Anatomy and functions of pigmentary epithelium

this presentation includes a detailed description of anatomy and functions of retinal pigment epithelium.

1. Dr. RAVULA HASIKA M.S.OPHTHALMOLOGY ( 1 ST YR) ANATOMY AND FU NCTIONS OF PIGMENTARY EPITHELIUM AND BIOCHEMISTRY OF VISUAL PIGMENTS

2. Retinal pigment epithelial ( RPE ) cells form a monolayer of highly specialized pigmented cells critically located between the neural retina and the vascular choroid, which play a critical role in the maintenance of visual function.

3.  Light micrograph of the human retinal pigment epithelium (left) with the choroids above and the retina below. Cartoon of the retinal pigment epithelium (RPE) (right) aligned alongside the micrograph. CC, choirocapillaris ; BM, Bruchs membrane; RPE, retinal pigment epithelium; ap , apical processes; os , outer segments; C, cones; R, rods; M, Muller cells

4.  Transmission electron micrograph of the RPE cells and the RPE-choroid interface in a normal human donor eye. CC, choirocapillaris ; BM, Bruchs membrane; RPE, retinal pigment epithelium; ros , outer segments; ph, phagosomes ; pg, pigment granules

5. Retina is developed from the two parts of the optic cup : Neurosensory retina from the inner wall Retinal Pigment Epithelium from the outer wall EMBRYOLOGY

6. At day 27 post-conception - Optic vesicles invaginate to form the optic cup and the neuroepithelium undergoes early differentiation and thickening. By day 30 – the neural retina lies closely apposed to the future RPE later. By day 35 - melanin pigment granules are identified in the presumptive RPE; this is the earliest site of pigmentation in the body. By 6 th week of gestation - RPE cells elaborate basement membrane material that participate in the formation of the first recognizable Bruch’s membrane.

7. 8 th week

8. By 10 weeks - RPE cells developed apical projections that extend into the subretinal space. Between 4weeks and 6 months of gestation RPE cells exhibit a high rate of proliferation that peaks at 4 months of gestation ONCE THE EYE IS FULLY GROWN, IN ABSENCE OF DISEASE, RPE CELLS ARE STATIONARY, DONOT PROLIFERATE AND UNDERGO GROWTH ONLY BY CELL ENLARGEMENT.

9. Initiation of RPE and retina development: frontal section through the center of the optic cup (region of the optic fissure). The arrows indicate how neuroepithelium designated to become the RPE and neuroepithelium designated to become retina move into an opposed position. The space between the two layers will then be filled with interphotoreceptor matrix, the important interface for cross-talk between these two tissues and proper development. The ocular field of the neural plate shows the fate of various regions.

10. ANATOMY AND HISTOLOGY

11. Normal morphology of a retinal pigment epithelial cell (RPE) and its association with the choriocapillaris (CH) and the photoreceptor outer segments (POS). Note Bruch’s membrane (B), melanosomes (M), lysosomes (L), apical microvilli (V), and cell nucleus (N).

12. Approx. 3.5million RPE cells Begins at the optic nerve, extends to ora serrata and continues as the pigment epithelium of the ciliary body. RPE cell density decreases from the fovea centralis to the periphery. Apical surface of the RPE cells - outer segments of the photoreceptors. Basal surface – attaches firmly to the underlying Bruch’s membrane

13. Brown color of the RPE layer - melanin granules; and the typical pattern of the fundus results from variations in the pigmentation of the RPE layer. Highest concentration of the pigment is found in peripheral retina, the lowest in the macular area

14. The RPE is a monolayer of cells that are cuboidal in cross section and hexagonal when viewed from above. S urface characteristics

15. The cell shape varies throughout the fundus. Macular area - tall and narrow; periphery - flatter, more spread out and are often binucleated . Apical cell membrane is characterized by numerous microvilli that interdigitate with the outer segments of the retinal photoreceptors Between 30-45 photoreceptors are in contact with each RPE cell . RPE basal membrane domain is characterized by infoldings that are approximately 1µm in length.

