Ceramide Triggers Weibel–Palade Body Exocytosis
Rinky Bhatia,* Kenji Matsushita,* Munekazu Yamakuchi, Craig N. Morrell,
Wangsen Cao, Charles J. Lowenstein
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Abstract—The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and
thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing von Willebrand factor (vWF)
and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide
induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. We added ceramide
to human aortic endothelial cells and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF
released into the media. Exogenous ceramide induces vWF release from endothelial cells in a dose-dependent manner.
Activators of endogenous ceramide production, neutral sphingomyelinase, or tumor necrosis factor-␣ also induce
Weibel-Palade body exocytosis. We next studied NO effects on ceramide-induced Weibel-Palade body exocytosis
because NO can inhibit vascular inflammation. The NO donor S-nitroso-N-acetylpenicillamine decreases ceramideinduced vWF release in a dose-dependent manner, whereas the NO synthase inhibitor NG-nitro-L-arginine methyl ester
increases ceramide-induced vWF release. In summary, our findings show that endogenous ceramide triggers
Weibel-Palade body exocytosis, and that endogenous NO inhibits ceramide-induced exocytosis. These data suggest a
novel mechanism by which ceramide induces vascular inflammation and thrombosis. (Circ Res. 2004;95:319-324.)
Key Words: nitric oxide 䡲 sphingomyelin 䡲 granule 䡲 endothelial cells 䡲 N-ethylmaleimide sensitive factor
I
tory molecules within endothelial cells.36 We showed recently
that NO inhibits Weibel-Palade body exocytosis.37
Sphingolipids found in plasma membranes of all eukaryotic cells play an important signaling role in vascular inflammation and thrombosis.38 – 41 The sphingolipid ceramide mediates a variety of stress responses.41 Ceramide is produced de
novo from serine condensation with palmitoyl-coenzyme A
in the endoplasmic reticulum or from hydrolysis of sphingomyelin by sphingomyelinase in the plasma membrane, cytosol, lysosomes, or endoplasmic reticulum.42 A variety of
factors induce ceramide production, including tumor necrosis
factor-␣ (TNF-␣), IL-1␤, interferon-␥, Fas ligand, B7, CD40
ligand, oxidized low-density lipoprotein (LDL), ischemia,
radiation, and chemotherapeutic agents.38 – 41 Ceramide and its
metabolites serve as intracellular second messengers in pathways mediating inflammation, thrombosis, apoptosis, cell
differentiation, proliferation, and vasomotor regulation by
acting on a variety of phosphatases, proteases, kinases,
phospholipases, and transcription factors (protein phosphatase [PP] 1, PP2A, cathepsin, protein kinases, endothelial
differentiation gene receptors, NF-␬B, mitogen-activated protein kinase, plasminogen activator inhibitor-1 (PAI-1), nicotinamide adenine dinucleotide phosphate oxidase, and endothelial NO synthase [NOS]).36 – 41,43– 49 Ceramide can induce
nflammation and thrombosis play major roles in atherosclerosis pathogenesis. Activated endothelial cells release
Weibel-Palade bodies, granules that contain procoagulant and
proinflammatory substances including multimeric von Willebrand factor (vWF), P-selectin, and CD63.1–5 Interleukin-8
(IL-8), thromboplastinogen, calcitonin gene–related peptide, and
endothelin have been reported in Weibel-Palade bodies as
well.6 –9 Exocytosis of Weibel-Palade bodies is triggered by a
variety of agonists, including thrombin, histamine, complement,
leukotrienes, superoxide anions, epinephrine, adenosine, and
vasopressin.10 –15 Weibel–Palade body exocytosis is mediated by
calcium, cAMP, and G-proteins.10,11,15,16 After Weibel–Palade
body exocytosis, P-selectin is translocated to the endothelial
surface, where it facilitates leukocyte rolling, and vWF is
released into the vessel lumen, where it mediates platelet
adhesion and aggregation.17,18
NO is a messenger molecule that mediates vasodilation and
inhibits vascular inflammation.19 –24 NO inhibits atherogenesis in mice, and lack of NO synthesis is associated with
atherosclerosis in humans.25–28 NO inhibits atherogenesis by
multiple mechanisms such as blocking smooth muscle cell
proliferation, decreasing leukocyte adhesion, and inhibiting
platelet adhesion and aggregation.29 –35 NO blocks nuclear
factor ␬B (NF-␬B)– directed transcription of proinflamma-
Original received September 22, 2003; resubmission received May 3, 2004; revised resubmission received June 3, 2004; accepted June 11, 2004.
