BackgroundCitrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. ObjectiveTo investigate features and patterns of citrullination in atherosclerotic plaques. MethodsWe collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. ResultsProteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. ConclusionThe development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Supraphysiological concentrations of calciprotein particles (CPPs), which are indispensable scavengers of excessive Ca2+ and PO43− ions in blood, induce pro-inflammatory activation of endothelial cells (ECs) and monocytes. Here, we determined physiological levels of CPPs (10 μg/mL calcium, corresponding to 10

A significant role in the pathogenesis of CAVD is played by innate immunity cells, such as macrophages. In stenotic valves, macrophages have enhanced inflammatory activity, and the population’s balance is shifted toward pro-inflammatory ones. Pro-inflammatory macrophages release cytokines, chemokines, and microRNA, which can directly affect the resident valvular cells and cause valve calcification. In CAVD patients, macrophages may have more pronounced pro-inflammatory properties, enhanced not only by paracrine signals but also by juxtacrine Notch signaling and epigenetic factors, which influence the maturation of macrophages’ progenitors. In this review, we observe the accumulated data on the involvement of macrophages in CAVD development via paracrine and juxtacrine interactions.

Highlights When modeling mineral stress, a closed biochemical system shows the following calcium distribution ratio among biochemical compartments: freely circulating ions (Ca2+) – 50%, bound to albumin (CPM) - 20%, bound to and phosphorus (CPC) - 30%. The ratio of ionized to protein- or phosphorus-bound calcium was 1:1 and corresponded to the physiological ratio of ionized to bound calcium in circulating blood, indicating the physiological relevance of the simulations performed. Even under supraphysiological mineral stress, natural mineral depots (CPM and HRC) maintain the calcium-binding capacity of the biochemical system at the physiological level (50%), and the ratio of the relative calcium capacity of the CPM and HRC under conditions of supraphysiological mineral stress indicates the priority role of the HRC as a buffer system limiting the uncontrolled increase in ionized calcium in case of disturbance of the mineral balance of the blood. Abstract Aim. To conduct a comparative analysis of calcium content in various biochemical compartments: 1) ionized (freely circulating, unbound) calcium; 2) calcium-albumin CPM (protein-bound calcium); 3) calcium phosphorus complexes (CPC). Methods. In order to prepare a biochemical system for the parallel synthesis of CPM and CPC, supraphysiological mineral stress was modeled by supersaturating a NaCl saline solution containing a physiological concentration of albumin with calcium (CaCl2) and phosphorus (Na2HPO4) ions. Separation of calcium-containing biochemical compartments was carried out by ultracentrifugation (to isolate CPM) and ultrafiltration (to separate CPM and the pool of free ions). Calcium concentration was measured using a colorimetric method based on the reaction of orthocresolphthalein complexone with calcium ions in an alkaline medium. Results. When modeling mineral stress, a closed biochemical system was obtained in which calcium ions were freely distributed in three states: circulating in free form (Ca2+) or being part of the CPM (colloidal primary depot) or PSC (corpuscular secondary depot). The distribution of calcium in the form of freely circulating ions (Ca2+) and when bound to albumin (CPM) and phosphorus (CPC) was 50%: 20%: 30% (5: 2: 3), respectively. The ratio of ionized to protein- or phosphorus-bound calcium was 1:1 and corresponded to the physiological ratio of ionized to bound calcium in circulating blood, indicating the physiological relevance of the simulations performed. The formation of HRC absorbed 10 to 20% of the total albumin. Conclusion. Even under supraphysiological mineral stress, natural mineral depots (CPM and HRC) maintain the calcium-binding capacity of the biochemical system at the physiological level (50%), and the ratio of the relative calcium capacity of the CPM and HRC under conditions of supraphysiological mineral stress indicates the priority role of the HRC as a buffer system limiting the uncontrolled increase in ionized calcium in case of disturbance of the mineral balance of the blood.

Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca2+ and PO43− ions in blood, being internalised and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Here, we performed a pathway enrichment analysis of cellular compartment-specific proteomes in primary human coronary artery ECs (HCAEC) and human internal thoracic artery ECs (HITAEC) treated with primary (amorphous) or secondary (crystalline) CPPs (CPP-P and CPPs, respectively). Exposure to CPP-P and CPP-S induced notable upregulation of: (1) cytokine- and chemokine-mediated signaling, Ca2+-dependent events, and apoptosis in cytosolic and nuclear proteomes; (2) H+ and Ca2+ transmembrane transport, generation of reactive oxygen species, mitochondrial outer membrane permeabilisation, and intrinsic apoptosis in the mitochondrial proteome; (3) oxidative, calcium, and endoplasmic reticulum (ER) stress, unfolded protein binding, and apoptosis in the ER proteome. In contrast, transcription, post-transcriptional regulation, translation, cell cycle, and cell–cell adhesion pathways were underrepresented in cytosol and nuclear compartments, whilst biosynthesis of amino acids, mitochondrial translation, fatty acid oxidation, pyruvate dehydrogenase activity, and energy generation were downregulated in the mitochondrial proteome of CPP-treated ECs. Differentially expressed organelle-specific pathways were coherent in HCAEC and HITAEC and between ECs treated with CPP-P or CPP-S. Proteomic analysis of mitochondrial and nuclear lysates from CPP-treated ECs confirmed bioinformatic filtration findings.

HighlightsTo achieve a 10% (i.e., 1.1-fold) increase in ionized calcium level in the serum-free cell culture medium and Wistar rat serum, 10 µg/mL calcium (e.g. with CaCl2) should be added; this corresponds to the upper quartile of ionized calcium in the human population.Incubation with ionized calcium or calciprotein monomers (10 µg/mL calcium) does not induce any pathological effects in primary arterial endothelial cells, although calciprotein monomers are internalised by endothelial cells similar to calciprotein particles.Incubation of primary human coronary artery endothelial cells with calciprotein particles (10 µg/mL calcium) triggers an increased expression of VCAM1, ICAM1, and SELE genes (i.e., those encoding cell adhesion molecules) as well as IL6, CXCL8, and CXCL1 genes (i.e., those encoding pro-inflammatory cytokines), together indicating endothelial activation. Aim. To compare the pathological effects of ionized calcium, calciprotein monomers (CPMs), and calciprotein particles (CPPs) after their addition to endothelial cells.Methods. CPMs and CPPs were synthesized by the supersaturation of albumin-supplemented NaCl solution with calcium (by adding CaCl2) and phosphate ions (by adding Na2HPO4). CPMs and CPPs were separated by sequential ultracentrifugation (to isolate CPPs) and ultrafiltration (to separate CPMs and free mineral ions). Calcium concentration in CPMs and CPPs was measured by an o-cresolphthalein complexone-based colorimetric assay. Internalization of fluorescent-labeled CPMs and CPPs by endothelial cells was interrogated by confocal microscopy after their 1-hour co-incubation under flow. Primary coronary artery and internal thoracic artery endothelial cells were incubated with ionized calcium (CaCl2), CPMs, or CPPs (10 µg/mL calcium) upon reaching confluence (n = 6 wells per group) to simulate a 10% increase in ionized calcium concentration. After 24 hours, we collected RNA and analyzed the gene expression pattern by reverse transcription-quantitative polymerase chain reaction.Results. CPMs and CPPs were internalized by endothelial cells as soon as after 1 hour of co-incubation under flow. Treatment with ionized calcium or CPMs (10 µg/mL calcium) did not cause any pathological effects in arterial endothelial cells. In contrast, CPPs promoted an expression of VCAM1, ICAM1, and SELE genes (i.e., those encoding cell adhesion molecules) as well as IL6, CXCL8, and CXCL1 genes (i.e., those encoding pro-inflammatory cytokines) in primary human coronary artery endothelial cells, together indicating their pro-inflammatory activation.Conclusion. Pathological effects of calcium stress are defined by the calcium source and not simply by its concentration.

Mitomycin C (MMC)-induced genotoxic stress can be considered to be a novel trigger of endothelial dysfunction and atherosclerosis—a leading cause of cardiovascular morbidity and mortality worldwide. Given the increasing genotoxic load on the human organism, the decryption of the molecular pathways underlying genotoxic stress-induced endothelial dysfunction could improve our understanding of the role of genotoxic stress in atherogenesis. Here, we performed a proteomic profiling of human coronary artery endothelial cells (HCAECs) and human internal thoracic endothelial cells (HITAECs) in vitro that were exposed to MMC to identify the biochemical pathways and proteins underlying genotoxic stress-induced endothelial dysfunction. We denoted 198 and 71 unique, differentially expressed proteins (DEPs) in the MMC-treated HCAECs and HITAECs, respectively; only 4 DEPs were identified in both the HCAECs and HITAECs. In the MMC-treated HCAECs, 44.5% of the DEPs were upregulated and 55.5% of the DEPs were downregulated, while in HITAECs, these percentages were 72% and 28%, respectively. The denoted DEPs are involved in the processes of nucleotides and RNA metabolism, vesicle-mediated transport, post-translation protein modification, cell cycle control, the transport of small molecules, transcription and signal transduction. The obtained results could improve our understanding of the fundamental basis of atherogenesis and help in the justification of genotoxic stress as a risk factor for atherosclerosis.

