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(Circulation. 1995;91:1525-1532.)
© 1995 American Heart Association, Inc.

Articles

Protection From Oxidized LDL–Induced Leukocyte Adhesion to Microvascular and Macrovascular Endothelium In Vivo by Vitamin C but Not by Vitamin E

Hans-Anton Lehr, MD, PhD; Balz Frei, PhD; A. Maria Olofsson, PhD; Thomas E. Carew, PhD; Karl-E. Arfors, PhD

From the Institute for Surgical Research (H.-A.L.), University of Munich, Germany; Whitaker Cardiovascular Institute (B.F.), Boston University Medical Center, Boston, Mass; Department of Medical and Physiological Chemistry (A.M.O.), University of Lund, Sweden; Experimental Medicine Inc (K.-E.A.), Princeton, NJ; and San Diego Regional Cancer Center (K.-E.A.), San Diego, Calif.

Correspondence to Hans-Anton Lehr, MD, PhD, Department of Pathology, University of Washington Medical Center, RC-72, 1959 NE Pacific St, Seattle, WA 98195.

	Abstract

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Background The ability of oxidized LDL (oxLDL) to stimulate leukocyte–endothelium interaction is considered to be an important aspect of its proatherogenic action. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in hamsters, we have previously shown that systemic administration of oxLDL stimulates leukocyte adhesion to microvascular endothelium through a mechanism that involves the generation and action of reactive oxygen species (ROS).

Methods and Results Through the combined use of scanning electron microscopy and intravital microscopy in the same animal model, we demonstrate that oxLDL-induced leukocyte adhesion is not confined to the microcirculation but can also be observed on aortic endothelium. OxLDL-induced leukocyte adhesion to both microvascular and macrovascular endothelium was almost entirely prevented by pretreatment of the hamsters with dietary or intravenous vitamin C, which has the capacity to scavenge and neutralize ROS (arterioles: 20.5±16.4 cells/mm² [diet] and 16.3±23.8 cells/mm² [IV] versus 74.2±47.5 cells/mm² [control, P<.01]; aorta: 1.0±0.4 cells/mm² [diet] and 1.1±0.5 cells/mm² [IV] versus 14.7±6.0 cells/mm² [control, P<.01], 15 minutes after oxLDL, n=7 animals per group). Vitamin C pretreatment also completely prevented oxLDL-induced leukocyte-platelet aggregate formation in the bloodstream but did not affect leukocyte rolling along the microvascular endothelium. No inhibitory effect on any of the studied parameters was observed as a result of pretreatment of the animals with the lipid-soluble antioxidants vitamin E and probucol.

Conclusions The protective effects of vitamin C on oxLDL-induced leukocyte adhesion and aggregate formation were seen at vitamin C plasma levels that can easily be reached in humans by diet or supplementation, suggesting that this could be one of the mechanisms by which vitamin C contributes to the well-documented protraction of atherogenesis as observed in large epidemiological surveys.

Key Words: platelets • probucol • atherogenesis • lipoproteins

	Introduction

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The oxidation of LDL has been proposed to be a key event during early atherogenesis.¹ One of the most prominent properties of oxidized LDL (oxLDL) is its ability to stimulate the adhesion of leukocytes to endothelial cells.^{2 3} Experiments with phospholipase or lipoxygenase inhibitors or with superoxide dismutase (SOD) have suggested that the process of cell-mediated LDL oxidation involves the generation and action of leukotrienes and of reactive oxygen species (ROS), in particular, of superoxide anion.^{4 5} These mediators are involved not only in the process of LDL oxidation but also in the biological changes observed after the introduction of oxLDL into the organism, as suggested from intravital microscopic studies on hamsters and mice: Treatment of the animals with inhibitors of leukotriene biosynthesis or with SOD effectively attenuates oxLDL-induced leukocyte adhesion to microvascular endothelium.^{3 6} The generation and action of superoxide anion and other ROS can be counteracted with SOD as well as with vitamin C (ascorbate), which scavenges aqueous-phase ROS by very rapid electron transfer and thus can prevent the initiation of lipid peroxidation,^{7 8 9 10} and with vitamin E and probucol, both of which can terminate the chain reactions of lipid peroxidation in biomembranes and lipoproteins.^{11 12} Indeed, the capacity of these antioxidants to interfere with the process of LDL oxidation^{11 12 13 14 15 16 17 18} may explain the antiatherogenic effects of antioxidants observed in laboratory animals^{19 20 21 22 23} and in humans in large epidemiological studies.^{24 25 26 27}

Because ROS are involved not only in the process of LDL oxidation^{1 4} but also in oxLDL-induced leukocyte adhesion in vivo,⁶ we were interested in whether the antioxidant vitamins C and E, as well as the antioxidant drug probucol, have the capacity to attenuate the leukocyte adhesion–promoting effects of oxLDL on microvascular and macrovascular endothelium.

