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(Circulation Research. 1996;78:780-789.)
© 1996 American Heart Association, Inc.

Articles

Lysophosphatidylcholine Promotes P-Selectin Expression in Platelets and Endothelial Cells

Possible Involvement of Protein Kinase C Activation and Its Inhibition by Nitric Oxide Donors

Toyoaki Murohara, Rosario Scalia, Allan M. Lefer

From the Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Allan M. Lefer, PhD, Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107-6799.

	Abstract

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Abstract Lysophosphatidylcholine (LysoPC), an atherogenic lysophospholipid contained in oxidized low-density lipoprotein (LDL), has been shown to stimulate protein kinase C (PKC). Since PKC activators are suggested to elicit rapid P-selectin expression in platelets and endothelial cells, we examined whether LysoPC promotes P-selectin expression in platelets and P-selectin–mediated leukocyte adherence to endothelial cells via a mechanism involving PKC activation. LysoPC, but not phosphatidylcholine (PC), which is a major phospholipid component in native LDL, significantly upregulated P-selectin on cat platelets by flow cytometric analysis. This P-selectin upregulation by LysoPC was significantly attenuated by two PKC inhibitors, 7-hydroxystaurosporine (UCN-01) and N,N,N-trimethylsphingosine, and by two NO donors, CAS1609 and sodium nitroprusside. Submicellar concentrations of LysoPC significantly activated PKC in platelets, and this was inhibited by either UCN-01 or CAS1609. LysoPC, but not PC, significantly increased adherence of autologous cat polymorphonuclear leukocytes to coronary vascular endothelium, which was also markedly attenuated by UCN-01 and by CAS1609. LysoPC induced P-selectin expression on the surface of cat coronary vascular endothelium as assessed by immunohistochemical analysis. These results suggest that LysoPC, an atherogenic lysophospholipid contained in oxidized LDL, rapidly induces P-selectin expression in both platelets and endothelial cells at least partially via PKC activation. Furthermore, NO-generating agents may inhibit P-selectin upregulation by LysoPC. Since P-selectin may play an important role in initiating atherosclerosis, our data provide further insight into the mechanism of early stages of atherogenesis and of NO-mediated inhibition of atherosclerosis.

Key Words: flow cytometry • cell adhesion molecules • platelets • leukocyte adherence • endothelium

	Introduction

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Oxidized LDL plays an important role in vascular atherogenesis.^{1 2} Also, recruitment of inflammatory cells into atherosclerotic sites is an essential event in the development of atherosclerosis.³ From these points of view, ox-LDL–induced interaction between inflammatory leukocytes and endothelial cells has received considerable attention.^{4 5} Lehr et al⁵ recently reported that human ox-LDL increases the number of rolling and adherent leukocytes in the hamster mesenteric microcirculation in vivo, and these effects were reduced by an anti–P-selectin antibody. During oxidative modification of LDL, intrinsic phospholipase A₂ substantially degrades PC to LysoPC.⁶ Ox-LDL and LysoPC indeed have been shown to accumulate in the atherosclerotic arterial wall,^{7 8} impair endothelial NO release,^{9 10 11} upregulate cell adhesion molecules (ie, ICAM-1 and VCAM-1),^{12 13} and act as a monocyte chemoattractant.¹⁴

Although Lehr et al⁵ reported that ox-LDL promotes P-selectin expression, the precise mechanisms remain unclear. LysoPC in ox-LDL has been shown to activate PKC.^{15 16 17} This function is relevant, since LysoPC inhibits endothelial NO release and increases ICAM-1 expression at least partially by PKC activation.^{13 18} Ohara et al¹⁹ further reported that LysoPC stimulates vascular superoxide production by PKC activation. These studies suggest that LysoPC stimulates the signal transduction pathway involving PKC and elicits significant pathophysiological effects.

The role of PKC in cell adhesion biology is emerging. Geng et al²⁰ showed that activation of PKC induces rapid P-selectin expression and neutrophil adherence to endothelium. P-Selectin is normally stored in Weibel-Palade bodies of endothelial cells and in -granules of platelets and is rapidly translocated to the cell surface after activation by inflammatory mediators.^{21 22} P-Selectin supports leukocyte rolling on the endothelial surface. Thrombin, a stimulator of P-selectin, also activates phospholipase C, promoting phosphoinositide turnover and PKC activation.²³ Conversely, PKC inhibitors have been shown to inhibit P-selectin expression in thrombin-stimulated platelets.^{24 25} Moreover, Hannun et al²⁶ suggested that PKC activation is a necessary and common event for platelet activation. These studies collectively support the concept that PKC activation may be involved in rapid P-selectin upregulation on the cell surface. Since LysoPC has been shown to activate PKC,¹⁸ we reasoned that LysoPC may induce P-selectin expression. This issue is relevant, since both LysoPC and P-selectin may play an important role in early atherogenesis.^{1 27 28} Although LysoPC has been shown to promote ICAM-1 and VCAM-1 expression, leukocyte adhesion normally requires an initial rolling step that is mainly mediated by selectins, and little is known about the effects of LysoPC on rapid P-selectin expression in endothelial cells.

