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Am J Physiol Cell Physiol. Apr 2011; 300(4): C872–C879.
Published online Jan 12, 2011. doi:  10.1152/ajpcell.00289.2010
PMCID: PMC3074629

Activation of group VI phospholipase A2 isoforms in cardiac endothelial cells

Abstract

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2 (iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2 (PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2 release and increased PAF production. Selective inhibition of iPLA2β with (S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI2 release. Stimulation of cardiac endothelial cells from group VIB (iPLA2γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI2 release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA2β activation and that both iPLA2β and iPLA2γ may be involved in PGI2 release.

Keywords: endothelium, platelet-activating factor, prostacyclin

inflammation is a component of several cardiovascular diseases. The endothelium lines the luminal surface of blood vessels and is an integral part of the inflammatory system. In the heart, the endothelium forms a critical barrier between circulating cells and underlying myocardium, and an interaction between endothelial cells and cardiomyocytes is physiologically and pathologically important (22). The endothelium contributes to cardiac homeostasis by regulating vascular permeability and resistance and by releasing mediators such as nitric oxide, endothelin, pro- and anticoagulants, growth factors, and prostaglandins that affect numerous vascular and cardiac functions (26). Owing to its close proximity to both circulating cells and cardiac myocytes, the endothelium can facilitate signal transduction between these cell types (26). The endothelium contributes to this process during inflammation by regulating inflammatory cell transmigration into cardiac tissue and by releasing mediators that are cardioprotective during acute inflammation, but chronic exposure to these mediators could be detrimental (17).

Under physiologic conditions, leukocytes flow across the vascular wall, but a key step in inflammation is leukocyte adherence to endothelium (16). An important regulatory molecule in this process is platelet-activating factor (PAF), which is a potent inflammatory mediator that is active at concentrations as low as 10−12 M (13). Activated endothelium produces PAF, which is expressed on the endothelial cell surface and interacts with circulating inflammatory cells via their surface PAF receptors. This interaction tethers the leukocytes to the endothelium and activates them, which results in their degranulation, chemotaxis, and increased expression of other cell surface adherence molecules (16).

PAF production is tightly regulated by the action of phospholipase A2 (PLA2), which comprises a large family of enzymes that hydrolyze the sn-2 ester bond of phospholipids to yield a free fatty acid, e.g., arachidonic acid, and a 2-lysophospholipid, e.g., lyso-PAF. Arachidonic acid can be converted to eicosanoids via the action of oxygenases, and acetylation of lyso-PAF yields PAF (30). Our previous studies have demonstrated an increase in calcium-independent PLA2 (iPLA2) activity in human coronary artery endothelial cells (HCAECs) following stimulation of protease-activated receptor (PAR) by thrombin or tryptase, and this is associated with corresponding increases in production of PAF and prostaglandins (19, 30).

Of iPLA2 enzymes identified to date, iPLA2γ and iPLA2β are prominent constituents of mammalian cells. Racemic bromoenol lactone (BEL) inhibits iPLA2 activity (6) and has been widely used to examine the potential physiologic and pathologic roles of these enzymes. Of BEL enantiomers, (R)-BEL inhibits iPLA2γ at concentrations tenfold lower than those required to inhibit iPLA2β, and the converse is true for (S)-BEL (7). This permits pharmacologic discrimination between these iPLA2 isoforms, but BEL can also inhibit serine hydrolases and other enzymes in addition to iPLA2 (24). This has motivated the development of iPLA2 isoform-specific knockout (KO) mice to permit genetic discrimination between iPLA2 isoforms to study their potential participation in physiologic and pathologic processes (3, 12).

In the studies described here, we have used cardiac endothelial cells isolated from wild-type (WT) mice and compared them to cells isolated from iPLA2γ-KO and iPLA2β-KO mice to evaluate the roles of these enzymes in production of inflammatory mediators derived from endothelial cell phospholipids, including PAF and prostaglandin I2 (PGI2).

MATERIALS AND METHODS

HCAECs.

