MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma.

Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis.

directed against the respective primary antibody, incubated with ECL-plus (GE Healthcare) for 5 min, and visualized with an Op-tiChemHR Imaging System (UVP).

Microplate-based immunoassay
In Lp(a) binding experiments, Microfl uor 96-well microtiter plates (Thermo Scientifi c) were coated with 5 g/ml anti-apo(a) antibody LPA4 ( 20 ) overnight at 4°C. Plates were washed and blocked with 1% BSA/TBS for 45 min. Plasma samples (diluted 1:50 for human or 1:100 for mouse plasma) were plated in triplicates and incubated for 75 min at room temperature. Plates were washed three times and incubated with 50 ng/ml biotinylated goat anti-MCP-1 antibody (R and D Systems) for 60 min at room temperature. Plates were washed three times, incubated with alkaline phosphatase-conjugated NeutrAvidin (Thermo Scientifi c, 1:40,000 dilution) for 60 min at room temperature, washed, and incubated with Lumi-Phos-530 (Lumigen, 1:1 dilution in water) for 75 min at room temperature. The plates were read with an MLX Microtiter Plate Luminometer (Dynex Technologies) and results were displayed as relative light units (RLU) per 100 ms.

Lipoproteins and human plasma samples
Native LDL (nLDL) (density = 1.019-1.063 g/ml) was isolated from plasma of normolipidemic donors by sequential ultracentrifugation ( 12 ). Contamination of native and modifi ed LDL preparations by endotoxin was assessed with a LAL QCL-1000 kit (Lonza). LDL preparations with LPS higher than 50 pg/mg protein were discarded. To produce OxLDL, 0.1 mg/ml of nLDL was incubated with 10 M CuSO 4 for 18 h at 37°C ( 13 ). The extent of LDL oxidation was assessed by measuring thiobarbituric acidreactive substances (typically, more than 30 nmol/mg protein), and OxLDL was concentrated to 1 mg/ml using a 100 kDa cut off centrifugal concentrator (Millipore) and sterile fi ltered (0.22 m).
Plasma samples (n = 127) were collected from patients presenting with chest pain and suspected acute coronary syndromes (ST-segment elevation MI; non-ST-segment elevation MI and unstable angina) on admission to the Veteran's Affairs Medical Center San Diego. Patients that ultimately ruled out for MI by clinical criteria and myocardial enzyme biomarkers were included as controls. The blood was immediately spun down in EDTA and the plasma separated and stored at Ϫ 70°C. The collection of human plasma and the assays on these samples were approved by the Veteran's Affairs Medical Center and the University of California, San Diego Human Research Subjects Protection Programs, respectively , and all participants gave written informed consent.

Transgenic mice
C57BL6/J mice were wild type or transgenic expressing human apoB-100, human apo(a), or lipoprotein(a) [Lp(a)], i.e., both apoB-100 and apo(a), as previously reported (14)(15)(16). Mice were housed in a barrier facility with a 12 h light/12 h dark cycle, and fed normal mouse chow containing 4.5% fat (Harlan Teklad). All animal experiments were approved by the University of California, San Diego Institutional Animal Care and Use Committee.

Size exclusion chromatography
nLDL and OxLDL samples (30 g/ml) were incubated with 380 ng/ml MCP-1 (wild type) for 30 min at 37°C before they were loaded (200 l) on a Superdex 200 column (GE Healthcare) and eluted at 0.5 ml/min using an FPLC system (Pharmacia). Twenty fractions of 1.5 ml each were collected and assayed for MCP-1 and apoB-100 concentrations using ELISA as described below.

Native gel electrophoresis and immunoblotting
Samples of OxLDL, preincubated with either wild-type MCP-1, mutant MCP-1, E06 ( 18 ) and/or isotype control, nonspecifi c IgM (eBioscience), were run on a 3-8% precast Tris-acetate polyacrylamide gel (Invitrogen) with Tris-glycine buffer for 18 h at 100 mV. No SDS was present in the sample buffer or the gel. The proteins were transferred to a PVDF membrane, the membrane was blocked with 5% dry milk in PBS, washed, and subsequently incubated with an anti-MCP-1 antibody (R & D Systems) or an anti-apoB-100 antibody [mouse monoclonal antibody (mAb) MB47 ( 19 ) specifi c for human apoB-100]. The membrane was then washed and incubated with a secondary HRP-conjugated antibody inserts were fi xed in ice-cold methanol and stained with crystal violet. Cells were counted in 150 fi elds of view, covering the whole insert. Experiments were performed in biological triplicates and repeated three to fi ve times.
In a separate set of experiments, Lp(a) was isolated from human plasma using the apo(a)-specifi c antibody LPA4 ( 20 ) immobilized on agarose beads. Some plasma samples were spiked with 400 ng/ml of recombinant MCP-1 before the Lp(a) pull down. In brief, Protein A/G beads (GE Healthcare) were added to human plasma and incubated for 2 h at 4°C to remove endogenous immunoglobulins. Immunoglobulin-depleted plasma (500 l; 1:1 diluted in chemotaxis buffer) was then incubated with 2 g of the monoclonal anti-human apo(a) antibody LPA4 ( 20 ) overnight at 4°C, with gentle shaking, followed by a 1 h incubation with 50 l of Protein A/G beads at 4°C. The beads were washed and used in the migration assay as a chemoattractant in the bottom well. To maintain beads in suspension, they were gently stirred every 10 min for the duration of the migration assay. Because of this interruption in the migration process, the number of cells migrated toward MCP-1 (positive control) was different in Figs. 2 and 5B .

