© 2001 American Heart Association, Inc.
Cellular Biology |
Cellular Pathology of Atherosclerosis
Smooth Muscle Cells Prime Cocultured Endothelial Cells for Enhanced Leukocyte Adhesion
From the Department of Physiology, The Medical School, The University of Birmingham, UK.
Correspondence to Dr G.E. Rainger, Department of Physiology, The Medical School, The University of Birmingham, B15 2TT, UK. E-mail g.e.rainger{at}bham.ac.uk
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Abstract |
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Abstract— During the development of an atherosclerotic plaque, mononuclear leukocytes infiltrate the artery wall through vascular endothelial cells (ECs). At the same time, arterial smooth muscle cells (SMCs) change from the physiological contractile phenotype to the secretory phenotype and migrate into the plaque. We investigated whether secretory SMCs released cytokines that stimulated ECs in a manner leading to increased leukocyte recruitment and thus might accelerate atheroma formation. SMCs and ECs were established in coculture on the opposite sides of a porous membrane, and the cocultured cells were incorporated into a flow-based assay for studying leukocyte adhesion. We found that coculture primed ECs so that their response to the inflammatory cytokine tumor necrosis factor-
was amplified. ECs
cocultured with SMCs supported greatly increased adhesion of flowing
leukocytes and were sensitized to respond to tumor necrosis factor-
at concentrations 10 000 times lower than ECs cultured alone. In
addition, coculture altered the endothelial selectin
adhesion molecules used for leukocyte capture. EC priming was
attributable to the cytokine transforming growth
factor-ß1, which was proteolytically
activated to a biologically active form by the serine protease
plasmin. These results suggest a new role for secretory SMCs in the
development of atheromatous plaque. We propose that
paracrine interaction between ECs and SMCs has the potential to amplify
leukocyte recruitment to sites of atheroma and exacerbate
the inflammatory processes believed to be at the heart of disease
progression.
Key Words: endothelial cells • smooth muscle cells • leukocyte adhesion • transforming growth factor-ß1 • tumor necrosis factor-
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Introduction |
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Atherosclerotic plaque typically contains abnormal deposits of mononuclear leukocytes and secretory smooth muscle cells (SMCs).1 Ordinarily, SMCs are confined to the medial layer of the artery wall, where, as contractile cells, their physiological role is the maintenance of mechanical structure and vascular tone. Early in the genesis of atherosclerotic plaque, medial SMCs undergo mitogenic stimulation and change phenotype, losing contractile elements and acquiring the ability to replicate and migrate within the artery wall.2 In consequence, SMCs invade the intimal layer of the diseased artery, where they contribute to progressive arterial hyperplasia by inappropriate replication and deposition of a fibrotic connective tissue matrix.1 2 Intimal SMCs also acquire a well-developed rough endoplasmic reticulum and alter their secretory profile, generating several proinflammatory agents.1 2 We wished to examine the possibility that secretory SMCs could release agents that promote the recruitment of mononuclear leukocytes to the developing plaque.
Adhesion between leukocytes in the arterial
blood and activated endothelial cells (ECs)
overlying atherosclerotic plaque seems to represent the major
route of entry into the artery wall for T lymphocytes and
monocytes.1 The recruitment
of blood-borne leukocytes to inflamed tissue normally occurs across the
ECs of postcapillary
venules.3 4 When
activated by cytokines, such as tumor necrosis
factor- (TNF-), ECs express specialized adhesion receptors (E-
and P-selectin and vascular cell adhesion molecule-1 [VCAM-1]), which
have rapid forward kinetics for bond formation essential for binding
flowing cell.3 4
These molecules also support subsequent rolling
adhesion,5 6
allowing the slow-moving leukocytes to assimilate signals from the
EC-borne chemoattractants that promote immobilization and
transendothelial migration via integrin adhesion
receptors.3 4 ECs
of the healthy human artery do not express selectins or VCAM-1, but
their expression has been demonstrated on the ECs overlying
atherosclerotic
plaque.7 8 9 10
Secretory SMCs generate several agents that could promote
leukocyte infiltration of the artery
wall.1 2 11
They release potent leukocyte chemoattractants, such as interleukin-8
(IL-8) and monocyte chemotactic protein-1
(MCP-1).12 They also release
the proinflammatory cytokines
TNF-13 and
IL-1,14 which induce the
expression of adhesion molecules and leukocyte chemoattractants on
ECs.3 4 The
cytokine transforming growth factor-ß1
(TGF-ß1) is also generated in biologically
active form when SMCs and ECs are cultured
together.15 16
Although TGF-ß1 does not induce leukocyte
adhesion to ECs in a manner comparable to TNF or IL-1, it can modulate
the effects of other proinflammatory
agents.17 18 The
ability of TGF-ß1 to either
inhibit19 20 21
or promote22 23
leukocyte adhesion to ECs has been reported depending on the type and
conditions of growth of the ECs.
