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Inflammatory Components of Atherosclerosis

L.K. Curtiss, W.A. Boisvert, N. Kubo, M. Marsh, V. Walker, C.L. Banka

THE INTEGRIN CD11B

Mac-1 is an integrin heterodimer that contains CD11b. Mac-1 is expressed on the surface of leukocytes, where it can mediate their binding and extravasation into the vessel wall. Because this event is critical in atherosclerosis, we measured expression of CD11b on circulating peripheral blood leukocytes in normal and hyperlipidemic mice. Wild-type (normal) C57BL/6 mice and a commonly used murine model for atherosclerosis, C57BL/6 mice deficient in receptors for low density lipoprotein (LDL), were fed either chow or a high-fat diet. In the wild-type mice, plasma cholesterol levels and the number of CD11b⁺ cells did not change regardless of the diet. In the atherosclerosis-prone mice fed chow, plasma cholesterol levels were approximately 3-fold higher than those in normal mice, and CD11b was expressed on a larger percentage of the leukocytes. When these atherosclerosis-prone animals were fed a high-fat diet for 4 weeks, they had even greater increases in both plasma cholesterol levels and the percentage of the leukocytes that expressed CD11b.

To examine the role of CD11b in the formation of atherosclerotic lesions in vivo, we irradiated mice deficient in LDL receptors and repopulated the animals with bone marrow cells isolated from either CD11b-deficient mice or wild-type mice. Fluorescence-activated cell sorting was used to detect expression of CD11b. Four weeks after transplantation of bone marrow cells, less than 2% of the peripheral blood leukocytes in mice transplanted with cells from CD11b-deficient mice expressed CD11b, whereas levels of CD11b on leukocytes in mice transplanted with cells from wild-type animals were nearly normal (~25% of total leukocytes). When all mice were fed a high-fat diet for the next 16 weeks, the total plasma cholesterol levels increased and remained similar in the 2 groups at all times. Histologic examination of frozen sections of the aortic valves for atherosclerotic lesions at 16 weeks revealed that the mean lesion areas were similar in the 2 groups. Furthermore, no differences in the degree of macrophage infiltration were apparent. Therefore, these studies indicated that the hypercholesterolemia induced by a high-fat diet in mice deficient in LDL receptors led to an increase in the percentage of peripheral leukocytes expressing CD11b. However, expression of CD11b was not required for macrophage accumulation and lesion development in the aortic valves of these mice.

THE CHEMOKINE RECEPTOR CXCR2

We have reported that CXCR2, a CXC receptor for chemokines IL-8 and GRO, is expressed by macrophages in human aortic lesions and in advanced fatty streak lesions in atherosclerosis-prone mice. Moreover, we have shown that the absence of this receptor in mice markedly inhibits lesion formation. To further understand the role of macrophage CXCR2 in atherosclerosis, we compared the macrophage content of early and late lesions. We again used bone marrow transplantation of mice deficient in LDL receptors. However, in this instance, the irradiated mice were repopulated with bone marrow cells from mice deficient in CXCR2. The mice were fed a high-fat diet, and frozen sections of the aortic valves were examined for lesions. As expected, CXCR2 was detected only in the lesions in mice transplanted with bone marrow cells from wild-type mice, and it colocalized with macrophages. Interestingly, no differences in mean lesion area in the 2 groups of mice were observed at 3 or 6 weeks. However, by 16 weeks, both the size of the lesions and the number of macrophages in the 2 groups differed markedly. The lesions in the mice transplanted with bone marrow cells from CXCR2-deficient mice were smaller and contained fewer macrophages. In addition, the macrophages were localized only immediately below the endothelium and were not found dispersed throughout the intima. Therefore, we have shown that upregulated expression of CXCR2 by macrophages is not required for recruitment of macrophages to lesions. Instead, it is critical for retention and/or expansion of macrophages during the progression of fatty streak lesions.

THE APOLIPOPROTEIN E

Apolipoprotein E (apoE), a 34-kD protein found on cholesterol-rich plasma lipoproteins, is a ligand for receptor-mediated hepatic clearance of these lipoproteins from the circulation. The importance of apoE for cholesterol homeostasis is clearly shown in apoE-deficient mice, in which advanced atherosclerosis develops as a consequence of severe hypercholesterolemia. Although most of the apoE in circulation is synthesized by the liver, many extrahepatic tissues, including macrophages, produce this apolipoprotein. As shown by replacing the bone marrow cells of apoE-deficient mice with cells from wild-type mice, apoE derived exclusively from macrophages reduces both the hypercholesterolemia and the atherosclerosis in the recipient mice.