Statistical analysis
Each experiment was repeated at least three times. ELISA and migration assays were performed in triplicates, and the results are presented as mean ± SD. Results of migration assays were analyzed by Student's t -test, and results of dose-dependent MCP-1 binding were analyzed by two-way ANOVA, with Bonferroni posttest. Differences with P < 0.05 were considered statistically signifi cant.

OxLDL binds MCP-1
To test the hypothesis that MCP-1 binds to OxLDL, MCP-1 was combined with OxLDL or nLDL, loaded onto a size exclusion column, and eluted fractions were analyzed for apoB-100 and MCP-1 content. As shown in Fig. 1 , MCP-1 bound OxLDL to a greater degree compared with nLDL. MCP-1 associated with OxLDL also retained its capacity to induce migration of THP-1 monocytes ( Fig. 2 ), microtiter well plates as described ( 18 ) and MCP-1 binding was measured as detailed above. PAPC was purchased from Avanti Polar Lipids and OxPAPC was produced by air oxidation of PAPC as previously described ( 21 ).

Migration assay
A transmigration assay was performed with human monocytic THP-1 cells using Transwell inserts (Costar, 5 m pore size) and following the manufacturer's protocol. In brief, MCP-1 and/or nLDL or OxLDL in chemotaxis buffer (RPMI without phenol red, 10 mM HEPES) were placed in the bottom well, and 1 × 10 6 THP-1 cells were added to the insert. In some experiments THP-1 cells were preincubated with 100 nM BMS CCR2 22 (Tocris), a highly specifi c C-C chemokine receptor type 2 (CCR2) antagonist ( 22,23 ), for 30 min before the start of the migration assay, and the antagonist was present in the media for the duration of the assay. The cells were allowed to migrate for 2 h at 37°C, and Chemotaxis induced by OxLDL-associated MCP-1. nLDL and OxLDL (50 g/ml) were used in a migration assay with THP-1 cells as described in Methods. Some nLDL and OxLDL samples were preincubated with recombinant MCP-1 (300 g LDL + 0.5 g MCP-1) for 30 min at 37°C, followed by extensive dialysis (100 kDa cut-off) to remove the MCP-1 that was not bound to LDL; 50 g/ml of LDL was then used for migration assay. Recombinant MCP-1 at 50 ng/ml was used as a positive control. Some THP-1 cells were preincubated with 100 nM BMS CCR2 22 (CCR2 antagonist) for 30 min before the start of the migration assay. Results are presented as mean ± SD (n = 3); ** P < 0.005. Fig. 3. A: Role of basic amino acids in mediating binding of MCP-1 to OxPLs in OxLDL. OxLDL (100 g/ml) was preincubated with or without 10 g/ml of wild-type (wt) or mutant (mu) MCP-1 for 30 min at 37°C and the samples were run on native gel (no SDS), transferred, and probed with antibodies to apoB-100 or MCP-1. Some samples were preincubated with 500 g/ml of E06 or IgM isotype control. B: MCP-1 binding to OxPLs. Binding of MCP-1 (500 ng/ml) to nonoxidized PAPC or OxPAPC (50 g/well) was tested in a plate-based assay. Results are presented as mean ± SD (n = 3); a.u., arbitrary units; * P < 0.05.

