Vamsi K. Gullapalli,
Ilene K. Sugino,
Marco A. Zarbin
MÜLLER CELLS
INTRODUCTION
Müller cells, first described by Heinrich Müller in 1851,[1] often are thought of as providing a structural framework for the retina. They are large cells spanning the entire width of the retina, from the internal limiting membrane (ILM) to processes extending into the subretinal space. Müller cells belong to the class of glial cells called astrocytes, which are characterized by a large nucleus, prominent cellular processes, and participation in neural homeostasis. However, Müller cells also exhibit functions of another type of glia, oligodendrocytes, which supply energy to neurons. Müller cells are truly retinal cells, as they originate in the retina, are found exclusively in the retina, and are the major glial cells of the retina.
OVERVIEW OF THE MÜLLER CELL AND ITS FUNCTIONS
Based on the intricate and extensive processes emanating from a single cell body, it is easy to imagine the Müller cell having the function of support, as Müller cells fill all extracellular spaces in the retina. Besides providing support for retinal neurons, Müller cells define outer and inner boundaries of the retina. Their endfeet terminate at the ILM, and their junctional complexes form the outer limiting membrane (OLM). The elaborate structure of the Müller cell involves contact with all parts of the retina, which enables Müller cells to provide functions other than support to the various cells they contact. During retinal development, Müller cells show extreme plasticity; they localize to areas of the retina undergoing development and differentiation and show localized development of processes in parallel with development of the adjacent neuroblast. In the mature retina, Müller cells provide support for adjacent cells, maintaining control of the extracellular environment and providing nutrients to maintain the high metabolic rate of the retina. Additionally, Müller cells are very important in the visual cycle of cones, providing the substrate for regeneration of cone opsin. Regional specializations to support these functions are present within each Müller cell.
EMBRYOLOGY AND DEVELOPMENT OF MÜLLER CELLS
The earliest morphologically distinct step in the development of the eye is the bilateral evagination of the diencephalon region of the neural tube on day-25 postconception.[2] However, it is likely that the cells in this region are already committed to the eye long before this event. Continued evagination of the diencephalon leads to the formation of the optic vesicle that abuts against the surface ectoderm. The cornea and lens develop from the surface ectoderm. The optic vesicle then invaginates on day-27 to form a bilayered optic cup. The inner layer comprises progenitor cells that give rise to the cells of the neural retina; the outer layer develops into retinal pigment epithelium (RPE). As in other parts of nervous system, the neural retina initially consists of a neural epithelium and inner marginal zone. The neural epithelium develops into an inner and outer neuroblastic layer and a transient layer of Chievitz. By the fourth month, all the layers of retina are discernible.[3]
Cell division generating the cells of the neural retina occurs in two phases. In phase one, ganglion, cone, and horizontal cells develop. In phase two, rod, bipolar, and Müller cells develop. Amacrine cells develop during both phases.[4] Animal studies suggest that a single progenitor cell gives rise to a column of cells containing one Müller cell ensheathing a defined number of rod, bipolar, and amacrine cells.[5]
Immunohistochemical studies of fetal human tissue show that the progenitor cell itself may differentiate into a Müller cell.[6] Müller cells are thought to be a key component in this columnar arrangement of cells, providing nutrients, guiding associated neurons in migration to their final positions, and acting as a substrate for neurite outgrowth.[5,7] In cell culture experiments utilizing stratospheroids of retinal cells of two different species (chick and quail), Willbold and coworkers demonstrated that spheroids composed of species-specific radial sectors or columns form, surrounded by a Müller cell forming a barrier to cross species cell lateral migration.[8] Selective damage to Müller cells showed their continued presence was necessary to maintain the columnar arrangement.[8] Cell recognition markers such as L1/NgCAM, 5A11, and F11 are present in Müller cells, which supports their role in migration and formation of radial columns. In addition to these migration cues, Müller cells also express barrier proteins providing negative cues that may constitute a repulsive barrier (e.g., EAP-300; embryonic avian polypeptide and clustrin).[4,8] Of note, these experiments showed that while cross species cell migration between radial columns is inhibited, lateral processes from Müller cells and their associated neurons can cross the species-specific border, suggesting their role in neurite outgrowth. Studies of neonatal rat retinal cells show that Müller cells can support long neurite extensions in rods while other retinal neurons are less supportive.[7]
Morphological studies of the human fetus have demonstrated the anatomic distribution of Müller cells during development.[9,10] Müller cells are present at gestation week-7 (20 mm) and are present in both neuroblastic layers although they are more common in the inner half of the inner neuroblastic layer. Dense processes extend from the Müller cells to the surface of the nerve fiber layer (NFL), and radial processes are found between ganglion cell axons. At gestation week-9 (40-43 mm), Müller cells are the major component of the inner neuroblastic layer, forming columns and extending across the full thickness of the retina. Dense inner processes are present near ganglion cells and are thought to guide the ganglion cells into the inner retina. At 66 mm, Müller cell nuclei and outer processes are observed between differentiating cones. By gestation week-13 (96 mm), Müller cells are present in all layers of the retina. At 159-200 mm, when the rods and cones are present, Müller cell nuclei are still present in the outer nuclear layer (ONL), are numerous in the inner nuclear layer (INL), and are still present in the ganglion cell layer and NFL. At week-20 (180 mm), Müller cell processes extend microvilli into the subretinal space. Observations from these studies show that Müller cell processes differentiate sequentially from the inner (vitread) to the outer (sclerad) retina, differentiating in synchrony with the cell on which the process abuts. Different parts of the same cell can differentiate at different times while other processes can differentiate at the same time but in morphologically different ways. It is not clear whether the differentiation of different portions of the Müller cell is in response to the demands of adjacent cells or if Müller cell process differentiation leads to an induction of neuronal differentiation.[10] The apparent shifts in location of Müller cell nuclei are believed to be due to migration of the nucleus along the cell length, cell loss, and, possibly, cell division.[9]
ANATOMY
Generally, Müller cells are recognized histologically and ultrastructurally by their relatively dense cytoplasm, which contains numerous glycogen particles, abundant filaments measuring 100å in diameter, and well-developed smooth endoplasmic reticulum consisting of vesicles of varying sizes and shapes.[11] In the adult eye, Müller cells span the entire width of the retina with their cell bodies located in the INL and nuclei located in the middle of this layer. Radial processes (Fig. 125.1a) extend vitread to terminate near the ILM and sclerad to contribute to the OLM, and lateral processes extend out at all levels of the neural retina. In the nuclear layers, lamellae form a network surrounding cell bodies (Fig. 125.1b, 'honeycomb meshwork'). In the plexiform layers, processes are interwoven between synaptic processes of neurons, ensheath dendrites, and surround axons in the NFL (Fig. 125.1c, 'horizontal fibers'). Müller cells cover most, but not all, of the neuronal surfaces. Small branches extend out to contact and surround blood vessels along with astrocytes, forming alternate layers of astrocyte and Müller cell processes, completely isolating vessels from the rest of the neural retina.[12] Radial trunk extensions thin in the inner plexiform layer (IPL), pass through the ganglion cell layer, and intertwine with astrocytes and microglia forming an endfoot expansion terminating at the ILM. Viewed en face, the endfeet form a mosaic pattern. Particle arrays, referred to as intramembranous orthogonal arrays of particles (OAPs), are prominent in the endfeet near the vitreous surface and in areas where processes contact blood vessels. The function of OAPs is not certain, although based on their location, they have been proposed to function as sites of K+ conductance.[13]
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FIGURE 125.1 Schematic drawing illustrating the complex structure of Müller cells. Müller cells (shown with dark cytoplasm and dark nuclei) extend radial processes (a) from the cell body to form the internal limiting membrane of the retina surface and the external limiting membrane facing the retinal pigment epithelium (RPE). Lateral processes (b, honey comb meshwork) extend from the radial processes to ensheath cell bodies of the ganglion cells, cells of the internal (inner) nuclear layer, and cell bodies of the photoreceptors in the external (outer) nuclear layer. Horizontal fibers (c) are found in the plexiform layers and the nerve fiber layer where they ensheath neurons and their processes. |
At their outermost ends, Müller cells exhibit junctions (zonula adherens) between other Müller cells and photoreceptors to form the OLM, which is thought to have a supportive function in maintaining the alignment and orientation of the photoreceptors.[14] Extending past the OLM into the subretinal space are long, thin apical Müller cell processes filling the spaces between photoreceptor inner segments (Figure 125.1d, 'fiber baskets').[11]
In the fovea centralis, Müller cell processes occupy most of the inner third of the retinal thickness. The processes form an inverted cone-shaped zone whose apex faces the ONL (Fig. 125.2). In the 50 ?m diameter apex of the Müller cell cone, Müller cell processes are present with atypical watery cytoplasm separating the foveal cone cells.[15] The Müller cell cone is hypothesized to be a reservoir for retinal xanthophylls in addition to providing structural support for the receptor cells of the fovea.[16] The Müller cell cone is thought to be involved in idiopathic age-related macular holes.[16] The role of Müller cells in creating structural support in the retina is illustrated by the condition, juvenile X-linked retinoschisis (JXRS). JXRS is caused by mutations in RS1, which encodes the protein, retinoschisin. Retinoschisin is secreted by photoreceptor and bipolar cells.[17-19] JXRS is a congenital disease characterized by foveal schisis in 100% of patients and peripheral retinoschisis in ?50% of patients. The electroretinogram demonstrates reduced b-wave amplitude and relative a-wave preservation. Visual acuity usually is in the range of 20/40-20/60. Three dimensional optical coherence tomography demonstrates a large cystic space in the fovea (probably in the outer plexiform layer (OPL)) as well as cleavage planes in the NFL, GCL, and OPL (Fig. 125.3).[20] Bridging columns spanning these cystic spaces probably represent Müller cells and demonstrate the mechanical effect of these cells on retinal architecture.
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FIGURE 125.2 Details of the fovea centralis. The nuclei of the photoreceptors are displaced internally (a) and the curving internal receptor fibers are seen at (b). The lighter cytoplasm of the Müller cells (arrow) is seen in the center of the fovea. |
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FIGURE 125.3 B-scans of 3-D OCT from a patient with juvenile X-linked retinoschisis. Two wide hyporeflective spaces split the retina. Anteroposterior or oblique linear columns form a bridge across a superficial wide hyporeflective space. In the same layer, there is a large cystoid space in the fovea (line N). This layer is probably the outer plexiform layer. Deeper cleavage is seen in the parafoveal area but not in the fovea (line P). This layer is probably the outer nuclear layer. Small cystoid spaces (arrowhead) are seen in the superficial parafoveal retina that split the retina (line M). This layer is probably the nerve fiber layer or the ganglion cell layer. |
FUNCTIONS
The complex structure of Müller cells, spanning the width of the retina with processes extending laterally, places them in a position to perform many different functions in the retina. Across the width of the retina, Müller cells are thought to control and regulate molecules and ions. In localized areas, Müller cells can control the extracellular microenvironment of neuronal cell bodies and synapses. Müller cells play an important role in retinal signaling processes by removing neurotransmitters rapidly from the extracellular space. Also, due to intimate association with the retinal vasculature, Müller cells are implicated in movement of substances into and out of the retinal vessels. Specialized regions of Müller cells, located adjacent to the regulated region of the neuron or endothelial cell, contain the specific molecules needed to carry out these support functions. The following sections describe some of the functions attributed to Müller cells in the adult eye.
