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CARDIOVASCULAR

Inhibition of Platelet-Activating Factor (PAF) Acetylhydrolase by Methyl Arachidonyl Fluorophosphonate Potentiates PAF Synthesis in Thrombin-Stimulated Human Coronary Artery Endothelial Cells

Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri

Received June 5, 2003; accepted September 5, 2003.

	Abstract

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We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca²⁺-independent phospholipase A₂ (iPLA₂) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA₂-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA₂ inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA₂ isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5–25 µM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA₂ activity, possibly due to the localization of PLA₂ activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 µM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA₂ activity.

In a recent study, Bernatchez et al. (2001) demonstrated that PAF production in endothelial cells in response to stimulation with vascular endothelial cell growth factor (VEGF) was dependent upon both PLA₂ and lyso-PAF acetyltransferase activities. Using several pharmacological inhibitors designed to be selective for different PLA₂ isoforms, the authors concluded that secretory PLA₂ activity was responsible for the increased PAF production and that there was minimal contribution from the intracellular cytosolic, Ca²⁺-activated PLA₂ (cPLA₂) or iPLA₂. The discrepancy between the results found in the two studies could arise from the different agents used for stimulation or the use of endothelial cells from different vascular sources. Because these authors had used methyl arachidonyl fluorophosphonate (MAFP), in addition to BEL, to examine inhibition of endothelial PAF production, we evaluated the role of MAFP in PAF production in thrombin-stimulated HCAECs.

MAFP was originally developed as a specific inhibitor for cPLA₂ but was subsequently found to inhibit cytosolic iPLA₂ at similar concentrations (Balsinde and Dennis, 1996a; Lio et al., 1996). MAFP has an arachidonyl tail coupled to a fluorophosphonate group that reacts with activated serine groups. The compound competes with endogenous phospholipid molecules for the active catalytic site on the PLA₂ enzyme. MAFP has been shown to irreversibly inhibit soluble cytosolic cPLA₂ and iPLA₂, possibly by phosphorylation of the active site serine residue (Ghomashchi et al., 1999). Although the relatively polar MAFP has direct access to the catalytic site of soluble PLA₂ isoforms, the active site serine residue of membrane-associated iPLA₂ may be "protected" from this inhibitor. In addition to PLA₂ inhibition, MAFP has been shown to inhibit anandamide amidase, the enzyme responsible for the hydrolysis of arachidonyl ethanolamide (Deutsch et al., 1997) and has been shown to be an irreversible cannabinoid receptor antagonist (Deutsch et al., 1997; Fernando and Pertwee, 1997).

In this study, we demonstrate that pretreatment of HCAECs with MAFP before thrombin stimulation did not inhibit membrane-associated endothelial cell PLA₂ activity and instead resulted in a significant augmentation of PAF production as a result of inhibition of PAF-AH. Phospholipase A₂ inhibitors have been proposed to be potential anti-inflammatory agents because PLA₂-catalyzed hydrolysis of membrane phospholipids is the rate-limiting step for the generation of inflammatory phospholipid metabolites such as eicosanoids and PAF. Our data demonstrate the importance of identifying the PLA₂ isoforms involved in phospholipid metabolite production to guide the development of specific PLA₂ inhibitors that could be used therapeutically.

	Materials and Methods

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Materials. HCAECs were obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). BEL was a gift from F. Hoffmann-La Roche (Nutley, NJ). MAFP was obtained from Cayman Chemicals (Ann Arbor, MI). [¹⁴C]Lysophosphatidylcholine and [¹⁴C]acetic anhydride were purchased from Amersham Biosciences Inc. (Piscataway, NJ). [³H]PAF, [³H]acetic anhydride, and [³H]arachidonic acid were obtained from PerkinElmer Life Sciences (Boston, MA). P-selectin antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and horseradish peroxidase-conjugated anti-goat IgG antibody was obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Endothelial Cell Cultures. Endothelial cells were grown to confluence in MCDB-131 medium with 5% fetal calf serum, 10 ng/ml epidermal growth factor, 1 µg/mg hydrocortisone, 200 µg/ml endothelial cell growth supplement, and 90 µg/ml heparin. Cells were allowed to grow to confluence achieving a contact-inhibited monolayer of flattened, closely apposed endothelial cells in 4 to 5 days. After achieving confluence, cells were passaged in a 1:3 dilution and cells from passages 3 to 4 used for experiments.

Phospholipase A₂ Activity. Confluent HCAEC cultures were stimulated with thrombin with or without MAFP or BEL for the allocated time intervals. At the end of the stimulation period, iPLA₂ activation was arrested by the removal of the surrounding buffer and immediate replacement with ice-cold buffer containing 250 mmol/l sucrose, 10 mmol/l KCl, 10 mmol/l imidazole, 5 mmol/l EDTA, 2 mmol/l dithiothreitol with 10% glycerol, pH 7.8. Cells were removed from the tissue culture well using a cell scraper, and the suspension was sonicated on ice for six bursts of 10 s each. After sonication, the suspension was centrifuged at 14,000g at 4°C for 20 min, and the resultant supernatant was centrifuged at 100,000g to separate the cytosolic (supernatant) and membrane (pellet) fractions. Phospholipase A₂ activity was assessed by incubating 50 µg of cytosolic protein or 8 µg of membrane protein with 100 µM (16:0, [³H]18:1) plasmenylcholine in assay buffer containing 100 mM Tris, 4 mM EGTA, 10% glycerol, pH 7.0 at 37°C for 5 min in a total volume of 200 µl. Reactions were initiated by adding the radiolabeled phospholipid substrate as a concentrated stock solution in ethanol. Specific enzyme activity was expressed as the rate of radiolabeled fatty acid production determined after separation from the labeled phospholipid substrate using thin layer chromatography and liquid scintillation spectrometry with activity normalized to protein content as described previously (McHowat and Creer, 1997, 1998a,b).

Separation and Quantification of Individual Choline and Ethanolamine Glycerophospholipid Molecular Species. Cellular phospholipids were extracted from HCAECs by the method of Bligh and Dyer (1959). The chloroform layer was dried under N₂ and the lipid residue resuspended in 1 ml of chloroform/methanol [1:1 (v/v)]. Phospholipids were separated into different classes by HPLC using gradient elution with a mobile phase comprised of hexane/isopropanol/water as described previously (McHowat et al., 2001). Individual choline and ethanolamine glycerophospholipid molecular species were separated by reverse-phase HPLC using a gradient elution system with a mobile phase comprised of acetonitrile/methanol/water with 20 mM choline chloride (McHowat et al., 2001). Quantification of individual phospholipid molecular species was achieved by determination of lipid phosphorus in reverse-phase HPLC column effluents (McHowat et al., 2001).

PAF Production. Confluent HCAEC monolayers were washed twice with Hanks' balanced salt solution containing 135 mM NaCl, 0.8 mM MgSO₄, 10 mM HEPES (pH 7.4), 1.2 mM CaCl₂, 5.4 mM KCl, 0.4 mM KH₂PO₄, 0.3 mM Na₂HPO₄, and 6.6 mM glucose and incubated with 50 µCi of [³H]acetic acid for 20 min. After thrombin stimulation for the selected time interval, lipids were extracted from the cells by the method of Bligh and Dyer (1959). The chloroform layer was concentrated by evaporation under N₂, applied to a silica gel 60 thin layer chromatography plate, and developed in chloroform/methanol/acetic acid/water [50:25:8:4 (v/v)]. The region corresponding to PAF was scraped and radioactivity was quantified using liquid scintillation spectrometry. Loss of PAF during extraction and chromatography was corrected by adding a known amount of [¹⁴C]PAF as an internal standard. [¹⁴C]PAF was synthesized by acetylating the sn-2 position of lyso-PAF with [¹⁴C]acetic anhydride using 0.33 M dimethylaminopyridine as a catalyst (McHowat et al., 2001) and was purified by HPLC.

