Roles and Regulation of Extracellular Vesicles in Cardiovascular Mineral Metabolism

Cardiovascular calcification is a multifaceted disease that is a leading independent predictor of cardiovascular morbidity and mortality. Recent studies have identified a calcification-prone population of extracellular vesicles as the putative elementary units of vascular microcalcification in diseased heart valves and vessels. Their action is highly context-dependent; extracellular vesicles released by smooth muscle cells, valvular interstitial cells, endothelial cells, and macrophages may promote or inhibit mineralization, depending on the phenotype of their originating cells and/or the extracellular environment to which they are released. In particular, emerging roles for vesicular microRNAs, bioactive lipids, metabolites, and protein cargoes in driving this pro-calcific switch underpin the necessity of innovative strategies to employ next-generation sequencing and omics technologies in order to better understand the pathobiology of these nano-sized entities. Furthermore, a recent body of work has emerged that centers on the novel re-purposing of extracellular vesicles and exosomes as potential therapeutic avenues for cardiovascular calcification. This review aims to highlight the role of extracellular vesicles as constituents of cardiovascular calcification and summarizes recent advances in our understanding of the biophysical nature of vesicle accumulation, aggregation, and mineralization. We also comprehensively discuss the latest evidence that extracellular vesicles act as key mediators and regulators of cell/cell communication, osteoblastic/osteoclastic differentiation, and cell/matrix interactions in cardiovascular tissues. Lastly, we highlight the importance of robust vesicle isolation and characterization when studying these phenomena, and offer a brief primer on working with cardiovascular applications of extracellular vesicles

Small entities with large impact: microcalcifications and atherosclerotic plaque vulnerability

Purpose of review Atherosclerotic 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 findings The 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. Summary Microcalcifications 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

A Novel Quantitative Approach for Eliminating Sample-To-Sample Variation Using a Hue Saturation Value Analysis Program

Objectives: As computing technology and image analysis techniques have advanced, the practice of histology has grown from a purely qualitative method to one that is highly quantified. Current image analysis software is imprecise and prone to wide variation due to common artifacts and histological limitations. In order to minimize the impact of these artifacts, a more robust method for quantitative image analysis is required. Methods and Results: Here we present a novel image analysis software, based on the hue saturation value color space, to be applied to a wide variety of histological stains and tissue types. By using hue, saturation, and value variables instead of the more common red, green, and blue variables, our software offers some distinct advantages over other commercially available programs. We tested the program by analyzing several common histological stains, performed on tissue sections that ranged from 4 µm to 10 µm in thickness, using both a red green blue color space and a hue saturation value color space. Conclusion: We demonstrated that our new software is a simple method for quantitative analysis of histological sections, which is highly robust to variations in section thickness, sectioning artifacts, and stain quality, eliminating sample-to-sample variation

Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol

Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles