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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1047-1051.)
© 1996 American Heart Association, Inc.

Articles

Probucol Downregulates E-Selectin Expression on Cultured Human Vascular Endothelial Cells

Masayo Kaneko; Joichiro Hayashi; Ichiro Saito; Nobuyuki Miyasaka

the Division of Immunological Diseases (M.K., I.S.), Medical Research Institute; the Department of Periodontology (J.H.), School of Dentistry; and the First Department of Medicine (N.M.), School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.

Correspondence to Nobuyuki Miyasaka, MD, First Department of Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan. E-mail miyasaka.med1@med.tmd.ac.jp.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Probucol, which inhibits monocyte adhesion, is a potent antioxidant to vascular endothelium in the cholesterol-fed rabbit. The accumulation of macrophages in the lesion is influenced by increased expression of specific adhesion molecules on vascular endothelial cells. We investigated the effect of probucol on the expression of cell adhesion molecules in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with lipopolysaccharide in the presence or absence of probucol (0 to 5 µmol/L) and assayed for the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin by cell–enzyme-linked immunosorbent assay. Probucol significantly downregulated the expression of E-selectin on HUVECs in a dose-dependent manner. In contrast, the expression of ICAM-1 was not affected. E-selectin but not ICAM-1 mRNA expression on HUVECs was also significantly inhibited by probucol in a dose-dependent manner. We also examined whether probucol affects cellular binding between the human monocytic cell line U937 and lipopolysaccharide-stimulated HUVECs by using an in vitro binding assay and found that probucol significantly suppressed their mutual binding in a dose-dependent manner. These data indicate a novel mechanism of action for probucol to reduce the development of atherosclerotic lesions in hyperlipidemic states.

Key Words: probucol • cell adhesion molecule • E-selectin • endothelial cell • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
There are two main hypotheses on the pathogenesis of atherogenesis: the response to injury hypothesis and the modified LDL hypothesis. In both hypotheses, the attachment of mononuclear cells to vascular endothelium is essential to the initiation of the atherosclerotic lesion. Exactly how these cells are recruited and retained in the artery wall remains unclear, but researchers speculate that cell adhesion molecules play a key role in the development of atherosclerosis.

Various multiple adhesion molecules have been identified on ECs, including E-selectin^{1 2 3} and ICAM-1.^{4 5 6} Expression of E-selectin on vascular ECs mediates binding with neutrophils, monocytes, and memory T cells.^{1 7} ICAM-1 binds to lymphocyte function-associated antigen-1, which is present on all leukocytes. These molecules are inducible on the endothelial surface in response to inflammatory cytokines such as interleukin-1, tumor necrosis factor-, and LPS, suggesting that these molecules are important in the selective recruitment of leukocytes, including monocytes.^{1 8 9 10 11} In addition, these molecules are expressed in human atherosclerotic lesions.^{12 13}

Probucol [bis(3,5-di-tert-butyl-4-hydroxyphenyl)(thio)propane], a potent lipid-soluble antioxidant, shows a marked antiatherogenic effect in Watanabe heritable hyperlipidemic rabbits^{14 15 16} and also inhibits lesion progression in fat-fed rabbits.^{17 18 19} Probucol therapy diminishes tendinous xanthomas in humans.²⁰ Furthermore, mononuclear cell adherence to vascular endothelium is decreased by probucol both in vitro and in vivo in the cholesterol-fed rabbit.^{21 22} However, its precise mechanism remains to be elucidated. We examined whether probucol modulates the expression of adhesion molecules on human vascular ECs in vitro. Our study clearly demonstrated that probucol downregulates expression of E-selectin in a dose-dependent manner not only at the protein level but also at the mRNA level. We also found that probucol significantly inhibited the cellular binding between the human monocytic cell line U937 and LPS-stimulated HUVECs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Cultures
HUVECs were isolated after collagenase digestion of human umbilical cord vein within 12 hours after delivery.²³ The cells were grown to confluence in RPMI 1640 supplemented with 20% fetal calf serum (both GIBCO), 30 µg/mL EC growth supplement (Collaborative Research), 10 U/mL porcine mucosal heparin, 100 U/mL penicillin, and 100 µg/mL streptomycin in gelatin-coated plastic dishes and harvested.²⁴ No cells were used for the study after more than three serial passages. The viability of cells was always examined morphologically by using an inverted microscope and by the trypan blue dye exclusion test within 4 to 24 hours of probucol treatment.

Reagents and Antibodies
Probucol (Otsuka Pharmaceutical Co) was dissolved in ethanol and kept as a stock solution. LPS (Escherichia coli O55:B5) was purchased from Sigma Chemical Co. Murine monoclonal antibodies against human adhesion molecules, including E-selectin (BBA1) and ICAM-1 (BBA3), were obtained from British Bio-technology.

