© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1465-1472 doi:10.1242/dev.104539
RESEARCH REPORT
Dynamin 2 regulation of integrin endocytosis, but not VEGF
signaling, is crucial for developmental angiogenesis
Monica Y. Lee1,2,*, Athanasia Skoura1,2,*, Eon Joo Park1,2, Shira Landskroner-Eiger1,2, Levente Jozsef1,2,
Amelia K. Luciano1,2, Takahisa Murata3, Satish Pasula4, Yunzhou Dong4, Mohamed Bouaouina2,
David A. Calderwood1,2,5, Shawn M. Ferguson4, Pietro De Camilli5,6 and William C. Sessa1,2,‡
KEY WORDS: Angiogenesis, Endocytosis, Endothelium, Mouse
INTRODUCTION
Dynamin (Dnm) is a GTPase essential for membrane fission leading
to clathrin-mediated endocytosis (Ferguson and De Camilli, 2012).
There are three isoforms of Dnm in mammalian cells, Dnm1, Dnm2
and Dnm3, which are encoded by distinct genes that are
differentially expressed in various tissues (Ferguson et al., 2009).
Mice deficient in Dnms have been generated and the loss of
neuronally enriched isoforms, Dnm1 and Dnm3, results in impaired
neurotransmission due to a defect in synaptic vesicle endocytosis
(Ferguson et al., 2007; Raimondi et al., 2011). The loss of Dnm2
leads to early embryonic lethality and the loss of Dnm1/2 in
podocytes regulates renal glomerular filtration (Soda et al., 2012).
Recent evidence largely based on pharmacological inhibition of
Dnm function (Gourlaouen et al., 2013; Lanahan et al., 2010;
Sawamiphak et al., 2010; Wang et al., 2006; Wang et al., 2010)
suggests that clathrin-mediated endocytosis is necessary for vascular
endothelial growth factor (VEGF) signaling in vitro and
angiogenesis in vivo. The VEGF receptor 2 (Vegfr2; Kdr – Mouse
Genome Informatics) receptor complex interacts with the Eph
receptor ligand ephrin B2, and this complex is internalized via
1
Vascular Biology and Therapeutics Program, Yale University School of Medicine,
New Haven, CT 06520, USA. 2Department of Pharmacology, Yale University
School of Medicine, New Haven, CT 06520, USA. 3Department of Veterinary
Pharmacology, Graduate School of Agriculture and Life Sciences, The University
of Tokyo, Tokyo 113-8657, Japan. 4Cardiovascular Biology Program, Oklahoma
Medical Research Foundation, Oklahoma City, OK 73104, USA. 5Cell Biology, Yale
University School of Medicine, New Haven, CT 06520, USA. 6Howard Hughes
Medical Institute, Yale University School of Medicine, New Haven, CT 06520,
USA.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Received 4 October 2013; Accepted 5 February 2014
clathrin to promote angiogenesis (Wang et al., 2011b). Moreover,
the cytoplasmic domain of ephrin B2 interacts with a complex
containing clathrin, the general clathrin adaptor complex (AP2),
Dab2, Dnm2 and myosin VI (Nakayama et al., 2013). The
endothelial-specific loss of Dab2 attenuates Vegfr2 internalization
and angiogenesis, and loss of aPKC increases endocytosis and
signaling, implying that Vegfr2 internalization is essential for
signaling and angiogenesis. However, there are additional data
suggesting that the cargo-specific endocytic clathrin adaptor
molecules epsin 1 and epsin 2 can terminate VEGF signaling as the
genetic loss of epsins impedes Vegfr2 endocytosis, increases VEGF
signaling and promotes abnormal pro-angiogenic functions (Pasula
et al., 2012). Indeed, the interaction of epsins with ubiquitylated
Vegfr2 allows for its clathrin-mediated internalization and
destruction. Clearly, the role of endocytosis in the angiogenesis
response is complex and is influenced by diverse routes of
endocytosis and multiple cargo-specific clathrin adaptor molecules.
