This Article Abstract Full Text (PDF) All Versions of this Article: 95/3/319    most recent 01.RES.0000136519.84279.7av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bhatia, R. Articles by Lowenstein, C. J. Search for Related Content PubMed PubMed Citation Articles by Bhatia, R. Articles by Lowenstein, C. J. Related Collections Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors Cell signalling/signal transduction

(Circulation Research. 2004;95:319.)
© 2004 American Heart Association, Inc.

Cellular Biology

Ceramide Triggers Weibel–Palade Body Exocytosis

Rinky Bhatia^*, Kenji Matsushita^*, Munekazu Yamakuchi, Craig N. Morrell, Wangsen Cao, Charles J. Lowenstein

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.

Correspondence to Charles J. Lowenstein, 950 Ross Bldg, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205. E-mail clowenst{at}jhmi.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 ceramide-induced vWF release in a dose-dependent manner, whereas the NO synthase inhibitor N^G-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.

Key Words: nitric oxide • sphingomyelin • granule • endothelial cells • N-ethylmaleimide sensitive factor

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inflammation 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 proinflammatory 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-1ß, 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 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
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), N^G-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-N-acetylpenicillamine (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 µL 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 µL 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 µmol/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 ceramide-triggered release of IL-8. HAECs were stimulated with 10 µmol/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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ceramide Triggers Weibel-Palade Body Exocytosis
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 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).

View larger version (39K): [in this window] [in a new window]   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 µmol/L; this experiment was repeated 3x with similar results). B, Time course; 1 U/mL thrombin or 10 µmol/L ceramide or its negative control 10 µmol/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 µmol/L). C, Ceramide activates IL-8 exocytosis over time; 1 U/mL thrombin or 10 µmol/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 µmol/L). D, Comparison with other agonists of exocytosis; 10 µmol/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-2-phenylindole 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 µmol/L ceramide, 1 U/mL thrombin, or 10 µmol/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).

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).

View larger version (14K): [in this window] [in a new window]   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).

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.

View larger version (24K): [in this window] [in a new window]   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 µmol/L 3-O-methylsphingomyelin (an inhibitor of neutral sphingomyelinase), or 5 µmol/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).

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 µmol/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.

View larger version (20K): [in this window] [in a new window]   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 µmol/L ceramide for 1 hour, and vWF release was measured as above (n=3±SD; *P<0.01 vs ceramide or ceramide+Ca-free).

NO Blocks Ceramide-Induced Weibel-Palade Body Exocytosis
To measure the effects of exogenous NO on ceramide-induced 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 µmol/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).

View larger version (23K): [in this window] [in a new window]   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 µmol/L ceramide, and released vWF was measured as above (n=2±SD; *P<0.01 vs 0 µmol/L SNAP). B, Endogenous NO. HAECs were pretreated with 1 mmol/L L-NAME for 16 hours and stimulated with 1 µmol/L ceramide, and released vWF was measured as above (n=2±SD; *P<0.01 for Ceramide vs Ceramide+L-NAME).

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 µmol/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

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of this study is that endogenous ceramide activates Weibel-Palade body exocytosis. Furthermore, NO inhibits ceramide-induced exocytosis.

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.).

	Footnotes

 
*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.

