Fig 2: Atheroprone flow mediated inflammatory signalling is regulated by P2X7 responses. qPCR analysis of E-selectin (A) and IL-8 (B) in HUVEC conditioned with atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) (n = 5, * indicates P = 0.05 using a two-way ANOVA). (C) IL-8 release measured by ELISA in the supernatant of HUVEC conditioned under atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) (n = 9, * indicates P = 0.05, *** indicates p = <0.001 and **** indicates P = 0.0001 using a two-way ANOVA). (D) Representative western blot and densitometry for p38 phosphorylation following 5- or 15-min treatment of BzATP (300 µM) in atheroprotective or atheroprone flow conditioned HUVEC (n = 4, * indicates P = 0.05, ** indicates P = 0.01, *** indicates P = 0.001 and **** indicates P = 0.0001 using a two-way ANOVA). (E) Representative western blot and densitometry of p38 phosphorylation in atheroprone or atheroprotective flow conditioned HUVEC using the orbital shaker system ± 30-min treatment with the P2X7 inhibitor 10 µM A438079 (P2X7i) (n = 5, ** indicates P = 0.01, **** indicates P = 0.0001 using a two-way ANOVA). Values are mean ± S.E.M.

Fig 3: P2X7 regulates E-selectin expression at sites susceptible to atherosclerosis in vivo. Representative en face immunostaining for E-selectin (red) on wildtype or P2X7-/- BALB/c mice at atheroprotective (outer curvature) and atheroprone (inner curvature) sites of the aorta (n = 4–5) (scale = 25 µm). Endothelial cells were identified by staining with CD31 (green) and cell nuclei were stained using TO-PRO (blue). E-selectin expression was analysed by measuring the relative fluorescent intensity at sites of protected or prone to atherosclerosis (n = 4–5, * indicates P = 0.05, ** indicates P = 0.01 and *** indicates P = 0.001 using a two-way ANOVA). Relative fluorescent intensities for P2X7 were corrected against the relative fluorescent intensity of IgG. Values are mean ± S.E.M.

Fig 4: Vaspin inhibits TNFa-induced expression of adhesion molecules. The effect of vaspin on TNFa-induced expression of ICAM-1 (A), VCAM-1 (A), E-selectin (B), and MCP-1 (B). The protein level and mRNA expression of each adhesion molecule was measured at 24 hr after treatment with 100 ng/mL vaspin in the presence or absence of 10 ng/mL TNFa. Data shown are representative Western blots (top panels) and mean values ± SEM of three independent experiments (bottom panels). The fold change in mRNA expression compared to levels in untreated cells is shown. *p < 0.05 vs. untreated cells, and †p < 0.05 vs. cells treated with TNFa alone.

Fig 5: Adhesion protein expression in BMT endothelial GSK3α/β knockout mice. (A) Representative images of stained cross sections of aortic sinus from 15 week old BMT endothelial GSK3α/β knockout and BMT control mice. Red arrow indicates atherosclerotic plaque. Scale bar represents 100 μm. Quantification of percentage of endothelium showing immunofluorescent staining of P-selectin (B) E-selectin (C) and VCAM-1 (D). (E) Quantification of immunohistochemistry staining of ICAM-1 expression on the endothelium. n = 5–8 (B–E), ** p < 0.005, *** p < 0.0005. ns = not significant.