In this article, we advance a hypothesis for the rupture of thin fibrous cap atheroma, namely that minute (10-m-diameter) cellular-level microcalcifications in the cap, which heretofore have gone undetected because they lie below the visibility of current in vivo imaging techniques, cause local stress concentrations that lead to interfacial debonding. New theoretical solutions are presented for the local stress concentration around these minute spherical inclusions that predict a nearly 2-fold increase in interfacial stress that is relatively insensitive to the location of the hypothesized microinclusions in the cap. To experimentally confirm the existence of the hypothesized cellular-level microcalcifications, we examined autopsy specimens of coronary atheromatous lesions using in vitro imaging techniques whose resolution far exceeds conventional magnetic resonance imaging, intravascular ultrasound, and optical coherence tomography approaches. These high-resolution imaging modalities, which include confocal microscopy with calcium-specific staining and micro-computed tomography imaging, provide images of cellular-level calcifications within the cap proper. As anticipated, the minute inclusions in the cap are very rare compared with the numerous calcified macrophages observed in the necrotic core. Our mathematical model predicts that inclusions located in an area of high circumferential stress (>300 kPa) in the cap can intensify this stress to nearly 600 kPa when the cap thickness is <65 m. The most likely candidates for the inclusions are either calcified macrophages or smooth muscle cells that have undergone apoptosis. cellular-level calcification ͉ stress concentration ͉ thin-cap fibroatheroma T he rupture of the thin fibrous cap overlying the necrotic core of a vulnerable plaque is the principal cause of acute coronary syndrome. It has been widely assumed that plaque morphology is the major determinant of clinical outcome (1-6). Several pathological studies of ruptured plaques have provided morphological descriptions of the high-risk, or vulnerable, coronary plaque that is prone to rupture or erosion as a positively remodeled lesion rich in vasa-vasorium, containing a lipid-rich core with an overlying thin fibrous cap infiltrated by macrophages (7-10). Virmani et al. (6) described thin-cap fibroatheroma with a large necrotic core and a fibrous cap of Ͻ65 m as a more specific precursor of plaque rupture due to tissue stress.Despite the above observations, the mechanism of vulnerable plaque rupture has remained a mystery because ruptures often occur in regions where computational finite element (FEM) and fluid structure interaction (FSI) models do not predict maximal stress. Forty percent of ruptures occur in the central part of the cap rather than regions of high curvature at the shoulders of the lipid core where FEM models predict maximum tissue stresses (11-13). Similarly, the latest study by Tang et al. (14), using an FSI model applied to 3D MRI images of sample plaques, predicts that maximal stress often ...

Objective We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques it has become appreciated that ~40% of cells classified as macrophages by histological markers may be of VSMC origin. We therefore sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins were reduced and of macrophage markers increased. Concurrent was down regulation of miR-143/145, which positively regulate the master VSMC-differentiation transcription factor myocardin (MYOCD). Mechanisms were further probed in mouse VSMC. Maintaining the expression of MYOCD or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions Cholesterol loading of VSMC converts them to a macrophage–appearing state by downregulating the miR-143/145-myocardin axis. Though these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

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Rationale Plasma HDL levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is due to the ability of HDL to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results We developed MAC-ABCDKO mice with efficient deletion of the ATP Binding Cassette Transporters A1 and G1 (ABCA1 and ABCG1) in macrophages but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr-/- recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared to controls. On the Western type diet (WTD), MAC-ABCDKO BM transplanted Ldlr-/- mice had disproportionate atherosclerosis, considering they also had lower VLDL/LDL cholesterol levels than controls. ABCA1/G1 deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, WTD-fed MAC-ABCDKO BM transplanted Ldlr-/- mice displayed monocytosis and neutrophilia in the absence of HSPC proliferation. Mechanistic studies revealed increased expression of M-CSF and G-CSF in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusion These studies 1) show that macrophage deficiency of ABCA1/G1 is pro-atherogenic likely by promoting plaque inflammation and 2) uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.