Original received September 22, 2003; resubmission received May 3, 2004; revised resubmission received June 3, 2004; accepted June 11, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol. 1982; 95: 355–360.[Abstract/Free Full Text]
2. Bonfanti R, Furie BC, Furie B, Wagner DD. PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells. Blood. 1989; 73: 1109–1112.[Abstract/Free Full Text]
3. Vischer UM, Wagner DD. CD63 is a component of Weibel-Palade bodies of human endothelial cells. Blood. 1993; 82: 1184–1191.[Abstract/Free Full Text]
4. Datta YH, Ewenstein BM. Regulated secretion in endothelial cells: biology and clinical implications. Thromb Haemost. 2001; 86: 1148–1155.[Medline] [Order article via Infotrieve]
5. Wagner DD. The Weibel-Palade body: the storage granule for von Willebrand factor and P-selectin. Thromb Haemost. 1993; 70: 105–110.[Medline] [Order article via Infotrieve]
6. Utgaard JO, Jahnsen FL, Bakka A, Brandtzaeg P, Haraldsen G. Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells. J Exp Med. 1998; 188: 1751–1756.[Abstract/Free Full Text]
7. Ueda H, Doi Y, Sakamoto Y, Hamasaki K, Fujimoto S. Simultaneous localization of histamine and factor VIII-related antigen in the endothelium of the human umbilical vein. Anat Rec. 1992; 232: 257–261.[CrossRef][Medline] [Order article via Infotrieve]
8. Ozaka T, Doi Y, Kayashima K, Fujimoto S. Weibel-Palade bodies as a storage site of calcitonin gene-related peptide and endothelin-1 in blood vessels of the rat carotid body. Anat Rec. 1997; 247: 388–394.[CrossRef][Medline] [Order article via Infotrieve]
9. Schaumburg-Lever G, Gehring B, Kaiserling E. Ultrastructural localization of factor XIIIa. J Cutan Pathol. 1994; 21: 129–134.[CrossRef][Medline] [Order article via Infotrieve]
10. Birch KA, Pober JS, Zavoico GB, Means AR, Ewenstein BM. Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. J Cell Biol. 1992; 118: 1501–1510.[Abstract/Free Full Text]
11. Hamilton KK, Sims PJ. Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1. J Clin Invest. 1987; 79: 600–608.[Medline] [Order article via Infotrieve]
12. Hattori R, Hamilton KK, McEver RP, Sims PJ. Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface. J Biol Chem. 1989; 264: 9053–9060.[Abstract/Free Full Text]
13. Datta YH, Romano M, Jacobson BC, Golan DE, Serhan CN, Ewenstein BM. Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells. Circulation. 1995; 92: 3304–3311.[Abstract/Free Full Text]
14. Vischer UM, Jornot L, Wollheim CB, Theler JM. Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells. Blood. 1995; 85: 3164–3172.[Abstract/Free Full Text]
15. Vischer UM, Wollheim CB. Epinephrine induces von Willebrand factor release from cultured endothelial cells: involvement of cyclic AMP-dependent signaling in exocytosis. Thromb Haemost. 1997; 77: 1182–1188.[Medline] [Order article via Infotrieve]
16. Klarenbach SW, Chipiuk A, Nelson RC, Hollenberg MD, Murray AG. Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases. Circ Res. 2003; 92: 272–278.[Abstract/Free Full Text]
17. Ramos CL, Huo Y, Jung U, Ghosh S, Manka DR, Sarembock IJ, Ley K. Direct demonstration of P-selectin- and VCAM-1-dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E-deficient mice. Circ Res. 1999; 84: 1237–1244.[Abstract/Free Full Text]
18. Andre P, Denis CV, Ware J, Saffaripour S, Hynes RO, Ruggeri ZM, Wagner DD. Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins. Blood. 2000; 96: 3322–3328.[Abstract/Free Full Text]
19. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest. 1997; 100: 2146–2152.[Medline] [Order article via Infotrieve]
20. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.[CrossRef][Medline] [Order article via Infotrieve]
21. Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994; 63: 175–195.[CrossRef][Medline] [Order article via Infotrieve]
22. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med. 1994; 120: 227–237.[Abstract/Free Full Text]
23. Cirino G, Fiorucci S, Sessa WC. Endothelial nitric oxide synthase: the Cinderella of inflammation? Trends Pharmacol Sci. 2003; 24: 91–95.[CrossRef][Medline] [Order article via Infotrieve]
24. Foster MW, McMahon TJ, Stamler JS. S-nitrosylation in health and disease. Trends Mol Med. 2003; 9: 160–168.[CrossRef][Medline] [Order article via Infotrieve]
25. Kauser K, da Cunha V, Fitch R, Mallari C, Rubanyi GM. Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol. 2000; 278: H1679–H1685.[Abstract/Free Full Text]
26. Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, Picard MH, Huang PL. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation. 2001; 104: 448–454.[Abstract/Free Full Text]
27. Quyyumi AA, Dakak N, Andrews NP, Husain S, Arora S, Gilligan DM, Panza JA, Cannon RO III. Nitric oxide activity in the human coronary circulation. Impact of risk factors for coronary atherosclerosis. J Clin Invest. 1995; 95: 1747–1755.[Medline] [Order article via Infotrieve]
28. Quyyumi AA, Dakak N, Mulcahy D, Andrews NP, Husain S, Panza JA, Cannon RO III. Nitric oxide activity in the atherosclerotic human coronary circulation. J Am Coll Cardiol. 1997; 29: 308–317.[Abstract]
29. Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res. 2002; 90: 21–28.[Abstract/Free Full Text]
30. Radomski MW, Palmer RM, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun. 1987; 148: 1482–1489.[CrossRef][Medline] [Order article via Infotrieve]
31. Alheid U, Frolich JC, Forstermann U. Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb Res. 1987; 47: 561–571.[CrossRef][Medline] [Order article via Infotrieve]
32. Busse R, Luckhoff A, Bassenge E. Endothelium-derived relaxant factor inhibits platelet activation. Naunyn-Schmiedebergs Arch Pharmacol. 1987; 336: 566–571.[Medline] [Order article via Infotrieve]
33. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991; 88: 4651–4655.[Abstract/Free Full Text]
34. Gauthier TW, Scalia R, Murohara T, Guo JP, Lefer AM. Nitric oxide protects against leukocyte-endothelium interactions in the early stages of hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1995; 15: 1652–1659.[Abstract/Free Full Text]
