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Lipoprotein(a) Enhances the Expression of Intercellular Adhesion Molecule-1 in Cultured Human Umbilical Vein Endothelial Cells

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Lipoprotein(a) Enhances the Expression of Intercellular Adhesion Molecule-1 in Cultured Human Umbilical Vein Endothelial Cells Shigeki Takami, MD; Shizuya Yamashita, MD, PhD; Shinji Kihara, MD, PhD; Masato
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Lipoprotein(a) Enhances the Expression of Intercellular Adhesion Molecule-1 in Cultured Human Umbilical Vein Endothelial Cells Shigeki Takami, MD; Shizuya Yamashita, MD, PhD; Shinji Kihara, MD, PhD; Masato Ishigami, MD, PhD; Kaoru Takemura, MD, PhD; Noriaki Kume, MD, PhD; Toru Kita, MD, PhD; Yuji Matsuzawa, MD, PhD Background We reported an increase in serum lipoprotein(a) [Lp(a)] levels in patients with thromboangiitis obliterans, suggesting that Lp(a) could also contribute to the pathogenesis of cardiovascular diseases by a mechanism different from atherosclerosis. Adhesion molecules were shown to contribute to the development of not only atherosclerotic but also inflammatory vascular diseases. Methods and Results We evaluated the effect of Lp(a) on the expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin in human umbilical vein endothelial cells by a cell ELISA. Lp(a) dramatically enhanced the levels of ICAM-1 in a dose-dependent manner. A discernible increase in ICAM-1 expression was observed at a physiological concentration of 0.26 mmol cholesterol/l Lp(a) after 48-hour incubation. A 1.8-fold increase in ICAM-1 expression was observed 48 hours after the addition of Lp(a) (1.04 mmol cholesterol/l). Northern blot analysis demonstrated that the amount of ICAM-1 mrna was increased after treatment with Lp(a). In contrast to ICAM-1, the expression of VCAM-1 and E-selectin was not significantly affected by Lp(a). Lp(a ) [apolipoprotein(a)- removed Lp(a) by reduction with dithiothreitol] and LDL had no significant effect on the expression of ICAM-1. In contrast, recombinant apolipoprotein(a) protein alone significantly enhanced ICAM-1 expression. Lp(a) decreased the level of active transforming growth factor (TGF)- in the conditioned medium. Furthermore, recombinant TGF- significantly decreased the Lp(a)-induced ICAM-1 expression. These findings suggested that Lp(a) may enhance the ICAM-1 expression by decreasing active TGF- level. Conclusions Lp(a) could contribute to the development of cardiovascular diseases by enhancing the expression of ICAM-1 in endothelial cells. (Circulation. 1998;97: ) Key Words: cardiovascular diseases cells growth substances leukocytes lipoproteins Serum Lp(a) was first identified in 1963 by Berg. 1 A high concentration of Lp(a) has been demonstrated to be one of the major risk factors for premature development of atherosclerosis. 2,3 Lp(a) is a particle with an unusual structure consisting of apo(a), which is linked to apo B-100 of an LDL-like particle through disulfide bonds. 4 6 cdna nucleotide sequence analyses have shown that apo(a) has a high degree of homology to plasminogen, 7 one of the important factors in fibrinolysis system. Therefore it is suggested that the pathophysiological effects of Lp(a), including those on fibrinolysis, may be attributable to apo(a). Lp(a) was shown to bind to vascular endothelial cells and macrophages and to extracellular components such as fibrin and inhibits cell-associated plasminogen activation in vitro. 8,9 One of the earliest events in atherogenesis in cholesterol-fed animals is an increased binding of monocytes to endothelial cells and their entry into vessel walls. 10,11 It is hypothesized that these monocytes contribute in several ways to plaque formation. 12,13 Although the molecular mechanism is not completely understood, in vitro studies have identified three molecules, ICAM-1, 14,15 E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1] 16,17 ), and VCAM These adhesion molecules are inducible on the endothelial cell surface and can support the adhesion of various leukocytes, including monocytes. 