From the Division of Cardiology, Department of Medicine (R.B., K.M., M.Y., C.N.M., W.C., C.J.L.) and the Departments of Comparative Medicine
and Pathology (C.N.M.), The Johns Hopkins University School of Medicine, Baltimore, Md.
*These authors contributed equally to this work.
This manuscript was sent to Donald Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Charles J. Lowenstein, 950 Ross Bldg, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205.
E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000136519.84279.7a
319
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Circulation Research
August 6, 2004
vascular inflammation by activating transcriptional pathways,
but ceramide can also rapidly trigger vascular inflammation
by nontranscriptional pathways, although the precise mechanism by which it does so is unclear.
We hypothesized that ceramide promotes rapid vascular
inflammation in part by triggering Weibel-Palade body exocytosis. We furthermore proposed that NO will block
ceramide-induced exocytosis.
Materials and Methods
Materials
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Ceramide was purchased from Matreya; dihydroceramide and sphingosine 1-phosphate from Biomol; neutral sphingomyelinase from
Bacillus cereus, thrombin, histamine, acetylpenicillamine (AP), vascular endothelial growth factor (VEGF), NG-nitro-L-arginine methyl
ester ( L -NAME), 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘tetraacetic acid tetrakis (acetoxymethyl ester; BAPTA-AM), and
TNF-␣ from Sigma; DMEM, DMEM without calcium, PBS, and
trypsin-EDTA from GIBCO; vWF ELISA kit from American Diagnostica; dimethyl sulfoxide from J.T. Baker; S-nitroso-Nacetylpenicillamine (SNAP) from Cayman Chemical; and endothelium-based medium (EBM-2) with growth factors (FBS,
hydrocortisone, VEGF, R3-insulin-like growth factor 1, ascorbic
acid, and heparin) and EBM-2 without growth factors
from Clonetics.
Cell Culture
Human aortic endothelial cells (HAECs) were obtained from Clonetics (Walkersville, Md) and grown in EGM-2 media with growth
factors until 80% confluent. Cells were then washed with PBS,
treated with trypsin-EDTA, and centrifuged at 1200 rpm for 5
minutes. The supernatant was removed, the cell pellet was resuspended in EBM-2 with growth factors, and 100 ␮L of the cell
suspension was placed into each well of a 96-well plate and
incubated for 24 hours at 37°C. The cells were then washed and
reincubated in 100 ␮L of EBM-2 without growth factors.
Measurement of Ceramide-Induced Weibel-Palade
Body Exocytosis
HAECs were pretreated or not with the NOS inhibitor L-NAME
1 mmol/L for 16 hours at 37°C, or with the NO donor SNAP or its
control AP for 4 hours at 37°C, or with VEGF for 2 hours. Some
HAECs were pretreated with BAPTA-AM or calcium-free media for
30 minutes, or with desipramine 5 ␮mol/L for 10 minutes.35 HAECs
were then treated with various amounts of ceramide, neutral sphingomyelinase, or TNF-␣ for 1 hour. Supernatants were harvested,
stored at ⫺20°C, and later analyzed for vWF concentration using an
ELISA (American Diagnostica). To confirm that the process of
exocytosis was being studied, we also examined the ceramidetriggered release of IL-8. HAECs were stimulated with 10 ␮mol/L
ceramide for 0 to 60 minutes, and the amount of IL-8 released into
the media was measured by an ELISA (R&D Systems).
