Small oxidative changes in atherogenic LDL concentrations irreversibly regulate adhesiveness of human endothelial cells: effect of the lazaroid U74500A

Atherosclerosis 149 (2000) 295 – 302 www.elsevier.com/locate/atherosclerosis

Small oxidative changes in atherogenic LDL concentrations irreversibly regulate adhesiveness of human endothelial cells: effect of the lazaroid U74500A Cristina Colome´, Jose´ Martı´nez-Gonza´lez, Francisco Vidal, Conxita de Castellarnau, Lina Badimon * Cardio6ascular Research Center, IIBB/CSIC-Institut de Recerca del Hospital de la Santa Creu i Sant Pau, A6da. Sant Antoni Maria Claret c 167, 08025 Barcelona, Spain Received 22 March 1999; received in revised form 20 July 1999; accepted 18 August 1999

Abstract The adherence of monocytes to the endothelium is an early event in atherogenesis which is modulated by low density lipoproteins (LDL). We analyzed the effect of atherogenic LDL levels (180 mg cholesterol/dl, for 24 h) with minimal oxidative modifications (thiobarbituric-acid-reactive-substances (TBARS) concentration between 1.2 90.1 and 2.5 9 0.3 nmol of malonaldehyde bis-diethyl acetal (MDA) per mg protein) on human umbilical vein endothelial cell (HUVEC) adhesive properties. We used native LDL (n-LDL), and LDL exposed to spontaneous oxidation without antioxidants (mox-LDL) or with 20 mmol/l of the antioxidant butylated hydroxytoluene (BHT-LDL) or 10 mmol/l U74500A (U74500A-LDL), a scavenger of free radicals. Thiobarbituric-acid-reactive-substances (TBARS) levels were significantly higher in mox-LDL (2.5 9 0.3 nmol MDA/mg protein) than in BHT-LDL (1.6 90.2), U74500A-LDL (1.2 9 0.1) or in n-LDL (1.3 9 0.1). mox-LDL induced the greatest adhesion of U937 cells to HUVEC (103 99% over controls) followed by BHT-LDL (75 9 10%), U74500A-LDL (36 99%) and n-LDL (359 3%). The lazaroid U74500A efficiently protected U74500A-LDL against oxidative damage and prevented endothelial adhesiveness associated with this LDL modification, inducing adhesion effects similar to those of n-LDL. However, U74500A could not reverse the adhesion induced by previously oxidized LDL (mox-LDL). LDL did not induce the expression of the intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) or E-selectin, but it produced a downregulation of endothelial nitric oxide synthase (NOS III) mRNA levels. Thus, adhesiveness of human endothelial cells (EC) exposed to atherogenic concentrations of LDL is closely modulated by minimal changes in LDL oxidative state, and could be related to a downregulation of NOS III. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human umbilical vein endothelial cells; Low density lipoproteins; Lipid peroxides; Adhesion molecules; Nitric oxide synthase

1. Introduction High levels of plasma low density lipoproteins (LDL) are a known risk factor for premature development of atherosclerosis and coronary artery disease (CAD) [1– 3]. LDL particles are highly susceptible to oxidative damage [4] and oxidative modifications increase LDL atherogenicity [5]. Recently, epidemiologic studies have shown that increased plasma levels of oxidized LDL (ox-LDL) are associated with CAD [6,7]. In addition, * Corresponding author. Tel./fax: +34-93-2919285. E-mail address: [email protected] (L. Badimon)

the role of ox-LDL on these processes is emphasized by the anti-atherogenic effect of antioxidants suggested in different studies [8,9], although the protective effect of antioxidants against CAD is controversial [10–12]. Monocyte binding to endothelial cells (EC), one of the earliest events in the development of atherosclerotic plaques [2], is potentiated by both atherogenic concentrations of native LDL (n-LDL) and low concentrations of medium-highly ox-LDL [13,14]. Different factors have been shown to be regulated by LDL, among them expression of chemotactic factors [15,16], cell adhesion molecules (CAM) [17–20], nitric oxide (NO) and free radical production [21,22].

