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The Biology of Lipid Trafficking in Macrophages

The Freeman laboratory's research centers on the role of lipid uptake and egress in macrophages and the contribution those processes make to atherosclerotic lesion development and progression. Coronary artery disease as well as peripheral vascular disease account for much of the morbidity and mortality associated with diabetes and diabetes is a major risk factor for the development of accelerated atherosclerosis. The macrophage plays a critical role in the initiation of atherosclerotic plaques and increasing evidence suggests that macrophages also contribute to complex lesion development and plaque rupture. Genetically engineered mice are employed to examine the role of macrophage receptors that bind modified forms of lipoproteins, leading to atherosclerotic foam cell formation. As glycated lipoproteins have also recently been demonstrated to bind to these macrophage scavenger receptors, their role in diabetic atherosclerosis may be significant. LDL receptor deficient and apo E deficient mice in the C57/BL6 background form atherosclerotic lesions in the proximal aorta that share many characteristics of human atheroma. Early foam cell lesions followed by the development of complex, calcific plaques are generated in these animals. Current studies are examining the following questions:

Is scavenger receptor uptake of modified lipoproteins essential for lesion formation and progression ? This issue is addressed through the generation of mice lacking the two major scavenger receptors known to be expressed in atherosclerotic lesions (ie, SR-A , and CD 36). Knock-outs for both receptors are in hafd and have been bred into hyperlipidemic mice.

We are also characterizing the role of scavenger receptors in macrophage activation and stimulation of pro-inflammatory cytokines following uptake of modified lipoproteins. We recently identified an important role for the innate immunity signaling pathways that link through the toll receptor adaptor molecule known as MyD88. Our studies of macrophage activation exploit microarray expression profiling to globally examine gene expression. Finally, the lipid egress mechanisms are being examined through a detailed structure/function analysis of ABCA1 and other members of the A class of ABC transporters.

Through studies of this kind the investigators are exploring the mechanisms by which lipids initiate macrophage activation and inflammation in the artery wall. These studies have the potential to provide insights into atherosclerotic lesion development that could open up new anti-inflammatory strategies for the prevention and treatment of atherosclerosis. The laboratory currently has transgenic and homologous recombinant technology in place and has created a vascular pathology lab that permits characterization and quantification of atherosclerosis using video microscopy and quantitative image analysis. Participation in the Diabetes Research Center would enhance the laboratory's ability to investigate signal transduction pathways that are involved in the activation of lesional macrophages.

References:

1. Chiang, N., K. Gronert, C. B. Clish, J. A. O'Brien, M. W. Freeman, and C. N. Serhan. 1999. Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion. J Clin Invest 104:309.

2. Freeman, M. W. 1999. Effluxed lipids: Tangier Island's latest export. Proc Natl Acad Sci U S A 96:10950.

3. Brousseau, M. E., E. J. Schaefer, J. Dupuis, B. Eustace, P. Van Eerdewegh, A. L. Goldkamp, L. M. Thurston, M. G. FitzGerald, D. Yasek-McKenna, G. O'Neill, G. P. Eberhart, B. Weiffenbach, J. M. Ordovas, M. W. Freeman, R. H. Brown, Jr., and J. Z. Gu. 2000. Novel mutations in the gene encoding ATP-binding cassette 1 in four tangier disease kindreds. J Lipid Res 41:433.

4. Brousseau, M. E., G. P. Eberhart, J. Dupuis, B. F. Asztalos, A. L. Goldkamp, E. J. Schaefer, and M. W. Freeman. 2000. Cellular cholesterol efflux in heterozygotes for Tangier disease is markedly reduced and correlates with high density lipoprotein cholesterol concentration and particle size. J Lipid Res 41:1125.

5. Fabunmi, R. P., K. J. Moore, P. Libby, and M. W. Freeman. 2000. Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic mice. Atherosclerosis 148:375.

6. Fitzgerald, M. L., K. J. Moore, M. W. Freeman, and G. L. Reed. 2000. Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages. J Immunol 164:2692.

7. Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg. 2000. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nature Immunol 1:398.

