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Retinal pigment epithelium in the pathogenesis of age‐related macular degeneration and photobiomodulation as a potential therapy?

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The retinal pigment epithelium (RPE) comprises a monolayer of cells located between the neuroretina and the choriocapillaries. The RPE serves several important functions in the eye: formation of the blood retinal barrier, protection of the retina from oxidative stress, nutrient delivery and waste disposal, ionic homeostasis, phagocytosis of photoreceptor outer segments, synthesis and release of growth factors, reisomerization of all-trans-retinal during the visual cycle, and establishment of ocular immune privilege. Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Dysfunction of the RPE has been associated with the pathogenesis of AMD in relation to increased oxidative stress, mitochondrial destabilization and complement dysregulation. Photobiomodulation or near infrared light therapy which refers to non-invasive irradiation of tissue with light in the far-red to near-infrared light spectrum (630-1000 nm), is an intervention that specifically targets key mechanisms of RPE dysfunction that are implicated in AMD pathogenesis. The current evidence for the efficacy of photobiomodulation in AMD is poor but its safety profile and proposed mechanisms of action motivate further research as a novel therapy for AMD.
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Review
Retinal pigment epithelium in the pathogenesis
of age-related macular degeneration and
photobiomodulation as a potential therapy?
Jack Ao MBBS,
1
John PM Wood DPhil,
1
Glyn Chidlow DPhil,
1
Mark C Gillies PhD FRANZCO
2
and
Robert J Casson DPhil FRANZCO
1
1
South Australian Institute of Ophthalmology, University of Adelaide, Adelaide, South Australia, and
2
The Save Sight Institute, Sydney
Medical School, The University of Sydney, Sydney, New South Wales, Australia
ABSTRACT
The retinal pigment epithelium (RPE) comprises a
monolayer of cells located between the neuroretina and
the choriocapillaries. The RPE serves several important
functions in the eye: formation of the blood-retinal bar-
rier, protection of the retina from oxidative stress, nutri-
ent delivery and waste disposal, ionic homeostasis,
phagocytosis of photoreceptor outer segments, synthe-
sis and release of growth factors, reisomerization of all-
trans-retinal during the visual cycle, and establishment
of ocular immune privilege. Age-related macular
degeneration (AMD) is the leading cause of blindness
in developed countries. Dysfunction of the RPE has
been associated with the pathogenesis of AMD in rela-
tion to increased oxidative stress, mitochondrial
destabilization and complement dysregulation. Photo-
biomodulation or near infrared light therapy which
refers to non-invasive irradiation of tissue with light
in the far-red to near-infrared light spectrum
(6301000 nm), is an intervention that specically tar-
gets key mechanisms of RPE dysfunction that are impli-
cated in AMD pathogenesis. The current evidence for
the efcacy of photobiomodulation in AMD is poor but
its safety proleandproposedmechanismsofaction
motivate further research as a novel therapy for AMD.
Key words: age-related macular degeneration (AMD),
low light therapy, near infrared light, photobiomodula-
tion, retinal pigment epithelium (RPE).
INTRODUCTION
The retinal pigment epithelium (RPE) comprises a
monolayer of cells located between the retinal pho-
toreceptors and the fenestrated choriocapillaris,
which is basally bordered by the elastin and
collagen-rich Bruchs membrane (BM). It serves sev-
eral important functions in maintaining photorecep-
tor health. Its tight junctions protect the neuroretina
from blood-borne toxins and its light-absorbing pig-
ments mitigate photo-oxidative stress. The RPE
delivers nutrients and disposes waste products
including phagocytosis of photoreceptor outer seg-
ments (POS). It is fundamental to the visual cycle
by recycling of retinoids. Furthermore, the RPE con-
tributes to the production and secretion of growth
factors and provides ocular immune privilege.
Age-related macular degeneration (AMD)
accounts for 8.7% of worldwide blindness and is
the leading cause of blindness in developed coun-
tries.
1
Although the pathogenesis of AMD remains
incompletely understood, there is evidence that dys-
function of the RPE resulting from oxidative stress,
impairment of nutrient delivery as well as energy
metabolism and complement dysregulation, contrib-
utes to the pathogenesis of AMD.
Photobiomodulation, also known as near infrared
(NIR) light therapy, is known to increase cellular
metabolism, energy supply and metabolic repair
processes.
2,3
Hence, photobiomodulation of dysfunc-
tional RPE could conceivably restore function and
attenuate the pathogenesis of AMD.
Correspondence: Dr Jack Ao, Ophthalmic Research Laboratory, WS7062.46, Level 7, Adelaide Health and Medical Sciences Building, University of
Adelaide, North Terrace, Adelaide, SA 5000, Australia. E-mail: jack.ao36@gmail.com
Received 11 September 2017; accepted 28 November 2017.
Conict of interest: None declared
Funding sources: None declared.
Clinical and Experimental Ophthalmology 2018; 46: 670686 doi: 10.1111/ceo.13121
© 2017 Royal Australian and New Zealand College of Ophthalmologists
The aim of this review is to summarize the struc-
ture and functions of the RPE and its role in the
pathogenesis of age-related macular degeneration,
and to discuss the current evidence and potential of
photobiomodulation in AMD.
STRUCTURE
The RPE comprises a polygonal monolayer of cells,
the size and shape of which vary as a function of ret-
inal eccentricity, from a diameter of 14 μm at the
fovea to considerably larger (60 μm) in the periph-
eral retina.
49
The height of RPE cells is a 1015 μm
at the fovea and approximately 7.5 μm at the periph-
ery.
10
The cell density of the RPE is greater in the
fovea compared to the equator.
11
With age, periph-
eral RPE density declines, but this is preserved at
the fovea due to inward migration of peripheral
RPE.
12
Approximately 3040 photoreceptors overlie
each RPE cell.
11
The RPE projects microvilli apically
that enshroud the POS. Long thin apical microvilli
measuring 57μm in length are projected from the
RPE and enshroud the POS, facilitating transepithe-
lial and retina-to-RPE transport. Similarly, the basal
surface of the RPE contains complex infoldings to
support molecular movement. This apical-basal
polarity is also reected in the organization of RPE
organelles and membrane proteins. Melanosomes
are situated apically whilst the nucleus, mitochon-
dria, endoplasmic reticulum, lysosomes, Golgi appa-
ratus, lipofuscin and melanolipofuscin granules are
located towards the basal side.
7,13
The polar distri-
bution of different combinations of ion channels and
pumps on either the apical or basal membrane
enables the diffusion of certain ions in specic direc-
tions in order to transport nutrients to photorecep-
tors and remove retinal waste products as well as
the maintenance of subretinal ionic homeostasis as
elucidated below.
BLOOD-RETINAL BARRIER
Tight junctions between adjacent RPE cells are
attached to the actin cytoskeleton and form part of
the outer blood-retinal barrier.
14
This prevents para-
cellular transport of large molecules, toxins, blood-
borne products and water.
15
As a result of the tight
junctional barrier produced by the RPE, resistance
to paracellular movement is 10 times higher than
resistance to transcellular movement.
16,17
However,
this resistance is plastic, as permeability of the tight
junctions can change in the presence of diffusible
factors produced from the neural retina.
18
One such
factor is vascular endothelial growth factor (VEGF)
which has been implicated in the breakdown of the
outer blood-retinal barrier in diabetic macular
oedema.
19,20
Hypoxia and hyperglycaemia may
upregulate hypoxia inducible factor 1 alpha and
therefore VEGF expression, resulting in altered tight
junction proteins.
2022
During the development of
the RPE, tight junctions tend to be leaky, but these
become progressively less permeable due to secre-
tion of these diffusible factors from the neural
retina.
18,23
PROTECTION FROM OXIDATIVE DAMAGE
The RPE is responsible for the absorption of
reected and scattered light. This helps to both opti-
mize image quality and protect the retina from oxi-
dative damage caused by reactive free radicals,
which result from the combination of constant RPE
exposure to light, the high RPE metabolic activity,
the elevated local oxygen tension and the photo-
oxidation of lipofuscin.
6,7
The pigment melanin,
which is stored and synthesized in melanosomes,
absorbs and lters harmful light, particularly in the
blue wavelength region of the spectrum which is
known to cause photo-oxidation of lipofuscin, a
lipid-protein pigment that accumulates with age
and from phagocytosis of the POS.
24,25
Melanin has
been identied as having additional antioxidative
properties, including counteracting singlet oxygen
and scavenging reactive oxygen species (ROS).
26,27
Melanin density increases towards the centre of the
retina, reaching its peak at the fovea, contributing to
the clinically darker appearance of the fovea and
surrounding macula. Melanin within the RPE
decreases uniformly after the age of 40.
7
Another
mechanism of defence against oxidative damage is
the intrinsic antioxidant defence system of the RPE,
which comprises both enzymes, such as catalase and
superoxide dismutase, and non-enzymatic antioxi-
dants such as α-tocopherol, β-carotene and
ascorbate.
2832
Glutathione has been implicated as
another antioxidant in the RPE with functions
including perioxidase-reduction of lipid hydroper-
oxides, maintenance of ascorbate in its reduced form
as well as detoxication of reactive products of lipid
peroxidation such as 4-hydroxynonenol.
33,34
TRANSPORT OF NUTRIENTS,WASTES
AND WATER
The close anatomical apposition of the RPE to the
photoreceptors is necessary for facilitating the trans-
cellular movement of nutrients, wastes, uid
and ions.