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In this study, we have analyzed how minimal oxidative modifications of pathophysiological levels of LDL (180 mg cholesterol/dl) affect endothelial adhesive properties. In addition, we have assessed the efficacy of U74500A, a member of the 21-aminosteroid family (lazaroids), to reduce LDL oxidative processes affecting EC adhesiveness. U74500A is a first-generation lazaroid; lazaroids are compounds derived from methylprednisolone without corticoid side-effects [23]. Lazaroids are scavengers of oxygen-based and nitrogen-based free radicals and inhibit lipid peroxidation [23]. Our results indicate that the hyperadhesiveness of human EC induced by atherogenic LDL concentrations is closely modulated by minimal changes in LDL oxidative state (B 0.3 nmol of malonaldehyde bis-diethyl acetal (MDA) per mg of protein). U74500A prevented both oxidative damage of LDL and the endothelial hyperadhesiveness induced by this LDL modification. However, once LDL were modified, U74500A addition did not reduce the monocytic cell adhesion induced by LDL exposed to spontaneous oxidation without antioxidants (moxLDL). In addition, we show that the increase in monocyte binding to EC promoted by atherogenic LDL concentrations correlated with a downregulation of nitric oxide synthase (NOS III).

apolipoprotein B [28], free amino groups of LDL were analyzed by 2,4,6-trinitrobenzenesulfonic acid method, using alanine as a standard as described previously [29]. The purity of n-LDL and the effect of oxidative modification on LDL electrophoretic mobility were assessed by electrophoresis on agarose gels (Paragon™ Electrophoresis system; Beckman) and SDS–PAGE (4–15% precast polyacrylamide gels, Bio-Rad).

2.2. Cell culture HUVEC were extracted by collagenase digestion as described [27,30]. Cells were cultured in medium 199 (Gibco) supplemented with 20 mmol/l Hepes pH 7.4 (Gibco), 30 mg/ml endothelial cell growth supplement (Sigma), 100 mg/ml heparin (Sigma), 20% fetal calf serum (FCS; Biological Industries) and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). HUVEC were used between passages 2 and 5. The human monocytic cell line U937 was obtained from the American Type Culture Collection and maintained in RPMI 1640 (Sigma) as described [20]. U937 cells have been well characterized as a model to study monocytic cell adhesion [20,31].

2.3. Cell-adhesion studies 2. Materials and methods

2.1. Isolation and characterization of LDL Human LDL (d1.019 – d1.063 g/ml) were obtained from pooled plasma of normocholesterolemic donors from the Barcelona area, as described previously [24]. LDL were dialyzed against four dosages of 200 vol. buffer (150 mmol/l NaCl, 1 mmol/l EDTA and 20 mmol/l Tris pH 7.4) and one last change against saline solution. LDL were sterilized by filtration. LDL protein concentration was determined by the Lowry method [25] and cholesterol concentration by a commercial kit (Boehringer). n-LDL used in the experiments was B 48 h old. n-LDL (7 mg/ml) was exposed to spontaneous oxidation during 30 days at 4°C in the dark without antioxidants (mox-LDL) or with 20 mmol/l butylated hydroxytoluene (BHT; Sigma), a BHT concentration used to prevent oxidation [26], or 10 mmol/l U74500A (UP-A, Upjohn, Kalamazoo, MI). U74500A- and BHT-protected LDL (U74500A-LDL, BHT-LDL) were dialyzed against saline before incubation with human umbilical vein endothelial cells (HUVEC). After dialysis, the oxidative state of LDL was determined by TBARS assay as described previously [27]. TBARS were expressed as nmol MDA/mg protein. To estimate derivatization of lysine residues of

Confluent HUVEC were exposed to LDL (180 mg cholesterol/dl) for 24 h. Afterwards, culture media were collected to measure TBARS, and monocytic cell adhesion to HUVEC monolayers was assessed. U937 cells were labeled by incubation at 37°C with 47 mmol/l of the fluorescent dye 7-amino-4chloromethylcoumarin (CMAC, Molecular Probes) for 30 min in plain RPMI medium. Labeled U937 cells (1×106/well) were added to control and LDLtreated HUVEC and were incubated for 1 h at 37°C (5% CO2). Following incubation, plates were sealed with a plastic wrap and centrifuged at 1000 × g for 10 min at 4°C in a Beckman TJ-6 centrifuge as described previously [32]. To quantify U937 cell adherence, CMAC was released from cells by adding lysis buffer (PBS/1% Triton X-100) and fluorescence was measured by using a spectrofluorimeter (Perkin Elmer LS50). Results were expressed as a percentage of U937 cells bound to untreated cells. The time-dependent effect of mox-LDL on monocytic adhesion was analyzed in HUVEC incubated with LDL (180 mg cholesterol/dl) for 1–4 days. The culture medium was changed daily. In these conditions the effect of 10 mmol/l U74500A on TBARS and monocytic cell adhesion was determined. As a control, LDL were incubated in cell-free medium and TBARS concentration was measured.