Lp(a) is a carrier of MCP-1 in human plasma
Whereas OxLDL occurs in vivo in the artery wall, it is unlikely that such extensively OxLDL occurs in the plasma. However, we have shown that E06-detectable OxPLs are found in plasma and that Lp(a) is the lipoprotein that carries a major fraction of OxPLs in human plasma ( 24,25 ). Because the anti-OxPL antibody E06 blocked MCP-1 binding to OxLDL and MCP-1 directly bound to OxPLs ( Fig. 3 ), we tested to determine whether Lp(a) binds MCP-1 as well. First, we tested plasma samples from transgenic mice expressing human apoB-100, human apo(a), or Lp(a) [i.e., both apoB-100 and apo(a)], using a sandwich ELISA with an apo(a) capture mAb and an MCP-1 detection antibody. It was previously shown that the human and mouse apoB particles of these mice carry negligible amounts of OxPLs and that most of the OxPLs circulating in plasma are carried by apo(a)/Lp(a) (14)(15)(16), similar to humans ( 26 ). There was no endogenous MCP-1 detected on Lp(a), which was expected given that the mice were normolipidemic. and the number of cells migrating in response to OxLDLbound MCP-1 was signifi cantly higher than in response to nLDL-bound MCP-1. The specifi city of MCP-1-induced monocyte migration was validated in experiments with an antagonist of the MCP-1 receptor CCR2.
MCP-1 binding to OxLDL was confi rmed in a native gel electrophoresis experiment in which MCP-1 migrated with the apoB band ( Fig. 3A ). In contrast to wild-type MCP-1, an MCP-1 mutant in which the basic amino acids Arg-18 and Lys-19 were replaced with nonpolar Ala, did not bind to OxLDL. Furthermore, the mAb E06, which binds and neutralizes oxidized phospholipids (OxPLs) on the surface of OxLDL ( 18 ), diminished binding of wild-type MCP-1 to OxLDL. To directly demonstrate that MCP-1 binds to OxPLs, we added MCP-1 to microtiter wells in which either PAPC or OxPAPC were plated. As shown in Fig. 3B , MCP-1 directly bound to OxPAPC, representing a mixture of several OxPL molecular species ( 21 ), but not to nonoxidized PAPC. much as 1,000-fold, ranging from 3 to 3,335 (measured in RLU of the chemiluminescent immunoassay) ( Fig. 5A ). We next measured to determine whether the Lp(a)-associated endogenous MCP-1 induced monocyte migration. Lp(a) was pulled from plasma with agarose beads coated with an apo(a) mAb, and the beads were used in a THP-1 monocyte migration assay. Migration of monocytes toward Lp(a) isolated from a high MCP-1/Lp(a) sample was signifi cantly higher than the migration toward Lp(a) from a low MCP-1/Lp(a) sample ( Fig. 5B ). Adding a CCR2 antagonist to THP-1 cells inhibited monocyte migration toward Lp(a) isolated from the high MCP-1/Lp(a) plasma. Conversely, adding recombinant MCP-1 to the low MCP-1/ Lp(a) sample increased monocyte migration. The MCP-1 bound to the Lp(a) beads was able to dissociate from the beads under the conditions of the assay ( Fig. 6 ) and thus establish a gradient necessary for monocyte migration. These results suggest that MCP-1 carried by Lp(a) in human plasma is an active chemokine.

