Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis

Abstract

Rationale: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation.

Objective: To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis.

Methods and results: Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression.

Conclusions: Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

Keywords: atherosclerosis; immunology; interferon-α; neutrophils; protein-arginine deiminase; thrombosis.

Figure 1. Peptidylarginine deiminase (PAD) inhibition with Cl-amidine reduces atherosclerosis and arterial thrombosis in Apoe^−/− mice

Apoe^−/− mice exposed to high-fat chow were treated with vehicle or Cl-amidine from 7 to 18 weeks of age (n=10/group). A, Atherosclerotic lesions were quantified in arterial trees after en face Oil Red O staining. B, Representative arterial trees from vehicle-treated (left) and Cl-amidine-treated (right) mice. C, Atherosclerosis was also scored by quantifying the lipid-rich region of the intima (containing foam cells and cholesterol clefts) in cross-section as a percentage of total intimal area. D, Carotid artery thrombosis was induced by photochemical injury, and time to occlusion was determined. E, Correlation between arterial lesion area and time to carotid occlusion (n=20); the best fit line and 95% confidence intervals are plotted. For A, C, and D, boxes represent the median, 25th percentile, and 75th percentile; whiskers delineate the minimum and maximum values. *P<0.05; **P<0.01; ***P<001.

Figure 2. Apoe^−/− mice demonstrate enhanced neutrophil extracellular trap (NET) formation and develop autoantibodies to NETs

A and B, Representative immunofluorescence staining of nonpermeabilized Apoe^−/− neutrophils for citrullinated histone H3 (H3-Cit; A) and MPO (B). DNA is stained blue, and the indicated protein green. Scale bars=10 μm. C, Bone marrow neutrophils were isolated from 8-week-old Apoe^−/− mice. Neutrophils were incubated in the presence of 10% serum from the indicated mice for 4 hours (n=5 mice per group). ***P<0.001. D, Apoe^−/− NETs were fixed and incubated with 1% serum from 18-week-old Apoe^−/− mice (top) or 8-week-old Apoe^−/− mice (bottom). Detection of bound antibodies was with Texas-Red-conjugated anti-immunoglobulin G; DNA is stained blue. Scale bars=50 μm. E, NET proteins were prepared as described in Methods and used to coat plates for the enzyme-linked immunosorbent assay (ELISA). Optical density (OD) index normalizes data to the average value for C57BL/6 mice. Box-and-whisker plots show data for 8 mice per group, with boxes representing the median, 25th percentile, and 75th percentile; whiskers delineate the minimum and maximum values. **P<0.01; ***P<0.001. F, An ELISA for anti-cathelicidin-related antimicrobial peptide (CRAMP) was performed as described in Methods. Some Apoe^−/− mice were placed on high-fat chow (HF) beginning at 8 weeks of age; others remained on regular chow (reg). OD index normalizes data to the average value for control mice. Mean and SEM are plotted, with n≥8 per group. *P<0.05 and ***P<0.001 when compared with C57BL/6 control mice.

Figure 3. Interferon expression and histone citrullination are upregulated in atherosclerotic lesions

A, Apoe^−/− mice were placed on high-fat chow beginning at 8 weeks of age. RNA was isolated from aortic arches of 8- and 18-week-old Apoe^−/− mice. Fold change in gene expression was calculated for 18-week-old mice, relative to 8-week-old mice (n=5). Mean and SEM are plotted. *P<0.05, **P<0.01, and ***P<0.001; P values that did not reach significance are indicated. B, Protein was prepared from aortic arches of the indicated Apoe^−/− mice. Protein from 5 mice per group was pooled and 20 μg of total protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before Western blotting with the indicated antibodies. C, Low magnification view of an atherosclerotic lesion with a phase-contrast image showing intima (I), media (M), and adventitia (A). DNA is stained blue, and MPO is stained green, with an overlay to the far right. Scale bar=100 μm. D, A higher magnification view of the media/adventitia interface shows an MPO-positive cell in more detail. Extracellular MPO (green) juxtaposed with decondensed DNA (blue) is seen (red arrowhead); in the middle, the DNA channel is shown in grayscale to improve contrast. Scale bar=20 μm.

Figure 4. Cl-amidine abrogates neutrophil extracellular trap (NET) formation, but not H₂O₂ production or L-selectin shedding

A, Bone marrow neutrophils were isolated from 8-week-old Apoe^−/− mice. Neutrophils were stimulated with phorbol-12-myristate-13-acetate (PMA) in the presence or absence of 200 μmol/L Cl-amidine, and NET formation was quantified by immunofluorescence microscopy. B, Cl-amidine treatment does not alter H₂O₂ production. Apoe^−/− bone marrow neutrophils were stimulated with PMA in the presence of inhibitors as indicated. The PMA-stimulated sample was arbitrarily set at 100% H₂O₂ production; statistical comparisons are to this group. DPI=NADPH oxidase inhibitor. C, Cl-amidine treatment does not alter L-selectin shedding. Neutrophils were stimulated with PMA in the presence of inhibitors as indicated. Surface staining was then with anti-Ly-6G (to confirm the identity of neutrophils) and anti-L-selectin, before analysis by flow cytometry. Data are presented as the percentage of Ly-6G+ that are also L-selectin+. All experiments were repeated at least 3 times. **P<0.01; ***P<0.001.

