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RESEARCH COMMUNICATION |
a Department of Cardiothoracic Surgery, Imperial College of Science, Technology and Medicine, Royal Brompton and Harefield NHS Trust Hospital, Harefield Hospital, Harefield, Middlesex UB9 6JH, United Kingdom
b Department of Biochemistry, Queens University, Kingston, Ontario, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog 
-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.—Allen, S., Khan, S., Tam, S.-P., Koschinsky, M., Taylor, P., Yacoub, M. Expression of adhesion molecules by Lp(a): a potential novel mechanism for its atherogenicity. FASEB J. 12, 1765–1776 (1998)
Key Words: calcium signaling • VCAM-1 • E-selectin • endothelial cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Lp(a) particle closely resembles low density lipoprotein (LDL) but is distinguishable by the presence of apolipoprotein (a) apo(a), which is covalently linked to the apolipoprotein B100 (apoB100) of LDL by a single disulfide bridge (3). Apolipoprotein (a) [apo(a)] has extensive structural homology to the fibrinolytic pre-enzyme plasminogen (4). By molecular mimicry, Lp(a) can compete with glu-plasminogen for the annexin II plasminogen binding domain on the cell surface (5) and therefore directly influence endothelial cell plasmin generation. By interfering with the fibrinolytic pathway via direct competition with plasminogen binding, Lp(a) down-regulates endothelial cell plasmin generation and shifts the vessel surface to a more thrombogenic phenotype. In addition, Lp(a) may act as a competitive inhibitor of tissue plasminogen activator in the presence of fibrinogen and also interferes with the binding of plasminogen to fibrin clots (6). This interaction also inhibits TGFß activation (7), resulting in increased smooth muscle cell proliferation and migration. Both inhibition of clot lysis and enhancement of cell migration could contribute to the process of atherogenesis.
By virtue of its similarity with LDL, it has been proposed that Lp(a) can effectively compete with LDL for binding and/or uptake by the LDL receptor. In addition to the LDL receptor, several other members of the LDL receptor family have been identified and may function in Lp(a) catabolism. The LDL receptor-related protein/2-macroglobulin receptor (LRP/2MR) is a recently described multifunctional receptor that binds to apoE-enriched lipoproteins (8, 9), as well as to nonlipoprotein ligands such as activated 2-macroglobulin (10) and type-1 plasminogen activator inhibitor/urokinase plasminogen activator (PAI-1/uPA) complexes (11). Owing to its structural similarity with the LDL receptor, a role for the LRP in Lp(a) binding has been postulated; recent evidence suggests that LRP may bind to a high molecular weight isoform of Lp(a) (12). Recent data have also suggested that the very low density lipoprotein (VLDL) receptor can bind and internalize Lp(a) (13). All LDL receptor family members bind a 39 kDa receptor-associated protein (RAP) that antagonizes ligand binding (14–17), although binding of RAP to the LDL receptor occurs with low affinity.
Lp(a) also appears to modulate vascular function. Lp(a) has been demonstrated in several acute inflammatory and vasculopathic states, including Crohn's disease, granulomatous lymph nodes, pericarditis, and thrombotic and thrombocytopenic purpura (5). The deposition of Lp(a) can further modify the state of vascular activation by decreasing production of endothelium-derived growth factor (18) and release of plasminogen activator inhibitor 1 (19).