DISCUSSION
LDL cholesterol is considered a major causal risk factor in development of atherosclerosis ( 2,10 ). Realization that LDL can be oxidized in vivo and that oxidation changes the mode of LDL interaction with vascular cells led to subsequent studies of specifi c proatherogenic effects of OxLDL ( 27,28 ). These effects include the excessive accumulation of OxLDL in macrophages and vascular smooth muscle cells, and the activation of proinfl ammatory signaling pathways ( 29,30 ). These properties of Ox-LDL are predominantly attributed to the presence of oxidized lipids and oxidized lipid-modifi ed apoB-100. The present study suggests that, in addition to oxidized lipids, OxLDL can also be a carrier of other proinfl ammatory molecules, such as the chemokine MCP-1. We demonstrate that in vitro generated OxLDL is capable of binding MCP-1 and becomes a monocyte-attracting lipoprotein. Our laboratory had previously reported that in vitro generated OxLDL had some monocyte chemoattractant activity, due in part to the content of lysophosphatidylcholine generated in the OxLDL ( 31,32 ). However, MCP-1 binding to OxLDL results in a signifi cantly stronger chemotactic effect on monocytes than that induced by OxLDL alone.
Fully oxidized LDL is not thought to exist in signifi cant quantities in plasma but instead is present mainly in the vessel wall ( 28 ). However, our studies demonstrate that the major lipoprotein carrier of OxPLs in human plasma, Lp(a), also binds MCP-1. As with OxLDL, addition of recombinant MCP-1 to Lp(a) turns it into a chemoattractant. More remarkable is the observation that endogenous MCP-1 is present on circulating Lp(a) in human plasma and that Lp(a) isolated from the plasma with a high MCP-1/Lp(a) value is a stronger chemoattractant than the Lp(a) from low MCP-1/Lp(a) plasma. These results suggest that Lp(a) in plasma may serve as a carrier for However, after adding recombinant MCP-1 to mouse plasma samples, we detected MCP-1 only on apo(a)-containing lipoproteins ( Fig. 4A ). Using the same assay with human plasma samples, we also detected binding of recombinant wild-type MCP-1, but not mutant R18A/K19A MCP-1, to Lp(a) ( Fig. 4B ). Addition of mAb E06 inhibited MCP-1 binding to Lp(a) in the human plasma ( Fig. 4C ).
Because MCP-1 binds to GAGs ( 1 ), we tested to determine whether MCP-1 binding to Lp(a) can be completed by unfractionated heparin, a soluble GAG . Adding heparin to human plasma inhibited MCP-1 binding to Lp(a) in a dose-dependent manner ( Fig. 4D ). This result suggests that GAGs and Lp(a) may compete for an MCP-1 pool and together determine compartmentalization of the chemokine.
Endogenous MCP-1 associated with Lp(a) was assayed in a cohort of patients presenting with chest pain, using the same assay in which we captured Lp(a) from human plasma and then examined for the presence of MCP-1. The values of MCP-1/Lp(a) in individual samples varied as chemokine transcytosis, provide protection from hydrolysis, and coreceptor and signaling functions ( 1 ). Although in vitro the GAG binding defi cient MCP-1 mutant R18A/ K19A, which was also used in this study, has only minimal reduction in the CCR2 (MCP-1 receptor) binding, in vivo it is unable to recruit monocytes when administered intraperitoneally ( 7 ). These results support the importance of GAG binding for in vivo MCP-1 function. Our results showing that GAGs and Lp(a) compete for the MCP-1 binding add to the complexity of MCP-1 in vivo compartmentalization and function. Future studies will demonstrate whether binding of MCP-1 to Lp(a) and OxLDL is important for the function of MCP-1 in the development of atherosclerosis, as suggested in the present study.
MCP-1 and that once it has entered the arterial intima with its associated MCP-1, it may subsequently enhance the traffi cking of monocytes to the vascular wall. Lp(a) is found in the human atheroma and a higher content of Lp(a) correlates with the severity of the clinical presentation ( 33,34 ). Upon penetration into the vascular wall, Lp(a) binds to lysine groups of various proteins via its multiple high-affi nity lysine binding sites. This results in retention and concentration of Lp(a) in the vascular wall ( 25 ), where its OxPL content may mediate a number of major proinfl ammatory effects and also may induce apoptosis of endoplasmic reticulum-stressed macrophages ( 35 ). In addition, Lp(a)-associated MCP-1 may also contribute to recruitment of additional monocytes to the lesion site and thereby exacerbate lesion progression. Thus, the Lp(a) content of MCP-1 may be added to the list of biological properties of Lp(a) that make it a proatherogenic lipoprotein ( 36 ). In the small clinical cohort that we evaluated, there was no clear relationship between MCP-1/Lp(a) levels and clinical presentation. We plan to conduct large event-powered studies to evaluate the role of MCP-1/Lp(a) in cardiovascular disease and test the hypothesis that this novel biomarker predicts clinical outcomes.
The mechanism of MCP-1 binding with OxLDL and Lp(a) is likely similar to that with GAGs and relies on ionic interactions. Both GAGs and OxLDL are electronegative and the replacement of basic Arg-18 and Lys-19 in MCP-1 with nonpolar Ala renders it incapable of binding to either GAGs or OxLDL. Many if not all chemokines, which tend to be highly basic proteins, bind to GAGs ( 1 ). This provides a mechanism to localize and present chemokines on cell surfaces and/or on the extracellular matrix and thus to support directional motility of monocytes and other leukocytes. The GAG interactions also facilitate  Fig. 5B , MCP-1 can dissociate from the Lp(a) immobilized on agarose beads and thus form a gradient necessary for monocyte migration. Recombinant MCP-1 (500 ng) was added to two plasma samples (300 l each), this corresponds to a 100% "starting" concentration shown on graphs. After Lp(a) isolation from plasma and its immobilization on agarose beads, as described in Methods, and following three rounds of washing the beads, MCP-1 was measured in the supernatant by immunoassay (on graphs: "isolated beads", "1st wash", "2nd wash", and "3rd wash"). The beads were then incubated in a buffer for 2 h at 37°C, with stirring every 10 min, and the supernatant ("following 2 h incubation") was analyzed for MCP-1. Results are presented as mean ± SD of technical triplicates; *** P < 0.001 versus 3rd wash. Note, plasma samples in this experiment were different from those tested in Fig. 5B .