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ARVO 2015 Annual Meeting Abstracts
407 Molecular biology of AMD
Wednesday, May 06, 2015 8:30 AM–10:15 AM
4AB Mile High Blrm Paper Session
Program #/Board # Range: 3986–3992
Organizing Section: Biochemistry/Molecular Biology
Program Number: 3986
Presentation Time: 8:30 AM–8:45 AM
The Cu/Zn+ superoxide dismutase knockout mouse (Sod1-/-), a
model of age-related macular degeneration (AMD), exhibits antiretinal autoantibodies (AAbs) and marked signs of intraretinal
inflammation prior to onset of an AMD-like phenotype
David New1, TJ Hollingsworth1, Francesco Giorgianni2, Nataliya
Lenchik3, Ivan Gerling3, Sarka Beranova-Giorgianni2, Marko
Radic4, Alessandro Iannaccone1. 1Ophthalmology, University of TN
Health Science Center, Memphis, TN; 2Pharmaceutical Sciences,
University of TN Health Science Center, Memphis, TN; 3Medicine/
Endocrinology, University of TN Health Science Center, Memphis,
TN; 4Molecular Biology, Immunology and Biochemistry, University
of TN Health Science Center, Memphis, TN.
Purpose: To understand the potential role of anti-retinal AAbs and
intraretinal inflammation in the Sod1-/- mouse model of AMD,
and to test the hypothesis that anti-retinal AAbs and intraretinal
inflammation could develop early in the disease and precede the onset
of the overt phenotype exhibited by these mice, typically at or after
12 mo. of age.
Methods: We examined differences between 6-9 mo. old Sod1-/- and
wild type (WT, C57BL/6J) mice at the serological, molecular, cellular
and tissue by several methods: retinal immunohistochemistry (IHC)
for markers of inflammation and oxidative stress (carbonylation),
Western blots (WB) and IHC for serum AAb binding.
Results: Before the onset of visible retinal disease, Sod1-/- mice
show marked upregulation of the glial fibrillary acidic protein
(GFAP) and glutamine synthase (GS) Müller cell reactivity (Fig. 1A),
increased protein carbonylation (Fig. 1B). and serum AAbs that bind
multiple retinal targets (Fig. 2).
Conclusions: Sod1-/- mice exhibit signs of intraretinal inflammation
and oxidation and display anti-retinal serum AAbs well in advance of
developing a retinal degenerative phenotype. These findings strongly
suggest that inflammation and an autoimmune component can
play at least a con-causal role in the pathogenesis of the phenotype
observed in Sod1-/- mice, feeding forward into the late-onset retinal
degeneration of this mouse model of AMD. Since AAbs recognizing
macular tissue antigens are found also in patients with early stage
AMD, our findings support the notion that similar mechanisms may
be at play early on, and thus be a target for intervention, in human
disease.
Fig. 1. Retinal gliosis and carbonylation in 9-mo Sod1-/- vs.
WT mice. Sod1-/- retinas show (A) marked upregulation of two
Müller cell markers, glial fibrillary acidic protein (GFAP, green)
and glutamine synthase (GS, red) and (B) marked increase in
carbonylation (dark grey stain).
©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 [email protected].
ARVO 2015 Annual Meeting Abstracts
Fig. 2. Immunostaining of WT retinas with WT or 9-mo Sod1-/sera. Sod1-/- serum stained discretely the GCL (white arrowheads),
diffusely but faintly the IPL (asterisks), intensely the OPL (arrows),
and in a linear and punctate pattern along the OLM (green
arrowheads).
Commercial Relationships: David New, None; TJ Hollingsworth,
None; Francesco Giorgianni, None; Nataliya Lenchik, None; Ivan
Gerling, None; Sarka Beranova-Giorgianni, None; Marko Radic,
None; Alessandro Iannaccone, None
Support: Support from NEI/NIH grant 1R01EY022706
(AI) and Research to Prevent Blindness, Inc. New York, NY
(Physician Scientist Award to AI and unrestricted grant to UTHSC
Ophthalmology/Hamilton Eye Institute), a UTHSC College of
Pharmacy Seed Grant (FG), NIH instrumentation grant S10RR16679
and UTHSC College of Pharmacy for LTQ mass spectrometer.
Program Number: 3987
Presentation Time: 8:45 AM–9:00 AM
In-silico model of retinal cholesterol dynamics: Insights into the
pathophysiology of dry AMD
Seyedeh M. Zekavat1, James Lu2, Cyrille Maugeais3, Norman Mazer2.