However, the role of the central GTPase in endocytosis, Dnm2, has
not been genetically dissected in endothelium.
Here, we show that Dnm2, the major isoform of Dnm expressed
in endothelial cells (ECs), is essential for developmental
angiogenesis. In cultured ECs depleted of Dnm2, VEGF receptor
endocytosis is impaired and VEGF signaling is enhanced. Despite
augmented VEGF signaling, VEGF-induced cell migration and
tubular morphogenesis is impaired, implying that pathways
downstream of Vegfr2 signaling are regulated by Dnm2.
Mechanistically, the loss of Dnm2 or clathrin increases focal
adhesion size and impairs integrin internalization thereby reducing
cell motility and morphogenesis. Moreover, the loss of Dnm2 in
endothelium in vivo attenuates branching angiogenesis and promotes
the accumulation of β1 integrin at sites of blunted angiogenic
sprouts, replicating the data in cultured ECs. Thus, Dnm-dependent
endocytosis uncouples VEGF signaling from angiogenic pathways
and is crucial for the endocytic turnover of integrins and
organization of EC during angiogenesis in vivo.
RESULTS AND DISCUSSION
To examine the role of endocytosis during angiogenesis, we
investigated the expression of all three Dnm proteins in mouse lung
ECs (MLECs) and found that Dnm2 is the major isoform expressed
(supplementary material Fig. S1A). Indeed, reducing DNM2 or
clathrin heavy chain (CHC; CLTC – Human Gene Nomenclature
Database) levels by 80-90% via siRNA in human umbilical vein
ECs (HUVECs) significantly reduced the endocytosis of
fluorescently labeled human transferrin, a well-characterized cargo
molecule dependent on Dnm and clathrin (Ferguson et al., 2009)
(Fig. 1A). DNM2 knockdown increased the surface accumulation of
actin nucleating protein p34 subunit of the Arp2/3 complex and the
clathrin adaptor adaptin 2, consistent with data in fibroblasts
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Development
ABSTRACT
Here we show that dynamin 2 (Dnm2) is essential for angiogenesis
in vitro and in vivo. In cultured endothelial cells lacking Dnm2,
vascular endothelial growth factor (VEGF) signaling and receptor
levels are augmented whereas cell migration and morphogenesis are
impaired. Mechanistically, the loss of Dnm2 increases focal adhesion
size and the surface levels of multiple integrins and reduces the
activation state of β1 integrin. In vivo, the constitutive or inducible loss
of Dnm2 in endothelium impairs branching morphogenesis and
promotes the accumulation of β1 integrin at sites of failed angiogenic
sprouting. Collectively, our data show that Dnm2 uncouples VEGF
signaling from function and coordinates the endocytic turnover of
integrins in a manner that is crucially important for angiogenesis in
vitro and in vivo.
