Involvement of Plasmalogens in Post-Natal Retinal
Vascular Development
Sarah Saab1,2,3, Be´ne´dicte Buteau1,2,3, Laurent Lecle`re1,2,3, Alain M. Bron1,2,3,4,
Catherine P. Creuzot-Garcher1,2,3,4, Lionel Bretillon1,2,3, Niyazi Acar1,2,3*
1 CNRS, UMR6265 Centre des Sciences du Gouˆt et de l’Alimentation, Dijon, France, 2 INRA, UMR1324 Centre des Sciences du Gouˆt et de l’Alimentation, Dijon, France,
3 Universite´ de Bourgogne, UMR Centre des Sciences du Gouˆt et de l’Alimentation, Dijon, France, 4 Department of Ophthalmology, University Hospital, Dijon, France
Objective: Proper development of retinal blood vessels is essential to ensure sufficient oxygen and nutrient supplies to the
retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A
congenital deficiency in ether-phospholipids, also termed ‘‘plasmalogens’’, was shown to lead to abnormal ocular
vascularization. Because plasmalogens are considered to be reservoirs of PUFAs, we wished to improve our understanding
of the mechanisms by which plasmalogens regulate retinal vascular development and whether the release of PUFAs by
calcium-independent phospholipase A2 (iPLA2) could be involved.
Methods and Results: By characterizing the cellular and molecular steps of retinal vascular development in a mouse model
of plasmalogen deficiency, we demonstrated that plasmalogens modulate angiogenic processes during the early phases of
retinal vascularization. They influence glial activity and primary astrocyte template formation, endothelial cell proliferation
and retinal vessel outgrowth, and impact the expression of the genes involved in angiogenesis in the retina. These early
defects led to a disorganized and dysfunctional retinal vascular network at adult age. By comparing these data to those
obtained on a mouse model of retinal iPLA2 inhibition, we suggest that these processes may be mediated by PUFAs
released from plasmalogens and further signalling through the angiopoietin/tie pathways.
Conclusions: These data suggest that plasmalogens play a crucial role in retinal vascularization processes.
Citation: Saab S, Buteau B, Lecle`re L, Bron AM, Creuzot-Garcher CP, et al. (2014) Involvement of Plasmalogens in Post-Natal Retinal Vascular Development. PLoS
ONE 9(6): e101076. doi:10.1371/journal.pone.0101076
Editor: Erica L. Fletcher, The University of Melbourne, Australia
Received February 28, 2014; Accepted June 3, 2014; Published June 25, 2014
Copyright: ß 2014 Saab et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: A PhD fellowship to Sarah Saab-Aoude´ was funded by a grant from the French National Institute for Agricultural Research (INRA, France) and the
Regional Council of Burgundy (France). This work was supported by a French Government grant managed by the French National Research Agency (ANR) under
the program ‘‘Investissements d’Avenir’’ with reference ANR-11-LABEX-0021, and by grants from Abbott Laboratories (Dijon, France), the Regional Council of
Burgundy (France), and the FEDER (European Funding for Regional Economic Development). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
While developing, the retinal vasculature associates several cell
types. The first stage of retinal vascular development is the
formation of the astrocytic bed [4]. The migration of astrocytes
from the optic nerve to the retinal periphery is closely followed by
the formation of the primary vascular network by endothelial cells
[5]. Distinct microglial populations also migrate across the retina
prior to or concomitantly with the vessels [6]. Finally, the
stabilization of immature vessels by pericytes appears either
simultaneously with blood flow or soon thereafter [7].
Dysregulation of retinal vascularization is a common feature of
several blinding diseases including retinopathy of prematurity
(ROP), diabetic retinopathy (DR) and age-related macular
degeneration (AMD) [8–10]. In DR and ROP, neovascular events
occur at the level of retinal vessels and result in complications such
as vitreous haemorrhages, torsional retinal detachment and
subsequent blindness [9,10], whereas choroidal neovascularization
is responsible for vision loss in patients with neovascular AMD [8].
Since vascular development is tightly regulated by complex
molecular interactions stimulating or inhibiting vasculogenesis
and angiogenesis, the pathophysiological mechanisms involved in
Vascular growth occurs through two complementary mechanisms: vasculogenesis and angiogenesis [1]. Vasculogenesis corresponds to the initial vascular tree formation by differentiation of
vascular endothelial lineage precursor cells, whereas fine endothelial cell extensions arise by sprouting from pre-existing vessels
during angiogenesis. In primates, the retina vascularizes as laminar
networks that sequentially radiate peripherally from the optic
nerve head [2]. Whereas all vascular laminae emerge post-natally
in several mammal species, the innermost plexus arises at
gestational age in humans, while the deeper vascular laminae
are formed at around 24 weeks of gestation and continue
developing after birth [2]. During retinal vascular development,
nutrients are supplied to the anterior eye by hyaloid vessels
extending from the optic disc. In the growing eye, the
development of the retinal vasculature coincides with hyaloid
vasculature regression [3]. The hyaloid vascular system fully
regresses before birth in humans and during the first post-natal
weeks in mice.
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Regulation of Retinal Angiogenesis by Plasmalogens
iPLA2, influence retinal vascular growth. These data demonstrated a temporary delay in retinal vessel outgrowth associated with
glial activation, then a secondary sub-numerous and defective
development of retinal capillaries, and subsequent scarring
processes involving glial and inflammatory cells.
these diseases may include an imbalance between pro- and antiangiogenic compounds.
Within the different factors influencing vascular growth,
polyunsaturated fatty acids (PUFAs) are drawing interest. In the
retina, the major PUFAs are found primarily in neuronal and
vascular cell membrane phospholipids from which they are
released by phospholipases A2 (PLA2) [11]. Recent discoveries
include in vitro data showing that PUFAs or their metabolites
control the expression of pro-angiogenic growth factors in vascular
cells [12–14], in vivo animal studies where dietary omega-3 PUFAs
reduced pathological angiogenesis [15], and a large-scale human
studies associating a higher dietary intake in omega-3 PUFAs with
a slower progression of neovascular AMD [16–18].
