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(Circulation. 1997;96:2514-2519.)
© 1997 American Heart Association, Inc.

Articles

Apolipoprotein(a) Induces Monocyte Chemotactic Activity in Human Vascular Endothelial Cells

Michael Poon, MD; Xiaoxia Zhang, MD, PhD; Kevin G. Dunsky, MD; Mark B. Taubman, MD; ; Peter C. Harpel, MD

From the Cardiovascular Institute (M.P., K.G.D., M.B.T.) and the Division of Hematology (X.Z., P.C.H.), Department of Medicine, and the Brookdale Center for Molecular Biology (M.B.T.), Mount Sinai School of Medicine, New York, NY.

Correspondence to Michael Poon, MD, Box 1030, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. E-mail mpoon{at}smtplink.mssm.edu

	Abstract

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
Background Elevated levels of lipoprotein(a) [Lp(a)] are associated with premature atherosclerosis; however, the mechanisms are not known. Recruitment of monocytes to the blood vessel wall is an early event in atherogenesis.

Methods and Results This study has found that unoxidized Lp(a) induced human umbilical vein endothelial cells (HUVECs) to secrete monocyte chemotactic activity (MCA), whereas LDL under the same conditions did not. In the absence of HUVECs, Lp(a) had no direct MCA. Endotoxin was shown not to be responsible for the induction of MCA. Actinomycin D and cycloheximide inhibited the HUVEC response to Lp(a), indicating that protein and RNA synthesis were required. The apolipoprotein(a) [apo(a)] portion of Lp(a) was identified as the structural component of Lp(a) responsible for inducing MCA. Lp(a) and apo(a) also stimulated human coronary artery endothelial cells to produce MCA. Granulocyte-monocyte colony–stimulating factor (GM-CSF) antigen was not detected in the Lp(a)-conditioned medium, nor was monocyte chemoattractant protein-1 mRNA induced in HUVECs by Lp(a).

Conclusions These findings suggest that Lp(a) may be involved in the recruitment of monocytes to the vessel wall and provide a novel mechanism for the participation of Lp(a) in the atherogenic process.

Key Words: lipoproteins • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
An elevated blood concentration of Lp(a) is associated with an increased risk for myocardial infarction,^{1 2} stroke,^{3 4} restenosis of saphenous vein coronary grafts,⁵ and restenosis after balloon angioplasty.⁶ Lp(a) consists of an LDL particle that is linked to apo(a),^{7 8} a glycoprotein of variable size that shares partial homology with plasminogen.^{9 10} Although both immunohistochemical and biochemical studies document the presence of Lp(a) in the atheromatous plaque,^{11 12 13} the mechanisms underlying its atherogenicity are not known. Lp(a) has been shown to colocalize with fibrin in the plaque^{14 15 16} and to bind to plasmin-degraded fibrin in vitro.¹⁷ The atherogenic amino acid homocysteine was found to increase the affinity between fibrin and Lp(a).¹⁸ Lp(a) has also been found to be internalized by macrophages¹⁹ and can be identified in macrophage-rich areas in the atheromatous plaque.¹²

The attraction of blood monocytes to the blood vessel wall is an important early event in human atherogenesis.²⁰ Lipoproteins may participate in this process: minimally oxidized LDL, but not native LDL, has been documented to induce MCA in both endothelial and vascular smooth muscle cells.²¹ Because Lp(a) is associated with macrophages in the plaque, we have examined the effect of Lp(a) on inducing MCA in both HUVECs and coronary artery endothelial cells. Our findings indicate that Lp(a), when incubated with endothelial cells, induces the accumulation of MCA in the culture medium but has no direct effect on stimulating monocyte chemotaxis. Furthermore, the apo(a) portion of Lp(a) is responsible for inducing the MCA.

	Methods

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
All chemicals used were reagent grade or better. Human coronary artery endothelial cells were obtained from Clonetics Corp. Human GM-CSF immunoassay (ELISA) kits were obtained from R&D Systems, Inc. Actinomycin D, BSA, cycloheximide, fMLP, and -amino-n-caproic acid were obtained from Sigma Chemical Co.

