Applying Patient-Specific Induced Pluripotent Stem Cells to Create a Model of Hypertrophic Cardiomyopathy.

Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.

[Visualisation and Radiofrequency Ablation of Sympathetic Innervation Loci in the Left Atrium in Patients with Paroxysmal Atrial Fibrillation].

INTRODUCTION: A novel cardiac gamma camera utilizes the radiopharmaceutical Iodine-123-Meta-iodobenzylguanidine (123I-MIBG) to visualize cardiac sympathetic innervation. Physiological accumulation of 123I-mIBG provides an anatomical quantitative determination of the structures of the autonomic nervous system (ANS) with discrete uptake areas (DUA) of sympathetic activity located in the left atrium (LA) corresponding to the main ganglionic plexi (GP) clusters that could not previously be visualized. AIM: to visualize the DUA of the heart in patients with paroxysmal atrial fibrillation (AF) and to assess the effect of radiofrequency ablation (RFA) on DUA in LA. MATERIALS AND METHODS: Computed tomography (CT) of the heart and radionuclide imaging with 123I-mIBG were performed in 15 patients with paroxysmal AF. The results of the study were combined with preliminary taken CT images to create a detailed anatomical map of the sympathetic activity of the heart. The processed images were combined with the 3D reconstruction of the LA, obtained with the navigation system (CARTO 3, CARTO RMT). In DUA, high-frequency stimulation (HFS) followed by RF ablation was performed using the current recommended parameters. RESULTS: Forty-eight DUA (median 3 [3; 3]) were identified. Average activity of DUA was 1315 [1171; 1462] cnt / sec / ml. Positive response to HFS in the DUA was obtained in 8 (53.3 %) patients. Prior to ablation, no response was received to HFS in areas of LA outside the DUA. After ablation, there was no response to HFS in the DUA sites. At repeated scans 3 DUA (median 0 [0; 0]; p<0.001 compared with preoperative data) were observed. Activity of DUA significantly decreased to 819 [684; 955] cnt / sec / ml (p<0.001 as compared with preoperative data). Thirteen of 13 of 15 patients (87 %) had no AF / AT / AFL recurrences for 6 month follow up. CONCLUSION: In patients with AF, the areas of sympathetic activity in LA can be visualized by physiological localized uptake of 123I-mIBG. Radiofrequency catheter ablation can target the identified sympathetic innervation structures in AF patients precisely and effectively.

Recanalization of Chronic Total Occlusions Using Modern Endovascular Techniques.

PURPOSE: to assess results of percutaneous coronary intervention (PCI) with contemporary endovascular techniques of recanalization of chronic total coronary artery occlusions (CTO) in patients with ischemic heart disease (IHD). Occlusion (CTO) he procedural and in-hospital outcomes of consecutive patients undergoing chronic total occlusion percutaneous coronary intervention. MATERIALS AND METHODS: We retrospectively analyzed data from 456 consecutive patients (mean age 59.9±7.1 years, 18.2 % women) who underwent CTO PCI procedures (n=477) during 2014-2016 in the E. N. Meshalkin National Medical Research Center. CTO was localized in the right (61.2 %), left anterior descending (23.2 %) and left circumflex (15.3 %) coronary arteries. In one patient CTO was located in the left main coronary artery. According to the J-CTO score, 30 % of lesions were classified as easy, 36.4 % intermediate, 23.7 % difficult, and 18.9 % very difficult. RESULTS: Technical and procedural successes were achieved in 374 (78.4 %) and 366 patients (76.7 %), respectively. Antegrade approach was used in 378 (79.2 %), retrograde approach - in 99 (20.7 %) cases. Retrograde approach as primary strategy was used in 27 cases (5.7 %). Most frequent access for CTO PCI was radial artery, contralateral injection was used in 151 cases (31.6 %). Total number of stents per lesion was 1.6±0.98. The mean fluoroscopy time was 36.2±31 min. CONCLUSIONS: The rate of procedural adverse events in our study was low and similar to the non-CTO PCI series. However, despite the large number of CTO PCIs, the procedural success rate was still lower than in centers with dedicated programs for the management of such patients. Thus, further work is required to overcome this difference. Possible solution of this problem might be development and introduction in clinical practice of an algorithm for CTO recanalization.

Effect of Polyethylene Terephthalate on Functional Properties of Endothelial and Mesenchymal Cells.