8. Li, Z., Y. Kako, L. Pang, M. W. Freeman, J. M. Glick, X. Wang, and I. J. Goldberg. 2000. Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production. J Lipid Res 41:1912.

9. Moore, K. J., L. P. Andersson, R. R. Ingalls, B. G. Monks, R. Li, M. A. Arnaout, D. T. Golenbock, and M. W. Freeman. 2000. Divergent response to LPS and bacteria in CD14-deficient murine macrophages. J Immunol 165:4272.

10. Wang, X., W. Zeng, M. Murakawa, M. W. Freeman, and B. Seed. 2000. Episomal segregation of the adenovirus enhancer sequence by conditional genome rearrangement abrogates late viral gene expression. J Virol 74:11296.

11. Bobryshev, Y. V., T. Taksir, R. S. Lord, and M. W. Freeman. 2001. Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice. Histol Histopathol 16:801.

12. Fitzgerald, M. L., A. J. Mendez, K. J. Moore, L. P. Andersson, H. A. Panjeton, and M. W. Freeman. 2001. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space. J Biol Chem 276:15137.

13. Moore, K. J., E. D. Rosen, M. L. Fitzgerald, F. Randow, L. P. Andersson, D. Altshuler, D. S. Milstone, R. M. Mortensen, B. M. Spiegelman, and M. W. Freeman. 2001. The role of PPAR-gamma in macrophage differentiation and cholesterol uptake. Nature Med 7:41.

14. Moore, K. J., M. L. Fitzgerald, and M. W. Freeman. 2001. Peroxisome proliferator-activated receptors in macrophage biology: friend or foe? Curr Opin Lipidol 12:519.

15. Fitzgerald, M. L., A. L. Morris, J. S. Rhee, L. P. Andersson, A. J. Mendez, and M. W. Freeman. 2002. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J Biol Chem 277:33178.

16. Henneke, P., O. Takeuchi, R. Malley, E. Lien, R. R. Ingalls, M. W. Freeman, T. Mayadas, V. Nizet, S. Akira, D. L. Kasper, and D. T. Golenbock. 2002. Cellular activation, phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathways. J Immunol 169:3970.

17. Kunjathoor, V. V., M. Febbraio, E. A. Podrez, K. J. Moore, L. Andersson, S. Koehn, J. S. Rhee, R. Silverstein, H. F. Hoff, and M. W. Freeman. 2002. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem 277:49982.

18. Moore, K. J., J. El Khoury, L. A. Medeiros, K. Terada, C. Geula, A. D. Luster, and M. W. Freeman. 2002. A CD36-initiated signaling cascade mediates inflammatory effects of beta-amyloid. J Biol Chem 277:47373.

19. Rosen, E. D., C. H. Hsu, X. Wang, S. Sakai, M. W. Freeman, F. J. Gonzalez, and B. M. Spiegelman. 2002. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 16:22.

20. Yang, K. K., B. G. Dorner, U. Merkel, B. Ryffel, C. Schutt, D. Golenbock, M. W. Freeman, and R. S. Jack. 2002. Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice. J Immunol 169:4475.

21. El Khoury, J. B., K. J. Moore, T. K. Means, J. Leung, K. Terada, M. Toft, M. W. Freeman, and A. D. Luster. 2003. CD36 mediates the innate host response to beta-amyloid. J Exp Med 197:1657.

22. Kim, W. S., C. M. Ordija, and M. W. Freeman. 2003. Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A. Biochem Biophys Res Commun 310:542.

23. Fitzgerald, M.L. et al. ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux. J Lipid Res 45, 287-94 (2004).

24. Chroni, A., Liu, T., Fitzgerald, M.L., Freeman, M.W. & Zannis, V.I. Cross-linking and lipid efflux properties of ApoA-I mutants suggest direct association between ApoA-I helices and ABCA1. Biochemistry 43, 2126-39 (2004).

25. Bjorkbacka H, Kunjathoor V, Moore K, Koehn S, Ordija C., Lee MA, Means T, Halmen K, Luster AD, Golenbock DT, and MW Freeman. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol to activation of innate immunity signaling pathways. Nature Med. In press.