RESEARCH REPORT
Development (2014) doi:10.1242/dev.104539
(Fig. 1B). The loss of DNM2 had no effect on cell viability or
apoptosis (supplementary material Fig. S1B). As DNM2-dependent
endocytosis has been implicated in VEGF signaling, HUVECs
treated with scrambled or DNM2 siRNA were stimulated with
VEGF165 (50 ng/ml) and downstream pathways examined. VEGF
signaling was increased in HUVECs lacking DNM2, as
demonstrated by enhanced phosphorylation of VEGFR2 (KDR –
1466
Human Gene Nomenclature Database), PLCγ, ERK and AKT
(Fig. 1C) and by VEGF-induced changes in intracellular calcium
(Fig. 1D). Moreover, the loss of DNM2 increased surface VEGFR2
levels as quantified by fluorescence-activated cell sorting (FACS)
(Fig. 1E). Interestingly, despite enhanced VEGF signaling, serumand VEGF-driven chemotaxis (Fig. 1F), and cord formation
(Fig. 1G) are impaired whereas F-actin rich stress fibers are
Development
Fig. 1. Loss of Dnm2 regulates VEGF signaling but reduces angiogenesis. (A) Loss of DNM2 or CHC (50 nM of each) reduces transferrin endocytosis in
HUVECs. n=4 experiments. Inset shows knockdown efficiency of corresponding siRNAs. (B) Localization of Arp2/3 complex and AP2 (adaptin 2) in control and
Dnm2-deficient HUVECs. Scale bars: 7.5 μm. (C-E) Effect of VEGF stimulation (50 ng/ml) on signaling. (C) Representative blot and quantification (from four
individual experiments) showing enhanced VEGF signaling via analysis of phospho-proteins. (D,E) Calcium (D) and surface Vegfr2 (E) levels in Dnm2depleted HUVECs (from four individual experiments). (F,G) Basal, serum-driven and VEGF-driven HUVEC migration (F) and cord formation (G) was markedly
impaired by Dnm2 siRNA. Data are from three independent experiments repeated in duplicates. SCR, scrambled siRNA. Error bars represent s.e.m. *P<0.05,
***P<0.001.
increased (supplementary material Fig. S1C) in cells that lack
DNM2.
The reduction in cell motility, impaired morphogenesis and
increased stress fibers triggered by the loss of DNM2 suggested that
DNM2 may be regulating integrin-dependent adhesive properties.
Previous work in other cell types have shown that clathrin-mediated
endocytosis is essential for integrin recycling and focal adhesion
disassembly and that Dnm2 can interact with clathrin adaptors or
Development (2014) doi:10.1242/dev.104539
focal adhesion kinase (FAK; Ptk2 – Mouse Genome Informatics) to
regulate focal adhesions (Arjonen et al., 2012; Chao et al., 2010;
Chao and Kunz, 2009; Ezratty et al., 2009; Ezratty et al., 2005;
Nishimura and Kaibuchi, 2007; Wang et al., 2011a). Therefore, we
tested whether Dnm2 could regulate adhesion dynamics and integrin
endocytosis in primary ECs. Immunofluorescence staining for
paxillin and FAK (Fig. 2A) showed increased size of focal adhesions
(quantified in Fig. 2B) in cells depleted of Dnm2 or CHC
Fig. 2. Dnm2 regulates focal adhesion size and integrin internalization. (A,B) Representative images of paxillin and FAK accumulation in DNM2-deficient
cells (A) and quantification of these data (B; triplicate transfections of siRNA from an experiment, repeated three additional times). (C) Loss of DNM2 or CHC
increases surface, but not total, levels of β1 integrin as detected by surface biotinylation (as described in Materials and Methods). Blot shown is representative
of four experiments. (D,E) FACS analysis of surface αvβ5, αvβ3 and β1 integrins after DNM2 knockdown in HUVECs (D) and quantification of these data (E).
n=3-4 experiments. (F) Levels of active β1 integrin (via binding FN 9-11) relative to total β1 levels (integrin activation index) in HUVECs treated with DNM2
siRNA were reduced in the absence of DNM2. Data represent results from three to four independent experiments. (G) Adhesion to fibronectin is reduced in
DNM2 siRNA-treated cells. O.D., optical density. Data are from four independent experiments. SCR, scrambled siRNA. Error bars represent s.e.m. *P<0.05,
