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Postprandial, but not postabsorptive low-density lipoproteins increase the expression of intercellular adhesion molecule-1 in human aortic endothelial cells
Atherosclerosis 186 (2006) 101–106
Postprandial, but not postabsorptive low-density lipoproteins increase the expression of intercellular adhesion molecule-1 in human aortic endothelial cells Peter Marschang, Claudia G¨otsch, Rudolf Kirchmair, Susanne Kaser, Christian M. K¨ahler, Josef R. Patsch ∗ Clinical Division of General Internal Medicine, Clinical Department of Internal Medicine, Innsbruck Medical University, Anichstr. 35, A-6020 Innsbruck, Austria Received 15 April 2005; received in revised form 9 July 2005; accepted 15 July 2005 Available online 24 August 2005
Abstract The magnitude of postprandial lipemia has been identified as independent risk factor for the development of coronary artery disease. To test the effect of postprandial versus postabsorptive low-density lipoproteins (LDL) on the expression of adhesion molecules, LDL were isolated from healthy subjects before and 4 h after ingestion of a standardized fatty test meal. We used flow cytometry and Northern blotting to quantify cell adhesion molecules in human aortic endothelial cells (HAEC). The adherence of leukocytes to HAEC was analyzed using a monocyte adhesion assay. Incubation of HAEC with postprandial, but not postabsorptive LDL induced a two-fold increase in the surface expression of intercellular adhesion molecule-1 (ICAM-1), but not of E-selectin or vascular cell adhesion molecule-1. In addition, increased amounts of ICAM-1 transcripts were found in HAEC treated with postprandial LDL. The adhesion of monocytes to HAEC was enhanced after pretreatment with postprandial, but not with postabsorptive LDL. We conclude that postprandial, but not postabsorptive LDL increase the surface expression of ICAM-1 in HAEC apparently by de novo protein synthesis leading to increased adhesion of monocytes. The upregulation of ICAM-1 by postprandial LDL may explain part of the proatherogenic effect of high postprandial lipemia. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Postprandial lipemia; Low-density lipoproteins; Cell adhesion molecules; Intercellular adhesion molecule-1; Human aortic endothelial cells; Monocyte adhesion
1. Introduction Pronounced postprandial lipemia has been identified as an independent risk factor for the development of coronary artery disease [1]. In the postprandial state, the rise of triglyceride-rich lipoproteins is followed by a temporary increased triglyceride content of low-density lipoproteins (LDL) and high-density lipoproteins (HDL) [2,3]. After hydrolysis of triglycerides, e.g. by hepatic lipase, small LDL (pattern B) and HDL (HDL3 ) are generated representing an atherogenic lipoprotein phenotype [4–6].
∗
Corresponding author. Tel.: +43 512 504 23251; fax: +43 512 504 23317. E-mail address: [email protected] (J.R. Patsch).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.07.014
One of the earliest events in the pathogenesis of atherosclerosis is the adherence of leukocytes to the endothelium and their subsequent transmigration into the arterial wall [7]. This process is mediated by cell adhesion molecules (CAM) expressed on the surface of endothelial cells and leukocytes. The endothelial cell-expressed selectins (E-selectin, P-selectin) mediate the initial weak interaction allowing leukocytes to roll along the vessel wall. Tight adhesion is then mediated by adhesion molecules of the immunoglobulin superfamily (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)) on endothelial cells and their counter receptors of the integrin family on leukocytes. In the third and final step, the transmigration of the leukocytes across the endothelial layer, platelet endothelial cell adhesion
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molecule-1 (PECAM-1) as well as other CAM are involved [8–10]. Numerous findings support the role of CAM in atherosclerosis. The expression of the endothelial cell-expressed adhesion molecules ICAM-1, E-selectin, VCAM-1, P-selectin and PECAM-1 is higher on endothelial cells overlying atherosclerotic lesions compared to endothelial areas outside plaques [11–15]. Elevated soluble isoforms of these CAM are present in the blood of patients suffering from coronary artery disease [16]. In knockout mice lacking ICAM-1 or P-selectin, the formation of atherosclerotic lesions is markedly delayed compared to wild-type animals [17,18]. The mechanisms responsible for the increased expression of CAM in regions prone to plaque development are only partially understood [19]. In addition to shear stress, elevated LDL have been suggested to be responsible for the upregulation of CAM on the surface of endothelial cells. Most studies have found an effect of LDL only after oxidation [20–22] although native LDL have also been described to increase the expression of ICAM-1 on endothelial cells [23]. In contrast to P-selectin [24] and PECAM-1 [13], the surface expression of ICAM-1, E-selectin and VCAM-1 appears to be regulated by LDL concentrations [19]. In the present study, we tested the hypothesis according to which postprandial low-density lipoproteins may be more effective than postabsorptive LDL in upregulating these adhesion molecules on the surface of endothelial cells.