Cell-ELISA
HUVECs (10⁴) were seeded onto gelatin-coated 96-well culture plates (Costar). Confluent cultures of HUVECs were stimulated with varying concentrations of LPS in the presence or absence of varying doses of probucol. Cells were subsequently fixed with 3% paraformaldehyde/8% saccharose/PBS. Nonspecific binding was blocked by the sequential addition of Block Ace (Yukijirushi)/PBS and 5% goat serum/PBS for 1 hour. Either anti–ICAM-1 or anti–E-selectin antibody and alkaline phosphatase–conjugated goat anti-mouse IgG antibody (Tago Inc) were used as the first and second antibodies, respectively, followed by the addition of phosphatase substrate (Sigma). The OD of each well was determined by using a microplate reader (Bio-Rad Laboratories) at 405 nm.²⁴ The percent inhibition of adhesion was calculated by using the following formula: (1-adhesion in the presence of probucol [OD]/adhesion in the absence of probucol [OD])x100. Data are expressed as mean±SD of three experiments.

Cell Binding Assay
HUVECs (10⁴) were seeded into each well of gelatin-coated 96-well culture plates. Confluent cultures of HUVECs were stimulated by LPS and varying concentrations of probucol. U937 cells were labeled with 10 µmol/L 2',7'-bis(carboxyethyl)-5(6') carboxyfluorescein tetraacetoxymethyl ester (Dojindo Laboratories)²⁴ for 1 hour at 37°C in RPMI 1640 with 10% fetal calf serum and then washed three times with serum-free RPMI 1640. Labeled cells (2x10⁴) were added to each well and incubated with HUVECs for 1 hour. Cells that were nonspecifically bound to HUVECs were removed by inverting the plates for 30 minutes. Wells were subsequently washed once with serum-free RPMI 1640, and the remaining cells were lysed with 1% Nonidet P-40 (Nakarai Chemicals). Fluorescence intensity in cell lysates was measured by using an automated microplate fluorometer (Nihon Bunko) at 490 nm. The percentage inhibition of cellular binding was calculated by using the following formula (1-adhesion in the presence of probucol [OD]/adhesion in the absence of probucol [OD])x100. Data are expressed as mean±SD of three experiments.

Northern Blot and Slot Blot Analyses
HUVECs were cultured in a plastic dish under the same conditions described above. Total RNA was isolated from HUVECs by using guanidine isothiocyanate extraction.²⁵ RNA (20 µg per lane) was separated in 1.2% agarose gel for Northern blot analysis and transferred to nylon membranes for Northern blot and slot blot analyses. Hybridization was performed by using a ³²P-labeled cDNA probe for E-selectin (British Bio-technology), ICAM-1 (kindly provided by Dr T.A. Springer), and GAPDH (Ambion Inc) in 5x SSPE (sodium chloride/sodium phosphate/EDTA buffer), 5x Denhardt's solution, 0.5% sodium dodecyl sulfate, and 50% formamide at 42°C overnight. Membranes were washed in 6x SSPE and 0.1% sodium dodecyl sulfate at 42°C for 90 minutes. The blots were then washed until the background counts were insignificant and subjected to autoradiography at -80°C.

Statistical Analysis
Data were analyzed by using ANOVA from each individual experiment.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LPS Induces Adhesion Molecule Expression on HUVECs in a Dose-Dependent Fashion
We first performed dose-response studies of LPS-induced adhesion molecule expression on HUVECs by using cell-ELISA to obtain optimal conditions for further study. HUVECs were cultured with LPS (0 to 1000 ng/mL) for different times. A small amount of ICAM-1 was constitutively expressed on HUVECs and there was a dose-dependent increase of ICAM-1 when HUVECs were stimulated with LPS. Stimulation with 1 µg/mL LPS showed a maximum ICAM-1 expression after 24 hours (Fig 1) and reached a plateau thereafter. E-selectin expression was not expressed on HUVECs without stimulation and was maximally induced by 1 µg/mL LPS at 4 to 6 hours (Fig 1). We therefore decided to use a suboptimal dose of LPS for the later experiments.

View larger version (51K): [in this window] [in a new window]   Figure 1. Bar graphs show dose-dependent increases induced by LPS in (top) ICAM-1 and (bottom) E-selectin expression as assessed by cell-ELISA. HUVECs were cultured with varying concentrations of LPS for 24 (ICAM-1) or 4 (E-selectin) hours.

