P2X4 deficiency reduces atherosclerosis and plaque inflammation in mice

Abstract

Extracellular adenosine-5′-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X₄ and promotes inflammation via increased expression of pro-inflammatory cytokines. Because of the central role of inflammation, we assumed a functional contribution of the ATP-P2X₄-axis in atherosclerosis. Expression of P2X₄ was increased in atherosclerotic aortic arches from low-density lipoprotein receptor-deficient mice being fed a high cholesterol diet as assessed by real-time polymerase chain reaction and immunohistochemistry. To investigate the functional role of P2X₄ in atherosclerosis, P2X₄-deficient mice were crossed with low-density lipoprotein receptor-deficient mice and fed high cholesterol diet. After 16 weeks, P2X₄-deficient mice developed smaller atherosclerotic lesions compared to P2X₄-competent mice. Furthermore, intravital microscopy showed reduced ATP-induced leukocyte rolling at the vessel wall in P2X₄-deficient mice. Mechanistically, we found a reduced RNA expression of CC chemokine ligand 2 (CCL-2), C-X-C motif chemokine-1 (CXCL-1), C-X-C motif chemokine-2 (CXCL-2), Interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) as well as a decreased nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-inflammasome priming in atherosclerotic plaques from P2X₄-deficient mice. Moreover, bone marrow derived macrophages isolated from P2X₄-deficient mice revealed a reduced ATP-mediated release of CCL-2, CC chemokine ligand 5 (CCL-5), Interleukin-1β (IL-1β) and IL-6. Additionally, P2X₄-deficient mice shared a lower proportion of pro-inflammatory Ly6C^high monocytes and a higher proportion of anti-inflammatory Ly6C^low monocytes, and expressend less endothelial VCAM-1. Finally, increased P2X₄ expression in human atherosclerotic lesions from carotid endarterectomy was found, indicating the importance of potential implementations of this study’s findings for human atherosclerosis. Collectively, P2X₄ deficiency reduced experimental atherosclerosis, plaque inflammation and inflammasome priming, pointing to P2X₄ as a potential therapeutic target in the fight against atherosclerosis.