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2.4. Determination of cell adhesion molecules (CAM) by immunocytochemistry HUVEC were grown on human fibronectin-coated coverslips (5 mg/ml) and incubated with the lipoprotein preparations (180 mg cholesterol/dl) for 24 h. As positive controls, cells were incubated with interleukin 1-b (IL-1b, 10 U/ml; Boehringer Mannheim) for 6 h to induce the expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) [33]. Cells were fixed with 4% paraformaldehyde/0.1 mol/l PBS pH 7.4 for 15 min at room temperature, blocked with PBS/1% BSA, and then permeabilized or not with PBS/1% BSA/0.4% Triton X-100 for 4 min. The following mouse monoclonal antibodies specific for human proteins were used: antiICAM-1 (CD54; 1:25 dilution; clone 6.5B5, Dako), anti-platelet endothelial cell adhesion molecule (PECAM-1; CD31; 1:20 dilution; clone JC/70A, Dako), anti-VCAM-1 (CD106; 1:100 dilution; Genzyme) and anti-E-selectin (ELAM-1; CD62E; 1:20 dilution; Genzyme). Afterwards, slides were washed and incubated with secondary antibodies for 1 h in the dark. As secondary antibodies, fluorescein isothiocianate (FITC) conjugated goat anti-mouse IgG (1:50 dilution; Dako) were used. After secondary antibody incubations, slides were washed (3×10 min) in 0.1 mol/l PBS. Incubations with only primary or secondary antibodies were simultaneously performed as control of the immunostaining. Controls using non-immune sera from mouse were also carried out. Results were examined with an Olympus Vanox AHBT3 microscope and photographs were taken with Kodak Ektachrome (ASA 400) daylight films. Fluorescence linked to adhesion molecules was evaluated by an image analysis system (Visolog version 4.1.5).

2.5. Determination of soluble adhesion molecules Levels of sICAM-1, sVCAM-1, sE-selectin and sP-selectin were determined by specific ELISA (Boehringer Ingelheim), as previously described [34], in cell-free media from control and LDL-treated (180 mg cholesterol/dl, for 24 h) cells.

2.6. Northern blot analysis Total RNA from control and LDL-treated cells was isolated by Ultraspec™ (Biotecx) according to the manufacturer’s recommendations. RNA samples were fractionated in 1% agarose gels containing formaldehyde. RNA was transferred by capillarity to Hybond-N™ (Amersham) membranes and UV-crosslinked. Filters were prehybridized and hybridized as described previously [27]. A cocktail of oligonucleotides that recognize specific domains of human ICAM-1 mRNA (R&D

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Systems), labeled with [g-32P]dATP (3000 Ci/mmol, Amersham), and the human NOS III cDNA [35], labeled with [a-32P]dCTP (3000 Ci/mmol, Amersham), were used as probes. Filters were exposed to Agfa Curix RP2 X-ray films at − 70°C.

2.7. Other methods To assess the possible cytotoxic effect of LDL, lactate dehydrogenase (LDH) activity in the media from LDL-treated cells was determined as previously described [36], using a commercial kit (LD-L 10, Sigma), and was expressed as a percentage of that found in control medium from untreated cells. Cell viability was analyzed by trypan blue exclusion test. Internucleosomal DNA fragmentation was evaluated as a index of cell apoptosis [37]. Total DNA was extracted, using the kit Kristal™ (Cambridge Molecular Technology), and was fractionated on 0.8% agarose/ TAE/ethidium bromide gels. The typical apoptotic tell-tale ladder pattern was not observed in any treatment.

2.8. Statistics Results are expressed as mean 9 S.E.M., unless otherwise stated. A Statview™ II (Abacus Concepts) statistical package for the Macintosh computer system, was used for all the analyses. Multiple groups were compared by one-factor ANOVA, followed by Fisher PLSD and Scheffe F-test to assess specific group differences. Differences between any two groups were evaluated by the two-tailed t-test.

3. Results

3.1. Modulation of LDL oxidation by U74500A and BHT The concentration of TBARS in mox-LDL (2.590.3 nmol MDA/mg protein) was significantly higher (PB 0.05) than in n-LDL (1.39 0.1). This minimal oxidation of LDL was associated with a mild change in electrophoretic mobility (Fig. 1), but it did not affect the LDL content of free amino groups or total protein and cholesterol content. LDL oxidation was attenuated by 20 mmol/l BHT (1.6 90.2 nmol MDA/mg protein) and completely prevented by 10 mmol/l U74500A (1.29 0.1 nmol MDA/mg protein).

