2018Journal of Lipid Research Volume 51, 2010
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
RETINAL DEGENERATIVE DISEASES
Retinal degenerative diseases are a complex group of
conditions with different etiologies that result in a com-
mon outcome: photoreceptor apoptotic cell death ( 1–5 ).
Accordingly, there are differences in how these conditions
evolve. For instance, in retinitis pigmentosa (RP), rod
photoreceptor death initially occurs in the periphery,
whereas in age-related macular degeneration (AMD),
death is initiated in the macular zone and spreads in later
phases throughout the retina ( 2, 5 ). RP is a collection of
inherited blinding diseases caused by the mutation of a
wide variety of genes resulting in more than 150 abnor-
malities of photoreceptor-specifi c proteins, including mu-
tations of rhodopsin, peripherin, the ? -subunit of cGMP
phosphodiesterase, and retinal outer-segment membrane
protein 1 ( 6–8 ).
Conversely, the etiology of AMD, which is the leading
cause of blindness over the age of 65, is not as clear as that
of RP. AMD is also a heterogeneous group of disorders,
but the causes are proposed to be multifactorial, and the
main known risk factors are both genetic and environmen-
tal ( 2, 5 ). There are two forms of AMD: the dry and the wet
form. In the dry form, photoreceptors degenerate slowly
Abstract Retinal degenerative diseases result in retinal pig-
ment epithelial (RPE) and photoreceptor cell loss. These
cells are continuously exposed to the environment (light)
and to potentially pro-oxidative conditions, as the retina’s
oxygen consumption is very high. There is also a high fl ux
of docosahexaenoic acid (DHA), a PUFA that moves through
the blood stream toward photoreceptors and between them
and RPE cells. Photoreceptor outer segment shedding and
phagocytosis intermittently renews photoreceptor mem-
branes. DHA is converted through 15-lipoxygenase-1 into
neuroprotectin D1 (NPD1), a potent mediator that evokes
counteracting cell-protective, anti-infl ammatory, pro-sur-
vival repair signaling, including the induction of anti-apop-
totic proteins and inhibition of pro-apoptotic proteins.
Thus, NPD1 triggers activation of signaling pathway/s that
modulate/s pro-apoptotic signals, promoting cell survival.
This review provides an overview of DHA in photoreceptors
and describes the ability of RPE cells to synthesize NPD1
from DHA. It also describes the role of neurotrophins as
agonists of NPD1 synthesis and how photoreceptor phago-
cytosis induces refractoriness to oxidative stress in RPE
cells, with concomitant NPD1 synthesis. — Bazan, N. G., J. M.
Calandria, and C. N. Serhan. Rescue and repair during
photoreceptor cell renewal mediated by docosahexaenoic
acid-derived neuroprotectin D1. J. Lipid Res . 2010. 51:
Supplementary key words age-related macular degeneration • retinal
pigment epithelial cells • oxidative stress • neurotrophins • 15-
This work was supported by National Institutes of Health, National Eye Insti-
tute Grant EY-005121, and National Center for Research Resources grant P20
RR-016816, by the Eye, Ear, Nose, and Throat Foundation, New Orleans, LA,
and by the Ernest C. and Yvette C. Villere Endowed Chair. C.N.S. acknowledges
support from National Institutes of Health Grant GM-38765 for the work re-
viewed herein. Its contents are solely the responsibility of the authors and do not
necessarily represent the offi cial views of the National Institutes of Health.
Manuscript received 13 August 2009 and in revised form 9 April 2010.
Published, JLR Papers in Press, April 9, 2010
Thematic Review Series: Lipids and Lipid Metabolism in the Eye
Rescue and repair during photoreceptor cell
renewal mediated by docosahexaenoic acid-derived
Nicolas G. Bazan , 1, * Jorgelina M. Calandria, * and Charles N. Serhan †
Neuroscience Center of Excellence and Department of Ophthalmology,* School of Medicine, Louisiana
State University Health Sciences Center , New Orleans, LA 70112; and Center for Experimental Therapeutics
and Reperfusion Injury, † Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and
Women’s Hospital and Harvard Medical School , Boston, MA 02115
Abbreviations: AA, arachidonic acid; AD, Alzheimer’s disease;
AMD, age-related macular degeneration; C2, component 2; CNTF, cili-
ary neurotrophic factor; COX-2, cycloogenase-2; DHA, docosa-
hexaenoic acid, 22:6,n-3; GDNF, glial-derived neurotrophic factor;
HETE, 15( S )-hydroxyeicosatetraenoic acid; IL, interleukin; NPD1,
neuroprotectin D1; PBMC, peripheral blood mononuclear cell; PD1,
protectin D1; PEDF, pigment epithelium derived factor; PLA 2 , phos-
pholipase A 2 ; PMN, polymorphonuclear leukocyte; RP, retinitis pig-
mentosa; RPE, retinal pigment epithelial; TNF ? , tumor necrosis
factor- ? , VEGF, vascular endothelial growth factor.
1 To whom correspondence should be addressed.
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2019
choriocapillaris, Bruch’s membrane, and the RPE ( 21 ).
The outer blood-retinal barrier mediates the exchange
of small molecules and solutes and other metabolites
from the blood stream to the photoreceptor layer ( 22 ).
RPE cells are the most restrictive layer of the three com-
ponents of the outer blood-retinal barrier, preventing
the passage of biomolecules based on size and charge
and thus preserving a controlled environment for photo-
receptors. The retinal pigment epithelium, like other
epithelia, is a compact structure where cells communi-
cate laterally through tight junctions. In addition, the
retinal pigment epithelium presents an elaborate trans-
cellular transport system and a high polarization, allow-
ing it to have different functions ( 22 ). The selective
permeability of the outer blood-retinal barrier depends
on RPE integrity. RPE cells are involved in the preserva-
tion of these structures by interacting reciprocally in
their formation and maintenance.
While the retinal pigment epithelium plays a role in
regulating nutrition, intercellular and intra/inter-tis sue
communication and remodeling, scavenging of sub-prod-
ucts, retinal functions, and promotion of neurotrophic
signaling, it is also responsible for the recycling of the
photobleached pigments produced by the photorecep-
tor activity of rods and, to some extent, the cones ( 23 ).
Rhodopsin is a light-sensitive molecule that transduces
the photon into a biochemical signal in rods. It is com-
posed of the G-coupled receptor protein opsin and
of the chromophore 11- cis -retinal. When rhodopsin
interacts with a photon, 11- cis -retinal is isomerized to
all- trans -retinal. The regeneration of 11-cis-retinal from
all- trans -retinal takes place between the photoreceptor
outer segment and the RPE ( 24 ). All- trans -retinol is es-
terifi ed with a fatty acid to be isomerized to 11- cis -retinol
and hydrolyzed from the ester bond in RPE cells. Then
11- cis retinol is oxidized to 11- cis -retinal, and the active
chromophore then leaves the RPE apical surface and
moves to the photoreceptor to regenerate rhodopsin;
afterward, the cycle begins again (reviewed in 25 and
26 ). Failure of the retinal pigment epithelium to accom-
plish its function in the retinoid cycle leads to retinal
degeneration. For instance, autosomal-recessive inher-
ited retinitis punctata albescens has been associated
with mutations in the RLBP1 gene that encodes the cel-
lular retinaldehyde-binding protein, which carries
11- cis -retinol and 11- cis -retinaldehyde in the RPE and
Müller cells. When cellular retinaldehyde-binding pro-
tein is mutated, it loses the ability to bind the second
ligand, and, as a consequence, all- trans -retinyl esters ac-
cumulate and evolve to produce RPE atrophy, retinal
pigmentary changes, and decreased blood vessel devel-
opment ( 27 ).
Integral to the fragility of photoreceptor cells is their
close relationship with RPE cells. In Stargardt’s disease, a
juvenile form of AMD, RPE cell functional integrity is ini-
tially compromised, and in turn photoreceptors are dam-
aged. Once RPE cells die, photoreceptor cells then
succumb ( 28 ). In other words, similar to what was proposed
to occur in the blood-brain barrier in the neurovascular
and progressively, producing a thinning in the retinal lay-
ers and leaving deposits known as drusen. In the wet form,
the less common of the two AMDs, the predominant fea-
ture is invasive choroidal neovascularization, which leads
to severe vision loss ( 9 ).
AMD also affects the choriocapillaris and Bruch’s mem-
brane, which separates the retinal pigment epithelium
(RPE) from the blood vessels ( 10 ). The results from the
Age-Related Eye Disease Study (NEI) show that the intake
of high amounts of antioxidants and zinc can reduce the
risk of developing advanced AMD by about 25%, implying
an indirect role for oxidative stress in the pathogenesis.
More specifi cally, the presence of oxidative damage mark-
ers in postmortem retinas of patients with geographic atro-
phy shows, at least in the dry form, oxidative stress is
involved in the pathogenic mechanisms of AMD ( 11 ). In
this manner, oxidative stress is enhanced and exaggerated
and mitochondrial function is compromised ( 8–13 ), which
leads to apoptotic cell death. Tunel studies performed on
postmortem human retinas of patients presenting geo-
graphic atrophy and exudative forms of the disease show
that apoptosis is the main mechanism of degeneration,
not only for photoreceptors, but also for the RPE and in-
ner retinal layers ( 12 ).
Initiation and progression of AMD involves the unsuc-
cessful resolution of the infl ammatory response. Single
nucleotide polymorphisms occurring in the gene encod-
ing factor H (CFH/HF1) ( 14–17 ) were proposed to be a
major risk factor for AMD. Factor H is an inhibitor of the
alternative pathway of complement system activation that,
as a result, has the ability to limit cell injury and infl amma-
tion ( 18, 19 ). Conversely, studies that focused on other
regulatory proteins of the complement pathway, such as
factor B and complement component 2 (C2) ( 18, 20 ), ex-
hibit protective effects and reduce the risk of AMD to some
extent. For example, the E318D variant of C2 (H10) as
well as a variant in intron 10 of C2 and the R32Q variant of
factor B (H7) confer a reduced risk of AMD.
Therefore, the identifi cation of early pro-survival, anti-
infl ammatory signaling critical for the maintenance of photo-
receptor cell integrity may be applicable for novel therapeutic
intervention/s for slowing or halting disease progression.
THE PROTECTIVE ROLE OF THE RETINAL
As in the brain, retinal function relies on glycolysis and
oxidative phosphorylation, which are coupled to the citric
acid cycle. These processes require a sustained delivery of
oxygen and glucose as well as adequate control of the en-
zymes that allow for equilibrated formation and consump-
tion of ATP. The blood-retinal barrier actively promotes
homeostasis by tightly controlling the stoichiometry and
activity of a network of proteins that maintain ionic gradi-
ents and metabolic transport systems and preserve the en-
vironment through detoxifying mechanisms that scavenge
and remove toxic molecules ( Fig. 1 ) ( 21 ).
