ARVO 2015 Annual Meeting Abstracts
283 AMD basic and retinal cell biology
Monday, May 04, 2015 3:45 PM–5:30 PM
Exhibit Hall Poster Session
Program #/Board # Range: 2351–2388/C0001–C0038
Organizing Section: Retina
Contributing Section(s): Biochemistry/Molecular Biology,
Physiology/Pharmacology, Retinal Cell Biology, Visual
Psychophysics/Physiological Optics
Program Number: 2351 Poster Board Number: C0001
Presentation Time: 3:45 PM–5:30 PM
Immunohistochemical Assessment of Proteins Associated with
Complement Activation and Inflammation in Human Maculas
with Homozygous Risk of AMD at Chromosome 1
Tiarnan D. Keenan2, 1, Marc Toso2, Chris Pappas2, Lisa Nichols2, Paul
N. Bishop1, Gregory S. Hageman2. 1Centre for Hearing and Vision
Research, Institute of Human Development, Faculty of Medicine
and Human Sciences, University of Manchester and Manchester
Royal Eye Hospital, Central Manchester University Hospitals NHS
Foundation Trust, Manchester Academic Health Science Centre,
Manchester, United Kingdom; 2Center for Translational Medicine,
Moran Eye Center, University of Utah, Salt Lake City, UT.
Purpose: To determine the effects of chromosome 1 genotype
and cigarette smoking on levels of complement activation and
inflammation in the human macula.
Methods: Eye tissue was obtained from consenting human
donors. Donors were stratified into two groups based on diplotype
at the AMD-associated CFH/CFHR locus: (i) homozygous risk
diplotype (tagged by CC at rs1061170) (n=9, ages 56-78 years)
and (ii) homozygous ‘protective’ diplotype (tagged by AA at
rs1410996) (n=6, 61-78 years). Importantly, all donors were
homozygous non-risk at the ARMS2/HTRA1 locus on chromosome
10. Immunohistochemistry was performed on macular tissue from
all donors, using 26 antibodies against markers of inflammation,
complement regulation and activation, followed by confocal
microscopy and immunofluorescence quantification (all masked to
donor status).
Results: Donors homozygous risk at the CFH/CFHR locus exhibited
significantly higher levels of the complement membrane attack
complex (MAC) in macular retinal pigment epithelium (RPE;
p=0.003), Bruch’s membrane (BM; p=0.002), choriocapillaris (CC;
p=0.001), CC intercapillary septa (IS; p=0.0001) and choroidal
stroma (CS; p=0.001) compared to homozygous protected donors.
Smoking was also associated with increased levels of MAC (e.g.
p=0.006 in CC IS) and CRP (e.g. p=0.0002 in CC), but only in the
risk group. Smoking, but not diplotype, was associated with higher
levels of the oxidative stress marker 4-hydroxynonenal in macular
retina (p=0.02) and RPE (p=0.05).
Conclusions: Genetic risk at the CFH/CFHR locus (without risk
at the ARMS2/HTRA1 locus) is associated with higher levels of
complement- and inflammation-associated proteins at the RPEchoroid interface. Levels of these proteins are higher yet in CFH/
CFHR risk donors with histories of cigarette smoking, which is also
associated with increased oxidative stress in both risk and non-risk
donors. Examination of human macular tissue from donors with
‘pure’ diplotypes allows assessment of AMD-associated pathways
that are driven solely by the CFH/CFHR gene locus. These
findings have important implications related to the identification of
chromosome 1-directed pathways and therapeutic targets.
Commercial Relationships: Tiarnan D. Keenan, None; Marc
Toso, None; Chris Pappas, None; Lisa Nichols, None; Paul N.
Bishop, None; Gregory S. Hageman, AGTC (C), Allergan Inc. (F),
Gerson Lehrman (C), Novartis Pharmaceuticals (C), Optherion Inc.
(I), Sequenom Inc. (C), Voyant Biotherapeutics (I)
Support: Fulbright / Fight For Sight UK-US Scholarship 2013/14
Program Number: 2352 Poster Board Number: C0002
Presentation Time: 3:45 PM–5:30 PM
Fluorescence and mass spectrometric imaging of monkey
(Cynomolgus) retinal pigment epithelium confirms the
mismatching of lipofuscin and A2E distributions observed in
human tissue
Patrick Pallitto1, Zsolt Ablonczy1, John E. Donello2, Yiannis
Koutalos1, Julia E. Herrmann2, Rosalie K. Crouch1. 1Department of
Ophthalmology, Medical University of South Carolina, Charleston,
SC; 2Department of Biological Sciences, Allergan Inc., Irvine, CA.
Purpose: Lipofuscin and its bisretinoid components have been
implicated in the development of age-related macular degeneration
(AMD). In cadaveric human eyes we have found a complete
mismatch between lipofuscin fluorescence and the bisretinoid
distributions. The goal of this project was to examine this relationship
in a non-human primate model with a retinal organization similar to
that of humans.
Methods: Ophthalmologically naive young (<10 yrs, N=4) and old
(>10 yrs, N=4) cynomolgus monkey eyes were enucleated, dissected
to yield RPE/choroid tissue layers, which were then flat-mounted
on indium-tin-oxide-coated conductive slides. To compare the
distributions of lipofuscin and bisretinoids, fluorescence and mass
spectrometric (MALDI) imaging were carried out sequentially on the
same tissues. First, fluorescence measurements were carried out in
a Maestro2 imaging system (Caliper Life Sciences), the slides were
then coated with 40mL of sinapinic acid in 70% ethanol for MALDI
imaging. Mass spectra over the entire tissue were collected using an
Autoflex II spectrometer (Bruker) in positive linear acquisition mode
at 350 nm spatial resolution in the range of m/z 510-1500 with gating
suppression set to m/z 490.
Results: Lipofuscin fluorescence intensity in the cynomolgus RPE
was highest in the posterior pole, tapering off toward the periphery.
In the posterior pole it exhibited an emission maximum of ~600nm
(λex=488nm). On the other hand, bisretinoid levels (including A2E)
were highest in the periphery, showing a lack of correlation between
lipofuscin fluorescence and bisretinoid distributions.
Conclusions: Multimodal imaging in the RPE of the cynomolgus
primate model reproduced the lipofuscin and bisretinoid distributions
previously obtained for human cadaveric eyes, with A2E distribution
highest in the periphery and completely lacking correlation with
fluorescence. We conclude that the mismatch between lipofuscin and
bisretinoids, which has not been observed in mice, is due to specific
anatomical features of the primate eye, such as the macula.
Commercial Relationships: Patrick Pallitto, Allergan Inc. (F);
Zsolt Ablonczy, Allergan Inc. (F); John E. Donello, Allergan Inc.
(E); Yiannis Koutalos, Allergan Inc. (F); Julia E. Herrmann,
Allergan Inc. (E); Rosalie K. Crouch, Allergan Inc. (F)
Support: Supported in part by a grant from Allergan, Inc.,
Irvine, CA; and an unrestricted grant to MUSC, Department of
Ophthalmology, from Research to Prevent Blindness, New York, NY.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Program Number: 2353 Poster Board Number: C0003
Presentation Time: 3:45 PM–5:30 PM
Hydroxyapatite in Sub-RPE Deposits of Ageing Japanese and
Rhesus Macaques
Richard Thompson1, Hilary Baruch1, Jacob Tatum1, 2, Trevor J.
McGill3, Martha Neuringer3, Imre Lengyel4. 1Dept of Biochemistry
and Molecular Biolo, University of Maryland School of Medicine,
Baltimore, MD; 2School of Medicine, University of Virginia,
Charlottesville, VA; 3National Primate Center, Portland, OR;
4
Institute of Ophthalmology, University College London, London,
United Kingdom.
Purpose: Sub-RPE deposits such as drusen are widely accepted as
precursors to age-related macular degeneration (AMD), although
the mechanism(s) of their development and exact relationship to
AMD remain to be elucidated. We recently discovered that sub-RPE
deposits in aging human retina contain microscopic (0.3 – 20 mm)
spherules of hydroxyapatite (HAP), the mineral form of calcium
phosphate found in bone and teeth; and that these spherules are
coated with proteins enriched in drusen such as complement factor H,
amyloid beta, and vitronectin. Aging Rhesus and Japanese macaques
also develop drusen similar to those in humans. Here, we examined
whether drusen in the aging macaques also contain HAP deposits,
and compared these to the HAP spherules found in humans.
Methods: We used multiple HAP-specific fluorescent dyes to stain
post-mortem fixed retinal slices and flatmounts from male and female
Japanese and Rhesus macaques of ages ranging from 16 to 38 years
in which drusen had been documented by fundus photography in
vivo. They were imaged by confocal fluorescence and brightfield
microscopy, which were compared to color fundus photographs
collected in vivo.
Results: Drusen in aged macaque retinas were confirmed by
brightfield and autofluorescence microscopy. Multiple drusen from
both sexes and species stained positive for HAP, but in comparison
to humans the deposits were generally smaller and more amorphous
in form, without distinguishable shell-like appearance. Older retinas
generally had more drusen, all of which exhibited HAP-positive
staining.
Conclusions: We conclude that drusen in the two macaque species
and humans contain HAP deposits that share important similarities as
well as some differences. The smaller size and differing morphology
of the macaque HAP deposits compared with the hollow spherules
in humans suggest clear differences in how the these deposits are
nucleated and their ability to grow.
Commercial Relationships: Richard Thompson, None; Hilary
Baruch, None; Jacob Tatum, None; Trevor J. McGill, None;
Martha Neuringer, None; Imre Lengyel, None
Support: Bright Focus Foundation (RBT, IL, HB), Foundation
Fighting Blindness and NIH P51 OD011092 (MN), NIH T35 DK
905737-02 (SPORT Program: JT), and RPB (TM)
Program Number: 2354 Poster Board Number: C0004
Presentation Time: 3:45 PM–5:30 PM
BEHAVIOURAL VALIDATION OF A TRANSGENIC MOUSE
MODELING SPECIFIC IMPAIRMENT OF DETAILED
VISION
Rachel Bryant1, Emma Stephens2, Yves Sauve2. 1Neuroscience,
University of Alberta, Edmonton, AB, Canada; 2Physiology,
University Of Alberta, Edmonton, AB, Canada.
Purpose: To test whether retinal degeneration in a mouse model
of monogenic maculopathy (Stargardt-like Dystrophy; STGD3)
initially leads to the specific impairment of detailed vision (a stage
at which movement detection remains spared) as occurs in its human
counterpart.
Methods: We relied on the TG1-2 ELOVL4 mouse model of
STGD3; WT littermates served as controls. Visual acuity thresholds
were determined in TG and WT mice (n=8 at 5 months of age, per
group) by relying on two distinct behavioural tests: one based on
movement detection (optokinetic reflex, OKR), the other based on
detailed vision (cortically based forced decision, Y water maze).
Results: Firstly, we confirmed previous findings that WT mice
have higher visual acuity thresholds with the Y-water maze test
(0.3457±0.0239) as opposed to the OKR test (0.3182±0.0104).
Secondly, we found that TG mice had reduced visual acuity
thresholds in the detailed vision test (0.2694±0.0149) when compared
to WT mice (0.3457±0.0239). However, visual acuity thresholds
of TG mice (0.3348±0.0078) were similar to those of WT mice
(0.3182±0.0104), when assessed with the OKR test.
Conclusions: While there is no anatomical way to directly assess
whether retinal dystrophies such as STGD3 affect movement-based
visual reflexes versus conscious detailed vision, our results imply
that retinal degeneration in mouse models, such as in STGD3, can
specifically impact on one aspect of vision. Furthermore, our results
indicate that despite the fact that mice do not have a macula, they
have distinct visual pathways established at the retina level, which
can be differentially affected by specific mutations.
Commercial Relationships: Rachel Bryant, None; Emma
Stephens, None; Yves Sauve, None
Support: Brain Canada
Program Number: 2355 Poster Board Number: C0005
Presentation Time: 3:45 PM–5:30 PM
High-effect induction of human iPS cells into retinal pigment
epithelial cells with small molecules
Qingjian Ou1, 2, Caixia Jin1, 2, Haibin Tian1, 2, Furong Gao1, 2, Weiye
Li3, Lixia Lu1, 2, Guo-Tong Xu1, 2. 1Laboratory of Clinical Visual
Sciences and Stem Cell Research Center, Tongji University School
of Medicine, Shanghai, China; 2Department of Ophthalmology of
Shanghai Tenth People’s Hospital, Tongji University School of
Medicine, Shanghai, China; 3Department of Ophthalmology, Drexel
University Collegeof Medicine, Philadelphia, PA.
Purpose: This study is aimed to establish a more effective method to
induce the differentiation of iPS cells into retinal pigment epithelial
(RPE) cells with small molecules in serum free medium, in order to
facilitate the clinical application of iPS cells derived donor cells.
Methods: Human iPS (hiPS) cells were cultured on the matrigelcoated dish in serum free conditioned medium. The iPS cells were
first treated with two small molecules, SB431542 and DMH1, for
7days (dual-Smad) and then with other groups of small molecules at
the appropriate time. The medium was changed every two days and
the cells were separated with accutase before 18 days once the cells
were confluence. RPE-like cells derived from hiPS cells (hiPS-RPE)
were collected after 24 days induction. The hiPS-RPE cells were
confirmed by Q-PCR, immunohistochemistry and western blot for the
expression of RPE related genes and proteins.
