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Yana G Toropova,1 Alexey S Golovkin,2 Anna B Malashicheva,3,4 Dmitry V Korolev,5,6 Andrey N Gorshkov,7 Kamil G Gareev,8 Michael V Afonin,9 Michael M Galagudza10,11 1Laboratory of Cardioprotection, Institute of Experimental Medicine, Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation; 2Gene and Cell Engineering Group, Institute of Molecular Biology and Genetics, Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation; 3Laboratory of Molecular Cardiology, Institute of Molecular Biology and Genetics, Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation; 4Department of Embryology, Faculty of Biology, Saint Petersburg State University, Saint Petersburg, Russian Federation; 5Laboratory of Nanotechnology, Institute of Experimental Medicine, Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation; 6Department of Photonics and Optical Information Technology ITMO University, Saint Petersburg, Russian Federation; 7Laboratory of Intracellular Signaling and Transport Research Institute of Influenza, Saint Petersburg, Russian Federation; 8Department of Micro and Nanoelectronics, Faculty of Electronics, Saint Petersburg Electrotechnical University LETI, Saint Petersburg, Russian Federation; 9Department of Inorganic Chemistry Saint Petersburg State Technological Institute (Technical University), Saint Petersburg, Russian Federation; 10Institute of Experimental Medicine, Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation; 11Departament of Pathophysiology, First Pavlov State Medical University of Saint Petersburg, Saint Petersburg, Russian Federation Abstract: Over the last decade, magnetic iron oxide nanoparticles (IONPs) have drawn much attention for their potential biomedical applications. However, serious in vitro and in vivo safety concerns continue to exist. In this study, the effects of uncoated, FemOn-SiO2 composite flake-like, and SiO2-FemOn core-shell IONPs on cell viability, function, and morphology were tested 48 h postincubation in human umbilical vein endothelial cell culture. Cell viability and apoptosis/necrosis rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin V-phycoerythrin kit, respectively. Cell morphology was evaluated using bright-field microscopy and forward and lateral light scattering profiles obtained with flow cytometry analysis. All tested IONP types were used at three different doses, that is, 0.7, 7.0, and 70.0 µg. Dose-dependent changes in cell morphology, viability, and apoptosis rate were shown. At higher doses, all types of IONPs caused formation of binucleated cells suggesting impaired cytokinesis. FemOn-SiO2 composite flake-like and SiO2-FemOn core-shell IONPs were characterized by similar profile of cytotoxicity, whereas bare IONPs were shown to be less toxic. The presence of either silica core or silica nanoflakes in composite IONPs can promote cytotoxic effects. Keywords: iron oxide nanoparticles, composite nanoparticles, silica coating, silica nanoflakes, cytotoxicity

The development of drug therapy for the pathological calcification of the aortic valve is still an open issue due to the lack of effective treatment strategies. Currently, the only option for treating this condition is surgical correction and symptom management. The search for models to study the safety and efficacy of anti-calcifying drugs requires them to not only be as close as possible to in vivo conditions, but also to be flexible with regard to the molecular studies that can be applied to them. The ex vivo model has several advantages, including the ability to study the effect of a drug on human cells while preserving the original structure of the valve. This allows for a better understanding of how different cell types interact within the valve, including non-dividing cells. The aim of this study was to develop a reproducible ex vivo calcification model based on valves from patients with calcific aortic stenosis. We aimed to induce spontaneous calcification in valve tissue fragments under osteogenic conditions, and to demonstrate the possibility of significantly suppressing it using a calcification inhibitor. To validate the model, we tested a Notch inhibitor Crenigacestat (LY3039478), which has been previously shown to have an anti-calcifying effect on interstitial cell of the aortic valve. We demonstrate here an approach to testing calcification inhibitors using an ex vivo model of cultured human aortic valve tissue fragments. Thus, we propose that ex vivo models may warrant further investigation for their utility in studying aortic valve disease and performing pre-clinical assessment of drug efficacy.

Cardiac fibrosis, characterized by the excessive deposition of extracellular matrix proteins, significantly contributes to the morbidity and mortality associated with cardiovascular diseases. This article explores the complex interplay between Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and Notch signaling pathways in the pathogenesis of cardiac fibrosis. Each of these pathways plays a crucial role in the regulation of cellular functions and interactions that underpin fibrotic processes in the heart. Through a detailed review of current research, we highlight how the crosstalk among RUNX2, BMP2, and Notch not only facilitates our understanding of the fibrotic mechanisms but also points to potential biomolecular targets for intervention. This article delves into the regulatory networks, identifies key molecular mediators, and discusses the implications of these signaling pathways in cardiac structural remodeling. By synthesizing findings from recent studies, we provide insights into the cellular and molecular mechanisms that could guide future research directions, aiming to uncover new therapeutic strategies to manage and treat cardiac fibrosis effectively.