	Methods

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Animal Model
The study was performed using the dorsal skinfold chamber preparation of Syrian golden hamsters (body weight, 50 to 70 g; age, 6 to 8 weeks). Animals were maintained on a diet of standard rodent chow and water ad libitum. For in vivo fluorescence microscopy, a dorsal skinfold chamber and indwelling catheters were implanted in pentobarbital-anesthetized hamsters as previously described.³ A recovery period of 48 to 72 hours between chamber implantation and the experiments was allowed to eliminate the effects of anesthesia and surgical trauma on the microvasculature. The study was performed according to a protocol approved by the local ethics committee. All experiments were performed in a blinded fashion.

Treatment Regimen
Vitamins were administered by supplementation of the homogenized standard laboratory chow (Ralston Purina rodent chow; basal vitamin E content, 60 mg/kg of chow) with vitamin C (l-ascorbic acid, 10 g/kg of chow, Sigma Chemical Co) or vitamin E (dl--tocopherol acetate, 10 000 IU/kg of chow, Omega Pharma). In the same way, probucol (10 g/kg of chow; Marion Merrell Dow) was added to homogenized chow. To facilitate absorption of the lipid-soluble vitamin E and the lipid-soluble drug probucol, the chow (including the vitamin C–supplemented chow and the unsupplemented control chow) was supplemented with 2% olive oil (Bertolli Classico). Vitamin plasma levels were assessed from EDTA-anticoagulated blood by high-performance liquid chromatography (HPLC) with electrochemical detection in control animals as well as in vitamin C– and E–treated animals after 1 week of dietary supplementation.²⁸ Probucol levels were assessed by HPLC after extraction with methanol-acetone.²⁰

Intravital Fluorescence Microscopy
Quantitative measurements of the microcirculation were evaluated in the striated skin muscle contained within the observation window by intravital microscopy as previously described in detail.³ The analyses included the quantification of the leukocyte–endothelium interaction in four to six collecting and postcapillary venules (, 20 to 60 µm) per observation chamber, as well as in four to six arterioles (, 20 to 60 µm). For contrast enhancement, leukocytes were stained in vivo with acridine orange (0.5 mg · kg^-1 · min^-1 IV; Sigma Chemical Co) and classified by fluorescence microscopy according to their interaction with the endothelial lining as adherent, rolling, or free-flowing cells.³ Acridine orange intercalates with DNA and thus stains all circulating leukocytes without discriminating individual leukocyte subpopulations. Adherent leukocytes were defined in each vessel segment as cells that did not move or detach from the endothelial lining within an observation period of 30 seconds and are given in the figures as the number of cells per square millimeter of vessel surface, as calculated from the diameter (in µm) and length (200 µm) of the vessel segment studied. Both single leukocytes and leukocytes involved in small aggregates were included in this cell count. In postcapillary venules, rolling leukocytes are given as a percentage of the nonadherent leukocytes passing through the observed vessel segment within 30 seconds. In arterioles, where the high erythrocyte velocity did not permit the quantification of free-flowing leukocytes within the central bloodstream, rolling leukocytes were defined as the number of cells slowly traversing the observed vessel segment along the endothelial wall within 30 seconds, expressed in proportion to the inner vessel circumference (in mm). At all time points before and after the injection of oxLDL, microvessel diameters were assessed with the use of a computer-assisted microcirculation analysis system (CAMAS),²⁹ and centerline red blood cell velocities were assessed by dual-slit cross-correlation.