Results: Combination of different small molecules can induce hiPS
cells differentiate into RPE cells after 24 days, and more than 90
percent induced cells differentiate into RPE cells. The hiPS-RPE cells
have the typical polygonal morphology and pigments. Meanwhile,
they express the RPE cells specific markers.
Conclusions: We can induce hiPS Cells into RPE-like cells under
chemically defined conditions with the combination of small
molecules in about 3weeks.
Commercial Relationships: Qingjian Ou, None; Caixia Jin, None;
Haibin Tian, None; Furong Gao, None; Weiye Li, None; Lixia Lu,
None; Guo-Tong Xu, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Support: the National Key Basic Research Program of China
(2013CB967501)
Program Number: 2356 Poster Board Number: C0006
Presentation Time: 3:45 PM–5:30 PM
Evaluation of the morphology of the mouse retinal pigment
epithelium/bruch’s membrane/choroid (RPE/BrM/Ch) using
quick-freeze, deep-etch transmission electron microscopy
(QFDE-TEM) and conventional (cTEM)
Ebraheim Ismail1, Jeffrey Ruberti1, Goldis Malek2, 3. 1Bioengineering,
Northeastern University, Sharon, MA; 2Ophthalmology, Duke
University School of Medicine, Durham, NC; 3Pathology, Duke
University School of Medicine, Durham, NC.
Purpose: The RPE/BrM/Ch is particularly vulnerable in retinal
diseases involving the posterior pole such as age-related macular
degeneration (AMD), which is the leading cause of vision loss in the
elderly in the Western World. Mice are frequently used as a platform
to model AMD pathologies because they are easily geneticallymodified. QFDE-TEM, a label-free, high-resolution, morphologypreserving imaging method permits the visualization of greater
morphological detail than can cTEM. We have previously shown
that QFDE-TEM is particularly good at resolving lipid particles in
the BrM of human eyes from aged donors. In the present study we
ask if QFDE can provide us with important additional information in
evaluating the pathology of mouse eyes.
Methods: Eyes from aged C57BL/6J mice (control or fed a high-fat
diet-HFD) were enucleated and the anterior segments and retinas
were removed. In select eyes the RPE layer was also removed. Eyes
were fixed in 2% glutaraldehyde. For cTEM: eyes were post-fixed in
1% osmium tetroxide and embedded in Spurrs resin (n=6). Ultrathin
sections were stained with uranyl acetate/lead citrate. For QFDETEM: tissues (n=17) were “touch” frozen on copper blocks (-196
o
C), transferred to a modified Cressington CFE-40 freeze fracture
system, fractured, etched and rotary shadowed with Pt/C. Replicas
were isolated in 25% bleach solution, washed in DIH2O and imaged on
a JEOL JEM1010 TEM.
Results: In the RPE, we catalogued characteristic features in the
apical (phagosomes, dense melanosomes), central (cell body,
mitochondria) and basal (basal infolding membrane) regions. In
BrM, we identified the basement membranes, inner/outer collagenous
and elastin layers. Variations in the density of the melanosomes
and associated collagen bundles indicated depth in the choroid. In
the HFD-fed mice, there were observable differences in baseline
morphology due to the presence of lipid particles.
Conclusions: QFDE-TEM enabled the generation of striking,
large area mosaic images of the mouse RPE/BrM/CCC complex,
complementary and additive to that seen with conventional TEM.
These images provide an excellent reference for our ongoing
investigations of mouse eyes which demonstrate pathological changes
associated with AMD.
Large area mosaic image of en face view of the choroid.
Commercial Relationships: Ebraheim Ismail, None; Jeffrey
Ruberti, None; Goldis Malek, None
Support: NEI EY02868 (GM), NIH P30 EY005722 (Duke Eye
Center), and RPB core grant (Duke Eye Center)
Program Number: 2357 Poster Board Number: C0007
Presentation Time: 3:45 PM–5:30 PM
Survival and efficacy of xenogeneic cell transplants in the RCS
rat in the absence of immunosuppression
Trevor J. McGill3, 1, Shaomei Wang2, Bin Lu2, Stephen Huhn4,
Raymond D. Lund5, Alexandra Capela4. 1Neuroscience, Oregon
National Primate Research Center, Beaverton, OR; 2Biomedical
Sciences, Cedar Sinai, Los Angeles, CA; 3Casey Eye Institute,
Oregon Health and Science University, Portland, OR; 4StemCells,
Inc., Newark, CA; 5Moran Eye Center, Salt Lake City, UT.
Purpose: Age-related Macular Degeneration (AMD) is the primary
cause of visual impairment in industrialized countries for which no
cure is available. Transplantation of cells into the subretinal space
(SRS) is a promising experimental neuroprotective treatment for
dry AMD which is currently under clinical investigation. Although
the normal SRS is considered to be relatively immunoprivileged, it
is unclear how this privilege is affected by degeneration; therefore,
ongoing clinical studies with allogeneic cells currently include
immunosuppression. Preclinical studies in the RCS rat have
demonstrated that, with the aid of continuous immunosuppression,
donor xenogeneic cells survive transplantation, rescue photoreceptors
and limit the associated loss of vision. The purpose of this study
was to examine the survival and efficacy of human neural stem cells
(HuCNS-SC) following transplantation under non continuous or even
absent immunosuppression.
Methods: HuCNS-SC (1x105) were transplanted into the SRS of
one eye of 38 RCS rats on post-natal day (P) 21. Thirteen (13)
contralateral eyes served as medium-injected controls, and twentyfive (25) eyes were maintained as unoperated controls. The 38
animals were divided into four groups: no immunosuppression
(Group A; n=8); two weeks of dexamethasone (DEX) (Group B;
n=12); 2 weeks of DEX and 30 days of cyclosporine (Group C;
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
n=11); and 2 weeks of DEX and continuous cyclosporine (Group
D; n=7), the immunosuppression used in our previous preclinical
studies. Visual acuity was measured through optokinetic tracking
(OKR) at 30 day intervals. Retinal histology was performed
following sacrifice at either P90 or P150.
Results: OKRs were maintained in all cell treated eyes at all time
points. Control eyes degenerated consistent with previously reported
values. Surprisingly, transplanted cells survived in all groups despite
the lack of immunosuppression in some animals. One animal in
Group C exhibited graft rejection evident by a mononuclear response.
Photoreceptor rescue was observed in all groups as evidenced by an
outer nuclear layer thickness of 6-10 nuclei at both P90 and P150,
consistent with our previously published data.
Conclusions: Our results demonstrate that xenogeneic HuCNS-SC
can survive and rescue function in the RCS rat in the absence of
immunosuppression.
Commercial Relationships: Trevor J. McGill, None; Shaomei
Wang, None; Bin Lu, None; Stephen Huhn, StemCells Inc (E);
Raymond D. Lund, None; Alexandra Capela, StemCells Inc (E)
Support: NIH P30EY010572, RPB Unrestricted Funds
Program Number: 2358 Poster Board Number: C0008
Presentation Time: 3:45 PM–5:30 PM
Inhibition of subretinal neovascularization in Vldlr knockout
mice by anti-VEGF agents
Haoyu Chen, Shaofen Huang, Zhen Li, Zhihao Lu. Joint Shantou
International Eye Center, Shantou, China.
Purpose: Vldlr knockout mice is characterized by subretinal
neovascularization which mimics wet age-related macular
degeneration in human. The purpose of this study is to investigate the
effects of two anti-VEGF agents, Conbercept and Ranibizumab on
subretinal neovascularization in Vldlr knockout mice.
Methods: Six vldlr knockout mice were treated with intravitreal
administration of 10ug/1ul conbercept or 10ug/1ul Ranibizumab
in the right eyes. The left eyes served as control. Fluorescein
angiography was performed before, 3 days and 7 days after
administration of drugs. The area of fluorescein leakage was
quantified using Image J software and compared.
Results: The area of subretinal neovascularization reduced to 73%
and 36% in the eyes treated with Conbercept at 3 days and 7 days.
The area of subretinal neovascularization in the eyes treated with
Ranibizumab reduced to 42% and 33% at 3 and 7 days respectively.
Conclusions: Both Conbercept and ranibizumab inhibits subretinal
neovascularization in Vldlr knockout mice.
Commercial Relationships: Haoyu Chen, None; Shaofen Huang,
None; Zhen Li, None; Zhihao Lu, None
Support: National Scientific Foundation of China 81170853
Program Number: 2359 Poster Board Number: C0009
Presentation Time: 3:45 PM–5:30 PM
Systemic Administration of Anti-Angiopoietin-2 (Ang2) Antibody Inhibits Matrigel Induced Choroidal
Neovascularization (CNV) in Rats
Yang Liu, zida li, Dan reef, Jingtai Cao, Carl Romano, Stanley
J. Wiegand. Ophthalmology, Regeneron Pharmaceuticals Inc,
Tarrytown, NY.
Purpose: To assess the inhibitory effects of REGN910-mediated
pharmacological inhibition of Ang-2 on CNV using a rat model of
choroidal neovascularization (CNV).
Methods: CNV was induced in Sprague Dawley (SD) rats by a
subretinal injection of Matrigel on Day 0. One group of animals
(animals: N=4-6; eyes: N=8-12) was used to establish a baseline
and animals were perfused with a dye [1,1’-Dioctadecyl-3,3,3’,3’-
Tetramethylindocarbocyanine Perchlorate (DiI)] to stain vessels
10 days after CNV induction. The other animals were divided into
two groups (animals: N=5-6, and eyes: N=10-12, in each group)
and treated with masked solutions by subcutaneous injection of 25
mg/kg REGN910, or 6.5 mg/kg hFc at equimolar amount relative
to REGN910, respectively, on days 10, 13, and 16 followed by
perfusion with DiI to stain vessels on Day 20. Subretinal lesion and
CNV vessel volumes were quantified from 50 mm sections throughout
the entire lesion. The results from two identical experiments were
combined for lesion volume, vessel volume, and vessel density
comparison among three groups. A two-tailed Student t-test was used
to compare the differences of lesion volume, vessel volume, and
vessel density between groups treated with REGN910 versus hFc.
Results: Systemic administration of REGN910 (25 mg/kg) to SD rats
produced a suppression of the total lesion volume and vessel volume
compared to hFc-treated control. In the hFc treated group, subretinal
lesion volume, neovessel volume, and neovessel density increased
by 28.2%, 62.5%, and 28.6%, respectively, by Day 20, relative to
the baseline at Day 10. The REGN910 treated group showed 26.5%
reduction of total lesion volume, and 26.9% reduction of vessel
volume, compared to hFc treated group, though the vessel density
was unchanged compared to the hFc treated group. Compared to
hFc-treated controls, the REGN910 treated group showed statistically
significant reduction of total lesion volume (p=0.0034), and a trend
(26.9%) towards neovessel volume reduction, but this trend did not
achieve statistical significance (p=0.1658, which may have been due
to variation within the group).
Conclusions: The results indicate that systemic (subcutaneous)
treatment with REGN910 can significantly inhibit Matrigel induced
CNV lesion in rats.
Commercial Relationships: Yang Liu, Regeneron Pharmaceuticals
INC (E); zida li, Regeneron Pharmaceuticals INC (E); Dan reef,
Regeneron Pharmaceuticals INC (E); Jingtai Cao, Regeneron
Pharmaceuticals INC (E); Carl Romano, Regeneron Pharmaceuticals
INC (E); Stanley J. Wiegand, Regeneron Pharmaceuticals INC (E)
Program Number: 2360 Poster Board Number: C0010
Presentation Time: 3:45 PM–5:30 PM
The Effect of Collagen Vitrigel Nanostructure on Stem Cell
Derived Retinal Pigment Epithelial Maturation
Xiaokun Wang1, Julien Maruotti1, Jose Roman2, Haiquan Mao2,
Donald J. Zack1, Jennifer Elisseeff1, 2. 1Wilmer Eye Institute, Johns
Hopkins University, Baltimore, MD; 2Biomedical Engineering, Johns
Hopkins University, Baltimore, MD.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of blindness in the US in individuals over the age of 65 years.
One uprising approach to obtain human RPE cells is to generate them
from human pluripotent stem cells. These stem cell-derived RPEs
could serve as cell resource for RPE transplantation. An optimal
cell scaffold for tissue engineered RPE monolayer should simulate
their natural microenvironment, support RPE maturation, along
with favorable surgical properties, and biocompatibility. With the
aim to develop an optimized tissue-engineered RPE monolayer, we
investigated the influence of chemistry and morphology of collagen
based materials to stem cell-derived RPE maturation in the current
research.
Methods: Retinal pigment epithelial-like cells were differentiated
from embryonic stem cells. In this study frozen differentiated cells
were thawed and passed for 3 times before seeding to materials.
For different collagen vitrigel (CV) groups, vitrification time varied
from 3 days to 3 weeks at 40 in order to obtain different nanofibril
structure. Beta-cyclodextrin collagen vitrigel (Beta CV) was prepared
by mixing 5% β-CD solution with collagen solution then go through
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
vitrification. PCL electrospun fibers were also prepared for RPE
maturation evaluation. Cell morphology, gene expression were tested
in all groups.
Results: Longer vitrification time resulted in thicker collagen fibril
(~100nm) and denser fibril density. CV3day has average fibril
diameter of ~60-70nm, while CV3week has >100 nm fibril diameter.
