Molecular Cardiology
Cyclic Stretch Controls the Expression of CD40 in
Endothelial Cells by Changing Their Transforming
Growth Factor–␤1 Response
Thomas Korff, PhD; Karin Aufgebauer, MD; Markus Hecker, PhD
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Background—CD40 is a costimulatory molecule that acts as a central mediator of various immune responses,
including those involved in the progression of atherosclerosis. Correspondent to its function, CD40 is present not
only on many immune cells, such as antigen-presenting cells and T cells, but also on nonimmune cells, such as
endothelial cells.
Methods and Results—Ex vivo analyses in mice revealed that CD40 is strongly expressed in distinct venous and capillary
but not arterial endothelial cell populations. Therefore, we analyzed to what extent determinants of an arterial
environment control CD40 expression in these cells. In vitro studies indicated that the presence of smooth muscle cells
or exposure to cyclic stretch significantly downregulates CD40 expression in human endothelial cells. Interestingly,
endothelial cells cocultured with smooth muscle cells upregulated CD40 expression in response to cyclic stretch through
a transforming growth factor–␤1/activin-receptor–like kinase-1 (Alk-1)– dependent mechanism. To corroborate that this
mechanism also operates in arteries in vivo, we analyzed the expression of Alk-1 and CD40 at atherosclerosis-prone
sites of the mouse aorta that also appear to be exposed to increased stretch. In wild-type mice, both Alk-1 and CD40
revealed a comparably heterogeneous expression pattern along the aortic arch that matched those sites in low-density
lipoprotein–receptor– deficient mice where atherosclerotic lesions develop.
Conclusions—Cyclic stretch thus increases the abundance of CD40 in endothelial cells through transforming growth
factor–␤1/Alk-1 signaling. This mechanism in turn may be responsible for the heterogeneous expression of CD40 at
arterial bifurcations or curvatures and would support a site-specific proinflammatory response that is typical for the early
phase of atherosclerosis. (Circulation. 2007;116:000-000.)
Key Words: endothelial cells 䡲 CD40 antigens 䡲 TGF-beta-1 䡲 smooth muscle cells
T
he costimulatory molecule CD40 plays a central role in
controlling inflammatory responses, including autoimmune syndromes, allograft rejection, and atherosclerosis. It is
expressed by numerous cell types dependent on their activation. Although CD40 appears crucial for the proinflammatory
phenotype of genuine immune-competent cells such as B
cells, this costimulatory molecule is also expressed in vascular cells such as endothelial and smooth muscle cells.1
Experiments in low-density lipoprotein (LDL)–receptor–
deficient mice have shown that neutralization of the CD40
ligand CD154 significantly reduces the size and the lipid,
macrophage, T-cell, and vascular cell adhesion molecule-1
(VCAM-1) content of atherosclerotic lesions,2– 4 hence pointing toward a role for CD40 in atherogenesis. Likewise,
analyses of human cultured endothelial cells revealed that
CD40 expression is upregulated by interleukin-1␤, tumor
necrosis factor-␣, and interferon-␥, proinflammatory cyto-
kines that abound in atherosclerotic plaques.1 Moreover,
ligation of CD40 in these cells enhances the expression of
intercellular adhesion molecule-1, VCAM-1, and E-selectin.5
Taken together, these findings have prompted the hypothesis
that CD40 signaling in endothelial cells contributes to the
recruitment and activation of circulating T cells and monocytes, thus amplifying the chronic inflammatory response in
the vessel wall that leads to atherosclerosis.6
Clinical Perspective p ●●●
Although CD40 expression in plaque-associated vascular
cells has been verified,7 it is not known whether biomechanical forces that are implicated not only in the early but also in
the progression phase of atherosclerosis also affect CD40
expression in these cells. Two biomechanical forces strongly
influence the functional and structural phenotype of vascular
cells. Laminar shear stress, which mainly acts on endothelial
Received July 25, 2007; accepted August 27, 2007.
From the Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, University Hospital Heidelberg, Heidelberg, Germany.
The online-only Data Supplement, consisting of figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/
CIRCULATIONAHA.107.730309/DC1.
