The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and choroid that plays a critical role in visual function. RPE cells originate from the outer wall of the optic cup during development. In adults, RPE cells form a tightly packed monolayer that acts as the outer blood-retinal barrier and performs several functions including phagocytosis of photoreceptor outer segments, retinoid metabolism, secretion of growth factors, and regulation of the immune response in the eye.

1. Dr. RAVULA HASIKA
M.S.OPHTHALMOLOGY ( 1ST YR)

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

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 6th week of gestation - RPE cells elaborate basement
membrane material that participate in the formation of the
first recognizable Bruch’s membrane.

7. 8th 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.

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

14.  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

15.  The RPE is a monolayer of cells that are cuboidal in cross
section and hexagonal when viewed from above.

16.  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.

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

19. LOCATION PROTEIN FUNCTION
Apical membrane Na⁺, K⁺-ATPase Na⁺ flux
N-CAM Adhesion to retina
ανβ5 integrin Phagocytosis
CD 36 Phagocytosis
Lateral membrane Occluden Tight junction
Cadherin Adherens junction
Connexin Gap junction
Basolateral membrane α3β1, α6β1, ανβ3 integrins Attachment to
ECM/Bruch’s membrane

20.  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.

21.  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.

22.  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.

23.  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

25.  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.

26.  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

27.  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.

28.  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.

29.  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

30.  In macula – RPE are smaller and columnar
 In periphery – larger and cuboidal

32. 1. Physiologic adhesion of neural retina
2. Phagocytosis of shed outer rod segments
3. Blood-retinal barrier, transport and ionic pumps
4. Retinol metabolism
5. Melanin pigment
6. Immune function
7. Growth factors and cytokines
8. RPE activation
9. Gene expression profiles of the RPE
10. Senescence and cell death

33.  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

34.  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 )

37.  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.

38.  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.

39.  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 .

40.  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.

41.  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.

42. 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

43.  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.

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

45.  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.

46. 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.

47. 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

48.  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.

49.  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.

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

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

52.  The RPE cells play an important role in the synthesis of
many immuno-modulating molecules ( Fcγ receptors, CR3
receptors and C5a receptors ), a host of immuno-modulatory
cytokines (eg. IL-1β, 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.

53.  Chemokines and inflammatory cytokines are secreted in
significant amounts only after RPE activation.
 RPE monolayer reveal expression of TGF-β2, 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

54.  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

55.  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.

56.  Activated and proliferating RPE cells express higher levels
of the α5 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.

57.  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.

58.  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.

59.  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.

60.  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.

61.  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.

62. 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.

64.  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.

65.  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

66.  It is the photosensitive visual pigment present in the discs
of the rod outer segments

68.  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

70.  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.

71.  2 palmitate molecules are linked with cysteines via
thioester linkages at the intracellular C-terminal.
 Oligosaccharide residues are located on the extracellular
N-terminal.

72.  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.

73.  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.
The peak absorbance wavelength
of the ‘blue’, ‘green’, and ‘red’
sensitive cones lie at about
435,535 and 580 nm , respectively

74.  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.

76.  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

77.  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.

78.  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.

80.  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.

81.  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.

82.  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.

83.  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.

84.  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.

85.  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.