Therefore, we examined the effects of LysoPC on rapid P-selectin expression on platelets and the mechanism of the LysoPC-induced P-selectin upregulation in platelets with special reference to PKC activation. We also studied the effects of LysoPC on immunohistochemical localization of P-selectin on the endothelium and on neutrophil-endothelium interaction. Since NO has been shown to inhibit vascular atherogenesis,^{29 30} we also examined whether NO donors can modulate LysoPC-induced P-selectin expression in these cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Flow Cytometric Determination of P-Selectin Expression on Cat Platelets
Flow cytometric analysis of P-selectin expressed on cat platelets was performed by previously described methods.³¹ Fifteen adult male cats (2.6 to 3.4 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). Arterial blood (100 mL) was drawn and anticoagulated with sodium citrate (Sigma Chemical Co). Platelet-rich plasma was obtained by centrifuging the blood at 200g for 15 minutes at room temperature. The platelet-rich plasma was recentrifuged at 2000g for 10 minutes to form a platelet-rich pellet, which was washed twice in Ca²⁺-free Tyrode's solution containing 0.2% BSA. The final cell pellet was resuspended in PBS containing 1 mmol/L Ca²⁺.

Aliquots of platelet suspensions were incubated with either thrombin (2 U/mL), PMA (100 nmol/L), palmitoyl LysoPC (10 µmol/L), egg yolk LysoPC (10 µmol/L), or dipalmitoyl PC (10 µmol/L) at 37°C for 10 minutes without stirring. In another experiment, aliquots of platelets were first treated with either of two PKC inhibitors, UCN-01 (100 nmol/L)³² or TMS (10 µmol/L),^{24 25} or one of two NO donors, CAS1609 (10 µmol/L) or sodium nitroprusside (100 µmol/L), for 10 minutes. Platelets were subsequently stimulated with palmitoyl LysoPC (10 µmol/L) for 10 minutes at 37°C without stirring. In a preliminary study, we tested the effects of an inactive non–NO-donating agent, C93-4845, on LysoPC-induced P-selectin expression; however, C93-4845 had no effect on any parameter studied.

After incubation, platelets were fixed by 1% paraformaldehyde in PBS at pH 7.2 and washed twice with PBS containing 0.2% BSA. The platelet suspensions were treated with the primary anti–P-selectin MAb, PB 1.3 (20 µg/mL), and were kept at 4°C for 60 minutes. After the incubation, platelets were washed in PBS with 0.2% BSA. F(ab')₂ fragments of a goat polyclonal anti-mouse IgG-phycoerythrin conjugate (Tago Inc) were used as the secondary antibody at a 1:100 dilution, and the cells were kept at 4°C for 30 minutes. The stained platelets were washed twice, fixed in 1% paraformaldehyde, and immediately analyzed by flow cytometry (FACScan, Becton-Dickinson).

Measurement of PKC Activity of Platelet Membrane Fraction
The platelets were suspended in modified Tyrode's/HEPES buffer, pH 7.4, containing (mmol/L) NaCl 134, KCl 2.9, NaHCO₃ 12, CaCl₂ 1, HEPES 5, and glucose 5. In the first experiment, aliquots of platelet suspensions were treated with graded concentrations of either palmitoyl LysoPC (1, 10, 30, and 100 µmol/L) or dipalmitoyl PC (1, 10, 30, and 100 µmol/L) for 10 minutes at 37°C. After incubation, the cells were lysed by sonication eight times for 2 seconds on ice. Additional platelet aliquots were treated with graded concentrations of the PKC inhibitor UCN-01 (1, 10, 100, and 1000 nmol/L) for 10 minutes. Subsequently, these aliquots were treated with a fixed concentration of palmitoyl LysoPC (10 µmol/L) for an additional 10 minutes. After this incubation, cells were lysed as described above. In the second experiment, the time course of PKC activation in platelet membranes was examined. Aliquots of platelet suspensions were treated with either PMA (100 nmol/L), LysoPC (10 µmol/L), or LysoPC+CAS1609 (10 µmol/L). Immediately before (0 minutes [control]) and after 1, 5, 10, and 20 minutes of incubation at 37°C, platelet suspensions were lysed on ice as described above. In the third experiment, platelet suspensions were treated with a fixed concentration of either PMA (100 nmol/L), thrombin (2 U/mL), palmitoyl LysoPC (10 µmol/L), or dipalmitoyl PC (10 µmol/L) in parallel at 37°C for 10 minutes. Cells were then lysed, and the lysates were immediately ultracentrifuged at 87 000g for 60 minutes at 4°C to separate the membrane fraction. The membrane of the pellet was then resuspended in 200 µL glycerol/Tris buffer, and PKC activity was measured.

The PKC activity of the platelet membrane suspension was measured by a method previously described,³¹ which is a colorimetric assay modified from Toomik et al³³ (Pierce). The relative PKC activity was expressed as a percentage of the PMA (100 nmol/L)–induced maximum phosphorylation of the PKC substrate. Protein concentration was assayed using the biuret method of Gornall et al.³⁴

Determination of NO Concentrations in Platelet Suspensions Treated With NO Donors
The selectivity of the Iso-NO electrode (World Precision Inc) to NO was previously determined by measurement of NO from authentic NO gas.³⁵ Calibration of the NO-specific electrode was performed daily just before use. Twenty milliliters of calibration solution containing 0.1 mol/L KI and 0.1 mol/L H₂SO₄ was purged with nitrogen gas for 20 minutes. After purging, graded concentrations of KNO₂ were added to the calibration solution to generate NO. KNO₂ reacts with KI and H₂SO₄ to generate NO according to the following equation:

In the present study, the standard calibration curve was generated by adding graded concentrations of KNO₂ at 0, 5, 10, 25, 50, 100, 250, and 500 nmol/L into the measuring bottle containing the calibration solution.