2.2. Stimulation assay HAEC cells were seeded in six well plates at 2.5 × 104 cells/well and grown to approximately 70% confluence in EGM-2 medium. The culture medium was then removed and replaced with fresh EGM medium supplemented with 2% lipoprotein deficient FCS. Low-density lipoproteins were isolated by rate zonal centrifugation from the plasma of healthy volunteers (five male and one female, age 22–35) before and 4 h after ingestion of a standardized fatty test meal as described previously [3]. The exact composition of the test meal (730 kcal per square meter of body surface, comprising 65 g fat (polyunsaturated to saturated fat ratio 0.06), 5.3 g protein, 24 g carbohydrates and 240 mg cholesterol) has been described [27]. LDL were dialyzed against phosphate buffered saline (PBS), stored under liquid nitrogen and used within 1 week after preparation in cell culture experiments. LDL were added at protein concentrations of 0.01–0.5 mg/ml to the culture medium of endothelial cells. Control wells were incubated with equal volumes of PBS (negative control) or with TNF-alpha at a final concentration of 10 ng/ml. Pilot experiments were set up to determine the time needed for maximal upregulation of CAM by the positive control (TNF-alpha). In subsequent experiments, stimulation for 8 h was used to measure E-selectin expression and 24 h were used in the case of ICAM-1 and VCAM-1. 2.3. Flow cytometry
2. Materials and methods 2.1. Cells and antibodies Primary human aortic endothelial cells (HAEC, Clonetics) were purchased from Cambrex (Verviers, Belgium) and cultured in EGM-2 medium containing 2% fetal calf serum (FCS) according to the instructions of the manufacturer. HAEC were used between passage four to seven for all experiments. The human monocytic cell line Mono Mac 6 [25] was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultured in RPMI-1640 medium supplemented with 10% FCS, 100 IU/ml penicillin and 100 g/ml streptomycin. Where indicated, lipoprotein-deficient FCS (Sigma, Vienna, Austria) was used instead of normal FCS. The anti-ICAM-1 monoclonal antibody 7F7 was a kind gift from M.P. Dierich, Innsbruck Medical University, Innsbruck, Austria [26]. Monoclonal antibodies against E-selectin (R&D Systems, Wiesbaden, Germany) and VCAM-1 (Serotec, Oxford, UK) were purchased. Recombinant human tumor necrosis factor-alpha (TNF-alpha) was purchased from Genzyme (Cambridge, MA, USA). F(ab )2 antibodies against mouse immunoglobulins (IgG and IgM) were purchased from An der Grub (Kaumberg, Austria).
Following stimulation, HAEC were washed twice with PBS and then mobilized gently with a cell scraper (Falcon 3058, Becton-Dickinson, Le Pont de Claix, France). After centrifugation, cells were resuspended in PBS containing 1% bovine serum albumin. The cells were stained with specific monoclonal antibodies at saturating concentrations and analyzed by flow cytometry as described [28]. Dead cells were excluded from analysis by counterstaining with propidium iodide (Sigma, Vienna, Austria) during the final wash steps. 2.4. Northern blot Total RNA was isolated from HAEC using RNA clean solution (Hybaid, Heidelberg, Germany) according to the instructions of the manufacturer. Total RNA (10 g per lane) was denatured in formaldehyde, separated by agarose gel electrophoresis and transferred to nylon membranes. The ICAM-1 cDNA probe and a control housekeeping gene (glyceraldehyd-3-phosphate dehydrogenase, GAPDH) cDNA probe were labeled with [␣-32 P]dCTP by the random primer method. The ICAM-1 specific cDNA probe was kindly provided by B. Seed, Harvard Medical School, Boston, MA, USA [29]. Hybridization was performed according to standard protocols [30]. Bands were visualized by autoradiography at −70 ◦ C.
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2.5. Monocyte adhesion assay The monocytes adhesion assay was performed as described [31]. Briefly, HAEC were seeded in 96 well plates at a density of 5 × 103 per well and grown to confluence. Mono Mac 6 cells were labeled with a fluorescent dye (calcein-acetomethylester, Molecular Probes, Eugene, OR, USA) and added to the endothelial cells for 30 min. After washing, the number of bound Mono Mac 6 cells was measured using a Cytofluor 2350 fluorometer (Millipore, Bedford, MA, USA). 2.6. Statistical analyses Plasma lipids of the study participants were compared with Student’s paired t-test. Since triglycerides were not distributed normally, logarithmic transformation of triglyceride values was performed prior to analysis. The expression of adhesion molecules and adhesive properties of HAEC after pretreatment with LDL and controls were compared using one way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test as a post test. Throughout, a p-value of 0.05 was considered statistically significant. Calculations were performed with Graph Pad Prism or Graph Pad InStat software (Version 3.0, Graph Pad software, San Diego, CA, USA).
3. Results Plasma lipids of the study subjects before and 4 h after ingestion of a standardized fatty test meal are shown in Table 1. Consistent with previous observations [32], a marked rise of plasma triglycerides was observed in the postprandial period without significant alterations of total cholesterol, LDL cholesterol and HDL cholesterol. HAEC cells were incubated with LDL isolated in the postprandial and postabsorptive state and the surface expression of cell adhesion molecules was measured by flow cytometry. As shown in Figs. 1 and 2, incubation with LDL of the postprandial state resulted in a two-fold increase of the surface expression of ICAM-1 compared to postabsorptive LDL and the negative control (PBS) (p < 0.05, ANOVA). Conversely, no significantly different surface expression of VCAM-1 and ETable 1 Plasma lipids of study subjects before and 4 h after ingestion of a standardized fatty test meal Parameter
Postabsorptive state
Total cholesterol (mg/dl) Triglycerides (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)
181 139 93 59
± ± ± ±
24 66 18 2
Postprandial state 193 250 89 56
± ± ± ±
27 64 21 2
p-Value n.s. 0.03 n.s. n.s.