The outer blood-retinal barrier is composed of three
distinctive structures: the fenestrated endothelium of the
by guest, on December 25, 2015
2020 Journal of Lipid Research Volume 51, 2010
THE MAINTENANCE OF PHOTORECEPTOR
Photoreceptor cells shed outer segment tips, which are
then phagocytized by RPE cells in a daily, intermittent,
and circadian fashion in mammals ( 34–36 ). RPE cells are
the most active phagocytes of the body. In mammals, the
circadian shedding and phagocytosis has been calculated
to be complete after 10 days for one photoreceptor outer
segment ( 36, 37 ). In rhesus monkeys, every RPE cell inter-
acts with 20–45 photoreceptor tips ( 38 ), and the human
macula support the RPE/photoreceptor interaction in a
ratio of 1 to 23 ( 39 ).
The constant light reception activity by photorecep-
tors exhausts pigments and other molecules involved in
the photo-transduction process. As a consequence, there
is a need to replace these molecules. In this context, the
reconstruction of the outer segments requires molecular
building blocks and energy. In fact, the length of the
outer segments remains constant as a consequence of a
well-regulated biogenesis of photoreceptor membranes
in the inner segments coupled to phagocytosis of the
shed tips. During photoreceptor outer segment renewal,
proteins turn over and are continually replaced ( 37 ). In
contrast, docosahexaenoic acid (DHA) and vitamin A
from the opsin chromophore are recycled back from the
RPE to inner segments through the interphotoreceptor
matrix. The retinoid in the rods includes the reisomer-
ization of all- trans retinal back to 11- cis retinal in the RPE.
Furthermore, the interdependence between RPE cells
and photoreceptors is notable in Usher type 1B syn-
drome. The lack of myosin VIIa in this progressive dis-
ease affects the ability of RPE cells to phagocytize
photoreceptor outer segments, leading to retinal degen-
eration in a mouse model ( 40 ).
The RPE-photoreceptor outer segments are potentially
highly susceptible to oxidative stress because of the high
oxygen consumption of the retina, active fl ux of PUFAs
hypothesis of Alzheimer’s disease (AD) ( 29 ), defective
clearance of certain molecules across the blood-retinal
barrier may initiate a series of faulty maintenance func-
tions that could lead to a retino-vascular infl ammatory re-
sponse, contributing to the development of AMD.
RPE CELLS AND VASCULAR REMODELING
The signifi cance of RPE cells in vascular remodeling is
highlighted in several studies. For example, absence of
RPE cells in mice expressing fi broblast growth factor
(FGF) 9 directed by the tyrosinase-related protein 2 pro-
moter (FGF9 transgenic mice) is due to forcing embry-
onic RPE cells to become neural retinal cells through the
ectopic expression of FGF9 ( 30 ). These mice fail to form
blood vessels in the choroidal layers adjacent to regions
where RPE cells are absent; however, vessels are found
near the patch where RPE cells are present at postnatal
day 7, indicating the importance of these cells in vessel
formation. Moreover, dependency between RPE and en-
dothelial vascular cells continues during adulthood
through regulation of neovascularization. Compelling
evidence links RPE cells with the secretion of angiogenic-
related factors. In particular, RPE from transgenic apoli-
poprotein E2 mice, which express human apolipoprotein
E2 protein and whose eyes present features common to
AMD patients, shows reciprocal unbalanced expression
of pigment epithelium-derived factor (PEDF) and vascu-
lar endothelial growth factor (VEGF), indicating that
neovascularization may be increased ( 31 ). Furthermore,
autocrine VEGF signaling in RPE cells stimulates VEGF-
related gene expression as well as PEDF modulation ( 32 ),
which is a potent angiogenic inhibitor ( 33 ). Taken to-
gether, the balanced production and secretion of these
factors contribute to the formation, maintenance, and
remodeling of cells that surround the RPE layer and/or
are in their vicinity.
Fig. 1. RPE/photoreceptor interactions and NPD1
bioactivity. A: RPE control of the permeability of the
outer blood-retinal barrier involving remodeling of
the blood vessels and selective fl ux of nutrients
and catabolites. B: NPD1 pro-survival enhances the
expression of Bcl-2 proteins and decreases COX-2
expression. C: Shedding and phagocytosis of the pho-
toreceptor outer segments.
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2021
DHA RELEASE AND NPD1 FORMATION
RPE cells respond to oxidative stress by activating syn-
thesis of NPD1 from DHA ( 46 ). The name NPD1 was sug-
gested based upon its neuroprotective bioactivity in
oxidative stressed RPE cells and the brain ( 46, 47 ) and its
potent ability to inactivate pro-apoptotic and pro-infl am-
matory signaling. D1 refers to its being the fi rst identifi ed
stereoselective mediator derived from DHA. NPD1 can be
formed from free (unesterifi ed) DHA released from mem-
brane phospholipids by a phospholipase A 2 (PLA 2 ) upon
stimulation ( Fig. 3A ). DHA belongs to the essential
omega-3 essential fatty acid family (derived from linolenic
acid, 18:3, n-3). Photoreceptor cells are highly enriched in
DHA, and they tenaciously retain DHA even during very
prolonged periods of omega-3 fatty acid deprivation ( 41,
48, 49 ).
The amount of unesterifi ed DHA simultaneously mea-
sured in RPE cells and in incubation media by MS/MS was
found to be increased as a function of time during expo-
sure to oxidative stress in RPE cells. Specifi cally, the free
intracellular DHA pool size showed a moderate increase
after 6 h when cells were subjected only to photoreceptor
outer segment phagocytosis ( 44 ). Oxidative stress, how-
ever, strongly enhanced free DHA accumulation in a time-
dependent fashion, peaking at 16 h ( 44 ). Interestingly,
although the overall increase reached 10-fold, photore-
ceptor outer segment phagocytosis kept the DHA pool size
at a constant 2.4-fold increased level. This implies that
NPD1 synthesis does not result from the simple enhance-
ment of the overall availability of free DHA upon phagocy-
tosis. There is a general correlation between increases in
free DHA pool size and in NPD1 synthesis. Photoreceptor
outer segment phagocytosis stimulates NPD1 synthesis at
3–6 h in cells and accumulation in media after 16 h, while
free DHA increases earlier and keeps accumulating up to
16 h. These enhancements in DHA and NPD1 pool size
are much larger when photoreceptor outer segment
phagocytosis takes place on RPE cells exposed to oxidative
stress. Interestingly, microsphere phagocytosis does not
cause enhanced changes in DHA and NPD1 ( Fig. 2B ). As
such, a very specifi c free DHA pool may be the precursor
Therefore, the supply of DHA and the induction of NPD1
synthesis during photoreceptor outer segment phagocytosis
represents a homeostatic regulatory event for RPE cell pro-
tection in conditions of oxidative stress challenge and, as a
consequence, the fostering of photoreceptor cell integrity
( 44 ). In this context, not only is photoreceptor outer seg-
ment phagocytosis in RPE cells essential for photoreceptor
cell function, but the survival of the RPE promoted by this
process correlates with NPD1 synthesis.
ON THE STRUCTURE OF NPD1, BIOSYNTHESIS,
AND STEREOCHEMICAL ASSIGNMENT
The results discussed in this review provide the biologi-
cal basis for the important actions of NPD1 derived from
DHA. In this section, we shall consider the results obtained
(e.g., omega-3 and also omega-6), and exposure to light
( 41, 42 ). Recently it was shown that phagocytosis (24–48
h) of oxidized photoreceptor outer segments containing
high oxidative products induces the downregulation of
complement factor H in RPE cells, similar to the effect of
pro-infl ammatory cytokines tumor necrosis factor- ?
(TNF ? ) and interleukin (IL)-6 ( 43 ). The RPE comple-
ment regulatory system, in this manner, may be sup-
pressed by pro-infl ammatory conditions as well as
phagocytosis of oxidized photoreceptor outer segments.
Surprisingly, the process enhances refractoriness to oxi-
dative stress-induced apoptosis in RPE cells ( Fig. 2A )
( 44 ). The protective effect of photoreceptor outer seg-
ments is specifi c, because the phagocytosis of polystyrene
microspheres by RPE cells does not lead to a protective
response against oxidative stress. Furthermore, polysty-
rene microspheres failed to induce DHA release and ac-
tivate synthesis of neuroprotectin D1 (NPD1); this will be
discussed in the following section. Interestingly, photore-
ceptor outer segment-mediated RPE cell protection
against oxidative stress, with concurrent activation of
NPD1 synthesis, was shown in ARPE-19 cells ( 44 ), a spon-
taneously immortalized human cell line ( 45 ), as well as in
low passage primary human RPE cells prepared from Na-
tional Disease Research Interchange-supplied eyes (un-
Fig. 2. Photoreceptor outer segment phagocytosis elicits protec-
tion in RPE cells subjected to oxidative stress. A: Quantitative analy-
sis of Hoechst stained ARPE-19 cells indicates that photoreceptor
outer segment phagocytosis signifi cantly decreases the amount of
apoptosis observed during oxidative stress. Phagocytosis of polysty-
rene microspheres during oxidative stress did not alter the amount
of apoptosis observed during oxidative stress alone. Results repre-
sent averages ± SEM of repeats of two independent experiments. B:
NPD1 changes as a function of time after photoreceptor outer seg-
ment phagocytosis or microspheres: effect of oxidative stress. NPD1
has been quantifi ed in cells as well as in incubation media. Data
represents average ± SEM of two independent studies. Statistical
analysis is Student’s t -test. NS, not statistically signifi cant.
by guest, on December 25, 2015
2022 Journal of Lipid Research Volume 51, 2010
Fig. 3. A: NPD1 biosynthesis. Representation of the oxygenation of DHA to form NPD1. PLA 2 releases DHA from the second carbon
position of the phospholipids upon stimulation. 15-Lipoxygenase-1 catalyzes the synthesis of 17S-H(p)DHA, which is converted to a 16(17)-
epoxide and then is enzymatically converted to NPD1. B: Comparison of NPD1/PD1 biosynthesis with that of 10S,17S-diHDHA isomer (see
detailed discussion in the text).
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2023
construct the potential biosynthetic routes involved in the
biosynthesis of these mediators. In this context, hypoxic
endothelial cells exposed to infl ammatory stimuli in vitro
converted DHA and eicosapentaenoic acid to intermedi-
ates that were taken up by human leukocytes and further
converted to bioactive products that showed potent activi-
ties relevant to the control of infl ammation ( 51, 59, 60 ).
Of interest to the present review, in these investigations
without aspirin treatment, 17S-HDHA and corresponding
17S-hydroxy-containing di- and trihydroxy products were
reported in murine exudates and isolated human cells
( 51 ). The formation of some of these compounds was
modeled in vitro and formed by sequential lipoxygenation
reactions. These products were investigated with 15-lipoxy-
genase and included the double dioxygenation products,
namely 7S,17S-diHDHA and 10,17S-diHDHA, which were
identifi ed along with trihydroxy-containing products
formed via epoxide-containing intermediates from DHA
( 51 ). The well-established lipoxygenase reaction mecha-
nism suggested that new products 7S,17S-diHDHA and
10,17S-diHDHA, which could easily be made in vitro, each
contained two diene conjugated double bond systems
both in a trans , cis geometry. For example, the well-known
5S,12S-diHETE formed from arachidonate via double di-
oxygenation is an isomer of the potent chemoattractant
LTB4 ( 50 ). This isomer 5S12S-diHETE separates in SP-
HPLC from LTB4 and are very similar to each other, but
5S12S-diHETE shows little chemotactic activity compared
with LTB4 ( 61 ).