Beta-CV has no distinctive fibril structure and PCL electrospun fibers
has diameter around 1 mm
After 4-6 weeks culture, ES-RPE became pigmented and polarized,
microvilli can be observed under SEM and TEM among all groups.
qPCR results showed significant enhancement of ES-RPE maturation
on CV-3D group, which indicates the nanofibril scale of CV3day
group is essential for RPE maturation
Conclusions: In this study, we evaluated the ES-derived RPE
maturation on different substrates, including synthetic polymer and
collagen-based materials. RPE proliferate well on different substrates
and became mostly pigmented and polarized between 4~6 weeks.
Quantitative study by qPCR showed RPE characteristic marker
expression significantly increased in CV3day group. This study
provided an insight of how nanofibril in collagen based material
influence RPE maturation.
Commercial Relationships: Xiaokun Wang, None; Julien
Maruotti, None; Jose Roman, None; Haiquan Mao, None; Donald
J. Zack, None; Jennifer Elisseeff, None
Support: Research to Prevent Blindness
Program Number: 2361 Poster Board Number: C0011
Presentation Time: 3:45 PM–5:30 PM
Nicotine Enhances Oxidative Stress Mediated Retinal
Degeneration In The Aging Eye
Heping Xu, Rosana Penalva, Mei Chen. Centre for Vision and
Vascular Science, Queen’s University Belfast, Belfast, United
Kingdom.
Purpose: Cigarette smoking is a major environmental risk factor to
age-related macular degeneration. Nicotine is an important chemical
component of cigarette. The aim of this study was to investigate
the effect of nicotine in the development of age-related retinal
degeneration.
Methods: Adults C57BL/6J mice (6 months old) were divided into
four treatment groups: Nicotine (12mg/kg/day); blue light (460 nm,
8h/day); Nicotine + blue light; control (2% saccharine). Retinal
lesions were examined at 6 and 12 months after the treatment
by fundus imaging, electroretinography (ERG), and Spectral
Domain Optical Coherence Tomography (SD-OCT). Eyes were
collected at the end of the study for histological and immunological
investigations.
Results: Compared to age-matched control animals, Nicotine alone
did no induce retinal degeneration. Blue light treatment resulted
in patches of retinal damage in 40% of animals, whereas the
combination of blue light and nicotine resulted in retinal damage in
100% of mice. ERG analysis revealed significant reduction in a- and
b-waves in light-treated and more in nicotine + light treated mice.
Retinal lesions were characterized by patches of RPE death and
photoreceptor degeneration accompanied by increased microglial
activation and subretinal accumulation in these mice.
Conclusions: Although nicotine alone is not toxic to the retina, it
enhances blue light induced retinal damage during aging. Nicotine is
the main chemical component of the E-cigarette. Our results suggest
that smoking E-cigarette may not be as safe as we thought. Further
study on the effect of nicotine in oxidative stress mediated damage in
the aging eye is necessary.
Commercial Relationships: Heping Xu, None; Rosana Penalva,
None; Mei Chen, None
Support: The project is funded by Fight for Sight (1361/1362;
1425/1426)
Program Number: 2362 Poster Board Number: C0012
Presentation Time: 3:45 PM–5:30 PM
A SEMA3E MUTANT RESISTANT TO CLEAVAGE BY
FURINS (UNCL-SEMA3E) INHIBITS LASER INDUCED
CHOROIDAL NEOVASCULARIZATION
Yoreh Barak1, Shira Toledano2, Gilad Alon1, Boaz Kigel2, Ofra
Kessler2, Shira Hagbi-Levi3, Liran Tiosano3, Shlomit Schaal4, Gera
Neufeld2. 1Ophthalmology, Rambam Medical Center, Haifa, Israel;
2
Cancer Research and vascular Biology Center, The Bruce Rappaport
Faculty of Medicine,, Technion, Israel Institute of Technology,, Haifa,
Israel; 3Department of Ophthalmology, Hadassah-Hebrew University
Medical Centerl, Jerusalem, Israel; 4Department of Ophthalmology
and Visual Sciences,Department of Ophthalmology and Visual
Sciences, University of Louisville, Louisville, KY.
Purpose: Abnormal subretinal choroidal neovascularization (CNV)
is a major blinding consequence of the exudative form of age-related
macular degeneration (AMD). Anti-angiogenic agents may be useful
in preventing CNV formation. A point mutated form of semaphorin3E resistant to cleavage by furin like pro-protein convertases (UNCLSema3E) is known to display potent anti-angiogenic properties.
UNCL-Sema3E is unique by its action of counteracting activities
of angiogenic growth factors other than VEGF, such as affecting
receptors of neuropilin and plexin. The purpose of this study is to
determine if UNCL-Sema3E may be used in-vivo to inhibit CNV
formation.
Methods: Cultured vascular endothelial cells were stimulated with
VEGF (10 ng/ml) in the presence or absence of UNCL-Sema3E/Fc
(1.5 μg/ml). After 10 min. at room temperature the cells were lysed
and ERK1/2 phosphorylation determined. CNV was induced in the
eyes of Evans-Long rats by laser photocoagulation (n=128) followed
by an intravitreal injection of either UNCL-Sema3E (125 μg/5 μl),
avastin (125 μg5 μl), or vehicle (5 μl) as control. After a week flat
whole mounts of retinas where used to determine CNV frequency
and size. Results were assessed by the staining of blood vessels with
isolectin and calculating the area of stained blood vessels using the
Image-J morphometric software.
Results: UNCL-Sema3E inhibits efficiently both VEGF and bFGF
induced signal transduction in cultured vascular endothelial cells.
UNCL-Sema3E injected into the vitreous cavity reduced the area of
laser induced CNV (n=65) by 50% (64040 ± 7321 μm2 for controls
(n=61) vs 32720 ±- 2369 μm2, P<0.0001) displaying efficacy similar
to that of bevacizumab[SS3] (n=54).
Conclusions: UNCL-Sema3E inhibits laser induced CNV formation
in the rat model as efficiently as bevacizumab. This suggest that
UNCL-Sema3E may be considered as a possible therapeutic agent
for the treatment of exudative AMD that is resistant to current
anti-VEGF treatments because it acts on a different anti-angiogenic
pathway.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
founders showed obvious deletions by PCR using primers covering
the IGFBP domain. DNA sequencing showed that four mice had
in-frame deletions (6.1%), which deleted the IGFBP domain from
HTRA1. Inheritance of IGFBP-domain specific deletion was
confirmed by DNA sequencing of the F1 offspring.
Conclusions: We showed that IGFBP domain of HTRA1 can be
deleted efficiently using CRISPR/Cas9 technology, which provides
a powerful gene modification tool for research in Vision and
Ophthalmology.
Commercial Relationships: HUI XU, None; Sandeep Kumar,
None; Yingbin Fu, None
Support: NIH Grant 1R01EY022901 and EY022614. The Career
Development Award from Research to Prevent Blindness (RPB) and
an unrestricted grant to the Department of Ophthalmology at the
University of Utah from RPB.
Commercial Relationships: Yoreh Barak, None; Shira Toledano,
None; Gilad Alon, None; Boaz Kigel, None; Ofra Kessler, None;
Shira Hagbi-Levi, None; Liran Tiosano, None; Shlomit Schaal,
None; Gera Neufeld, None
Support: This work was suported by the United States Israel
Binational Science Foundation
Program Number: 2363 Poster Board Number: C0013
Presentation Time: 3:45 PM–5:30 PM
Efficient deletion of the IGFBP domain of multifunctional serine
protease HTRA1 by CRISPR/Cas9
HUI XU1, 2, Sandeep Kumar2, Yingbin Fu1, 2. 1Interdepartmental
Program in Neuroscience, University of Utah, Salt Lake City, UT;
2
Ophthalmology & Visual Sciences, University of Utah, Salt Lake
City, UT.
Purpose: Two synonymous single nucleotide polymorphisms
(SNPs) rs1049331 and rs2293870 in IGFBP domain of HTRA1
were reported to be associated with increased risk to neovascular
(Nv) type age-related macular degeneration (AMD). The proposed
mechanism is that the two SNPs convert common codons for Ala34
and Gly36 to less frequently used codons and affect the folding of the
IGFBP domain, which in turn reduced HTRA1’s ability to antagonize
insulin-like growth factor 1 (IGF-1)-stimulated signaling events
and cellular responses. To investigate the role of the IGFBP domain
of HTRA1 in AMD pathogenesis, we generated mice in which
the IGFBP domain of HTRA1 was successfully deleted using the
CRISPR/Cas9 technology.
Methods: We first designed four single-guide RNAs (sgRNAs)
targeting the IGFBP domain of HTRA1. Mouse embryonic fibroblasts
(MEFs) from C57Bl6 were electroporated with the plasmids encoding
sgRNAs and Cas9. The efficiency of each of four sgRNAs were
evaluated by SURVEYOR mutation detection kit (Transgenomic).
The most efficient three sgRNAs targeting the beginning, middle, and
the end of the IGFBP domain were selected for pronuclear injection
in C57Bl6 background with either 5 ng or 10 ng of sgRNA plasmids.
PCR and DNA sequencing of PCR products were used to determine
the modifications of the three sgRNA targets of sixty-five founder
mice and their offspring.
Results: Thirty-nine mice from 5 ng and twenty-six from 10 ng
injection (total sixty-five) were generated. DNA Sequencing showed
highly efficient genomic modification by CRISPR/Cas9, sixty out
of sixty-five (92%) founders have modifications in sgRNA targets
in IGFBP domain. Moreover, Thirteen out of thirty-nine (33%, 5
ng plasmids) and seven out of twenty-six (26%, 10 ng plasmids)
Program Number: 2364 Poster Board Number: C0014
Presentation Time: 3:45 PM–5:30 PM
A Highly Potent Inhibitor of PI3K and the Mammalian Target
of Rapamycin, GSK2126458, Inhibits Laser-Induced Choroidal
Neovascularization
Yu Sun1, Jie Ma1, Gianna C. Teague1, Francisco J. Lopez2, Peter
S. Adamson2, Edit Kurali3, Kameran Lashkari1. 1Ophthalmology,
Schepens Eye Research Institute, Harvard Medical School, Boston,
MA; 2Ophthalmology IPE US, RD Alternative Discovery &
Development, GSK, King of Prussia, PA; 3Quantitative Sciences, RD
Alternative Discovery & Development, GSK, King of Prussia, PA.
Purpose: Choroidal neovascularization (CNV) is the major cause
of severe visual loss in subjects with AMD. A significant number
of patients with wet AMD exhibit some degree of resistance to
anti-VEGF monotherapy. The PI3K/Akt/mTOR pathway plays an
important role in downstream cellular processes from the VEGF
receptor, including growth, survival and angiogenesis. In this
study, we investigated whether blockade of phosphatidylinositol-3kinase (PI3K) and mTOR pathway using a highly potent blocker,
GSK2126458, was able to effectively inhibit laser-induced CNV
lesions.
Methods: Laser-induced CNV was created in C57BL/6 mice using
a 532 nm laser under direct observation with the Micron III fundus
camera. Mice were treated with daily oral gavage of GSK2126458
or vehicle for 14 days after laser injury. Alternatively, mice received
intravitreal injections of GSK2126458, Eylea™ (aflibercept) or
vehicle on day 0 and 7 after laser injury. CNV areas and extent of
leakage were determined by fluorescein angiography followed by
intracardiac perfusion of FITC-dextran in gelatin (10%) on day 14.
Choroidal flatmounts were examined by immunohistochemistry for
Iba-1 expression
Results: Fluorescence intensities of laser-induced CNV lesions
in mice treated with oral GSK2126458 were significantly lower
than control vehicle. Vehicle-treated group exhibited significantly
increased number of leaking lesions (≈ 88%) and larger CNV areas.
Intravitreal injections of GSK2126458 had strong inhibitory effects
on CNV leakage (21%) and size (~ 6000 mm2/eye), and significantly
lower than Eylea™ (36%, ~6500 mm2/eye) and vehicle (47%, ~7000
mm2/eye) groups.
Conclusions: The PI3K/mTOR pathway participates in laser-induced
CNV formation. GSK2126458 a highly potent inhibitor of the PI3K/
mTOR pathway is more effective than Eylea™ in inhibiting laserinduced CNV lesions through its downstream signaling effects when
given orally. Compared to Eylea™, intravitreal GSK2126458 showed
a trend toward more effective blockade of CNV lesions. Our findings
suggest that inhibition of the PI3K/mTOR pathway may be an
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
advantageous approach for inhibiting CNV lesions on its own right
and potentially in CNV clinically resistant to VEGF monotherapy.
Commercial Relationships: Yu Sun, None; Jie Ma, None; Gianna
C. Teague, None; Francisco J. Lopez, Ophthalmology IPE US, RD
Alternative Discovery & Development, GSK (F); Peter S. Adamson,
None; Edit Kurali, None; Kameran Lashkari, None
Program Number: 2365 Poster Board Number: C0015
Presentation Time: 3:45 PM–5:30 PM
A Mouse Model of Geographic Atrophy Resembling Human
Geographic Age-Related Macular Degeneration
Srini Goverdhan1, Maureen Gatherer1, Elizabeth Angus1, Robert
F. Mullins2, Andrew J. Lotery1. 1Southampton Eye Unit, SGH,
Southampton Univ Hospitals, Southampton, United Kingdom; 2Univ
of Iowa, Iowa, IA.