One of the earliest signs of endothelial cell activation is a rise in intracellular calcium. We have recently shown that LDL binding to the LDL receptor can induce a rise in intracellular calcium in human endothelial cells, which triggers the induction of low levels of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin (20). These adhesion molecules can increase the binding of leukocytes to the vascular endothelium, which is thought to be an important factor in the early development of atherosclerosis. It is possible that Lp(a) could also induce adhesion molecules and influence atherogenesis because it shares global homology with LDL. We initiated studies to investigate the role of the LDL receptor family members in the activation of cultured human coronary artery endothelial cells (HCAEC) by Lp(a) and a recombinant form of apo(a) [r-apo(a)]. Our results have identified a calcium-dependent interaction of Lp(a) with HCAEC that does not appear to involve any of the known potential Lp(a) binding sites that induce potent surface expression of VCAM-1 and E-selectin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-aminocaproic acid (-APA), heparinase III (H8891), chondroitinase ABC (C2905), sodium chlorate, and 2-macroglobulin were purchased from Sigma Chemicals (Poole, U.K.). The LDL receptor antibody IgGC7 was purchased from Amersham International (Amersham, U.K.). Human VCAM-1 [clone BBA 6] and E-selectin [clone BBA 2] antibodies were purchased from R&D systems (Oxford, U.K.). Simvastatin, the Sandoz compound 58–035, and r-apo (a) were kindly provided by Dr. Tam and Dr. Koschinsky, Queens University, Canada. RAP was kindly provided by Dr. Gonias, University of Charlottesville,Virginia, and by Dr. Owen, University Department of Medicine, The Royal Free Hospital School of Medicine, Rowland Hill St., London. All other reagents were analar grade and purchased from BDH Chemicals, Poole, U.K.
Purification of proteins
Lipoprotein (a) and LDL (Sigma Chemicals) were isolated sequentially from plasma under sterile conditions to avoid endotoxin contamination as previously described (20). Briefly, Lp(a) was in a solution of 10 mM Tris-HCl containing 1 mg/ml Na2 EDTA, pH 7.2, and LDL was extensively dialyzed against 0.15 M NaCl, 0.01% EDTA, pH 7.4–7.5. Both lipoproteins were checked for their characteristic electrophoretic mobility and chemical composition. The lipoproteins were essentially free from contamination by other lipoproteins as determined by cellulose acetate electrophoresis using fat red 7B staining for lipid. Our own sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Lp(a) confirmed its purity. Lipoproteins were stored at 4°C and used within 2 wk after isolation. A recombinant form of apolipoprotein (a) [r-apo(a)] containing 17 kringle IV domains, as well as the kringle V and protease domains, was purified from conditioned media harvested from stably transfected human embryonic kidney cells by lysine-Sepharose affinity chromatography, as previously described (21).
Cell culture
The HCAEC were cultured in medium 199 containing 10% human AB serum, 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) as previously described (20). Cells were grown to confluency in 24-well tissue culture plates in serum-supplemented media; before each experiment, cells were preconditioned by an 24 h incubation in serum-free media plus various supplements, as indicated. To down-regulate the LDL receptor, some experiments were performed in HCAEC that were cultured in serum-containing medium. Cell viability was routinely monitored by trypan blue exclusion. In all experiments, the number of nonviable cells never exceeded 5% of the total cell number and there was no difference in viability between cells grown in the presence or absence of serum.
Calcium measurements
The HCAEC were plated on glass coverslips at 105 cells/ml for 18–24 h and then loaded with fura-2/AM (1 µM for 30 min at room temperature) in Krebs-HEPES medium (pH 7.4) containing 120 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 4.8 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, 25 mM glucose, and 0.1% bovine serum albumin. The cells were then washed twice with Krebs-HEPES and left for 15 min to allow complete hydrolysis of the fura-2 ester. In experiments involving pharmacological agents, endothelial cells were preincubated for a given time and temperature (as described) with the agent before the endothelial cells were loaded with fura-2/AM. The coverslips were then secured between two plates of a 3 ml-volume coverslip holder, mounted in a temperature-controlled incubation holder (34°C), and placed onto a microscope stage of a Zeiss Axiovert 35 inverted epifluorescence microscope. Intracellular calcium measurements were performed on individual cells using an ionVision dual excitation system (ImproVision, Coventry, U.K.). Absolute calcium levels were calculated as previously described (20).
