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(Circulation. 1995;92:3304-3311.)
© 1995 American Heart Association, Inc.

Articles

Peptido-Leukotrienes Are Potent Agonists of von Willebrand Factor Secretion and P-Selectin Surface Expression in Human Umbilical Vein Endothelial Cells

Yvonne H. Datta, MD; Mario Romano, MD; Brian C. Jacobson, MD; David E. Golan, MD, PhD; Charles N. Serhan, PhD; Bruce M. Ewenstein, MD, PhD

From the Hematology-Oncology Division, Brigham and Women's Hospital, and Departments of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Mass.

Correspondence to Bruce M. Ewenstein, MD, PhD, Hematology-Oncology Division, or Charles N. Serhan, PhD; new address: Center for Experimental Therapeutics and Reperfusion Injury, 75 Francis St, Boston, MA 02115.

	Abstract

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Background The peptido-leukotrienes (LTs) and lipoxins (LX) are produced by platelets through the transcellular conversion of leukocyte-derived LTA₄ at sites of vascular inflammation and injury, such as during coronary artery balloon angioplasty. We studied the actions of these eicosanoids on vascular endothelium.

Methods and Results We found that stimulation of cultured human umbilical vein endothelial cells (EC) with LTC₄ and LTD₄ resulted in the release of high-molecular-weight multimers of von Willebrand factor (vWF) in a concentration- and time-dependent fashion, as measured by ELISA. Neither LXA₄ nor LXB₄ stimulated vWF release. LTC₄ and LTD₄ also stimulated a rapid increase in the surface expression of P-selectin indicated by increased binding of anti–P-selectin monoclonal antibody–coated beads. Fluorescence cytometry detected prolonged peaks of [Ca²⁺]_i in EC in response to concentrations of thrombin and LTD₄ that induce near-maximal vWF secretion. In contrast, concentrations of LTC₄ that induce similar levels of vWF secretion produced only asynchronous oscillations of [Ca²⁺]_i in most EC and rarely induced prolonged peaks of [Ca²⁺]_i. Depletion of external Ca²⁺ had no apparent impact on LT-stimulated [Ca²⁺]_i transients and vWF secretion, implicating an intracellular pool as the source of this response. Staurosporine, sphingosine, and H-7 each had only modest effects on peptido-LT–induced vWF secretion, suggesting that protein kinase C is not a primary mediator of peptido-LT–induced exocytosis. Inhibitors of cyclooxygenase and platelet-activating factor had no effect on peptido-LT–mediated vWF secretion.

Conclusions Through the induction of vWF secretion and P-selectin surface expression, peptido-LTs are likely to play an important role in the interrelated processes of hemostasis and inflammation.

Key Words: von Willebrand factor • secretion • leukotriene • eicosanoid • transcellular metabolism

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
VWF is an adhesive glycoprotein that plays a major role in the initial adhesion of platelets to vascular subendothelium¹ and regulates the plasma level of coagulant factor VIII.^{2 3} vWF is synthesized exclusively by EC and megakaryocytes. In plasma, vWF circulates as a series of disulfide-linked subunits with an aggregate molecular weight, varying from 0.5x to 20x10⁶. Physiological stimuli such as exercise and epinephrine and pharmacological agents such as the vasopressin analogue desmopressin acetate⁴ result in a rapid increase in plasma levels of vWF. The source of this increase is believed to be a releasable pool of vWF that is stored within EC secretory granules called Weibel-Palade bodies.⁵ In culture, EC also rapidly release Weibel-Palade body stores of vWF in response to a number of naturally occurring agonists, including thrombin,^{6 7} histamine,⁸ complement membrane attack complex (C5b-9),⁹ and fibrin.^{10 11} The vWF that is stored within Weibel-Palade bodies consists primarily of high-molecular-weight multimers^{12 13} that avidly bind to the extracellular matrix¹⁴ and to platelet receptors.¹⁵

Also associated with the Weibel-Palade body is a leukocyte-binding protein, P-selectin (CD62),^{16 17} an integral membrane protein that is rapidly translocated to the plasma membrane of the cell during exocytosis.¹⁸ EC surface expression of P-selectin promotes the binding and "rolling" of monocytes and neutrophils in a ß₂-integrin–independent process, which precedes leukocyte migration into sites of inflammation.¹⁹ Moreover, neutrophils that adhere to endothelium expressing P-selectin become "primed" for enhanced secretion in response to chemotactic peptides.²⁰

