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(Circulation. 2001;104:588.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Oxidized Low-Density Lipoprotein Downregulates Endothelial Basic Fibroblast Growth Factor Through a Pertussis Toxin-Sensitive G-Protein Pathway

Mediator Role of Platelet-Activating Factor-Like Phospholipids

Po-Yuan Chang, MD, PhD; Sherry Luo, BS; Tao Jiang, PhD; Yuan-Teh Lee, MD, PhD; Shao-Chun Lu, PhD; Philip D. Henry, MD; Chu-Huang Chen, MD, PhD

From the Department of Medicine, Baylor College of Medicine, Houston, Tex (P.-Y.C., S.L., T.J., P.D.H., C.-H.C.); and the Departments of Internal Medicine (P.-Y.C., Y.-T.L.) and Biochemistry (S.-C.L.), National Taiwan University Medical School, Taipei, Taiwan.

Correspondence to Chu-Huang Chen, MD, PhD, MS A-601, Department of Medicine, Baylor College of Medicine, 6565 Fannin, Houston, TX 77030. E-mail cchen{at}bcm.tmc.edu

	Abstract

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Background— Oxidized LDL (oxLDL) inhibits angiogenesis in part by downregulating endothelial basic fibroblast growth factor (bFGF). To determine the mechanism of the downregulation, we investigated the signal transduction pathway involving potential phospholipid mediators.

Methods and Results— Cultured bovine aortic endothelial cells were incubated with PBS (lipoprotein-free control), LDL, or copper oxLDL under serum-free conditions. At 24 hours, oxLDL (50 µg/mL) decreased bFGF mRNA (Northern blot), bFGF protein (Western blot and ELISA), and concomitant DNA synthesis, all by 40% to 50% compared with PBS. LDL had no effect. Pretreating the cells with 100 ng/mL pertussis toxin (PTX) for 18 hours before oxLDL exposure almost completely blocked the inhibitory effects of oxLDL. In contrast, inhibiting other major cellular signal transduction pathways with PD-98059 (mitogen-activated protein kinase kinase inhibitor), HA-1004 (inhibitor of cGMP- and cAMP-dependent protein kinase), or Ro-31-8220 (protein kinase C inhibitor) or chelating intracellular Ca²⁺ with BAPTA-AM failed to attenuate any of the oxLDL effects assayed. Addition to the cultures of WEB 2086, a specific antagonist of the PTX-sensitive G protein–coupled platelet-activating factor (PAF) receptor, blocked the action of oxLDL. Whereas PAF dispersed in the culture medium failed to produce oxLDL-like effects, degradation of PAF and PAF-like phospholipids accumulated in oxLDL with a recombinant human PAF acetylhydrolase eliminated the inhibitory effects of oxLDL on bFGF expression and DNA synthesis.

Conclusions— OxLDL suppresses endothelial bFGF expression and DNA synthesis through a PTX-sensitive heterotrimeric G-protein pathway involving mediator phospholipids similar, but not identical, to PAF.

Key Words: phospholipids • lipoproteins • growth substances • endothelium • genes

	Introduction

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The cell growth and survival factor basic fibroblast growth factor (bFGF) directly or, by inducing other genes, indirectly regulates a wide range of functions in vascular endothelial cells (ECs).^1–3 Massive apoptosis and greatly impaired angiogenic responses occur in ECs deprived of bFGF.^4–6 In arterial explants, EC proliferation and growth of capillary-like microtubules are inhibited by oxidized LDL (oxLDL)–induced bFGF downregulation.^6,7 Delineation of the signal transduction pathway mediating the downregulation may reveal clues to the nature of the components in oxLDL responsible for the inhibition. Characterization of the mediator components in copper oxLDL may suggest a method useful for the identification of similar mediators in the oxLDL-like lipoproteins present in the plasma of patients with hypercholesterolemia.