HCAECs were obtained from Lonza Walkersville (Walkersville, MD). Cells were grown to confluence in EGM-2MV media obtained from Lonza, with 5% fetal bovine serum. 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 were used for experiments.

Mouse endothelial cell isolation and culture.

Animal protocols were in strict accordance with the National Institutes of Health guidelines for humane treatment of animals and were reviewed and approved by the Animal Care and Use Committee of Saint Louis University. iPLA2β-KO and iPLA2γ-KO mice were generated by Dr. John Turk's and Dr. Richard Gross's research groups, respectively, and have been described in detail previously (3, 12). Mice were bred as heterozygous trios, and WT and KO littermates were used for endothelial cell isolation. Genotyping for each animal was performed by PCR as described previously (3, 12). Male and female WT, iPLA2β-KO and iPLA2γ-KO mice on the C57BL6/J background were used at 10–12 wk of age for endothelial cell isolation. Endothelial cells were isolated from WT and iPLA2-KO mouse hearts by collagenase digestion. The diced heart muscle was incubated in 2 mg/ml collagenase for 1 h at 37°C, and the digested tissue was passed through a cell strainer. Cells were incubated with murine immunoglobulins to block Fc receptors and then incubated with anti-mouse platelet/endothelial cell adhesion molecule 1 (PECAM-1) coupled to magnetic beads. Cells obtained were cultured until they reached confluence and sorted again using ICAM-2 antibodies coupled with magnetic beads. The eluted cells were washed, resuspended in cell culture medium, and plated in culture. Nonadherent cells were removed the next day, and cells were grown to confluence and passaged at a 1 to 3 dilution. Genotype of each cell culture was verified by real-time RT-PCR analysis (Table 1). Relative quantitation of each iPLA2 isoform was performed compared with 18s RNA. There was no detectable iPLA2β and no significant compensation in iPLA2γ expression in iPLA2β-KO endothelial cells (Table 1). Likewise, we did not detect iPLA2γ expression or change in iPLA2β expression in iPLA2γ-KO endothelial cells (Table 1).

Table 1.
Real-time RT-PCR analysis of iPLA2β, iPLA2γ, and 18s RNA expression in endothelial cells isolated from the hearts of wild type, iPLA2β and iPLA2γ knockout mice

Stimulation of confluent endothelial cells.

All experiments were carried out with confluent monolayers of endothelial cells. Human recombinant skin β-tryptase (Promega, Madison, WI) or thrombin (Sigma Chemical, St. Louis, MO) were diluted with medium (iPLA2 assay, arachidonic acid release, prostaglandin release) or Hanks' balanced salt solution (HBSS; PAF production) to the working concentration. Tryptase or thrombin was added to the cell culture plate, and the plate was gently rotated to ensure thorough mixing and even distribution on the monolayer. Where appropriate, stock solutions of bromoenol lactone in DMSO were diluted with HEPES buffer and added before the thrombin or tryptase stimulation. At the end of the stimulation period, chloroform and methanol were added directly to the endothelial cell monolayer for measurement of PAF production. For measurement of PLA2 activity, the surrounding buffer was removed and immediately replaced with ice-cold PLA2 activity buffer. For measurement of arachidonic acid or 6-keto-PGF release, the surrounding medium was removed and centrifuged and the supernatant removed for assay of arachidonic acid radioactivity or 6-keto-PGF content.

Phospholipase A2 activity.

