Anna Malashicheva

Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve.

Bicuspid aortic valve is the most common congenital heart malformation and the reasons for the aortopathies associated with bicuspid aortic valve remain unclear. NOTCH1 mutations are associated with bicuspid aortic valve and have been found in individuals with various left ventricular outflow tract abnormalities. Notch is a key signaling during cardiac valve formation that promotes the endothelial-to-mesenchymal transition. We address the role of Notch signaling in human aortic endothelial cells from patients with bicuspid aortic valve and aortic aneurysm. Aortic endothelial cells were isolated from tissue fragments of bicuspid aortic valve-associated thoracic aortic aneurysm patients and from healthy donors. Endothelial-to-mesenchymal transition was induced by activation of Notch signaling. Effectiveness of the transition was estimated by loss of endothelial and gain of mesenchymal markers by immunocytochemistry and qPCR. We show that aortic endothelial cells from the patients with aortic aneurysm and bicuspid aortic valve have down regulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch ligands. Our findings support the idea that bicuspid aortic valve and associated aortic aneurysm is associated with dysregulation of the entire Notch signaling pathway independently on the specific gene mutation.

Variants in the NOTCH1 Gene in Patients with Aortic Coarctation

BACKGROUND AND OBJECTIVE Malformations of the left ventricular outflow tract are one of the most common forms of congenital heart disorders. Recently, it has been shown that mutations in the NOTCH1 gene can lead to bicuspid aortic valve, aortic aneurysm, and hypoplastic left heart syndrome. The aim of our study was to estimate the frequency of NOTCH1 gene mutations/substitutions in patients with aortic coarctation, isolated or combined with bicuspid aortic valve. DESIGN AND PATIENTS The study included 51 children with coarctation. Detailed family history was obtained for every study subject, and echocardiographic data were obtained for the relatives when available. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Control DNA was obtained from 200 healthy donors. RESULTS In more than half of the cases, coarctation was combined with bicuspid aortic valve, and in approximately half of the cases, it was combined with hypoplasia of the aortic arch or descending aorta. Familial history of congenital heart disease was observed in 34.3% of the cases. In total, 29 variants of the NOTCH1 gene were identified in the patient group and in the control subjects. Four of those variants led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. This variant was significantly overrepresented in the patients with aortic coarctation compared with those in the control group (P < .05). We conclude that the R1279H substitution in the NOTCH1 gene is significantly overrepresented in patients with aortic coarctation and, therefore, may represent a disease-susceptibility allele.

Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms

Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin) and smooth muscle (SMA, SM22α, calponin, and vimentin) cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis.

Regulation of nutrition-associated receptors in blood monocytes of normal weight and obese humans.

Obesity, type 2 diabetes and associated metabolic diseases are characterized by low-grade systemic inflammation which involves interplay of nutrition and monocyte/macrophage functions. We suggested that some factors such as nutrient components, neuropeptides involved in the control of gastrointestinal functions, and gastrointestinal hormones might influence immune cell functions and in this way contribute to the disease pathogenesis. The aim of this study was to investigate the mRNA expression of twelve nutrition-associated receptors in peripheral blood mononuclear cells (PBMC), isolated monocytes and monocyte-derived macrophages and their regulation under the switching from the high-carbohydrate low-fat diet to the low-carbohydrate high-fat (LC/HFD) isocaloric diet in healthy humans. The mRNA expression of receptors for short chain fatty acids (GPR41, GPR43), bile acids (TGR5), incretins (GIPR, GLP1R), cholecystokinin (CCKAR), neuropeptides VIP and PACAP (VIPR1, VIPR2), and neurotensin (NTSR1) was detected in PBMC and monocytes, while GPR41, GPR43, GIPR, TGR5, and VIPR1 were found in macrophages. Correlations of the receptor expression in monocytes with a range of metabolic and inflammatory markers were found. In non-obese subjects, the dietary switch to LC/HFD induced the increase of GPR43 and VIPR1 expression in monocytes. No significant differences of receptor expression between normal weight and moderately obese subjects were found. Our study characterized for the first time the expression pattern of nutrition-associated receptors in human blood monocytes and its dietary-induced changes linking metabolic responses to nutrition with immune functions in health and metabolic diseases.