Values are mean ± standard deviation from six healthy volunteers. n.s.: not significant.
Fig. 1. LDL were isolated from healthy volunteers in the postabsorptive (LDLpa) and postprandial (LDLpp) state. HAEC were incubated with these LDL preparations and with controls (TNF-alpha, PBS) for 24 h. The surface expression of ICAM-1 was measured by flow cytometry. The histograms of a typical experiment repeated six times with identical results are shown.
selectin was observed after preincubation with either of the two LDL preparations (Fig. 2). The upregulation of ICAM1 expression by postprandial LDL was dose-dependent and marked increases of the surface expression of ICAM-1 were seen after incubation with concentrations equaling or exceeding 0.1 mg/ml LDL (see Fig. 3). To test whether the upregulation of ICAM-1 expression was caused by de novo synthesis of ICAM-1 in endothelial cells, we performed Northern blotting of RNA isolated from HAEC preincubated with postprandial and postabsorptive LDL and the controls (TNFalpha and PBS). As can be seen in Fig. 4, pretreatment with postprandial LDL resulted in increased ICAM-1 specific mRNA transcripts compared to postabsorptive LDL. To test whether the increased surface expression of ICAM-1 resulted in increased binding of leukocytes to endothelial cells, we performed a monocyte adhesion assay using Mono Mac 6 cells. This monocytic cell line is known to have adhesion characteristics similar to freshly isolated human blood monocytes [33]. As shown in Fig. 5, significantly more Mono Mac 6 cells bound to HAEC after pretreatment with 0.5 mg/ml postprandial LDL compared to the control (PBS) (p < 0.05, ANOVA).
4. Discussion Almost all studies of the effect of LDL on endothelial cells have been performed with LDL isolated from plasma in the postabsorptive state. However, under physiological conditions, the endothelium is exposed to postprandial lipoproteins for prolonged periods of time. An impairment of endothelial function after ingestion of a standardized fatty meal was observed in vivo by measuring the endothelium—dependent dilation of the brachial artery [32]. The magnitude of postprandial lipemia has been identified as an independent risk factor for the development of coronary artery disease and was found to correlate with the intima media thickness of the carotid arteries [1,34]. Thus, studies addressing the role of
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Fig. 3. Dose dependence of the effect of LDL on ICAM-1 surface expression in HAEC. HAEC were incubated with different concentrations (given in mg/ml) of LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state for 24 h. The surface expression of ICAM-1 was measured by flow cytometry and is shown compared to the positive (TNF-alpha) and negative (PBS) control. Values are shown as percent mean fluorescence intensity ±standard error from at least three independent experiments.
of the earliest steps in atherosclerosis leading to the subsequent migration of blood leukocytes into the subendothelial space, the increase of the adhesive properties of endothelial cells may be an important mechanism by which postprandial LDL exert their proatherogenic effect. However, additional mechanisms including the preferred uptake and degradation
Fig. 2. Effect of LDL on the surface expression of cell adhesion molecules in HAEC. HAEC were incubated with TNF-alpha, LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state, or PBS for 8 h (E-selectin) or 24 h (ICAM-1, VCAM-1). The surface expression of cell adhesion molecules was quantified by flow cytometry. Values are shown as percent mean fluorescence intensity ±standard error from at least four independent experiments. *p < 0.05 compared to the negative control (PBS), ANOVA, Dunnett’s multiple comparisons test.
lipoproteins in the postprandial state appear to be needed to identify the mechanisms by which high postprandial lipemia confers a higher risk for the development of atherosclerotic lesions. In the present study, we found that LDL isolated from postprandial, but not from postabsorptive human plasma induce a two-fold increase in the surface expression of ICAM-1 in HAEC. The higher surface expression of ICAM-1 caused by postprandial LDL results in an increased adhesion of human monocytes to endothelial cells. Since adhesion is one
Fig. 4. ICAM-1 transcripts in HAEC treated with LDL. RNA was isolated from HAEC after preincubation with LDL from the postprandial (LDLpp, lane 1) or postabsorptive (LDLpa, lane 2) state, PBS (lane 3), or TNF-alpha (lane 4) for 24 h. Total RNA (10 g per lane) was separated by electrophoresis, transferred to nylon membranes and probed with cDNA probes specific for ICAM-1 (top panel) or GAPDH (lower panel).
