Background

[PubMed]

Chemotactic cytokines, such as monocyte chemoattractant protein-1 (chemotactic protein-1; MCP-1), play a pivotal role in the inflammatory process associated with the development and progression of atherosclerosis (1, 2). MCP-1, a low molecular weight monomeric peptide (9–15 kDa), is initially upregulated at the site of vessel wall injury by vascular smooth muscle cells (SMC) and endothelial cells (3). MCP-1, in interaction with several cytokines and cell adhesion molecules, mediates the transendothelial migration of monocytes to the subendothelial layer via cysteine-cysteine motif chemokine receptor-2 (CCR-2) receptor (4-7). As a response to the chemotactic signal, monocytes upregulate the number of CCR-2 receptors on their cell surface (8). The recruited monocytes then express MCP-1 by themselves, which accelerates the further influx of mononuclear cells. Once resident within the neointima, the monocytes differentiate to macrophages, express matrix metalloproteinases (MMPs), transform to foam cells by digesting oxidized low-density lipoproteins via expression of scavenger receptors, and form a necrotic core by undergoing apoptosis or necrosis (1). The MMPs expressed by macrophages and foam cells induce dissolution of SMCs and collagen, which causes the plaque cap to thin and makes the cap susceptible to rupture (9, 10). Because the extent of macrophage inflammation in atherosclerotic plaque is positively related to plaque instability, and because the receptor for MCP-1 is only expressed by infiltrating monocytes, the detection of MCP-1 receptors should allow monitoring of the extent of inflammation in atherosclerotic plaque. The use of radiolabeled MCP-1 for non-invasive imaging of the CCR-2 receptor density in atherosclerotic plaque might provide to be a useful clinical tool for the detection of plaques vulnerable to rupture.

Ohtsuki et al. (11) demonstrated the feasibility of using ¹²⁵I-MCP-1 to identify lesional monocytes and macrophages in experimental atherosclerosis with autoradiography. Blankenberg et al. (12) successfully labeled recombinant human MCP-1 with ^99mTc using the nicotinic acid analog hydrazinonicotinamide (HYNIC) as the chelator. The study demonstrated that ^99mTc-MCP-1 could detect abnormally increased numbers of perivascular mononuclear cells in native and grafted hearts in prediabetic rats. Kown et al. (13) reported that ^99mTc-MCP-1 could be used to assess graft coronary artery disease in rats. Hartung et al. (14) evaluated ^99mTc-MCP-1 for detection of inflammation in rabbits with induced atherosclerotic lesions. The study concluded that non-invasive detection of inflammation in experimental atherosclerotic lesions was feasible using ^99mTc-MCP-1.

Synthesis

[PubMed]

Recombinant human MCP-1, expressed in Escherichia coli, was derivatized with HYNIC for ^99mTc chelation (12, 15). MCP-1 was gently mixed with 5.5 mol succinimidyl-6-HYNIC per mol MCP-1 and incubated for 3 h in the dark at room temperature. The reaction was quenched with glycine, and the preparation was dialyzed. Blankenberg et al. (12) described the labeling of MCP-1 with ^99mTc with the use of a tin/tricine reduction and exchange reagent (16). The reagent contained 200 mmol/l tricine and 40 mg/l stannous chloride (pH 7.1); ^99mTc-pertechnetate (^99mTcO₄^-) was added to HYNIC-MCP-1, and then the tin/tricine reagent was added. The reaction mixture was incubated for 20 min at room temperature. The radiochemical yield was 96–99%, and the specific activity was 7.4 GBq/mg (200 mCi/mg), or 0.067–0.111 GBq/nmol (1.8–3.0 mCi/nmol) on the basis of a molecular weight of 9–15 kDa for MCP-1.

Hartung et al. (14) used commercially prepared protein vials and stannous tricine vials. For radiolabeling, 50–100 μl of ^99mTc-pertechnetate sodium (1.110 MBq (30mCi)) was added to the protein vial. Then, 1 ml of 0.9% sodium chloride solution was added to the stannous tricine vial, and the vial was gently swirled. After dissolving, 20 μl were withdrawn from the stannous tricine solution and added to the protein vial. After an incubation of 30 minutes at room temperature, instant thin-layer chromatography was performed. The radiochemical purity was >95%; the specific activity of ^99mTc-MCP-1 was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Hartung et al. (14) reported the results of a ligand-binding analysis. THP-1 monocytes (human monocytic leukemia cell line) and SMC cultured from the median layer of the aorta of New Zealand White (NZW) rabbits were used for the binding assay. Samples of 6 x 10⁵ THP-1 and SMC were placed in 12-well culture plates. Phorbol 12-myristate 13-acetate (PMA) (10 ng/mL) was added to half of the THP-1 and SMC wells for cell activation, but not to the other half. After incubation and resuspension in serum-free medium, ^99mTc-MCP-1 was added. After 15 minutes, 1 h, and 4 h incubation at 37°C, the samples were washed and solubilizing radioactivity was counted. MCP-1 radioactivity binding in the untreated THP-1 cells was significantly higher than that of the SMC at 4 h (20,493 ± 256 counts per min (cpm) versus 10,312 ± 266 cpm; P = 0.007). Compared to untreated cells, PMA-treated THP-1 cells showed a two-fold increase in MCP-1 radioactivity uptake at 4 h (43,988 ± 1.084 cpm; P < 0.003), suggesting that the MCP-1 uptake in culture occurs preferentially after the conversion of monocytes to macrophages. Further, it has been reported that monocyte-derived macrophages have the ability to increase the number of chemotactic receptors on their surface from about 3,000/cell to >60,000/cell in response to activation (17). A 20% increase of CCR-2 receptors on the cell surfaces doubles the chemotactic response of THP-1 monocytes or macrophages to MCP-1. MCP-1 has a high affinity (10^-9 M) for the CCR-2 chemokine receptor at the surface of THP-1 cells (8).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH Grant # RO1 (HL 078681), HL-47151, HL-61717.

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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.