16. The functional polarity of RPE cells is expressed in the differential distribution of membrane proteins along the apical - basal axis.

17. LOCATION PROTEIN FUNCTION Apical membrane Na⁺, K⁺- ATPase Na⁺ flux N-CAM Adhesion to retina ανβ 5 integrin Phagocytosis CD 36 Phagocytosis Lateral membrane Occluden Tight junction Cadherin Adherens junction Connexin Gap junction Basolateral membrane α 3 β 1, α 6 β 1, ανβ 3 integrins Attachment to ECM/Bruch’s membrane

18. The lateral domains of adjacent RPE cells are connected by apical zonulae occludens (tight junctions) and adjacent zonulae adherentes ( adherens junctions ) These junctions seal off the subretinal space where the exchange of macromolecules with the choriocapillaris takes place, and form the so-called Verhoeff’s membrane . C ellular Junctions

19. The zonulae occludens between adjacent RPE cells form a ‘tight’ intercellular junction due to interaction between the extracellular domains of adjacent occludin molecules leading to high transepithelial resistance and an intact blood-retinal barrier. Tight junctions are also responsible for the sequestration of molecules into the apical and basal plasma membrane domains.

20. The zonulae adherentes ( adherens junction ) form a junction with a separation of 200 A˚ and are associated with circumferential microfilament bundles. The adherens junctions play a role in maintenance of the polygonal shape of the RPE cell and in the organization of actin cytoskeleton.

21. Melanin granules – ovoid or spherical in shape, 2-3µm in length & 1 µm in dia – Apical part of the cell Endoplasmic reticulum – Apical part of the cell Nucleus – dia of 8-12µm – Basal part of the cell Most of the Mitochondria - Basal part of the cell C ytoplasmic Organelles

22. Composed of three major elements : - Actin microfilaments ( dia 7nm ) - Microtubules ( dia 25 nm ) - Intermediate filaments ( dia 10 nm ) Microtubules and microfilaments are dynamic structures that undergo polymerization and depolymerization & are critical for intracellular transport. C ytoskeleton

23. Microtubules play a role in mitosis, and the movement of subcellular organelles and pigment granules. Actin microfilaments – located in the microvilli and throughout the cytoplasm – play an important role in the generation and maintenance of cellular shape and cell migration. Intermediate filaments provide a structural framework – type I (acidic keratins ), type II (basic/neutral keratins ) and type V ( lamin ) are found

24. RPE cells actively synthesize and degrade extracellular matrix ( ECM ) components. RPE Cells elaborate a basal basement membrane, which constitutes the innermost layer of Bruch’s membrane and contains type IV collagen, a specific basement membrane- associated heparan sulfate proteoglycan , and laminin . The apical domain of the RPE cells is embedded in the interphotoreceptor matrix ( IPM ), which is produced by the RPE and the inner segments of the photoreceptors. E xtracellular Matrix

25. Major protein components of IPM that are involved in retinoid transport between the photoreceptors and the RPE include the interphotoreceptor binding protein (IRBP), retinol binding protein (RBP ) and transthyretin (TTR). Components of ECM can influence cell behavior by activating cell surface receptors. Integrins play a critical role in cell – ECM interaction by mediating bidirectional signals between the cell and the external environment.