We measured NO release in platelet suspensions from two NO donors, CAS1609 and sodium nitroprusside. Cat platelet suspensions (1x10⁸ cells per milliliter) in 2 mL modified Tyrode's/HEPES buffer were treated with either CAS1609 (10 µmol/L) or sodium nitroprusside (100 µmol/L) for 10 minutes at room temperature. Maximum NO concentrations in platelet suspensions were subsequently measured by an amperometric method using an NO-specific electrode, Iso-NO, which has been previously described in detail.³⁵

Cat Neutrophil Isolation
Cat neutrophils were isolated by a Percoll-density gradient method from peripheral blood (100 mL) collected in citrate-phosphate-dextrose solution, which has been previously described.³¹ PMN preparations obtained by this method were >95% pure and >95% viable by trypan blue exclusion. The PMN pellet was finally suspended in 2 mL of Dulbecco's PBS, and the number of cells was counted using a hemocytometer.

Preparation of Cat Coronary Artery Segments
Immediately after drawing 100 mL of blood, the heart was rapidly excised, and coronary artery segments were isolated and prepared according to the method previously reported.³¹ Arteries were cut into rings 2 to 3 mm in length and cut open for studies of PMN-endothelium adherence.

Autologous Cat PMN Adherence to Coronary Endothelium
PMNs were labeled with a fluorescent dye, PKH2-GL (Sigma), according to the method previously described.³⁶ This labeling procedure does not affect the normal morphology or function of cat PMNs.^{31 36}

Coronary segments were placed with their endothelial surface up in culture dishes filled with 3 mL oxygenated K-H buffer (37°C). In the first experiment, coronary segments were incubated with either 2 U/mL thrombin, 10 µmol/L palmitoyl LysoPC, 10 µmol/L dipalmitoyl PC, or the PKC activator PMA (100 nmol/L) for 10 minutes. After this incubation, segments were replaced into other dishes filled with fresh K-H solution, and then labeled autologous PMNs (4x10⁵ cells per milliliter) were added and incubated for an additional 20 minutes. During this period, the culture dishes were agitated in a shaker bath at 37°C. After the incubation, coronary segments were removed, placed onto glass slides, and covered with a coverslip. Labeled PMNs adherent to the coronary endothelial surface were counted using an epifluorescence microscopy (Nikon Diaphot). Adherent neutrophils on five regions of each segment were randomly counted and expressed as the number of PMNs per square millimeter of endothelial surface.

In the second experiment, coronary segments were first incubated with either the PKC inhibitor UCN-01 (100 nmol/L) or the NO donor CAS1609 (10 µmol/L) or their vehicle for 10 minutes. After this, palmitoyl LysoPC (10 µmol/L) was further added to each bath and coincubated with coronary segments for 10 minutes. In some segments, the NO donor CAS1609 was incubated for 10 minutes either before or after LysoPC treatment. In other coronary segments, PMA (100 nmol/L) was added under sterile conditions for 24 hours to downregulate PKC activity. After this incubation, arterial segments were treated with LysoPC for 10 minutes. Subsequently, coronary segments were removed to fresh K-H solution, and labeled PMNs were added and incubated for 20 minutes. Adherent PMNs were counted as described above.

To test whether LysoPC-mediated PMN-endothelium interaction is dependent on P-selectin on endothelial cells, coronary segments were first incubated with palmitoyl LysoPC (10 µmol/L) for 10 minutes. Subsequently, segments were transferred to cell culture dishes with fresh K-H solution containing either the anti–P-selectin MAb PB 1.3 (20 µg/mL) or a nonblocking control anti–P-selectin MAb NBP 1.6 (20 µg/mL) and were incubated for 5 minutes. PMNs were then added and incubated for 20 minutes. Adherent PMNs were counted as described above.

Immunohistochemistry
To test the effects of LysoPC on the coronary endothelial expression of P-selectin, coronary rings were isolated from an additional three control cats. Rings were incubated with either K-H solution or K-H solution containing either thrombin (2 U/mL), palmitoyl LysoPC (10 µmol/L), LysoPC plus UCN-01 (100 nmol/L), or LysoPC plus CAS1609 (10 µmol/L) at 37°C for 10 minutes. After this incubation, rings were placed into 4% paraformaldehyde (Sigma) in PBS at 4°C, and the rings were fixed for 2 hours on ice. After dehydration, rings were embedded in plastic embedding solution (Polysciences Inc), and 4-µm-thick sections from the tissue blocks were prepared by a glass-cutting microtome.

Immunohistochemical procedures with these plastic sections were performed using the methods of Beckstead et al.³⁷ Sections were first treated with the primary anti–P-selectin monoclonal antibody PB 1.3 overnight at room temperature at a dilution of 1:100 and then by the avidin-biotin immunoperoxidase technique (Vectastain ABC reagent, Vector Laboratory). The sections were counterstained with Gill's hematoxylin 3 (Sigma) and examined using a Zeiss Axioplan microscope. MAb PB 1.3 cross-reacts with feline endothelial cells and platelets.^{31 38 39}

Antibodies and Reagents
Thrombin, palmitoyl L--LysoPC, egg yolk L--LysoPC, dipalmitoyl L--PC, D-sphingosine, and PMA were purchased from Sigma. Solutions of LysoPC, PC, sphingosine, and phosphatidylserine (PKC activator) were sonicated before use. The selective PKC inhibitor UCN-01 was a gift from Kyowa Hakko Kogyo. TMS was a gift from Dr S. Hakomori, Biomembrane Institute. CAS1609 and control compound C93-4845 were gifts from Cassella AG. MAb PB 1.3 binds to P-selectin and blocks the interaction between P-selectin and its ligands, whereas control MAb NBP 1.6 binds to P-selectin but does not inhibit P-selectin–mediated adhesive interactions. MAbs PB 1.3 and NBP 1.6 are murine IgG₁ monoclonal antibodies raised against human P-selectin and were kindly provided by Dr J.C. Paulson, Cytel Corp. Our previous studies demonstrated that MAb PB 1.3 and NBP 1.6 bind avidly to feline platelets and endothelial cells.^{31 38}

