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Cell-cell interaction between platelets and IL-1β-stimulated vascular smooth muscle cells in synthesis of thromboxane A2
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91 © Pearson Professional Ltd 1997
Cell-cell interaction b e t w e e n platelets and IL.l .stimulated vascular smooth muscle cells in synthesis of t h r o m b o x a n e A2 M. Zou, C. Anges Institut Biomedical des Cordeliers and INSERM, 15, rue de I'@cole de M@decine, 75005, Paris, France
Summary Transcellular biosynthesis of thromboxane (Tx) A2 between vascular smooth muscle cells (SMC) and platelets has been investigated by using 14C-arachidonic acid (AA) radiolabeled rat SMC (or platelets) and the fate of the label in phospholipids and eicosanoid fractions was studied using radioimmunoassay (RIA) and thin-layer chromatography (TLC). Stimulation of SMC with interleukin-ll3 (IL-113) resulted in production of cyclooxygenase metabolites (e.g. 6-keto-PGFl~, PGE2, PGF2~, PGD2), 15-, 11-, 5-HETE, and free AA, with a coincident decline of phosphatidylcholine (PC) in SMC. IL-113did not induce TXB2 production, a stable metabolite of TXA2 measured by TLC and radioimmunoassay, either in human platelets from 0.01-100 U/ml for 1 h or in SMC for 24 h. However, human platelets converted exogenous PGH2 to TXA2 despite cyclooxygenase inhibition or PGH2 receptor blockade. Furthermore, TXB2 was produced in large quantities during co-incubation of IL-113-stimulated SMC with human platelets for 30 min in concert with a significant decrease of 6-keto-PGFl~ and eicosanoids (PGE2, PGF2~ and PGD2) compared with control (P < 0.01). Pretreatment of SMC with cycloheximide and actinomycin not only inhibited IL-1 ~induced eicosanoid synthesis and phospholipid breakdown but also diminished TXB2 production when co-incubated with platelets. These data suggest that a cell-cell interaction, i.e. platelet utilizing SMC-derived endoperoxides for its TXA2 production, might cause an excess thromboxane A2 synthesis. INTRODUCTION
The interaction of activated platelets with the arterial wall, culminating in eicosanoid production, has been addressed by several investigators utilizing a variety of approaches. ~-4 The common precursors for both thromboxane (TX) A2 and prostaglandin (PC) I2 are endoperoxides (PGG2, PGH2) generated from arachidonic acid (AA) by cyclooxygenase present in both platelets and v a s c u l a r tissues, 4'5 The ability of a cell to obtain endoperoxide substrates formed by an adjacent cell of a different type provides the potential for a transcellular regulatory scheme. Several laboratories have demonstrated the ability of platelets to transfer endoperoxide intermediates to vessel wall segments, 2,3,6 cultured endothelial cells z,8 or lymphocytes9'~° for PGI2 synthesis. There
is additional evidence that unidirectional transfer of PG endoperoxides occurs, i.e., cultured human endothelial cells are not capable of providing PGH2 to platelets. 7 It is not known whether vascular tissue can supply PGH2 to platelets. Interleukin-1 (IL-I) has potent amplifying effects on the inflammation responses and may modulate the atherogenic responses of vascular tissues to injury. However, precise mechanisms by which this occurs are not known. 10-12The present investigation was designed to demonstrate that: (a) IL-I[~ activates AA metabolism in vascular smooth muscle cells (SMC), and (b) that platelets can utilize endoperoxides from IL-l~-stimulated vascular SMC for its biosynthesis of TXA2. MATERIALS AND METHODS
Received 26 September 1995 Accepted 27 November 1996 Correspondence to: M. Zou, Institut Biomedical des Cordeliers and INSERM, 15 rue del'@cole de M@decine, 75005, Paris, France. Fax. 49-07531-883099
Reagents
Human recombinant rIL-1[3-specific activity 10 7U/mg of protein), cycloheximide, actinomycin D, acetylsalicytic 85
86
Zou and Anges
acid (ASA) and thrombin were obtained from Sigma (Saint Quentin Fallavier, France). Labeled compound 14CAA (specific activity 55.7 mCi/mmol) was from Amersham UK. PG endoperoxide PGH2 (in hexane thylacetate 10:1) and thromboxane B2 were from Cayman Chemical (Ann Arbor, MI, USA), 13-azaprostanoic acid (13-APA) from Biomol Research Laboratories (Philadelphia, PA, USA), and culture medium (Ham F 10) and additives (antibiotics, glutamine and trypsin) were purchased from Gibco (Paisley UIO Rat SMC culture and IL-lp stimulation
Rat vascular SMC were isolated from the rat abdominal aorta by techniques previously described. 2 During the first weeks, the cells were cultured at 37°C, 5% C Q in HAM F-10 medium supplemented with 20% fetal calf serum. When a monolayer was reached, the cells were removed by trypsin-EDTA (0.05-0.02%) and cultured in a 25 cm2 flask in Ham F10 medium supplemented with 10% calf serum and 1% penicillin-streptomycin. At confluence, the cells were trypsinized and passaged. Before experiments, the cell lines (10-25 passages) were plated in a fresh culture medium and allowed to grow for 24 h. The cell viability (> 990/0) was assessed by the Trypan blue dye-exclusion test. The cells were incubated with 2 mlVI ~4C-AA for 18 h, the medium was harvested, and the cells were washed and incubated in fresh serumfree medium. As a control, 14C-AA was incubated under identical conditions in the same medium in the absence of cells to determine whether non-enzymatic metabolism occurred during the incubation period. Under these conditions, greater than 85% of initial radiolabel was incorporated into the ceils. IL-I~ (5 U/ml), which is optimal for stimulation of PGI2 synthesis, was added and the cells were further incubated at 37°C for 24 h in an atmosphere of 5% CO2 for studies of eicosanoid synthesis from endogenous AA. Incubation were also conducted with IL-1B in the presence or absence of the protein synthesis inhibitors actinomycin D or cycloheximide. These substances were added to evaluate whether the synthesis of new proteins by SMC was required for the increased prostaglandin biosynthesis in response to IL-1[3. Next, SMC were pretreated for 30 rain with 200 mM acetylsalicytic acid (ASA). This was carried out to irreversibly inactivate existing fatty acid cyclooxygenase. The cells were then washed and treated with IL-I~ or control media. If IL-I~ stimulates prostaglandin production in SMC after pretreatment with ASA, it would have to induce new fatty acid cyclooxygenase activity. For the test of co-incubation, the SMC that had been stimulated by IL-113 for 2 4 h were further incubated with human platelets at a ratio of 10 platelets to one SMC for 30 min.
