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VASCULAR BIOLOGY
Deletion of Adam10 in endothelial cells leads to defects in organ-specific vascular
structures
Krzysztof Glomski,1,2 Sébastien Monette,3 Katia Manova,4 Bart De Strooper,5,6 Paul Saftig,7 and Carl P. Blobel1,8
1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY, 2Weill Cornell/Rockefeller/Sloan-Kettering, Tri-Institutional MD-PhD
Program, New York, NY; 3Center of Comparative Medicine and Pathology and 4Molecular Cytology Core Facility, Sloan-Kettering Institute, New York, NY;
5Center for Human Genetics, KU Leuven, Leuven, Belgium; 6Department for Developmental and Molecular Genetics, VIB, Leuven, Belgium; 7Institute for
Biochemistry, Christian-Albrechts-University, Kiel, Germany; and 8Departments of Medicine and of Physiology, Systems Biology and Biophysics, Weill Medical
College of Cornell University, New York, NY
During vertebrate angiogenesis, Notch
regulates the cell-fate decision between
vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored
ligands triggers proteolytic processing,
first by Adam10 and then presenilins.
This liberates the Notch intracellular domain, allowing it to enter the nucleus and
activate Notch-dependent genes. Here we
report that conditional inactivation of
Adam10 in endothelial cells (A10⌬EC)
recapitulates the increased branching and
density of the retinal vasculature that is
also caused by interfering with Notch
signaling. Moreover, A10⌬EC mice have
additional vascular abnormalities, including aberrant subcapsular hepatic veins,
enlarged glomeruli, intestinal polyps con-
taining endothelial cell masses, abnormal
endochondral ossification, leading to
stunted long bone growth and increased
pathologic neovascularization following
oxygen-induced retinopathy. Our findings support a model in which Adam10 is
a crucial regulator of endothelial cell-fate
decisions, most likely because of its essential role in canonical Notch signaling.
(Blood. 2011;118(4):1163-1174)
Introduction
In the developing vasculature, appropriate vessel sprouting and
branching is thought to be regulated by Notch signaling.1-10
Interference with Notch signaling, such as through postnatal loss of
Notch-1 in endothelial cells, or hemizygosity of the Notch ligand
Delta-like–4, or inhibition of ␥-secretase, an enzyme that releases
the Notch intracellular domain from its membrane anchor and
allows it to activate Notch-dependent transcription in the nucleus,
leads to increased branching and density in the developing retinal
vasculature.11 Moreover, conditional inactivation of Notch-1 in
endothelial cells leads to an arrest of prenatal developmental at
embryonic day E9.5.5 Taken together, these findings point toward
an essential role of Notch-1 in endothelial cells during early mouse
development.
Canonical Notch signaling depends on sequential proteolytic
events.12 The first is processing of Notch by furin in the secretory
pathway.13 This generates the mature Notch heterodimer that can
interact with a membrane-anchored Notch ligand on an adjacent
cell. This, in turn, stimulates processing of Notch by Adam10,
thereby generating a membrane anchored stub14,15 that is recognized by the presenilin complex.16,17 Intramembrane processing by
presenilin releases the intracellular domain of Notch from its
membrane anchor, allowing it to enter the nucleus and activate
Notch-dependent gene transcription programs.12 Adam10 belongs
to a family of membrane-anchored metalloproteinases containing
“a disintegrin and metalloprotease” domains that serve to proteolytically process a variety of transmembrane proteins, including
growth factors, cytokines, structural proteins, and receptors.18,19
Both Adam10 (Kuzbanian) and Adam17 (TACE) have been
reported to process Notch-1 in cell-based assays, although the most
recent reports clearly implicated Adam10 as the enzyme responsible for ligand-dependent processing of Notch.14,15,20 Moreover,
studies in Drosophila,21 C elegans22,23 and mice24-27 have demonstrated that Adam10 is essential for Notch signaling in development. For example, mice lacking Adam10 die at E9.5 with defects
resembling those observed in Notch-deficient mice,24 and lymphocyte-specific and neuronal-specific conditional inactivation of
Adam10 in mice has recapitulated Notch dysfunction in those
tissues, further implicating Adam10 as the functionally relevant
Notch proteinase.25,26 The goal of this study was to generate
conditional knockout mice lacking Adam10 in endothelial cells to
evaluate the role of Adam10 in vascular development as well as in
retinal angiogenesis and pathologic retinal neovascularization.
Submitted April 12, 2011; accepted May 19, 2011. Prepublished online as
Blood First Edition paper, June 7, 2011; DOI 10.1182/ blood-2011-04-348557.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of TWs article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Methods
Reagents
All reagents were from Sigma-Aldrich, unless indicated otherwise. Rabbit
anti–mouse Adam10 antibody (MAB946) was from R&D Systems, rat
anti–mouse CD31 antibody (MEC 13.3) from BD Biosciences, rabbit
anti–mouse Adam17 cytoplasmic domain antibody has been described
previously,28 and rat anti–mouse MECA-32 monoclonal IgG2a antibody29
was from Developmental Studies Hybridoma Bank. Fluorescein conjugated
Isolectin B4 was from Vector Labs and fluorescent mounting media from
Dako. Sheep anti–rat conjugated magnetic beads (Dynabeads) were from
Dynal Biotech ASA, and protease 3 was from Ventana Medical System.
1163
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1164
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
GLOMSKI et al
Isolation of lung endothelial cells and Western blotting
Oxygen Induced Retinopathy
Primary lung endothelial cells were isolated from 12-day-old mice and
subjected to Western blot analysis as previously described.28,30,31
Oxygen-induced retinopathy experiments were performed as described.33
Subsequently, animals were euthanized and their eyes removed, and the size
of the central avascular area and the development of neovascular tuft was
analyzed as described.31 A Wilcoxon-Mann-Whitney test was used to assess
statistical signifigance.
Mouse strains, genotyping and breeding
To generate mice lacking Adam10 in endothelial cells, Adam10flox/flox mice25
were mated with Tie2-Cre transgenic mice31,32 (provided by Dr Tom Sato).