1
Biological Engineering, Massachusetts Institute of Technology,
Cambridge, MA; 2Clinical Pharmacology, Roche Innovation
Center Basel, Basel, Switzerland; 3Department of Neuroscience,
Ophthalmology and Rare Diseases, Roche Innovation Center Basel,
Basel, Switzerland.
Purpose: AMD is the leading cause of blindness in the elderly and
begins with the currently un-treatable “dry form,” characterized by
cholesterol (Ch) deposits beneath the retinal pigment epithelium
(RPE). To better understand the normal physiology of retinal
Ch dynamics (RCD) and the pathophysiology of dry AMD, we
developed a novel in-silico model based on a quantitative analysis of
literature data and mechanistic hypotheses of RCD.
Methods: Our model (Figure) uses a transit-chain to represent the
turnover and recycling of Ch in Rod Outer Segment (ROS) discs;
permeability coefficients (P1 and P2) to characterize transcytosis of
LDL across the choroid; Michaelis-Menten (MM) kinetics to describe
LDL-receptor mediated Ch uptake by the RPE; mass-balance for the
rate of Ch deposition in Bruch’s membrane (BrM); and MM kinetics
for the rate of macrophage-mediated removal of Ch deposits (via
ABCA1/ApoA-I). Calculations are made using MATLAB.
Results: Based on the shedding rate of ROS discs, their Ch content
and the rod density, we calculate that the retinal Ch turnover rate will
range from 1 pg/min/mm^2 retina (complete Ch recycling) to 6 pg/
min/mm^2 retina (no Ch recycling). From in-vitro studies of LDL
transcytosis across capillary endothelia, we estimate P1 to be ~1x10^7 cm/sec, which is sufficient to allow LDL-receptor-mediated uptake
by the RPE to provide Ch at a rate comparable to the estimated Ch
turnover in the ROS. Assuming that the rate of Ch secretion from
RPE to BrM is equal to the estimated turnover rate in the retina,
and that the secreted Ch is retained in the BrM, the thickness of the
deposited Ch layer is calculated to increase by 0.7 - 4.2 um per year,
consistent with the slow formation of drusen over decades. Finally,
the rate at which deposited Ch can be removed by macrophages
is proportional to their density in the BrM. Assuming 10,000
macrophages/mm^2, a 125 um-thick druse could be cleared in ~10
months, consistent with the maximum rates observed in OCT studies.
Conclusions: Our model suggests that retinal Ch turnover in the
ROS is central to understanding the normal physiology of RCD and
the slow rate of Ch deposition in dry AMD. Our model also suggests
that the Ch removal rate in drusen is consistent with macrophagemediated ABCA1/ApoA-1 transport. Further data would be useful to
support and test these concepts.
Model of retinal cholesterol dynamics and cartoon of retinal
structures.
Commercial Relationships: Seyedeh M. Zekavat, Roche (F);
James Lu, Roche (E), Roche (I); Cyrille Maugeais, Roche (E),
Roche (I); Norman Mazer, Roche (E), Roche (I)
Support: Summer Internship, Roche Innovation Center Basel
Program Number: 3988
Presentation Time: 9:00 AM–9:15 AM
Rare variants in complement genes associated with age-related
macular degeneration result in a lower age at onset and higher
familial occurrence
Maartje Geerlings1, Nicole T. Saksens1, Bjorn Bakker1, Tina Schick2,
Sascha Fauser2, Camiel J. Boon3, Eiko de Jong1, Carel C. Hoyng1,
Anneke I. Den Hollander1. 1Ophthalmology, Radboudumc, Nijmegen,
Netherlands; 2Ophthalmology, University Hospital of Cologne,
Cologne, Germany; 3Ophthalmology, Leiden University Medical
Center, Leiden, Netherlands.
Purpose: Recently, rare variants in the CFH (Arg1210Cys), CFI
(Gly119Arg), C3 (Lys155Gln) and C9 (Pro167Ser) genes were
found to be highly associated with age-related macular degeneration
(AMD). The aim of the current study was to determine the
contribution of these rare variants in the development of AMD in 22
multiplex families. In addition, we aimed to describe differences in
clinical characteristics in carriers versus non-carriers of rare genetic
variants, in these multiplex families and in a retrospective casecontrol cohort.