DHA is also precursor to a novel family of endogenous
docosatrienes formed in blood, leukocytes, brain, and glial
cells ( 46, 47, 52 ). The main bioactive member of the do-
cosatrienes from the 17S-hydroxy-containing docosanoids
proved to be 10,17S-docosatriene, in addition to the re-
solvins ( 47, 52 ). Also, human polymorphonuclear leuko-
cytes (PMN) convert 10,17S-docosatriene to its omega-22
hydroxy product with DHA as the precursor; this is likely
an inactivation route for this compound ( 47 ). As with the
new bioactive products from omega-3 precursors, the ba-
sic structures and proposed stereochemical assignments
reported were based on results of biosynthesis studies and
given as tentative stereochemical assignments, because
matching studies with synthetic reference compounds of
known stereochemistry were still underway (see below). At
the time, some of the newly identifi ed compounds were
matched to reference compounds prepared with plant li-
poxygenases, e.g., 17S-HDHA and 7S,17S-diHDHA, which
matched to those profi led by LC-MS-MS in exudates and
with isolated cells. It was clear that the 10,17S-docosatriene
from exudates did not coelute with the major 10,17S-do-
cosatriene produced in vitro with plant enzymes, suggest-
ing it was an isomer; however, this system could be used to
prepare related docosatrienes via the LTA4 synthase activ-
ity of lipoxygenases to further probe the bioactions of the
10,17S-docosatriene while complete matching and total
synthesis were in progress.
Of note, glial cells generate both 17S series resolvins
and the 10,17S-docosatriene. Importantly, evidence for a
from several independent lines of investigation required
to address the structure of the potent bioactive NPD1. As
with other bioactive mediators, such as the eicosanoids
( 50 ), it is important to establish the stereochemistry of the
compound and/or mediator, because many structurally
related products can be less active, inactive, or even in
some cases display opposing biologic actions as a result of
subtle changes in stereochemistry that are recognized in
biologic systems. To confi rm the proposed basic structure
and establish the complete stereochemistry, these studies
on the 10,17S-docosatriene termed NPD1 included results
from biosynthesis studies, matching of materials prepared
by total organic synthesis with defi ned stereochemistry,
and the actions of these and related compounds in bio-
logical systems ( 42, 44, 46, 47, 51–55 ). We also considered
the chronology in which these fi ndings appear in the lit-
erature with the goal of providing a clear and rigorous ac-
count of the evidence that supports the structure and
bioactions of NPD1/protectin D1 (PD1) for the reader-
ship of The Journal of Lipid Research . As interested JLR read-
ers will surmise, investigations along these lines were
essential to establish the complete structure of the potent
NPD1/PD1 and related endogenous products biosynthe-
sized from DHA in vivo/in situ because of the small
amounts of NPD1 attainable from biological systems at the
time, which precluded direct stereochemical analyses of
the products identifi ed in RPE cells. Thus, in this section,
we focus on the evidence for NPD1/PD1 structural eluci-
dation. JLR readers interested in the structural elucidation
of the resolvins and their complete stereochemical assign-
ments are directed to other recent reviews ( 56, 57 ).
In 1984, the fi rst evidence was obtained for the conver-
sion of DHA to mono-, di-, and tri- DHA-derived products,
named docosanoids, in the retina (an integral part of the
central nervous system) ( 58 ). Use of available inhibitors of
the time suggested a role for lipoxygenase in the biosyn-
thesis of these compounds. An initial step in docosanoid
synthesis was envisioned to be the release of DHA from
membrane phospholipids by PLA/sA 2 , early demonstrated
to be rapidly activated by ischemia or seizures ( 41 ). The
structure of 10,17-docosatriene was fi rst disclosed while re-
porting on the characterization of the novel bioactive re-
solvins that were identifi ed using a systems approach with
resolving infl ammatory exudates and LC-MS-MS-based
lipidomics ( 51 ). These new compounds (resolvins and do-
cosatrienes) were biosynthesized from omega-3 essential
fatty acids during the resolution phase of acute infl amma-
tory reactions in vivo that promote resolution of infl amma-
tion in vivo [see Fig. 8 in reference (51) and related text].
Since the DHA-derived compounds we identifi ed in resolv-
ing infl ammatory exudates, additional evidence was ob-
tained for their biosynthesis from murine brain and
vascular endothelial cells for the new bioactive compounds
( 47 ). These investigations focused on aspirin and its im-
pact in the biosynthesis of 17R-hydroxy-containing re-
solvins and related structures. The initial results indicated
that DHA-derived products reduced cytokine IL-1 ? pro-
duction by human glioma cells stimulated with TNF ? . In
parallel, studies with human cells were carried out to re-
by guest, on December 25, 2015
2024 Journal of Lipid Research Volume 51, 2010
dogenous compound and those of the synthetic with
established stereochemistry. In recognition of its wide
scope of formation and uncovered actions, PD1 was intro-
duced and used to denote the structure of this chemical
mediator in the immune system. The prefi x Neuro before
PD1 (NPD1) was proposed to denote its biosynthesis and
potent neuroprotective actions ( 53 ). It was also apparent
that NPD1/PD1 was a member of a larger family of 17-hy-
droxy-containing docosatrienes, termed protectins.
Biosynthesis and function studies were undertaken with
human T H 2-skewed peripheral blood mononuclear cells
(PBMC) that specifi cally express 15-lipoxygenase type 1
and convert DHA to the 10,17S-docosatrienes by serving as
a 17-lipoxygenase with DHA as a substrate. When pro-
duced by these cells, PD1 promotes T cell apoptosis via the
formation of lipid raft-encoded signaling complexes and
reduces T-cell traffi c in vivo. These results were consistent
with the physical properties of NPD1. Matching materials
prepared by total organic synthesis determined the com-
plete stereochemistry of the PBMC DHA-derived product.
NPD1/PD1 generated by human PBMC carried the
complete stereochemistry of (10R,17S)-dihydroxydocosa-
4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid and was matched
to the most potent bioactive product using several dihy-
droxytriene-containing, DHA-derived products isolated
from human PBMC, human PMN, and murine exudates
( 53, 54 ).
During the course of these investigations, Butovich et al.
( 64 ) reported that NPD1 had the complete structure of
10 S ,17 S -diHDHA. This was based on results obtained with
isolated lipoxygenase enzymes incubated with DHA with-
out mammalian cell/tissue biosynthesis or authentic
NPD1, as defi ned earlier in the literature ( 46, 51, 52 ). Im-
portantly, neither the bioactivity of the product nor appro-
priate comparisons with authentic NPD1 was presented to
support the conclusions in this report ( 64 ) in regards to
the complete structure of NPD1. Note that 10 S ,17 S -diH-
DHA is an isomer of NPD1/PD1 ( Fig. 3B ).
With the preparation of six stereochemically defi ned,
10,17-dihydroxy-containing geometric isomers by total or-
ganic synthesis that had been initiated earlier, it was pos-
sible to match the stereochemistry and biological actions
of the endogenously produced materials ( 54 ). In addition
to PD1 formed from human leukocytes, additional isomers
were identifi ed in infl ammatory exudates, including
? 15-trans-PD1 (isomer III), 10 S ,17 S -dihydroxy-docosa-4 -
Z ,7 Z ,11 E ,13 Z ,15 Z ,19 Z -hexaenoic acid (isomer IV), and the
expected double dioxygenation product 10 S ,17 S -dihy-
droxy-docosa-4 Z ,7 Z ,11 E ,13 Z ,15 E ,19 Z -hexaenoic acid (iso-
mer I), which are present in infl ammatory exudates
obtained from mice. 18 O-labeling results provided evi-
dence that this isomer I was a double dioxygenation prod-
uct and that the 10 position of NPD1/PD1 originated from
enzymatic conversion. Also, the rank order of activities was
established between these isomers, and NPD1/PD1 proved
to be most potent ( 55 ), with doses as low as 1–10 ng re-
ducing murine peritonitis, followed by ? 15-trans-PD1 >
10S,17S-diHDHA (isomer I). Hence, although the double
dioxygenation product 10 S ,17 S -diHDHA was generated in
was obtained from these cells and human PMN, which
suggest that the 10,17S-docosatriene biosynthesis is via a
16(17S)-epoxide-containing intermediate, because the
identifi ed vicinal diol could be a product of this epoxide
intermediate. Hence, a series of alcohol trapping studies
were undertaken to address the potential role of epoxide-
containing intermediates in the biosynthesis of the bioac-
tive compounds, in particular the 10,17S-docosatriene.
Indeed, evidence for epoxide-containing intermediates in
the biosynthesis of docosatrienes and 17S series resolvins
was obtained from human PMN ( 52 ). In these incubations
with human cells, two 16-OCH3 and two 10-OCH3 meth-
oxy-trapping products, likely all- trans in their triene conju-
gation, were obtained, which implicates production of a
16(17S)-epoxide intermediate that was proposed in the
biosynthetic pathway for the bioactive 10,17S-docosatriene
( 52 ). Also, 10,17S-docosatriene proved to display potent
actions with human glial cells and produced 10,17S-do-
cosatriene, which reduced IL-1 ? production at 1–50 nM
and evoked ligand-operated extracellular acidifi cation
with glial cells in a microphysiometer ( 52 ). These fi ndings
indicated that DHA is precursor to novel potent protective
mediators and that 10,17S-docosatriene carries potent
anti-infl ammatory activity in mice in vivo and with human
cells in vitro as well as activated surface receptors present
on human glial cells to regulate their function. Thus, the
basic structure of the novel 10,17S-docosatriene, later
coined NPD1/PD1 (see below) due to its potent actions in
vivo and in vitro in cell cultures, was established ( 47, 51,
52 ). This docosatriene also displayed potent anti-infl am-
matory actions, i.e., reducing PMN numbers in vivo and
reducing the production of infl ammatory cytokines by
glial cells in vitro. Moreover, during the resolution phase
of peritonitis, unesterifi ed DHA levels increase in resolv-
ing exudates, where it appears to promote catabasis or the
return to homeostasis following tissue insult via conver-
sion to D-series resolvins and also 10,17S-docosatrienes
( 62 ) by shortening the resolution interval of an infl amma-
tory response in vivo ( 63 ).
We then found that the DHA-derived 10,17S-docosa-
triene proved to be generated in vivo during experimental
stroke in the ipsilateral cerebral hemisphere following fo-
cal ischemia and also demonstrated potent bioactions in
this system, where it limits the entry of leukocytes, down-
regulates cyclooxygenase-2 expression and nuclear factor
? B activation, and decreases infarct volume ( 47 ). Next, we
found that 10,17S-docosatriene is formed in the human
retinal pigment epithelial cell line, ARPE-19, and intro-
duced the term NPD1 based on its neuroprotective bioac-
tivity ( 46, 47 ).
Taken together, these fi ndings in ARPE-19 cells ( 46 ),
infl ammatory murine exudates, human PMN, glial cells,
and the brain ( 47, 51, 52 ) underscored the need to estab-
lish the complete stereochemistry of endogenous, biologi-
cally generated, active 10,17S-docosatriene, namely the
chirality of its carbon 10 position alcohol and its triene
double geometry, which remained to be established ( Fig.