3.2. Effect of minimally oxidized LDL on monocytic cell adhesion HUVEC were incubated with 180 mg cholesterol/dl of n-LDL, minimally oxidized LDL (mox-LDL and

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Fig. 1. Electrophoretic mobility of LDL. (A) Polyacrylamide gel electrophoresis of LDL (10 mg protein/lane) with BSA as internal standard (lanes 1 – 4, and 6) and without BSA (lane 5); n-LDL (lanes 5 and 6); mox-LDL (lanes 3 and 4); U74500A-protected LDL (lanes 1 and 2). (B) Agarose gel electrophoresis of two different preparations of LDL (6 mg protein/lane); n-LDL (lanes 1 and 2) and mox-LDL (lanes 3 and 4).

BHT-LDL) and U74500A-protected LDL for 24 h. Afterwards, culture medium was removed for TBARS analysis, and HUVEC monolayers were incubated with labeled-U937 cells to assess cell adhesion. No differences in TBARS concentrations were detected in media from n-LDL and U74500A-LDL treated cells with respect to untreated cells (Fig. 2A). In contrast, TBARS concentrations from mox-LDL- and BHT-LDL-treated cells were significantly higher than those from control cells. n-LDL (180 mg cholesterol/dl) incubated in cell-free medium under the same conditions showed 6.369 1.4 nmol MDA/ml. Neither cell morphology nor cell viability, assessed by trypan blue exclusion test (\ 95%), were affected by mox-LDLtreatment. LDH activity levels in the supernatants from LDL-treated cells (108911%) were similar to those in supernatants from control cells (1009 12%).

Monocytic cell adhesion to HUVEC was significantly increased (PB 0.05) by all LDL particles (Fig. 2B). mox-LDL induced the highest monocytic cell adhesion (10399% over control), followed by BHT-LDL (759 10% over control). U74500A completely prevented LDL oxidation, and U74500A-LDL-induced U937 cell adhesion to HUVEC (3699% over control) was similar to that of n-LDL (359 3% over control). Thus, unmodified LDL at atherogenic concentrations significantly promoted monocyte adhesion to HUVEC, and the increased adhesiveness induced by mox-LDL and BHT-LDL correlated with their oxidative degree.

3.3. Effect of U74500A on monocytic cell adhesion promoted by mox-LDL Since U74500A was more effective than BHT in preventing the increase in adhesion linked to LDL oxidative modifications, we further analyzed the effect of U74500A on TBARS and monocytic cell adhesion to HUVEC incubated with mox-LDL (45–180 mg cholesterol/dl for 24 h, or 180 mg cholesterol/dl for 1–4 days). mox-LDL significantly increased TBARS concentration in culture media in a dose- (P= 0.0001) and timedependent (P =0.0001) manner, that was abolished by the addition of U74500A to the culture medium (Fig. 3A and B). TBARS concentration in supernatants of control cells did not change over time. mox-LDL (180 mg cholesterol/dl) incubated in cell-free medium under the same conditions showed 10.89 1.8 nmol MDA/ml and did not significantly change during the studied time intervals.

Fig. 2. Effect of LDL on TBARS concentration in culture media and monocyte adhesion to HUVEC monolayers. Confluent HUVEC were exposed to LDL (180 mg cholesterol/dl) for 24 h. Afterwards, TBARS concentration in culture media (A) and adhesion of U937 cells to HUVEC (B) was assessed. TBARS concentration is expressed as nmol MDA/ml of media. Cell adhesion is expressed as a percentage of U937 cells bound to LDL-untreated cells (control). BHT-LDL, BHT-protected LDL; mox-LDL, minimally oxidized LDL; n-LDL, native LDL; U74500A-LDL, U74500A-protected LDL. Results are the mean 9 S.E.M. of five independent experiments performed in triplicate. P B 0.05; *, versus control; +, versus mox-LDL-treated HUVEC; c, versus BHT-LDL-treated HUVEC.

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Fig. 3. Effect of U74500A on TBARS concentration in culture media from HUVEC treated with mox-LDL. Confluent HUVEC were incubated with mox-LDL (45, 90 or 180 mg cholesterol/dl) for 24 h (A) (TBARS in control cells 5.58 9 0.30 nmol MDA/ml), or with mox-LDL (180 mg cholesterol/dl) during 1 –4 days (B). TBARS were measured in culture media from HUVEC incubated without LDL addition (control, open circles), and from cells incubated with mox-LDL alone (closed squares) or with mox-LDL and 10 mmol/l U74500A (open squares). Results are the mean 9S.E.M. of five independent experiments performed in triplicate. PB 0.05; *, versus untreated HUVEC (control); +, versus mox-LDL-treated HUVEC.