Both apoE mRNA and antigen are absent in normal vessels, but they are abundant in the macrophage-rich fatty streak lesions of the atherosclerosis-prone mice deficient in LDL receptors. However, the role of macrophage apoE in the microenvironment of the lesion is not clear. This apoE could be proatherogenic by promoting cellular uptake and degradation of cholesterol-rich lipoproteins. ApoE secreted by macrophages and acting in an autocrine fashion enhances macrophage uptake of cholesterol-rich lipoproteins. Alternatively, apoE secreted by lesion foam cells could be atheroprotective by facilitating cellular cholesterol efflux. Cholesterol-loaded macrophages secrete apoE that can be transferred onto high density lipoprotein and promote free cholesterol efflux from the vessel wall. According to these in vitro findings, apoE could exacerbate the development of lesions in vivo by promoting macrophage uptake of atherogenic lipoproteins, or it could protect the vessel wall by promoting cholesterol efflux out of the lesion.

After we verified that bone marrow replacement of mice deficient in LDL receptors did not change either plasma cholesterol levels or atherosclerosis in the mice, this mouse model was used to examine the role of macrophage-specific apoE in atherosclerosis. Again, mice deficient in LDL receptors were irradiated and then reconstituted with bone marrow cells from either wild-type or apoE-deficient mice. Beginning 4 weeks after bone marrow transplantation, the animals were fed a high-fat diet for 16 weeks. As expected, no differences between the 2 experimental groups in plasma cholesterol levels were observed at any time during the study. Moreover, both groups of animals had comparable levels of circulating plasma apoE. However, the lesions in animals that received bone marrow cells from apoE-deficient mice were significantly larger and more extensive than the lesions in the mice that received cells from wild-type mice. Furthermore, after the lipids were extracted from the dissected aortas (arch to just above the right renal artery), a highly significant difference (P < .001) in aortic cholesteryl ester levels was revealed. We showed previously that the cholesterol content of the dissected thoracic aorta is a strong indicator of the lesion area of aortic valve tissues. Therefore, these studies confirm that the apoE produced locally by macrophages in the lesions in mice deficient in LDL receptors is protective and can reduce both the extent of lesion area and the content of cholesteryl esters within atherosclerotic lesions.

PUBLICATIONS

Banka, C.L. Non-genomic actions of estrogens: Estrogens as antioxidants. In: Estrogen and the Vessel Wall. Rubanyi, G. (Ed.). Harwood Academic, Reading, England, 1998, p. 91.

Boisvert, W.A., Santiago, R., Curtiss, L.K., Terkeltaub, R.A. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL-receptor-deficient mice. J. Clin. Invest. 101:353, 1998.

DeMattos, R.B., Curtiss, L.K., Williams, D.L. A minimally lipidated form of cell-derived apolipoprotein E exhibits isoform-specific stimulation of neurite outgrowth in the absence of exogenous lipids or lipoproteins. J. Biol. Chem. 273:4206, 1998.

Hill, J.S., Yang, D., Nikazy, J., Curtiss, L.K., Sparrow, J.T., Wong, H. Subdomain chimeras of hepatic lipase and lipoprotein lipase: Localization of heparin and cofactor binding. J. Biol. Chem., in press.

Kerjaschki, D., Exner, M., Ullrich, R., Susani, M., Curtiss, L.K., Witztum, J.L., Farquhar, M.G., Orlando, R.A. Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/GP330 in passive Heymann nephritis. J. Clin. Invest. 100:2303, 1997.

Lindholm, E.M., Bielicki, J.K., Curtiss, L.K., Rubin, E.M., Forte, T.M. Deletion of amino acids glu146arg160 in human apolipoprotein A-I (apo A-I Seattle) alters lecithin:cholesteryl acyltransferase activation and recruitment of cell phospholipid. Biochemistry 37:4863, 1998.

Miles, L.A., Sebald, M.T., Fless, G.M., Scanu, A.M., Curtiss, L.K., Plow, E.F., Hoover-Plow, J.L. Interaction of lipoprotein (a) with the extracellular matrix. Fibrinolysis Proteolysis, in press.

Nikoulin, I.R., Curtiss, L.K. An apolipoprotein E synthetic peptide targets to lipoproteins in plasma and mediates both cellular lipoprotein interactions in vitro and acute clearance of cholesterol-rich lipoproteins in vivo. J. Clin. Invest. 101:223, 1998.

Terkeltaub, R., Baird, S., Sears, P., Santiago, R., Boisvert, W. The murine homolog of the interleukin-8 receptor CXCR-2 is essential for the occurrence of neutrophilic inflammation in the air pouch model of acute urate crystal-induced gouty synovitis. Arthritis Rheum. 41:900, 1998.