Endothelial Expression of Guidance Cues in
Vessel Wall Homeostasis
Dysregulation Under Proatherosclerotic Conditions
Janine M. van Gils,* Bhama Ramkhelawon,* Luciana Fernandes, Merran C. Stewart, Liang Guo,
Tara Seibert, Gustavo B. Menezes, Denise C. Cara, Camille Chow, T. Bernard Kinane,
Edward A. Fisher, Mercedes Balcells, Jacqueline Alvarez-Leite, Adam Lacy-Hulbert, Kathryn J. Moore
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Objective—Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved
in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance
cues may regulate leukocyte trafficking into the vascular wall during atherogenesis.
Approach and Results—We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules
are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are
expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes.
Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory
shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show
using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte
adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under
proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of
additional chemokines.
Conclusions—The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of
the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously
unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking
during atherogenesis. (Arterioscler Thromb Vasc Biol. 2013;33:911-919.)
Key Words: atherosclerosis ◼ axonal guidance ◼ endothelial–leukocyte interaction ◼ migration
A
therosclerosis is a chronic inflammatory disease of
the large arteries, in which monocyte recruitment
into the vessel wall plays an essential role in both the
initiation and progression of disease. Atherosclerotic
plaques form predominantly at sites of disturbed laminar
flow, notably, arterial branch points and bifurcations,
and are initiated by the subendothelial accumulation of
apolipoprotein B-containing lipoproteins.1,2 The key early
inflammatory response to these atherogenic lipoproteins is
activation of overlying endothelial cells in a manner that
leads to recruitment of blood-borne monocytes.3 Research
in the last decades has provided a rich description of the
molecules involved in the recruitment of leukocytes into
the artery wall, and highlighted the importance of the
coordinated action of chemokines, integrins, and other
adhesion molecules in directing this pathological process.4
The chemoattractant and adhesive forces that recruit
leukocytes to the vessel wall have been well studied in this
context.5 However, our understanding of other aspects of
immune cell migration remains incomplete, particularly
the mechanisms by which these cells are excluded from
the artery in the absence of inflammation. In addition to
the well-known positive migration cues that promote
atherosclerotic plaque formation, it is reasonable to assume
that chemorepulsive forces, or negative guidance cues, also
exist to inhibit leukocyte–endothelial interactions under
homeostatic conditions that may become dysregulated
during disease.
Received on: August 21, 2012; final version accepted on: February 4, 2013.
From the Marc and Ruti Bell Vascular Biology and Disease Program, Leon H. Charney Division of Cardiology, Department of Medicine, New
York University School of Medicine, New York, NY (J.M.V.G., B.R., M.C.S., L.G., E.A.F., K.J.M.); Department of Biochemistry and Immunology,
Federal University of Minas Gerais, Belo Horizonte, MG, Brazil (L.F., G.B.M., D.C.C., J.A.-L.); University of Ottawa Heart Institute, Ottawa, Ontario,
Canada (T.S.); Program of Developmental Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, MA (C.C., B.K., A.L.-H.);
Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA (M.B.); and Institut Quimic de Sarria, IQS School of
Engineering, Ramon Llull University, Barcelona, Spain (M.B.).
*These authors contributed equally to this study.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.301155/-/DC1.
Correspondence to Kathryn J Moore, New York University School of Medicine, 522 First Ave, Smilow 705, New York, NY 10016. E-mail Kathryn.
[email protected]
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
911
DOI: 10.1161/ATVBAHA.112.301155
912 Arterioscler Thromb Vasc Biol May 2013
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The integration of chemoattractive and chemorepulsive
signals is essential for controlling neuronal migration during development, and this paradigm is evolutionarily conserved from Caenorhabditis elegans to mammals.6 Netrins,
slits, semaphorins, and ephrins compose the 4 major families
of conserved neuronal guidance cues that control neuronal
migration and vascular patterning through a complex interplay of signals. However, accumulating evidence points to
additional functions for these guidance molecules in regulating cell migration outside of the nervous system, including
roles in the physiological and pathological regulation of the
immune response.7–9 Such studies have shown that members
of the netrin, slit, semaphorin, and ephrin families of guidance cues can regulate immune cell activation or differentiation, and have both chemoattractive and chemorepulsive
effects on leukocyte migration.9–12 For example, recent work
from our group identified netrin-1 as a leukocyte guidance cue
expressed by the endothelium, where its expression was modulated during acute inflammation because of Staphylococcus
aureus infection.11 In this model, netrin-1 was found to be
expressed on the luminal surface of lung endothelial cells,
where it acted to block the migration of monocytes to such
bacterial factors as the N-formylated peptide N-formylmethionine-leucine-phenylalanine, suggesting that it may
play a role in endothelial barrier function. At the onset of
S. aureus infection or on treatment with tumor necrosis
factor-α (TNF-α) in vitro, endothelial expression of netrin-1
was rapidly downregulated consistent with a lowering of this
barrier to leukocyte infiltration of the tissues.11
Given the emerging roles for neuronal guidance cues in
regulating inflammation, we hypothesized that these molecules may also have roles in early atherosclerosis, when,
as alluded to above, endothelial dysfunction attributable to
injury or lipoprotein retention precedes the development of
atherosclerotic plaques. To perform a systematic evaluation
of the 4 major classes of evolutionarily conserved neuronal
guidance molecules, we used a microarray approach using
RNA samples harvested from atherosclerosis-prone and
-resistant aortic sites in a mouse model of early atherogenesis. We identified key members of the netrin, semaphorin, and
ephrin families that are regulated in the initial stages of plaque
formation, and confirmed these changes in arterial endothelial cells exposed to atherogenic factors in vitro. Notably, we
identified guidance cues that both block (netrin-1 and semaphorin3A) or promote (ephrinB2) monocyte migration, and
showed that their expression by endothelium is concurrently
regulated by proatherosclerotic conditions in a manner that
would facilitate leukocyte recruitment. Furthermore, using
intravital microscopy, we demonstrated that blocking peptides targeting these molecules alters leukocyte adhesion to
the endothelium in vivo, as would have been predicted by
the array and in vitro results. Together, these data suggest an
emerging paradigm for the coordination of leukocyte–endothelial interactions in vessel wall homeostasis by neuroimmune guidance cues.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Results
Neuronal Guidance Molecules Are Differentially
Regulated by Flow
It is well established that atherosclerotic lesions develop in
areas of branching or high curvature that are associated with
oscillatory or turbulent flow, so-called atheroprone regions.13
We therefore investigated the expression of neuronal guidance
molecules in 2 different regions of the vessel wall: (1) the inner
curvature (atheroprone region) and (2) the outer curvature (atheroprotected or homeostatic region) of the aortic arch (Figure
1A). We placed Ldlr–/– mice on a Western diet for 2 weeks to
model conditions for the early initiation of atherosclerosis, and
isolated the inner and outer curvature from their aortic arches.