3B ). These involved the total organic synthesis and match-
ing of both bioactivity and physical properties of the en-
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair 2025
during the resolution of Lyme disease infections in mice
( 70 ). NPD1 inhibits retinal ganglion cell death ( 71 ), is renal
protective ( 72 ), and regulates adiponectin ( 73 ). Of inter-
est, the double dioxygenation product 10 S ,17 S -diHDHA
isomer of NPD1/PD1 was recently shown to have actions
on platelets, reducing platelet aggregation at 0.3 ? M,
1 ? M, and higher concentrations ( 74 ). In peritonitis, this
isomer also showed biological activity but was less potent
than NPD1/PD1 ( 54, 74 ). It is noteworthy that NPD1/
PD1 and the resolvins also are produced by trout tissues,
including trout brain, from endogenous DHA, suggesting
that these structures are highly conserved from fi sh to hu-
mans ( 75 ). This shows we still have much to learn regard-
ing the bioactions and functions of NPD1/PD1, the
D-series and E-series resolvins, and related products in hu-
man physiology and pathophysiology as well as in biologi-
cal systems such as fi sh, where the actions of NPD1/PD1
remain to be fully appreciated.
NEUROTROPHINS INDUCE THE SYNTHESIS AND
RELEASE OF NPD1 FROM HUMAN RPE CELLS
RPE cells also are capable of producing a wide variety of
growth factors ( 76 ). These trophic factors support sur-
rounding cells by paracrine and autocrine signaling and
hence promote the communication and structure of the
retina as well as photoreceptor survival ( 77, 78 ). It is im-
portant to note that neurotrophins enhance the produc-
tion of NPD1 in RPE cells. In turn, NPD1 is released and
serves as a lipid signaling messenger in its surroundings.
This observation was made in human RPE cells grown to
confl uence using a specialized culture ( 79 ) that allows the
cells to develop a high degree of differentiation, preserv-
ing the apical-basolateral polarization ( Fig. 4A, C ). Neu-
rotrophins [pigment epithelium derived factor (PEDF),
BDNF, ciliary neurotrophic factor, FGF, glial-derived neu-
rotrophic factor (GDNF), leukemia inhibitory factor, NT3,
or persephin], which have bioactivities that promote neu-
ronal and/or photoreceptor cell survival, are also agonists
of NPD1 synthesis ( Fig. 4B ), favoring the release of this
lipid messenger through the cell’s apical surface ( 80 ).
Among all the growth factors tested, PEDF is by far the
most potent stimulator of NPD1 synthesis. PEDF, a mem-
ber of the serine protease inhibitor (serpin) family, was
identifi ed in human RPE cells. If PEDF or ciliary neu-
rotrophic factor are added to the incubation media, bath-
ing the basolateral side in increasing concentrations, they
evoke a lesser degree of NPD1 release on the apical side.
Conversely, if these neurotrophins are added to the apical
side, they exert concentration-dependent increases in
NPD1 release only on the apical side ( Fig. 4B, C ) ( 80 ).
NPD1 BIOSYNTHESIS IS AN ENDOGENOUS
RESPONSE TO OXIDATIVE STRESS
Previous studies have shown that the retina forms
mono-, di-, and trihydroxy derivatives of DHA and that
murine exudates, it was far less active than PD1/NPD1
both in vitro and in vivo.
The proposed biosynthetic route for NPD1/PD1 is
shown in Fig. 3B from results previously reported ( 54 ).
Following 17 S -HpDHA formation from 15-lipoxygenase
action on DHA, an epoxide intermediate is formed that
requires enzymatic transformation to obtain the correct
double bond geometry, namely cis,trans,trans present in
NPD1/PD1. This double bond geometry and chirality of
the carbon 10 position and the R confi guration were estab-
lished from the matching of synthetic compounds of de-
fi ned chirality. Of interest, both 7 S ,17 S -diHDHA (resolvin
D5) and 10 S ,17 S -diHDHA are double dioxygenation prod-
ucts, and at this point they appear to be less active than
endogenous or synthetic NPD1/PD1. Without endoge-
nous biosynthesis studies or assessment of biological ac-
tions, it is surprising that a claim could be made for
assessment of the complete stereochemistry of NPD1 based
only on nuclear magnetic resonance results obtained for
10S,17S-diHDHA from plant lipoxygenase-prepared mate-
rial with DHA ( 64 ).
With the stereochemistry of NPD1/PD1 established, its
identifi cation in human material was sought and found to
be present in breath condensates from human asthmatics
( 65 ) and in the human brain, both under basal conditions
and from patients with AD ( 66 ). In addition, PD1 was
found to be a major product in bone marrow of female
rats fed eicosapentaenoic acid and DHA ( 67 ). PD1 was
generated in vivo during ischemia-reperfusion of renal tis-
sues, where it has profound actions, namely reversing the
deleterious consequences of ischemia-reperfusion in renal
tissues ( 68 ), in agreement with our previous observations
in the brain ( 47 ).
With the complete stereochemistry and synthetic com-
pound in hand, it was possible to demonstrate for the fi rst
time that PD1 activates resolution programs in vivo and
that it shortens the resolution time of experimental in-
fl ammation in animal models ( 63 ). With the total organic
synthesis route of NPD1/PD1 in place, it was possible to
radiolabel and purify 3 H-NPD1/PD1 made from the syn-
thetic intermediate. With this radiolabel, it was then pos-
sible to defi ne for the specifi c binding sites present with
ARPE-19 cells ( K d ? 31 pM/mg cell protein) for 3 H-NPD1/
PD1 as well as specifi c binding to human neutrophils that
gave a K d of ? 25 nM ( 55 ). Most importantly, critical infor-
mation on NPD1 biosynthesis with ARPE-19 cells was ob-
tained, namely identifi cation of alcohol-trapping products,
indicating the formation of a 16,17 S -epoxide-containing
intermediate from DHA in the biosynthesis of NPD1 ( 55 ).
These results, as well as the rank order of potency estab-
lished for NPD1/ PD1 of defi ned stereochemical analysis,
indicated that the most potent of the isomers prepared
was NPD1/PD1. Also in the ARPE-19 cells, NPD1 was more
potent than either resolvin D1 or resolvin E1. In other sys-
tems, resolvin E1 and resolvin D1 established higher po-
tencies than PD1 ( 69 ).
Along with the complete stereochemical identifi cation
of NPD1/PD1, recent studies using an unbiased LC-MS-
MS profi ling approach demonstrated that PD1 is made
by guest, on December 25, 2015
2026 Journal of Lipid Research Volume 51, 2010
synthesis. Then this stereospecifi c mediator is synthesized
by 15-lipoxygenase-1 (15-LOX-1) ( 91 ) ( Fig. 3A ). In AD
brain (short postmortem time), cPLA 2 ? and 15-LOX-1
expression changed in concert with NPD1-decreased
content and DHA-enhanced pool size in the CA1 area of
the hippocampus ( 66 ). In ARPE-19 cells (spontaneously
transformed human RPE cells), IL-1 ? , oxidative stress, or
the Ca 2+ ionophore A23187 activates the synthesis of
NPD1 ( 46 ). In turn, NPD1 might act in an autocrine fash-
ion and/or diffuse through the interphotoreceptor ma-
trix to act in a paracrine mode on photoreceptor cells
and/or Müller cells ( 41 ).
15-LOX-1 DEFICIENCY IN RPE CELLS PROMOTES
APOPTOSIS AND IT IS RESCUED SELECTIVELY
The increased availability of DHA is followed by NPD1
synthesis. This is of particular interest in the process of
phagocytosis of photoreceptor outer segments, given
that the endogenous pool of DHA is augmented upon
activation of the RPE cell phagolysosomal system. In this
context, it is known that NPD1 production and release is
increased ( Fig. 2 ). Recent evidence shows 15-LOX-1 as
the enzyme that oxygenates DHA into NPD1 ( 91 ). In
ARPE-19 cells where 15-LOX-1 protein expression was
knocked down by 70% posttranscriptionally, the produc-
tion of NPD1 was not increased, in contrast with normal
cells ( Fig. 5 ) . In normal cells, NPD1 production was in-
duced as early as 4 h after oxidative stress was applied. In
this way, preexisting pools of 15-LOX-1 are activated
upon stress stimulation ( Fig. 5B ). Thus, phagocytosis
triggers the activation of 15-LOX-1 to oxygenate DHA
15-LOX-1, a nonheme iron-containing dioxygenase, ste-
reospecifi cally inserts oxygen into AA, dually forming
15( S )-hydroxyeicosatetraenoic acid (HETE) and 12( S )-
HETE as well as lipoxin A 4 , a product of its joint activity
with 5-LOX. 15-LOX-1 also has the capability to oxygenate
linoleic acid into 13-hydroxyoctadecadienoic acid ( 92 ).
Human 15-LOX-1 and 12-LOX are highly homologous
proteins (65% identity) encoded by different genes, and
their mRNAs are similar (70% identity). On the other
hand, 15-LOX-2 is a different lipoxygenase that shares
only 39% identity with human 15-LOX-1 ( 93 ). 15-LOX 2
and human 12-LOX differ from 15-LOX-1 not only in the
ratio of 15-HETE and 12-HETE produced from AA but
also in their localization ( Fig. 5A ). This means that they
possess different selective product formations, and thus
their activities contribute to different lipid mediators as
well as display different substrate availability.
Silencing 15-LOX-1 also leads to enhanced susceptibil-
ity to apoptosis ( Fig. 5C ). It is worth noting that only
NPD1 could rescue these cells from the exaggerated
apoptosis experienced under oxidative stress conditions.
This indicates that cells failing to produce this lipid me-
diator have an increased sensibility to oxidative stress
caused by lack of the pro-survival signaling elicited by
lipoxygenase inhibitors block this synthesis, indicating
an enzymatic process of a lipoxygenase nature ( 42 ). Al-
though the stereochemistry and bioactivity of DHA-oxy-
genated derivatives were not defi ned at the time of these
observations, it was suggested that these lipoxygenase
products might be neuroprotective (and therefore the
name docosanoids was introduced) ( 42, 81 ). Liquid
chromatography-photodiode array-electrospray ioniza-
tion MS/MS-based lipidomic analysis was used to identify
oxygenation pathways for the synthesis of the docosanoid
NPD1 during brain ischemia-reperfusion ( 47 ) and the
retinal pigment epithelium ( 46 ). Moreover, it was also
found that RPE cells have the ability to synthesize NPD1
( 46 ). Photoreceptors and RPE cells, although they con-
tain phospholipids richly endowed with DHA (as docosa-
hexaenoyl- or DHA-elongated fatty acyl-chains), display
an undetectable quantity of unesterifi ed (free) DHA [as
is the case with unesterifi ed arachidonic acid (AA)] un-
der basal, nonstimulated conditions ( 82–86 ). This means
that the pool size of unesterifi ed DHA is tightly regulated
by production (PLA 2 ), by its removal (e.g., by reacyla-
tion), and by peroxidation. Free DHA incorporated into
membrane phospholipids fi rst becomes the substrate
of docosahexaenoyl-CoA synthesis for its channeling
through acyltransferases, which incorporate this fatty
acid into phospholipids ( 87–90 ). The RPE cell thus mod-
ulates the uptake, conservation, and delivery of DHA to
photoreceptors ( 81 ). In addition, RPE cells utilize a spe-
cifi c DHA-phospholipid pool as a precursor for NPD1
Fig. 4. Neurotrophins activate NPD1 synthesis in cultured pri-
mary human RPE cells. A: Zonula occludens-1 (ZO-1) antibody im-
munoreactivity (green) illustrates confl uence of the monolayer
polyhedric-shape of the cells. B: Differential ability of growth fac-
tors to selectively release NPD1 through the apical surface of the
cell. Growth factors (20 ng/ml) were added to the apical medium.