	Freeman, Mason, M.D The Biology of Lipid Trafficking in Macrophages The Freeman laboratory's research centers on the role of lipid uptake and egress in macrophages and the contribution those processes make to atherosclerotic lesion development and progression. Coronary artery disease as well as peripheral vascular disease account for much of the morbidity and mortality associated with diabetes and diabetes is a major risk factor for the development of accelerated atherosclerosis. The macrophage plays a critical role in the initiation of atherosclerotic plaques and increasing evidence suggests that macrophages also contribute to complex lesion development and plaque rupture. Genetically engineered mice are employed to examine the role of macrophage receptors that bind modified forms of lipoproteins, leading to atherosclerotic foam cell formation. As glycated lipoproteins have also recently been demonstrated to bind to these macrophage scavenger receptors, their role in diabetic atherosclerosis may be significant. LDL receptor deficient and apo E deficient mice in the C57/BL6 background form atherosclerotic lesions in the proximal aorta that share many characteristics of human atheroma. Early foam cell lesions followed by the development of complex, calcific plaques are generated in these animals. Current studies are examining the following questions: Is scavenger receptor uptake of modified lipoproteins essential for lesion formation and progression ? This issue is addressed through the generation of mice lacking the two major scavenger receptors known to be expressed in atherosclerotic lesions (ie, SR-A , and CD 36). Knock-outs for both receptors are in hafd and have been bred into hyperlipidemic mice. We are also characterizing the role of scavenger receptors in macrophage activation and stimulation of pro-inflammatory cytokines following uptake of modified lipoproteins. We recently identified an important role for the innate immunity signaling pathways that link through the toll receptor adaptor molecule known as MyD88. Our studies of macrophage activation exploit microarray expression profiling to globally examine gene expression. Finally, the lipid egress mechanisms are being examined through a detailed structure/function analysis of ABCA1 and other members of the A class of ABC transporters. Through studies of this kind the investigators are exploring the mechanisms by which lipids initiate macrophage activation and inflammation in the artery wall. These studies have the potential to provide insights into atherosclerotic lesion development that could open up new anti-inflammatory strategies for the prevention and treatment of atherosclerosis. The laboratory currently has transgenic and homologous recombinant technology in place and has created a vascular pathology lab that permits characterization and quantification of atherosclerosis using video microscopy and quantitative image analysis. Participation in the Diabetes Research Center would enhance the laboratory's ability to investigate signal transduction pathways that are involved in the activation of lesional macrophages. References: 1. Chiang, N., K. Gronert, C. B. Clish, J. A. O'Brien, M. W. Freeman, and C. N. Serhan. 1999. Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion. J Clin Invest 104:309. 2. Freeman, M. W. 1999. Effluxed lipids: Tangier Island's latest export. Proc Natl Acad Sci U S A 96:10950. 3. Brousseau, M. E., E. J. Schaefer, J. Dupuis, B. Eustace, P. Van Eerdewegh, A. L. Goldkamp, L. M. Thurston, M. G. FitzGerald, D. Yasek-McKenna, G. O'Neill, G. P. Eberhart, B. Weiffenbach, J. M. Ordovas, M. W. Freeman, R. H. Brown, Jr., and J. Z. Gu. 2000. Novel mutations in the gene encoding ATP-binding cassette 1 in four tangier disease kindreds. J Lipid Res 41:433. 4. Brousseau, M. E., G. P. Eberhart, J. Dupuis, B. F. Asztalos, A. L. Goldkamp, E. J. Schaefer, and M. W. Freeman. 2000. Cellular cholesterol efflux in heterozygotes for Tangier disease is markedly reduced and correlates with high density lipoprotein cholesterol concentration and particle size. J Lipid Res 41:1125. 