Not only PUFAs but also their phospholipid origin may be
important in the control of vascular growth. Indeed, phospholipids
in cell membranes can have different sub-types: conventional
phospholipids on which fatty acids are connected through ester
linkages or specific phospholipids termed ‘‘plasmalogens’’ where a
vinyl–ether bond replaces an ester linkage (Figure 1). We have
shown that plasmalogens accounts for 13% of retinal phospholipids and about 30% of retinal ethanolamine phospholipids [19].
Given that plasmalogens are also considered to be reservoirs of
PUFAs in membranes, they are suspected of having signalling
functions by releasing these PUFAs through a specific calciumindependent PLA2 (iPLA2) [20]. This hypothesis is reinforced by
studies showing higher iPLA2 activities in various pathologic
conditions involving plasmalogen metabolism [21,22]. The
importance of plasmalogens in retinal vascular development was
previously suggested in a mouse model of plasmalogen deficiency
(DAPAT2/2 mice). DAPAT2/2 mice are characterized by a
targeted disruption of the gene encoding for dihydroxyacetonephosphate acyltransferase, the first enzyme of plasmalogen
biosynthesis. The main phenotypic characteristics of DAPAT2/2
mice consisted of reduced levels of docosahexaenoic acid (DHA) in
neural tissue and complex and severe developmental defects of the
central nervous system, the testis and the eye. The ophthalmologic
examination of DAPAT2/2 mice elicited abnormalities including
persistent hyaloid vessels [23]. This feature is likely to be associated
with other defects in retinal vasculature that have not been
adequately investigated so far.
We therefore characterized blood vessel development in the eye
of DAPAT2/2 mice and compared the morphologic defects to
those of a mouse model of retinal iPLA2 inhibition we developed
previously [24]. By correlating these observations with the gene
expression of important angiogenic factors, we collected data
elucidating the processes by which plasmalogens, and subsequently
Materials and Methods
Experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology statements and
with French legislation (authorization number 21CAE086 for N.A.
and animal quarters agreement number A21231010 EA), after
approval by the local ethics committees (#105 Comite´ d’Ethique de
l’Expe´rimentation Animale Grand Campus Dijon). C57BL/6 mice (12
weeks old, 20–25 g) were obtained from Elevage Janvier (LeGenest-Saint-Isle, France). DAPAT heterozygous (DAPAT+/2)
mutants were kindly provided by Prof. W.W. Just (Heidelberg,
Germany). The animals were housed in animal quarters under
controlled temperature (2261uC) and light conditions (12-h light,
12-h dark cycle). Animals were fed ad libitum with standard
laboratory chow and water.
DAPAT+/2 mice were backcrossed with C57Bl/6 mice to
provide DAPAT wild-type (DAPAT+/+), and DAPAT+/2 animals.
DAPAT+/2 couples were further crossed to generate DAPAT+/+,
DAPAT+/2 and DAPAT knock-out (DAPAT2/2) mice. The
genotype of heterozygous, DAPAT+/+ and DAPAT2/2 mice
was determined according to Rodemer et al. (2003) with slight
modifications [23]. The genotype was determined using nested
PCR of genomic tail DNA using the primers neomycin-forward
Operon, Ebersberg, Germany), exon7-forward (CGATACCTACTTTGTCCCAATTAGC, Eurofins) and exon7-reverse
extraction was performed using the Archive Pure DNA Cell/
Tissue kit (5 Prime GmbH, Catalog no 2300820; Gaithersburg,
MD). Pure genomic DNA was dosed on the nanodrop spectrophotometer (ND 1000, Labtec; Palaiseau, France). For the
amplification step, 2 ng of genomic DNA, 100 pmol of each
primer in reaction buffer and 2.5 U of Taq polymerase (BiotaqTM
DNA Polymerase, BIO-21040, Bioline, Paris, France) were used in
a total volume of 25 ml. After 2 min of denaturation at 95uC, PCR
was performed on a C1000TM Thermel Cycler (Biorad Laboratories, Hercules, CA) through 35 cycles at 94uC for 30 s and 57uC
for 1 min, followed by a final extension step at 72uC for 1 min.
This resulted in a 650-bp product for the wild-type gene and an
860-bp product for the neomycin-recombinant DAPAT gene.
Figure 1. Structure of conventional phospholipids and plasmalogens. Conventional phospholipids such as phosphatidyl-ethanolamine
contain ester bonds to link R1 and R2 acyl-moieties at the sn-1 and sn-2 positions of glycerol, respectively. Ethanolamine-plasmalogens (also termed
plasmenyl-ethanolamine) are characterized by the presence of a vinyl-ether bond at the sn-1 position of the glycerol backbone to link alkenylmoieties and an ester bond at the sn-2 position to link acyl-residues.
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Regulation of Retinal Angiogenesis by Plasmalogens
PCR products were analysed using agarose gel electrophoresis and
gels were visualized on Gel doc 2000 (Biorad).
and then perfused with a FITC-dextran solution (molecular weight
2,000,000, Sigma Aldrich) through the left ventricle. Flat-mounted
retinas were visualized under a Leica confocal laser-scanning
microscope SP2, AOBS (Leica Microsystemes SAS), and processed with Leica LCSlite. After drawing a circle centred on the
optic nerve head and covering the entire retina, two additional
concentric circles with a radius equivalent to one and two-thirds of
the first circle were drawn to delimit retinal central, mid-peripheric
and far-peripheric areas. The number of capillaries was determined by drawing an additional concentric circle in the middle of
each zone and counting the number of capillaries crossing this ring
over 360u. The evaluations were made manually by two operators
on unlabelled pictures displayed at the same magnification.
Inhibition of retinal iPLA2 in DAPAT+/+ mice
Retinal iPLA2 was inhibited in vivo according to previously
described procedures [24]. Briefly, retinal iPLA2 was inhibited by
45–55% in DAPAT+/+ pups from birth to post-natal days 7 (PN7),
14 (PN14) or 21 (PN21), by repeated intraperitoneal administration of a bromoenol lactone solution (BEL, B1552, Sigma-Aldrich,
Saint-Quentin-Fallavier, France) at a concentration of 6 mg/g of
body weight in DMSO/saline (1:10, v:v). Controls were injected
with vehicle (DMSO/saline (1:10, v:v)) only.