Acid-Catalyzed Hydrolysis of [³H]PAF. [³H]PAF produced in thrombin-stimulated HCAEC with or without MAFP pretreatment were divided into two equal samples and one sample was incubated with 0.1 M HCl for 20 min at 37°C to hydrolyze the vinyl ether linkage at the sn-1 position of PAF species derived from plasmalogen lysophospholipids (McHowat et al., 2001). After acid-catalyzed hydrolysis, both samples were extracted using the Bligh and Dyer (1959) method and [³H]PAF separated by HPLC using a mobile phase comprised of hexane/isopropanol/water (465:465:70). Radioactivity in the eluate was collected at 1-min intervals and counted by liquid scintillation spectrometry.

PAF-Acetylhydrolase Activity. HCAECs were removed from the tissue culture plate in 1.2 mM Ca²⁺ HEPES buffer and sonicated on ice. Cellular protein (25 µg) was incubated with 0.1 mM [acetyl-³H]PAF (10 mCi/mmol) for 30 min at 37°C. The reaction was stopped by the addition of acetic acid and released [³H]acetic acid was separated from the [³H]PAF substrate by passing the reaction mixture through a C18 gel cartridge (Baker Chemical Co., Phillipsburg, NJ). Released [³H]acetic acid was quantified by liquid scintillation spectrometry.

Statistics. Statistical comparison of values was performed by the Student's t test or analysis of variance with the Fisher multiple-comparison test as appropriate. All results are expressed as means ± S.E.M. Statistical significance was considered to be p < 0.05.

	Results

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We have demonstrated previously that thrombin-stimulated PAF production in HUAECs involves iPLA₂-catalyzed hydrolysis of membrane plasmalogen phospholipids. Pretreatment of HUAEC with the iPLA₂-selective inhibitor BEL resulted in complete inhibition of thrombin-stimulated iPLA₂ activity and increased PAF production. Because MAFP inhibits the cytosolic isoforms of both cPLA₂ and iPLA₂, we measured PAF production in HCAECs pretreated with MAFP before thrombin stimulation. Contrary to the results we expected, we observed that, at concentrations of 5 µM or greater, MAFP pretreatment resulted in significant increases in PAF production in response to thrombin stimulation (Fig. 1). In addition, incubation of HCAECs with 25 µM MAFP without thrombin stimulation resulted in a significant increase in PAF production (Fig. 1). Thus, our initial data demonstrate that MAFP pretreatment augments, rather than inhibits, thrombin stimulated PAF production in HCAECs.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effect of pretreatment with increasing concentrations of MAFP (10-min incubation) on control (X) and thrombin-stimulated (0.1 IU/ml, 10 min, ) PAF production in HCAECs. *, p < 0.05 between unstimulated cells in the presence and absence of MAFP. **, p < 0.01 when comparing control to thrombin-stimulated values in the presence and absence of each concentration of MAFP. Values shown represent the mean ± S.E.M. for results derived from 6 different cell cultures.