Adam10flox/flox/Tie2-Cre mice (A10⌬EC) were maintained on a mixed genetic
background (129P2/OlaHsd/C57BL6). Adam10flox/flox mice displayed no histopathologic abnormalities and were therefore used as controls for experiments
with A10⌬E individuals. A previous study demonstrated that Tie2-Cre mice had
no evident histopathologic defects.31 R26R reporter mice (The Jackson Laboratory, B6.129S4-Gt(ROSA)26Sortm1Sor/J) were used to monitor the expression
of Cre-recombinase in Tie2-Cre individuals. Adam10flox/flox mice were mated to
R26R individuals, and the offspring crossed to generate Adam10flox/flox,
R26R/R26R mice. Adam10flox/flox, R26R/R26R mice, in turn, were mated to
Adam10flox/flox, Tie2-Cre individuals and their offspring were used in X-gal
staining analysis. All animal experiments were approved by the Internal Animal
Care and Use Committee of the Hospital for Special Surgery.
Whole-mount Lac-Z Staining A10⌬EC and A10⌬EC/R26R mice were
euthanized and their eyes removed and fixed for 15 minutes in 4%PFA/
0.5% glutaraldehyde. Then the retinas were isolated, washed 3 times in
wash buffer (PBS, 2mM MgCl2, 5mM EGTA, 0.01% sodium deoxycholate,
0.02% Nonident P-40) and then stained for 3 hours at 37°C in staining
buffer (wash buffer with 0.1% wt/vol bromo-chloro-indolyl-galactopyranoside, 5mM K3Fe(CN)6, and 5mM K4Fe(CN)6䡠6H2O). After staining, retinas
were fixed in 4% PFA overnight and imaged on a Zeiss Axiovision SteREO
Lumar.V12 Microscope.
Developmental Retinal Angiogenesis
Retinas were isolated from 5-day-old mice and incubated in blocking buffer
(1% BSA, 100mM glycine, 0.5% vol/vol Triton-X 100 in PBS) for 1 hour at
4°C and then incubated in blocking buffer with 2␮g/mL Isolectin B4
overnight at 4°C. A Zeiss AxioVert Observer.Z1 microscope was used in
conjunction with a motorized stage to generate approximately 40 images at
200⫻ magnification to capture the entire retina and Zeiss Axiovision V4.7
software was used to generate a contiguous retinal image. For quantification, lines bisecting each retinal “leaf” from the center of the optic disk to
the angiogenic front were generated and measured, with 4 measurements
made on each retina. These values were used to generate an average value
of angiogenic front progression. To measure the coverage of vessels at the
angiogenic front, rectangular fields (8 fields of 500␮m ⫻ 200␮m per retina
for 17-21 retinas) were imported into Adobe Photoshop (see supplemental
Figure 1, available on the Blood Web site; see the Supplemental Materials
link at the top of the online article). To quantify branching, loops generated
by the developing vasculature were counted in these fields as shown in
supplemental Figure 1B (red dots mark individual loops). To determine
endothelial cell coverage, images were processed in Photoshop by setting a
minimum signal threshold such that Isolectin B4 fluorescence registered at
saturating levels (255) while background was set to minimum signal (0; see
supplemental Figure 1C). This processing step was performed to standardize the variable background fluorescence present between retinal images
and to aid in endothelial cell coverage calculation in ImageJ. After
processing, ImageJ was used to determine the area of vessel coverage by
measuring the integrated intensity of pixels across each image and image
area, then applying the following formula: % coverage ⫽ ([integrated
intensity])/[area ⫻ 255]) ⫻ 100. Eight fields from each retina were
quantified. To determine tip cell density at the vascular front, tip cells
(defined as endothelial cells possessing bursts of filopodia,11 marked by red
dots in supplemental Figure 1D) were counted based on morphologic
presence of filopodial collections (arrows) in 4 microscopic fields of
200␮m ⫻ 200␮m from each retina (15 retinas per group). Filopodia density
at the vascular front was quantified by demarcating 100␮m of vascular front
(yellow line in supplemental Figure 1E) and counting filopodia protrusions11 (marked by red dots in supplemental Figure 1E) along that front (4
measurements per retina; 15 retinas per group). Statistical analysis was
performed using a 2-tailed Student t test.
Pathologic analysis
After euthanasia by CO2 (according to the guidelines of the American
Veterinary Medical Association), all organs were examined grossly and
fixed in 10% neutral buffered formalin or 4% paraformaldehyde. Tissues
were processed routinely for histology and embedded in paraffin, sectioned
at 4 microns thickness stained with H&E and examined. Selected tissues
were stained with the Masson trichrome. The following tissues were
examined: diaphragm, semimembranosus and semitendinosus muscles,
sciatic nerve, heart, thymus, lungs, kidneys, salivary glands, lymph nodes
(submandibular, mesenteric), stomach, duodenum, jejunum, ileum, cecum,
colon, pancreas, thyroid gland, esophagus, trachea, adrenal glands, liver,
gallbladder, spleen, testes, epididymides, seminal vesicles, prostate gland,
urinary bladder, skin (trunk, head, digits), femur, tibia (with stifle joint),
humerus (with shoulder joint), tarsus, metatarsus and digit, sternum
(including bone marrow), brain, pituitary gland, ears, eyes, nose, mouth,
teeth, vertebrae with discs, spinal cord. Smears of femoral bone marrow
were prepared, stained with Wright-Giemsa and examined.
Serum chemistry
For serum chemistry, blood was collected in tubes containing a serum
separator, centrifuged, and the serum was analyzed by an Olympus AU400
analyzer. The following parameters were measured: alkaline phosphatase
(ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST),
␥ glutamyltransferase (GGT), albumin, total protein, globulin, total bilirubin, blood urea nitrogen (BUN), creatinine, cholesterol, glucose, calcium,
phosphorus, total carbon dioxide (TCO2), chloride, potassium, sodium,
albumin/globulin (A/G) ratio, BUN/creatinine (B/C) ratio, sodium/
potassium (Na/K) ratio, osmolality (calculated), and anion gap.
