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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:585-590.)
© 1996 American Heart Association, Inc.

Articles

Native LDL Increases Endothelial Cell Adhesiveness by Inducing Intercellular Adhesion Molecule–1

Presented in part at the 66th American Heart Association Scientific Sessions, Atlanta, Ga, November 8-11, 1993 and published in abstract form (Circulation. 1993;88[pt 2]:I-367).

David M. Smalley; Jane H.-C. Lin; Michelle L. Curtis; Yukage Kobari; Michael B. Stemerman; Kirkwood A. Pritchard, Jr

From the Department of Experimental Pathology (J.H.-C.L., Y.K., M.B.S.), New York Medical College, Valhalla, NY, and the Cardiovascular Research Center and Departments of Pathology (D.M.S., M.L.C., K.A.P.) and Pharmacology and Toxicology (K.A.P.), Medical College of Wisconsin, Milwaukee.

Correspondence to Kirkwood A. Pritchard, Jr, PhD, Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226. E-mail kpritch@post.its.mcw.edu.

	Abstract

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Abstract Native LDL (n-LDL) increases human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL–treated ECs bound nearly threefold more phorbol myristate acetate (PMA)–stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti–intercellular adhesion molecule–1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL–treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n-LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule–1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis.

Key Words: native LDL • intercellular adhesion molecule–1 • E-selectin • vascular cell adhesion molecule–1 • adhesion

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Prolonged exposure to elevated levels of LDL is a major risk factor for the premature development of atherosclerosis.¹ EC perturbation is considered to be a critical initiating event in the pathogenesis of this disease.^{1 2 3} One of the earliest changes in hypercholesterolemia-induced atherosclerosis is increased monocyte adherence to the vessel wall.^{4 5} Alterations in endothelial adhesiveness for monocytes develop through increased production or redistribution of CAMs, ⁶ but the mechanisms by which n-LDL increases the adhesive properties of the endothelium remain unclear. To date, VCAM-1, E-selectin, and ICAM-1 have been implicated in playing key but distinct roles in adherence, both in vivo and in vitro.⁷ Of these three CAMs, only ICAM-1 is constitutively expressed, yet all are induced by agonist activation with IL-1, tumor necrosis factor, or endotoxin.⁶ The leukocyte counterligands to these CAMs, VLA-4, sLex, and activated LFA-1, bind to VCAM-1, E-selectin, and ICAM-1, respectively.⁸ U937 cells, a monocyte-like cell line, have been used as indicators in monitoring changes in EC adherent properties.^{8 9 10} Unstimulated U937 cells express VLA-4, sLex, and an inactive LFA-1.⁸ Incubating U937 cells with PMA (2 ng/mL for 3 days) differentiates them by decreasing VLA-4 expression and activating LFA-1 receptors.⁸

Our laboratories routinely use pathophysiological n-LDL levels (>160 mg cholesterol/dL) to perturb EC function without causing cytotoxicity. To date, several critical changes in EC function in response to n-LDL have been identified, including increased endocytosis,¹¹ increased generation of cytochrome P450–dependent epoxyeicosatrienoic acids,¹² generation of superoxide anion (O₂^-) from eNOS,¹³ and increased monocyte and U937 cell adherence.^{9 14} The purpose of the present study was to determine the mechanisms by which n-LDL increases the adhesive properties of the endothelium by examining how this lipoprotein modulates CAM expression. Our data indicate that chronic n-LDL exposure increases adherence by an ICAM-1–dependent mechanism. Although n-LDL also increased E-selectin and VCAM-1 transcripts, such increases did not lead to statistically significant differences in immunogenic-detectable protein.