Cell Culture
HUVECs were harvested and cultured as described.²² The fresh umbilical cord was washed with a cold solution of (mmol/L) HEPES 10, NaCl₂ 137, KCl 4, and glucose 11, pH 7.4. Endothelial cells were isolated from the umbilical cord vein with 0.1% type 1 collagenase and seeded onto 0.2% gelatin-coated 100-mm Petri dishes. Cells were cultured at 37°C in 5% CO₂ in Medium 199 with 50 U/mL penicillin, 50 mg/mL streptomycin, 2.5 mg/mL amphotericin B, 2 mmol/L L-glutamine, 20 mg/mL endothelial cell growth factor, 4% (vol/vol) pooled human serum, and 16% (vol/vol) heat-inactivated FCS. Human coronary artery endothelial cells were grown according to the methods recommended by Clonetic Co. For MCA assays, cells from passages 2 through 5 were grown in six-well plates. Aliquots (100 µL) of medium were removed at the indicated time points and stored at -80°C for further analysis. For analysis of translation and transcription, HUVECs were incubated in medium for 15 minutes with either cycloheximide or actinomycin D (10 µmol/L final concentration), respectively. Lp(a) was then added directly to the medium, and the incubation was continued for 4 hours. Aliquots (100 µL) were stored at -80°C.

Purification of Lipoproteins
Lp(a) and LDL were purified from fresh plasma in the presence of EDTA and stored under nitrogen to prevent lipid oxidation.¹⁷ The concentration of Lp(a) and LDL was measured with the bicinchoninic acid assay (Pierce) with BSA as the standard.²³ Phenotyping was kindly performed by Dr Santica Marcovina as described.²⁴ Endotoxin contamination of Lp(a) was monitored with an E-Toxate (limulus amoebocyte lysate) test kit (Sigma). Standard curves were constructed with a preparation of LPS from Escherichia coli, serotype 026:B6; the minimum detectable level was 1 ng/mL. The maximum LPS concentration of the Lp(a) preparations used was 8 ng/100 µg Lp(a) protein. To decrease the LPS content of Lp(a) preparations, Lp(a) was incubated 18 hours at 4°C with an equal volume of immobilized polymyxin B gel (Affi-prep Polymyxin, Bio-Rad Laboratories). After centrifugation and filtering (0.22-mm membrane), the Lp(a) was equilibrated in serum-free medium with Sephadex G-25. This affinity procedure reduced LPS in Lp(a) to undetectable levels [<1 ng/600 µg Lp(a)]. Lipid hydroperoxides, a measure of the oxidation of Lp(a) and LDL, were determined by use of the cholesterol color reagent from Merck as described.²⁵ Only lipoprotein preparations with undetectable lipoperoxides were used [<2 nmol lipid hydroperoxides/mg Lp(a) or LDL protein].

Purification of Apo(a)
Apo(a) was purified from Lp(a) prepared as above. Apo(a) was dissociated from Lp(a) by reduction with 2 mmol/L dithioerythritol followed by ultracentrifugation in 30% sucrose containing 0.2 mol/L -amino-n-caproic acid as described.²⁶ The purified apo(a) was stored at -80°C in sucrose. Before incubation with the endothelial cell monolayer in culture, the sucrose was exchanged for tissue culture medium by gel filtration chromatography on Sephadex G-25 (Pharmacia). The preparations were analyzed by SDS-PAGE and Western blotting as detailed.²⁷

Monocyte Chemotaxis Assay
MCA was measured with a modified Boyden chamber housing a polycarbonate filter with 5-µm pores (Nucleopore). This pore size has previously been shown to limit migration exclusively to monocytes.²⁸ For each assay, 27 µL of HUVEC-conditioned culture medium was loaded into the wells of the bottom chamber. Monocytes (50 µL; 4x10⁶ cells/mL) were loaded into each well of the upper chamber. After a 90-minute incubation in 5% CO₂ at 37°C, nonmigrating cells were scraped from the upper surface of the filter. Migrated cells on the lower surface were fixed with methanol and stained with Diff-Quik (Baxter Healthcare Co). The number of monocytes on the lower surface of the filter was determined microscopically by counting five high-power (x400) fields of constant area per well. For normalization, MCA in HUVEC-conditioned medium was expressed as the percentage of MCA induced by the positive control, 10 nmol/L fMLP, after subtraction of the negative control (0.2% BSA in serum-free medium). Experiments were performed in triplicate, in duplicate wells.

Northern Blot Analysis
RNA preparation and blot hybridization were performed as previously described²⁹ with the full-length MCP-1 cDNA.³⁰ Final washes were in 0.1xSSC (1xSSC=0.15 mol/L NaCl and 0.015 mol/L sodium citrate, pH 7.0) and 0.1% SDS at 65°C for 1 hour. Equal loading of total RNA was verified by ethidium bromide staining of the 18S and 28S ribosomal RNA.

