–14. Hartnett, M. E., Lappas, A., Darland, D., et al. (2003). Retinal pigment

epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism. Experimental Eye Research 77(5): 593–599.

Ivert, L., Kong, J., and Gouras, P. (2003). Changes in the choroidal circulation of rabbit following RPE removal. Graefe’s Archive for Clinical and Experimental Ophthalmology 241(8): 656–666.

Korte, G. E., Reppucci, V., and Henkind, P. (1984). RPE destruction causes choriocapillary atrophy. Investigative Ophthalmology and Visual Science 25(10): 1135–1145.

Mancini, M. A., Frank, R. N., Keirn, R. J., Kennedy, A., and Khoury, J. K. (1986). Does the retinal pigment epithelium polarize the choriocapillaris? Investigative Ophthalmology and Visual Science

27(3): 336–345.

Marneros, A. G., Fan, J., Yokoyama, Y., et al. (2005). Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.

American Journal of Pathology 167(5): 1451–1459. Ramrattan, R. S., van der Schaft, T. L., Mooy, C. M., et al. (1994).

Morphometric analysis of Bruch’s membrane, the choriocapillaris,

and the choroid in aging. Investigative Ophthalmology and Visual Science 35(6): 2857–2864.

Rousseau, B., Larrieu-Lahargue, F., Bikfalvi, A., and Javerzat, S. (2003). Involvement of fibroblast growth factors in choroidal angiogenesis and retinal vascularization. Experimental Eye Research 77(2): 147–156.

Saint-Geniez, M. and D’Amore, P. A. (2004). Development and pathology of the hyaloid, choroidal and retinal vasculature.

International Journal of Developmental Biology 48: 1045–1058. Saint-Geniez, M., Maldonado, A. E., and D’Amore, P. A. (2006). VEGF

expression and receptor activation in the choroid during development and in the adult. Investigative Ophthalmology and Visual Science 47(7): 3135–3142.

Saint-Geniez, M., Maharaj, A. S., Walshe, T. E., et al. (2008). Endogenous VEGF is required for visual function: Evidence for a survival role on Mu¨ller cells and photoreceptors. PLoS ONE 3(11): e3554.

Steinberg, R. H. (1985). Interactions between the retinal pigment epithelium and the neural retina. Documenta Ophthalmologica 60: 327–346.

Strauss, O. (2005). The retinal pigment epithelium in visual function.

Physiological Reviews 85(3): 845–881.

Zhao, S. and Overbeek, P. A. (2001). Regulation of choroid development by the retinal pigment epithelium. Molecular Vision 2(7): 277–282.

Glossary

Cytokines – A large and diverse family of polypeptide regulators that are used in cell regulation.

ELISA – The enzyme-linked immunosorbent assay is a biochemical technique that uses an enzymelinked antibody to detect its corresponding ligand within a sample. It can be used to assay for chemokines and cytokines.

Subretinal space (SRS) – The extracellular space between the retinal photoreceptors and the apical surface of the retinal pigment epithelium.

Tight junctions – A structure of specialized proteins that form a seal between neighboring cells and regulate ion and small molecule selectivity and resistance in the paracellular pathway between epithelial cells. Tight junction proteins include occluding, claudins, and junction adhesion molecules (JAMs).

Introduction

In the back of the vertebrate eye, the apical membrane of the retinal pigment epithelium (RPE) and the photoreceptor outer segments form a very tight anatomical relationship (Figure 1). This structural feature supports a whole host of mechanical, electrical, and metabolic interactions that maintain the health and integrity of the neural retina throughout the life of the organism. Like all epithelia, the RPE plasma membrane contains a wide variety of proteins, enzymes, and small molecules that are specifically segregated to the apical or basolateral sides of the epithelium, which face the neural retina and choroidal blood supply, respectively (Figure 2).

The asymmetrical distribution of these functionally distinct molecules is maintained by junctional complexes that surround each cell and by the continuous synthesis and regulated traffic of these molecules to each membrane. Epithelial polarity is defined by the steady-state maintenance of this asymmetric distribution and is critical for the ongoing vectorial transport of ions, metabolites, fluid, and waste products across the RPE. Epithelial polarity is also fundamentally important for controlling

changes in the volume and chemical compositions of the extracellular spaces on either side of the RPE, following transitions between light and dark. In the distal retina, the extracellular or subretinal space (SRS) separates the photoreceptor outer segments and the RPE apical processes. The chemical composition of this space is tightly buffered by the cells which surround it (Mu¨ller cells, photoreceptors, and RPE). On the opposite side of the RPE, an extracellular space is formed between its basolateral membrane and Bruch’s membrane, which is adjacent to the choriocapillaris. The physiological and pathophysiological states of the RPE/distal retina complex are significantly affected by changes in the chemical composition of these extracellular spaces as evidenced in disease processes such as age-related macular degeneration (AMD) or uveitis. AMD develops within the RPE/distal retina complex and eventually leads to RPE impairment and loss of photoreceptor function. The RPE’s ability to control and respond to varying levels of oxidative insult from light quanta, outer segment phagocytosis, vitamin A uptake and delivery, and oxygen consumption diminishes with age. These changes significantly affect the chemical composition of the surrounding extracellular spaces, SRS and choroid, and are a major factor in disease pathogenesis. In recent years, significant advances have been made in identifying the role of the immune system in neurodegenerative disease in general and in AMD, in particular (summarized by Hageman and colleagues and by Nussenblatt and Ferris). This article summarizes recent experiments from our lab and others, which show that inflammation induced changes in the environment surrounding human RPE can significantly alter intracellular signaling and physiology. This study provides a basis for understanding disease progression and regression.