In the present study, we have developed an in vitro model of the artery wall to investigate interactions between secretory SMCs, endothelial cells, and flowing purified leukocytes in a physical milieu resembling the circulation.3 4 Our studies show that secretory SMCs can greatly potentiate adhesion of leukocytes to ECs. The results suggest a new role for SMCs in the cellular pathology of atherosclerosis.
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Materials and Methods |
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Leukocyte Isolation
Mononuclear leukocytes (29% monocytes and 71% lymphocytes), purified monocytes (91% monocytes and 9% lymphocytes), peripheral blood lymphocytes (PBLs) (95% lymphocytes and 5% monocytes), and purified neutrophils (>95% granulocytes) were prepared from EDTA anticoagulated blood by density gradient centrifugation,24 cell panning, or magnetic bead separation (for more information, see the online data supplement available at http://www.circresaha.org). To aid visualization, leukocytes were loaded with the fluorochrome calcein-AM (5 µg/mL), which did not affect adhesion.25
Primary Culture of ECs, SMCs, and
Fibroblasts
Human ECs were isolated from the veins of umbilical
cords, as described
elsewhere.24 26 27
Each experiment used first-passage ECs from a different donor. Human
SMCs were explanted from the arteries of umbilical cords. SMCs from 8
different donors were used. Skin fibroblasts were explanted from normal
human foreskin. For details, see the online data
supplement.
ECs and SMCs Coculture on Porous
Inserts
SMCs were cultured for 24 hours on the inside of
culture inserts (Becton Dickinson). ECs were then added to the outside
face of the membrane and cultured alone or with SMCs for 24 hours.
Coculture inserts or inserts bearing ECs alone were treated with
TNF- at 0 to 100 U/mL for an additional 24 hours before adhesion
assay. See the online data supplement for details. In some experiments,
inserts bearing ECs were treated for 24 hours with 1 or 10 ng/mL
recombinant TGF-ß1 before addition of
TNF.
Flow Adhesion Assay
Adhesion assays were conducted in a novel
parallel-plate flow chamber engineered to incorporate the insert and
allow perfusion of leukocytes over the ECs on the outer face of the
culture membrane at 37°C. The chamber was placed on the stage of a
fluorescence microscope and connected to a withdrawal syringe
pump. Fluorescently labeled leukocytes were perfused over the
ECs at a wall shear stress of 0.05 or 0.1 Pa, and adhesion was assessed
by fluorescence microscopy. See the online data supplement for
details.
Antibodies and Inhibitors
ECs or mononuclear leukocytes were treated with
monoclonal adhesion-blocking antibody (mAb) against E-selectin,
P-selectin, VCAM-1, or 4 integrin for 15
minutes before adhesion assay. See the online data supplement for
details.
TGF-ß1 (100 µg/mL) function
neutralizing antibody or control antibody or inhibitors of
plasmin activity aprotinin, used at 50 µg/mL, or
2-antiplasmin, used at 2.5 µg/mL, (both
from Sigma) were added at the start of EC/SMC coculture.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Secretory Smooth Muscle Cells Prime but Do Not Activate Cocultured Endothelial Cells for Leukocyte Adhesion
When ECs and secretory SMCs were cultured on the opposite sides of porous culture membranes for 48 hours, the ECs barely supported the adhesion of isolated mononuclear leukocytes from flow (Figure 1
). The level of binding to cocultured ECs was no
greater than for ECs cultured alone. Thus, secretory SMCs did not
activate endothelial cells.