Regulation of the Extracellular Space
Potassium
Light-evoked neuronal activity causes an increase in extracellular K+ concentration ([K+]0) in the synaptic layers while [K+]0 decreases adjacent to the photoreceptor nuclei. One very important function of Müller cells is the regulation of [K+]0 since fluctuations alter neuronal excitability. In a process termed 'K+ siphoning',[21] movement of K+ from areas of high [K+]0 to low concentration is thought to be achieved by nonuniform distribution of potassium channels.[22] Several types of potassium channels have been identified in Müller cells, and it appears that the type of channel may vary considerably among different species.[14] An important K+ channel thought to contribute significantly to the Müller cell K+ buffering system is the Kir channel, found in the plasma membrane. Studies performed in mice identified two Kir channels with very different subcellular concentrations.[23] Kir2.1, a strongly inward rectifying channel, was identified in regions of high K+ flow into Müller cells (i.e., membrane domains localized near retinal neurons, including the synaptic layers). These channels are such that K+ enters more readily than it can exit; thus, these channels are described as inwardly rectifying potassium channels. A weakly rectifying channel (i.e., movement of K+ inward or outward depends upon the K+ concentration), Kir4.1, was detected in Müller cells throughout the retinal layers and was highly concentrated in the membrane domains of the endfeet facing the vitreous humor and in membranes of processes ensheathing blood vessels. Thus, the vitreous humor and blood vessels may act as potassium sinks. A third Kir channel, Kir5.1, was found in rats to be present diffusely in Müller cell bodies and appeared to form a heteromeric channel with Kir4.1 in the distal processes (fiber baskets, Fig. 125.1) in the subretinal space and in processes of Müller cells located in the IPL, INL, OPL, and ONL but not in the endfeet and processes surrounding blood vessels where homomeric Kir4.1 channels are found. The properties of the two channels are different (e.g., pH sensitivity of the Kir4.1/Kir5.1 channel), and therefore, they are thought to play different functional roles in K+ buffering.[24]
Studies in rodents have shown co-localization of Kir4.1 channels with the water channel, aquaporin-4, in membrane domains facing the vitreous humor and blood vessels, indicating that potassium and water movement are closely linked in Müller cells. The localized distribution of aquaporin in Müller cell membranes indicates that Müller cells mediate osmotic gradients associated with K+ movement and function to direct water flux to specific extracellular compartments. The tight co-localization of the two channels may support the movement of K+ while preventing osmotic imbalances in the retina.[25]
Another type of potassium channel, the tandem pore channels TASK-1 and TASK-2, has been localized in the cell body and fiber baskets of mammalian Müller cells (e.g., rat, mouse, and guinea pig). These channels, along with Kir 4.1 channels, are thought to maintain the negative resting potential of Müller cells in nonpathological conditions and could play a minor role in K+ siphoning. Skatchkov et al. suggest these channels are important in maintaining membrane hyperpolarization in areas where Kir4.1 channels are sparse and strongly rectifying channels, such as Kir2.1, are located (e.g., photoreceptor cell bodies). TASK channels may become important in potassium regulation when Kir4.1 channels are downregulated (see section on Müller cell gliosis).[26]
CO2 and pH regulation
The high metabolic rate of the retina leads to the generation of large amounts of CO2. Müller and RPE cells contain the sodium-bicarbonate co-transporter, pNBC1,[27] and large stores of carbonic anhydrase, an enzyme that converts CO2 to bicarbonate in a reaction helping to maintain the extracellular pH.[28] One form of carbonic anhydrase, CAXIV, is located in the membrane domains of Müller cells facing the photoreceptors and is also enriched in the Müller cell endfeet. The presence of CAXIV in the Müller cell endfeet has led to the notion that the endfeet are a clearance route for CO2 to the vitreous cavity and retinal blood vessels.[28]
Neurotransmitter uptake and conversion
Müller cells are important in regulating the extracellular levels of glutamate, the major excitatory transmitter of neurons of the retina, and ?-aminobutyric acid (GABA), the major inhibitory transmitter. Potent uptake systems and degradative pathways exist for both transmitters in Müller cells. Glutamate is also taken up by neurons where it is recycled, but in Müller cells it is thought to be inactivated by conversion to glutamine in a reversible reaction catalyzed by glutamine synthetase, which in the retina is present only in Müller cells. The conversion of glutamate to glutamine involves ammonia fixation, ammonia production being associated with increased synaptic activity. Thus, another important function of Müller cells may be extracellular ammonia detoxification.[29] Both glutamine synthetase and the Müller cell-specific glutamine transporter, GLAST (L-glutamate-L-aspartate transporter), are co-localized in the fine horizontal processes of Müller cells that ensheath rod photoreceptor synaptic terminals.[30]
Müller cells contain GABA transporters (GAT-1 and GAT-3) and the GABA-degrading enzymes, GABA-transaminase (GABA-t) and succinic semialdehyde dehydrogenase (SSADH), located in mitochondria, indicating their role in the regulation of GABA concentration in the extracellular space. In mammalian Müller cells, the predominant transporter is GAT-3, which is found in the plasma membrane throughout the cell.[31] Once transported into the cell, GABA and ?-ketoglutarate are catalyzed by GABA-t to glutamate and succinic semialdehyde. Succinic semialdehyde is then degraded by SSADH to succinate, which can then enter the tricarboxylic acid cycle.[14]
Glycogen Metabolism
Müller cells are thought to play a central role in maintaining the nutritional needs of the retina (see Winkler et. al and references therein[32]). Glucose is thought to be transported from the blood by Müller cells via facilitated diffusion mediated by the glucose transporter, GLUT 1, converted into glycogen, and stored.[33] GLUT 1 has been demonstrated in the plasma membrane of human Müller cells.[33] (GLUT 2 has been localized to the apical processes of rat Müller cells (fiber baskets of Fig. 125.1), indicating one possible function for these structures.[34]) Müller cells contain the metabolic pathways for glycogen deposition and are the sole site of glycogen storage in the retina. Glucose uptake is thought to be stimulated by synaptically released glutamate.[29] Similarly, glycogen breakdown and release of metabolic substrates is thought to be regulated by neuroactive substances released by neurons.[14]
Visual Pigment Recycling
In studies of cone-dominant eyes of chicken and ground squirrel, Müller cells were found to contain an alternate visual cycle for cone opsin regeneration that is distinct from the visual cycle in RPE cells (Fig. 125.4, cycle indicated with black text and black arrows). All-trans-retinol, which is released into the extracellular space by rods and cones following light activation of an opsin pigment molecule, can be taken up by Müller cells, directly converted to 11-cis-retinol by a unique all-trans-retinol isomerase, and bound by cellular retinaldehyde binding protein (CRALBP). The 11-cis-retinol is released into the extracellular space where it can be taken up by cones for regeneration of visual pigment utilizing the cone-specific 11-cis-dehydrogenase. Both 11-cis-retinol and all-trans-retinol are bound to interphotoreceptor retinoid binding protein (IRBP) during translocation between the cones and Müller cells.[35] The energy source for this isomerization may be a palmitoyl CoA-dependent esterification of 11-cis-retinol and all-trans-retinol.[35,36]
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FIGURE 125.4 Cone opsin recycling pathway in Müller cells. Generation of 11-cis-retinol from all-trans-retinol (released into the extracellular space) (black text).[35,36] Proposed generation of 11-cis-retinal from all-trans-retinol involving the enzymes, retinal G protein-coupled receptor (RGR) and all-trans retinol dehydrogenase (RDH10) (blue text).[37] |
In RPE cells, RDH10 (an all-trans-retinol dehydrogenase) oxidizes all-trans-retinol to all-trans-retinal in the photic visual cycle (see section on Role in Visual Cycle in The Retinal Pigment Epithelium). When exposed to light, the RPE retinal G protein-coupled receptor (RGR) isomerizes all-trans-retinal to 11-cis-retinal. Both RDH10 and RGR have been found in Müller cells (although in lower levels than found in RPE), indicating the possibility that Müller cells can generate 11-cis-retinal from all-trans-retinol utilizing mechanisms similar to those found in RPE cells (Fig. 125.4, proposed cycle indicated by blue text and blue arrows).[37]
Neuronal Activation of Müller Cells
As noted above, Müller cells affect the function of retinal neurons by regulating the extracellular environment. However, neurons seem to be able to affect Müller cells directly by neuronal signaling. Light stimulation of the retina, for example, results in a transient increase in Ca2+ in the Müller cell processes located in the IPL, which spreads to the cell endfeet at the vitreal surface. The resultant release of ATP by Müller cells is thought to enhance or depress neuronal activity in a neuronal excitability feedback mechanism.[38]
Müller Cell Contribution to the ERG
Early studies by Witkovsky[39] on isolated retina showed that Müller cells contribute to the negative slow PIII of the c-wave of the ERG. The Müller cell response was attributed to the photoreceptor-dependent light-evoked decrease in subretinal extracellular [K+]0.[40] (RPE contributes to the positive component of the c-wave; please see section, Ion Transport in The Retinal Pigment Epithelium). Further evidence indicating that K+ movement by Müller cells contributes to the ERG is the loss of the PIII wave in Kir4.1 knockout animals.[41] Some studies indicate that K+ buffering by Müller cells may also contribute to the b-wave although conflicting studies attribute the b-wave to depolarizing bipolar cells.[14] The lack of change in the b-wave response in the Kir4.1 knockout mice supports the neuronal origin of the b-wave.[41]
Phagocytosis
Müller cells can phagocytose a variety of substances (e.g., latex beads, carbon, copper particles, blood cells) and are thought to assist the RPE cells in phagocytosis of outer segments.[14] In development and in retinal disease, Müller cells can phagocytose debris from dying neurons.[42] Also, the phagocytotic capacity of Müller cells may aid in maintaining the clarity of the vitreous humor.[42]
PATHOLOGY
Müller cells play a prominent role in injury and diseases of the retina. They exhibit the ability to respond to subtle insults by modulation of normal functions (e.g., moderating the rise in extracellular K+ and glutamate following plasma leakage into the retina[43]) as well as the ability to undergo marked functional and morphological change in response to more severe insults. Müller cells can undergo cell division and migration.[44] Müller cells can respond to a number of growth factors, cytokines, and neurotransmitters derived from photoreceptors and other neurons and RPE cells. In pathological conditions, inflammatory cells, platelets, and plasma can activate Müller cells.[45] Some Müller cell responses are related to tissue repair (e.g., resurfacing of Bruch's membrane following RPE and photoreceptor loss to maintain the blood-retina barrier in atrophic macular lesions[14]) and wound healing (e.g., Müller cell wound closure of the OLM following macular hole repair by vitrectomy with cortical vitreous and epicortical vitreous membrane peeling[46]) while other responses are related to neuroprotection (see section on Growth Factor, Cytokine, and Neurotrophic Factor Secretion) and detoxification (e.g., Müller cell response to elevated ammonia levels in hepatic retinopathy[47,48]).