Apical and basal media were collected separately after 72 h and
subjected to lipidomic analysis. Each bar is an average ± SEM of
four or fi ve independent wells. Values are averages ± SEM of fi ve
independent wells. Statistical analysis was performed using Stu-
dent’s t -test shows * P < 0.05. C: Schematic representation of the
monolayer orientation within the insert. [ Fig. 4 A, C, modifi ed with
permission from reference ( 40 )].
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2027
PHOTORECEPTOR OUTER SEGMENT
PHAGOCYTOSIS INDUCES RPE CELL SURVIVAL:
The novel signaling pathway, resulting from the NPD1
action, may be initiated through the photoreceptor outer
segment phagocytosis. A signifi cant body of evidence has
been accumulated that supports this idea.
FGF2 promotes bovine RPE cell survival through a sus-
tained adaptive phenomenon that involves both FGF1-
mediated activation of extracellular signal-regulated
kinase andextracellular signal-regulated kinase 2-depen-
dent Bcl-xL production ( 94 ). Bcl-xL may play a key role
in integrating and transmitting exogenous FGF2 signals
for RPE cell survival. Moreover, a well-organized signal-
ing regulatory mechanism on the apical side of the RPE
cell is refl ected by the ability of neurotrophins to induce
NPD1 synthesis and release ( 80 ). The response of human
RPE cell monolayers in culture with NPD1 synthesis and
release upon addition of certain neurotrophins to the
apical side suggests sidedness of receptors for these li-
gands ( 80 ). Persephin is a novel neurotrophin with ho-
mology to GDNF ( 95, 96 ). Both persephin and GDNF are
agonists of NPD1 synthesis and activators of its release
from the apical surface of RPE cells ( 80 ). The same was
true for leukemia inhibitory factor and FGF2 as well as
for other neurotrophins ( 80 ). The fi nding that there is
polarized (apically) neurotrophin/mediated NPD1 re-
lease has relevance for the initiation and progression of
retinal degenerations. This is because when RPE cell po-
larization in the plane of the epithelium is disrupted, dys-
regulated growth factor secretion and pro-infl ammatory
signaling arises ( 2, 97, 98 ), thereby setting in motion
pathological changes that include the proliferative com-
ponent of macular degeneration: choroidal neovascular-
ization ( 99–101 ).
Bcl-2 family proteins regulate apoptotic signaling at the
mitochondrial level and at the endoplasmic reticulum. As
a consequence, cytochrome c is released from mitochon-
dria and caspase-3, a downstream effector of pro-apoptotic
and anti-apoptotic Bcl-2 proteins, is activated ( 102 ). Also,
oxidative stress-induced activation of caspase-3 in RPE cells
is decreased by NPD1 ( 46 ). Apoptosis is an outcome of
excessive oxidative stress in RPE cells, and NPD1 is effec-
tive in counteracting this oxidative stress-induced cell
death ( 46 ). It is interesting that DHA itself inhibits apop-
tosis, concomitant with a remarkable, time-dependent for-
mation of NPD1. Signifi cantly, the potency of DHA for
cytoprotection is much higher than that of added NPD1
( 46 ), suggesting that NPD1 might exert its action close to
the subcellular site of its synthesis. Importantly, these ac-
tions of DHA cannot be mimicked by other PUFAs (e.g.,
Fig. 5. NPD1 synthesis is mediated by 15-lipoxygenase-1. A: Im-
munocytochemistry showing localization of 15-LOX-1 in ARPE-19
cells. Right column shows normal cells and left column shows 15-
LOX-1 silenced cells. The four upper panels depict the localization
of 15-LOX1 (green) relative to the nuclei (blue) and Actin (red).
The four lower panels display nuclear localization of 15-LOX-2
(red) in relationship with nuclei (blue) and Actin (green). B: His-
tograms showing the differential production of NPD1 upon differ-
ent strength of oxidative stress treatment (0, 400, 600, 800 ? M
H 2 O 2 and 10 ng/ml TNF ? ). In each cell, as in the medium, the
production of NPD1 was almost completely abolished (* P < 0.01).
C: Apoptosis percentage measured by Hoechst staining of ARPE-19
cultures of control and silenced cells subjected to oxidative stress
and treated with different metabolites of 15-LOX-1 and NPD1 pre-
cursor DHA. In silenced cells, apoptosis was augmented by oxida-
tive stress. In normal cells, PEDF/DHA, NPD1, and lipoxin A4 did
prevent apoptotic cell death, but in the silenced cells, neither
DHA nor lipoxin A4 had any effect. Only NPD1 was able to rescue
these cells from apoptosis. Data represents average ± SEM of two
independent studies; statistical analysis is Student’s t -test (* P < 0.01,
** P < 0.001, *** P < 0.0001 and **** P < 0.00001). Modifi ed with
permission from reference ( 91 ).
by guest, on December 25, 2015
2028 Journal of Lipid Research Volume 51, 2010
plays increased expression under oxidative stress treat-
ment (unpublished observations). In RPE cells, NPD1
downregulates the expression of pro-infl ammatory genes,
such as cycloxygenase 2 (COX-2), which is induced by cy-
tokines such as IL-1 ? ( 46, 109 ). In ischemia reperfusion-
injured hippocampus, as well as in neural progenitor cells
stimulated by IL-1 ? , NPD1 also inhibits COX-2 induction
( 46, 47 ). In brain ischemia reperfusion, NPD1 decreases
infarct size and inhibits PMN infi ltration ( 47 ). Moreover,
our laboratory, through a genome-wide screen in human
brain progenitor cells in culture ( 66 ), has identifi ed other
NPD1-targeted pro-infl ammatory genes; they include IL-
1 ? , cytokine exodus protein-1, and TNF ? -inducible pro-
infl ammatory element (B94, TNFAIP2). NPD1 bioactivity
acts as a modulatory signal that counteracts pro-infl amma-
tory injury to the RPE, a condition involved in pathoangio-
genic signaling in the wet form of AMD and in proliferative
vitreoretinopathy of diabetic retinopathy.
Oxidative stress enhances pro-infl ammatory gene ex-
pression that leads to RPE cell injury. The inducible en-
zyme COX-2 is the rate-limiting step in prostaglandin
synthesis and is involved in oxidative stress as well as cell
function. COX-2 expression is regulated in RPE cells by
photoreceptor outer-segment phagocytosis and by growth
factors ( 110 ), and IL-1 ? activates expression of the proxi-
mal human COX-2 promoter. In the latter case, NPD1 po-
tently counteracts the activation of the transcription
mediated by IL-1 ? , displaying an IC 50 of <5 nM ( 46 ).
20:4, n-6). Alternatively, it implies that other NPD-like me-
diators participate in promoting RPE cell survival, even
though DHA fails to exert protection in 15-LOX-1-defi -
cient cells, confi rming that its pro-survival effect is medi-
ated via NPD1. In RPE cells, cleavage of endogenous
substrates by caspase-3 is enhanced by oxidative stress, as
indicated by increased accumulation of poly(ADP-ribose)
polymerases. NPD1 inhibits caspase-3 activation when
added at the onset of oxidative stress ( 46 ), likely refl ecting
a downstream consequence of NPD1 modulation of Bcl-2
A consequence of RPE cell damage and apoptosis is im-
paired photoreceptor cell survival, a dominant factor in
AMD ( 103 ). The pigment lipofuscin, which increases in
the RPE during aging, accumulates further during AMD.
The progressively greater onslaught of photooxidative
damage to the RPE affects photoreceptor survival. For ex-
ample, in the juvenile form of macular degeneration
known as Stargardt’s disease, oxidative stress mediated by
the lipofuscin fl uorophore N -retinylidene- N -retinyletha-
nolamine produces RPE damage; caspase-3 is part of the
damaging cascade, whereas Bcl-2 exerts cellular protec-
tion ( 104 ). NPD1 downregulates lipofuscin fl uorophore
N -retinylidene- N -retinylethanolamine-mediated apoptosis
induced by oxidative stress, restoring the integrity of the
RPE and its relationship with the photoreceptor ( 80 ).
ANTI-APOPTOTIC AND ANTI-INFLAMMATORY
BIOACTIVITY OF NPD1 IS MEDIATED IN PART
THROUGH MODULATION OF GENE EXPRESSION
Photoreceptor outer segment phagocytosis is a mecha-
nism that initiates NPD1 signaling. This signaling pro-
motes modulation of protein expression and activity
involved in counteracting apoptosis at the cell fate deci-
sion level. For instance, NPD1 promotes differential
changes in expression of Bcl-2 family proteins, upregulat-
ing protective Bcl-2 proteins (Bcl-2, Bcl-xL, and Bfl -1/A1)
and attenuating expression of proteins that challenge cell
survival (e.g., Bax, Bad, Bid, and Bik). Thus, an NPD1-me-
diated and coordinated regulation of Bcl-2 protein avail-
ability for subsequent downstream signaling may be crucial
for cell survival ( 46, 80 ). NPD1 regulates expression of the
genes encoding death repressors and effectors of the Bcl-2
family of proteins, e.g., Bcl-2-associated athanogene do-
main 3, whose mRNA is upregulated in mouse retinas sub-
jected to light damage (105, unpublished observations).
Bcl-2-associated athanogene domain 3 is a co-chaperone
protein involved in release of the refolded protein by the
chaperone Hsp70 ( 106–108 ).
NPD1 activity lessens the effects of stressors such as oxi-
dative stress not only by its production and release upon
stimulation but also because its down-stream signaling
modulates gene transcription by compensating the activa-
tion or inhibition of gene expression. Thus, the conse-
quence of the signaling is to return the system to normal
levels. This is the case with NPD1 downregulation of the
pro-apoptotic death-associated protein kinase 1, which dis-
Fig. 6. Diagram outlining 15-LOX-1 activity of NPD1 synthesis
and bioactivity in RPE cells. Noxious stimuli activate 15-LOX-1,
promoting the synthesis of NPD1 from DHA. NPD1 signaling mod-
ulates the activity and gene expression of proteins involved in pro-
and anti-infl ammatory signaling in apoptosis, ultimately fostering
photoreceptor cell integrity and overall homeostasis.
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2029
12 . Dunaief , J. L. , T. Dentchew , G. S. Ying , and A. H. Milam . 2002 .
The role of apoptosis in age-related macular degeneration. Arch.
Ophthalmol. 120 : 1435 – 1442 .
13 . Feher , J. , I. Kovacs , M. Artico , C. Cavallotti , A. Papale , and C.
Balacco Gabrieli . 2006 . Mitochondrial alterations of retinal pig-
ment epithelium in age-related macular degeneration. Neurobiol.
Aging . 27 : 983 – 993 .