We studied the influence of vascular prostheses made of polytetrafluoroethylene and polyethylene terephthalate on the proliferation, migration, and NO production by bone marrow mesenchymal stem cells, human endothelial progenitor cells, and EA.hy926 endothelial cells, colonization of the prosthesis surface by endothelial and mesenchymal cells was also analyzed. Synthetic prostheses have a negative effect on cell proliferation and migration, while surface treatment with proteins (fibronectin or gelatin) promotes colonization of the prostheses with cells.

Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts.

BACKGROUND: Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. METHODS: Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function. RESULTS: Endothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate angiogenesis in vivo. Endothelial cells proliferated most effectively on membranes made of polycaprolactone and chitosan blended in a 25:75 ratio, neutralized by a mixture of alkaline and ethanol. Endothelial and smooth muscle cells retained their functional properties when seeded on the blended membranes. CONCLUSIONS: We established endothelial and smooth muscle cell cultures from human right atrial appendage and right ventricle post-operative explants. The isolated cells revealed angiogenic potential and may be a promising source of patient-specific cells for regenerative medicine.

[Efficacy of intramuscular administration of stem/progenitor cells in experiment on the model of lower limb ischaemia]. / Effektivnost' vnutrimyshechnogo vvedeniya stvolovykh/progenitornykh kletok v eksperimente na modeli ishemii nizhnei konechnosti.

An experimental model of lower limb ischaemia induced by ligation of the femoral artery in CBA-line mail rats was used to study efficacy and effect of intramuscular administration of autologous bone-marrow stem/progenitor cells on the process of angiogenesis. Based on the findings of laser Doppler flowmetry it was shown that administration of autologous stem/progenitor cells promotes acceleration of the processes of neoangiogenesis, which was confirmed by the results of histological study.

Fluorescence-free flow cytometry for measurement of shape index distribution of resting, partially activated, and fully activated platelets.

Whereas commercially available hematological analyzers measure volume of individual platelets, angle-resolved light-scattering provides unique ability to additionally measure their shape index. We utilized the scanning flow cytometer to measure light-scattering profiles (LSPs) of individual platelets taken from 16 healthy donors and the solution of the inverse light-scattering problem to retrieve the volume and shape index of each platelet. In normal conditions, the platelet shape index distribution (PSID) demonstrates three peaks, which relate to resting, partially activated, and fully activated platelets. We developed an algorithm, based on fitting PSID by a sum of three peak functions, to determine the percentage, mean platelet shape index, and distribution width of each platelet fraction. In total, this method gives eight additional parameters of platelet morphology and function to be used in clinical hematological analysis. We also stimulated the platelets with adenosine diphosphate (ADP) and measured the dependence of equilibrium PSID, including the total percentage of activated platelets, on ADP concentration. © 2016 International Society for Advancement of Cytometry.

Effect of Vascular Endothelial Growth Factor and Erythropoietin on Functional Activity of Fibroblasts and Multipotent Mesenchymal Stromal Cells.

The study examined the effect of VEGF and erythropoietin on proliferative and migratory activities of skin fibroblasts and multipotent mesenchymal stromal cells of human adipose tissue. VEGF stimulated proliferation and migration of fi broblasts, but produced no significant effect on functional activity of multipotent mesenchymal stem cells. Erythropoietin stimulated proliferation of both cell types, but did not affect their migration.

Lymph cytokines as markers oncogenesis and effective treatment of experimental breast cancer Wistar rat.

The purpose of this paper is to examine the levels of cytokines in the lymph involved in the pathogenesis of breast cancer. Methods: Breast cancer was induced by introducing n-methyl-N-nitrosourea rats Wistar breed. Some of the animals subjected to surgery alone or chemotherapy alone (cyclophosphamide, methotrexate, 5-fluorouracil). Some animals combine both types of therapy, as well as a separate group to the administration of chemotherapy added Panagene drag presenting a fragmented DNA. To investigate the concentration of cytokines used in lymph test system Bio-Plex Pro Rat Cytokoness 24-Plex Assay (Bio-Rad, USA). Results: In rats with breast cancer content of most studied cytokines such as, IL-1b, IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, IL-17A, MIP-1a, MIP-3a, RANTES, TNF-a, MCP-1 was significantly higher than in intact animals. Surgical removal of the tumor resulted in a significant decrease in the content in the lymph as a pro-inflammatory cytokine. Comparative performance study cytokine content in the lymph after tumor removal from intact animals showed that the content of cytokines such as IL-10, IL-18, GRO / KC, RANTES were significantly higher in the control animals group. Conducting chemotherapy has led to a significant decrease in the content of IL-1b, IL-4, IL-6, IL-7, IL-10, MIP-1a, MIP-3a, RANTES in rat breast cancer lymph. Comparative study of cytokine content in the lymph operated animals after the administration of chemotherapy and Panagene revealed that most of the content indicators cytokines such as IL-5, IL-6, IL-7, IL-10, IL-13, IL-17A, IL- 18, GRO / KC, IFNg, MIP-3a in the lymph was higher after administration of the drug Panagene. Conclusion: In a comparative study cytokine profile lymph Wistar rats found that cytokine content depended on the therapy in animals with induced breast cancer. Lymph cytokine levels may serve as a diagnostic criterion for tumor growth, as well as the predictor of the effectiveness of the therapy and the risk of metastasis of breast cancer.