	Methods

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Chemicals
The reagents employed for the experiments were as follows. Human fibronectin was from the New York Blood Center. BCECF–acetometoxymethyl ester was from Molecular Probes. Primeria flasks, 100-mm dishes, six-well plates, and IL-1 were from Becton-Dickinson. M-199 with Earl's salts, RPMI 1640, FBS, antibiotics/micotics, heparin (H-7005), nonspecific antibodies (mouse IgG1, Kappa [MOPC-31c]), FITC-conjugated rabbit anti-mouse IgG, and PMA were from Sigma Chemical Co. Antibodies against ICAM-1, E-selectin, and VCAM-1 were obtained from Harlan. DPBS and all RNA isolation and Northern blotting chemicals were molecular biology grade or better and were obtained from GIBCO. Zeta Probe nylon membranes were from Bio-Rad. Stratalinker, a UV light source, was obtained from Stratagene. Random primer reagents for labeling cDNA probes were from Boehringer Mannheim. All other reagents were reagent grade or better and were obtained from standard commercial sources.

Cell Culture and Isolation
ECs were extracted from human umbilical veins after collagen digestion and cultured on human fibronectin–coated plates.^{9 14} ECs were maintained in EC medium, ie, M-199 containing 16.7% FBS, 20 mmol/L HEPES (pH 7.4), antibiotics/micotics, and 10 ng/mL recombinant human basic fibroblast growth factor–ß; the latter was kindly provided by John Anthony Thompson, University of Alabama, Birmingham. For all studies ECs were used at passages 3 through 5. U937 cells were obtained from the American Type Culture Collection and maintained in RPMI 1640 supplemented with 5% FBS. Neutrophils and mononuclear cells were isolated from peripheral blood.¹⁵

Isolation and Characterization of n-LDL
Fresh nonfrozen human plasma from two to five donors was obtained, and BHT (a lipid-soluble antioxidant) and EDTA were immediately added to the plasma at final concentrations of 20 µmol/L and 0.01%, respectively. The plasma was then mixed for 15 minutes at 4°C before ultracentrifugation. Sterile techniques, reagents, and dialysis solutions were employed for isolation of n-LDL by sequential density ultracentrifugation (1.019<d<1.063 g/mL).^{9 12 14} Cholesterol levels were determined by a cholesterol oxidase colorimetric kit from Sigma. Endotoxin levels were determined by using the colorimetric limulus amebocyte lysate kit from BioWhitaker.⁹ n-LDL was stored at 4°C and used for experiments within 2 weeks. The protocols for isolating n-LDL were designed to specifically examine the effects of atherogenic concentrations of n-LDL on EC function.^{9 11 12 13 14 16} ECs have been incubated with n-LDL in concentrations that exceed >160 mg cholesterol/dL for 4 days, with media changes every 24 hours.^{9 12 13 14} The protocols for protecting n-LDL with BHT essentially eliminated nonspecific oxidation of the LDL particles during isolation and culture.^{9 13} In the present study, the oxidation state of n-LDL particles conditioned by standard culture (with and without ECs) was characterized by a modified¹³ thiobarbituric acid–reactive substances assay.^{9 12 16} Briefly, apoB-containing lipoproteins were isolated by precipitation with phosphotungstate-MnCl₂ prior to adding the thiobarbituric acid. Malonyldialdehyde equivalents were quantified on a CytoFluor II (PerSeptive Biosystems). As before,¹³ we found that n-LDL did not experience significant changes in malonyldialdehyde equivalents during culture (0.62±0.04 versus 0.56±0.05 nmol malonyldialdehyde/mg cholesterol before and after culture, respectively).