35. Jeremy JY, Rowe D, Emsley AM, Newby AC. Nitric oxide and the proliferation of vascular smooth muscle cells. Cardiovasc Res. 1999; 43: 580–594.[Free Full Text]
36. Kupatt C, Weber C, Wolf DA, Becker BF, Smith TW, Kelly RA. Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NFkappaB. J Mol Cell Cardiol. 1997; 29: 2599–2609.[CrossRef][Medline] [Order article via Infotrieve]
37. Qian Z, Gelzer-Bell R, Yang Sx SX, Cao W, Ohnishi T, Wasowska BA, Hruban RH, Rodriguez ER, Baldwin WM III, Lowenstein CJ. Inducible nitric oxide synthase inhibition of Weibel-Palade body release in cardiac transplant rejection. Circulation. 2001; 104: 2369–2375.[Abstract/Free Full Text]
38. Hannun YA, Obeid LM. The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J Biol Chem. 2002; 277: 25847–25850.[Free Full Text]
39. Auge N, Negre-Salvayre A, Salvayre R, Levade T. Sphingomyelin metabolites in vascular cell signaling and atherogenesis. Prog Lipid Res. 2000; 39: 207–229.[CrossRef][Medline] [Order article via Infotrieve]
40. Levade T, Auge N, Veldman RJ, Cuvillier O, Negre-Salvayre A, Salvayre R. Sphingolipid mediators in cardiovascular cell biology and pathology. Circ Res. 2001; 89: 957–968.[Abstract/Free Full Text]
41. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science. 1996; 274: 1855–1859.[Abstract/Free Full Text]
42. Huwiler A, Kolter T, Pfeilschifter J, Sandhoff K. Physiology and pathophysiology of sphingolipid metabolism and signaling. Biochim Biophys Acta. 2000; 1485: 63–99.[Medline] [Order article via Infotrieve]
43. Igarashi J, Thatte HS, Prabhakar P, Golan DE, Michel T. Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci U S A. 1999; 96: 12583–12588.[Abstract/Free Full Text]
44. Dantas AP, Igarashi J, Michel T. Sphingosine 1-phosphate and control of vascular tone. Am J Physiol Heart Circ Physiol. 2003; 284: H2045–H2052.[Abstract/Free Full Text]
45. Gonzalez E, Kou R, Lin AJ, Golan DE, Michel T. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase. J Biol Chem. 2002; 277: 39554–39560.[Abstract/Free Full Text]
46. Kou R, Igarashi J, Michel T. Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase. Biochemistry. 2002; 41: 4982–4988.[CrossRef][Medline] [Order article via Infotrieve]
47. Igarashi J, Michel T. Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways. J Biol Chem. 2001; 276: 36281–36288.[Abstract/Free Full Text]
48. Igarashi J, Bernier SG, Michel T. Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells. J Biol Chem. 2001; 276: 12420–12426.[Abstract/Free Full Text]
49. Igarashi J, Michel T. Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction. J Biol Chem. 2000; 275: 32363–32370.[Abstract/Free Full Text]
50. Auge N, Andrieu N, Negre-Salvayre A, Thiers JC, Levade T, Salvayre R. The sphingomyelin-ceramide signaling pathway is involved in oxidized low density lipoprotein-induced cell proliferation. J Biol Chem. 1996; 271: 19251–19255.[Abstract/Free Full Text]
51. Auge N, Escargueil-Blanc I, Lajoie-Mazenc I, Suc I, Andrieu-Abadie N, Pieraggi MT, Chatelut M, Thiers JC, Jaffrezou JP, Laurent G, Levade T, Negre-Salvayre A, Salvayre R. Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein. J Biol Chem. 1998; 273: 12893–12900.[Abstract/Free Full Text]
52. Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem. 2001; 276: 7614–7620.[Abstract/Free Full Text]
53. Xia P, Gamble JR, Rye KA, Wang L, Hii CS, Cockerill P, Khew-Goodall Y, Bert AG, Barter PJ, Vadas MA. Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc Natl Acad Sci U S A. 1998; 95: 14196–14201.[Abstract/Free Full Text]
54. Modur V, Zimmerman GA, Prescott SM, McIntyre TM. Endothelial cell inflammatory responses to tumor necrosis factor alpha. Ceramide- dependent and -independent mitogen-activated protein kinase cascades. J Biol Chem. 1996; 271: 13094–13102.[Abstract/Free Full Text]
55. Xu XX, Tabas I. Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages. J Biol Chem. 1991; 266: 24849–24858.[Abstract/Free Full Text]
56. Tabas I, Li Y, Brocia RW, Xu SW, Swenson TL, Williams KJ. Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. J Biol Chem. 1993; 268: 20419–20432.[Abstract/Free Full Text]
57. Chatterjee S. Sphingolipids in atherosclerosis and vascular biology. Arterioscler Thromb Vasc Biol. 1998; 18: 1523–1533.[Abstract/Free Full Text]
58. Tabas I. Secretory sphingomyelinase. Chem Phys Lipids. 1999; 102: 123–130.[CrossRef][Medline] [Order article via Infotrieve]
59. Bhunia AK, Han H, Snowden A, Chatterjee S. Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. J Biol Chem. 1997; 272: 15642–15649.[Abstract/Free Full Text]
60. Yatomi Y, Yamamura S, Ruan F, Igarashi Y. Sphingosine 1-phosphate induces platelet activation through an extracellular action and shares a platelet surface receptor with lysophosphatidic acid. J Biol Chem. 1997; 272: 5291–5297.[Abstract/Free Full Text]
61. Soeda S, Honda O, Shimeno H, Nagamatsu A. Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells. Thromb Res. 1995; 80: 509–518.[CrossRef][Medline] [Order article via Infotrieve]
62. Hirokawa M, Kitabayashi A, Kuroki J, Miura AB. Induction of tissue factor production but not the upregulation of adhesion molecule expression by ceramide in human vascular endothelial cells. Tohoku J Exp Med. 2000; 191: 167–176.[CrossRef][Medline] [Order article via Infotrieve]
63. Kolesnick R, Hannun YA. Ceramide and apoptosis. Trends Biochem Sci. 1999; 24: 224–225;author reply 227.[CrossRef][Medline] [Order article via Infotrieve]
64. Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK III, Schuchman EH, Fuks Z, Kolesnick R. Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation. J Exp Med. 1997; 186: 1831–1841.[Abstract/Free Full Text]
65. Mallat Z, Tedgui A. Current perspective on the role of apoptosis in atherothrombotic disease. Circ Res. 2001; 88: 998–1003.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. J. Metcalf, T. D. Nightingale, H. L. Zenner, W. W. Lui-Roberts, and D. F. Cutler Formation and function of Weibel-Palade bodies J. Cell Sci., January 1, 2008; 121(1): 19 - 27. [Abstract] [Full Text] [PDF]

