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Originally published online as doi:10.1189/jlb.0205101 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2005;78:1166-1174.)
© 2005 by Society for Leukocyte Biology

PAF-receptor antagonists, lovastatin, and the PTK inhibitor genistein inhibit H2O2 secretion by macrophages cultured on oxidized-LDL matrices

Indra Sethy-Coraci*,1, Lara W. Crock*,2 and Samuel C. Silverstein*,{dagger},3

* Departments of Physiology and Cellular Biophysics and
{dagger} Medicine, Columbia University, New York, New York

3Correspondence: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032. E-mail: scs3{at}columbia.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesion of mononuclear phagocytes (Macs) to extracellular matrices containing oxidized low-density lipoproteins (oxLDL) stimulates these cells to secrete reactive oxygen species (e.g., O2–, H2O2) that are believed to promote atherogenesis. Current in vitro systems designed to measure Mac H2O2 secretion in response to oxLDL show that these cells secrete H2O2 for only a few hours after plating. The slow onset and chronicity of atherogenesis, however, suggested to us that Mac ROS secretion might be sustained for much longer periods when Macs are maintained in an environment resembling that in the intima of arteries undergoing atherogenesis. The findings reported here confirm this suggestion. They show that Macs maintained on collagen IV matrices containing oxLDL in medium containing human plasma-derived serum secrete H2O2 continuously and in large amounts for at least 11 days. Using this system we tested the effects of compounds known to attenuate atherogenesis in vivo. Platelet-activating factor (PAF) receptor antagonists, lovastatin, and the isoflavone protein tyrosine kinase (PTK) inhibitor genistein each reduced H2O2 secretion by Macs maintained on oxLDL-containing matrices by ~60%. Lovastatin’s inhibitory effect was blocked completely by addition of geranylgeranyl pyrophosphate to the medium. We conclude that matrix-bound and oxidized lipoproteins stimulate Macs to produce H2O2 continuously and in large quantities via a pathway that involves PAF receptors and PTK and is reversibly blocked by inhibitors of protein prenylation.

Key Words: hydrogen peroxide • mononuclear phagocytes • platelet-activating factor • statins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherogenesis is a chronic inflammatory process in which lipoproteins accumulate and are oxidized within the artery wall. Products of oxidized low-density lipoprotein [oxLDL; e.g., platelet-activating factor (PAF)-like substances] and chemokines [e.g., monocyte chemoattractant protein-1 (MCP-1)] produced by endothelial cells in response to products of oxLDL stimulate monocytes to emigrate from the vasculature into the subendothelial space. Monocytes that enter this compartment are signaled to remain in it by products of oxLDL and by interactions of their integrins and scavenger receptors with oxLDL-containing matrix proteins [1 2 3 4 ]. These monocytes mature into macrophages (Macs) and secrete proteases, cytokines, growth factors, and reactive oxygen species (ROS; e.g., H2O2) [3 , 5 ]).

ROS secretion was first recognized as a component of the antimicrobial armamentarium of neutrophils. Indeed, the term "respiratory burst" was coined to describe the brief burst of O2 consumption and ROS production that accompanies phagocytosis of microbial pathogens by these cells. The discovery that neutrophil adhesion to certain extracellular matrix proteins increased the duration and magnitude of their respiratory burst [6 ] provided the first indication that matrix proteins affect ROS secretion by phagocytic cells. We reported previously that monocytes matured into Macs in vitro in Teflon beakers produce a burst of H2O2 lasting 2–3 h when they adhere acutely to collagen IV (CIV) matrices containing oxLDL [1 ]. We reasoned that monocytes that matured in an environment more like that in the intima of arteries undergoing atherogenesis might be stimulated to secrete H2O2 for much longer periods. To test this hypothesis, we plated human monocytes on CIV matrices containing oxLDL in medium containing human plasma-derived serum. We report here that Macs maintained under these conditions secrete H2O2 continuously and in relatively large amounts for at least 11 days. Using this system, we tested the effect of known antiatherogenic drugs (e.g., PAF receptor antagonists; genistein, a phytoestrogen/isoflavone protein tyrosine kinase (PTK) inhibitor; and lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Mac H2O2 secretion. We found that all three types of antiatherogenic compounds markedly attenuated H2O2 secretion by Macs maintained on these matrices.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lipoproteins
LDL (Intracell, Baltimore, MD) was oxidized with CuSO4 and dialyzed at 4°C versus phosphate-buffered saline (PBS; without Ca+2/Mg+2) to remove CuSO4, as described [1 ]. The oxLDL contained 18.5 ± 0.004 nmoles malondialdehyde/mg protein (average of nine measurements using three different LDL preparations) versus 2.1 ± 0.004 nmoles malondialdehyde/mg protein for non-oxLDL, as measured using Oxi-Tek thiobarbituric acid assay kit according the manufacturer’s directions (ZeptoMetrix, Buffalo, NY).