Correspondence to Dr Markus Hecker, University Hospital Heidelberg, Institute of Physiology and Pathophysiology, Division of Cardiovascular
Physiology, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.107.730309
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Figure 1. Immunohistochemical detection of CD40 in endothelial cells ex
vivo. Representative photomicrographs
of cross-sectioned mouse tissues
showing endothelial cell CD40 expression in veins (B, mesenteric vein; D,
femoral vein) and capillaries (E, hindlimb muscle; F, heart muscle) but not
arteries (A, mesenteric artery; C, femoral artery; scale bars⫽25 ␮m).
cells, is generally viewed as an atheroprotective force that
upregulates both the activity and expression of endothelial
cell nitric oxide synthase, with the resulting enhanced production of nitric oxide in turn blocking proatherosclerotic
gene expression in these cells.8,9 In contrast, cyclic stretch,
which affects endothelial and vascular smooth muscle cells
alike, is thought to promote atherosclerosis through upregulation of endothelial cell reactive oxygen species formation
and hence proatherosclerotic gene expression.10 –12
Here, we report that in vascular homeostasis, CD40 is
preferentially expressed in venous and capillary endothelial
cells that are typically involved in the recruitment and
activation of leukocytes during inflammatory processes. Although generally not detectable in arterial endothelial cells,
CD40 is also expressed at atherosclerosis-prone sites along
the mouse aortic arch. This surprising finding prompted us to
analyze biomechanical and cellular determinants that may be
responsible for this nonuniform expression pattern of CD40.
Methods
Materials
Transforming growth factor–␤1 (TGF-␤1), activin-receptor–like
kinase-1 (Alk-1) chimeric protein, and anti-human TGF-␤1 and
anti-human Alk-1 antibodies were purchased from R&D Systems
GmbH (Wiesbaden, Germany). Anti-phospho-Smad 1/5 and antiphospho-Smad 1/5/8 antibodies were from Cell Signaling (Danvers,
Mass).
Animal Studies
All animal studies were performed with permission of the Regional
Council of Karlsruhe and conformed to the “Guide for the Care and
Use of Laboratory Animals” published by the US National Institutes
of Health (National Institutes of Health publication No. 85-23,
Korff et al
revised 1996). Adult (⬎6 months old) C57/BL6 or LDL-receptor–
deficient mice were euthanized, and the aortic arch was perfused
with zinc fixative, excised, and processed for histological
examination.
Cell Culture
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Human umbilical vein endothelial cells (HUVECs) were isolated
from freshly collected umbilical cords and cultured in M199 medium
(Invitrogen, Karlsruhe, Germany) containing 20% fetal bovine serum
(Life Technologies, Karlsruhe, Germany), 50 U/mL penicillin, 50
␮g/mL streptomycin, and 5 mmol/L HEPES, supplemented with
endothelial cell growth supplement (PromoCell, Heidelberg, Germany). Human smooth muscle cells were isolated from human
thymus glands after thymectomy and cultured in DMEM (Invitrogen) supplemented with 50 U/mL penicillin, 50 ␮g/mL streptomycin, and 10% fetal bovine serum. The phenotype of these cells was
confirmed by ␣-smooth muscle actin and desmin-specific immunofluorescence analysis. Human aortic and saphenous vein endothelial
cells were purchased from PromoCell and cultured according to the
manufacturer’s instructions. Cells were cultured at 37°C, 5% CO2,
and 100% humidity. Only cells cultured up to passage 4 were used
for the experiments. All types of cells were cultured on plastic dishes
or BioFlex collagen type I 6-well plates (Flexcell, Hillsborough, NC)
coated with gelatin (2 mg/mL gelatin in 0.1 mol/L HCl for 30
minutes at ambient temperature). Stretching was performed with a
FX-3000 FlexerCell strain unit with 15% cyclic elongation at 0.5 Hz,
which corresponds to the physiological maximum deformation in the
human carotid artery.9
Coculture of Endothelial and Smooth Muscle Cells
Before cells were seeded onto a BioFlex collagen type I 6-well plate,
a silicon wall (self-made with biologically inert medical silicone
from Wacker, Munich, Germany) was inserted into each well,
equally dividing it into 2 areas. If HUVECs were cultured alone, they
were seeded into one half of the well only to ensure that they were
grown to confluence at the beginning of each experiment. For
coculture conditions, equal numbers of HUVECs and human smooth
muscle cells were simultaneously seeded in individual halves of 1
well. Both cell types were cultured in M199 medium containing 20%
fetal bovine serum, 50 U/mL penicillin, 50 ␮g/mL streptomycin, and
5 mmol/L HEPES. The silicon wall was removed after cellular
adhesion, followed by changing of the medium. Thereafter, cells
were grown overnight before experiments were started. In all
experimental setups, the same number of endothelial cells was used.