**P<0.01, ***P<0.001.
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(supplementary material Fig. S2). As seen in Fig. 2C, reducing
levels of Dnm2 or CHC (Cltc – Mouse Genome Informatics) via
siRNA-enhanced β1 integrin levels at the cell surface (surface
fraction) without altering the total levels of this integrin in whole
cell lysates. This was confirmed by FACS in non-permeabilized
cells using β1-, αvβ3- or αvβ5 integrin selective antibodies
(Fig. 2D,E). To examine if the loss of Dnm2 has a net effect on
integrin activation, FACS (see supplementary material Fig. S3 for
profiles and gating) was performed using a GST fusion protein
composed of fibronectin type III repeats 9 through 11 (FN 9-11),
which selectively binds to activated integrins (Bouaouina et al.,
2012). As seen in Fig. 2F, the loss of Dnm2 reduced the ratio of
active to total pools of β1 integrin, implying that despite an elevation
of surface integrins by the loss of Dnm2, the net activation state was
reduced. Finally, we examined whether the loss of Dnm2 affected
integrin-matrix interactions by quantification of EC interactions with
fibronectin, a ligand for β1 and β3 integrins. As seen in Fig. 2G, the
loss of Dnm2 reduced EC adhesion to fibronectin, consistent with
increased levels of inactive integrins and this effect was also seen
using electric cell-substrate impedance sensing (ECIS) resistance
measurements (supplementary material Fig. S4).
To study the role of Dnm2 function in angiogenesis in vivo, we
inactivated Dnm2 in ECs via Cre/loxP-mediated recombination of
floxed Dnm2 using a Tie2 CRE mouse (supplementary material
Fig. S5A,B). EC deletion of Dnm2 caused gross morphological
defects at embryonic day (E) 9.5 and no live embryos remained at
E11.5 (Fig. 3A-D). Major blood vessels, such as dorsal aortae, were
formed in Dnm2flox/flox, Tie2Cre mice, suggesting that Dnm2 is not
required for the initial stages of vasculogenesis. However,
Dnm2flox/flox; Tie2Cre embryos showed pale, abnormally wrinkled
yolk sacs with a small number of blood-filled vessels at E9. Vessels
in the head and trunk exhibited defective vascular patterning
(Fig. 3C,D) and the yolk sac was strikingly abnormal (Fig. 3E,F).
As early as E9.0, tail intersomitic vessels (ISVs) of Dnm2flox/flox;
Tie2Cre embryos (identical in somite number and size to littermates)
failed to elongate properly resulting in reduced vessel length
(supplementary material Fig. S5C). These data indicate that Dnm2
function in ECs is required for mouse embryonic vascular
morphogenesis and remodeling.
Next, we used inducible loss of function in ECs to selectively
excise the Dnm2 gene by breeding Cdh5-CreERT2 mice (Benedito
et al., 2009) with floxed Dnm2 mice (Ferguson et al., 2009).
Downregulation of Dnm2 expression was achieved by oral gavage
of tamoxifen (2 mg) into pregnant Dnm2flox/flox females at either E9.5
or E10.5 (Benedito et al., 2009; Pitulescu et al., 2010). Two days
after the initiation of gene inactivation in ECs (E11.5-E12.5),
Dnm2iΔEC embryos were macroscopically indistinguishable from
littermate controls; however, detailed examination revealed the
formation of subcutaneous hemorrhages (Fig. 3I). Histological serial
cross-sections of E12.5 embryos revealed the presence of dilated
vessels filled with blood that diffused into the abluminal space
(Fig. 3J, top right panel). Finally, deletion of Dnm2 at E12.5 led to
significant impairments in branching in the hindlimb (Fig. 3J,
middle panels) and yolk sac (Fig. 3J, bottom panels). These
observations demonstrate that the absence of Dnm2 impairs
developmental angiogenesis and vascular patterning in mouse
embryo.
Postnatal inactivation [initiated at postnatal day (P) 1 and
analyzed at P5] of Dnm2 in Dnm2iΔEC mice (P5) displayed impaired
retinal vascular development demonstrated by reduced endothelial
cell coverage, delayed radial growth towards the periphery
(Fig. 4A,D), reduced branching (Fig. 4E,F) and impaired
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Development (2014) doi:10.1242/dev.104539
filopodia/tip cells at the sprouting angiogenic front of the retina, but
no significant differences in the thickness of the vessels and
proliferation (supplementary material Fig. S6A,B) based on
phospho-histone-3 staining (Fig. 4C). Dnm2iΔEC pups (P5) were a
similar weight to littermate controls (supplementary material
Fig. S6C). The loss of Dnm2 had no effect on the distribution of
NG2 (Cspg4 – Mouse Genome Informatics)-positive pericytes and
GFAP-positive astrocytes (supplementary material Fig. S6D,E).