Statistical Analysis
Results are presented as mean±SEM, based on independent experiments. All data were subjected to ANOVA followed by Fisher's t test for evaluation of the difference among groups. Values of P<.05 were considered to be significant. The procedures and protocols in the present study were approved by the Thomas Jefferson University Committee on the Use and Care of Experimental Animals.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Flow Cytometric Analysis of P-Selectin Expression on Platelets Stimulated With Either Thrombin, PMA, or LysoPC
We first examined P-selectin expression in thrombin-stimulated cat platelets. Platelets incubated with only secondary phycoerythrin-conjugated anti-mouse IgG (ie, omission of the primary MAb PB 1.3) revealed only 3% positive staining for platelets and a mean channel fluorescence of 3.5, indicating a very small extent of nonspecific background antibody binding. Table 1 shows the mean channel fluorescence and percent positive staining for platelets without stimulation and after stimulation with either thrombin (2 U/mL), the PKC activator PMA (100 nmol/L), palmitoyl LysoPC (10 µmol/L), egg yolk LysoPC (10 µmol/L), a control phospholipid (dipalmitoyl PC, 10 µmol/L), which is mainly contained in native LDL, or a control lysolipid (sphingosine, 10 µmol/L). The percent positive staining and mean channel fluorescence of nonspecific background fluorescence (ie, omission of the primary MAb) were subtracted from each value. Ten-minute poststimulation was found to result in maximum expression of P-selectin on platelets. P-selectin expression was markedly enhanced after stimulation with either thrombin (Fig 1a) or PMA for 10 minutes (Table 1). After incubation with thrombin (2 U/mL), the binding of PB 1.3 to cat platelets increased by 3.5-fold compared with unstimulated platelets. Pretreatment with the PKC inhibitor UCN-01 (100 nmol/L) significantly attenuated both the percent P-selectin–positive cells and the mean channel fluorescence on thrombin-stimulated platelets (Table 1), suggesting that PKC activation significantly participates in the thrombin-induced upregulation of P-selectin.

View this table: [in this window] [in a new window]   Table 1. Flow Cytometric Analysis of P-Selectin Expression on Cat Platelets

View larger version (16K): [in this window] [in a new window]   Figure 1. Representative fluorescence histograms of P-selectin expression on cat platelets determined by flow cytometry (FACScan). Thrombin (2 U/mL) and palmitoyl LysoPC (10 µmol/L) induced significant rightward shifts of the fluorescence histograms (ie, P-selectin expression) compared with nonstimulated control platelets (a and b). The increased P-selectin expression induced by LysoPC was significantly attenuated by a selective PKC inhibitor, UCN-01 (100 nmol/L) (c) or an NO donor, CAS1609 (10 µmol/L) (d).

After treatment with PMA (100 nmol/L), the binding of PB 1.3 to platelets was markedly increased 5.5-fold compared with nonstimulated platelets (Table 1). Pretreatment with the PKC inhibitor UCN-01 (100 nmol/L) significantly inhibited P-selectin upregulation on PMA-stimulated platelets. Thus, PKC activation appears to mimic the effects of thrombin on platelet P-selectin expression (Table 1).

Both palmitoyl LysoPC (10 µmol/L) and egg yolk LysoPC (10 µmol/L) also significantly increased P-selectin expression on platelets 4.5-fold and 3.5-fold, respectively, compared with nonstimulated platelets. However, dipalmitoyl PC (10 µmol/L), which is mainly contained in the lipid fraction of native LDL but not in ox-LDL, failed to stimulate P-selectin expression (Table 1). To determine whether this effect of LysoPC is a nonspecific action of lysolipid, we also tested the effects of sphingosine, another control lysolipid (ie, lysosphingolipid), on platelet P-selectin expression. However, sphingosine failed to upregulate P-selectin.

Flow Cytometric Analysis of P-Selectin Expression on LysoPC-Stimulated Platelets With PKC Inhibitors or NO Donors
We then investigated whether a PKC-dependent signal transduction pathway is involved in LysoPC-induced P-selectin expression by using two PKC inhibitors. Table 2 shows the mean channel fluorescence and percent positive staining for P-selectin on platelets after incubation with palmitoyl LysoPC (10 µmol/L) for 10 minutes in the absence or presence of PKC inhibitors (ie, UCN-01 and TMS) or NO donors (ie, CAS1609 and sodium nitroprusside). After incubation with LysoPC, the binding of anti–P-selectin MAb PB 1.3 to platelets was significantly increased compared with nonstimulated platelets. In contrast, LysoPC-stimulated P-selectin expression was significantly attenuated by coincubation of platelets with either of the PKC inhibitors UCN-01 (100 nmol/L) (Fig 1c) or TMS (10 µmol/L) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Flow Cytometric Analysis of P-Selectin Expression on Cat Platelets

Since NO donors have been shown to attenuate adhesion molecule expression, we tested whether NO donors have any effect on the LysoPC-induced P-selectin expression. Coincubation of platelets with either CAS1609 (10 µmol/L) (Fig 1d) or sodium nitroprusside (100 µmol/L) significantly attenuated P-selectin expression induced by LysoPC (10 µmol/L) (Table 2).