Cell Adherence Studies
ECs were passaged onto 12-well plates, brought to confluence, and treated with n-LDL or control media for 4 days. Unstimulated U937 cells were incubated in RPMI 1640 containing 2.5% FBS and BCECF/acetometoxymethyl ester (2 µg/10⁶ cells) at 37°C for 30 minutes. BCECF-labeled U937 cells were resuspended in EC medium at 1x10⁶ cells/mL. The EC monolayers were washed twice with M-199 and then incubated with 2 mL BCECF-labeled U937 cells at 37°C (100% humidity and 5% CO₂) for 30 and 10 minutes for unstimulated and PMA-stimulated U937 cells, respectively. Nonadherent and adherent cells were separated by washing the monolayers with M-199 at room temperature. This was accomplished by inverting the test plate over an empty plate and immediately placing the inverted test plate on a second plate with the wells filled with M-199 that contained 10 mmol/L HEPES, pH 7.45. The test plate (top) and the wash plate (bottom) were carefully and slowly inverted to allow the M-199–HEPES wash to gently drain onto the monolayers and U937 cells. The plates (test plate on bottom, empty wash plate on top) were gently swirled and then slowly inverted again to carry away nonadherent U937 cells with the M-199 wash. These steps were repeated a second time with a second plate containing M-199–HEPES wash.

The number of U937 cells bound per well was determined by comparing the fluorescence level in each well to the fluorescence level of increasing aliquots of BCECF-loaded U937 cells (0.05 to 0.5 mL). To quantify U937 cell adherence, the BCECF was released from the cells by adding lysis buffer (0.1% Triton X-100 in 100 mmol/L Tris, pH 8.0), and the fluorescence of each aliquot was measured by using a fluorescent concentration analyzer (IDEXX) or a CytoFluor II.⁹ Because PMA stimulation increases the adhesive characteristics of U937 cells, PMA-stimulated U937 cells (2 ng PMA/mL for 3 days) were incubated with the test cultures for only 10 minutes. Human neutrophil- and monocyte-binding studies were performed by using similar protocols. The cells were incubated with BCECF/acetometoxymethyl ester in PBS rather than RPMI 1640. Results for the binding studies are expressed as cells bound per well.

Antibody blocking of PMA-stimulated U937 cell binding was performed as follows. Thirty minutes before the addition of PMA-stimulated U937 cells to the ECs, 50 µg/mL R3.1 (an ICAM-1 blocking antibody) was added to control and n-LDL–treated EC cultures. R3.1 was generously provided by Dr Robert Rothlein, Department of Pharmacology and Immunology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Conn. Unbound antibody was washed away before PMA-stimulated U937 cells were added. To determine any nonspecific effects of IgG antibodies on binding, 50 µg/mL of a nonspecific IgG1 (MOPC-31c) was added to separate wells on the same plate.

RNA Isolation and Northern Blot Analysis
Total RNA was isolated by the CsCl-guanidinium-isothiocyanate method.¹⁷ Total EC RNA (15 to 20 µg) was fractionated in a 1.2% agarose gel containing 0.66 mol/L formaldehyde and 0.02 mol/L 3-(N-morpholino)propanesulfonic acid (pH 7). Fractionated RNA was transferred to nylon membranes by capillary blotting and cross-linked by UV irradiation or baking at 80°C for 2 hours. The cDNA probes were labeled by a random primer hexamer protocol to a specific activity of 10⁹ cpm/µg. Hybridizations and washes were performed,¹⁸ and signals were detected by exposing the blot to x-ray film with intensifying screens at -80°C.

cDNA Probes
The ICAM-1 cDNA probe was provided by Dr Timothy Springer, Department of Pathology, Harvard Medical School, Boston, Mass. The von Willebrand factor cDNA was provided by Esther Sabban, Department of Biochemistry, New York Medical College, Valhalla, NY. E-selectin was from Tucker Collins and Michael Bevilacqua, Department of Pathology, Harvard Medical School, Boston, Mass. The VCAM-1 cDNA probe was cloned by Jane H.-C. Lin by using reverse transcription–polymerase chain reaction of RNA from human umbilical vein ECs treated with 1 µg/mL lipopolysaccharide for 4 hours. The upstream primer SB1, 5'-ACCACAGGCTGTGAGTCC-3', was derived from nucleotide sequence 251 through 268. The downstream primer SB2, 5'-TGTGTCTCCTGTCTCCGC-3', was derived from nucleotide sequence 1745 through 1762. Both primers were synthesized by Oligos Etc. The reaction conditions were essentially as described¹⁹ with the exception of a 56°C annealing temperature for the polymerase chain reaction. The 1.5-kb reverse transcription–polymerase chain reaction product was cloned by TA Cloning (Invitrogen), sequenced²⁰ to confirm its VCAM-1 identity, and used for Northern blot hybridization.