Methods: We included 707 AMD patients and 518 control
individuals (>50 years) from the European Genetic Database
©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 [email protected].
ARVO 2015 Annual Meeting Abstracts
(EUGENDA) database, including 114 affected and 60 unaffected
members of 22 multiplex AMD families. All individuals underwent
an ophthalmic examination including grading according to the
standard protocol of the Cologne Image Reading Center and
Laboratory, and completed a questionnaire on non-genetic risk
factors, family history for AMD and age at onset of first symptoms.
Venous blood was obtained for genetic analysis and measurement of
complement activation levels.
Results: Rare variants CFI Gly119Arg, C9 Pro167Ser and C3
Lys155Gln were identified in five out of 22 multiplex families, but
did not completely segregate with the disease phenotype. AMD
patients who carried rare variant CFI Gly119Arg, C9 Pro167Ser
or C3 Lys155Gln had a significantly lower age at first symptoms
(65.7 vs 71.8 years; p = 0.011), and more often had a positive family
history for AMD (52.5% vs 19.8%; p < 0.001) than patients who
did not carry these rare variants. Advanced AMD patients with
geographic atrophy carried these rare variants more frequently than
patients with neovascular AMD (p = 0.03).
Conclusions: Rare genetic variants CFI Gly119Arg, C9 Pro167Ser
and C3 Lys155Gln are more prevalent in patients with a positive
family history for AMD, but do not completely segregate within
families. Patients who carry one of these rare variants differ clinically
from patients without this rare variant, as they have a lower age at
first symptoms and more often progress to geographic atrophy.
Commercial Relationships: Maartje Geerlings, None; Nicole T.
Saksens, None; Bjorn Bakker, None; Tina Schick, None; Sascha
Fauser, None; Camiel J. Boon, None; Eiko de Jong, None; Carel
C. Hoyng, None; Anneke I. Den Hollander, None
Support: European Research Council ERC Grant Agreement n.
310644 (MACULA) & Foundation Fighting Blindness USA - Grant
C-GE-0811-0548-RAD04
Program Number: 3989
Presentation Time: 9:15 AM–9:30 AM
miRNA-34a down-regulates the triggering receptor and
phagocytosis sensor TREM2 in age-related macular degeneration
(AMD)
Walter J. Lukiw1, Surjyadipta Bhattacharjee2, Yuhai Zhao2, Maire
E Percy3, Evgeny I. Rogaev4, Prerna Sethi-Dua5. 1Neurology,
Neuroscience & Ophthalmology, Louisiana State Univ Hlth Sci
Ctr, New Orleans, LA; 2LSU Neuroscience Center, Louisiana
State University Health Sciences Center, New Orleans, LA;
3
Neurogenetics, University of Toronto, Toronto, ON, Canada;
4
University of Massachusetts, Worcester, MA; 5Bioinformatics and
Health Information Management, LA Technical University, Ruston,
LA.
Purpose: Pathogenic aggregation of Aβ42-peptides & drusen
formation in AMD is due in part to the inability of phagocytic
mechanisms to clear neurotoxic & self-aggregating Aβ42-peptides
from the extracellular space. One key participant in Aβ42 clearance
is the triggering receptor expressed in microglial cells-2 (TREM2),
a transmembrane sensor-receptor of the immune-globulin gene
superfamily. Here we have examined microRNA (miRNA)-mediated
amyloid peptide clearance mechanisms involving TREM2 in aging
5xFAD retina, in AMD & in Aβ42-peptide- or cytokine-stressed
microglial cells.
Methods: Aβ42 analysis, bioinformatics, DNA & miRNA
arrays, ELISA, microglial culture, Northern & Western
immunocytochemistry, RNA sequencing, RT-PCR, transfection
Results: Parallel DNA & miRNA array, RT-PCR, Northern &
Western analysis indicated up-regulation of an NF-B-sensitive
miRNA-34a & down-regulation of TREM2 in the same samples.
Aging 5xFAD mice exhibited progressive decreases in retinal
TREM2; transfection using luciferase reporters showed that miRNA34a targets the TREM2 mRNA 3’UTR to down-regulate TREM2
expression. C8B4 microglial cells challenged with Aβ42 were able to
phagocytose these neurotoxic peptides, while miRNA-34a downregulated both TREM2 abundance and the ability of microglial
cells to phagocytose. Treatment of stressed microglial cells with the
NF-kB inhibitor/resveratrol analog CAY10512, the anti-inflammatory
caffeic-acid phenethyl ester (CAPE) or the natural phenolic
antioxidant diferuloylmethane (curcumin) abrogated these responses;
anti-miRNA-34a strategies were found to normalize induced miRNA34a levels & restore homeostatic TREM2 expression.