Statistical Analysis
Results were expressed as mean⫾SD. Significance between mean
values was determined by the Student t test, with a value of P⬍0.05
considered significant.
Figure 1. Exogenous ceramide activates
Weibel-Palade body exocytosis. A,
Dose-response. Increasing amounts of
ceramide were added to HAECs for 1
hour, and the concentration of vWF
released into the media was measured
by an ELISA (n⫽2⫾SD; *P⫽0.01 compared with 0 ␮mol/L; this experiment
was repeated 3⫻ with similar results). B,
Time course; 1 U/mL thrombin or
10 ␮mol/L ceramide or its negative control 10 ␮mol/L dihydroceramide was
added to HAECs for 1 hour, and the
concentration of vWF released into the
media was measured by an ELISA
(n⫽2⫾SD; *P⬍0.01 compared with
0 ␮mol/L). C, Ceramide activates IL-8
exocytosis over time; 1 U/mL thrombin
or 10 ␮mol/L ceramide was added to
HAECs for 1 hour, and the concentration
of IL-8 released into the media was
measured by an ELISA (n⫽3⫾SD;
*P⬍0.01 compared with 0 ␮mol/L). D,
Comparison with other agonists of exocytosis; 10 ␮mol/L ceramide, 1 mmol/L
histamine, 1 U/mL thrombin, or media
alone was added to HAECs for 1 hour,
and the concentration of vWF released
into the media was measured by an
ELISA (n⫽2⫾SD; *P⬍0.01 compared
with media). E, Ceramide has an insignificant effect on endothelial apoptosis.
HAECs were treated with ceramide for
24 hours, and apoptosis levels were
measured by 4⬘,6-diamidino-2phenylindole staining for fragmented
chromatin (black bars; n⫽4⫾SD). As a
positive control, HAECs were treated with 1 mmol/L hydrogen peroxide for 24 hours (gray bars; n⫽2⫾SD; *P⬍0.01 compared with
media). F, Ceramide effects on total vWF in endothelial cells. HAECs were treated with 10 ␮mol/L ceramide, 1 U/mL thrombin, or
10 ␮mol/L cycloheximide (CHX) for 1 hour. Cells were washed, and vWF concentrations in cell lysates were measured by an ELISA
(n⫽3⫾SD; *P⬍0.05 vs Control).
Bhatia et al
Figure 2. Endogenous ceramide induces Weibel-Palade body
exocytosis. Neutral sphingomyelinase was added in increasing
doses (0, 0.01, 0.1, and 1 U/mL) to HAECs for 1 hour, and vWF
release was measured as above (n⫽3⫾SD; *P⬍0.01 vs 0 U/mL).
Results
Ceramide Triggers Weibel-Palade Body Exocytosis
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To explore the effect of ceramide on Weibel-Palade body
exocytosis, we treated HAECs with ceramide for 1 hour and
measured the concentration of vWF in the media by an
ELISA. Ceramide activates vWF release from endothelial
cells in a dose-dependent manner (Figure 1A). Ceramide
induces vWF release over time, starting within 5 minutes of
treatment and continuing through 60 minutes after treatment
(Figure 1B). To confirm that ceramide activates exocytosis,
we studied the effect of ceramide on the release of IL-8,
another component of Weibel-Palade bodies. Ceramide activates endothelial release of IL-8 (Figure 1C). Ceramide is
almost as effective in stimulating endothelial cell exocytosis
as classical triggers of Weibel-Palade body release such as
Ceramide and Endothelial Exocytosis
321
thrombin or histamine (Figure 1D). To confirm that ceramide
does not damage endothelial cells, we treated HAECs with
increasing amounts of ceramide for 24 hours and measured
apoptosis by counting the number of cells with fragmented
chromatin. Ceramide does not activate endothelial cell apoptosis (Figure 1E).
The effect of endogenous ceramide on Weibel-Palade body
exocytosis was evaluated by adding to HAECs various
concentrations of neutral sphingomyelinase, which hydrolyzes sphingomyelin to ceramide. The concentration of vWF
in the media was measured 1 hour later using an ELISA.
Sphingomyelinase activates vWF release from endothelial
cells in a dose-dependent manner (Figure 2).
To examine the effect of endogenous sphingomyelinase on
ceramide-induced exocytosis, we added various doses of
TNF-␣, an activator of sphingomyelinase, to HAECs for 1
hour. TNF-␣ stimulates vWF release in a dose-dependent
manner (Figure 3A). This effect was partially blocked by
desipramine, an inhibitor of acidic sphingomyelinase (Figure
3B). The addition of an inhibitor of neutral sphingomyelinase
has no effect. Together, these data suggest that endogenous
ceramide synthesis activates endothelial exocytosis.
Calcium plays a role in exocytosis of Weibel-Palade
bodies. To determine whether ceramide induces exocytosis of
Weibel-Palade bodies in a calcium-dependent manner, we
pretreated HAECs with BAPTA or calcium-free media for 30
minutes and then incubated HAECs with 1 ␮mol/L ceramide
for 1 hour. Pretreatment with BAPTA partially decreases
ceramide-induced exocytosis, whereas pretreatment with
calcium-free media does not affect Weibel-Palade body
exocytosis (Figure 4). Pretreatment with BAPTA and
calcium-free media also decreases Weibel-Palade body exocytosis (Figure 4). These data suggest that intracellular
calcium pools mediate ceramide induction of endothelial cell
exocytosis.
NO Blocks Ceramide-Induced Weibel-Palade
Body Exocytosis
Figure 3. TNF-␣ activates endogenous ceramide-induced
Weibel-Palade body exocytosis. A, Dose-response. Increasing
amounts of TNF-␣ were added to HAECs for 1 hour, and vWF
release was measured as above (n⫽2⫾SD; *P⬍0.01 vs 0
ng/mL). B, Inhibitors of sphingomyelinase. HAECs were pretreated or not for 30 minutes with no drug, 100 ␮mol/L 3-Omethylsphingomyelin (an inhibitor of neutral sphingomyelinase),
or 5 ␮mol/L desipramine (an inhibitor of acidic sphingomyelinase), and then 10 ng/mL TNF-␣ was added for 1 hour, and
released vWF was measured as above (n⫽3⫾SD; *P⬍0.01 vs
TNF⫹thrombin).
To measure the effects of exogenous NO on ceramideinduced exocytosis of Weibel-Palade bodies, HAECs were
pretreated with the NO donor SNAP or its negative control
AP at different concentrations for 4 hours before 1 ␮mol/L
ceramide was added. SNAP decreases ceramide-induced
vWF release in a dose-dependent manner (Figure 5A). Different classes of NO donors inhibit ceramide-induced
Weibel-Palade body exocytosis (data not shown).
To measure endogenous NO effects on ceramide-induced
exocytosis, we pretreated HAECs with the NOS inhibitor
L-NAME for 16 hours before the addition of 1 ␮mol/L
ceramide. L-NAME increases ceramide-induced vWF release
(Figure 5B). Together, these data suggest that endogenous
NO inhibits exocytosis of Weibel-Palade bodies induced by
ceramide.
Discussion
The major finding of this study is that endogenous ceramide
activates Weibel-Palade body exocytosis. Furthermore, NO
inhibits ceramide-induced exocytosis.
322
Circulation Research
August 6, 2004
Figure 4. Calcium and ceramide activation of Weibel-Palade
body exocytosis. HAECs were pretreated with BAPTA-AM or
incubated in calcium-free media and then stimulated with
1 ␮mol/L ceramide for 1 hour, and vWF release was measured
as above (n⫽3⫾SD; *P⬍0.01 vs ceramide or
ceramide⫹Ca-free).
