Figure 1.
Eosinophils promote atherosclerotic plaque formation. (A) Atherosclerotic lesion formation (white dotted lines) assessed by lipid staining with Oil-Red O in the aortic root of eosinophil-deficient ApoE−/−;ΔdblGATA1−/− mice (n = 14: 7 female, 7 male) compared with littermate controls (ApoE−/−;ΔdblGATA+/+ (n = 13: 6 female, 7 male) after 13 weeks on an HFD. (B) Quantification of macroscopic lesion formation by Sudan III staining in the aortic arch of ApoE−/−;ΔdblGATA1−/− mice (n = 19: 9 female, 10 male) compared with ApoE−/−;ΔdblGATA+/+ littermate controls (n = 19: 9 female, 10 male). Scale bar, 200 µm. (C) Eosinophils were absent in plaques (white dotted line) of ApoE−/−;ΔdblGATA+/+ mice as assessed by staining for Siglec-F (green), nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Image representative of 3 experiments. (D) ApoE−/−;ΔdblGata+/+ mice (n = 10) showed enlarged areas of macrophages (green) relative to plaque area as well as areas of smooth muscle cells (red) compared with the ApoE−/−;ΔdblGATA1−/− mice (n = 9). (E) Left: Elevated CCL11 serum levels in ApoE−/− mice in response to 12 weeks on an HFD compared with a chow diet (n = 5 each). Right: Representative staining for CCL11 (green) and DAPI (nuclei in blue) in the aortic root of ApoE−/− mice after 12 weeks on an HFD. Dotted line indicates endothelium; solid line indicates internal elastic lamina. Scale bar, 50 µm. (F) Formation of eosinophil-platelet aggregates given as CD41+Siglec-F+ eosinophils of all eosinophils assessed by FACS (n = 5 with an HFD; n = 4 with a chow diet) in ApoE−/− mice after 12 weeks. (G) Platelets of ApoE−/−;ΔdblGATA1−/− mice (n = 5) adhered less often at the uninjured endothelium of the carotid artery under steady-state conditions compared with platelets of ApoE−/−;ΔdblGATA1+/+ mice (n = 6) as assessed by intravital microscopy. (H) Exposure of VWF on the endothelial surface of the intact carotid artery quantified by immunohistology in ApoE−/−;ΔdblGATA1−/− (n = 16) and ApoE−/−;ΔdblGATA1+/+ (n = 13) littermates after 13 weeks on an HFD. (I) Plasma levels of VWF in ApoE−/−;ΔdblGATA1−/− and ApoE−/−;ΔdblGATA1+/+ (n = 5 each) littermates after 13 weeks on an HFD. (J) Endothelial cells exposed VWF in response to supernatant of eosinophils activated with IL-5 and CCL11 compared with supernatant from resting eosinophils or medium with IL-5 and CCL11 (n = 4 each). Tumor necrosis factor α (TNF-α) was used as a positive control. For panels A-B and D-J, data are mean ± standard deviation (SD). *P < .05; **P < .01.

Eosinophils promote atherosclerotic plaque formation. (A) Atherosclerotic lesion formation (white dotted lines) assessed by lipid staining with Oil-Red O in the aortic root of eosinophil-deficient ApoE−/−;ΔdblGATA1−/− mice (n = 14: 7 female, 7 male) compared with littermate controls (ApoE−/−;ΔdblGATA+/+ (n = 13: 6 female, 7 male) after 13 weeks on an HFD. (B) Quantification of macroscopic lesion formation by Sudan III staining in the aortic arch of ApoE−/−;ΔdblGATA1−/− mice (n = 19: 9 female, 10 male) compared with ApoE−/−;ΔdblGATA+/+ littermate controls (n = 19: 9 female, 10 male). Scale bar, 200 µm. (C) Eosinophils were absent in plaques (white dotted line) of ApoE−/−;ΔdblGATA+/+ mice as assessed by staining for Siglec-F (green), nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Image representative of 3 experiments. (D) ApoE−/−;ΔdblGata+/+ mice (n = 10) showed enlarged areas of macrophages (green) relative to plaque area as well as areas of smooth muscle cells (red) compared with the ApoE−/−;ΔdblGATA1−/− mice (n = 9). (E) Left: Elevated CCL11 serum levels in ApoE−/− mice in response to 12 weeks on an HFD compared with a chow diet (n = 5 each). Right: Representative staining for CCL11 (green) and DAPI (nuclei in blue) in the aortic root of ApoE−/− mice after 12 weeks on an HFD. Dotted line indicates endothelium; solid line indicates internal elastic lamina. Scale bar, 50 µm. (F) Formation of eosinophil-platelet aggregates given as CD41+Siglec-F+ eosinophils of all eosinophils assessed by FACS (n = 5 with an HFD; n = 4 with a chow diet) in ApoE−/− mice after 12 weeks. (G) Platelets of ApoE−/−;ΔdblGATA1−/− mice (n = 5) adhered less often at the uninjured endothelium of the carotid artery under steady-state conditions compared with platelets of ApoE−/−;ΔdblGATA1+/+ mice (n = 6) as assessed by intravital microscopy. (H) Exposure of VWF on the endothelial surface of the intact carotid artery quantified by immunohistology in ApoE−/−;ΔdblGATA1−/− (n = 16) and ApoE−/−;ΔdblGATA1+/+ (n = 13) littermates after 13 weeks on an HFD. (I) Plasma levels of VWF in ApoE−/−;ΔdblGATA1−/− and ApoE−/−;ΔdblGATA1+/+ (n = 5 each) littermates after 13 weeks on an HFD. (J) Endothelial cells exposed VWF in response to supernatant of eosinophils activated with IL-5 and CCL11 compared with supernatant from resting eosinophils or medium with IL-5 and CCL11 (n = 4 each). Tumor necrosis factor α (TNF-α) was used as a positive control. For panels A-B and D-J, data are mean ± standard deviation (SD). *P < .05; **P < .01.

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