An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca²⁺-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

Calciprotein particles (CPPs), which increasingly arise in the circulation during the disorders of mineral homeostasis, represent a double-edged sword protecting the human organism from extraskeletal calcification but potentially causing endothelial dysfunction. Existing models, however, failed to demonstrate the detrimental action of CPPs on endothelial cells (ECs) under flow. Here, we applied a flow culture system, where human arterial ECs were co-incubated with CPPs for 4 h, and a normolipidemic and normotensive rat model (10 daily intravenous injections of CPPs) to simulate the scenario occurring in vivo in the absence of confounding cardiovascular risk factors. Pathogenic effects of CPPs were investigated by RT-qPCR and Western blotting profiling of the endothelial lysate. CPPs were internalised within 1 h of circulation, inducing adhesion of peripheral blood mononuclear cells to ECs. Molecular profiling revealed that CPPs stimulated the expression of pro-inflammatory cell adhesion molecules VCAM1 and ICAM1 and upregulated transcription factors of endothelial-to-mesenchymal transition (Snail, Slug and Twist1). Furthermore, exposure to CPPs reduced the production of atheroprotective transcription factors KLF2 and KLF4 and led to YAP1 hypophosphorylation, potentially disturbing the mechanisms responsible for the proper endothelial mechanotransduction. Taken together, our results suggest the ability of CPPs to initiate endothelial dysfunction at physiological flow conditions.

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca²⁺ and PO₄³⁻ ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca²⁺ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Background: Whereas the risk factors for structural valve degeneration (SVD) of glutaraldehyde‐treated bioprosthetic heart valves (BHVs) are well studied, those responsible for the failure of BHVs fixed with alternative next‐generation chemicals remain largely unknown. This study aimed to investigate the reasons behind the development of SVD in ethylene glycol diglycidyl ether–treated BHVs. Methods and Results: Ten ethylene glycol diglycidyl ether–treated BHVs excised because of SVD, and 5 calcified aortic valves (AVs) replaced with BHVs because of calcific AV disease were collected and their proteomic profile was deciphered. Then, BHVs and AVs were interrogated for immune cell infiltration, microbial contamination, distribution of matrix‐degrading enzymes and their tissue inhibitors, lipid deposition, and calcification. In contrast with dysfunctional AVs, failing BHVs suffered from complement‐driven neutrophil invasion, excessive proteolysis, unwanted coagulation, and lipid deposition. Neutrophil infiltration was triggered by an asymptomatic bacterial colonization of the prosthetic tissue. Neutrophil elastase, myeloblastin/proteinase 3, cathepsin G, and matrix metalloproteinases (MMPs; neutrophil‐derived MMP‐8 and plasma‐derived MMP‐9), were significantly overexpressed, while tissue inhibitors of metalloproteinases 1/2 were downregulated in the BHVs as compared with AVs, together indicative of unbalanced proteolysis in the failing BHVs. As opposed to other proteases, MMP‐9 was mostly expressed in the disorganized prosthetic extracellular matrix, suggesting plasma‐derived proteases as the primary culprit of SVD in ethylene glycol diglycidyl ether–treated BHVs. Hence, hemodynamic stress and progressive accumulation of proteases led to the extracellular matrix degeneration and dystrophic calcification, ultimately resulting in SVD. Conclusions: Neutrophil‐ and plasma‐derived proteases are responsible for the loss of BHV mechanical competence and need to be thwarted to prevent SVD.

Although saphenous veins (SVs) are commonly used as conduits for coronary artery bypass grafting (CABG), internal thoracic artery (ITA) grafts have significantly higher long-term patency. As SVs and ITA endothelial cells (ECs) have a considerable level of heterogeneity, we suggested that synergistic paracrine interactions between CA and ITA ECs (HCAECs and HITAECs, respectively) may explain the increased resistance of ITA grafts and adjacent CAs to atherosclerosis and restenosis. In this study, we measured the gene and protein expression of the molecules responsible for endothelial homeostasis, pro-inflammatory response, and endothelial-to-mesenchymal transition in HCAECs co-cultured with either HITAECs or SV ECs (HSaVECs) for an ascending duration. Upon the co-culture, HCAECs and HITAECs showed augmented expression of endothelial nitric oxide synthase (eNOS) and reduced expression of endothelial-to-mesenchymal transition transcription factors Snail and Slug when compared to the HCAEC–HSaVEC model. HCAECs co-cultured with HITAECs demonstrated an upregulation of HES1, a master regulator of arterial specification, of which the expression was also exclusively induced in HSaVECs co-cultured with HCAECs, suggestive of their arterialisation. In addition, co-culture of HCAECs and HITAECs promoted the release of pro-angiogenic molecules. To conclude, co-culture of HCAECs and HITAECs results in reciprocal and beneficial paracrine interactions that might contribute to the better performance of ITA grafts upon CABG.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.