The delivery of nutrients, including glucose, ascor-
bate and fatty acids, from the choroidal vasculature to
the photoreceptors occurs through transporters within
the RPE membranes. Glucose is passively transported
through numerous GLUT1 (glucose transporter 1) and
GLUT3 (glucose transporter 3) transporters on both
RPE in AMD and the role of photobiomodulation 671
© 2017 Royal Australian and New Zealand College of Ophthalmologists
the apical and basal membranes.
35,36
More recently,
additional glucose transporters GLUT2 (glucose trans-
porter 2) and GLUT5 (glucose transporter 5) have
been identied on the apical and basolateral mem-
branes in cultured human RPE cells.
37
Ascorbate is a carbohydrate vitamin that is vital
in hydroxylation reactions and biosynthetic path-
ways, especially in the production of collagen and
glycosaminoglycans which are components of the
interphotoreceptor matrix (IPM) and BM.
3840
It is
also an effective antioxidant and scavenger of super-
oxide radicals. The transcellular movement of ascor-
bic acid across the RPE occurs through active
sodium-dependent transport that is reduced by pres-
ence of ouabain, 2,4-dinitrophenol and dehydroas-
corbic acid.
40
Photoreceptor membranes, which comprise a sig-
nicant portion of the omega 3 fatty acid docosahex-
anoic acid (DHA), are continuously renewed and
added to the POS as photoreceptor tips are shed and
phagocytized by the RPE.
41
Thus, photoreceptors
require a constant supply of DHA. Initially, DHA is
produced in the liver from the dietary precursor
α-linelonic acid before being carried by lipoproteins
in the circulation to the RPE. Transport through RPE
membranes occurs through a concentration-
dependent manner.
4143
DHA acid has also been
identied as a precursor to neuroprotectin 1, which
protects RPE from oxidative damage and may also
play a role in RPE retinoid transport by modulating
binding between interphotoreceptor binding protein
(IRPB) and 11-cis-retinal.
44
Lactic acid, a metabolic end product produced in
excess by the retina and thought to be specically
released from POS and to a lesser extent, photore-
ceptor inner segments has been proposed to act as a
nutrient for RPE.
45,46
Analysis of bovine eyes
revealed the distribution of lactate on the retinal sur-
face was seven times greater than at the RPE whilst
glucose was 50 times greater at the RPE than the ret-
inal surface. The distribution of these fuels suggests
RPE consumes more lactate and releases more glu-
cose from the retina.
46
However no comparison has
been made in vivo and the mechanism of lactate
metabolism in RPE remains unclear. From the
subretinal space, lactic acid enters the RPE apical
membrane by active transport through lactate-H+
co-transporter monocarboxylate transporter
1 (MCT1) and the Na
+
-dependent co-transporter.
Irrespective of whether lactate acts as a fuel source
for RPE cells or not, excess levels of this compound
are removed through the RPE basolateral membrane
via monocarboxylate transporter 3 (MCT3) and the
Na
+
-lactate exchanger.
47,48
Constant transport of water away from the subret-
inal space to the choriocapillaris is required to main-
tain close apposition of the RPE and photoreceptors.
Large volumes of subretinal uid are generated from
metabolic turnover in photoreceptors as well as
intraocular uid ux from the vitreous body towards
the retina. Water travels across the RPE to the chor-
iocapillaris by active transport through the aqua-
porin 1 channel, identied in studies of cultured rat
and human RPE.
49,50
Water transport is also facili-
tated by the transport of ions such as Cl
and K
+
which will be elaborated below.
TRANSEPITHELIAL TRANSPORT AND
MAINTENANCE OF SUBRETINAL ION LEVELS
The RPE plays a major role in preserving excitability
of photoreceptors by careful regulation of subretinal
ionic concentration through transepithelial transport
of ions (Fig. 1). The apical Na
+
/K
+
ATPase pump,
which results in outow of Na
+
and inux of K
+
,
provides the energy gradient that is vital to transe-
pithelial transport.
5153
The Na
+
/K
+
ATPase,
together with the polarized distribution of other
channels and transporters, produce a high subretinal
sodium concentration and high potassium concen-
tration in the RPE cytosol. The elevated subretinal
sodium is critical to the dark current which is pro-
duced by the entry of sodium ions through cGMP
(cyclic guanosine monophosphate) gated channels,
resulting in depolarization of photoreceptors. Apical
entry of sodium occurs through the Na
+
K
+
2Cl
co-
transporter and exit occurs basolaterally through
mechanisms that are unclear.
54
However, most intra-
cellular sodium ions are recycled through the apical
Na
+
/K
+
ATPase to maintain the high subretinal
sodium concentration.
The RPE also effectively compensates for ionic
changes such as uctuation in potassium levels dur-
ing phototransduction. Potassium can enter the RPE
apically through Na
+
/K
+
ATPase or Na
+2
K
+
2Cl co-
transporter and leave basolaterally or apically
through K
+
channels.
54
Whilst in the dark, the
potassium concentration is 5 mM with net transe-
pithelial efux of potassium from the subretinal
space to the choroid. Exposure to light causes the
hyperpolarization of photoreceptors leading to the
closure of cGMP gated Na
+
channels in the outer
segment membranes and reduced K
+
efux from the
inner segment. Subsequently, the subretinal potas-
sium concentration decreases to 2 mM which trig-
gers hyperpolarization of the RPE apical membrane
and activation of inward rectifying K
+
channels on
the apical membrane, causing K
+
ow into the sub-
retinal space. In the early phase of RPE hyperpolari-
zation, the apical Na
+
K
+
2Cl
co-transporter
reverses direction, further increasing K
+
movement
to the subretinal space, resulting in the restoration
of subretinal potassium concentration.
55
672 Ao et al.
© 2017 Royal Australian and New Zealand College of Ophthalmologists
Chloride, a vital facilitator for uid movement, enters
through the RPE apical membrane via the Na
+
K
+
2Cl
co-transporter before exiting basolaterally via voltage-
dependent chloride and CIC-2 channels. The basolat-
eral Cl
/HCO
3
exchanger, an important channel in pH
regulation, transports chloride back into the RPE. An
additional efux channel for chloride in RPE cells is the
CFTR channel on the basolateral aspect.
54
CFTR chan-
nels along with the CIC family channels have been
found to be highly susceptible to oxidative stress in
human foetal and adult RPE cultures.
56,57
Chloride
channel dysfunction and altered uid transport second-
arytooxidativestressmayplayaroleinAMD.
58
Chlo-
ride transport, similar to potassium transport in the
RPE is regulated by calcium, cAMP (cyclic adenosine
monophosphate), epinephrine and ATP (adenosine
triphosphate).
57,59
Transepithelial transport of HCO
3
is intrinsically
associated with pH regulation of the subretinal
space and the RPE cell itself. At high pH, HCO
3
travels apically into the RPE via the Na
+
/HCO
3
transporter and leaves basolaterally in exchange
with Clthrough the HCO
3
/Cl
exchanger. Con-
versely, at low pH HCO
3
enters basolaterally
through the HCO
3-
/Cl
exchanger before travelling
to the subretinal space through the Na
+
/HCO
3
transporter.
60
PHAGOCYTOSIS OF POS
Photoreceptor membranes undergo a renewal pro-
cess whereby the outer segment tips are shed and
subsequently phagocytosed by RPE before new
outer segments are constructed at the cilum. This is
Apical side
Basolateral side
Glucose
Na+
Na+
Na+
HCO3-
GLUT transporter (1,2,3,5)
Na+-K+-2Cl-
Na+-Lactate exchanger
Aquaporin 1 CFTR
K+ channel
CIC channel
Voltage dependent
Cl- channel
HCO3-/Cl-
H3
exchanger*
Aquaporin 1
Na+-K+
ATPase pump
Inward
Rectifying
K+ channel
MCT1
MCT3
Lactate Na+
dependent cotransporter
cotransporter*
GLUT transporter (1,2,3,5)
Lactate
Lactate
Lactate
Lactate
H+
Na+
K+
K+
K+
Cl-
Cl-
Cl-
Cl-
K+ 2Cl-
Na+
Glucose
Na+ Na+
Na+-HCO3
contransporter°
HCO3-
H2O
Figure 1. Glucose enters and exits through GLUT transporters 1,2,3 and 5. Lactate enters apically through MCT1 and lactate Na
+
co-transporter before exiting basolaterally via MCT3 and Na
+
lactate exchanger. The apical Na
+
/K
+
ATPase pump, which results in out-
ow of Na
+
in exchange for K
+
, provides the energy gradient that for transepithelial transport. Sodium enters apically through Na
+
K
+
2Cl
co-transporter and exits basolaterally through mechanisms that are unclear. Potassium can enter apically through Na
+
/K
+
ATPase or Na
+2
K
+
2Cl co-transporter and leave basolaterally or apically through K
+
channels. Chloride enters the RPE apically via the
Na
+
K
+
2Cl
co-transporter and exits basolaterally via voltage-dependent chloride and CIC-2 channels. The basolateral Cl
/HCO
3
exchanger, which is involved in pH regulation, facilitates chloride entry into the RPE cell. CFTR channels on the basolateral aspect facil-
itates the exit of chloride. Transport of water occurs through aquaporin channels distributed apically and basolaterally.
RPE in AMD and the role of photobiomodulation 673
© 2017 Royal Australian and New Zealand College of Ophthalmologists
necessary for several reasons: to maintain photore-
ceptor excitability, to recycle nutrients, including
DHA and retinal and to prevent oxidative damage
from photo-oxidation of damaged POS lipid and
protein components.
