Fig 2: Pharmacological MAP4K4 inhibition ameliorates atherosclerosis.(a–c) ApoE−/− mice were administered PF-06260933 or vehicle twice daily for 6 weeks. (a) Dosing regimen (b) Oil red-O-stained en face aortic preparations from vehicle and PF-06260933-treated animals. (c) Quantification of the Oil Red-O-stained area. Data represent the mean±s.e.m. (*P<0.05, N=14–15). (d–f) Ldlr−/− mice were fed HFD for 10 weeks followed by PF-06260933 or vehicle administration daily for 10 additional weeks. (d) Dosing regimen. (e) Oil Red-O-stained en face aortic preparations from vehicle and PF-06260933-treated animals. (f) Quantification of the Oil Red-O-stained area. Data represent the mean±s.e.m. (**P<0.005, N=6).

Fig 3: Endothelial MAP4K4 promotes EC activation via NF-κB.HUVECs were treated with scrambled or MAP4K4 siRNA, and cells were stimulated with 1 or 10 ng ml−1 TNF-α for the indicated times. (a) Cells were seeded onto transwell chambers. Confluent cells were treated overnight with 10 ng ml−1 TNF-α or left untreated, and FITC-labelled dextran that migrated through the HUVEC monolayer was measured. The data represent the mean fluorescence intensity±s.e.m. (analysis of variance (ANOVA) *P<0.05, N=4). (b) THP-1 monocytes were stained with calcein green and adhered to activated endothelium for 30 min. Fluorescence microscopy was performed to determine the number of adherent THP-1 monocytes per microscopic field (× 100). The data represent the mean±s.e.m. as normalized to the unstimulated control time point (ANOVA **P<0.01, ****P<0.0001, N=5). (c–f) Messenger RNA was extracted and quantitative RT–PCR was performed for (c) MAP4K4, (d) ICAM-1, (e) VCAM-1 and (f) SELE. The data represent the mean±s.e.m. as normalized to RPLP0 (*P<0.05, **P<0.005, #P<0.0001, N=5–7). (g) Immunoblots were performed for ICAM-1, VCAM-1 and E-selectin. (h) Densitometric analyses from g. The data represent the means±s.e.m. as normalized to VE-cadherin (*P<0.05, N=4–6). (i) Biochemical fractionations were performed, and nuclear fractions were immunoblotted for MAP4K4, p-p65, total p65, p50 and lamin-β1. (j) Densitometric analyses represent the mean±s.e.m. for the 60-min time point as normalized to lamin-β1 (*P<0.05, **P<0.005, N=3). (k) NFκB-luciferase and SV40-Renilla were transfected into HUVECs after treatment with scrambled or MAP4K4 siRNA. Cells were left unstimulated or stimulated with 10 ng ml−1 TNF-α overnight before luciferase and Renilla measurement. The data represent the mean±s.e.m. from four independent experiments. (l–n) HUVECs were transfected with MAP4K4 siRNA and stimulated or not with 1 ng ml−1 TNF-α for 1 h. IgG, p65 and histone antibodies were used to immunoprecipitate chromatin and RT–PCR was used to amplify (l) E-selectin, (m) VCAM-1 and (n) IκBα promoters. The data represent the mean±s.e.m. as normalized to input (*P<0.05, N=3–4).

Fig 4: Reduced atherosclerosis in M4K4 KD mice.(a) Schematic of the transgenic construct used to generate MAP4K4 KD animals. The U6 promoter becomes reconstituted after cre-mediated recombination to drive tissue-specific shRNA expression. (b,c) Primary lung endothelial cells (MLECs) were derived from control and MAP4K4 KD animals. (b) Messenger RNA (mRNA) was extracted and quantitative RT–PCR was performed for Map4k4 in immune-selected or unselected cells. The data represent the mean±s.e.m. as normalized to 36b4 expression (*P<0.05, N=7). (c) Primary endothelial or unselected cell lysates were immunoblotted for Map4k4 and Vegfr2. Data represent the mean±s.e.m. as normalized to tubulin expression (*P<0.05, N=3–6). (d–g) MAP4K4 KD mice were crossed with Apoe−/− mice and fed a WD for 16 weeks. (d) Aortic root sections of control and MAP4K4 KD animals stained with haematoxylin and eosin (H&E) (top) and Oil Red-O (bottom). Scale bar, 250 μm. (e) Quantification of aortic root lesion area. Data represent the mean±s.e.m. (**P<0.005, N=9,11). (f) Oil Red-O-stained en face aortic preparations from control and MAP4K4 KD animals. (g) Quantification of Oil red-O-stained area. Data represent the mean±s.e.m. (*P<0.05, N=5–7). (h–i) mRNA was prepared from whole aortas, and qPCR was performed. (h) Macrophage markers F4/80 and Cd68. (i) Chemokines Ccl2, Cxcl1, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl9 and Cxcl10. Data represent the mean±s.e.m. as normalized to 36b4 (*P<0.05, **P<0.005, N=9–10).

Fig 5: Less macrophages in plaques from M4K4 ECKO and KD mice.(a,b) Messenger RNA was prepared from whole aortas, and qPCR was performed. (a) Macrophage markers F4/80 and Cd68. (b) Chemokines Ccl2, Cxcl1, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl9 and Cxcl10. Data represent the mean±s.e.m. as normalized to 36b4 (*P<0.05, **P<0.005, N=9–10). (c,d) Homing of GFP leukocytes into atherosclerotic lesions 48 h after intravenous injection into control or MAP4K4 KD mice that were fed WD for 16 weeks. (c) Fluorescence micrograph of atherosclerotic plaque demonstrating four GFP leukocytes within the aortic arch. The dashed line indicates the plaque border. Inset, magnification of three GFP leukocytes. Left, 4,6-diamidino-2-phenylindole; middle, GFP; right, merge. Scale bars, 100 μm. (d) Quantification of GFP leukocytes per square millimetre of plaque. Data represent the mean±s.e.m. (**P<0.005, N=4,7).