Concentration-Response Effects of LysoPC on PKC Activity of Platelet Membrane Fraction
We examined the effects of LysoPC on platelet PKC activity. Since LysoPC has been shown to stimulate PKC in intact cells and since PKC activation by phorbol esters has been shown to elicit P-selectin expression, we examined the concentration-response effects of palmitoyl LysoPC (1 to 100 µmol/L) on cat platelet membrane PKC activity. Palmitoyl LysoPC at 1 µmol/L did not significantly alter platelet PKC activity. However, at 10 and 30 µmol/L, LysoPC significantly stimulated PKC (186±10% and 185±20% of control, respectively), whereas this stimulatory effect on PKC was overridden with LysoPC (100 µmol/L) (Fig 2a). We also tested the effects of dipalmitoyl PC, a phospholipid that is mainly contained in native LDL but not in ox-LDL. Dipalmitoyl PC at 1 to 100 µmol/L did not alter cat platelet PKC activity (Fig 2a).

View larger version (22K): [in this window] [in a new window]   Figure 2. a, Effects of palmitoyl LysoPC and dipalmitoyl PC on PKC activity of cat platelet membrane fractions. Different concentrations of LysoPC and PC were incubated with platelets for 10 minutes at 37°C. The enzyme activity in the absence of LysoPC or PC was set at 100%. b, Effects of the PKC inhibitor UCN-01 on PKC activity stimulated by a fixed concentration of LysoPC (10 µmol/L). The LysoPC-stimulated enzyme activity in the absence of UCN-01 was set at 100%. Data are mean±SEM of two independent experiments, each consisting of triplicate determinations. *P<.05 vs control PKC activity in the absence of phospholipids.

We then examined the concentration-response effects of the PKC inhibitor UCN-01 (1 to 1000 nmol/L) on PKC activity stimulated by a fixed concentration of palmitoyl LysoPC (10 µmol/L). UCN-01 significantly attenuated LysoPC (10 µmol/L)–mediated PKC stimulation in a concentration-dependent manner (Fig 2b). The IC₅₀ of this inhibitory effect was 5.0±0.3 nmol/L UCN-01 in the present study.

Time Course of Platelet Membrane PKC Activation by LysoPC
Since P-selectin translocation onto the cell surface is rapid (ie, within 10 minutes), we examined the time course of PKC activation by either LysoPC (10 µmol/L), PMA (100 nmol/L), or LysoPC plus CAS1609 (10 µmol/L). Both PMA and LysoPC initiated PKC activation within 1 minute. PMA stimulated PKC activity 300% above control values, continuing for at least 20 minutes. LysoPC also significantly stimulated PKC, peaking at 5 minutes and lasting until 10 minutes after its addition and then declining to almost control values by 20 minutes (Fig 3). However, coincubation with the NO donor CAS1609 significantly attenuated the PKC activation mediated by LysoPC (Fig 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Time course of PKC activity of platelet membrane fractions after stimulation with either palmitoyl LysoPC (10 µmol/L) or PMA (100 nmol/L). The enzyme activities before the addition of LysoPC or PMA (ie, 0-minute incubation) were set at 100%. An NO donor, CAS1609 (10 µmol/L), significantly suppressed LysoPC-mediated PKC activation. Data are mean±SEM of two independent experiments, each consisting of triplicate determinations. *P<.05 vs control PKC activity (time point=0).

PKC Activity of Platelet Membrane Fraction After Stimulation With Various Agonists
We measured and compared platelet PKC activity after incubation with various agonists and control lipids in parallel. Platelet PKC activity was measured after a 10-minute incubation with either thrombin (2 U/mL), PMA (100 nmol/L), palmitoyl LysoPC (10 µmol/L), or dipalmitoyl PC (10 µmol/L). Fig 4 illustrates platelet membrane PKC activity expressed as a percentage of maximum PKC activity in response to PMA (100 nmol/L). Thrombin significantly increased platelet membrane PKC activity to 52±5% of the PMA value compared with control untreated platelets (19±2%, P<.05). Moreover, LysoPC also increased PKC activity to 72±5% of the PMA value (P<.05 versus control). In contrast, PC, which is mainly contained in native LDL but not in ox-LDL, failed to activate PKC. Furthermore, the PKC inhibitor UCN-01 and the NO donor CAS1609 significantly attenuated LysoPC-induced PKC activation.

View larger version (23K): [in this window] [in a new window]   Figure 4. PKC activity of cat platelet membrane fraction, expressed as a percentage of PMA (100 nmol/L)–induced PKC activity set at 100%. Each substrate was incubated for 10 minutes at 37°C. UCN-01 (100 nmol/L) and CAS1609 (10 µmol/L) were first incubated for 10 minutes before addition of LysoPC and were present for the LysoPC incubation period. Heights of bars are means, brackets indicate ±SEM, and numbers of experiments are shown inside the bars.

NO Concentrations in Platelet Suspensions Treated With Two NO Donors
We measured NO concentrations in platelet suspensions (1x10⁸ cells per milliliter) after incubation with the two NO donors (CAS1609 and sodium nitroprusside) for 10 minutes. After calibration, the tip of the NO-specific electrode was dipped in 2 mL of platelet suspensions, and NO was measured amperometrically. CAS1609 (10 µmol/L), the furoxan class NO donor, and sodium nitroprusside (100 µmol/L) released 36±3 nmol/L and 55±4 nmol/L (n=4) of NO, respectively. Thus, administration of the two NO donors used in the present study released significant quantities of NO in the platelet suspensions.