The thrombin-stimulated acceleration of membrane plasmalogen phospholipid hydrolysis results in increased production of both lysoplasmenylcholine (LysoPlsCho) and lysoplasmenylethanolamine (LysoPlsEtn). However, we subsequently determined that there was little, if any, PAF produced by the subsequent acetylation of LysoPlsCho. We hypothesized that the generation of LysoPlsEtn promotes PAF synthesis through a transacylation pathway by acting as an acyl group acceptor (McHowat et al., 2001) and proposed that LysoPlsEtn production by iPLA₂ was the rate-controlling step in PAF production rather than the activation of PAF acetyltransferase. To demonstrate the validity of this hypothesis, we incubated HCAECs with LysoPlsEtn and measured PAF production. Incubation of HCAEC with 5 µM LysoPlsEtn for 10 min resulted in a significant increase in PAF production that was comparable with that observed in thrombin-stimulated cells (Fig. 2, filled columns), demonstrating that basal PAF acetyltransferase activity is sufficient to support PAF synthesis and that availability of LysoPlsEtn is rate-limiting. Pretreatment with MAFP (25 µM, 10 min) resulted in a significant increase in PAF production in unstimulated-, thrombin-stimulated, and LysoPlsEtn-stimulated HCAECs (Fig. 2, open columns). In further studies, we determined that incubation of HCAECs with 5 µM lyso-PAF for 10 min also resulted in a 3.3-fold increase in PAF production that was comparable with the increase in thrombin-stimulated cells (data not shown). Thus, incubation of HCAECs with either LysoPlsEtn or lyso-PAF results in increased PAF production. In contrast, increased PAF production was not observed when HCAECs were incubated with LysoPlsCho or lysophosphatidylcholine (data not shown). Together, these data support our hypothesis that thrombin-stimulated PAF production occurs primarily via the remodeling pathway. However, to ensure that the increase in PAF production by MAFP proceeded through the transacylase pathway and was not due to increased acetylation of lysoplasmalogens produced in response to thrombin stimulation, we incubated the PAF extracted from thrombin-stimulated and MAFP-pretreated HCAECs with 0.1 M HCl before separation by HPLC (Fig. 3). Acid pretreatment of HCAEC-derived PAF did not result in a significant loss of radioactivity in PAF production in thrombin-stimulated HCAECs with or without MAFP pretreatment, demonstrating that MAFP does not increase acetylation of plasmalogen lysophospholipids generated by thrombin-stimulated PLA₂. Accordingly, MAFP augments PAF production by a mechanism that does not result from alterations in PLA₂ activity or increased acetylation of acid-labile (plasmalogen) lysophospholipids.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Incubation of HCAECs with thrombin (0.1 IU/ml, 10 min) and LysoPlsEtn (5 µM, 10 min) results in a significant increase in PAF production (filled columns, *, p < 0.05 when comparing stimulated and unstimulated PAF production). Pretreatment with MAFP (5 µM, 10 min) significantly potentiated both thrombin- and LysoPlsEtn-induced PAF production (open columns, **, p < 0.01 compared with corresponding values in the absence of MAFP). Values shown represent the mean ± S.E.M. for results derived from three different cell cultures.

View larger version (17K): [in this window] [in a new window]   Fig. 3. HPLC separation of PAF produced in thrombin-stimulated HCAECs (left, 0.1 IU/ml, 10 min), in HCAECs pretreated with MAFP (5 µM, 10 min), and stimulated with thrombin (right, 0.1 IU/ml, 10 min). Cells were stimulated and PAF extracted as described. Aliquots of the extract were equally divided, and one aliquot was incubated with 0.1 M HCl for 20 min at 37°C to hydrolyze the vinyl ether linkage at the sn-1 position. Acid-catalyzed hydrolysis (dotted lines) did not significantly alter the radioactivity present in the peak corresponding to the PAF produced in thrombin-stimulated HCAECs with or without MAFP pretreatment; thus, little, if any, PAF is derived directly from plasmalogen lysophospholipid precursors.