Matrices
Wells of Falcon 96-well flat-bottom plates (Becton Dickinson, Franklin Lakes, NJ), were treated with 50 µl distilled-deionized water (ddH2O) containing CIV (Fluka, Milwaukee, WI; 50 µg/ml) overnight at 4°C, washed twice with ddH2O, and further incubated with 10 µg/well oxLDL, all as described [1 ].

Monocytes
Human mononuclear cells were isolated from leukocyte concentrates (New York Blood Center, New York) on the day following donation by centrifugation over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) as described [1 ] and then enriched by Percoll gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) as described [7 ]. The entire isolation procedure was performed at room temperature. Purified cells {~80% monocytes, as measured by flow cytometry using Immunotech (Marseille, France) IOTest mouse monoclonal antibodies against CD45 [immunoglobulin G1 (IgG1)-fluorescein isothiocyanate] and CD14 (IgG2a-phycoerythrin) were resuspended in RPMI complete medium {phenol red-free RPMI 1640 (Life Technologies BRL, Rockville, MD) containing 10% v/v heat-inactivated human AB serum (Gemini Bio-Products, Calabasas, CA), penicillin (140 units/ml), and streptomycin (140 µg/ml, Gibco BRL, Grand Island, NY)} and used immediately for experiments (see Figs. 1 3 4 5 ), or maintained at 1–2 x 106 cells/ml in Teflon jars at 37°C in a 95% air, 5% CO2 atmosphere for the period indicated (see Fig. 4 ). For experiments, 200,000 cells (~160,000 monocytes) in 200 µl RPMI complete medium were added to each CIV- or CIV-oxLDL-coated well and incubated at 37°C in a 95% air, 5% CO2 atmosphere for the period indicated. Every other day, 50 µl medium was removed from each well and replaced with 50 µl RPMI complete medium, with or without inhibitors or other compounds as indicated.



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  		Figure 1. Macs maintained on CIV matrices containing ox-LDL secrete H2O2 continuously for 11 days. (A) Wells were precoated with 2 µg CIV and 10 µg oxLDL (•). Mononuclear cells (200,000) in complete medium were added to each well; plates were incubated at 37°C; and H2O2 production was measured at the indicated times as described in Materials and Methods. (B) Cumulative H2O2 production by cells maintained for 11 days on matrices containing oxLDL (•). Data are from the experiments shown in (A). (C) Mononuclear cells (200,000) were plated in wells precoated with CIV and oxLDL, as in (A), and incubated for 4 days. The medium (supernatant) was removed and assayed for H2O2 as described. The cells were subjected to 3 cycles of freezing and thawing (Dead), and assayed for H2O2by addition of 100 µl complete medium and 50 µl Amplex Red buffer. Data for A and C are mean values for H2O2 produced/3 h ± SEM in three experiments, each done in triplicate.

 


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  		Figure 3. PTK, not C-type protein kinases, participate in signaling H2O2 secretion by Macs maintained on CIV matrices containing oxLDL. Mononuclear cells (200,000) were plated in wells precoated with CIV and 10 µg oxLDL and incubated at 37°C for 1 day (A) or 4 days, (B, C). (A, B) Effect of C-type protein kinase inhibitors. Cells maintained for 1 day (A) or 4 days (B) on matrices containing oxLDL were incubated for 1 h in medium containing 1 µM stauroporine (ST), 1 µM bisindolylmalamide (BM), 5 µM celerythrine chloride (CC), or vehicle alone (Con). Amplex Red containing medium without (open bars) or with 80 ng/ml PMA (solid bars) was added to each well, and the plates were further incubated for 30 min at 37°C to assess spontaneous and PMA-stimulated H2O2 production in the absence and presence of the indicated inhibitors. Data are the mean values for H2O2produced/30 min ± SEM of 3 experiments done in triplicate. Student’s t-tests confirmed that stauroporine and bisindolylmalamide significantly inhibited PMA-stimulated H2O2 production by Macs on matrices containing oxLDL, but that none of the inhibitors had a significant effect on Mac H2O2 production stimulated by matrices containing ox-LDL in the absence of PMA. (C) Effect of genistein. Cells maintained for 4 days on matrices containing oxLDL were incubated for 30 min in medium containing vehicle alone (open bars) or 150 µM genistein (solid bars). Amplex Red-containing medium was added and the cells were further incubated for 3 h. Data are the mean values for H2O2 produced/3 h ± SEM of three experiments done in triplicate.