LTs are 5-lipoxygenase–derived products of arachidonic acid that are believed to have a central role in inflammation.²¹ First characterized as major components of the "slow-reacting substance of anaphylaxis,"²² the peptido-LTs LTC₄ and LTD₄ are generated in eosinophils, mast cells, and macrophages. LTC₄ and LTD₄ are also generated in substantial quantities by activated platelets (and to a lesser extent by vascular EC) through the transcellular conversion of neutrophil-derived LTA₄.^{23 24 25 26 27 28} LX are a class of biologically active arachidonic acid derivatives that are generated in a number of cell types, including platelets and leukocytes, by the sequential interactions of 5- and 15-lipoxygenases or 5- and 12-lipoxygenases.²⁹ Activation of platelets and leukocytes triggers the production of both peptido-LTs and LXs, and we have found that both classes of lipoxygenase products are generated intraluminally during coronary artery balloon angioplasty.³⁰ The intraluminal origin of these lipoxygenase products prompted us to characterize more fully their influence on vascular endothelium.

In the present study, we demonstrate that among the lipoxygenase products examined, the peptido-LTs are uniquely potent agonists of regulated secretion in cultured human umbilical vein EC. The ability of LTs to stimulate exocytosis is independent of the previously reported LT-induced production of prostacyclin³¹ and PAF.³² Moreover, the stimulus-secretion pathways used by LTs and thrombin are likely to be different.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
LTB₄, LTC₄, LTD₄, LTE₄, LXA₄, and LXB₄ were purchased from Biomol Research Laboratories. Human -thrombin was generously provided by Dr John W. Fenton II (Department of Health, Albany, NY) or was purchased from Enzyme Research Laboratories, Inc. SKF 104353 was a gift from Martin Wasserman (Smith Kline & French Laboratories). WEB 2086 was a gift from Boehringer Ingelheim Pharmaceutical, Inc. Staurosporine and H-7 were purchased from LC Laboratories. Indo-1-AM and BAPTA-AM were purchased from Molecular Probes Inc. Fibronectin was purchased from the New York Blood Center, and EC growth factor was obtained from Upstate Biotechnology. Other tissue culture reagents were purchased from GIBCO-BRL. All other reagents were purchased from Sigma Chemical Co.

Cell Culture
Endothelial cells were isolated from two to four human umbilical vein segments by collagenase digestion and serially subcultured (two or three passages) in M199 containing 20% heat-inactivated fetal calf serum, 100 µg/mL of porcine heparin, and 50 µg/mL of EC growth factor. Final plating was onto gelatin-coated C-24 or C-6 wells of plastic tissue culture plates or onto ultrasonically cleaned, fibronectin-coated, 31-mm No. 1 glass coverslips (Biophysica Technologies) in C-6 wells. For radiolabeling experiments, EC were maintained for 3 days in C-6 wells in M199; each well was supplemented with 0.5 mCi of [³⁵S]cysteine (NEN-DuPont) and then chased for 4 hours with unlabeled medium.

vWF Immunoisolation and Agarose Gel Electrophoresis
To examine the composition of secreted vWF, we metabolically labeled EC with [³⁵S]cysteine and then stimulated them with agonist or vehicle control for 20 minutes at 37°C. The conditioned media were harvested and treated to final concentrations of 1 mmol/L PMSF and 10 µg/mL leupeptin. Radiolabeled vWF was isolated using rabbit anti-vWF antibody (Dako Corporation) previously conjugated to Affi-Gel 10 (Bio-Rad Laboratories). After a 16-hour incubation at 4°C, the beads were washed four times in a buffer containing 10 mmol/L Tris · HCl, pH 7.2, 1 mmol/L EDTA, 150 mmol/L NaCl, 0.5% Triton X-100, and 0.5% sodium deoxycholate. vWF was eluted from the beads by being heated at 60°C for 20 minutes in 1.5 vol of sample buffer (20 mmol/L Tris · HCl, pH 7.2, 2% SDS, 7.7 mol/L urea, 2 mmol/L EDTA, and 5% glycerol). Electrophoretic separation of multimers was performed on 1% agarose (FMC BioProducts) as previously described.³³