The process of POS phagocytosis is triggered
by interactions with ligand receptors including
CD36(Cluster of Differentiation 36),
61,62
the
mannose-6-phosphate receptor,
63
alpha V beta5
integrin,
64
MerTK (C-MER proto-oncogene Tyrosine
Kinase),
65,66
CD81 (cluster of differentiation 81)
67
and
L-type calcium channels.
68
Initially, specic binding
of the POS to the RPE apical membrane leads to
induction of a secondary messenger cascade. This
results in the ingestion of the POS before undergoing
lysosomal digestion. The components from lysosomal
digestion are either recycled back to photoreceptors as
nutrients or transported into the choroid vasculature
as waste.
PRODUCTION AND SECRETION OF GROWTH
FACTORS
In order to preserve the neural retina and choroid
architecture, the RPE produces and secretes several
protein mediators, cytokines and growth factors,
including platelet-derived growth factor (PDGF),
pigment epithelium-derived growth factor (PEDF),
vascular endothelial growth factor, broblast
growth factors (FGF), transforming growth factor β
(TGF β), insulin-like growth-I (IGF-I), ciliary neu-
rotrophic factor (CNTF), tissue inhibitor of metal-
loprotease (TIMP), lens epithelium-derived growth
factor (LEDGF) and members of the interleukin
family.
6975
Photoreceptors are protected by the
neuroprotectant growth factors PEDF, FGF, CNTF,
LEDGF and IGF-I whilst PDGF modulates cell
growth and healing. PEDF, which is secreted from
the RPE apical membrane, has anti-angiogenic
properties and helps maintain the fenestrations of
the choriocapillaris endothelium. TGF βmoderates
inammation and regulates extracellular matrix
synthesis and turnover, a function shared by
TIMP. VEGF, which is secreted from basal RPE,
basically has an opposite role to PEDF and pro-
motes angiogenesis and regulates the permeability
of the choriocapillaris. VEGF overexpression has
been implicated as a key player in the pathogene-
sis of choroidal neovascularisation (CNV) in
AMD.
76,77
The expression of VEGF is regulated by
numerous factors, including mechanical stress,
hypoxia advanced glycation end products (AGE),
cytokines (interleukin 1 and TNF), vasopressor
hormones (angiotensin II), vasopressin, growth
factors (TGFβ, FGF and PDGF) and L-type Ca
2+
channels.
78
REISOMERIZATION OF ALL-TRANS-RETINAL
AND TRANSPORT OF RETINOIDS IN THE
VISUAL CYCLE
The visual cycle is the fundamental process by
which a photon is converted into an electrical signal
in the retina. The triggering event of the visual cycle
is the absorption of light by rhodopsin in the POS
which results in one of its components, the chromo-
phore 11-cis-retinal, to be converted to all-trans-reti-
nal. This results in a conformational change in
opsin, a G-protein coupled receptor component of
rhodopsin, which activates the regulatory protein
transducin to initiate signal transduction cascades
leading to closure of cyclic GMP-gated cation chan-
nels, hyperpolarization of the photoreceptor cell
membrane and, ultimately, decreased glutamate
release. The RPE plays an integral role in the trans-
port of retinoids as well as reisomerization of all-
trans-retinal to 11-cis-retinal in the visual cycle.
Following light exposure, all-trans-retinal is con-
verted into all-trans-retinol by retinal dehydroge-
nase in the POS before being transported to the RPE
by interstitial retinol-binding protein 3 (RBP3).
79,80
All-trans-retinol can also originate from the blood
circulation, being carried by serum retinol-binding
protein, before entering the RPE cell basolaterally
through the STRA6 (stimulated by retinoic acid 6)
protein.
81,82
Whilst in the RPE, all-trans-retinol is
bound to cellular retinol-binding protein and esteri-
ed to all-trans-retinyl-ester in the presence of the
catalyst lecithin retinol acyl transferase. All-trans-
retinyl-ester undergoes isomerization through
RPE65 to produce 11-cis-retinol before being con-
verted by 11-cis-retinol dehydrogenase to produce
11-cis-retinal. 11-cis-retinal is then transported by
RBP3 to the POS where it combines with opsin to
reform rhodopsin. The cycle is repeated following
light exposure whereby the resulting all-trans-
retinal is reconverted to its all-trans-retinol and
cycled back to the RPE for reisomerization and
oxidation.
8,81
IMMUNE PRIVILEGE
The RPE has a central role in maintaining immune
privilege in the eye. Firstly, the outer blood-retinal
barrier formed by tight junctions between RPE cells
creates a microenvironment which allows inltra-
tion into the retina by immune system components
to be carefully regulated. Secondly, the RPE itself is
capable of secreting immunosuppressive factors,
including TGF β, interleukin 11 and interferon β,to
downregulate T-cell activity.
83
Thirdly, the presence
of Fas ligand on the membrane allows RPE cells to
induce apoptosis in Fas expressing effector
674 Ao et al.
© 2017 Royal Australian and New Zealand College of Ophthalmologists
leukocyte cells.
84,85
Furthermore, RPE cells which
express mass histocompatibility complex (MHC)
class I and II are able to act as antigen-presenting
cells in the eye.
86,87
RPE cells can also synthesize or
express numerous complement proteins and regula-
tors. PCR analysis of RPE cells revealed that the
RPE was capable of local production of complement
3(C3), complement factor B (CFB), complement fac-
tor H (CFH), complement factor D (CFD) and com-
plement factor I (CFI). Complementary regulatory
proteins membrane cofactor protein (MCP), decay
accelerating factor (DAF) and CD59 are also
expressed on the RPE membrane.
88
AGE-RELATED MACULAR DEGENERATION
AMD is an ocular disease associated with aging and
characterized symptomatically by impairment of
central vision. Classically, AMD is divided into two
subtypes: earlyand lateAMD. In the early stages,
AMD is marked by increasing and abnormal deposi-
tion of extracellular debris between Bruchs mem-
brane and the RPE, known as drusen. Yellowish
subretinal lesions known as subretinal drusenoid
deposits or pseudodrusen located initially in the
outer superior macula are markers for progression of
AMD.
89,90
The progression of early AMD is charac-
terized by large drusen formation and/or clinically
visible RPE abnormalities. Late AMD is character-
ized by the development of geographic atrophy and
increasing impairment of central vision. There is
currently no treatment for the atrophic form of
AMD. Neovascular AMD is the other clinical variant
of late AMD and causes more rapid and dramatic
loss of central vision and is characterized by choroi-
dal neovascularisation, the development of new
blood vessels from the choroid and rarely the retina
that often leak into the retina, causing haemorrhage,
retinal detachment and eventually disciform scars.
Anti-VEGF intravitreal injections are currently the
main form of treatment for CNV. Although the path-
ogenesis of AMD remains unclear, there is evidence
that degeneration or dysfunction of RPE cells often
occurs in the early AMD pathogenesis and can be a
predictor of photoreceptor loss although in cases
such as outer retinal atrophy associated with pseu-
dodrusen, photoreceptor loss can precede RPE
disease.
9195
OXIDATIVE STRESS
Oxidative stress has been suggested as a potential
integral mechanism of RPE injury in the pathogene-
sis of AMD. In vitro experiments where RPE cells are
exposed to oxidative stress, that is, H
2
O
2
have
shown increased apoptosis, decreased proliferation
and features of senescence.
9698
Apoptotic RPE cells
have also been identied in CNV membranes from
AMD donor eyes.
99
The underlying mechanism of oxidative stress-
induced apoptosis in AMD is thought to be an inter-
ference in the protective epidermal growth factor
receptor/protein kinase B pathway as well as an
activation of apoptosis-promoting factors such as
caspase 3 and 9, as triggered by the release of cyto-
chrome c from mitochondria.
97,100
AMD donor eyes
have histological evidence of established wide-
spread oxidative damage in patients with advanced
GA and also in eyes with CNV to a lesser extent.
101
Genetic evidence of the importance of RPE oxidative
stress in the pathogenesis of AMD is supported by
transgenic mice having a deletion of the superoxide
dismutase 1 (SOD 1) gene. These mice develop clin-
ical features of AMD, including drusen deposition,
RPE atrophy and CNV.
102
Carboxyethyl pyrole (CEP) residues, which are
oxidized remnants of DHA, have been used as bio-
markers in patients with AMD who have increased
plasma CEP autoantibodies and accumulation of
CEP within the RPE and outer retina.
103
Mice that
have been immunized with mouse serum albumin
adducted with CEP developed AMD-like lesions
similar to GA.
104
Subretinal injections of CEP modi-
ed human serum album have also been found to
exacerbate angiogenesis in laser-induced CNV
mouse models.
105
A major source of oxidative stress in RPE cells is
the formation of ROS during phagocytosis of POS.
Extracellular H
2
O
2
is presumably generated from
the action of NAPH (nicotinamide adenine dinucleo-
tide phosphate) oxidase in a phagosome or from
perioxidation of ingested POS lipids. Downregula-
tion of the antioxidant enzyme catalase also occurs
during phagocytosis, leading to increased suscepti-
bility of RPE cells to ROS.
29
Lipofuscin, autouorescent collections of unde-
gradable protein and lipids associated with aging,
accumulate in lysosomes of postmitotic cells includ-
ing RPE cells. Lipofuscin within RPE cells has been
postulated to originate from insufcient RPE phago-
cytosis of POS and it is thought that this may also
contribute to the decline in RPE phagocytic function
with aging.