26. Degradation of ECM is regulated, in part, by the equilibrium between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) Normal RPE express the membrane bound type I (MT1-MMP) as well as type 2 (MMP-2) metalloproteinase, as well as the metalloproteinase inhibitors, TIMP-1 and TIMP-3. TIMP-3 mutations - Sorsby fundus dystrophy TIMP-3 accumulation in Bruch’s membrane - ARMD

27. In macula – RPE are smaller and columnar In periphery – larger and cuboidal P henotypic Heterogenecity of the RPE

28. FUNCTIONS OF THE RPE

29. Physiologic adhesion of neural retina Phagocytosis of shed outer rod segments Blood-retinal barrier, transport and ionic pumps Retinol metabolism Melanin pigment Immune function Growth factors and cytokines RPE activation Gene expression profiles of the RPE Senescence and cell death

30. Adhesion of retina to RPE – passive & active forces Passive forces – endotamponade of the vitreous gel - transretinal fluid gradients - the interphotoreceptor matrix - osmotic pressure of the choroid Active forces – actively pumping water and electrolytes out of the subretinal space by the apically segregated Na⁺K ⁺ pump & secondarily by HCO₃ transport system. Apical expression of N-CAM 1.Physiologic Adhesion Of Neural Retina

31. One of the critical functions of the RPE is to phagocytose and degrade ROS which are shed diurnally from the distal end of the photoreceptors. ROS phagocytosis is a highly specialized receptor mediated multistep process including Recognition- attachment (receptor- ligand interactions) Internalization ( transmembrane signaling and contractile proteins ) Degradation ( hydrolytic enzymes ) 2.Phagocytosis Of Shed Outer Rod Segments ( ROS )

32. Anatomically the outer blood-retinal barrier is formed by the RPE, which controls the exchange of fluid and molecules between the fenestrated capillaries and the outer retina. Two major components of RPE barrier function are : the tight junctions between the RPE cells , and the polarized distribution of RPE membrane proteins. 3. Blood-retinal barrier, Transport and Ionic pumps

33. Since tight junctions inhibit intercellular diffusion, molecular exchanges predominantly occur across the RPE cells themselves. The regulation of transepithelial transport is dependent on the asymmetric distribution of cellular proteins. In RPE, Na⁺, K⁺- ATPase is localized at the apical cell membrane, and cytoskeletal proteins ( ankyrin and fodrin ) are also localized apically. Important for the transport functions is the presence of apical microvilli and basal plasma membrane infoldings, which increase the surface area available for exchange of nutrients and catabolites.

34. Human RPE express 2 proton coupled monocarboxylate transporters : MCT-1 in the apical membrane and MCT-3 in the basolateral membrane. Coordinated activity of these 2 transporters could facilitate the flux of lactate from the retina to choroid. P-glycoprotein – contribute to normal transport function of RPE cells. Aquaporin – facilitates water movement across the RPE monolayer .

35. Breakdown of the blood-retinal barrier has serious consequences for the health of the eye and is present in many types of retinopathies. HEPATOCYTE GROWTH FACTOR is one important growth factor that regulates the barrier function of RPE cells.

36. Phototransduction begins by absorption of a photon by a highly sensitive analog of vitamin A, 11- cis -retinal that is bound to opsin proteins in the photoreceptors. 4.R etinol metabolism

37. Retinol is transported to the retina via the circulation, where it moves into retinal pigment epithelial cells. There, retinol is esterified to form a retinyl ester that can be stored. When needed, retinyl esters are broken apart (hydrolyzed) and isomerized to form 11- cis -retinol, which can be oxidized to form 11- cis -retinal. 11- cis -retinal can be shuttled to the rod cell, where it binds to a protein called opsin to form the visual pigment, rhodopsin (also known as visual purple). Absorption of a photon of light catalyzes the isomerization of 11- cis -retinal to all- trans -retinal and results in its release. This isomerization triggers a cascade of events, leading to the generation of an electrical signal to the optic nerve. The nerve impulse generated by the optic nerve is conveyed to the brain where it can be interpreted as vision. Once released, all- trans -retinal is converted to all- trans -retinol, which can be transported across the interphotoreceptor matrix to the retinal epithelial cell to complete the visual cycle

38. Provision of a continuous source of 11- cis retinal is accomplished by the recycling of vitamin A analogs between the photoreceptors and the RPE. Thus the metabolism of retinol is one of the most important and highly specialized functions of the RPE.