HighlightsThe creation of a domestic bank of standardized endothelial cell cultures (arterial, venous, microvascular, valvular, and lymphatic lineages) and the development of an import-substituting technological process for working with them is a key task to enable next-generation endothelial physiology studies.The cultural and molecular justification for selecting the best source of autologous endothelial cells with the highest regenerative potential (in particular in context of their proliferative and angiogenic capacities) represents a primary task for endothelial physiology.The research methodology for studying endothelial cell physiology under normal and pathological conditions includes assessing their proliferative and angiogenic properties and molecular profiling. AbstractOne of the primary goals in studying endothelial physiology is the development of a biomedical cell product in the form of autologous endothelial cells with high regenerative potential. These cells would be used to stimulate angiogenesis in ischemic tissues and to coat tissue-engineered constructs (such as vascular grafts) for their endothelialization prior to implantation into the human body. This review examines the key applied aspects of endothelial physiology aimed at achieving these objectives, including the selection of endothelial cell sources for modeling various pathological processes and regenerative medicine, the creation of a bank of standardized endothelial cell lines with various differentiation directions to enhance the effectiveness of this modeling, methods to ensure adhesion, sustained proliferation, and physiological functioning of primary endothelial cells in culture, as well as the methodology for assessing proliferative and angiogenic activity in the context of analyzing endothelial cell resistance to external influences. The discussion emphasizes the need for a comparative analysis of the regenerative properties of microvascular endothelial cells from subcutaneous adipose tissue, colony-forming endothelial cells from peripheral venous blood, and endothelial cells differentiated from induced pluripotent stem cells. Additionally, the current state of import substitution for various components of endothelial cell technology is reviewed, including standardized cell lines, functional protein coatings, and three-dimensional matrices for angiogenesis assessment, culture media for isolation, cultivation, and experimentation with endothelial cells, as well as other reagents for the isolation and subculturing of endothelial cells, culture dishes, and antibodies for flow cytometric and fluorescent microscopic immunophenotyping. The review also analyzes the prospects for import substitution of missing components, such as certain endothelial cell lines, magnetic beads with immobilized antibodies for their isolation, and antibodies conjugated with various fluorophores.

Calciprotein particles (CPPs) are essential circulating scavengers of excessive Ca2+ and PO43- ions, representing a vehicle that removes them from the human body and precludes extraskeletal calcification. Having been internalised by endothelial cells (ECs), CPPs induce their dysfunction, which is accompanied by a remarkable molecular reconfiguration, although little is known about this process's extracellular signatures. Here, we applied ultra-high performance liquid chromatography-tandem mass spectrometry to perform a secretome-wide profiling of the cell culture supernatant from primary human coronary artery ECs (HCAECs) and internal thoracic artery ECs (HITAECs) treated with primary CPPs (CPP-P), secondary CPPs (CPP-S), magnesiprotein particles (MPPs), or Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline (DPBS) for 24 h. Incubation with CPP-P/CPP-S significantly altered the profiles of secreted proteins, delineating physiological and pathological endothelial secretomes. Neither pathway enrichment analysis nor the interrogation of protein-protein interactions detected extracellular matrix- and basement membrane-related molecular terms in the protein datasets from CPP-P/CPP-S-treated ECs. Both proteomic profiling and enzyme-linked immunosorbent assay identified an increased level of protectin (CD59) and reduced levels of osteonectin (SPARC), perlecan (HSPG2), and fibronectin (FN1) in the cell culture supernatant upon CPP-P/CPP-S treatment. Elevated soluble CD59 and decreased release of basement membrane components might be considered as potential signs of dysfunctional endothelium.