19,20 Of these, ICAM-1 was shown to be expressed in human atherosclerotic plaques by an immunohistochemical method 21 and may be a candidate that plays an important role in mediating the localization of monocytes in the intima of arteries. ICAM-1 (CD54) is a markedly glycosylated adhesion molecule belonging to the immunoglobulin gene superfamily. Its expression is restricted on resting cells but is highly inducible by activation such as exposure to IL-1 or TNF-. 14,15 ICAM-1 Received August 6, 1997; revision received October 1, 1997; accepted October 20, From the Second Department of Internal Medicine, Osaka University Medical School, Suita, Osaka, and the Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University Medical School, Kyoto, Japan. Correspondence to Shizuya Yamashita, MD, PhD, Second Department of Internal Medicine, Osaka University Medical School, 2 2, Yamadaoka, Suita, Osaka 565, Japan American Heart Association, Inc. 721 722 Lp(a) and ICAM-1 Expression in HUVEC Selected Abbreviations and Acronyms apo apolipoprotein BSA bovine serum albumin DTT dithiothreitol FCS fetal calf serum HBSS Hanks balanced salt solution HUVEC human umbilical vein endothelial cells ICAM-1 intercellular adhesion molecule-1 IFN- interferon- IL-1 interleukin-1 Lp(a) lipoprotein(a) MDA malondialdehyde TBARS thiobarbituric acid reactive substance TGF- transforming growth factor- TNF- tumor necrosis factor- VCAM-1 vascular cell adhesion molecule-1 binds to its counterreceptor leukocyte function-associated-1 molecule (LFA-1, or CD11a/CD18) as well as to Mac-1 (CD11b/CD18). We previously reported an increase in Lp(a) levels in patients with thromboangiitis obliterans, 22 suggesting that Lp(a) could also contribute to the pathogenesis of cardiovascular disorders by a mechanism different from atherosclerosis. Adhesion molecules including ICAM-1 were shown to contribute to the development of not only atherosclerotic but also inflammatory vascular disorders by regulating cell adhesion between leukocytes and endothelial cells. To date, there has been no report dealing with the effect of Lp(a) on cell adhesion. In the current study, to investigate the effects of Lp(a) on the expression of adhesion molecules in endothelial cells, cultured HUVEC were subjected to human plasma-derived Lp(a). We evaluated the effects of Lp(a) on the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC by cell ELISA and Northern blotting. We demonstrated an enhanced expression of ICAM-1 but not VCAM-1 or E-selectin by addition of Lp(a) to culture medium. Moreover, to clarify the mechanism of Lp(a)-induced ICAM-1 enhancement, we investigated the effects of TGF- and anti TGF- antibody on the enhanced expression of ICAM-1 by Lp(a). Methods Reagents Medium 199, HBSS, and FCS were purchased from Gibco Laboratories; lysine-sepharose was purchased from Pharmacia-LKB; mouse anti-human Lp(a) monoclonal antibodies from Chemicon International; mouse anti-human ICAM-1 monoclonal antibodies, BBA3, from British Bio-technology; mouse anti-human VCAM-1 monoclonal antibodies, mouse anti-human E-selectin monoclonal antibodies, recombinant active human TGF-, and mouse anti-human TGF- monoclonal antibodies from Genzyme; peroxidase-conjugated goat anti-mouse IgG from Organon Teknika-Cappel; and endotoxinspecific limulus amoebocyte lysate, LAL-ES from Wako Pure Chemical Industries. Recombinant apo(a) protein was a generous gift from Prof Gert M. Kostner (Kaar-Franzens-University of Graz, Graz, Austria). All other chemicals were of reagent grade. Isolation of Lp(a), Lp(a ), and LDL Lp(a) was prepared from the plasma of a male donor whose apo(a) had a high affinity for lysine-sepharose as reported by Fless and Sydney. 