Study of Cytokine Profile of Cultured "Early" and "Late" Endothelial Progenitor Cells in Peripheral Blood of Chronic Heart Failure Patients after Mobilization Course with Granulocyte Colony-Stimulating Factor.

The effect of cell culturing protocol with various adhesion proteins and different culture time on the profile of cytokine and growth factors produced by endothelial progenitor cells harvested after mobilization with granulocyte colony-stimulating factor was examined in patients with chronic heart failure. The endothelial progenitor cells cultured on fibronectin or gelatin produced a broad and overall similar spectrum of cytokines and growth factors, the levels of which depended on the culture time. On culture day 16, the cells grown on fibronectin diminished the production of cytokines and growth factors (IL-10, IL-18, IL-8, erythropoietin, and VEGF), while the cells grown on gelatin down-regulated the synthesis of TNF-α, IL-8, and erythropoietin, although they up-regulated the production of IL-10, IL-18, and VEGF.

Therapy of Chronic Cardiosclerosis in WAG Rats Using Cultures of Cardiovascular Cells Enriched with Cardiac Stem Cell.

We developed a protocol for preparing cardiac cell culture from rat heart enriched with regional stem cells based on clonogenic properties and proliferation in culture in a medium with low serum content. Experiments on WAG rats with experimental ischemic myocardial damage showed that implantation of autologous regional stem cells into the left ventricle reduced the volume of cicatricial tissue, promoted angiogenesis in the damaged zone, and prevented the risk of heart failure development.

Effect of morphofunctional properties of mobilized progenitor cells of patients with chronic heart failure on the efficiency of autologous intramyocardial cell transplantation.

We studied the phenotype and cytokine production in peripheral blood mononuclear cells mobilized from the bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) and the relationship of these parameters with clinical efficacy of autologous intramyocardial injection of donor cells in patients with chronic heart failure. We found that mononuclear cells contain endothelial progenitor cells and are capable of producing pleiotropic cytokines exhibiting angiogenic and cardioprotective properties. High concentration of cells with of CD34+ and CD34+/CD133+ phenotype in the cell transplant and enhanced production of IL-10, TNF-α, and granulocyte-macrophage-CSF by blood mononuclear cells mobilized from the bone marrow can contribute to improvement of myocardial perfusion and increase in left ventricular ejection fraction after intramyocardial transplantation and can serve as predictors of high efficiency of cell therapy. Peripheral blood is an available source of progenitor cells, while mononuclear cells after administration of granulocyte-CSF can produce a reparative effect on ischemic myocardium.

[Analysis of genotype combinations at the polymorphic points of the promoter regions of the genes of three matrix metalloproteinases and the gene of vascular endothelial growth factor (VEGF) in patients with history of acute myocardial infarction].

AIM: To analyze the association of the promoter polymorphism of the genes of the matrix metalloproteinases (MMP) MMP2 (-1306), MMP3 (-1171), and MMP9 (-1562) and two vascular endothelial growth factor (VEGF) gene regulatory regions (-2578, +936) with the development of myocardial infarction (MI). MATERIALS AND METHODS: DNA was analyzed in 251 patients with a history of MI. Five polymorphic positions were genotyped by restrictase analysis of amplification products, by using specific primers. RESULTS: In addition to the MMP3 5A5A monogenotype, there were 4 complex genotypes that were significantly different between two analyzed groups and positively associated with acute coronary syndrome. Among them, each of two genotypes included 2 polymorphic positions; two genotypes did 3 analyzed polymorphic positions. Four complex (two-locus (n = 1), three-locus (n = 2), four-locus (n = 1) genotypes that were negatively associated with MI were also identified. CONCLUSION: These findings are evidence in favor of our assumption that the increasing number of genotypes as part of the analyzed combined genetic complexes detectable in one patient considerably enhances the clinical significance of the results of immunogenetic analysis.