Adherence of Unstimulated PMNs to Cat Coronary Endothelium Stimulated With Thrombin, PMA, or LysoPC
Another important site of P-selectin expression is endothelial cells, where P-selectin is rapidly translocated to the cell surface after stimulation with inflammatory mediators and facilitates PMN adherence to the endothelium. Therefore, we examined the effects of LysoPC on PMN adherence to cat coronary vascular endothelium compared with thrombin and the PKC activator PMA. Fig 5 summarizes these results. PMN adherence was markedly enhanced by endothelial stimulation with either thrombin (2 U/mL), PMA (100 nmol/L), or palmitoyl LysoPC (10 µmol/L) but not in response to dipalmitoyl PC (10 µmol/L). The PMA-induced increase in adherence was significantly attenuated by the selective PKC inhibitor UCN-01 (100 nmol/L). The specific anti–P-selectin MAb PB 1.3 (20 µg/mL) also significantly attenuated PMN adherence to coronary endothelium stimulated with either PMA (48±6 cells per square millimeter, P<.05) or thrombin (38±4 cells per square millimeter, P<.01).

View larger version (25K): [in this window] [in a new window]   Figure 5. Autologous cat PMN adherence to cat coronary endothelium after incubation with various stimulators for 10 minutes. Heights of bars are means; brackets indicate ±SEM. Numbers of coronary segments examined are shown inside the bars. In each coronary segment, the number of adherent PMNs were counted in five microscopic fields and expressed as cells per square millimeter.

Adherence of Unstimulated PMNs to LysoPC-Stimulated Cat Coronary Endothelium: Effects of PKC Inhibition and NO Donor CAS1609
We further examined the mechanism of PMN adherence to cat coronary endothelium stimulated with LysoPC (10 µmol/L). Fig 6 summarizes the adherence of unstimulated autologous PMNs to LysoPC-stimulated coronary endothelium. PMN adherence was markedly enhanced by treatment with LysoPC compared with nonstimulated control endothelium (P<.01). This increase in PMN adherence to the endothelium was significantly attenuated by treatment with the specific anti–P-selectin MAb PB 1.3 (P<.01 versus vehicle) but not with MAb NBP 1.6 (20 µg/mL), a nonblocking control MAb (Fig 6). These results indicate that P-selectin expression on the endothelial surface significantly contributes to the LysoPC-stimulated increase in PMN-endothelium interaction after 10 minutes of stimulation.

View larger version (26K): [in this window] [in a new window]   Figure 6. Autologous cat PMN adherence to cat coronary endothelium stimulated by palmitoyl LysoPC (10 µmol/L). Effects of the anti–P-selectin MAb PB 1.3, the control nonblocking MAb NBP 1.6, the PKC inhibitor UCN-01, PKC downregulation with 24-hour PMA, and the NO donor CAS1609 are shown. Heights of bars are means; brackets indicate ±SEM. Numbers of coronary segments examined are shown inside the bars. In each coronary segment, the number of adherent PMNs were counted in five microscopic fields and expressed as cells per square millimeter.

This increased autologous PMN adherence to the LysoPC-stimulated coronary endothelium was significantly attenuated by either treatment with the selective PKC inhibitor UCN-01 or by incubating coronary segments with PMA (100 nmol/L) for 24 hours (ie, downregulation of PKC activity) (Fig 6). Thus, LysoPC-mediated P-selectin expression may be at least partially mediated by a PKC-involving mechanism. Further examination showed that the increased PMN adherence to LysoPC-stimulated coronary endothelium was significantly attenuated by coincubation with the NO donor CAS1609 (10 µmol/L) (Fig 6). This inhibitory effect was also observed (50±7 cells per square millimeter, n=7; P<.05 versus LysoPC-stimulated segments) when CAS1609 was preincubated with segments and did not coexist with LysoPC. However, when CAS1609 was added to the chambers after stimulation with LysoPC, CAS1609 no longer inhibited PMN adherence to the coronary endothelium (87±6 cells per square millimeter, n=7; P=NS versus LysoPC-stimulated segments). These results suggest that the PKC inhibitor and the NO donor can significantly inhibit LysoPC-induced PMNendothelium interaction mediated by P-selectin.

Immunohistochemical Localization of P-Selectin on Cat Coronary Endothelium After Stimulation With LysoPC
P-Selectin localization on cat coronary endothelium was examined by immunohistochemical analysis. Omission of the primary antibody showed no staining of P-selectin in endothelial cells (Fig 7A). There was very little positive staining for P-selectin in nonstimulated control coronary endothelium, which was incubated in K-H solution for 10 minutes (Fig 7B). In contrast, incubation of coronary rings with 2 U/mL thrombin for 10 minutes increased immunostaining of P-selectin on coronary endothelium (Fig 7C). Incubation of segments with palmitoyl LysoPC (10 µmol/L) for 10 minutes also upregulated P-selectin expression on coronary endothelium (Fig 7D). However, pretreatment of coronary vascular segments with either the NO donor CAS1609 (10 µmol/L) or the PKC inhibitor UCN-01 (100 nmol/L) in the presence of palmitoyl LysoPC (10 µmol/L) attenuated the LysoPC-induced P-selectin positive immunostaining (Fig 7E and 7F).