23 The donor had a plasma Lp(a) concentration of 1000 mg/l and an apo(a) phenotype B (single band) by the typing system of Utermann et al 24 Briefly, total lipoproteins (d g/ml) were isolated by ultracentrifugation and extensively dialyzed against 0.1 mol/l phosphate buffer (ph 7.4) containing 0.01% Na 2 -EDTA, 0.01% NaN 3, and 1 mmol/l benzamidine. These lipoproteins were passed through a column containing lysine-sepharose. The column was washed with 0.5 mol/l NaCl, 0.1 mol/l NaHCO 3, 1 mmol/l benzamidine, ph 8.3. Lp(a) was eluted with 20 mmol/l 6-aminohexanoic acid dissolved in 0.1 mol/l phosphate buffer (ph 7.4), 1 mmol/l benzamidine, and 0.01% Na 2 -EDTA and NaN 3. The unbound lipoproteins obtained after lysine-sepharose chromatography were dialyzed against EDTA-saline (d g/ml), and then LDL (d to g/ml) was isolated by sequential ultracentrifugation according to Havel s method. 25 The isolated Lp(a) and LDL were extensively dialyzed against 0.15 mol/l NaCl, 0.01% Na 2 -EDTA, and 0.01% NaN 3. The Lp(a) particles were separated into two products: apo(a) and a floating apob-100 containing lipoprotein, Lp(a ). To make Lp(a ), 1 mol/l DTT was added to the Lp(a) solution to give a final concentration of 0.01 mol/l. Reduction of Lp(a) by DTT was achieved by incubation at 37 C for 15 minutes. After reduction with DTT, apo(a) and Lp(a ) were separated by ultracentrifugation at rpm for 18 hours in a Beckman 50.2 Ti rotor. The obtained Lp(a ) fraction did not contain apo(a) as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis or by Western blot analysis using a monoclonal anti-human Lp(a). Lp(a ) was dialyzed against 0.15 mol/l NaCl, 0.01% Na 2 -EDTA, and 0.01% NaN 3. Isolated samples were dialyzed finally against medium 199 and sterilized by ultrafiltration before use. Cell Culture HUVEC were purchased from Kurabo Industries, Ltd. HUVEC were seeded in plastic plates precoated with collagen (type I) (Becton & Dickinson Labware) and cultured in medium 199 supplemented with 20% FCS, 12.5 g/ml endothelial cell growth supplement, 1 g/ml hydrocortisone, 100 U/mL penicillin, and 100 g/ml streptomycin. The cells were cultured at 37 C in humidified 5% CO 2 and 95% air. HUVEC were used for experiments at passages 3 to 4. Incubation of HUVEC With Lp(a), Lp(a ), LDL, and Recombinant Apo(a) Protein HUVEC were seeded at a concentration of cells/well into 96-well collagen (type I)-coated microplates (Becton & Dickinson Labware) and cultured in the medium as described previously. After reaching confluence (after 24 hours), the culture medium was replaced with medium 199 supplemented with 5% FCS designated as culture medium. After 24 hours at 37 C, the medium was replaced again with the culture medium containing varying concentrations of Lp(a), Lp(a ), LDL, and recombinant apo(a) protein. These samples were added to the culture medium at the indicated final concentrations just before incubation with HUVEC. Before the incubation, the concentration of endotoxin in the culture medium containing Lp(a), Lp(a ), LDL, and recombinant apo(a) protein was determined by a turbidimetric kinetic assay using a commercially available kit (Wako Pure Chemical Industries). The endotoxin levels of the culture medium used in the current study were all less than 1 pg/ g protein; these contaminated endotoxins were shown not to affect the expression of adhesion molecules in HUVEC at the present experimental concentrations. The cells were cultured for various times at 37 C in 5% CO 2 incubator before assays were performed. Cell ELISA of ICAM-1, VCAM-1, and E-Selectin on HUVEC The protocols used for cell ELISA of ICAM-1, VCAM-1, and E-selectin were modified from that of Rothlein et al 26 Briefly, HUVEC in 96-well microplates, which had been treated with Lp(a), Lp(a ), or LDL, were washed with warm HBSS containing 0.1% BSA. The monolayers were washed and then incubated with mouse anti-human ICAM-1, VCAM-1, or E-selectin monoclonal antibodies at a final concentration of 0.5 g/ml in HBSS containing 0.1% BSA to detect the surface expression of these adhesion molecules. After Takami et al 723 incubation of cells at room temperature for 30 minutes, the plates were washed five times with HBSS containing 0.1% BSA and then treated with 0.1 ml/well of peroxidase-conjugated goat anti-mouse IgG (1:1000 dilution in HBSS containing 0.1% BSA). After 1-hour incubation at room temperature, the