This article is divided into three main parts. We begin with a description of our development of a robust and welldefined primary cell culture model of human fetal retinal pigment epithelium (hf RPE). We use this model to analyze how metabolic waste products, produced in the retina following light/dark transitions, can be disposed of by CO2/ HCO3 and lactate transporters located in the apical and basolateral cell membranes. In the second part, we use this cell culture model to analyze RPE antioxidant mechanisms that are protective against disease processes, such as AMD or uveitis. In the third part, we describe a series of experiments that use this model to define the impact of cytokines on human RPE function. Finally, we briefly focus on the role of interferon gamma (INFg) in controlling RPE physiology.

Human RPE: Morphology, Polarity, and

Function

The availability of native human tissue, fetal or adult, is limited and extant models of cultured human RPE have been, in varying degrees, less than adequately characterized or understood, not reproducible or available in large quantities, and lacking expression of melanin pigment and key functional proteins such as bestrophin (Best1), and RPE-specific protein 65 (RPE65). Therefore, we developed a set of standard procedures for producing confluent monolayers of hf RPE cells and demonstrated that they have the morphology, polarity, and function of the native tissue from which they were derived (Figure 3). Light and electron microscopy (EM) studies confirmed the presence of apical processes (microvilli) and basal infoldings that increased the elaboration of the apical and basolateral membranes, respective, in cultured hfRPE cells. We carried out immunoblot and immunofluorescence experiments for a variety of proteins to help define the polarity of this model. In the course of these experiments, we discovered that human RPE tight junctions contain a variety of

membrane proteins (claudins) that are important for regulating the selectivity and conductance of the paracellular pathway, and we also confirmed the presence of several visual cycle and cytoskeleton proteins. Intracellular recordings confirmed many membrane physiological properties and demonstrated the polarity of purinergic and adrenergic receptors at the apical membrane, which serve to regulate cell calcium and transepithelial fluid absorption (Figure 2). By enzyme-linked immunosorbent assay (ELISA), we showed that these monolayers constitutively secrete pigment epithelium-derived factor (PEDF) to the apical bath and vascular endothelial growth factor (VEGF) to the basal bath; the former provides neuroprotection for the retina and the latter would allow active regulation of endothelial cell fenestration, a structural feature critical for choroidal circulation.

pHi – Induced Changes in Fluid Absorption

In previous experiments we showed that acetazolamide, a carbonic anhydrase (CA) inhibitor, reduced net 36Cl flux across frog RPE. Subsequent animal models and clinical

trials have utilized CA inhibitors to reduce diseaseinduced abnormal accumulation of retinal fluid. CAs catalyze the reversible hydration of CO2 to HCO3 and protons, which are transported across the plasma membrane (e.g., Na/HCO3 or H/lactate co-transporters) to regulate cell pH. As a first step, we have identified and localized several highly expressed CAs in human RPE and begun study of their physiology. A total of 16 CAs have been identified in human tissues. In human fetal RPE cell cultures, 14 of the 16 known isozymes have been confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Immunocytochemical studies indicate that CA II is localized intracellularly, as in many other cell types. CA IV, XII, and XIV are localized to the apical surface, while CA IX, the most abundantly expressed isozyme in hfRPE cultures, is expressed apically and laterally (Figure 4). However, it should be noted that CA IX messenger RNA (mRNA) and proteins are not expressed in native adult or fetal human RPE. CA inhibitors have had limited success in alleviating the effects of retinal disease, partly because of systemic side effects, but mainly because of their lack of specificity. The positive clinical outcomes for some patients with retinal edema suggest that nonspecific CA inhibitors, such as acetazolamide, may be affecting multiple CAs or other transportrelated mechanisms that can either increase or decrease

net fluid absorption across the RPE in varying degrees in different patients. This is supported by in vivo animal studies, in which intravenous administration of acetazolamide to rabbits increased fluid clearance from the SRS. The regulatory role of CAs is potentially important in human RPE, which critically depends on HCO3 transport to maintain fluid absorption ( JV) out of the SRS (Figure 5).

Modulation of SRS Metabolic Load and

Chemical Composition

In the intact eye (cat/monkey), the transition from light to dark causes significant alterations in SRS pH, Ca2+, and K. In addition, the transition from light to dark increases photoreceptor O2 consumption by 2-fold as measured in situ in cat and nonhuman primate retina. The rates of retinal O2 consumption in light and in dark were used by Linsenmeier and Winkler and their colleagues to estimate the associated changes in glucose metabolism that leads to the concomitant release of carbon dioxide, lactic acid, and water from the photoreceptor inner segments into the SRS. This metabolic acid load is potentially damaging to all of the cells that surround the SRS (i.e., photoreceptors, Mu¨ller cells, and RPE). It raises the