Victoria Markova

Background: Citrullination 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. Objective: To investigate features and patterns of citrullination in atherosclerotic plaques. objective: To investigate features and patterns of citrullination in atherosclerotic plaques. Methods: We 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. Results: Proteomic 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. Conclusions: The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Palmitic acid (PA) is the most common dietary saturated fatty acid, and is abundant in palm and cottonseed oil, butter, and cheese, whereas oleic acid (OA) is a monounsaturated omega-9 fatty acid found in olive oil. The differences in the cytotoxic and pro-inflammatory effects of PA and OA across endothelial cells (ECs) isolated from different vascular beds have not been investigated in detail. Here, we incubated primary human aortic valve (HAVEC), saphenous vein (HSaVEC), internal thoracic artery (HITAEC), and microvascular (HMVEC) ECs with albumin-bound PA or OA for 24 h and found that PA induced a considerable cytotoxic response, accompanied by an elevated expression of the genes encoding cell adhesion molecules (VCAM1, ICAM1, SELE, and SELP) and pro-inflammatory cytokines (MIF, PTX3, CSF2, CSF3, IL1A, IL6, CCL2, CCL5, CCL20, CSF2, CSF3, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CXCL10), followed by an increased release of interleukin-6 and interleukin-8. HAVEC and HSaVEC were more susceptible to PA, whereas OA had mild-to-moderate cytotoxic effects on HAVEC and HMVEC but did not induce generalized EC activation. Compared with other EC types, HITAEC was the most resistant to PA and OA treatment. Collectively, these results indicate considerable heterogeneity across the ECs of distinct origin in response to PA.

Previous studies suggested a certain efficiency of proteinogenic branched-chain amino acid (BCAA) and magnesium supplementations in reducing cardiovascular risk and increasing quality of life. This investigation assessed the anti-atherogenic and anti-calcific effects of BCAA (55 mg/day, corresponding to a human equivalent dose of 13.5 g/day) and magnesium citrate (MgCit, 1.85 mg/day, corresponding to a human equivalent dose of 450 mg/day) intake in male and female ApoE-knockout mice, with the treatment initiation at either 1, 3, or 6 months of age. At the 12-month time point, lipid retention and calcium deposition in the aortic valve, lipid burden in the aorta, and serum ionized calcium were evaluated. The early BCAA intake (from 1/3 to 12 months of age) significantly reduced lipid retention in the aortic valve, whilst MgCit decreased ionized calcium. Both of these protective effects were higher in male than in female mice. Furthermore, it was tested whether human serum albumin (HSA) or MgCit can be applied to decrease the serum calcification propensity in 100 patients with myocardial infarction. A dual supplementation with HSA and MgCit reduced serum calcification propensity in 68% of cases. Collectively, these results highlight the potential benefits of BCAA/HSA and magnesium supplementations for cardiovascular prevention and justify further clinical trials in this regard.

Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here, we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC), human internal thoracic artery endothelial cells (HITAEC), and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC, ≈50% for HITAEC, and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC), ≈2–3 weeks (HITAEC), and ≈3–4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation, as well as real-time bioimpedance monitoring using the xCELLigence instrument, demonstrated high proliferative activity in HSaVEC, HITAEC, and HMVEC, whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC, HITAEC, and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity, to interrogate endothelial responses to various stresses, and to pinpoint the optimal approaches for restoring endothelial homeostasis, thereby reproducing them within the bedside-to-bench-to-bedside concept.

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% increase in Ca²⁺ in the serum or medium) and investigated whether the pathological effects of calcium stress depend on the calcium delivery form, such as Ca²⁺ ions, albumin- or fetuin-centric calciprotein monomers (CPM-A/CPM-F), and albumin- or fetuin-centric CPPs (CPP-A/CPP-F). The treatment with CPP-A or CPP-F upregulated transcription of pro-inflammatory genes (VCAM1, ICAM1, SELE, IL6, CXCL8, CCL2, CXCL1, MIF) and promoted release of pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, and MIP-3α/CCL20) and pro- and anti-thrombotic molecules (PAI-1 and uPAR) in human arterial ECs and monocytes, although these results depended on the type of cell and calcium-containing particles. Free Ca²⁺ ions and CPM-A/CPM-F induced less consistent detrimental effects. Intravenous administration of CaCl₂, CPM-A, or CPP-A to Wistar rats increased production of chemokines (CX3CL1, MCP-1/CCL2, CXCL7, CCL11, CCL17), hepatokines (hepassocin, fetuin-A, FGF-21, GDF-15), proteases (MMP-2, MMP-3) and protease inhibitors (PAI-1) into the circulation. We concluded that molecular consequences of calcium overload are largely determined by the form of its delivery and CPPs are efficient inducers of mineral stress at physiological levels.

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.

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.

Highlights To 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.

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. AbstractAim. 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.

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.