Scanning Electron Microscopy
Before (n=7 animals per group) and 15 minutes after (n=7 animals per group) intravenous injection of oxLDL, EDTA-anticoagulated blood was taken through aortic puncture in pentobarbital-anesthetized hamsters. Buffy coat cells were isolated by density gradient centrifugation and fixed in a medium containing 2.5% glutaraldehyde and 0.05% CaCl₂ in 0.1 mol/L sodium cacodylate buffer (pH 7.4, 21°C). After dehydration in graded ethanol, buffy coat samples were placed on aluminum stubs, critically point-dried under liquid CO₂ (model CPD030, Baltec), sputter-coated with a layer of gold, and examined with a scanning electron microscope (model 35CF, JEOL) at 15 kV. Likewise, aortas were removed by laparatomy in pentobarbital-anesthetized hamsters before (n=7 animals per group) and 15 minutes after (n=7 animals per group) injection of oxLDL. For this purpose, aortas were first flushed retrograde with normal saline and then by perfusion with the above-described fixation medium. The abdominal part as well as major portions of the thoracic aorta were excised, opened longitudinally along the plane of the celiac and mesentery arteries, and pinned onto cork mounts. These samples were subsequently dehydrated and examined by electron microscopy as described above. The number of leukocytes were counted and expressed as cells per square millimeter of the aortic segment in each animal.

Lipoproteins
Isolation and oxidative modification of LDL were performed as previously described in detail.³ Briefly, LDL was isolated by density gradient ultracentrifugation from EDTA-anticoagulated blood of healthy humans. The density cut was 1.045 to 1.065 g/mL. LDL stock solutions were stored (4°C under argon in the dark) for a maximum of 7 days. Before oxidative modification of LDL, EDTA was removed by chromatography on Sephadex columns (PD-10, Sephadex G-25M, Pharmacia Fine Chemicals). Cholesterol content was determined by Cholesterin Monotest (Boehringer Mannheim GmbH). LDL was diluted with phosphate-buffered saline ([PBS] without Ca²⁺ and Mg²⁺, pH 7.3 at 21°C) to reach a final concentration of 0.85 mg/mL LDL cholesterol. Oxidative modification was achieved by incubation (18 hours, 37°C) of the LDL suspension (1 to 1.5 mL) with 7.5 µmol/L Cu²⁺. Cu²⁺ was not removed from the oxLDL suspension before injection into the animals. In control experiments, we had observed that neither free Cu²⁺ (7.5 µmol/L CuSO₄ in PBS) nor a freshly prepared LDL-Cu²⁺ suspension stimulated the interaction of fluorescently stained leukocytes with the microvascular endothelium. Conversely, removal of Cu²⁺ from oxLDL by column chromatography was found not to affect the stimulation of leukocyte–endothelium interaction. LDL oxidation was verified by determinations of fatty acid composition and lipoperoxide levels in native LDL and oxLDL as well as by assessment of the electrophoretic mobility of LDL particles on agarose gel electrophoresis.³ Immediately after oxidation, oxLDL (4 mg LDL cholesterol/kg body wt) was injected intravenously as a bolus into the hamsters via a permanent jugular catheter. It has been pointed out by Chisolm³⁰ that this dose of oxLDL transiently exposes the endothelium to approximately 8 mg/dL of oxLDL cholesterol. He reasoned that if a human lesion were to contain LDL at concentrations similar to those in plasma, then less than 10% of the arterial LDL would need to be present in oxidized form to expose the endothelium to a concentration comparable to that used in the present study. Such a scenario certainly seems possible, particularly in patients with severe atherosclerosis³¹ or diabetes mellitus.³²

Statistical Analysis
Values were tested for parametric distribution. Although parametric distribution was not uniformly found in all data sets, the data in the figures are given as mean±SD values to facilitate interpretation. P values were calculated using the Mann-Whitney U test or the Wilcoxon test with Bonferroni's correction. Values of P<.05 or <.01 were considered statistically significant and are indicated in the figures with one or two asterisks, respectively.