Purpose: Blindness from geographic atrophy (GA) in Age-related
macular degeneration (AMD) remains unaddressed with distinct lack
of animal models to evaluate new treatments. GA is thought to result
primarily from loss of the retinal pigment epithelium (RPE) and
photoreceptors. Current models of AMD like ELOVL4 mutant mice
have no visible GA type lesions. Sodium iodate induced RPE/retinal
degeneration is non-specific with wide spread damage. Using 810nm
diode laser (low xanthophyll and nerve fibre layer absorption), we
aimed to recreate the age related loss of RPE/photoreceptors seen in
GA in a mouse model.
Methods: C57Bl6 wild mice (female ex-breeders aged 11-13 months,
n=24) were used. Confluent diode laser spots (sub-threshold) were
applied in 1 quadrant of the retina at 1.3mw power and 60 sec
duration each. Color photographs with fluorescein angiography
(Micron 3 imaging) and ERG at weeks 8 and 12 were collected.
Peanut agglutinin, anti-rhodopsin, histology and transmission electron
microscopy (TEM) were used to document the photoreceptor/RPE
atrophy. Histological features were compared to human donor eyes
with GA.
Results: All laser exposed mouse retinal lesions progressed towards
GA type lesions by week 12 (fig 1). The phenotype and histology
resembled donor human GA AMD sections (with RPE atrophy and
photoreceptor degeneration) (fig 1). ERG was marginally reduced in
amplitude with larger GA lesions compared to control eyes. Known
features of GA such as atrophy of RPE cells with loss of normal basal
infoldings, increase in RPE lysosomes especially at junctional zones
(TEM images, fig 2), photoreceptor loss, loss of outer nuclear layer &
choroidal thinning could all be demonstrated in mouse study sections
(figs 1,2).
Conclusions: Using long wavelength laser, we have created a novel
and reproducible mouse model of GA. Such models are crucial for
developing and testing therapeutic interventions of autologous or
stem cell derived treatments for GA in AMD.
A- Donor human GA section showing RPE atrophy, photoreceptor
cell loss and loss of outer nuclear layer, B-Retinal appearance, C,D,ESections showing GA features in study, F,G- Sections showing
advanced GA features.
TEM images showing GA features such as RPE atrophy (white arrow,
B), photoreceptor loss (black arrow), loss of RPE basal infoldings
(white arrow, C), RPE atrophy with lysosomes (white arrow, D),
white star - Bruch’s membrane.
Commercial Relationships: Srini Goverdhan, None; Maureen
Gatherer, None; Elizabeth Angus, None; Robert F. Mullins, None;
Andrew J. Lotery, None
Support: National Eye Research Council (NERC), Gift of Sight
(GOS)
Program Number: 2366 Poster Board Number: C0016
Presentation Time: 3:45 PM–5:30 PM
Effects of oxidized phospholipids on formation of laser induced
choroidal neovascularization in mice
Hongjun Du. Department of Ophthalmology, Xijing Hospital, Xi’an,
China.
Purpose: The wet form of age-related macular degeneration
(AMD) is characterized by choroidal neovascularization (CNV).
Drusen is highly enriched with polyunsaturated fatty acid, which
can be modified to oxidized phospholipids (Ox-PLs) under the high
oxidative condition. Our former experiments shown that Ox-PLs on
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
oxidized low density lipoprotein (Ox-LDL) could induce CNV in
mice when injected subretinally. However, due to the unevenness
and difficulties of subretinal injection, this model is not ideal for a
quantitative research. As laser induced CNV model is now the most
commonly used one in quantitative research, the purpose of this
experiment is to test whether Ox-PLs injection intravitreally can
stimulate CNV formation in this model.
Methods: Male C57Bl/6 mice of 6 to 8 weeks old were anesthetized
by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg
xylazine. Laser burns were performed two disk diameters from the
optic disk in each quadrant with the following parameters: 120 mW
power, 75 μm spot size, and 100 ms duration. For Ox-LDL effects
on CNV formation experiments, mice were given an intravitreal
injection of 1 μl of Ox-LDL (100 μg/ml in PBS) right after laser
treatment. An equal volume of native LDL (Nat-LDL) (100 μg/
ml in PBS) or PBS was given by intravitreal injection as control.
Each group had 4 mice (4*4 laser spots, n=16). Seven days after
laser treatment, mice were sacrificed and choroidal flat mounts
were generated. FITC-conjugated isolectin was used to perform
immunolabelling of CNV. Images were taken and areas of CNV
were calculated by using a Zeiss AX10 fluorescence microscope
(Carl Zeiss, Inc.). Two-tailed Student’s t-test was used for statistical
analysis.
Results: At day 7, the mean CNV areas per lesion were
54596.25±9516.09 μm2, 33635.94±6234.57μm2, and
36598.50±10553.75 μm2 in the Ox-LDL, PBS and Nat-LDL injected
eyes respectively. The mean CNV area of Ox-LDL injected eyes
was larger than that of PBS (p=8.19E-8) or Nat-LDL injected eyes
(p=1.93E-5). There had no significant difference between PBS and
Nat-LDL injected eyes (p=0.34).
Conclusions: As we estimated, Ox-PLs intravitreal injection can
stimulate laser induced CNV formation in mice. This Laser&Ox-PLs
Intravitreal Injection model may be an ideal model to quantitatively
study the influence of Ox-PLs on AMD development, and how
impaired lipid metabolism and intense oxidative stress influence such
activity.
Commercial Relationships: Hongjun Du, None
Support: National Natural Science Foundation of China (No.
30973253, 81470654)
Program Number: 2367 Poster Board Number: C0017
Presentation Time: 3:45 PM–5:30 PM
Oxidized lipoproteins induce RPE cell death through NLRP3inflammasome activation
Gopalan Gnanaguru1, Ariel Choi2, Dhanesh Amarnani1, Wen Tseng3,
Leo A. Kim1, Patricia A. D’Amore1. 1Department of Ophthalmology,
Schepens Eye Research Institute,MEEI,HMS, Boston, MA; 2Program
in Liberal Medical Education, Brown University, Providence, RI;
3
Department of Biological Engineering, massachusetts institute of
technology, Cambridge, MA.
Purpose: Oxidized phospholipids/lipoproteins have been shown
to accumulate with age and in age-related macular degeneration.
We hypothesize that the uptake of oxidized lipoproteins by RPE
cells leads to lysosomal destabilization and cell death via NLRP3inflammasome.
Methods: Primary human fetal RPE (hf-RPE) cells were treated with
LDL or oxidized LDL (ox-LDL) at different doses (300, 400, and 500
mg/ml). Cell death was assessed by measurement of LDH release.
Transepithelial resistance was assayed as a measure of RPE barrier
function. Lysosomal integrity was revealed by immunostaining for
lysosomal associated membrane protein-1 (LAMP-1). RNA and cell
lysates were prepared from control, LDL (500 mg/ml), and ox-LDL
(500 mg/ml)-treated cells and the expression of NLRP3, PYCARD,
and caspase-1 examined by RT-PCR and western blot. IL-1b release
was quantified by ELISA.
Results: The results show that the treatment of RPE with 500 mg/
ml of ox-LDL for 48 hr altered RPE cytoskeleton as evidenced
by abnormal actin cytoskeleton organization. Further analysis of
RPE barrier properties showed a significant decreased (P< 0.01)
in transepithelial resistance following ox-LDL treatment for 48 hr.
Analysis of media conditioned by RPE treated with ox-LDL for 48 hr
revealed a dose-dependent increase in LDH release (over 50% LDH
release at 500 mg/ml), a reflection of cell death. ox-LDL co-localized
with LAMP-1, indicating its presence in the lysosomes. ox-LDL
treatment for 24 hr led to a 4-fold increase in NLRP3 mRNA level
and a 2-fold increase in PYCARD mRNA. Pro-caspase-1 and cleaved
active caspase-1 protein levels were upregulated by treatment with
ox-LDL for 48 hr. IL-1ß secretion was elevated were following 48 hr
of exposure to ox-LDL.
Conclusions: Our results indicate that ox-LDL accumulation in the
lysosome of hfRPE activates NLRP3 mediated cell death. Thus,
normalization of lipid levels and/or inhibition of NLRP3 activation
may attenuate/prevent RPE degeneration.
Commercial Relationships: Gopalan Gnanaguru, None; Ariel
Choi, None; Dhanesh Amarnani, None; Wen Tseng, None;
Leo A. Kim, None; Patricia A. D’Amore, AGTC (C), Eleven
Biotherapeutics (S)
Support: NEI EY015435, NIH/NEI K12EY016335
Program Number: 2368 Poster Board Number: C0018
Presentation Time: 3:45 PM–5:30 PM
Monomeric C-reactive protein (mCRP) in human donor
choroids: relationship with CFH genotype and effects on
endothelial cell function
Kathleen R. Chirco1, 2, Miles Flamme-Wiese1, 2, Jeaneen Andorf1, 2,
Rebecca Johnston1, 2, Lawrence Potempa3, Megan Riker1, 2, Grefachew
Workalemahu1, 2, Edwin M. Stone1, 2, Budd Tucker1, 2, Robert F.
Mullins1, 2. 1The Stephen A. Wynn Institute for Vision Research,
University of Iowa, Iowa City, IA; 2Department of Ophthalmology
and Visual Sciences, University of Iowa, Iowa City, IA; 3College of
Pharmacy, Roosevelt University, Schaumburg, IL.
Purpose: Previous reports showed an increase in overall levels
of C-reactive protein (CRP) in the choroids of human donor eyes
with AMD (Bhutto et al., 2011) and eyes homozygous for the CFH
(Y402H) gene polymorphism (Johnson et al., 2006). In this study,
we employed immunohistochemical analysis and functional assays
to study the localization of pro-inflammatory mCRP versus antiinflammatory pCRP in the human macula and to determine the effect
these two conformations of CRP have on endothelial cells and retinal
pigment epithelium (RPE) cells in vitro.
Methods: Cryostat sections of genotyped human donor maculas
(n=12) were labeled using antibodies specific for mCRP (3H12) or
pCRP (1D6), and their pattern of labeling was compared to sections
incubated with secondary antibody only. Scratch closure assays were
performed to quantify migration of the chorioretinal endothelial
cell line RF/6A and ARPE-19 cells during incubation with: media
only, pCRP (20mg/mL), boiled pCRP, mCRP (20mg/mL), or boiled
mCRP. VEGF (5mg/mL) was used as a positive control for RF/6A
cell migration. To study the effect of CRP on cell permeability,
transepithelial resistance (TER) assays were performed on both cell
types using the same conditions.
Results: mCRP was found to localize mainly to the choriocapillaris
and Bruch’s membrane, with qualitatively increased staining in tissue
from donors with a high-risk CFH (Y402H) genotype compared to
those with a low-risk genotype. pCRP was not detected in any of
the evaluated samples regardless of genotype. In functional assays,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
mCRP markedly promotes RF/6A cell migration after 24 hours
(p<0.05), while it increases the permeability of confluent endothelial
cell layers within 15 minutes (p<0.05). Migration and TER of ARPE19 cells were not significantly altered by mCRP or pCRP treatment.
Conclusions: Our data suggests that pro-inflammatory mCRP is the
major conformation of CRP present in the aging macula. The elevated
CRP in the choroids of high-risk CFH donor eyes appears to be
predominantly due to mCRP. In addition, mCRP, but not pCRP, has a
significant effect on endothelial cell phenotypes in vitro, suggesting
a potential role for mCRP in choroidal vascular dysfunction in AMD
pathogenesis.
mCRP increases RF/6A cell migration 24 hours after treatment
Commercial Relationships: Kathleen R. Chirco, None; Miles
Flamme-Wiese, None; Jeaneen Andorf, None; Rebecca Johnston,
None; Lawrence Potempa, None; Megan Riker, None; Grefachew
Workalemahu, None; Edwin M. Stone, None; Budd Tucker, None;
Robert F. Mullins, None
Support: NIH Grant EY024605; NIH Grant EY016822; DP2OD007483-01; Elmer and Sylvia Sramek Charitable Foundation
Program Number: 2369 Poster Board Number: C0019
Presentation Time: 3:45 PM–5:30 PM
Role of Homocysteine in Retinal Neurodegeneration
Giulia Malaguarnera2, 1, Shereen Nizari2, Lisa Turner2, James
Brodie2, Benjamin Davis2, Li Guo2, Eduardo M. Normando2,
4
, Claudio Bucolo3, Filippo Drago3, M Francesca Cordeiro2,
4 1
. Department of Ophthalmology, Int’l PhD Prog in
Neuropharmacology, Catania, Italy; 2Visual Neuroscience, Institute
of Ophtalmology, London, United Kingdom; 3Department of
Clinical and Molecular Biomedicine, Section of Pharmacology and
Biochemistry, University of Catania, Catania, Italy; 4Western Eye,
London, United Kingdom.
Purpose: Epidemiological studies have linked type 2 diabetes
mellitus (T2DM) with an increased risk of Alzheimer’s disease (AD).
Histopathological, molecular, and biochemical abnormalities have
commonalities in boh these major disease, which has lead to AD
recently termed as “Type 3 Diabetes”. Several neurodegenerative
conditions that affect the brain have manifestations in the eye, and
ocular symptoms often precede conventional diagnosis of such CNS
disorders. High levels of Homocysteine (Hcy) in the plasma has
been associated with both AD and T2DM. The aim of this study
was to investigate whether retinal Hcy is associated with retinal
neurodegeneration in animal models of AD (TASTPM transgenic
mice) and T2DM (Goto-Kakizaki (GK) rats).