Reverse-Transcription Polymerase Chain Reaction
Total RNA was isolated from the cultured cells by solid-phase
extraction with an RNeasy kit (Qiagen, Düsseldorf, Germany)
according to the manufacturer’s instructions. Reverse transcription
and polymerase chain reaction for human TGF-␤1 and 60S ribosomal protein L32 (RPL32) cDNA as an internal standard was
performed as described previously.12 The following primers were
used for amplification: human TGF- ␤ 1 forward, 5⬘GCCCTGGACACCAACTATT-3⬘; human TGF-␤1 reverse, 5⬘TCAGCTGCACTTGCAGGAG-3⬘; human RPL32 forward, 5⬘GTTCATCCGGCACCAGTCAG-3⬘; human RPL32 reverse, 5⬘ACG-TGCACATGAGCTGCCTAC-3⬘; human CD40 forward, 5⬘CAGAGTTCACTGAAACGGAAT-GCC-3⬘; human CD40 reverse,
5⬘-TGCCTGCCTGTTGCACAACC-3⬘; human VCAM-1 forward,
5⬘-CATGACCTGTTCCAGCGAGG-3⬘; human VCAM-1 reverse,
5⬘-CATTCACGAGGCCAC-CACTC-3⬘; human Alk-1 forward, 5⬘CGACGGGAGGCAGGAGAAGCAG-3⬘; human Alk-1 reverse:
5⬘-TGAAGTCGCGGTGGGCAATGG-3⬘.
Western Blotting
Cells cultured as spheroids or a monolayer were lysed with sample
buffer containing 1% Triton X-100. Samples were resolved on a 10%
SDS-PAGE gel and blotted. The blots were probed with a polyclonal
rabbit anti-human CD40 antibody (1:2000 dilution, Research Diagnostics, Concord, Mass) or rabbit anti-human VCAM-1 antibody
Control of CD40 Expression in Endothelial Cells
3
Table. Distribution of CD40 Expression as Judged by
Immunohistochemistry in Endothelial Cells of Different
Mouse Organs
Arteries
Veins
Main blood vessels
⫺
⫹
Capillaries
Skeletal muscle
⫺
⫹
⫹
Heart
⫺
⫺
⫹
Lung
⫺
⫺
⫺
Kidney
⫺
⫺
⫹/⫺
lleum
⫺
⫺
⫺
Liver
⫺
⫺
⫺
Pancreas
⫺
⫹/⫺
⫹/⫺
Spleen
⫺
⫺
⫺
Lymph node
⫺
⫺
⫺
⫹ Indicates strong expression; ⫹/⫺, partial expression; and ⫺, no
expression detected.
(1:2000 dilution, Santa Cruz Biotechnology, Heidelberg, Germany)
and reprobed for CD31 (goat polyclonal, sc-1506, Santa Cruz
Biotechnology) and ␤-actin (1:3000 dilution, Sigma-Aldrich, Munich, Germany) after stripping.
Morphological, Immunocytochemical, and
Immunohistochemical Analyses
Cells were fixed with buffered 4% paraformaldehyde, blocked with
1% BSA/PBS (albumin bovine fraction V, Sigma-Aldrich), and
incubated with a monoclonal mouse anti-human CD40 antibody
(Dianova, Hamburg, Germany), a polyclonal goat anti-mouse Alk-1
antibody, or a monoclonal mouse anti-human CD31 antibody (Dako,
Glostrup, Denmark). Binding of primary antibodies was detected by
an anti-mouse or anti-goat–specific antibody conjugated to Cy3
(Invitrogen). Nuclei were visualized by Hoechst dye 33258.
Mouse tissue specimens were fixed for 24 hours in zinc fixative,
dehydrated, and embedded in paraffin. Immunohistochemical staining for CD40 or Alk-1 and phosphorylated Smad 1/5/8 was performed on 4-␮m sections with a polyclonal rabbit anti-mouse CD40
antibody (Dianova) or a polyclonal anti-mouse Alk-1 antibody in
combination with a 3,3-diaminobenzidine (DAB)– based enhanced
detection method (EnVision, Dako) according to the manufacturers’
instructions. Localization of atherosclerotic lesions in the aortic arch
of LDL-receptor– deficient mice was macroscopically and microscopically determined by the white appearance of these plaques,
which could be differentiated easily from healthy vessel segments.
Statistical Analysis
All results are expressed as mean⫾SD. Differences between experimental groups were analyzed by unpaired Student t test with
P⬍0.05 considered statistically significant.
The authors had full access to and take full responsibility for
the integrity of the data. All authors have read and agree to the
manuscript as written.