Additionally, since Müller cells play a prominent role in retinal homeostasis, any dysfunction involving Müller cells can have a profound effect on retinal function. For example, long-term replacement of the vitreous with a fluid, such as perfluorocarbon or silicon oil, incapable of acting as a K+ sink could interfere with Kir function and might lead to outer retina degeneration especially when little vitreous fluid remains.[49] Similarly, damage to the Müller cell endfeet, such as occurs in retinoschisis, would impair spatial K+ buffering capabilities of damaged cells.[42] In a study comparing healthy versus diseased eyes, Müller cell ion channels appeared to be affected in the diseased eye. For example, changes in Müller cell membrane properties (e.g., down-regulation of Kir current density) have been found in diseased retinae from human eyes with secondary glaucoma, mechanical injury, choroidal melanoma, and retinal tissue obtained from partial retinectomies to relieve traction.[50,51] Loss or diminished activity of ion channels can lead to a change in the Müller cell membrane potential and affect the ability of Müller cells to take up glutamate. Since glutamate is highly toxic to the photoreceptors, extracellular regulation of glutamate is critical for vision preservation.[50,52,53]
Although there are no diseases of the retina in which Müller cells are known to be the primary site affected, Müller cells appear to be involved in many retinal disorders.[14] The role of Müller cells in specific diseases is addressed elsewhere (Chapters 182 and 183). Müller cell responses in general are discussed in the following sections.
Müller Cell Gliosis
Pathologies involving neuronal degeneration in the retina appear to be associated with some level of Müller cell activation (gliosis).[44] Müller cell response or activation can be detrimental or beneficial, depending on the magnitude of the response. For example, Müller cell activation can be beneficial if modest, as activation could be helpful due to increased growth factor secretion by the cells (see section on Neuroprotection). If gliosis results in proliferation and/or is long-lasting, gliosis can lead to neuronal degeneration (e.g., subretinal fibrosis following retinal detachment and epiretinal membrane formation leading to retinal detachment).[54] Müller cell activation can be detected by upregulation of the intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP). Since GFAP expression is not detected in healthy retinas, GFAP detection can be used as an early indicator of reactive Müller cells.[14] Another early indicator of Müller cell activation is activation of ERKs (extracellular signal-regulated kinases) and release of neuroprotective growth factors (see section on Growth Factor, Cytokine, and Neurotrophic Factor Secretion).[55] Gliosis can lead to changes in cell shape including changes to or loss of processes.[56] In the detached retina, gliotic responses also include downregulation of glutamine synthetase, cellular retinaldehyde-binding protein, K+ channel expression, and carbonic anhydrase.[57] Studies performed in rats (ischemia model), pigs, and rabbits (detachment models) show that one of the consequences of Müller cell activation can be the loss or relocation of Kir4.1 channels. Impairment of potassium buffering capabilities through loss of Kir channels could lead to the inability of the cells to maintain their hyperpolarized state and thus lead to breakdown of normal homeostasis (e.g., K+ buffering and water regulation) resulting in edema and retinal degeneration.[57-59] In the rabbit and pig detachment models, and as sometimes occurs in patients following retinal reattachment, loss of photoreceptors (or visual function) can be seen in attached areas adjacent to the reattached retina. This loss is attributed to activation of Müller cells adjacent to the area of detachment.[57,59]
As previously mentioned, Müller cells can respond to a number of stimuli, in particular mitogenic growth factors.[45] Blood-derived factors (e.g., thrombin) released from the breakdown of the blood-brain barrier may trigger the proliferative response through downregulation of Kir channels.[45,60] In addition to Kir channels, which mediate potassium levels during light-activated K+ elevation in the extracellular space (K+ siphoning), Müller cells express other ion channels including Ca2+-activated K+ channels.[60] Studies comparing Müller cells from diseased and healthy retinae show impaired K+ conductance due to loss of Kir channels in cells from diseased retinae although Ca2+-dependent K+ channels are activated.[50,52] The Ca2+-dependent K+ channels are activated when the membrane potential of the cell is markedly reduced (as in pathological conditions when the Kir channels are lost) and are not thought to play an important role in potassium siphoning in the normal retina.[61] Müller cell proliferation can be triggered by mechanisms activating these Ca2+-dependent K+ channels.[60] The mechanism of Ca2+-dependent K+ channels activation is thought to be through activation of P2 receptors. The P2Y receptor, minimally active in healthy retinae, is activated by ATP. Following P2Y receptor activation, the cells become unresponsive to ATP and cease to proliferate. The receptors can be re-sensitized to ATP following exposure to growth factors (e.g., platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and nerve growth factor (NGF).[57,62]
Müller cell proliferation also can be initiated by mitogenic growth factors. Basic fibroblast growth factor (bFGF), which has been shown to be released intraretinally following injury (mechanical or light-induced) and retinal detachment, is a potent growth factor in Müller cell activation and proliferation. Other growth factors shown to evoke Müller cell proliferation include PDGF, heparin-binding epidermal growth factor (HB-EGF), and transforming growth factors (TGF).[63]
Neuroprotection
Elevation of the extracellular glutamate concentration is highly toxic to retinal neurons.[56] Extracellular glutamate concentrations are highly regulated through Müller cell uptake involving the GLAST transporter. Excess glutamate can be generated in a variety of retinal conditions including ischemia, hypoglycemia, glaucoma, diabetes, and trauma.[14,56] Accordingly, some studies have correlated increases in GLAST activity with elevated glutamate levels.[56] However, in diabetic retinopathy, GLAST levels appear to decrease (possibly due to oxidative damage since the effects can be reversed by exposure to a disulfide-reducing agent) leading to elevated glutamate levels and resultant glutamate excitotoxicity.[64,65]
Studies in rodents have shown that intraocular injection of neurotrophic factors can promote photoreceptor survival in retinal degenerations caused by genetic or environmental factors.[66-68] Neuroprotection of photoreceptors following injection of growth factors (e.g., brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), or bFGF) is thought to be mediated by activation of Müller cells, since photoreceptors are not activated by exposure to these growth factors as indicated by lack of intracellular signaling pathway activation.[69,70] Sustained growth factor delivery by viral vectors to activate Müller cells offers a possible treatment to prevent retinal degeneration.[71,72] The mechanism by which activated Müller cells affect photoreceptor survival is not known.
Nitric Oxide Synthetase
Nitric oxide synthetase (NOS) catalyzes the formation of highly reactive nitric oxide (NO) from L-arginine. Nitric oxide has been implicated in the regulation of retinal blood flow, in visual transduction, and is toxic to microorganisms.[73] As such, NO may also contribute to retinal pathology and has been implicated in reperfusion injury following ischemia, diabetic vascular damage, HIV retinitis, and endotoxin-induced uveitis.[73,74] Histochemical staining for NOS has shown its presence throughout the retina;[73] mRNA for inducible and constitutive forms of nitric oxide synthetase (iNOS and cNOS, respectively) have been found in human retinal tissue.[75] cNOS is calcium-dependent; iNOS is calcium-independent. iNOS is expressed in Müller cells following stimulation by interferon (IFN)-? and lipopolysaccharide[76]and, following upregulation, can produce sustained levels of NO. cNOS (found in mammalian Müller cells of mice[77] and the tree shrew,[78]) is relatively short-lived and produces picomolar quantities of NO.[73](iNOS has also been detected in human RPE following stimulation by interleukin-1 and IFN.[73]) mRNA for iNOS found in Müller cells of mice is similar in size to that of macrophage iNOS and thus may be important as a reactive mediator in inflammation and infection.[79] However, the exact role of iNOS in pathology and regulation of NO by Müller cells is not understood fully.