Previous studies in P388D₁ macrophages have indicated that multiple PLA₂ isoforms in cells may interact with each other by generating products (e.g., eicosanoids) that modulate PLA₂ activity by specific receptor-mediated events and also possibly by competing for the same substrate pools. Although we have determined that the majority of endothelial cell PLA₂ activity is inhibited by BEL, is selective for plasmalogen substrates, and is maximal in the absence of Ca²⁺ and thus represents iPLA₂, we cannot rule out the possibility that MAFP inhibits a PLA₂ isoform in HCAECs that potentially regulates iPLA₂ activity. To determine whether MAFP alters HCAEC iPLA₂ activity, we pretreated cells with MAFP before thrombin stimulation and measured cellular PLA₂ activity in the absence of Ca²⁺ using (16:0, [³H]18:1) plasmenylcholine as substrate (Fig. 4). Pretreatment with MAFP did not significantly alter iPLA₂ activity in unstimulated HCAECs (Fig. 4, open squares, dotted lines) or inhibit thrombin-stimulated iPLA₂ activity (Fig. 4, open squares, solid lines) compared with activity measured in cells without MAFP. In contrast, pretreatment with BEL resulted in a significant decrease in iPLA₂ activity measured in both unstimulated (Fig. 4, filled circles, dotted lines) and thrombin-stimulated HCAEC (Fig. 4, filled circles, solid lines). Thus, MAFP does not affect the activity of thrombin-stimulated, membrane-associated iPLA₂ despite previous reports demonstrating MAFP inhibition of soluble iPLA₂ isoforms. Mass measurements of individual molecular species in choline and ethanolamine phospholipids demonstrate that MAFP pretreatment does not significantly alter accelerated membrane phospholipid hydrolysis in response to thrombin stimulation (Table 1). This supports our hypothesis that MAFP pretreatment does not augment PAF production via alterations in membrane phospholipid hydrolysis, resulting in increased lysophospholipid production.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of pretreatment with MAFP (open squares, 25 µM, 10 min) or bromoenol lactone (filled circles, 10 µM, 10 min) on membrane-associated PLA2 activity in HCAECs in the absence (dotted lines) or presence (solid lines) of thrombin (0.1 IU/ml). X represents control PLA2 activity measurements made in the absence of either PLA2 inhibitor. PLA2 activity was measured using 100 µM (16:0, [3H]18:1) plasmenylcholine substrate in the absence of Ca2+ (4 mM EGTA). *, p < 0.05; **, p < 0.01 compared with untreated HCAECs. Values shown represent the mean ± S.E.M. for results derived from four different cell cultures.

Endothelial cell PAF synthesis is tightly controlled by rapid, inducible synthesis initiated by iPLA₂ and equally efficient degradation mediated by PAF-AH. PAF-AH is a unique, soluble, cytosolic PLA₂ isoform that does not have a Ca²⁺ requirement for activity and possesses a preference for substrates containing short acyl chains at the sn-2 position. We proposed that because MAFP has been demonstrated to inhibit other PLA₂ isoforms, it may also inhibit endothelial cell PAF-AH activity. Inhibition of PAF-AH activity would augment PAF generation in HCAECs as a result of inhibition of PAF catabolism rather than increased PAF production.

Although PAF-AH activity has been characterized previously in human umbilical vein endothelial cells (Blank et al., 1986) it has not been characterized in HCAECs. Consequently, we performed initial studies to ensure our assay conditions would result in linear reaction velocities with respect to time and protein content. Incubation of HCAEC protein with [³H]PAF resulted in linear reaction velocities for PAF-AH activity for up to 120 min, after which time the rate of acetate production decreased (Fig. 5). In previously published assay systems, investigators have routinely used an incubation time of 15 min, so we chose to use the same time interval for subsequent experiments. Varying the amount of cellular protein used, linear reaction velocities were observed with protein content between 10 and 100 µg, thus we chose to use 25 µg of protein in subsequent assays. Maximal reaction velocities were consistently achieved with [³H]PAF substrate concentrations greater than 50 µM, thus we used 100 µM substrate concentration for our assays.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Release of [3H]acetic acid from 0.1 mM [3H]PAF substrate after incubation with 25 µg of HCAEC protein for increasing time intervals at 37°C. The rate of release of free acetic acid was found to be linear for up to 120 min.