Results
Venous and Capillary but Not Arterial Endothelial
Cells Express CD40 During Vascular Homeostasis
CD40 has been shown to be expressed in vascular cells under
proinflammatory conditions.5,13 To get an idea of its distribution in native nonactive blood vessels, we analyzed CD40
expression by immunohistochemistry in different organs of
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November 13, 2007
HUVEC
hAoEC
-
-
hSaVEC
CD40
β -actin
Cyclic stretch:
+
D
E
F
G
+
Figure 2. CD40 expression in different endothelial cells on exposure to cyclic stretch or
smooth muscle cells. Lysates from control and
stretch-stimulated (15% elongation, 0.5 Hz)
HUVECs, aortic endothelial cells (hAoEC), and
saphenous vein endothelial cells (hSaVEC)
were subjected to Western blot analysis (A),
which uniformly revealed a decrease in CD40
protein abundance after exposure for 3 days
to cyclic stretch. B and C, Representative
photomicrographs of the control (B) and
stretched cell morphology (C; scale bar⫽100
␮m). D, No CD40 expression was detected by
immunocytochemistry in serial sections of
umbilical veins (E, CD31 staining; scale
bar⫽100 ␮m), whereas CD40 expression was
readily detected in cultured HUVECs (F; red
staining; scale bar⫽50 ␮m). G, Western blot
analysis of these cells (EC) also visualized the
downregulation of CD40 protein on coculture
with human smooth muscle cells (SMC). CD31
protein was used as an endothelial cell–specific loading control, and the CD40/CD31 ratio
under control conditions was set to 100%
(*P⬍0.05).
CD40/CD31 ratio [%]
50
EC
NMRI mice. Surprisingly, CD40 was strongly expressed in
distinct venous and capillary but not arterial endothelial cells
(Figure 1; Table).
Endothelial Cell CD40 Abundance Is
Downregulated by Interaction With Smooth
Muscle Cells or Exposure to Cyclic Stretch
To test whether CD40 expression is affected by environmental parameters, we cultured arterial and venous endothelial
cells and exposed them to cyclic stretch. Although CD40
protein was readily detected in all types of cells, its abundance decreased markedly on prolonged (18 hours to 3 days)
exposure to cyclic stretch (Figure 2A through 2C) but
returned to baseline within 3 hours after cessation of stretch
(Data Supplement Figure I). In contrast to cyclic stretch,
exposure to arterial shear stress (30 dyne/cm2) did not affect
n= 5
*
EC
+SMC
100
C
0
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B
-
+
endothelial cell CD40 expression (data not shown). Further
analyses of HUVECs revealed that these usually CD40negative cells (Figure 2D and 2E) rapidly upregulate CD40 in
culture (Figure 2F) if the influence of smooth muscle cells is
lacking. When both cell types were cocultured, however,
endothelial cell CD40 protein abundance declined significantly (Figure 2G).
Cyclic Stretch Upregulates CD40 Expression
in Endothelial Cells Cocultured With
Smooth Muscle Cells
Next, we investigated whether cyclic stretch and the presence
of smooth muscle cells complement each other in terms of
their effect on endothelial cell CD40 expression. To this end,
a coculture system was set up (Figure 3A and 3B) in which
Korff et al
Control of CD40 Expression in Endothelial Cells
5
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Figure 3. CD40 protein and mRNA abundance in stretch-stimulated HUVECs (EC) cocultured with human smooth muscle cells (SMC).
ECs were cultured alone (A, red fluorescence, CD31 staining; blue fluorescence, Hoechst 33298 nuclear staining) or together with
SMCs (B, formation of EC islands; scale bar⫽50 ␮m). A weak CD40 signal was detected in the EC islands under control conditions (C),
which was increased (D; scale bar⫽50 ␮m) after 18 hours of exposure to cyclic stretch. Likewise, CD40 protein was detected in cell
lysates by Western blot analysis (E, statistical summary; **P⬍0.01; representative blots are shown at the bottom). F, Semiquantitative
reverse-transcription polymerase chain reaction analysis of CD40 mRNA levels (statistical summary; representative gels are shown at
the bottom) in stretch-stimulated ECs cocultured with SMCs showing that CD40 expression is upregulated only in EC/SMC cocultures
on exposure to cyclic stretch. RPL32 mRNA was used as a loading control, with the CD40/RPL32 ratio under control conditions set to
100% (*P⬍0.05; n.s. indicates not significant).
HUVECs and human smooth muscle cells were simultaneously exposed to cyclic stretch followed by analysis of
CD40 protein and mRNA expression (Figure 3E and 3F). In
contrast to the stretch-dependent downregulation of CD40
protein in solo endothelial cell cultures (Figure 3E), this was
surprisingly upregulated under conditions in which both cell
types had been cultured in close proximity (Figure 3C
through 3E). Distant coculture of both cell types (separated
by a defined space), conversely, fell short of upregulating
endothelial cell CD40 abundance but prevented its downregulation (Data Supplement Figure III). Even though Western
blot analyses have shown that human cultured smooth muscle
cells do not express CD40 in vitro (data not shown), we
verified by immunohistochemistry that the observed upregulation of CD40 abundance was limited to the endothelial cells
(Figure 3C and 3D). RNA expression analyses further revealed that downregulation of endothelial cell CD40 protein
is not accompanied by significant changes in CD40 mRNA
(Figure 3F), and only on interaction with the smooth muscle
cells was upregulation of endothelial cell CD40 protein
accompanied by a significant rise in mRNA abundance
(Figure 3F).