Müller Cell Immune Modulation
Experimental evidence from in vitro cell culture studies indicates that Müller cells may provide an immune modulation function in the retina. Müller cells, along with RPE and vascular endothelial cells, have been shown to express MHC class II (MHCII) antigens.[14] Müller cells from rat retina can be induced to proliferate following addition of conditioned media from activated spleen cells or activated lymphocytes and can express MHCII antigens.[80] Müller cells in the human eye of a patient with hypopigmented subretinal fibrosis and uveitis syndrome were shown to express MHCII antigens by immunohistochemical staining.[81] However, when Müller cells expressing MHCII antigens were cultured with activated T cells, proliferation of the latter was inhibited. The prevention of T cell proliferation required physical contact between Müller cells and T cells, indicating that the inhibition was through a membrane bound protein present in the cells.[82] When this membrane bound protein is inhibited (e.g., using glucocorticoids), Müller cells can function as an antigen presenting cell.[83] Thus, Müller cells have the potential to inhibit or stimulate T cell proliferation. Since lymphocytes migrating out of retinal capillaries contact Müller cells, the interaction between the two cells could either suppress retinal inflammation or enhance it.[14]
Growth Factor, Cytokine, and Neurotrophic Factor Secretion
Müller cells and RPE can up-regulate the expression of growth factors, cytokines, and neurotrophic factors in response to retinal injury or other stimuli (Table 125.1).[84] In addition to acting on photoreceptors, Müller cells also may be involved in ganglion cell and bipolar cell neuroprotection.[56] Secreted growth factors such as bFGF, VEGF (vascular endothelial growth factor), and PEDF (pigment epithelium-derived factor) appear to provide constitutive trophic support to the photoreceptors and vascular endothelia.[85-87] Changes in the endogenous concentration of these growth factors can affect surrounding cells. For example, PEDF downregulation results in increased Müller cell secretion of VEGF and TNF-? (tumor necrosis factor), which may contribute to inflammation and vascular changes associated with diabetic retinopathy.[86,87] Müller cells contribute to the endogenous levels of VEGF, PEDF, TGF-?2, TSP-1 (thrombospondin), BDNF, and bFGF.[88-90] Changes in bFGF secretion by Müller cells can result in either protection (increased bFGF secretion) or increased apoptosis (decreased bFGF secretion) in adjacent photoreceptors.[91] Microglia are an endogenous source of growth factors leading to Müller cell stimulation resulting in increased (e.g., NT-3) or decreased (e.g., NGF) bFGF secretion.[92]
TABLE 125.1 -- Müller Cell Growth Factor, Cytokine, and Neurotrophin Secretion Following Stimulation
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Growth Factor Secreted |
Stimulus |
Model |
Associated Retina Pathology or Function |
References |
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BDNF, NGF, NT-3, NT-4, GDNF |
Glutamate |
Cultured rat MC |
Neuroprotection during glutamate toxicity |
95 |
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VEGF |
TGF-?, hypoxia |
Cultured rat MC |
Hypoxia-induced angiogenesis |
96 |
|
TNF-?, NO |
IFN-? and LPS |
RCS cultured rat MC |
PR degeneration in retinal dystrophies |
97 |
|
TNF-?, IL-6, IFN-?, IFN-? |
HSV-1, IFN-? |
Mouse and cultured mouse MC |
Immune and inflammatory responses |
93,94 |
|
bFGF, CNTF, GDNF |
Light, mechanical injury |
Light damaged rat |
Neuroprotection |
44 |
|
VEGF, TSP-1 |
Hypoxia |
Cultured human |
TSP-1 angiogenesis inhibition; VEGF angiogenesis induction |
88 |
|
VEGF |
HB-EGF |
MIO-M1 |
PVR |
100 |
|
VEGF, TNF-? |
Reduced PEDF level |
Cultured rat MC |
Diabetic retinopathy |
87 |
|
VEGF |
Advanced glycation endproducts (AGE) |
Cultured human MC |
Diabetic retinopathy |
101 |
|
bFGF |
bFGF |
Cultured rat MC |
Injury response to secreted bFGF (neuroprotection) |
102 |
|
bFGF |
NT-3 |
Cultured rat MC |
Neuroprotection |
92 |
|
CNTF |
Optic nerve damage |
rat |
Neuroprotection |
103 |
|
BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT, neurotrophin; GDNF, glial cell line-derived neurotrophic factor; MC, Müller cells; VEGF, vascular endothelial growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor; NO, nitric oxide; IFN, interferon; LPS, lipopolysaccharide; RCS, Royal College of Surgeons; PR, photoreceptor; IL, interleukin; HSV-1, herpes simplex virus type 1; bFGF, basic fibroblast growth factor; CNTF, ciliary neurotrophic factor; TSP, thrombospondin; MIO-M1, spontaneously immortalized human Müller cell line; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PVR, proliferative vitreoretinopathy; PEDF, pigment epithelium-derived factor. |
Both in vivo and in vitro studies show that Müller cells respond to many different stimuli and can secrete different growth factors, depending on the stimulus or culturing conditions (Table 125.1). In addition to providing neuroprotective growth factors and factors capable of modulating blood flow and vascular permeability, Müller cells can produce several pro-inflammatory cytokines in response to viral infections.[93,94]
Two types of receptors, Trk (high-affinity neurotrophin tyrosine kinase receptor) and p75 (low-affinity neurotrophin receptor), have been identified in pro-survival (Trk) and antisurvival (p75) signaling by neurotrophins.[104] In a light damaged mouse model, enhanced TrK and p75 labeling was localized to different parts of Müller cells.[92] These studies showed photoreceptor survival following light damage with p75 blockade or p75 absence in p75 knockout mice p75 activation was hypothesized to suppress growth factor release. Based on the results of experimental studies in different model systems, it may be that p75 signaling is not the main pathway mediating cell death by Müller cells.[105-107] Additional stress pathways or impairment of survival signals such as those initiated by TrK receptors may be required for p75 mediated activation of cell death.[104]
FUTURE DEVELOPMENTS
Müller cells have complex involvement in a variety of functions in the developing, normal, and pathological retina. Much work remains to be done to characterize and clarify the mechanisms involved in retinal homeostasis, particularly in the human eye. Additionally, the mechanisms underlying the morphological, cellular, and molecular changes Müller cells undergo as part of the retina's pathophysiological responses are not understood fully. Future studies will lead to insights to these unanswered questions, which should foster the development of therapies specifically targeting Müller cell reactivity or dysfunction in retinal pathology.
THE RETINAL PIGMENT EPITHELIUM
OVERVIEW OF THE RETINAL PIGMENT EPITHELIAL CELL AND ITS FUNCTIONS
Retinal pigment epithelium (RPE) is a simple monolayer of postmitotic, melanin-laden cells lying between the photoreceptors and the choroid but serving functions that are critical to the eye. These include the formation of a blood-retinal barrier, phagocytosis of the photoreceptor outer segments (OS), recycling of the vision-mediating visual pigments of the photoreceptors, transport from the systemic circulation of molecules necessary for the structure and function of photoreceptors and maintenance of a dry, pH-balanced, and immune-privileged subretinal space. Unlike Müller cells, RPE are easy to study, isolate, and culture. This fact has resulted in a vast and rich database of knowledge on RPE structure and function.[108,109] This chapter provides a broad introduction to the RPE.
EMBRYOLOGY AND DEVELOPMENT OF RPE
RPE progenitors originate from the dorsal portion of the optic vesicle (Fig. 125.5). Cells in the early stages of optic vesicle development are similar morphologically and molecularly and express numerous transcription factors (e.g., Otx2, Pax6, Rx1, Six3)[110] that initiate eye development (Fig. 125.5). At this stage, all the cells of the optic vesicle can give rise to the neural retina, optic stalk, or RPE.[111-113]Signals from the surrounding tissues determine the fate of different regions of the optic vesicle, e.g., whether the cells develop into RPE or neural retina. The surface ectoderm generates signals that promote neural retinal development and suppress RPE development.[114] These signals are molecules that belong to the FGF family. Thus, the outer rim of optic vesicle adjacent to the surface ectoderm (that later invaginates to form the inner layer of the optic cup) eventually develops into the neural retina rather than into RPE.[115,116] Conversely, the mesenchyme surrounding the posterior aspect of the optic vesicle (the prospective RPE) generates signals that activate the RPE development pathway and suppress neural retinal pathway.[117] Members of TGF-? superfamily have been shown to play a role.[117,118] These signals act on the target cells and activate or suppress transcription factors like Mitf (microphthalmia-associated transcription factor),[119,120] Otx (orthodenticle-related transcription factors),[121,122] and Pax6 (paired-box transcription factors).[123,124] Mitf, for example, binds and transactivates the genes involved in terminal differentiation of RPE, including tyrosinase.[119] Further differentiation of RPE may be influenced by a different set of signaling factors, including bone morphogenetic protein (BMP)[125] and hedgehog (Hh)[126,127] families and cell-cycle regulators. The factors mentioned above are not unique to RPE. How these factors promote formation of RPE in the optic vesicle and different structures in different parts of the forebrain is not clearly understood.
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FIGURE 125.5 Diagrammatic representation of the development of the RPE. A. The optic vesicle develops as an outpouching of the diencephalon (A-1). The vesicle invaginates to form an optic cup (A2, and A3). Signals from the surface ectoderm (e.g., FGF and others) (A-1) promote neural retinal development (A3) from the ventral part of the optic vesicle while the dorsal part of the optic vesicle develops into the RPE following activation by signals (e.g., TGF-?) from the adjacent mesenchyme. B. Until the activation of signals specifying the fate, the cells of the optic vesicle are indistinguishable (multiple colors). TGF-? from extraocular mesenchyme favors development of RPE while FGF from the lens placode suppresses RPE development and favors neural retinal development (B1). Different transcription factors activated by the extracellular signals become confined to prospective RPE and prospective neural retina (B2). The interaction of the activated transcription factors with other regulatory molecules consolidates the identity of the cells and promotes differentiation. Otx: orthodenticle-related transcription factor; Pax6: paired-box transcription factor; Hh: hedgehog; BMP: bone morphogenetic protein; Wnt: wingless and integration site gene; Six: sine oculis homeobox; Chx10: C. elegans ceh-10 homeo domain containing homolog; Mitf: microphthalmia-associated transcription factor; Rx: retina homeobox. |
By day-30, the invagination is complete, and the two layers of the optic vesicle are apposed. RPE initially is pseudostratified but matures into a monolayer.[3] On day-35, melanin granules appear in RPE cells. This is the earliest site of pigmentation in the body. By 6-weeks gestation, RPE basement membrane becomes evident, and by 10-weeks, the apical processes develop. Further differentiation results in apicobasal polarity and formation of tight junctions to establish the outer blood-retinal barrier. Development of tight junctions occurs in three stages. In the first stage, specific proteins, ?-tubulin in the apical portions of the cells, and ?1?3 integrin in basal portions are expressed, but tight junctions are rudimentary. In the second stage, Na+-K+-ATPase is apically polarized. In the final stage, mature tight junctions composed of ZO-1, occludin, and claudin are formed.[128]
In accordance with a general theme of development, interaction of the RPE with the surrounding structures is important for RPE development and maturation. Conversely, RPE is important for development of the adjacent neural retina, choroid, and sclera.[129,130]
The presence of RPE is necessary for the formation of photoreceptor discs[131] and photoreceptor differentiation.[132] Loss of RPE leads to improper retinal development.[129,131] Neural retinal development (generation of cells and differentiation) occurs along a gradient starting in the center and extending to periphery.[133,134] RPE maturation precedes neural retinal maturation[135] but retinal development does not depend solely on RPE (e.g., hedgehog signaling from ganglion cells is important for the development of laminar organization of neural retina[127]). Photoreceptors also influence development of RPE. Neural cell adhesion molecule (NCAM) localization to apical regions of RPE, for example, requires the presence of photoreceptors; absence of photoreceptors leads to basolateral redistribution.[136] One possible mechanism by which RPE may be influencing development of neural retina is by release of a soluble factor.[129,137] This factor is believed to be ATP, and it increases cell proliferation in the neural retina.[138] Melanin-related agents in RPE appear to be responsible for some aspects of retinal development. Human albino eyes can exhibit foveal hypoplasia and have abnormal chiasmal projections.[139,140]Lack of pigment results in temporal delay in neural retinal differentiation as well as abnormalities in ganglion cell axon decussation (i.e., greater numbers of crossed fibers compared to pigmented eyes). These retinae are underdeveloped with abnormally thin INLs and ONLs. There is a rod cell deficit (30%), but the cone cell numbers and cone mosaic are unaffected.[140]
RPE cells also may play a role in melanocyte differentiation and vascular development in the choroid.[130] RPE secretes growth factors that influence endothelial cell and photoreceptor differentiation.[141-144]Through its transport capabilities, RPE may relay growth regulating signals from the neural retina that influence choroidal and scleral growth, thereby potentially modulating the development of the latter structures.[145] RPE is capable of secreting all the components of Bruch's membrane.[146] Bruch's membrane layers develop sequentially from inside-out, i.e., the RPE basement membrane forms first, followed by progressive development of the outer laminae. The choriocapillaris basement membrane is the last layer to form.[147,148]
ANATOMY AND CELL BIOLOGY
RPE forms a monolayer of cells sclerad to the neural retina, extending from the edge of the optic disc, posteriorly, to the ora serrata, anteriorly. When viewed en face, the cells appear hexagonal. In cross section, cells are cuboidal and contain a basally located nucleus and apically located pigment granules. The RPE size varies with the location in the eye; submacular RPE cells have a diameter of 14 ?m and are 10-14 ?m tall. RPE in the peripheral regions, especially near the ora serrata, are flatter and can have a diameter of up to 60 ?m.[149,150] The RPE apical surface is characterized by numerous microvilli, which are of two different lengths; fine, filopodia-like microvilli of 5-7 ?m in length interdigitate with the photoreceptor outer segments; shorter, broader, lamellopodia-like microvilli of ?3 ?m length enclose the tips of the OS (Fig. 125.6). The basal RPE surface exhibits complex infoldings that may extend 1 ?m or more into the cytoplasm. The RPE rests on Bruch's membrane, which is a pentalaminar structure that includes the RPE basement membrane (on which RPE rests directly), the inner collagenous layer, the elastic layer, the outer collagenous layer, and the choriocapillaris basement membrane.