Endothelial cells were suspended in 1 ml buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 dithiothreitol (DTT) with 10% glycerol, pH 7.8 (PLA2 activity buffer). The suspension was sonicated on ice six times for 10 s (using microtip probe at 20% power output, 500 Sonic Dismembrator, Fisher Scientific), and the sonicate was centrifuged at 20,000 g for 20 min to remove cellular debris and nuclei. The pellet was resuspended in activity buffer. PLA2 activity was assessed by incubation of enzyme (50 μg protein) with 100 μM (16:0, [3H]18:1) plasmenylcholine substrate in assay buffer containing 10 mmol/l Tris, 4 mmol/l EGTA, and 10% glycerol, pH 7.0, at 37°C for 5 min in a total volume of 200 μl. The radiolabeled phospholipid substrate was introduced into the incubation mixture by injection in 5 μl ethanol to initiate the assay. Reactions were terminated by the addition of 100 μl butanol, and released radiolabeled [3H]oleic acid was isolated by the application of 25 μl of the butanol phase to channeled Silica Gel G plates, development in the petroleum ether/diethyl ether/acetic acid (70/30/1, vol/vol), and subsequent quantification by liquid scintillation spectrometry. Protein content of each sample was determined by the Lowry method utilizing freeze-dried bovine serum albumin as the protein standard.

Real-time RT-PCR analysis.

RNA from confluent endothelial cells was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was synthesized using the TaqMan Reverse Transcription Gene Expression Assay kit (Applied Biosystems, Carlsbad, CA). Real-time PCR analysis of iPLA2 isoforms and 18s RNA was performed using iPLA2 and 18s RNA-specific Taqman primer/probes and the ABI 7500 Real-Time PCR System (Applied Biosystems). Relative quantitation of each iPLA2 isoform in endothelial cells was determined by comparing ΔCT (where CT is cycle threshold) between iPLA2 isoforms and 18s RNA for each isolation.

Measurement of total arachidonic acid release.

Endothelial cells were incubated at 37°C with 3 μCi [3H]arachidonic acid for 18 h. This incubation resulted in >70% incorporation of radioactivity into membrane phospholipids. After incubation, endothelial cells were washed three times with Tyrode solution containing 0.36% bovine serum albumin to remove unincorporated [3H]arachidonic acid. Endothelial cells were incubated at 37°C for 15 min before being subjected to experimental conditions. At the end of the stimulation period the supernatant was removed. Endothelial cells were lysed in 10% sodium dodecyl sulfate, and radioactivity in both supernatant and pellet was quantified by liquid scintillation spectrometry.

PAF assay.

Endothelial cells grown in 12-well culture dishes were washed twice with HBSS containing (in mM) 135 NaCl, 0.8 MgSO4, 10 HEPES (pH 7.4), 1.2 CaCl2, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4 and 6.6 glucose and were incubated with 50 μCi [3H]acetic acid for 20 min. After the selected time interval for incubation with the appropriate agents, the synthesis and degradation of PAF were terminated by the addition of ice-cold methanol. The cells and surrounding buffer were removed from the tissue culture plate using a cell scraper and added to Teflon tubes containing 7 nmol carrier PAF (β-acetyl-γ-O-hexadecyl-l-α-phosphatidylcholine, Sigma Chemical) to improve recovery and visualize PAF zones on thin-layer chromatography (TLC) plates. The lipids, including PAF, were isolated by chloroform-methanol extraction. The chloroform layer was concentrated by evaporation under N2, applied to a silica gel 60 TLC plate, and developed in chloroform-methanol-acetic acid-water (50/25/8/4 vol/vol). The region corresponding to PAF was scraped, and radioactivity was quantified using liquid scintillation spectrometry. Loss of PAF during extraction and chromatography was corrected for by the addition of 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. The synthesized [14C]PAF was purified by HPLC. Analysis of PAF production was validated by the addition of a known amount of hexadecyl-2-acetyl-sn-glyceryl-3-phosphoryl choline, 1-0-[acetyl-3H(N)] (Perkin Elmer, Boston, MA) to representative samples.

Prostaglandin release.

Endothelial cells were grown to confluence in 16-mm tissue culture dishes. Cells were washed twice with HBSS containing (in mmol/l) 135 NaCl, 0.8 MgSO4, 10 HEPES (pH 7.6), 1.2 CaCl2, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, and 6.6 glucose. After the cells were washed, 0.5 ml HBSS with 0.36% bovine serum albumin was added to each culture well. Following stimulation, the surrounding buffer was removed after selected time intervals, and prostaglandin release was measured immediately using enzyme-linked immunoassay kits. PGI2 release was measured as its stable metabolite 6-keto-prostaglandin F (Oxford Biomedical Research, Oxford, MI). PGE2 release was measured directly (R&D Systems, Minneapolis, MN).