Flow cytometry analysis
Changes in VCAM-1 and E-selectin protein in control (TNF 10 ng/ml, positive regulator of these adhesion molecules) and Lp(a)-treated endothelial cell cultures were determined using a Flow Cytometer. Endothelial cells were passaged into 24-well plates, allowed to grow to confluence, transferred to serum-free medium for 24 h and preincubated with Lp(a) (10 to 400 µg/ml) or normal cell culture media for 5 min, 30 min, 5 h, and 12 h. In experiments to investigate whether Lp(a)-induced changes in intracellular calcium correlate with adhesion molecule expression, endothelial cells were preincubated with the intracellular calcium chelator BAPTA/AM (5 µM; 30 min) before the addition of Lp(a). At the end of each Lp(a) incubation time, cells were left for 5 h, at which time control wells [no Lp(a)] were treated with tumor necrosis factor (TNF) for 5 h. At the end of this period the cells were washed, trypsinized, and assayed for adhesion molecule expression. Expression of cell-surface molecules was measured as fluorescence intensity by use of an EPICS XL-MCL Flow Cytometer (Coulter Electronics). Each sample counted 5 x 103 cells. Controls for each assay included the absence of the primary antibody and, in some cases, incubation of cells with an isotype-matched, irrelevant antibody.
Role of the LDL receptor
At 24 h prior to the experiment, the medium of the HCAEC cultures was replaced with serum-free, lipoprotein-deficient medium. In some experiments, the cells were preincubated overnight in serum free medium containing one of the following supplements—Simvastatin (0.1 to 10 µM), the Sandoz compound 58–035 (5 µg/ml), or serum-containing medium—before the addition of Lp(a). To block the LDL receptor, cells were preincubated for 30 min at 37°C with either the monoclonal anti-LDL receptor antibody IgGC7 (22) at a concentration of 50 µg/ml or an irrelevant IgG antibody as a control, before Lp(a) was added. Other cells were treated with a combination of Lp(a) ± LDL or LP(a) + r-apo(a) for competition studies, or treated with different concentrations of r-apo(a) (10 to 100 µg/ml) alone.
LRP/2MR and VLDL receptor interactions with Lp(a)
To investigate whether Lp(a) was interacting with other LDL receptor family members such as the LRP/2MR and VLDL receptor, we incubated HCAEC with RAP (100 nM), a molecule that binds to these receptors and antagonizes ligand binding (14–17) before the addition of the Lp(a). Coincubation of cells with activated 2-macroglobulin (10 µM) and 100 µg/ml Lp(a) was also performed in order to determine whether or not competitive binding to the LRP2MR receptor could block Lp(a) activation of HCAEC.
Plasminogen and Lp(a) interactions
The role of plasminogen binding sites was also determined. Before the addition of Lp(a), HCAEC were incubated in serum-free media containing either 50 mM -ACA or 10 mM tranexamic acid for 30 min. Competition experiments were performed by the simultaneous addition of 100 µg/ml Lp(a) and glu-plasminogen (0.1 to 1 µM) to each multiwell.
Role of proteoglycan binding sites in Lp(a) function
Experiments were also performed to evaluate proteoglycan binding sites on HCAEC. The HCAEC were cultured as described above. Conditions for the use of heparinase III (Sigma H8891) and chondroitinase ABC (Sigma C 2905) were based on the procedures described by Ji et al. (23) and Oike et al. (24), respectively. Briefly, the cells were washed four times at 37°C in phosphate-buffered saline and then incubated for 2 h at 37°C with either heparinase (2 units/ml) or chondroitinase (0.25 units/ml) in serum-free medium. The cells were washed an additional four times with phosphate-buffered saline at 37°C and Lp(a) was added as described above. In some experiments, the cells were preincubated for 48 h with 30 mM sodium chlorate prior to cell activation with Lp(a).
Immunofluoresence
Lipoprotein (a) (100 µg/ml) or TNF (10 ng/ml was added to HCAEC grown on glass coverslips 12–18 h previously. Some coverslips were preincubated with BAPTA/AM (5 M for 30 min) before cells were treated with Lp(a). Coverslips were washed after 5 h and fixed in 1% (v/v) para-formaldehyde. E-selectin and VCAM-1 were visualized by indirect immunofluorescence using either a monoclonal anti E-selectin (clone BBA2) or anti-VCAM-1 antibody (clone BBA 6), followed by biotinylated rabbit antimouse IgG and then Streptavidin-Texas red. Cells were visualized with a Laser-Scanning Confocal Microscope (Noran Instruments, U.K.).
