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.

These in vitro observations and analyses of human plaques indicate that TLR2 stimulation followed by neutrophil participation may render smooth muscle cell-rich plaques susceptible to superficial erosion and thrombotic complications by inducing ER stress, apoptosis, and favouring detachment of EC.

Purpose of review This review explores the mechanisms of superficial intimal erosion, a common cause of thrombotic complications of atherosclerosis. Recent findings Human coronary artery atheroma that give rise to thrombosis due to erosion differ diametrically from those associated with fibrous cap rupture. Eroded lesions characteristically contain few inflammatory cells, abundant extracellular matrix, and neutrophil extracellular traps (NETs). Innate immune mechanisms such as engagement of Toll-like receptor 2 (TLR2) on cultured endothelial cells (EC) can impair their viability, attachment, and ability to recover a wound. Hyaluronan fragments may serve as endogenous TLR2 ligands. Mouse experiments demonstrate that flow disturbance in arteries with neointimas tailored to resemble features of human eroded plaques disturbs EC barrier function, impairs EC viability, recruits neutrophils, and provokes EC desquamation, NET formation, and thrombosis in a TLR2-dependent manner. Summary Mechanisms of erosion have received much less attention than those that provoke plaque rupture. Intensive statin treatment changes the characteristic of plaques that render them less susceptible to rupture. Thus, erosion may contribute importantly to the current residual burden of risk. Understanding the mechanisms of erosion may inform the development and deployment of novel therapies to combat the remaining atherothrombotic risk in the statin era.

Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to 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 TLR2 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 (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era.

Evidence has linked collagen loss with the onset of acute coronary events. Objective This study tested the hypothesis that selective MMP-13 collagenase inhibition increases collagen content in already established and nascent mouse atheromata. Methods and Results In vitro and in situ experiments documented the selectivity and efficacy of an orally available MMP-13 inhibitor (MMP13i-A). In vivo observations monitored macrophage accumulation and MMP-13 activity using molecular imaging. After 10 weeks of MMP13i-A treatment, apoE-/- mice with evolving or established lesions exhibited reduced MMP-13 activity without affecting macrophage content, measured either by intravital microscopy or fluorescence reflectance imaging. Histological analysis indicated that MMP13-iA did not affect plaque size, or macrophage or smooth-muscle cell accumulation. Administration of MMP13i-A to mice with evolving or established atheromata substantially increased plaque interstitial collagen content in the intima and locally in the fibrous cap, compared to vehicle-treated controls. Analysis of collagen revealed thicker collagen fibers within the plaques of treated groups. Conclusions Pharmacological MMP-13 inhibition yields collagen accumulation in plaques (a feature associated in humans with resistance to rupture), even in established plaques. This study of considerable clinical relevance furnishes new mechanistic insight into regulation of the plaque's extracellular matrix, and validates molecular imaging for studying plaque biology.

Rationale Inflammation drives atherogenesis. Animal and human studies have implicated interleukin (IL)-1β in this disease. Moderate hypoxia, a condition that prevails in the atherosclerotic plaque, may conspire with inflammation and contribute to the evolution and complications of atherosclerosis through mechanisms that remain incompletely understood. Objective This study investigated the links between hypoxia and inflammation by testing the hypothesis that moderate hypoxia modulates IL-1β production in activated human macrophages. Methods and Results Our results demonstrated that hypoxia enhances pro-IL-1β protein — but not mRNA — expression in LPS-stimulated human macrophages. We show that hypoxia limits the selective targeting of pro-IL-1β to autophagic degradation, thus prolonging its half-life and promoting its intracellular accumulation. Furthermore, hypoxia increased the expression of NLRP3, a limiting factor in NLRP3 inflammasome function, and augmented caspase-1 activation in LPS-primed macrophages. Consequently, hypoxic human macrophages secreted higher amounts of mature IL-1β than did normoxic macrophages after treatment with crystalline cholesterol, an endogenous danger signal that contributes to atherogenesis. In human atherosclerotic plaques, IL-1β localizes predominantly to macrophage-rich regions that express activated caspase-1 and the hypoxia markers hypoxia-inducible factor 1α (HIF-1α) and hexokinase-2 (HK-2), as assessed by immunohistochemical staining of carotid endarterectomy specimens. Conclusions These results indicate that hypoxia potentiates IL-1β expression in cultured human macrophages and in the context of atheromata, therefore unveiling a novel pro-inflammatory mechanism that may participate in atherogenesis.

Although the involvement of the Notch pathway in several areas of vascular biology is now clearly established, its role in vascular inflammation at the endothelial level remains to be elucidated. In this study, we demonstrated that proinflammatory cytokines drive a specific

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