ELISA Determination of GM-CSF
GM-CSF concentrations in the conditioned medium were determined by ELISA according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as mean±SEM with the exception of the time-course data, which were reported as mean±SD. Results represented the mean of three or more experiments, with multiple data determinations in each experiment. Experimental groups were compared by ANOVA with repeated measures or one-group variance test, followed, when appropriate, by a Dunn's test for multiple comparisons. All statistical analysis was performed with StatView 4.5 for PowerMac (Abacus Concepts). A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
Lp(a) but Not LDL Induced Significant MCA in HUVECs
HUVECs were treated with 50 µg/mL of Lp(a) or LDL that had no assayable lipid hydroperoxides, and at various intervals, the conditioned medium was assayed for MCA (Fig 1). Lp(a) induced a significant increase in MCA secreted by HUVECs at 30 minutes, reached a peak at 2 to 3 hours, and persisted for at least 10 hours. In contrast, LDL prepared from the same donor had no effect when incubated with HUVECs. Lp(a) and LDL were also incubated in medium in the absence of HUVECs. These preparations did not stimulate migration of monocytes. Thus, the effect of Lp(a) on monocyte migration required incubation with endothelial cells. The induction of MCA in HUVECs by Lp(a) was concentration dependent, with no activity at 12.5 µg/mL but with increasing activity beginning at 25 µg/mL. Lp(a) at 100 µg Lp(a) protein/mL induced levels of MCA that were 85% of that seen with the potent monocyte chemoattractant fMLP (data not shown). This concentration of Lp(a) represents the lower limit of the pathological range of Lp(a).²

View larger version (19K): [in this window] [in a new window]   Figure 1. Lipoprotein(a) induces monocyte chemotactic activity when incubated with HUVECs. Cultured HUVECs were incubated with medium 199 alone (, n=6), medium containing 50 µg/mL Lp(a) (, n=6), or 50 µg/mL of LDL (, n=4). At indicated times, 100-µL aliquots of culture medium were removed and tested for MCA as described in "Methods." Medium alone (, n=6), or medium containing the same concentrations of LDL (, n=4) or Lp(a) (, n=6) without exposure to HUVECs were also tested. Results are expressed as percent of monocytes migrating in response to positive control, fMLP, and represent mean±SD. *P<.0001 vs medium alone.

Endotoxin Was Not Responsible for the MCA Induced by Lp(a)
Because the lipoprotein preparations used had traces of LPS as measured by the limulus amoebocyte lysate assay, it was important to rule out the possibility that LPS was responsible for the generation of endothelial cell MCA. Polymyxin B, a potent LPS inhibitor, had no effect on the induction of MCA in HUVECs by Lp(a) (Fig 2). Lp(a) was also treated with immobilized polymyxin B, a procedure that decreased the LPS concentration by >48-fold. This LPS-depleted Lp(a) induced the same level of MCA in HUVECs as did the untreated Lp(a). The LDL preparation contained a concentration of LPS similar to that of native Lp(a) and yet was inactive in inducing MCA (Fig 1). Thus, LPS appeared not to be responsible for the MCA induced in HUVECs by Lp(a).

View larger version (55K): [in this window] [in a new window]   Figure 2. Polymyxin B does not affect induction of MCA by Lp(a). HUVECs were treated for 6 hours with medium alone (CON), Lp(a) alone (L/H), or both polymyxin B and Lp(a) (L/P/H). Controls include 50 µg/mL Lp(a) alone (L) or Lp(a)+20 µg/mL polymyxin B (L/P). Aliquots of culture medium were removed and tested for MCA. There was no statistical difference between CON, L, and L/P. n=3; *NS, P>.05 vs control.

MCA Induced by Lp(a) in HUVECs Required Both RNA and Protein Synthesis
Incubation of HUVECs with Lp(a) in the presence of the transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide completely abolished the accumulation of MCA in the culture medium (Fig 3). In control studies, neither actinomycin D nor cycloheximide affected fMLP-mediated monocyte migration when added directly to the bottom wells of the Boyden chamber (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 3. Role of RNA transcription and protein synthesis in HUVEC-mediated monocyte chemotaxis. HUVECs were incubated with serum-free medium (CON), with Lp(a) (L), or with Lp(a) in presence of cycloheximide (L/Cyc) or actinomycin D (L/Act) as detailed in "Methods." Aliquots of postculture media were removed and tested for MCA. n=3; *NS, P>.05 vs control.