Immunostainings on flat-mounted whole retinas
Microscope characteristics
The pups were euthanized by CO2 exposure, the eyeballs were
isolated and fixed in 4% paraformaldehyde. The corneas were
incised, the lenses taken out, and four radial cuts were made on the
eyecups. Vitreous bodies were removed with forceps, and retinas
were delicately isolated and flattened on microscope slides.
Flat-mounted retinas were stained to visualize endothelial cells,
astrocytes, pericytes and extracellular matrix, as described
elsewhere [25]. Briefly, retinas (n = 7–8) were incubated in a
blocking solution (1% BSA, PBS-Tween (0.5%), pH 6.8), washed
in PBLec solution (PBS, 0.1 mM CaCl2, 0.1 mM MgCl2, pH 6.8)
and then incubated in a biotin conjugated-isolectin-B4 solution
(ILB4, Lectin from Bandeiraea simplicifolia BS-I, L2140, SigmaAldrich) for endothelial cell labelling. ILB4 solution was prepared
at a concentration of 1 mg/ml in 0.9% saline and further diluted in
PBLec solution (1:50, v:v) prior to assay. The retinas were then
washed in PBS and incubated in a 0.01 mg/ml streptavidin-Alexa
568 solution (pH 7.2) (S11226, Invitrogen, Saint Aubin, France)
prepared in the blocking solution which in turn was prediluted to
50% in PBS. For astrocyte labelling, polyclonal rabbit anti-mouse
glial fibrillary acidic protein antibody was used (GFAP, 1:200,
Neomarkers, RB-087-A0,-A1, Illkirch, France). For pericyte and
extracellular matrix labelling, polyclonal rabbit anti-chondroitin
sulfate proteoglycan antibody (NG-2, 1:200 Chemicon, Molsheim,
France) and polyclonal goat anti-fibronectin antibody (1:100,
Santa Cruz Biotechnology, Le Perray-en-Yvelines, France) were
used, respectively. Secondary antibodies were Alexa 488-labelled
goat anti-rabbit (1:200, Invitrogen A11008) and Alexa 488labelled donkey anti-goat (1:200, Invitrogen A11055). Following
the labelling steps, the retinas were rinsed in PBS and coverslipped
using a fluorescence-mounting medium. Controls for these
experiments consisted of removing primary antibodies. Fluorescence microphotographs were taken using a Nikon microscope
(model Eclipse E600, Nikon, Champigny-sur-Marne, France) and
a Nikon digital camera (model OXm 1200C, Nikon) equipped
with the Nikon Nis-element BR V2.2 software. Confocal
fluorescent micrographs were obtained using a Leica scanning
laser confocal microscope SP2 AOBS (Leica Microsystemes SAS,
Nanterre, France) and processed with Leica LCSlite.
For the Leica confocal laser-scanning microscope SP2, Acousto
Optical Beam Splitters (AOBS), we used the following objectives:
10 HC PL APO CS 1060.4 dry, 20 HC PL APO CS 2060.7 dry
and 40 HC PL APO CS 4061.25 oil. The light source excitation
for the Alexa 488 and the Alexa 568 fluorochromes was an Argon
laser set at 488 (emission spectrum, 507–547 nm) and a helium/
xenon laser set at 543 (emission spectrum, 574–657 nm, 600–
701 nm or 608–686 nm), respectively. The Nikon microscope
(Eclipse E600, Champigny-sur-Marne, France) was equipped with
a Nikon digital camera (Nikon OXm 1200C equipped with the
Nikon Nis-element BR software V2.2). We used the 460.1 dry,
1060.3 dry and 2060.5 dry objectives. A mercury lamp was used
as a light source, with an excitation spectrum of 450–490 nm with
DM 505 and BA 520 for Alexa 488 fluorochrome and 510–
560 nm with DM 575 and BA 590 for Alexa 568 fluorochrome.
Gene expression analyses in retinas
Extraction of total RNAs. After the animals were deeply
sedated with an intraperitoneal injection of a ketamine (70 mg/g of
body weight) and xylazine (14 mg/g of body weight) solution,
retinas were isolated from the eyeballs and pooled for one animal
(n = 4–6 per group). Total RNAs were isolated using the Ambion
RNAqueous kit (AM1912, Life Technologies) according to the
manufacturer’s instructions. The quantity and the quality of RNAs
were evaluated on a nanodrop spectrophotometer (ND1000,
Thermo Fisher, Illkirch, France).
cDNAs synthesis and quantitative real-time polymerase
chain reaction (PCR). For quantitative real-time PCR, RNAs
were reverse-transcripted to cDNA using the Invitrogen SuperScript VILOTM Master Mix (No. 11755, Life Technologies). The
expression of genes coding for known pro- and anti-angiogenic
factors [25], inflammatory factors, the glial cell marker and
endogenous control genes (Table 1) was quantified using 5 ng of
total cDNAs in 10 ml of 1X TaqMan Fast Advanced Master Mix
(No. 4444557, Applied Biosystems, Life Technologies). Quantitative real time-PCR was performed on TaqMan Array 96-well
FAST Plates (Applied Biosystems, Life Technologies), using the
StepOnePlus Real-Time PCR System equipped with the StepOne
software V2.2.2 (Applied Biosystems, Life Technologies). Data
were analysed using DataAssist software V3.0 (Applied Biosystems,
Life Technologies). Genes coding for glucuronidase-beta, beta-2microglobulin and hypoxanthine guanine phosphoribosyl-transferease-1 were used as endogenous controls for normalization and
relative quantification (RQ) with the Cycle Threshold (CT)-method.
Capillary morphometry on flat-mounted whole retinas
Retinal vascular network outgrowth was evaluated on ILB4stained flat-mounted retinas by calculating the ratio of outgrowth
distance to retinal radius. ILB4-positive angiogenic sprouts were
counted manually by two operators on unlabelled pictures of flatmounted retinas displayed at the same magnification and taken
from DAPAT+/+, DAPAT2/2 mice, and mice with retinal iPLA2
inhibition (n = 10–12 per group).