Aleksandra S [...]

The NOTCH-signaling pathway is responsible for intercellular interactions and cell fate commitment. Recently, NOTCH pathway genes were demonstrated to play an important role in aortic valve development, leading to an increased calcified aortic valve disease (CAVD) later in life. Here, we further investigate the association between genetic variants in the NOTCH pathway genes and aortic stenosis in a case–control study of 90 CAVD cases and 4723 controls using target panel sequencing of full-length 20 genes from a NOTCH-related pathway (DVL2, DTX2, MFNG, NUMBL, LFNG, DVL1, DTX4, APH1A, DTX1, APH1B, NOTCH1, ADAM17, DVL3, NCSTN, DTX3L, ILK, RFNG, DTX3, NOTCH4, PSENEN). We identified a common intronic variant in NOTCH1, protecting against CAVD development (rs3812603), as well as several rare and unique new variants in NOTCH-pathway genes (DTX4, NOTCH1, DTX1, DVL2, NOTCH1, DTX3L, DVL3), with a prominent effect of the protein structure and function.

Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.

Aim. To assess the association between pathological ascending aortic (AA) wall changes and its planimetric characteristics in non-syndromic non-familial (sporadic) aneurysm and dilation of the AA.Material and methods. The study included 174 patients with sporadic aneurysms and dilation of the AA, who underwent elective surgery between January 2010 and July 2015 and were divided into 2 groups: patients with AA aneurysm (AAA) (AA diameter >50 mm) and tricuspid aortic valve (AV) with significant aortic stenosis (AS) or regurgitation (AR) (AAA group, n=120), and persons with borderline AA dilatation (AA diameter 45-50 mm), associated with a bicuspid aortic valve (BAV) and significant AS (BD group, n=54). Standard paraclinical investigations and pathological examination of the VA wall were used. Statistical processing was carried out in the SPYDER 4.1.5 environment (Python 3.8), and included univariate correlation analysis, logistic regression analysis, as well as supervised machine learning (ML) methods (support vector machine, k-nearest neighbor method, random forest).Results. Logistic regression revealed positive associations between AA atherosclerosis and age, cystic medial necrosis (CMN) and sinus of Valsalva (SV) diameters. The support vector machine method demonstrated a tendency towards AA expansion at the SV level in individuals with CMN (accuracy, 60,5%), as well as towards expansion of the tubular AA in atherosclerosis (accuracy, 79,2%). During the random forest analysis, the first stage was to construct decision trees to predict three following outcomes: the presence of CMN, atherosclerosis, or normal aortic structure. The model accuracy was 64,2%. Next, the variables "CMN" and "atherosclerosis" were combined, and prediction was made for the outcomes "normal AA wall structure" and "pathological AA wall structure". The model accuracy was 73,5%.Conclusion. The use of ML opens up new opportunities for predicting aortopathy and a patient-centered approach to treatment. In AR, a more aggressive AA intervention is warranted. To predict aortopathies, thoracic aorta diameters indexed to body surface area should not be used. Aortic wall sampling (circular section) followed by a continuous pathological examination may be promising.

Fibrosis is a major medical challenge, as it leads to irreversible tissue remodeling and organ dysfunction. Its progression contributes significantly to morbidity and mortality worldwide, with limited therapeutic options available. Extensive research on the molecular mechanisms of fibrosis has revealed numerous factors and signaling pathways involved. However, the interactions between these pathways remain unclear. A comprehensive understanding of the entire signaling network that drives fibrosis is still missing. The TGF-β and Notch signaling pathways play a key role in fibrogenesis, and this review focuses on their functional interplay and molecular mechanisms. Studies have shown synergy between TGF-β and Notch cascades in fibrosis, but antagonistic interactions can also occur, especially in cardiac fibrosis. The molecular mechanisms of these interactions vary depending on the cell context. Understanding these complex and context-dependent interactions is crucial for developing effective strategies for treating fibrosis. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis, characterized by the excessive deposition of extracellular matrix proteins, significantly contributes to the morbidity and mortality associated with cardiovascular diseases. This article explores the complex interplay between Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and Notch signaling pathways in the pathogenesis of cardiac fibrosis. Each of these pathways plays a crucial role in the regulation of cellular functions and interactions that underpin fibrotic processes in the heart. Through a detailed review of current research, we highlight how the crosstalk among RUNX2, BMP2, and Notch not only facilitates our understanding of the fibrotic mechanisms but also points to potential biomolecular targets for intervention. This article delves into the regulatory networks, identifies key molecular mediators, and discusses the implications of these signaling pathways in cardiac structural remodeling. By synthesizing findings from recent studies, we provide insights into the cellular and molecular mechanisms that could guide future research directions, aiming to uncover new therapeutic strategies to manage and treat cardiac fibrosis effectively. [ABSTRACT FROM AUTHOR]