Hematology
For hematology, blood was collected in tubes containing EDTA, and
analyzed by a Sysmex XTV analyzer. Blood smears were prepared, stained
with Wright-Giemsa, and examined. The following parameters were
measured: total white blood cell count, differential and absolute counts for
neutrophils, lymphocytes, monocytes, eosinophils, and basophils, hematocrit, red blood cell count, hemoglobin, mean corpuscular volume (MCV),
mean corpuscular hemoglobin concentration (MCHC), and platelet count.
Manual relative reticulocyte counts were performed on new methylene
blue-stained blood smears, and absolute counts were calculated.
Analysis of embryos at different stages of development
After timed matings, embryos were isolated at the indicated time points by
dissecting the uterine horns in ice-cold PBS. For CD31 staining, embryos
were fixed in 4% PFA overnight and dehydrated with an increasing series of
methanol. Six percent H2O2 in methanol was used to saturate endogenous
peroxidase activity, followed by rehydration with a decreasing series of
methanol in PBS. After blocking for 2 hours in normal goat serum, 5␮g/mL
of rat anti–mouse CD31 was added overnight and subsequently washed for
6 hours in PBS. Biotinylated goat anti–rat IgG antibody was added to
embryos overnight and washed for 6 hours in PBS. Finally, embryos were
incubated with streptavidin-HRP overnight followed by thorough washing
with PBS. Embryos were developed using DAB according to the manufacturer’s instructions (Vector). For X-gal staining, embryos were fixed in 2%
PFA, 0.5% glutaraldehyde for 1 hour at 4°C. Embryos were washed in wash
buffer (2mM MgCl2, 5mM EGTA, 0.01% sodium deoxycholate, 0.02% Nonident P-40) 3 times for 10 minutes, and were incubated for ⬃ 5 hours in staining
solution (washing solution with 50␮g/mL X-gal, 5mM K3Fe(CN)6, and 5mM
K4Fe(CN)6). After staining, embryos were post-fixed in 4% PFA overnight.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Immunhistochemical detection of bound MECA-32 antibodies (used at 3
␮g/mL) was carried out at the Molecular Cytology Core Facility of MSKCC,
using a protocol established in the Core Facility, with a Discovery XT processor
(Ventana Medical Systems). Briefly, the samples were blocked for 30 minutes
with 10% normal rabbit serum, 2% BSA, then Protease 3 was added for antigen
retrieval for 4 minutes. The incubation with the primary antibody was for 7 hours,
followed by 60 minutes incubation with biotinylated rabbit anti–rat IgG (Vector,
BA-4000, in 1:200 dilution), Blocker D, Streptavidin-HRP and DAB detection
kit (Ventana Medical Systems) following the manufacturers instructions.
Results
Generation of mice lacking Adam10 in endothelial cells
Mice carrying floxed alleles of Adam1025 were crossed with
Tie2-Cre mice31,32 to generate animals lacking Adam10 in endothelial cells (Adam10flox/flox/Tie2-Cre, referred to as A10⌬EC).
A10⌬EC mice were born at the expected Mendelian ratio from
crosses of A10⌬EC mice with Adam10flox/flox controls (n ⫽ 123;
A10⌬EC: 64 [52%], controls: 59 [48%]) and were viable and
fertile. However, an increased lethality of A10⌬EC mice became
evident starting at 10 weeks of age, with only ⬃ 25% of A10⌬EC
still surviving at 50 weeks, compared with 100% of controls
(supplemental Figure 2). The endothelial-specific expression of
Tie2-Cre was corroborated using a Rosa-26–reporter (R26R) that
drives expression of Lac-Z after Cre-mediated recombination.
X-gal staining of whole-mounted retinas from A10⌬EC/R26R mice
highlighted the retinal vascular tree in blue, demonstrating effective recombination of LoxP sites in endothelial cells by Tie2-Cre
(Figure 1A) whereas no staining was seen in retinas from
Adam10flox/flox/R26R mice lacking Tie2-Cre (Figure 1B). Western
blot analysis of endothelial cells isolated from lungs of A10⌬EC
animals showed a strong reduction of Adam10 compared with
controls, with comparable levels of Adam17 (Figure 1C). To
corroborate that the Cre-mediated deletion of the floxed alleles of
Adam10 inactivates Adam10, we crossed Adam10wt/flox mice with
animals expressing Cre under control of the ubiquitously expressed
EIIa-promoter to delete Adam10 in the germ line. Matings of the
resulting Adam10⫹/⫺ individuals produced Adam10⫺/⫺ embryos
that died at E9.5, and thus resembled the previously reported
Adam10⫺/⫺ mice24 (supplemental Figure 3A,B).
Evaluation of developmental retinal angiogenesis
Notch signaling serves as a negative regulator of vessel branching
during developmental retinal angiogenesis.3,4,6-9,11 When vascular
branching was assessed on whole-mounted FITC-isolectin B4stained retinas from 5-day-old A10⌬EC mice, a higher vascular
density with increased numbers of branching tip cells and increased
vascular loops was observed in the leading third of the developing
vascular tree compared with controls (Figure 1D-L). In addition,
A10⌬EC tip cells had a greater number of filopodia than controls
(Figure 1K-M). The distance between the leading edge of the retinal
vascular tree and its origin at the optic nerve was comparable in
A10⌬EC mice and littermate controls (1.05mm2 ⫾ 0.09mm2, n ⫽ 21
and 1.02mm2 ⫾ 0.07mm2, n ⫽ 17) so there was no significant difference in the growth of the retinal vasculature. A similar rise in vascular
density was seen in the deep retinal vascular plexus in 12-day old mice,
while it had largely resolved in superficial vascular beds (supplemental
Figure 4A-D). The retinal vasculature in adult A10⌬EC mice appeared
normal (supplemental Figure 4G-J), suggesting that the abnormalities
during vascular development are transient and later remodel.