Fig. 4. Effect of U74500A on monocyte adhesion to HUVEC treated with mox-LDL. Confluent HUVEC were incubated with mox-LDL (45, 90 or 180 mg cholesterol/dl) for 24 h (A), or with mox-LDL (180 mg cholesterol/dl) during 1 – 4 days (B). U937 cell adhesion to HUVEC was measured in cells cultured without LDL addition (control, open circles), and in cells incubated with mox-LDL alone (closed squares) or with mox-LDL and 10 mmol/l U74500A (open squares). Cell adhesion is expressed as a percentage of U937 cells bound to LDL-untreated cells (control). Results are the mean9 S.E.M. of five independent experiments performed in triplicate. PB 0.05; *, versus untreated HUVEC (control); + , versus mox-LDL-treated HUVEC.

HUVEC treated with 45 or 90 mg cholesterol/dl of mox-LDL for 24 h, did not significantly increase U937 cells adhesion over controls (Fig. 4A). The enhanced adhesiveness of HUVEC exposed to mox-LDL (180 mg cholesterol/dl for 24 h) was not modified by the addition of 10 mmol/l U74500A to the culture medium. Similar results were obtained in HUVEC treated with mox-LDL (180 mg cholesterol/dl) during 1 – 4 days: the time-dependent increase (P =0.001) in monocyte adhesion promoted by mox-LDL was not prevented by U74500A (Fig. 4B).

3.4. Expression of CAM The effect of n-LDL and mox-LDL (180 mg cholesterol/dl, for 24 h) on the expression of CAM in HUVEC was assessed by immunocytochemistry. HUVEC monolayers constitutively expressed PECAM-1. Neither control nor LDL-treated cells expressed detectable levels of VCAM-1 or E-selectin; however, IL-1b (10 U/ml for 6 h) significantly induced the expression of both adhesion molecules. ICAM-1 expression levels, quantitated by fluorescent microscopy, were similar in control

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and LDL-treated cells (: 0.14-fold those of IL-1b-stimulated cells). Permeabilization of HUVEC with Triton X-100 significantly increased the levels of ICAM-1 in IL-1b-stimulated cells, however, it did not modify the ratio between controls and LDL-treated cells. In addition, ICAM-1 mRNA levels were similar in controls and in HUVEC treated with LDL (Fig. 5A). sICAM-1, sVCAM-1, sE-selectin and sP-selectin levels were measured in cell-free supernatants by ELISA. No differences were observed with any LDL treatment (data not shown).

3.5. NOS III mRNA le6els Northern blot analyses were performed to determine whether LDL (180 mg cholesterol/dl) regulate NOS III mRNA expression in HUVEC. NOS III mRNA levels did not change during 2, 4 or 8 h of incubation with either native or mox-LDL, but they were significantly downregulated after 24 h of incubation (Fig. 5B). Similar results were obtained using BHT-LDL or U74500ALDL. 4. Discussion The present study shows that adhesiveness of HUVEC induced by atherogenic LDL levels (180 mg cholesterol/dl, for 24 h) is closely modulated by minimal changes in LDL oxidative state. U74500A, a scavenger of peroxyl radicals, prevented both oxidative damage of LDL and the hyperadhesiveness associated with this modification. Quantitative immunocytochemical analysis did not show significant effect of LDL on HUVEC ICAM-1, VCAM-1 or E-selectin expression

Fig. 5. Effect of LDL on ICAM-1 and NOS III mRNA levels. Confluent HUVEC were treated with LDL (180 mg cholesterol/dl), total RNA was extracted and analyze by Northern blot (10 mg/lane). (A) ICAM-I mRNA levels after 24 h of incubation without LDL (lanes 1 and 2), with n-LDL (lanes 3 and 4), with U74500A-protected LDL (lanes 5 and 6) or with mox-LDL (lanes 7 and 8). (B) Time-dependent effect on NOS III mRNA levels of n-LDL and mox-LDL. The integrity and relative amounts of RNA in each sample were checked by ethidium bromide staining (bottom panels). Representative from three independent experiments.