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In contrast, cocultured ECs markedly altered their response
to the cytokine TNF-. When ECs were treated alone with
TNF- for 24 hours at concentrations between 0 and 100 U/mL, a
dose-dependent increase in their ability to capture mononuclear
leukocytes from flow between 1 and 100 U/mL was observed
(Figure 1
). A qualitatively similar response to TNF- was
observed on endothelial cells cocultured with SMCs
(Figure 1). However, the presence of SMCs greatly sensitized
the ECs for response to TNF-. Significant adhesion of leukocytes was
inducible with a concentration of TNF- as low as 0.0001 U/mL on
cocultured ECs, whereas the response of ECs alone was extinct below 1
U/mL TNF
(Figure 1). With TNF- at or above 1 U/mL, ECs cocultured
with SMCs supported levels of mononuclear cell adhesion between 100%
and 200% higher than ECs cultured alone
(Figure 1).
Secretory SMCs Prime Cocultured ECs for
Enhanced Adhesion of Purified Neutrophils, Monocytes, and
Peripheral Blood Lymphocytes
When ECs were cocultured with SMCs and then treated
with either 0.1 or 100 U/mL TNF, the adhesion of purified neutrophils,
monocytes, or PBL was increased compared with adhesion on ECs cultured
alone
(Figure 2). As was the case for the mixed mononuclear cell
preparation, adhesion to ECs cultured alone was negligible with 0.1
U/mL TNF, whereas adhesion of all cell types could be readily observed
for ECs cocultured with SMCs and 0.1 U/mL TNF. At 100 U/mL TNF, there
was an 
2-fold increase in monocyte and neutrophil adhesion and a
3-fold increase in lymphocyte adhesion for cocultured ECs compared with
ECs cultured alone. Thus, the increase in adhesion observed in mixed
populations of mononuclear cells was not attributable solely to the
recruitment of a particular leukocyte
subset.
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Contractile SMCs or Skin Fibroblasts Do Not
Prime Cocultured ECs
We cocultured ECs with contractile SMCs to determine
whether SMCs in their physiological
phenotype had any priming effect. To culture contractile SMCs,
they must be maintained in medium devoid of growth factors and
containing only 0.5% serum. ECs cannot ordinarily be maintained under
this regimen and have been reported to undergo apoptosis on
exposure to TNF after serum-free
culture.28 In only 1 of 4
experiments did ECs maintain a monolayer suitable for adhesion studies.
When cultured with contractile SMCs and 0.1 or 100 U/mL TNF, ECs showed
no priming in that experiment (data not shown). In the other 3
experiments, >50% of ECs detached from the coculture inserts and
attached cells had rounded up.
To ensure that EC priming was not a characteristic of all
stromal cells, we cocultured skin fibroblasts with ECs. In the presence
of 0.1 or 100 U/mL TNF, ECs cocultured with skin fibroblasts showed no
increase in the ability to bind flowing purified PBL compared with ECs
cultured alone
(Figure 3). Thus, although secretory SMCs could prime ECs for
sensitized responses to TNF, this was not a characteristic of all
stromal cells.
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Coculture of ECs With SMCs Alters the Patterns
of EC Adhesion Molecules Used to Recruit Flowing Mononuclear
Cells
We used a panel of function-blocking antibodies to
investigate the roles of the endothelial receptors
E-selectin, P-selectin, and VCAM-1 and the leukocyte integrin
4ß1 integrin (ligand
for VCAM-1) in adhesion observed in our model. In studies on ECs
cultured alone and stimulated with 100 U/mL TNF-, antibodies against
P-selectin, VCAM-1, or 4 integrin inhibited
leukocyte attachment by 50% whereas antibody against E-selectin had
no significant effect
(Figure 4a). Antibodies against 4
integrin and P-selectin had an additive effect and blocked >80% of
adhesion
(Figure 4a). Thus, mononuclear leukocytes used a combination
of VCAM-1 and P-selectin to bind to ECs cultured alone.