	Results

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Leukocyte Rolling, Aggregation, and Adhesion to Microvascular and Macrovascular Endothelium
As observed in previous studies, injection of oxLDL elicited the rolling and adhesion of fluorescently stained leukocytes to the endothelial lining of both arterioles and postcapillary venules (Figs 1 and 2). Likewise, oxLDL elicited the formation of loose leukocyte aggregates that tumbled down the microvessels and firmly adhered to the microvascular lining (Fig 3, inset). These leukocyte aggregates were further characterized by scanning electron microscopy and found to consist of leukocytes and activated dendritic platelets, forming either broad or threadlike bridges between individual adjacent leukocytes (Fig 3). In agreement with previous observations,³ the injection of oxLDL resulted in a rapid drop in circulating leukocyte counts, suggesting that oxLDL-stimulated leukocyte–endothelium interaction was not confined to the striated muscle tissue under observation in the skinfold chamber but rather constituted a generalized event throughout the organism (Fig 4). This assumption was confirmed by scanning electron micrographs, which revealed a significant increase in the number of adherent leukocytes on the aortic endothelium 15 minutes after the injection of oxLDL. Leukocytes adhered either singly (Fig 5, top) or in small aggregates of three and more cells, involving the participation of activated dendritic platelets in aggregate formation (Fig 5, middle). Platelets also interacted directly with aortic endothelium after stimulation with oxLDL, as indicated by the observation of mural thrombi (Fig 5, bottom). Adherent leukocytes and mural thrombi were distributed over the entire aorta, involving the abdominal segment and parts of the thoracic aorta. Leukocytes tended to adhere to aortic endothelium predominantly in the vicinity of interendothelial junctions (Fig 5, middle). Although the process of perfusion fixation may have flushed a considerable number of loosely adherent leukocytes from the endothelial lining, leaving only leukocytes that were engaged in strong adhesive interaction or in the process of emigration (Fig 5, top and middle), an effort was made to quantify the extent of leukocyte adhesion: a mean value of 14.7±6.0 leukocytes/mm² of aortic surface were counted in control animals 15 minutes after oxLDL injection. Scanning electron microscopy did not, however, allow us to discern whether the adherent leukocytes were neutrophils, monocytes, or lymphocytes.

View larger version (27K): [in this window] [in a new window]   Figure 1. Plots of leukocyte rolling along the endothelium of postcapillary venules (top) and arterioles (bottom) before and 5, 15, 30, and 60 minutes after intravenous injection of oxidized LDL (oxLDL). Leukocyte rolling was assessed in four to six postcapillary venules (top) and four to six arterioles (bottom) per observation window before injection of oxLDL (0') and in the time course thereafter. Measurements were performed in control hamsters (n=7), vitamin C–fed (n=7) and –injected hamsters (n=7), and hamsters pretreated with vitamin E (n=7) or probucol (n=7). Rolling leukocytes are expressed as a percentage of nonadherent leukocytes (in percent, venules) or as number of leukocytes slowly rolling through the vessel segment under investigation within 30 seconds, normalized for the inner vessel circumference (in 1/mm, arterioles). Values are given as mean±SD. #P<.05 and ##P<.01 vs baseline values (0').

View larger version (26K): [in this window] [in a new window]   Figure 2. Plots of leukocyte adhesion to the endothelium of postcapillary venules (top) and arterioles (bottom) before and 5, 15, 30, and 60 minutes after intravenous injection of oxidized LDL (oxLDL). Leukocyte adhesion was assessed in four to six postcapillary venules (top) and four to six arterioles (bottom) per observation window before injection of oxLDL (0') and in the time course thereafter. Measurements were performed in control hamsters (n=7), vitamin C–fed (n=7) and –injected hamsters (n=7), and hamsters pretreated with vitamin E (n=7) or probucol (n=7). Adherent leukocytes are expressed as number per square millimeter of endothelial surface. Values are given as mean±SD. #P<.05 and ##P<.01 vs baseline values (0'), *P<.05, **P<.01 vs comparable values in control animals.

View larger version (97K): [in this window] [in a new window]   Figure 3. Scanning electron micrograph of leukocyte aggregates in hamster bloodstream 15 minutes after injection of oxidized LDL showing the involvement of platelets forming broad and threadlike bridges between individual leukocytes. Inset, Intravital fluorescence microscopic demonstration of leukocyte aggregates tumbling down the endothelial lining of a small venule; for contrast enhancement, leukocytes were stained in vivo with acridine orange. Note the formation of leukocyte aggregates involving two or more cells. Magnification x7500; space bar in inset indicates 100 µm.

View larger version (16K): [in this window] [in a new window]   Figure 4. Plots of the number of circulating leukocytes in the peripheral blood of hamsters before and 5, 15, 30, and 60 minutes after intravenous injection of oxidized LDL (oxLDL). Measurements were performed in control hamsters (n=7), vitamin C–fed (n=7) and –injected hamsters (n=7), and hamsters pretreated with vitamin E (n=7) or probucol (n=7). Values are given as mean±SD. #P<.05 and ##P<.01 vs baseline values (0'), *P<.05, **P<.01 vs comparable values in control animals.