levels. However, the increased monocyte binding to HUVEC induced by LDL correlated with a downregulation of NOS III mRNA levels. We show that atherogenic concentrations of LDL significantly induced monocytic cell adhesion, in a timedependent manner. At low concentrations neither nLDL nor mox-LDL induced cell binding to HUVEC. The induction of monocyte attachment by low concentrations of LDL has been reported, however, these LDL exhibited higher oxidative modification than those tested in our study [13,31,38,39]. Minimally oxidized LDL (mox-LDL and BHT-LDL) induced higher monocyte adhesion than n-LDL or U74500A-protected LDL. The pro-adhesive effect of LDL on HUVEC closely correlates with minimal changes in its oxidative state. U74500A, a scavenger of peroxyl radicals, efficiently prevented oxidative damage of LDL and the endothelial hyperadhesiveness associated with this deleterious process, but it did not affect adhesion attributable to the LDL-atherogenic-concentration-effect. In addition, once LDL were modified, U74500A addition did not reduce the monocytic cell adhesion induced by moxLDL, although there was a reduction in the levels of TBARS in the supernatants. Thus, HUVEC adhesiveness induced by minimally oxidized LDL (vs. n-LDL) could be basically attributable to minimal non-reversible oxidative changes suffered by LDL during its spontaneous oxidation, but unrelated to a modification during the incubation with the cells. In addition, we observed that LDL oxidation is not increased by incubation with HUVEC in the presence of serum during 24 h, in agreement with Smalley et al. [26]. Multiple factors modulate monocyte recruitment by the vessel wall, including opposite forces such as the modification of the LDL particle and the presence of antioxidants [4,40]. Lazaroids were developed for the acute treatment of post-traumatic and ischemic lesions of the central nervous system, where peroxidative injury seems to be involved [41]. Recently, a strong cardioprotective effect of these drugs in animal models of ischemia [42,43] as well as an improvement in the ventricular and endothelial functions have been reported [44,45]. Our results, in addition, suggest that 21-aminosteroids significantly block LDL modifications that induce monocytic cell adhesion to endothelium. It has been reported that long time exposure of HUVEC to atherogenic concentrations of n-LDL upregulate ICAM-1 [20] and VCAM-1 [46] expression. However, we did not observe significant induction of ICAM-1, VCAM-1 or E-selectin after 24 h of cells exposition to atherogenic levels of n-LDL or moxLDL, in agreement with previous data [31,38,47]. Our results did not exclude the induction of these molecules at times that did not match the end-point analyses in our study, neither did they rule out the involvement of

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other CAM such as P-selectin [19,48]. On the other hand, we did not investigate the role of integrins involved in monocyte-EC interactions. Hypercholesterolemia impairs endothelium-dependent dilatation both in animal models of atherosclerosis [49] and in humans [50,51], and enhances monocyte adhesion to EC [21]. Hypercholesterolemia decreases basal NO release and increases endothelium adhesive properties in vivo [52]. NO synthase inhibitors increase monocyte adhesion to endothelium [53,54] and Larginine, the physiological substrate for NO synthesis, reduces monocyte adhesion to EC [21,54,55]. In addition, both ox-LDL [56] and atherogenic concentrations of n-LDL [27] decrease NOS III expression. In the present work, we found that increased monocytic cell adhesion to EC, promoted by atherogenic concentrations of both native and minimally oxidized LDL, correlates with a downregulation of NOS III mRNA levels. Although in this in vitro study we used a model of cellular adhesion (HUVEC and the U937 cell line) and a method to assess LDL oxidation (TBARS) commonly used by different authors, the results can not be extrapolated to an in vivo situation. However, these findings suggest that NO could be an endogenous antiatherogenic molecule [57] and that downregulation of NO synthase mRNA/protein levels or activity participates, at least in part, in the process of monocyte recruitment. The mechanism by which atherogenic concentrations of LDL induce monocyte-endothelium interaction is not completely understood. More studies, including differential analysis of transcripts expressed by EC exposed to lipoproteins, are needed to identify new gene products involved in the process.

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements This study has been possible thanks to funds partially provided by FIS 96/2046, FIS 98-715, CDTI-BMS 96/ 035 and Mapfre-Medicine. Dr Colome´ is a fellow of MEC-Almirall-Prodesfarma. The authors thank M. Berrozpe and J. Llenas for their help.

