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. 2021 Apr;19(4):1001-1017.
doi: 10.1111/jth.15236. Epub 2021 Feb 10.

Pharmacological targeting of coagulation factor XI mitigates the development of experimental atherosclerosis in low-density lipoprotein receptor-deficient mice

Anh T P Ngo  1 , Kelley R Jordan  2 , Paul A Mueller  2 , Matthew W Hagen  2 , Stéphanie E Reitsma  1 , Cristina Puy  1 , Alexey S Revenko  3 , Christina U Lorentz  1   4 , Erik I Tucker  1   4 , Quifang Cheng  5 , Monica T Hinds  1 , Sergio Fazio  2 , Brett P Monia  3 , David Gailani  5 , András Gruber  1   4 , Hagai Tavori  2 , Owen J T McCarty  1
Affiliations

Pharmacological targeting of coagulation factor XI mitigates the development of experimental atherosclerosis in low-density lipoprotein receptor-deficient mice

Anh T P Ngo et al. J Thromb Haemost. 2021 Apr.

Abstract

Background: Human coagulation factor (F) XI deficiency, a defect of the contact activation system, protects against venous thrombosis, stroke, and heart attack, whereas FXII, plasma prekallikrein, or kininogen deficiencies are asymptomatic. FXI deficiency, inhibition of FXI production, activated FXI (FXIa) inhibitors, and antibodies to FXI that interfere with FXI/FXII interactions reduce experimental thrombosis and inflammation. FXI inhibitors are antithrombotic in patients, and FXI and FXII deficiencies are atheroprotective in apolipoprotein E-deficient mice.

Objectives: Investigate the effects of pharmacological targeting of FXI in experimental models of atherogenesis and established atherosclerosis.

Methods and results: Low-density lipoprotein receptor-knockout (Ldlr-/- ) mice were administered high-fat diet (HFD) for 8 weeks; concomitantly, FXI was targeted with anti-FXI antibody (14E11) or FXI antisense oligonucleotide (ASO). 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas when compared with controls, and 14E11 also reduced aortic sinus lesions. In an established disease model, in which therapy was given after atherosclerosis had developed, Ldlr-/- mice were fed HFD for 8 weeks and then administered 14E11 or FXI-ASO weekly until 16 weeks on HFD. In this established disease model, 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas, but not in aortic sinus. In cultures of human endothelium, FXIa exposure disrupted VE-Cadherin expression and increased endothelial lipoprotein permeability. Strikingly, we found that 14E11 prevented the disruption of VE-Cadherin expression in aortic sinus lesions observed in the atherogenesis mouse model.

Conclusion: Pharmacological targeting of FXI reduced atherogenesis in Ldlr-/- mice. Interference with the contact activation system may safely reduce development or progression of atherosclerosis.

Keywords: atherosclerosis; contact activation; factor XI; obesity; vascular permeability.

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Conflict of interest statement

CONFLICT OF INTEREST

Dr. Revenko and Dr. Monia are employees of Ionis Pharmaceuticals. Dr. Gruber, Dr. Lorentz, Dr. Tucker, and the Oregon Health & Science University have a significant financial interest in Aronora, Inc., a company that may have a commercial interest in the results of this research. This potential conflict of interest has been reviewed and managed by the Oregon Health & Science University Conflict of Interest in Research Committee. The remaining authors declare no competing financial interests.