Furthermore, Dnm2 inactivation (P5-P9) showed that remodeling of
established vessels in the superficial capillary plexus at P12 is
strongly altered (Fig. 4G) and this difference was not due to growth
impairment (supplementary material Fig. S6F). Centrifugal
outgrowth and sprouting of the nerve fiber layer (NFL) vasculature
into the outer plexiform layer (OPL) was compromised in Dnm2iΔEC
retinas resulting in cyst-like outgrowths (Fig. 4H, asterisks).
Collectively, these data demonstrate that the postnatal lack of Dnm2
in endothelium diminishes sprouting angiogenesis.
Finally, to examine whether Dnm2 regulates the localization and
distribution of Vegfr2 and β1 integrin in vivo, Dnm2 was inactivated
by injection of tamoxifen at P1-P3 and FACS was carried out for
surface β1 integrins and Vegfr2 levels in retinal single cell isolates
at P6. The loss of Dnm2 increased the surface expression of both
proteins in vivo as seen in Fig. 4I,J. Moreover, β1 integrin was
diffusely distributed throughout the growing vascular networks and
at this time point did not show preference for expression in cells at
the growing front versus mature stalk cells (Fig. 4K). However, in
retinal ECs lacking Dnm2, there was a marked accumulation of β1
integrin in blunted tip cells near the angiogenic front. Thus, Dnm2dependent endocytosis is crucial for the proportional turnover of
Vegfr2 and β1 integrin needed for sprouting angiogenesis in vivo.
Collectively, the data reveal a major role for Dnm2 in endothelial
cell functions in vitro and developmental angiogenesis in vivo. Our
genetic data in conjunction with results from endothelial-specific
epsin-deficient mice (Pasula et al., 2012) indicates that endocytosis
negatively regulates VEGF signaling. Epsins selectively bind to
ubiquitylated Vegfr2, but not integrins, permitting increased Vegfr2
levels, signaling and function, whereas the loss of Dnm2 blocks
endocytosis of both Vegfr2 and integrins, resulting in reduced
angiogenesis. These data imply that a dominant function of Dnm2
during angiogenesis is to regulate the assembly/disassembly of cellmatrix adhesions, key molecular events that promote endothelial
morphogenesis and vessel stability.
Upon first glance, our data may appear to contradict previous
work showing that Vegfr2 endocytosis is necessary for signaling and
angiogenesis. Previously, Lanahan et al. (Lanahan et al., 2010)
documented that the loss of synectin or myosin VI did not affect the
movement of Vegfr2 from the cell surface to the endosome but
indeed reduced, not eliminated, intracellular VEGF signaling.
Lampugnani et al. (Lampugnani et al., 2006) showed that the loss
of clathrin reduced Vegfr2 endocytosis, but VEGF signaling was
only suppressed in cells lacking VE-cadherin (also known as
cadherin 5), not in normal ECs. In Wang et al. (Wang et al., 2010),
the authors injected dynasore, a Dnm GTPase inhibitor, into the
developing retina to show that Dnm was required for Vegfr2
signaling; however, a recent paper now shows this class of inhibitors
block endocytosis in cells lacking all Dnms, showing that these
inhibitors are non-specific (Park et al., 2013). Our work and the
work on epsins clarifies that blocking endocytosis using discrete
molecular tools increases VEGF signaling, consistent with a recent
paper showing that Dnm2 negatively regulates epidermal growth
factor receptor (EGFR) signaling (Sousa et al., 2012). However,
once signaling is initiated at the cell surface, the subsequent
Development
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Development (2014) doi:10.1242/dev.104539
endocytosis of Vegfr2 can promote additional post-plasma
membrane signaling.