References [1] Fuster M, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242–50. [2] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801–9. [3] Grundy SM. Role of low-density lipoproteins in atherogenesis and development of coronary heart disease. Clin Chem 1995;41:139 – 46. [4] Reaven PD, Ferguson E, Navab M, Powell FL. Susceptibility of human LDL to oxidative modification. Arterioscler Thromb Vasc Biol 1994;14:1162–9. [5] Steinberg D, Lewis A. Conner Memorial Lecture. Oxidative

[17]

[18]

[19]

[20]

[21]

[22]

301

modification of LDL and atherogenesis. Circulation 1997;95:1062 – 71. Holvoet P, Stassen JM, Van Cleemput J, Collen D, Vanhaecke J. Correlation between oxidized low density lipoproteins and coronary artery disease in heart transplant patients. Arterioscler Thromb Vasc Biol 1998;18:100 – 7. Holvoet P, Vanhaecke J, Janssen S, Van der Werf F, Collen D. Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation 1998;98:1487 – 94. Sasahara M, Raines EW, Chait A, Carew TE, Steinberg D, Wahl PW, et al. Inhibition of hypercholesterolemia-induced atherosclerosis in the non-human primate by probucol. J Clin Invest 1994;94:155 – 64. Kritchevsky SB, Shimakawa T, Tell GS, Dennis B, Carpenter M, Eckfeldt JH, et al. Dietary antioxidants and carotid artery wall thickness. The ARIC Study. Circulation 1995;92:2142 –50. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993;328:1444 – 9. Stephens NG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ, et al. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996;347:781 – 5. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, et al. Effect of the combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996;334:1150 – 5. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, et al. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest 1990;85:1260 – 6. Pritchard KA Jr, Tota RR, Lin JH-C, Danishefsky KJ, Kurilla BA, Holland JA, et al. Native low density lipoprotein: endothelial cell recruitment of mononuclear cells. Arterioscler Thromb 1991;11:1175 – 81. Navab M, Imes SS, Hama SY. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest 1991;88:2039 – 46. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, et al. Minimally modified LDL induces monocyte chemotactic protein 1 in human endothelial and smooth muscle cells. Proc Natl Acad Sci USA 1990;87:5134 – 8. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherosclerosis. Science 1991;251:788 – 91. Li H, Cybulsky MI, Gimbrone MA, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatible mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb 1993;13:197 – 204. Gebuhrer V, Murphy JL, Bordet JC, Reck MP, McGregor JL. Oxidized low-density lipoprotein induces the expression of P-selectin (GMP140/PADGEM/CD62) on human endothelial cells. Biochem J 1995;306:293 – 8. Smalley DM, Lin JH, Curtis ML, Kobari Y, Stemerman MB, Pritchard KA Jr. Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol 1996;16:585 – 90. Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation 1994;89:2176 – 82. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, et al. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res 1995;77:510 – 8.

302

C. Colome´ et al. / Atherosclerosis 149 (2000) 295–302

[23] Hall ED, McCall JM. Antioxidant action of lazaroids. Methods Enzymol 1994;234:548–55. [24] Llorente-Corte´s V, Martı´nez-Gonza´lez J, Badimon L. Esterified cholesterol accumulation induced by aggregated LDL uptake in human vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitors. Arterioscler Thromb Vasc Biol 1998;18:738 – 46. [25] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265 – 75. [26] Smalley DM, Hogg N, Kalyanaraman B, Pritchard KA Jr. Endothelial cells prevent accumulation of lipid hydroperoxides in low-density lipoprotein. Arterioscler Thromb Vasc Biol 1997;17:3469 – 74. [27] Vidal F, Colome´ C, Martı´nez-Gonza´lez J, Badimon L. Atherogenic concentrations of native low-density lipoproteins down-regulate nitric-oxide-synthase mRNA and protein levels in endothelial cells. Eur J Biochem 1998;252:378–84. [28] Steinbrecher UP. Oxidation of low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem 1987;262:3603 – 8. [29] Go´mez V, Colome´ C, Reig F, Rodrı´guez L, Alsina MA. Effect of reductive alkylation on transferrin conformation and physicochemical properties. Anal Chim Acta 1994;290:65–74. [30] Lo´pez S, Vila L, Breviario F, de Castellarnau C. Interleukin g increases 15-hydroxyeicosatetraenoic acid formation in cultured human endothelial cells. Biochem Biophys Acta 1993;1170:17 – 24. [31] Kim JA, Territo MC, Wayner E, Carlos TM, Parhami F, Smith CW, et al. Partial characterization of leukocyte binding molecules on endothelial cells induced by minimally oxidized LDL. Arterioscler Thromb 1994;14:427–33. [32] Charo IF, Yuen C, Goldstein IM. Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay. Blood 1985;65:473 – 9. [33] Scholz D, Devaux B, Hirche A, Po¨zsch B, Kropp B, Schaper W, et al. Expression of adhesion molecules is specific and time-dependent in cytokine-stimulated endothelial cells in culture. Cell Tissue Res 1996;284:415 – 23. [34] Gurbel PA, Serebruany VL. Soluble vascular cell adhesion molecule-1 and E-selectin in patients with acute myocardial infarction treated with thrombolytic agents. Am J Cardiol 1998;81:772 – 5. [35] Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD. Cloning and expression of a cDNA encoding human endotheliumderived relaxing factor/nitric oxide synthase. J Biol Chem 1992;267:14519– 22. [36] Vin˜als M, Martı´nez-Gonza´lez J, Badimon JJ, Badimon L. HDLinduced prostacyclin release in smooth muscle cells is dependent on cyclooxygenase-2 (Cox-2). Arterioscler Thromb Vasc Biol 1997;17:3481 – 8. [37] Cotter TG, Al-Rubeai M. Cell death (apoptosis) in cell culture systems. Trends Biotechnol 1995;13:150–5. [38] Parhami F, Fang ZF, Fogelman AM, Andalibi A, Territo MC, Berliner JB. MM-LDL induced inflammatory responses in endothelial cells are mediated by cAMP. J Clin Invest 1993;92:471 – 8. [39] Maier JA, Barenghi L, Pagani F, Bradamante S, Comi P, Ragnotti G. The protective role of high density lipoprotein on oxidized low density lipoprotein induced U937/endothelial cell oxidation. Eur J Biochem 1994;221:35–41. [40] Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, et al. The Yin and Yang of oxidation in the development of the fatty streak. Arterioscler Thromb Vasc Biol 1996;16:831 – 42. [41] Hall ED. Novel inhibitors of iron-dependent lipid peroxidation for neurodegenerative disorders. Ann Neurol 1992;32:137–42. .