plates were washed five times with HBSS containing 0.1% BSA and incubated at room temperature in 0.1 ml/well of the substrate solution (10 ml 0.1 mol/l phosphatecitrate buffer [ph 5] 4 mgo-phenylenediamine 15 L 30% H 2 O 2 ; mixed immediately before incubation). After an incubation for 15 minutes in a dark place, 50 L/well of 2 mol/l H 2 SO 4 was added and spectrophotometric readings were made at 492 nm, using a microplate reader (model 450, Bio-Rad). Northern Blot Analysis Total cellular RNA from cultured HUVEC was extracted by acidguanidinium phenol-chloroform method 27 and electrophoresed through 1% agarose gels containing formaldehyde and transferred onto nitrocellulose membranes. Northern blots were hybridized with human ICAM-1 cdna probes labeled with [ - 32 P]dCTP using random hexanucleotide primers. 28 A 1.3-kb Xho I fragment of human ICAM-1 cdna, 14 kindly provided by Dr Brian Seed (Massachusetts General Hospital, Boston, Mass), was used. The blots were rehybridized with radiolabeled human -actin cdna probe for comparison. TGF- Assay The amount of active TGF- in the medium was determined by a modification of the mink lung epithelial cell assay. 29 Briefly, the conditioned media and TGF- standards were diluted 1:100 in serum-free DMEM. DNA synthesis was determined by [ 3 H]thymidine (1 Ci/mL) incorporation during 1 hour plus 23 hours after the addition of the conditioned media with or without neutralizing TGF- antibody. TGF- activity was calculated as the proportion of the inhibition of DNA synthesis that was reversed in the presence of the neutralizing antibody. The TGF- samples and conditioned media both contained 5% FCS. The amount of total (active plus latent) TGF- in the media was determined by an ELISA, using a commercial kit (Quantikine; R&D Systems). Briefly, to activate latent TGF- to the immunoreactive form, 1N HCl was added to the conditioned medium to give a final concentration of 0.167N HCl. After incubation for 10 minutes at room temperature, the acidified medium was neutralized with 1.2N NaOH/0.5 mol/l HEPES free acid (ph 7.2 to 7.6). Ninety-six well microplates coated with recombinant TGF- soluble receptor type II were incubated for 1 hour with the samples and the TGF- standards on a horizontal orbital microplate shaker at 500 rpm, with peroxidase-conjugated antibody to TGF- at room temperature on a horizontal orbital microplate (1 hour), and then with the chromogenic substrate tetramethylbenzidine at room temperature on the benchtop (20 minutes). Absorbances at 450 nm were converted into quantities with a standard curve. Chemical Analyses Protein content was determined by the method of Lowry et al, using BSA as a standard. 30 Total cholesterol was measured enzymatically using a commercial kit (Kyowa Medex). Lp(a) was measured by an ELISA kit (Biopool, Umeå). The content of lipid peroxides in the conditioned medium from the control and treated HUVEC was determined as TBARS by the modified method of Yagi, 31 using a commercial kit (Wako Pure Chemical Industries). Statistical Analysis The statistical significance of the differences between the means of groups was determined by Student s paired t test or ANOVA. Results Expression of ICAM-1 in HUVEC Treated With Lp(a) Cultured HUVEC monolayers were treated with varying concentrations of Lp(a) in medium 199 containing 5% FCS, Figure 1. Dose response of ICAM-1 induction by Lp(a) in HUVEC. Endothelial cell monolayers were treated for 48 hours with the indicated concentrations of Lp(a) in medium 199 supplemented with 5% FCS, and cell surface expression of ICAM-1 was measured by a cell ELISA, as described in Methods. Values represent mean SD of quadruplicate determinations. *P.05, **P.01 vs control. and cell surface expression of ICAM-1 was measured by the cell ELISA method. We found that cultured HUVEC without Lp(a) treatment constitutively expressed low levels of ICAM-1. This expression was essentially constant over the time course of all experiments (data not shown). As shown in Fig 1, a physiological level of Lp(a) consistently and dramatically