Measurement of vWF Secretion
Confluent monolayers of EC were washed four times with M199/0.1% gelatin or HBSS/25 mmol/L HEPES, pH 7.4/0.1% gelatin and then incubated with various agonists. In experiments involving pharmacological agents, EC were preincubated for 15 to 20 minutes at 37°C with the agent before the addition of an agonist. Conditioned media from treated EC were transferred to separate tubes and stored at -25°C. In early experiments, the amounts of vWF in the media were assayed with an inhibition ELISA¹³ ; in later experiments, vWF was quantified with a simpler protocol that uses polyclonal goat F(ab')₂ anti-human vWF (American Diagnostica) as the coating antibody and anti-human vWF peroxidase conjugate as the detecting antibody. The quantity of vWF released per well of confluent EC varied among different cultures and passage levels; in some cases, these values were normalized to maximal agonist-stimulated vWF release measured in the same experiment.

Analysis of Eicosanoids
To assess possible conversion or further metabolism of LTC₄ by EC, confluent monolayers of EC in 75-cm² flasks were incubated with [³H]LTC₄ (4.1x10⁴ cpm/mL tracer; 0.5 µmol/L final LTC₄ concentration) for indicated intervals. The incubations were terminated by the addition of 6 mL of ice-cold methanol. The monolayers were immediately washed with methanol/PBS (1:2 v/v). For each time point, materials from three culture flasks were pooled, known amounts of PGB₂ were added as an internal standard, and the samples were taken to dryness by rotoevaporation. Materials were resuspended in methanol/H₂O (1:45 v/v), rapidly acidified to pH 3.5, and loaded into individual C₁₈ reverse-phase cartridges containing silica (C₁₈ Sep-Paks, Waters Associates). After elution, materials from the C₁₈ columns were concentrated and chromatographed on a system consisting of a Beckman gradient HPLC (pump model 11A and gradient liquid HPLC model 332), an Altrex Ultrasphere-ODS (4.6 mmx25 cm) column, an injector, and a Perkin-Elmer LC-75 spectraphotometer detector. The column was eluted with methanol/H₂O/acetic acid (65:35:0.01, pH 5.7) at a flow rate of 1 mL/min, and 1-mL fractions were analyzed separately by ß-scintillation counting. Peptido-LTs (LTC₄, LTD₄, and LTE₄) and radioactive peptido-LTs were identified by their retention times and coelution with synthetic standards.

Detection of P-Selectin on the Surface of EC
Detection of cell surface expression of P-selectin was performed as previously described.³⁴ Magnetic 2.8-µm polystyrene beads were coated with anti–P-selectin monoclonal antibody (AC1.2) (a generous gift of Dr Bruce Furie, Tufts Medical School, Boston, Mass) or with an irrelevant IgG₁ control antibody, K16/16. EC were grown to confluence on fibronectin-coated Lab Tek eight-chamber glass coverslips. The cells were washed four times with M199/gelatin and then incubated with agonist in the presence of antibody-coated beads (1.5x10⁹/mL) in M199/0.1% gelatin for 20 minutes at room temperature with gentle rocking. The chambers and rubber gaskets were then removed, and the slide was rinsed manually in a beaker containing M199/gelatin. The cells were visualized on a Nikon Diaphot inverted microscope and photographed with a Nikon FX-35DX camera.

