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.

Figure 1

P2X₄ is expressed murine atherosclerosis. LDL-R^−/− mice consumed either a high cholesterol diet (n = 16) or a chow diet (n = 14) for 16 weeks. After diet, RNA was isolated from aortic arches and P2X₄ expression was assessed by quantitative polymerase chain reaction (A). Aortic roots (n = 10) were stained with anti-P2X4, and P2X₄-positive staining area were quantified within the aortic sections (B, C). Distribution of P2X₄ in atherosclerotic lesions from LDLR^−/− mice (n = 15) was analyzed by 3-colour immunofluorescence staining for cell nuclei (DAPI, blue), endothelial cells (CD 31, green) and P2X₄ (red). Representative sections are shown in the merged images (D). Results are presented as mean ± SEM. Statistical significance was calculated using unpaired t-test (parametric data) or Mann–Whitney-U-test (non-parametric data). *p < 0.05; ****p < 0.0001.

Figure 2

P2X₄-deficiency reduces atherosclerosis. P2X₄^−/− LDLR^−/− mice (n = 24) and P2X₄^+/+ LDLR^−/− mice (n = 14) were fed a high-cholesterol diet for 16 weeks. Atherosclerotic lesion size in aortic roots (A), aortic arches (P2X₄^−/− LDLR^−/− n = 21, P2X₄^+/+ LDLR^−/− n = 15) (B) and abdominal aortas (P2X₄^−/− LDLR^−/− n = 20, P2X₄^+/+ LDLR^−/− n = 11) (C) was determined by histochemistry, representative images are shown. Results are presented as mean ± SEM. Statistical significance was calculated using unpaired t-test (parametric) or Mann–Whitney-U-test (non-parametric data). *p < 0.05; **p < 0.01.

Figure 3

P2X₄-deficiency limits leukocyte rolling and reduces endothelial VCAM-1 expression. P2X₄-deficient (n = 6) and P2X₄–competent mice (n = 5) were intraperitoneally stimulated with ATP. 2 h after stimulation, leukocyte rolling and adhesion was assessed by intravital microscopy (A, B). Representative images of leukocyte rolling are presented (C). Endothelial expression of VCAM-1 (red) (D) and ICAM-1 (red) (E) was assessed by 3-colour-immunofluorescence with additional stainings for cell nuclei (DAPI, blue) and endothelial cells (anti-CD31, green), representative images of merged sections are shown (D, E). Statistical significance was calculated using unpaired t-test. **p < 0.01.

Figure 4

P2X₄-deficiency reduces expression release of inflammatory cytokines. Atherosclerotic aortic roots from P2X₄^−/− LDLR^−/− mice (n = 24) and P2X₄^+/+ LDLR^−/− mice (n = 15) after 16 weeks of high-cholesterol diet were stained for FLICA-positive cells (FLICA-fmk, green) and cell nuclei (DAPI, blue). Representative images are shown (A). Frequency of FLICA-positive cells per DAPI-positive cells was analyzed (B). RNA was isolated from atherosclerotic lesions from aortic arches of P2X₄^−/− LDLR^−/− mice (n = 12) and P2X₄^+/+ LDLR^−/− mice (n = 13). Two-step multiplex TaqMan RT-PCR was performed to determine expression of cytokines and adhesion molecules. Expression fold change was calculated by ddCt method, results were referred to β-Actin as the housekeeping gene (C). BMDMs were isolated from the bone marrow of 8-week-old P2X4-competent (n = 5) and P2X4-deficient mice (n = 5). Fully differentiated BMDMs were first stimulated with 100 ng/mL LPS for 4 h, followed by stimulation with either 100 µM or 5 mM ATP for 1 h. Multiplex fluorescence-encoded beads assay was performed to determine concentrations of inflammatory cytokines (D). Results are presented as mean ± SEM. Statistical significance was calculated using Shapiro–Wilk Test followed by an unpaired t-test for parametric or Mann–Whitney-U-test for non-parametric data. *p < 0.05; **p < 0.01; ***p < 0.001.