Using custom mRNA arrays that covered all 4 families of neuronal guidance molecules, we compared the expression profile
between these 2 vascular sites. In the aortic arch, we found that
31 of the 36 neuronal guidance molecules we profiled were
expressed (Figure 1B and 1C). Comparing the expression of
neuronal guidance molecules from the inner curvature relative
to the outer curvature, we found that 10 were >25% downregulated in the inner compared with the outer curvature, and
only 2 were >25% upregulated (Figure 1C). From these, we
selected candidate molecules from the netrin, semaphorin, slit,
and ephrin families and confirmed the array findings by independent quantitative real-time PCR (Figure 1D). Expression
of netrin-1 (Ntn1), which we previously showed can limit the
migration of monocytes, granulocytes, and lymphocytes,11 was
reduced by 48% in the inner curvature (atheroprone) compared
with the outer curvature. Like Ntn1, another reported repulsive
cue of the semaphorin family, semaphorin3A (Sema3a), was
downregulated in the inner curvature compared with the outer
curvature (–53%), consistent with roles in endothelial barrier
function for these 2 guidance molecules. From the slit family, Slit1 was not detectable, whereas Slit2 was expressed at
very low levels. However, Slit3 was confirmed to be expressed
at similar levels between the inner and outer curvature. From
the ephrin family, the ephrinB2 gene (Efnb2) was selected for
validation, as ephrinB2 has been demonstrated to be expressed
in vascular endothelial cells and may be sufficient to initiate
monocyte–endothelial interactions.12,14 However, we did not
detect differential expression of Efnb2 mRNA between the
inner curvature and outer curvature of the aorta (Figure 1D).
To directly investigate whether it was the endothelial cells
of the aorta that contributed to the observed differences in
Ntn1, Sema3a, and Efnb2 gene expression, and to extend those
differences to the protein level, we performed immunostaining
of longitudinal sections of the aortic arch of the Ldlr–/– mice
fed a Western diet for 2 weeks. At this time point, changes
in endothelial cell morphology are observed in the disturbed
flow regions of the lesser curvature of the aorta, before the
subendothelial accumulation of monocytes.15 Netrin-1
immunostaining was detected in endothelial cells (positive for
CD31) of the outer curvature, but was highly downregulated
on endothelial cells of the inner curvature of the aortic
arch (Figure 2). Similarly, semaphorin3A was expressed
by endothelial cells in the atheroprotected outer curvature,
whereas there was little to no semaphorin3A expression by
endothelial cells of the inner aortic curvature. In contrast,
van Gils et al Expression of Guidance Cues in Atherosclerosis 913
A
Outer Inner
1 2 3 1 2 3
B
Outer Inner
1 2 3 1 2 3
CD31
Outer
Inner
Magnitude of gene expression
min
C
max
average
Inner versus outer curvature
1.75
D
1.8
Fold change mRNA
1.25
1
0.75
0.5
0.25
0
ND
ND
ND ND
ND
Ntn1
Ntn3
Ntn4
Ntn5
Ntng1
Ntng2
Slit1
Slit2
Slit3
Efna1
Efna2
Efna3
Efna4
Efna5
Efnb1
Efnb2
Efnb3
Sema3a
Sema3b
Sema3c
Sema3d
Sema3e
Sema3f
Sema3g
Sema4a
Sema4b
Sema4c
Sema4d
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Sema5b
Sema6a
Sema6b
Sema6c
Sema6d
Sema7a
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Fold change mRNA
1.5
1.6
outer curvature
inner curvature
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Ntn1 Sema3a Efnb2
Slit3
Figure 1. Differential expression of neuronal guidance molecules in the inner vs outer curvature of the aortic arch. A, Ldlr–/– mice were
fed a Western diet for 2 weeks, after which their aortas were harvested and the atheroprotected outer curvature or atheroprone inner
curvature isolated. CD31 staining confirmed disruption of endothelial integrity suggestive of early atherosclerosis in the inner curvature
compared with outer curvature. B and C, mRNA expression profiling of these aortic regions for netrin, slit, ephrin, and semaphorin family
members using custom quantitative real-time PCR arrays (n=3 mice); (B) heat-map; (C) relative fold change in mRNA expression in the
inner curvature compared with outer curvature. D, Validation of candidate guidance cue expression in the inner and outer curvature by
quantitative real-time PCR. Data are mean±SEM. *P<0.05.
endothelial cells of the inner aortic curvature expressed
ephrinB2; however, staining for ephrinB2 was not detected in
the atheroprotected outer curvature.
Endothelial Expression of Netrin-1, Semaphorin3A,
and EphrinB2 Are Regulated by Proatherosclerotic
Conditions In Vitro
Given the difference in netrin-1, semaphorin3A, and ephrinB2
mRNA, and protein levels in atheroprotected versus atheroprone
regions of the aorta, we postulated that hemodynamic
proatherosclerotic forces regulate the expression of these
guidance cues on endothelial cells. We therefore seeded human
coronary artery endothelial cells (HCAECs) onto fibronectincoated gas-permeable laboratory tubes and exposed them to
arterial (atheroprotective), oscillatory (atheroprone) flow, or
no flow (static) for 6 hours. As a positive control, we measured
endothelial nitric oxide (Nos3), an atheroprotective factor
known to be expressed under arterial laminar flow conditions,
and found that its mRNA abundance was upregulated 3-fold
compared with static control but was not significantly changed
by oscillatory flow conditions compared with static control
(Figure 3A). Similarly, Ntn1 mRNA was almost 5-fold
upregulated by arterial flow, but unchanged in oscillatory flow
conditions when compared with static control (Figure 3A). A
different pattern emerged for Sema3a; expression of this gene
was unchanged between static and arterial flow conditions,
however it was highly downregulated by oscillatory flow
(≈50%; Figure 3B). Notably, Efnb2 gene expression was
also regulated by oscillatory flow, but unlike Sema3a, it was
significantly upregulated under these proatherosclerotic flow
conditions (1.7-fold; Figure 3B). Slit3 mRNA and protein
were not affected by either atheroprotective or atheroprone
flow conditions, and, therefore, was not investigated further
(Figure IA in the online-only Data Supplement).