To evaluate retinal capillary density, adult DAPAT+/+ and
DAPAT2/2 mice were deeply sedated using a ketamine (70 mg/g
of body weight) and xylazine (14 mg/g of body weight) solution
SLO imaging of hyaloid vasculature
The regression of hyaloid vasculature was followed between
PN21 and PN27 using a Heidelberg Retina Angiograph confocal
scanning laser ophthalmoscope (cSLO, Heidelberg Engineering,
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Regulation of Retinal Angiogenesis by Plasmalogens
Table 1. Symbol, name, assay ID, and GenBank reference of assayed genes.
Gene name;
celera annotation
angiopoietin 1; mCG113640
angiopoietin 2; mCG1200
ephrin B2; mCG17314
Eph receptor B4; mCG6855
fibroblast growth factor 2;
FMS-like tyrosine kinase 1;
fibronectin 1; mCG121782
frizzled homolog 4 (Drosophila);
glial fibrillary acidic protein;
integrin alpha V; mCG7872
integrin beta 3; mCG11220
kinase insert domain protein receptor;
platelet-derived growth factor;
B polypeptide; mCG11519
platelet-derived growth factor
receptor; beta polypeptide;
phospholipase A2; group VI;
NM_001199025.1; NM_016915.4
endothelial-specific receptor
tyrosine kinase; mCG122568
thrombospondin 1; mCG14570
tyrosine kinase with
and EGF-like domains 1; mCG120003
vascular endothelial growth factor A;
NM_001110267.1; NM_009505.4;
NM_001110266.1; NM_001025250.3
Assay ID
Control genes
beta 2 microglobulin
glucuronidase beta
hypoxanthine guanine
phosphoribosyl transferase 1
Consult, Brandenburg, Germany). The electroretinograms (ERGs)
(n = 6 per group) were obtained according to previously published
procedures [27].
Dossenheim, Germany) as previously described [25,26]. Prior to
cSLO angiography, animals were anaesthetized with a ketamine
(70 mg/g of body weight) and xylazine (14 mg/g of body weight)
solution. After the pupils were dilated with tropicamide (Mydriaticum, Thea Laboratories, Clermont-Ferrand, France), a custommade contact lens was placed on the cornea using methylcellulose
solution (Methocel 2%, OmniVision, Puchheim, Germany).
Fluorescein angiography was performed using the Argon laser of
the HRA (488 nm; barrier filter: 500 nm), and after a subcutaneous injection of a fluorescein-Na solution in 0.9% NaCl at a
dose of 75 mg/kg of body weight.
Statistical analysis
The results are expressed as the mean 6 standard deviation
(SD) or standard error of the mean (SEM). Statistical analyses were
performed using the Statistical Analysis System (SAS Institute,
Cary, NC). The non-parametric Kruskal-Wallis test was used
between the different groups. Statistical significance was accepted
at P,0.05. For the statistical analysise of gene expression, the
DataAssist software V3.0 (Applied Biosystems, Life Technologies)
was used to compare groups two by two through a two-tailed
Students t-test on the DeltaCT values.
The electroretinography equipment consisted of a Ganzfeld
bowl, a DC amplifier and a computer-based control and recording
unit (RETI port/scan 21, Stasche & Finger GmbH, Roland
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Regulation of Retinal Angiogenesis by Plasmalogens
Based on previous observations [30,31], these lesions might result
from the down-regulation of tie1, tie2 and kdr genes that was
observed at an earlier age.
The functional analysis of the retina of plasmalogen-deficient
mice using electroretinography revealed a specific alteration of the
ERG b-wave, thus resulting in a negative ERG waveform
(Figure 3D). Except for the oscillatory potentials, the ERG traces
of plasmalogen-deficient mice closely resembled those obtained
from retinas displaying retinal hypoxia [25,32], thus suggesting
that the increased diameter of large vessels may be a secondary
consequence of reduced retinal oxygenation, as previously
described [33].
Temporary delayed retinal vascular outgrowth associated
with modifications in the expression of genes involved in
Morphometric evaluation of ILB4-stained retinal vasculature
revealed a significantly reduced outgrowth of retinal capillaries at
PN7 in mice with plasmalogen deficiency (69% of radius) and
iPLA2 inhibition (76% of radius) when compared to control mice
(84% of radius) (Figures 2A and 2B). These observations were
correlated to a significant up-regulation of the anti-angiogenic
gene thrombosporin-1 (thbs1) in both plasmalogen-deficient mice and
retinal iPLA2-inhibited mice. The delay in vascular outgrowth was
also associated with down-regulation of endothelial transcripts
coding for the angiopoietin1/2 receptor tie2/tek, and the orphan
receptor tie1 in both plasmalogen-deficient and iPLA2-inhibited
mice. Among the pro-angiogenic genes, only angiopoietin-1 (angpt1)
and ephrin-B2 (efnb2) genes were significantly over-expressed in
both mice models (Figure 2C). Angpt1 protein is a critical actor
involved in vessel maturation since it mediates migration, adhesion
and survival of endothelial cells, whereas Efnb2 protein is involved
in angio-proliferative retinopathy [28,29]. We also observed a
significant over-expression of the angiopoietin-2 (angpt2) gene in
plasmalogen-deficient mice. Angpt2 protein is known to disrupt
the connections between the endothelium and perivascular cells,
thus promoting cell death and vascular regression [28].
The dysregulation in the expression of these pro- and antiangiogenic genes was completely abolished in plasmalogendeficient mice at PN14 (Figure 2D). Only beta-3 integrin (itgb3)
and fibroblast growth factor 2 (fgf2) genes 2 which are known to
promote angiogenesis and wound healing 2 were up-regulated in
plasmalogen deficiency conditions. These modifications in gene
expression at PN14 were associated to complete centrifuge
development of retinal vessels in both mice models (Figure 2B).
To check whether the recovery of retinal vascular outgrowth was
due to greater angiogenic activity, we further quantified the ILB4stained angiogenic sprouts at PN14 (Figures 2E and 2F). The
number of angiogenic sprouts was significantly higher in retinas of
plasmalogen-deficient mice (350 AU621.72) and mice with retinal
iPLA2 inhibition (290 AU612.2) compared to controls
(247 AU614.47), suggesting ongoing active angiogenesis processes. This suggests the existence of a secondary increase of proangiogenic activity in these animal models that was consistent with
the up-regulation of the itgb3 and fgf2 genes in retinas of
plasmalogen-deficient mice.