To examine whether the PLA₂ inhibitors BEL and MAFP inhibited PAF-AH, we pretreated HCAECs with either BEL or MAFP and measured activity under our linear maximal rate conditions (Fig. 6). PAF-AH activity was significantly inhibited by MAFP concentrations greater than 1 µM but was unaffected by BEL concentrations as high as 20 µM (Fig. 6). Incubation of HCAEC cellular protein with increasing concentrations of MAFP demonstrated that the inhibition of PAF-AH by MAFP was found to be rapid and both concentration- and time-dependent (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Inhibition of PAF acetylhydrolase in HCAECs incubated with increasing concentrations of the PLA2 inhibitors MAFP (open squares) and BEL (filled circles) for 10 min. **, p < 0.01 compared with untreated activity. Values shown represent the mean ± S.E.M. for results derived from eight different cell cultures.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Effect of inhibition of PAF acetylhydrolase activity with increasing concentrations of MAFP over a 60-min time course. Inhibition of PAF acetylhydrolase by MAFP is both time- and concentration-dependent. Aliquots of HCAEC suspensions were incubated with the indicated concentration of MAFP for increasing time intervals. PAF-acetylhydrolase activity was then measured by the addition of 0.1 mM [acetyl-3H]PAF for 30 min at 37°C and subsequent quantitation of released [3H]acetic acid. Values shown are for a single cell culture.

Together, these data indicate that the increase in thrombin-stimulated PAF production by pretreatment of HCAECs with MAFP is due to inhibition of PAF catabolism by PAF-AH. PAF-AH activity in HCAECs was found to be approximately equal for 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (3.4 ± 0.4 nmol/mg protein/min, n = 4), 1-O-alkenyl-2-acetyl-sn-glycero-3-phosphocholine (2.9 ± 0.7 nmol/mg protein/min, n = 4), and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine (2.8 ± 0.6 nmol/mg protein/min, n = 4). Pretreatment of HCAECs with 5 µM MAFP resulted in complete inhibition of PAF-AH activity using each of the three classes of PAF species as substrate. Conversely, BEL pretreatment did not inhibit PAF-AH activity with any substrate used.

	Discussion

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Inhibitors of PLA₂ activity have been proposed to be anti-inflammatory agents for many years because PLA₂-catalyzed hydrolysis of membrane phospholipids is the rate-limiting event in the generation of proinflammatory lipid mediators such as prostaglandins, leukotrienes, and PAF. However, after their development, almost all selective PLA₂ inhibitors have been subsequently demonstrated to inhibit other enzymes that are involved in phospholipid hydrolysis or remodeling. For example, arachidonyl trifluoromethyl ketone (AACOCF₃), a selective cytosolic PLA₂ inhibitor, has been shown to inhibit both 5-lipoxygenase and CoA-independent transacylase (Fonteh, 2002), and BEL has been shown to inhibit phosphatidate phosphohydrolase (Balsinde and Dennis, 1996b). MAFP has been shown previously to induce cyclooxygenase-2 expression in murine macrophages, leading to an increase in prostaglandin E₂ release (Lin and Chen, 1999). Thus, the use of selective PLA₂ inhibitors in the context as potential anti-inflammatory agents has to be treated with caution.

The inflammatory actions of PAF include activation of polymorphonuclear leukocytes and increased vascular permeability (Prescott et al., 2002). The concentration of PAF in plasma and tissues is tightly regulated by the balance of synthesis and degradation (Snyder, 1995). PAF-AH, the enzyme that catalyzes the hydrolysis of biologically active PAF into biologically inactive lyso-PAF, is a member of the PLA₂ family of enzymes that selectively hydrolyze phospholipids with short acyl chains at the sn-2 position (Snyder, 1995; Stafforini et al., 1997). Thus, a PLA₂ inhibitor that inactivates PAF-AH could act as a proinflammatory agent, prolonging the inflammatory response and increasing the recruitment of inflammatory cells to areas of injury (Prescott et al., 2002).