The observed changes in the expression of Ntn1, Sema3a,
and Efnb2 under differential flow conditions led us to consider
whether other proatherogenic factors could modulate the
expression of these molecules. To examine this, we stimulated
HCAECs with chemokines that have been implicated in
the recruitment of monocytes during early atherosclerosis,
including CCL2 (monocyte chemotactic protein-1 [MCP-1]),
CX3CL1 (Fractalkine; FKN), or interleukin (IL)-8.5 Analysis
of HCAEC mRNA expression revealed that MCP-1, FKN, and
IL-8 all significantly reduced Ntn1 after 6 hours of treatment
(Figure 4A). Similarly, MCP-1 and FKN, but not IL-8,
reduced Semaphorin3A expression under these conditions
914 Arterioscler Thromb Vasc Biol May 2013
Outer aortic arch
L
Inner aortic arch
500µm
Inner aortic arch
CD31
Outer aortic arch
Netrin-1
Merge
CD31
Netrin-1
Merge
CD31
Sema3a
Merge
CD31
EphrinB2
Merge
L
L
CD31
Sema3a
Merge
L
L
EphrinB2
L
Merge
L
100µm
Figure 2. Immunofluorescent staining of netrin-1, semaphorin3A, and ephrinB2 in atheroprotected and atheroprone regions of the aorta.
Longitudinal sections of the aortic arch of the Ldlr–/– mice fed a Western diet for 2 weeks were stained for CD31 (green), netrin-1, semaphorin3A, or ephrinB2 (red), and DAPI (blue) in the atheroprone inner curvature (left) or atheroprotected outer curvature (right) of the aortic arch. Areas of colocalization (yellow) are shown in the merged image. Images are representative of 5 mice (L=lumen).
(Figure 4A). In contrast, ephrinB2, which we found to be
induced by oscillatory flow conditions, was not affected by
atherosclerotic cytokines/chemokines (Figure 4A). Similar
effects on Ntn1 and Sema3a mRNA were seen with other
classical proatherogenic activators of the endothelium, such as
IL-1β and TNF-α, as well as with bacterial lipopolysaccharide
(Figure 4B). Notably, these proinflammatory stimuli increased
Efnb2 mRNA in HCAEC (Figure 4B). To ascertain whether
these changes were reflected at the protein level, we treated
HCAECs with MCP-1 or TNF-α for 24 hours, and performed
Western blotting for netrin-1, semaphorin3A, and ephrinB2.
Expression of netrin-1 and semaphorin3A by HCAECs was
7
*
6
5
B
Arterial flow
Oscillatory flow
*
4
3
2
1
0
Ntn1
Nos3
2.5
Fold change relative to static
A
Fold change relative to static
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CD31
*
2.0
Arterial flow
Oscillatory flow
1.5
1.0
0.5
0
*
Sema3a
Efnb2
Slit3
Figure 3. Regulation of netrin-1, semaphorin3A, and ephrinB2
expression by arterial flow conditions. (A) Ntn1, Nos3 (endothelial
nitric oxide) and (B) Sema3a, Efnb2, and Slit3 mRNA in human
coronary artery endothelial cells (HCAECs) subjected to arterial
(17±1 dynes/cm2 continuous flow in one direction) or oscillatory (17±1 dynes/cm2 biphasic flow in opposing directions) for 6
hours. Data are expressed as the fold change in mRNA in each
condition compared with static flow. Data are the mean±SD of
quadruplicate samples in a single experiment and are representative of an experimental n of 4. *P<0.05.
decreased after treatment with MCP-1 and TNF-α, whereas
ephrinB2 was increased under similar conditions (Figure 4C).
Netrin-1, Semaphorin3A, and EphrinB2 Regulate
Leukocyte Migration
Because we observed lower expression of netrin-1 and semaphorin3A in regions of the aorta subject to proatherosclerotic/
oscillatory flow and in endothelial cells exposed to oscillatory
flow conditions or proatherosclerotic cytokines, we hypothesized that endothelial expression of netrin-1 and semaphorin3A may act as a barrier to prevent monocyte migration into
the arterial intima under basal conditions. To test this hypothesis, we investigated the effects of netrin-1 and semaphorin3A
on monocyte migration to 2 chemokines implicated in monocyte recruitment during early atherogenesis; FKN (CX3CL1)
and MCP-1 (CCL2). Cultured THP-1 monocytes or freshly
isolated human peripheral blood mononuclear cells were used
in modified Boyden chamber assays to examine monocyte
migration to FKN or MCP-1 by adding recombinant netrin-1
to the lower chamber in the absence or presence of chemoattractants. As we previously reported,11 recombinant netrin-1
did not alter monocyte migration in the absence of chemokine
(data not shown). However, addition of recombinant netrin-1
inhibited peripheral blood mononuclear cell migration to
FKN and MCP-1 by up to 50% (Figure 5A and 5B). This
inhibitory effect of netrin-1 was reversed by preincubation
of peripheral blood mononuclear cells with an antibody that
binds to the extracellular domain of Unc5b, a receptor for
netrin-1 expressed by monocytes11 (Figure 5C). Similarly,
when semaphorin3A was added in combination with FKN or
MCP-1, it was found to be a potent inhibitor of peripheral
van Gils et al Expression of Guidance Cues in Atherosclerosis 915
A
1.4
Ntn1
1.0
0.8
*
*
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*
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0.0
unstim MCP-1 FKN
Sema3A
1.0
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*
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IL-8
unstim MCP-1 FKN
Efnb2
1.4
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Fold change in mRNA
1.2
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1.4
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1.0
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IL-8
unstim MCP-1 FKN
C
Ntn1
*
*
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unstim
*
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unstim LPS
unstim
TNFα
MCP-1
*
IL-1b
unstim
Efnb2
3.5
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unstim LPS IL-1b TNFα
unstim
Sema3A
1.2
TNFα
Fold change in mRNA
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Fold change in mRNA
B
IL-8
*
3
2.5
*
2
*
1.5
1
0.5
0
TNFα
Netrin-1
Sema3A
EphrinB2
GAPDH
GAPDH
GAPDH
unstim LPS IL-1b
unstim
MCP-1
TNFα
unstim
TNFα
Figure 4. Proatherosclerotic chemokines regulate netrin-1, semaphorin3A, and ephrinB2 expression in human coronary artery
endothelial cells (HCAECs). A and B, Ntn1, Sema3a, and Efnb2 mRNA in HCAECs treated with proatherosclerotic chemokines/cytokines
(A; MCP-1, 5 nmol/L; Fractalkine, 5 nmol/L; and IL-8, 5 nmol/L) or (B) lipopolysaccharide (LPS; 1μg/mL), IL-1b (20ng/mL), or tumor necrosis factor-α (TNF-α) (10ng/mL). Data are the mean±SD of triplicate samples in a single experiment and are representative of an experimental n of 3. *P<0.05. C, Western blot analysis of netrin-1, sema3A, ephrinB2, or GAPDH (internal control) in HCAECs stimulated with
MCP-1 or TNF-α for 24 hours.
blood mononuclear cell chemotaxis to both FKN (Figure
5D) and MCP-1 (Figure 5E). In dose–response experiments,
semaphorin3A-dependent effects on migration produces
a bell-shaped curve (also typically seen with chemokines),
with 60% to 90% inhibition observed at 250 ng/mL and
reduced effects observed at the highest doses (1000 ng/mL;
Figure 5D and E).