ROLE OF ENDOTHELIAL CELL ADAM10 IN ANGIOGENESIS
1165
Oxygen-induced retinopathy
To investigate the role of Adam10 in pathologic retinal neovascularization, A10⌬EC mice and littermate controls were subjected to
oxygen-induced retinopathy (OIR), a mouse model for proliferative retinopathies.33,34 In this model, 7-day-old mice are placed in a
chamber with 75% oxygen and then returned to room air 5 days
later (p12). The resulting relative hypoxia triggers a neovascular
response that leads to growth of vessels into the central avascular
area and also to the development of pathologic neovascular tufts at
p17. Retinas from A10⌬EC mice had a significantly increased
number of endothelial cells that had crossed the internal limiting
membrane compared with controls (Figure 2A-C). In a wholemount analysis, these resembled thin layers of endothelial cells
instead of the typical compact neovascular tufts observed in
controls (Figure 2D,E). Moreover, the revascularization of the
central avascular area at p17 was increased in A10⌬EC mice
(Figure 2F-H), even though the size of the avascular area at the end
of oxygen treatment at p12 was comparable to controls (Figure
2I-K), suggesting that Adam10 serves to limit both the revascularization of the avascular area as well as the extent of pathologic
neovascularization.
Vascular development at E9.5
The development of the retinal vasculature is considered to be a
model system for embryonic vascular development. However, the
vascular tree in the brain, cerebellum and somites of A10⌬EC
embryos at E9.5 stained with the endothelial cell marker CD31 was
indistinguishable from that of littermate controls. Figure 3A-D
shows representative examples of a total of 8 embryos (A10⌬EC: 4;
controls: 4 analyzed in a blinded manner). Moreover, the distribution of
vessels in hindbrain sections of E9.5 embryos was comparable in
A10⌬EC embryos and littermate controls (Figures 3E-F; A410⌬EC:
8.7 per 400 ␮m ⫾ 0.9, n ⫽ 9 sections and controls: 8.3 per
400 ␮m ⫾ 1.0, n ⫽ 9 sections). Staining of E8.5 embryos carrying
Tie2-Cre and the R26R reporter with X-gal demonstrated an irregular
and patchy expression of Tie2-Cre (Figure 3G), suggesting that the lack
of an evident branching defect in the developing vasculature of E9.5
A10⌬EC embryos could be explained by the incomplete expression of
the Tie2-Cre transgene in endothelial cells at this stage.
Analysis of the vasculature in liver, heart and diaphragm
Histopathologic evaluation of 6-week-old A10⌬EC animals uncovered a variety of vascular abnormalities (n ⫽ 8, A10⌬EC: 4;
controls: 4). The most evident defect on macroscopic examination
of the abdominal cavity was the presence of large-caliber vessels
on the liver surface and marked pallor of the liver that were not
visible in control animals (Figure 4A-B). The enlarged vessels
communicated with hepatic sinusoids, which were often moderately dilated (Figure 4C-D). The abnormal vessels were thinwalled, lined by a single layer of MECA-32–positive endothelial
cells,29 and without a visible tunica media, suggesting that they are
veins or venules (Figures 4E-F). Moreover, there were randomly
distributed foci of hepatic coagulative necrosis in the adjacent
parenchyma, which correlated with elevation of liver enzymes in
all A10⌬EC individuals (n ⫽ 4) compared with controls (n ⫽ 4)
(alanine transaminase, 2-10 fold elevation, aspartate aminotransferase, 1.5-9 fold elevation; Figures 4G-H and data not shown).
Similar thin-walled vessels lined by MECA-32–positive cells were
seen in the subepicardial myocardium (Figure 4I-L). In the
diaphragm, there was diffuse endomysial hypercellularity, characterized by MECA-32–positive cells forming smaller, capillary-like
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A
C
Co
nt
ro
l
B
100
Adam10(p)
75
Adam10(m)
Adam17(p)
Adam17(m)
A10∆EC, R26R
F
G
Control
A10∆EC
Control
**
I
5.0
2.5
0.0
A10∆EC Control
K
A10∆EC
60
***
J
50
40
30
20
10
0
A10∆EC Control
L
A10lox/lox
M
Vascular Loops per Field
Tip Cells per Field
H 7.5
E
1000
***
750
Filopodia per Vessel Length
A10∆EC
100
Control, R26R
Leading Edge Density (%)
D
0∆
EC
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
GLOMSKI et al
A1
1166
500
250
0
30
A10∆EC Control
**
20
10
0
A10∆EC Control
Figure 1. Adam10 deficiency in endothelial cells causes increased vascular density at the front of the developing retinal vasculature. (A,B) Lac-Z staining of retinas
from A10⌬EC-R26R or A10flox/flox-R26R control mice. (C) Immunoblot of Adam10 and Adam17 in primary endothelial cells isolated from lungs of A10flox/flox control or
A10⌬EC mice (p: pro-form, m: mature form). (D-G) Isolectin-B4 staining of retinas from 5-day-old A10⌬EC or control mice. Panels F and G correspond to the area marked by
insets in panels D and E, respectively. (H-J) Quantitative analysis of tip cell density (H, A10⌬EC: 6.9 ⫾ 1.8 per 100 ␮m of vascular front, n ⫽ 15; controls: 4.9 ⫾ 1.4, n ⫽ 15),
endothelial cell coverage at the front of the retinal vascular tree (I, A10⌬EC: 54.8% ⫾ 4.42, n ⫽ 17; controls: 35.3% ⫾ 3.8, n ⫽ 21), and vascular loops per field (J, A10⌬EC:
980 loops per mm2 ⫾ 65, n ⫽ 17, controls 628 ⫾ 68 per mm2, n ⫽ 21). (K,L) Micrographs of tip cells at the leading edge of the developing retinal vascular tree in A10⌬EC and
control mice, and quantitative analysis of filopodia density (M, A10⌬EC: 26.4 ⫾ 6.8 per 100 ␮m vessel length, n ⫽ 15, controls: 18.6 ⫾ 5.6, n ⫽ 15). Please see “Evaluation of
developmental retinal angiogenesis” and supplemental Figure 1 for details. Error bars represent mean ⫾ SEM. ** indicates P ⬍ .01, and *** indicates P ⬍ .001 in a 2-tailed
Student t test. Scale bars in panels A-B: 300 ␮m; D-E: 400 ␮m; F-G: 100 ␮m, K-L: 20 ␮m.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