Contribution of extracellular matrix and astrocytes to
endothelial cell proliferation during early steps of
vascular development
Fibronectin is an extra-cellular matrix protein known to
promote endothelial cell proliferation and migration during
vascular development [34]. In physiologic conditions, fibronectin
is expressed in the zone of vasculogenesis immediately prior to
vessel formation, and it was shown to be over-expressed in
pathological retinal microvessels [35]. In the animal models used
herein, the delayed vascular outgrowth observed at PN7 was
associated with up-regulation of fibronectin gene expression
(Figure 4A). At the protein level, fibronectin immuno-reactivity
was more intense in the retina of animals with plasmalogen
deficiency and iPLA2 inhibition. The protein was particularly
expressed around vessels situated at the front of outgrowth,
suggesting more active angiogenesis processes in this area
(Figure 4B). Fibronectin up-regulation was correlated to an
increased expression of the gfap gene coding for the glial fibrillary
acidic protein (GFAP) in the retina (Figure 4A). This is consistent
with previous studies reporting fibronectin-induced endothelial cell
proliferation by gfap over-expressing activated astrocytes [34,36].
These data suggest that astrocytes may be at the origin of the
mechanisms promoting angiogenesis in mice with plasmalogen
deficiency and iPLA2 inhibition at PN7.
Influence of astrocyte template on vessel architecture of
adult mice
As previously reported, a proper astrocyte template is required for
retinal vascular development and remodelling [34,37]. To check
whether the vascular tortuosity observed in adult plasmalogendeficient mice is related to irregularities in astrocyte template
formation, we observed ILB4-stained endothelial cells and GFAPstained astrocytes on retinal flat-mounts from adult DAPAT2/2 mice
(Figure 5A). The co-localization of ILB4- and GFAP-positive cells
suggests that endothelial cells have passively followed the defective
astrocytic template (arrows on Figure 5A). Then, the abnormal
spatial positioning of endothelial cells might be the secondary
consequence of a defective arrangement of the astrocytic bed.
To better understand the origin of the localized vascular lesions,
we further investigated astrocyte template formation and retinal
vasculature at PN14 and PN21. In addition to the well-shaped
astrocyte bed, we observed several localized, sharply outlined and
strongly GFAP-immuno-reactive astrocyte accumulation areas in
plasmalogen-deficient mice and in iPLA2-inhibited mice at PN14.
The co-localization of ILB4- and GFAP-positive cells showed that
these areas corresponded to the sites of vascular lesion development. At PN21, astrocytes appeared to be less accumulated and/
or less immuno-reactive than at PN14, to be less sharpened and to
have a more fibrous aspect (Figure 5B). The areas where
astrocytes accumulated were not present at PN7 and seemed to
Defects in fully grown retinal vessels at adult age
To check whether these early metabolic and cellular abnormalities affect the final organization of retinal vessels, we further
investigated the retinal vascular phenotype of adult plasmalogendeficient mice. We quantified retinal capillaries on FITC-stained
flat-mounted retinas in adult plasmalogen-deficient mice (aged of
more than 6 months) to check whether the increased sprouting
activity and the expression of the itgb3 and fgf2 genes at post-natal
ages would have consequences on capillary density at adulthood.
The number of capillaries was strongly increased in plasmalogendeficient mice over the entire retinal surface (+49% in the central
retina, +40% in the mid-periphery and +68% in the far periphery)
(Figures 3A and 3B). Moreover, microphotographs of ILB4stained retinal blood vessels taken from adult plasmalogendeficient mice showed significant vascular defects compared to
controls (Figure 3C). These included dilated arteries and veins
(stars on Figure 3C), tortuous large vessels (arrows on Figure 3C)
and localized punctuated vascular lesions (circle on Figure 3C).
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Regulation of Retinal Angiogenesis by Plasmalogens
Figure 2. Temporarily delayed outgrowth and increased retinal angiogenic activity in Pls-deficient and iPLA2-inhibited animals.
A. Representative fluorescence microscopy pictures of isolectin-B4-labelled (ILB4) endothelial cells on retinal whole mounts of control, Pls-deficient
and iPLA2-treated mice at PN7. Dashed and solid arrows indicate the outgrowth distance and the retinal radius, respectively. B. Quantitative analysis
of retinal outgrowth represented by the ratio (%) of outgrowth distance (l) to retinal radius length (L) from the optic nerve to the retinal periphery
(R = l/L6100). A delay in retinal vascular outgrowth was observed in Pls-deficient mice and iPLA2-inhibited mice at PN7 (n = 6–18 per group) but not
at PN14 (n = 6–10 per group). *: statistically significant difference when compared to control group (Kruskal-Wallis test, P,0.05); **: statistically
significant difference when compared to control group (Kruskal-Wallis test, P,0.01). C. and D. Transcriptional analysis of angiogenic factors in
retinas of control, Pls-deficient and iPLA2-inhibited mice (n = 6–8 per group) at PN7 (C.) and PN14 (D.). The relative expression of angiogenic genes
was normalized to gusb, hprt and b2m genes and compared to control levels (set as 1). Pls-deficient and iPLA2-treated mice displayed fluctuations in
the expression of genes encoding for pro- and anti-angiogenic proteins that were related to vascular phenotype. *: Statistically significant difference
when compared to control group (Student’s t-test, P,0.05); **: statistically significant difference when compared to control group (Student’s t-test,
P,0.01); ***: statistically significant difference when compared to control group (Student’s t-test, P,0.001). E. Representative pictures of ILB4labelled retinal wholemounts showing angiogenic sprouts (arrowheads) in control, Pls-deficient and iPLA2-inhibited mice at PN14. F. Quantitative
evaluation of angiogenic sprouts in retinas of control, Pls-deficient, and iPLA2-inhibited mice at PN14 (n = 10–12 per group). The number of
angiogenic sprouts was significantly increased in mice deficient in Pls and in animals displaying a chemical inhibition of iPLA2 when compared to
control mice (set as 100), suggesting greater sprouting activity. ***: Statistically significant difference when compared to control group (Kruskal-Wallis
test, P,0.001).