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FIGURE 125.6 Ion channels in RPE cells. |
There are ?4-6 million RPE cells in each human eye. The cell density decreases from center (4000 cells/mm) to mid-periphery (3000 cells/mm) to far periphery (1600 cells/mm).[151] This distribution is likely due to the growth of the eye being confined to the ora-equatorial region from infancy to adolescence and the lack of RPE cell proliferation after development. RPE cells increase in size in the peripheral region near the ora serrata from infancy to adolescence. In the periphery, RPE cell density is highest in the nasal quadrant; all other quadrants have a lower packing density. Cell density decreases by ?0.3% per year with increasing age, most marked under the fovea and mid-peripheral retina.[151] This rate of loss is more in blacks than in whites.[152] Cell number or cell density is not affected by gender, but it correlates with rod cell density. The rod:RPE ratio is 0 in the fovea and increases to ?19 rods for every RPE in the periphery.[151] In the periphery, ratio of RPE to rods is highest in the nasal followed by the superior quadrant. The other two quadrants have similar densities. The cone:RPE ratio is highest under the fovea (?40 cones/RPE), declines toward mid-periphery (?1), and increases toward the far periphery because of high cone density in that region.[151,153] The ratio of RPE cells to photoreceptors does not change with age because the cone and rod numbers also decline with age.[152,154-158]Hexagonal shape is lost under the foveal area with age.[159]
In addition to the topographic variation, there is cell-to-cell heterogeneity in the RPE monolayer. This heterogeneity has been termed cellular mosaicism-patches of cells of varying phenotype forming the monolayer, cells within a patch being identical but differing from cells in an adjacent patch.[160]
The RPE nucleus is 5-12 ?m in diameter and is somewhat oval with the long axis parallel to the basal surface. The nucleus contains one or two nucleoli and diffusely distributed chromatin. Many RPE are bi- or multinucleated (one out of 30 RPE have multiple nuclei), more commonly in peripheral RPE cells.[149,161] Mitosis has not been seen in adult RPE cells in situ. If there is a defect resulting from cell loss due to any reason, the neighboring cells spread to fill in the defect.[162] However, RPE are capable of proliferating if cells are isolated and grown in culture.
RPE cell cytoplasm contains mitochondria (present mostly between the nucleus and the basal surface) and extensive smooth endoplasmic reticulum (characteristic of only one other cell in the eye, the Müller cell). In the periphery, the amount of smooth endoplasmic reticulum is lower, but cells have more rough endoplasmic reticulum (present in the apical portion of the cell). RPE cells contain lysosomal granules, melanin granules, and lipofuscin. Pigment granules measure 2-3 ?m in length and 1 ?m in diameter. They are present mostly in the apical portion of the cell. The granules are either ovoid or needle shaped, the former located in perinuclear cytoplasm and the latter near the apical region. Melanin granules may fuse with lysosomal granules or lipofuscin to form complex melanolysosomal or melanolipofuscin granules. The content of 'pure' melanin declines with age, whereas the number of complex melanin granules increases. Submacular RPE cells contain more complex granules than RPE elsewhere, particularly in young eyes. With age, lipofuscin granules increase in RPE cells, the largest increase occurring between the first and second decade of life. Due to the increase in granules, cytoplasmic volume not occupied by pigments ('free space') decreases with age.[163]
The RPE is polarized with a distinct distribution of molecules and ion channels on the apical and basal surfaces. For example, the apical membrane contains ?v?5 integrin and Na+-K+-ATPase while the basal membrane contains ?6?1 integrin. The polarity is achieved by a specific distribution of molecules to either surface and is maintained by the presence of tight junctions that prevents intra-membrane diffusion of molecules between the apical and basal domains of the cell membrane.[164] Organization of the cytoskeletal elements also helps to maintain the polarity.[165]
Laterally, RPE connect with each other via tight junctions (zonula occludens [ZO]), adherens junctions (zonula adherens), and gap junctions.[365] Tight junctions are responsible for the barrier function of RPE monolayer. In freeze-fracture images, tight junctions appear as 'sealing strands' encircling the cell near the apex. By transmission electron microscopy, the tight junctions appear as close apposition of the plasma membrane of adjacent cells near their apices. These strands are extracellular domains of tight junction proteins claudin and occludin.[164] The cytoplasmic side of the cell membrane contains ZO proteins, ZO-1 and ZO-2, that connect to the actin filaments present in the cytoplasm as well as activate signal transduction pathways.[164]
The zonula adherens is formed by interaction of cadherin molecules. These junctions appear as a 200å separation between the plasma membranes of adjacent cells. The cytoplasmic domain of cadherins interacts with vinculin, catenins, ?-actinins, and actin filaments. Actin filaments are arranged circumferentially and maintain the hexagonal shape of RPE cells. Gap junctions are present toward the base of the cell and are formed by connexins.[166] These junctions allow intercellular communication and transport of ions and metabolites.
FUNCTIONS
Role in Retinal Adhesiveness
The development of the retina by invagination of the optic cup results in a potential space between the neural retina and the RPE. Yet in a fully developed eye the neural retina is attached to the RPE. A force of ?100 dynes is needed to detach one centimeter of retina in live rabbits; ?180 dynes in cats, and ?140 dynes in monkeys.[167] Numerous factors are responsible for keeping the retina attached. The most important contributions to retinal adhesiveness come from interphotoreceptor matrix (IPM), which passively glues the neural retina to RPE; the metabolic activity of the RPE; and fluid transport across the RPE.[168,169]
Induction of retinal detachment by experimentally induced perturbations demonstrates the role of RPE metabolic activity in retinal adhesiveness. Removal of Ca2+ and Mg2+, for example, leads to reversible decrease in retinal adhesiveness; reducing the pH also leads to a decrease in adhesiveness.[170,171] A decrease in oxidative metabolism resulting from ischemia leads to reduced retinal adhesiveness.[168]
Due to the resistance to free fluid movement across RPE resulting from the tight junctions, there is a hydrostatic pressure differential across the retina. This pressure differential forces fluid movement from the retina into the choroid. This differential, which could be as little as 0.001 mmHg, may be sufficient to keep the retina attached.[172] If the direction of fluid movement is reversed by injection of hyperosmolar solution into the vitreous cavity, retinal detachment occurs.[173] Similarly, if choroidal osmolarity is increased, there is an increase in retinal adhesiveness.[174] Vitreous gel plays some role in retinal attachment by physically keeping the retina apposed to RPE. Of uncertain importance to retinal-RPE adhesion is the physical ensheathment of OS by RPE microvilli as well as the frictional and electrostatic resistance resulting from the ensheathment. Recently, integrin ?v?5 has been shown to play a role in retinal-RPE adhesion, and, interestingly, the strength of adhesion mediated by ?v?5 was maximal at 3.5 hr after light onset when the phagocytosis is complete.[175]
Phagocytosis
While RPE cells are capable of nonspecific phagocytosis, a property shared with other phagocytic cells like macrophages, RPE cells show a strong preference for photoreceptor OS.[176] Nonspecific phagocytosis and phagocytosis in vitro is not rhythmic while phagocytosis of OS is. By the time a human RPE cell is 80 years old, it has phagocytosed ?108 OS.[177] Dysfunctional phagocytosis can lead to accumulation of debris in the subretinal space and cause loss of vision as is evident in Royal College of Surgeons (RCS) rats. These rats have a genetic mutation in the MerTK receptor (vide infra) that mediates binding to OS (vide infra), and, therefore, RCS RPE cells have defective OS phagocytosis. Rod OS are maintained at a fixed length,[178] and there is a need to create new discs/opsin in photoreceptors to replace damaged OS due to light exposure.[179,180] The constant length is achieved by RPE phagocytosis of OS.[179] Phagocytosis also allows for recycling of retinoids and fatty acids present in photoreceptor OS (discussed earlier).
The nature of structural interface between RPE and photoreceptor OS is different in rods and cones. Rod OS (ROS) contact the apical surface of RPE while cone OS (COS) do not reach the surface. In the case of COS, RPE cells extend one or more processes of 10-20 ?m length to reach the tip. Each process expands near the COS tip to ensheath it. These processes surround the COS to form a supracone space. COS discs are shed into this space and guided to RPE.[181,182] Since the COS is shorter than ROS and has a narrow tip, the resulting phagosomes are smaller, and there are fewer phagosomes in the RPE that contact cones vs. rods.
Phagocytosis can be divided into the following stages: shedding of OS, binding of OS to RPE, internalization, migration to the basal cytoplasm, and digestion.[183] Photoreceptors shed their OS cyclically, based on the circadian rhythm, in a process mediated by dopamine (which provides the light signal) and melatonin (which provides the dark signal). It is not clear whether the initiation of disc shedding occurs in the photoreceptors (e.g., shedding can sometimes occur in the absence of RPE in vitro[184]), whether RPE cells play a role (i.e., do RPE processes 'bite' off the tips?), or whether both processes occur during the initiation. Regardless, the interdigitation of OS with RPE microvilli is important for phagocytosis to occur. When there is no interdigitation, as occurs in retinal detachment[185] or in mutant vitiligo mice that have RPE with short apical villi that do not ensheath the OS,[186] phagocytosis does not occur even though the RPE is capable of phagocytosis. The trigger may be the length of OS,[184] or some other signal that has not yet been identified. Light-induced oxidized tips of OS have been proposed as a signal for phagocytosis by Sun and co-workers.[187]
Recognition and binding of OS to the RPE apical surface occurs by receptor-ligand interactions. The receptors on RPE that have been implicated include mannose receptor,[188] CD36, MerTK,[189] integrin ?v?5,[190,191] and a gp55 glycoprotein. Blocking the mannose receptor inhibits RPE phagocytosis of ROS.[188,192] The identity of the natural ligand for this receptor is not known. CD36 is a macrophage receptor that is involved in phagocytosis and binds to Toll-like receptor, TLR4.[193] CD36 may also bind to light-induced oxidative products of photoreceptor lipids, and the latter may be a physiological signal to initiate phagocytosis.[187] CD36 binding is a necessary and sufficient signal for activation of phagocytosis.[190]
MerTK is tyrosine kinase receptor, i.e., the cytoplasmic domain of the receptor is a tyrosine kinase that becomes activated when an appropriate ligand binds to the receptor on the cell surface. Cells lacking MerTK are capable of binding to OS but are not able to ingest them.[194] The cognate receptor for MerTK is believed to be a vitamin K-dependent growth arrest-specific protein, Gas6.[195,196] The role of Gas6 in phagocytosis has been demonstrated in vitro,[197] but its role in vivo is not known. Integrin ?v?5 present on the apical membrane of RPE binds to vitronectin. However, vitronectin may not be involved in OS phagocytosis in situ, and there may be other proteins involved;[191] ?v?5 interacts with MerTK to mediate phagocytosis. It activates MerTK via focal adhesion kinase.[198]
OS binding to RPE surface receptors activates second messenger systems that lead to reorganization of cytoskeletal elements to cause ingestion. MerTK activation causes an increase in inositol triphosphate (IP3) which, in turn, leads to an increase in intracellular free calcium concentration.[199] Together, these two initiate phagocytosis by influencing the reorganization of cytoskeletal filaments. Increased calcium also results in activation of protein kinase C that shuts off phagocytosis.[200] Cyclic AMP (cAMP) modulates the rate of phagocytosis: increased cAMP results in decreased phagocytosis.[201,202]
Following phagocytosis, the phagosomes are guided to the basal region by microtubules[203] and fuse with lysosomes in the basal region of the cell. Initially, small lysosomes fuse with phagosomes. Subsequently, large lysosomes fuse with the phagosomes via pore-like structures through which lysosomal contents may enter the phagosome.[204] Cathepsin D is the major lysosomal enzyme involved in degradation of OS with possible involvement of cathepsin S.[205-207] The degraded products are either reused or eliminated. The rapid and transient increase in gene and protein expression that follows OS phagocytosis does not occur following nonspecific phagocytosis of latex beads in vitro. These genes include c-fos, zif-268, tis-1, and peroxisome proliferator-activated receptor ? that is a regulator of lipid metabolism.[208,209]
Molecules that have been implicated in regulation of phagocytosis include ?-adrenergic agonists, adenosine A2 receptors,[210] serotonin, bFGF, acetylcholine, glutamate dopamine, and melatonin.[183] The latter two are responsible for the circadian regulation of shedding and phagocytosis.[211] Melatonin is a hormone secreted rhythmically by the pineal gland and also by photoreceptors at night. It activates light-induced OS shedding in dark. During daytime, dopamine synthesis results in inhibition of melatonin and inhibition of OS shedding. RPE cells express receptors for both dopamine and melatonin so that shedding and phagocytosis are synchronized. Recently, the role of ?v?5 integrin in synchronized phagocytosis has been demonstrated.[212]
Secretion
RPE cells secrete numerous growth factors.[213] These include PDGF, PEDF, FGF, TGF, CNTF, VEGF, insulin-like growth factor (IGF), lens-epithelium derived growth factor, cytokines (interleukins), and tissue inhibitor of metalloproteinases (TIMP). Some of these are secreted by cultured RPE while others like VEGF, TGF-?, IGF, and FGF are secreted in vivo as well.