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A different pattern was seen when ECs were cocultured with
SMCs. When cocultured ECs were stimulated with 100 U/mL TNF-,
antibodies against E-selectin, VCAM-1, or 4
integrin inhibited 50% of adhesion. Combination of antibodies
against 4 integrin and E-selectin blocked
>90% of adhesion
(Figure 4b). Antibody against P-selectin had no effect. Thus,
VCAM-1 provided a common route for leukocyte adhesion under both
culture regimes, but EC/SMC coculture altered the pattern of selectin
adhesion molecules used for leukocyte adhesion. We determined whether
the same adhesive mechanisms were functional in cocultures at lower
concentrations of TNF-. Using 0.1 U/mL TNF- (a concentration that
was stimulatory for ECs only when cocultured with SMCs), leukocytes
used the same combination of receptors, ie, VCAM-1 and E-selectin
(Figure 4c) as with 100 U/mL TNF-. Antibody studies also
showed that purified monocytes and PBL both used VCAM-1 and P-selectin
on ECs cultured alone but VCAM-1 and E-selectin on cocultured ECs (data
not shown) (see the online data supplement for details). Thus, in
coculture, both E-selectin and VCAM-1 supported adhesion at TNF-
concentrations, where they were not functional on ECs cultured
alone.
Immunofluorescence labeling of ECs with antibodies against E-selectin or VCAM-1 and flow cytometry demonstrated that E-selectin and VCAM-1 expression were significantly increased on cocultured ECs compared with ECs cultured alone when both were stimulated with 100 U/mL (data not shown) (see the online data supplement for details). This strongly indicated that increased expression of EC adhesion receptors contributed to the adhesion of leukocytes to primed ECs.
Endothelial Cell Priming Is
Dependent on Proteolytically Activated
TGF-ß1
Modulation of EC responses to TNF has been observed by
some laboratories using recombinant, biologically active
TGF-ß1.19 20 21 22 23
To investigate whether TGF-ß1 primed ECs in
our model, we used a TGF-ß1 function
neutralizing antibody in cocultures of ECs and SMCs. The antibody
greatly inhibited increases in adhesion in cocultures exposed to
TNF-
(Figure 5). Adhesion to ECs cocultured in the presence of 0.1
U/mL TNF was nearly abolished
(Figure 5a), whereas the adhesion to ECs cocultured in the
presence of 100 U/mL TNF was reduced to the level seen on ECs cultured
alone
(Figure 5b).
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TGF-ß1 is secreted in a
biologically inert form bound to latency-associated peptide.
Biologically active TGF-ß1 is released from
this complex by an acid environment or by proteolytic
cleavage.17 18
The serine protease plasmin, derived from plasminogen,
processes TGF-ß1 from the inert to the
biologically active form in cocultures of ECs with pericytes or with
SMCs.16 When we added the
serine protease inhibitor aprotinin or the plasmin
inhibitor 2-antiplasmin to
cocultures, EC priming was completely inhibited
(Figure 5). Thus, at 0.1 U/mL TNF, the adhesion of
mononuclear cells to cocultured ECs was virtually abolished by both
agents
(Figure 5a). In the presence of 100 U/mL TNF, mononuclear
cell adhesion to cocultured ECs was reduced nearly to the levels seen
on ECs alone
(Figure 5b).
To confirm that ECs could be primed in coculture by
TGF-ß1, we inhibited endogenous
plasmin activity (and thus the activation of endogenous
TGF-ß1) with
2-antiplasmin but also added biologically
active recombinant TGF-ß1. Recombinant
TGF-ß1 reconstituted priming of cocultured ECs
in a dose-dependent manner
(Figure 6). In the presence of 0.1 U/mL TNF
(Figure 6a) or 100 U/mL TNF
(Figure 6b), 2-antiplasmin reduced
the level of mononuclear cell adhesion to cocultured ECs to the levels
seen on ECs cultured alone. When
2-antiplasmin and 1 ng/mL of recombinant
TGF-ß1 were simultaneously added
to the cocultures, EC priming was partially restored and adhesion was
higher than on ECs cultured alone
(Figures 6a and 6b). The addition of
2-antiplasmin and 10 ng/mL recombinant
TGF-ß1 fully restored EC priming, and
mononuclear cell adhesion was equivalent to that on EC/SMC cocultures
(Figures 6a and 6b).