upregulated the levels of ICAM-1 expression in a dose-dependent manner. Lp(a) at a concentration of as low as 0.26 mmol cholesterol/l suspended in culture medium caused a significant increase in ICAM-1 expression detected at 48 hours of treatment. After 48-hour treatment with 1.56 mmol cholesterol/l Lp(a), cell surface expression of ICAM-1 was increased about twofold of the basal expression. The concentrations of Lp(a) used in the current experiment were similar to those commonly observed in vivo. This dose-response experiment in Fig 1 was a representative of two independent experiments. Fig 2 shows the time course of Lp(a)-induced ICAM-1 upregulation. A significant increase in ICAM-1 expression was observed 24 hours after the addition of Lp(a) (1.04 mmol cholesterol/l); the expression of ICAM-1 reached a plateau by 72 hours and remained stable up to at least 96 hours. In contrast, cell surface expression of ICAM-1 in HUVEC was Figure 2. Time course of ICAM-1 induction by Lp(a) in HUVEC. Endothelial cell monolayers were treated with Lp(a) (F) in medium 199 supplemented with 5% FCS for the indicated times, and cell surface expression of ICAM-1 was measured by a cell ELISA, as described in Methods. The effect of addition of LDL (E) is also shown for comparison. Values represent mean SD of quadruplicate determinations. *P.05, **P.01 vs control (0 hours). 724 Lp(a) and ICAM-1 Expression in HUVEC Figure 3. Northern blot analysis of ICAM-1 mrna content in unstimulated, LDL-stimulated, and Lp(a)-stimulated HUVEC. Endothelial cell monolayers were treated for 24 hours with medium 199 supplemented with 5% FCS in the presence or absence of added LDL or Lp(a) (1.04 mmol cholesterol/l, respectively), and then total cellular RNA was isolated for Northern blot analysis as described in Methods. Each lane contained 20 g of total RNA. The amount of -actin mrna was also presented for comparison. not significantly affected by LDL treatment (1.04 mmol cholesterol/l) within 96 hours. Comparable results were obtained in two additional experiments under the same conditions. These results suggest that Lp(a) but not LDL enhances the expression of cell surface ICAM-1. Northern Blot Analysis To determine whether the enhancement of ICAM-1 expression occurred at the transcriptional level, the amount of ICAM-1 mrna was evaluated by Northern blot analysis. Fig 3 shows the Northern blot for ICAM-1 mrna abundance from HUVEC maintained for 24 hours in medium containing 1.04 mmol cholesterol/l lipoproteins. The addition of Lp(a) caused an apparent increase in the ICAM-1 mrna level, whereas LDL had no obvious effect. These results were confirmed by an additional experiment. Expression of VCAM-1 and E-Selectin in HUVEC Treated With Lp(a) In addition, the effect of Lp(a) on the expression of VCAM-1 and E-selectin in HUVEC was examined. Cultured HUVEC monolayers were treated with 1.04 mmol cholesterol/l Lp(a) in medium 199 containing 5% FCS for 72 hours, and cell surface expression of VCAM-1 and E-selectin was measured Figure 4. Effects of Lp(a) on the expression of VCAM-1 and E-selectin in HUVEC: Expression of VCAM-1 and E-selectin in untreated (open bars) and Lp(a)-treated (1.04 mmol cholesterol/l; solid bars) HUVEC in medium 199 supplemented with 5% FCS. After 72-hour incubation, cell surface expression of VCAM-1 and E-selectin was measured by cell ELISA, as described in Methods. The effect of Lp(a) on the expression of ICAM-1 is also shown for comparison. Values represent mean SD of quadruplicate determinations. *P.01. Figure 5. Effects of LDL, Lp(a), and Lp(a ) on the expression of ICAM-1 in HUVEC. Endothelial cell monolayers were treated for 48 hours with LDL, Lp(a), or Lp(a ) (1.04 mmol cholesterol/l, respectively) in medium 199 supplemented with 5% FCS, and the cell surface expression of ICAM-1 was measured by a cell ELISA, as described in Methods. Data are expressed as mean SD of quadruplicate determinations. *P.01 vs control. by the cell ELISA method. The results are presented in Fig 4. In
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