P. Marschang et al. / Atherosclerosis 186 (2006) 101–106
Fig. 5. Monocyte adhesion to HAEC pretreated with LDL. HAEC were pretreated with LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state in the concentrations indicated for 24 h. Mono Mac 6 cells were labeled with calcein-acetomethylester and binding to the endothelial cells was quantified with a fluorometer. Values are shown as percent adherence (mean ± standard error) of six replicates in three separate experiments. *p < 0.05 compared to the negative control (PBS), ANOVA, Dunnett’s multiple comparisons test.
of postprandial when compared to postabsorptive LDL by macrophages and foam cells are likely to be of importance [3]. The effect of postprandial LDL on the surface expression of ICAM-1 was seen with LDL protein concentrations starting from 0.1 mg/ml and most pronounced with the highest concentration tested (0.5 mg/ml). This corresponds to LDL cholesterol concentrations of approximately 20–100 mg/dl, well within the physiological range of LDL lipoproteins in vivo. In a previous study, comparable concentrations of native LDL were shown to increase the expression of ICAM-1 on the surface of human umbilical vein endothelial cells [23]. Conversely, LDL modified by oxidation can induce the expression of ICAM-1 in endothelial cells already at concentrations as low as 0.01 mg/ml [20,22,35]. In addition, oxidized LDL were also reported to upregulate the expression of VCAM1 and E-selectin in some studies [21,36]. In contrast to our results, one study also described the induction of VCAM-1 and E-selectin by native LDL [37]. An increased intracellular accumulation of P-selectin without direct changes in surface expression has been described after incubation with minimally modified LDL [24]. Postprandial LDL were characterized in detail in a previous report [3]. As expected, postprandial LDL contain more triglycerides (and phospholipids) compared to postabsorptive LDL. There is no difference in the electrophoretic mobility in agarose gels, particle size, or the fatty acid composition between the two LDL species from the two absorptive states. However, postprandial LDL are more susceptible to oxidation by CuSO4 , while cell-mediated oxidation is not substantially different [3]. The cellular receptors and intracellular pathways responsible for the induction of ICAM-1 expression by LDL have only been partly characterized. For oxidized LDL, the lectin-like LOX-1 receptor is believed to mediate the upregulation of ICAM-1 by activation of nuclear fac-
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tor kappa B (NF-B) [36]. Conversely, in the case of native LDL, the LDL receptor, intracellular calcium and activator protein-1 (AP-1) have been implied [37,38]. Further studies are necessary to determine whether the increased triglyceride and/or phospholipid content of postprandial versus postabsorptive LDL is responsible for the different activity of these two LDL preparations. In addition, it remains to be clarified which lipoprotein receptors are involved and which intracellular mechanisms are triggered by postprandial LDL. Our observation that postprandial LDL directly alter the adhesive properties of endothelial cells provide a novel mechanism for the proatherogenic effect of postprandial LDL. With respect to the atherosclerotic risk conferred by LDL, it has been suggested that a compositional characteristic, i.e. a high triglyceride content of LDL, may reflect better a proatherogenic state of LDL when compared to high LDL cholesterol [39]. Therefore, not only the concentration but also the composition of LDL appears to be important, as is certainly the case with small dense LDL present in hypertriglyceridemic states [6]. Similarly, high postprandial LDL may be deleterious because of their higher triglyceride content and exert their proatherogenic potential by various mechanisms, including the induction of ICAM-1 on the surface of endothelial cells.
Acknowledgements This work was supported by grants (P 11693-MED and P16121-B05) from the Austrian Science Fund (FWF) and by the Austrian National Bank (grant Nr. 6442), all to Josef R. Patsch. We are grateful to Drs. Manfred P. Dierich and Brian Seed for valuable reagents. We wish to thank Karin Salzmann, Isa Hauser and Barbara Fuchsberger for expert technical assistance.