Background and objectives: Idiopathic pulmonary fibrosis is a chronic, progressive, incurable lung disease, leading to irreversible lung tissue remodeling. The Notch signaling pathway, essential for lung development, has gained attention for its role in pulmonary fibrosis. While Notchl and Notch3 have been extensively studied, the involvement of other Notch receptors, especially Notch4, remains less explored. This study aimed to evaluate the impact of Notch4 on lung fibroblast activation and its potential interaction with the transforming growth factor-beta 1 (TGFpi) signaling. Methods: Primary human lung fibroblasts were transduced with lentivirus containing the intracellular domain of NOTCH4 (N4ICD). Changes in gene expression in transduced cells were assessed using real-time polymerase chain reaction, immunoflu-orescence staining, and Western blotting. Transcriptomic analysis was also performed on N4ICD-transduced lung fibroblasts. Results: N4ICD overexpression significantly upregulated key fibrotic markers such as ACTA2 and COL1A1. It also induced the TGFpi pathway, as evidenced by SMAD2 phosphorylation and elevated TGFfll mRNA level. Transcriptomic analysis revealed that N4ICD-induced cells exhibited characteristics of highly invasive myofibroblasts. Conclusions: This study establishes Notch4 as a novel contributor to pulmonary fibrosis, by demonstrating its ability to induce myofibroblast differentiation and interact with the TGFpi pathway. [ABSTRACT FROM AUTHOR]

Biocompatible poly(lactide-co-glycolide) scaffolds fabricated via electrospinning are having promising properties as implants for the regeneration of fast-growing tissues, which are able to degrade in the body. The hereby-presented research work investigates the surface modification of these scaffolds in order to improve antibacterial properties of this type of scaffolds, as it can increase their application possibilities in medicine. Therefore, the scaffolds were surface-modified by means of pulsed direct current magnetron co-sputtering of copper and titanium targets in an inert atmosphere of argon. In order to obtain different amounts of copper and titanium in the resulting coatings, three different surface-modified scaffold samples were produced by changing the magnetron sputtering process parameters. The success of the antibacterial properties’ improvement was tested with the methicillin-resistant bacterium Staphylococcus aureus. In addition, the resulting cell toxicity of the surface modification by copper and titanium was examined using mouse embryonic and human gingival fibroblasts. As a result, the scaffold samples surface-modified with the highest copper to titanium ratio show the best antibacterial properties and no toxicity against mouse fibroblasts, but have a toxic effect to human gingival fibroblasts. The scaffold samples with the lowest copper to titanium ratio display no antibacterial effect and toxicity. The optimal poly(lactide-co-glycolide) scaffold sample is surface-modified with a medium ratio of copper and titanium that has antibacterial properties and is non-toxic to both cell cultures.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)‐2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP‐2/9, and both total and phosphorylated forms of Smad2/3 (P‐Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate–dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)‐β signaling pathway to further investigate the role of TGF‐β. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P‐Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd‐induced SDC4 downregulation, and hyperpermeability. MMP‐2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF‐β/Smad pathway in low‐dose Cd‐induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure. This study aims to explore the effects of low‐dose cadmium on syndecan‐4 expression. The results revealed that Cd exposure decreases SDC4 expression in both mice kidney tissues and human renal glomerular endothelial cells (HRGECs). Cd exposure induces hyperpermeability in HRGECs and increases the levels P‐Smad2/3 and MMP‐2/9. TGF‐β signaling inhibitor SB431542 blocks Cd‐induced SDC4 reduction and MMP‐2/9 upregulation. The study revealed that low‐dose Cd reduces expression of SDC4 through activation of the TGF‐β signaling. [ABSTRACT FROM AUTHOR]

Purinergic signaling is associated with a vast spectrum of physiological processes, including cardiovascular system function and, in particular, its pathological calcifications, such as aortic valve stenosis. Aortic valve stenosis (AS) is a degenerative disease for which there is no cure other than surgical replacement of the affected valve. Purinergic signaling is known to be involved in the pathologic osteogenic differentiation of valve interstitial cells (VIC) into osteoblast-like cells, which underlies the pathogenesis of AS. ATP, its metabolites and related nucleotides also act as signaling molecules in normal osteogenic differentiation, which is observed in pro-osteoblasts and leads to bone tissue development. We show that stenotic and non-stenotic valve interstitial cells significantly differ from each other, especially under osteogenic stimuli. In osteogenic conditions, the expression of the ecto-nucleotidases ENTPD1 and ENPP1, as well as ADORA2b, is increased in AS VICs compared to normal VICs. In addition, AS VICs after osteogenic stimulation look more similar to osteoblasts than non-stenotic VICs in terms of purinergic signaling, which suggests the stronger osteogenic differentiation potential of AS VICs. Thus, purinergic signaling is impaired in stenotic aortic valves and might be used as a potential target in the search for an anti-calcification therapy.