Signal transduction pathways described to date in oxLDL effects include involvement of G protein–coupled receptors, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), cGMP- and cAMP-dependent protein kinase (PK), and PKC.^8–12 Some oxLDL activities have been associated with excessive release of Ca²⁺ in the cytoplasm.¹³ The lack of uniformity in the signaling pattern suggests that different LDL components modified during oxidation may participate in mediating cellular effects. Our data suggest that oxLDL-induced bFGF downregulation is sensitive to pertussis toxin (PTX). One of the PTX-sensitive G protein–coupled membrane receptors implicated in the mediation of oxLDL signals is platelet-activating factor (PAF) receptor (PAFR).^14–17 Some oxLDL effects have been attributed to mediator phospholipids accumulated in the modified lipoproteins.^14,18–20 The phospholipids may share structural similarities with PAF, because they exhibit sensitivity to PAFR antagonists.^14–17 Here, we addressed the question of whether oxLDL downregulates endothelial bFGF via a signal pathway that involves PAFR.

	Methods

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Cells and LDL Preparations
Primary cultures of bovine aortic ECs (BAECs) were derived and purified as described.⁷ Cells at 8 to 12 passages, maintained in DMEM supplemented with 10% FBS and antibiotics (streptomycin 100 µg/mL, penicillin 100 IU/mL, amphotericin B 0.25 µg/mL), were used. LDL particles (d=1.019 to 1.063 g/mL) from pooled human plasma anticoagulated with EDTA (0.5 mg/mL) were isolated by sequential ultracentrifugation.¹⁸ For oxLDL preparation, LDL was exposed at 37°C for 24 hours to 5 µmol/L CuSO₄ in PBS. After the incubation was terminated by EDTA (0.5 mg/mL), the preparation was dialyzed and preserved in nitrogen-filled tubes.⁶ Precautions previously described were taken to prevent endotoxin contamination.⁶ Protein in LDL preparations was estimated by the Lowry method, and thiobarbituric acid–reactive substances (TBARS) contained in LDL preparations were assayed as a measure of oxidative lipid modification.¹⁸ At 24 hours, the TBARS values in LDL were 18 to 22 and 1 to 2 nmol/mg protein with and without CuSO₄ exposure, respectively.

Protocol
Cell cultures grown to subconfluence were washed 3 times with serum-free medium and maintained under serum-free conditions for 6 hours before treatment with various signal transduction inhibitors, PAF receptor blockers, or PBS, according to protocols determined by preliminary experiments. After another 18 hours, cells were incubated with PBS (lipoprotein-free control), native LDL (50 µg/mL), or oxLDL (50 µg/mL) for 24 hours.

Inhibitors of Signal Transduction Pathways
To determine the involvement of major signal transduction pathways, cells were treated with PTX (a G_i protein inhibitor), PD-98059 (an MEK/MAPK inhibitor), HA-1004 (an inhibitor of cGMP- and cAMP-dependent PK), Ro-31-8220 (a PKC inhibitor), and BAPTA-AM (an intracellular Ca²⁺ chelator) before exposure to oxLDL. All agents were purchased from Calbiochem. Protocols for individual agents were determined on the basis of the maximal doses and durations tolerable by the cells; tolerability was defined as <5% reduction in DNA synthesis.

PAFR Antagonists, PAF-16, and PAF-Acetylhydrolase
To determine whether the signal was transmitted through PAFR, the competitive PAFR antagonist WEB 2086 (10 µmol/L; gift from Boehringer Ingelheim) was added to the medium and allowed to equilibrate for 1 hour before addition of oxLDL. To test whether PAF was equivalent to oxLDL in inhibiting bFGF expression and DNA synthesis, some cultures were incubated with 0.1 to 1 µmol/L PAF-16 (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; Calbiochem), a synthetic, water-soluble PAF, instead of oxLDL. To degrade the sn2 acetyl group of phospholipids accumulated in oxLDL, some oxLDL preparations were preincubated with 50 to 200 µg/mL of a recombinant PAF-acetylhydrolase (rPAF-AH; gift from ICOS, Bothell, Wash) for 1 hour at 37°C.