View larger version (100K): [in this window] [in a new window]   Figure 5. Scanning electron micrographs of aortic endothelium of hamsters 15 minutes after injection of oxidized LDL. Leukocytes adhere either singly (top) or as clusters of three and more cells (middle) to the aortic endothelium. Top, Impression of the adhesive forces required to maintain firm leukocyte adhesion to aortic endothelium against the dispersal shear forces exerted by the bloodstream and the stream of the fixation medium during perfusion fixation. Middle, Localization of adherent leukocytes in the vicinity of an interendothelial junction. Bottom, Mural thrombus on the aortic endothelium, involving red blood cells. Note that in all three panels, the endothelial lining is intact. Space bars indicate 1 µm (top) and 10 µm (middle and bottom).

Pretreatment With Water- and Lipid-Soluble Antioxidants
Vitamin C and E supplementation (10 g vitamin C or 10 000 IU vitamin E per kg of chow, supplemented with 2% olive oil for better absorption of the lipid-soluble antioxidant vitamin E) resulted in a 3.2- and 3.9-fold increase in vitamin C and E plasma levels, respectively, over levels measured in control hamsters (baseline: 18.0±7.2 µmol vitamin C per liter of plasma, 10.3±4.2 µmol vitamin E per liter of plasma; after supplementation: 58.5±24.8 µmol vitamin C per liter of plasma, 40.7±14.3 µmol vitamin E per liter of plasma; n=7 animals in each group). The increase was significant at P<.01 for both vitamins C and E. A comparable increase in vitamin C plasma levels was reached by injecting vitamin C as a bolus intravenously at a dose of 5 mg/kg body wt (59.7±18.6 µmol vitamin C per liter of plasma, n=7 animals, P<.01 versus control). Supplementation of the homogenized chow with 10 g probucol per kg of laboratory chow resulted in a probucol level of 5.3±1.4 µg/mL of plasma (n=7 animals), which is lower than values measured in previous studies involving other animal species.²⁰

Pretreatment of the hamsters with vitamin C for 1 week significantly attenuated oxLDL-induced leukocyte adhesion to microvascular endothelium (Fig 2). However, vitamin C treatment did not affect oxLDL-induced leukocyte rolling along the endothelial lining (Fig 1). The changes in leukocyte adhesion were not secondary to alterations in local shear force conditions since we observed no differences in microhemodynamic parameters (microvessel diameter and red blood cell velocity) between animals of different treatment groups (data not shown). We no longer observed the formation of leukocyte–platelet aggregates after injection of oxLDL into vitamin C–treated animals. A comparable inhibition of oxLDL-induced leukocyte adhesion and aggregate formation was obtained by acutely raising vitamin C plasma levels to comparable plasma levels (see above) through intravenous bolus injection (Fig 2). Finally, a significant inhibition by vitamin C diet and injection was observed on oxLDL-induced leukocyte adhesion to aortic endothelium, where only 1.0±0.4 leukocytes/mm² (vitamin C diet) and 1.1±0.5 leukocytes/mm² (intravenous vitamin C) were counted (n=7 animals per group). The data for both groups were significantly different from the values observed in control animals (14.7±6.0 cells/mm², P<.01). The inhibition of oxLDL-induced leukocyte adhesion by vitamin C was also reflected by the circulating leukocyte counts, which remained virtually unchanged after the injection of oxLDL (Fig 4).

In contrast to these striking inhibitory effects of vitamin C pretreatment, no inhibition of oxLDL-induced leukocyte rolling or adhesion was observed in animals pretreated with vitamin E or probucol (Figs 1 and 2). We also did not observe a reduction in leukocyte aggregate formation. In addition, oxLDL-induced leukocyte adhesion to aortic endothelium in vitamin E– and probucol-treated animals was not different from that of control animals (vitamin E: 13.5±7.2 cells/mm²; probucol: 12.8±4.3 cells/mm²; n=7 animals per group), and the drop in circulating leukocyte count after oxLDL injection was similar in vitamin E– or probucol-treated animals and control animals (Fig 4).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous intravital microscopic studies on the dorsal skinfold chamber model in hamsters have shown that oxLDL induces rolling and subsequent adhesion of leukocytes to the endothelium of small venules and arterioles.^{3 6 33} In the present study, we provide the first electron microscopic evidence that oxLDL-induced leukocyte adhesion is not confined to the microcirculation but also affects large blood vessels such as the aorta (Fig 5, top and middle). This is of crucial importance because, with the exception of accelerated allograft atherosclerosis,³⁴ the clinical effects of atherogenesis are mainly confined to medium-sized and large arteries, in particular, the aorta.