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Nuclei Per Section
A
B
C
A10∆EC
Control
50
25
A10∆EC
Percent Dropout area
Percent Dropout area
aa
I
1167
***
75
0
F
ROLE OF ENDOTHELIAL CELL ADAM10 IN ANGIOGENESIS
Control
D
E
A10∆EC
Control
G
H
A10∆EC
Control
J
K
A10∆EC
Control
30
***
20
10
0
A10∆EC
Control
60
50
40
30
20
10
0
A10∆EC
Control
Figure 2. Oxygen Induced Retinopathy (OIR) in A10⌬EC mice. (A) Quantitative analysis of cell nuclei found vitreal to the internal limiting membrane after OIR in A10⌬EC
and control mice (A10⌬EC: 68.9 ⫾ 4.2, n ⫽ 12; controls: 33.3 ⫾ 2.6, n ⫽ 8). (B,C) Histologic analysis revealed an abnormal sheet-like vascular structure (marked by an arrow)
in an A10⌬EC retina (B) and a typical neovascular tuft (pointed by an arrow) in a control retina (C). (D-E) Micrographs of a segment of an Isolectin B4-stained retina from an
A10⌬EC mouse showed a sheet-like vascular structure (outlined by a dotted line in panel D), whereas typical neovascular tufts were visible in a retina from a control mouse
(marked by arrows in panel E). (F) Quantification of the size of the central avascular area in A10⌬EC eyes compared with controls after a 5-day period at room air (p17;
A10⌬EC: 11.3 ⫾ 1.5%, n ⫽ 9; controls: 25.7% ⫾ 1.1% n ⫽ 8), and corresponding representative micrographs of A10⌬EC (G) or control (H) retinas. (I) Quantification of the
avascular area in A10⌬EC (J) and control (K) retinas after 5 days at 75% oxygen (p12; A10⌬EC: 43.2 ⫾ 2.0%, n ⫽ 6; controls: 43.8% ⫾ 0.6%, n ⫽ 5). The central avascular
area is bounded by a yellow line in panels G,H,J and K. ***P ⬍ .001 in a 2-tailed Student t test. Scale bars in panels B-C: 50 ␮m; D-E: 400 ␮m; G-H,J-K: 750 ␮m.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
GLOMSKI et al
A
B
A10∆EC
Control
C
D
Figure 3. Analysis of developmental angiogenesis at
E9.5. (A-D) Micrographs of CD31-stained whole-mounted
A10⌬EC (A,C) and control (B,D) embryos at E9.5 (superficial cranial vessels are marked by arrows, and intersomitic vessels by arrowheads). (E,F) Representative
micrographs of CD31-stained sections of the vasculature
of the fourth ventricular floor in A10⌬EC (E) and control
(F) embryos at E9.5 showed a similar distribution of small
caliber vessels (arrowheads). (G) Whole-mounted LacZ
staining of a Tie2-Cre/R26R control E8.5 embryo showed
patchy ␤-galactosidase staining within cranial vessels
(arrow). Scale bars in panels A-B,G: 600 ␮m; C-D: 400
␮m; E-F: 30 ␮m.
A10∆EC
Control
E
G
A10∆EC
F
Control, R26R
Control
vascular structures (Figure 4M-P). These abnormalities were not
visible in control animals and had not yet developed in A10⌬EC
embryos at E15.5, where the distribution of MECA-32–stained
cells in sections of the liver, diaphragm or heart were comparable
with controls (supplemental Figure 5A-D).
Intestinal polyps and enlarged glomeruli in A10⌬EC mice
Analysis of the small intestine of A10⌬EC mice revealed numerous
hyperplastic sessile or pedunculated mucosal polyps containing
multifocal clusters of atypical cells in the lamina propria (Figure
5A-B), which were not observed in control mice (Figure 5C-D).
The atypical cellular masses in the polyps of A10⌬EC mice were
composed of densely arranged plump MECA-32–positive spindle
cells, with scattered single cell necrosis, and small clefts containing
red blood cells, suggesting formation of poorly differentiated
vascular structures (Figure 5B,E). By comparison, only a fraction
of the cells within the lamina propria of control mice stained with
MECA-32 (Figure 5F, please note strong background staining of
plasma cells, arrow heads). Moreover, kidney sections from
A10⌬EC mice revealed enlarged and hypercellular glomeruli with
occasionally dilated peripheral capillaries compared with controls
(Figure 6A-D) and an increased amount of mesangial collagenous
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ROLE OF ENDOTHELIAL CELL ADAM10 IN ANGIOGENESIS
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Figure 4. Histopathologic analysis of the liver, heart and diaphragm in adult mice. (A-D) Livers from A10⌬EC animals had enlarged vessels under the surface in gross
(A) and histologic (C) specimens that were not present in livers from control animals (B,D). (E-F) MECA-32 immunohistochemistry of liver sections from A10⌬EC (E) and
control (F) individuals showed positive staining for endothelial cells lining vascular structures. Arrows mark the abnormally enlarged vessels in panels A,C and E. (G,H) Liver
sections from A10⌬EC mice demonstrated areas of coagulative necrosis (G, arrow) not present in controls (H). (I,J) A heart from an A10⌬EC animal showed numerous, dilated,
superficial blood-filled vascular structures (marked by an arrow in panel I) that were not present in a heart from a control animal (J). (K,L) MECA-32 immunohistochemistry of a
heart section from an A10⌬EC mouse (K) showed positive staining for endothelial cells surrounding the enlarged vascular structures (pointed by an arrow in panel K) that were
not present in a control heart (I). (M-N) Histopathologic analyses of muscular diaphragm specimens revealed increased cellularity between myofibers in A10⌬EC animals
(M, arrow) compared with controls (N). (O-P) MECA-32 immunohistochemistry of diaphragm sections from A10⌬EC (O) and control (P) individuals showed positive staining of
cells present between myofibers of A10⌬EC diaphragms (arrow in panel O). The micrographs of vascular abnormalities on H&E-stained sections are representative of all
animals analyzed for each genotype (n ⫽ 6). Scale bars in panels A-B: 1000 ␮m; C-D: 100 ␮m; E-H,K-P: 20 ␮m; I-J: 500 ␮m.