Washed human platelet suspension preparation and 14C-AA metabolism in platelets
Human venous blood anticoagulated with ACD (trisodium citrate 2.5%, citric acid 1.5%, dextrose (+) 2.0%) (1/9, v/v) was drawn from healthy volunteers who had not ingested aspirin for at least 2 weeks before donation. Blood was then centrifuged at 100 x gfor 20 man to obtain platelet-rich plasma (PRP). The PRP was then adjusted to pH 6.5 with additional trisodium citrate and citric acid, and was centrifuged at 1500 x gfor 10 min. The resulting platelet pellets were then resuspended in Tyrode buffer containing (in mM): 0.412 Na2HPO4, 137 NaC1, 2 MgC12 6 H20, 2.6 KC1, 11.9 NaI-ICO3, 5.6 glucose D(+), 5 N-2hydroxyethyliperazine-/V-2-ethanesulfonic acid (HEPES) and 150 mM trisodium citrate as well as 1 mM EDTA plus 0.01% (w/v) albumin and incubated for 3 h at 37°C. After incubation, the platelets were centrifuged at 1500 x g for 10 min and platelet pellets were washed with the same buffer plus 0.05% albumin. Above 85% of radioactivity was incorporated into cells determined by liquid scintillation counting of the combined fraction. The platelets were finally suspended in phosphate-buffered saline (PBS) for release. Platelets were counted in microscope and were adjusted to 3 x 108 platelets/ml. Extraction and analysis of 14C-AA metabolites
After incubation, the cells were centrifuged at 1500 x g for 20 rain, and the supernatants were collected and acidified at pH 2.5-3.5 by addition of hydrochloric acid (1 N). The supernatants were extracted twice with 3 volumes of ethyl acetate. The combined organic phases were evaporated to dryness under a stream of nitrogen and the residues reconstituted in 200ml ethyl acetate were spotted in the TLC silica G plate (Whatman). The plates were developed in the organic phase of the solvent system: ethyl acetate/isooctane/acetic acid/water (220:100:40:200) for 75 rain and were read by a Berthold TLC scanner. Analysis of membrane phospholipid composition
To analyse membrane phospholipid composition, the adherent cells resuking from culture were scraped, harvested and resuspended in a mixture of chloroform/ methanol (1:2, v/v). Lipids were extracted as previously described 2 and spotted on TLC silica gel G plates (Whatman). Separation of different phospholipids was carried out by migration in a solvent system of chloroform/ methanol/acetic acid/water (195:129:3:9) for 80 min. The radioactive spots were read by a Berthold TLC scanner. Radioimmunoassay of thromboxane B2
The supernatants (1 ml) acidified to pH 3.5 were extracted
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 55(2), 85-91
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Transcellular thromboxane A2 biosynthesis
twice with 3 volumes of ethyl acetate. The organic phases were pooled and evaporated to dryness under nitrogen. The residue was then dissolved in PBS delatin buffer and appropriate aliquots were used for RIA of TXB2 as described./3 TXB2 levels were evaluated in duplicated serial dilutions of the samples. The results were expressed in ng/ml.
87
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RESULTS Effect of I1-1~ on AA metabolism in rat SMC
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Rat vascular SMC prelabeled with 14C-AAwere incubated with 5 U/ml IL-I[3 or thrombin (1 U/ml) for 30 min to 24 h. As expected, thrombin caused a time-dependent increase of eicosanoids in the stimulated cells. The addition of IL-I[~ to the cultured SMC stimulated the production of PGI2 (measured as 6-keto-PGF~ its stable degradation product in vitro), which accounted for 25% by the appearance of radioactivity in the supernatant of IL-1 [3 stimulated cells after 24 h incubation (Fig. 1). A similar activation was observed for the other eicosanoJds and AA release (Figs 2 & 3). Kinetic studies revealed that there were two periods of stimulation in IL-l[~-treated cells. An obvious, but transient increase of 6-keto-PGF~ and PGs (PGF2~, PGE2, PGD2) was observed at the first 2 h of stimulation. Following a lag period from 2 h to 8 h, there was a much more significant increase of 6-keto-PCF~ and PGs in the stimulated cells (Figs 1, 2 & 3). In contrast to IL-I[3, thrombin led to a regular and continued increase in 6-keto-PGF~, PGs and AA in ~4C-AA prelabeled SMC with incubation time (P < 0.01). In addition, preincubation of SMC with cycloheximJde (10 mM), or actinomycin D (10 mM) inhibited IL- 1[Mnduced 6-keto-PGF~ production (87% and 91% inhibition, respectively) (Table 1), but without any significant inhibitory action on thrombin-
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Fig. 2 The time course of prostagtandin endoperoxide (PG) formation upon stimulation by IL-1 [3 and thrombin. Prelabeled rat vascular smooth muscle cells (7 x 106 cells/7 ml) were incubated with 5 U/ml and 1 U/ml IL-11~. Date is presented as the mean _+SEM dpm of five separate assays.
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Fig. 3 The time course of arachidonic acid (AA) release upon stimulation by IL-113 and thrombin. Prelabled rat vascular smooth muscle cells (7 x 106 cells/7 m[) were incubated with medium or 5 U/ml IL-113 or 1 U thrombin. Date is presented as the mean +- SEM dpm of five separate experiments.
Table 1 Effects of aspirin, cycloheximide and actinomycin D on ILl-induced eicosanoid formation in adherent rat aortic smooth muscle cells.
Sample
6-keto-PGFl~ (dmp/106cells)
PGF~, PGE2, PGD (dpm/106cells)
Control
622.1 ~ 94.6
1395.3 + 267.4
Aspirin pretreatment 10 -5 M 10-4 M
589.4 _+ 137.5 569.5 + 71.3
1418.5 _+ 117.6 1141,6 _+_+57.3
Cycloheximide
83.5 _+ 17.6
185.1 + 41.3
Actinomycin D
57.9 _+ 17,8
127.5 _+51.4
t i m e (h)
Fig. 1 The time course of 6-keto-PGFl,, formation upon stimulation by IL-1 J3and thrombin. Prelabeled rat vascular smooth muscle cells (7 x 106 cells/7 ml) were incubated with medium or 5 U/ml IL-I~ or 1 U/ml thrombin. Date is presented as the mean + SEM dpm of five separate experiments.
© Pearson Professional Ltd 1997
, 2
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o 1.20-
, 1
Adherent smooth muscle cells (7 x 10 Gcells) were incubated with IL-113 (5 U/ml) after preincubation with aspirin, cycloheximide (10 -s M) and actinomycin (104 M) for 1 h. The whole incubation mixture was extracted with ethyl acetate and analyzed by TLC as described in the Materials and methods. Results are expressed as means dpm/106 cells _+SEM from five experiments.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
88
Zou a n d A n g e s
Table 2 IL-I~ and thrombin-induced 14C arachidonic acid incorporation and effects of aspirin, cycloheximide (CH), and actinomycin D (AM) on IL-l-induced 14C arachidonic acid incorporation in lipids of rat vascular smooth muscle cells. % of total 14Carachidonic acid incorporated Sample
PC
PI
PE
PS
AA
Control IL-1 IL-1 + aspirin IL-1 + C H IL-1 + A M Thrombin
17.6_+4.6 11.3_+1.8 14.1 _+2.7 17.1 _+3.9 18.3_+5.1 15.3 _+5.1
21.8_+5.7 19.2_+5.1 22.5 _+4.8 23.5_+6.7 22.7_+5.2 14.4 _+3.2**
20.9_+ 11.1 20.7_+4.8 20.1 _+3.9 21.3_+4.4 19.5_+5.7 38.1 _+6.5**
9.1 _ + 3 . 1 8.0_+2.6 9.4 _+3.7 9.7_+5.7 9.4_+3.1 4.7 _+0.8
9.9_+4.5 20.7_+8.7 26.3 _+5.7 11.4_+4.1 14.3_+4.5 26.7 _+9.3**
Adherent vascular smooth muscle cells (5 x 106 cells) were incubated with buffer (control) or with IL-1 (5 U/ml) for 24 h after preincubation with aspirin (10 -6 M), cycloheximide (10 -6 M) and actinomycin D (10 -6 M) for 1 h. The cell layers were extracted with chloroform-methanol as described in the Materials and methods. Results are expressed as mean _+SEM from five experiments. PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol, PE, phosphatidylethanolamine; AA, arachidonic acid. * represents the P value compared with control * P < 0.05; P < 0.01; + represents the Pvalue compared with IL-1 + P < 0.05, ++ P < 0.01.
induced prostanoid production in SMC (data not shown). Finally, I1-113 significantly accelerated the return of prostaglandin production following elimination of active fatty cyclooxygenase by ASA (Table 1), which suggested that prostaglandin production stimulated by IL-113 probably involved induction of cyclooxygenase synthesis.
with II- 113 (0.01-100 U/ml) did not induce an obvious platelet activation. TXB2, the major metabolite of platelet AA metabolism in platelets and the specific radioactivity of ~4C-arachidonate in platelet phospholipid classes remained constant after incubation for 1 h (data not shown).