Mechanistically, impairments in integrin internalization and
inactivation are likely to explain the defective morphogenesis and
remodeling observed in Dnm2iΔEC mice. Indeed, endothelial cell
deletion of the β1 integrin subunit leads to early lethality (E9.5)
with defects in vascular branching phenocopying the Dnm2flox/flox;
Tie2Cre vascular defects (Carlson et al., 2008; Lei et al., 2008;
Tanjore et al., 2008; Zovain et al., 2010). In the present paper, the
loss of Dnm2 impedes endocytosis of the three integrins tested;
thus, the loss of Dnm2 retards the endocytosis and recycling of
integrins, resulting in the net accumulation of inactive integrins.
However, Dnm2 regulation of integrins is not likely to be an
exclusive mechanism because endocytosis of ephrin B2, Notch and
Notch ligands or VE-cadherin may also contribute to the Dnm2
knockout phenotypes (Alghisi et al., 2009; Wang et al., 2006;
Wang et al., 2011b). In summary, our results provide the first
definitive molecular insights into the role of Dnm-mediated
endocytosis in angiogenesis in vivo and show that VEGF signaling
can be uncoupled from pro-angiogenic endothelial cell functions,
suggesting that endocytosis may be an ‘Achilles heel’ to attack
pathological angiogenesis in disease.
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Fig. 3. Dnm2 function in ECs is required for vascular morphogenesis. (A,B) Deletion of Dnm2 in ECs caused severe embryo growth retardation at E9.5.
(C,D) Dnm2flox/flox; Tie2Cre embryos (E9.5) showed defective vascular patterning. (E,F) Dnm2flox/flox; Tie2Cre yolk sacs exhibited abnormal vascular structures
(E9.5). (G,H) At E9.0, tail intersomitic vessels (ISVs) of Dnm2flox/flox; Tie2Cre embryos showed significantly reduced vessel length. (I) Dnm2iΔEC embryos
develop subcutaneous hemorrhages at E9.5-E11.5 and at E10.5-E12.5. (J) Histological serial cross-sections of E12.5 after tamoxifen (TMX) administration at
E10.5. Dnm2iΔEC embryos showed dilated vessels and blood diffusion (top panel) and an impairment in vessel morphogenesis in the hindlimb (middle panel)
and yolk sac vascular beds (bottom panel E12.5). A, aorta, V, cardinal Vein. Scale bars: 2 mm (A,B,I); 240 μm (C,D); 100 μm (E-H); 50 μm (J).
RESEARCH REPORT
Development (2014) doi:10.1242/dev.104539
MATERIALS AND METHODS
RNAi experiments and immunofluorescence
HUVECs (P2-P6) were transfected by Oligofectamine (Invitrogen)
according to the manufacturer’s instructions with siRNA of control
(luciferase), human DNM2 or clathrin heavy chain (CHC) designed by
Qiagen. For endocytosis experiments, human Transferrin Alexa 568
1470
(Invitrogen, T13343) was added to cells and incubated at 4°C for 15
minutes (5 mg/ml stock, 1:400). Plates were transferred back to 37°C and
incubated for 30 minutes. To remove cell surface-bound transferrin, cells
were washed with cold PBS, pH 2.7, containing 25 mM glycine and 3%
bovine serum albumin and fixed with 4% paraformaldehyde for 15
minutes, on ice.
Development
Fig. 4. Dnm2 is crucial for retinal angiogenesis and β1 integrin levels in vivo. (A) Representative images of reduced radial retinal EC growth in Dnm2iΔEC
pups (P5). (B) Emerging tip cell front of angiogenic vessels (P5). (C) Lack of Dnm2 in EC does not influence growth, as determined by PH3 staining.
(D) Quantification of data shown in A. (B,E,F) Loss of Dnm2 in ECs leads to reduced branching (E,F; control, n=4 and Dnm2iΔEC, n=11) and impaired filopodia.