View larger version (149K): [in this window] [in a new window]   Figure 7. Immunohistochemical staining of P-selectin on cat coronary endothelium. The brown reaction product located along the endothelial lining represents P-selectin (arrows). Omission of the primary antibody showed no staining in endothelial cells (A). There was very little positive staining for P-selectin in nonstimulated control coronary endothelium (B). Thrombin increased immunostaining of P-selectin on coronary endothelium (C). Palmitoyl LysoPC also increased P-selectin expression on coronary endothelium as shown (D). However, pretreatment of coronary rings with either the NO donor CAS1609 (E) or the PKC inhibitor UCN-01 (F) markedly attenuated the LysoPC-induced P-selectin expression. Bar=50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
LysoPC is an atherogenic lysophospholipid that has at least two sources in atherosclerotic lesions. First, oxidation of native LDL is associated with the hydrolysis of PC to LysoPC via the action of LDL-associated phospholipase A₂.^{1 6} Henry and coworkers^{9 40} showed that the LysoPC content in oxidatively modified LDLs closely correlated with the degree of their oxidation status. Another source of LysoPC is the plasma, in which lecithin/cholesterol acyltransferase catalyzes LysoPC formation.^{41 42} This enzyme activity has been shown to increase with hypercholesterolemia.^{41 42} LysoPC is transferred and incorporated into endothelial cell membrane in an apoprotein-independent manner.^{9 43} Recently, LysoPC has been shown to mediate the ox-LDL–induced impairment of endothelium-dependent vasorelaxation⁹ and induce cell adhesion molecule expression (eg, VCAM-1 and ICAM-1).^{12 13}

Although the LysoPC-mediated intracellular signaling pathway is not totally clear, Oishi et al¹⁵ and Sasaki et al¹⁷ showed that LysoPC can activate PKC in intact cells. These findings were further extended by Kugiyama et al,¹⁸ who reported that ox-LDL and LysoPC impair NO release and ICAM-1 expression via a PKC-dependent mechanism in endothelial cells. More recently, Ohara et al¹⁹ demonstrated that LysoPC stimulates vascular superoxide anion production via PKC activation. These studies provide clear evidence that LysoPC stimulates cells at least partially by a PKC-mediated mechanism. In the present study, 10 µmol/L LysoPC significantly activated platelet PKC, which was markedly attenuated by the PKC inhibitor UCN-01. The concentration of UCN-01 used in the present study (100 nmol/L) has been shown to sufficiently inhibit conventional PKC (ie, -, ß-, and -PKC) isozymes, although cAMP-dependent protein kinase (protein kinase A) was also slightly inhibited.⁴⁴ These results are consistent with the previous work by Sasaki et al,¹⁷ who found that LysoPC selectively stimulates conventional PKC isozymes.

Although Lehr et al⁵ demonstrated that in vivo administration of human ox-LDL increased P-selectin–mediated leukocyte rolling in the microcirculation, the precise mechanism for this upregulation is unclear. Berliner et al⁴⁵ reported that minimally modified LDL enhanced monocyte adhesion in endothelial cells. This activity of minimally modified LDL was shown to reside in the phospholipid fraction (lipoprotein fraction) and not in the aqueous fraction. Berliner et al further indicated that much of this active phospholipid was LysoPC. More recently, the same group⁴⁶ suggested that ox-LDL induces rapid P-selectin expression and that minimally modified LDL also increases P-selectin mRNA expression in cultured endothelial cells. However, the role of LysoPC in ox-LDL–induced rapid P-selectin expression is unknown. In the present study, we first report that incubation of platelets with LysoPC (10 µmol/L) for 10 minutes elicits a rapid P-selectin expression on platelets. Furthermore, LysoPC increases PMN adherence to the endothelium, which is significantly attenuated by an anti–P-selectin MAb PB 1.3 but not by a nonblocking control MAb, NBP 1.6, indicating that LysoPC-induced rapid PMN-endothelium interaction is mainly mediated by P-selectin. Increased P-selectin expression by LysoPC as well as thrombin on the endothelial cells was also confirmed by immunohistochemistry. These results demonstrate that LysoPC rapidly induces P-selectin expression in both platelets and endothelial cells.

LysoPC significantly stimulated platelet PKC activity within 1 minute in the present study. This time course of LysoPC-mediated PKC activation is similar to the study by Kugiyama et al,¹⁸ who showed PKC stimulation by LysoPC in cultured endothelial cells. The rapid stimulation of PKC by LysoPC correlates closely with the generally accepted time course of P-selectin expression on the cell surface (ie, 5 minutes) after stimulation. Furthermore, LysoPC-induced P-selectin expression in platelets and endothelial cells was significantly attenuated by the PKC inhibitor UCN-01, and PMN-endothelium adherence was inhibited either by UCN-01 or by downregulation of PKC after 24 hours of incubation with PMA. These results support our primary hypothesis that LysoPC promotes P-selectin expression at least partially by PKC activation. In this regard, Hannun et al²⁶ showed that PKC activation is both a necessary and sufficient event for agonist-induced platelet activation. We examined the effect of sphingosine as a control lysolipid but as having PKC inhibitory activity on platelet P-selectin expression. However, sphingosine failed to induce P-selectin expression. Therefore, the observed P-selectin induction by LysoPC is not mediated by a nonspecific effect of lysolipids. More over, the N-methyl derivative of sphingosine, TMS, a potent PKC inhibitor, significantly inhibited LysoPC-induced P-selectin expression on platelets. This is consistent with previous observations that TMS inhibits P-selectin expression in stimulated platelets.^{24 25} Geng et al²⁰ further reported that the PKC activator PMA significantly induces P-selectin expression and facilitates PMN adhesion to endothelial cells. The present results and these previous studies support a crucial role of PKC activation in the process of rapid P-selectin expression both in platelets and in endothelial cells.