Lp(a)-Induced Monocyte Migration Was Chemotactic
To establish that the activity induced by Lp(a) was chemotactic, a "checkerboard" analysis was performed. The medium from Lp(a)-treated HUVECs was placed in both the top and bottom wells, only in the top wells (containing the monocytes), or only in the bottom wells. As shown in Fig 4, significant monocyte migration was observed when Lp(a)-conditioned medium was placed only in the bottom wells and was abolished in the absence of a concentration gradient. This demonstrates that the migration of monocytes was due to chemotaxis rather than chemokinesis.

View larger version (41K): [in this window] [in a new window]   Figure 4. Checkerboard analysis of MCA induced by Lp(a) incubated with HUVECs. Medium from HUVECs treated with 50 µg/mL Lp(a) for 6 hours was placed in both top and bottom (BTM) wells, only in top well (containing monocytes), or only in bottom wells. - indicates 0.2% BSA; L, medium from HUVECs treated with Lp(a). n=3; *NS, P>.05.

Apo(a) Portion of Lp(a) Induced MCA in Human Endothelial Cells
To identify which structures of the Lp(a) particle were responsible for the induction of endothelial cell MCA, the apo(a) portion was isolated from Lp(a). Fig 5 (inset) shows the preparations of LDL, Lp(a), and apo(a) purified from the same individual (donor 1) and used for the present studies as analyzed by SDS-PAGE (4% to 16% gradient gel). This donor had two isoforms of apo(a) containing either 18 or 27 copies of kringle IV, representing 90% and 10%, respectively, of the total apo(a) (see the Table). The purified apo(a), which was also free of apo B-100, as analyzed by a Western immunoblotting study, induced the secretion of MCA by HUVECs (Fig 6). As shown previously for Lp(a), apo(a) had no chemoattractant activity when added directly to the bottom wells of the Boyden chamber.

View larger version (81K): [in this window] [in a new window]   Figure 5. Apo(a) induces HUVECs to produce MCA. HUVECs were treated for 6 hours with serum-free medium (M+H); with two different concentrations of Lp(a), 50 µg/mL [L(50)+H] or 100 µg/mL [L(100)+H]; or with apo(a) 30 µg/mL [A(30)+H] or 60 µg/mL [A(60)+H]. Lp(a) 100 µg/mL [L(100)] or apo(a) 60 µg/mL [A(60)] was added directly to bottom wells of Boyden chamber (n=5, *NS, P>.05 vs M+H). Inset, SDS-PAGE analysis of LDL, Lp(a), and apo(a). LDL, Lp(a), and apo(a) were isolated from donor 1. Unreduced samples (2 µg/lane) in 1% SDS were analyzed by 4% to 16% gradient SDS-PAGE. After electrophoresis, gels were stained with Coomassie brilliant blue. Lane 1, LDL; lane 2, Lp(a); lane 3, apo(a) purified from donor 1 Lp(a) demonstrating two apo(a) isoforms with a distribution of 10% and 90%.

View this table: [in this window] [in a new window]   Table 1. Effect of Different Lp(a) Phenotypes on MCA and GM-CSF Production

View larger version (42K): [in this window] [in a new window]   Figure 6. Lp(a) and apo(a) induce MCA in human coronary endothelial cells. Human coronary endothelial cells were treated for 6 hours exactly like HUVECs in Fig 5. n=4; *NS, P>.05 vs M+H.

Lp(a) and Apo(a) Induced MCA in Human Coronary Artery Endothelial Cells
Coronary endothelial cells were incubated for 6 hours with either Lp(a) or apo(a) (Fig 7). Similar to their effects on HUVECs, Lp(a) and apo(a) induced high levels of MCA when incubated with human coronary artery endothelial cells.

View larger version (63K): [in this window] [in a new window]   Figure 7. RNA blot analysis of MCP-1 mRNA in HUVECs. HUVECs were treated for the times indicated with (1) Medium 199 (Med), (2) LPS (100 ng/mL), or (3) Lp(a) (50 µg/mL). Aliquots (10 µg) of total RNA were size-fractionated on agarose gels and hybridized to 32P-labeled human MCP-1 cDNA as described in "Methods." Ethidium bromide staining of 18S and 28S ribosomal RNA is shown as control for equal loading of samples. Experiment was performed in duplicate.