Aim. To assess gene expression of enzymes of the sphingomyelinase pathway of ceramide biosynthesis and degradation in fat depots of various localization in patients with cardiovascular diseases. Material and methods. A total of 38 patients were examined: 20 with coronary artery disease (CAD) and 18 with aortic stenosis/insufficiency. Biopsies of subcutaneous, epicardial, perivascular adipose tissue (AT) (SAT, EAT, PVAT, respectively) were obtained during surgery. The gene expression of sphingomyelinase pathway enzymes (acid and neutral sphingomyelinase SMPD1 and SMPD3 ) and the degradation of ceramides (acid ceramidase ASAH1 ; sphingomyelin synthase 1 and 2 SGMS1 and SGMS2 ) was assessed using a quantitative polymerase chain reaction. Analysis of the level of corresponding proteins was carried out using immunoblotting (western blotting). Statistical processing was performed using GraphPad Prism 8 (GraphPad Software). Results. In CAD, the maximum expression of SMPD1 was observed in subcutaneous and epicardial adipocytes. In acquired heart defects (AHD), the level of SMPD1 mRNA in the SAT was higher than in the PVAT. Expression of the SMPD1 gene in the EAT of patients with CAD was more pronounced than in patients with heart defects. PVAT was characterized by minimal expression of SMPD1 regardless of disease. Expression of SMPD3 had no tissue features in studied groups, while SMPD1 was more expressed in cardiac AT adipocytes than SMPD3 . ASAH1 in the EAT of patients with CAD was maximal relative to adipocytes of other localizations. Persons with AHD were characterized by a high expression of ASAH1 , regardless of AT localization, exceeding the values of patients with CAD. In CAD, the level of SGMS1 in EAT was higher than in SAT and PVAT, while no differences were found in patients with AHD depending on AT location. SGMS1 gene expression in EAT of patients with CAD was higher than in the group of AHD. Expression of SGMS2 significantly exceeded SGMS1 in both study groups and was maximal in SAT and PVAT adipocytes compared to EAT in the CAD group and in PVAT in the AHD group. Coronary pathology was characterized by a higher level of SGMS2 mRNA in SAT and EAT. The level of ceramide metabolism enzymes in AT of patients corresponded to the expression of their genes. Conclusion. In coronarogenic disease, cardiac AT (mainly epicardial) is characterized not only by increased expression of gene ceramide synthesis enzymes via the sphingomyelinase pathway, but also by activation of ceramide utilization with sphingosine formation. The observed changes may contribute to the accumulation of ceramides and sphingomyelin associated with atherosclerotic processes.

Current techniques for the detection of vasa vasorum (VV) in vascular pathology include staining for endothelial cell (EC) markers such as CD31 or VE-cadherin. However, this approach does not permit an objective assessment of vascular geometry upon vasospasm and the clinical relevance of endothelial specification markers found in developmental biology studies remains unclear. Here, we performed a combined immunostaining of rat abdominal aorta (rAA) and human saphenous vein (hSV) for various EC or vascular smooth muscle cell (VSMC) markers and found that the latter (e.g., alpha smooth muscle actin (α-SMA) or smooth muscle myosin heavy chain (SM-MHC)) ensure a several-fold higher signal-to-noise ratio irrespective of the primary antibody origin, fluorophore, or VV type (arterioles, venules, or capillaries). Further, α-SMA or SM-MHC staining allowed unbiased evaluation of the VV area under vasospasm. Screening of the molecular markers of endothelial heterogeneity (mechanosensitive transcription factors KLF2 and KLF4, arterial transcription factors HES1, HEY1, and ERG, venous transcription factor NR2F2, and venous/lymphatic markers PROX1, LYVE1, VEGFR3, and NRP2) have not revealed specific markers of any lineage in hSV (although KLF2 and PROX1 were restricted to venous endothelium in rAA), suggesting the need in high-throughput searches for the clinically relevant signatures of arterial, venous, lymphatic, or capillary differentiation.

Here, we examined the expression of ceramide metabolism enzymes in the subcutaneous adipose tissue (SAT), epicardial adipose tissue (EAT) and perivascular adipose tissue (PVAT) of 30 patients with coronary artery disease (CAD) and 30 patients with valvular heart disease (VHD) by means of quantitative polymerase chain reaction and fluorescent Western blotting. The EAT of patients with CAD showed higher expression of the genes responsible for ceramide biosynthesis (SPTLC1, SPTLC2, CERS1, 5, 6, DEGS1, and SMPD1) and utilization (ASAH1, SGMS1). PVAT was characterized by higher mRNA levels of CERS3, CERS4, DEGS1, SMPD1, and ceramide utilization enzyme (SGMS2). In patients with VHD, there was a high CERS4, DEGS1, and SGMS2 expression in the EAT and CERS3 and CERS4 expression in the PVAT. Among patients with CAD, the expression of SPTLC1 in SAT and EAT, SPTLC2 in EAT, CERS2 in all studied AT, CERS4 and CERS5 in EAT, DEGS1 in SAT and EAT, ASAH1 in all studied AT, and SGMS1 in EAT was higher than in those with VHD. Protein levels of ceramide-metabolizing enzymes were consistent with gene expression trends. The obtained results indicate an activation of ceramide synthesis de novo and from sphingomyelin in cardiovascular disease, mainly in EAT, that contributes to the accumulation of ceramides in this location.

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.