5. Fabunmi, R. P., K. J. Moore, P. Libby, and M. W. Freeman. 2000. Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic mice. Atherosclerosis 148:375. 6. Fitzgerald, M. L., K. J. Moore, M. W. Freeman, and G. L. Reed. 2000. Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages. J Immunol 164:2692. 7. Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg. 2000. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nature Immunol 1:398. 8. Li, Z., Y. Kako, L. Pang, M. W. Freeman, J. M. Glick, X. Wang, and I. J. Goldberg. 2000. Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production. J Lipid Res 41:1912. 9. Moore, K. J., L. P. Andersson, R. R. Ingalls, B. G. Monks, R. Li, M. A. Arnaout, D. T. Golenbock, and M. W. Freeman. 2000. Divergent response to LPS and bacteria in CD14-deficient murine macrophages. J Immunol 165:4272. 10. Wang, X., W. Zeng, M. Murakawa, M. W. Freeman, and B. Seed. 2000. Episomal segregation of the adenovirus enhancer sequence by conditional genome rearrangement abrogates late viral gene expression. J Virol 74:11296. 11. Bobryshev, Y. V., T. Taksir, R. S. Lord, and M. W. Freeman. 2001. Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice. Histol Histopathol 16:801. 12. Fitzgerald, M. L., A. J. Mendez, K. J. Moore, L. P. Andersson, H. A. Panjeton, and M. W. Freeman. 2001. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space. J Biol Chem 276:15137. 13. Moore, K. J., E. D. Rosen, M. L. Fitzgerald, F. Randow, L. P. Andersson, D. Altshuler, D. S. Milstone, R. M. Mortensen, B. M. Spiegelman, and M. W. Freeman. 2001. The role of PPAR-gamma in macrophage differentiation and cholesterol uptake. Nature Med 7:41. 14. Moore, K. J., M. L. Fitzgerald, and M. W. Freeman. 2001. Peroxisome proliferator-activated receptors in macrophage biology: friend or foe? Curr Opin Lipidol 12:519. 15. Fitzgerald, M. L., A. L. Morris, J. S. Rhee, L. P. Andersson, A. J. Mendez, and M. W. Freeman. 2002. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J Biol Chem 277:33178. 16. Henneke, P., O. Takeuchi, R. Malley, E. Lien, R. R. Ingalls, M. W. Freeman, T. Mayadas, V. Nizet, S. Akira, D. L. Kasper, and D. T. Golenbock. 2002. Cellular activation, phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathways. J Immunol 169:3970. 17. Kunjathoor, V. V., M. Febbraio, E. A. Podrez, K. J. Moore, L. Andersson, S. Koehn, J. S. Rhee, R. Silverstein, H. F. Hoff, and M. W. Freeman. 2002. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem 277:49982. 18. Moore, K. J., J. El Khoury, L. A. Medeiros, K. Terada, C. Geula, A. D. Luster, and M. W. Freeman. 2002. A CD36-initiated signaling cascade mediates inflammatory effects of beta-amyloid. J Biol Chem 277:47373. 19. Rosen, E. D., C. H. Hsu, X. Wang, S. Sakai, M. W. Freeman, F. J. Gonzalez, and B. M. Spiegelman. 2002. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 16:22. 20. Yang, K. K., B. G. Dorner, U. Merkel, B. Ryffel, C. Schutt, D. Golenbock, M. W. Freeman, and R. S. Jack. 2002. Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice. J Immunol 169:4475. 21. El Khoury, J. B., K. J. Moore, T. K. Means, J. Leung, K. Terada, M. Toft, M. W. Freeman, and A. D. Luster. 2003. CD36 mediates the innate host response to beta-amyloid. J Exp Med 197:1657. 22. Kim, W. S., C. M. Ordija, and M. W. Freeman. 2003. Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A. Biochem Biophys Res Commun 310:542. 23. Fitzgerald, M.L. et al. ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux. J Lipid Res 45, 287-94 (2004). 24. Chroni, A., Liu, T., Fitzgerald, M.L., Freeman, M.W. & Zannis, V.I. Cross-linking and lipid efflux properties of ApoA-I mutants suggest direct association between ApoA-I helices and ABCA1. Biochemistry 43, 2126-39 (2004). 25. Bjorkbacka H, Kunjathoor V, Moore K, Koehn S, Ordija C., Lee MA, Means T, Halmen K, Luster AD, Golenbock DT, and MW Freeman. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol to activation of innate immunity signaling pathways. Nature Med. In press.