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Alma Zernecke E-mail: [email protected] Phone: +49(0)931 31 80373 Fax: +49(0)931 31 83255 http://www.rudolf-virchow-zentrum.de/forschung/zernecke.html Atherosclerosis, with its clinical manifestations of myocardial infarction, stroke and peripheral artery disease, is imminently becoming the leading cause of death worldwide. Inflammation has emerged as a crucial force driving the initiation and progression of atherosclerotic lesion formation. Initiated by the activation and dysfunction of endothelial cells, leukocyte subsets are recruited and accumulate in atherosclerotic lesions. Details are emerging regarding the involvement of different leukocyte subpopulations in the pathology of this disease. While mononuclear cells found in the lesions are predominantly comprise monocyte-derived macrophages, which transform into foam cells characteristic for fatty-streak lesions, T-lymphocytes and dendritic cells have also been revealed in close proximity. Moreover, immune responses are described to participate in all phases of atherosclerosis, and pro-atherogenic and atheroprotective cytokines and T cell subpopulations have been defined. The delicately adjusted two-edged immune balance and the exact function of these cell types remain elusive to date. The non-random attraction of mononuclear cells to specific tissue targets is governed by sequential, and also mutually overlapping steps in the interaction with the vessel wall: namely rolling interactions, followed by integrin-dependent arrest and chemokine-triggered transendothelial diapedesis. We previously identified several adhesion molecules and chemokines/receptors as well as their regulation that are important in the accumulation of leukocytes at sites of inflammation. Recently, we could show that the transmembrane adhesive glycoprotein junctional adhesion molecule A (JAM-A) is not only expressed as a cellular variant, but is also released after cleavage, predominantly by the disintegrin and metalloproteinase (ADAM) 17. Functionally, soluble JAM-A inhibited the transendothelial migration of isolated neutrophils in vitro, and decreased the recruitment of neutrophils in a murine air pouch (Koenen et al.). In addition, we addressed the function of JAM-C, another member of the immunoglobulin superfamily, in leukocyte recruitment. Treatment with a blocking anti-JAM-C antibody inhibited the accumulation of monocytes in carotid arteries and reduced neointima formation after arterial injury in Apolipoprotien-E deficient (Apoe-/-) mice (Shagdarsuren et al.). Moreover, in this study we could unravel that JAM-C, in contrast to findings in human platelets, was not expressed by murine platelets. 24 Fig. 1: Reduced numbers of Gr1low monocytes in cx3cr1-deficient mice under steady-state conditions. Flow cytometric analysis of blood monocytes and comparison between monocyte population size of wt, cx3cr1gfp/+ and cx3cr1gfp/gfp mice (left panel). cx3cr1-deficient atheromas show changes in cellular composition. Irradiated Apoe-/- recipients of indicated bone marrow cells were subjected to high-fat diet for 12 weeks, after which their plaque content was analyzed. cx3cr1/GFP expression is shown in green; DAPI staining in blue (right panels). Activated platelets contribute to the exacerbation of atherogenesis; for example, by depositing the chemokines platelet factor4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by an enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. We have deter- mined the structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt pro-inflammatory CCL5-CXCL4 interactions. This results in attenuating monocyte recruitment and reducing atherosclerosis, establishing the in vivo relevance of chemokine heteromers in inflammatory leukocyte recruitment (Koenen et al.). Besides their function in leukocyte recruitment, chemokines are also important in controlling the survival and cell homeo- stasis of different cell subpopulations. For example, we could show that absence of the receptor cx3cr1 resulted in reduced monocyte survival. In this study, we used knock-in mice that carry a targeted replacement of the cx3cr1 gene by a gene encoding green fluorescent protein in order to visualize monocytes/macrophages by their GFP-expression. cx3cr1 is a chemokine receptor with a single ligand, the membrane-tethered chemokine CX3CL1 (fractalkine). All blood monocytes express cx3cr1, but its expression levels differ between the two main subsets, with human CD16+ and murine Gr1low monocytes being CX3CR1high. In