106
N-retinyl-N-retinylidene ethanol-
amine (A2E), a component of lipofuscin, is theoreti-
cally susceptible to photo-oxidation from exposure
to blue light, generating toxic A2E-epitopes that
cause oxidative damage and subsequent caspase 3-
mediated apoptosis of RPE cells.
107,108
However, the
role of A2E in the pathogenesis of AMD has recently
been questioned. Recent mass spectrometry studies
of human RPE have shown that greater amounts of
A2E were located in the periphery rather than the
central region, incongruent with the spatial distribu-
tion of lipid uorescence.
109,110
In addition, clinical
RPE in AMD and the role of photobiomodulation 675
© 2017 Royal Australian and New Zealand College of Ophthalmologists
evaluation of lipofuscin in early AMD patients
showed no signicant increase compared to age-
matched controls.
111
Epidemiological studies associating cigarette
smoking and sunlight to increased risk of develop-
ing AMD support the role of oxidative stress as a
key factor in the pathogenesis of AMD.
112
Wang
et al.
113
demonstrated exposure to benzo(a)pyrene,
the toxic element in cigarette smoke, resulted in
apoptosis of cultured RPE cells as well as ultrastruc-
tural changes akin to oxidative damage in mice.
The role of oxidative stress in AMD pathogenesis
is also supported by the AREDS study which dem-
onstrated high-dose antioxidants (vitamin C, vita-
min E and β-carotene) as well as zinc, which
increases the activity of enzymes such as SOD and
catalase, signicantly reduced the progression of
intermediate AMD to advanced AMD.
114
The
follow-up AREDS2 study in 2013 concluded that
the antioxidants lutein and zeaxanthin were effec-
tive and safe alternatives to β-carotene which had,
as a side-effect, been found to increase lung cancer
in smokers.
115
MITOCHONDRIAL DYSFUNCTION
Impaired mitochondrial function in the RPE appears
to be a critical factor in AMD. Mitochondria are an
endogenous source of oxidative stress. Mitochondrial
injury or dysfunction results in impaired respiration
which leads to the increased accumulation of
ROS.
116
In a continuous cycle, oxidative stress in
mitochondria can exacerbate the generation of ROS,
eventually overwhelming RPE cells resulting in apo-
ptosis. Mitochondrial DNA (MtDNA) is a more vul-
nerable target to oxidative damage than nuclear
DNA (nDNA) given its proximity to the ROS-
producing inner mitochondrial membrane, relatively
poor repair capabilities and its lack of protective his-
tones or other DNA-associated proteins. Further-
more, the coding regions of MtDNA are more
predisposed to oxidative damage since there exist a
large number of intronless regions with high tran-
scription rates.
100
In vitro experiments, where RPE
cells were exposed to oxidative stress demonstrated
preferential damage to MtDNA over nDNA along
with a decline in mtDNA repair and compromised
mitochondrial redox function.
98,117
In human AMD
donor eyes, there is evidence of increased macula-
specic mtDNA damage, mitochondrial heteroplas-
mic mutations and diminished mtDNA repair capac-
ity in the RPE.
118
The association between AMD and
RPE mitochondrial dysfunction was further illus-
trated in a proteomic analysis of AMD donor eyes
demonstrating altered mitochondrial translation fac-
tors, decreased ATP synthase subunits and reduced
mitochondrial heat shock protein 70 expression
(mtHsp70).
119
Important functions of mtHSP70
include regulation of p53-mediated apoptosis, iron
sulphur cluster biogenesis, mitochondrial calcium
regulation and ATP-dependent transport of nuclear-
encoded proteins into the mitochondrial matrix.
119
A2E in lipofuscin has been shown to induce apo-
ptosis of RPE cells in a light-dependent manner
through a mitochondria-related mechanism. Upon
exposure to blue light, A2E binds non-covalently to
cytochrome oxidase C (COX), an essential mito-
chondrial enzyme in oxidative phosphorylation.
Upon attachment, A2E exhibits its toxic effect by
inhibiting COX causing impaired mitochondrial res-
piration, generation of ROS and formation of pro-
apoptotic complexes involving cytochrome C.
120122
There is also genetic evidence for the association
between mitochondrial dysfunction and AMD. In
human AMD patients, mtDNA lesions were
increased signicantly in all regions of the mito-
chondrial genome compared to age-matched controls
where age-related mtDNA lesions only occurred in
the common deletion regions.
123
In addition, poly-
morphisms of ARMS2 gene, which encodes for an
outer mitochondrial protein in RPE, is associated
with signicantly increased predisposition to
AMD.
124,125
Macular dystrophy patients resulting
from the A3243G mtDNA mutation display clinical
phenotypes similar to AMD including varying levels
of RPE atrophy and subretinal deposits.
126,127
COMPLEMENT DYSREGULATION
Inappropriate activation of the complement system
has been implicated in AMD pathogenesis. Bio-
chemical analysis of drusen constituents reveals a
signicant number of complement activators (A2E,
amyloid β, immunoglobulins, CRP, advanced glyca-
tion end products and cholesterol), complement
components and inhibitors.
88
The alternate complement pathway (AP) has been
the main complement pathway implicated in AMD
pathogenesis. Although there have been reports of
classical pathway involvement, with variants of C2
genes and SERPING1 gene encoding c1 inhibitor
being associated with AMD, this has not been con-
rmed in larger case-controlled studies.
128130
Genetic variations of the genes encoding AP pro-
teins CFH (inhibitor of AP),
131133
CFB
128
,
CFI
134,135
as well as C3
136
component are associated
with higher susceptibility to AMD, indicating the
signicance of AP dysregulation in AMD pathogen-
esis. Haplotypes of CFH with deletion of CFH-
related proteins protects against AMD.
137
Genetic
mutations associated with altered extracellular
matrix such as the EFEMP1 and TIMP3 mutations
have been associated with inherited macular dis-
eases, displaying local inammation as well as
676 Ao et al.
© 2017 Royal Australian and New Zealand College of Ophthalmologists
complement factor deposition. Patients display
AMD characteristics such as sub-RPE drusen-like
deposits as well as eventual atrophy and
CNV.
138,139
Rodent models with genetic knockout of
CFH display structural and functional retina degen-
erations similar to AMD. Increasing expression of
human CFH in this model resulted in the inhibition
of AP complement pathway, reduction of sub-RPE
deposits as well as prevention of retinal and kidney
damage caused by CFH deletion in a dose-
dependent manner.
140,141
The process of AP-
mediated RPE cell death which occurs in GA
involves lysis of cells marked with C3b by mem-
brane attack complex (MAC) that is modulated by
extracellular calcium.
142
The AP also has a critical role in the modulation
of CNV in laser-induced models.
143
Complement
components C3a, C5a, CFB and MAC induce CNV
by upregulating RPE secretion of angiogenic factors
including VEGF, TGF-β2 and β-broblast growth
factor.
144,145
Consequently, specic inhibition of AP
reduced angiogenesis in CNV mouse models.
146
The complement system may have a role in pro-
moting the chronic local inammatory process in
AMD. C3a and C5a are known to have chemotactic
properties and can increase RPE expression of the
inammatory cytokines interleukin-1β, interleukin-
6, interleukin-8, MCP-1 and granulocyte-
macrophage colony-stimulating factor (GM-CSF).
147
There is evidence that oxidative stress can render
RPE more susceptible to complement-mediated
damage.
142,148
RPE cells exposed to oxidative stress
exhibited reduced expression of the membrane
bound complement inhibitors DAF, CD55 and
CD59 and downregulation of CFH.
149151
Addition-
ally, mice immunized with CEP display increased
deposition of C3d below the RPE.
104
In cultured
human RPE cells, complement and oxidative stress
synergistically increased VEGF secretion up to 100-
fold.
149
PHOTOBIOMODULATION
Photobiomodulation refers to the non-invasive irra-
diation of tissue with light in the far-red to near-
infrared light spectrum (6301000 nm) with deliv-
ery methods varying from laser sources to light
emitting diode (LED) devices.
152
Photobiomodula-
tion originated in the 1960s shortly after the advent
of the laser. Mester et al.
153
were the rst to demon-
strate the positive effects of photobiomodulation.
Using NIR lasers they were able to induce increased
hair growth and wound healing in mice and treat
non-healing skin ulcers in human patients. Since
then the eld of photobiomodulation has broadened
to include treatment of a range of conditions includ-
ing wound healing, diabetic ulcers, neurological
pain, peripheral nerve injury, stroke and myocardial
infarction.
154
More recently, focus has turned to the
potential benecial effects of photobiomodulation as
a treatment for blinding retinal diseases. Treatment
effects from photobiomodulation has been demon-
strated in various retinal disease animal models
including light-induced retinal degeneration,
155,156
AMD,
157
retinitis pigmentosa,
158
diabetic retinopa-
thy
159
and retinopathy of prematurity
160,161
as evi-
denced by improved ERG, decreased inammatory
markers and diminished cell loss.
Photobiomodulation has been reported to have
positive results in patients with AMD,
162,163
dia-
betic retinopathy,
164
amblyopia
165
and retinitis
pigmentosa,
166
reected in improvement of visual
acuity and decreased visual eld loss. However,
these clinical studies range from interventional case
series to a single case report. In addition, the mecha-
nism of photobiomodulation particularly on ambly-
opia is unclear. Hence, the current evidence for the
efcacy of photobiomodulation in retinal disease is
poor. Nevertheless the mechanism of photobiomo-
dulation and its relative safety should motivate more
robust clinical studies.