PAF-acetylhydrolase assay.

Endothelial cells were grown to confluence and incubated with BEL for 10 min and were then removed from the culture plates 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 sodium acetate. Released [3H] acetic acid was isolated by passing the reaction mixture through a C18 silica gel column (J. T. Baker, Phillipsburg, NJ), and eluted radioactivity was measured using a liquid scintillation counter.

Statistical analysis.

All studies were repeated with at least four separate cell cultures. Data were analyzed using Student's t-test or one-way analysis of variance followed by post hoc analysis using Dunnett's test. Differences were regarded as significant at P < 0.05 and highly significant at P < 0.01. Error bars in the figures represent the standard error of the mean.

RESULTS

PLA2 activity in cardiac endothelial cells.

PLA2 activity was measured in cardiac endothelial cells isolated from WT and iPLA2-KO mice, using (16:0,[3H]18:1) plasmenylcholine or phosphatidylcholine substrates in the presence (1 mM Ca2+) or absence (4 mM EGTA) of Ca2+ (Table 1), and most cardiac endothelial cell PLA2 activity did not require the presence of Ca2+. Mouse iPLA2β- and iPLA2γ-KO endothelial cells exhibited less PLA2 activity than WT cardiac endothelial cells under all conditions studied (Table 2). When enzyme (50 μg of cellular protein) was incubated with increasing concentrations of (R)-BEL (0.05 to 1.0 μM for 10 min), the iPLA2 activity in WT cardiac endothelial cells and HCAECs was not inhibited at concentrations of 1 μM or less (Fig. 1), which indicates that iPLA2γ does not contribute significantly to their total iPLA2 under these conditions. Endothelial cells isolated from iPLA2γ-KO mice demonstrated a lower iPLA2 activity than cells from WT mice or HCAECs, but they also demonstrated no inhibition of iPLA2 activity at (R)-BEL concentrations below 1 μM. iPLA2β-KO endothelial cells had significantly less iPLA2 activity than WT endothelial cells or HCAECs, but the residual iPLA2β-KO endothelial cell iPLA2 activity was sensitive to inhibition by (R)-BEL at concentrations below 1 μM, suggesting that the residual activity is attributable to iPLA2γ that may be upregulated to compensate for the absence of iPLA2β. These data indicate that cardiac endothelial cell PLA2 activity is largely Ca2+-independent and that iPLA2β is the predominant iPLA2 isoform in WT cells.

Table 2.
PLA2 activity in cell lysate from WT and iPLA2-KO cardiac endothelial cells under control, thrombin- (0.05 IU/ml, 10 min), or tryptase-stimulated (20 ng/ml, 10 min) conditions
Fig. 1.
Calcium-independent phospholipase A2 (iPLA2) activity measured in human coronary artery endothelial cells (HCAECs), wild-type (WT) cardiac and iPLA2β- or iPLA2γ-knockout (KO) cardiac endothelial cells in the presence of (R)-bromoenol lactone ...

Thrombin (0.05 IU/ml, 10 min) or tryptase (20 ng/ml, 10 min) stimulation of mouse cardiac endothelial cells resulted in a significant increase in iPLA2 activity (Table 2). Increased thrombin- or tryptase-stimulated iPLA2 activity was similar between WT and iPLA2γ-KO endothelial cells, whereas that in iPLA2β-KO endothelial cells was smaller. Activation of iPLA2 activity by thrombin (0.05 IU/ml, 10 min) did not result in a significant change in iPLA2 mRNA expression (Table 1), suggesting that the increase in iPLA2 activity is a result of activation of a latent enzyme in endothelial cells. These findings caused us to hypothesize that iPLA2β might play a central role in lipid signaling in cardiac endothelial cells, and we next measured arachidonic acid release and production of PGI2 and PAF in WT and iPLA2-KO mouse cardiac endothelial cells.