It has been previously demonstrated that class III secreted
semaphorins can signal through neuropilins and plexins to
mediate their effects on target cells.16,17 We therefore investigated whether neuropilin-1 was required for semaphorin3A-mediated inhibition of monocyte chemotaxis. THP-1
monocytes were preincubated with a blocking antibody to
neuropilin-1 or an IgG isotype control antibody. Preincubation
with the control antibody did not restore monocyte chemotaxis to MCP-1 (Figure 5F). However, preincubation with the
neuropilin-1–blocking antibody restored the ability of THP-1
monocytes to respond to MCP-1 (Figure 5F).
In contrast to netrin-1 and semaphorin3A, ephrinB2
expression was observed to be upregulated in endothelial cells
exposed to oscillatory flow conditions. Thus, we hypothesized
that the upregulation of ephrinB2 may act to recruit monocytes
into the arterial intima. To test this hypothesis, we examined
the ability of ephrinB2 to act as a monocyte chemoattractant.
Indeed, we found that ephrinB2 was a potent monocyte
chemoattractant when added to the lower chamber of Boyden
migration assays (Figure 5G). Migration was induced in a
dose-dependent manner, with maximal chemotaxis observed
at 250 ng/mL of ephrinB2. When ephrinB2 was added with
FKN or MCP-1 to the lower well, chemotaxis was similar to
that seen with ephrinB2 alone (data not shown), indicating
that the effects on chemotaxis were not additive.
Netrin-1 and Semaphorin3A Inhibit Leukocyte
Adhesion
We next investigated whether netrin-1 and semaphorin3A
could alter leukocyte adhesion. Treatment with netrin-1 or
semaphorin3A, both at 250 ng/mL, reduced the adhesion
of RAW264.7 cells to surfaces coated with BSA by 50% to
60% (Figure 6A). As netrin-1 and semaphorin3A inhibit both
leukocyte migration and adhesion in vitro, we next determined
whether enhancing or blocking these 2 negative guidance
molecules would alter leukocyte–endothelial dynamics. We
find that netrin-1 and semaphorin3A actively inhibit THP-1
monocyte binding to HCAEC treated with TNF-α (Figure 6B
and 6C) or lipopolysaccharide (Figure IB in the online-only
Data Supplement). Conversely, blocking peptides to netrin-1
and semaphorin3A increased THP-1 adhesion to quiescent
HCAEC (Figure 6D). Hence, the expression of netrin and
semaphorin3A by the endothelium would be expected to
actively prevent leukocyte arrest and tissue entry.
To examine this we used intravital microscopy, a technique
that allows the observation of leukocyte–endothelial cell interactions in vivo.18 Similar to areas of disturbed blood flow in the
conduit artery, the venous system is exposed to low shear stress
ranging from 1 to 6 dynes/cm2. Thus, intravital microscopy
916 Arterioscler Thromb Vasc Biol May 2013
*
6
*
*
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2
C
10 nM MCP-1
6
35
30
25
* * *
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*
15
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10 100 250 500 1000
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12
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*
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0
0
0
0
E
10 100 250 500 1000
Sema3a (ng/ml)
0
10 100 250 500 1000
Netrin-1 (ng/ml)
25
*
20
15
*
*
*
10
5
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3
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Sema3a (ng/ml)
_
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+
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25
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10 nM MCP-1
Control
30
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MCP-1
Netrin-1
Control Ab
Unc5b Ab
Chemotactic Index
0
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0
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40
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*
B
Chemotactic Index
Chemotactic Index
8
0
Chemotactic Index
1 nM Fractalkine
Chemotactic Index
Control
10
Chemotactic Index
A
MCP-1
Sema3a
Control Ab
NRP-1 Ab
_
_
_
_
+
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+
+
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+
+
+
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+
+
_
*
12
10
*
8
6
*
*
*
4
2
0
0
10 100 250 500 1000
EphrinB2 (ng/ml)
+
Figure 5. Netrin-1, semaphorin3A, and ephrinB2 alter leukocyte migration. Migration of peripheral blood mononuclear cells (PBMCs) to
(A) fractalkine (FKN; 1 nmol/L) or (B) MCP-1 (10 nmol/L) with or without recombinant netrin-1 at the concentrations indicated. C, Preincubation of PBMCs with an Unc5b-blocking antibody (5 μg/mL) blocks the inhibitory effect of netrin-1 (250 ng/mL) on migration to MCP-1
(10 nmol/L). D and E, Migration of THP-1 monocytes to fractalkine (1 nmol/L; D) or MCP-1 (10 nmol/L; E) with or without recombinant
semaphorin3A at the concentrations indicated. F, Preincubation of THP-1 monocytes with a blocking neuropilin-1 antibody (5 μg/mL),
but not a control antibody (5 μg/mL), reverses the inhibitory effect of semaphorin3A on migration to 10 nmol/L MCP-1. G, Migration of
PBMCs to ephrinB2 added at the indicated concentrations in the absence of a chemotactic stimulus. Data are the mean±SD of triplicate
samples in a single experiment and are representative of an experimental n=3. *P<0.05.
of the cremaster vein has proven to be a useful in vivo model
for assessing leukocyte–endothelial cell interactions relevant
to atherosclerosis.19–21 Intravenous infusion of netrin-1– or
semaphorin3A–blocking peptides to C57BL/6J mice did not
alter leukocyte rolling but significantly increased leukocyte
adhesion to the endothelium compared with control peptides
(Figure 7A and 7B). The number of leukocytes adherent to the
endothelium increased approximately 2-fold after the addition
of netrin-1– or semaphorin3A–blocking peptides (Figure 7B),
further supporting our hypothesis that these guidance cues act
to impair this earliest interaction with endothelial cells in vivo.