Conclusions:
For the first time we report a miRNA-34a-mediated down-regulation
of TREM2 in AMD, in aging 5xFAD retina & in stressed microglia.
Our data support 4 novel observations: (i) that a NF-kB-sensitive,
miRNA-34a-mediated modulation of TREM2 regulates a phagocytic
response; (ii) that gene products encoded on 2 different chromosomes
(TREM2 at chr6p21.1 and miRNA-34a at chr1q36.22) orchestrate
an Aβ42-clearance system in the retina; (iii) that this NF-kBmiRNA-34a-TREM2 system is inducible & effectively clears Aβ42
peptide monomers from the extracellular medium; & (iv) that these
results underscore the potential for anti-NF-kB/anti-miRNA-based
therapeutic strategies against deficits in phagocytic signaling that
drive amyloidogenesis.
Commercial Relationships: Walter J. Lukiw, None; Surjyadipta
Bhattacharjee, None; Yuhai Zhao, None; Maire E Percy, None;
Evgeny I. Rogaev, None; Prerna Sethi-Dua, None
Support: Sincere thanks are extended to Drs. L. Carver, E. Head,
W. Poon, H. LeBlanc, F. Culicchia, C. Eicken, S. Bhattacharjee
and C. Hebel for short post-mortem interval (PMI) human brain
and retinal tissues or extracts, miRNA array work and initial data
interpretation, and to D Guillot and AI Pogue for expert technical
assistance. Thanks are also extended to the many neuropathologists,
physicians and researchers of Canada and the USA who have
provided high quality, short post-mortem interval (PMI) human CNS
tissues or total brain and retinal RNA for scientific study; additional
human CNS tissues were provided by the Memory Impairments
and Neurological Disorders (MIND) Institute at the University of
California, Irvine Alzheimer’s Disease Research Center (UCI-ADRC;
NIA P50 AG16573). Research on miRNA in the Lukiw laboratory
involving the innate-immune response in AD and in retinal disease,
amyloidogenesis and neuro-inflammation was supported through a
COBRE III Pilot Project NIH/NIGMS Grant P30-GM103340, an
unrestricted grant to the LSU Eye Center from Research to Prevent
Blindness (RPB); the Louisiana Biotechnology Research Network
(LBRN) and NIH grants NEI EY006311, NIA AG18031 and NIA
AG038834.
©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 [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 3990
Presentation Time: 9:30 AM–9:45 AM
A genetic variant in NRP1 is associated with worse response
to ranibizumab treatment in neovascular age-related macular
degeneration
Laura Lorés de Motta1, Freekje Van Asten1, Philipp S. Muether2,
Dzenita Smailhodzic1, John C. Chen3, Robert K. Koenekoop4, Sascha
Fauser2, Carel C. Hoyng1, Anneke I. Den Hollander1, 5, Eiko de
Jong1. 1Department of Ophthalmology, Radboud university medical
center, Nijmegen, Netherlands; 2Department of Ophthalmology,
University Hospital of Cologne, Cologne, Germany; 3Department
of Ophthalmology, McGill University Health Center, Montreal,
QC, Canada; 4Departments of Pediatric Surgery, Human Genetics,
and Ophthalmology, McGill University Health Centre, Montreal,
QC, Canada; 5Department of Human Genetics, Radboud university
medical center, Nijmegen, Netherlands.
Purpose: The highly variable response to anti-vascular endothelial
growth factor (VEGF) drugs in neovascular age-related macular
degeneration (nvAMD) patients is, in part, due to genetic
predisposition. Several studies have implicated genetic variability in
genes associated with VEGF signaling, such as KDR (VEGFR2), in
this process but the exact mechanisms remain elusive. The aim of this
study was to investigate the role of single nucleotide polymorphisms
(SNPs) located in neuropilin-1 (NRP1), a co-receptor for VEGFA,
in treatment response to anti-VEGF therapy in a cohort study of
nvAMD patients treated with ranibizumab (Lucentis).