Measurement of Intracellular Ca²⁺ Concentrations
Concentrations of cytosolic free calcium ([Ca²⁺]_i) were measured in individual EC with an ACAS 570 Interactive Laser Cytometer (Meridian Instruments) as previously described.³⁴ Briefly, second- or third-passage EC were grown to confluence on fibronectin-coated glass coverslips and then loaded with 1 µmol/L of the acetoxymethyl ester of indo-1 (indo-1–AM) in M199/0.1% gelatin for 1 hour at 37°C. The cells were washed three times with HBSS supplemented with 1 mmol/L MgCl₂, 0.1% gelatin, and 25 mmol/L HEPES, pH 7.4, with or without 2 mmol/L Ca²⁺ and then mounted on the laser cytometer stage. Fields of 6 to 14 cells were visualized with an Olympus IMT-2 inverted fluorescence microscope and illuminated with 355-nm light from an argon laser. The ratio of fluorescence emission at 400 nm (bound Ca²⁺) to that at 480 nm (unbound Ca²⁺) was taken as a measure of [Ca²⁺]_i. Absolute values for [Ca²⁺]_i were not calculated because indo-1 ratios within cells cannot be accurately calibrated by comparison with indo-1 in solution. Cells were monitored at room temperature for 5 to 10 minutes after the addition of agonist. MERIDIAN software was used to draw polygons around individual EC, and the integrated signal from all pixels within each cell boundary was taken as the relative measure of [Ca²⁺]_i for that cell.

Statistical Analysis
Each ELISA experiment was composed of at least three replicates. Comparisons between patterns of agonist-induced [Ca²⁺]_i transients were analyzed with the Pearson ² test. Statistical significance among experimental groups in vWF release was determined by Student's t test for paired values, except for the PKC inhibitor experiments (see Fig 6), which were analyzed with the Tukey multiple-comparisons model. In all experiments, P<.05 was considered to be statistically significant.

View larger version (49K): [in this window] [in a new window]   Figure 6. Bar graphs of effect of protein kinase C inhibitors on vWF secretion. EC cultured on 24-well plates were preincubated for 15 minutes at 37°C with staurosporine (30 nmol/L), sphingosine (10 µmol/L), or H-7 (100 µmol/L). Agonists were then added, and incubation continued for 30 minutes at 37°C in the continued presence of the inhibitor. Supernatants were removed, and vWF was quantified by ELISA. Results are representative of three separate experiments performed in triplicate and are reported as ng/mL (±SEM). *P<.05 vs no inhibitor controls.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Peptido-LTs Stimulate vWF Secretion in EC
Twenty-minute exposure of confluent monolayers of EC to different LTs (500 nmol/L) induced up to fourfold increases in vWF in conditioned medium with the following order of activity: LTC₄>LTD₄>>LTE₄>>>LTB₄ (Table 1). LTC₄-stimulated vWF secretion was 60% more than that elicited by thrombin (1 U/mL). In contrast, the same concentration of two other platelet-derived lipoxygenase products, LXA₄ and LXB₄, resulted in vWF release that was <10% of that released by LTC₄ over the experimental time period.

View this table: [in this window] [in a new window]   Table 1. Peptido-LTs Stimulate vWF Secretion by Cultured EC

LT Stimulation of vWF Secretion Is Both Concentration and Time Dependent
The concentration dependence of LT-stimulated vWF secretion was next examined. The addition of 3 nmol/L LTC₄ or 30 nmol/L LTD₄ produced half-maximal secretion of vWF, with threshold concentrations of 0.1 and 1 nmol/L for LTC₄ and LTD₄, respectively. Plateau levels of stimulated secretion were not observed in response to LTE₄ (or LTB₄; data not shown) at concentrations up to 1 µmol/L (Fig 1A). Small increases in vWF secretion could be detected within 2 minutes after the addition of 50 nmol/L LTC₄ or 500 nmol/L LTD₄ and reached plateau levels by 30 minutes (Fig 1B). However, the appearance of vWF in the conditioned media after peptido-LT addition was slow in onset compared with thrombin. Half-maximal secretion occurred at 5 minutes in response to thrombin and at 17 and 20 minutes after exposure to LTD₄ and LTC₄, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 1. Plots of dose- and time-dependent secretion of vWF in response to peptido-LTs. A, Confluent monolayers of human umbilical vein EC were incubated with the indicated concentrations of LTC4, LTD4, or LTE4 for 20 minutes and the quantity of vWF released into the culture medium determined by ELISA. Data points are the mean±SEM of three separate experiments performed in triplicate and are expressed relative to maximal LTC4-stimulated vWF secretion. (Mean maximum concentrations of vWF secretion were LTC4=12.4±1.8 [ng/mL±SEM], and control=1.3±0.1.) B, EC were incubated with vehicle, 300 nmol/L LTD4, 30 nmol/L LTC4, or 1 U/mL thrombin for the indicated times, and the quantity of vWF released into the conditioned media was determined by ELISA. Data are the mean±SEM of three separate experiments performed in triplicate and are expressed relative to maximal agonist-induced vWF secretion at 30 minutes. (Mean maximal concentrations of vWF were control=3.8±0.9 [ng/mL±SEM], LTD4=39.1±13.6, LTC4=25.7±9.4, and thrombin=28.2±7.7). The time to half-maximal secretion (t1/2) was significantly less for thrombin than for either LT (P<.001).