Arachidonic acid release.

Arachidonic acid released from WT and iPLA2-KO mouse cardiac endothelial cells was measured following treatment with tryptase (20 ng/ml) or thrombin (0.05 IU/ml) for up to 30 min (Fig. 2). WT endothelial cells released significantly greater amounts of arachidonic acid after stimulation, whereas both iPLA2β- and iPLA2γ-KO cardiac endothelial cells released smaller amounts of arachidonic acid, which indicates that PAR stimulation-induced hydrolysis of arachidonic acid from endothelial cell membrane phospholipids is catalyzed predominantly by both iPLA2β and iPLA2γ.

Fig. 2.
Arachidonic acid release from tryptase (20 ng/ml; top) or thrombin (0.05 IU/ml; bottom)-stimulated WT, iPLA2β-KO, or iPLA2γ-KO cardiac endothelial cells. Results represent means ± SE of 4 independent experiments. **P < ...

PGI2 production.

Prostacyclin (PGI2) is the predominant prostaglandin generated by endothelial cells as a result of sequential arachidonic acid release from membrane phospholipids and subsequent hydrolysis by cyclooxygenases and prostacyclin synthase. Since iPLA2 isoforms catalyze arachidonic acid release in cardiac endothelial cells, they may also be involved in PGI2 production. To examine the contributions of iPLA2 isoforms in cardiac endothelial cell PGI2 production, we compared the time course of PGI2 release from WT, iPLA2β-KO, and iPLA2γ-KO cardiac endothelial cells stimulated with thrombin (0.05 IU/ml) or tryptase (20 ng/ml) for up to 30 min (Fig. 3). WT endothelial cells exhibited increased PGI2 production after stimulation, and iPLA2γ-KO endothelial cells exhibited a similar response. In contrast, iPLA2β-KO cardiac endothelial cells exhibited significantly attenuated thrombin-stimulated PGI2 production compared with WT and iPLA2γ-KO endothelial cells (Fig. 3), and the majority of thrombin-stimulated PGI2 release appears to require iPLA2β activity. Tryptase-stimulated PGI2 release was similar for iPLA2β-KO and iPLA2γ-KO cardiac endothelial cells but was significantly less than that from WT hearts (Fig. 3). These data suggest that the signaling pathways for coupling PAR-1 receptor activation and iPLA2 production differ from those for PAR-2 receptors.

Fig. 3.
PGI2 release from thrombin (0.05 IU/ml; top) or tryptase (20 ng/ml; bottom)-stimulated WT, iPLA2β-KO, or iPLA2γ-KO cardiac endothelial cells. Results represent means ± SE of 4 independent experiments. **P < 0.01 compared ...

We next determined the effects of BEL enantiomers on thrombin- or tryptase-stimulated WT cardiac endothelial cell PGI2 production and found that both (R)-BEL (5 μM, 10 min) and (S)-BEL (5 μM, 10 min) inhibited PGI2 production and that the latter was more effective, which indicates that both iPLA2β and iPLA2γ are involved in PGI2 production (Fig. 4). When endothelial cells were incubated with racemic BEL (10 μM, 10 min), PGI2 production was inhibited completely following stimulation with thrombin or tryptase (Fig. 4). Prostaglandin E2 release from WT cardiac endothelial cells was significantly increased by thrombin (0.05 IU/ml, 10 min) or tryptase (20 ng/ml, 10 min) stimulation (Fig. 5). Cardiac endothelial cells isolated from iPLA2γ or iPLA2β-KO mice demonstrated smaller increases in PGE2 production in response to protease stimulation (Fig. 5). Thus PGI2 and PGE2 production are reduced similarly in iPLA2-KO endothelial cells, suggesting that this is a direct result of decreased arachidonic acid release.