Effects of IL-I~ on phospholipid metabolism in SMC
Platelet TXA~ production from exogenously added PGH2
Rat vascular SMC cells were labeled for 18 h with 14C-AA as described in the Materials and methods. The specific radioactivity of 14C arachidonate in the major phospholipid classes is shown in Table 2. By using this labelling strategy, experiments were performed to determine which arachidonate-containing phosphotipid classes were broken down during cell activation. After the incubation with 5 U/ml IL- 113for 24 h, the specific radioactivity of I4C arachidonate in phosphatidylcholine (PC) was decreased by 35.8% as compared with control (P< 0.01) along with an obvious increase of 14C arachidonate in the supernatants (P < 0.01). The specific activity of 14C arachidonate in phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylserine (PS) remained constant in the IL-113 treated cells. Thrombin did induce a decrease of x4C arachidonate in PI (33.9%) and PS (48.4%) respectively (P < 0.01) along with a significant increase of radioactivity in PE (P < 0.01). Cycloheximide(10-hM) and actinomycin D (10-~M), did inhibit IL-l[Mnduced phosphatidylcholine breakdown (Table 2), but had no effect on thrombin-induced phospholipid breakdown in SMC (data not shown).
The ability of human platelets to utilize exogenous AA intermediates was initially examined by incubating washed platelet suspensions with exogenously added PGH2. Washed human platelets suspensions (5 x 108) or buffer blanks or washed aspirin-pretreated platelets were incubated with PGH2 in a shaking 37°C water bath. The supernatants were collected for RIA. The production of TXA2, measured by RIA of its stable breakdown product, TXB2, was increased by the addition of PGH2. Platelets converted PGH2 to TXB2 in a dose-dependent manner (Fig. 4). The inhibition of platelet cyclooxygenase by preincubation with aspirin caused no significant change in TXB2 production from PGH2 (Fig. 4). 13-APA, the endoperoxide receptor antagonist, did not alter TXB2 production after the addition of exogenous PGH2, which suggested that endoperoxide receptor stimulation might not be involved in the platelet TXA2 synthesis.
Effect of I1-1J3on 14C-AA prelabeled human platelets
The treatment of 14C-AA prelabeled human platelets
Transcellular synthesis of TXA2 in co-culture of SMC cells and platelets
14C-arachidonate-prelabeled SMC, pretreated with I1-113 (5 U/ml) for 24 h, were incubated for 30 min with washed platelets at a ratio of 10 platelets to one SMC, which was found to induce a maximal transfer of endoperoxides in cells in our previous experiments. As shown in Fig. 5, the
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
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Transcellular thromboxane A~ biosynthesis
Table 3 Thin layer chromatography of ~4C-arachidonic acid (AA)
70 T platelets 56
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buffer blank 1
89
metabolites released by Interleukin ll~-stimulated rat vascular smooth muscle cells (SMC) with or without human platelets.
Eicosanoid
SMC (dpm/lO6cells)
SMC + platelets (dpm/lO6cells)
6-keto-PGF~ PGE2 PGD PGF2~ AA
622.1 304.6 517.1 574.8 652.3
423.3 224.8 357.1 392.7 765.7
_+94.6 _+78.6 __.72.9 + 171.8 + 103.3
+_69.6** +- 46.6* + 55.5* _+97.1" _+ 147.8
1'0
Fig. 4 Platelet production of thromboxane B2 from exogenously added PGH2, Washed human platelets (5 x 107/ml) or aspirinpretreated platelets were incubated for 3 min with PGH 2. Control represents incubation without SMC. Data is presented as the mean _+SEM of replicate determinations representing three experiments,
Adherent smooth muscle cells (7 x 106 cells) were incubated with or without platelets (7 x 107 cells) for 30 min after preincubation with IL-1 ~ (5 U/ml) for 24 h. The whole incubation mixture was extracted with ethyl acetate and analyzed as described in the Materials and methods. Results were expressed as dpm/106 cells. Values are the mean + SEM present in the supernatants of five separate experiments. *P < 0.05 ** P < 0.01 (paired Student's t-test.
Table 4 TXB2 production in co-culture of platelets (PLA) with
o
o
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PGD2
F2~
X
E
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Fig. 5 Radiochromatogram of metabolites derived from ~4C arachidonic acid generated in the IL-1 stimulated rat vascular smooth muscle cells and during co-incubation of IL-1 treated vascular smooth muscle cells and platelets. Extraction and separation were described in the Materials and methods. The positions of authentic prostaglandin (PG), thromboxane B2, and monohydroxylated compounds are as described.
Sample
TXB2 (pg/ml)
PLA+IL-1 SMC+IL-1 PLA+SMC SMC+IL-1 +PLA SMC+iL-1 +CH SMC+IL-I+AM ASA pretreated SMC+IL-I+PLA CH pretreated SMC+IL-I+PLA AM pretreated SMC+IL-I+PLA ASA pretreatment PLA+SMC+IL-1
26.3 +_7.6 Non-detectable Non-detectable 237.5 _+41.6 51.5 + 17.1 46.4 _+ 13.5 225.4 + 48.9 31.5 + 5.3 25.6 -+ 4.9 218.7 +_51.3
Adherent smooth muscle cells (5 x 10 e cells) were incubated with 5 x 107 platelets in the presence of IL-11~ (5 U/ml) with aspirin (10 -5 M), NDGA (10 -5 M), cycloheximide (10-2 M) and actinomycin (10 -5 M) for 1 h. The incubation mixture (1 ml) was extracted and analyzed by radioimmunoassay as described in the Materials and methods. Results are expressed as means + SEM from four experiments.