Peptido-LTs Stimulate the Release of vWF From Weibel-Palade Bodies
We next investigated the process by which LTs stimulate vWF release. The multimeric composition of vWF released in response to thrombin (1 U/mL), LTD₄ (500 nmol/L), or vehicle alone was evaluated by immunoisolation and agarose gel electrophoresis as described in "Methods." It was observed that vWF released in response to either agonist consisted principally of the high-molecular-weight multimeric species, which have been previously shown to reside exclusively in EC Weibel-Palade bodies^{12 13} (Fig 2).

View larger version (53K): [in this window] [in a new window]   Figure 2. Multimeric composition of vWF released in response to thrombin or LTD4. EC were metabolically labeled with [35S]cysteine in M199 for 3 days and then chased for 4 hours with unlabeled medium. The washed monolayers were incubated with control vehicle, LTD4 (500 nmol/L) or thrombin (1 U/mL) for 20 minutes, and the released vWF was immunopurified using a rabbit anti-vWF antibody conjugated to Affi-Gel. Radiolabeled vWF was electrophoresed on 1% agarose gels under nonreducing conditions, dried, and autoradiographed. Constitutively secreted vWF (lane 1) consists primarily of dimeric forms, whereas stimulation with either LTD4 (lane 2) or thrombin (lane 3) results in the release of high-molecular-weight (HMW) multimers of vWF, indicating that they are derived from Weibel-Palade body pools.

In addition to the release of high-molecular-weight forms of vWF, the process of regulated secretion in EC results in the rapid translocation of P-selectin from Weibel-Palade body–associated membranes to the apical surface of EC.¹⁸ To determine whether LTs stimulated an increase in P-selectin surface expression, EC were incubated with polystyrene beads coated with anti–P-selectin mAb and with LTs, thrombin, or vehicle-alone controls as described in "Methods."³⁴ In three separate experiments, the mean number of beads bound per field of cells in response to LTC₄ (50 nmol/L) or LTD₄ (500 nmol/L) was increased by 3.8- to 6.5-fold compared with vehicle controls and was similar to that induced by thrombin (1 U/mL) (Fig 3). Uncoated beads or beads coated with an irrelevant antibody, K16/16, demonstrated minimal binding to EC. The concomitant surface expression of P-selectin and release of high-molecular-weight vWF provide further evidence that the LTs are stimulating exocytosis of the Weibel-Palade body pools rather than promoting the constitutive release of vWF from EC.

View larger version (169K): [in this window] [in a new window]   Figure 3. Photomicrographs of surface expression of P-selectin in response to peptido-LTs or thrombin. Human umbilical vein EC cultured on fibronectin-coated glass coverslips were incubated for 20 minutes with anti–P-selectin mAb-coated beads and control buffer (A), 50 nmol/L LTC4 (B), 500 nmol/L LTD4 (C), or 1 U/mL thrombin (D). The cells were then gently washed to remove unbound beads, visualized by a Nikon Diaphot microscope, and photographed. Shown are representative fields chosen from five experiments performed in duplicate. The mean numbers of beads per cell (±SD) from this representative experiment are control=4.5±1.4, LTC4=16.8±2.6, LTD4=19.9±2.5, and thrombin=22.9±3.1.