Northern Blot Analysis
After isolation from cultured cells, 50 µg total RNA was subjected to electrophoresis in each lane of 1% agarose/2.2 mol/L formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell) as described.⁷ The bFGF cDNA insert was excised from plasmid constructs with EcoRI and gel-purified (Geneclean Kit, Bio 101, Inc) for use as templates. After labeling with [-³²P]dCTP to a high specific activity (10⁹ cpm/µg) by a random-priming method, probes were hybridized to the blots (10⁶ cpm/mL) overnight at 42°C in a solution containing 40% formamide, 5x SSC, 5x Denhardt’s solution, 0.5% SDS, 250 µg/mL salmon sperm DNA, and 10% dextran sulfate. The blots were washed under high stringency at 65°C in 0.2x SSC plus 0.1% SDS. For autoradiographic detection, films were exposed at -70°C for 3 days with 2 intensifying screens. A 1.2-kb PstI fragment of human GAPDH cDNA, cloned according to a described method, ²¹ was ³²P-labeled by random priming and used as an internal control. A PhosphorImager was used to determine bFGF expression.

Western Blot Analysis
Protein concentrations were determined according to the method of Bradford (BioRad) in cells lysed in a pH 7.5 buffer containing 20 mmol/L Tris-HCl, 0.5% SDS (wt:vol), 10% glycerol, and 50 mmol/L dithiothreitol. Protein samples (180 µg) were resolved by electrophoresis on denaturing 15% SDS–polyacrylamide gels for 2 hours at 100 V with a Mighty Small Gel System (Hoefer). After electrophoresis, the samples were transferred to a polyvinylidine difluoride membrane in a buffer containing 25 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol, pH 8.3. Nonspecific binding sites were blocked with nonfat skim milk, and the membranes were incubated with a monoclonal antibody against bovine bFGF (Upstate Biotechnology). Antigen-antibody complexes were visualized with a horseradish peroxidase chemiluminescent system (ECL kit, Pharmacia). PhosphorImager quantification was used to determine bFGF expression.

ELISA and DNA Synthesis
For ELISA and DNA synthesis, 100x10⁴ cells were inoculated in each well of 12-well Corning cell culture plates. Intracellular bFGF concentrations in cell lysates prepared with Nonidet P-40 (Sigma) were measured by ELISA with a Quantikine kit (R&D Systems) and estimated spectrophotometrically at 450 nm, as described.⁷ To evaluate treatment effects on DNA, DNA synthesis was assayed by addition of 3 µCi/mL [³H]thymidine (Moravek Biomedicals) to the medium during the final 4 hours of incubation. Incubation was terminated by decanting the medium and fixing the cells with 1 mL of 10% (wt:vol) cold trichloroacetic acid for 15 minutes at 4°C. [³H]Thymidine incorporated in extracted DNA was assayed by scintillation spectrometry.^6,7

Statistical Analysis
The significance of differences between the means of treatment and control (PBS) groups was assessed by a 2-sided Student’s t test with Bonferroni correction. Values of P<0.05 were considered significant. Results are expressed as mean±SEM values. A GB-STAT program (Dynamic Microsystems) was used.

	Results

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Signal Transduction of oxLDL-Induced bFGF Downregulation
At 24 hours, oxLDL (50 µg/mL) decreased bFGF mRNA by 40% to 50% compared with PBS, whereas LDL had no effect (Figure 1), findings similar to those reported previously.⁷ The inhibitory effect was almost completely blocked in cells incubated with the G-protein inhibitor PTX (100 ng/mL) for 18 hours before oxLDL exposure. In contrast, pretreating the cells with the MEK/MAPK inhibitor PD-98059 (20 µmol/L) for 18 hours or the PKC inhibitor Ro-31-8220 (3.5 µmol/L) for 1 hour failed to exhibit any inhibitory effects (Figure 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of signal transduction inhibitors on oxLDL-induced reduction in bFGF mRNA assessed by Northern blot analysis and densitometry in cultured BAECs. Cells were incubated with 50 µg/mL oxLDL for 24 hours alone or with preceding exposure to MEK/MAPK inhibitor (PD-98059), PKC inhibitor (Ro-31-8220), or G-protein inhibitor (PTX) according to protocols described in text. PBS and LDL (50 µg/mL) were used as lipoprotein-free and oxidation-free controls. Data are representative of 3 separate experiments with similar results, normalized to GAPDH standards.

