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.