 


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  		Figure 4. Effect of oxLDL and of lovastatin on Mac morphology. (A, C) Mononuclear cells maintained on CIV-oxLDL-matrices for 3 or 7 days, respectively, in lovastatin-free medium. (B) Macs maintained on CIV-oxLDL-matrices for 3 days in medium containing 5 µM lovastatin. (D) Macs maintained on CIV-oxLDL-matrices for 3 days in medium containing 5 µM lovastatin, washed, and incubated for 4 additional days in lovastatin-free medium. Arrows indicate cells with spindle-like morphology. Original scale bar (A) = 50 µm.

 


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  		Figure 5. Lovastatin inhibits H2O2 production by macrophages maintained on CIV matrices containing oxLDL. Mononuclear cells (200,000), cultured for 24 h on CIV matrices containing 10 µg oxLDL, were treated with lovastatin for the period indicated and assayed for H2O2 secretion. (A) Cells incubated in medium containing 0, 2, 5, or 10 µM lovastatin for 4 days. (Cells in lovastatin-free medium produced 2.76 nmoles H2O2/3 h.) (B) Cells incubated for 3 days in medium without or with 5 µM lovastatin, washed 5 times as indicated in Materials and Methods, incubated in fresh medium without or with 5 µM lovastatin for an additional 4 days, and assayed for H2O2 secretion on Day 7. Con = 7 days in medium without lovastatin. Lov = 7 days in medium with lovastatin. Reversal = 3 days in lovastatin-containing medium followed by 4 days in lovastatin-free medium. (C) Cells incubated on matrices containing oxLDL for 3 days in medium containing DMSO alone (Con), 5 µM lovastatin (Lov), 5 µM lovastatin, and 10 mM mevalonate (Mev), 5 µM lovastatin and 10 mM farnesylpyrophosphate (FPP), or 5 µM lovastatin and 10 mM geranylgeranyl pyrophosphate (GPP). On Day 7, Amplex Red/HRP was added to all cultures, and H2O2 production was measured as described in Materials and Methods. (A, B) Mean values for H2O2 produced/3 h ± SEM for three experiments, each done in triplicate. (C) Mean values for H2O2 produced/3 h ± SD for two experiments, each done in triplicate.

 
Lovastatin reversal
Lovastatin was from A.G. Scientific (San Diego, CA) and mevalonate, farnesylpyrophosphate, and geranylgeranyl pyrophosphate were from Sigma-Aldrich. Cultures in 96-well plates were washed five times to remove lovastatin. To prevent loss of loosely adherent cells, plates were centrifuged at 1500 rpm prior to each wash and 150 µl medium was removed from each well and replaced with 150 µl of RPMI complete medium with or without lovastatin as indicated. Subsequently, cells were fed every 24 h by removal of 50 µl of medium and replacement with 50 µl of fresh RPMI complete medium with or without lovastatin.

H2O2 assay
H2O2 was assayed at 37°C using AmplexTM Red/horseradish peroxidase (HRP; Molecular Probes, Eugene, OR). Medium (100 µl) was removed from each well and replaced with 50 µl of Krebs Ringer Buffer (KRB) containing 5 mM glucose and 1% BSA (KRBGA), 1.2 mM Amplex Red, 0.8 U HRP, and, where indicated, 80 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich). Thus, the final assay mixture contained cells in 1/3 KRBGA and 2/3 RPMI complete medium, 0.4 mM Amplex Red, and 0.266 U HRP. Fluorescence was measured using a Cytofluor II reader (Biosearch, Bedford, MA). A standard curve was constructed for each assay using H2O2 in phenol red-free RPMI complete medium. Control experiments in which HRP was omitted from the assay mixture showed no oxidation of Amplex Red, indicating that H2O2 was the principal ROS detected in this assay.

Cumulative H2O2 secretion
H2O2 secreted/day = nmoles H2O2/3 h x 8. H2O2 secretion on Days 3, 5, 7, 9, and 10 was estimated using Excel Forecast assuming H2O2 secreted by the cells increased linearly between preceding and succeeding days.

Effects of inhibitors
PAF receptor inhibitors FR-49175, PCA-4248, and CV-6209 (Biomol Research Laboratories Inc., Plymouth Meeting, PA) were dissolved in ethanol at 20 and 25 mg/ml and in water at 25 mg/ml, respectively, and used at final concentrations of 20, 4, and 1 µM, respectively. These concentrations are 2.5, 4, and 5 times their respective IC50s as reported by the supplier. Genistein, staurosporine, bisindolylmaleimide, chelerythrine chloride (Sigma-Aldrich), and PP1 (Calbiochem, San Diego, CA) were dissolved in DMSO at 300, 1, 1, 5, and 36 mM, respectively. Inhibitors were added 1 h prior to assays by replacing 100 µl medium with 100 µl of complete medium containing ethanol or DMSO with or without twice the indicated final concentration of inhibitor.

Cell number
Cells in wells of 96-well Falcon plates were washed twice with 150 µl PBS (without Ca+2/Mg+2). To prevent cell loss, the plates were centrifuged at 1500 rpm prior to each change of medium or buffer. Cell number was measured using the CyQuant® Cell Proliferation Assay Kit (Molecular Probes), according to the manufacturer’s protocol, using a Cytofluor II plate reader. DNA content of cultures did not vary significantly over 11 days, indicating no significant change in cell number (data not shown).

Protein content of cells
Mononuclear cells (~80% monocytes) were incubated in RPMI complete medium at a concentration of 1 x 106/ml for 1 day and then in RPMI complete medium containing lovastatin (5 µM) for 3 days, all in Teflon beakers. Cells (1.5x106) were removed, washed twice with PBS, lysed, and then assayed using the BCA Protein Assay Kit (Pierce, Rockford, Il). Data were analyzed using a PowerWaveX Select microplate reader (Bio-Tek Instruments Inc., Winooski, VT).

Light microscopy
Cells were photographed with an Axiocam high-resolution camera on a Zeiss Axiovert 200M microscope with a 40 x objective.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Macs maintained on matrices containing oxLDL secrete H2O2 continuously
Monocytes isolated from human blood as described in Materials and Methods were plated in fresh medium in wells coated with CIV alone or CIV and oxLDL and assayed for H2O2 secretion 2, 4, 6, 8, and 11 days following plating (Fig. 1A ). Macs secreted H2O2 on all days and at high levels. Macs maintained for 2 days on CIV matrices containing oxLDL secreted >6 nmoles H2O2/3 h. This very high level of H2O2 secretion declined to a nadir of ~4 nmoles H2O2/3 h on Day 4. Thereafter, the amount of H2O2 secreted by these cells increased as the length of time they were maintained on CIV-oxLDL matrices increased. By Day 11 after plating they were again producing ~6 nmoles H2O2/3 h.

Figure 1B shows cumulative H2O2 secretion by Macs maintained for 2–11 days on CIV-oxLDL matrices. Although the rate of H2O2 secretion per 3 h was linear at all time points examined, the amount of H2O2 secreted per 3 h by Macs maintained on CIV-oxLDL matrices increased continuously between Days 4 and 11. This increase in apparent rate of H2O2 secretion was observed in all three 11-day-long experiments and in experiments using different preparations of CIV and oxLDL.

To confirm that matrix-bound oxLDL was responsible for stimulating Mac H2O2 secretion, we plated these cells on CIV alone. Macs maintained for 4 days on these matrices secreted <0.1 nmole H2O2/3h (data not shown), indicating that the matrix-bound oxLDL, not the CIV matrix or the underlying plastic, stimulated Macs to secrete H2O2.

Approximately 20% of the cells in the experiments reported above were lymphocytes. To determine whether lymphocytes affected Mac H2O2 secretion, we incubated 200,000 Mac-enriched cells (~80% Macs) on CIV-oxLDL matrices for 1 h at 37°C to allow the Macs to adhere, and then washed some cultures to remove non-adherent cells (lymphocytes). Washed and unwashed cultures were incubated for four additional days and assayed for H2O2 secretion. Washed cultures (containing only Macs) produced nearly twice as much H2O2 as unwashed cultures (data not shown), suggesting the presence of lymphocytes or of their secretory products, diminished H2O2 production by Macs maintained on oxLDL-containing matrices. Consistent with this suggestion, we have found that addition of interferon-{gamma} (IFN-{gamma}) to these cultures reduced H2O2 secretion by Macs maintained on oxLDL-containing matrices (data not shown).

As oxLDL and dead cells contain lipid peroxides [8 ], it was conceivable that dead cells could be responsible for some of the Amplex Red oxidation observed. To test this, we maintained Macs on oxLDL-containing matrices in complete medium for 4 days, removed the medium, and then subjected half of the cultures to three cycles of freezing and thawing. Virtually all of the frozen and thawed cells were killed, as measured by Trypan blue dye uptake (data not shown). We then compared H2O2 production by viable vs. frozen and thawed cultures. Only the viable cells produced significant H2O2 above background (Fig. 1C , dead cells), indicating that dead cells and matrix-bound oxLDL made little or no contribution to oxidation of Amplex Red.

In the experiments described in Figure 1A we removed only half (100 µl) of the total (200 µl) culture medium bathing the cells before measuring H2O2 secretion. Thus, it was possible that substances that accumulated in the medium contributed to Amplex Red oxidation. To test this, we assayed media conditioned for 4 days by Macs maintained on CIV-oxLDL matrices. These media contained virtually no Amplex Red oxidizing activity (Fig. 1C , supernatant). We conclude that Amplex Red oxidation reflects H2O2 secreted by viable Macs.

Effects of antiatherogeneic drugs
The findings reported in Figure 1 indicate that matrix-bound oxLDL stimulates Macs to secrete H2O2 continuously and in amounts sufficient to bring the H2O2 concentration in the volume of medium within 1 µm of the Macs’ plasma membrane to ~1 mM in 2 min. Because this concentration of H2O2 can exert deleterious effects on cells and tissues, we inquired whether drugs (e.g., PAF-receptor antagonists, lovastatin) and natural products (e.g., genistein, an isoflavone) known to inhibit atherogenesis [9 10 11 12 ] affect H2O2 secretion by Macs maintained on oxLDL containing matrices.

OxLDL contains substances that are agonists for PAF receptors, and PAF receptor antagonists slow the development [10 , 11 ] and speed the regression [4 ] of atherosclerotic lesions in experimental animals. PAF receptor antagonists attenuated H2O2 secretion by Macs on oxLDL-containing matrices by 54–69% (Fig. 2 ). Because PAF receptors activate C-type and tyrosine kinases [13 ], we tested whether inhibitors of these kinases affected H2O2 secretion by Macs on CIV-oxLDL matrices. The protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide had no effect on H2O2 production by Macs on oxLDL-containing matrices. To confirm that these inhibitors were having the expected effect on PKC-activated H2O2 production, we treated parallel cultures with PMA, a known PKC activator. As anticipated, staurosporine and bisindolylmaleimide completely blocked PMA-stimulated H2O2 production, confirming their inhibitory effect on PKC in these cells (Fig. 3A and 3B ). Celerythrine chloride, a PKC {zeta} inhibitor, had no effect on either spontaneous or PMA-stimulated H2O2 production by Macs (Fig. 3A and 3B) . Genistein, an isoflavone that inhibits a broad spectrum of PTK and slows the development of atherosclerotic lesions in mice [9 ], reduced H2O2 secretion by Macs maintained on CIV-oxLDL matrices by ~64% (Fig. 3C) . PP1, a src kinase inhibitor, had no effect (data not shown). Taken together, the results reported in Figures 2 and 3 suggest that interactions of Mac PAF receptors with PAF-like substances released from or present on oxLDL stimulate these cells to secrete H2O2 via pathways involving one or more PTK.



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  		Figure 2. PAF inhibitors block H2O2 production by Macs maintained for 4 days on CIV matrices containing oxLDL. Monocytes were cultured for 24 h in Teflon jars, plated on matrices containing CIV and oxLDL, and incubated on these matrices for 4 days, as in Figure 1 . PAF receptor inhibitors (FR49175 ({blacksquare}) at 20 µM; PCA-4248 ({blacktriangleup}) at 2 µM; or CV-6209 (•) at 1 µM] or DMSO ({diamondsuit}) alone, were added, and cells were incubated at 37°C for an additional 1 h. H2O2 production was assayed as described in Materials and Methods. Data are the mean values for H2O2 produced/3 h ± SEM. in three experiments, each done in triplicate.

 
Effects of HMG-CoA inhibitors
Statins slow the onset and progression, and may even facilitate regression, of atherosclerotic lesions [3 ]. One of the mechanisms by which they exert this effect is by blunting pro-inflammatory effector functions of leukocytes. To determine whether ox-LDL-stimulated ROS production is one of these functions, we examined the effect of lovastatin in our system.