At the Western blot–assayed protein level as well, the inhibitory effect of oxLDL (40% to 50% reduction) was sensitive to PTX but not to PD-98059 or Ro-31-8220 (Figure 2). Blocking the cGMP- and cAMP-dependent PK with HA-1004 (10 µmol/L) for 18 hours or chelating the intracellular Ca²⁺ with BAPTA-AM (16 µmol/L) for 1 hour failed to attenuate the reduction caused by oxLDL. In parallel, the effect of oxLDL in reducing intracellular bFGF concentration measured by ELISA was preventable only by PTX pretreatment, among the treatments tested (Figure 3). The concomitant 50% reductions in DNA synthesis (n=3; P<0.05) at 24 hours were also sensitive to PTX but not other signal inhibitors or the Ca²⁺ chelator (data not shown).

View larger version (6K): [in this window] [in a new window]   Figure 2. Effects of signal transduction inhibitors on oxLDL-induced reduction in bFGF protein assessed by Western blot analysis in cultured BAECs. Cells were incubated with 50 µg/mL oxLDL for 24 hours alone or with preceding exposure to MEK/MAPK inhibitor (PD-98059), PKC inhibitor (Ro-31-8220), PK inhibitor (HA-1004), Ca2+ chelator (BAPTA-AM), or G-protein inhibitor (PTX) according to protocols described in text. PBS was used as a lipoprotein-free control. Data are representative of 3 separate experiments with similar results.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effects of signal transduction inhibitors on oxLDL-induced reduction in intracellular bFGF protein concentrations assessed by ELISA in cultured BAECs. Cells were incubated with 50 µg/mL oxLDL for 24 hours alone or with preceding exposure to MEK/MAPK inhibitor (PD-98059), inhibitor of cGMP- and cAMP-dependent PK (HA-1004), PKC inhibitor (Ro-31-8220), Ca2+ chelator (BAPTA-AM), or G-protein inhibitor (PTX) according to protocols described in text. PBS and LDL (50 µg/mL) were used as lipoprotein-free and oxidation-free controls. Values are mean±SEM (n=3). Each well contained 100x104 cells at inoculation. *P<0.05 vs PBS.

Mediator Role of PAF-Like Phospholipids
Compared with PBS, WEB 2086 (10 µmol/L) pretreatment for 1 hour prevented the reduction in bFGF mRNA in cells incubated with oxLDL (Figure 4). In addition, WEB 2086 pretreatment greatly attenuated oxLDL-induced reductions in intracellular bFGF concentrations and DNA synthesis (Table).

View larger version (28K): [in this window] [in a new window]   Figure 4. Effects of WEB 2086 (WEB+oxLDL), pretreatment of oxLDL with rPAF-AH (rPAF-AH+oxLDL), and PTX (PTX+oxLDL) on oxLDL-induced reduction in bFGF mRNA assessed by Northern blot analysis and densitometry in cultured BAECs. PBS was used as a lipoprotein-free control. Data are representative of 3 separate experiments with similar results, normalized to GAPDH standards.

View this table: [in this window] [in a new window]   Table 1. Effects of LDL Preparations, PAF-16, rPAF-AH, OxLDL+rPAF-AH, and WEB 2086+OxLDL on Intracellular bFGF Protein Levels and DNA Synthesis at 24 Hours

PAF-16 (1 µg/mL) did not induce significant reductions in intracellular bFGF concentrations or DNA synthesis (Table). Cells incubated with rPAF-AH alone (200 µg/mL) exhibited no differences in bFGF concentrations or DNA synthesis compared with PBS at 24 hours. The inhibitory effects of oxLDL on bFGF expression and DNA synthesis, however, were abolished by pretreatment with rPAF-AH (Figure 4 and Table). Sham pretreatment of oxLDL with PBS did not alter its effects on bFGF expression or DNA synthesis (data not shown).

