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.

Using 2.1-μm high-resolution microcomputed tomography, we have examined the spatial distribution, clustering, and shape of nearly 35,000 microcalcifications (μCalcs) ≥ 5 μm in the fibrous caps of 22 nonruptured human atherosclerotic plaques. The vast majority of these μCalcs were <15 μm and invisible at the previously used 6.7-μm resolution. A greatly simplified 3D finite element analysis has made it possible to quickly analyze which of these thousands of minute inclusions are potentially dangerous. We show that the enhancement of the local tissue stress caused by particle clustering increases rapidly for gap between particle pairs (h)/particle diameter (D) < 0.4 if particles are oriented along the tensile axis of the cap. Of the thousands of μCalcs observed, there were 193 particle pairs with h/D ≤ 2 (tissue stress factor > 2), but only 3 of these pairs had h/D ≤ 0.4, where the local tissue stress could increase a factor > 5. Using nondecalcified histology, we also show that nearly all caps have μCalcs between 0.5 and 5 μm and that the μCalcs ≥ 5 μm observed in high-resolution microcomputed tomography are agglomerations of smaller calcified matrix vesicles. μCalcs < 5 μm are predicted to be not harmful, because the tiny voids associated with these very small particles will not explosively grow under tensile forces because of their large surface energy. These observations strongly support the hypothesis that nearly all fibrous caps have μCalcs, but only a small subset has the potential for rupture.finite element analysis of fibrous caps | microcomputed tomography imaging of microcalcifications | vulnerable plaque | clustered microcalcifications

The role of microcalcifications (μCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44), using a very limited tissue sample, that μCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-μm resolution and undecalcified histology with special emphasis on calcified particles <50 μm in diameter. Our results reveal the presence of thousands of μCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 μCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these μCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the μCalc and surrounding tissue, μCalc size, and clustering. We found that local tissue stress could be increased fivefold when μCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 μm) if no μCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous μCalcs < 15 μm in the caps, not visible at 6.7-μm resolution, and that our failure to find any nonruptured caps between 30 and 66 μm is a strong indication that many of these caps contained μCalcs.

Rationale Mitochondrial changes occur during cell differentiation and cardiovascular disease. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission. We hypothesized that DRP1 plays a role in cardiovascular calcification, a process involving cell differentiation and a major clinical problem with high unmet needs. Objective To examine the effects of osteogenic promoting conditions on DRP1, and whether DRP1 inhibition alters the development of cardiovascular calcification. Methods and Results DRP1 was enriched in calcified regions of human carotid arteries, examined by immunohistochemistry. Osteogenic differentiation of primary human vascular smooth muscle cells (SMCs) increased DRP1 expression. DRP1 inhibition in human SMCs undergoing osteogenic differentiation attenuated matrix mineralization, cytoskeletal rearrangement, mitochondrial dysfunction, and reduced type 1 collagen secretion and alkaline phosphatase activity. DRP1 protein was observed in calcified human aortic valves, and DRP1 RNA interference reduced primary human valve interstitial cell calcification. Mice heterozygous for Drp1 deletion did not exhibit altered vascular pathology in a PCSK9 gain-of-function atherosclerosis model. However, when mineralization was induced via oxidative stress, DRP1 inhibition attenuated mouse and human SMC calcification. Femur bone density was unchanged in mice heterozygous for Drp1 deletion, and DRP1 inhibition attenuated oxidative stress-mediated dysfunction in human bone osteoblasts. Conclusions We demonstrate a new function of DRP1 in regulating collagen secretion and cardiovascular calcification, a novel area of exploration for the potential development of new therapies to modify cellular fibrocalcific response in cardiovascular diseases. Our data also support a role of mitochondrial dynamics in regulating oxidative stress-mediated arterial calcium accrual and bone loss.