In addition to the adhesion of leukocytes to the aortic endothelium, we observed the formation of mural thrombi in response to oxLDL injection (Fig 5, bottom). This finding is in agreement with previous in vitro studies demonstrating that oxLDL induces a procoagulant state of endothelial cells, presumably through inhibition of prostacyclin and nitric oxide synthesis.^{35 36} Under physiological conditions, prostacyclin and nitric oxide act together to prevent the adhesion of platelets and leukocytes to intact endothelium.^{37 38}

One objective of the present study was to investigate the effects of antioxidants on oxLDL-induced leukocyte adhesion. The presumed threshold for effective protection from cardiovascular diseases in humans by vitamins C and E has been estimated at 40 to 50 and 27.5 to 30 µmol/L, respectively.²⁷ These threshold levels, which must be surpassed in humans to obtain statistically significant protection from cardiovascular diseases,²⁷ were surpassed by all of the supplemented hamsters in the present study, suggesting that the vitamin plasma levels measured in the hamsters of the present study translate into data found in humans, with low baseline vitamin levels in control hamsters corresponding to a predicted high risk of cardiovascular disease and high levels in vitamin-supplemented hamsters corresponding to a predictive low risk of cardiovascular diseases.^{24 25 26 27}

Pretreatment of hamsters for 1 week with vitamin C almost completely prevented leukocyte adhesion to microvascular endothelium as well as the formation of leukocyte–platelet aggregates in response to oxLDL injection (Figs 2 and 3). A comparable extent of protection was obtained by acutely raising vitamin C plasma levels through a single bolus injection of vitamin C just 5 minutes before oxLDL challenge, suggesting that vitamin C does not need to be incorporated into cells to be effective but that it merely needs to be circulating in the bloodstream. The inhibitory effect of vitamin C on oxLDL-induced leukocyte–endothelium interaction was not confined to the striated muscle microcirculation of the skinfold chamber (Fig 2) but rather represents a systemic phenomenon, as suggested (1) by the observed reduction in the number of adherent leukocytes on the aortic endothelium of vitamin C–treated hamsters and (2) by the fact that vitamin C treatment effectively prevented the drop in circulating leukocyte counts that was observed in control animals in response to oxLDL-induced systemic leukocyte–endothelium interaction (Fig 4). However, vitamin C treatment did not affect oxLDL-induced leukocyte rolling. This finding is in agreement with earlier observations in the same animal model, in which SOD was found to inhibit oxLDL-induced leukocyte adhesion but not rolling along the microvascular endothelium,⁶ and suggests that ROS-independent mechanisms are involved in the stimulation of leukocyte rolling by oxLDL. These mechanisms could include the action of leukotrienes and/or of platelet-activating factor (PAF) or PAF-like lipid (PAF-LL), as implied in experiments in which inhibition of leukotriene generation³⁹ or PAF receptor blockade³³ significantly attenuated not only leukocyte adhesion but also leukocyte rolling in response to oxLDL injection.

The demonstration in the present study that vitamin C inhibits leukocyte adhesion and aggregate formation is in agreement with reports demonstrating that vitamin C exerts direct antiadhesive and antiaggregatory effects on leukocytes, platelets, and/or endothelial cells in response to diverse stimuli in vitro,^{40 41} decreases leukocyte infiltration in an animal model of localized adjuvant arthritis,⁴² and corrects the pathologically increased aggregation and adhesion of platelets in patients with diabetes mellitus or coronary artery disease and in subjects on a high-fat diet.^{43 44}

The mechanism by which vitamin C exerts its inhibitory effects on the interaction among platelets, leukocytes, and endothelium remains a matter of speculation. Vitamin C could interfere with the biological response to oxLDL by reducing the formation of PAF or of PAF-LL. Recent in vitro evidence suggests that an ROS attack on membrane phospholipids results in the generation of fragmented phospholipids that activate leukocytes via the PAF receptor and have thus been called PAF-LL.⁴⁵ Previous studies on the hamster skinfold chamber model have shown that pharmacological blockade of the PAF receptor significantly attenuates oxLDL-induced leukocyte–platelet aggregate formation and adhesion of leukocytes to microvascular endothelium, demonstrating the involvement of PAF, or rather PAF-LL, in this event.³³ Furthermore, it is conceivable that oxLDL favors the interaction among platelets, leukocytes, and endothelial cells through ROS-mediated inhibition of nitric oxide synthase⁴⁶ and/or through nitric oxide inactivation,³⁵ steps that could well be prevented by the administration of vitamin C⁴⁷ under the conditions of our experiment. Nitric oxide has been identified as a powerful antiadhesive and antiaggregatory mediator in vivo.⁴⁸

