Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

Abstract

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Time-dependent Role of Bisphosphonates on Atherosclerotic Plaque Calcification

“…However, our data suggest that timing of BiP intervention in relation to ongoing atherosclerotic remodeling could influence mineral morphology and thus plaque stability. Previous studies using electron microscopy revealed that timing of BiP regimen affects the morphology and topography of plaque microcalcifications (less than 5 μm 2 ) [1]; starting BiP treatment in early plaque remodeling led to bigger individual mineral aggregates with higher surface roughness, while starting at later time points reduced both mineral aggregates size and surface roughness [1]. Furthermore, mineral aggregates associated with later BiP treatment beginning showed qualitatively disorganized and loose morphologies [1].…”

“…Previous studies using electron microscopy revealed that timing of BiP regimen affects the morphology and topography of plaque microcalcifications (less than 5 μm 2 ) [1]; starting BiP treatment in early plaque remodeling led to bigger individual mineral aggregates with higher surface roughness, while starting at later time points reduced both mineral aggregates size and surface roughness [1]. Furthermore, mineral aggregates associated with later BiP treatment beginning showed qualitatively disorganized and loose morphologies [1]. Finite element-based predictions of these previous data suggested that the microcalcifications associated with all BiP treatment groups would reduce plaque stress compared to non-treated controls.…”

“…Atherosclerosis represents the most common cause of cardiovascular disease, and atherosclerotic plaque rupture results in arterial thrombosis, leading to heart attacks and strokes [1, 2]. Vascular smooth muscle cell migration and osteogenic differentiation promote calcification within the atherosclerotic plaque [3].…”

“…BiPs prevented medial calcinosis in a male renal failure rat model; however, it was parallel to reduction in bone mass density [26]. BiPs altered the mineral microstructure in Apolipoprotein E-deficient (Apoe -/- ) mice fed an atherogenic diet, indicating a direct interaction between BiPs and mineral within the plaque [1]. Given the long term administration of BiPs for osteoporosis treatment [27], further studies are needed to assess how BiPs affect vascular calcification over the course of plaque development.…”

EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues

“…The mentioned drawbacks can be resolved by TEM, which is incompatible with large samples (e.g., most of the blood vessels) due to a critically reduced amount of analysable tissue, needs difficult sample preparation, and the final sample represents a single tissue snapshot rather than serial sections evenly distributed along or across the specimen. Notably, the visualisation of calcified tissues is often performed by means of density-dependent scanning electron microscopy (SEM), which implies the superimposing of images acquired by secondary and backscattered electron detectors and further assigning them to the distinct colour channels (e.g., green and red), thereby permitting the coloured mapping of calcific nodules and distinguishing them from soft tissues (51)(52)(53). The main advantage of this technique is that it allows the simultaneous visualisation of both topography and density in a single image (51).…”