				A. C.M. Mulders, S. L.M. Peters, and M. C. Michel Sphingomyelin metabolism and endothelial cell function Eur. Heart J., April 1, 2007; 28(7): 777 - 779. [Full Text] [PDF]

				D. J. Dietzen, K. L. Page, T. A. Tetzloff, A. Bohrer, and J. Turk Inhibition of 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG CoA) Reductase Blunts Factor VIIa/Tissue Factor and Prothrombinase Activities via Effects on Membrane Phosphatidylserine Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 690 - 696. [Abstract] [Full Text] [PDF]

				M. G. Rondaij, R. Bierings, A. Kragt, J. A. van Mourik, and J. Voorberg Dynamics and Plasticity of Weibel-Palade Bodies in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1002 - 1007. [Abstract] [Full Text] [PDF]

				S. A. Summers and D. H. Nelson A Role for Sphingolipids in Producing the Common Features of Type 2 Diabetes, Metabolic Syndrome X, and Cushing's Syndrome Diabetes, March 1, 2005; 54(3): 591 - 602. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 95/3/319    most recent 01.RES.0000136519.84279.7av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bhatia, R. Articles by Lowenstein, C. J. Search for Related Content PubMed PubMed Citation Articles by Bhatia, R. Articles by Lowenstein, C. J. Related Collections Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors Cell signalling/signal transduction