(G,H) Remodeling of established vessels in the superficial capillary plexus at P12 is strongly altered in Dnm2iΔEC pups (100 μg TMX, P5-P12). P12 Dnm2iΔEC
pups with compromised centrifugal outgrowth (H). Centrifugal outgrowth of the nerve fiber layer (NFL) and compromised sprouting into the outer plexiform layer
(OPL). Zooming into the NFL shows cyst-like outgrowths in Dnm2iΔEC pups. (I,J) FACS plots of retinal cells isolated from P6 mice showing elevated surface
Vegfr2 (I) and β1 integrin (J) in Dnm2iΔEC pups. (K) Distribution of β1 integrin is abnormal in Dnm2iΔEC retinas. *P<0.05, **P<0.001. Scale bars: 5 mm (A); 50
μm (B); 100 μm (C); 2 mm (G,H,J); 250 μm (K).
RESEARCH REPORT
Analysis of integrin activation and expression
siRNA-treated HUVECs were used for analysis of surface expression and
integrin activation by measuring the binding of a fibronectin fragment (FN911) as described previously (Bouaouina et al., 2012). Total β1, αVβ3 and
αVβ5 integrin expression levels were measured in parallel by staining,
respectively, with anti-β1 (clone TS2/16, Biolegend), anti-αVβ3 (clone
23C6, Biolegend) and anti-αVβ5 (clone 15F11, Millipore) antibodies. Bound
FN9-11 and integrin expression were detected with Allophycocyanin (APC)conjugated streptavidin (Thermoscientific) or AlexaFluor647-conjugated
donkey anti-mouse IgG, respectively, as described in supplementary material
Fig. S3.
Generation of Dnm2 conditional deletion in the endothelium
Dnm2 conditional knockout targeting strategy was previously reported
(Ferguson et al., 2009). EC Dnm2-deficient mice were generated by crossing
Dnm2flox/flox females with Dnm2flox/+; Tie2Cre males (Jackson Laboratory).
Tamoxifen (Sigma, T5648)-inducible Dnm2iΔEC were obtained by breeding
Dnm2flox/flox females with Dnm2flox/flox;Cdh5-CreERT2 male mice. Pregnant
females were treated with TMX by oral gavage (2 mg, Sigma T5648) and
embryos (E11.5-E12.5) were fixed in 4% PFA/PBS, embedded in OCT and
cut into 8 µm serial cross-sections. All experimental procedures were
approved by the Yale University Institutional Animal Care Use Committee.
Immunohistochemistry and immunofluorescence of mouse
tissues
Hematoxylin and Eosin staining was performed with Mayer’s Hematoxylin
(10 minutes) and counterstained with Eosin (1 minute). Images were
processed with a Nikon Eclipse 80i microscope and using the Photoshop CS
software. For whole-mount staining, freshly isolated embryos and yolk sacs
were fixed for 10 minutes in 4% PFA/PBS at 4°C, washed in PBS and
incubated in blocking/permeabilization solution [PBS (pH 7.2), 1% BSA,
0.1% Triton X-100] for 1 hour. Embryos were incubated overnight at 4°C
in the presence of PE-conjugated rat anti-mouse CD31 (BD Pharmingen
553373). Microscopy was carried out with a confocal laser scanning Leica
TCS SP5 microscope. ISV length quantification was performed using the
Image J software (National Institutes of Health, NIH).
Adhesion assay
Ninety-six-well plates were coated with fibronectin (5 μg/ml, 354008,
Becton Dickinson) at 4°C overnight. Plates were blocked with 10 mg/ml
heat-denatured bovine serum albumin for 30 minutes. A set volume (100 μl)
of HUVECs in cell suspension (5×105 cells/ml in basal medium with 0.1%
BSA) were added onto plates and incubated for 30 minutes at 37°C. After
incubation, non-adherent cells were removed by rinsing with PBS. Attached
cells were fixed in 4% PFA/PBS for 20 minutes and stained with 0.1%
Crystal Violet. Crystal Violet was dissolved in 10% acetic acid, and plates
were read at an absorbance reading of 575 nm.