mice, the absence of either cx3cr1 or cx3cl1 resulted in a significant reduction of Gr-1low blood monocyte levels under both steady-state (Fig. 1) and inflammatory conditions. Apoe-/- mice transplanted with cx3cr1-deficent bone marrow displayed a diminished accumulation of plaque cx3cr1+ cells (Fig. 1) and were protected against development of atherosclerosis. Introduction of a Bcl2 transgene to enforce the survival of monocytes restored the wild-type phenotype, suggesting that the CX3C-axis provides an essential survival signal (Landsman et al.). These findings provided a mechanistic explanation for the role played by the previously recognized function of the cx3cr1 chemokine family in atherogenesis. Chemokines are induced in the context of inflammation and in the course of apoptosis, as previously observed for the induction of CXCL12. This chemokine and its receptor CXCR4 have been implicated in mechanisms counteracting apoptosis. Apoptosis involves shedding of membranous microvesicles called apoptotic bodies. We could now show that endothelial cell-derived apoptotic bodies are generated during the development of atherosclerosis, and convey paracrine alarm signals to recipient vascular cells, inducing CXCL12 expression. This is mediated by microRNA-126, enriched in apoptotic bodies, repressing the negative regulator of G-protein signaling, RGS16, and unlocking CXCR4 to trigger an auto-regulatory feedback loop that increases production of CXCL12. Transfer of apoptotic bodies, microRNA-126, or local intraluminal exposure of carotid arteries of Apoe-/- mice to endothelial AB isolated from miR-126+/+ but not miR-126-/- mice limited atherosclerosis (Fig. 2) and conferred features of plaque stability (Zernecke et al.), highlighting the important functions of microRNAs in atherosclerosis. Less is known about the recruitment and function of T cells and dendritic cell subsets in atherosclerosis. By targeting specific chemokines/cytokines and their receptors in Apoe-/- mice, we will address the functions of different immune cell subpopulations in atherosclerosis. A particular focus will not only be on their interactions at sites of inflammation, but also within lymphatic tissue, and their role in shaping specialized immune responses that control the development of atherosclerosis. Furthermore, we will investigate the localization of these cells in the vessel wall and their routes of entry during lesion formation. Given the remarkable role of adaptive and innate immunity in atherosclerosis, targeting of its cellular constituents and understanding the complex equilibrium and interplay between immune cell subpopulations that contribute to the process of atherosclerosis will be important to identify new therapeutic approaches for treating this disease. Fig. 2: PBS or apoptotic bodies protect against early atherosclerotic lesion formation. Apoe-/- mice were fed a high-fat diet for 6 weeks and injected twice weekly with PBS or AB. Representative images of aortic roots stained by Oil-red-O to detect atherosclerotic lesion areas. Extramural Funding DFG (SFB688, TP A12, ZE 827/1-1, ZE827/4-1) Selected Publications Koenen, R.R., Pruessmeyer, J., Soehnlein, O., Fraemohs, L., Zernecke, A., Schwarz, N., Reiss, K., Sarabi, A., Lindbom, L., Hackeng, T.M., and Weber, C. (2009) Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases. Blood, 113, 4799-809. Shagdarsuren, E., Djalali-Talab, Y., Aurrand-Lions, M., Bidzhekov, K., Liehn, E.A., Imhof, B.A., Weber, C., and Zernecke, A. (2009) Importance of junctional adhesion molecule-C for neointimal hyperplasia and monocyte recruitment in atherosclerosis-prone mice. Arterioscler Thromb Vasc Biol, 29, 1161-3. Koenen, R.R., von Hundelshausen, P., Nesmelova, I.V., Zernecke, A., Liehn, E.A., Sarabi, A., Kramp, B.K., Piccinini, A.M., Paludan, S.R., Kowalska, M.A., Kungl, A.J., Hackeng, T.M., Mayo, K.H., and Weber, C. (2009) Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med, 15, 97-103. Landsman, L.*, Bar-On, L.*, Zernecke, A.*, Kim, K.W., Krauthgamer, R., Shagdarsuren, E., Lira, S.A., Weissmann, I.L., Weber, C., and Jung, S. (2009) cx3cr1 is required for monocyte homeostasis and atherogenesis by promoting cell survival. Blood, 113, 963-72. *equal contribution. Zernecke, A., Bidzhekov, K., Noels, H., Shagdarsuren, E., Gan, L., Denecke, B., Hristov, M., Köppel, T., Nazari Jahantigh, M., Lutgens, E., Wang, S., Olson, E.N., Schober, A., and Weber, C. (2009) Delivery of microRNA-126 apoptotic bodies induces CXCL12-dependent vascular protection . Science Sign, 2; ra 81. 25