The lack of suitable autologous grafts and the impossibility of using synthetic prostheses for small artery reconstruction make it necessary to develop alternative efficient vascular grafts. In this study, we fabricated an electrospun biodegradable poly(ε-caprolactone) (PCL) prosthesis and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) (PHBV/PCL) prosthesis loaded with iloprost (a prostacyclin analog) as an antithrombotic drug and cationic amphiphile with antibacterial activity. The prostheses were characterized in terms of their drug release, mechanical properties, and hemocompatibility. We then compared the long-term patency and remodeling features of PCL and PHBV/PCL prostheses in a sheep carotid artery interposition model. The research findings verified that the drug coating of both types of prostheses improved their hemocompatibility and tensile strength. The 6-month primary patency of the PCL/Ilo/A prostheses was 50%, while all PHBV/PCL/Ilo/A implants were occluded at the same time point. The PCL/Ilo/A prostheses were completely endothelialized, in contrast to the PHBV/PCL/Ilo/A conduits, which had no endothelial cells on the inner layer. The polymeric material of both prostheses degraded and was replaced with neotissue containing smooth-muscle cells; macrophages; proteins of the extracellular matrix such as type I, III, and IV collagens; and vasa vasorum. Thus, the biodegradable PCL/Ilo/A prostheses demonstrate better regenerative potential than PHBV/PCL-based implants and are more suitable for clinical use.

Aim. To evaluate the initial concentration of calciprotein particles (CPPs), which are scavengers of excessive calcium and phosphate, in patients with cardiovascular disease and in patients with chronic kidney disease as compared with the healthy volunteers. Material and methods . The study included 308 individuals as follows: 1) 88 participants of the PURE study without hemodynamically relevant carotid athero scle rosis and symptomatic coronary atherosclerosis; 2) 88 patients with cere brovascular disease (CVD) who required carotid endarterectomy; 3) 88 pa tients with coronary artery disease (CAD) who required percutaneous coronary intervention or coronary artery bypass graft surgery; 4) 63 patients with stage 5 chronic kidney disease (CKD). We measured following mineral homeostasis parameters: total and ionized calcium, phosphate, total protein, albumin, and fetuin-A. Then, we determined a baseline serum CPP concentration by flow cytometry using a fluorescent-labeled bisphosphonate OsteoSense 680EX. Results. In comparison with other patients, healthy volunteers had the highest serum CPP concentration (249 CPPs/µL), indicating the retained ability to compensate mineral homeostasis disturbances by aggregation of excessive calcium and pho sphate with acidic proteins (mineral chaperones). Reduced serum CPP concentration in patients with CVD (170 CPPs/µL), CAD (139 CPPs/µL), and stage 5 CKD (193-203 CPPs/µL) showed impaired aggregation of excessive serum calcium and phosphate, which was also reflected by an increased level of blood ionized calcium. Conclusion. Patients with CVD, CAD, and stage 5 CKD have lower serum CPP concentration than healthy individuals. In combination with elevated ionized calcium and reduced albumin, this suggests the depletion of calcium binding buffers in the serum of patients with cardiovascular and renal diseases.

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.

Here, we discuss pathophysiological approaches to the defining of endothelial dysfunction criteria (i.e., endothelial activation, impaired endothelial mechanotransduction, endothelial-to-mesenchymal transition, reduced nitric oxide release, compromised endothelial integrity, and loss of anti-thrombogenic properties) in different in vitro and in vivo models. The canonical definition of endothelial dysfunction includes insufficient production of vasodilators, pro-thrombotic and pro-inflammatory activation of endothelial cells, and pathologically increased endothelial permeability. Among the clinical consequences of endothelial dysfunction are arterial hypertension, macro- and microangiopathy, and microalbuminuria. We propose to extend the definition of endothelial dysfunction by adding altered endothelial mechanotransduction and endothelial-to-mesenchymal transition to its criteria. Albeit interleukin-6, interleukin-8, and MCP-1/CCL2 dictate the pathogenic paracrine effects of dysfunctional endothelial cells and are therefore reliable endothelial dysfunction biomarkers in vitro, they are non-specific for endothelial cells and cannot be used for the diagnostics of endothelial dysfunction in vivo. Conceptual improvements in the existing methods to model endothelial dysfunction, specifically, in relation to the blood–brain barrier, include endothelial cell culturing under pulsatile flow, collagen IV coating of flow chambers, and endothelial lysate collection from the blood vessels of laboratory animals in situ for the subsequent gene and protein expression profiling. Combined with the simulation of paracrine effects by using conditioned medium from dysfunctional endothelial cells, these flow-sensitive models have a high physiological relevance, bringing the experimental conditions to the physiological scenario.