Figures

FIGURE 1
FIGURE 1
The anti-FXI mAb 14E11 and FXI-ASO reduce FXI levels in mice. (A) clotting time of C57BL/6 mice injected with a single dose of 14E11, measured by APTT assay. FXI levels in plasma from Ldlr−/− mice after 4 weeks (B) and 8 weeks (C) of HFD and treatments with vehicle, 14E11, or FXI-ASO. (D) Clotting time of human FXI−/− plasma supplemented with increasing concentrations of purified human FXI, measured by APTT assay. Dotted lines indicate 2.75-fold increase in clotting times compared to baseline (day 0) or 30 nM FXI
FIGURE 2
FIGURE 2
Atherosclerosis assessment in 14E11- and FXI-ASO-treated Ldlr−/− mice on 8 weeks of HFD. (A) 14E11 (4 mg/kg) and FXI-ASO (7.5 mg/kg) were administered weekly while Ldlr−/− mice were fed HFD for 8 weeks. (B) Atherosclerotic lesion area in the proximal aortas, quantified under light microscopy. Scale bar =1 mm. (C) Cross-sections of aortic sinus were obtained and atherosclerotic lesion area was determined by ORO staining (red, ×104 μm2). Scale bar = 200 μm. Data were analyzed using Kruskal-Wallis with Dunn post hoc test. *p < .05; **p < .005; ***p < .0001
FIGURE 3
FIGURE 3
Body weight, total cholesterol, and lipid profiles in 14E11- and FXI-ASO-treated Ldlr−/− mice on 8 weeks of HFD. (A) Animal body weight recorded weekly and (B) total cholesterol levels (mg/dl) from Ldlr−/− mice (males and females) at baseline and following 8 weeks of HFD were measured. (C-D) Lipid profiles, performed by FPLC for total cholesterol (μg per FPLC fraction) from pooled plasma at 8 weeks of HFD. (E) Monocyte-platelet aggregates and (F) Ly6Chigh, shown as % of total monocytes at baseline and 8 weeks of HFD in vehicle-, 14E11-, and FXI-ASO-treated animals. Data were analyzed using anova with repeated measures and Kruskal-Wallis with Dunn post hoc test. *p < .05; ***p < .0001 vs. baseline
FIGURE 4
FIGURE 4
Atherosclerosis assessment in 14E11- and FXI-ASO-treated Ldlr−/− mice on 16 weeks of HFD. (A) At 8 weeks of HFD, hyperlipidemic Ldlr−/− mice were randomized and administered 14E11 (4 mg/kg) and FXI-ASO (7.5 mg/kg) weekly until 16 weeks of HFD. (B) Atherosclerotic lesion area in the proximal aortas, quantified under light microscopy. Scale bar = 1 mm. (D) Cross-sections of aortic sinus were obtained and atherosclerotic lesion area was determined by ORO staining (red, ×105 μm2). Scale bar = 200 μm. (D) Total plasma cholesterol at week 8 before randomization into treatment groups and 16 weeks of HFD in Ldlr−/− mice. (E) Monocyte-platelet aggregates and (F) Ly6Chigh, shown as % of total monocytes at 8 weeks of HFD before treatments and 8 weeks of HFD in vehicle-, 14E11-, and FXI-ASO-treated animals. Data were analyzed using Kruskal-Wallis with Dunn post hoc test. **p < .005. ***p < .0001
FIGURE 5
FIGURE 5
Macrophage accumulation and fibrin deposition into atherosclerotic lesions in 14E11- and FXI-ASO-treated Ldlr−/− mice. (A-B) Cross-sections of aortic sinus were obtained and macrophage accumulation was determined by CD68 staining (red) together with nuclei (blue) in the atherogenesis model (A) and established atherosclerosis model (B). (C-D) Cross-sections of aortic sinus were obtained and fibrin deposition was determined by fibrin staining (red) together with nuclei (blue) in the atherogenesis model (C) and established atherosclerosis model (D). Area fraction was calculated as percent lesion area threshold positive after using a Phansalkar local autothreshold with radius of 5 px using Fiji software. Data were analyzed using Kruskal-Wallis with Dunn post hoc test. *p < .05. Scale bar = 200 μm
FIGURE 6
FIGURE 6
VE-Cadherin expression on the surface of endothelial cells following exposure to FXIa. HUVECs were seeded onto gelatin-coated glass coverslips and grown to confluence in a 24-well plate before exposure to vehicle, FXIa (30 nM), FXI (30 nM), α-thrombin (10 nM), or FXIa or thrombin in the presence of PPACK. Cells were fixed and stained for VE-Cadherin (green), together with actin (red), nuclei (blue), and visualized by immunofluorescence microscopy. Scale bar = 50 μm
FIGURE 7
FIGURE 7
Endothelial permeability to lipoproteins in vitro and aortic lesion VE-Cadherin expression in vivo. HUVECs were seeded onto gelatin-coated Transwell devices (upper chamber) before exposure to vehicle, α-thrombin (10 nM), FXIa (30 nM), FXI (30 nM), or FXIa or thrombin in the presence of PPACK for 3 h. (A) Absorbance of labeled BSA measured at 650 nm every 10 min from the lower chamber media. (B) Alexa Fluor 488-conjugated acLDL was added in 0.3% BSA to the upper chamber and media from the lower chamber was collected at 24 h after incubation. Fluorescence was measured at 495/519 Ex/Em and acLDL concentration (ng/ml) was calculated based on standard curve. (C) Cross-sections of aortic sinus were obtained and lesion endothelial barrier integrity was determined by VE-Cadherin (red) and CD31 (green) staining together with nuclei (blue). Scale bar = 200 μm. White arrows indicate regions with disrupted VE-Cadherin staining pattern, White boxes highlight regions chosen for zoomed images. Data were analyzed using one-way anova with Tukey’s post hoc tests to compare treatment groups. *p < .05 thrombin vs. vehicle. #p < .05 FXIa vs. vehicle. **p < .005 FXIa vs. vehicle
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