For Electrical Cell Impedance System (ECIS, Applied Biophysics)
measurements, HUVECs in suspension treated with either SCR or siDnm2
were plated in suspension onto polycarbonate wells containing gold electrodes
(1×105 cells/well, in normal growth medium). Endothelial resistance
measurements as a function of time were recorded as described by
manufacturers. In brief, a 4000 Hz current was applied across the electrodes
where the in-phase and out-of-phase voltages were monitored in real-time and
subsequently converted to measurements of trans-endothelial resistance.
Mouse retina vascular system analysis
For retina staining, procedures were followed as described (Pitulescu et
al., 2010) with minor modifications. Pups (P1-P3) were injected with 50
μg tamoxifen and for later stages (P12) a 100 μg was used (P5-P9).
Briefly, retinas were dissected out, fixed, permeabilized and stained with
Alexa-594-conjugated isolectin B4 (Invitrogen, I21413; 1:200), rabbit
anti-phospho-histone H3 (Cell Signaling, 9701; 1:200), rabbit anti-GFAP
(Neomarkers, RB-087-A0; 1:200) and rabbit anti-NG2 (Millipore,
AB5320; 1:200).
Whole retina FACS
Retinas of control or Dnm2iEC mice were excised at P6 (n=5-7 per group)
and digested in Type I Collagenase for 45 minutes at 37°C (Sigma), filtered
via a 40-μm cell strainer and washed with PBS/0.5% fetal bovine serum, pH
7.4. Cells were blocked with mouse BD Fc Block (BD Pharmingen,
553142), followed by staining for either phycoerythrin (PE)-conjugated
VEGFR-2 (BD Pharmingen) or PE-conjugated Integrin β1 (eBiosciences).
Cells were measured by FACSCalibur flow cytometer and analyzed using
FlowJo software.
Statistical analyses
Statistical analyses were performed with Prism 5 software (GraphPad) using
the two-tailed, unpaired Student’s t-test or one-way ANOVA, when
appropriate. P values <0.05 were considered statistically significant.
Acknowledgements
The authors would like to acknowledge Drs Anne Eichmann, Martin Schwartz and
David Cheresh for helpful comments and suggestions.
Competing interests
The authors declare no competing financial interests.
Author contributions
Concept development: W.C.S., A.S., P.D.C. and S.M.F.; performance and analysis
of data: M.L., E.P., L.J., S.L.-E., A.L., T.M., S.P., Y.D. and M.B.; preparation of
manuscript: W.C.S., A.S., M.L., D.A.C., S.M.F. and P.D.C.
Funding
This work was supported by the National Institutes of Health [RO1 grants
HL64793, HL61371, HL081190, HL096670 and PO1 1070205 to W.C.S.; RO1
GM068600 and GM088240 to D.A.C.]. Deposited in PMC for release after 12
months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.104539/-/DC1
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Biotin surface labeling
Biotin cell surface labeling was performed on HUVECs 72 hours postsiDnm2 transfection. In brief, cells were incubated with a 1 mM EZ-linksulfo-NHS-S-S-biotin solution (Pierce #21331) for 30 minutes on ice. Cells
were washed with a 50 mM glycine/PBS solution, pH 7.2 and subsequently
harvested in protein lysis buffer. Following centrifugation, an aliquot was
collected for protein levels in total cell lysates, and the biotin-labeled surface
proteins were incubated with Neutravidin Protein agarose beads (Pierce
#29200) at 4°C overnight. Flow-through fractions were collected after the
incubation period following a 3000 rpm (960 g) spin. The beads were then
washed several times in lysis buffer prior to addition of Laemmli Buffer for
immunoblot analysis of β1 integrin (BD Biosciences).
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