PEDF is a 50 kD protein present in RPE and secreted into the adjacent IPM.[214] PEDF is neurotrophic, prevents cell proliferation, and promotes differentiation in retina and other tissues.[215] PEDF has neuroprotective effects against ischemic damage,[216] glutamate excitotoxicity, oxidative stress, and retinal degeneration. It has antiangiogenic effects, stabilizing the choriocapillaris endothelium.[217,218]VEGF[219] is secreted in low concentrations in healthy eyes and prevents apoptosis of choriocapillaris endothelium. PEDF is secreted apically from RPE cells and VEGF basally.[220,221] PEDF also influences glial cell maturation. RPE removal results in failure of Müller cells to form adherens junctions with photoreceptors.[222]
TGF-? was initially demonstrated in eyes with proliferative vitreoretinopathy (PVR). Vitreous samples from these eyes contained three times more TGF-? than eyes with uncomplicated retinal detachment. RPE cells can synthesize and secrete TGF-? normally. RPE cells display increased production of predominantly TGF-?2 in response to photocoagulation,[223] decreased oxygen tension,[224] PVR,[225] age-related macular degeneration,[226] myopia, and sickle cell retinopathy.[227] In PVR, RPE cells are a major component of the proliferating cells. In addition, the TGF-? produced by RPE increases the fibrotic response.[228] PDGF is chemotactic and mitogenic to RPE and glial cells.[229] PDGF receptors are also expressed by RPE.[230]
Growth factors like FGF and CNTF play a neurotrophic role in maintenance of photoreceptors.[68]
Lipofuscin Accumulation and other Aging Changes
Lipofuscin ('age pigment') is an intralysosomal complex of protein, lipid, carbohydrate, metals, and other compounds that accumulates in postmitotic, metabolically active cells.[231] The amount accumulated in these cells has a linear correlation with age, hence the label, age pigment, and has a negative correlation with longevity. Accumulation in RPE seems to be inversely related to melanin content.[232] Lipofuscin content of RPE cells depends on the location in the eye. Cells in the posterior pole have a higher amount of lipofuscin with subfoveal RPE showing a lower accumulation relative to cells in the subparafoveal region.[233] Lipofuscin is nondegradable and cannot be eliminated from the cell via exocytosis.[231] Formation and accumulation of lipofuscin may occur by autophagy of cytoplasmic organelles and contents and subsequent fusion with lysosomes or by phagocytosis of material from a different cell (heterophagy). Phagocytosed photoreceptor OS are a major source of lipofuscin; if RPE that are unable to phagocytose photoreceptor OS or have mutations in RPE 65 (and therefore lack 11-cis-retinal and vitamin A aldehyde), they do not accumulate lipofuscin as much as native RPE.[234,235] If photoreceptors are eliminated by light exposure, lipofuscin accumulation is not significant.[236] Thus, phagocytosed photoreceptors probably are a major source of lipofuscin in RPE. RPE cells phagocytose 6000-8000 discs every day, and the digestion is not complete.[183] The residual structures are fluorescent and can be seen as early as 16 months after birth.[237] Autophagy also contributes to the debris.[238-240] Since many RPE cells persist through the life span, this debris can occupy up to 70% of the cytoplasmic volume, which leads to decreased RPE phagocytic activity.[241] Lipofuscin deposition also depends on dietary vitamin A.[242]
The natural autofluorescence of the fundus (between 500 and 750 nm with maximum emission at ?590-630 nm) is attributed to RPE lipofuscin. The autofluorescence increases with age and declines after age 70.[243] The latter could be either due to loss of RPE or due to decreased fluorescence of A2E as a result of photo-oxidation.[244]
A prominent component of lipofuscin is A2E (C42H58NO, molecular weight 592): vitamin A aldehyde (generated upon photoisomerization of 11-cis-retinal) + ethanolamine in 2:1 ratio. A2E is often named as N-retinylidene-N-retinylethanolamine, but the name does not reflect the pyridinium ring present in the structure.[245] Sparrow hypothesizes that since A2E structure is unprecedented, it may not be recognized by RPE lysosomal enzymes.[245] A2E formation starts with condensation of phosphatidylethanolamine (from the photoreceptor membrane) and light-generated 11-cis-retinal to form N-retinylidene-phosphatidylethanolamine (NRPE) (NRPE is the substrate for ABCA4; vide infra), followed by a series of intermediate products, including A2PE, the immediate precursor of A2E.[246,247] A2E is formed by cleavage of A2PE in RPE.[247] The cyclical phagocytosis of OS prevents accumulation of A2PE in the photoreceptor OS. A2PE is the fluorescent pigment in photoreceptor OS. Thus, accumulation of A2PE due to defects in phagocytosis contributes to the fluorescence in photoreceptors (e.g., RCS rats, Stargardt disease). A2E undergoes further photoisomerization to generate iso-A2E and other cis-isomers that are not as abundant as A2E.[248]
One hypothesis for age-related accumulation of lipofuscin was age-related decline in activity of lysosomal enzymes like cathepsins.[249] However, lipofuscin accumulation itself appears to alter lysosomal degradation.[231] What makes lipofuscin resistant to degradation? It is believed that oxidative damage to cellular components results in cross-linking of proteins and other biomolecules,[250] rendering them indigestible. Thus, cells exposed to oxidative stress tend to accumulate lipofuscin. Accumulation of lipofuscin in the cell affects cell function and health. Lipofuscin-containing cells are more susceptible to oxidative damage.[251] Lipofuscin is a photoinducible generator of oxygen radicals, e.g., superoxide anion, singlet oxygen, and hydrogen peroxide.[252] These free radical species, in turn, react with biomolecules to cause lipid peroxidation, protein oxidation, loss of lysosomal integrity, and RPE cell death.[253,254] A2E is one of the photosensitizers in lipofuscin, and there may be other as yet unidentified molecules in lipofuscin that are also photosensitizers. Even though lipofuscin is compartmentalized, there is increased production of reactive oxygen species within the lipofuscin-containing lysosomes due to the presence of iron that reacts with H2O2.[255] Lipofuscin accumulation results in decreased phagocytosis by RPE, including that of photoreceptor OS.[241] Proteins in lipofuscin granules are modified by peroxidation; the presence of mitochondrial proteins indicate autophagy and a possible role in lipofuscin formation.[256] Nonenzymatic oxidative modification of amino acids may result in inhibition of proteolysis, and incomplete proteolysis of proteins may promote lipofuscin formation.[257] The presence of A2E in RPE results in delayed degradation of phospholipids as well.[258] Melanosomes are photoprotective, but with age melanosomes become photoreactive. (This change could be due to association of A2E with melanosomes although contamination of the melanosome preparation with A2E in the experiments cannot be ruled out.) Aggregates of A2E have a detergent-like property of compromising membrane integrity leading to membrane blebbing.[259]
Ion Transport
RPE cells express ion channels on their apical and basolateral surfaces that mediate transport of Na+, K+, HCO3?, Cl?, etc. One of the consequences of RPE cell fluid transport is dehydration of the subretinal space and maintenance of neural retina-RPE apposition (vide infra). Fluid movement is linked to the ion movement. In RPE, H+/lactate, and Cl? movement are the main ions that are linked to fluid movement.[260,261] The ion differences between the cytoplasm and the surrounding tissues result in a potential difference across the RPE cell monolayer. This transepithelial potential (TEP) is ?5-15 mV.[262]
Ion channels present in RPE cells are summarized in Figure 125.6. Sodium is secreted actively into the subretinal space by a Na+/K+ ATPase pump present in the apical surface.[263,264] This pump creates a sodium gradient from the subretinal space to the RPE cytoplasm that facilitates as well as provides energy for the transepithelial transport. HCO3?, K+, and Cl? are co-transported with Na+ (vide infra). The mechanism of Na+ entry from the basal plasma membrane is not known. Cl? is absorbed actively by apical Na+K+-2Cl? co-transport present in the apical membrane,[265,266] Ca+-dependent Cl? channels,[267,268] ClC-2 channels (a family of chloride conducting, voltage-gated channels),[269] and cystic fibrosis transmembrane conductance regulator (CFTR)[270] present in the basolateral membrane. Na+/HCO3?enters the cell via an apical Na+/HCO3? cotransporter[271,272] and leaves the RPE through a basal Cl?/HCO3? exchanger and possibly a Na+/HCO3? co-transporter. The transport of HCO3? regulates the pH of subretinal space and the intracellular pH of RPE cells. If the pH is high in the subretinal space or in RPE, HCO3? is moved out of RPE into the choroid via a NaHCO3 co-transporter in the apical membranes and Cl?/HCO3? exchanger in the basal membranes. When pH is low, the direction is reversed so that HCO3? moves through RPE from choroid into the subretinal space. There is a net secretion (i.e., movement of ion across the RPE into the subretinal space) of Ca+, with movement out of the apical membrane via a Na+/Ca2+ exchanger and Ca2+-ATPase. K+ enters RPE on the apical side via a Na+K+-ATPase and a Na+K+-2Cl co-transporter and leaves via apical or basal K+ channels. There is a net transepithelial transport of K+ from the subretinal space to choroid. Movement of Cl? and K+ drives the movement of water.