39. The re- isomerisation of all- trans -retinol to 11- cis -retinal in the RPE is a crucial aspect of the visual cycle.

40. Melanosomes , the organelles responsible for the biosynthesis of melanin, appear in the human RPE beginning at 6 weeks of gestational age. With in the adult RPE cell, the melanin granules are located in the apical portion of the cell, adjacent to ROS. 5. M elanin Pigment

41. Retinal pigment epithelium (RPE) contains numerous elongated melanin granules that are aggregated in the apical portion of the cell, where the microvilli extend from the surface toward the outer segments of the rod and cone cells.

42. Melanin granules Reduce light scattering & block light absorption via the sclera – better image Absorbs radiant energy and dissipates the energy in heat Can bind redox -active metal ions and sequester them in an inactive state – preventing oxidative damage to the retina RPE cells high in melanin content exhibit significantly less formation of lipofuscin than cells low or devoid of melanin

43. Melanin concentration of RPE cells decreases between the periphery and posterior pole and increases in the macular region With age – melanin content decreases in RPE cells The melanin in RPE cells represents single most important source for heat in thermal photocoagulation Congenital disorder in melanin production – albinism – associated with decreased vision, photophobia and nystagmus. Since albino individuals show foveal hypoplasia , it is suspected that melanin plays an important role in retinal development.

44. The RPE is located at the critical interface between the systemic circulation and the neural retina, and part of its role at this site may be as a regulator of the local immune response. Immunosuppressive mechanisms - includes both the passive barrier provided by the RPE and the active secretion of immunosuppressive cytokines such as transforming growth factor-beta (TGF- β ). In the presence of inflammatory response RPE may inhibit the action of the inflammatory mediators. 6. I mmune Function

45. RPE cells are able to contribute to immunosuppressive and inflammatory responses in the eye by secretion of cytokines, antagonists, and soluble cytokine receptors.

46.  Secretion of cytokines by stimulated RPE cells and the immune cells targetted by these factors.

47. The RPE cells play an important role in the synthesis of many immuno -modulating molecules ( Fc γ receptors, CR3 receptors and C5a receptors ), a host of immuno-modulatory cytokines ( eg . IL-1 β , IL-6,IL-8), their ability to phagocytoze T-lymphocytes, their resistance to attack, in vivo and invitro , by sensitized T-cells and their suppression of T-cell activation.

48. Chemokines and inflammatory cytokines are secreted in significant amounts only after RPE activation. RPE monolayer reveal expression of TGF- β 2, bFGF , aFGF , FGF-5, HGF and PDGF-A as well as their corresponding receptors . Production of TGF- β by RPE – maintenance of an anti-inflammatory state, inhibition of cellular proliferation and stimulation of phagocytosis. FGF’s – enhance RPE cell proliferation and migration. VEGF and its receptors are expressed by RPE; however expression of VEGF in the resting monolayer appears to be very low 7. G rowth Factors And Cytokines

49. In response to injury or certain alterations in the microenvironment, RPE cells, which in situ do not proliferate, may detach from their substratum, migrate, proliferate and aquire a macrophage like or fibroblast like morphology. These morphologic and functional changes, associated with alterations on gene expression, may be reffered to as ACTIVATION 8.RPE activation

50. Activation events include physical trauma with displacement to a new environment, accumulation of intraocular blood, breakdown of the blood-retinal barrier with inflammatory cell infiltration, alteration of components of extracellular matrix, or alterations in choroidal circulation or diffusion of oxygen to the RPE layer. Mediators of activation include vitreous and ECM components such as fibronectin or TGF- β , blood derived products such as thrombin or PDGF, macrophage or lymphocyte derived inflammatory cytokines, accumulated products in Bruch’s membrane or Drusen or hypoxia.

51. Activated and proliferating RPE cells express higher levels of the α 5 integrin than quiescent RPE. Production of ECM components, such as collagen and fibronectin is also stimulated in activated RPE. Proliferation of RPE cells occurs after stimulation with a number of factors including PDGF, TNF- α ,IGF and VEGF.