SMC cells used alone in these studies did not produce TXB2 in response to IL-I~3. However, an obvious TXB2 peak, which accounted for 10% of the radioactivity in the supernatants, was found after 30 min co-incubation with platelets (Fig. 5). Together with an increase of TXB2, there was an obvious coincident decrease of 6-keto-PGFl~ and PGs (PGE2, PGF2~ and PGD2) in the mixture after coincubation as compared with control (P < 0.01); however, free AA, which might be used by platelets for its TXA~ synthesis, remained constant after co-incubation (P> 0.05) (Table 3). In addition, the pretreatment of ~4C-arachidonatelabeled SMC with cycloheximide (10mM) and actinomycin D (10mM) before IL-1 stimulation not only inhibited eicosanoid production of SMC in response to IL-1B but also diminished TXB2 production when co© Pearson Professional Ltd 1997
Interleukin 1 (IL-1)-stimulated rat vascular smooth muscle cells (SMC) in the presence or absence of cycloheximide (CH), actinomycin D (AM), and aspirin (AP).
incubated with platelets (Table 4). However, a similar amount of TXB2 was found when aspirin-pretreated platelets (10mM, 15min) were incubated with IL-1stimulated SMC. DISCUSSION
Prostaglandin production by vascular cells plays an important role in the development and progression of atherosclerosis and arterial hypertension. ~°,11 The eicosanoids appear to cause a local release of cytokines, including IL-I~ from local tissues. 1°,11 These cytoldnes cause a significant release of prostaglandins from vascular cells? 4-~6 In our study, we have demonstrated for the first time that IL-I~3, a major proinflammatory agent, effectively induced eicosanoid production in SMC and
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
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Zou and Anges
endoperoxide transfer to platelets and that platelets could utilize endoperoxides originated from SMC for synthesis of TXA> Different mechanisms might be involved in the activation of eicosanoid metabolism by IL-I[3 and thrombin. Although IL-113, like thrombin, induced an immediate increase of cyclooxygenase metabolites (including 6-ketoPGFI~, PGE2, PGF2~ and PGD2), lipoxygenase metabolite HETEs, and free AA from 30 rain to 2 h, there was a lag period between 2 and 8 h in IL-I~ induced eicosanoid formation and 14C-arachidonate degradation in phosphatidylcholine. Actinomycin D and cycloheximide, which have been reported to block transcription and translation, respectively, selectively inhibited the prolonged increase of IL-l~-induced eicosanoid formation and phosphatidylcholine breakdown, but they did not inhibit thrombin-induced eicosanoid formation. These data suggested that the synthesis of new enzyme proteins were required for increased prostaglandin production in response to IL-1[5. This is consistent with the fact that IL-I[3 caused a rapid recovery from ASA pretreatment, which irreversibly inhibits existing cyclooxygenase. Therefore, our study suggested that prostaglandin production by SMC in response to IL-I[5 stimulation was likely via synthesis of cyclooxygenase protein. Recently, investigators have described two forms of cyclooxygenase (COX) in mammalian cells including cultured endothelial cells: COX-l, which is present constitutively, and COX-2, which is inducible. The relative distribution of these two forms of cyclooxygenase has not been clearly delineated in cultured SMC cells, lz~3,~z Based on our results, it is likely that IL-lJ] stimulated prostaglandin production primarily via induction of COX-2. Nevertheless, the possibility existed that there might be actions on isomerase activities in the conversion of prostaglandin endoperoxides to prostaglandins. Co-incubation of IL-l[3-stimulated vascular SMC with washed platelets induced obvious TXA2 production in which a cell-cell interaction might be involved. In agreement with others. 2,18,19 we found that IL-l[3-stimulated SMC did not synthetise TXA2 because of a lack of TXA2 synthetase. Therefore, TXB2 in the supernatants of coculture could be only originated from human platelets. Human platelets dose-dependently converted exogenous PGH2 to TXB2, and the inhibition of platelet cyclooxygenase by preincubation with aspirin caused no significant change in TXB2 production from PGH2. Mayaux et al6 have shown that aspirin-treated platelets (unable to generate their own PGH2) can use PGH2 generated by the bovine coronary artery for TXA2 synthesis, and Bourgain et al 8 have demonstrated a transfer of endoperoxides from vessel wall to platelets for TXA2 production. In this study, our data showed that together with TXBz production, there was a significant decrease of eicosanoids
(PGE2, PGF2~, PGD2 and PGA) in the co-culture supernatants, whereas the free AA, which could also be used by platelets, was constant after co-incubation. The pretreatmerit of SMC with cycloheximide, and actinomycin D, which blocked IL-l~-stimulated eicosanoid synthesis in SMC, abolished IL-l[3-induced TXB2 synthesis in the co-culture; Aspirin-treated platelets (unable to produce endoperoxides) were able to synthesize TXB2 in the presence of IL-l[5-pretreated CML. These resuks indicate that TXB2 formation is dependent on SMC activation and transfer of SMC-derived endoperoxides to platelets. There is a discrepancy with the report of Schafer. 7 The explanation of this difference is uncertain; however, it may be related to the use of cultured endothelial cells rather than vascular smooth muscle cells and to the ratio of platelets to tested vascular cells that has been reported as an important factor in determining the transcellular AA metabolism. 1 This study provides evidence that platelets are capable of utilizing vascular cell-derived endoperoxide intermediates for further metabolism to TXA2. The transcellular synthesis of TXA2 might be involved in many pathophysiological processes such as inflammaton and atherosclerosis, in which endothelium is damaged and SMC have a potential to interact simultaneously with the blood components.iS In summary, in this study, we have demonstrated for the first time that II-l[t influences vascular tissues metabolism not only through eliciting a direct activation but also through promoting transcellular metabolism of AA betwen different vascular cells. REFERENCES
1. Hechtman D. H., KrollH. M., Gimbrone M. A., SchaferA. I. Platelet interaction with vascular smooth muscle in synthesis of prostacyclin. AmJPhysio11990; 260: H1544-1551. 2. Zou M., Anges C. Cell-cell interaction of macrophages and vascular smooth muscle cells in the synthesis of leukotriene B4. Mediat Inflamm 1994; 3: 297-302. 3. Marcus A.J., Wesksler B.B.,Jafe E. A., BroekmanM.J. Synthesis of prostacyclin from platelet derived endoperoxides by cultured human endothelial cells. J Clin Invest 1980; 66: 979-986. 4. Marcus A.J. Transcellularmetabolism of eicosanoids. Prog Hemost Thromb 1986; 8: 127-142. 5. Maghni K., CarrierJ., Cloutier S., Sirois P. Cell-cell interactions between platelets, macrophages, eosinophils and natural killer cells in thromboxane A2biosynthesis.JLipid Mediat 1993; 6: 321-332. 6. Mayeux P. R., KadowitzP.J., McNamara D. B. Evidencefor a biodirectional prostaglandin endoperoxide shunt between platelets and the bovine coronary artery. Biochim Biophys Acta 1979; 1011: 18-24. 7. SchaferA.J., CrawfordD. D., Gimbrone M. A. Unidirectional transfer of prostaglandin endoperoxide between platelets and endothelial cells. J Clin Invest 1984; ?3:1105-1112. 8. Bourgain R. H., Andries R., Decuyper K., Braquet P. Exchange of cyclooxygenasedependent metabolites between vessel wall and
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
© Pearson Professional Ltd 1997
Transcellular thromboxane A2 biosynthesis
platelets in arterial thrombosis. Thromb Res 1988; B0: 401-408. 9. Wu I52K., Papp A. C., Manner C. E., Hall E. R. Interaction between lymphocytes and platelets in the synthesis of prostacyclin. J Clin Invest 1987; 79: 1601-1606. 10. Pomerantz K., Hajjar D. Signal transduction in atherosclerosis: Integration of cytokines and the eicosanoid network. FASEBJ 1992; 6: 2933-2991. 11. Pomerantz K., Hajjar D. Eicosanoids in regulation of arterial smooth muscle cell phenotype, proliferative capacity, and cholesterol metabolism. Atherosderosis 1992; 9: 713-429. 12. Rzymkiewicz D. M., Dumaine J., Morrison A. K IL-1 beta regulates rat mesangial cyclooxygenase II gene expression by tyrosine phosphorylation. Kidney Int 1995; 47: 1354-1363. 13. Vane J. R., Botting R. M. New insights into the mode of action of anti-inflammatory drugs. Inflamm Res 1995; 44: 1-10. 14. Bolmalaski J. S., Steiner M. R., Simon P. L., Clark M. A. IL-1 increase phospholipase A2 activity, expression of phospholipase A2 activity protein and release of linoleic acid from the murine T helper cell line EL-4. J Immunol 1992; 148:155-160. 15. Geng Y., Blanco F.J., Cornelisson M., Lotz M. regulation of cyclooxygenase-2 expression in normal human articular chondrocytes. J Immuno11995; 15~: 796-801. 16. Corbett J. A., Kwon G., Turk J., McDaniel M. L. IL-I[3 induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of langerhans: activation of cyclooxygenase by nitric oxides. Biochemistry 1993; ]2: 13767-13770.