Discussion
The pioneering studies from the Gimbrone laboratory showed
that the properties of atheroprone and susceptible regions of
the aorta varied in a number of significant ways that could be
attributed to the different flow characteristics at specific sites.13
For example, endothelial cells in the lesser (inner) curvature
of the aortic arch or at branch points, where the laminar flow
tended to be lower, were relatively activated. These observations were subsequently extended by a number of investigators to show that before there was evidence of monocyte
infiltration, a host of molecules, such as P-selectin, vascular
cell adhesion molecule-1, intercellular adhesion molecule-1,
and endothelial nitric oxide, were already adversely affected
in the susceptible areas.3,13 Based on the results of the present
studies, we can now add certain neuronal guidance molecules
as early-disease stage, atheroregulatory, factors that also operate at the level of the endothelium.
Based on the microarray gene expression results (Figure 1),
we focused on netrin-1 and semaphorin3A, because of their
decreased expression in the atheroprone, lesser curvature in
the aortae of prelesional, but diet-stimulated, Ldlr–/– mice. We
also included analyses of eprinB2 and slit3 as members of
their respective classes of neuronal guidance molecules. In our
previously published studies, we reported that netrin-1 inhibits
monocyte migration in vitro in response to bacterial N-formylmethionine-leucine-phenylalanine.11 In that same study, we
found that netrin-1 was expressed in the vascular endothelium
of the lung, as well as in human umbilical vein endothelial
cells, and that its expression was rapidly downregulated
during acute inflammation. Based on these findings, we
hypothesized that the higher expression of netrin-1 in the
endothelium of the atheroresistant greater curvature reflected
an important and novel mechanism by which leukocyte
interactions with endothelial cells are limited in homeostatic
conditions. Furthermore, we considered that there could
also be regulation of leukocyte entry into susceptible arterial
regions by other neuronal guidance molecules, given some
overlap in properties among the family members.
Importantly, we found that the expression of netrin-1
and semaphorin3A was increased under conditions that
recapitulated the steady flow found in healthy arterial
segments relative to the oscillatory pattern found at arterial
regions prone to development of atherosclerotic plaques, as
would be expected if these molecules were atheroprotective
at the endothelial cell level. Endothelial expression of
netrin-1 and semaphorin3A was also decreased by treatment
with proinflammatory cytokines or chemokines implicated
6
4
3
2
1
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Fold change in adhesion
_
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
3
2
1
0
rNetrin-1
*
Peptide: Control Netrin-1
blocking
_
Fold change in adhesion
5
0
C
B
4
Adherent Cells (x 105)
Adherent Cells (x 105)
A
2.5
2
*
1.5
1
*
0.5
0
TNFα _
rNetrin-1 _
rSema3A _
Sema3A
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Fold change in adhesion
van Gils et al Expression of Guidance Cues in Atherosclerosis 917
_
_
+
_
_
*
+
+
_
_
+
+
_
+
_
+
Figure 6. Netrin-1 and semaphorin3A inhibits
leukocyte adhesion. A, Adhesion of RAW264.7
cells, treated without or with netrin-1 (250 ng/mL)
and sema3A (250 ng/mL), to BSA-coated tissue
culture plates. B, Adhesion of THP-1 monocytes
to untreated or tumor necrosis factor-α (TNF-α)–
(10ng/mL) treated human coronary artery endothelial cells (HCAECs) in the presence/absence of
recombinant netrin-1 or sema3A (250 ng/mL). Data
are the mean±SEM of 4 experiments. C, Adhesion of THP-1 monocytes to HCAECs treated with
either netrin-1– or semaphorin3A-blocking peptide
or control peptides. Data are the mean±SEM of 6
experiments. *P<0.05.
Peptide: Control Sema3A
blocking
in leukocyte recruitment to atheroprone regions. We had
previously demonstrated that most leukocyte subclasses,
including monocytes/macrophages and neutrophils, express
the chemorepulsive receptor for netrin1, Unc5b.11,22 This
suggests that when circulating monocytes encounter an
endothelial cell expressing relatively more netrin-1, a barrier
mechanism would be triggered either because secreted netrin-1
inhibited monocyte chemotaxis by creating a diffusible
netrin-1 gradient across endothelial cell layers (similar to
that created by endothelial cell-secreted MCP-1), or through
presentation of netrin-1 on the surface of endothelial cells. In
this regard, there is evidence of netrin-1 binding to α6β4 and
α3β1 integrins on pancreatic epithelial cells,23 and this is one
mechanism by which netrin-1 could be presented on arterial
endothelial cells.
Consistent with either scenario, our results show that (1)
netrin-1 inhibits leukocyte migration to the proatherogenic
chemokines, MCP-1 and FKN, in vitro, in a manner that
involves the Unc5b receptor; (2) netrin-1 inhibits monocyte binding to endothelial cells; and (3) peptides that block
netrin-1 increase leukocyte adhesion to endothelium in vitro.
In addition to supporting an atheroprotective role for netrin-1
at the endothelial level, the current study also suggests a
similar role for semaphorin3A. For example, semaphorin3A
inhibited monocyte migration to MCP-1 in vitro in a process
dependent on its receptor neuropilin, and a semaphorin3Ablocking peptide also reduced leukocyte adhesion in vitro.
Notably, using intravital microscopy of the cremaster vein,
we show that administration of netrin-1 and semaphorin3Ablocking peptides similarly increase leukocyte adhesion
in vivo.