52. The production of growth factors by the RPE may affect not only the immediate microenvironment but may affect adjacent compartments. VEGF expression is enhanced by hypoxia as well as several other cytokines and may play a role in induction of choroidal neo vascularization . Enhanced collagen production, and induction of smooth muscle actin expression in RPE cells by TGF- β , may play a role in contraction of cellular retinal membranes leading to retinal detachment.

53. RPE shows specific features associated with aging. The proportion of apoptotic human RPE cells increase significantly with age and these apoptotic cells are confirmed mainly to the macula. Furthurmore , cells become more irregular in size and shape, deposits of RPE derived material accumulate in Bruch’s membrane and lipofuscin appears in the cell’s cytoplasm. 9.Senescence And Cell Death

54. LIPOFUSCIN GRANULES , the second most prominent pigment in the RPE cell, appear after birth as the residue of phagocytosis and metabolism of the RPE cells. Uniform in size (1.5µ); basal portion of cell; yellowish in color and exhibit auto fluorescence. Number increases with age. Can lead to RPE dysfunction by reducing functional cytoplasmic space and distorting the cellular architecture. In human RPE, lipofuscin load appears associated with age-related macular degeneration.

55. Age-related changes are also found in the enzyme content of the RPE cells. Activities of cathepsin D, acid phosphatase and β - glucuronidase increase with age, whereas other enzymes such as α - mannosidase decrease. Glycosaminoglycans distribution are altered with age . With age, there is thickening of Bruch’s membrane – leads to impaired macromolecular exchange between choroidal and RPE compartments, decreased amino acid permeability and very likely a decrease in all metabolites and waste products.

56. With increasing age a gradual loss of RPE cells is seen, and the remaining cells increase in size. The entry of RPE cells into senescence is probably controlled by loss of telomerase expression, progressive telomerase shortening and the crossing of a threshold telomerase length.

57. Electron microscopic images of apoptotic and necrotic RPE cells. ( A ,  B ) Normal nucleus and cytoplasm. ( C ) Nuclear chromatin condensation characteristic of apoptosis. ( D ) Cytoplasmic vacuoles characteristic of necrosis.

58. BIOCHEMISTRY OF VISUAL PIGMENTS

59. Visual pigments – property of absorbing light. Most of the pigments in the visual cells are not limited in their absorption to one small band of wavelengths but rather absorb, to a greater or lesser extent, over a broad range of spectrum. The peak of each pigment’s absorption curve is called its Absorption Maximum .

60. The visual pigments in the eyes of humans and most other mammals are made up of a protein called opsin and retinene, the aldehyde of vitamin A. Retinene = retinal

61. It is the photosensitive visual pigment present in the discs of the rod outer segments RHODOPSIN ( VISUAL PURPLE )

62. It consists of protein opsin ( called as scotopsin ) and a carotenoid called retinal ( the aldehyde of vitamin A ) Membrane bound glycolipid . Molecular weight – 40,000 Serpentine receptor coupled to G proteins

63. Insoluble in water but can be taken into solution if detergent is added. Sensitive to heat and chemical agents. Opsin – 348 amino acid protein that crosses the disc membrane seven times.

64. 2 palmitate molecules are linked with cysteines via thioester linkages at the intracellular C-terminal. Oligosaccharide residues are located on the extracellular N-terminal.

65. The absorption spectrum of rhodopsin depicts that its peak sensitivity to light lies within the narrow limits of 493-505 nm which means light of that wavelength ( deep green ) is most effective for bleaching and is the color to which dark adapted eye is most sensitive. It absorbs primarily yellow wavelength of light, transmitting violet and red to appear purple by transmitted light; it is, therefore called Visual purple .