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[24] Vora DK, Fang ZT, Liva SM, et al. Induction of P-selectin by oxidized lipoproteins. Separate effects on synthesis and surface expression. Circ Res 1997;80:810–8. [25] Ziegler-Heitbrock HW, Thiel E, Futterer A, et al. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer 1988;41:456–61. [26] Schulz TF, Mitterer M, Vogetseder W, et al. Identification and characterization of a novel membrane activation antigen with wide cellular distribution. Eur J Immunol 1988;18:7–11. [27] Patsch JR, Karlin JB, Scott LW, Smith LC, Gotto Jr AM. Inverse relationship between blood levels of high density lipoprotein subfraction 2 and magnitude of postprandial lipemia. Proc Natl Acad Sci USA 1983;80:1449–53. [28] Marschang P, Gurtler L, Totsch M, et al. HIV-1 and HIV-2 isolates differ in their ability to activate the complement system on the surface of infected cells. AIDS 1993;7:903–10. [29] Simmons D, Makgoba MW, Seed B. ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature 1988;331:624–7. [30] Sambrook J, Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press; 2001. [31] Kahler CM, Kaufmann G, Kahler ST, Wiedermann CJ. The neuropeptide secretoneurin stimulates adhesion of human monocytes to arterial and venous endothelial cells in vitro. Regul Pept 2002;110:65–73. [32] Gaenzer H, Sturm W, Neumayr G, et al. Pronounced postprandial lipemia impairs endothelium-dependent dilation of the brachial artery in men. Cardiovasc Res 2001;52:509–16. [33] Erl W, Weber C, Wardemann C, Weber PC. Adhesion properties of Mono Mac 6, a monocytic cell line with characteristics of mature human monocytes. Atherosclerosis 1995;113:99–107. [34] Sharrett AR, Chambless LE, Heiss G, Paton CC, Patsch W. Association of postprandial triglyceride and retinyl palmitate responses with asymptomatic carotid artery atherosclerosis in middle-aged men and women. The Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol 1995;15:2122–9. [35] Takei A, Huang Y, Lopes-Virella MF. Expression of adhesion molecules by human endothelial cells exposed to oxidized lowdensity lipoprotein. Influences of degree of oxidation and location of oxidized LDL. Atherosclerosis 2001;154:79–86. [36] Li D, Chen H, Romeo F, et al. Statins modulate oxidized low-density lipoprotein-mediated adhesion molecule expression in human coronary artery endothelial cells: role of LOX-1. J Pharmacol Exp Ther 2002;302:601–5. [37] Allen S, Khan S, Al Mohanna F, Batten P, Yacoub M. Native lowdensity lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells. J Clin Invest 1998;101:1064–75. [38] Wang N, Verna L, Liao H, et al. Adenovirus-mediated overexpression of dominant-negative mutant of c-Jun prevents intercellular adhesion molecule-1 induction by LDL: a critical role for activator protein-1 in endothelial activation. Arterioscler Thromb Vasc Biol 2001;21:1414–20. [39] Marz W, Scharnagl H, Winkler K, et al. Low-density lipoprotein triglycerides associated with low-grade systemic inflammation, adhesion molecules, and angiographic coronary artery disease: the Ludwigshafen Risk and Cardiovascular Health study. Circulation 2004;110:3068–74.
Postprandial, but not postabsorptive low-density lipoproteins increase the expression of intercellular adhesion molecule-1 in human aortic endothelial cells Peter Marschang, Claudia G¨otsch, Rudolf Kirchmair, Susanne Kaser, Christian M. K¨ahler, Josef R. Patsch ∗ Clinical Division of General Internal Medicine, Clinical Department of Internal Medicine, Innsbruck Medical University, Anichstr. 35, A-6020 Innsbruck, Austria Received 15 April 2005; received in revised form 9 July 2005; accepted 15 July 2005 Available online 24 August 2005
Abstract The magnitude of postprandial lipemia has been identified as independent risk factor for the development of coronary artery disease. To test the effect of postprandial versus postabsorptive low-density lipoproteins (LDL) on the expression of adhesion molecules, LDL were isolated from healthy subjects before and 4 h after ingestion of a standardized fatty test meal. We used flow cytometry and Northern blotting to quantify cell adhesion molecules in human aortic endothelial cells (HAEC). The adherence of leukocytes to HAEC was analyzed using a monocyte adhesion assay. Incubation of HAEC with postprandial, but not postabsorptive LDL induced a two-fold increase in the surface expression of intercellular adhesion molecule-1 (ICAM-1), but not of E-selectin or vascular cell adhesion molecule-1. In addition, increased amounts of ICAM-1 transcripts were found in HAEC treated with postprandial LDL. The adhesion of monocytes to HAEC was enhanced after pretreatment with postprandial, but not with postabsorptive LDL. We conclude that postprandial, but not postabsorptive LDL increase the surface expression of ICAM-1 in HAEC apparently by de novo protein synthesis leading to increased adhesion of monocytes. The upregulation of ICAM-1 by postprandial LDL may explain part of the proatherogenic effect of high postprandial lipemia. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Postprandial lipemia; Low-density lipoproteins; Cell adhesion molecules; Intercellular adhesion molecule-1; Human aortic endothelial cells; Monocyte adhesion
1. Introduction Pronounced postprandial lipemia has been identified as an independent risk factor for the development of coronary artery disease [1]. In the postprandial state, the rise of triglyceride-rich lipoproteins is followed by a temporary increased triglyceride content of low-density lipoproteins (LDL) and high-density lipoproteins (HDL) [2,3]. After hydrolysis of triglycerides, e.g. by hepatic lipase, small LDL (pattern B) and HDL (HDL3 ) are generated representing an atherogenic lipoprotein phenotype [4–6].