Fluorescent-Linked Immunoassay of CAMs
Changes in ICAM-1, E-selectin, and VCAM-1 protein on control and n-LDL–treated EC cultures were determined using an immunofluorescent assay based on protocols established by Pober et al.²¹ Briefly, washed ECs from control and n-LDL–treated EC cultures were resuspended in 1% BSA in DPBS (BSA/DPBS). The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in BSA/DPBS, divided into two equal aliquots, and centrifuged again. The supernatant was discarded, and nonspecific antibody was added to the cells in one tube and anti–ICAM-1 (1:100), anti–VCAM-1 (1:100), or anti–E-selectin (1:500) was added to the cells in the other tube. The tubes were gently shaken to ensure complete mixing of antibody with cells, and the mixture was incubated for 1 hour on ice. The cells labeled with primary antibody were then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed with BSA/DPBS. These last two steps were repeated two more times. To detect changes in the amount of primary antibody bound to the cells, FITC-labeled secondary antibody was added (1:256). The tubes were gently shaken and then incubated for 1 hour on ice. Unbound FITC-labeled antibody was removed by washing the cells three times with BSA/DPBS and two times with DPBS alone as described above. After the final centrifugation, the FITC-labeled cells were resuspended in 100 µL DPBS, and 85 µL was transferred to a Fluoricon assay plate that was precoated with 85 µL of a 1:20 dilution of 0.84-µm polystyrene beads (5% wt/vol; IDEXX). This plate is a self-contained filtration unit that is specifically designed for FITC-based assays. When working with cells, a cushion of polystyrene beads is used to prevent cell trapping, which could clog the membrane. This system of analysis decreases background fluorescence resulting from nonspecific binding and increases the fluorescence signal by collecting the FITC-labeled cells in a single point in the bottom of the well. After sample filtration, fluorescence was measured (excitation, 485 nm; emission, 535 nm). Results are expressed as a percentage of the amount of fluorescence detected for each adhesion molecule in IL-1–stimulated control ECs (5 U/mL for 5 hours).

Statistics
Results are presented as mean±SEM. Student's t test was used to determine significance (P<.05).

	Results

Top Abstract Introduction Methods Results Discussion References

 
n-LDL Increases Adherence of PMA-Stimulated U937 Cells
Control and n-LDL–treated ECs exhibited a low affinity for U937 cells. The amount of U937 cell binding was <2% of the levels induced by IL-1 (5 U/mL for 4 hours, data not shown). Because U937 cells express VLA-4 and sLex, the counterligands for VCAM-1 and E-selectin, respectively, this low level of basal binding suggested that n-LDL may not have altered the expression of VCAM-1 and E-selectin protein. U937 cells also express LFA-1, which does not participate in binding until it is activated. Treating U937 cells with PMA (2 ng/mL for 3 days) activates LFA-1, thus allowing the PMA-activated U937 cells to adhere to ICAM-1. n-LDL–treated ECs bound almost three times more PMA-activated U937 cells than control ECs (Fig 1). Moreover, the anti-ICAM antibody R3.1 decreased control and n-LDL–treated EC binding of PMA-treated U937 cells to nearly equal levels (approximately 3000 cells per well). These data indicate that ICAM-1 plays a critical role in increasing the adhesive properties of the endothelium after exposure to n-LDL. n-LDL appeared to increase PMA-treated U937 cell binding in a dose- and time-dependent fashion (Fig 2). Statistically significant increases in adherence of PMA-treated U937 cells, however, occurred only after prolonged exposure (>2 days) to high levels of n-LDL (>180 mg cholesterol/dL).