Methods: Eyes of 3, 12 and 18 months GK-rats and 2,4,8, and
12 months TASTPM mice and age-matched controls were fixed
in 4% paraformaldehyde following their termination. The GKrats eyes were dissected and the cornea, lens and vitreous were
removed, whereas the eyes of TASTPM were directly embedded
in paraffin. 4um sections were stained for homocysteine, amyloid
beta (Aβ), Amyloid Precursor Protein (APP), and caspase-3, using
immunofluorescence. Confocal microscopy images were analysed
in a masked fashion for the mean intensity staining in the Retinal
Ganglion Cells Layer.
Results: When compared with aged-matched control, increased
hcy fluorescence was found in 12-month and 18-month GK-rats
retinas (p>0.05 and p>0.005 respectively). Hcy was significantly
increased in TASTPM mice at 4 months (p<0.05), preceding the
cognitive impairment onset at 6 months. In both models, this was
found to occur at smilar time points for increased levels of Aβ, APP,
and cleaved caspase-3 expression, with TASTPM mice showing
significant increases at 4 months in Aβ, APP (p<0.05), and cleaved
caspase-3 ( p<0.005).
Conclusions: As far as we are aware, this is the first study that shows
a role of homocysteine in retinal neurodegeneration in GK-rats and
TASTPM mice. Hcy is increased with aging and the worsening of
the diabetes in GK-rats, and in both models appears to be linked with
RGC apoptosis and Aβ expression. This highlights the association of
T2DM with AD, and provides further evidence that the GK-rat may
be a good model of AD.
Commercial Relationships: Giulia Malaguarnera, None;
Shereen Nizari, None; Lisa Turner, None; James Brodie, None;
Benjamin Davis, None; Li Guo, None; Eduardo M. Normando,
None; Claudio Bucolo, None; Filippo Drago, None; M Francesca
Cordeiro, None
Program Number: 2370 Poster Board Number: C0020
Presentation Time: 3:45 PM–5:30 PM
Quantified autofluorescence maps of human retinal pigment
epithelium in age-related macular degeneration (AMD)
Thomas Ach1, Anna V. Zarubina1, Kristen M. Hammack2, Jeffrey
D. Messinger1, Theodore Smith3, Kenneth R. Sloan2, 1, Christine A.
Curcio1. 1Dept of Ophthalmology, Univ of Alabama at Birmingham,
Birmingham, AL; 2Computer and Information Sciences, University
of Alabama at Birmingham, Birmingham, AL; 3Department of
Ophthalmology, NYU Langone Medical Center, New York, NY.
Purpose: To map quantified autofluorescence (AF) of human retinal
pigment epithelium (RPE) at different stages of AMD relative to agematched controls with healthy maculas.
Methods: Human chorioretinal tissue (25 donors (69-95 years)
were fixed in paraformaldehyde <4 hr after death. Internal fundus
examination revealed different AMD stages: incipient and early
(n=18), late atrophic (2), and neovascular AMD (5). Incipient
and early AMD had qualitatively similar RPE pathology (Ach
ARVO2014). RPE flat-mounts preserved the foveal position. RPE
cytoskeleton (labeled with AlexaFluor647-phalloidin) and AF
(exc. 460-490 nm, em. > 505 nm) were imaged on a spinning disk
fluorescence microscope. AF was normalized to a fluorescence
reference (Delori, PMID 22016060). Custom FIJI plugins compared
normalized AF to 9 age-matched healthy RPE flatmounts (Ach,
PMID 25034602). Difference maps plotted mean log pairwise ratios
vs. all controls for each eye (i.e., difference of logs).
Results: Normalized AF is variable among AMD donor eyes, as in
age-matched normal eyes. AF levels in AMD tissues are below levels
measured for controls (Figure for example). Only a few eyes show
similar or higher AF levels, presumably independent from AMD
stage. In 9 of 20 eyes with incipient, early, or late atrophic AMD,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
AF was particularly lower on the inner slope of the rod ring (< 4 mm
eccentricity from the fovea, Curcio PMID 2324310). In remaining
eyes no specific sub-regional predilection was discerned. AF was
independent of AMD stage.
Conclusions: This is the first systematic analysis of quantified
histological AF in AMD eyes. Normal aging leads to increased
AF in the perifovea and near periphery (1.25-2.75 and 2.75-4.25
mm eccentricity per Polyak; Ach, PMID 25034602) reflecting high
density of rods. In AMD eyes the perifovea is characterized by
noticeably reduced AF, indicating the spatial non-correspondence
between areas of high lipofuscin-attributable AF and RPE loss.
Unchanged or high AF in some tissues suggests additional factors
regulating AF that may be best characterized in longitudinal imaging
of genetically defined patient populations.
cytoskeleton). Cell nuclei, devoid of AF, were manually marked using
a custom FIJI plug-in. Cell borders were defined by Voronoi regions.
Poisson regression models using generalized estimating equations
(GEE) were used to compare the mean number of nuclei per cell
among eccentricity/quadrant groups and by age.
Results: A total of 11403 RPE cells at 200 locations were analyzed:
94.66 % mono-, 5.31% bi-, 0.02% trinucleate, and 0.01% with 5
nuclei. Age had no effect on number of nuclei. There were significant
spatial differences. Highest frequencies of MN cells were found in
the perifovea (9.9%) and periphery (6.8%), while the fovea lacked
MN cells almost entirely (Figure). Nasal quadrant had significantly
more MN cells compared to other quadrants, at all eccentricities.
Conclusions: This is the first study to demonstrate that the
human macula contains MN RPE cells, which might be due to
endoreplication, cell fusion, or incomplete cell division. Their
near-absence in the fovea suggests that this seemingly homogenous
epithelium actually reflects the specialized needs of foveal cones
in sustaining high-acuity vision. The perifovea is where rod
photoreceptor density and AF are both high. Nasal quadrant contains
the papillomacular area and closure of optic fissure. High frequency
of MN cells in perifovea might reflect specific requirements of retinal
metabolism, markers of past developmental processes, and other
mechanisms to be explored in further studies.
Representative AF map of a donor with incipient AMD (right) shows
reduced AF at the perifoveal region. AMD tissues showed reduced
AF throughout the flatmount, as compared to control eyes (left).
Commercial Relationships: Thomas Ach, None; Anna V.
Zarubina, None; Kristen M. Hammack, None; Jeffrey D.
Messinger, None; Theodore Smith, None; Kenneth R. Sloan,
None; Christine A. Curcio, None
Support: DFG (German Research Foundation) AC265/1-1 (TA);
NEI EY06109 (CAC); EyeSight Foundation of Alabama; Research to
Prevent Blindness
Program Number: 2371 Poster Board Number: C0021
Presentation Time: 3:45 PM–5:30 PM
Multinucleate RPE cells in normal human macula exhibit
exquisite regional specificity
Austin Starnes1, Carrie E. Huisingh1, Kristen M. Hammack2, Jeffrey
D. Messinger1, Gerald McGwin3, Kenneth R. Sloan1, 2, Christine A.
Curcio1, Thomas Ach1. 1Department of Ophthalmology, University
of Alabama at Birmingham, Birmingham, AL; 2Department of
Computer and Information Sciences, University of Alabama at
Birmingham, Birmingham, AL; 3Department of Epidemiology,
University of Alabama at Birmingham, Birmingham, AL.
Purpose: Human retinal pigment epithelium (RPE) is reportedly 3%
bi-nucleated, determined in a limited series of eyes (PMID 4963693).
To assess the potential importance to human vision of multinucleated
(MN) RPE cells, we report frequencies and spatial distribution from
RPE flatmounts.
Methods: Nineteen RPE flatmounts (PMID 25034602; 9<51yrs,
10>80 yrs) were imaged at 12 predefined locations: 3 eccentricities
(fovea, perifovea, near periphery) in 4 quadrants (superior, inferior,
temporal, and nasal). At each location, image stacks were taken using
a confocal fluorescence microscope (488 nm excitation for lipofuscin
AF and 647 nm excitation for the phalloidin labeled F-actin
Frequencies (%) of MN RPE in the human macula at different
eccentricities (fovea, perifovea, near periphery) and quadrants
(nasal, temporal). MN cells are almost completely absent from the
fovea. Highest frequencies of MN RPE cells are found in the nasal
perifovea.
Commercial Relationships: Austin Starnes, None; Carrie
E. Huisingh, None; Kristen M. Hammack, None; Jeffrey D.
Messinger, None; Gerald McGwin, None; Kenneth R. Sloan,
None; Christine A. Curcio, None; Thomas Ach, None
Support: German Research Foundation (DFG) AC265/1-1, NIH
grant R01 EY06109, Research to Prevent Blindness, EyeSight
Foundation of Alabama.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Program Number: 2372 Poster Board Number: C0022
Presentation Time: 3:45 PM–5:30 PM
Subretinal CD14+ monocytes, observed in geographic atrophy,
induce loss of photoreceptor outer segments and lead to rod
apoptosis.
Chiara M. Eandi1, 2, Elisa Dominguez2, Sophie Lavalette2, Shulong
Hu2, Michael Housset2, Cheryl M. Craft3, Michel Pâques2, José
Sahel2, Xavier P. Guillonneau2, 4, Florian Sennlaub2, 4. 1Eye Clinic,
University Torino, Torino, Italy; 2Institut National de la Santé et de
la Recherche Médicale, Institut de la Vision, Paris, France; 3Mary
D. Allen Lab for Vision Research, CSA 215, Los Angeles, CA;
4
Université Pierre et Marie Curie, Paris, France.
Purpose: Rod photoreceptor cell loss has been shown to be
particularly prominent in geographic atrophy (GA), and residual
cones and rods within the geographic lesion lack their outer segment.
GA is also associated with CCR2+subretinal inflammatory monocyte
accumulation in and around the GA lesion and inflammatory
monocytes have been shown to induce RPE cell death in vitro.
It is not clear if or how the chronic subretinal monocyte accumulation
participates in the pathological changes of photoreceptors in
GA. We here evaluate the subretinal monocyte accumulation
and photoreceptor phenotype in GA patients and investigated the
influence of CD14+monocytes on photoreceptors ex vivo.
Methods: CD14, rhodopsin, cone arrestin, L/M opsin and S opsin
were visualized using immunohistochemistry on donor eyes from
control subjects and GA patients. CD14+cells, cones and rods were
quantified within the GA lesion and at increasing distances from the
lesion. Adherent CD14+ human blood-derived monocytes were cocultured with an overlying mouse retinal explant and photoreceptor
apoptosis (TUNEL) and cone morphology (peanut agglutinine (PNA)
immunohistochemistry) was analyzed after 18h.
Results: Our results show the association of subretinal CD14+
monocytes accumulation with a 90% of the rod population in donor
eyes with GA lesions. Residual cones within the lesion were 50% of
the extrafoveal normal cone count. However residual cones and rods
within the lesion were not distributed equally and lacked their outer
segments. In retinal explants co-cultured with CD14+monocytes,
apoptosis of rod photoreceptors dramatically increased (1600%),
but the number of PNA+ cone photoreceptors was not affected.
Cones and rods invariably had lost their outer segments compared to
controls.
Conclusions: Our experiments confirm the subretinal accumulation
of monocytes and photoreceptor changes in GA lesions. Our results
suggest that subretinal inflammatory monocytes contribute to rod cell
loss and might participate in the loss of cone and rod outer segments
observed in the residual photoreceptors within the lesion. Inhibition
of subretinal monocyte accumulation in GA might help inhibit rod
apoptosis.
Commercial Relationships: Chiara M. Eandi, None; Elisa
Dominguez, None; Sophie Lavalette, None; Shulong Hu, None;
Michael Housset, None; Cheryl M. Craft, None; Michel Pâques,
None; José Sahel, None; Xavier P. Guillonneau, None; Florian
Sennlaub, None
Program Number: 2373 Poster Board Number: C0023
Presentation Time: 3:45 PM–5:30 PM
TLR3 stimulation leads to ATP release through lysosomal
exocytosis from optic nerve head astrocytes and RPE cells
Claire H. Mitchell1, Nestor Mas Gomez1, Jason C. Lim1, Wennan
Lu1, Jonathan Beckel2, Alan M. Laties3. 1Anatomy and Cell Biology,
University of Pennsylvania, Philadelphia, PA; 2University of
Pittsburgh, Pittsburgh, PA; 3University of Pennsylvania, Philadelphia,
PA.
Purpose: The release of transmitters by secretory lysosomes
represents a novel method of cell signaling. Here we asked whether
lysosomal stores of ATP were released after TLR3 stimulation in
optic nerve head astrocytes and RPE cells.
Methods: Experiments were performed on mouse RPE cells, ARPE19 cells, and rat optic nerve head astrocytes. Released ATP was
detected using the luciferase assay, acid phosphatase was measured
with an Elisa, and TLR3-phosphorylation with a Western blot.
Results: Stimulation of TLR3 with agonist poly(I:C) released ATP
from astrocytes and RPE cells. Extracellular ATP levels peaked
after 15-30 min poly(I:C) in astrocytes, but over an hour in RPE
cells. Poly(I:C) led to TLR3 phosphorylation, while ATP release
was triggered by 21-mer but not 16-mer siRNA. ATP release was
blocked by vesicular transport inhibitor NEM, but not pannexin
inhibitor carbenoxelone. Fluorescent MANT-ATP showed particulate
staining which colocalized with lysosomal stains. Poly(I:C) released
lysosomal enzyme acid phosphatase with ATP, implying lysosomal
exocytosis upon TLR3 activation. Both ATP and acid phosphatase
were released upon stimulation of cells with scrambled siRNA.