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Ceramide mediates a variety of stress responses including
vascular inflammation.38,41 Multiple physical and biological
stimuli activate ceramide synthesis, including ischemia and
reperfusion, oxidized LDL, and inflammatory cytokines.38 –
41,50,51 Ceramide and its metabolites mediate vascular inflammation by increasing endothelial cell expression of
E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule, thereby inducing leukocyte and monocyte adhesion to the vascular wall.52–54 These activated
leukocytes and macrophages secrete cytokines, which further
induce inflammation and ceramide production. Ceramide also
mediates lipoprotein accumulation in the vascular wall, and
ceramide levels are increased in atherosclerotic plaques
compared with the normal vessel wall.55–58 In addition,
ceramide has been implicated in pathways leading to proliferation of smooth muscle cells.50,51,59 Ceramide is also
involved in generation of reactive oxygen species, which
leads to further inflammation and tissue injury.59 Our data
show a novel mechanism by which ceramide might rapidly
activate vascular inflammation. By triggering Weibel-Palade
body exocytosis, ceramide increases endothelial P-selectin
expression, which can induce leukocyte adhesion and aggregation to the vascular wall.
Ceramide also activates multiple pathways leading to
vascular thrombosis. The ceramide metabolite sphingosine
1-phosphate induces platelet aggregation.60 In addition, ceramide induces endothelial cell release of prothrombotic tissue
factor and PAI-1.61,62 Ceramide and its metabolites promote
apoptosis of endothelial cells, which may play a role in
plaque erosion and further thrombosis.63– 65 Our data show a
novel mechanism by which ceramide might induce thrombosis. By activating Weibel-Palade body exocytosis, ceramide
induces vWF release, which can lead to platelet adhesion and
aggregation.
Ceramide has 2 opposing effects on endothelial cells. In
addition to triggering proinflammatory and prothrombotic
pathways, ceramide and its metabolite sphingosine
1-phosphate also activate endothelial NOS (NOS3).43– 49 NO
has multiple effects on the vasculature, including inhibition of
vascular inflammation and thrombosis.19 –23 One mechanism
by which NO decreases vascular inflammation is blocking
Weibel-Palade body exocytosis.37 We found that ceramide
activates a greater release of vWF when NOS is inhibited
(Figure 5B). One possible explanation for this phenomenon is
that ceramide normally activates exocytosis and NOS, and
NO then inhibits further exocytosis. Our results might partially explain why patients with endothelial dysfunction who
cannot synthesize normal levels of NO have higher levels of
vascular inflammation and a greater risk for atherosclerosis
than patients with normal endothelial function and normal
NO production.
In conclusion, our study shows that ceramide triggers
Weibel-Palade body exocytosis and that NO blocks
ceramide-induced exocytosis. These data suggest a novel
mechanism by which ceramide induces vascular inflammation and thrombosis.
Acknowledgments
This research was supported in part by American Heart Association
Established Investigator Grant EIG 0140210N (C.J.L.); National
Institutes of Health grants P01 HL56091, P01 HL65608, NIH R01
HL53615, R01 HL63706 (C.J.L.); RR07002, and HL074945 (to
C.M.); the Ciccarone Center for the Prevention of Heart Disease
(C.J.L.); and the Cora and John H. Davis Foundation (C.J.L.).
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Figure 5. NO inhibits ceramide-induced Weibel-Palade body
exocytosis. A, Exogenous NO dose response. HAECs were pretreated with the NO donor SNAP for 4 hours and stimulated
with 1 ␮mol/L ceramide, and released vWF was measured as
above (n⫽2⫾SD; *P⬍0.01 vs 0 ␮mol/L SNAP). B, Endogenous
NO. HAECs were pretreated with 1 mmol/L L-NAME for 16
hours and stimulated with 1 ␮mol/L ceramide, and released
vWF was measured as above (n⫽2⫾SD; *P⬍0.01 for Ceramide
vs Ceramide⫹L-NAME).
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Ceramide Triggers Weibel−Palade Body Exocytosis
Rinky Bhatia, Kenji Matsushita, Munekazu Yamakuchi, Craig N. Morrell, Wangsen Cao and
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Circ Res. 2004;95:319-324; originally published online June 24, 2004;
doi: 10.1161/01.RES.0000136519.84279.7a
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