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17. Tazawa K, Xu X., Wu K K., Wang L. H. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun 1994; 203: 190-199. 18. Pomerantz K., Hajjar D. Eicosanoid in regulation of arterial smooth muscle cell phenoty-pe, proliferative capacity, and cholesterol metabolism. Arteriosclerosis 1989; 9:413-429. 19. Larrue J., Beroux C., Daret D., Bricaud H. Decreased prostacyclin production in cultured smooth muscle cells from atherosclerosis. Biochim Biophys Acta 1988; 710: 257-262. 20. Goppeltstruebe M. Regulation of prostaglandin endoperoxide synthase isozyme expression. Prostaglandins Leukot Essent Fatty Acids 1995; 52: 213-222. 21. Feng L., Xia X., Garcia G. E., Hwang D., Wilson C. Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor, and lipopolysaccharide. 1995; 95: 1669-1675. 22. Gryglewski R.J., Dembinska-Kiec A., Zmuda A., Gryglewski T. Prostacyclin and thromboxane A2 biosynthesis capacities of heart arteries and platelets at various stage of experimental atherosclerosis in rabbits. Atherosderosis 1986; 31: 385-393. 23. Larrue J., Rigaud M., Daret D., Demond J., Durand J., Bicaud H. Prostcyclin production by cultured smooth muscle cells from atherosclerosis rabbit aorta. Nature 1980; a85: 480-482. 24. Ross R. The pathogenesis of atherosclerosis-an update. NEnglJ Med 1986; 314: 488-500.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
Cell-cell interaction b e t w e e n platelets and IL.l .stimulated vascular smooth muscle cells in synthesis of t h r o m b o x a n e A2 M. Zou, C. Anges Institut Biomedical des Cordeliers and INSERM, 15, rue de I'@cole de M@decine, 75005, Paris, France
Summary Transcellular biosynthesis of thromboxane (Tx) A2 between vascular smooth muscle cells (SMC) and platelets has been investigated by using 14C-arachidonic acid (AA) radiolabeled rat SMC (or platelets) and the fate of the label in phospholipids and eicosanoid fractions was studied using radioimmunoassay (RIA) and thin-layer chromatography (TLC). Stimulation of SMC with interleukin-ll3 (IL-113) resulted in production of cyclooxygenase metabolites (e.g. 6-keto-PGFl~, PGE2, PGF2~, PGD2), 15-, 11-, 5-HETE, and free AA, with a coincident decline of phosphatidylcholine (PC) in SMC. IL-113did not induce TXB2 production, a stable metabolite of TXA2 measured by TLC and radioimmunoassay, either in human platelets from 0.01-100 U/ml for 1 h or in SMC for 24 h. However, human platelets converted exogenous PGH2 to TXA2 despite cyclooxygenase inhibition or PGH2 receptor blockade. Furthermore, TXB2 was produced in large quantities during co-incubation of IL-113-stimulated SMC with human platelets for 30 min in concert with a significant decrease of 6-keto-PGFl~ and eicosanoids (PGE2, PGF2~ and PGD2) compared with control (P < 0.01). Pretreatment of SMC with cycloheximide and actinomycin not only inhibited IL-1 ~induced eicosanoid synthesis and phospholipid breakdown but also diminished TXB2 production when co-incubated with platelets. These data suggest that a cell-cell interaction, i.e. platelet utilizing SMC-derived endoperoxides for its TXA2 production, might cause an excess thromboxane A2 synthesis. INTRODUCTION
The interaction of activated platelets with the arterial wall, culminating in eicosanoid production, has been addressed by several investigators utilizing a variety of approaches. ~-4 The common precursors for both thromboxane (TX) A2 and prostaglandin (PC) I2 are endoperoxides (PGG2, PGH2) generated from arachidonic acid (AA) by cyclooxygenase present in both platelets and v a s c u l a r tissues, 4'5 The ability of a cell to obtain endoperoxide substrates formed by an adjacent cell of a different type provides the potential for a transcellular regulatory scheme. Several laboratories have demonstrated the ability of platelets to transfer endoperoxide intermediates to vessel wall segments, 2,3,6 cultured endothelial cells z,8 or lymphocytes9'~° for PGI2 synthesis. There
is additional evidence that unidirectional transfer of PG endoperoxides occurs, i.e., cultured human endothelial cells are not capable of providing PGH2 to platelets. 7 It is not known whether vascular tissue can supply PGH2 to platelets. Interleukin-1 (IL-I) has potent amplifying effects on the inflammation responses and may modulate the atherogenic responses of vascular tissues to injury. However, precise mechanisms by which this occurs are not known. 10-12The present investigation was designed to demonstrate that: (a) IL-I[~ activates AA metabolism in vascular smooth muscle cells (SMC), and (b) that platelets can utilize endoperoxides from IL-l~-stimulated vascular SMC for its biosynthesis of TXA2. MATERIALS AND METHODS
Received 26 September 1995 Accepted 27 November 1996 Correspondence to: M. Zou, Institut Biomedical des Cordeliers and INSERM, 15 rue del'@cole de M@decine, 75005, Paris, France. Fax. 49-07531-883099
Reagents
Human recombinant rIL-1[3-specific activity 10 7U/mg of protein), cycloheximide, actinomycin D, acetylsalicytic 85
86
Zou and Anges
acid (ASA) and thrombin were obtained from Sigma (Saint Quentin Fallavier, France). Labeled compound 14CAA (specific activity 55.7 mCi/mmol) was from Amersham UK. PG endoperoxide PGH2 (in hexane thylacetate 10:1) and thromboxane B2 were from Cayman Chemical (Ann Arbor, MI, USA), 13-azaprostanoic acid (13-APA) from Biomol Research Laboratories (Philadelphia, PA, USA), and culture medium (Ham F 10) and additives (antibiotics, glutamine and trypsin) were purchased from Gibco (Paisley UIO Rat SMC culture and IL-lp stimulation
Rat vascular SMC were isolated from the rat abdominal aorta by techniques previously described. 2 During the first weeks, the cells were cultured at 37°C, 5% C Q in HAM F-10 medium supplemented with 20% fetal calf serum. When a monolayer was reached, the cells were removed by trypsin-EDTA (0.05-0.02%) and cultured in a 25 cm2 flask in Ham F10 medium supplemented with 10% calf serum and 1% penicillin-streptomycin. At confluence, the cells were trypsinized and passaged. Before experiments, the cell lines (10-25 passages) were plated in a fresh culture medium and allowed to grow for 24 h. The cell viability (> 990/0) was assessed by the Trypan blue dye-exclusion test. The cells were incubated with 2 mlVI ~4C-AA for 18 h, the medium was harvested, and the cells were washed and incubated in fresh serumfree medium. As a control, 14C-AA was incubated under identical conditions in the same medium in the absence of cells to determine whether non-enzymatic metabolism occurred during the incubation period. Under these conditions, greater than 85% of initial radiolabel was incorporated into the ceils. IL-I~ (5 U/ml), which is optimal for stimulation of PGI2 synthesis, was added and the cells were further incubated at 37°C for 24 h in an atmosphere of 5% CO2 for studies of eicosanoid synthesis from endogenous AA. Incubation were also conducted with IL-1B in the presence or absence of the protein synthesis inhibitors actinomycin D or cycloheximide. These substances were added to evaluate whether the synthesis of new proteins by SMC was required for the increased prostaglandin biosynthesis in response to IL-1[3. Next, SMC were pretreated for 30 rain with 200 mM acetylsalicytic acid (ASA). This was carried out to irreversibly inactivate existing fatty acid cyclooxygenase. The cells were then washed and treated with IL-I~ or control media. If IL-I~ stimulates prostaglandin production in SMC after pretreatment with ASA, it would have to induce new fatty acid cyclooxygenase activity. For the test of co-incubation, the SMC that had been stimulated by IL-113 for 2 4 h were further incubated with human platelets at a ratio of 10 platelets to one SMC for 30 min.