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Recombinant TGF-ß1 Can
Prime ECs in the Absence of Secretory SMCs
We conducted a series of experiments to determine if
recombinant TGF-ß1 could prime ECs cultured
alone for increased levels of leukocyte adhesion. ECs were cultured for
24 hours on inserts in the absence of SMCs but in the presence of
recombinant TGF-ß1 at concentrations of 1 or
10 ng/mL. TGF-ß1 dose-dependently primed ECs
for increased adhesion of purified flowing mononuclear cells in
response to stimulation with TNF at 100 U/mL
(Figure 7). ECs also responded to TNF at 0.1 U/mL after
priming with 1 ng/mL recombinant TGF-ß1. ECs
bound 36±3 compared with 2±0.4 leukocytes on primed and unprimed ECs,
respectively (mean±SEM of 3 matched experiments;
P<0.05 by Student’s paired
t test).
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In separate experiments, we cultured ECs in microtiter
plates in the presence of TGF-ß1. ECs cultured
with 1 or 10 ng/mL TGF-ß1 demonstrated
increased expression of both VCAM-1 and E-selectin after stimulation
with TNF at 100 or 0.1 U/mL
(Figure 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a novel, multicellular, flow-based model of the artery wall, we demonstrated a paracrine interaction between ECs and secretory SMCs, which may influence the progression of atherosclerosis. ECs cultured with secretory SMCs were primed for increased responsiveness to the inflammatory cytokine TNF-
. At equal concentrations of TNF-, ECs
cocultured with SMCs supported greatly increased adhesion of flowing
leukocytes compared with ECs cultured alone. Cocultures were also
sensitized to respond to much lower concentrations of TNF-. In
addition, coculture of ECs with SMCs altered the selectin adhesion
molecules used by leukocytes to bind to ECs. ECs were primed by the
cytokine TGF-ß1, which was
enzymatically processed from an inert to biologically active form by
the serine protease plasmin in coculture.
The present study improves our understanding of how
interactions between cells of the artery wall may promote recruitment
of leukocytes to developing atheroma. During inflammation,
leukocytes adhere to selectins or VCAM-1 expressed by
cytokine-stimulated ECs of postcapillary venules and migrate
under the action of local
chemoattractants.3 4
Leukocytes do not usually adhere to the wall of healthy arteries, but
monocytes and T lymphocytes are found in atherosclerotic
plaque.1 Studies using animal
models or human tissue have revealed the cellular composition of plaque
and the local patterns of expression of adhesion molecules and
chemoattractants.7 8 9 10 11
However, in these systems, it is difficult to dissect the complex
interactions between cellular components and cytokines leading
to leukocyte recruitment. To overcome these limitations, we developed
an in vitro model incorporating chosen cellular elements of the
atherosclerotic lesion. Flow was included both to mimic the in vivo
requirement for fast-acting capture receptors (selectins or VCAM-1) and
enable washout of secreted chemotactic agents that may otherwise
accumulate in static systems. Thus, we identified a novel pattern of
adhesion molecule usage on human umbilical vein ECs (HUVECs) responding
to TNF- in the presence of SMCs.
Ordinarily, E-selectin expression on HUVECs peaks after 4
hours and is absent after 24 hours of exposure to
TNF-.29 30 In
the present study, E-selectin was functional after 24 hours on ECs
cocultured with SMCs, whereas P-selectin was functional on ECs cultured
alone. VCAM-1 was functional for either type of culture stimulated with
TNF-. A switch from P-selectin to E-selectin could influence the
leukocytes found in atherosclerotic plaque, for example, because the
ability to use P- or E-selectin differs between subsets of T
lymphocytes.31 We have not
described the intracellular mechanisms that underlie prolonged
E-selectin expression. Others, using dermal microvascular ECs that
demonstrate prolonged expression of E-selectin in response to
TNF,32 have described a role
for sustained activity of nuclear factor-
B (NF-B), the nuclear
factor responsible for transcriptional activity of the E-selectin
gene.29 NF-B activity was
sustained by reduction of cytoplasmic levels of IB-ß, an
inhibitor of NF-B function. Alterations in the membrane
turnover and intracellular degradation of E-selectin protein may also
contribute to prolonged E-selectin expression in dermal microvascular
ECs.33
ECs cocultured with SMCs were primed for increased adhesion of all leukocyte types tested, including neutrophils. However, neutrophils are not present in atherosclerotic plaque, even though receptors that support neutrophil capture and adhesion are known to be expressed on diseased arteries.7 8 9 10 The inability of arterial ECs to generate chemotactic stimuli, which activate neutrophils and drive migration into the artery wall, could account for the observed lack of plaque resident neutrophils. Alternatively, neutrophils may infiltrate the artery wall but undergo rapid clearance, for example, by apoptosis.