Statistical analysis
Statistical analysis were carried out using the commercial program Unistat version 4 for Windows (Unistat Ltd, U.K.). The results are expressed as mean ± SEM. Calcium measurements were analyzed by an unpaired Student's t test when appropriate. Adhesion molecule data was analyzed using repeated measures ANOVA (analysis of variance) coupled with a post hoc Bonferroni correction analysis. Differences were considered to be significant when the probability value was <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1a).
The calcium transients in HCAEC were dependent on the concentration of Lp(a) and were saturable ( Fig. 1b). Furthermore, the responses of the cells to Lp(a) were heterogeneous and asynchronous, with some endothelial cells not responding and some exhibiting oscillations in calcium transients ( Fig. 1c). Lp(a)-induced calcium signaling in HCAEC cultured in the presence of serum (500±80 nM; mean±SEM, n=12) was not significantly different from that triggered in the absence of serum. Elevation of intracellular calcium by Lp(a) was not a sole characteristic of HCAEC, as both human aortic and human heart microvascular endothelial cells also responded to Lp(a) with a rise in intracellular calcium (data not shown). The average calcium response induced by the calcium ionophore A23187 in HCAEC was 7.0 ± 3 µM, which is 13-fold greater than that triggered by Lp(a).
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Lp(a) induces VCAM-1 and E-selectin expression on HCAEC
Further studies were performed to elucidate the nature of the rise in intracellular calcium by Lp(a). Incubation of endothelial cells with Lp(a) (10–400 µg/ml) resulted in a dose-dependent increase in VCAM-1 and E-selectin ( Fig. 2A, B)
expression, which stabilized after 100 µg/ml Lp(a). In all experiments Lp(a) at a concentration of 100 µg/ml induced significantly more E-selectin than VCAM-1 expression: 61 ± 8% vs. 35 ± 10%, P < 0.01, respectively. The expression of VCAM-1 and E-selectin was inhibited in the HCAEC that were preloaded with the intracellular calcium chelator BAPTA/AM prior to Lp(a) treatment ( Fig. 2A, B),
suggesting that calcium transients are involved in Lp(a)-induced adhesion molecule production. However, as BAPTA also has IP3 antagonist properties, we cannot rule out that IP3 antagonism with BAPTA, which would result in the inhibition of release of calcium, could also contribute to the effect observed. Figure 3
shows the effects of exposure to Lp(a) (100 µg/ml) over time. Both VCAM-1 and E-selectin showed similar kinetics, with significant levels of each protein occurring after 2 h incubation with Lp(a) and peaking at 5 h. Indirect immunofluorescence confirmed the flow cytometry data, showing VCAM-1 (data not shown) and E-selectin ( Fig. 4)
surface staining induced by TNF and Lp(a), which was inhibited by treating the HCAEC with BAPTA/AM.
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It seems unlikely that the effects of Lp(a) are due to the mild oxidation of the lipoprotein since experiments performed in the presence of 20 µmol/l butylated hydroxytoluene (BHT), which is known to eliminate nonspecific oxidation of lipoprotein particles during endothelial cell culture experiments (25, 26), had no significant effect on the levels of adhesion molecules expressed in the HCAEC compared to non-BHT treated cells (data not shown).