matrix, as detected by H&E and Masson’s trichrome staining
(Figure 6E-F). There was stronger MECA-32 staining in glomeruli of adult A10⌬EC mice than in controls, consistent with an
increase in endothelial cells (Figure 6G-H). At embryonic stage
E14.5, the MECA-32 staining in the developing intestine and
kidney where similar in A10⌬EC mice and littermate controls
(supplemental Figure 5E-H), suggesting that the expansion of
MECA-32–positive cells in the intestinal polyps and enlarged
glomeruli occurs at a later stage.
Defects in long bone growth and increased erythropoiesis
Live adult A10⌬EC mice could be visually distinguished from their
littermate controls because of their shorter hind limbs, caused by
significantly shortened femurs (Figure 7A-C). The tibiae and
humeri were also slightly shorter in A10⌬EC mice than in controls
(Figures 7A-B,D and data not shown), whereas the length of the
vertebral column and the size and appearance of other skeletal
structures such as tarsals, metatarsals and ribs were not detectably
affected (supplemental Figure 6 and data not shown). Sections of
the distal femoral growth plate of A10⌬EC mice uncovered
abnormalities such as discontinuous and poorly organized zones of
proliferating and hypertrophic chondrocytes in which the overall
thickness of these zones varied substantially (Figure 7E-H). The
primary spongiosa showed areas of increased and decreased bone
density, as well as multifocal replacement of hematopoietic cells in
intertrabecular spaces by loosely arranged atypical spindle cells
(Figure 7I-J). Finally, histologic analysis of the spleen revealed
marked expansion of the red pulp by increased numbers of
erythroid precursors in A10⌬EC mice (supplemental Figure 7), and
cytologic evaluation of the bone marrow showed expansion of the
immature and mature erythroid pools, with normal cellular morphology and maturation sequence (supplemental Table 1 and data not
shown). The hemoglobin, hematocrit, and red blood cell counts
were lower in A10⌬EC mice compared with controls, and relative
reticulocyte counts were elevated (supplemental Figure 8), raising
the possibility of hemolysis accompanied by a regenerative erythroid response, perhaps caused by flow through the disorganized
vascular beds of mutant glomeruli and intestinal polyps.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
GLOMSKI et al
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Discussion
The principal goal of this study was to elucidate the function of the
metalloproteinase Adam10 in endothelial cells during mouse
development and adult homeostasis. Inactivation of Adam10 in
endothelial cells resulted in severe vascular abnormalities in the
developing mouse retina and in various specialized vascular
compartments. The phenotype of the developing retinal vascular
tree in A10⌬EC mice at postnatal day P5 had a strong resemblance
with that described for mice that are heterozygous for the endothelial Notch-ligand Dll4 or after a conditional temporal inactivation
of Notch1, or after treatment with ␥-secretase inhibitors.11 However, mice lacking Notch1 in endothelial cells die at E9.5 with
severe defects in cardiovascular development,5 whereas the A10⌬EC
mice described here are viable and fertile, despite their vascular
abnormalities in the liver, heart, kidney and intestine. Interestingly,
a recently reported conditional deletion of Adam10 using a
different Tie2-Cre driver line resulted in early embryonic lethality.35 Because it is now well established that some Tie2-Cre
transgenes can induce germ line deletion of floxed alleles,36 one
possible cause for the difference in phenotypes could be a more
ubiquitous expression of the Tie2-Cre driver used by Zhang et al,
although a contribution of different genetic backgrounds cannot be
ruled out. Interestingly, the activation of an R26R Lac-Z reporter at
embryonic day E8.5 by the Tie2-Cre used in our study had a mosaic
pattern, raising the possibility that a later onset of expression of this
Figure 5. A10⌬EC mice develop intestinal polyps.
(A-D) H&E staining of intestine from an A10⌬EC (A-B)
and control (C-D) mouse revealed hyperplastic intestinal
polyps (indicated by arrows in panel A) with marked
hypercellularity within the lamina propria of A10⌬EC
individuals (marked by arrows in panel B). (E-F) Almost
all cells within the lamina propria of the enlarged polyps
in A10⌬EC intestine stained positively for MECA-32
(E, arrow), while less than half the cells were MECA-32–
positive in the corresponding area in control villi (F).
Please note that the round dark-staining cells marked by
arrowheads most likely represented plasma cells and
not endothelial cells. Scale bars in panels A,C: 500 ␮m;
B,D-F: 20 ␮m.
Tie2-Cre, perhaps coupled with a lack of germ line expression
could provide an explanation for the survival of the A10⌬EC mice
used here. We excluded that recombination of the floxed Adam10
allele we used could lead to a hypomorphic phenotype by
generating Adam10⫺/⫺ mice after germ line deletion of floxed
Adam10. These animals displayed severe defects in somitogenesis
and heart development at E9.5, and therefore resembled previously
described Adam10⫺/⫺ knockout mice.24 The viability of the A10⌬EC
mice generated in this study thus provided a unique opportunity to
study how deleting Adam10 in endothelial cells affects vascular
structures in developing and adult mice.