[42] Tanoue Y, Morita S, Ochiai Y, Hisahara M, Masuda M, Kawachi Y, et al. Inhibition of lipid peroxidation with the lazaroid U74500A attenuates ischemia-reperfusion injury in a canine heart transplantation model. J Thorac Cardiovasc Surg 1996;112:1017– 26. [43] Nediami C, Perna AM, Liguori P, Formigli I, Ibba-Manneschi L, Zecchi-Orlandini S, et al. Beneficial effects of the 21-aminosteroid U74389G on the ischemia-reperfusion damage in pig hearts. J Mol Cell Cardiol 1997;29:2825 – 35. [44] Mosher M, Hurst TS, Mayers Y, Johson DH. Lazaroids — not nitric oxide synthetase inhibitors — improve hemodynamics after thermal injury in anesthetized guinea pigs. J Burn Care Rehabil 1996;17:294 – 301. [45] Nishida T, Morita S, Miyamoto K, Masuda M, Tominaga R, Kawachi Y, et al. Lazaroid (U74500A) prevents vascular and myocardial dysfunction after 24-h heart preservation. A study based on cross-circulated blood-perfused rabbit hearts. Circulation 1996;94:326 – 31. [46] Lin JH, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB. Induction of vascular cell adhesion molecule-1 by lowdensity lipoprotein. Atherosclerosis 1996;127:185 – 94. [47] Khan BV, Parthasarathy SS, Alexander RW, Medford RM. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest 1995;95:1262 – 70. [48] Johson-Tidey RR, McGregor JL, Taylor PR, Poston RN. Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1. Am J Pathol 1994;144:952 – 61. [49] Andrews HE, Bruckdorfer KR, Dunn RC, Jacobs M. Low density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta. Nature 1987;327:237 – 9. [50] Zeiher AM, Drexler H, Wollschlager H, Just H. Modulation of coronary vasomotor tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 1991;83:391 – 401. [51] Alonso R, Badimon L, de Andre´s R, Villacastin B, Campos T, Mata P. Effect of different doses of simvastatin on soluble cell adhesion molecule (sCAM) levels in patients with heterozygous familial hypercholesterolemia (hFH). Atherosclerosis 1999;144:32. [52] Lefer AM, Ma XI. Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Thromb 1993;13:771 – 6. [53] Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991;88:4651 – 5. [54] Adams MR, Jessup W, Hailstones D, Celermajer DS. LArginine reduces human monocyte adhesion to vascular endothelium and endothelial expression of cell adhesion molecules. Circulation 1997;95:662 – 8. [55] Adams MR, McCredie R, Jessup W, Robinson J, Sullivant D, Celermajer DS. Oral L-arginine improves endothelium-dependent dilatation and reduces monocyte adhesion to endothelial cells in young men with coronary artery disease. Atherosclerosis 1997;129:261 – 9. [56] Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem 1995;270:319 – 24. [57] Cooke JP, Tsao PS. Is NO an endogenous antiatherogenic molecule? Arterioscler Thromb 1994;14:653 – 5.