Phosphate in Cardiovascular Disease: From New Insights Into Molecular Mechanisms to Clinical Implications.

Hyperphosphatemia is a common feature in patients with impaired kidney function and is associated with increased risk of cardiovascular disease. This phenomenon extends to the general population, whereby elevations of serum phosphate within the normal range increase risk; however, the mechanism by which this occurs is multifaceted, and many aspects are poorly understood. Less than 1% of total body phosphate is found in the circulation and extracellular space, and its regulation involves multiple organ cross talk and hormones to coordinate absorption from the small intestine and excretion by the kidneys. For phosphate to be regulated, it must be sensed. While mostly enigmatic, various phosphate sensors have been elucidated in recent years. Phosphate in the circulation can be buffered, either through regulated exchange between extracellular and cellular spaces or through chelation by circulating proteins (ie, fetuin-A) to form calciprotein particles, which in themselves serve a function for bulk mineral transport and signaling. Either through direct signaling or through mediators like hormones, calciprotein particles, or calcifying extracellular vesicles, phosphate can induce various cardiovascular disease pathologies: most notably, ectopic cardiovascular calcification but also left ventricular hypertrophy, as well as bone and kidney diseases, which then propagate phosphate dysregulation further. Therapies targeting phosphate have mostly focused on intestinal binding, of which appreciation and understanding of paracellular transport has greatly advanced the field. However, pharmacotherapies that target cardiovascular consequences of phosphate directly, such as vascular calcification, are still an area of great unmet medical need.

Serum total testosterone is associated with phosphate and calcium excretion in response to oral phosphate loading in healthy middle-aged males.

Androgen receptors are expressed in the kidney and serum testosterone is negatively associated with serum phosphate in males, suggesting a role of testosterone in renal phosphate handling. In this cross-sectional study, we examined the association of serum total and free testosterone with acute phosphate and calcium excretion in males in response to an oral phosphate challenge. Thirty-five healthy adult males with normal baseline testosterone levels consumed a 500 mg phosphorus drink and the urinary excretion of minerals, as well as levels of relevant circulating parameters, were assessed at baseline and hourly for 4 h. Serum total testosterone was positively associated with overall phosphate excretion (r = 0.35, p = 0.04) and calcium excretion (r = 0.44, p = 0.00) in response to the challenge. Serum free testosterone was positively associated with post-challenge calcium excretion (r = 0.34, p = 0.048), but significance was not reached for phosphate excretion (r = 0.31, p = 0.07). Serum total and free testosterone were not associated with parathyroid hormone, fibroblast growth factor-23, or vitamin D-key factors implicated in phosphate and calcium regulation. Overall, higher serum total testosterone levels in healthy middle-aged males are associated with a greater capacity to acutely excrete phosphate and calcium after a single oral phosphate challenge, suggesting potential ramifications of testosterone deficiency related to mineral homeostasis.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Multi-omic Landscape of Extracellular Vesicles in Human Carotid Atherosclerotic Plaque Reveals Endothelial Communication Networks.

Background: Carotid atherosclerosis is a multifaceted disease orchestrated by a myriad of cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed cell-derived nanoparticles that represent a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into plaque and marginal zones (n= 29 patients, paired plaque and marginal zone; symptomatic n=16, asymptomatic n=13), with further density gradient ultracentrifugation for proteomic analysis. EV cargoes were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with publicly available bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Human carotid plaques contained greater quantities of EVs than adjacent marginal zones. EV-miRNA and protein content was different in diseased plaque versus adjacent marginal zones, with differential functions in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Furthermore, EV signatures from SMCs and immune cells were most enriched in the marginal and plaque zones, respectively. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication strategies that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated a strong endothelial signature with potential roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was corroborated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV-vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed processes mediated by EVs creates new avenues for novel therapeutics in atherosclerosis.