Upregulation of CD40 Expression in Endothelial
Cells Cocultured With Smooth Muscle Cells Is
Controlled by TGF-␤1
On the basis of the fact that TGF-␤1 is a key regulator of
endothelial cell/smooth muscle cell communication in vivo
and in vitro, we analyzed to what extent this cytokine might
be involved in the control of CD40 expression in the
coculture setup. To this end, TGF-␤1 activity was blocked
with a neutralizing antibody, and this prevented the stretchinduced increase in CD40 mRNA and protein outright (Figure 4A and 4B; Data Supplement Figure III). The stretchinduced downregulation of CD40 expression in solo
endothelial cell cultures, on the other hand, was not affected
by the neutralizing anti-TGF-␤1 antibody. Interestingly, additional polymerase chain reaction and cytokine array analyses revealed only a modest increase in TGF-␤1 expression in
stretch-stimulated cocultures of endothelial and smooth muscle cells (Data Supplement Figure II).
Stretch-Induced Upregulation of Endothelial Cell
CD40 Expression Is Mediated by Alk-1
Assuming that the observed stretch-induced increase in endothelial cell CD40 expression cannot be based solely on an
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Figure 4. TGF-␤1– dependent regulation of CD40 expression. A, Solo endothelial cells (EC) and EC/SMC cocultures were treated with a
neutralizing anti-TGF-␤1 antibody (2 ␮g/mL) and exposed to cyclic stretch for 18 hours. Western blot analyses revealed that CD40 is
downregulated in both experimental settings (**P⬍0.01). B, Reverse-transcription polymerase chain reaction analyses showed that
CD40 mRNA expression is not significantly affected under these conditions (n.s. indicates not significant).
increase in biologically active TGF-␤1 but may also be due to
a heightened endothelial cell responsiveness to TGF-␤1, we
analyzed whether TGF-␤ receptor expression was changed
under the chosen experimental conditions. In contrast to the
TGF-␤ type I receptor Alk-5 and the TGF-␤ type II receptor
(data not shown), endothelial cell expression of Alk-1,
another TGF-␤ type I receptor, was strongly upregulated in
the coculture setup after exposure to cyclic stretch (Figure 5A
through 5C). Again, close proximity between the endothelial
and smooth muscle cells appeared to be mandatory for this
effect to occur (Data Supplement Figure IV). Furthermore,
the disturbance of Alk-1 function by the employment of
soluble Alk-1 receptor molecules blocked the increase in
CD40 protein under these conditions (Figure 5D). In addition
to cyclic stretch, exposure to TGF-␤1 per se resulted in an
upregulation of endothelial cell Alk-1 expression (data not
shown).
Heterogeneous Expression of CD40 in Endothelial
Cells at Distinct Sites of the Aortic Arch Coincides
With the Distribution of Alk-1
On the basis of the aforementioned findings, we hypothesized
that arterial segments that are continuously exposed to cyclic
stretch upregulate CD40 in endothelial cells as the result of an
Alk-1– dependent alteration of their TGF-␤1 responsiveness.
To corroborate this notion, we analyzed CD40 and Alk-1
protein abundance at sites of the mouse aortic arch that are
supposed to be exposed to an increased level of cyclic
stretch.9 In contrast to the heterogeneous expression of the 2
proteins among the endothelial cells lining the aortic arch
(Figure 6B through 6D), straight segments of the abdominal
aorta that are thought to be exposed to a comparatively
smaller biomechanical strain were devoid of any CD40 or
Alk-1 staining (Figure 6F through 6H). By semiquantitatively
mapping the expression pattern of both proteins on an
anatomic scheme of the aortic arch, endothelial cell CD40
and Alk-1 expression appeared to colocalize (Figure 6A and
6E). A corresponding staining pattern was also found for
phosphorylated Smad 1 and 5 in endothelial cell nuclei
(Figure 6I through 6M), indicative of increased TGF-␤1
activity at these sites. Furthermore, the expression pattern of
CD40, Alk-1, and phosphorylated Smads 1/5 in wild-type
mice largely coincided with sites along the aortic arch of
low-density–lipoprotein–receptor– deficient mice at which atherosclerotic plaques develop (cf Figure 6A, 6E, 6I, and 6N).