14 . Hageman , G. S. , D. H. Anderson , L. V. Johnson , L. S. Hancox ,
A. J. Taiber , L. I. Hardisty , J. L. Hageman , H. A. Stockman , J. D.
Borchardt , K. M. Gehrs , et al . 2005 . A common haplotype in the
complement regulatory gene factor H (HF1/CFH) predisphoto-
receptor outer segment es individuals to age-related macular de-
generation. Proc. Natl. Acad. Sci. USA . 102 : 7227 – 7232 .
15 . Klein , R. J. , C. Zeiss , E. Y. Chew , J. Y. Tsai , R. S. Sackler , C. Haynes ,
A. K. Henning , J. P. SanGiovanni , S. M. Mane , S. T. Mayne , et al .
2005 . Complement factor H polymorphism in age-related macu-
lar degeneration. Science . 308 : 385 – 389 .
16 . Edwards , A. O. , R. Ritter III , K. J. Abel , A. Manning , C. Panhuysen ,
and L. A. Farrer . 2005 . Complement factor H polymorphism and
age-related macular degeneration. Science . 308 : 421 – 424 .
17 . Haines , J. L. , M. A. Hauser , S. Schmidt , W. K. Scott , L. M. Olson ,
P. Gallins , K. L. Spencer , S. Y. Kwan , M. Noureddine , J. R. Gilbert ,
et al . 2005 . Complement factor H variant increases the risk of age-
related macular degeneration. Science . 308 : 419 – 421 .
18 . Gold , B. , J.E. Merriam , J. Zernant , L.S. Hancox , A.J. Taiber , K.
Gehrs , K. Cramer , J. Neel , J. Bergeron , G.R. Barile , et al . 2006 .
Variation in factor B (BF) and complement 2 (C2) genes is as-
sociated with age-related macular degeneration. Nat. Genet. 38 :
458 – 462 .
19 . Bok , D. 2005 . Evidence for an infl ammatory process in age-related
macular degeneration gains new support. Proc. Natl. Acad. Sci.
USA . 102 : 7053 – 7054 .
20 . Spencer , K. L. , M. A. Hauser , L. M. Olson , S. Schmidt , W. K. Scott ,
P. Gallins , A. Agarwal , E. A. Postel , M. A. Pericak-Vance , and J. L.
Haines . 2007 . Protective effect of complement factor B and com-
plement component 2 variants in age-related macular degenera-
tion. Hum. Mol. Genet. 16 : 1986 – 1992 .
21 . Pournaras , C. J. , E. Rungger-Brändle , C. E. Riva , S. H. Hardarson ,
and E. Stefansson . 2008 . Regulation of retinal blood fl ow in health
and disease. Prog. Retin. Eye Res. 27 : 284 – 330 .
22 . Strauss , O. 2005 . The retinal pigment epithelium in visual func-
tion. Physiol. Rev. 85 : 845 – 881 .
23 . Wang , J. S. , and V. J. Kefalov . 2009 . An alternative pathway
mediates the mouse and human cone visual cycle. Curr. Biol. 19 :
1665 – 1669 .
24 . Navid , A. , S. C. Nicholas , and R. D. Hamer . 2006 . A proposed role
for all-trans retinal in regulation of rhodopsin regeneration in hu-
man rods. Vision Res. 46 : 4449 – 4463 .
25 . Saari , J. C. 2000 . Biochemistry of visual pigment regeneration: the
Friedenwald lecture. Invest. Ophthalmol. Vis. Sci. 41 : 337 – 348 .
26 . Lamb , T. D. , and E. N. Pugh , Jr . 2004 . Dark adaptation and the
retinoid cycle of vision. Prog. Retin. Eye Res. 23 : 307 – 380 .
27 . Besch , D. , H. Jägle , H. P. N. Scholl , M. W. Seeliger , and E. Zrenner .
2003 . Inherited multifocal RPE-diseases: mechanisms for local
dysfunction in global retinoid cycle gene defects. Vision Res. 43 :
3095 – 3108 .
28 . Cideciyan , A. V. , T. S. Aleman , M. Swider , S. B. Schwartz , J. D.
Steinberg , A. J. Brucker , A. M. Maguire , J. Bennett , E. M. Stone ,
and S. G. Jacobson . 2004 . Mutations in ABCA4 result in accumula-
tion of lipofucsin before slowing the retinoid cycle: a reappraisal
of the human disease sequence. Hum. Mol. Genet. 13 : 525 – 534 .
29 . Zlokovic , B. V. 2005 . Neurovascular mechanisms of Alzheimer’s
neurodegeneration. Trends Neurosci. 28 : 202 – 208 .
30 . Zhao , S. , and P. A. Overbeek . 2001 . Regulation of choroid
development by the retinal pigment epithelium. Mol. Vis. 2 :
277 – 282 .
31 . Lee , S. J. , J. H. Kim , J. H. Kim , M. J. Chung , Q. Wen , H. Chung , K.
W. Kim , and Y. S. Yu . 2007 . Human apolipoprotein E2 transgenic
mice show lipid accumulation in retinal pigment epithelium and
altered expression of VEGF and bFGF in the eyes. J. Microbiol.
Biotechnol. 17 : 1024 – 1030 .
32 . Gao , G. , Y. Li , D. Zhang , S. Gee , C. Crosson , and J. Ma . 2001 .
Unbalanced expression of VEGF and PEDF in ischemia-induced
retinal neovascularization. FEBS Lett. 489 : 270 – 276 .
33 . Dawson , D. W. , O. V. Volpert , P. Gillis , S. E. Crawford , H. Xu , W.
Benedict , and N. P. Bouck . 1999 . Pigment epithelium-derived fac-
tor: a potent inhibitor of angiogenesis. Science . 285 : 245 – 248 .
NPD1 is a pro-survival, anti-infl ammatory, and homeo-
static mediator that, by acting on RPE cells, promotes pho-
toreceptor cell integrity ( 42 ). NPD1 synthesis agonists
include neurotrophins and oxidative stress, and thus this
lipid mediator modulates signaling pathways related with
the pro- and anti-apoptotic balance ( Fig. 6 ). As a conse-
quence, NPD1 tilts the equilibrium toward cell survival.
Recently, the presence of stereoselective specifi c binding
of NPD1 was shown in ARPE-19 cells and in human leuko-
cytes, suggesting a specifi c receptor in immune and retinal
cells ( 55 ).
A proof of principle of the in vivo bioactivity of NPD1
was obtained in a mouse model of choroidal neovascular-
ization by clinically grading retinal laser-induced lesions,
measuring leakage area, and volumetrically quantifying
vascular endothelial cell proliferation. NPD1 injected
intraperitoneally was found to inhibit choroidal neovas-
cularization, thus suggesting a sustained protection and
highlighting the potential applicability of this lipid media-
tor in preventing or ameliorating endothelial cell growth
in pathoangiogenesis ( 111 ).
Several questions remain: What are the molecular
mechanisms through which NPD1 synthesis is stimulated?
What are the early molecular NPD1 interactors/effectors
in the signaling pathway? The evolving information on the
bioactivity of NPD1 in RPE/photoreceptor interactions
suggests that DHA/NPD1 signaling may contribute to pre-
venting and/or halting progression of retinal degenera-
1 . Papermaster , D. S. 2002 . The birth and death of photoreceptors:
the Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 43 : 1300 –
2 . Rattner , A. , and J. Nathans . 2006 . Macular degeneration: recent
advances and therapeutic opportunities. Nat. Rev. Neurosci. 7 :
860 – 872 .
3 . Chang , G. Q. , Y. Hao , and F. Wong . 1993 . Apoptosis: fi nal com-
mon pathway of photoreceptor death in rd, rds, and rhodopsin
mutant mice. Neuron . 11 : 595 – 605 .
4 . Portera-Cailliau , C. , C. H. Sung , J. Nathans , and R. Adler . 1994 .
Apoptotic photoreceptor cell death in mouse models of retinitis
pigmentosa. Proc. Natl. Acad. Sci. USA . 91 : 974 – 978 .
5 . Bird , A. C. 2003 . The Bowman lecture. Towards an understanding
of age-related macular disease. Eye (Lond.) . 17 : 457 – 466 .
6 . Liu , Q. , J. Zuo , and E. A. Pierce . 2004 . The retinitis pigmentosa
1 protein is a photoreceptor microtubule-associated protein. J.
Neurosci. 24 : 6427 – 6436 .
7 . Dryja , T. P. , T. L. McGee , E. Reichel , L. B. Hahn , G. S. Cowley , D.
W. Yandell , M. A. Sandberg , and E. L. Berson . 1990 . A point muta-
tion of the rhodopsin gene in one form of retinitis pigmentosa.
Nature . 343 : 364 – 366 .
8 . Mendes , H. F. , J. van der Spuy , J. P. Chapple , and M. E. Cheetham .
2005 . Mechanisms of cell death in rhodopsin retinitis pigmentosa:
implications for therapy. Trends Mol. Med. 11 : 177 – 185 .
9 . de Jong , P. T. 2006 . Age-related macular degeneration. N. Engl. J.
Med. 355 : 1474 – 1485 .
10 . Sreekumar , P. G. , R. Kannan , J. Yaung , C. K. Spee , S. J. Ryan ,
and D. R. Hinton . 2005 . Protection from oxidative stress by me-
thionine sulfoxide reductases in RPE cells. Biochem. Biophys. Res.
Commun. 334 : 245 – 253 .
11 . Shen , J. K. , A. Dong , S. F. Hackett , W. R. Bell , W. R. Green , and P.
A. Campochiaro . 2007 . Oxidative damage in age-related macular
degeneration. Histol. Histopathol. 22 : 1301 – 1308 .
by guest, on December 25, 2015
2030Journal of Lipid Research Volume 51, 2010
55 . Marcheselli , V. L. , P. K. Mukherjee , M. Arita , S. Hong , R. Antony ,
K. Sheets , N. Petasis , C. N. Serhan , and N. G. Bazan . 2010 .
Neuroprotectin D1/protectin D1 stereoselective and specifi c
binding with human retinal pigment epithelial cells and neutro-
phils. Prostaglandins Leukot. Essent. Fatty Acids . 82 : 27 – 34 .
56 . Serhan , C. N. 2007 . Resolution phases of infl ammation: novel
endogenous anti-infl ammatory and pro-resolving lipid mediators
and pathways. Annu. Rev. Immunol. 25 : 101 – 137 .
57 . Serhan , C. N. , N. Chiang , and T. E. Van Dyke . 2008 . Resolving
infl ammation: dual anti-infl ammatory and pro-resolution lipid
mediators. Nat. Rev. Immunol. 8 : 349 – 361 .
58 . Bazan , N. G. , D. L. Birkle , and T. S. Reddy . 1984 . Docosahexaenoic
acid (22:6, n-3) is metabolized to lipoxygenase reaction products
in the retina. Biochem. Biophys. Res. Commun. 125 : 741 – 747 .
59 . Serhan , C. N. , C. B. Clish , J. Brannon , S. P. Colgan , N. Chiang ,
and K. Gronert . 2000 . Novel functional sets of lipid-derived medi-
ators with antiinfl ammatory actions generated from omega-3 fatty
acids via cyclooxygenase 2-nonsteroidal antiinfl ammatory drugs
and transcellular processing. J. Exp. Med. 192 : 1197 – 1204 .