LTD₄-Stimulated vWF Secretion Is Receptor Mediated
To determine whether LT-stimulated vWF secretion was receptor mediated, EC were exposed for 20 minutes to 50 nmol/L LTD₄ or 1 U/mL thrombin in the presence of increasing concentrations of SKF 104353, an LTD₄ receptor-level antagonist. SKF 104353 inhibited LTD₄-mediated vWF secretion in a dose-dependent manner but had no effect on secretion in response to thrombin within the concentration range tested (Fig 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. Plot showing SKF 104353 inhibits LTD4-mediated vWF secretion in a dose-dependent manner. Cultured human umbilical vein EC were preincubated with SKF 104353 (50 nmol/L, 500 nmol/L, 5 µmol/L, and 10 µmol/L) for 15 minutes at 37°C and then treated with 1 U/mL thrombin (open circles) or 50 nmol/L LTD4 (closed squares) for 30 minutes at 37°C in the continued presence of inhibitor. Supernatants were then removed, and vWF was quantified by ELISA. Results are the mean±SEM of four separate experiments performed in triplicate and are expressed relative to maximal agonist-induced vWF secretion. (Mean maximum vWF concentrations were control=9.2±1.2 [ng/mL±SEM], thrombin=87.1±13.5, and LTD4=82.2±8.5.) *P<.001 vs no inhibitor.

Minimal [³H]LTC₄ Is Converted to LTD₄ After Incubation With Human Umbilical Vein EC
Previous reports have documented the -glutamyl transpeptidase–mediated conversion of exogenous LTC₄ to LTD₄ and LTE₄ in several tissues, including vascular EC.³⁵ To examine the extent of LTC₄ metabolism during the course of vWF secretion, confluent monolayers of human umbilical vein EC were incubated with [³H]LTC₄ for up to 20 minutes. After methanol extraction, radioactive material from conditioned media was analyzed by C₁₈ reverse-phase HPLC, and [³H]peptido-LTs were identified by comparison of their retention times with those of synthetic standards. As shown in Table 2, <7% of LTC₄ was converted to LTD₄ within the time course examined, suggesting that the action of LTC₄ was not due to conversion to LTD₄ by human umbilical vein EC.

View this table: [in this window] [in a new window]   Table 2. Time Course of [14,15-3H]LTC4/LTC4 Conversion to LTD4 with EC

LT-Induced vWF Secretion Requires Only Oscillations Rather Than Sustained Peaks of [Ca²⁺]_i
We next examined LTC₄, LTD₄, and thrombin for their effects on [Ca²⁺]_i in individual EC at concentrations that produce maximal vWF secretion. Similar to thrombin³⁴ and histamine,³⁶ LTs produced heterogeneous responses in individual ECs but with significantly different patterns (Fig 5A). Thrombin (1 U/mL) produced a prolonged peak of [Ca²⁺]_i in every cell examined, and LTD₄ (300 nmol/L) produced chiefly a prolonged peak of [Ca²⁺]_i followed by asynchronous oscillations, whereas LTC₄ (30 nm) produced mainly an oscillatory pattern, with only rare cells demonstrating a prolonged peak of [Ca²⁺]_i (Fig 5B). Removal of external Ca²⁺ by washing the cells with Ca²⁺-free medium before stimulation with agonist did not affect the pattern of [Ca²⁺]_i responses or affect LT-induced vWF secretion. In contrast, preincubation of the cells with BAPTA-AM (25 µmol/L), an intracellular Ca²⁺ chelator, eliminated the [Ca²⁺]_i transients induced by LTC₄ or LTD₄ and decreased LT-induced vWF secretion by >75% (data not shown). Thus, although prolonged elevation of [Ca²⁺]_i is required for thrombin-induced vWF secretion,³⁴ a comparable degree of exocytosis in response to LTC₄ is achieved under conditions for which only an oscillatory pattern of [Ca²⁺]_i is produced.

View larger version (18K): [in this window] [in a new window]   Figure 5. A, Tracings of [Ca2+]i transients in response to the peptido-LTs and thrombin. [Ca2+]i transients were recorded from individual cells in fields of adherent EC as described in Methods. For clarity, only three of six to 14 representative [Ca2+]i transients from one of 10 fields are displayed. B, Bar graph showing tabulation of [Ca2+]i transients in individual EC in response to agonists. Between 150 and 350 individual cells were analyzed under each condition. Oscillations of [Ca2+]i were defined as rapid elevations lasting <80 seconds and typically followed by one or more similar transients; prolonged peaks were defined as rapid increases in [Ca2+]i transients, which remain above basal levels for >80 seconds. Peak responses may return to basal levels, slowly decline (plateau), or assume an oscillatory behavior. The pattern of [Ca2+]i in response to each LT was significantly different from that observed in response to thrombin (P<.01).