Discussion
The surface receptor CD40 is an important costimulatory
molecule for both the humoral and cellular immune response
that can be expressed in leukocytes and in vascular cells.14
Gene-targeting studies support the pivotal role of CD40 for
T-cell– dependent immunoglobulin class switching and germinal center formation.15 Furthermore, CD40 signaling appears critical for T-cell– dependent activation of macrophages16 and endothelial cell stimulation of extravasating
monocytes.17 Likewise, small interfering RNA silencing of
endothelial cell CD40 expression has been shown to prevent
CD154-activated leukocyte adhesion.18 These results underline the importance of CD40 expression in endothelial cells
for proinflammatory responses.
Despite this, little is known about CD40 distribution and
the molecular mechanisms that control its expression in
vascular homeostasis. Here, we report that CD40 is expressed
in distinct venous and capillary endothelial cell populations
but usually not in arterial endothelial cells, which suggests
that it may be affected by microenvironmental conditions.
Artery-specific determinants may prevent CD40 expression,
whereas CD40 expression is promoted by venous- or
capillary-specific determinants, the latter representing a vascular entity that is typically involved in the recruitment and
activation of leukocytes during inflammatory responses. Expression of specific molecules on distinct endothelial cell
Korff et al
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Figure 5. Expression of Alk-1 in stretch-stimulated HUVECs
(EC) cocultured with human smooth muscle cells (SMC). A,
Semiquantitative reverse-transcription polymerase chain reaction analyses with the Alk-1/RPL32 ratio under control conditions set to 100% revealed that after exposure to cyclic
stretch for 18 hours, Alk-1 mRNA is upregulated only in
stretch-stimulated EC/SMC cocultures (*P⬍0.05; n.s. indicates not significant). B, A weak Alk-1–specific fluorescence
signal was detected in the EC islands under control conditions, which was increased after 18 hours of exposure to
cyclic stretch (C; scale bar⫽50 ␮m). D, Treatment with chimeric Alk-1 protein (2 ␮g/mL) did not affect the stretch-
Control of CD40 Expression in Endothelial Cells
7
subpopulations is well established throughout the literature.19 –21 Similar to the present findings that CD40, contrary
to the situation in the native vasculature, is expressed in all
cultured endothelial cells irrespective of their origin, most
endothelial cell–specific molecules are not part of an imprinted phenotype but are regulated by microenvironmental
parameters such as the local hemodynamics or communication with other cell types.22–24 The present results suggest that
endothelial cell CD40 protein abundance is affected by at
least 3 determinants, ie, cyclic stretch, smooth muscle cells,
and TGF-␤1.
First, we identified cyclic stretch as a biomechanical force
that downregulates endothelial cell CD40 protein abundance.
This effect was critically dependent on the microenvironment. For instance, if the cells were cultured on top of a
Matrigel-coated surface, CD40 expression was increased on
exposure to cyclic stretch (data not shown). Similarly, coculture with smooth muscle cells transformed the observed
downregulation of CD40 abundance into an increase. In line
with these results, additional in vivo analyses indicated that
baseline CD40 expression in arteries or veins is not affected
by acute alterations of the main biomechanical forces to
which the endothelial cells are exposed (data not shown).
Likewise, varying the frequency and/or intensity of cyclic
stretch did not affect CD40 downregulation in human cultured endothelial cells. Moreover, the decline in CD40
protein content was not preceded or accompanied by a
significant change in CD40 mRNA abundance, which suggests that it may be caused by a posttranslational mechanism
such as degradation of the protein, a hypothesis that is
currently under investigation (A.H. Wagner, PhD, oral communication, March 2007). On the basis of these results, cyclic
stretch can be viewed as an important modulator of baseline
CD40 expression in endothelial cells.
Second, we demonstrated that smooth muscle cells affect
CD40 expression in endothelial cells. The influence of
smooth muscle cells on the proinflammatory and arteriovenous differentiation of endothelial cells has been demonstrated in various experimental setups9,22 and may also be
responsible for the observed decline in CD40 protein content.
However, the complex nature of this cellular interplay is still
obscure, and the present protein array studies did not identify
a particular cytokine that may be responsible for the smooth
muscle cell– dependent decrease in endothelial cell CD40
abundance. Coculture of both cell types at close proximity
appears to be essential for intercellular communication,9 and
this was the case under the present experimental conditions.
In contrast, a coculture setup in which both cell types were
separated by a defined space did not induce downregulation
of endothelial cell CD40 expression.
