Grégory Franck

Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked.

Matrix Metalloproteinase-13 Predominates Over Matrix Metalloproteinase-8 as the Functional Interstitial Collagenase in Mouse Atheromata

Objective—Substantial evidence implicates interstitial collagenases of the matrix metalloproteinase (MMP) family in plaque rupture and fatal thrombosis. Understanding the compensatory mechanisms that may influence the expression of these enzymes and their functions, therefore, has important clinical implications. This study assessed in mice the relative effect of the 2 principal mouse collagenases on collagen content and other plaque characteristics. Approach and Results—Apolipoprotein E–deficient (apoE−/−) mice, MMP-13−/− apoE−/−, MMP-8−/− apoE−/− double knockout mice, and MMP-13−/− MMP-8−/− apoE−/− triple knockout mice consumed a high-cholesterol diet for 10 and 24 weeks. Both double knockout and triple knockout mice showed comparable atherosclerotic lesion formation compared with apoE−/− controls. Analysis of aortic root sections indicated that lesions of MMP-8/MMP-13–deficient and MMP-13–deficient mice accumulate more fibrillar collagen than apoE−/− controls and MMP-8−/− apoE−/− double knockout. We further tested the relative effect of MMPs on plaque collagenolysis using in situ zymography. MMP-13 deletion alone abrogated collagenolytic activity in lesions, indicating a predominant role for MMP-13 in this process. MMP-13 and MMP-13/MMP-8 deficiency did not alter macrophage content but associated with reduced accumulation of smooth muscle cells. Conclusions—These results show that among MMP interstitial collagenases in mice, MMP-13 prevails over MMP-8 in collagen degradation in atheromata. These findings provide a rationale for the identification and selective targeting a predominant collagenase for modulating key aspects of plaque structure considered critical in clinical complications, although they do not translate directly to human lesions, which also contain MMP-1.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice - Implications for Superficial Erosion

Rationale: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). Conclusions: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice

Rationale: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). Conclusions: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.

Chemokine (C-X-C Motif) Receptor 4 Blockade by AMD3100 Inhibits Experimental Abdominal Aortic Aneurysm Expansion Through Anti-Inflammatory Effects

Objective— Inflammation plays a critical role in the development of abdominal aortic aneurysms (AAAs). Because stromal cell–derived factor 1 (SDF-1) is known for its ability to attract inflammatory cells, we investigated whether SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis is expressed in aneurysmal aortic wall and plays a role in AAA physiopathology and asked whether its blockade modulates AAA formation and expansion. Approach and Results— Quantitative real-time polymerase chain reaction analysis showed that SDF-1&agr; and CXCR4 mRNA levels are increased in both human and CaCl2-induced mouse AAA wall and are positively correlated to the aortic diameter in mice. ELISA quantification and immunostaining demonstrated that, in mice, aortic SDF-1&agr; is rapidly induced during AAA formation, first by apoptotic vascular smooth muscle cells in the injured media and then by adventitial macrophages once AAA is fully established. Using green fluorescent protein-positive (GFP+/−) bone marrow transplantation experiments, we demonstrated that aortic SDF-1 overexpression is implicated in the recruitment of bone marrow–derived macrophages within the AAA wall. Furthermore, in mice, blockade of CXCR4 by AMD3100 decreases the infiltration of adventitial macrophages, inhibits AAA formation, and prevents aortic wall destruction. AMD3100 reduces the mRNA levels of MMP-12 and MMP-14 as well as that of inflammatory effectors MCP-1, MIP-1&bgr;, MIP-2&agr;, RANTES, IL-1&bgr;, IL-6, TNF-&agr;, and E-selectin. Finally, AMD3100 stabilizes the diameter of formed, expanding AAAs in 2 experimental models. Conclusions— SDF-1/CXCR4 axis is upregulated in human and mouse AAAs. Blockade of CXCR4 with AMD3100 suppresses AAA formation and progression in two rodent models. Blockade of SDF-1/CXCR4 axis may represent a new strategy to limit progression of small human AAAs.

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10

Thrombogenic and inflammatory mediators, such as thrombin, induce NF‐kB–mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti‐inflammatory microRNA‐181b (miR‐181b) on thrombosis remains unknown. Our previous study demonstrated that miR‐181b inhibits downstream NF‐kB signaling in response to TNF‐a. Here, we demonstrate that miR‐181b uniquely inhibits upstream NF‐kB signaling in response to thrombin. Overexpression of miR‐181b inhibited thrombin‐induced activation of NF‐kB signaling, demonstratedbyreductionofphospho‐IKK‐b,‐IkB‐a, and p65 nuclear translocation in ECs. MiR‐181b also reduced expression of NF‐kB target genes VCAM‐1, intercellular adhesion molecule‐1, E‐selectin, and tissue factor. Mechanistically, miR‐181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease‐activated receptor‐1. miR‐181b reduced expression of Card10 mRNA and protein, but not protease‐activated receptor‐1. 39‐Untranslated region reporter assays, argonaute‐2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR‐181b target. Small interfering RNA–mediated knockdown of Card10 expression phenocopied effects of miR‐181b on NF‐kB signaling and targets. Card10 deficiency did not affect TNF‐a–induced activation of NF‐kB signaling, which suggested stimulus‐specific regulation of NF‐kB signaling and endothelial responses by miR‐181b in ECs. Finally, in response to photochemical injury‐induced arterial thrombosis, systemic delivery of miR‐181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6‐fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR‐181b and Card10 are important regulators of thrombin‐induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA‐dependent targets in response to ligand‐specific signaling in ECs.—Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA‐181b inhibits thrombin‐mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10. FASEB J. 30, 3216–3226 (2016). www.fasebj.org

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

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.

Diameter and thickness-related variations in mechanical properties of degraded arterial wall in the rat xenograft model

The purpose of this study was to evaluate the diameter and thickness-related variations in mechanical properties of degraded arterial wall. To this end, ring tests were performed on 31 samples from the rat xenograft model of abdominal aortic aneurysm (AAA) and failure properties were determined. An inverse finite element method was then employed to identify the material parameters of a hyperelastic and incompressible strain energy function. Correlations with outer diameter and wall thickness of the rings were examined. Furthermore, we investigated the changes in mechanical properties between the grafts, which consist in guinea pig decellularized aortas, native murine aortas and degraded aortas (AAAs). Decellularized aortas presented a significantly lower ultimate strain associated with a higher stiffening rate compared to native aortas. AAAs exhibited a significantly lower ultimate stress than other groups and an extensible-but-stiff behavior. The proposed approach revealed correlations of ultimate stress and material parameters of aneurysmal aortas with outer diameter and thickness. In particular, the negative correlations of the material parameter accounting for the response of the non-collagenous matrix with diameter and thickness (r=-0.67 and r=-0.73, p<0.001) captured the gradual loss of elastin with dilatation observed in histology (r=-0.97, p<0.001). Moreover, it exposed the progressive weakening of the wall with enlargement and thickening (r=-0.64 and r=-0.69, p<0.001), suggesting that wall thickness and diameter may be indicators of rupture risk in the rat xenograft model.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in MiceNovelty and Significance

Rationale: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). Conclusions: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in MiceNovelty and Significance: Implications for Superficial Erosion

Rationale: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). Conclusions: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.