Probucol Significantly Inhibits E-Selectin but Not ICAM-1 Expression on LPS-Stimulated HUVECs
We next examined the effect of probucol on the expression of E-selectin and ICAM-1 on HUVECs. HUVECs were treated with 100 ng/mL LPS in the presence of probucol (0 to 5 µmol/L) for 4 hours and assayed for their expression by cell-ELISA. The expression of E-selectin on HUVECs was significantly downregulated by probucol in a dose-dependent manner (P<.001) (Fig 2). Cell viability was >98% as assessed by the trypan blue dye exclusion test, and this inhibition could not be explained by detachment of the cells from the culture plates. In contrast, probucol (0 to 5 µmol/L) did not significantly affect LPS-induced ICAM-1 expression (Fig 2), nor was constitutive expression of ICAM-1 on HUVECs modulated by probucol. In addition, changing the length of the culture period with probucol also did not change the results in ICAM-1 expression (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 2. Bar graphs show inhibition of (top) E-selectin and (bottom) ICAM-1 expression by probucol. Varying concentrations of probucol were added to the HUVEC culture system together with LPS (100 ng/mL for 6 hours for E-selectin and 10 ng/mL for 24 hours for ICAM-1). E-selectin expression was measured by cell-ELISA. Each experiment is representative of three separate experiments. *P<.001.

Probucol Inhibits Cellular Binding of Human Monocytic Cell Line U937 to LPS-Stimulated HUVECs
To assess whether probucol really affects mononuclear cell binding with activated endothelium, an in vitro cellular binding assay was performed between the human monocytic cell line U937 and LPS-stimulated HUVECs in the presence or absence of probucol. HUVECs were treated with probucol (0 to 10 µmol/L) for either 4 or 12 hours in the presence of 100 ng/mL LPS, after which the binding of U937 cells to HUVECs was measured. The adhesion of U937 cells to LPS-stimulated HUVECs was significantly inhibited by the 4-hour treatment with probucol (P<.001) (Fig 3), but the 12-hour treatment did not affect their mutual binding (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 3. Bar graph shows inhibition of cellular binding between U937 cells and LPS-stimulated HUVECs with probucol. Varying concentrations of probucol were added to the HUVEC culture system together with LPS (100 ng/mL) for 4 hours. Binding between U937 and LPS-stimulated HUVECs was measured by the cell binding assay described in "Methods." The experiment is representative of three separate experiments. *P<.001.

Probucol Reduces E-Selectin mRNA Expression in LPS-Stimulated HUVECs
Northern blot analysis was performed to determine whether the LPS-induced E-selectin mRNA expression was affected by probucol treatment. E-selectin mRNA was not detected under basal conditions in the presence or absence of probucol. LPS stimulation of HUVECs resulted in a significant induction of E-selectin mRNA after 3 hours of culture; this induction was significantly inhibited by treatment with probucol in a dose-dependent manner (Fig 4). This inhibition corresponded to the decreased E-selectin expression shown by cell-ELISA. However, LPS-induced ICAM-1 mRNA expression was hardly affected by probucol (Fig 5).

View larger version (60K): [in this window] [in a new window]   Figure 4. Northern blot analysis for E-selectin mRNA. HUVECs were stimulated with or without 100 ng/mL LPS in the presence or absence of 5 µmol/L probucol for 3 hours. Total RNA (20 µg) (as confirmed by measurement of mRNA) was applied to each lane. Lane 1, HUVECs with medium alone; lanes 2 through 5, HUVECs stimulated with 100 ng/mL LPS, 100 ng/mL LPS and 1 µmol/L probucol, 100 ng/mL LPS and 2.5 µmol/L probucol, and 100 ng/mL LPS and 5 µmol/L probucol, respectively. The experiment is representative of three separate experiments.

View larger version (42K): [in this window] [in a new window]   Figure 5. Slot blot analysis for ICAM-1 mRNA and GAPDH mRNA. HUVECs were stimulated with or without 1 ng/mL LPS in the presence or absence of 5 µmol/L probucol for 3 hours. Total RNA (20 µg) was applied to each lane. Lane 1, HUVECs with medium alone; lanes 2 through 5, HUVECs stimulated by 100 ng/mL LPS, 1 ng/mL LPS and 1 µmol/L probucol, 1 ng/mL LPS and 2.5 µmol/L probucol, and 1 ng/mL LPS and 5 µmol/L probucol, respectively. The experiment is representative of three separate experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our study demonstrated that probucol downregulates E-selectin expression in a dose-dependent manner on LPS-stimulated HUVECs not only at the protein level but also at the level of mRNA. Probucol also suppressed the cellular binding between the human monocytic cell line U937 and LPS-stimulated HUVECs. ICAM-1 expression was not affected by probucol.