∗
Corresponding author. Tel.: +43 512 504 23251; fax: +43 512 504 23317. E-mail address: [email protected] (J.R. Patsch).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.07.014
One of the earliest events in the pathogenesis of atherosclerosis is the adherence of leukocytes to the endothelium and their subsequent transmigration into the arterial wall [7]. This process is mediated by cell adhesion molecules (CAM) expressed on the surface of endothelial cells and leukocytes. The endothelial cell-expressed selectins (E-selectin, P-selectin) mediate the initial weak interaction allowing leukocytes to roll along the vessel wall. Tight adhesion is then mediated by adhesion molecules of the immunoglobulin superfamily (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)) on endothelial cells and their counter receptors of the integrin family on leukocytes. In the third and final step, the transmigration of the leukocytes across the endothelial layer, platelet endothelial cell adhesion
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molecule-1 (PECAM-1) as well as other CAM are involved [8–10]. Numerous findings support the role of CAM in atherosclerosis. The expression of the endothelial cell-expressed adhesion molecules ICAM-1, E-selectin, VCAM-1, P-selectin and PECAM-1 is higher on endothelial cells overlying atherosclerotic lesions compared to endothelial areas outside plaques [11–15]. Elevated soluble isoforms of these CAM are present in the blood of patients suffering from coronary artery disease [16]. In knockout mice lacking ICAM-1 or P-selectin, the formation of atherosclerotic lesions is markedly delayed compared to wild-type animals [17,18]. The mechanisms responsible for the increased expression of CAM in regions prone to plaque development are only partially understood [19]. In addition to shear stress, elevated LDL have been suggested to be responsible for the upregulation of CAM on the surface of endothelial cells. Most studies have found an effect of LDL only after oxidation [20–22] although native LDL have also been described to increase the expression of ICAM-1 on endothelial cells [23]. In contrast to P-selectin [24] and PECAM-1 [13], the surface expression of ICAM-1, E-selectin and VCAM-1 appears to be regulated by LDL concentrations [19]. In the present study, we tested the hypothesis according to which postprandial low-density lipoproteins may be more effective than postabsorptive LDL in upregulating these adhesion molecules on the surface of endothelial cells.
2.2. Stimulation assay HAEC cells were seeded in six well plates at 2.5 × 104 cells/well and grown to approximately 70% confluence in EGM-2 medium. The culture medium was then removed and replaced with fresh EGM medium supplemented with 2% lipoprotein deficient FCS. Low-density lipoproteins were isolated by rate zonal centrifugation from the plasma of healthy volunteers (five male and one female, age 22–35) before and 4 h after ingestion of a standardized fatty test meal as described previously [3]. The exact composition of the test meal (730 kcal per square meter of body surface, comprising 65 g fat (polyunsaturated to saturated fat ratio 0.06), 5.3 g protein, 24 g carbohydrates and 240 mg cholesterol) has been described [27]. LDL were dialyzed against phosphate buffered saline (PBS), stored under liquid nitrogen and used within 1 week after preparation in cell culture experiments. LDL were added at protein concentrations of 0.01–0.5 mg/ml to the culture medium of endothelial cells. Control wells were incubated with equal volumes of PBS (negative control) or with TNF-alpha at a final concentration of 10 ng/ml. Pilot experiments were set up to determine the time needed for maximal upregulation of CAM by the positive control (TNF-alpha). In subsequent experiments, stimulation for 8 h was used to measure E-selectin expression and 24 h were used in the case of ICAM-1 and VCAM-1. 2.3. Flow cytometry
2. Materials and methods 2.1. Cells and antibodies Primary human aortic endothelial cells (HAEC, Clonetics) were purchased from Cambrex (Verviers, Belgium) and cultured in EGM-2 medium containing 2% fetal calf serum (FCS) according to the instructions of the manufacturer. HAEC were used between passage four to seven for all experiments. The human monocytic cell line Mono Mac 6 [25] was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultured in RPMI-1640 medium supplemented with 10% FCS, 100 IU/ml penicillin and 100 g/ml streptomycin. Where indicated, lipoprotein-deficient FCS (Sigma, Vienna, Austria) was used instead of normal FCS. The anti-ICAM-1 monoclonal antibody 7F7 was a kind gift from M.P. Dierich, Innsbruck Medical University, Innsbruck, Austria [26]. Monoclonal antibodies against E-selectin (R&D Systems, Wiesbaden, Germany) and VCAM-1 (Serotec, Oxford, UK) were purchased. Recombinant human tumor necrosis factor-alpha (TNF-alpha) was purchased from Genzyme (Cambridge, MA, USA). F(ab )2 antibodies against mouse immunoglobulins (IgG and IgM) were purchased from An der Grub (Kaumberg, Austria).
Following stimulation, HAEC were washed twice with PBS and then mobilized gently with a cell scraper (Falcon 3058, Becton-Dickinson, Le Pont de Claix, France). After centrifugation, cells were resuspended in PBS containing 1% bovine serum albumin. The cells were stained with specific monoclonal antibodies at saturating concentrations and analyzed by flow cytometry as described [28]. Dead cells were excluded from analysis by counterstaining with propidium iodide (Sigma, Vienna, Austria) during the final wash steps. 2.4. Northern blot Total RNA was isolated from HAEC using RNA clean solution (Hybaid, Heidelberg, Germany) according to the instructions of the manufacturer. Total RNA (10 g per lane) was denatured in formaldehyde, separated by agarose gel electrophoresis and transferred to nylon membranes. The ICAM-1 cDNA probe and a control housekeeping gene (glyceraldehyd-3-phosphate dehydrogenase, GAPDH) cDNA probe were labeled with [␣-32 P]dCTP by the random primer method. The ICAM-1 specific cDNA probe was kindly provided by B. Seed, Harvard Medical School, Boston, MA, USA [29]. Hybridization was performed according to standard protocols [30]. Bands were visualized by autoradiography at −70 ◦ C.