Figure 5. Peptidylarginine deiminase (PAD) inhibition abrogates neutrophil extracellular trap (NET) formation and alters anti-NET autoantibody profiles in Apoe^−/− mice

High-fat chow-fed Apoe^−/− mice were treated with vehicle or Cl-amidine from 7 to 18 weeks of age. A, Body weight was recorded at the indicated time points. B, Serum was collected at 18 weeks of age and total cholesterol, triglycerides, and HDL were determined by direct measurement; LDL was calculated. *P<0.05; no other comparison of vehicle versus Cl-amidine was significant C, Bone marrow neutrophils were isolated at 18 weeks of age and stimulated with phorbol-12-myristate-13-acetate (PMA). **P<0.01. D, Neutrophil-platelet aggregates (NPA) were determined in blood at 18 weeks of age. NPA were defined as events positive for both Ly-6G and CD61. The percentage is calculated relative to total Ly-6G-positive cells. E, Serum cytokine levels were measured by multiplex assay; ns=not significant. A-E, n=10 per group. For A, C, D, and E, the mean and SEM are plotted. For B, boxes represent the median, 25th percentile, and 75th percentile; whiskers delineate the minimum and maximum values.

Figure 6. Peptidylarginine deiminase (PAD) inhibition reduces the recruitment of netting neutrophils to the media and adventitia of Apoe^−/− aortic sinus lesions

A, Aortic sinuses from the 18-week-old mice presented in Figure 1 were sectioned and stained by immunohistochemistry for neutrophil extracellular traps (citrullinated histone H3 [H3-Cit]), neutrophils (Ly-6G), and macrophages (F4/80). Representative staining is shown, with a neutrophil and H3-Cit-rich infiltrate at the interface between the intima and the media/adventitia (arrows). The inset shows a relatively intact H3-Cit-positive cell with polymorphonuclear morphology (black arrowhead) as well as a cell with decondensed morphology (red arrowhead). Scale bar=250 μm. B, Quantification of H3-Cit-positive cells in the media/adventitia. C, Correlation between the number of H3-Cit-positive cells and arterial lesion area; the best fit line and 95% confidence intervals are plotted. D and E, Quantification of Ly-6G-positive cells in the media/adventitia (D), and F4/80-positive area in the intima (E). B, D, and E, n=10 per group, and mean and SEM are plotted. *P<0.05; ***P<0.001. F, PAD inhibition reduces IFN-α expression in the aortic arch of Apoe^−/− mice. RNA was prepared from aortic arches at 18 weeks of age. The data are expressed as fold change (positive value indicates activation; and negative value, repression) for Cl-amidine-treated mice relative to vehicle-treated mice (n=10 per group). *P<0.05; no other Cl-amidine versus vehicle comparison reached statistical significance. G, Protein was prepared from aortic arches at 18 weeks of age. Each lane represents protein pooled from 5 similarly treated mice, with H3-Cit and α-tubulin detected by Western blotting. Data are plotted as the ratio of H3-Cit density to α-tubulin density for each sample. The Western blot portion of the experiment was performed twice with similar results.

Figure 7. Cl-amidine does not protect against atherosclerosis in neutropenic mice

A, Apoe^−/− mice exposed to high-fat chow were treated with vehicle or Cl-amidine from 7 to 18 weeks of age as indicated. Mice were also treated with either anti-Ly-6G or a control antibody from 8 to 18 weeks of age as indicated. At 18 weeks of age, peripheral blood was collected and anti-Ly-6G-positive cells were determined by flow cytometry as a percentage of total circulating leukocytes. B, Mice were treated as in A, and atherosclerotic lesions were quantified in arterial trees by en face Oil Red O staining. C, Apoe^−/− Ifnαβr^−/− mice lack the type I IFN receptor. These mice were exposed to high-fat chow, and were treated with vehicle or Cl-amidine from 7 to 18 weeks of age as indicated. Atherosclerotic lesions were quantified in arterial trees by en face Oil Red O staining. For all experiments, box- and-whisker plots show data for 10 mice per group, with boxes representing the median, 25th percentile, and 75th percentile; whiskers delineate the minimum and maximum values. *P<0.05; **P<0.01; ns indicates not significant. One P value that approaches significance is denoted.