While it is generally accepted that ruptures in fibrous cap atheromas cause most acute coronary deaths, and that plaque rupture occurs in the fibrous cap at the location where the tissue stress exceeds a certain critical peak circumferential stress, the exact mechanism of rupture initiation remains unclear. We recently reported the presence of multiple microcalcifications (μCalcs) < 50μm diameter embedded within the fibrous cap, μCalcs that could greatly increase cap instability by introducing up to a 5-fold increase in local tissue stress. Here, we explore the hypothesis that, aside from cap thickness, μCalc size and interparticle spacing are principal determinants of cap rupture risk. Also, we propose that cap rupture is initiated near the poles of the μCalcs due to the presence of tiny voids that explosively grow at a critical tissue stress and then propagate across the fibrous cap. We develop a theoretical model based on classic studies in polymeric materials by Gent (1980), which indicates that cavitation as opposed to interfacial debonding is the more likely mechanism for cap rupture produced by μCalcs < 65μm diameter. This analysis suggests that there is a critical μCalc size range, from 5μm to 65μm, in which cavitation should be prevalent. This hypothesis for cap rupture is strongly supported by our latest μCT studies in which we have observed trapped voids in the vicinity of μCalcs within fibrous caps in human coronaries.

Purpose of reviewAtherosclerotic plaque rupture and subsequent acute events, such as myocardial infarction and stroke, contribute to the majority of cardiovascular-related deaths. Calcification has emerged as a significant predictor of cardiovascular morbidity and mortality, challenging previously held notions that calcifications stabilize atherosclerotic plaques. In this review, we address this discrepancy through recent findings that not all calcifications are equivalent in determining plaque stability.Recent findingsThe risk associated with calcification is inversely associated with calcification density. As opposed to large calcifications that potentially stabilize the plaque, biomechanical modeling indicates that small microcalcifications within the plaque fibrous cap can lead to sufficient stress accumulation to cause plaque rupture. Microcalcifications appear to derive from matrix vesicles enriched in calcium-binding proteins that are released by cells within the plaque. Clinical detection of microcalcifications has been hampered by the lack of imaging resolution required for in-vivo visualization; however, recent studies have demonstrated promising new techniques to predict the presence of microcalcifications.SummaryMicrocalcifications play a major role in destabilizing atherosclerotic plaques. The identification of critical characteristics that lead to instability along with new imaging modalities to detect their presence in vivo may allow early identification and prevention of acute cardiovascular events.

Approximately half of all cardiovascular deaths associated with acute coronary syndrome occur when the thin fibrous cap tissue overlying the necrotic core in a coronary vessel is torn, ripped or fissured under the action of high blood pressure. From a biomechanics point of view, the rupture of an atheroma is due to increased mechanical stresses in the lesion, in which the ultimate stress (i.e. peak circumferential stress (PCS) at failure) of the tissue is exceeded. Several factors including the cap thickness, morphology, residual stresses and tissue composition of the atheroma have been shown to affect the PCS. Also important, we recently demonstrated that microcalcifications (µCalcs) > 5 µm are a common feature in human atheroma caps, which behave as local stress concentrators, increasing the local tissue stress by at least a factor of two surpassing the ultimate stress threshold for cap tissue rupture. In the present study, we used both idealized µCalcs with spherical shape and actual µCalcs from human coronary atherosclerotic caps, to determine their effect on increasing the circumferential stress in the fibroatheroma cap using different hyperelastic constitutive models. We have found that the stress concentration factor (SCF) produced by µCalcs in the fibroatheroma cap is affected by the material tissue properties, µCalcs spacing, aspect ratio and their alignment relative to the tensile axis of the cap.

Asymptomatic vulnerable plaques (VP) in coronary arteries accounts for significant level of morbidity. Their main risk is associated with their rupture which may prompt fatal heart attacks and strokes. The role of microcalcifications (micro-Ca), embedded in the VP fibrous cap, in the plaque rupture mechanics has been recently established. However, their diminutive size offers a major challenge for studying the VP rupture biomechanics on a patient specific basis. In this study, a highly detailed model was reconstructed from a post-mortem coronary specimen of a patient with observed VP, using high resolution micro-CT which captured the microcalcifications embedded in the fibrous cap. Fluid-structure interaction (FSI) simulations were conducted in the reconstructed model to examine the combined effects of micro-Ca, flow phase lag and plaque material properties on plaque burden and vulnerability. This dynamic fibrous cap stress mapping elucidates the contribution of micro-Ca and flow phase lag VP vulnerability independently. Micro-Ca embedded in the fibrous cap produced increased stresses predicted by previously published analytical model, and corroborated our previous studies. The 'micro-CT to FSI' methodology may offer better diagnostic tools for clinicians, while reducing morbidity and mortality rates for patients with vulnerable plaques and ameliorating the ensuing healthcare costs.