Although the present study cannot elucidate the exact mechanism by which vitamin C prevents oxLDL-induced leukocyte–platelet–endothelium interactions, the above-listed potential mechanisms involve direct attacks by aqueous-phase ROS and not the sequelae of lipid peroxidation. It was thus not entirely unexpected that neither vitamin E nor the lipid-soluble antioxidant drug probucol affected oxLDL-induced adhesion-promoting events. Although the water-soluble vitamin C has the capacity to scavenge and neutralize aqueous-phase ROS by very fast electron transfer and thus effectively prevent the initiation of lipid peroxidation,^{7 8 9 10} both vitamin E and probucol are lipid-soluble compounds, which intercalate with biomembrane and lipoprotein phospholipids and thus serve primarily to terminate radical chain reactions of lipid peroxidation.^{11 12} However, both of these lipid-soluble antioxidants contribute little to plasma antioxidative activity and cannot prevent the initiation of lipid peroxidation, as suggested from experiments using human blood plasma.⁹ In contrast, the capacity of plasma to prevent the initiation of lipid peroxidation is directly related to its vitamin C content.¹⁰

The observation that vitamin C, but not vitamin E or probucol, inhibits leukocyte–platelet aggregation and leukocyte adhesion to endothelial cells in response to oxLDL is also in agreement with a similar study in which we demonstrated that cigarette smoke exposure induces similar changes in the hamster microcirculation and macrocirculation and that these cigarette smoke–induced effects are inhibited by dietary vitamin C but not by vitamin E or probucol.⁴⁹ Similarly, vitamin C was found to scavenge ozone in the lung lining fluid layer before it can interact with the lung epithelial lining, whereas vitamin E can prevent only lipid peroxidation initiated by ozone-induced free radicals.⁵⁰ The fact that under the conditions of our experiment lipid-soluble antioxidants did not prevent leukocyte aggregation and adhesion does not, of course, conflict with previous reports demonstrating that these antioxidants do interfere with other steps of atherogenesis, such as LDL oxidation,^{4 11} oxLDL-induced endothelial cell cytotoxicity,⁵¹ the accumulation of lipid peroxidation products in the vessel wall,²³ or dysfunction of endothelium-dependent vasomotor activity.⁴⁷

In summary, we have demonstrated in the present study that oxLDL induces leukocyte–platelet aggregation and leukocyte adhesion to the endothelium, not only of small venules and arterioles but also of the aorta. The effects of oxLDL on the microcirculation and macrocirculation were almost completely prevented by pretreatment of the hamsters with dietary or intravenously injected vitamin C but not with the lipid-soluble antioxidants vitamin E or probucol, emphasizing the role of aqueous-phase ROS in this event. These findings may contribute to our understanding of the mechanisms by which antioxidants—in particular, vitamin C—confer effective prophylaxis from chronic diseases like atherosclerosis as well as from other pathophysiological conditions that involve leukocyte adhesion, such as ischemia-reperfusion damage^{52 53} and cigarette smoke–induced vascular damage.⁴⁹

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-46022 (Dr Arfors) and HL-49954 (Dr Frei) and by a grant from Svenska Tobaks AB. Dr Frei was supported by a Future Leader Award from the International Life Sciences Institute–Nutrition Foundation. The excellent technical assistance of E. Berger (La Jolla), S. Green (La Jolla), J. Pattison (La Jolla), B. Sehringer-Mansour (Hamburg), U. Weinhardt (Munich), and I. Wernicke (Hamburg) is gratefully acknowledged. The scanning electron microscopy was made possible through the generous help of Prof Dr U. Welsch, Anatomic Institute, University of Munich.

This paper is dedicated to the memory of Dr Thomas E. Carew, who died on May 7, 1993.

	Footnotes

 
¹ Deceased.

Received June 16, 1994; revision received August 8, 1994; accepted October 2, 1994.

	References

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