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Figure 3. Defects in fully developed retinal vasculature of Pls-deficient mice. A. Quantification of retinal capillaries on confocal microscopy
pictures of whole-mounted and FITC-dextran-perfused retinas from control and Pls-deficient mice. Three concentric circles (yellow dashed circles)
centred on the optic nerve head were drawn to delimit central, mid-peripheric and far peripheric areas. The number of capillaries crossing a ring
situated in the middle of each area (red dashed circles) was counted over 360u. B. Quantitative evaluation of retinal capillaries in adult control and Plsdeficient mice. The capillary density was significantly increased in central, mid-peripheric and far-peripheric areas of the retina of Pls-deficient mice
(n = 6/group). C. Representative fluorescence microscopy pictures of isolectin-B4-labelled (ILB4) endothelial cells on retinal wholemounts of control
and Pls-deficient mice. Retinal vasculature of adult Pls-deficient mice was characterized by tortuous large vessels (arrows), dilated arteries and veins
(stars) and vascular lesions (circle). D. Representative electroretinographic response of Pls-deficient and control mice. The ERG traces of Pls-deficient
mice (red trace) exhibited a specific alteration of the positive b-wave (arrow) that is typical of retinal hypoxia. Scale bar = 75 mm.
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Regulation of Retinal Angiogenesis by Plasmalogens
Figure 4. Greater retinal astroglial activity in Pls-deficient and iPLA2-inhibited animals at PN7. A. Relative expression of genes encoding
for two markers of astroglial activity in control animals (n = 4–5), Pls-deficient mice (n = 5) and iPLA2-treated mice (n = 6) examined by RT-qPCR. The
relative expression of gfap and fn1 genes encoding for GFAP and fibronectin, respectively, was normalized to gusb, hprt and b2m genes and
compared to the control level (set at 1). The expression of gfap and fn1 genes was significantly increased in Pls-deficient and iPLA2-treated mice at
PN7, suggesting increased astroglial activity. *: Statistically significant difference when compared to control group (Student’s t-test, P,0.05);
**: statistically significant difference when compared to control group (Student’s t-test, P,0.01). B. Confocal microscopy of anti-fibronectin (green)
and ILB4 (red) labelled retinal whole mounts of control, Pls-deficient and iPLA2-inhibited mice at PN7 (n = 3–6 per group). The secretion of fibronectin
protein by retinal astrocytes at the front of vascular outgrowth was more pronounced in Pls-deficient and iPLA2-inhibited animals when compared to
controls, confirming greater astroglial activity. Scale bar = 150 mm.
result from secondary scarring mechanisms. Gene expression
analysis confirmed up-regulation of retinal GFAP mRNAs at
PN14 and a return to control levels at PN21 (Figure 5C).
No impact of plasmalogen deficiency and iPLA2
inhibition on pericyte recruitment
We speculated that a defective vessel maturation through
pericyte recruitment would be involved in vessel dilation and
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Regulation of Retinal Angiogenesis by Plasmalogens
Figure 5. Influence of astrocyte template on vessel architecture of adult mice. A. Fluorescence microscopy pictures of anti-GFAP- (green)
and isolectin-B4- (ILB4, red) labelled retinal whole mounts of control and Pls-deficient mice and mice at adult age. ILB4-positive cells (endothelial cells)
and GFAP-positive cells (astrocytes) were co-localized, suggesting that vessel tortuosity is a secondary consequence of an abnormal arrangement of
the astrocytic bed. B. Confocal microscopy pictures of anti-GFAP- (green, labelling astrocytes) and isolectin-B4- (ILB4, red, labelling endothelial cells)
labelled retinal whole mounts of control, Pls-deficient and iPLA2-inhibited mice at PN14 and PN21. The retinal vasculatures of Pls-deficient and iPLA2inhibited mice were characterized by vascular lesions that co-localized with activated astrocytes. These activated-astrocyte areas were sharply
outlined at PN14, whereas they were less immuno-reactive to GFAP and had a fibrous aspect at PN21. C. Relative expression of GFAP, a marker of
astroglial activity in control animals, Pls-deficient mice and iPLA2-treated mice examined by RT-qPCR (n = 4–6 per group) at PN14 and PN21. The
relative expression of the gfap gene was normalized to gusb, hprt and b2m genes and compared to the control level (set at 1). The expression of the
gfap gene was significantly increased in Pls-deficient and iPLA2-treated mice at PN14, suggesting increased astroglial activity at this age.
*: Statistically significant difference when compared to control group (Student’s t-test, P,0.05). Scale bar = 75 mm.
tortuosity, and in the formation of vascular lesions. Because
platelet-derived growth factor-beta (PDGF-b) signalling is required
for pericyte recruitment and migration [38], we wanted to know
whether the expression of pdgfb and pdgfrb genes is modified in the
retinas of our mice model. Slight but significant down-regulation
of pdgfb and pdgfrb genes was observed at PN21 (Figures 6A).
However, immuno-stainings of retinal pericytes with anti-NG2
antibody did not reveal any impact of plasmalogen deficiency or
iPLA2 inhibition on pericyte recruitment and positioning next to
vessels, at any stage of development (Figure 6B).
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Regulation of Retinal Angiogenesis by Plasmalogens
Figure 6. Pericyte recruitment and vessel stabilization in retinas of control, Pls-deficient and iPLA2-inhibited mice. A. Relative
expression of PDGF and PDGFR in control animals, Pls-deficient mice and iPLA2-treated mice examined by RT-qPCR (n = 4–6 per group) at PN7, PN14
and PN21. The relative expression of the genes was normalized to gusb, hprt and b2m genes and compared to the control level (set at 1). The
expression of pdgfb and pdgfrb genes was significantly reduced in mice with iPLA2 inhibition at PN7 and in Pls-deficient and iPLA2-inhibited mice at
PN21. *: Statistically significant difference when compared to control group (Student’s t-test, P,0.05); **: statistically significant difference when
compared to control group (Student’s t-test, P,0.01); ***: statistically significant difference when compared to control group (Student’s t-test,
P,0.001). B., C. and D. Confocal microscopy pictures of anti-NG2- (green, labelling pericytes) and isolectin-B4- (ILB4, red, labelling endothelial cells)
labelled retinal whole mounts of control and Pls-deficient mice and mice at PN7 (B.), PN14 (C.) and PN21 (D.). No abnormality was observed in retinas
of Pls-deficient and iPLA2-inhibited mice, suggesting normal vessel stabilization by pericytes. Scale bar = 75 mm.