Washed human platelet suspension preparation and 14C-AA metabolism in platelets
Human venous blood anticoagulated with ACD (trisodium citrate 2.5%, citric acid 1.5%, dextrose (+) 2.0%) (1/9, v/v) was drawn from healthy volunteers who had not ingested aspirin for at least 2 weeks before donation. Blood was then centrifuged at 100 x gfor 20 man to obtain platelet-rich plasma (PRP). The PRP was then adjusted to pH 6.5 with additional trisodium citrate and citric acid, and was centrifuged at 1500 x gfor 10 min. The resulting platelet pellets were then resuspended in Tyrode buffer containing (in mM): 0.412 Na2HPO4, 137 NaC1, 2 MgC12 6 H20, 2.6 KC1, 11.9 NaI-ICO3, 5.6 glucose D(+), 5 N-2hydroxyethyliperazine-/V-2-ethanesulfonic acid (HEPES) and 150 mM trisodium citrate as well as 1 mM EDTA plus 0.01% (w/v) albumin and incubated for 3 h at 37°C. After incubation, the platelets were centrifuged at 1500 x g for 10 min and platelet pellets were washed with the same buffer plus 0.05% albumin. Above 85% of radioactivity was incorporated into cells determined by liquid scintillation counting of the combined fraction. The platelets were finally suspended in phosphate-buffered saline (PBS) for release. Platelets were counted in microscope and were adjusted to 3 x 108 platelets/ml. Extraction and analysis of 14C-AA metabolites
After incubation, the cells were centrifuged at 1500 x g for 20 rain, and the supernatants were collected and acidified at pH 2.5-3.5 by addition of hydrochloric acid (1 N). The supernatants were extracted twice with 3 volumes of ethyl acetate. The combined organic phases were evaporated to dryness under a stream of nitrogen and the residues reconstituted in 200ml ethyl acetate were spotted in the TLC silica G plate (Whatman). The plates were developed in the organic phase of the solvent system: ethyl acetate/isooctane/acetic acid/water (220:100:40:200) for 75 rain and were read by a Berthold TLC scanner. Analysis of membrane phospholipid composition
To analyse membrane phospholipid composition, the adherent cells resuking from culture were scraped, harvested and resuspended in a mixture of chloroform/ methanol (1:2, v/v). Lipids were extracted as previously described 2 and spotted on TLC silica gel G plates (Whatman). Separation of different phospholipids was carried out by migration in a solvent system of chloroform/ methanol/acetic acid/water (195:129:3:9) for 80 min. The radioactive spots were read by a Berthold TLC scanner. Radioimmunoassay of thromboxane B2
The supernatants (1 ml) acidified to pH 3.5 were extracted
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 55(2), 85-91
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Transcellular thromboxane A2 biosynthesis
twice with 3 volumes of ethyl acetate. The organic phases were pooled and evaporated to dryness under nitrogen. The residue was then dissolved in PBS delatin buffer and appropriate aliquots were used for RIA of TXB2 as described./3 TXB2 levels were evaluated in duplicated serial dilutions of the samples. The results were expressed in ng/ml.
87
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RESULTS Effect of I1-1~ on AA metabolism in rat SMC
0.00
Rat vascular SMC prelabeled with 14C-AAwere incubated with 5 U/ml IL-I[3 or thrombin (1 U/ml) for 30 min to 24 h. As expected, thrombin caused a time-dependent increase of eicosanoids in the stimulated cells. The addition of IL-I[~ to the cultured SMC stimulated the production of PGI2 (measured as 6-keto-PGF~ its stable degradation product in vitro), which accounted for 25% by the appearance of radioactivity in the supernatant of IL-1 [3 stimulated cells after 24 h incubation (Fig. 1). A similar activation was observed for the other eicosanoJds and AA release (Figs 2 & 3). Kinetic studies revealed that there were two periods of stimulation in IL-l[~-treated cells. An obvious, but transient increase of 6-keto-PGF~ and PGs (PGF2~, PGE2, PGD2) was observed at the first 2 h of stimulation. Following a lag period from 2 h to 8 h, there was a much more significant increase of 6-keto-PCF~ and PGs in the stimulated cells (Figs 1, 2 & 3). In contrast to IL-I[3, thrombin led to a regular and continued increase in 6-keto-PGF~, PGs and AA in ~4C-AA prelabeled SMC with incubation time (P < 0.01). In addition, preincubation of SMC with cycloheximJde (10 mM), or actinomycin D (10 mM) inhibited IL- 1[Mnduced 6-keto-PGF~ production (87% and 91% inhibition, respectively) (Table 1), but without any significant inhibitory action on thrombin-
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Fig. 2 The time course of prostagtandin endoperoxide (PG) formation upon stimulation by IL-1 [3 and thrombin. Prelabeled rat vascular smooth muscle cells (7 x 106 cells/7 ml) were incubated with 5 U/ml and 1 U/ml IL-11~. Date is presented as the mean _+SEM dpm of five separate assays.
8.00 T 6.40 -
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Fig. 3 The time course of arachidonic acid (AA) release upon stimulation by IL-113 and thrombin. Prelabled rat vascular smooth muscle cells (7 x 106 cells/7 m[) were incubated with medium or 5 U/ml IL-113 or 1 U thrombin. Date is presented as the mean +- SEM dpm of five separate experiments.
Table 1 Effects of aspirin, cycloheximide and actinomycin D on ILl-induced eicosanoid formation in adherent rat aortic smooth muscle cells.
Sample
6-keto-PGFl~ (dmp/106cells)
PGF~, PGE2, PGD (dpm/106cells)
Control
622.1 ~ 94.6
1395.3 + 267.4
Aspirin pretreatment 10 -5 M 10-4 M
589.4 _+ 137.5 569.5 + 71.3
1418.5 _+ 117.6 1141,6 _+_+57.3
Cycloheximide
83.5 _+ 17.6
185.1 + 41.3
Actinomycin D
57.9 _+ 17,8
127.5 _+51.4
t i m e (h)
Fig. 1 The time course of 6-keto-PGFl,, formation upon stimulation by IL-1 J3and thrombin. Prelabeled rat vascular smooth muscle cells (7 x 106 cells/7 ml) were incubated with medium or 5 U/ml IL-I~ or 1 U/ml thrombin. Date is presented as the mean + SEM dpm of five separate experiments.