Different Isoforms of Lp(a) Induced Similar Levels of MCA in HUVECs
The studies presented above used Lp(a) isolated from the plasma of a single donor (donor 1, Table). To determine whether phenotypes of Lp(a) different from donor 1 would also stimulate HUVECs to produce MCA, Lp(a) from three additional subjects was isolated and the phenotypes were determined as described.²⁴ Including donor 1, three of the Lp(a) preparations demonstrated two different isoforms, and one preparation possessed a single isoform (Table). Incubation of these Lp(a) preparations (50 µg/mL) for 6 hours with HUVECs induced similar amounts of MCA (Table). These isoforms did not demonstrate MCA in the absence of incubation with HUVECs.

	GM-CSF and MCP-1 Appear Not to Be Responsible for the MCA Induced in HUVECs

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
GM-CSF and MCP-1 are potent monocyte chemoattractants that are produced by endothelial cells^{31 32} and have been implicated in atherogenesis. As shown in Fig 6, unstimulated HUVECs expressed low levels of MCP-1 mRNA. Incubation of HUVECs with LPS (100 ng/mL) caused a marked induction of MCP-1 mRNA. In contrast, treatment of HUVECs with 50 µg/mL of Lp(a) for 8 hours did not induce accumulation of MCP-1 mRNA. In additional experiments, the concentration of GM-CSF in the Lp(a)-induced HUVEC-conditioned medium was determined by ELISA. No GM-CSF antigen was detected (Table).

	Discussion

Top Abstract Introduction Methods Results GM-CSF and MCP-1 Appear... Discussion References

 
This report demonstrates that exposure of endothelial cells to Lp(a), an atherogenic lipoprotein, results in the accumulation of MCA in the culture medium. Most significantly, the accumulation of MCA is attributable to the apo(a) portion of Lp(a). Neither Lp(a) nor apo(a) displayed direct MCA; incubation with endothelial cells was an absolute requirement. The accumulation of MCA was dependent on new transcription and translation in that it was blocked by actinomycin D and cycloheximide, respectively. These data suggest that the accumulation of MCA in the culture medium is due to the de novo synthesis of a chemoattractant by endothelial cells in response to Lp(a) and apo(a), rather than the release of a chemoattractant from intracellular stores.

Cushing et al²¹ demonstrated that LDL did not induce MCA when incubated with endothelial cells; however, minimally oxidized LDL induced chemotactic activity. The Lp(a) used for these studies had undetectable lipid hydroperoxides when assayed by a sensitive chemical assay. Lp(a) stimulated HUVECs to produce MCA, whereas under similar conditions, LDL purified from the same donor did not. In addition, apo(a) purified from Lp(a) and free of apo B-100 was at least as potent in stimulating HUVECs and coronary artery endothelial cells to produce MCA as was the intact Lp(a). Lipid peroxidation would not contribute to the activity of the apo(a). These data strongly argue against the oxidation of Lp(a) being responsible for the observed induction of MCA.

Because LPS is a ubiquitous contaminant known to induce the synthesis of chemoattractants, we determined its potential contribution to the induction of MCA in Lp(a)-treated HUVECs. Polymyxin B, a strong inhibitor of LPS, did not attenuate the action of Lp(a) on HUVECs. Furthermore, treatment of Lp(a) with immobilized polymyxin B, a procedure that decreased the LPS concentration >48-fold, did not affect the MCA-inducing activity of Lp(a). Thus, we feel confident that contaminating LPS was not responsible for the production of endothelial cell MCA.

The production of MCA by cultured endothelial cells was not dependent on a single phenotype of Lp(a). The molecular size of the apo(a) portion of Lp(a) is highly variable, depending on the number of kringle IV type 2 repeats, which vary in different individuals.^{33 34 35} In addition, an individual may have two different isoforms of apo(a). Some studies have found that there is an inverse relationship between kringle number and plasma concentration of Lp(a)^{33 34 35 36} and that the numbers of kringle repeats are correlated with carotid arterial vascular disease³⁷ and with coronary artery disease.³⁸ In the present study, four individuals with different phenotypes were examined. There was no apparent difference in the MCA-inducing capacity of these Lp(a) preparations; however, the number tested was too few to conclude that kringle number may not influence MCA induction.