HAs and radiating in the vitreous (Figure 7A) [39]. With the
confocal module of the angiograph, we identified and quantified
individual VHPs at the level of the lens and HAs in the posterior
eye. As expected, HAs were present in greater quantities in iPLA2inhibited mice at PN21 (mean 6 SEM, 4.0760.19 and 3.3660.24
in treated and control animals, respectively; Figure 7B) and were
persistent in iPLA2-inhibited mice, while they regressed in controls
(25% (non-significant) and 231% (P,0.05) between PN21 and
PN27 in treated and control animals, respectively). The effect of
iPLA2 inhibition was even more striking for VHPs, whose number
was greater in iPLA2-inhibited animals (mean 6 SEM, 2.1560.16
and 0.4560.15 in treated and control animals, respectively) at
Delayed regression of hyaloid arteries in mice with retinal
iPLA2 inhibition
A persistent hyaloid vasculature was previously reported in
plasmalogen-deficient mice [23]. As evidence of the implication of
plasmalogens and iPLA2 in retinal vascular development, the
regression of hyaloid vasculature in animals with iPLA2 inhibition
was observed (Figure 7). We performed fluorescein angiography
on iPLA2-inhibited mice at PN21 and PN27 to visualize and
quantify the functional hyaloid vessels, namely hyaloid arteries
(HAs) arising from the optic nerve and entering the vitreous and
vasa hyaloidea propria (VHP), which are small vessels branched to
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Regulation of Retinal Angiogenesis by Plasmalogens
Figure 7. Defects in hyaloid vasculature regression in mice with iPLA2 inhibition. A. Quantification of hyaloid arteries (HA) and vasa
hyaloidea propria (VHP) vessels on depth-scan images from confocal cSLO angiography. VHP vessels (stars) were visualized and quantified at the level
of the posterior lens, whereas HAs (arrowheads) were counted in the posterior eye in control and iPLA2-inhibited animals (n = 11 per group).
B. Quantitative evaluation of hyaloid arteries (HA) and vasa hyaloidea propria (VHP) vessels in control and iPLA2-inhibited mice at PN21 and PN27.
The numbers of HAs and VHPs were significantly higher in iPLA2-inhibited mice at PN21 and PN27 when compared to controls, thus confirming that
the control of hyaloid vessel regression by Pls involves the iPLA2 enzyme. *: Statistically significant difference when compared to control group
(Kruskal-Wallis test, P,0.05); **: statistically significant difference when compared to control group (Kruskal-Wallis test, P,0.01); ***: statistically
significant difference when compared to control group (Kruskal-Wallis test, P,0.001).
PN21 and still persistent at PN27, whereas they were fully
regressed in control animals. This evidence could also explain the
delay in retinal vessel outgrowth at earlier stages, as was previously
hypothesized [25].
Based on a preliminary description of the ocular phenotype of a
mouse model of plasmalogen deficiency [23] and to the wellknown implication of PUFAs in angiogenesis [15,40,41], we
hypothesized that plasmalogens may participate in the control of
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Regulation of Retinal Angiogenesis by Plasmalogens
retinal vascular development through PUFA release by a
phospholipase belonging to iPLA2 family. To test this hypothesis,
we characterized the steps of retinal vascular development in
plasmalogen-deficient mice and in a previously developed mouse
model of retinal iPLA2 inhibition [24].
We found that plasmalogens were involved in the control of the
early steps of retinal vascular development. During these phases,
endothelial cell proliferation results in the formation of the
primary vascular bed on an astrocyte template. As shown by the
present data, plasmalogen deficiency results in delayed vascular
outgrowth and in an imbalance in the retinal expression of proand anti-angiogenic genes. These anti-angiogenic conditions were
associated with high macroglial activity and the resulting overproduction of fibronectin. Astrocytes seem to be the principal
actor, since fibronectin production was reported to be linked to
astrocyte activity. Fibronectin is known to promote endothelial cell
proliferation and vessel formation in the retina [34,42] and was
found to be over-expressed in retinal capillaries of patients with
proliferative DR, a disease displaying an abnormal proliferation of
retinal vessels [35]. Nevertheless, further studies are needed to
decipher any involvement of Mu¨ller cells, which are also known to
promote endothelial cell proliferation and to be immuno-reactive
to anti-GFAP antibodies. Although the production of fibronectin
was enhanced, it was not sufficient, at least at PN7, to balance the
lack of development of retinal vessels observed at this age. One
hypothesis would be that this greater astroglial activity is a
secondary reaction of the retina to delayed vascular development.
This process might have been mediated by transient hypoxia, since
some data demonstrated an increased secretion of fibronectin in
hypoxic astrocytes in the brain [43] and the retina [36].
Whereas the expression of VEGF was not affected by
plasmalogen deficiency or iPLA2 inhibition, the only up-regulated
pro-angiogenic factors at PN7 belong to the angiopoietin family.
Angiopoietins are involved in vessel formation through the control
of endothelial cell migration, adhesion and survival. Angiopoietins
have been found to be ligands of tie receptors [28]. The present
results indicate that the retinal expression of endothelial
transcripts, such as tie2 and tie1 [44], does not follow that of
angiopoietins at PN7. On the contrary, the mRNAs of tie
receptors decreased, whereas those of angiopoietins increased.
These findings may indicate either a lower relative number of
endothelial cells in the retina or an altered activity of the
angiopoietin/tie pathway, which may contribute to the delayed
formation of the endothelial network. Since angiopoietin production was previously shown to be negatively regulated by omega-3
PUFAs [13], one may assume that the over-expression of
angiopoietins is a consequence of the lack of plasmalogens and
the subsequent PUFA signalization. Further analyses of the kinases
involved in these pathways would be helpful to confirm this
hypothesis and to elucidate the molecular mechanisms involved.