Probucol has been used with efficacy in the treatment of patients with atherosclerosis. It was originally developed as a hypolipidemic drug,²⁶ but recent interest has been focused on its potent antioxidant properties, which block the oxidative modification of LDL.^{15 20 27} Probucol also inhibits lipid storage in a macrophage-like cell line, resulting in the prevention of the development of foam cells.¹⁵ Probucol may exert its antiatherogenic effect through different mechanisms, such as inhibition of interleukin-1 secretion from macrophages^{28 29} and stimulation of reverse cholesterol transport transfer.^{27 30 31} However, its mode of action in vivo remains unclear.

Endothelial-mononuclear cell binding is a crucial step in the development of atherosclerosis. This process involves sequential and multiple receptor-ligand interactions of adhesion molecules. Monocyte adhesion to activated ECs in vitro therefore serves as a useful model of an early step in atherosclerosis. Probucol can suppress agonist-induced monocytic cell adhesion to HUVECs in vitro.³² However, it is not clear from this report³² whether probucol acted directly on ECs to alter the expression of adhesion molecules or modified augmented production of proinflammatory cytokines. E-selectin and ICAM-1 are expressed on ECs in atherosclerotic lesions.^{12 13 33} Among mononuclear cells, T cells and monocytes initially attach to vascular endothelium in the early phase of atherosclerosis.^{34 35} T cells infiltrated within plaques simultaneously express CD4 and CD45RO antigens,³⁶ which are the hallmarks for memory T cells. Memory T cells employ E-selectin to attach to ECs.^{7 36} E-selectin, a 110-kD glycoprotein and receptor for sialyl Lewis^x and sialyl Lewis^a, also mediates in vitro monocyte adhesion to cytokine-activated ECs.^{33 37 38} Although multiple pathways are operative in this process, monocyte adhesion to cytokine-activated HUVECs is inhibited by various monoclonal antibodies.^{39 40 41} Some reports demonstrate that antibodies directed against E-selectin block the binding of U937 cells to activated HUVECs.^{3 11 42} It is therefore possible that E-selectin could be one of the key molecules in the formation of atherosclerotic lesions.^{36 43 44} In vitro, E-selectin expression reaches a maximum 4 to 6 hours after stimulation and rapidly disappears thereafter.^{3 37 43} In our study, probucol substantially abrogated LPS-induced expression of E-selectin but not ICAM-1 on HUVECs. E-selectin mRNA expression on HUVECs induced by LPS was also inhibited by probucol. These results indicate that probucol might inhibit the cell-surface expression of E-selectin on LPS-stimulated HUVECs through the suppression of E-selectin mRNA. Furthermore, probucol significantly inhibited the cellular binding between U937 and LPS-stimulated HUVECs after 4 but not 12 hours of treatment, suggesting that probucol affects the quantitative and/or qualitative changes of rapidly and transiently inducible adhesion molecules, including E-selectin, on HUVECs. These results suggest that probucol may prevent the progression of atherosclerosis by inhibiting induced monocyte adhesion to activated endothelium.

Regulation of adhesion molecule expression is now becoming one of the innovative treatments for inflammatory diseases in animal models. We have reported⁴⁵ that gold sodium thiomalate, which has been used with marked efficacy in the treatment of rheumatoid arthritis, significantly inhibits ICAM-1 expression on vascular ECs and suppresses cellular binding between human monocytic cell lines and interleukin-1–stimulated HUVECs. 3-Deazaadenosine, a structural analogue of adenosine with immunomodulatory and anti-inflammatory activity, inhibits the binding of polymorphonuclear cells to tumor necrosis factor-–stimulated HUVECs and ICAM-1 synthesis by HUVECs.⁴⁶ E-selectin expression is also reduced by 3-deazaadenosine.⁴⁷ Faruqi et al³² report that -tocopherol (vitamin E), an antioxidant and antiatherosclerotic agent, inhibits monocytic cell adhesion to HUVECs in vitro. The inhibition correlated with a decrease in E-selectin mRNA and cell-surface expression of E-selectin in their study.

In conclusion, ours is the first study to show that probucol downregulates E-selectin expression on HUVECs in vitro. Further development of therapeutic agents to regulate the expression of adhesion molecules on vascular endothelium may create a new mode of treatment for atherosclerosis.

	Selected Abbreviations and Acronyms

 

EC = endothelial cell ELISA = enzyme-linked immunosorbent assay HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule-1 LPS = lipopolysaccharide OD = optical density PBS = phosphate-buffered saline

	Acknowledgments

 
This work was partly supported by a grant-in-aid from the Ministry of Health and Welfare, Japan. Dr Hayashi received a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.

Received October 6, 1995; revision received March 5, 1996;

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