PAF acetylhydrolases are maximally active in the basal state and do not require Ca²⁺ for activity, thus providing an immediate mechanism for inactivation of PAF (Stafforini et al., 1996, 1997). Several studies have determined that the level of PAF accumulation is determined by the PAF-AH activity (Touqui et al., 1985; Suzuki et al., 1988; Elstad et al., 1989). In addition, the magnitude of the inflammatory response to PAF may function in an autoregulatory manner because increased production of PAF stimulates the expression of PAF-AH (Satoh et al., 1991). Thus, it follows that inhibition of PAF-AH would propagate the PAF-mediated inflammatory response. Indeed, a decrease in plasma PAF-AH activity has been observed in asthma (Miwa et al., 1988; Stafforini et al., 1999), systemic lupus erythematosus (Tetta et al., 1990) and septic shock (Graham et al., 1994).

The intracellular cPLA₂ and iPLA₂ isoforms do not require Ca²⁺ for catalysis, use a central Ser for catalysis, and operate via the formation of an acyl-enzyme intermediate (Six and Dennis, 2000). Because their catalytic features are similar and MAFP acts at the catalytic site of the enzyme, it is not surprising that MAFP inhibits both iPLA₂ and cPLA₂ isoforms. PAF-AH resembles many neutral lipases, because its active site is composed of a Ser-Asp-His catalytic triad (Tjoelker et al., 1995); thus, it might be expected that MAFP would inhibit this enzyme. However, the selectivity of this enzyme for short-chain sn-2 acyl groups suggests that a nonhydrolyzable analog of arachidonic acid may not be recognized and targeted for hydrolysis by PAF-AH. This study is the first to demonstrate inhibition of PAF-AH by MAFP, suggesting that MAFP may be a nonselective inhibitor of multiple serine-dependent lipases. The failure of MAFP to inhibit PAF production would suggest that the serine-dependent intracellular PLA₂ isoforms inhibited by MAFP do not play a role. This finding may explain the reason that a role for endothelial PAF production by intracellular PLA₂ isoforms was ruled out previously (Bernatchez et al., 2001). However, in addition to inhibition of PAF-AH activity, we measured PLA₂ activity in HCAECs and found that MAFP had little effect on membrane-associated, BEL-inhibitable PLA₂ activity measured in the absence of Ca²⁺. These results are consistent with the conclusion that the majority of thrombin-stimulated iPLA₂ activity in HCAECs is membrane-associated rather than cytosolic and that the MAFP inhibitor cannot gain access to the enzyme when it is present as an integral membrane protein. In a previous study, we have demonstrated that inhibition of membrane-associated iPLA₂ activity with BEL results in inhibition of PAF production in thrombin-stimulated endothelial cells (McHowat et al., 2001). In this study, we show that BEL does not inhibit PAF-AH, even though this enzyme is a serine-dependent intracellular PLA₂. Thus, BEL is a selective, active site-directed inhibitor that can inhibit both soluble, cytosolic and membrane-associated iPLA₂ isoforms. In a separate study, we found that BEL pretreatment would inhibit the MAFP-induced increase in PAF production in response to thrombin stimulation; however, this required total inhibition of PLA₂ activity and a significant reduction in lysophospholipid content before it could be achieved (data not shown).