MECHANISMS OF PHOTOBIOMODULATION
In the far-red and near-infrared light spectrum,
COX is the primary photoacceptor. By targeting
COX, photobiomodulation modulates electron trans-
fer in the reduction of oxygen during mitochondrial
respiration, hence increasing the mitochondrial
membrane potential and ATP synthesis (Fig. 2).
Ultimately, this triggers and enhances cellular repair
processes and metabolism in photoreceptors, cho-
roid and retinal pigment epithelium.
162
Evidence of
COX as the primary target comes from Eels who
reported photobiomodulation improved rat retinal
function following methanol intoxication which is
known to inhibit cytochrome oxidase activity.
167
NIR light treatment of primary cultured neurones
reversed the effect of tetrodotoxin by upregulating
COX activity.
168
Desmettre et al.
169
found in choroi-
dal layers, that transpupillary application of laser
therapy-induced increased expression of heat shock
proteins, which are known to stimulate cellular
metabolism and prevent premature cell death.
Another potential mechanism of photobiomodula-
tion is the unbinding of nitric oxide (NO) from COX
as demonstrated by Karu et al.
170
Since NO inhibits
mitochondrial respiration, its dissociation from COX
would restore mitochondrial oxygen consumption,
therefore increasing energy production and boosting
cellular metabolic processes.
152
In vitro experiments
have reported that photobiomodulation therapy
increases phagocytosis and lysosomal activity, cellu-
lar processes important in the reduction of
RPE in AMD and the role of photobiomodulation 677
© 2017 Royal Australian and New Zealand College of Ophthalmologists
inammation and enhancing repair of the retina.
154
These processes occur from photobiomodulation
triggering downstream signalling cascades via ATP,
cAMP, NO, ROS and Ca
2+
resulting in eventual
gene expression that promotes protein synthesis,
anti-inammatory processes, antioxidants, anti-
apoptotic proteins as well as cell migration and pro-
liferation.
171
Photobiomodulation usually stimulates
mitochondrial ROS production at low levels how-
ever it may decrease ROS production during oxida-
tive stress.
172
In addition, photobiomodulation has
observed to have a biphasic dose response in vitro in
particular relation to ATP levels and mitochondrial
potential.
173
APPLICATIONS IN AMD
The pathogenesis of AMD as discussed above
encompasses a complex interplay of oxidative stress,
mitochondrial dysfunction and complement dysre-
gulation, likely involving the RPE. Photobiomodu-
lation may prove to be an effective treatment for
AMD as there is evidence that it can signicantly
modulate all of these pathological processes
(Table 1.).
Exposure of NIR light with a dose of 2.88 J/cm
2
to human RPE cells in vitro stimulated a 56%
increase in ATP and twofold increase in intracellular
NO production at 5 h post-exposure, presumably
via unbinding of NO from COX leading to an
enhancement in mitochondrial oxidative phosphory-
lation.
174
Photobiomodulation resulted in a six-fold
increase in levels of growth promoting nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-
KB), an 11-fold increase in levels of apoptosis sup-
pressor protein Bcl-2 and a 70% decrease in levels of
the apoptotic effector protein Bax. In RPE cultures
exposed to the oxidative stressor H
2
O
2
and chal-
lenged with POS, Fumar et al.
175
reported improve-
ment of phagocytosis, increased expression of
MerTK and reduced ROS production following treat-
ment with 250 s of 670 mm light at 3.89 mW/cm
2
for
four consecutive days. Interestingly, mitochondrial
membrane potential was not affected by NIR light in
this study. Photobiomodulation has also been dem-
onstrated to protect RPE cells from the lethal effects
of thermal laser.
176
There is also considerable evidence of a benecial
effect of photobiomodulation in animal AMD
models. In an aged mice model of AMD, Kokkino-
poulos
177
reported that photobiomodulation could
improve mitochondrial function of RPE cells and
reduce inammation. Following ve treatments of
NIR light exposure at 40 mW/cm
2
lasting 90 s, there
was signicantly increased mitochondrial mem-
brane potential as well as reduced component C3d,
proinammatory cytokine TNF-αand reduced mac-
rophage numbers. This was conrmed by Begum
et al.
157
who reported similar results in CFH knock-
out mice. Mice exposed to NIR light at 20 mW/cm
2
Near infrared light
Nitric Oxide
ATP
cAMP
ROS
Ca
Activation of transcription factors
Altered expression of genes
2+
Mitochondria
Protein synthesis
Anti-apoptotic protein production
Cell metabolism, repair, proliferation
and migration
Anti-oxidants
Mitochondrial respiration Downstream signalling pathways
Cytochrome Oxidase C
Dissociation
Anti-inflammatory process
Figure 2. Schematic diagram of the mechanisms of photobiomodulation near infrared light within wavelengths (6301000 nm) tar-
gets the mitochondrial enzyme cytochrome oxidase C resulting in (i) direct stimulation in mitochondrial respiration and (ii) dissociation
of nitric oxide which indirectly increases mitochondrial respiration. These processes result in elevation of ATP, cAMP, reactive oxygen
species and intracellular calcium which impact downstream signalling pathways that triggers increase in anti-inammatory processes,
protein synthesis, anti-apoptotic protein production, cellular repair/metabolism/proliferation/migration and antioxidants.
678 Ao et al.
© 2017 Royal Australian and New Zealand College of Ophthalmologists
for 6 min twice daily for 2 weeks had signicantly
increased COX, decreased C3 complement deposi-
tion in the outer retina as well as reduced inamma-
tory markers vimentin and glial brillary acidic
protein (GFAP). There were also notable changes in
the morphology of RPE macrophages and dendritic
cells however there was no signicant change in
numbers. The relatively indirect delivery of
photobiomodulation through supplemental environ-
mental lighting in this study demonstrated the effec-
tiveness of NIR light in penetrating tissue to reach
the target. Calaza et al.
178
reported improved ATP
levels in CFH mice following NIR light exposure at
40 mW/cm
2
supporting evidence that photobiomo-
dulation improves oxidative phosphorylation. In a
separate experiment, Kokkinopoulos
179
reported
Table 1. Applications of photobiomodulation in AMD
Experiment Delivery Dose Frequency Results
hTERT-RPE culture (Lavey
et al.)
174
671 and 637 nm light via
LED, unspecied duration
2.88 J/cm
2
Unspecied x2 "NO
56% "ATP 5 h post
x6 "NF-KB
x11 "BCl-2
70% #Bax
Human RPE cultures exposed
to H
2
O
2
and POS (Fumar
et al.)
175
670 nm light via LED for
250 s
3.89 mW/cm
2
Twice daily for 4 days "phagocytosis
"MerTK expression
#ROS production
C57BL/six mice
(Kokkinopoulos et al.)
177
670 nm light via LED for 90 s 40 mW/cm
2
Five times over 35 h "mitochondrial membrane
potential
#C3d
#TNF-α
#macrophage numbers
CFH knockout mice (Begum
et al.)
157
670 nm light via LED
environmental lighting for
6 min
20 mW/cm
2
Twice daily for
14 days
"COX
#C3 complement deposition
in outer retina
#vimentin and GFAP
CFH knockout mice (Calaza
et al.)
178
670 nm light via LED for 90 s 40 mW/cm
2
Daily for 5 days "ATP
CFH knockout mice
(Kokkinopoulos et al.)
179
670 nm light via LED for 90 s 40 mW/cm
2
Four times over
2 days for 1 and
8 weeks
Shift of C3 deposition from
neural retina to RPE/BM
#C3b in RPE/BM.
#TLR 2 and TLR 4 expression
inner nuclear layer
#calcitonin in all layers
Light-induced rat model of
atrophic AMD (Rutar
et al.)
180
670 nm light via LED for
3 min
9 J/cm
2
(50 mW/cm
2
)
Daily for 5 days #C1s, C2, C3, C4b, C3aR1,
and C5r1 gene expression
#C3 deposition in ONL
#C3 expression in subretinal
microglia/monocytes
#oxidative damage marker
4-hydroxynonenal (4-HNE)
Prospective study of dry and
wet AMD patients (Ivandic
and Ivandic)
162
780 nm light via
semiconductor laser diode
to the macula for 40 s
0.3 J/cm
2
(7.5 mW/cm
2
)
Twice weekly for
2 weeks
improvement in VA of two
lines in 94.597% treated
patients up to 36 months
#pigment accumulations and
cystic drusen as well as
#metamorphopsia,
#dyschromatopsia and
#relative scotomas
#oedema and bleeding in
wet AMD
TORPA II study
interventional longitudinal
case series (Merry et al.)
181
670 nm light via LED for 88 s
590 and 790 nm light via
LED for 35 s
5080 mW/cm
2
(670 nm)
4 mW/cm
2
(590 nm)
0.6 mW/cm
2
(790 nm)
Nine sessions over
3 weeks
"BCVA of 5.90 letters at 3-
week treatment and 5.14
letters at 3 months
"contrast sensitivity at
3 weeks and 3 months at
three cycles per degree
#Drusen volume decreased
by 0.024 mm
3
#central drusen thickness
mean of3.78 μm
RPE in AMD and the role of photobiomodulation 679
© 2017 Royal Australian and New Zealand College of Ophthalmologists
that photobiomodulation can drastically ameliorate
inammation from innate immunity. CFH mice
exhibited decreased C3b expression in RPE/BM fol-
lowing regular 90 s NIR light exposure at
40 mW/cm
2
over 8 weeks. Interestingly, TLR 2 and
TLR 4 expression decreased in the inner nuclear but
not in the in RPE layer. Expression of the systemic
inammatory marker calcitonin was also signi-
cantly in all layers. In a light-induced model of atro-
phic AMD, 3 min of daily NIR light exposure at
9 J/cm
2
(50 mW/cm
2
) over 5 days was reported to
reduce complement propagation, C3 deposition,
photoreceptor death and oxidative stress.