	Discussion

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Induced angiogenesis has the potential to become an important therapy for ischemia from atherosclerotic vascular disease. Key to its pursuit is understanding factors that may render the ECs unresponsive to angiogenic stimuli. Copper oxLDL inhibits EC proliferation and angiogenesis in vitro,^6,7 and modified LDL occurring in arterial walls and circulating in plasma may have similar effects.^22–24 The angiostatic effects of oxLDL are attributable to suppressed expression of bFGF, a growth factor involved in all phases of angiogenesis: EC proliferation, EC migration, and vascular differentiation.²⁵ Here, we demonstrated that oxLDL downregulates endothelial bFGF through a PTX-sensitive heterotrimeric G-protein pathway. The mediators—culprit components accumulated in oxLDL—are characterized by their sensitivity to rPAF-AH, an enzyme that degrades phospholipids at the glycerol sn2 position.^14–16 Their actions are attenuated by WEB 2086,¹⁷ a selective antagonist of PAFR, a heptahelical G protein–coupled receptor.²⁶

Angiogenesis involves multiple growth factors/cytokines. Like bFGF,^5,6 vascular endothelial growth factor (VEGF) improves collateral-dependent tissue perfusion in hypercholesterolemic rabbits.²³ Previously, we showed that oxLDL downregulates endothelial bFGF without affecting the FGF receptor FGFR-1; the associated reductions in DNA synthesis and capillary-like microtubule growth in arterial explants are reversed by exogenous bFGF, but not by VEGF or transforming growth factor-ß.^6,7 Because concentrations of circulating material reactive to anti-oxLDL monoclonal antibody are found to be highly increased in subjects with coronary events,²⁷ bFGF downregulation by oxLDL may be an important mechanism in impaired angiogenic responses in atherosclerosis.

Signal transduction pathways in oxLDL-induced growth factor modulation have not been demonstrated. In this study, the inhibitory effect of oxLDL on endothelial bFGF expression at both mRNA and protein levels was nearly completely abolished by PTX, suggesting a role of G_i and probable involvement of a PTX-sensitive G protein–coupled receptor. he signal pathway for this particular action of oxLDL did not involve the activation of MEK/MAPK, cGMP- and cAMP-dependent PK, or PKC: their respective inhibitors—PD98059, HA-1004, and Ro-31-8220—failed to alter the effect of oxLDL. In addition, the inhibition was largely independent of intracellular Ca²⁺ release, because chelating the cytosolic Ca²⁺ with BAPTA-AM failed to stop the action of oxLDL.

Our results complicate understanding of how oxLDL exerts various bioactivities through different signaling pathways. In contrast to our finding in bFGF modulation, it was recently reported that oxLDL upregulates endothelial monocyte chemoattractant protein 1 via a lectin-like receptor pathway that involves activation of MAPK but not PTX-sensitive G proteins.¹⁰ Yet in canine vascular smooth muscle cells, mitogenic effects of oxLDL are mediated through a PTX-sensitive G protein–coupled receptor mechanism that involves activation of the Ras/Raf/MEK/MAPK pathway.⁸ Lysophosphatidylcholine, a lipolytic product that accumulates in oxLDL, regulates endothelial nuclear factor-B activity through a PKC-mediated pathway.¹² In renal mesangial cells, minimally modified LDL (MM-LDL) upregulates platelet-derived growth factor through a cAMP-dependent, PKA-mediated pathway without PKC involvement.¹¹ The different findings may in part reflect the use of different animals and cell types. In the present study, however, the effects of oxLDL in downregulating bFGF and reducing DNA synthesis appeared to be mediated by a common transduction pathway.