Turning to ephrinB2, in contrast to netrin-1 and semaphorin 3A, it was upregulated in HCAECs in vitro by proatherogenic (oscillatory) flow (Figure 3), and at the protein
level, it was more highly expressed in the inner curvature
endothelium (Figure 2). Another contrast was that it acted to
stimulate leukocyte recruitment. These findings are supported
by Goettsch et al,24 who found that ephrinB2 expression was
downregulated by arterial, but not venous, laminar shear stress
in HUVECs,24 suggesting that this downregulation may keep
endothelial cells in a nonactivated state. Other supporting data
for a role of ephrinB2 in vivo are contained in a report that
showed that monocytes express EphB2, one of the possible
receptors for ephrinB2, and that the expression of EphB2
in monocytes is increased on their adhesion.14 In agreement
with the present results, in that report, there was also greater
expression of ephrinB2 in the endothelium of the lesser curvature, which correlated with an increased abundance of macrophages in the underlying neointima.14
Although we have shown that there is concurrent regulation
of netrin-1, semaphorin3A, and ephrinB2 in the endothelium,
resulting in concerted atheroprotective effects, whether this
represents a common coordinating regulatory mechanisms or
a more complex process remains to be determined. In contrast
to Ntn1 and Sema3a, we did not detect a difference in EfnB2
mRNA expression in the atheroprone and atheroresistant sites
of the aorta, which implies that a combination of transcriptional and posttranscriptional mechanisms may regulate the
expression of these factors. Similarly, whereas TNF-α treatment of HCAECs markedly downregulated Sema3a mRNA,
as well as netrin-1 and semaphorin3A protein levels, this
cytokine increased Ntn1 mRNA in vitro. There are scant data
about the regulation of endothelial expression for any of the
factors we studied. There is some evidence for netrin-1 that
NF-κB and hypoxia-inducible factor-1α may be involved in
its transcriptional upregulation,25,26 but on the surface, this
would be counter to the present results in that (1) treatment
with lipopolysaccharide or TNF-α (activators of NF-κB)
or MCP-1 (a classic target of NF-κB) was associated with
decreased netrin-1 expression (Figure 4); and (2) in areas of
918 Arterioscler Thromb Vasc Biol May 2013
B
Peptide:
D
Control
Netrin-1
blocking
Adhesion
% adhesion
200
180
160
140
120
100
80
60
40
20
0
0 min
10 min
Peptide: Control
270
240
210
180
150
120
90
60
30
0
0 min
10 min
*
Peptide: Control
E
*
C
Adhesion
0 min
10 min
% adhesion
% rolling
Rolling
200
180
160
140
120
100
80
60
40
20
0
% rolling
A
Netrin-1
blocking
Peptide:
0 min
10 min
Control
Sema3a
blocking
leukocyte
Oscillatory flow
Laminar flow
Netrin-1
leukocyte
EphrinB2
Sema3a
endothelium
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Sema3a
blocking
Rolling
200
180
160
140
120
100
80
60
40
20
0
endothelium
Atheroprotective
Atheroprone
Figure 7. Blocking netrin-1 or semaphorin3A increases leukocyte adhesion to endothelium in vivo. Leukocyte rolling and adhesion before
and after administration of a (A and B) netrin-1– or (C and D) semaphorin3A–blocking peptide or control peptide measured by intravital
microscopy in C57BL/6 mice. Data are the mean±SEM, n=5 to 7 mice/group. *P<0.05. E, Schematic diagram of the regulated expression
of neuroimmune guidance cues under atheroprotective and atheroprone (right) conditions. Under conditions of laminar flow (left), netrin-1
and semaphorin3A are expressed on endothelial cells and inhibit leukocyte recruitment, maintaining an atheroprotective state. In contrast,
oscillatory flow characteristic of atheroprone regions of the vasculature downregulates expression of netrin-1 and semaphorin3A, while
increasing levels of ephrinB2, thereby facilitating leukocyte recruitment.
disrupted laminar flow (as in the lesser curvature of the aortic
arch), hypoxia-inducible factor-1α is induced independent of
hypoxia. Thus, although the phenomenon of intersecting protective changes in the 3 guidance molecules is clear, a dissection of the underlying regulatory mechanisms awaits detailed
analyses beyond the scope of the present report.
Our data suggest that netrin-1 and semaphorin3A may
reduce leukocyte migration and recruitment to atherosclerotic plaque by inhibiting firm adhesion to the vessel wall.
In vivo, leukocyte recruitment to inflamed sites is mediated by a combination of adhesion and signaling molecules.
Selectins, expressed at inflamed sites, slow leukocytes and
initiate rolling; next, chemokine signaling to leukocytes
activates integrins to mediate firm adhesion and subsequent
extravasation. Our observations that netrin-1 and semaphorin3A inhibit the initiation of firm adhesion are consistent
with known roles for neuronal guidance molecules. For
example, semaphorin3A inhibits the activation and adhesive
function of β1 and β3 integrins in endothelial cells,27 and
similar effects might be expected to regulate activity of β2
integrins on leukocytes, which are primarily responsible for
firm adhesion of leukocytes to the vessel wall. Netrin-1 has
also been shown to bind to integrins (α3 and α6) and to activate integrin ligand binding through focal adhesion kinase
and small GTPases.23
In summary, our data support a model (Figure 7) in which
the inhibitory guidance molecules netrin-1 and semaphorin3A are more highly expressed by endothelial cells in
atheroresistant aortic regions, where they help to maintain
tissue homeostasis by preventing leukocyte influx. In atheroprone regions, however, they become downregulated by
disturbed laminar flow and local inflammation, which lowers the endothelial barrier to leukocyte entry. Consistent
with this model, systemic delivery of netrin-1 to Ldlr–/– mice
using adenovirus-associated virus reduced atherosclerosis,28
presumably by increasing its expression on endothelial cells.
Adding to the increased predilection for leukocyte recruitment in the prone regions is the upregulation of another
guidance molecule, eprinB2, which increases monocyte
migration in vitro. In addition to these guidance cues, which
we selected for in-depth study, our microarray profiling data
suggest that additional family members, including Ntng2,
Sema3g, Sema4d, and Sema7a, may also be relevant to atherosclerotic processes. During development, the various
families of guidance molecules have been shown to have
overlapping effects and to also coregulate each other positively or negatively. Further exploration of the expression
of neuronal guidance molecules and their receptors under
resting and inflammatory conditions, and their functional
interactions, will likely identify new regulatory mechanisms
and therapeutic targets in atherosclerosis and other inflammatory disorders.
Sources of Funding
Support for this work came from the American Heart Association
(Grant-in-aid 0655840T to K.J.M.; 09POST2080250 to J.M.V.G.),
Fondation de la recherche Medicale to B.R., National Institutes of
Health (RC1HL100815 to K.J.M.; R01 HL084312 to E.A.F.; R01/
GM049039 to M.B.), Canadian Institute of Health Research (CGSMSFSS to T.S.), Conselho Nacional de Desenvolvimento Científico
e Tecnológico (01558/2007-6 to L.R.F. and J.I.A.-L.), Ministerio
de Innovación Plan Nacional BFU2009-09804 (M.B.), Fundació
Empreses IQS (M.B.), and POSIMAT (M.B.).