180
There
have been several clinical studies investigating the
effects of photobiomodulation on AMD patients
including a prospective study by Ivandic and Ivan-
dic
162
where 348 eyes of 203 patients with dry and
wet type AMD were exposed to 40 s of transconjunc-
tival 780 nm light from a semiconductor laser diode
with 0.3 J/cm
2
delivered to the macula four times
over 2 weeks. The treatment group included 146 with
cataracts with 182 without. The remaining 20 eyes of
10 patients received sham treatments and served as
the control group. There was substantial improve-
ment in both treated groups with 97% of patients
with cataracts improving in visual acuity by a mean
of two lines and 94.5% of the non-cataract patients
by the same amount up to 36 months. The authors
also reported reduced pigmentation and cystic dru-
sen as well as improvement in metamorphopsia, dys-
chromatopsia and relative scotomas. Patients with
wet AMD had reduced oedema and bleeding. No
change was observed in the control group. The
TORPA II study, which examined 42 eyes in
24 patients with dry AMD by Merry et al.
181
con-
sisted of delivering 88 s of 670 nm light at
47.7 J/cm
2
through LED-based devices as well as
590 and 790 nm for 35 s delivering 0.1 J/cm
2
in nine
treatments over 3 weeks. Following photobiomodu-
lation, there were positive functional changes indi-
cated by improvement of contrast sensitivity and
visual acuity at 3 weeks and 3 months as well as ana-
tomical improvement exhibited by decrease in dru-
sen volume and central drusen thickness. However,
there are signicant aws in the methodologies of
both studies: lack of control arm or signicant num-
ber discrepancies between treatment groups, large
ranges in total dosages delivered in addition to the
patient demographic with varying stages of disease.
Hence, it is difcult to conclude clinical efcacy of
photobiomodulation in these studies. It is notewor-
thy to mention that in both studies there were no
reported adverse effects or subjective discomfort by
patients, illuminating an excellent safety prole and
suggesting that NIR light treatment is well tolerated.
There is also signicant variance of power settings
and delivery methods of photobiomodulation
between in vitro, animal studies and clinical studies.
Thus, the optimal treatment parameters of photobio-
modulation are poorly dened which poses a prob-
lem in its translation to the clinical setting.
CONCLUSION AND FUTURE DIRECTIONS
The RPE is a vital component of the eye and its dys-
function has been implicated in the pathogenesis of
AMD. Photobiomodulation is a promising non-
invasive therapy that has demonstrated the capacity
to ameliorate oxidative stress, mitochondrial dys-
function and complement dysregulation, all of
which are key mechanisms of RPE dysfunction lead-
ing to AMD.
Although clinical studies of photobiomodulation
in AMD patients have reported positive functional
and anatomical improvements, these studies suffer
from signicant aws in methodology including
the lack of a comparative control group. As a result,
conclusions regarding clinical efcacy cannot be
established. Hence, there is a need for randomized
control trials. The LIGHTSITE study which is cur-
rently enrolling in Canada, will be a 30-subject,
randomized, double-masked, sham-controlled clini-
cal trial examining similar outcomes from the
TORPA II study.
181
As previous studies have used
varying dosages, duration and frequency of NIR
light with varying effectiveness, there is currently
no consensus on treatment regimens. Therefore fur-
ther studies are needed to establish effective treat-
ment parameters.
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... One RPE cell takes care of ca. 30-40 photoreceptors (Ao et al., 2018;Kaarniranta et al., 2013). The chronic presence of oxidative stress and the postmitotic phenotype of RPE cells are estimated to be key factors behind the accumulation of lipofuscin in lysosomes . ...
... Postmitotic RPE cells are located as a single cell layer between Bruch's membrane and the photoreceptor cells (Figures 4 and 5;Ao et al., 2018;Kaarniranta et al., 2013;Lakkaraju et al., 2020). RPE cells take care of the retina, for example, by recycling photoreceptor outer segments in a process called heterophagy, by forming the bloodretinal barrier and by ensuring a chemical balance via the transfer of nutrients, oxygen and waste materials between the choroid and photoreceptor cells Lakkaraju et al., 2020). ...
... RPE cells take care of the retina, for example, by recycling photoreceptor outer segments in a process called heterophagy, by forming the bloodretinal barrier and by ensuring a chemical balance via the transfer of nutrients, oxygen and waste materials between the choroid and photoreceptor cells Lakkaraju et al., 2020). RPE cells protect the retina from oxidative stress and function as antioxidative protectors, for example, by absorbing and reflecting light and by reducing the production of ROS (Ao et al., 2018). Each RPE cell takes care of approximately 30-40 photoreceptor cells (Ao et al., 2018;Kaarniranta et al., 2013). ...
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Age-related macular degeneration (AMD) is an eye disease, which causes impaired vision that can lead to blindness. The incidence of AMD increases with age. Retinal pigment epithelial (RPE) cells maintain retinal homeostasis and support the functionality of photoreceptors. In the pathogenesis of AMD, the degeneration of the RPE cells precedes photoreceptor cell death. RPE cells are susceptible to oxidative stress, and chronic inflammation involving nucleotide-binding domain, leucine-rich repeat and pyrin domain 3 (NLRP3) inflammasome activation and impaired autophagy are challenges faced by aged RPE cells in AMD. There are two types of AMD, dry (85-90%) and wet (10-15%) disease forms. Choroidal neovascularization is typical for wet AMD, and anti-vascular endothelial growth factor (anti-VEGF) injections are used to prevent the progression of the disease but there is no curative treatment. There is no cure for the dry disease form, but antioxidants have been proposed as a potential treatment option. Ageing is the most important risk factor of AMD, and tobacco smoke is the most important environmental risk factor that can be controlled. Hydroquinone is a cytotoxic, immunotoxic, carcinogenic and pro-oxidative component of tobacco smoke. The aim of this PhD thesis was to study hydroquinone-induced oxidative stress and NLRP3 inflammasome activation in human RPE cells (ARPE-19 cells). An age-related eye disease study (AREDS) formulation (incl. omega-3 fatty acids, vitamin C and E, copper, zinc, lutein and zeaxanthin), which is clinically investigated p.o. dosing combination of dietary supplements for AMD patients, has been evaluated as a possible treatment and restraining option for AMD. Resvega (4.1.1, Table 2) is a similar kind of product to AREDS with added resveratrol, and many of the components incorporated within Resvega can be considered as belonging to the normal antioxidative defence system of the retina. Another aim was to evaluate the effects of Resvega on hydroquinone-induced oxidative stress or NLRP3 inflammasome activation induced by impaired protein clearance. The results of this study reveal that hydroquinone elevated the activity of NADPH oxidase which subsequently mediated the production of reactive oxygen species (ROS) and predisposed RPE cells to degeneration by reducing levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Hydroquinone induced an NLRP3-independent IL-18 release and NLRP3 accumulation inside the IL-1α-primed cells. Resvega treatment reduced the extent of hydroquinone-induced ROS production and NLRP3 inflammasome activation evoked by impaired protein clearance. Thus, Resvega alleviated hydroquinone- and impaired protein clearance-induced stress in human RPE cells, but more studies are needed, for example, to reveal the most optimal route of administration for targeting the cells in the retina, since both oxidative stress and NLRP3 inflammasome activation are important contributors to the development of AMD and represent significant treatment targets.
... Growing evidence has revealed the vital role of complement signaling in retinal physiopathology (9,10). Complement components, such as C2, C4b, C5/C5a and C9, have been indicated to be elevated in the vitreous humor of patients with RRDCD (11). ...
... Complement components, such as C2, C4b, C5/C5a and C9, have been indicated to be elevated in the vitreous humor of patients with RRDCD (11). Of note, RPE cells become more susceptible to complement-mediated damage (10). Complement and its receptor signaling may activate oxidative stress, contribute to transcriptional and metabolic homeostasis and promote inflammation in RPE (12,13). ...
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Retinal detachment (RD) and its special form of rhegmatogenous RD associated with choroidal detachment (RRDCD) feature similar pathological alterations, including enhanced retinal cell inflammation. Although the importance of the complement components C3a and C5a and their corresponding receptors in retinal maintenance has been demonstrated, the relevance of these molecules to the pathogenesis of RD or RRDCD remains to be investigated. The contents of C3a, C5a and inflammatory factors, such as TNF-α, IL-1β, IL-6 and prostaglandin (PG)E2, in related clinical samples were examined by ELISA. Subsequently, human retinal pigment epithelial (HRPE) cells were subjected to challenge with the C3a and C5a recombinant proteins with or without C3a and C5a antagonists and NF-κB inhibitor, and the cell viability and inflammatory cytokines were then determined by a Cell Counting Kit-8 assay and ELISA, respectively. In addition, reverse transcription-quantitative PCR and western blot analyses were utilized to examine the mRNA or/and protein levels of C3a and its receptor C3aR, as well as C5a and its receptor C5aR, and NF-κB. In addition, the correlation of C3a and C5a with the aforementioned inflammatory factors was analyzed. The inflammatory factor levels of C3a and C5a were considerably elevated in patients with RRDCD compared to those in the controls. Consistently, C3a and C5a treatment led to increased cell viability and aggravated inflammation in HRPE cells. Accordingly, C3a and C5a induced upregulation of their corresponding receptors C3aR and C5aR, which was in turn observed to be linked to the activation of the NF-κB signaling pathway. Furthermore, there was a positive correlation of the complements C3a and C5a with individual TNF-α, IL-1β, IL-6 and PGE2. Taken together, the C3a-C3aR and C5a-C5aR pathways were indicated to promote cell viability and inflammation of HRPE cells by targeting the NF-κB signaling pathway.