Fig. 4.
WT endothelial cells were stimulated with tryptase (20 ng/ml) or thrombin (0.05 IU/ml) for 10 min. PGI2 release was measured with and without pretreatment with (R)- and (S)-BEL (5 μM, 10 min) or racemic BEL (10 μM, 10 min). Results represent ...
Fig. 5.
PGE2 release from thrombin (0.05 IU/ml, 10 min) or tryptase (20 ng/ml, 10 min)-stimulated WT, iPLA2β-KO, or iPLA2γ-KO cardiac endothelial cells. Results represent means ± SE of 4 independent experiments. **P < 0.01 compared ...

PAF production.

In WT endothelial cells a significant increase in PAF production in response to thrombin or tryptase was observed (Fig. 6). Pretreatment with (R)-BEL did not affect PAF production, but pretreatment with (S)-BEL significantly reduced PAF production to a level similar to that for the untreated control, suggesting that iPLA2β activity is required for protease-stimulated cardiac endothelial cell PAF production.

Fig. 6.
Platelet-activating factor (PAF) production in WT, iPLA2β-KO, and iPLA2γ-KO cardiac endothelial cells unstimulated or stimulated with tryptase (try, 20 ng/ml, 10 min) or thrombin (thr, 0.05 IU/ml, 10 min), with or without (R)- or (S)-BEL ...

To exclude a significant contribution of iPLA2γ, PAF production by tryptase- or thrombin-stimulated iPLA2γ-KO cells was compared with that of WT and iPLA2β-KO endothelial cells (Fig. 6). As with WT endothelial cells, iPLA2γ-KO cells exhibited increased PAF production upon protease stimulation, and this was unaffected by pretreatment with (R)-BEL. In contrast, pretreatment with (S)-BEL inhibited PAF production completely. Similarly, tryptase or thrombin stimulation of iPLA2β-KO endothelial cells resulted in no increase in PAF production (Fig. 6).

PAF-acetylhydrolase activity.

To determine whether variations in PAF-acetylhydrolase (PAF-AH), the enzyme responsible for PAF catabolism, might constrain net PAF production by cardiac endothelial cells, we measured PAF-AH activity in WT and iPLA2β-KO cells without or with BEL pretreatment (Fig. 7) and observed no significant difference in basal PAF-AH activity between WT and iPLA2β-KO cells and no effect of BEL on their PAF-AH activity (Fig. 7). These data demonstrate that the absence of PAF production in iPLA2β-KO cardiac endothelial cells does not result in increased PAF-AH activity and PAF catabolism.

Fig. 7.
PAF-acetylhydrolase (AH) activity in WT and iPLA2β-KO cardiac endothelial cells without or with BEL (5 μM, 10 min) pretreatment.

DISCUSSION

In this study we have demonstrated that the majority of cardiac endothelial cell iPLA2 activity is attributable to iPLA2β, an enzyme that has been cloned from hamster (25), rat (10), mouse (2), and human cells (9), inter alia. This isoform is classified as the group VIA PLA2 (11, 23) that is expressed in various tissues as splice variants, including 84-kDa and 88-kDa species, and as proteolysis products, including a 70-kDa variant, inter alia (9). Each of these variants contains a GXSXG lipase consensus motif and a stretch of seven to eight ankyrin-like repetitive sequence motifs that mediate protein interactions in other proteins (25). Studies involving pharmacological inhibition of iPLA2β with BEL have suggested its involvement in modulating arachidonic acid release from vascular cells and vasomotor tone, among other processes (5, 14). BEL, like other pharmacologic inhibitors, has a number of recognized off-target effects and probably also has such effects that are not yet recognized. This has motivated the preparation of genetically modified cell lines and animals with altered iPLA2 expression to characterize the participation of the enzyme in various biological processes (3, 12, 14). In this study we have demonstrated that iPLA2β is the predominant Ca2+-independent PLA2 in WT and iPLA2-KO mouse cardiac endothelial cells and that iPLA2β activity is responsible for most of the protease-stimulated PAF, PGE2, and PGI2 production by these cells. However, cardiac endothelial cells from iPLA2γ-KO mice indicate that iPLA2γ can also contribute to prostaglandin production. These findings with cells from genetically modified mice were corroborated by experiments with BEL enantiomers to strengthen the hypothesis that iPLA2β is involved in protease-stimulated cardiac endothelial cell production of PAF, PGE2, and PGI2.