Disclosures
None.
van Gils et al Expression of Guidance Cues in Atherosclerosis 919
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Significance
We identified key members of the netrin, semaphorin, and ephrin families that are regulated on the endothelium in the initial stages of atherosclerotic plaque formation. We identified guidance cues that both block (netrin-1 and semaphorin3A) or promote (ephrinB2) monocyte migration, and
showed that their expression by endothelium is concurrently regulated by proatherosclerotic conditions in a manner that would facilitate leukocyte
recruitment. Furthermore, blocking peptides targeting these molecules alter leukocyte adhesion to the endothelium in vivo, as would have been
predicted by the array and in vitro results. Together, these data suggest an emerging paradigm for the coordination of leukocyte–endothelial
interactions in vessel wall homeostasis by neuroimmune guidance cues.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Endothelial Expression of Guidance Cues in Vessel Wall Homeostasis Dysregulation
Under Proatherosclerotic Conditions
Janine M. van Gils, Bhama Ramkhelawon, Luciana Fernandes, Merran C. Stewart, Liang Guo,
Tara Seibert, Gustavo B. Menezes, Denise C. Cara, Camille Chow, T. Bernard Kinane, Edward
A. Fisher, Mercedes Balcells, Jacqueline Alvarez-Leite, Adam Lacy-Hulbert and Kathryn J.
Moore
Arterioscler Thromb Vasc Biol. 2013;33:911-919; originally published online February 21,
2013;
doi: 10.1161/ATVBAHA.112.301155
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/33/5/911
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MATERIALS AND METHODS
Mice. 6-8 week old C57BL/6J mice and Ldlr–/– mice were purchased from Jackson Laboratories. All
mice were maintained in a pathogen-free facility. Ldlr–/– mice were fed a western diet for 2 weeks and
then anesthetized with Tribomoethanol (0.4mg/g i.p.) and ex-sanguinated by cardiac puncture. Aortas
were flushed with PBS and perfused with RNAlater® (Ambion). The aortic arches were dissected clean
and the inner and outer curvature separated for RNA isolation. Experimental procedures were done in
accordance with the USDA Animal Welfare Act and the PHS Policy for the Human Care and Use of
Laboratory Animals and the New York University School of Medicine’s and Universidade Federal de
Minas Gerais’s Subcommittees on Research Animal Care and Use.
Cell Culture. Human coronary artery endothelial cells (HCAECs) (Cambrex Bio Sciences, Walkersville,
Maryland) were cultured in growth factor supplemented EBM media. At 70-80% confluency, cells were
trypsinized and plated in a 6-well plate for 24 h prior to use. Cells were either unstimulated (control) or
stimulated with 5 nM IL-8, 10 ng/ml IFN-γ, 5 nM MCP-1, or 5 nM Fractalkine for 6 or 24 hours. THP-1
cells were maintained in RPMI 1640 media (Sigma-Aldrich) supplemented with 10% fetal bovine serum
(FBS) and 2% penicillin-streptomycin.
Leukocyte Isolation. Whole blood was obtained from healthy volunteers and PBMC were isolated
using Histopaque centrifugation (Sigma-Aldrich, St. Louis, MO) as we previously described1. Cells were
resuspended in RPMI-1640 media containing 1% bovine serum albumin (BSA) at a cell density of
1x106 cells per ml. Cells were kept at 4ºC until use and were used within 3 hours of isolation.
Gene Expression Profiling. The inner and outer curvature of the aortic arch were homogenized in
TRIzol reagent (Invitrogen) using the Bullet BlenderTM (New Englang Bio Group) and total RNA
isolated as we described2. Aortic RNA (0.4 µg) from 3 mice was reverse transcribed and quantitative
RT-PCR analysis of 36 axonal guidance molecules (Supplemental Table 1) was performed using RT2
Custom Profiler PCR Arrays (Qiagen) according to manufacturer’s protocol. Data analysis was
performed using the manufacturer’s integrated web-based software package of the PCR Array System
using ΔΔCt based fold-change calculations. Gene expression was normalized to Gapdh and Actinb.
Real-time Quantitative RT-PCR Analysis. RNA (0.5-1 µg) was reverse transcribed using iScript™
cDNA Synthesis Kit (Biorad) and RT-PCR analysis was conducted using iQ SYBR green Supermix
(Biorad) and a Mastercycler Realplex (Eppendorf). Gene expression was normalized to Gapdh. Fold
change in mRNA expression was calculated using the comparative cycle method (ΔΔCt).
Immunofluorescence Staining. HCAEC were cultured on coverslips and stimulated with 100 ng/µl
MCP-1 or PBS 24 hours. Cells were then fixed with 4% paraformaldehyde and made permeable with
0.1% Triton X-100. Endothelial cells were blocked with 5% BSA for 30 minutes and stained with antiNetrin1 chicken polyclonal (Abcam, ab39370), or rabbit polyclonal anti-Semaphorin3A antibody
(Abcam, ab23393) or anti-EphrinB2 (LifeSpan Biosciences, LS-C33253). Secondary fluorescent
antibodies used were Alexafluor 488-coupled anti-chicken antibody (Molecular Probes, A11039), or
Alexafluor 488-coupled anti-rabbit antibody respectively (Molecular Probes, A11008). DAPI was used to
visualize the nucleus. Longitudinal-sections of the aortic arch from Ldlr–/– mice (7 µm) were fixed with
cold acetone for 10 min and non-specific binding sites were blocked with 5% BSA for 30 min at room
temperature. Thereafter, either goat anti-Netrin1 antibody (R&D, AF 1109), or rabbit polyclonal antiSemaphorin3A antibody (Abcam, ab23393) or anti-EphrinB2 (LifeSpan Biosciences, LS-C33253)
followed by rat polyclonal anti-CD31 (BD Pharmigen, 553370) were successively applied to the
sections. After PBS washes, Alexafluor 568-coupled to either anti-goat or anti-rabbit antibody
(Molecular Probes, A11079, A11011), were applied for 1 h each at room temperature. CD31 was
detected by using the secondary Alexafluor 488-coupled anti-rat antibody (Molecular Probes, A11006).
DAPI was used to visualize the nucleus and sections were mounted with Dako fluorescent mounting
medium (Dako, S3023). The slides were visualized under Nikon Eclipse microscope and images
captured under x20 objective
Laminar Shear Stress Experiments. HCAECs were seeded onto the inner surface of sterilized
fibronectin-coated gaspermeable Silastic laboratory tubes by axially rotating the tubes at 10 rotations
per hour at 37°C for 18 h as described3. Closed loops of tubing were created using the section with the
confluent cell monolayer and placed in a perfusion bioreactor at 37°C under 5% CO2. Cells were
exposed to static conditions or shear stress of 18 dynes/cm2 with either continuous laminar flow in one
direction (steady flow) or biphasic laminar flow in opposing directions equaling no net flow (oscillatory
flow) for 6 h before harvest by trypsinization. RNA was isolated using TRIzol reagent (Invitrogen).