66. The cone pigments in humans have not been chemically isolated but are presumed to be similar to rhodopsin. Three cone pigments have been identified : erythrolabe , chlorolabe , and cyanolabe . Erythrolabe is most sensitive to red light waves; chlorolabe is most sensitive to green light waves; and cyanolabe is most sensitive to blue light waves. CONE PIGMENTS The peak absorbance wavelength of the ‘blue’, ‘green’, and ‘red’ sensitive cones lie at about 435,535 and 580 nm , respectively

67. The light falling upon the retina is absorbed by the photosensitive pigments in the rods and cones and initiates photochemical changes which in turn initiate electrical changes and in this way the process of vision sets in. The photochemical changes occur in the outer segments of both the rods and the cones. The photochemical reactions in the rod outer segments can be described under three headings : Rhodopsin bleaching Rhodopsin regeneration and Visual cycle . LIGHT INDUCED CHANGES

68. R hodopsin Bleaching

69. Rhodopsin – protein called opsin and a carotenoid called retinene ( vitamin A aldehyde or 11-cis-retinal ) The light absorbed by the rhodopsin converts its 11-cis-retinal into all-trans-retinal. These are isomers having same chemical composition but different shapes

70. This light induced isomerization of 11-cis-retinal into all-trans-retinal occurs through formation of many intermediates which exist for a transient period. One of the intermediate compounds ( metarhodopsin II ) of the above isomerization chain reaction acts as an enzyme to activate many molecules of transducin.

71. The transducin is a GTP/GDP exchange protein present in an inactive form bound to GDP in the membranes of discs and cell membrane of the rods. The activated transducin (bound to GTP) in turn activates many more molecules of phosphodiesterase (PDE) which catalyses conversion of cyclic guanosine monophosphate ( cGMP ) to GMP, leading to a reduction in concentration of cyclic GMP ( cGMP ) within the photoreceptor. The reduction in cGMP is responsible for producing the electrical response, which marks the beginning of the nerve impulse.

72. The all-trans-retinal (produced from light-induced isomerization of 11-cis retinal) can no longer remain in combination with the opsin and thus there occurs separation of opsin and all-trans-retinal. This process of separation is called photodecomposition & the rhodopsin is said to be bleached by the action of light.

73. The all-trans-retinal separated from the opsin, subsequently enters into the chromophore pool existing in the photoreceptor outer segment and the pigment epithelial cells. The all-trans-retinal may be further reduced to retinol by alcohol dehydrogenase , then esterified to re-enter the systemic circulation. R hodopsin Regeneration

74. The first stage in the reformation of rhodopsin, is isomerization of all-trans-retinal back to 11-cis-retinal. The process is catalyzed by retinal isomerase. The 11-cis-retinal in the outer segments of photoreceptors reunites with the opsin to form rhodopsin. This whole process is called Regeneration of the rhodopsin. Thus, the bleaching of the retinal photopigments occurs under the influence of light, whereas the regeneration process is independent of light.

75. In the retina, under constant light stimulation, a steady state must exist under which the rate at which the photochemicals are bleached is equal to the rate at which they are regenerated. This equilibrium between the photodecomposition and regeneration of visual pigments is referred to as visual cycle. V isual Cycle

76. Like rhodopsin, cone pigments also consist of the protein opsin ( called photopsin ) and the retinene (11-cis-retinal). Photopsin differs slightly from the scotopsin ( rhodopsin ). There are three classes of cone pigments : red sensitive ( erythrolabe ), green sensitive ( chlorolabe ) and blue sensitive ( cyanolabe ), which have different absorption spectra. PHOTOCHEMISTRY OF PHOTOPIC VISION

77. It has been assumed that when light strikes the cones, the photochemical changes occur in the cone pigments which are very similar to those of rhodopsin. However, it has been noted that, nearly total rod bleaching occurs before significant bleaching can be observed in cones. This differential bleaching quality sets aside the scotopic rod portion of the visual system from the photopic portion which functions during brightly lighted conditions.

78. THANK YOU …