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2.5. Monocyte adhesion assay The monocytes adhesion assay was performed as described [31]. Briefly, HAEC were seeded in 96 well plates at a density of 5 × 103 per well and grown to confluence. Mono Mac 6 cells were labeled with a fluorescent dye (calcein-acetomethylester, Molecular Probes, Eugene, OR, USA) and added to the endothelial cells for 30 min. After washing, the number of bound Mono Mac 6 cells was measured using a Cytofluor 2350 fluorometer (Millipore, Bedford, MA, USA). 2.6. Statistical analyses Plasma lipids of the study participants were compared with Student’s paired t-test. Since triglycerides were not distributed normally, logarithmic transformation of triglyceride values was performed prior to analysis. The expression of adhesion molecules and adhesive properties of HAEC after pretreatment with LDL and controls were compared using one way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test as a post test. Throughout, a p-value of 0.05 was considered statistically significant. Calculations were performed with Graph Pad Prism or Graph Pad InStat software (Version 3.0, Graph Pad software, San Diego, CA, USA).
3. Results Plasma lipids of the study subjects before and 4 h after ingestion of a standardized fatty test meal are shown in Table 1. Consistent with previous observations [32], a marked rise of plasma triglycerides was observed in the postprandial period without significant alterations of total cholesterol, LDL cholesterol and HDL cholesterol. HAEC cells were incubated with LDL isolated in the postprandial and postabsorptive state and the surface expression of cell adhesion molecules was measured by flow cytometry. As shown in Figs. 1 and 2, incubation with LDL of the postprandial state resulted in a two-fold increase of the surface expression of ICAM-1 compared to postabsorptive LDL and the negative control (PBS) (p < 0.05, ANOVA). Conversely, no significantly different surface expression of VCAM-1 and ETable 1 Plasma lipids of study subjects before and 4 h after ingestion of a standardized fatty test meal Parameter
Postabsorptive state
Total cholesterol (mg/dl) Triglycerides (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)
181 139 93 59
± ± ± ±
24 66 18 2
Postprandial state 193 250 89 56
± ± ± ±
27 64 21 2
p-Value n.s. 0.03 n.s. n.s.
Values are mean ± standard deviation from six healthy volunteers. n.s.: not significant.
Fig. 1. LDL were isolated from healthy volunteers in the postabsorptive (LDLpa) and postprandial (LDLpp) state. HAEC were incubated with these LDL preparations and with controls (TNF-alpha, PBS) for 24 h. The surface expression of ICAM-1 was measured by flow cytometry. The histograms of a typical experiment repeated six times with identical results are shown.
selectin was observed after preincubation with either of the two LDL preparations (Fig. 2). The upregulation of ICAM1 expression by postprandial LDL was dose-dependent and marked increases of the surface expression of ICAM-1 were seen after incubation with concentrations equaling or exceeding 0.1 mg/ml LDL (see Fig. 3). To test whether the upregulation of ICAM-1 expression was caused by de novo synthesis of ICAM-1 in endothelial cells, we performed Northern blotting of RNA isolated from HAEC preincubated with postprandial and postabsorptive LDL and the controls (TNFalpha and PBS). As can be seen in Fig. 4, pretreatment with postprandial LDL resulted in increased ICAM-1 specific mRNA transcripts compared to postabsorptive LDL. To test whether the increased surface expression of ICAM-1 resulted in increased binding of leukocytes to endothelial cells, we performed a monocyte adhesion assay using Mono Mac 6 cells. This monocytic cell line is known to have adhesion characteristics similar to freshly isolated human blood monocytes [33]. As shown in Fig. 5, significantly more Mono Mac 6 cells bound to HAEC after pretreatment with 0.5 mg/ml postprandial LDL compared to the control (PBS) (p < 0.05, ANOVA).
4. Discussion Almost all studies of the effect of LDL on endothelial cells have been performed with LDL isolated from plasma in the postabsorptive state. However, under physiological conditions, the endothelium is exposed to postprandial lipoproteins for prolonged periods of time. An impairment of endothelial function after ingestion of a standardized fatty meal was observed in vivo by measuring the endothelium—dependent dilation of the brachial artery [32]. The magnitude of postprandial lipemia has been identified as an independent risk factor for the development of coronary artery disease and was found to correlate with the intima media thickness of the carotid arteries [1,34]. Thus, studies addressing the role of
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Fig. 3. Dose dependence of the effect of LDL on ICAM-1 surface expression in HAEC. HAEC were incubated with different concentrations (given in mg/ml) of LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state for 24 h. The surface expression of ICAM-1 was measured by flow cytometry and is shown compared to the positive (TNF-alpha) and negative (PBS) control. Values are shown as percent mean fluorescence intensity ±standard error from at least three independent experiments.
of the earliest steps in atherosclerosis leading to the subsequent migration of blood leukocytes into the subendothelial space, the increase of the adhesive properties of endothelial cells may be an important mechanism by which postprandial LDL exert their proatherogenic effect. However, additional mechanisms including the preferred uptake and degradation
Fig. 2. Effect of LDL on the surface expression of cell adhesion molecules in HAEC. HAEC were incubated with TNF-alpha, LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state, or PBS for 8 h (E-selectin) or 24 h (ICAM-1, VCAM-1). The surface expression of cell adhesion molecules was quantified by flow cytometry. Values are shown as percent mean fluorescence intensity ±standard error from at least four independent experiments. *p < 0.05 compared to the negative control (PBS), ANOVA, Dunnett’s multiple comparisons test.
