The role of the purinergic receptor P2X7 in the pathogenesis of atherosclerosis

S. von Garlen, A. Heidenreich, P. Stachon, J. Merz, S. Geis, B. Dufner, N. Hoppe, N. Anto Michel, C. Bode, M. Idzko, A. Zirlik (Freiburg, Germany)

Vascular Wall
Date: 17.02.2017,
Time: 08:00 - 09:15

Objective: The P2X7 receptor plays a crucial role during inflammasome assembly and immune responses and might show a direct correlation to the pathophysiology in the chronic disease atherosclerosis. The objective of this study was to determine whether the ubiquitous knock out (ko) of P2X7 influences the outcome of atherosclerosis in mice to further investigate its role in future therapeutic target.

Methods: The P2X7-LDLR double ko (DKO) mice and LDLR ko mice were fed a high cholesterol or chow diet for 16 weeks. The P2X7 expression in atherosclerosis was determined by qPCR and immunohistochemistry. Plaque size was assessed by histological quantification via ImagePro. Intravital microscopy was performed after intra-peritoneal (i.p.) stimulation with ATP. Boyden Chamber assay was used to assess leukocyte migration. To evaluate Caspase-1 activity in macrophages we used an in vitro luciferin based Caspase-1 Kit and an FLICA fluorescence microscopy staining of atherosclerotic lesions. P2X7 expression in human plaques was assessed by immunohistochemistry.

Results: P2X7 expression was higher mostly in plaque resident macrophages. The atherosclerotic lesions in the aortic arches were smaller in the P2X7 ko compared to P2X7 competent mice (lesions mean P2X7-LDLR DKO = 0.084cm^2, lesions mean LDLR ko = 0.162cm^2; n=20, p=0.004). No Caspase-1 activity could be detected in isolated bone-marrow derived macrophages of P2X7 ko animals upon 4h 1µg/ml LPS stimulation followed by 1h stimulation with 5mM ATP (wt = 11053±1986 RFU (n=3) versus P2X7 ko = 192±131 RFU (n=4), p < 0.01). Fluorescence microscopy revealed a decreased relative proportion of FLICA+ plaque resident cells in P2X7 ko animals (P2X7-LDLR DKO = 2.65%±0.84 (n=9), LDLR-/- = 17.41%±3.87 (n=9), p < 0.01). Upon i.p. ATP administration P2X7 competent mice showed more leukocytes rolling and adhering on the vessel wall compared to P2X7 ko mice. In vitro leukocyte migration was reduced in P2X7 ko compared to P2X7 competent mice after ATP-stimulation. Human plaques showed a strong P2X7 signal via immunohistochemistry.

Conclusion: The ubiquitous depletion of P2X7 ameliorates the outcome of atherosclerosis in mice with reduced plaque size, decreased inflammasome activation and diminished immune cell infiltration. Our current study supports a potential promising effect in the treatment of atherosclerosis by P2X7 antagonism.