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Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice

Abstract

Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment1,2,3,4. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium5,6,7,8,9. 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 a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions10,11,12,13. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis9,14; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions15,16. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.

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Figure 1: Role of blood cell–derived CCL5 and CXCL4 in diet-induced atherogenesis.
Figure 2: NMR spectroscopic analysis of CXCL4 and CKEY2 interactions with CCL5.
Figure 3: In vitro characterization of the peptide antagonists.
Figure 4: Disruption of CCL5-CXCL4 heteromer formation inhibits atherosclerosis in mice.

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Acknowledgements

This study was supported by grants from the Deutsche Forschungsgemeinschaft (WE1913/5-2, WE1913/7-1 to C.W., KO2948/1-1 to R.R.K., HU1618/1-1 to P.v.H., ZE827/1-1 to A.Z. and FOR809 to R.R.K., P.v.H., A.Z. and C.W.), the Interdisciplinary Center for Clinical Research “Biomat” within the Medical Faculty of RWTH Aachen University (TV-B112 and TV-B113 to R.R.K. and C.W.), Nederlandse Organisatie voor Wetenschappelijk Onderzoek (VIDI 917.36.372 to T.M.H.) and the US National Institutes of Health (National Research Service Award training grant HL 07062 to I.V.N.). We thank S. Meiler, S. Winkler, J. Tupiec, S. Knarren, M. Garbe, S. Wilbertz, D. Suylen and W. Adriaens for technical assistance. Computer resources were provided by the Minnesota Supercomputing Institute (University of Minnesota). NMR instrumentation was provided with funds from the US National Science Foundation (BIR-961477), the University of Minnesota Medical School and the Minnesota Medical Foundation. Met-RANTES was provided by P. Nelson (University of Munich). NMR chemical-shift assignments for the CCL5 monomer state were provided by S. Grzesiek (University of Basel).

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Authors

Contributions

R.R.K. designed the peptides; designed, supervised and conducted experiments; and wrote the paper. P.v.H. designed and conducted experiments. I.V.N. designed, conducted and analyzed NMR studies. A.Z. designed, conducted and supervised animal experiments. E.A.L. conducted animal experiments. A.S. and B.K.K. expressed and purified recombinant proteins. A.M.P. conducted biophysical experiments. M.A.K. supplied transgenic mice. S.R.P. conducted viral clearance experiments in mice. A.J.K. designed, supervised and analyzed biophysical experiments. T.M.H. synthesized peptide inhibitors. K.H.M. designed, supervised and analyzed NMR studies and wrote the paper. C.W. designed and supervised the study and wrote the paper.

Corresponding author

Correspondence to Christian Weber.

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Koenen, R., von Hundelshausen, P., Nesmelova, I. et al. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med 15, 97–103 (2009). https://doi.org/10.1038/nm.1898

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