lipoproteins in the postprandial state appear to be needed to identify the mechanisms by which high postprandial lipemia confers a higher risk for the development of atherosclerotic lesions. In the present study, we found that LDL isolated from postprandial, but not from postabsorptive human plasma induce a two-fold increase in the surface expression of ICAM-1 in HAEC. The higher surface expression of ICAM-1 caused by postprandial LDL results in an increased adhesion of human monocytes to endothelial cells. Since adhesion is one
Fig. 4. ICAM-1 transcripts in HAEC treated with LDL. RNA was isolated from HAEC after preincubation with LDL from the postprandial (LDLpp, lane 1) or postabsorptive (LDLpa, lane 2) state, PBS (lane 3), or TNF-alpha (lane 4) for 24 h. Total RNA (10 g per lane) was separated by electrophoresis, transferred to nylon membranes and probed with cDNA probes specific for ICAM-1 (top panel) or GAPDH (lower panel).
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Fig. 5. Monocyte adhesion to HAEC pretreated with LDL. HAEC were pretreated with LDL from the postprandial (LDLpp) or postabsorptive (LDLpa) state in the concentrations indicated for 24 h. Mono Mac 6 cells were labeled with calcein-acetomethylester and binding to the endothelial cells was quantified with a fluorometer. Values are shown as percent adherence (mean ± standard error) of six replicates in three separate experiments. *p < 0.05 compared to the negative control (PBS), ANOVA, Dunnett’s multiple comparisons test.
of postprandial when compared to postabsorptive LDL by macrophages and foam cells are likely to be of importance [3]. The effect of postprandial LDL on the surface expression of ICAM-1 was seen with LDL protein concentrations starting from 0.1 mg/ml and most pronounced with the highest concentration tested (0.5 mg/ml). This corresponds to LDL cholesterol concentrations of approximately 20–100 mg/dl, well within the physiological range of LDL lipoproteins in vivo. In a previous study, comparable concentrations of native LDL were shown to increase the expression of ICAM-1 on the surface of human umbilical vein endothelial cells [23]. Conversely, LDL modified by oxidation can induce the expression of ICAM-1 in endothelial cells already at concentrations as low as 0.01 mg/ml [20,22,35]. In addition, oxidized LDL were also reported to upregulate the expression of VCAM1 and E-selectin in some studies [21,36]. In contrast to our results, one study also described the induction of VCAM-1 and E-selectin by native LDL [37]. An increased intracellular accumulation of P-selectin without direct changes in surface expression has been described after incubation with minimally modified LDL [24]. Postprandial LDL were characterized in detail in a previous report [3]. As expected, postprandial LDL contain more triglycerides (and phospholipids) compared to postabsorptive LDL. There is no difference in the electrophoretic mobility in agarose gels, particle size, or the fatty acid composition between the two LDL species from the two absorptive states. However, postprandial LDL are more susceptible to oxidation by CuSO4 , while cell-mediated oxidation is not substantially different [3]. The cellular receptors and intracellular pathways responsible for the induction of ICAM-1 expression by LDL have only been partly characterized. For oxidized LDL, the lectin-like LOX-1 receptor is believed to mediate the upregulation of ICAM-1 by activation of nuclear fac-
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tor kappa B (NF-B) [36]. Conversely, in the case of native LDL, the LDL receptor, intracellular calcium and activator protein-1 (AP-1) have been implied [37,38]. Further studies are necessary to determine whether the increased triglyceride and/or phospholipid content of postprandial versus postabsorptive LDL is responsible for the different activity of these two LDL preparations. In addition, it remains to be clarified which lipoprotein receptors are involved and which intracellular mechanisms are triggered by postprandial LDL. Our observation that postprandial LDL directly alter the adhesive properties of endothelial cells provide a novel mechanism for the proatherogenic effect of postprandial LDL. With respect to the atherosclerotic risk conferred by LDL, it has been suggested that a compositional characteristic, i.e. a high triglyceride content of LDL, may reflect better a proatherogenic state of LDL when compared to high LDL cholesterol [39]. Therefore, not only the concentration but also the composition of LDL appears to be important, as is certainly the case with small dense LDL present in hypertriglyceridemic states [6]. Similarly, high postprandial LDL may be deleterious because of their higher triglyceride content and exert their proatherogenic potential by various mechanisms, including the induction of ICAM-1 on the surface of endothelial cells.
Acknowledgements This work was supported by grants (P 11693-MED and P16121-B05) from the Austrian Science Fund (FWF) and by the Austrian National Bank (grant Nr. 6442), all to Josef R. Patsch. We are grateful to Drs. Manfred P. Dierich and Brian Seed for valuable reagents. We wish to thank Karin Salzmann, Isa Hauser and Barbara Fuchsberger for expert technical assistance.
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