PKC Is Not a Primary Mediator in LTC₄- or LTD₄-Induced vWF Secretion
Activation of PKC has been found to be the primary determinant of secretion in several cell types,³⁷ and phorbol esters stimulate vWF secretion in human umbilical vein EC.^{13 38} The observation that peptido-LTs stimulate vWF secretion in conjunction with submaximal calcium responses suggested that PKC activation might play a synergistic role. Pharmacological inhibitors of PKC were therefore examined for their effects on vWF secretion in response to peptido-LTs, thrombin, and PMA. EC were preincubated for 15 minutes at 37°C with staurosporine (30 nmol/L), sphingosine (10 µmol/L), or H-7 (100 µmol/L) and then were treated with PMA, thrombin, LTC₄, or LTD₄ for 30 minutes at 37°C in the continued presence of the inhibitor. Staurosporine almost completely inhibited PMA-induced vWF secretion (4% to 16% of maximal secretion) as previously described,^{39 40} whereas LT-induced secretion was inhibited to a much smaller degree. Sphingosine was modestly inhibitory against all agonists tested. In contrast, H-7 treatment yielded enhanced vWF secretion in response to thrombin (as previously reported⁴⁰ ) as well as to each of the peptido-LTs examined (Fig 6).

Prostacyclin and PAF Do Not Mediate Peptido-LT–Induced vWF Secretion
LTC₄ and LTD₄ induce the formation of prostacyclin and PAF in EC.^{31 32} We thus tested the hypothesis that LT-induced vWF secretion was mediated through these phospholipid-derived mediators. Pretreatment of EC for 15 minutes at 37°C with 10 µmol/L indomethacin, a cyclooxygenase inhibitor, had no significant effect on LT-induced vWF secretion (Table 3). Similarly, pretreatment with 5 µmol/L WEB 2086, a PAF receptor antagonist,²⁰ did not inhibit LT-induced vWF secretion (data not shown). Thus, LT-stimulated exocytosis is not dependent on either prostacyclin or PAF production.

View this table: [in this window] [in a new window]   Table 3. Effects of Indomethacin on Thrombin and LT-stimulated vWF Secretion

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Through the secretion of vWF and the expression of P-selectin, the process of regulated exocytosis in EC has been proposed to play an important role in integrating the early phases of hemostatic and inflammatory responses. In the present report, we demonstrated that the peptido-LTs LTC₄ and LTD₄ are potent secretagogues of regulated exocytosis in EC. Previous reports that LTC₄ and LTD₄ stimulate EC production of prostacyclin,^{31 41} a potent antiplatelet aggregatory agent, suggested that these compounds would exert a predominantly "antithrombotic" effect at the intravascular sites at which they are generated. The observation that the peptido-LTs are also potent stimuli for the release of high-molecular-weight vWF from Weibel-Palade bodies suggests a previously unappreciated action that may promote a "prothrombotic" state in inflammatory conditions.

McIntyre et al³² observed that LTC₄ and LTD₄ stimulate PAF production in human umbilical vein EC and suggested that this activity was responsible for enhanced leukocyte adhesion observed in response to these agonists. More recently, Hashemi et al⁴⁴ demonstrated that PAF induces vWF release from cultured human umbilical vein EC. We thus postulated that PAF generated in response to LTs might act in an autocrine fashion to stimulate exocytosis. However, treatment of EC with a PAF receptor antagonist, WEB 2086, did not inhibit vWF secretion by LTs. We also hypothesized that prostacyclin produced by EC in response to LT stimulation may play a role in vWF secretion. Indomethacin, which blocks prostacyclin synthesis by reversibly inhibiting cyclooxygenase, did not significantly inhibit LT-stimulated vWF release. Therefore, exocytosis in response to LTs appears to be an independent action that does not result from increased PAF or prostacyclin production.