Migration Assays. Chemotaxis of PBMCs and THP-1 monocytes was measured using 96-well Boyden
chamber with a filter with a pore size of 5 µm (ChemoTx® Disposable Chemotaxis System Neuro
Probe) as decribed previously1. Cell migration toward 10 nM recombinant human monocyte
chemotactic protein (MCP-1; R&D Systems), 1 nM recombinant human CX3CL1/Fractalkine (FKN;
R&D Systems) or recombinant mouse EphrinB2 (R&D Systems) was measured in the absence or
presence of recombinant chicken Netrin-1 (R&D Systems, Inc., 128-N1) or recombinant human
Semaphorin3A (R&D Systems Inc, 1250S3). In some assays, cells were pretreated with NRP-1
blocking antibody (5 µg/ml, R&D Systems, AF566), Unc5b blocking antibody (5 µg/ml, R&D systems,
AF1006) or control goat IgG (5 µg/ml, SC2028; Santa Cruz) for 30 min at 4ºC, washed 3X with 1% BSA
RPMI, and resuspended in 1% BSA RPMI at 1 x 106 cells/mL prior to addition to the upper wells. After
cell migration for 1-3 h, cells in four randomly selected high-power fields for each well were counted to
determine the number of cells that had migrated into the lower chamber. Each condition was performed
in triplicate. Migration is presented as the chemotactic index, calculated by division of the number of
migrating cells in the treated groups by the number of migrating cells in the control well with the lowest
number of cells as we previously described1, 2.
Adhesion Assays. Confluent HCAECs monolayers (2x105 cells/well in a 96-well plate) were incubated
for 5 hours at 37oC without (control) or with LPS (1 µg/ml) or TNF (10 ng/ml). Recombinant human
netrin-1 (250 ng/ml) or semaphorin3A (250 ng/ml) were added one hour prior to the adhesion assays.
In other experiments, HCAECs were pretreated for 1 hour with either netrin-1- or semaphorin3Ablocking peptide (250 ng/ml) or corresponding control peptide. Fluorescently labeled THP- 1 monocytes
(CellTrackerTM Orange, 1x105 per well) were then added in fresh medium and incubated another 30
minutes. Unbound cells were removed by washing 3 times with PBS. The fluorescence of bound
monocytes was then read in a spectrometer at 576 nm. The experiments were carried out in parallel on
cover slips, and fluorescent cells were visualized under an inverted fluorescent microscope and images
(x4) were taken from 5 random fields in each condition. For experiments with RAW264.7 cells, cells
were resuspended in RPMI 1604 with 2% BSA, alone or with the addition of netrin-1 or Semaphorin3A,
both at 250 ng/ml. 1 x 106 cells/well were incubated in 96 well tissue culture plates previously treated
with RPMI/ 2% BSA at 37˚C for 1 hour, and then
washed 3 times with cold PBS. Adherent cells were then quantified using Cell Titer Reagent
(Promega) or Cell counting kit-8 (Dohindo, MD) and cell number determined by comparison to standard
curves with defined numbers of adherent cells.
Intravital Microscopy. C57BL/6J mice were anesthetized with a solution of 200 mg/kg of ketamine and
xylazine 10 mg/kg and after that, a small incision was made in the scrotal skin to expose the right
testicle and the cremaster muscle. The cremaster muscle was exposed and dissected free, opened and
fixed with surgical cotton yarn. The tissue was constantly irrigated with bicarbonate buffer (37° C, pH
7.4). An optical microscope (Olympus, Japan) with 20x objective was used to examine the muscle
microcirculation. A video camera (Sony, Japan) was used for projecting the images on a television, and
the images were recorded on a recorder on DVD for further analysis. Capillary venules with diameters
between 25 to 40 µm were monitored for 5 minutes before and after i.v. infusion of 80 µg of Netrin-1 or
Semaphorin3A blocking peptide (C-terminal 50 residues) or control peptide. The same vein was used
to observe leukocytes before and after injections. Rolling and adhering cells was counted within a 100
mm length of venule during 1 minute. Leukocyte rolling was defined as the number of cells passing by
fixed points on endothelium surface. A leukocyte was considered adhered when not moving for at least
30 seconds. The percentage of basal adhesion or rolling was calculated by dividing the observed
number of adherent or rolling leukocytes after infusion of the peptide by the number of adherent or
rolling leukocytes before the infusion of the peptide (set to 100%).
Western Blotting: Protein was extracted from HCAEC in RIPA buffer (50 mM Tris pH 7.4, 150 mM
NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40) and separated by SDS-PAGE (40-80 µg).
Proteins were transferred to nitrocellulose membranes, blocked in TBST-5% milk and incubated with
antibodies against netrin-1 (R&D Systems Inc) (1:500 dilution), ephrinB2 (LifeSpan Biosciences)
(1:1000 dilution), semaphorin3A (Abcam) (1:500 dilution) or GAPDH (1:10000 dilution). Horseradish
peroxidase conjugated secondary antibodies anti-mouse IgG (Sigma A9917), anti-rabbit IgG (Sigma
A0545), anti-goat IgG (Sigma A5420) and SuperSignal West Pico Chemiluminescent Substrate (Pierce
34080) were used, and the signals were detected by using LI-COR Odyssey Fc Imaging system.
Statistics. Statistical significance was assessed by an unpaired, two-tailed Student’s t-test for single
comparison or analysis of variance for multiple comparisons. A P value of less than 0.05 was
considered significant.
REFERENCES
1.
2.
3.
Ly NP, Komatsuzaki K, Fraser IP, Tseng AA, Prodhan P, Moore KJ, Kinane TB. Netrin-1 inhibits
leukocyte migration in vitro and in vivo. Proc Natl Acad Sci U S A. 2005;102:14729-14734
van Gils JM, Derby MC, Fernandes LR, Ramkhelawon B, Ray TD, Rayner KJ, Parathath S,
Distel E, Feig JL, Alvarez-Leite JI, Rayner AJ, McDonald TO, O'Brien KD, Stuart LM, Fisher EA,
Lacy-Hulbert A, Moore KJ. The neuroimmune guidance cue netrin-1 promotes atherosclerosis
by inhibiting the emigration of macrophages from plaques. Nature immunology. 2012;13:136143
Balcells M, Fernandez Suarez M, Vazquez M, Edelman ER. Cells in fluidic environments are
sensitive to flow frequency. Journal of cellular physiology. 2005;204:329-335
Supplement Material
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Supplemental Figure I
A
Slit3
Fold change in mRNA
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
unstim MCP-1 FKN IL-8
Fold change in adhesion
B
2.5
2
1.5
*
1
*
0.5
0
LPS _
_
Netrin-1
_
Sema3a
_
+
_
_
_
+
_
+
_
+
+
_
+
_
+
(A) Slit3 mRNA in HCAECs treated with MCP-1 (5 nM), Fractalkine (5 nM), and IL-8 (5 nM). Data are the
mean ± s.d. of triplicate samples in a single experiment and are representative of an experimental n of 3.
(B) Adhesion of THP-1 monocytes to untreated or LPS (1 µg/ml) treated HCAECs in the presence/absence
of recombinant netrin-1 or sema3A (250 ng/ml). Data are the mean ± sem of 4 experiments.
Supplementary Table I. Primer Sequences
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