We demonstrated that EC priming was abolished by a neutralizing mAb against TGF-ß1 or by inhibitors of plasmin-mediated activation of TGF-ß1. EC priming in cocultures could be reconstituted with exogenous recombinant TGF-ß1 when the activation of endogenous cytokine was inhibited by antiproteases. Recombinant TGF-ß1 also effectively primed ECs cultured in the absence of SMCs. However, TGF-ß1 has previously been reported to inhibit as well as prime ECs for the recruitment of leukocytes. A series of studies described TGF-ß1–dependent inhibition of leukocyte adhesion to E-selectin expressed on subconfluent HUVECs.19 34 35 Studies using ECs specialized for lymphocyte trafficking during immune surveillance, eg, high endothelial venule ECs21 and dermal microvascular ECs,13 36 also showed inhibition of leukocyte adhesion in response to TGF-ß1. Alternatively, TGF-ß1–mediated priming of confluent HUVECs for increased VCAM-1 expression by lipopolysaccharide has been demonstrated.23 Also, injection of TGF-ß1 into the skin of rabbits greatly increased the infiltration of neutrophils in response to a secondary challenge with TNF 24 hours later.22 Thus, we believe that TGF-ß1 should be considered a regulator of leukocyte adhesion, with the nature of the response depending on the phenotype of the responding ECs. From our own studies and those of others,22 23 we believe that in the context of inflammation, preexposure of ECs to TGF-ß1 can sensitize ECs to subsequent exposure to TNF.
ECs or SMCs cultured alone do not generate biologically active TGF-ß1, but cocultures of these cells produce the bioactive cytokine, which has been demonstrated to influence cellular functions, such as EC proliferation and motility.15 16 TGF-ß1 is secreted in complex with a latency-associated peptide by both ECs and SMCs in monoculture.16 Thus, additional production of latent TGF-ß1 is unlikely to be a prerequisite for priming in coculture. Rather, it is the proteolytic processing of the latent cytokine to a biologically active form by plasmin that occurs in coculture but not in EC and SMC monocultures.15 16 It is probable that plasmin is activated in EC/SMC coculture by plasminogen activator (PA), because the inclusion of a function neutralizing antibody directed against urokinase-PA inhibits the functional activation of TGF-ß1 in EC/SMC cocultures.16 Both ECs and SMCs in monoculture produce PA,16 and it is thus unlikely that TGF-ß1 activation requires induction of this system in coculture. Why PA is not functional in EC and SMC monocultures is not clear, but alterations in the expression of receptors required to colocalize plasminogen and PA to the cell surface where plasmin activation occurs or alterations in the function or production of PA-inhibitor have been postulated.15 16 37
Conclusions
Our results suggest a new role for secretory SMCs in
the development of atheromatous plaque. SMCs contribute
to arterial thickening by invading the intima and
depositing a fibrotic connective tissue
matrix.1 2 They
have not previously been directly implicated in the recruitment of
inflammatory mononuclear leukocytes. These leukocytes may not normally
be recruited, because arterial endothelium
may be exposed only to low levels of cytokines or have low
responsiveness to them. We describe a mode of paracrine interaction
between ECs and SMCs whereby ECs are rendered highly sensitive to
TNF-. The priming, attributable to TGF-ß1,
has the potential to amplify leukocyte recruitment to sites of
established atheroma and, therefore, to exacerbate the
inflammatory processes believed to be at the heart of disease
progression.2 EC priming
could potentiate the inflammatory effects of cytokines
generated by the cells of the
lession.2 Moreover, primed
ECs would have a heightened sensitivity to cytokines released
elsewhere into the systemic circulation and typically found at
relatively low concentration in a range of disorders with an
inflammatory component.38
This heightened sensitivity could be an important factor in the
increased risk of developing atherosclerotic occlusive disease linked
with chronic infectious diseases.39 40
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Acknowledgments |
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This study was supported by a British Heart Foundation Non-Clinical Lectureship (BS/97001) awarded to G.E.R.
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Footnotes |
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Original received July 28, 2000; resubmission received December 28, 2000; revised resubmission received February 1, 2001; accepted February 1, 2001.
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