Characteristics of cardiac cell cultures derived from human myocardial explants.

Primary cell cultures derived from human myocardial explants were obtained and characterized. The explant cultures contained cardiac stem cells (c-kit(+); ≈ 4%), microvascular cells (endothelial cells and pericytes), fibroblasts, and myofibroblasts. It was demonstrated that culturing of cardiac cells in cardiospheres did not promote enrichment of the cell culture with stem cells. MACS-sorted c-kit(+) cells from the explant culture were characterized by limited proliferative capacity and were capable of cardiomyogenic differentiation. The presence of microvascular cells determined general angiogenic potential of the culture.

[The use of implantable devices for long-term monitoring of cardiac rhythm after surgical treatment of atrial fibrillation in patients with ischemic heart disease].

We present in this paper experience of the use of implantable devices for long-term monitoring of cardiac rhythm after one stage operation of coronary artery bypass grafting (CABG) and radiofrequency ablation of atrial fibrillation (AF) source and results of a prospective randomized study, in which we included patients (n=95) with persistent AF and ischemic heart disease. These patients were randomized into 3 groups: with radiofrequency isolation of ostia of pulmonary veins (group 1, n=31), radiofrequency modified mini-maze procedure (group 2, n=30); CABG without AF elimination (control group 3, n=34). Implantable devices Reveal XT were used in 53 patients (21, 25, and 7 in groups 1, 2, and 3, respectively). According to data obtained with these devices AF was absent in 86.7, 95.6, 53%, and in 80, 86.2, 44.1% of patients in groups 1, 2, 3 after 1 and 2 years after operation, respectively). In 24% of patients Reveal devices also registered asymptomati-c arrhythmias. The use of implantable devices for monitoring of rhythm allowed to detect such arrhythmia and to provide timely correction of therapy.

Characteristics of induced human pluripotent stem cells using DNA microarray technology.

We performed transcriptome analysis of some human induced pluripotent stem cells, embryonic stem cells, and human somatic cells using DNA microarrays. PluriTest bioinformatic system was used for evaluation of cell pluripotency. Changes in the genome structure and status of X-chromosome gene expression was analyzed using microarray technology.

Phenotype of peripheral blood cells mobilized by granulocyte colony-stimulating factor in patients with chronic heart failure.

Administration of granulocyte CSF preparation to patients with chronic heart failure produced a hemostimulating effect and increased the content of leukocytes and neutrophils in the peripheral blood. Granulocyte CSF induced mobilization of bone marrow progenitor cells into the peripheral blood. The content of hemopoietic CD34(+)progenitor cells, which attained 0.42% (0.25-0.64) by the end of mobilization, inversely correlated with the number of myocardial infarctions. Administration of granulocyte CSF not only led to mobilization of bone marrow hemopoietic cells, but also increased the pool of endothelial progenitor cells in the peripheral blood: the content of CD34(+)/CD133(+)and CD34(+)/KDR(+)attained 0.02% (0.013-0.075) and 0.1% (0.05-0.20), respectively. Peripheral blood is an available source of progenitor cells, while mononuclear cells after administration of granulocyte CSF can produce a reparative effect on ischemic myocardium.

Epigenetics of pluripotent cells.

Pluripotency is maintained by a complex system that includes the genetic and epigenetic levels. Recent studies have shown that the genetic level (transcription factors, signal pathways, and microRNAs) closely interacts with the enzymes and other specific proteins that participate in the formation of the chromatin structure. The interaction between the two systems results in the unique chromatin state observed in pluripotent cells. In this review, the epigenetic features of embryonic stem cells and induced pluripotent stem cells are considered. Special attention is paid to the interplay of the transcription factors OCT4, SOX2, and NANOG with the Polycomb group proteins and other molecules involved in the regulation of the chromatin structure. The participation of the transcription factors of the pluripotency system in the inactivation of the X chromosome is discussed. In addition, the epigenetic events taking place during reprogramming of somatic cells to the pluripotent state and the problem of "epigenetic memory" are considered.