Macs maintained on CIV-oxLDL matrices for 3 days in medium containing 5 µM lovastatin (Fig. 4B ) were round, smaller, and less well spread than Macs maintained for the same period on CIV-oxLDL-matrices in lovastatin-free medium (Fig. 4A) . Macs maintained on CIV-oxLDL matrices in medium containing 5 µM lovastatin for 3 days, and then placed in lovastatin-free medium for an additional 4 days (Fig. 4D) , returned to the well-spread, spindle-shaped morphology typical of Macs maintained in lovastatin-free medium on CIV-oxLDL-matrices for 7 days (compare Fig. 4C and 4D ). Trypan blue exclusion studies showed that >95% of cells maintained on oxLDL-containing matrices remained viable regardless of whether they were incubated in lovastatin-free or lovastatin-containing medium. However, Macs maintained for 3 days in medium with 5 µM lovastatin contained ~33% less protein than Macs maintained for the same time period in lovastatin-free medium (data not shown), suggesting that lovastatin slows Mac maturation in culture.

Macs maintained for 3 and 7 days on oxLDL-containing matrices in medium containing 2, 5, or 10 µM lovastatin produced significantly less H2O2 than Macs in lovastatin-free medium (Fig. 5A ). Lovastatin’s inhibitory effect on Mac H2O2 secretion was partially reversed by replacement of lovastatin-containing medium on Day 3 with lovastatin-free medium (Fig. 5B) . This coincided with their return to a spindle-like morphology (Fig. 4D) .

The finding that lovastatin inhibited Mac H2O2 production in medium containing human serum (a source of LDL) suggested that lovastatin affected the availability of HMG-CoA products other than cholesterol. Huang et al. [14 ] reported that lovastatin inhibits Mac protein prenylation. Because protein prenylation is required for activity of GTPases (e.g., Rac) that participate in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assembly, we tested whether addition of biosynthetic products of HMG-CoA reductase to the medium reduced lovastatin’s inhibitory effect on H2O2 production by Macs on oxLDL-containing matrices. Mevalonate and geranylgeranylpyrophosphate blocked lovastatin’s inhibitory effect by >95%, and farnesyl pyrophosphate was much less effective (Fig. 5C) . These findings suggest that lovastatin inhibits Mac H2O2 secretion in cholesterol-replete medium by blocking protein geranylgeranylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Macs in nascent and established atherosclerotic lesions in the arterial intima are adherent to matrix proteins containing native and oxLDL and are bathed in a protein-rich solution containing LDL at twice or more its concentration in plasma [15 ]. The culture system used in the experiments reported here was designed to reproduce certain aspects of the conditions in the arterial intima. That is, it uses monocyte-derived Macs adherent to CIV matrices containing oxLDL and bathed in lipoprotein-containing human serum. Using this system, we have made two observations that we believe are relevant to understanding the pathogenesis of atherosclerosis. First, Macs maintained on matrices containing oxLDL secrete H2O2 continuously. In the system described here, they did so at an ever-increasing rate for 11 days (Fig. 1A) . Sustained H2O2 secretion by monocytes/Macs for this period has not been reported previously. Second, three different classes of antiatherogenic compounds, PAF receptor antagonists, a PTK inhibitor, and a statin, reduced Mac H2O2 secretion to about the same extent. This finding suggests that they may affect different steps of a common pathway and that their capacity to slow atherogenesis may be due to their inhibitory effects on this pathway.

The athero-protective effects of PAF receptor antagonists have been attributed to their capacity to block MCP-1 secretion by oxLDL-stimulated endothelial cells [16 ], thereby inhibiting monocyte migration into atherosclerotic lesions [1 , 4 ] and the athero-protective effects of statins to their ability to reduce plasma LDL levels. Our findings that PAF receptor antagonists and statins attenuate H2O2 production by Macs maintained on oxLDL-containing matrices (Figs. 2 and 4) suggest another mechanism by which these drugs may inhibit atherogenesis, namely, by blocking oxLDL-stimulated H2O2 production. Moreover, our studies show that the pathway by which oxLDL-containing matrices stimulate Mac H2O2 secretion is independent of C-type protein kinases (Fig. 3) .

