Fig 1.
Physical and biological characterization of hr-anxA1.
(A) Hr-anxA1 induced concentration dependent calcium flux in FPR2 transfected HEK-293 cells, 1 μM ionomycin is taken as reference value (100%). (B) Ellipsometry analysis shows calcium dependent binding of 1 µg/ml purified annexin to a 20/80 mol% PS/PC bilayer. (C) Internalization of fluorescent annexin by apoptotic Jurkat cells in presence and absence of inhibitors of FPR1 (cyclosporin H, 1 μM) and FPR2/3 (WRW, 10 μM) as analyzed by flow cytometry. Mean fluorescence intensity (MFI) is normalized to MFI of annexin internalization on ice. (D) Hr-anxA1 internalization (green) as visualized by fluorescent confocal laser scanning microscopy (CLSM). Nuclei are stained with DAPI (blue) and PS-expression is stained with anxA5-AF568 (red). PS negative cells (indicated with white arrow) did not internalize hr-anxA1. (E) Pretreatment of PMN with 10 nM hr-anxA1 inhibited both rolling and adhesion of PMN on a TNF-α activated HUVEC monolayer. (F) One frame out of 32 is shown of a flow chamber model with rolling and adhering fluorescent THP-1 monocytes, flowing over TNF-α activated HUVEC monolayer. Rolling and adhering THP-1 monocytes are indicated by white and red arrows respectively. (G) Pretreatment of THP-1 cells with 10 nM hr-anxA1 has effect on neither rolling nor adhesion of THP-1 cells. All values are represented as mean ± SEM of 3 independent experiments.
Table 1.
Baseline characteristics of control and hr-anxA1 treated mice.
Fig 2.
Hr-anxA1 has no significant effect on early plaque development.
12 weeks old mice were fed WTD during 6 weeks. Hr-anxA1 treatment started at start of WTD. (A) Representative H&E staining of aortic arches after 6 weeks of treatment. (B) Treatment with hr-anxA1 did not affect plaque burden in the arch and subclavian (SA) brachiocephalic (BCA) and left common carotid artery (CA). (C) Individual plaque stability and progression was scored on following parameters: neutrophil and macrophage content, apoptosis and necrosis, cap thickness and calcification status (see S1 and S2 Tables). Hr-anxA1 treatment had no effect on early plaque stability and progression. (D, E) Hr-anxA1 treatment had no effect on endothelial ICAM-1 expression of IX and SL lesions. IX: intimal xanthoma; SL: small lesion; IL: intermediate lesion; AL: advanced lesion. All values are represented as mean ±SEM, n = 12 animals per group. Panel A: 40x magnification, scale bar represents 500μm. Panel D: 200x magnification, scale bar represents 100μm.
Fig 3.
Hr-anxA1 attenuates progression into advanced plaques.
(A) 12 weeks old mice were fed WTD 12 weeks. During the last 6 weeks mice were treated with hr-anxA1 or vehicle (ctrl). (A) Representative H&E staining of aortic arches. (B) Treatment with hr-anxA1 significantly reduced total plaque burden in the inner arch (arch) and subclavian artery (SA) but not in the brachiocephalic (BCA) and left common carotid artery (CA). (C) Individual plaque progression was scored on following parameters: neutrophil and macrophage content, apoptosis and necrosis, cap thickness and calcification status (see S1 and S2 Tables). (D, E) Endothelial ICAM-1 expression was reduced in early plaque development (IX/SL) after anxA1 treatment. (F, G) Macrophage and (H, I) smooth muscle cell content were comparable between anxA1 and vehicle treated controls. IX: intimal xanthoma; SL: small lesion; IL: intermediate lesion; AL: advanced lesion. All values are represented as mean ±SEM, n = 12 animals per group. Panel A: 40x magnification, scale bar represents 500μm; Panel D,F: 100x magnification, scale bar represents 200 μm; Panel H: 200x magnification, scale bar represents 100 μm.
Table 2.
Flow cytometry analysis of blood and bone marrow.
Fig 4.
Hr-anxA1 does not affect macrophage polarization in atherosclerotic plaque.
(A) Representative staining for M1 macrophage specific marker iNOS. Black arrows indicate fully commited M1 macrophages, white arrows indicate iNOS negative macrophages. (B) Quantification of iNOS-staining indicates no difference between control and anxA1 treated animals. (C) Representative staining of M2 specific marker Ym1. Both control and hr-anxA1 treated mice were negative for Ym1. (D) Staining of Ym1 positive cells in the spleen indicating functionality of anti-Ym1 antibody. All values are represented as mean ±SEM, n = 12 animals per group. Panel A: 200x magnification, scale bar represents 100μm; panel D: 40x and 400x magnification, scale bar represent 500μm and 50μm respectively.