Various lamin A/C mutations alter expression profile of mesenchymal stem cells in mutation specific manner

Various mutations in LMNA gene, encoding for nuclear lamin A/C protein, lead to laminopathies and contribute to over ten human disorders, mostly affecting tissues of mesenchymal origin such as fat tissue, muscle tissue, and bones. Recently it was demonstrated that lamins not only play a structural role providing communication between extra-nuclear structures and components of cell nucleus but also control cell fate and differentiation. In our study we assessed the effect of various LMNA mutations on the expression profile of mesenchymal multipotent stem cells (MMSC) during adipogenic and osteogenic differentiation. We used lentiviral approach to modify human MMSC with LMNA-constructs bearing mutations associated with different laminopathies--G465D, R482L, G232E, R527C, and R471C. The impact of various mutations on MMSC differentiation properties and expression profile was assessed by colony-forming unit analysis, histological staining, expression of the key differentiation markers promoting adipogenesis and osteogenesis followed by the analysis of the whole set of genes involved in lineage-specific differentiation using PCR expression arrays. We demonstrate that various LMNA mutations influence the differentiation efficacy of MMSC in mutation-specific manner. Each LMNA mutation promotes a unique expression pattern of genes involved in a lineage-specific differentiation and this pattern is shared by the phenotype-specific mutations.

Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification

Aortic valve stenosis due to calcification of the valve leaflets is the most common valve disease in the developed world. It is the third leading cause of cardiovascular disease.[1][1] Risk factors include male gender, smoking, diabetes mellitus, hypertension, high levels of circulating lipids, and

In vitro toxicity of Fe m O n , Fe m O n -SiO 2 composite, and SiO 2 -Fe m O n core-shell magnetic nanoparticles

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.

Modulation of insulin degrading enzyme activity and liver cell proliferation

Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.

Nuclear lamins regulate osteogenic differentiation of mesenchymal stem cells

AbsractNuclear lamins are the main proteins of the nuclear envelope providing nuclear-membrane strength. Recently, it became clear that lamins in cells play not only a structural role, but are also involved in regulation of gene expression. The LMNA gene encodes lamin A or C depending on the synthesizing splicing variant. The best-known LMNA mutation causes severe disorders in development known as progeria (premature aging syndrome). The disease is of rare occurrence. More frequently, point mutations in LMNA gene encoding lamin A/C result in so-called laminopathies, these diseases manifesting as tissue damage, mostly in tissues of mesenchymal origin. The mutations manifest in a tissue-specific manner: particular mutations always display the same disease phenotype. The nature of this phenomenon, as well as the mechanisms by which lamins regulate cell differentiation remain poorly understood. The aim of this study was to investigate the effect of different LMNA mutations on human mesenchimal stem cell (MSC) osteogenic differentiation and explore the possible interaction of lamins and Notch signaling pathway. We modified human MSCs with mutant LMNA bearing known mutations with tissue specific phenotype associated with different laminopathies. Differentiation was evaluated 21 days after its induction by number of differentiated cells, as well as by the expression level of specific osteogenic markers SPP, IBSP, and BGLAP. Some mutations enhance differentiation whereas others decrease its level. These findings support the notion that lamin A/C is involved in the regulation of MMSC differentiation. Introduction of mutant LMNA forms together with the activated Notch domain modified the expression of HEY1, a major target of Notch signaling. Thereby, one of the mechanisms involved in the regulation of MSC differentiation may be the interaction of lamins A/C with components of Notch signaling.

NOTCH1 Mutations in Aortic Stenosis: Association with Osteoprotegerin/RANK/RANKL

Background. The NOTCH pathway is known to be important in the pathogenesis of calcific aortic valve disease, possibly through regulators of osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK), and its ligand (RANKL) system. The purpose of the present study was to search for possible associations between NOTCH1 gene mutations and circulating levels of OPG and soluble RANKL (sRANKL) in patients with aortic stenosis (AS). Methods. The study was performed on 61 patients with AS including 31 with bicuspid and 30 with tricuspid aortic valves. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Serum OPG and sRANKL levels were assessed. Results. In total, 6 genetic variants of the NOTCH1 gene including two new mutations were identified in the study group. In an age- and arterial hypertension-adjusted multivariable regression analysis, the serum OPG levels and the OPG/sRANKL ratio were correlated with NOTCH1 missense variants. All studied missense variants in NOTCH1 gene were found in Ca(2+)-binding EGF motif of the NOTCH extracellular domain bound to Delta-like 4. Conclusion. Our results suggest that the OPG/RANKL/RANK system might be directly influenced by genetic variants of NOTCH1 in aortic valve calcification.