Effects of up- and down-regulation of the LDL receptor as well as an anti-LDL receptor antibody on Lp(a)-induced adhesion molecule expression
Figure 5
demonstrates that Lp(a)-induced adhesion molecule expression of both VCAM-1 and E-selectin do not alter significantly with up- and down-regulation of the LDL receptor activity induced by Simvastatin or the Sandoz compound 58–035, respectively. Experiments in the presence of serum-containing medium also did not affect adhesion molecule expression induced by Lp(a) (VCAM-1, 14.8±5.5%; E-selectin, 53±5.3%, n=4) when compared to experiments performed in serum-free medium (VCAM-1: 15.4±3.7%, E-selectin: 60±,1.3%, n=4). We also performed Lp(a) studies in the presence of LDL. LDL alone induced small changes in adhesion molecules (data not shown) as described previously (20). No competitive effect was observed in the coincubation experiments, with the levels of adhesion molecules not significantly different, suggesting there was no additive or inhibitive effect of LDL on Lp(a) function. There was also no effect of an anti-LDL receptor monoclonal antibody IgG-C7 on Lp(a)-induced adhesion molecule production (data not shown). Finally, since Lp(a) is distinguishable from LDL by the apo(a) moiety, we were interested to know whether r-apo(a) alone could induce adhesion molecule expression in HCAEC or whether coincubation of r-apo(a) + Lp(a) would inhibit adhesion molecule expression in HCAEC. Addition of r-apo(a) in a concentration range of 10 to 100 µg/ml was not able to induce VCAM-1 or E-selectin expression in HCAEC (data not shown). In contrast, coincubation of r-apo(a) with Lp(a) induced a significant attenuation of VCAM-1 [Lp(a): 51.5% vs. Lp(a)+r-apo(a) 19.8%, n=2] and, to a lesser extent, E-selectin [Lp(a): 55% vs. Lp(a)+r-apo(a): 44%, n=2], indicating that apo(a) is involved with the interaction of Lp(a) with HCAEC.
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Effects of receptor-associated protein (RAP), anti-LRP antibody, and 2-macroglobulin on Lp(a) responses
Treatment of HCAEC with RAP or competition with 2-macroglobulin failed to diminish levels of VCAM-1 or E-selectin induced by Lp(a) (data not shown).
Role of plasminogen receptors in Lp(a)-induced adhesion molecule expression in HCAEC
Figure 6
shows that preincubation with glu-plasminogen, -ACA, and tranexamic acid failed to block VCAM-1 or E-selectin expression in Lp(a)-stimulated HCAEC.
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Effects of addition of heparinase, chondroitinase ABC, or sodium chlorate on Lp(a)-induced adhesion molecule expression in HCAEC
Controlled digestion of the cells with either heparinase or chondroitinase to remove cell-surface proteoglycans or preincubation of the cells for 48 h with sodium chlorate, an inhibitor of proteoglycan sulfation, did not affect expression of adhesion molecules in Lp(a)-stimulated HCAEC ( Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have identified a calcium-dependent interaction of Lp(a) with HCAEC that does not appear to involve any of the known potential Lp(a) binding sites and can trigger the expression of VCAM-1 and E-selectin on the surface of HCAEC. This conclusion is supported by several lines of evidence. We have ruled out involvement of the LDL receptor in Lp(a)-stimulated expression of adhesion molecules in HCAEC. This conclusion is based on the inability of a monoclonal antibody specific for this receptor (IgGC7) to block VCAM-1 and E-selectin expression as well as the lack of competitive inhibition by LDL when coincubated with Lp(a). It has been demonstrated that lipoprotein depletion of cells (27, 28) as well as treatment of cells with 3-hydroxy-3-methyglutaryl-coenzyme A reductase inhibitors (statins) (28, 29) results in up-regulation of the LDL receptor. In contrast, normal serum medium (30) and the Sandoz compound 58–035 (31) are associated with a reduction in LDL receptor levels. Data presented here show that up-regulating the LDL receptor with LPDS medium or Simvastatin had little effect on levels of each adhesion molecule in Lp(a)-stimulated HCAEC. Similarly, down-regulation of the LDL receptor with the Sandoz compound 58–035 or culturing cells in normal serum-containing medium had no significant effects on the levels of VCAM-1 and E-selectin induced by LDL or Lp(a).
The presence of serum in the medium of these experiments reinforces the point that Lp(a) effects are specific and not due to artifacts such as oxidation or toxicity. While it is possible that in the absence of serum, fatty acids can be hydrolyzed from LDL and achieve concentrations that are toxic to the endothelial cells, it is unlikely that in serum-free conditions LDL will be hydrolyzed by lipoprotein lipase. Even though the endothelial cells can produce this enzyme, LDL is not a substrate for this enzyme. The actual substrates are VLDL or chylomicrons, which would not be present in the absence of serum. Further confirmation that serum-free media does not alter the function of Lp(a) is indicated by the fact that Lp(a)-induced calcium transients are not significantly different in the presence or absence of serum-containing medium. Finally, trypan blue staining of viability of HCAEC was not different between endothelial cells grown in serum-containing medium, serum-free medium, or incubated with BHT, clearly indicating that in our model, no detectable oxidation or toxicity is occurring that would affect the ability of Lp(a) to induce adhesion molecule expression.