The ion channels maintain the ion homeostasis in the subretinal space, especially the changes that occur in ion concentrations as a result of light stimulation of photoreceptors. In the dark, the apical RPE membrane is more hyperpolarized than the basal membrane. Voltage across the apical membrane in a resting RPE cell is ? -50 mV and across the basal membrane is -45 mV. Thus, a transepithelial potential, TEP (Vba-Vap= 5 mV), exists across the RPE.[262] Membrane potential across the apical membrane is primarily due to K+ channels with additional contributions from the Na+/K+ pump and a Na+/HCO3? cotransporter (Fig. 125.6). K+ and Cl? channels in the basal membrane contribute to the membrane potential. The difference in membrane potential between the apical and basal membrane is due to the large Cl? conductance. This difference manifests as the standing potential in a dark-adapted eye when an electrode is placed on the cornea and amplified, with the cornea positive.
Exposure to light induces a series of changes that are recorded in the electroretinogram (ERG) and electrooculogram (EOG). The ERG response begins with a rapid dip in voltage (a-wave) followed by a rapid upward deflection (b-wave) that does not quite return to the baseline (Fig. 125.7) a- and b-waves reflect electrical activity in the neural retina.[273] These waves are followed by a slow rising c-wave for which the RPE is responsible.[274] In the dark, Na+ flows into the photoreceptors via the cGMP-gated Na+ channels in the OS and is pumped out by the Na+/K+ pump in the inner segment. There is a passive efflux of K+ out of the inner segment. In response to light, cGMP-gated channels close, and the OS is hyperpolarized. Additionally, the passive efflux of K+ is also shut down. Decreased concentration of K+ in the subretinal space and the large conductance of K+ at the RPE apical membrane results in hyperpolarization of the latter.[262] Additionally, the distal ends of Müller cells hyperpolarize as well, resulting in a slow negative transretinal potential termed slow PIII.[275] Since the RPE contribution is larger, a positive deflection is recorded on the surface of the cornea. In summary, both RPE and Müller cells contribute to c-wave. The former results in a positive deflection, and the latter results in a negative deflection. Since RPE contribution is greater than Müller cell's, the net change is a positive c-wave in the EOG. The c-wave is followed by fast oscillation, which is a negative slow potential fall toward or below the dark-adapted baseline. This change is produced by the hyperpolarization of the RPE basal membrane as a consequence of the above-mentioned fall in subretinal K+.[276] The decrease in K+ slows the activity of Na+/K+/2Cl? activity causing a decrease in Cl? influx across the apical membrane of RPE. The resultant decrease in intracellular Cl? concentration produces a change in Cl? equilibrium across the basolateral Cl? channels leading to a hyperpolarization of basal membrane. The latter is termed fast because it is fast relative to the next response, the light peak. The light peak is the slowest and largest component of the EOG and refers to the slow increase in potential that follows the fast oscillation. It is generated by an increased conductance of Cl? in the basolateral membrane, depolarizing it, and increasing the transepithelial potential.[262]
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FIGURE 125.7 Electrical changes recorded from a corneal electrode in a dark-adapted eye after exposure to a bright light. The response has been depicted in four different time scales from top to bottom; a- and b-waves occur relatively rapidly and represent neural retinal responses. The light-induced drop in subretinal K+ concentration causes hyperpolarization of the RPE apical membrane and the distal ends of Müller cells resulting in the c-wave. The hyperpolarization of the RPE basal membrane is recorded as a fast oscillation. The depolarization of the RPE basolateral membrane by increased conductance of Cl? results in light peak generation. |
Transport of other molecules
The RPE also eliminates waste produced by photoreceptors. Lactic acid is an excellent example. Photoreceptor OS produce lactic acid, which is removed by RPE via a lactate-H+ co-transporter, MCT1, in the apical membrane, and MCT3, in the basal membrane.[277]
Glucose is transported from blood to photoreceptors by GLUT 1 and GLUT3. The latter transports glucose routinely, and the former functions when there is an increased metabolic demand.[278,279] Vitamin A is taken up on the basal surface by receptor binding to vitamin A bound to retinol-binding protein/transthyretin complex and enters the visual cycle.[280,281] RPE also transports docosahexaenoic acid synthesized in the liver and circulating in the blood.[282] Docosahexaenoic acid, which is enriched in photoreceptor membranes, is not synthesized in neuronal tissue.[283]
Mutations in the bestrophin molecule (product of VMD2 gene), which functions as Ca+-sensitive Cl? channel, are associated with three diseases: Best vitelliform macular dystrophy,[284] adult-onset vitelliform dystrophy, and autosomal dominant vitreoretinalchoroidopathy.[285] In the former two conditions, appearance of an egg yolk-like yellow lesion in the fundus is characteristic and results from accumulation of deposits, including lipofuscin, in the subretinal space and sub-RPE region as well as RPE hypertrophy. Vitreoretinalchoroidopathy is characterized by a circumferential zone of hyperpigmentation anterior to the equator along with punctate white pre-retinal opacities, fibrillar condensation of vitreous, and breakdown of the blood-retinal barrier.[286]
Patients with cystic fibrosis have defective Cl? channels in RPE cells as well.[270,287] However, retinal degeneration is not seen, probably because of the presence of other channels that compensate.
Fluid Transport
About 10-20% of aqueous fluid is absorbed posteriorly with the rate limiting structure being the neural retina. The RPE also limits passive fluid movement across Bruch's membrane, but it has an active pumping mechanism that has a large capacity and helps to keep the subretinal space dehydrated. The fluid permeability across dog retina is 0.03 ?1 min?1 mmHg?1 sqcm?1 and across dog RPE is 0.01 ?l min?1 mmHg?1 sqcm?1. Permeability across monkey RPE is 0.005 ?1 min?1 mmHg?1 sqcm?1.[288,289] The transretinal pressure of 0.5 × 10?3 mm Hg[172] that exists across the retina is sufficient to keep retina attached but has no effect on the fluid movement across it. In the monkey eye, a pressure drop of ?4 mmHg exists between the vitreous and choroid, which implies a possible fluid flow across the RPE of 0.3 ?1/min.[290]
The forces driving the fluid movement across the retina in the eye include intraocular pressure, osmotic/oncotic pressure, and the active solute-linked fluid transport by the RPE monolayer. A change in intraocular pressure from 0 to 38 mmHg leads to a 39% increase in rate of fluid absorption from subretinal space in rabbits.[291] Because of the higher protein content in the choroid and because osmotic pressure in vitreous and subretinal space is maintained at somewhat similar levels, there is a net osmotic pressure that drives fluid outwards only.[292,293] In a normal eye, active transport by RPE cells is the major force eliminating water from the subretinal space; intraocular pressure and osmotic pressure contribution to fluid transport is minor but effective when RPE cells are damaged. Müller cells may play a role in water transport in the inner retina.[25] RPE is estimated to transport fluid at a rate of 0.1 ?1 h?1 sqmm?1.[294] The ion channels responsible for solute-linked fluid movement are the apical Na+,K+,2Cl?channel, basal Cl? channels, and the H+/lactate co-transporter.[260,261] Recently aquaporin that forms "water channels" in secretory and absorptive epithelia,[295] have been shown to play a significant role in water transport across RPE cells.[296]
Melanin
Melanin granules are ovoid in shape and measure ?2-3 ?m in length, 1 ?m in diameter, and are distributed near the apical region of the cell. Melanin granules contain polymerized melanin in a protein matrix. The melanin can be pheomelanin or eumelanin, imparting yellowish/reddish or brown/black color, respectively. The human eye contains eumelanin.[297] While uveal melanin-containing cells are derived from melanoblasts originating from neural crest, RPE, ciliary epithelium, and iris pigment epithelium develop from the neurectoderm.[297,298] Melanin is produced during development. However, it is possible that melanin is synthesized in adult cells as well.[299,300] Tyrosinase synthesis can be induced in adult cultured RPE by feeding them photoreceptor OS.[301]
Melanin synthesis begins with the production of premelanosomes and synthesis of tyrosinase, the two processes being independent of one another (Fig. 125.8).[302,303] Premelanosomes are vesicular bodies that contain the structural proteins of melanin but no tyrosinase enzyme. They are assembled in smooth endoplasmic reticulum and released into the cytoplasm. Tyrosinase is synthesized in rough endoplasmic reticulum and transported through the Golgi apparatus during which the enzyme undergoes posttranslational modification, predominantly glycosylation. The enzyme is then assembled into coated vesicles that bud off and fuse with the premelanosomes to form melanosomes.
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FIGURE 125.8 Melanin synthesis begins with hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) followed by oxidation of DOPA to dopaquinone. Both reactions are catalyzed by tyrosinase. Spontaneous endocyclic ring formation results in formation of leucodopachrome from dopaquinone. Leucodopachrome undergoes oxidation to form dopachrome and DOPA. This oxidation is coupled with re-oxidation of DOPA to dopaquinone by tyrosinase. Dopachrome is converted to 5,6 dihydroxyindole-2-carboxylic acid (DHICA). A small portion of dopachrome undergoes spontaneous conversion to 5,6 dihydroxyindole (DHI). Oxidative polymerization of DHICA and DHI results in melanin formation. |
Tyrosinase catalyzes the first two steps of melanin synthesis: hydroxylation of the amino acid, L-tyrosine, to 3,4-dihydroxyphenylalanine (DOPA) and oxidation of DOPA to dopaquinone. Spontaneous endocyclic ring formation results in formation of leucodopachrome from dopaquinone. Leucodopachrome undergoes oxidation to form dopachrome and DOPA; this oxidation is coupled with re-oxidation of DOPA to dopaquinone by the tyrosinase enzyme. Dopachrome is converted to 5,6 dihydroxyindole-2-carboxylic acid (DHICA). A small portion of dopachrome undergoes spontaneous conversion to 5,6 dihydroxyindole (DHI). Oxidative polymerization of DHICA and DHI results in eumelanin polymer. Pheomelanin is formed by addition of cysteine to dopaquinone.[297]
The different stages of melanin granule maturation can be seen by electron microscopy.[149] Premelanosomes are membrane-bound, ovoid, nonpigmented organelles containing a protein matrix. Stage I granules have protein filaments pole to pole, in an ordered arrangement; melanin synthesis becomes evident at this stage. Increasing melanin synthesis become evident in stages II and III, and by stage IV, a fully melanized melanosome, i.e., melanin granule, is formed.
RPE cells are the first to synthesize melanin in the eye.[149] By 7-weeks gestation, immature melanosomes are visible, and in the subsequent few weeks (?7 or more weeks), continued melanin synthesis is evident by the appearance of melanosomes of various stages. Production of new melanosomes then stops, but the polymerization of melanin within the granules continues for up to two years.