Serum starvation, but not rapamycin, prevented the ATP release
triggered by poly(I:C).
Conclusions: Stimulation of TLR3 activates lysosomes in astrocytes
and RPE cells. The resulting lysosomal exocytosis leads to ATP
release into the surrounding extracellular space. This may identify
lysosomal activation, exocytosis and release of lysosomal ATP as a
widespread cellular response to double stranded RNA exposure and
TLR3 stimulation. The role of this released ATP and other lysosomal
constituents in the reaction to TLR3 stimulation remains to be
determined.
Commercial Relationships: Claire H. Mitchell, None; Nestor Mas
Gomez, None; Jason C. Lim, None; Wennan Lu, None; Jonathan
Beckel, None; Alan M. Laties, None
Support: EY013434, EY015537
Program Number: 2374 Poster Board Number: C0024
Presentation Time: 3:45 PM–5:30 PM
Green tea extract restores oxidative stress-induced retinal
pigment epithelial cell degeneration
Tsz Kin Ng, Jian Xiong Wang, Yaping Yang, Kai On Chu, Chi Pui
Pang. Department of Ophthalmology and Visual Sciences, The
Chinese University of Hong Kong, Kowloon, Hong Kong.
Purpose: Oxidative stress is the most critical risk factor for retinal
pigment epithelium (RPE) degeneration in age-related macular
degeneration (AMD). Current AMD treatments only focus on
relieving the symptoms, such as choroidal neovascularization, rather
than controlling the disease origin. Green tea extract (GTE) has been
shown to be anti-oxidative in different disease models. Here, we
hypothesized that GTE could protect the oxidative stress-induced
RPE degeneration. This study aims to determine the protective effect
of GTE against the sodium iodate-induced RPE degeneration.
Methods: Human RPE cell line, ARPE-19, was treated with sodium
iodate (10 mM) and GTE (Theaphenon®E; 100 mg/ml) for 5 days.
The cell viability was determined by the MTT assay. For animal
experiment, Sprague-Dawley rats were intravenously injected with
single dose of sodium iodate (40 mg/kg) and then fed intragastrically
with GTE (550 mg/kg) for 2 weeks. Markers for RPE (RPE65),
oxidative stress (8-OHdG), tight junction (ZO-1) and apoptosis
(caspase-3) were analyzed by immunohistochemistry. The study
protocol was approved by the Animal Experimentation Ethics
Committee of the Chinese University of Hong Kong, which is in
accordance with the guidelines of the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Results: Oxidative stress was induced by sodium iodate as confirmed
by the nuclear signal of 8-OHdG. Moreover, the viability of sodium
iodate-treated APRE-19 cells was reduced. For the sodium iodatetreated rats, photoreceptor rosettes were appeared along the whole
retina. RPE65 and ZO-1 expressions were drastically reduced in RPE
cells of sodium iodate-treated rats. In contrast, caspase-3 expression
was increased in the outer segment of photoreceptor cells. In reverse,
GTE rescued the cell death and oxidative stress in ARPE-19 cells as
well as the disorganization of outer nuclear layer in rats. Furthermore,
RPE65 and ZO-1 expressions in RPE were restored, whereas
caspase-3 expression was reduced in the outer segment.
Conclusions: Sodium iodate induces oxidative stress, photoreceptor
cell disorganization and apoptosis through RPE disintegration and
dysfunction. GTE treatment restored RPE function and integrity
as well as reduced photoreceptor cell death and disorganization.
Our results demonstrated the therapeutic effect and the possible
mechanism for GTE against sodium iodate-induced RPE changes.
Commercial Relationships: Tsz Kin Ng, None; Jian Xiong Wang,
None; Yaping Yang, None; Kai On Chu, None; Chi Pui Pang, None
Program Number: 2375 Poster Board Number: C0025
Presentation Time: 3:45 PM–5:30 PM
Oxidative stress induces microparticle release from retinal
pigment epithelial cells
Kyle Carver, Dongli Yang. Ophthalmology and Visual Sciences,
University of Michigan, Ann Arbor, MI.
Purpose: Oxidative stress is one of major factors involved in
the retinal pigment epithelium (RPE) dysfunction and death that
underlie age-related macular degeneration (AMD). Drusen and
subretinal deposits are extracellular lipid- and protein-containing
deposits that are found in early AMD and are strongly associated
with the development of late AMD. Microparticles (MPs) are
small membrane-bound vesicles that have been demonstrated to be
released from cells under both normal and pathologic conditions. The
purpose of this study was to determine if oxidative stress drives MP
release from RPE cells and to assess whether these MPs may carry
membrane complement regulatory proteins that are present in drusen
or subretinal deposits.
Methods: Primary RPE cells isolated from human donor eyes were
cultured and treated with 50-2000 mM hydrogen peroxide for 2-24
hours to induce oxidative stress. MPs were isolated with differential
centrifugation and stained for component analysis by flow cytometry
and confocal microscopy or fixed for electron microscopy.
Results: Transmission electron microscopy showed that the size of
MPs, released from both hydrogen peroxide- and vehicle-treated
RPE cells, ranged from 100 to 1000 nm. Hydrogen peroxide
treatment led to time- and dose-dependent elevations in MPs with
exposed phosphatidylserine and phosphatidylethanolamine, known
markers of MPs. These increases were strongly correlated (Pearson
correlation coefficient = 0.756 and 0.9173, respectively) to RPE cell
apoptosis, as measured by annexin V and propidium iodide labelling.
In addition, oxidative stress significantly increased the presence of
membrane complement regulatory proteins CD46 (10-fold increase, p
< 0.0001), CD55 (6-fold, p = 0.0015), and CD59 (6-fold, p < 0.0001)
on released MPs.
Conclusions: This is the first evidence that oxidative stress induces
primary cultured human RPE cells to release MPs, strongly correlated
with RPE cell apoptosis, which contain CD46, CD55, and CD59.
Together with previous detection of CD46 in drusen and CD59
in subretinal deposits in early AMD, as well as oxidative stressinduced loss of CD46, CD55, and CD59 from the RPE surface, we
suggest that oxidative stress may render RPE cells more sensitive to
complement activation by inducing loss of the complement inhibitors
and could trigger drusen or subretinal deposit formation by releasing
MPs from the RPE.
Commercial Relationships: Kyle Carver, None; Dongli Yang,
None
Support: University of Michigan Start-Up Funds (DY) and NIH
Grant P30EY007003 (core)
Program Number: 2376 Poster Board Number: C0026
Presentation Time: 3:45 PM–5:30 PM
High-resolution molecular imaging of hydroxyapatite associated
with sub-RPE deposits
Matthew G. Pilgrim1, 2, Lajos Csincsik1, Sarah Fearn3, David S.
McPhail3, Jonathan C. Knowles2, Imre Lengyel1. 1Department of
Ocular Biology and Therapeutics, UCL Institute of Ophthalmology,
London, United Kingdom; 2Department of Biomaterials and Tissue
Engineering, UCL Eastman Dental Institute, London, United
Kingdom; 3Department of Materials, Imperial College London,
London, United Kingdom.
Purpose: We have shown recently that sub-RPE deposits, a feature
of ageing and age-related macular degeneration (AMD), contain
hydroxyapatite (HAP), the mineral component of bone and teeth.
However, the molecular mechanism by which HAP deposition
occurs is not yet known. We hypothesize that the molecular events
underlying deposit formation will be the key to understanding the
early molecular events leading to AMD. Here, using high-resolution
molecular imaging, we analysed the composition of sub-RPE deposits
in sections of human eyes.
Methods: After obtaining local ethical permission, human donor
eyes with sub-RPE deposits were obtained from the UCL Institute of
Ophthalmology tissue depository. Multiple high-resolution imaging
modalities were used including scanning electron microscopy
(SEM), transmission electron microscopy (TEM) and time-offlight secondary ion mass spectrometry (TOF-SIMS). Molecular
analysis was performed using energy dispersive X-ray (EDX),
X-ray diffraction (XRD) and mass spectrometry. This was correlated
with Von Kossa staining and fluorescent HAP labelling of adjacent
tissue sections and visualized under light, fluorescence and confocal
microscopy.
Results: High-resolution correlative imaging identified spherules
of 0.5μm – 6.0μm in diameter. The spherules were numerous, were
observed in clusters and were distributed throughout all the examined
sub-RPE deposits. SEM images showed a wide variation in the
spherule surface structure; subsequent EDX analysis and SIMS
imaging confirmed the presence of calcium and phosphate within
these structures. XRD patterns obtained using TEM were consistent
with the presence of HAP.
Conclusions: Using multiple complementary high-resolution
methodologies, we confirmed that sub-RPE deposits contain
spherules with HAP shells. Determining how these HAP spherules
develop will be important for fully understanding the molecular
changes underlying deposit formation and progression to diseases
like AMD. This is the first step in curing, or at least slowing down,
progression of this age-related disease.
Commercial Relationships: Matthew G. Pilgrim, None; Lajos
Csincsik, None; Sarah Fearn, None; David S. McPhail, None;
Jonathan C. Knowles, None; Imre Lengyel, None
Program Number: 2377 Poster Board Number: C0027
Presentation Time: 3:45 PM–5:30 PM
Real-time imaging by spectral domain-optical coherence
tomography in albino and pigmented rabbits to visualize
subretinal injection-induced outer retinal degeneration
Hammurabi Bartuma. St. Erik’s Eye Hospital, Stockholm, Sweden.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Purpose: To study the effect on the neuroretina and the retinal
pigment epithelium (RPE) in albino rabbits with high-resolution realtime spectral domain-optical coherence tomography (SD-OCT) after
subretinal injection of isotonic salt solution.
Methods: Subretinal injections of phosphate buffered salt solution
(PBS) or balanced salt solution (BSS) were performed in albino or
pigmented rabbits by standard small-gauge vitrectomy. Real-time
SD-OCT, infrared confocal scanning laser ophthalmoscopy (IRcSLO) and blue light fundus autofluorescence (BAF) was done up to
12 weeks following subretinal injection. The effects on morphology
were compared to histologic hematoxylin eosin-stained sections of
the outer retina and flat-mounts of phalloidine-labeled RPE.
Results: SD-OCT of the normal albino and pigmented rabbit retina
revealed 11 distinct layers including the outer nuclear layer, the
ellipsoid zone, the photoreceptor outer segments and the retinal
pigment epithelium. Subretinal injection of PBS or BSS resulted in an
acute thickening followed by a significant thinning of the outer retinal
layers that remained stable for up to 12 weeks post-injection. The
thinning involved the loss of the outer segments, the ellipsoid zone
and thinning of the outer nuclear layer. IR-cSLO of the subretinal
bleb area showed a hyperreflective ring with a hyporeflective margin,
whereas analysis of the RPE by BAF revealed areas of both hyperand hypofluorescence. SD-OCT results were confirmed by histology
and on subretinal flat-mounts, which demonstrated photoreceptor loss
and disturbed RPE.
Conclusions: SD-OCT, IR-cSLO and BAF provide detailed
morphologic information of the posterior segment in rabbits, making
it suitable as a preclinical model. Subretinal injection of isotonic salt
solutions cause significant and long-lasting degeneration of the outer
retina, suggesting that care should be taken when using this route of
administration in treatments of retinal disease.
Commercial Relationships: Hammurabi Bartuma, None
Program Number: 2378 Poster Board Number: C0028
Presentation Time: 3:45 PM–5:30 PM
Plasma pentosidine and esRAGE concentrations in patients with
age-related macular degeneration
Eiichi Sato, Akira Takamiya, Atsushi Takahashi, Shinji Ono, Shinichi
Otani, Chiemi Matsumoto, Akitoshi Yoshida. Ophthalmology,
Asahikawa Medical University, Asahikawa, Japan.
Purpose: Advanced glycation end products (AGEs) have been
implicated in the pathogenesis of age-related macular degeneration
(AMD) through binding to their receptor, RAGE. However,
endogenous secretory RAGE (esRAGE) captures AGEs and protects
against AGE-induced effects. In the current study, we investigated the
relationship between plasma pentosidine (a well-defined AGE) and
plasma esRAGE and wet AMD.
Methods: Thirty-one patients with wet AMD and 15 control subjects
(age range, 58~90 years) were included. Of the patients with wet
AMD, 10 had typical AMD and 21 had polypoidal choroidal
vasculopathy (PCV). A commercially available competitive enzymelinked immunosorbent assay was used to measure the plasma
pentosidine and esRAGE concentrations in the patients with AMD
and the control subjects.
Results: The plasma pentosidine level in typical AMD (mean ±
standard deviation, 0.125 ± 0.0210 mg/mL) was significantly (P <
0.05) higher than in the control subjects (0.0245 ± 0.0153 mg/mL)
and the patients with PCV (0.0221 ± 0.0202 mg/mL). Although the
esRAGE level in the patients with PCV (0.177 ± 0.0991 ng/mL)
tended to be lower than in the control subjects (0.241 ± 0.0918 ng/
mL) and in the patients with typical AMD (0.259 ± 0.134 ng/mL), the
plasma esRAGE levels did not differ significantly among the groups.
Conclusions: Our results suggested that increased pentosidine levels
may be implicated in the pathogenesis of typical AMD, but systemic
levels of circulating esRAGE may not be associated with wet AMD.