Methods: The SNPs rs2229935, rs2247383, rs2070296 and
rs2804495 located in the NRP1 gene were genotyped in 377 nvAMD
patients who received the loading dose of three monthly ranibizumab
(Lucentis) injections. Treatment response was assessed as the change
in visual acuity after three monthly loading injections compared to
baseline. The association of the SNPs with the outcome variable was
evaluated using Mann-Whitney U and Kruskal-Wallis tests.
Results: Patients carrying the GA or AA genotypes of SNP
rs2070296 performed significantly worse than individuals carrying
the GG genotype (p=0.01) after three months of treatment. A
cumulative effect of rs2070296 in the NRP1 gene and rs4576072
located in the KDR gene, previously associated with treatment
response, was observed. Patients carrying two risk alleles performed
significantly worse than patients carrying zero or one risk allele
(p=0.03) and patients with more than two risk alleles responded even
worse to the therapy (p=3x10-3).
Conclusions: This study demonstrates that genetic variation in
NRP1, a key molecule in VEGFA-driven neovascularization,
influences treatment response to ranibizumab in nvAMD patients.
The results of this study may be used to generate prediction models
for treatment response, which in the future may help tailor medical
care to individual needs.
Commercial Relationships: Laura Lorés de Motta, None; Freekje
Van Asten, None; Philipp S. Muether, None; Dzenita Smailhodzic,
None; John C. Chen, None; Robert K. Koenekoop, None; Sascha
Fauser, Bayer (C), Novartis (C); Carel C. Hoyng, None; Anneke I.
Den Hollander, None; Eiko de Jong, None
Support: European Union’s Seventh Framework Programme for
research, technological development and demonstration under grant
agreement no 317472 (EyeTN). Foundation Fighting Blindness
Canada and the Canadian Institutes for Health Research.
Program Number: 3991
Presentation Time: 9:45 AM–10:00 AM
Intravitreally delivered neprilysin reduces amyloid-beta in the
mouse eye
Rajni Parthasarathy1, 2, K. Martin Chow3, Zahra Derafshi2, Michael
P. Fautsch4, John R. Hetling2, David W. Rodgers3, Louis B. Hersh3,
David R. Pepperberg1, 2. 1Ophthalmology and Visual Sciences,
University of Illinois at Chicago, Chicago, IL; 2Bioengineering,
University of Illinois, Chicago, Chicago, IL; 3Molecular and
Cellular Biochemistry, University of Kentucky, Lexington, KY;
4
Ophthalmology, Mayo Clinic, Rochester, MN.
Purpose: Amyloid-beta peptide (Aβ), generated in the eye and other
tissues, has been hypothesized to exert toxic effects that contribute to
progression and pathology of multiple retinal degenerative diseases.
Neprilysin (NEP), a native endopeptidase that cleaves Aβ into
inactive products, is a membrane-anchored protein. However, the
extracellular domain of NEP (sNEP) is soluble and retains catalytic
activity. We tested the ability of intravitreally injected recombinant
sNEP to reduce, in vivo, ocular levels of Aβ40 and Aβ42 (respectively,
40 and 42 amino acids in length), two principal Aβ forms.
Methods: Anesthetized 10-month wildtype (C57BL/6J) mice, and
2-3-month 5XFAD transgenic mice expressing human Aβ42, received
intravitreal injections (2 mL) of phosphate-buffered saline (PBS)
containing sNEP (0.004 to 10 mg). The fellow, untreated eye served
as control. Treated mice were maintained for 30 min up to 12 weeks.
Shortly before euthanasia, the treated eye intravitreally received
sNEP inhibitor (phosphoramidon; PA) to block further sNEP activity.
Harvested eye tissues (combined lens/vitreous, retina and RPE/
choroid) were homogenized and extracted with PBS. Extracts were
analyzed for protein (Bradford) and for Aβ40 and Aβ42 (ELISA).
sNEP activity remaining at defined post-treatment times (with no PA
delivery) was analyzed by a fluorometric assay. Retinal function in
sNEP-treated eyes was analyzed by electroretinography (ERG).