View larger version (19K): [in this window] [in a new window]   Figure 1. Bar graph shows PMA-treated U937 (PMA-U937) cell binding to control and n-LDL–treated (240 mg cholesterol/dL for 4 days) ECs. Results are expressed as number of U937 cells bound per well and are mean±SEM (n=3). R3.1 (blocking antibody for ICAM-1; hatched bars) blocked binding of PMA-stimulated U937 cells. *P<.05 vs control ECs.

View larger version (35K): [in this window] [in a new window]   Figure 2. Bar graph shows dose-response curve for increased PMA-stimulated U937 cell adherence to n-LDL–treated ECs (n-LDL-EC). PMA-stimulated U937 cell binding was determined after 2- and 4-day exposures to n-LDL; significant elevations in PMA-stimulated U937 cell adherence occurred after 4 days with elevated LDL levels (>180 mg cholesterol/dL). Values are mean±SEM (n=3). *P<.05 vs control ECs (C-EC).

n-LDL Increases Adherence of Neutrophils and Monocytes
n-LDL increased the affinity of the endothelium for freshly isolated human neutrophils and monocytes (Fig 3). Neutrophil binding increased from 3810±80 cells per well in control EC cultures to more than 6040±520 cells per well in n-LDL–treated EC cultures, representing a 60% increase. Monocyte binding increased from 3620±840 cells per well in control EC cultures to almost 8600±1760 cells per well in n-LDL–treated EC cultures, representing a nearly 150% increase.

View larger version (17K): [in this window] [in a new window]   Figure 3. Bar graph shows neutrophil and mononuclear cell binding to EC monolayers. n-LDL–treated (240 mg cholesterol/dL for 4 days) ECs bound 60% more neutrophils and almost 150% more monocytes than control ECs. Values are mean±SEM (n=3). *P<.05 vs control ECs. PMA-U937 indicates PMA-treated U937 cells; Chol, cholesterol.

n-LDL Perturbs Usual CAM mRNA and Protein Levels
n-LDL increased ICAM-1 mRNA expression by approximately three times the levels observed in control EC cultures (Fig 4). Such changes in message likely account for the nearly twofold increase in ICAM-1 protein levels (Fig 5). Although n-LDL increased both VCAM-1 and E-selectin mRNA levels, significant differences in VCAM-1 and E-selectin protein levels were not detected using existing protocols.

View larger version (54K): [in this window] [in a new window]   Figure 4. Northern blot analysis shows the mRNA expression of VCAM-1, ICAM-1, and E-selectin for control ECs (C) and n-LDL–treated ECs (LDL) after a 4-day exposure to LDL (240 mg cholesterol/dL). von Willebrand factor (vWF) and 18S RNA were analyzed to verify equivalent RNA loading.

View larger version (15K): [in this window] [in a new window]   Figure 5. Bar graph shows effects of prolonged exposure of ECs to atherogenic levels of n-LDL on CAM protein levels. ECs were incubated with (LDL-EC) or without (C-EC) n-LDL (240 mg cholesterol/dL) for 4 days before the levels of E-selectin, VCAM-1, and ICAM-1 were measured by using the fluorescence-linked immunoassay method. Results are expressed as percent stimulation; 100% was defined as the levels of CAM observed after ECs were exposed to IL-1 (5 U/mL). Values are mean±SEM (n=3). *P<.05 vs control ECs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that n-LDL increases the adhesive properties of the endothelium by increasing ICAM-1 expression. n-LDL induction of ICAM-1 was observed at the levels of protein, message, and binding activity. Exposing ECs to elevated levels of n-LDL induced marked increases in the adherence of human neutrophils and monocytes. It is important to note that this phenotypic change occurs only in ECs exposed to elevated concentrations of n-LDL for 4 days. Such changes suggest that n-LDL induces dysfunction by mechanisms that are influenced by the concentration and duration of n-LDL exposure. These in vitro observations parallel epidemiological data demonstrating that high plasma concentrations of LDL are a critical risk factor for premature development of atherosclerosis.¹ Increases in the adhesive properties of the endothelium are believed to be a crucial first step in the initiation and progression of atherogenesis.^{4 5} Although n-LDL–treated ECs exhibited slight increases in transcripts for E-selectin and VCAM-1, such changes did not translate into statistically significant differences in protein levels when assayed by existing analysis techniques. Regardless, n-LDL induction of ICAM-1 at both the message and protein levels suggests that this is one of the early changes in EC function that may play a critical role in atherogenesis.