Vascular endothelium plays important roles of vascular homeostatic processes and responses to injury, and phospholipid-derived mediators are involved in endothelial cell signaling events (21). In its basal state, the endothelium is required to control inflammation and coagulation, but in response to destructive stimuli, endothelial cells produce a variety of vasoactive and proinflammatory mediators, including the bioactive lipids PAF and PGI2 (27). These compounds are formed from the precursors arachidonic acid and lyso-PAF that are generated by the action of iPLA2 on membrane phospholipid substrates. The generation of these precursors is the rate-limiting step for formation of eicosanoids and PAF.

Human coronary artery endothelial cells (HCAECs) and cardiac endothelial cells isolated from WT mice have comparable levels of iPLA2 activity, and it is sensitive to inhibition by relatively high concentrations of (R)-BEL. In contrast, iPLA2β-KO mouse cardiac endothelial cells have much lower levels of iPLA2 activity that is sensitive to inhibition by low concentrations of (R)-BEL, suggesting that this residual activity is attributable to iPLA2γ. Our previous studies have demonstrated that tryptase or thrombin activate HCAEC iPLA2, resulting in arachidonate release, and production of PGE2 and PGI2 (19). Activation of endothelial cell iPLA2 and the resultant release of free arachidonic acid can lead to the formation of multiple eicosanoids that are involved in inflammation. Endothelial cell arachidonic acid is metabolized by cytochrome P450 to produce epoxyeicosatrienoic acids in response to shear stress, stretch, and bradykinin (4). Leukotrienes have been implicated in the inflammatory component of atherosclerosis (1) and can induce proinflammatory signaling via activation of specific leukotriene receptors (20). PGI2 is the predominant prostaglandin produced by endothelial cells and has been implicated in acute and chronic pain (18). PGI2 contributes to normal inflammatory responses by promoting vasodilation and increasing vascular permeability. PGI2 is also cardioprotective since it inhibits platelet and lymphocyte adhesion to endothelium, restricts vascular smooth muscle cell proliferation and migration, and prevents production of profibrotic growth factors (28).

Our data here demonstrate that iPLA2β activity contributes to tryptase- or thrombin-stimulated cardiac endothelial cell PGI2 production and thereby would exert a cardioprotective effect. iPLA2γ may also contribute to PGI2 production because its pharmacological inhibition resulted in a reduction in PGI2 production, and iPLA2γ-KO mouse cardiac endothelial cells exhibited a reduction in protease-stimulated PGI2 production that could reflect coupling with cyclooxygenases, PARs, or other PLA2 family members. That only a small amount of arachidonic acid is released from iPLA2β-KO endothelial cells suggests that the reduction in PGI2 production by these cells results from the low availability of free arachidonic acid substrate for cyclooxygenase, and it is unlikely that members of the PLA2 family other than iPLA2 make significant contributions to this substrate pool under these conditions. We have observed different patterns of PGI2 production here in response to thrombin compared with tryptase, and this suggests that distinct populations of PAR may be differentially coupled to signaling events involved in PLA2 activation, arachidonate release, and prostanoid generation. This sequence requires further scrutiny to clarify the mechanisms whereby distinct proteases couple to their cognate receptors to initiate the cascade of intracellular events that culminate in the production of lipid mediators that influence vascular biologic processes.