60 . Arita , M. , F. Bianchini , J. Aliberti , A. Sher , N. Chiang , S. Hong ,
R. Yang , N. A. Petasis , and C. N. Serhan . 2005 . Stereochemical
assignment, anti-infl ammatory properties, and receptor for the
omega-3 lipid mediator resolvin E1. J. Exp. Med. 201 : 713 – 722 .
61 . Serhan , C. N. , U. Lundberg , G. Weissmann , and B. Samuelsson .
1984 . Formation of leukotrienes and hydroxy acids by hu-
man neutrophils and platelets exposed to monosodium urate.
Prostaglandins . 27 : 563 – 581 .
62 . Bannenberg , G. L. , N. Chiang , A. Ariel , M. Arita , E. Tjonahen , K.
H. Gotlinger , S. Hong , and C. N. Serhan . 2005 . Molecular circuits
of resolution: formation and actions of resolvins and protectins. J.
Immunol. 174 : 4345 – 4355 .
63 . Schwab , J. M. , N. Chiang , M. Arita , and C. N. Serhan . 2007 .
Resolvin E1 and protectin D1 activate infl ammation-resolution
programmes. Nature . 447 : 869 – 874 .
64 . Butovich , I. A. 2005 . On the structure and synthesis of neuropro-
tectin D1, a novel anti-infl ammatory compound of the docosa-
hexaenoic acid family. J. Lipid Res. 46 : 2311 – 2314 .
65 . Levy , B. D. , P. Kohli , K. Gotlinger , O. Haworth , S. Hong , S. Kazani ,
E. Israel , K. J. Haley , and C. N. Serhan . 2007 . Protectin D1 is gen-
erated in asthma and dampens airway infl ammation and hyper-
responsiveness. J. Immunol. 178 : 496 – 502 .
66 . Lukiw , W. J. , J. G. Cui , V. L. Marcheselli , M. Bodker , A. Botkjaer ,
K. Gotlinger , C. N. Serhan , and N. G. Bazan . 2005 . A role for do-
cosahexaenoic acid-derived neuroprotectin D1 in neural cell sur-
vival and Alzheimer disease. J. Clin. Invest. 115 : 2774 – 2783 .
67 . Poulsen , R. C. , K. H. Gotlinger , C. N. Serhan , and M. C. Kruger .
2008 . Identifi cation of infl ammatory and pro-resolving lipid me-
diators in bone marrow and their profi le alteration with ovariec-
tomy and omega-3 intake. Am. J. Hematol. 83 : 437 – 445 .
68 . Duffi eld , J. S. , S. Hong , V. Vaidya , Y. Lu , G. Fredman , C. N. Serhan ,
and J. V. Bonventre . 2006 . Resolvin D series and protectin D1 miti-
gate acute kidney injury. J. Immunol. 177 : 5902 – 5911 .
69 . Sun , Y-P. , S. F. Oh , J. Uddin , R. Yang , K. Gotlinger , E. Campbell ,
S. P. Colgan , N. A. Petasis , and C. N. Serhan . 2007 . Resolvin D1
and its aspirin-triggered 17 R epimer: stereochemical assignments,
anti-infl ammatory properties and enzymatic inactivation. J. Biol.
Chem. 282 : 9323 – 9334 .
70 . Blaho , V. A. , M. W. Buczynski , C. R. Brown , and E. A. Dennis .
2009 . Lipidomic analysis of dynamic eicosanoid responses during
the induction and resolution of Lyme arthritis. J. Biol. Chem. 284 :
21599 – 21612 .
71 . Qin , Q. , K. A. Patil , K. Gronert , and S. C. Sharma . 2008 .
Neuroprotectin D1 inhibits retinal ganglion cell death fol-
lowing axotomy. Prostaglandins Leukot. Essent. Fatty Acids . 79 :
201 – 207 .
72 . Hassan , I. R. , and K. Gronert . 2009 . Acute changes in dietary
omega-3 and omega-6 polyunsaturated fatty acids have a pro-
nounced impact on survival following ischemic renal injury and
formation of renoprotective docosahexaenoic acid-derived pro-
tectin D1. J. Immunol. 182 : 3223 – 3232 .
73 . González-Périz , A. , R. Horrillo , N. Ferré , K. Gronert , B. Dong , E.
Morán-Salvador , E. Titos , M. Martínez-Clemente , M. López-Parra ,
V. Arroyo , et al . 2009 . Obesity-induced insulin resistance and he-
patic steatosis are alleviated by omega-3 fatty acids: a role for re-
solvins and protectins. FASEB J. 23 : 1946 – 1957 .
74 . Chen , P. , B. Fenet , S. Michaud , N. Tomczyk , E. Véricel , M. Lagarde ,
and M. Guichardant . 2009 . Full characterization of PDX, a neuro-
34 . Bok , D. 1993 . The retinal pigment epithelium: a versatile partner
in vision. J. Cell Sci. Suppl. 17 : 189 – 195 .
35 . Kolko , M. , J. Wang , C. Zhan , K. A. Poulsen , J. U. Prause , M. H.
Nissen , S. Heegaard , and N. G. Bazan . 2007 . Identifi cation of in-
tracellular phospholipases A2 in the human eye: involvement in
phagocytosis of photoreceptor outer segments. Invest. Ophthalmol.
Vis. Sci. 48 : 1401 – 1409 .
36 . LaVail , M. M. 1980 . Circadian nature of rod outer segment disc
shedding in the rat. Invest. Ophthalmol. Vis. Sci. 19 : 407 – 411 .
37 . Young , R. W. , and B. Droz . 1968 . The renewal of protein in retinal
rods and cones. J. Cell Biol. 39 : 169 – 184 .
38 . Young , R. W. 1971 . The renewal of the rod and cone outer seg-
ments in the rhesus monkey. J. Cell Biol. 49 : 303 – 318 .
39 . Gao , H. , and J. G. Hollyfi eld . 1992 . Aging of the human retina.
Differential loss of neurons and retinal pigment epithelial cells.
Invest. Ophthalmol. Vis. Sci. 33 : 1 – 17 .
40 . Gibbs , D. , J. Kitamoto , and D. S. Williams . 2003 . Abnormal phago-
cytosis by retinal pigmented epithelium that lacks myosin VIIa,
the Usher syndrome 1B protein. Proc. Natl. Acad. Sci. USA . 100 :
6481 – 6486 .
41 . Bazan , N. G. 2006 . Cell survival matters: docosahexaenoic acid sig-
naling, neuroprotection and photoreceptors. Trends Neurosci. 29 :
263 – 271 .
42 . Bazan , N. G. 2007 . Homeostatic regulation of photoreceptor cell
integrity: signifi cance of the potent mediator neuroprotectin D1
biosynthesized from docosahexaenoic acid: the Proctor Lecture.
Invest. Ophthalmol. Vis. Sci. 48 : 4866 – 4881 .
43 . Chen , M. , J. V. Forrester , and H. Xu . 2007 . Synthesis of comple-
ment factor H by retinal pigment epithelial cells is down-regu-
lated by oxidized photoreceptor outer segments. Exp. Eye Res. 84 :
635 – 645 .
44 . Mukherjee , P. K. , V. L. Marcheselli , J. C. de Rivero Vaccari , W.
C. Gordon , F. E. Jackson , and N. G. Bazan . 2007 . Photoreceptor
outer segment phagocytosis selectively attenuates oxidative stress-
induced apoptosis with concomitant neuroprotectin D1 synthesis .
Proc. Natl. Acad. Sci. USA . 104 : 13158 – 13163 .
45 . Dunn , K. C. , A. E. Aotaki-Keen , F. R. Putkey , and L. M. Hjelmeland .
1996 . ARPE-19, a human retinal pigment epithelial cell line with
differentiated properties. Exp. Eye Res. 62 : 155 – 169 .
46 . Mukherjee , P. K. , V. L. Marcheselli , C. N. Serhan , and N. G.
Bazan . 2004 . Neuroprotectin D1: a docosahexaenoic acid-derived
docosatriene protects human retinal pigment epithelial cells from
oxidative stress. Proc. Natl. Acad. Sci. USA . 101 : 8491 – 8496 .
47 . Marcheselli , V. L. , S. Hong , W. J. Lukiw , X. H. Tian , K. Gronert ,
A. Musto , M. Hardy , J. M. Gimenez , N. Chiang , C. N. Serhan , et al .
2003 . Novel docosanoids inhibit brain ischemia-reperfusion-medi-
ated leukocyte infi ltration and pro-infl ammatory gene expression.
J. Biol. Chem. 278 : 43807 – 43817 .
48 . SanGiovanni , J. P. , and E. Y. Chew . 2005 . The role of omega-3
long-chain polyunsaturated fatty acids in health and disease of the
retina. Prog. Retin. Eye Res. 24 : 87 – 138 .
49 . Marszalek , J. R. , and H. F. Lodish . 2005 . Docosahexaenoic acid,
fatty acid interacting proteins, and neuronal function: breast milk
and fi sh are good for you. Annu. Rev. Cell Dev. Biol. 21 : 633 – 657 .
50 . Samuelsson , B. , S. E. Dahlen , J. A. Lindgren , C. A. Rouzer , and C.
N. Serhan . 1987 . Leukotrienes and lipoxins: structures, biosynthe-
sis, and biological effects. Science . 237 : 1171 – 1176 .
51 . Serhan , C. N. , S. Hong , K. Gronert , S. P. Colgan , P. R. Devchand ,
G. Mirick , and R-L. Moussignac . 2002 . Resolvins: a family of bioac-
tive products of omega-3 fatty acid transformation circuits initi-
ated by aspirin treatment that counter pro-infl ammation signals.
J. Exp. Med. 196 : 1025 – 1037 .
52 . Hong , S. , K. Gronert , P. Devchand , R-L. Moussignac , and C. N.
Serhan . 2003 . Novel docosatrienes and 17S-resolvins generated
from docosahexaenoic acid in murine brain, human blood and
glial cells: autacoids in anti-infl ammation. J. Biol. Chem. 278 :
14677 – 14687 .
53 . Ariel , A. , P-L. Li , W. Wang , W-X. Tang , G. Fredman , S. Hong ,
K. H. Gotlinger , and C. N. Serhan . 2005 . The docosatriene
protectin D1 is produced by T H 2 skewing and promotes hu-
man T cell apoptosis via lipid raft clustering. J. Biol. Chem. 280 :
43079 – 43086 .
54 . Serhan , C. N. , K. Gotlinger , S. Hong , Y. Lu , J. Siegelman , T. Baer ,
R. Yang , S. P. Colgan , and N. A. Petasis . 2006 . Anti-infl ammatory
actions of neuroprotectin D1/protectin D1 and its natural stereo-
isomers: assignments of dihydroxy-containing docosatrienes. J.
Immunol. 176 : 1848 – 1859 .
by guest, on December 25, 2015
Neuroprotectin D1 in photoreceptor rescue and repair2031 Download full-text
protectin/protectin D1 isomer, which inhibits blood platelet ag-
gregation. FEBS Lett. 583 : 3478 – 3484 .
75 . Hong , S. , E. Tjonahen , E. L. Morgan , L. Yu , C. N. Serhan , and A.