Third, we identified the endothelial cell response to
TGF-␤1 as a third factor that determines CD40 expression
that may be due to a smooth muscle cell–mediated sensitization to or an increase in the local concentration of biologically
Figure 5 (Continued). induced downregulation of CD40 protein
in EC but abolished its upregulation in EC/SMC cocultures
exposed to cyclic stretch for 18 hours (***P⬍0.001; n.s. indicates not significant).
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Figure 6. Immunohistochemical detection
of CD40, Alk-1, and phosphorylated
Smad 1/5 (pSmad) at atherosclerosisprone sites of the mouse aortic arch. The
spatial frequency of CD40-positive (A),
Alk-1–positive (E), and pSmad-positive (I)
endothelial cells and the occurrence of
atherosclerotic plaques in LDL-receptor–
deficient mice (N) along the aortic arch of
individual animals (analysis of several
sections of at least 5 different mice) are
represented by the number of red dots.
The arrow indicates the direction of
blood flow. The gray-shaded areas were
not sectioned and therefore were not analyzed (cf the inset in Figure 6A displaying a CD31-stained longitudinal section
of the aortic arch). From panels A, E, I,
and N, the coincident staining of endothelial cell CD40, Alk-1, and pSmad with
the location of the atherosclerotic
plaques is readily apparent. Representative images of the aortic arch demonstrate that endothelial cell CD40 (B) and
Alk-1 (C) staining is detected in corresponding positions. The integrity of the
endothelial cell monolayer was checked
by CD31 staining (D). In contrast, neither
CD40 (F) nor Alk-1 (G) positive immunoreactivity was detected in straight segments of the abdominal aorta (H; CD31
staining; scale bar⫽50 ␮m). J through M,
Confocal microscopy was used to analyze sections of the aortic arch for
pSmad 1/5/8 immunofluorescence.
Autofluorescence of the elastic fibers is
clearly visible in the red and green channel (J and K, arrowhead), whereas
pSmad 1/5/8 –positive nuclei are specifically detected by the fluorescence signal
in the red channel (J and K, arrow).
Nuclei were visualized by Hoechst 33258
staining (L). Merging of the red and blue
channel verifies detection of pSmad
1/5/8 in an elongated endothelial cell
nucleus but not in the adjacent smooth
muscle cell nuclei (M, arrow; scale
bar⫽20 ␮m). Atherosclerotic lesions (N;
green box delineates the site of the cutouts shown in O and P) were detected predominantly at the inside of the main branches (O; plaque demarcations illustrated by red
dots) in LDL-receptor– deficient mice but not in wild-type mice (P; CD31 staining; scale bar⫽100 ␮m).
active TGF-␤1. Typically, CD40 expression is elevated if
endothelial cells are exposed to proinflammatory cytokines
that are abundantly expressed in atherogenesis.1,25 The present data show that TGF-␤1 is capable of upregulating CD40
expression in stretch-stimulated endothelial cells and thus
may act as an immunomodulatory molecule.
TGF-␤1 is a well-known vasculotropic cytokine, present at
high levels in the vessel wall,26 which has been shown to
inhibit proliferation and migration of smooth muscle
cells.27,28 Moreover, TGF-␤1 inhibits proinflammatory responses both in leukocytes and endothelial cells29,30 and
stabilizes atherosclerotic plaques.31,32 Conversely, TGF-␤1
has been shown to stimulate vascular remodeling processes
such as arteriogenesis,33 adhesion molecule expression in and
adhesion of leukocytes to endothelial cells,34,35 and leukocyte
chemotaxis.36 These somewhat contradictory findings may be
explained at least in part by the fact that TGF-␤1–mediated
inhibitory or stimulatory effects are strongly dependent on the
level of the biologically active cytokine and on the biological
context.37
We noted that only exposure to cyclic stretch unmasks the
stimulatory effect of TGF-␤1 on endothelial cell CD40
expression and that this was mediated through an Alk-1–
dependent signaling pathway, notwithstanding the fact that
additional stretch-dependent TGF-␤1–sensitizing mechanisms may exist (cf Data Supplement Figure V). Alk-1
appears to be critically involved in balancing the activation
state of endothelial cells,38 – 40 whereby its activation stimulates expression of Id-1 (inhibitor of differentiation/DNA
synthesis-1), which in turn promotes endothelial cell proliferation and migration.41 Moreover, the level of expression of
TGF-␤1 and its receptors in atherosclerotic lesions may play
an important role in controlling the proinflammatory and
profibrotic responses to this cytokine.32,40,42 Thus, our observation in the present study of increased Alk-1 expression and
the phosphorylation of its downstream transcription factors
Korff et al
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Smad 1 and 5 at atherosclerosis-prone sites in the aortic arch
of the mouse makes sense.