Although oxidized apolipoprotein B as a peptide mediator may contribute to oxLDL-induced macrophage proliferation,²⁸ most oxLDL bioactivities are probably mediated by modified lipid components in the lipoprotein. Possible lipid mediators include oxysterols (7ß-hydroxycholesterol, 7-ketocholesterol), free fatty acid–derived metabolites, and phospholipids (lysophosphatidylcholine, PAF, ceramide-sphingomyelin, lysophosphatidic acid).^22,29 In MM-LDL, the major biologically active lipids are derived from arachidonate-containing phospholipids, such as 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphorylcholine (PAPC) and derivatives of oxidized PAPC, including POVPC, GPGC, and an m/z 828.6 (M+H⁺) molecule.^14,30 Some of the lipid components, including lysophosphatidylcholine, PAF, and those derived from MM-LDL, have been shown to exert their effects by means of the PAFR, ^16,17,31 which is known to be coupled with PTX-sensitive G proteins.²⁶

The nearly complete suppression of oxLDL-induced bFGF downregulation in the presence of the PAFR-specific antagonist WEB 2086 substantiates the importance of the PAFR pathway. WEB 2086 and WEB 2170, both PAFR antagonists of pyrrolothiazole-derivative origin, have been shown to attenuate various oxLDL or MM-LDL bioactivities, such as monocyte-EC adhesion and smooth muscle cell proliferation.^15,17,32 The oxLDL effects may also be blocked by structurally different PAFR antagonists, such as L659,989, a naturally occurring compound.³²

Whereas the oxLDL effects were prevented by WEB 2086, PAF itself failed to elicit oxLDL-like effects. Although PAF-16, a potent, water-soluble PAF analogue,³³ was administered within the dose range (0.1 to 1 µg/mL) capable of inducing maximal gallbladder muscle contraction,³⁴ it failed to exhibit oxLDL-like effects in suppressing bFGF expression and DNA synthesis. The seemingly paradoxical results raise 2 possibilities: that WEB 2086 antagonizes oxLDL by a mechanism other than binding to PAFR or that PAFR acts as a receptor for phospholipids other than PAF.³¹ Although the issue warrants further investigation, complete inactivation of oxLDL by rPAF-AH suggests a mediator role of PAF-like phospholipids. PAF-AH is an enzyme that degrades PAF and PAF-like phospholipids at the glycerol sn2 position; a loss of PAF-AH activity in LDL during oxidative modification of such phospholipids may be important in generating lipoproteins with oxLDL-like properties.³⁵ Protective effects of HDL against atherosclerosis may be mediated by the PAF-AH activity of HDL acting on phospholipids accumulated in oxidatively modified LDL.¹⁴ Depleting apolipoprotein B–containing lipoproteins (LDL, IDL, VLDL) of their intrinsic PAF-AH activity increases their potency in stimulating monocyte chemotaxis and adhesion.¹⁶

Thus, many oxLDL bioactivities appear be to mediated by a common pathway that involves PAFR. The oxLDL pathway mediating bFGF downregulation exhibits a striking similarity to that mediating phospholipase D activity stimulation, which also goes through the PTX-sensitive G protein–coupled PAFR without involving PKC.⁹ Accordingly, we propose a possible signal transduction pathway (Figure 5) that mediates the inhibitory effect of oxLDL on endothelial bFGF expression. The schematic pathway includes the finding that oxLDL reduces intracellular bFGF concentration in part by destabilizing mRNA posttranscriptionally.⁷ The cytosolic messengers and the mechanisms of transcription factor–bFGF promoter complex modulation are yet to be identified or delineated. Because other mediators remain to be identified, other possible pathways cannot be excluded.

View larger version (48K): [in this window] [in a new window]   Figure 5. Schematic of signal transduction pathway of oxLDL-induced bFGF downregulation in endothelial cells. Arrows indicate stimulation or direction of transport. Lines with end bar indicate inhibition.

	Acknowledgments

 
This study was supported in part by Scientist Development Grant 9630095N from the American Heart Association. Drs Chang, Lee, and Lu were supported by grants NSC 89-2314-B-002-029 and NSC 89-2316-B-002-017M52 from the National Science Council, Taiwan. The authors are grateful to Suzanne Simpson for editorial assistance.

Received December 7, 2000; revision received March 27, 2001; accepted April 10, 2001.

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