... The freed nitric oxide also activates mechanisms increasing antioxidant production, antiapoptotic pathways and cellular metabolism. photobiomodulation treatment generates more mitochondrial energy via ATP activity and replication [25]. Moreover, it also increases the production of proteins and RNA. ...
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Background and Objectives: The purpose of this study is to describe the effects of photobiomodulation on drusen regression with patients presenting with reticular pseudodrusen (RPD). Materials and Methods: This study is a retrospective observational case series study including patients presenting with RPD who underwent treatment by photobiomodulation. All patients underwent a complete ophthalmic examination and multimodal imaging prior to treatment, including spectral-domain optical coherence tomography (SD-OCT). Eyes were treated two times per week for six consecutive weeks. Best corrected-visual acuity (BVCA) was measured prior and after treatment for all patients. The number of RPD on the SD-OCT scans centered on the macula and stages of RPD was noted at baseline and 6 months after the first treatment session. Results: Five eyes of five patients were included in the study. Mean BCVA did not change 6 months after treatment compared to baseline. Mean number of RPD per eye was 112.60 +/− 48.33 RPD at baseline and 111.6 +/− 49.29 in the same area 6 months after treatment. Changes in RPD distribution according to RPD classification were observed before and after treatment with photobiomodulation. Changes in distribution mostly concerned stages 1 and 3 RPD: Total number of stage 1 RPD was 289 and increased to 324 after treatment. Total number of stage 3 RPD was 97 at baseline and decreased to 67 6 months after treatment. Percentage of stage 1 RPD increased from 46% to 56% after treatment. Percentage of stage 3 RPD decreased from 20% to 13% after treatment. Conclusions: Changes in RPD distribution were observed before and after treatment with photobiomodulation. The number of stage 3 reticular pseudodrusen decreased while number of stage 1 reticular pseudodrusen increased after treatment.
... Retinal pigment epithelial is an integral part of the retina, both structurally and functionally. RPE cells are involved in the visual cycle, light absorption, nutrient transportation, and photoreceptor cell outer segment phagocytosis (Sparrow et al., 2010;Ao et al., 2017). Dysfunctional RPE is the major cause of age-related retinal degeneration observed in the elderly population (Fleckenstein et al., 2021;Lewandowski et al., 2021;Yang et al., 2021). ...
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Palmitoylation is a dynamic process that regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in the visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 55 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant enrichment of palmitoylated proteins in cell-matrix adhesion, cell-cell recognition, protein cellular localization, and translation, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900, and 816) of Niemann-Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys799 and 800 caused significant cholesterol accumulation in the late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. However, the mutation did not alter the cellular localization of the protein. Conclusion: PRE cells have an abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol export, but not the intracellular localization of NPC1.
... According to the International AMD Genomics Consortium (IAMDGC), 52 genetic versions are associated with the risk of AMD late stage and localized in 34 loci, 16 of which were not regarded as associated with AMD, have been identified so far [24]. Nowadays it is considered that the pathogenesis of AMD is based on dysfunction of RPE cells, which is caused by impaired metabolism in mitochondria, oxidative stress, inflammation, changes in the extracellular matrix, impaired lipid metabolism and angiogenesis in the retinal capillary network [25][26][27]. Previous studies have shown the regulatory role of SIRT1 in these pathological processes [28][29][30][31][32]. ...
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Purpose: Age-Related Macular Degeneration (AMD) is a complex multifactorial disease, which consists of various genetic, environ- mental and constitutional factors and is characterized by damage to the macular zone of the retina. The AMD is one of the most common causes of blindness and poor vision in people of a senior age group. Aging is regarded as one of the most significant factors predisposing to age-related macular generation. Sirtuins, in particular SIRT1, are a family of signaling proteins that play an important role in the aging process. The SIRT1 is the most studied protein in the topic of aging and its level of expression plays an important role in the AMD de- velopment. For these reasons, we evaluated the relationship of the rs12778366 polymorphism with the risk of AMD. Methods: The study used genomic DNA isolated and purified from buccal epithelial cells from 384 people (192 AMD patients and 192 non-AMD patients). Genotyping of the selected polymorphisms was conducted by real-time PCR using the TaqMan competing probe technology. Results: The С allele in the additive inheritance model and the TC heterozygous genotype in the сodominant and recessive models serve as the genetic factor predisposing to this disease (p<0,001, OR: 2,121, 95% of CI: 1.435-3.133; p<0.001, OR: 2.499, 95% of CI: 1.595-3.915; p<0.001, OR: 2.507, 95% of CI: 1.612-3.900). In addition, the C allele was more common in women with AMD and in AMD patients over 65 years of age, which may be associated with a higher risk of AMD. Conclusion: Our study discovered a significant association be- tween rs12778366 polymorph locus in SIRT1 gene with AMD. Keywords: Age-related macular degeneration; Gene polymor- phism; SIRT1
... There is a rich extracellular matrix (ECM) between them. RPE cells are also responsible for the daily phagocytosis of shed outer segment membrane discs, nutrients and ions' transepithelial transport, directional secretion of growth factors and visual cycle [20,21]. Retina degeneration is known to be a significant feature of RP, but its effect on the biological function of RPE needs to be further investigated. ...
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Pre-mRNA processing factors (PRPFs) are vital components of the spliceosome and are involved in the physiological process necessary for pre-mRNA splicing to mature mRNA. As an important member, PRPF6 mutation resulting in autosomal dominant retinitis pigmentosa (adRP) is not common. Recently, we reported the establishment of an induced pluripotent stem cells (iPSCs; CSUASOi004-A) model by reprogramming the peripheral blood mononuclear cells of a PRPF6-related adRP patient, which could recapitulate a consistent disease-specific genotype. In this study, a disease model of retinal pigment epithelial (RPE) cells was generated from the iPSCs of this patient to further investigate the underlying molecular and pathological mechanisms. The results showed the irregular morphology, disorganized apical microvilli and reduced expressions of RPE-specific genes in the patient’s iPSC-derived RPE cells. In addition, RPE cells carrying the PRPF6 mutation displayed a decrease in the phagocytosis of fluorescein isothiocyanate-labeled photoreceptor outer segments and exhibited impaired cell polarity and barrier function. This study will benefit the understanding of PRPF6-related RPE cells and future cell therapy.
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Diabetic retinopathy (DR) is the most common complication of diabetes. It is also the main cause of blindness caused by multicellular damage involving retinal endothelial cells, ganglial cells, and pigment epithelial cells in adults worldwide. Currently available drugs for DR do not meet the clinical needs; thus, new therapeutic targets are warranted. Noncoding RNAs (ncRNAs), a new type of biomarkers, have attracted increased attention in recent years owing to their crucial role in the occurrence and development of DR. NcRNAs mainly include microRNAs, long noncoding RNAs, and circular RNAs, all of which regulate gene and protein expression, as well as multiple biological processes in DR. NcRNAs, can regulate the damage caused by various retinal cells; abnormal changes in the aqueous humor, exosomes, blood, tears, and the formation of new blood vessels. This study reviews the different sources of the three ncRNAs—microRNAs, long noncoding RNAs, and circular RNAs—involved in the pathogenesis of DR and the related drug development progress. Overall, this review improves our understanding of the role of ncRNAs in various retinal cells and offers therapeutic directions and targets for DR treatment.
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Background The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods The cytoprotective consequence of CK in hydrogen peroxide (H2O2)-treated cells was evaluated by cell viability, DNA damage and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results H2O2-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H2O2-treated ARPE-19 cells. Conclusion This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.
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Background Loganin, a type of iridoid glycoside derived from Corni Fructus, is known to have beneficial effects various chronic diseases. However, studies on mechanisms related to antioxidant efficacy in human retinal pigment epithelial (RPE) cells have not yet been conducted.Objectives This study was to investigate whether loganin could inhibit oxidative stress-mediated cellular damage caused by hydrogen peroxide (H2O2) in human RPE ARPE-19 cells.Methods The preventive effect of loganin on H2O2-induced cytotoxicity, reactive oxygen species (ROS) generation, DNA damage and apoptosis was investigated. In addition, immunofluorescence staining and immunoblotting analysis were applied to evaluate the related mechanisms.ResultsThe loss of cell viability and increased ROS accumulation in H2O2-treated ARPE-19 cells were significantly abrogated by loganin pretreatment, which was associated with activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased expression of heme oxygenase-1 (HO-1). Loganin also markedly attenuated H2O2-induced DNA damage, ultimately ameliorating apoptosis. In addition, H2O2-induced mitochondrial dysfunction was reversed in the presence of loganin as indicated by preservation of mitochondrial integrity, decrease of Bax/Bcl-2 expression ratio, reduction of caspase-3 activity and suppression of cytochrome c release into the cytoplasm. However, zinc protoporphyrin, a selective inhibitor of HO-1, remarkably alleviated the preventive effect offered by loganin against H2O2-mediated ARPE-19 cell injury, suggesting a critical role of Nrf2-mediated activation of HO-1 in the antioxidant activity of loganin.Conclusion The results of this study suggest that loganin-induced activation of the Nrf2/HO-1 axis is at least involved in protecting at least ARPE-19 cells from oxidative injury.