Relevance of the effects of lovastatin on Mac H2O2 production in vitro to its effects in vivo
The minimal lovastatin concentration needed to reproducibly attenuate H2O2 production by Macs maintained on oxLDL-containing matrices was 2 µM, 2- to fivefold higher than the peak plasma lovastatin concentration in adults ingesting 80 mg of this drug/day. This difference in lovastatin concentrations in vitro and in vivo may lead one to question whether lovastatin’s inhibitory effects on Mac functions in vitro are relevant to its effects in vivo. The findings that lovastatin treatment of hypercholesterolemic patients decreased expression of CD14 [17 , 18 ], LFA-1, ICAM-1 [19 ], SR-AI, SR-AII, and CD36 [20 ], and CD40 ligand (CD154) [21 ] by their monocytes suggest they are. Taken together with the report that lovastatin’s inhibitory effects on Mac functions were most pronounced when these cells were cultured in lovastatin-containing medium immediately following harvest [20 ] and that monocytes maintained on oxLDL-containing matrices in the presence of lovastatin contained ~33% less protein than monocytes maintained on these matrices in lovastatin-free medium, these findings suggest that statins exert their most pronounced effects on Macs during their development in bone marrow and during the early stages of monocyte differentiation into Macs in tissues.

Statins block mevalonate synthesis with equal efficiency in LDL-deficient and LDL-sufficient medium. Their primary effect in LDL-sufficient medium appears to be to limit the supply of isoprenyl lipids such as farnesyl and geranylgeranyl pyrophosphate [22 ]. These lipids are required for the activity of small GTPases such as Rac, which participates in NADPH oxidase assembly. Our finding that geranylgeranyl pyrophosphate blocked lovastatin’s inhibitory effect on oxLDL-stimulated H2O2 production (Fig. 5) suggests that one or more geranylgeranylated proteins is involved in signaling H2O2 production by Macs adherent to oxLDL-containing matrices.

Role of ROS in atherogenesis
Substantial evidence indicates that oxidant stress promotes atherogenesis in experimental animals and in humans [3 , 23 ]. Although conflicting evidence exists regarding the contribution of NADPH oxidase to atherogenesis in mice [24 25 26 27 ], strong evidence indicates that Mac-generated ROS contribute to atherogenesis in humans [28 , 29 ]. What has been uncertain is whether Macs produce H2O2 in significant amounts for sustained periods and the nature of the substances that stimulate them to do so. The findings presented here show that Macs maintained on oxLDL-containing matrices in medium containing normal human serum secrete substantial amounts of H2O2 continuously for at least 14 days (the longest period examined, unpublished data) and that they are stimulated to do so, at least in part, by oxLDL-derived substances that interact with Mac PAF receptors.

Macs maintained on oxLDL-containing matrices secrete much more H2O2 than Macs acutely plated on these matrices
Macs maintained on oxLDL-containing matrices for 8 days produced 1.7 nmoles H2O2/160,000 Macs/h (Fig. 1A and 1B) , while Macs maintained in medium containing fetal bovine serum (deficient in LDL) for ≥8 days in Teflon jars prior to plating on oxLDL-containing matrices released only background levels of H2O2 [1 ] (i.e., ~0.1 nmoles/160,000 Macs/h). These findings suggest that long-term maintenance of Macs on oxLDL-containing matrices increases their capacity to produce ROS. Consistent with this suggestion are the findings that oxLDL up-regulates expression of CD36 by human monocyte-derived Macs (refs. [16 , 30 ], and data not shown) and that CD36 mediated signaling accounts for at least 50% of H2O2 secretion by Macs acutely plated on oxLDL-containing matrices [1 ].

Pathways by which oxLDL-containing matrices signal Macs to produce H2O2
Oxidation of LDL generates PAF-like substances that bind to Mac PAF receptors [31 ], and PAF stimulates human Macs to produce H2O2 [32 ]. OxLDL also binds to CD36 and CD36 stimulates Macs to secrete H2O2 [1 ]. PAF signals by activating heterotrimeric G-protein-coupled receptors [32 ], whereas CD36 signals by activating lyn and possibly other PTK [33 ]. Thus, it is not surprising that PAF receptor antagonists and genestein, a PTK inhibitor (Fig. 2) , inhibited oxLDL-stimulated Mac H2O2 production (Fig. 3) .

The present study shows that PAF receptor antagonists suppress H2O2 production by Macs maintained on oxLDL-containing matrices by ~60% (Fig. 2) . Maxeiner et al. [1 ] reported that antibodies against CD36 reduced H2O2 production by Macs acutely plated on oxLDL-containing matrices by ~60%. We suspect that if PAF and CD36 receptor antagonists were added simultaneously in the same assay, their effects would be additive and they would completely block oxLDL-stimulated H2O2 production. We are testing this hypothesis.