RPE cells exhibit regional variation in melanin content. Cells in the periphery have higher melanin content, and the amount decreases posteriorly.[304,305] With age, melanin content decreases in the periphery while there is no change under the macula. There is no difference in RPE melanin content in whites and blacks.[305]
Melanin absorbs light passing through the retina, preventing reflection of light within the fundus that might affect the clarity of the image formed by the photoreceptors and protecting the photoreceptors from excess light.[306] Absorption of excess light raises the possibility of an RPE photoprotective effect. However, there are conflicting data. In vitro studies show that intracellular melanin clearly protects the cell from light-induced DNA damage.[307] Some in vivo studies show that RPE pigmentation protects the overlying photoreceptors from light-induced damage in rodents.[308,309] In another study using chimeric mice expressing pigmented and nonpigmented RPE cells, light damage to photoreceptors was more severe in the central retina than in the periphery, regardless of the pigmentation of the underlying RPE.[310]
Even though synthetic melanin has been shown to have strong free-radical scavenging properties, natural melanin is not very effective.[306,311] Thus, it is unlikely that it plays as significant a role in RPE cells as other antioxidants, e.g., superoxide dismutase.[306] Melanin is also capable of generating free-radicals and was thought to be responsible for damage caused by light of shorter wavelength.[312,313]However, this blue light damage is now attributed to the presence of liposfuscin.[314]
Melanin traps radiation and dissipates energy, which can result in tissue damage if there is acute exposure to high intensity radiation as occurs in laser photocoagulation.[315] Hansen and Fine have demonstrated in melanin models that a temperature increase above 10°C will cause a zone of denaturation in the vicinity of the granule that would spread with the conduction of heat from the granule, or, if the energy is great enough, generation of steam at the granule cytoplasm interface. The heat and the steam lead to coagulative damage as well as disruption of the structures.[316]
Melanin can bind to numerous chemicals, thus becoming an effective sink or storage site responsible for toxicity.[317,318] Chlorpromazine and chloroquine can bind irreversibly to melanin and can lead to toxicity.[319] Since melanins are polyanions, ionic interaction is one mechanism by which they bind to chemicals.
The role of melanin in development is discussed in the embryology section. With increasing age, complex melanin-containing granules begin to appear. In addition to melanin, these contain a cortex of lipofuscin (melanolipofuscin) or a cortex of enzyme-reactive material (melanolysosomes).[163] The amount of complex granules increases with age while pure melanin declines. The highest density of complex granules is in the submacular region and declines toward periphery and fovea. In human eyes over 90 years old, there are no pure melanin granules.[163] Pigment-free cytoplasm in the RPE also declines with age.[163] The fluorescence of the melanosomes shows a shift toward red with increasing age.[300]
Role in Visual Cycle
Transduction of a light signal to vision begins with absorption of light by rhodopsin (or cone opsin). Rhodopsin (or cone opsin) is formed by binding opsin, a G-protein coupled protein that is present within the membranes of the photoreceptor discs, with the visual chromophore, 11-cis-retinal, in the intradiscal space (Fig. 125.9). Absorption of light results in irreversible isomerization of 11-cis-retinal to all-trans-retinal, which leads to a conformational change in the rhodopsin molecule, thus activating it.[320,321] Activated rhodopsin initiates the phototransduction cascade. Rhodopsin is phosphorylated and inactivated by rhodopsin kinase and subsequent binding to arrestin. Inactivated rhodopsin releases all-trans-retinal and binds to 11-cis-retinal so that it can become activated again by exposure to light. All-trans-retinal generated in the photoreceptor disk space cannot be converted back to 11-cis-retinal in the photoreceptor. This conversion occurs in RPE cells and possibly in Müller cells. Transport of all-trans-retinal to RPE, however, is not a matter of simple diffusion. All-trans-retinal in the disk space is transported to the extra-discal space by an ATP-binding cassette protein (ABCR), known as ABCA4.[322,323] This is a flippase for NRPE, flipping the molecule across the disk membrane. All-trans-retinal is converted to NRPE by reacting with phosphatidylethanolamine. Once in the photoreceptor extra-discal cytoplasm, all-trans-retinal is released. It is then reduced to all-trans-retinol (vitamin A) by a membrane-bound retinol dehdyrogenase (RDH) in the photoreceptor extra-discal cytoplasm. All-trans-retinol is then transported to RPE cells from the rods or to Müller cells from cones by interphotoreceptor-binding protein (IRBP) present in the interphotoreceptor space.[324,325]
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FIGURE 125.9 Schematic of the rod visual cycle. See text for details. ABCR: ATP-binding cassette, retina; CRBP: cellular retinol-binding protein; CRALBP: cellular retinaldehyde-binding protein; IMH: isomerohydrolase; IRBP: interphotoreceptor retinoid-binding protein; LRAT: lecithin:retinol acyltransferase; RDH: all-trans-retinol dehydrogenase; 11-RDH: 11-cis-retinol dehydrogenase; Rho: rhodopsin; Rho*: activated rhodopsin; 11-Ral: 11-cis-retinal; 11-Rol: 11-cis-retinol; at-Ral: all-trans-retinal; at-Rol: all-trans-retinol; at-RE: all-trans-retinyl ester. |
All-trans-retinol transport within the RPE cell is not well characterized. Water-insoluble all-trans-retinol in the RPE is bound to cellular retinol-binding protein (CRBP), thus trapping it within the RPE cell as well as protecting it from oxidation or isomerization. CRBP-bound all-trans-retinol is carried to the endoplasmic reticulum where it undergoes esterification to long-chain fatty acids derived from membrane phospholipids. This reaction, catalyzed by lecithin:retinol acyl transferase (LRAT), leads to formation of all-trans-retinyl esters. Hydrolysis of retinyl esters coupled with isomerization results in formation of 11-cis-retinol.[326] The coupling of hydrolysis is important because isomerization requires energy and is provided by the energy generated during hydrolysis. An additional protein involved in the isomerization step is RPE-65, which acts as a chaperone for all-trans retinyl esters.[327] RPE-65 exists in two forms: a membrane bound form that is triply palmitoylated and a soluble form that is not. The former has high affinity to all-trans retinyl esters and donates a palmitoyl group for the reaction mediated by LRAT. This ensures 11-cis-retinal production during light. The soluble form of RPE-65 reduces the production of 11-cis-retinal in the dark. The switch between the membrane and soluble forms of RPE-65 is controlled by LRAT and by the concentration of 11-cis-retinol in RPE.[328] 11-cis-retinol is either esterified by LRAT and stored in the cell or oxidized to 11-cis-retinal, a reaction catalyzed by 11-cis-retinol dehydrogenase or RDH5.[329] Cellular retinaldehyde-binding protein (CRALBP) accelerates the latter enzymatic reaction:[330] 11-cis-retinal is transported back to photoreceptors by IRBP.[324,325]
An alternate pathway for regeneration of 11-cis-retinal has been described.[331] This pathway is the reverse of the light-induced reaction occurring in the photoreceptors, i.e., all-trans-retinal is converted to 11-cis-retinal by RPE retinal G protein coupled receptor (RGR) using light energy. The source of all-trans-retinal is the RPE pool of all-trans-retinol acquired from the photoreceptors. The conversion is mediated by a RDH that is presumed to be different from RDH5. This alternate pathway maintains a constant level of 11-cis-retinal despite changes in ambient light, i.e., maintains the levels of 11-cis-retinal after onset of light. The main pathway is important for the acute changes in levels that occur during the process of vision. Therefore, loss of RDH5 results in delayed recovery from light exposure.
Why does a two-cell system involving Müller and RPE cells exist for 11-cis-retinal renewal? It is speculated that such a system separates molecules in different compartments so that thermodynamically unfavorable reactions can proceed by mass action.[332] An alternative explanation could be that there may be no room for all the molecules required due to the high packing density of visual pigments in the OS.[332]
Mutations in the molecules involved in visual cycle result in retinal degenerations. Total loss of ABCA4 function causes retinitis pigmentosa.[333] Partial loss results in Stargardt disease or fundus flavimaculatus.[323] Loss of ABCA4 may also play a role in age-related macular degeneration.[334-336] Loss of CRALBP causes retinitis punctata albescens and autosomal recessive retinitis pigmentosa due to decreased efficiency of regeneration of visual pigment.[337,338] Loss of RDH5 causes fundus albipunctatus,[339] and loss of RGR leads to retinitis pigmentosa.[340,341] Mutations in RPE-65 can cause Leber congenital amaurosis and autosomal recessive childhood-onset severe retinal dystrophy.[342,343] Finally, loss of function mutations in LRAT leads to early onset retinal dystrophy.[344]
An opsin that is localized exclusively to RPE has been identified. It is present in the microvilli of the RPE. The exact role has not been elucidated.[345]
Immune Functions
The subretinal space is an immune-privileged site and can accept allografts for prolonged periods compared to a nonimmune-privileged site (e.g., the subconjunctival space).[346] Immune-privileged tissue can survive for prolonged periods in nonimmune-privileged sites compared to nonimmune-privileged tissue. RPE is an immune-privileged tissue. Neonatal RPE sheets, when placed in heterotopic sites (i.e., anatomic locations in which the transplanted tissue is not found normally) do not undergo rejection when compared to nonprivileged tissue.[347] Constitutive expression of Fas ligand on the RPE may contribute to its status as immune-privileged tissue.[347,348] RPE contributes to the immune-privilege of subretinal space by maintaining a blood-retinal barrier; RPE cells create a local immune suppressive microenvironment through the production of immuno-modulatory factors such as TGF-?,[349-352] through suppression of T-cell activation,[353,354] and through the induction of activated-T cell apoptosis.[355-357]
RPE cells express transplantation antigens. Major and minor histocompatibility (MHC) class I antigens are present on the surface of fetal RPE cells.[358,359] They do not seem to express MHC class II antigens normally but do so if exposed to IFN-?.[360] Culturing RPE cells also can induce MHC antigen expression.[360] RPE cells can process antigen prior to antigen presentation, thereby potentially acting as antigen-presenting cells.[361] Higher levels of IFN-?-induced MHC class II antigen expression on RPE cells leads to increased activation of antigen-specific T-cells as manifested by the production of pro-inflammatory TNF-?.[362] This activation does not include T-cell proliferation or production of IL-2, however, as is the norm for T-cell activation.
FUTURE DEVELOPMENTS
Due to the critical functions performed by RPE cells, perturbations in cell function can have serious consequences that affect vision. Their central role in visual physiology is demonstrated by the numerous visually debilitating conditions that are caused by mutations in RPE. RPE cells already are the target of therapeutic interventions for conditions such as Leber congenital amaurosis.[363] The ease of RPE isolation, the straightforward surgical access to the subretinal space, and the facile interdigitation of transplanted RPE cells with host photoreceptors renders the RPE a logical choice for initial attempts at cell-based therapy in the retina.[364] Newer experimental techniques like gene chip array and protein chips will continue to add to our knowledge of structure and function of RPE.
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