The endothelium regulates trafficking of cells from blood to tissues via cell surface adherence molecules (16). In this setting, PAF is considered an important inflammatory mediator, and it is expressed on the surfaces of endothelial cells where it regulates inflammatory cell endothelial transmigration and causes increased vascular permeability. PAF is involved in cardiac anaphylaxis, ischemia-reperfusion cardiac injury, and atherogenesis, among other (patho)physiologic processes. PAF interacts with endothelial cell PAF receptors (PAFR) and with PAFR on inflammatory cells that include neutrophils, macrophages, and eosinophils. The interaction of PAF with circulating leukocyte PAFRs results in leukocyte activation, adhesion, and migration, resulting in leukocyte recruitment to sites of injury. PAF also facilitates NF-κB translocation to the nucleus and may thereby contribute to early inflammatory cytokine generation (13). Leukocyte activation also results in generation of reactive oxygen species, lipid mediators, cytokines, and degradative enzymes. These mediators are released to restrict and eradicate the inflammatory stimulus but can subsequently cause tissue injury in inflammatory diseases (15). Oxidants and free radicals modify proteins and nucleic acids and activate the immune and inflammatory systems to produce deleterious effects.

PAF synthesis and degradation are highly regulated processes. PAF-acetylhydrolase (PAF-AH) is a PLA2 family member that catalyzes hydrolysis of PAF to biologically inactive lyso-PAF (8). We have previously demonstrated that thrombin or tryptase stimulation of HCAECs results in PAF production by an iPLA2-dependent process, and inhibition of HCAEC PAF-AH activity increases the net amount of PAF produced (6). In the studies described here, stimulation of WT mouse cardiac endothelial cells with tryptase or thrombin resulted in increased PAF production. Experiments with BEL enantiomers to selectively inhibit iPLA2 isoforms indicated that iPLA2β is important for protease-stimulated cardiac endothelial cell PAF production but that iPLA2γ made little contribution. These findings are consistent with the observations that iPLA2β-KO mouse cardiac endothelial cells failed to respond to tryptase or thrombin stimulation by increasing PAF production. As expected, iPLA2γ-KO mouse cardiac endothelial cells did respond to protease stimulation by increasing PAF production but were unable to so when pretreated with the iPLA2β inhibitor (S)-BEL. Together these findings suggest that iPLA2β is almost exclusively responsible for PAF production in cardiac endothelial cells.

To determine whether variations in PAF-AH activity might explain the low PAF production by iPLA2β-KO mouse cardiac endothelial cells, PAF-AH activity was directly measured, and WT and iPLA2β-KO mouse cardiac endothelial cells exhibited similar levels PAF-AH activity. Pretreatment of the cells with BEL failed to affect PAF-AH activity, and together these findings indicate that net PAF production under these conditions is not governed by variations in the level of PAF-AH activity. Because PAF is responsible for inflammatory cell transmigration and activation and plays important roles in cardiac (patho)physiological processes, interventions targeted at affecting local PAF levels based on an understanding of factors modulating its production may prove to be therapeutically beneficial.

In summary, our studies demonstrate that iPLA2β and iPLA2γ have distinct roles in cardiac endothelial cell prostacyclin and PAF production in response to inflammatory stimuli. To the best of our knowledge, this is the first study to examine the role of iPLA2β in cardiac endothelial cell lipid mediator production that uses genetically modified mice and that does not rest primarily on pharmacologic inhibitors with known and potential off-target effects. Our data demonstrate that iPLA2β is the predominant iPLA2 isoform in cardiac endothelial cells, that iPLA2β is solely responsible for protease-stimulated cardiac endothelial PAF production, and that iPLA2β also accounts for majority of the PGI2 production under these conditions. These findings indicate that iPLA2β is an important component of the inflammatory signaling via participation in the production of two major lipid modulators of inflammation. Although inflammation can represent a protective response to remove injurious insults and to initiate healing, prolongation of the inflammatory response can cause tissue destruction and impair wound healing. Better understanding of the processes governing production and persistence of inflammatory mediators such as PAF and eicosanoids may permit development of means to manipulate these processes in therapeutically beneficial ways. Our studies suggest that interventions targeted at modulating the activity of iPLA2 isoforms could be useful in that regard.

GRANTS

This work was supported in part by United States Public Health Service Grants R37-DK34388, R01 HL041250, PPG P01 HL057278, P41-RR00954, P60-DK20579, and P30-DK56341.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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