In the "response-to-injury" hypothesis, the interaction between growth agonists and the dysfunctional endothelium plays an important role in early atherogenesis.²⁰ After the initial injury, endothelial cells secrete growth factors and cytokines, which lead to increased adherence of circulating monocytes and T lymphocytes to the vessel wall.²⁰ Recruitment of circulating monocytes into the intima is a critical step in the events leading to the formation of an atherosclerotic lesion.

Although a wide variety of agents have been shown to induce monocyte chemotaxis, particular attention has focused on MCP-1. A number of agents, including cytokines³² and oxidized LDL,²¹ have been shown to stimulate vascular endothelial cells to produce MCP-1 in vitro. The MCA induced by minimally modified LDL was found to be solely attributable to the secretion of MCP-1 by cultured human aortic endothelial cells and smooth muscle cells.²¹ In addition to in vitro studies, MCP-1 mRNA and protein have been found in human and rabbit atherosclerotic lesions.³⁹ In previous studies, the secretion of MCP-1 by cultured cells has been accompanied by new synthesis of MCP-1 mRNA and has not been thought to be due to release of MCP-1 protein from intracellular stores.^{21 32 40} In the present study, Lp(a) failed to induce MCP-1 mRNA. Under similar conditions, LPS markedly induced MCP-1 mRNA. In preliminary gel shift analyses, Lp(a) also failed to induce NF-B in HUVECs (unpublished observations, Poon et al, 1997). NF-B has been shown to be critical to the induction of MCP-1 in a variety of human cells.^{41 42 43} These data suggest that Lp(a) is inducing a monocyte chemoattractant distinct from MCP-1.

A second monocyte chemoattractant examined was GM-CSF. GM-CSF is produced in response to the direct adhesive interaction between monocytes and endothelial cells in culture.³¹ Human arterial endothelial cells secrete GM-CSF in response to minimally oxidized LDL.⁴⁴ GM-CSF is also essential for the in vitro growth and differentiation of monocytes and macrophages and may contribute to the initiation and progression of atherosclerotic lesions. Levels of GM-CSF mRNA are often significantly lower than those of MCP-1 and may not be detectable by RNA blot analysis. We therefore examined GM-CSF antigen. GM-CSF antigen was not present in Lp(a)-conditioned medium, which possessed potent MCA.

In summary, we have documented a novel property of Lp(a) that may contribute to its atherogenicity. Lp(a) stimulated cultured HUVECs to accumulate MCA, most likely by inducing the transcription and translation of a monocyte chemoattractant. The activity of Lp(a) was attributable to the apo(a) moiety of the particle. We propose that high levels of Lp(a) may actively contribute to recruiting circulating monocytes to the vascular endothelium, thereby promoting the formation of an atherosclerotic lesion. Although it is beyond the scope of this initial report to examine all of the known monocyte chemoattractants, MCP-1 and GM-CSF, which have received considerable attention as mediators of monocyte recruitment in atherosclerosis and which are inducible in endothelial cells by a variety of agonists, are apparently not responsible for the MCA. The identity of the Lp(a)- and apo(a)-inducible chemoattractant thus remains to be determined.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein fMLP = formyl-methionyl-leucyl-phenylalanine GM-CSF = granulocyte-macrophage colony–stimulating factor HUVEC = human umbilical vein endothelial cell Lp(a) = lipoprotein(a) LPS = lipopolysaccharide MCA = monocyte chemotactic activity MCP-1 = monocyte chemotactic protein-1 NF = nuclear factor

	Acknowledgments

 
This work was supported by a Clinician-Scientist Award from the American Heart Association and an Arthur Ross Scholarship in Cardiovascular Medicine to Dr Poon and NIH grant HL-54469 to Drs Taubman and Harpel. We thank Dr Bruce Gordon (The New York Hospital-Cornell Medical Center), who provided the Lp(a)-rich plasma, and Dr Valentin Fuster for his critical review of the manuscript. We acknowledge the kindness of Dr Santica Marcovina (The University of Washington Medical Center) for performing the phenotypes of the Lp(a) preparations used in this study. We also wish to thank Dr Joan Berman (Albert Einstein School of Medicine) for the MCP-1 cDNA probe and T.-S. Chang and Ariel Kohane for their excellent technical assistance.

	Footnotes

 
Presented by M.P. in the Young Investigators' Award Competition at the 45th Annual Scientific Sessions of the American College of Cardiology, Orlando, Fla, March 24, 1996.

Received March 26, 1997; revision received May 19, 1997; accepted May 22, 1997.

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