The large vessels under the liver capsule of adult A10⌬EC mice
are consistent with a defect in Notch signaling since they resemble
those described in transgenic mice overexpressing the Notch1
extracellular domain,37 which is likely to interfere with Notch
signaling in a dominant negative manner. However, unlike the
abnormal vessels throughout the liver that were reported by Li et al,
the enlarged vessels in A10⌬EC mice were confined to the
subcapsular region, and were not seen deeper within the parenchyma. In addition, we observed an increased vascularity in the
muscular diaphragm and epicardium, which, together with the liver
capsule, have developmental origins from the septum transversum
mesenchyme.38 The enlarged vascular structures in all 3 tissues
were thin-walled, surrounded by a single layer of endothelial cell
without a tunica media, suggesting that they are small veins or
venules, and therefore that Adam10 has a role in limiting the size of
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 6. A10⌬EC mice display marked glomerular
pathology. (A-D) H&E staining of kidney sections revealed enlarged glomeruli (A) with increased cellularity
(C) in A10⌬EC mice (A,C) compared with control mice
(B,D; arrows point to glomeruli in panels A and B, and to
glomerular microvessels in panels C and D). (E-F)
Masson trichrome staining revealed increased amounts
of blue-staining material consistent with increased collagen deposition within glomeruli of A10⌬EC individuals
(indicated by arrow in panel E) not present in control
individuals (F). MECA-32 immunohistochemistry of kidney sections showed positive staining of most cells
present within glomeruli of an A10⌬EC mouse
(H, arrows), with only few capillaries stained in a glomerulus of a control mouse (G, arrows). Scale bars in panels
A-B: 200 ␮m; C-H: 20 ␮m.
ROLE OF ENDOTHELIAL CELL ADAM10 IN ANGIOGENESIS
A
B
A10∆EC
Control
C
D
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Control
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Control
G
H
A10∆EC
Control
these veins during development, as has been previously demonstrated for Notch.6,8 Interestingly, there were no evident defects in
the brain, lung and appendicular muscle of A10⌬EC mice (data not
shown), suggesting that these tissues and their vascular beds
develop normally in the absence of Adam10. However, we cannot
rule out temporary defects that are later remodeled, as in the retinal
vasculature, where the transient nature of the increased branching
phenotype can most likely be attributed to vascular remodeling and
pruning by leukocytes, which also occurs during normal retinal
angiogenesis.39
Additional vascular defects were uncovered in renal glomeruli
and in the small intestine of adult A10⌬EC mice. Mutant glomeruli
were markedly enlarged and hypercellular, with increased staining
for the endothelial cell marker MECA-32,29 suggesting that this
morphologic abnormality resulted from an expansion of endothelial cells. Likewise, the numerous intestinal polyps in A10⌬EC
mice contained masses of cells expressing MECA-32, which
1171
appeared to have caused these polyps by expanding within the
underlying lamina propria. Because endothelial cells in glomeruli
and intestinal villi each represent vascular niches40 with specialized
functions (filtration or absorption, respectively), we hypothesize
that Adam10 has a role in regulating cell fate decisions in these
specialized vascular cells, leading to their abnormal expansion in
the absence of Adam10. Future studies will be required to better
understand the developmental origin and properties of these cells,
and to determine whether there is a functional equivalent of tip and
stalk cells in the development of the vasculature in glomeruli and
intestinal villi.
The significantly shortened femurs in A10⌬EC mice are presumably also caused by abnormalities in the specialized vasculature
invading the growth plate, as previous studies have uncovered
important roles for angiogenesis during bone growth by endochondral ossification.41-44 Interestingly, the growth of other long bones
was less strongly or not affected in A10⌬EC mice, even though
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
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*
*
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G
H
I
J
Figure 7. Long bone growth is impeded in A10⌬EC mice. (A,B) Radiographs of the hind limb of an A10⌬EC mouse (A) revealed a substantially shorter femur than in a
control mouse (B). (C-D) Quantification showed significant reduction in the length of the femur and tibia in A10⌬EC mice compared with controls (C; femur length, A10⌬EC:10.9
mm ⫾ 0.5 n ⫽ 6; controls: 14.7 mm ⫾ 0.3 n ⫽ 6; D; tibia length, A10⌬EC: 15.9 mm ⫾ 0.3 n ⫽ 6; controls: 17.4 mm ⫾ 0.2 n ⫽ 6,). (E-J) H&E staining of femoral condyles from
A10⌬EC (E,G,I) or control mice (F,H,J) revealed an abnormal growth plate (arrows in panel E), disturbed trabecular bone architecture (asterisk in E), abnormally oriented and
enlarged vessels (arrow in panel G) and aberrant spindle shaped stromal cells (arrowhead in panel I) in A10⌬EC specimens compared with the normal growth plate (arrows in
panel F), trabecular bone (asterisk in panel F), vessels (arrow in panel H) and compact hematopoietic cells within the marrow compartment (arrows in I,J) in controls. Data show
mean ⫾ SEM. Scale bars in panels A-B: 8 mm; E-F: 500 ␮m; G-H: 100 ␮m; I-J: 20 ␮m.
sections of all long bones analyzed exhibited abnormal morphology of the growth plate. Perhaps the growth of the femur is more
strongly affected than that of other long bones in A10⌬EC mice
because it has the largest diameter of all long bones, which could
conceivably further exacerbate the hypoxia gradient that develops
toward the center of growing long bones.44 A more hypoxic
environment is likely to affect the fate of chondrocytes in the
growth plate of A10⌬EC mice, as these cells require proper
oxygenation to undergo apoptosis.44 Interestingly, femur length is
normal in adolescent 20-day old A10⌬EC mice, which is consistent
with this hypothesis. An alternative explanation to the observed
shortened femoral length is that hematopoiesis in the femur could
lead to more severe hypoxia of its growth plate than in other bones,
although this explanation seems unlikely because the bone marrow
in A10⌬EC mice contains substantially less hematopoietic cells
than the bone marrow in controls. These results support the notion
that abnormal vascularization in developing long bones can make a
significant contribution to the pathogenesis of phocomelia in
humans,45 although additional studies will be required to understand the cause of the shortened femurs in A10⌬EC mice.