ß2 integrins also participate in oxLDL-stimulated H2O2 production by Macs and are the primary cell surface receptors that mediate adhesion to oxLDL-containing matrices [2 ]. Macs from SRA-I/II knockout mice (SRA-I/II–/–) secrete as much H2O2 as wild-type Macs when plated on ox-LDL matrices [2 ]. This finding suggests that ß2 integrins are required for oxLDL-matrix-mediated signaling of NADPH oxidase activation and/or assembly. Our finding that matrix-bound, but not soluble, oxLDL stimulates Mac H2O2 production [1 ] suggests a requirement for oligomerization of ß2 integrins by matrix-bound ligands in signaling and/or assembling the Macs’ NADPH oxidase. We suggest that interactions of PAF receptors and CD36 with matrix-bound oxLDL signal activation of Mac ß2 integrins, that activation of the ß2 integrins enables them to bind and oligomerize when they interact with matrix-bound oxLDL, and that signals generated by these activated and oligomerized ß2 integrins participate in the final common pathway by which PAF receptors and CD36 signal NADPH oxidase assembly and H2O2 secretion. Experiments are in progress to explore this hypothesis.

Removal of lymphocytes enhances H2O2 secretion by Macs maintained on oxLDL-containing matrices
A report by Nakagawa et al. [34 ] that IFN-{gamma} down-regulates Mac CD36 expression, a demonstration by Fong et al. [35 ] that T helper cell-derived IFN-{gamma} reduces the capacity of mouse Macs to oxidize human oxLDL, and our finding that IFN-{gamma} suppresses ox-LDL-stimulated Mac H2O2 secretion (data reported but not shown) suggest that removal of lymphocytes eliminated the primary source of this cytokine in our cultures, thereby relieving its inhibitory effect on Mac CD36 expression. The resulting increase in Mac CD36 expression likely increased the number of CD36 receptors engaged by matrix-bound oxLDL, thereby enhancing CD36-mediated signals that promote NADPH oxidase activation/assembly, and H2O2 secretion. The resulting increase in secreted H2O2 should have further oxidized matrix-bound LDL, thereby elevating the concentrations of PAF-like substances and matrix-bound ligands for CD36 and ß2 integrins, resulting in acceleration of the rate of Mac H2O2 production. Indeed, this is exactly what we observed (Fig. 1A and 1B) .

Does Mac ROS secretion also play a role in healing of atherosclerotic lesions?
Formation of reactive aldehydes, peroxides, and carbonyl groups is an unavoidable consequence of life in an oxygen-rich environment [36 ]. These substances alone, and in association with products of glyco-oxidation reactions, cross-link many biopolymers, thereby producing a variety of substances, many of which are insoluble and/or resistant to enzymatic digestion [37 ]. ROS have the capacity to fragment proteins, lipids, polynucleotides, and polysaccharides [36 ]. Fragmentation of these substances by ROS may increase their solubility and their susceptibility to enzymatic attack, thereby promoting their elimination from the body. As reported by Petersen et al. [38 ], in the absence of ROS such substances accumulate and are associated with heightened levels of inflammation and tissue damage [39 ]. Thus, although all cells produce ROS, Macs and granulocytes produce more of these substances. Viewed from this perspective, ROS secreted by Macs in atherosclerotic lesions may facilitate solubilization and digestion of insoluble intra- and extra-cellular debris. By this and other means [4 ], once atherogenesis-promoting conditions (e.g., high blood pressure, elevated plasma LDL) have been controlled, ROS secreted by lesion-associated Macs may play a role in healing of these lesions. If it does, Macs in regressing atherosclerotic lesions might be expected to produce more H2O2 than Macs in progressing lesions and regressing lesions might be expected to contain fewer IFN-{gamma} producing lymphocytes than progressing lesions.


   ACKNOWLEDGEMENTS
 
The work was supported by postdoctoral fellowship HL10196 (to I. S-C.) and Grant AI20516 (to S. C. S.). We thank Drs. Jens Husemann and John Loike for reading the manuscript.


   FOOTNOTES
 
1 Current address: Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT. Back

2 Current address: M.D./Ph.D. Program, Washington University, St. Louis, MO Back

Received February 2, 2005; revised June 10, 2005; accepted June 28, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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