Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity.

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.

Embedding and Backscattered Scanning Electron Microscopy (EM-BSEM) Is Preferential over Immunophenotyping in Relation to Bioprosthetic Heart Valves.

Hitherto, calcified aortic valves (AVs) and failing bioprosthetic heart valves (BHVs) have been investigated by similar approaches, mostly limited to various immunostaining techniques. Having employed multiple immunostaining combinations, we demonstrated that AVs retain a well-defined cellular hierarchy even at severe stenosis, whilst BHVs were notable for the stochastic degradation of the extracellular matrix (ECM) and aggressive infiltration by ECM-digesting macrophages. Leukocytes (CD45+) comprised ≤10% cells in the AVs but were the predominant cell lineage in BHVs (≥80% cells). Albeit cells with uncertain immunophenotype were rarely encountered in the AVs (≤5% cells), they were commonly found in BHVs (≥80% cells). Whilst cell conversions in the AVs were limited to the endothelial-to-mesenchymal transition (represented by CD31+α-SMA+ cells) and the formation of endothelial-like (CD31+CD68+) cells at the AV surface, BHVs harboured numerous macrophages with a transitional phenotype, mostly CD45+CD31+, CD45+α-SMA+, and CD68+α-SMA+. In contrast to immunostaining, which was unable to predict cell function in the BHVs, our whole-specimen, nondestructive electron microscopy approach (EM-BSEM) was able to distinguish between quiescent and matrix-degrading macrophages, foam cells, and multinucleated giant cells to conduct the ultrastructural analysis of organelles and the ECM, and to preserve tissue integrity. Hence, we suggest EM-BSEM as a technique of choice for studying the cellular landscape of BHVs.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Expression of Ceramide-Metabolizing Enzymes in the Heart Adipose Tissue of Cardiovascular Disease Patients.

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.

Neurohumoral Markers of Cardiac Autonomic Denervation after Surgical Ablation of Long-Standing Persistent Atrial Fibrillation.

Although the autonomic nervous system has an evident impact on cardiac electrophysiology and radiofrequency ablation (RFA) is the conventional technique for treating persistent atrial fibrillation, the specific effects of RFA have been insufficiently studied to date. Here, we investigated whether RFA affects neurohumoral transmitter levels and myocardial 123I-metaiodobenzylguanidine (123I-MIBG) uptake. To perform this task, we compared two groups of patients with acquired valvular heart disease: patients who had undergone surgical AF ablation and patients with sinus rhythm. The decrease in norepinephrine (NE) level in the coronary sinus had a direct association with the heart-to-mediastinum ratio (p = 0.02) and a negative correlation with 123I-MIBG uptake defects (p = 0.01). The NE level decreased significantly after the main surgery, both in patients with AF (p = 0.0098) and sinus rhythm (p = 0.0039). Furthermore, the intraoperative difference between the norepinephrine levels in the ascending aorta and coronary sinus (ΔNE) of -400 pg/mL was determined as a cut-off value to evaluate RFA efficacy, as denervation failed in all patients with ΔNE < -400 pg/mL. Hence, ΔNE can be utilized to predict the efficacy of the "MAZE-IV" procedure and to assess the risk of AF recurrence after RFA.

Comparison of the Patency and Regenerative Potential of Biodegradable Vascular Prostheses of Different Polymer Compositions in an Ovine Model.

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.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

BACKGROUND: Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. METHODS: CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). RESULTS: CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CONCLUSIONS: CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Cardiovascular and Renal Comorbidities Included into Neural Networks Predict the Outcome in COVID-19 Patients Admitted to an Intensive Care Unit: Three-Center, Cross-Validation, Age- and Sex-Matched Study.

Here, we performed a multicenter, age- and sex-matched study to compare the efficiency of various machine learning algorithms in the prediction of COVID-19 fatal outcomes and to develop sensitive, specific, and robust artificial intelligence tools for the prompt triage of patients with severe COVID-19 in the intensive care unit setting. In a challenge against other established machine learning algorithms (decision trees, random forests, extra trees, neural networks, k-nearest neighbors, and gradient boosting: XGBoost, LightGBM, and CatBoost) and multivariate logistic regression as a reference, neural networks demonstrated the highest sensitivity, sufficient specificity, and excellent robustness. Further, neural networks based on coronary artery disease/chronic heart failure, stage 3-5 chronic kidney disease, blood urea nitrogen, and C-reactive protein as the predictors exceeded 90% sensitivity and 80% specificity, reaching AUROC of 0.866 at primary cross-validation and 0.849 at secondary cross-validation on virtual samples generated by the bootstrapping procedure. These results underscore the impact of cardiovascular and renal comorbidities in the context of thrombotic complications characteristic of severe COVID-19. As aforementioned predictors can be obtained from the case histories or are inexpensive to be measured at admission to the intensive care unit, we suggest this predictor composition is useful for the triage of critically ill COVID-19 patients.