© Pearson Professional Ltd 1997
, 2
/"
o 1.20-
, 1
Adherent smooth muscle cells (7 x 10 Gcells) were incubated with IL-113 (5 U/ml) after preincubation with aspirin, cycloheximide (10 -s M) and actinomycin (104 M) for 1 h. The whole incubation mixture was extracted with ethyl acetate and analyzed by TLC as described in the Materials and methods. Results are expressed as means dpm/106 cells _+SEM from five experiments.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
88
Zou a n d A n g e s
Table 2 IL-I~ and thrombin-induced 14C arachidonic acid incorporation and effects of aspirin, cycloheximide (CH), and actinomycin D (AM) on IL-l-induced 14C arachidonic acid incorporation in lipids of rat vascular smooth muscle cells. % of total 14Carachidonic acid incorporated Sample
PC
PI
PE
PS
AA
Control IL-1 IL-1 + aspirin IL-1 + C H IL-1 + A M Thrombin
17.6_+4.6 11.3_+1.8 14.1 _+2.7 17.1 _+3.9 18.3_+5.1 15.3 _+5.1
21.8_+5.7 19.2_+5.1 22.5 _+4.8 23.5_+6.7 22.7_+5.2 14.4 _+3.2**
20.9_+ 11.1 20.7_+4.8 20.1 _+3.9 21.3_+4.4 19.5_+5.7 38.1 _+6.5**
9.1 _ + 3 . 1 8.0_+2.6 9.4 _+3.7 9.7_+5.7 9.4_+3.1 4.7 _+0.8
9.9_+4.5 20.7_+8.7 26.3 _+5.7 11.4_+4.1 14.3_+4.5 26.7 _+9.3**
Adherent vascular smooth muscle cells (5 x 106 cells) were incubated with buffer (control) or with IL-1 (5 U/ml) for 24 h after preincubation with aspirin (10 -6 M), cycloheximide (10 -6 M) and actinomycin D (10 -6 M) for 1 h. The cell layers were extracted with chloroform-methanol as described in the Materials and methods. Results are expressed as mean _+SEM from five experiments. PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol, PE, phosphatidylethanolamine; AA, arachidonic acid. * represents the P value compared with control * P < 0.05; P < 0.01; + represents the Pvalue compared with IL-1 + P < 0.05, ++ P < 0.01.
induced prostanoid production in SMC (data not shown). Finally, I1-113 significantly accelerated the return of prostaglandin production following elimination of active fatty cyclooxygenase by ASA (Table 1), which suggested that prostaglandin production stimulated by IL-113 probably involved induction of cyclooxygenase synthesis.
with II- 113 (0.01-100 U/ml) did not induce an obvious platelet activation. TXB2, the major metabolite of platelet AA metabolism in platelets and the specific radioactivity of ~4C-arachidonate in platelet phospholipid classes remained constant after incubation for 1 h (data not shown).
Effects of IL-I~ on phospholipid metabolism in SMC
Platelet TXA~ production from exogenously added PGH2
Rat vascular SMC cells were labeled for 18 h with 14C-AA as described in the Materials and methods. The specific radioactivity of 14C arachidonate in the major phospholipid classes is shown in Table 2. By using this labelling strategy, experiments were performed to determine which arachidonate-containing phosphotipid classes were broken down during cell activation. After the incubation with 5 U/ml IL- 113for 24 h, the specific radioactivity of I4C arachidonate in phosphatidylcholine (PC) was decreased by 35.8% as compared with control (P< 0.01) along with an obvious increase of 14C arachidonate in the supernatants (P < 0.01). The specific activity of 14C arachidonate in phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylserine (PS) remained constant in the IL-113 treated cells. Thrombin did induce a decrease of x4C arachidonate in PI (33.9%) and PS (48.4%) respectively (P < 0.01) along with a significant increase of radioactivity in PE (P < 0.01). Cycloheximide(10-hM) and actinomycin D (10-~M), did inhibit IL-l[Mnduced phosphatidylcholine breakdown (Table 2), but had no effect on thrombin-induced phospholipid breakdown in SMC (data not shown).
The ability of human platelets to utilize exogenous AA intermediates was initially examined by incubating washed platelet suspensions with exogenously added PGH2. Washed human platelets suspensions (5 x 108) or buffer blanks or washed aspirin-pretreated platelets were incubated with PGH2 in a shaking 37°C water bath. The supernatants were collected for RIA. The production of TXA2, measured by RIA of its stable breakdown product, TXB2, was increased by the addition of PGH2. Platelets converted PGH2 to TXB2 in a dose-dependent manner (Fig. 4). The inhibition of platelet cyclooxygenase by preincubation with aspirin caused no significant change in TXB2 production from PGH2 (Fig. 4). 13-APA, the endoperoxide receptor antagonist, did not alter TXB2 production after the addition of exogenous PGH2, which suggested that endoperoxide receptor stimulation might not be involved in the platelet TXA2 synthesis.
Effect of I1-1J3on 14C-AA prelabeled human platelets
The treatment of 14C-AA prelabeled human platelets
Transcellular synthesis of TXA2 in co-culture of SMC cells and platelets
14C-arachidonate-prelabeled SMC, pretreated with I1-113 (5 U/ml) for 24 h, were incubated for 30 min with washed platelets at a ratio of 10 platelets to one SMC, which was found to induce a maximal transfer of endoperoxides in cells in our previous experiments. As shown in Fig. 5, the
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
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Transcellular thromboxane A~ biosynthesis
Table 3 Thin layer chromatography of ~4C-arachidonic acid (AA)
70 T platelets 56
~[
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42
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f / /5~f j Tf51 0.1
~ "", f . J 0.5
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. . . PGH 2 (gM)
buffer blank 1
89
metabolites released by Interleukin ll~-stimulated rat vascular smooth muscle cells (SMC) with or without human platelets.
Eicosanoid
SMC (dpm/lO6cells)
SMC + platelets (dpm/lO6cells)
6-keto-PGF~ PGE2 PGD PGF2~ AA
622.1 304.6 517.1 574.8 652.3
423.3 224.8 357.1 392.7 765.7
_+94.6 _+78.6 __.72.9 + 171.8 + 103.3
+_69.6** +- 46.6* + 55.5* _+97.1" _+ 147.8
1'0
Fig. 4 Platelet production of thromboxane B2 from exogenously added PGH2, Washed human platelets (5 x 107/ml) or aspirinpretreated platelets were incubated for 3 min with PGH 2. Control represents incubation without SMC. Data is presented as the mean _+SEM of replicate determinations representing three experiments,
Adherent smooth muscle cells (7 x 106 cells) were incubated with or without platelets (7 x 107 cells) for 30 min after preincubation with IL-1 ~ (5 U/ml) for 24 h. The whole incubation mixture was extracted with ethyl acetate and analyzed as described in the Materials and methods. Results were expressed as dpm/106 cells. Values are the mean + SEM present in the supernatants of five separate experiments. *P < 0.05 ** P < 0.01 (paired Student's t-test.