Results: Untreated C57BL/6J eyes (n=78) exhibited Aβ40 at 42.8 ±
26.3 (mean ± SEM) pmol per g protein (pmol/g); those of 5XFAD
mice (n=10) exhibited substantial Aβ42 (14.6 ± 8.4 pmol/g) as well
as Aβ40 (73.4 ± 35.5 pmol/g). In C57BL/6J mice, increasing sNEP
yielded progressively greater Aβ40 reductions at 2 hr post-treatment
[reductions of 12% ± 3% (n=3) with 4 ng sNEP, and 85% ± 3% (n=5)
with 10 mg sNEP]. sNEP activity declined by 50% within ~8 hr after
delivery and was undetectable after 2 days, but Aβ40 remained low
(~80% reduction) for up to ~8 weeks. In 5XFAD mice at 24 hr after
10 mg sNEP treatment, the reduction of Aβ40 (99% ± 1%) exceeded
that for Aβ42 (42% ± 36%) (n=4; p=0.002). sNEP treatment of
C57BL/6J and 5XFAD eyes preserved robust ERG responsiveness.
Conclusions: sNEP delivery to the mouse eye yields substantial
in vivo reductions in Aβ40 and Aβ42 levels. The results encourage
further study of intravitreal sNEP treatment for investigational and,
potentially, therapeutic applications.
Commercial Relationships: Rajni Parthasarathy, None; K.
Martin Chow, None; Zahra Derafshi, None; Michael P. Fautsch,
None; John R. Hetling, None; David W. Rodgers, None; Louis B.
Hersh, None; David R. Pepperberg, None
Support: BrightFocus Foundation (Clarksburg, MD), Illinois Society
for the Prevention of Blindness (Chicago, IL), Beckman Initiative for
Macular Research (Los Angeles, CA); Research to Prevent Blindness
(New York, NY); NIH grants EY001792, EY023430, GM110787 and
EY021727.
©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 [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 3992
Presentation Time: 10:00 AM–10:15 AM
Multimodal molecular imaging of Bruch’s membrane can predict
age and macular degeneration
Hannah E. Bowrey1, E E. Jones2, Mark Fields1, Rosalie K.
Crouch1, Lucian V. Del Priore1, Zsolt Ablonczy1. 1Department
of Ophthalmology, The Medical University of South Carolina,
Charleston, SC; 2Department of Cell and Molecular Pharmacology,
The Medical University of South Carolina, Charleston, SC.
Purpose: During the course of aging the physiological functions of
Bruch’s membrane (BM) gradually deteriorate, in part due to drusen
accumulation between the retinal pigment epithelium (RPE) and BM.
A profound accumulation of large soft drusen in the posterior pole
is a key early clinical feature of age-related macular degeneration
(AMD). Identification of the molecular composition of BM, including
drusen deposits, is necessary for understanding AMD pathogenesis.
Therefore, we sought to determine molecules in BMs that underlie its
fluorescent lesions from a spectrum of ages, with- or without AMD.
Methods: Matrix-assisted laser desorption/ionization (MALDI)
imaging mass spectrometry (IMS) allows molecule-specific imaging
of biological surfaces and can determine the spatial localization
of thousands of molecules in a single experiment. We adapted an
established protocol developed for imaging RPE by MALDI-IMS
(Ablonczy et al. Proteomics 14: 936-944, 2014), used here on ex vivo
human BMs (n = 15; mass range: m/z 200-1500; spatial resolution:
350 mm; positive mode). Individual autofluorescence images were
co-registered, and correlated with each individual image in the
MALDI imaging dataset from the same tissue. The total abundance
of each molecular species was calculated and correlated with patient
age. Finally, principal component analysis (PCA) was carried out to
identify specific molecular patterns in BMs.
Results: Within each patient sample, at least 500 molecules exhibited
significant spatial correlation with autofluorescence images. In all,
311 molecules were shown to correlate with patient age. Of these,
three molecular species - 268 m/z, 410 m/z and 426 m/z - increased
with age (R = 0.77, 0.78 and 0.75, respectively; p < 0.05, all cases),
whilst all others decreased with patient age. Bisretinoids were not
found in high abundance and did not correlate with age. PCA analysis
could easily distinguish BMs of different ages and disease states.
Conclusions: Imaging of BM by MALDI-IMS can be utilized as
an important tool for the identification of molecular constituents
involved in age- and disease-related changes of BM, as these
methods can reliably predict the clinical diagnosis of the tissue. Our
results show that age-related changes are associated with the loss
of many small molecules. However, only three molecules exhibited
an increase with age, suggesting their potential importance in the
pathobiology of AMD.
Commercial Relationships: Hannah E. Bowrey, None; E E. Jones,
None; Mark Fields, None; Rosalie K. Crouch, None; Lucian V. Del
Priore, None; Zsolt Ablonczy, 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 [email protected].