Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion

Abstract

Rationale: Neutrophils likely contribute to the thrombotic complications of human atheromata. In particular, neutrophil extracellular traps (NETs) could exacerbate local inflammation and amplify and propagate arterial intimal injury and thrombosis. PAD4 (peptidyl arginine deiminase 4) participates in NET formation, but an understanding of this enzyme's role in atherothrombosis remains scant.

Objective: This study tested the hypothesis that PAD4 and NETs influence experimental atherogenesis and in processes implicated in superficial erosion, a form of plaque complication we previously associated with NETs.

Methods and results: Bone marrow chimeric Ldlr deficient mice reconstituted with either wild-type or PAD4-deficient cells underwent studies that assessed atheroma formation or procedures designed to probe mechanisms related to superficial erosion. PAD4 deficiency neither retarded fatty streak formation nor reduced plaque size or inflammation in bone marrow chimeric mice that consumed an atherogenic diet. In contrast, either a PAD4 deficiency in bone marrow-derived cells or administration of DNaseI to disrupt NETs decreased the extent of arterial intimal injury in mice with arterial lesions tailored to recapitulate characteristics of human atheroma complicated by erosion.

Conclusions: These results indicate that PAD4 from bone marrow-derived cells and NETs do not influence chronic experimental atherogenesis, but participate causally in acute thrombotic complications of intimal lesions that recapitulate features of superficial erosion.

Keywords: acute coronary syndrome; atherosclerosis; endothelial cells; granulocytes; thrombosis.

Figure 1. Localization of NETs in human atheromata

(A) A region of carotid artery plaque with a “rupture-prone” morphology contains neutrophils (CD66b) colocalizing with neutrophil elastase (NE) and citrullinated histone H4 (H4cit). The panels on the right show H4cit, NE and DAPI immunofluorescence within the intima. (B) Carotid artery plaques with erosion-prone morphology stained by immunohistochemistry for neutrophils (CD66b), neutrophil elastase (NE), and citrullinated histones (H4cit). The panels on the right show H4cit, NE and DAPI positive NET structures by immunofluorescence at the intima surface. Scale bar: 150 μm. (C) Shows a representative longitudinal section of a human left coronary artery stained for CD31, H4cit and DNA (DAPI). Elastin autofluorescence appears in white. The four insets show specific areas containing NETosing neutrophils in both plaques (D and E) and near eroded luminal endothelium (F and G), as shown with CD31, H4cit and DAPI staining, and an adjacent section stained for CD31, CD66b and DAPI. Arrows show flow orientation. Arrowheads show the limit of local endothelial denudation. Stars indicate lumen. NET visualization in space after 3D reconstruction from plaques (H) and eroded endothelium (I).

Figure 2. Bone marrow PAD4 deficiency does not influence plaque formation and development in LDLR deficient mice

(A) Experimental design summarizing the procedure. (B) En face visualization of Oil red O stained aortas isolated from Ldlr^−/− mice with WT or Pad4^−/− deficient bone marrow, after 5 or 10 weeks of high fat diet, and aortic root plaque size was quantified (C). Quantification of root plaque size (D). Staining and quantification of lipids (Oil red O, E,F), macrophages (Mac3, G,H), smooth muscle cells (αSMA, I,J), and collagen (Sirius red, K,L). Each data point represents one mouse. Data are expressed as mean (red bar) ±SEM.

Figure 3. Impaired NETosis in atheromata of mice lacking PAD4 in bone marrow-derived cells

(A) Experimental design summarizing the procedures. (B) Immunofluorescent staining for H4cit, neutrophils (ly6G), and DNA (DAPI) in the brachiocephalic artery (top), and in the aortic root (bottom), in mice reconstituted either with WT (left) or PAD deficient bone marrow (right). Arrows show neutrophils. Dashed lines delimit the intima in aortic roots. (C) 3D reconstruction of H4cit/DAPI positive NETs in aortic root from WT bone marrow mice. Quantification of H3cit (D and E) and Ly6G (F and G) in the brachiocephalic artery (D and F) or the aortic root (E and G). (F) or the aortic root (G). (H) Adhesion of neutrophils isolated from WT or Pad4^−/− mice to endothelial cells resting or activated with TNFα. The right panel shows the quantification of the neutrophil-related fluorescence. Each point represents one mouse. *P<0.05. ****P<0.0001. Mann–Whitney U test.

Figure 4. Arterial flow perturbation in experimentally expanded intimas promote the recruitment of neutrophils undergoing NETosis

(A) Experimental design summarizing the procedure to examine the effects of introduction of flow disturbance over an expanded intima “tailored” to replicate features of eroded human plaques to influence acute neutrophil recruitment to the luminal surface. In this and panel E the scale bars indicate 100μm. (B) Immunostaining showing luminal citrullinated Histone H3 (H3cit) and (C) quantification of the signal 1 hour after introduction of the flow disturbance. (D) Positive correlation between the number of adherent neutrophils and the H3cit signal 1 hour after flow perturbation. (E) Immunofluorescent staining localizing H3cit in recruited Ly6G⁺ cells. Scale bar: 25μm. (F) Transmission electron microscopy shows extracellular granulocytic content adjacent to luminal endothelial cells 1h after flow perturbation. Arrow shows disrupted endothelial junction, ni indicates neointima; ec, endothelial cell, and lum, lumen. (G) En face immunofluorescent staining for Ly6G and DNA (DAPI) shows extruding DNA from Ly6G⁺ cells on the luminal surface after flow perturbation. (H) Comparison between areas upstream and downstream of the partial stenosis showing NETs (delineated by H3cit/DAPI co-staining) in the vicinity of the endothelium (left), their release by Ly6G⁺ cells (center) and the colocalization of MPO with ly6G⁺ cells (right). Arrow shows NETs filaments. Scale bar: 10μm. (I) Circulating H3cit concentrations in mice from groups NC, CC, or from mice undergoing a sham procedure. *P<0.05 and **P<0.01. Mann–Whitney U test. Each point represents one mouse.

Figure 5. Abrogation of the capacity to form NETs decreases endothelial injury, death and thrombosis in arteries with expanded intimas exposed to flow disturbance

(A) Experimental protocol and time points studied: Mice underwent a LCCA injury and were lethally irradiated 4 weeks later. Mice were immediately reconstituted with either WT or Pad4^−/− bone marrow cells. After 2 weeks, flow perturbation was induced for 6 hours in the previously injured LCCA using a constrictive cuff (CC). (B) Circulating H3cit levels were quantified in mice from both groups. (C) LCCA isolated from Ldlr^−/− mice reconstituted with either WT or Pad4^−/− bone marrow cells were probed for endothelial permeability using Evans blue intravital staining after 6 h of flow perturbation. (D) Visualization by immunofluorescence of luminal NETosis using an anti-H3Cit and a DAPI staining with either CD31 (left panel) or Ly6G (right panel) immunolocalization, in Ldlr^−/− mice reconstituted with WT (top) or Pad4^−/− bone marrow (bottom). White arrows show accumulated neutrophils undergoing NETosis. (E) CD31 immunoreactivity shows endothelium. The graph (right) quantitates endothelial continuity. The star shows a neutrophil-rich fresh thrombus. (F) Fibrinogen staining shows preferential thrombus formation on the intimal surface in mice reconstituted with WT bone marrow compared to those with Pad4^−/− bone marrow. The graph (right) shows the quantification of intimal fibrinogen staining. *P<0.05. (G) Tissue factor (TF) staining shows higher content in tissue factor in the lumen of the LCCA subjected to flow perturbation in WT>Ldlr^−/− vs Pad4^−/−>Ldlr^−/−. (H) Experimental procedure showing the use of DNAseI in mice subjected to LCCA injury and flow perturbation. (I) Endothelial lining, neutrophil and DNA accumulation was probed in mice treated with DNAseI or vehicle, using a 3D reconstruction, and endothelial continuity was quantified in (J). (K) IF and quantification showing TUNEL+ luminal endothelial cells in DNaseI or vehicle treated mice. Each point represents one mouse. Arrows show TUNEL+ endothelial cells. Dashed lines show internal elastic laminae. *P<0.05 and **P<0.01. Mann–Whitney U test.