Bernatchez et al. (2001) have reported previously that MAFP and BEL pretreatment of bovine aortic endothelial cells and human umbilical artery endothelial cells resulted in a potentiation of VEGF-stimulated PAF production (Bernatchez et al., 2001). In their study, inhibition of endothelial cell PLA₂ activity by MAFP or BEL was only inferred from measurement of PAF accumulation, and the authors did not measure either endothelial cell PLA₂ or PAF-AH activities directly. Thus, from the data obtained, the authors concluded that iPLA₂ activity did not play a role in PAF production. We have found that BEL pretreatment results in specific inhibition of thrombin-stimulated, membrane-associated iPLA₂ activity, with no inhibition of PAF-AH (Fig. 8). Conversely, we find that although MAFP does not inhibit thrombin-stimulated, membrane-associated endothelial cell PLA₂ activity, MAFP is a potent inhibitor of PAF-AH, and results in a net potentiation of thrombin-stimulated PAF production (Fig. 8). In human umbilical vein endothelial cells, Bernatchez et al. (2001) observed a 6-fold increase in VEGF-stimulated PAF production by MAFP and a 2-fold increase in VEGF-stimulated PAF production by BEL. Thus, our results using MAFP are similar, whereas our finding with BEL are different and may be due to different endothelial cells or the stimulus used.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Activation of calcium-iPLA2 by thrombin stimulation of human coronary artery endothelial cells results in selective hydrolysis of membrane plasmalogen phospholipids. LysoPlsEtn acts as an acceptor for the sn-2 fatty acid from PakCho, resulting in production of lyso-platelet-activating factor (lyso-PAF), which is then acetylated with lyso-PAF acetyltransferase using acetylCoA to form biologically active PAF. Inhibition of iPLA2 activity with BEL results in inhibition of PAF production as a result of decreased lysoPlsEtn production. Pretreatment with MAFP augments PAF production by inhibiting PAF hydrolysis by PAF acetylhydrolase.

Inhibition of PAF-AH would suggest that MAFP pretreatment would potentiate PAF accumulation in endothelial cells regardless of the initiating event. We proposed previously that the majority of PAF produced in thrombin-stimulated HUAECs was synthesized via the remodeling pathway, involving the production of LysoPlsEtn that acts as an acceptor for the sn-2 fatty acid from alkylacyl glycerophosphocholine, resulting in the production of lyso-PAF that can be acetylated subsequently (McHowat et al., 2001). To further support this hypothesis, we incubated HUAECs with LysoPlsEtn but were not able to demonstrate an increase in PAF production (McHowat et al., 2001). However, in the present study we incubated HCAECs with LysoPlsEtn and demonstrated increased PAF production that was similar to that observed in thrombin-stimulated cells. It is not clear why increased PAF production is observed in HCAECs, but not HUAECs, when incubated with LysoPlsEtn; however, it may be that the uptake of LysoPlsEtn into HCAECs is greater or that the catabolism of LysoPlsEtn in HCAECs is slower than in HUAECs. The increase in PAF production after incubation with LysoPlsEtn demonstrates the involvement of the remodeling pathway for PAF synthesis in HCAECs. As would be expected, the inhibition of PAF-AH with MAFP resulted in increased PAF production in response to both thrombin and LysoPlsEtn incubation.

In summary, we have demonstrated that pretreatment of human endothelial cells with MAFP did not inhibit thrombin-stimulated, membrane-associated iPLA₂ activity, but paradoxically enhanced thrombin-stimulated PAF production by virtue of MAFP-induced inhibition of endothelial cell PAF-AH. Accordingly, MAFP may provide a useful reagent to further study the role of PAF-AH inhibition on atherosclerotic disease progression.

	Footnotes

 
This research was supported in part by the National Institutes of Health (HL-68588 to J.M.) and the American Heart Association (Missouri Affiliate to M.H.C. and J.M.).

DOI: 10.1124/jpet.103.055392.

ABBREVIATIONS: HUAEC, human umbilical artery endothelial cell; PakCho, alkyl acyl glycerophosphocholine; PakEtn, alkyl acyl glycerophosphoethanolamine; BEL, bromoenol lactone; DMAP, dimethylaminopyridine; HUAEC, human umbilical artery endothelial cell; LysoPlsCho, lysoplasmenylcholine; LysoPlsEtn, lysoplasmenylethanolamine; MAFP, methyl arachidonyl fluorophosphonate; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PLA₂, phospholipase A₂; PlsCho, plasmenylcholine; PlsEtn, plasmenylethanolamine; PAF, platelet-activating factor; VEGF, vascular endothelial cell growth factor.

Address correspondence to: Dr. Jane McHowat, Department of Pathology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. E-mail: mchowatj{at}slucare1.sluh.edu

	References

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