A recent study has shown that the interaction of Lp(a) with HepG2 cells can be mediated through the LDL receptor by a `hitchhiking' process. This pathway appears to involve binding of apo(a) to apoB-containing particles prior to their uptake by the LDL receptor (32). In the present study, we directly examined the ability of a recombinant form of apo(a) to activate cultured HCAEC. Incubation of HCAEC with increasing concentrations of r-apo(a) alone failed to stimulate VCAM-1 or E-selectin expression. In contrast, coincubation of Lp(a)+r-apo(a) produced a significant reduction in levels of VCAM-1 and E-selectin. These results suggest that apo(a) is responsible for mediating the interaction of Lp(a) with the HCAECs, and also confirms that nonspecific effects of Lp(a) such as oxidation or endotoxin contamination are unlikely to be responsible for Lp(a)-induced adhesion molecule expression.
Compared to our previous findings with LDL on adhesion molecule expression in HCAEC (20), Lp(a) is approximately fivefold more potent in inducing VCAM-1 and E-selectin. This suggests that linkage of apo(a) to apoB could be important for Lp(a) to induce greater expression of adhesion molecules compared to LDL. Also, unlike LDL, which only induced transient calcium rises, Lp(a) frequently induced calcium oscillations in the HCAEC. Whether different calcium signals produced by LDL and Lp(a) relate to the different abilities of both lipoproteins to induce VCAM-1 and E-selectin requires further investigation.
We have also provided data to exclude the LRP/2MR and VLDL receptor as the binding site for Lp(a)-induced adhesion molecule production. We performed Lp(a) experiments in the presence of activated 2-macroglobulin. No competition effect was observed with the levels of each adhesion molecule unchanged. In addition, incubation of HCAEC with RAP, a molecule known to inhibit ligand binding to LDL receptor family members such as LRP2MR and the VLDL receptor, failed to significantly alter levels of VCAM-1 or E-selectin in Lp(a)-stimulated HCAEC. These data suggest that adhesion molecule production after the interaction of Lp(a) with HCAEC is not mediated by the LRP or VLDL receptor.
It is also unlikely that the Lp(a) binding site on HCAEC responsible for VCAM-1 and E-selectin expression represents the plasminogen receptor. Lipoprotein (a) has been previously shown to compete with plasminogen for cellular binding sites, mediated by lysine affinity sites present in both apo(a) and plasminogen. We have demonstrated here that plasminogen did not compete with Lp(a) for induction of VCAM-1 and E-selectin since the levels of adhesion molecules in HCAEC were unaffected by its presence. Further confirmation that plasminogen binding sites were not involved in Lp(a) activation of HCAEC came from experiments with the lysine analog -ACA (33) and tranexamic acid, which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen (34). Neither inhibitor had an effect on the levels of each adhesion molecule after Lp(a) treatment.
It has been suggested that lipoprotein lipase enhances the binding of Lp(a) or lipoproteins to cells by acting as a bridge between lipoproteins in the medium and proteoglycans on the cell surface (35). No significant effect of Lp(a)-induced expression of adhesion molecules was observed in HCAEC either when the cells were depleted of heparan sulfate by heparinase treatment or when sulfation was inhibited by the addition of sodium chlorate treatment. Furthermore, removal of chondroitin sulfate by chondroitinase ABC treatment did not seem to influence Lp(a) effects on adhesion molecule levels. Our data demonstrate a lack of involvement of cell-surface glycosaminoglycans or proteoglycans in the expression of VCAM-1 and E-selectin by Lp(a). This is supported by a previous report that also failed to show a direct interaction between Lp(a) and cell-surface glycosaminoglycan chains (36). Together, these experiments provide evidence indicating that a novel binding site for Lp(a) may mediate the cellular activation and subsequent expression of VCAM-1 and E-selectin in cultured HCAEC. Bottalico et al. (37) showed that cholesterol loading of macrophages leads to an enhancement of Lp(a) internalization and degradation via induction of a specific receptor that has yet to be identified.