The splenic extramedullary hematopoiesis and peripheral reticulocytosis in A10⌬EC mice is presumably secondary to peripheral hemolysis,
a notion that is supported by the observation that hematocrit, hemoglobin and red blood cell count are lower than in controls. However,
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ROLE OF ENDOTHELIAL CELL ADAM10 IN ANGIOGENESIS
because Notch has been implicated in the regulation of apoptosis in
erythroblasts,46 and hematopoietic cells derive from aortic endothelial
cells,47 and express Tie2-Cre,48 it is possible that the inactivation of
Adam10 by Tie2-Cre also reduces apoptosis in erythroblasts, thereby
contributing to the increased number of reticulocytes in A10⌬EC mice.
However, there would have to be an additional accelerated hemolysis to
explain why other measurements related to red blood cells are decreased. Further studies will be necessary to distinguish between these
possible causes.
The viability of A10⌬EC mice provided the unique opportunity
to study how the lack of Adam10 in endothelial cells affects
pathologic neovascularization in a mouse model for oxygeninduced retinopathy (OIR), which depends on VEGF-A expression
triggered through relative hypoxia.33,34 OIR elicited a strong
increase in the number of neovascular endothelial cells that crossed
the internal limiting membrane in A10⌬EC mice, suggesting that
Adam10 normally limits the development of pathologic neovascular tufts. Previous studies of OIR in zebrafish also identified an
increase in neovascularization in the presence of ␥-secretase
inhibitors, which block Notch signaling.49 Interestingly, the endothelial cells that had crossed the internal limiting membrane in
retinas of A10⌬EC mice after OIR formed large sheets with lumens
containing red blood cells, whereas pathologic neovascularization
normally manifests itself in the form of small neovascular tufts that
often lack a lumen. These large vein-like structures could be caused
by an inability of Notch to limit vein formation6,8 in the absence of
Adam10. Finally, the increased revascularization of the central
avascular area in A10⌬EC mice suggests that Adam10 also serves
to restrict beneficial neovascularization.
In summary, the inactivation of Adam10 in endothelial cells led
to a variety of vascular abnormalities, including an increased
vascular density at the leading edge of the developing retinal
vasculature, enhanced pathologic neovascularization in the OIR
model, abnormal vessels in the liver, epicardium and diaphragm,
increased endothelial cells in glomeruli and intestinal polyps and
an abnormal growth of long bones. Because Adam10 has emerged
as a crucial regulator of Notch signaling in a variety of developmental systems, and because Notch is known to regulate cell fate
decisions, we hypothesize that the Adam10/Notch signaling axis is
crucial for the proper development and/or maintenance of the
specialized endothelial cells populating the organ-specific vascular
niches that are abnormal in A10⌬EC mice.40 Of the 4 mammalian
Notch receptors, Notch1 and Notch4 are highly expressed in
endothelial cells.2 It is therefore possible that interruption of
signaling through one or both of these receptors is responsible for
the multisystemic vascular pathology in A10⌬EC mice, although a
role for Adam10 in processing Notch4 remains to be established.
This hypothesis would provide a simple and unifying explanation
for the various distinct vascular defects in A10⌬EC mice, although
we cannot rule out that processing of other substrates by Adam10,50
or other roles of Adam10 that are not related to its catalytic activity,
contribute to these vascular phenotypes. Because the Notch1⌬EC
1173
mice5 were generated with a Tie2-Cre driver that is known to have
germ line expression,36 it will now be interesting to delete Notch1
in endothelial cells with the Tie2-Cre driver used here, and to
generate Notch1⌬EC mice that also lack Notch4 to assess vascular
development and pathologic neovascularization in these animals.
Moreover, it will be interesting to generate mice lacking both
Adam10 and Adam17, which has also been implicated in Notch
processing,20 in endothelial cells to determine whether Adam17 can
perhaps partially compensate for the loss of Adam10 in these cells.
Finally, from a clinical perspective, these findings have implications for the use of metalloproteinase inhibitors or Notch inhibitors
to treat cancer or proliferative retinopathies, which should be tested
for their effect on Adam10 and Notch signaling in endothelial cells
to avoid any increase in neovascularization or other unintended
consequences on the organ systems affected in A10⌬EC mice.
Acknowledgments
The authors thank Dr Mark Rosenblatt for helpful discussions
and use of his microscopy facility, Dr Tom Sato for providing
Tie2-Cre mice, and Francesca Cardello and Elin Mogollon for
their assistance in this project.
This work was supported by EY015719 to C.P.B., and was
conducted in a facility constructed with support from Research
Facilities Improvement Program Grant C06-RR12538-01 from the
National Center for Research Resources, National Institutes of
Health. K.G. was supported by a training grant from the National
Heart, Lung, and Blood Institute (NHLBI; T32 GM007739-31S1)
to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional
MD/PhD program. P.S. was supported by the Deutsche Forschungsgemeinschaft SFB877, and IUAP P6/58 of the Belgian Federal
Science Policy Office and the Center of Excellence “Inflammation
at Interfaces.” B.D.S. is supported by a Methusalem grant (KU
Leuven) of the Flemisch Government.
Authorship
Contribution: K.G. performed experiments, analyzed and interpreted the data, and wrote the paper; S.M. performed the histopathologic evaluations and contributed to writing the paper; K.M
performed the analysis of embryonic angiogenesis and contributed
to writing the paper; B.D-S. and P.S. contributed to the generation
of Adam10flox mice and provided intellectual input and corrections
on the paper; and C.P.B. supervised the project, analyzed the data,
and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Carl P. Blobel, Arthritis and Tissue Degeneration Program, Caspary Research Bldg, Rm 426, Hospital for
Special Surgery, 535 E 70th St, New York, NY 10021; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2011 118: 1163-1174
doi:10.1182/blood-2011-04-348557 originally published
online June 7, 2011
Deletion of Adam10 in endothelial cells leads to defects in
organ-specific vascular structures
Krzysztof Glomski, Sébastien Monette, Katia Manova, Bart De Strooper, Paul Saftig and Carl P.
Blobel
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