In the present study, 10 µmol/L LysoPC significantly activated platelet PKC, whereas 100 µmol/L LysoPC inhibited PKC to values below those occurring in untreated platelets (Fig 2); thus, LysoPC has a differential action on PKC that depends on the concentration. The present results are consistent with the data reported by Oishi et al,¹⁵ who suggested that the high concentration of LysoPC (100 µmol/L) inhibited PKC activity probably by a nonspecific detergent-like action. Sugiyama et al¹³ recently demonstrated that 10 µmol/L LysoPC stimulates ICAM-1 expression in pig coronary endothelium by activating PKC. Ohara et al¹⁹ also demonstrated that the same concentration of LysoPC stimulated vascular superoxide anion production via PKC activation. The levels of LysoPC present in human atherosclerotic lesions¹⁰ are considered to be similar to those used in the present study and in the studies of Sugiyama et al¹³ and Ohara et al.¹⁹ Taken together, effects of submicellar concentrations of LysoPC (ie, 10 µmol/L) on P-selectin expression in the present study appear to be mediated by a mechanism involving PKC activation.

P-Selectin plays a key role in platelet adherence and leukocyte rolling on the endothelium.^{21 22} P-Selectin may play an important pathophysiological role in the early stage of atherogenesis, since recruitment of macrophages into the atherosclerotic site is a key event for fatty streak formation.³ In this regard, Sakai et al²⁷ recently showed that endothelial P-selectin expression preceded accumulation of macrophages and T lymphocytes into the intima of hypercholesterolemic rabbits. Furthermore, we have recently demonstrated that P-selectin is significantly upregulated on the endothelium, which facilitates leukocyte rolling on the microvascular endothelium during mild hypercholesterolemia in rats.²⁸ Also, macrophage recruitment to chronic inflammatory sites was significantly attenuated in P-selectin–deficient mice.⁴⁷ Our present study first demonstrates an important link between LysoPC and P-selectin expression, thus providing new and important insight into the mechanisms of the early stages of vascular atherogenesis.

Recent studies suggest that NO, whether endogenous or exogenous, inhibits vascular atherogenesis. Cooke et al²⁹ have shown that dietary L-arginine, a precursor of endogenous NO, inhibits atherosclerosis development in cholesterol-fed rabbits. Also, an NO donor was shown to inhibit leukocyte rolling and adherence to the microvascular endothelium in cholesterol-fed rats.²⁸ Chronic inhibition of NO production by an L-arginine analogue accelerates neointimal formation in hypercholesterolemic rabbits.⁴⁸ Furthermore, NO has been shown to inhibit cytokine-induced expression of ICAM-1 and VCAM-1 on endothelium.³⁰ These studies provide strong evidence that NO protects against ox-LDL– mediated and atherosclerosis-related cellular injury. Therefore, we further tested whether NO donors have any effects on the LysoPC-mediated P-selectin expression and PMN-endothelium interaction. In the present study, NO donors (CAS1609 and sodium nitroprusside) significantly attenuated LysoPC-induced P-selectin expression in platelets. Moreover, CAS1609 significantly attenuated PKC activity in platelets and attenuated endothelial P-selectin expression and PMN adherence to the coronary endothelium stimulated by LysoPC. There are several possible mechanisms whereby the NO attenuated LysoPC-induced P-selectin expression. First, NO might inhibit PKC activity directly by nitrosylation of thiol.⁴⁹ Gopalakrishna et al⁴⁹ demonstrated that NO inhibited PKC activity in both the purified enzyme and intact cells. In the present study, CAS1609 significantly inhibited LysoPC-induced PKC activation in platelets (Fig 3). Thus, CAS1609 might attenuate P-selectin expression by a NO-mediated inhibition of PKC activity. Second, Takai et al⁵⁰ demonstrated that cGMP inhibited phosphatidylinositol hydrolysis, thus inhibiting diacylglycerol formation and PKC activation. Thus, NO might also attenuate PKC via a cGMP-dependent mechanism. Third, NO derived from CAS1609 possibly scavenged superoxide radicals.⁵¹ Oxygen radicals are known to stimulate P-selectin expression.⁵² Since LysoPC has been shown to reduce endothelial NO release^{9 10 18 53} and promote vascular superoxide radical production,¹⁹ CAS1609 might inhibit P-selectin expression also by scavenging superoxide.

In conclusion, we have demonstrated that LysoPC, an atherogenic lysophospholipid, elicits rapid P-selectin expression in platelets and endothelial cells and leukocyte adherence to the coronary endothelium. These effects appear to be at least partially mediated by PKC activation. Furthermore, LysoPC-induced P-selectin expression was significantly attenuated by NO donors. Since P-selectin may play an important role in initiation of atherosclerosis via monocyte recruitment and platelet deposition,^{27 28 47 54} our present data provide further insight into the mechanism of atherogenesis and of NO-mediated inhibition of atherosclerosis.²⁹

	Selected Abbreviations and Acronyms

 

CAS1609 = 4-hydroxymethyl-furazan-3-carboxylic acid-oxide ICAM-1 = intercellular adhesion molecule-1 K-H = Krebs-Henseleit LDL = low-density lipoprotein LysoPC = lysophosphatidylcholine MAb = monoclonal antibody ox-LDL = oxidized LDL PC = phosphatidylcholine PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate PMN = polymorphonuclear leukocyte TMS = N,N,N-trimethylsphingosine UCN-01 = 7-hydroxystaurosporine VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This study was supported by research grant GM-45434 from the National Institutes of Health (Dr Lefer). Dr Murohara was the recipient of a Japan Heart Foundation-Bayer Yakuhin Postdoctoral Fellowship (1993).

Received October 11, 1995; accepted February 5, 1996.

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