Table 4 TXB2 production in co-culture of platelets (PLA) with
o
o
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,!
.....
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- -
SMC+platelets
PGD2
F2~
X
E
o. "o
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l i TX
12 HETE HETE ~i
15HE E
Fig. 5 Radiochromatogram of metabolites derived from ~4C arachidonic acid generated in the IL-1 stimulated rat vascular smooth muscle cells and during co-incubation of IL-1 treated vascular smooth muscle cells and platelets. Extraction and separation were described in the Materials and methods. The positions of authentic prostaglandin (PG), thromboxane B2, and monohydroxylated compounds are as described.
Sample
TXB2 (pg/ml)
PLA+IL-1 SMC+IL-1 PLA+SMC SMC+IL-1 +PLA SMC+iL-1 +CH SMC+IL-I+AM ASA pretreated SMC+IL-I+PLA CH pretreated SMC+IL-I+PLA AM pretreated SMC+IL-I+PLA ASA pretreatment PLA+SMC+IL-1
26.3 +_7.6 Non-detectable Non-detectable 237.5 _+41.6 51.5 + 17.1 46.4 _+ 13.5 225.4 + 48.9 31.5 + 5.3 25.6 -+ 4.9 218.7 +_51.3
Adherent smooth muscle cells (5 x 10 e cells) were incubated with 5 x 107 platelets in the presence of IL-11~ (5 U/ml) with aspirin (10 -5 M), NDGA (10 -5 M), cycloheximide (10-2 M) and actinomycin (10 -5 M) for 1 h. The incubation mixture (1 ml) was extracted and analyzed by radioimmunoassay as described in the Materials and methods. Results are expressed as means + SEM from four experiments.
SMC cells used alone in these studies did not produce TXB2 in response to IL-I~3. However, an obvious TXB2 peak, which accounted for 10% of the radioactivity in the supernatants, was found after 30 min co-incubation with platelets (Fig. 5). Together with an increase of TXB2, there was an obvious coincident decrease of 6-keto-PGFl~ and PGs (PGE2, PGF2~ and PGD2) in the mixture after coincubation as compared with control (P < 0.01); however, free AA, which might be used by platelets for its TXA~ synthesis, remained constant after co-incubation (P> 0.05) (Table 3). In addition, the pretreatment of ~4C-arachidonatelabeled SMC with cycloheximide (10mM) and actinomycin D (10mM) before IL-1 stimulation not only inhibited eicosanoid production of SMC in response to IL-1B but also diminished TXB2 production when co© Pearson Professional Ltd 1997
Interleukin 1 (IL-1)-stimulated rat vascular smooth muscle cells (SMC) in the presence or absence of cycloheximide (CH), actinomycin D (AM), and aspirin (AP).
incubated with platelets (Table 4). However, a similar amount of TXB2 was found when aspirin-pretreated platelets (10mM, 15min) were incubated with IL-1stimulated SMC. DISCUSSION
Prostaglandin production by vascular cells plays an important role in the development and progression of atherosclerosis and arterial hypertension. ~°,11 The eicosanoids appear to cause a local release of cytokines, including IL-I~ from local tissues. 1°,11 These cytoldnes cause a significant release of prostaglandins from vascular cells? 4-~6 In our study, we have demonstrated for the first time that IL-I~3, a major proinflammatory agent, effectively induced eicosanoid production in SMC and
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(2), 85-91
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Zou and Anges
endoperoxide transfer to platelets and that platelets could utilize endoperoxides originated from SMC for synthesis of TXA> Different mechanisms might be involved in the activation of eicosanoid metabolism by IL-I[3 and thrombin. Although IL-113, like thrombin, induced an immediate increase of cyclooxygenase metabolites (including 6-ketoPGFI~, PGE2, PGF2~ and PGD2), lipoxygenase metabolite HETEs, and free AA from 30 rain to 2 h, there was a lag period between 2 and 8 h in IL-I~ induced eicosanoid formation and 14C-arachidonate degradation in phosphatidylcholine. Actinomycin D and cycloheximide, which have been reported to block transcription and translation, respectively, selectively inhibited the prolonged increase of IL-l~-induced eicosanoid formation and phosphatidylcholine breakdown, but they did not inhibit thrombin-induced eicosanoid formation. These data suggested that the synthesis of new enzyme proteins were required for increased prostaglandin production in response to IL-1[5. This is consistent with the fact that IL-I[3 caused a rapid recovery from ASA pretreatment, which irreversibly inhibits existing cyclooxygenase. Therefore, our study suggested that prostaglandin production by SMC in response to IL-I[5 stimulation was likely via synthesis of cyclooxygenase protein. Recently, investigators have described two forms of cyclooxygenase (COX) in mammalian cells including cultured endothelial cells: COX-l, which is present constitutively, and COX-2, which is inducible. The relative distribution of these two forms of cyclooxygenase has not been clearly delineated in cultured SMC cells, lz~3,~z Based on our results, it is likely that IL-lJ] stimulated prostaglandin production primarily via induction of COX-2. Nevertheless, the possibility existed that there might be actions on isomerase activities in the conversion of prostaglandin endoperoxides to prostaglandins. Co-incubation of IL-l[3-stimulated vascular SMC with washed platelets induced obvious TXA2 production in which a cell-cell interaction might be involved. In agreement with others. 2,18,19 we found that IL-l[3-stimulated SMC did not synthetise TXA2 because of a lack of TXA2 synthetase. Therefore, TXB2 in the supernatants of coculture could be only originated from human platelets. Human platelets dose-dependently converted exogenous PGH2 to TXB2, and the inhibition of platelet cyclooxygenase by preincubation with aspirin caused no significant change in TXB2 production from PGH2. Mayaux et al6 have shown that aspirin-treated platelets (unable to generate their own PGH2) can use PGH2 generated by the bovine coronary artery for TXA2 synthesis, and Bourgain et al 8 have demonstrated a transfer of endoperoxides from vessel wall to platelets for TXA2 production. In this study, our data showed that together with TXBz production, there was a significant decrease of eicosanoids
(PGE2, PGF2~, PGD2 and PGA) in the co-culture supernatants, whereas the free AA, which could also be used by platelets, was constant after co-incubation. The pretreatmerit of SMC with cycloheximide, and actinomycin D, which blocked IL-l~-stimulated eicosanoid synthesis in SMC, abolished IL-l[3-induced TXB2 synthesis in the co-culture; Aspirin-treated platelets (unable to produce endoperoxides) were able to synthesize TXB2 in the presence of IL-l[5-pretreated CML. These resuks indicate that TXB2 formation is dependent on SMC activation and transfer of SMC-derived endoperoxides to platelets. There is a discrepancy with the report of Schafer. 7 The explanation of this difference is uncertain; however, it may be related to the use of cultured endothelial cells rather than vascular smooth muscle cells and to the ratio of platelets to tested vascular cells that has been reported as an important factor in determining the transcellular AA metabolism. 1 This study provides evidence that platelets are capable of utilizing vascular cell-derived endoperoxide intermediates for further metabolism to TXA2. The transcellular synthesis of TXA2 might be involved in many pathophysiological processes such as inflammaton and atherosclerosis, in which endothelium is damaged and SMC have a potential to interact simultaneously with the blood components.iS In summary, in this study, we have demonstrated for the first time that II-l[t influences vascular tissues metabolism not only through eliciting a direct activation but also through promoting transcellular metabolism of AA betwen different vascular cells. REFERENCES
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