It is broadly accepted that atherosclerosis preferentially
develops at specific sites along the arterial tree comprising
branches, bifurcations, and curvatures.43,44 Here, the usually
laminar blood flow becomes turbulent, which results in a
major change of the biomechanical forces acting on the vessel
wall. Shear stress changes from laminar to oscillatory while
pulsatile alterations in wall strain are accentuated, which
results in increased cyclic stretch.9,45 In particular, cyclic
stretch is thought to promote atherogenesis by stimulating the
production of reactive oxygen species, which in turn activate
transcription factors that upregulate the expression of proatherosclerotic gene products, especially in endothelial
cells.10,11,46,47 Compatible with this, we continuously observed an expression of CD40 at atherosclerosis-prone sites
of the aortic arch that was not detectable in other arterial
endothelial cells of the mouse but that coincided with
expression of the TGF-␤ receptor Alk-1. We conclude,
therefore, that the increased level of cyclic stretch at these
sites changes the endothelial cell response to TGF-␤1 by
inducing Alk-1 expression, which in turn enables the upregulation of CD40 expression.
However, whether the heterogeneous expression pattern of
the 2 molecules is in fact also indicative of endothelial cell
dysfunction that precedes atherosclerotic plaque formation
remains to be established. In this context, it is important to
note that attributing the observed changes in gene expression
solely to changes in cyclic stretch would oversimplify the
complex local hemodynamics along the aortic arch and its
tributaries, with changes in shear stress (from laminar to
oscillatory) and blood flow per se accompanying those in
cyclic stretch. Moreover, the development of atherosclerotic
lesions along the aortic arch of LDL-receptor– deficient mice,
although it too presumably has a hemodynamic basis, most
likely will be governed by the excessive dyslipidemia in this
animal experimental model.
Taken together, the present findings suggest the following:
(1) the expression of CD40 in endothelial cells is highly
dependent on the local environment; (2) cyclic stretch upregulates CD40 abundance in endothelial cells under the
influence of smooth muscle cells; (3) this is dependent on
TGF-␤1; and (4) this is mediated though endothelial cell
Alk-1. This sequence of events may be responsible for the
expression of CD40 at atherosclerosis-prone sites of the
arterial tree and thus may act as an early site-specific
indicator of endothelial cell dysfunction.
Acknowledgments
The authors would like to acknowledge Professor Katrin Schäfer
(University of Göttingen) for providing the LDL-receptor– deficient
mice and the excellent technical assistance of Gudrun Scheib, Lorena
Urda, Sabine Krull, and Kathrin Schreiber.
Sources of Funding
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB TR23, project C5).
Disclosures
None.
Control of CD40 Expression in Endothelial Cells
9
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CLINICAL PERSPECTIVE
Atherosclerosis is a multistep process thought to be due to a local destabilization of vascular homeostasis that leads to
endothelial cell dysfunction. This process preferentially occurs at arterial bifurcations, branches, or curvatures, sites
subjected to major changes in hemodynamics best described by a decrease in laminar shear stress, considered to be
atheroprotective, and an increase in cyclic stretch, thought to be proatherogenic. Here, we report that endothelial cells
upregulate expression of the important immunomodulatory molecule CD40 in response to cyclic stretch through a
transforming growth factor–␤1/activin-receptor–like kinase (Alk)-1– dependent mechanism both in vitro and at atherosclerosis predilection sites in mice in vivo. Although the comparability of mouse models to human physiology may be
limited, these observations suggest that the altered hemodynamics at atherosclerosis-prone sites have an impact not only
on the activation state of infiltrating T cells and/or monocyte/macrophages but also on the responsiveness of the resident
cells of the vessel wall to cytokines such as transforming growth factor–␤1. In addition to interventional strategies such
as angioplasty, passivation or stabilization of atherosclerotic lesions is regarded as an important strategy to prevent plaque
rupture and hence the risk of an acute and potentially life-threatening ischemia. Another promising approach, therefore,
may be to identify agonists that compensate for the local disturbances in vascular homeostasis, thereby delaying the
development of vulnerable plaques. Such a strategy might comprise stabilization of Alk-5 expression in vascular cells,
which has been shown to inhibit endothelial cell and smooth muscle cell migration and proliferation and to trigger collagen
synthesis in vascular smooth muscle cells.
Cyclic Stretch Controls the Expression of CD40 in Endothelial Cells by Changing Their
Transforming Growth Factor−β1 Response
Thomas Korff, Karin Aufgebauer and Markus Hecker
Circulation. published online October 29, 2007;
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