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Retinal pigmented epithelium (RPE) has essential functions, such as nourishing and supporting the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE in aging remain poorly understood. Here, we isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10-67 years). A quantitative proteomic analysis was performed to analyze changes in their intracellular and secreted proteins. Age-group related subtypes and age-associated proteins were revealed and potential age-associated mechanisms were validated by ARPE-19 and hRPE cells. The results of proteomic data and verifications suggested that RNF123 and RNF149 related ubiquitination play an important role in protecting hRPE cells from oxidative damage in aging. In older hRPE cells, apoptotic signaling-related pathways were up-regulated, and endoplasmic reticulum organization was down-regulated both in the intra-cellular and secreted proteome. Our work paints a detailed molecular picture of hRPE in the aging process and provides new insights into the molecular characteristics of RPE in aging and related clinical retinal conditions.
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Diabetic retinopathy (DR) is one of the leading causes of blindness in the developed world. Characteristic features of DR are retinal neurodegeneration, pathological angiogenesis and breakdown of both the inner and outer retinal barriers of the retinal vasculature and RPE-choroid respectively. Vascular endothelial growth factor-A (VEGF), a key regulator of angiogenesis and permeability, is the target of most pharmacological interventions of DR. VEGF-A can be alternatively spliced at exon 8 to form 2 families of isoforms, pro- and anti-angiogenic. VEGF-A165a is the most abundant pro-angiogenic isoform, is pro-inflammatory and a potent inducer of permeability. VEGF-A165b is anti-angiogenic, anti-inflammatory, cytoprotective and neuroprotective. In the diabetic eye, pro-angiogenic VEGF-A isoforms are up-regulated such that they overpower VEGF-A165b. We hypothesized that this imbalance may contribute to increased breakdown of the retinal barriers and by redressing this imbalance, the pathological angiogenesis, fluid extravasation and retinal neurodegeneration could be ameliorated. VEGF-A165b prevented VEGF-A165a and hyperglycaemia-induced tight junction breakdown and subsequent increase in solute flux in RPE cells. In streptozotocin-induced diabetes, there was an increase in Evans' blue extravasation after both 1 and 8 weeks of diabetes, which was reduced upon intravitreal and systemic delivery of rhVEGF-A165b. 8-weeks diabetic rats also showed an increase in retinal vessel density, which was prevented by VEGF-A165b. These results show rhVEGF-A165b reduce DR-associated BRB dysfunction, angiogenesis and neurodegeneration and may be a suitable therapeutic in treating DR.
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Purpose: To evaluate the efficacy of photobiomodulation (PBM) treatment for patients with dry age-related macular degeneration (AMD). Methods: Assessments on 42 eyes with dry AMD (age related eye disease study (AREDS) 2-4) were conducted. Multiwavelength light emitting diode (LED) light comprising of yellow (590 nm), red (670 nm) and near-infrared (790 nm) bandwidths was applied to subjects' eyes for a treatment course of 3 weeks. Outcome measures were changes in best-corrected visual acuity (BCVA), contrast sensitivity (CS), drusen volume and central drusen thickness. Results: Significant improvement in mean BCVA of 5.90 letters (p < 0.001) was seen on completion of the 3-week treatment and 5.14 letters (p < 0.001) after 3 months. Contrast sensitivity improved significantly (log unit improvement of 0.11 (p = 0.02) at 3 weeks and 3 months (log unit improvement of 0.16 (p = 0.02) at three cycles per degree. Drusen volume decreased by 0.024 mm(3) (p < 0.001) and central drusen thickness was significantly reduced by a mean of 3.78 μm (p < 0.001), while overall central retinal thickness and retinal volume remained stable. Conclusion: This is the first study demonstrating improvements in functional and anatomical outcomes in dry AMD subjects with PBM therapy. These findings corroborate an earlier pilot study that looked at functional outcome measures. The addition of anatomical evidence contributes to the basis for further development of a non-invasive PBM treatment for dry AMD.
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The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated betagalactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.
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Photobiomodulation also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infrared region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential, and adenosine triphosphate production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium (Ca2+) to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO, and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, and antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.
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Purpose: Diabetic macular edema (DME), an accumulation of fluid in the subretinal space, is a significant cause of vision loss. The impact of diabetes on the breakdown of the inner blood-retina barrier (BRB) is an established event that leads to DME. However, the role of the outer BRB in ocular diabetes has received limited attention. We present evidence that the breakdown of normal RPE function in hyperglycemia facilitates conditions conducive to DME pathogenesis. Methods: Brown Norway rats (130-150 g) were injected intraperitoneally with streptozotocin (STZ; 60 mg/kg) to induce hyperglycemia. After 4 weeks, Evans blue (EB) dye was injected intravenously to determine whether there was leakage of albumin into the retina. Subretinal saline blebs (0.5-1 μL) were placed 4 and 9 weeks after STZ injection, and time-lapse optical coherence tomography tracked the resorption rate. In a subset of rats, intravitreal bevacizumab, a humanized monoclonal antibody targeted to VEGF, was given at 5 weeks and resorption was measured at 9 weeks. Results: The ability of the RPE to transport fluid was reduced significantly after 4 and 9 weeks of hyperglycemia with a reduction of over 67% at 9 weeks. No EB dye leakage from inner retinal vessels was measured in hyperglycemic animals compared to control. The intravitreal administration of bevacizumab at week 5 significantly increased the rate of fluid transport in rats subjected to hyperglycemia for 9 weeks. Conclusions: These results demonstrate that chronic hyperglycemia altered RPE fluid transport, in part dependent on the actions of VEGF. These results support the idea that RPE dysfunction is an early event associated with hyperglycemia that contributes to fluid accumulation in DME.
Article
Aim: To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. Methods: Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). Results: EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. Conclusion: Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.
Article
Importance: Increased lipofuscin accumulation is assumed to be an important factor in the pathogenesis of age-related macular degeneration (AMD), although direct evidence for this hypothesis is missing. Objective: To quantitatively investigate lipofuscin-associated fundus autofluorescence (AF) in patients with early and intermediate AMD. Design, setting, and participants: A prospective, single-center, case-control study was conducted from August 1, 2014, to October 31, 2015, at a university referral center. Participants included 40 patients aged 65 years or younger and 108 individuals without eye disease serving as controls. All participants underwent quantitative fundus AF (qAF) imaging with a modified scanning laser ophthalmoscope equipped with an internal fluorescent reference. Mean qAF values of an 8-segment circular ring centered on the fovea (qAF8) were measured and compared between patients and controls. For subgroup analysis, drusen were categorized as soft drusen, cuticular drusen, and/or reticular pseudodrusen (RPD). Main outcomes and measures: The qAF8 levels. Results: In the 40 patients with AMD, mean (SD) age was 54.8 (5.6) years, and 32 (80%) were women. None of the investigated patients had qAF8 values above the 95% prediction interval (PI) of the 108 controls. In the soft drusen (28 [70%]) and cuticular drusen (8 [20%]) groups, qAF8 levels within the 95% PI were noted in 22 patients (79%; 95% CI, 60% to 90%) and 7 patients (88%; 95% CI, 51% to 99%) respectively. The qAF8 values in the RPD group (4 [10%]) were below the 95% PI in 3 patients (75%; 95% CI, 29% to 97%). Compared with the controls, statistical analysis revealed lower qAF8 values in the overall AMD cohort after adjusting for age (difference, -19.9% [95% CI, -25.6% to -12.7%], P < .001) as well as in all subgroups (soft drusen, -17.1% [95% CI, -24.1% to -9.5%], P < .001; cuticular drusen, -19.6% [95% CI, -30.3% to -7.2%], P = .003; and RPD, -34.5% [95% CI, -47.1% to -21.3%]; P < .001). Conclusions and relevance: The qAF8 measurements in this sample showed no increased lipofuscin-related fundus AF in patients with early and intermediate AMD. Lower qAF levels in certain subgroups may point to subnormal lipofuscin levels in the retinal pigment epithelium or, alternatively, limitations to detection of true retinal pigment epithelial lipofuscin content. The results of this study might expand the understanding of the pathogenesis of AMD and may have an effect on upcoming treatment trials that aim to modify lipofuscin accumulation.
Chapter
The most important function of the blood—retinal barrier (BRB) is maintenance of the homeostasis of the retina environment by separating the retina from the systemic blood circulation. The organization of the BRB is such that the retina is protected from blood-borne compounds, since a strict homeostasis of the neuronal environment and an intact barrier are essential for optimal retina functioning. For treatment of diseases involving the retina, drugs must pass the BRB in a significant amount to have therapeutic effect. Drug entry into the retina, must take into account the plasma concentration profile of the drug, the volume of its distribution, the rate of metabolism of the drug, its plasma protein binding and the relative permeability of the BRB for that drug. All parameters affect the therapeutic efficacy of the drug and are also relevant for potential side effects.
Chapter
Age-related macular degeneration (ARMD) is the leading cause of severe visual loss in the western world in people over 50 years of age1–3 Unfortunately, no effective preventive or treatment strategy for the disease exists. The mechanistic basis for development and progression of ARMD is not clearly understood but may involve degenerative changes to Bruch’s membrane, damage to choroidal vasculature, or oxidative injury to the retinal pigment epithelial (RPE) photoreceptor complexe4,5.
Article
The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by ARNA had no effect on the, development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B ARNA. Inhibition of the alternative pathway by factor B ARNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta 2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.