An increased adherence of leukocytes to the endothelium appears to be a crucial event in the development of atherosclerosis. In this context, there is a growing body of evidence to suggest that modified and unmodified LDL molecules can induce adhesion molecules in vascular endothelial cells. Recently it has been shown that chylomicrons can induce VCAM-1 and E-selectin expression on endothelial cells, suggesting that the apoB moiety of these lipoproteins may be important for mediating these effects. The evidence to date suggesting a link between lipoprotein induction of adhesion molecules and the development of atherosclerosis is very important in light of the recent findings by Hackman et al. (38), who reported that patients with hypercholesterolemia had increased levels of soluble VCAM-1 and E-selectin compared to control subjects, which the authors propose may be a marker for atherosclerosis.
In summary, we have shown here that the expression of VCAM-1 and E-selectin on the surface of cultured HCAEC can be induced by Lp(a) in a dose- and time-dependent manner. The response is triggered by a rise in intracellular free calcium, and can be blocked by the calcium chelator BAPTA/AM and via competition with r-apo(a). Furthermore, substantial adhesion molecule expression by Lp(a) occurs at levels of Lp(a) that are considered to constitute a risk factor for cardiovascular disease (above 300 µg/ml) (39). Despite this risk, the pathogenic mechanism(s) of Lp(a) remains obscure. We suggest that Lp(a) may contribute to atherogenesis by inducing adhesion molecules on endothelial cells and thus promoting the interaction of leukocytes with the vessel wall. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: ANOVA, analysis of variance; -APA, -aminocaproic acid; apo(a), apolipoprotein (a); BHT, butylated hydroxytoluene; HCAEC, human coronary artery endothelial cells; LDL, low density lipoprotein; Lp(a), lipoprotein (a); LRP, LDL receptor-related protein; 2MR, 2-macroglobulin receptor; RAP, receptor-associated protein; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule 1; VLDL, very low density lipoprotein.
Received for publication February 25, 1998.
Revision received June 23, 1998.
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 G. Lippi, E. Arosio, M. Prior, and G. Guidi Biochemical Risk Factors for Cardiovascular Disease in an Aged Male Population: Emerging Vascular Pathogens Angiology, October 1, 2001; 52(10): 681 - 687. [Abstract] [PDF]
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 C. Buechler, H. Ullrich, M. Ritter, M. Porsch-Oezcueruemez, K. J. Lackner, S. Barlage, S. O. Friedrich, G. M. Kostner, and G. Schmitz Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes Blood, February 15, 2001; 97(4): 981 - 986. [Abstract] [Full Text] [PDF]
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 P. Stenvinkel, B. Lindholm, M. Heimburger, and O. Heimburger Elevated serum levels of soluble adhesion molecules predict death in pre-dialysis patients: association with malnutrition, inflammation, and cardiovascular disease Nephrol. Dial. Transplant., October 1, 2000; 15(10): 1624 - 1630. [Abstract] [Full Text] [PDF]
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 Z.-W. Yang, A. Gebrewold, M. Nowakowski, B. T. Altura, and B. M. Altura Mg2+-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2000; 278(3): R628 - R639. [Abstract] [Full Text] [PDF]
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 R. G. Collins, R. Velji, N. V. Guevara, M. J. Hicks, L. Chan, and A. L. Beaudet P-Selectin or Intercellular Adhesion Molecule (ICAM)-1 Deficiency Substantially Protects against Atherosclerosis in Apolipoprotein E-deficient Mice J. Exp. Med., January 3, 2000; 191(1): 189 - 194. [Abstract] [Full Text] [PDF]
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 K. K. Koh, A. Blum, L. Hathaway, R. Mincemoyer, G. Csako, M. A. Waclawiw, J. A. Panza, and R. O. Cannon III Vascular Effects of Estrogen and Vitamin E Therapies in Postmenopausal Women Circulation, November 2, 1999; 100(18): 1851 - 1857. [Abstract] [Full Text] [PDF]
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