Commercial Relationships: Eiichi Sato, None; Akira Takamiya,
None; Atsushi Takahashi, None; Shinji Ono, None; Shinichi
Otani, None; Chiemi Matsumoto, None; Akitoshi Yoshida, None
Program Number: 2379 Poster Board Number: C0029
Presentation Time: 3:45 PM–5:30 PM
Modeling Late-Onset Retinal Degeneration with Human iPSCs:
Insights into the Shared Pathogenesis of Macular Degenerations
Katharina Clore-Gronenborn1, Kiyoharu Miyagishima2, Congxiao
Zhang2, Vaisakh Rajan1, Jason Silver1, Qin Wan2, Ruchi Sharma1,
Catherine A. Cukras3, Sheldon S. Miller2, Kapil Bharti1. 1Unit on
Ocular Stem Cell and Translational Research, National Eye Institute,
National Institutes of Health, Bethesda, MD; 2Section on Epithelial
& Retinal Physiology & Disease, National Eye Institute, National
Institutes of Health, Bethesda, MD; 3Clinical Trials Branch, National
Eye Institute, National Institutes of Health, Bethesda, MD.
Purpose: Late-Onset Retinal Degeneration (L-ORD) is an autosomal
dominant disorder caused by a S163R mutation in CTRP5. The
disease is characterized by delayed dark adaptation, long anterior
lens zonules, and drusen-like deposits in the sub-retinal pigment
epithelium (RPE). The purpose of our study is to deduce the
mechanism by which mutated CTRP5 causes retinal degeneration
using RPE derived from induced pluripotent stem cells (iPSCs)
specific to L-ORD patients and their healthy siblings.
Methods: iPSCs were generated from skin biopsies obtained from
two affected and two unaffected siblings from a L-ORD family using
Sendai virus based transfection of OCT3/4, SOX2, c-MYC, and
KLF4 factors. iPSCs were characterized for expression of pluripotent
markers by immunofluorescence and karyotyped for genetically
stability. iPSCs were differentiated into RPE cells (iRPE) using a
developmentally-guided differentiation protocol. The iRPE cells were
authenticated by gene expression, immunofluorescence, TEM, and
physiology.
Results: We successfully generated and authenticated iPSCs and
iRPEs from a L-ORD family. We confirmed that patient iPSCs
retained the S163R mutation in CTRP5 and control iPSCs retained
the wild-type sequence. TEM images of healthy sibling- and L-ORD
patient- iRPE confirmed their epithelial morphology, presence of
apical microvilli, melanosomes, and tight junctions. iRPE monolayers
also show electrophysiological responses that are typical of native
RPE.
Preliminary findings indicate L-ORD Patient i-RPE have impaired
trans-epithelial resistance. This is consistent with the impaired
adhesion of L-ORD RPE to Bruch’s membrane reported in the
literature. Further investigation is currently underway to determine
the disease mechanism.
Conclusions: The retinal degeneration in L-ORD patients resembles
age-related macular degeneration (AMD), a multi-factorial late-onset
disease. However, unlike AMD, L-ORD is a monogenic disease
caused by a single missense (S163R) mutation in the CTRP5 protein,
making it a powerful tool to study more genetically complex diseases
such as AMD. Elucidating the mechanism by which mutated CTRP5
causes L-ORD will aid in understanding disease mechanisms in AMD
and other retinal degenerations.
Commercial Relationships: Katharina Clore-Gronenborn, None;
Kiyoharu Miyagishima, None; Congxiao Zhang, None; Vaisakh
Rajan, None; Jason Silver, None; Qin Wan, None; Ruchi Sharma,
None; Catherine A. Cukras, None; Sheldon S. Miller, None; Kapil
Bharti, None
Support: NEI Intramural Funds
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Program Number: 2380 Poster Board Number: C0030
Presentation Time: 3:45 PM–5:30 PM
Identification and evaluation of the levels of various age related
lipofuscin components in human macula
Ankita Kotnala1, Senthilkumari S2, Nabanita Halder1, B Jayaram3,
Atul Kumar4, Thirumurthy Velpandian1. 1Ocular Pharmacology
& Pharmacy, All India Institute of Medical Sciences, New Delhi,
India; 2Department of Ocular Pharmacology, Dr. G. Venkataswamy
Eye Research Institute, Madurai, India; 3Department of Chemistry,
Indian Institute of Technology, New Delhi, India; 4Department of
Ophthalmology, Dr. R. P. Centre, AIIMS, New Delhi, India.
Purpose: Accumulation of lipofuscin in human retinal pigment
epithelium is reported to be one of the causative factors in the
pathogenesis of Age related Macular Degeneration.Therefore, the
present study was undertaken to identify and evaluate the levels of
various lipofuscin components in normal age matched aging Indian
donor eyes.
Methods: Donor eyes (Group 4,above 80 yrs; N=36, Group 3, 6080 yrs; N=60, Group 2, 40-60 yrs ;N=13,Group 1, below 40 yrs ;
N=44) obtained after the removal of cornea for transplantation were
collected from the Eye Bank of Arvind Hospital, Marudai with prior
approval from the Human Ethics Committee. 8mm macular portion
was punched, extracted and sent to AIIMS for analysis. Lipofuscin
components (A2-glycerophosphoetahnolamine(A2GPE) All trans
retinal dimer (ATRD), A2-dihydrophosphoethanolamine (A2DHPE)),
of age matched samples were analyzed with a newly developed LCESI/MS/MS method.
Results: Several lipofusion components viz A2GPE, ATR-dimer,
ATRDHPE, A2E, isomers of A2E, monofuran A2E, mono-peroxy
A2E etc were found. Presence of A2GPE, ATRD and A2DHPE were
further confirmed by their fragmentation pattern. In the group 4,3
& 2, A2GPE levels were found to increase 41, 39, 7 % more than
the levels found in macula belong to >40 years group (group 1).
Increasing A2GPE levels between Group 4 and 1 were found to be
statistically significant. In age group 4,3 & 2 levels were found out to
be 11, 8,8 % more then that of levels below >40 years group(group
1). Increasing A2DHPE levels were found to be significant for group
1. Similar increasing trend found for ATRD. In the group 4, 3 & 2,
ATRD levels were found out to be 49, 73 and 24 % more then the
levels below>40 years age group (group 1). Increasing ATRD levels
were found to be significant for group 1, 2 and 3.
Conclusions: This study further confirmed the presence of several
lipofusion components such as A2GPE, ATR-dimer, ATRDHPE,
A2E, isomers of A2E, monofuran A2E, mono-peroxy A2E etc. in
the human macular extracts. Age related increase in A2E has already
been reported in the literature, however, for the first time the present
study shows that along with A2E increasing levels of A2GPE, ATRD
and A2DHPE can also contribute towards the pathogenesis of AMD.
Further studies are in progress to elucidate the levels of oxidized
products of lipofusion components.
Commercial Relationships: Ankita Kotnala, None; Senthilkumari
S, None; Nabanita Halder, None; B Jayaram, None; Atul Kumar,
None; Thirumurthy Velpandian, None
Support: CSIR (for providing Senior Research Fellowship)
Program Number: 2381 Poster Board Number: C0031
Presentation Time: 3:45 PM–5:30 PM
Components of human lipofuscin
Masahiro Kono1, John E. Oatis1, Zsolt Ablonczy1, Patrice W. Goletz1,
Joe G. Hollyfield2, Rosalie K. Crouch1. 1Ophthalmology, Medical
University of South Carolina, Charleston, SC; 2Ophthalmology,
Cleveland Clinic Lerner College of Medicine, Cleveland, OH.
Purpose: In the aging human eye, fluorescence in the retinal pigment
epithelium (RPE) increases due to accumulation of lipofuscin, which
is thought to be toxic to the RPE and to contribute to the pathology
of age-related macular degeneration. To date, the best-characterized
component of lipofuscin is the bisretinoid A2E. However, recent
studies indicated that areas of highest fluorescence in human RPE
do not correlate with A2E levels. Thus, other fluorescent compounds
must be responsible for a significant proportion of lipofuscin. The
purpose of this study is to identify new fluorophores in human
lipofuscin, with the ultimate goal of understanding their toxicity.
Methods: On the premise that established purification methods have
been optimized for bisretinoids, we are developing new extraction
and separation conditions. RPE samples were brushed from the
inner eye wall after removal of the retina from human donor eyes.
Molecules were extracted from the RPE homogentate with various
organic solvent/aqueous buffer mixtures and separated by thin layer
chromatography (TLC). Bands were isolated from the plate and
subjected to mass spectrometry (MS) with MS/MS information
collected on prominent MS peaks. Pure samples of A2E and retinyl
palmitate served as TLC controls.
Results: Fluorescent compounds that did not co-migrate with either
A2E or retinyl palmitate were resolved by TLC. The brightest
bands displayed multiple peaks by MS, but dominant peaks did
not correspond to A2E. MS-MS of these samples fragmented well.
Analysis from previously obtained MALDI-MS imaging of human
RPE tissue indicated that the abundances of at least four of these
newly identified compounds correlate spatially with lipofuscin
fluorescence.
Conclusions: Fluorescent compounds other than A2E are extractable
from human RPE. They have distribution patterns that match those of
lipofuscin. This study may provide new strategies into determining
the relative toxicity of the multiple fluorophores present in lipofuscin
associated with aging and age-related macular degeneration.
Commercial Relationships: Masahiro Kono, None; John E. Oatis,
None; Zsolt Ablonczy, None; Patrice W. Goletz, None; Joe G.
Hollyfield, None; Rosalie K. Crouch, None
Support: NIH Grants EY019515 (MK), EY019065 (ZA), EY014240
(JGH); Research to Prevent Blindness; Foundation Fighting
Blindness; Llura and Gordon Gund Foundation
Program Number: 2382 Poster Board Number: C0032
Presentation Time: 3:45 PM–5:30 PM
Investigating the Chemical Composition of Human Retinal
Lipofuscin from Age Related Macular Degeneration Donor
Tissue: Observation of Biomarkers of Inflammation
Jennifer Tournear1, Elizabeth R. Gaillard1, 2. 1Chemistry and
Biochemistry, Northern Illinois University, Dekalb, IL; 2Biological
Sciences, Northern Illinois University, Dekalb, IL.
Purpose: To investigate the chemical composition of Folch extracts
of human retinal lipofuscin from human donor tissue diagnosed as
neovascular (wet) or avascular (dry) age related macular degeneration
(AMD).
Methods: Human retinal lipofuscin is extracted from human donor
eyes diagnosed with either wet or dry AMD as previously described
by Feeney-Burns (Feeney-Burns and Eldred, 1983). The organic
soluble portion of lipofuscin is collected, dried, and reconstituted
with methanol for analysis by high resolution, high performance
liquid chromatography (LC/MS) tandem mass spectrometry. LC/MS
and tandem mass spectrometry data is analyzed in comparison to age
matched donors to determine the chemical composition specific to
wet and dry AMD. Biomarkers specific to inflammation have been
identified.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Results: Analysis of the mass chromatograms alongside photodiode
array and fluorescence chromatograms show unique components for
the samples from diseased tissue compared to those samples from age
matched donors.
Additional analysis of dry AMD diagnosed extracts have shown a
higher prevalence for L-homocitrulline compared to that of an age
matched donor. Increased levels of L-homocitrulline have been
associated with inflammation.
Conclusions: These data suggest that wet and dry AMD may be two
different diseases. Additionally, increased levels of L-homocitrulline
have been detected in the dry AMD diagnosed extracts supporting
inflammation as a possible contributor to the progression of the
disease. Understanding the chemical composition found in these
samples can aid in understanding the pathogenesis of wet and dry
AMD.
Commercial Relationships: Jennifer Tournear, None; Elizabeth
R. Gaillard, None
Program Number: 2383 Poster Board Number: C0033
Presentation Time: 3:45 PM–5:30 PM
Lipofuscin bodies decrease and melanosomes increase in number
progressively with retinal eccentricity in elderly rhesus monkey
RPE
Peter Gouras1, Maerta Lena Ingeborg Ivert2, Martha Neuringer3,
Takayuki Nagasaki1. 1Columbia University, New York, NY; 2St
Erik Eye Hospital, Stockholm, Sweden; 3Oregon National Primate
Research Center, Beaverton, OR.
Purpose: To determine how the cytoplasmic organelles, lipofuscin
bodies and melanosomes, change in number and size with retinal
eccentricity and age.
Methods: Transmission electron microscopy was used to identify
and measure the number and length of lipofuscin bodies and
melanosomes in the retinal pigment epithelium (RPE) along the
temporal horizontal meridian at the macula, peri-macula, equator,
periphery, and ora serrata in young and old monkeys.
Results: In elderly monkeys, lipofuscin bodies are maximum in
number and total area at the macula and decrease progressively with
eccentricity. There are more than 10 times as many lipofuscin bodies
in the macula than in the peripheral RPE. No lipofuscin bodies are in
the ora. Aside from a foveal dip, the results resemble the fluorescent
measurements of human eyes by Wing et al. (IOVS 17:601, 1978).
In elderly monkeys, melanosomes are minimal in number and total
area in the macula RPE and increase progressively with eccentricity.
There are more than 5 times as many melanosomes in the peripheral
than the macula RPE. In young monkeys there are no lipofuscin
bodies and more melanosomes and less of an increase in number with
eccentricity than in elderly RPE.