The role of [Ca²⁺]_i in thrombin-induced vWF secretion has been extensively characterized.^{34 38 39 40} We therefore compared the nature of the [Ca²⁺]_i transients associated with LT- and thrombin-stimulated vWF secretion. Most striking was the demonstration that concentrations of LTC₄ that induced significant vWF secretion produced only asynchronous oscillations in [Ca²⁺]_i in EC. In sharp contrast, significant vWF secretion in response to thrombin is observed only at concentrations that produce prolonged peaks of [Ca²⁺]_i; lower concentrations of thrombin, which produce [Ca²⁺]_i oscillations but not prolonged peaks, stimulate minimal vWF secretion.³⁴

The pharmacological PKC activator PMA induces vWF secretion by EC,^{13 38} and PKC is known to play a dominant role in exocytosis in other cell types.^{45 46 47 48} We therefore examined pharmacological inhibitors of PKC to determine their effect on peptido-LT–induced vWF secretion. The partial inhibition of both PMA- and peptido-LT–stimulated vWF secretion in the presence of sphingosine suggests that PKC may play a role in peptido-LT signaling. However, sphingosine also inhibits calmodulin-dependent enzymes in vitro and in GH3 cells,⁴⁹ and we have previously shown that calmodulin-mediated pathways play a dominant role in thrombin-stimulated vWF secretion in human umbilical vein EC.³⁹ Moreover, we found that staurosporine was far more inhibitory of PMA-induced secretion than of that promoted by LTC₄ or LTD₄. Although staurosporine is also known to inhibit other classes of protein kinases, the present results (Fig 6) argue that peptido-LT– and PMA-mediated secretion proceed through different pathways. The enhancement of vWF secretion in the presence of H-7 cannot be readily explained. Whether this effect is related to the known inhibitory actions of H-7 on cAMP- and cGMP-dependent kinases^{50 51} remains to be determined. Nevertheless, the results obtained with pharmacological agents suggest that PKC is not primarily involved in exocytosis in response to the peptido-LTs or thrombin and that PKC activation does not account for the differences in the requisite [Ca²⁺]_i profiles in response to each of these agonists.

LTC₄ and LTD₄ stimulate leukocyte binding to cultured human umbilical vein EC.³² Because peptido-LTs do not directly activate human neutrophils,^{32 42 43} their ability to promote neutrophil adherence to EC is believed to result from their effects on vascular endothelium. The present results (Figs 1 through 6) provide direct evidence that LTC₄ and LTD₄ stimulate the mobilization of Weibel-Palade bodies, resulting in vWF release and rapid upregulation of cell surface P-selectin. The concomitant increase in the local concentrations of high-molecular-weight vWF and surface P-selectin would be predicted to promote platelet adhesion to exposed subendothelium (and possibly to intact EC monolayers) while promoting leukocyte adhesion to EC. As the peptido-LTs are generated in large quantities by the sequential action of leukocytes and platelets, they may participate in a "positive feedback loop" that results in the further interaction of these formed elements with vascular endothelium, thus upregulating the inflammatory process. The clinical implications of these effects of LTs on EC remain to be fully elucidated. Nevertheless, increased production of peptido-LTs has been demonstrated during coronary artery balloon angioplasty³⁰ and may contribute to the development of abrupt vessel closure or later restenosis. On the basis of the present results, we propose that antagonists of peptido-LT biosynthesis or receptor function may be clinically useful in a variety of prothrombotic conditions.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular calcium concentration EC = endothelial cells H-7 = 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine-dihydrochloride HBSS = Hanks' balanced salt solution HPLC = high-performance liquid chromatography LT = leukotriene LX = lipoxin M199 = Medium 199 mAb = monoclonal antibody PAF = platelet-activating factor PBS = phosphate-buffered saline PG = prostaglandin PKC = protein kinase C PMSF = phenylmethylsulfonyl fluoride SDS = sodium dodecyl sulfate vWF = von Willebrand factor

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants HL-45629 (Dr Ewenstein), HL-15157 (Drs Ewenstein and Golan), HL-32854 (Dr Golan), and GM-38765 (Dr Serhan); Dr Serhan is an Established Investigator of the American Heart Association. We thank Patrick Yacono and Seth Narins for expert technical assistance, Kay Case and Andrew Ritchie for assistance with cell culture, and Fran Ross for assistance in the preparation of the manuscript.

Received April 10, 1995; revision received July 17, 1995; accepted July 23, 1995.

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