Endothelial Cell Markers Are Inferior to Vascular Smooth Muscle Cells Markers in Staining Vasa Vasorum and Are Non-Specific for Distinct Endothelial Cell Lineages in Clinical Samples.

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.

Proteolytic Degradation Is a Major Contributor to Bioprosthetic Heart Valve Failure.

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.

Calciprotein Particles Induce Cellular Compartment-Specific Proteome Alterations in Human Arterial Endothelial Cells.

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.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation.

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.

Controlled and Synchronised Vascular Regeneration upon the Implantation of Iloprost- and Cationic Amphiphilic Drugs-Conjugated Tissue-Engineered Vascular Grafts into the Ovine Carotid Artery: A Proteomics-Empowered Study.

Implementation of small-diameter tissue-engineered vascular grafts (TEVGs) into clinical practice is still delayed due to the frequent complications, including thrombosis, aneurysms, neointimal hyperplasia, calcification, atherosclerosis, and infection. Here, we conjugated a vasodilator/platelet inhibitor, iloprost, and an antimicrobial cationic amphiphilic drug, 1,5-bis-(4-tetradecyl-1,4-diazoniabicyclo [2.2.2]octan-1-yl) pentane tetrabromide, to the luminal surface of electrospun poly(ε-caprolactone) (PCL) TEVGs for preventing thrombosis and infection, additionally enveloped such TEVGs into the PCL sheath to preclude aneurysms, and implanted PCLIlo/CAD TEVGs into the ovine carotid artery (n = 12) for 6 months. The primary patency was 50% (6/12 animals). TEVGs were completely replaced with the vascular tissue, free from aneurysms, calcification, atherosclerosis and infection, completely endothelialised, and had clearly distinguishable medial and adventitial layers. Comparative proteomic profiling of TEVGs and contralateral carotid arteries found that TEVGs lacked contractile vascular smooth muscle cell markers, basement membrane components, and proteins mediating antioxidant defense, concurrently showing the protein signatures of upregulated protein synthesis, folding and assembly, enhanced energy metabolism, and macrophage-driven inflammation. Collectively, these results suggested a synchronised replacement of PCL with a newly formed vascular tissue but insufficient compliance of PCLIlo/CAD TEVGs, demanding their testing in the muscular artery position or stimulation of vascular smooth muscle cell specification after the implantation.

Excessive Adventitial and Perivascular Vascularisation Correlates with Vascular Inflammation and Intimal Hyperplasia.

Albeit multiple studies demonstrated that vasa vasorum (VV) have a crucial importance in vascular pathology, the informative markers and metrics of vascular inflammation defining the development of intimal hyperplasia (IH) have been vaguely studied. Here, we employed two rat models (balloon injury of the abdominal aorta and the same intervention optionally complemented with intravenous injections of calciprotein particles) and a clinical scenario (arterial and venous conduits for coronary artery bypass graft (CABG) surgery) to investigate the pathophysiological interconnections among VV, myeloperoxidase-positive (MPO+) clusters, and IH. We found that the amounts of VV and MPO+ clusters were strongly correlated; further, MPO+ clusters density was significantly associated with balloon-induced IH and increased at calciprotein particle-provoked endothelial dysfunction. Likewise, number and density of VV correlated with IH in bypass grafts for CABG surgery at the pre-intervention stage and were higher in venous conduits which more frequently suffered from IH as compared with arterial grafts. Collectively, our results underline the pathophysiological importance of excessive VV upon the vascular injury or at the exposure to cardiovascular risk factors, highlight MPO+ clusters as an informative marker of adventitial and perivascular inflammation, and propose another mechanistic explanation of a higher long-term patency of arterial grafts upon the CABG surgery.

Early Postoperative Immunothrombosis of Bioprosthetic Mitral Valve and Left Atrium: A Case Report.

A 72-year-old female patient with mixed rheumatic mitral valve disease and persistent atrial fibrillation underwent mitral valve replacement and suffered from a combined thrombosis of the bioprosthetic valve and the left atrium as soon as 2 days post operation. The patient immediately underwent repeated valve replacement and left atrial thrombectomy. Yet, four days later the patient died due to the recurrent prosthetic valve and left atrial thrombosis which both resulted in an extremely low cardiac output. In this patient's case, the thrombosis was notable for the resistance to anticoagulant therapy as well as for aggressive neutrophil infiltration and release of neutrophil extracellular traps (NETs) within the clot, as demonstrated by immunostaining. The reasons behind these phenomena remained unclear, as no signs of sepsis or contamination of the BHV were documented, although the patient was diagnosed with inherited thrombophilia that could impede the fibrinolysis. The described case highlights the hazard of immunothrombosis upon valve replacement and elucidates its mechanisms in this surgical setting.

EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues.

Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.

Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation.

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.

Tissue-Engineered Carotid Artery Interposition Grafts Demonstrate High Primary Patency and Promote Vascular Tissue Regeneration in the Ovine Model.

Tissue-engineered vascular graft for the reconstruction of small arteries is still an unmet clinical need, despite the fact that a number of promising prototypes have entered preclinical development. Here we test Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)Poly(ε-caprolactone) 4-mm-diameter vascular grafts equipped with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor 1α (SDF-1α) and surface coated with heparin and iloprost (PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo, n = 8) in a sheep carotid artery interposition model, using biostable vascular prostheses of expanded poly(tetrafluoroethylene) (ePTFE, n = 5) as a control. Primary patency of PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts was 62.5% (5/8) at 24 h postimplantation and 50% (4/8) at 18 months postimplantation, while all (5/5) ePTFE conduits were occluded within the 24 h after the surgery. At 18 months postimplantation, PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts were completely resorbed and replaced by the vascular tissue. Regenerated arteries displayed a hierarchical three-layer structure similar to the native blood vessels, being fully endothelialised, highly vascularised and populated by vascular smooth muscle cells and macrophages. The most (4/5, 80%) of the regenerated arteries were free of calcifications but suffered from the aneurysmatic dilation. Therefore, biodegradable PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts showed better short- and long-term results than bio-stable ePTFE analogues, although these scaffolds must be reinforced for the efficient prevention of aneurysms.

Aortography Keypoint Tracking for Transcatheter Aortic Valve Implantation Based on Multi-Task Learning.

Currently, transcatheter aortic valve implantation (TAVI) represents the most efficient treatment option for patients with aortic stenosis, yet its clinical outcomes largely depend on the accuracy of valve positioning that is frequently complicated when routine imaging modalities are applied. Therefore, existing limitations of perioperative imaging underscore the need for the development of novel visual assistance systems enabling accurate procedures. In this paper, we propose an original multi-task learning-based algorithm for tracking the location of anatomical landmarks and labeling critical keypoints on both aortic valve and delivery system during TAVI. In order to optimize the speed and precision of labeling, we designed nine neural networks and then tested them to predict 11 keypoints of interest. These models were based on a variety of neural network architectures, namely MobileNet V2, ResNet V2, Inception V3, Inception ResNet V2 and EfficientNet B5. During training and validation, ResNet V2 and MobileNet V2 architectures showed the best prediction accuracy/time ratio, predicting keypoint labels and coordinates with 97/96% accuracy and 4.7/5.6% mean absolute error, respectively. Our study provides evidence that neural networks with these architectures are capable to perform real-time predictions of aortic valve and delivery system location, thereby contributing to the proper valve positioning during TAVI.

Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells.

The nuclear factor of activated T-cells 5 (NFAT5) is a transcriptional regulator of macrophage activation and T-cell development, which controls stabilizing responses of cells to hypertonic and biomechanical stress. In this study, we detected NFAT5 in the media layer of arteries adjacent to human arteriosclerotic plaques and analyzed its role in vascular smooth muscle cells (VSMCs) known to contribute to arteriosclerosis through the uptake of lipids and transformation into foam cells. Exposure of both human and mouse VSMCs to cholesterol stimulated the nuclear translocation of NFAT5 and increased the expression of the ATP-binding cassette transporter Abca1, required to regulate cholesterol efflux from cells. Loss of Nfat5 promoted cholesterol accumulation in these cells and inhibited the expression of genes involved in the management of oxidative stress or lipid handling, such as Sod1, Plin2, Fabp3, and Ppard. The functional relevance of these observations was subsequently investigated in mice fed a high-fat diet upon induction of a smooth muscle cell-specific genetic ablation of Nfat5 (Nfat5(SMC)-/- ). Under these conditions, Nfat5(SMC)-/- but not Nfat5fl/fl mice developed small, focal lipid-rich lesions in the aorta after 14 and 25 weeks, which were formed by intracellular lipid droplets deposited in the sub-intimal VSMCs layer. While known for being activated by external stimuli, NFAT5 was found to mediate the expression of VSMC genes associated with the handling of lipids in response to a cholesterol-rich environment. Failure of this protective function may promote the formation of lipid-laden arterial VSMCs and pro-atherogenic vascular responses.