Furthermore, and as suggested by Hoffman et al., the overexpression of the angpt1 gene may also have influenced retinal
vessel maturation through mechanisms not involving pericytes
[45]. Although pericytes are known to participate in vessel
stabilization and maturation, these cells may not be involved in
the formation of mature vessels in animal models of ROP. These
data are concordant with our results, as plasmalogen-deficient
mice exhibit features resembling to those observed in ROP,
namely vascular growth retardation, proliferation of retinal
capillaries, and dilated and tortuous arteries and veins.
Several genetic and phenotypic changes occurred between PN7
and PN14. The retinal vascular outgrowth was boosted in
plasmalogen-deficient and iPLA2-inhibited animals, as confirmed
by the increased sprouting activity at PN14 and the higher number
of retinal capillaries at adulthood. This higher angiogenic activity
was confirmed at the molecular level by the return to control levels
of pro-angiogenic gene expression, whereas these were downregulated at PN7. Only the itgb3 and fgf2 genes remained upregulated in plasmalogen-deficient mice at PN14. The itgb3 gene
encodes for beta3-integrin protein, which is considered as a
marker of angiogenesis [46]. Although the pro-angiogenic
properties of beta-3-integin are still under debate in cancer
research [47], studies have shown that its activation enhances
tumour angiogenesis and metastatic growth in the brain [48].
Another particularity of integrins is their ability to interact with a
number of pro-angiogenic factors such as EGF, PDGF-beta and
IGF [47]. Such interactions may be important for the activation of
integrins as well as for the regulation of kinase activity of these
growth factors [49]. Even if this type of interaction has not yet
been demonstrated with FGF-2, several reports suggest that beta-3
integrin and FGF-2 are involved in common metabolic pathways
in vascular or ocular cells [50–52]. Whereas FGF-2 itself is known
to be an important actor in wound healing [53], it was
demonstrated that beta-3 integrin is also required for wound
angiogenesis [54]. Taken together, these data suggest that FGF-2
and beta-3 integrin may have promoted the scarring processes
observed in plasmalogen-deficient and iPLA2-inhibited mice at
PN14. These wound areas were characterized by strongly
activated astrocytes on which angiogenic processes take place.
These scarring processes were very transitory and incomplete at
PN21, thus leading to punctuated vascular lesions at later ages.
One of the vascular abnormalities observed in fully developed
retinas of plasmalogen-deficient and iPLA2-inhibited mice consisted of defects in large vessels. We observed an up-regulation of
genes encoding for angpt1, angpt2 and Efnb2 proteins, which are
known to be over-expressed in the retinas of animal models of
oxygen-induced retinopathy and in the vitreous of patients with
ROP. Oxygen exposure is also known to promote vessel regression
and up-regulate Efnb2 gene expression [29,45,55]. The angpt2 gene
was shown to be over-expressed in patients with highly vascularactive ROP [55] and was only up-regulated in plasmalogendeficient mice, suggesting a more severe phenotype in mice with
plasmalogen deficiency than in those with retinal iPLA2 inhibition.
The tortuosity of large vessels can result from either poor
positioning of astrocytes when forming the astroglial template or
a secondary modification of the extracellular matrix that
subsequently modified the vessel shape. Given that omega-3
PUFAs were shown to influence the tissue expression of several
proteins of the extracellular matrix [56,57], this second hypothesis
would be attractive.
As final evidence of the involvement of an iPLA2-dependent
control of vascular development by plasmalogens, we have shown
another phenotypic similarity between our mouse models, namely
the persistence of hyaloid vasculature. Based on previous
observations, one might assume that the defective regression of
hyaloid vasculature may also be related to alterations in astrocyte
metabolism, and particularly their ability to produce bA3/A1crystallin protein, further work being needed to document this
hypothesis [37].
There are a several limitations to acknowledge in this work.
First, and to better characterize the influence of plasmalogens on
the early steps of retinal vascular development, it would have been
useful to increase the number of time points for a complete time
course of retinal development. Second, and since this study is
mainly based on data obtained from mRNA analyses, the tissue
levels of the different proteins could have been evaluated by
western blotting or ELISA. Third, the inhibition rate of iPLA2
activity in our animal model is only 45%, meaning that about half
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Regulation of Retinal Angiogenesis by Plasmalogens
of the retinal iPLA2 activity is still persistent [24]. Moreover,
bromoenol lactone was shown to inhibit plasmalogen-specific
isoforms of iPLA2 as well as others that are not specifically related
to these phospholipids. Other approaches such as suppression of
plasmalogen-specific iPLA2 gene activity by siRNA or by
homologous recombination would have been more effective.
However, to the best of our knowledge, the nucleotide sequence of
the retinal isoform of this plasmalogen-specific enzyme is not
known, making these approaches impossible to use.
In summary, plasmalogen deficiency resulted in primary
vascular defects that led to a secondary tissue reaction that is
insufficient to ensure physiological vascular development. In
addition to confirming the need for plasmalogens for normal
retinal vascular development, this study provides evidence that
helps elucidate the cellular and molecular events involved.
Regulation of angiogenesis by plasmalogens can be mediated by
the action of iPLA2 and involves the angiopoietin/tie pathway.
The authors gratefully acknowledge Bruno Pasquis and Laurence Decocq
(from the animal facility of Centre des Sciences du Gouˆt et de
l’Alimentation, Dijon, France) for their assistance in animal handling,
Christine Arnould (INRA/uB Dimacell, Cell Imaging Platform, UMR
1347 Agroecology - AgroSup/INRA/uB - BP 86510-21065 Dijon Cedex,
France) for her assistance in using the confocal microscope, and Vale´rie
Febvret (Centre des Sciences du Gouˆt et de l’Alimentation, Dijon, France)
for her technical assistance. The authors gratefully acknowledge Linda
Northrup (PhD, ELS, English Solutions, Voiron, France) for editing the
Author Contributions
Conceived and designed the experiments: AMB CPC-G LB NA.
Performed the experiments: SS BB LL NA. Analyzed the data: SS AMB
CPC-G LB NA. Contributed reagents/materials/analysis tools: SS BB LL.
Wrote the paper: SS AMB CPC-G LB NA. Contributed to data acquisition
and analysis: SS BB LL NA. Contributed to drafting the manuscript: SS
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June 2014 | Volume 9 | Issue 6 | e101076