The search and study of endogenous heart repair remains an urgent issue in modern regenerative medicine. It is generally accepted that the human heart has a limited regenerative potential, but recent studies show that functionally significant regeneration is possible. However, the mechanisms underlying these processes remain poorly understood. In the heart, there are populations of resident mesenchymal cells that have some properties of stem cells that carry certain markers, such as c-kit+, Sca-1, etc. The ability of these cells to differentiate directly into cardiomyocytes remains controversial, but their use in clinical trials has shown improved cardiac function in patients with myocardial infarction. Currently, approaches are being developed to use, mainly, induced pluripotent stem cells as a promising regenerative therapy, but the cardioprotective role of cardiac mesenchymal cells remains the subject of active study due to their paracrine signaling.

Mutations in the LMNA gene cause developing of several phenotypes, both with isolated involvement of cardiac, muscle, adipose and bone tissues, and with their combination. The dominance of cardiovascular signs in the clinical performance and false clarity in nosology definition can cause underestimation of subclinical markers of other systems partaking. It leads to an incorrect interpretation of the true disease etiology, failure in genetic diagnostics, and untimely determination of the correct management and prognosis. The article presents clinical cases that demonstrate both the most typical manifestations of laminopathy and rare combinations of symptoms, which represent a certain diagnostic difficulty.

Human iPSC lines were generated from peripheral blood mononuclear cells of patient carrying LMNA mutation associated with Emery–Dreifuss muscular dystrophy accompanied by atrioventricular block and paroxysmal atrial fibrillation. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai virus transduction. iPSCs were characterized in order to prove the pluripotency markers expression, normal karyotype, ability to differentiate into three embryonic germ layers. Generated iPSC lines would be useful model to investigate disease development associated with genetic variants in LMNA gene.

Human iPSC cell lines (FAMRCi004-A and FAMRCi004-B) were generated from patient with progressive cardiac conduction disease and sick sinus syndrome carrying DSP p.His1684Arg genetic variant. Patient-specific adipose tissue-derived mesenchymal multipotent stromal cells were reprogrammed using non-integrative Sendai viruses. Established iPSC lines showed normal karyotype, expressed pluripotent markers and were able to differentiate toward three germ layers in vitro. The reported iPSC lines could be useful tool for in vitro modeling of progressive cardiac conduction disease associated with mutations in desmosomal genes.

Mutations in LMNA gene are known to cause a broad range of diseases called laminopathies. We have generated two induced pluripotent stem cell lines FAMRCi006-A and FAMRCi006-B from a patient carrying LMNA p. p.Arg527Pro mutation associated with Emery–Dreifuss muscular dystrophy and dilated cardiomyopathy. Patient-specific peripheral blood mononuclear cells were reprogrammed to iPSCs using Sendai virus reprogramming system. Characterization of iPSCs had revealed pluripotency marker expression, normal karyotype, ability to differentiate into three embryonic germ layers. The reported iPSC lines could be a useful tool for in vitro modeling of laminopathies associated with LMNA genetic variants. Keywords: Laminopathies, Induced pluripotent stem cells, Emery-Dreifuss muscular dystrophy, Dilated cardiomyopathy

LMNA mutations are often linked to laminopathies characterized by tissue-specific disorders. We generated two induced pluripotent stem cells lines from patient carrying genetic variant LMNA p.Asp357Val associated with paroxysmal ventricular tachycardia and myopathy. Reprogramming of patient's peripheral blood mononuclear cells was performed using Sendai viruses. Characterization of the FAMRCi005-A and FAMRCi005-B lines revealed that generated iPSC lines expressed pluripotent stem cell markers, had normal karyotype and demonstrated triliniage differentiation ability. Generated cell lines can be used to investigate the molecular links between LMNA genetic variants and cardiac disorders.

Aim. To evaluate the impact of genetics in development of thoracic aneurysm in patients with tricuspid valve (TAV) and bicuspid aortic valve (BAV) based on the analysis and search for mutations in NOTCH1.Material and methods. In the study, 60 patients included with the dilation of thoracic aorta more than 40 mm and 200 patients with no aortic pathology, included in the comparison group. All patients underwent echocardiographic assessment on Vivid 7 (GE, USA) equipment, by standard protocol. For molecular genetics we utilized the strategy of targeted mutation screening, including the analysis of 10 of 34 exones of the gene NOTCH1, performed with the direct sequencing.Results. Patients with BAV were younger than those with no inborn defect. Arterial hypertension was verified only in every second BAV patient. Also, maximal rates of blood pressure were significantly lower in patients with inborn defects (р