Conclusions: Lipofuscin bodies are mainly undigested lysosomal
degradation products resulting from the phagocytic turnover of
photoreceptor outer segments. If this turnover rate is the same across
the retina, the macular RPE of elderly monkeys must have a unique
defect in being less able to digest outer segment material, or has
a greater phagocytic load (Dory et al., IOVS 30:169, 1989). The
higher concentration of lipofuscin could be responsible for the lower
amounts of melanosomes in the macula.
Commercial Relationships: Peter Gouras, None; Maerta Lena
Ingeborg Ivert, None; Martha Neuringer, None; Takayuki
Nagasaki, None
Support: NIH EY015293, Research to Prevent Blindness
Program Number: 2384 Poster Board Number: C0034
Presentation Time: 3:45 PM–5:30 PM
Fundus Autofluorescence in exudative age-related macular
degeneration ( AMD)
Lorraine North, Manju Chandran, Geeta Menon. Ophthalmology,
Frimley Health Foundation Trust, Frimley, United Kingdom.
Purpose: To evaluate the characteristics of fundus auto-fluorescence
( FAF) patterns in patients with exudative AMD treated with
intravitreal ranibizumab.
Methods: Retrospective analysis of 33 patients (40 eyes) diagnosed
with exudative AMD was carried out at baseline with ranibizumab
intravitreal injections and 1 month after the third injection. All
patients had fundus fluorescein angiography and FAF imaging using
Heidelberg Spectralis. Patients were divided into 3 groups, classic
wet AMD, occult AMD, and mixed wet AMD. The characteristics of
the FAF were then described using previous recognised patterns.
Results: Of the 40 eyes evaluated 19 patients were classified as
classic wet AMD, 14 as occult and 6 mixed AMD. FAF results for
different patterns of exudative AMD were compared before treatment
and after 3 intravitreal injections. The 3 classifications were analysed.
Classic: At baseline of the 19 with classic wet AMD, 18 demonstrated
decreased FAF at the centre of the lesion but also increased signal
toward the lesion edge.1 patient with classic showed near normal
FAF. After 3 injections, 73.68% demonstrated less hypofluorescence
with increased hyperfluorescence at the lesion. 26.32 % remained
unchanged.
Occult: At baseline of the 16 patients with occult AMD, 75%
demonstrated a mottled FAF. 18.75% patients had a FAF pattern
the same as the classic of decreased FAF at the centre of the
lesion with increased signal at the edges and 1 FAF appeared near
normal. Following the third injection of ranibizumab 62.5% eyes
demonstrated less hypofluorescence and 37.5% were unchanged.
Mixed:100% of the mixed type exhibited characteristics of both
occult and classic AMD. After 3 injections some change in the pattern
was observed of either increased FAF or decreased FAF.
Conclusions: It has been suggested that the noninvasive imaging
technique of FAF could be used as a prognostic predictive marker
in AMD. FAF is a result of RPE cell metabolism and dysfunction
which leads to large amounts of lipofuscin accummulating in the RPE
cells.FAF can be used to visualize the extent of damage and stage
of disease progression. Others have described different patterns of
FAF in early non exudative AMD but our findings show that patients
with classic wet AMD, occult AMD, and mixed AMD can exhibit
different FAF patterns before and after intravitreal injections. FAF
could therefore be used to monitor disease development,severity and
prognosis of AMD.
Commercial Relationships: Lorraine North, None; Manju
Chandran, None; Geeta Menon, Alcon (F), Alcon (R), Allergan (F),
Allergan (R), Bayer (F), Bayer (R), Novartis (F), Novartis (R)
Program Number: 2385 Poster Board Number: C0035
Presentation Time: 3:45 PM–5:30 PM
Polarization properties of amyloid beta deposits in ex vivo
human retinas from those with Alzheimer’s disease differ from
surrounding retina
David DeVries1, Melanie C. Campbell1, Laura Emptage1, Chris
Cookson1, Marsha Kisilak1, Francisco J. Avila2, Juan M. Bueno2,
Rachel Redekop1, Matthew Wilson1. 1University of Waterloo,
Waterloo, ON, Canada; 2Universidad de Murcia, Murcia, Spain.
Purpose: Alzheimer’s disease (AD) is diagnosed from plaques
composed of insoluble fibrils of amyloid beta (Aβ) in the brain. Aβ
has been found near the optic nerve fibre layer of the retina by us in
those with AD using fluorescence staining. We have shown that some
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
polarization properties of presumed Aβ deposits and pure Aβ differ
from surrounding areas. The neural retina is optically accessible
so polarization properties could enable non-invasive detection
of Aβ deposits in the living eye. Here we characterize additional
polarization properties of Aβ deposits which produce contrast,
including retardation.
Methods: Retinas were dissected from eyes obtained following
informed consent and fixed in paraformaldehyde or formalin for
those with a diagnosis of AD and age matched normals without AD
or glaucoma. The retina was stained with 0.1% Thioflavin-S, (which
is polarization insensitive) and measured using a Nikon transmission
microscope fitted with a polarimeter. The Mueller matrix (MM)
describes the interaction of a sample with states of polarized light.
It is calculated from 16 images. Polarimetry images were taken of
regions, -ve and +ve for Aβ deposits. Spatially resolved MM of the
areas and then their false colour images of polarization properties
were calculated. These properties included degree of polarization,
diattentuation, polarizance, and retardance. Paired t-tests compared
the properties of +ve and -ve areas in AD +ve retinas and unpaired
t-tests compared them with areas in control retinas.
Results: 28 deposits and nearby -ve areas were characterised in
3 retinas, +ve for AD, as well as areas in 3 control retinas. As
previously reported by us, degree of polarization, diattentuation
and polarizance produced some contrast of the deposits. Across
all deposits, degree of polarization (p<0.0008) and retardation
(p<0.0005) gave significantly different mean values between areas
with and without deposits and compared to control retinal areas.
Retardation values of deposits were well above those reported for
the optic nerve fibre layer in ex vivo retinas. Related indices were
also explored for the sensitivity and specificity of using polarization
imaging to detect the deposits.
Conclusions: The measurement of polarization properties is a
promising non-invasive and inexpensive method of locating and
tracking Aβ deposits in the retina as a biomarker of AD.
Commercial Relationships: David DeVries, None; Melanie C.
Campbell, CA2832517 (P), CN201180033685 (P), EP20110777072
(P), US13/696238 (P), US61/282999 (P); Laura Emptage, None;
Chris Cookson, None; Marsha Kisilak, None; Francisco J. Avila,
None; Juan M. Bueno, None; Rachel Redekop, None; Matthew
Wilson, None
Support: NSERC Canada 052549, CIHR Canada 052509
Program Number: 2386 Poster Board Number: C0036
Presentation Time: 3:45 PM–5:30 PM
Comparing the Chemical Compositions of Human RPE
Lipofuscin and Melanolipofuscin
Michael Vega1, Elizabeth R. Gaillard1, 2. 1Chemistry and
Biochemistry, Northern Illinois University, DeKalb, IL; 2Biological
Sciences, Northern Illinois University, DeKalb, IL.
Purpose: To investigate and compare the chemical compositions of
human RPE lipofuscin and melanolipofuscin. Understanding their
chemical compositions will provide insight into the mechanisms or
pathways that generate these granules. As such, these mechanisms
or pathways could be targeted to prevent or retard pigment granule
accumulation in the RPE.
Methods: Human RPE lipofusicn and melanolipofuscin is extracted
from human donor eyes as previously described by Feeney-Burns.
Folch extraction is performed to obtain the organic soluble portion
of each pigment granule. The organic soluble lipofuscin and
melanolipofuscin is collected, dried under Argon, and reconstituted
in HPLC grade methanol for use in high performance liquid
chromatography tandem mass spectrometry (LC/MS/MS) coupled
a fluorescent detector (Surveyor LC with PDA, Thermo Finigan
LCQ Advantage MS, Surveyor FL). Tandem mass spectrometry
data is analyzed for the investigation of differences in chemical
compositions of human RPE lipofuscin and melanolipofuscin.
Results: Lipofuscin and melanolipofuscin extracts from human donor
tissue have been subjected to LC/MS/MS. Base peak chromatograms
observed from LC/MS/MS analysis suggest that both similarities and
differences exist between the chemical compositions of human RPE
lipofuscin and melanolipofuscin. This supports the working models
for their formation but also suggests that their respective formations
are more complex than originally perceived.
Conclusions: Human RPE melanolipofuscin is thought to form
as accreting human RPE lipofuscin fuses with human RPE
melanosomes. The similarities in chemical compositions observed
further support this working model. However, the differences
observed also suggest additional pathways or mechanisms. As such,
melanolipofusicn formation may be more complex than originally
perceived.
Commercial Relationships: Michael Vega, None; Elizabeth R.
Gaillard, None
Program Number: 2387 Poster Board Number: C0037
Presentation Time: 3:45 PM–5:30 PM
Examining the Role of Anthocyanins as an Antioxidant: Potential
Filtering Effects and Modifications to A2E
Sally Yacout1, Elizabeth R. Gaillard1, 2. 1Chemistry and Biochemistry,
Northern Illinois University, DeKalb, IL; 2Biological Sciences,
Northern Illinois University, Dekalb, IL.
Purpose: Previous studies on the protective effects of the
anthocyanins delphinidin galactoside and cyanidin galactoside on
blue light irradiated RPE cells indicated diminished light induced
damage due to the antioxidant properties of delphinidin galactoside
and cyanidin galactoside. However, it was not clear if the effects were
due to antioxidant behavior (chemical) or light filtering (physical)
effects. In addition the doses of delphinidin galactoside and cyanidin
galactoside used were non-physiological. This study distinguishes
between the filtering and anti-oxidative potential of anthocyanins
malvidin-3-O-glucoside (oenin) and pelargonidin-3-O-glucoside
(callistephin) at physiologically relevant concentrations in solution
and aims to assign structures to generated chemical product species.
Methods: Equal concentrations of A2E and anthocyanin were
subjected to blue light irradiation for 30 minutes, or A2E only was
irradiated for 30 minutes with subsequent addition of anthocyanin.
Electrospray ionization mass spectrometry (ESI-MS) and tandem
mass spectrometry (MS/MS) were used to analyze organic soluble
species. An optical filter transmitting light between 570 and 640
nanometers is placed over irradiated solution as a control.
Results: Irradiation of A2E and callistephin together resulted in
negligible A2E photo-oxidation. A2E oxidation products were not
observed with the subsequent addition of callistephin to irradiated
A2E. A decrease in oxidized A2E was observed after the addition of
oenin to irradiated A2E. These reactions generated a species with m/z
304, which was only present with anthocyanin. MS/MS of the 304
peak yields a fragmentation pattern of m/z peaks 212 and 91.
Conclusions: The addition of callistephin and oenin to irradiated
A2E results in a substantial decrease in A2E photo-oxidation
products, indicating antioxidant behavior of these anthocyanins. The
observed m/z 304 is unique to the anthocyanin-A2E reaction and this
species’ structure can be used to elucidate the interaction between
anthocyanins and A2E in addition to structural modifications to A2E.
Commercial Relationships: Sally Yacout, None; Elizabeth R.
Gaillard, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Program Number: 2388 Poster Board Number: C0038
Presentation Time: 3:45 PM–5:30 PM
Sub-lethal photodynamic stress mediated by phagocytized
lipofuscin granules induces oxidation of proteins in ARPE-19
cells
Anna K. Pilat, Magdalena M. Olchawa, Tadeusz J. Sarna.
Biophysics, Jagiellonian University, Krakow, Poland.
Purpose: Chronic oxidative stress in human RPE, induced by sublethal phototoxic reactions, mediated by lipofuscin, could contribute
to development of age related macular degeneration. To evaluate
photooxidizing capabilities of the age pigment, peroxidation of
proteins in ARPE-19 cells containing phagocytized lipofuscin
granules was determined by the use of a sensitive fluorescent probe.
Methods: Lipofuscin granules (LFG), isolated from human RPEs
from donors of different age, were enriched with a combination of
zeaxathin and alpha tocopherol (A). Control or antioxidant enriched
lipofuscin granules (A-LFG) were introduced to APRE-19 cells
by phagocytosis. Control cells, A-treated cells or cells with LFG
and A-LFG, irradiated with blue light for selected time intervals,
were analyzed for the presence of protein hydroperoxides using the
coumarin boronic acid (CBA) indicator. Photoreactivity of LFG and
A-LFG was tested in model systems by determining the granules
ability to photooxidize albumin.
Results: Irradiation of LFG containing cells with blue light induced
peroxidation of cellular proteins with the effect being light dose
dependent. The extent of protein oxidation mediated by lipofuscin
was higher for lipofuscin isolated from older donors (age: 30-39)
compared to younger donors (age: 20-29). Enrichment of lipofuscin
granules isolated from both age groups with zeaxathin and alpha
tocopherol reduced protein photoperoxidation. Consistent results
were also observed in model systems, in which photoperoxidation of
albumin was analyzed.
Conclusions: Photooxidation of proteins in ARPE-19 cells mediated
by phagocytized lipofuscin granules can be reproducibly analyzed
by CBA used as sensitive indicator of protein hydroperoxides. Such
analyses can be carried out on a moderate number of surviving
cells subjected to sub-lethal oxidative stress. Photoreactivity of
lipofuscin granules in cells and in model system can be modulated by
combination of antioxidants.
Commercial Relationships: Anna K. Pilat, None; Magdalena M.
Olchawa, None; Tadeusz J. Sarna, None
Support: Poland National Science Center (grant Maestro 2013/08/A/
NZ1/00194)
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.