F. Rowley . 2005 . Rainbow trout ( Oncorhynchus mykiss ) brain cells
biosynthesize novel docosahexaenoic acid-derived resolvins and
protectins–mediator lipidomic analysis. Prostaglandins Other Lipid
Mediat. 78 : 107 – 116 .
76 . Tanihara , H. , M. Inatani , and Y. Honda . 1997 . Growth factors and
their receptors in the retina and pigment epithelium. Prog. Retin.
Eye Res. 16 : 271 – 301 .
77 . LaVail , M. M. , D. Yasumura , M. T. Matthes , C. Lau-Villacorta ,
K. Unoki , C. H. Sung , and R. H. Steinberg . 1998 . Protection of
mouse photoreceptors by survival factors in retinal degenerations.
Invest. Ophthalmol. Vis. Sci. 39 : 592 – 602 .
78 . Politi , L. E. , N. P. Rotstein , and N. G. Carri . 2001 . Effect of GDNF
on neuroblast proliferation and photoreceptor survival: additive
protection with docosahexaenoic acid. Invest. Ophthalmol. Vis. Sci.
42 : 3008 – 3015 .
79 . Hu , J. , and D. Bok . 2001 . A cell culture medium that supports the
differentiation of human retinal pigment epithelium into func-
tionally polarized monolayers. Mol. Vis. 7 : 14 – 19 .
80 . Mukherjee , P. K. , V. L. Marcheselli , S. Barreiro , J. Hu , D. Bok ,
and N. G. Bazan . 2007 . Neurotrophins enhance retinal pigment
epithelial cell survival through neuroprotectin D1 signaling. Proc.
Natl. Acad. Sci. USA . 104 : 13152 – 13157 .
81 . Bazan , N. G. , D. L. Birkle , and T. S. Reddy . 1985 . Biochemical
and nutritional aspects of the metabolism of polyunsaturated fatty
acids and phospholipids in experimental models of retinal de-
generation. In Retinal Degeneration: Experimental and Clinical
Studies. M.M. LaVail, J.G. Hollyfi eld, and R.E. Anderson, editors.
Alan R. Liss, Inc. New York, NY. 159–187.
82 . Bazan , N. G. 2003 . Synaptic lipid signaling: signifi cance of polyun-
saturated fatty acids and platelet-activating factor. J. Lipid Res. 44 :
2221 – 2233 .
83 . Aveldano , M. I. , and N. G. Bazan . 1974 . Displacement into incu-
bation medium by albumin of highly unsaturated retina free fatty
acids arising from membrane lipids. FEBS Lett. 40 : 53 – 56 .
84 . Aveldano , M. I. , and N. G. Bazan . 1975 . Differential lipid deacyla-
tion during brain ischemia in a homeotherm and a poikilotherm.
Content and comphotoreceptor outer segment ition of free fatty
acids and triacylglycerols. Brain Res. 100 : 99 – 110 .
85 . Horrocks , L. A. , and A. A. Farooqui . 1994 . NMDA receptor-stim-
ulated release of arachidonic acid: mechanisms for the Bazan ef-
fect. In Cell Signal Transduction, Second Messengers, and Protein
Phosphorylation in Health and Disease. A.M. Municio and M.T.
Miras-Portugal, editors. Plenum Press, New York, NY. 113–128.
86 . Sun , G. Y. , J. Xu , M. D. Jensen , and A. Simonyi . 2004 . Phospholipase
A2 in the central nervous system: implications for neurodegenera-
tive diseases. J. Lipid Res. 45 : 205 – 213 .
87 . Reddy , T. S. , and N. G. Bazan . 1984 . Activation of polyunsaturated
fatty acids by rat tissues in vitro. Lipids . 19 : 987 – 989 .
88 . Reddy , T. S. , and N. G. Bazan . 1984 . Synthesis of arachidonoyl
coenzyme A and docosahexaenoyl coenzyme A in retina. Curr. Eye
Res. 3 : 1225 – 1232 .
89 . Reddy , T. S. , and N. G. Bazan . 1985 . Synthesis of arachidonoyl
coenzyme A and docosahexaenoyl coenzyme A in synaptic plasma
membranes of cerebrum and microsomes of cerebrum, cere-
bellum, and brain stem of rat brain. J. Neurosci. Res. 13 : 381 – 390 .
90 . Reddy , T. S. , and N. G. Bazan . 1985 . Synthesis of docosahexaenoyl-,
arachidonoyl- and palmitoyl-coenzyme A in ocular tissues. Exp. Eye
Res. 41 : 87 – 95 .
91 . Calandria , J. M. , V. L. Marcheselli , P. K. Mukherjee , J. Uddin , J.
W. Winkler , N. A. Petasis , and N. G. Bazan . 2009 . Selective survival
rescue in 15-lipoxygenase-1 defi cient retinal pigment epithelial
cells by the novel docosahexaenoic acid-derived mediator, neuro-
protectin D1. J. Biol. Chem. 284 : 17877 – 17882 .
92 . Kühn , H. , B. J. Thiele , A. Ostareck-Lederer , H. Stender , H. Suzuki ,
T. Yoshimoto , and S. Yamamoto . 1993 . Bacterial expression, puri-
fi cation and partial characterization of recombinant rabbit reticu-
locyte 15-lipoxygenase. Biochim. Biophys. Acta . 1168 : 73 – 78 .
93 . Brash , A. R. , W. E. Boeglin , and M. S. Chang . 1997 . Discovery of a
second 15S-lipoxygenase in humans. Proc. Natl. Acad. Sci. USA . 94 :
6148 – 6152 .
94 . Bryckaert , M. , X. Guillonneau , C. Hecquet , Y. Courtois , and F.
Mascarelli . 1999 . Both FGF1 and bcl-x synthesis are necessary for
the reduction of apoptosis in retinal pigmented epithelial cells by
FGF2: role of the extracellular signal-regulated kinase 2. Oncogene .
18 : 7584 – 7593 .
95 . Bilak , M. M. , D. A. Shifrin , A. M. Corse , S. R. Bilak , and R. W.
Kuncl . 1999 . Neuroprotective utility and neurotrophic action of
neurturin in photoreceptor outer segment tnatal motor neurons:
comparison with GDNF and persephin. Mol. Cell. Neurosci. 13 :
326 – 336 .
96 . Milbrandt , J. , F. J. de Sauvage , T. J. Fahrner , R. H. Baloh , M. L.
Leitner , M. G. Tansey , P. A. Lampe , R. O. Heuckeroth , P. T.
Kotzbauer , K. S. Simburger , et al . 1998 . Persephin, a novel neu-
rotrophic factor related to GDNF and neurturin. Neuron . 20 :
245 – 253 .
97 . Kannan , R. , N. Zhang , P. G. Sreekumar , C. K. Spee , A.
Rodriguez , E. Barron , and D. R. Hinton . 2006 . Stimulation of
apical and basolateral VEGF-A and VEGF-C secretion by oxida-
tive stress in polarized retinal pigment epithelial cells. Mol. Vis.
12 : 1649 – 1659 .
98 . Bhutto , I. A. , D. S. McLeod , T. Hasegawa , S. Y. Kim , C. Merges , P.
Tong , and G. A. Lutty . 2006 . Pigment epithelium-derived factor
(PEDF) and vascular endothelial growth factor (VEGF) in aged
human choroid and eyes with age-related macular degeneration.
Exp. Eye Res. 82 : 99 – 110 .
99 . Frank , R. N. , R. H. Amin , D. Eliott , J. E. Puklin , and G. W. Abrams .
1996 . Basic fi broblast growth factor and vascular endothelial
growth factor are present in epiretinal and choroidal neovascular
membranes. Am. J. Ophthalmol. 122 : 393 – 403 .
100 . Frank , R. N. 1997 . Growth factors in age-related macular degen-
eration: pathogenic and therapeutic implications. Ophthalmic Res.
29 : 341 – 353 .
101 . Frank , R. N. , R. H. Amin , and J. E. Puklin . 1999 . Antioxidant enzymes
in the macular retinal pigment epithelium of eyes with neovascular
age-related macular degeneration. Am. J. Ophthalmol. 127 : 694 – 709 .
102 . Mattson , M. P. , and N. G. Bazan . 2006 . Apoptosis and necrosis. In
Basic Neurochemistry: Molecular, Cellular and Medical Aspects.
7th ed. G.J. Siegel, R.W. Albers, S.T. Brady, and D.L. Price, edi-
tors. Elsevier Academic Press, Burlington, MA. 603–615.
103 . Hinton , D. R. , S. He , and P. F. Lopez . 1998 . Apoptosis in surgically
excised choroidal neovascular membranes in age-related macular
degeneration. Arch. Ophthalmol. 116 : 203 – 209 .
104 . Sparrow , J. R. , and B. Cai . 2001 . Blue light-induced apoptosis of
A2E containing RPE: involvement of caspase-3 and protection by
Bcl-2. Invest. Ophthalmol. Vis. Sci. 42 : 1356 – 1362 .
105 . Chen , L. , W. Wu , T. Dentchev , Y. Zeng , J. Wang , I. Tsui , J. W.
Tobias , J. Bennett , D. Baldwin , and J. L. Dunaief . 2004 . Light dam-
age induced changes in mouse retinal gene expression. Exp. Eye
Res. 79 : 239 – 247 .
106 . Stuart , J. K. , D. G. Myszka , L. Joss , R. S. Mitchell , S. M. McDonald , Z.
Xie , S. Takayama , J. C. Reed , and K. R. Ely . 1998 . Characterization
of interactions between the anti-apoptotic protein BAG-1 and
Hsc70 molecular chaperones. J. Biol. Chem. 273 : 22506 – 22514 .
107 . Takayama , S. , D. N. Bimston , S. Matsuzawa , B. C. Freeman , C.
Aime-Sempe , Z. Xie , R. I. Morimoto , and J. C. Reed . 1997 . BAG-1
modulates the chaperone activity of Hsp70/Hsc70. EMBO J. 16 :
4887 – 4896 .
108 . Takayama , S. , Z. Xie , and J. C. Reed . 1999 . An evolutionarily con-
served family of Hsp70/Hsc70 molecular chaperone regulators. J.
Biol. Chem. 274 : 781 – 786 .
109 . Belvisi , M. G. , M. A. Saunders , E. B. Haddad , S. J. Hirst , M. H.
Yacoub , P. J. Barnes , and J. A. Mitchell . 1997 . Induction of cyclo-
oxygenase-2 by cytokines in human cultured airway smooth mus-
cle cells: novel infl ammatory role of this cell type. Br. J. Pharmacol.
120 : 910 – 916 .
110 . Ershov , A. V. , and N. G. Bazan . 1999 . Induction of cyclooxyge-
nase-2 gene expression in retinal pigment epithelium cells by pho-
toreceptor rod outer segment phagocytosis and growth factors. J.
Neurosci. Res. 58 : 254 – 261 .
111 . Sheets , K. G. , Y. Zhou , M. E. Ertel , E. J. Knott , C. E. Regan , Jr .,
J. R. Elison , W. C. Gordon , P. Gjorstrup , and N. G. Bazan . 2010 .
Neuroprotectin D1 attenuates laser-induced choroidal neovascu-
larization in mouse. Mol. Vis. 16 : 320 – 329 .
by guest, on December 25, 2015