These observations are significant because they were obtained with LDL, which is resistant to the type of oxidation that occurs under standard tissue-culture conditions. Thiobarbituric acid–reactive substance analyses of n-LDL before and after incubation confirm that adding BHT to the plasma protects this atherogenic particle against oxidation not only during isolation but also culture. Furthermore, n-LDL-induced increases in ICAM-1 expression are not a consequence of endotoxin contamination since the levels detected (<0.01 EU/mL) are lower than those required for activation. These observations reinforce the concept that n-LDL plays a crucial role in EC activation that is distinct from the role of other altered LDL particles.

We have reported⁹ that n-LDL increases the adhesive properties of ECs for human monocytes and unstimulated U937 cells. These studies were performed in a tissue-culture system based on human serum. In addition, we used human lipoprotein–deficient serum to minimize the effects of human lipoproteins. We found⁹ that the human serum/human lipoprotein–deficient serum system increased the basal expression of ICAM-1, VCAM-1, and E-selectin in control cultures by a mechanism that was not related to endotoxin contamination or lipoprotein oxidation. On the basis of others' reports, we reasoned that FBS might decrease basal CAM expression in ECs. When FBS was substituted for human lipoprotein–deficient serum, unstimulated U937 cell binding decreased in control EC cultures. Furthermore, FBS reduced ICAM-1 expression in control ECs and eliminated basal expression of VCAM-1 and E-selectin (data not shown). Interestingly, n-LDL–treated ECs no longer exhibited adherence for unstimulated U937 cells. We repeated these studies to determine whether this change in culture conditions also decreased adherence for freshly isolated human monocytes. A 4-day exposure to atherogenic concentrations of n-LDL clearly increased EC adherence for monocytes and neutrophils (Fig 3). More importantly, changing serum sources brought the levels of adherence in line with others' results.⁸ Although this change in culture conditions alters the basal levels of adherence, the effects of n-LDL on the adhesive properties of the endothelium remain clearly evident.

Elevated levels of LDL have long been recognized as a major risk factor in the development of atherosclerosis. EC perturbation is believed to play a key role in the pathophysiology of this disease. One of the earliest events in the atherogenic process is the binding of mononuclear cells to the endothelium. Animals fed hypercholesterolemic diets exhibit increases in monocytic cell binding to the endothelium after 7 days.^{4 5} After only 1 week on atherogenic diets, rabbits exhibit focal increases in VCAM-1 before the first appearance of intimal macrophages.²² Immunohistochemical studies of normal human aortic and coronary arteries without lesions reveal weak ICAM-1 staining in intimal ECs and no staining of E-selectin or VCAM-1.²³ Specimens with diffuse intimal thickening, however, express higher ICAM-1 and E-selectin levels in both the endothelium of coronary arteries and the vasa vasorum, but VCAM-1 was not detected in the same sections.²³ In advanced plaques and occasionally in fibrofatty plaques, however, VCAM-1 was detected.^{23 24} Thus, in humans, one of the earliest changes in the adhesive properties of the vessel wall in early lesions is increased ICAM-1 expression. Differences between these studies in CAM spatial distribution or in which CAM is first induced or expressed in greatest quantity may simply be a consequence of differences between species. Regardless, temporal and spatial changes in differential CAM expression do develop during the sequential stages of plaque formation. Additional insight into the importance of CAM expression in atherosclerosis may be obtained by following plasma CAM levels. Increases in plasma ICAM-1 levels directly correlate with the onset and severity of peripheral vascular disease and ischemic heart disease.²⁵ Taken together, these findings indicate that CAMs play critical roles in the progression of atherogenesis and that an increase in ICAM-1 levels may be of particular importance since it appears to be one of the earliest changes in vascular adhesiveness.

The mechanisms by which n-LDL increases ICAM-1 expression remain unclear at this time. On the basis of our study¹³ on the effects of n-LDL on eNOS function, perturbations in the usual reactive oxygen species generation may play a central role in the activation mechanisms observed here. n-LDL uncouples arginine metabolism from eNOS activity, so that eNOS becomes a new source for superoxide anion production.¹³ Such changes enhance the production of peroxynitrite, a potent oxidant that can induce a wide variety of oxidative modifications of cellular proteins, lipids, and DNA.^{26 27 28 29} Thus, protracted exposure to n-LDL increases EC oxidative stress. In addition, increases in peroxynitrite formation decrease the levels of functional nitric oxide, an antiatherogenic molecule³⁰ that has been implicated in the induction and expression of Iß.³¹ It is important to note that this n-LDL–induced increase in oxidative stress is due to perturbations in eNOS function and oxidative arachidonic acid metabolism but not to the exogenous lipid peroxide insults resulting from LDL oxidation.^{13 16} Thus, perturbations in the usual biochemistry of the endothelium may enhance activation of transcription factors such as nuclear factor–ß³² or fos/jun complexes.³³ Interestingly, cis elements for both transcription factor families have been found in the promoters for ICAM-1, E-selectin, and VCAM-1.^{32 34 35 36 37 38} Clearly, additional work is necessary to sort out the molecular signaling mechanisms by which n-LDL induces transcription factor activation to preferentially increase ICAM-1 expression.

In conclusion, n-LDL increased EC adhesiveness primarily by increasing ICAM-1 expression. Although n-LDL increased transcript levels for VCAM-1 and E-selectin, significant differences in protein levels were not observed when existing assay protocols were used. Increases in adherence of PMA-differentiated U937 cells and human neutrophils and monocytes to the endothelium developed when n-LDL that was protected against oxidation with BHT was used. Furthermore, increases in the adhesiveness of the endothelium were observed with elevated n-LDL concentrations. Finally, these studies begin to shed new light on the early mechanisms by which n-LDL promotes atherogenesis by establishing a direct link between elevated concentrations of n-LDL, induction of ICAM-1, and mononuclear cell adherence.

	Selected Abbreviations and Acronyms

 

BCECF = 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein BHT = butylated hydroxytoluene CAM = cell adhesion molecule DPBS = Dulbecco's phosphate-buffered saline EC = endothelial cell eNOS = endothelial nitric oxide synthase FBS = fetal bovine serum ICAM-1 = intercellular adhesion molecule–1 IL-1 = interleukin-1 LFA-1 = lymphocyte function–associated antigen–1 M-199 = medium 199 n-LDL = native LDL PMA = phorbol 12-myristate 13-acetate sLex = sialylated Lewis X glycoprotein VCAM-1 = vascular cell adhesion molecule–1 VLA-4 = very late antigen–4

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants 33742, 48251, and 1-PO-HL-43023R1, and by American Heart Association and New York Heart Association Affiliate grant 90-082G. We also recognize the contributions of John A. Thompson, Robert Rothlein, Timothy Springer, Esther Sabban, Tucker Collins, and Michael Bevilacqua for providing the recombinant human basic fibroblast growth factor, R3.1 and the cDNA probes as described in the Methods section. We would like to thank Patricio Villalon for his technical assistance and Susan Koethe for her assistance in preparing this manuscript.

Received August 29, 1995; accepted December 1, 1995.

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