Aortic valve calcification is mediated by valve interstitial cells (VICs). We hypothesized that valve endothelial cells (VECs) stimulate pro-osteogenic changes in VICs, potentially via extracellular vesicles (EVs). We investigated VECs' pro-osteogenic effects on VICs, compared proteomes of healthy versus calcified VECs, and examined VECs-derived EVs effects on calcification. Human VECs and VICs were co-cultured in 2D and 3D (collagen gel) formats with osteogenic induction. Proteomic analysis compared VECs from healthy and calcified valves. Pro-osteogenic markers were assessed by qPCR, gel contractility was measured, and VECs-derived EVs were added to VICs cultures. In 2D co-cultures, induced calcification almost doubled versus VICs monocultures (p = 0.008). In 3D co-cultures, VECs from calcified valves induced strongest pro-osteogenic marker transcription in healthy VICs and reduced gel contraction. Proteomic analysis identified 3378 proteins with 27 and 8 proteins exclusive to healthy or calcified VECs, respectively. Nitric oxide synthase 3 and Hedgehog-interacting protein increased in calcified VECs (p = 0.005 and 0.002). VECs-derived EVs did not influence VICs calcification. VECs from calcified valves possess enhanced pro-osteogenic properties and promote VICs calcification independently of EVs. Pro-calcification EVs likely originate from other valve cell types.

Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo, in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

Одной из наиболее важных социальных проблем является сохранение демографического потенциала нации. Ученые всего мира пытаются найти наиболее перспективные направления для решения проблем, связанных с женским и мужским факторами бесплодия. Тяжелой формой мужской инфертильности является азооспермия, которая в зависимости от вида (обструктивная, секреторная) характеризуется прогнозом на возможность иметь детей. В данной статье изложен наш опыт ведения, диагностики и лечения мужчин с азооспермией в условиях отделения вспомогательных репродуктивных технологий клиники акушерства и гинекологии Военно-медицинской академии им. С.М. Кирова. ЦЕЛЬ ИССЛЕДОВАНИЯ Изучить результаты спермограмм 1564 мужчин в возрасте от 17 до 30 лет (средний возраст 21,2 года) и выявить частоту азооспермии. Провести дополнительное гормональное и генетическое обследование мужчин с азооспермией. Определить возможность получения живых сперматозоидов из семенных канальцев, полученных путем открытой биопсии яичка (TESE). МАТЕРИАЛ И МЕТОДЫ В период с 2018 по 2020 г. в отделении вспомогательных репродуктивных технологий Военно-медицинской академии им. С.М. Кирова на кафедре акушерства и гинекологии проходило обследование 1564 военнослужащих МО РФ. Возраст колебался от 17 до 30 лет и в среднем составил 21,2 года. РЕЗУЛЬТАТЫ У 9 (0,5%) обследованных пациентов диагностирована азооспермия. Исследование гормонального статуса пациентов с азооспермией показало наличие гипопролактинемии у 4 (44,4%) больных, снижение в крови уровня общего тестостерона (гипогонадизм) у 5 (55,6%). Полученные у 5 (56,6%) мужчин с азооспермией сперматозоиды успешно подвергнуты процедуре криоконсервации. ЗАКЛЮЧЕНИЕ Азооспермия является одной из наиболее тяжелых разновидностей мужского бесплодия. Несмотря на кажущуюся простоту диагностики, определение разновидности патологии и возможности получения здорового потомства целесообразно проводить в специализированных центрах репродуктивного здоровья.

Today, cesarean scar pregnancy is a rare type of ectopic pregnancy (1:18001: 2200). Moreover, the mortality rate in cesarean scar pregnancy is 191.2 per 100 000 cases, that 12 times exceeds the mortality rate in tubal pregnancy. In this article, we present a clinical observation of a rare case of cesarean scar pregnancy, as diagnosed by ultrasonography, in a 23-year-old patient after the cesarean surgery. The diagnosis was based on clinical, biochemical and ultrasound studies. Ultrasound played a crucial role in the determination of the fetus localization. Pathological pregnancy was eliminated by means of excision of the old scar and restoration of the uterine wall integrity. Such cases are becoming more common in connection with the increase in the frequency of caesarean sections, the improvement of technology, continuing medical education and professional development in the field of ultrasound. Knowledge of differential diagnosis can save the patient's life since the outcomes of cesarean scar pregnancy include severe complications with a possible loss of patients fertile function and immediate risk of death.

Introduction Although surgical treatment of carpal tunnel syndrome (CTS) is known to be highly effective outcomes may not be equally satisfactory for the patients due to severity of clinical presentation and objectifying assessment of the condition. Purpose Provide clinical evaluation of outcomes of surgical treatment of CTS using questionnaires and electroneuromyography (ENMG) findings depending on baseline severity of the condition. Material and methods The review included 161 patients who underwent 189 operations of open decompression of the median nerve using mini-access. The patients were assigned to three groups with mild (Group I), moderate (Group II) and severe (Group III) CTS. Evaluations were produced at 6 weeks, 3, 6 and 12 months following the surgery. Results The majority of patients showed positive dynamics, and delayed recovery of the wrist function was noted in Group III at a 12-month follow-up. Patients of Group III exhibited spasmodic improvement of the wrist function at 6-week-to-3-month follow-up. Conclusion Open decompression of the median nerve performed for patients with CTS using mini-approach facilitated substantial clinical and functional improvement in most cases. However, the most favorable results could be provided for mild and moderate CTS.

Background: Identification of novel biomarkers could provide prognostic information and improve risk stratification in patients with aortic stenosis (AS). YKL-40 (chitinase-3-like protein 1), a protein involved in atherogenesis, is upregulated in human calcific aortic valves. We hypothesized that circulating YKL-40 would be elevated and associated with the degree of AS severity and outcome in patients with symptomatic AS. Methods: Plasma YKL-40 was analyzed in 2 AS populations, one severe AS (n=572) with outcome measures and one with mixed severity (n=67). YKL-40 expression in calcified valves and in an experimental pressure overload model was assessed. Results: We found (1) patients with AS had upregulated circulating YKL-40 compared with healthy controls (median 109 versus 34 ng/mL, P <0.001), but levels were not related to the degree of AS severity. (2) High YKL-40 levels (quartile 4) were associated with long-term (median follow-up 4.7 years) all-cause mortality (adjusted hazard ratio, 1.93 [95% CI, 1.37–2.73], P <0.001). (3) YKL-40 protein expression in human calcific valves co-localized with its putative receptor IL-13rα2 in close proximity to valve interstitial cells. (4) Myocardial YKL-40 increased in experimental pressure overload (6-fold in decompensated versus sham mice). Conclusions: YKL-40 levels were elevated in AS and associated with mortality but not with other metrics of disease severity including the degree of AS severity. Despite scientific rationale for its role in AS, the clinical utility of circulating YKL-40 as a biomarker is limited. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01794832.

Osteogenic differentiation is a tightly regulated process realized by progenitor cell osteoblasts. Notch signaling pathway plays a critical role in skeletal development and bone remodeling. Controversial data exist regarding the role of Notch activation in promoting or preventing osteogenic differentiation. This study aims to investigate the effect of several Notch components and their dosage on osteogenic differentiation of mesenchymal stem cells of adipose tissue. Osteogenic differentiation was induced in the presence of either of Notch components (NICD, Jag1, Dll1, Dll4) dosed by lentiviral transduction. We show that osteogenic differentiation was increased by NICD and Jag1 transduction in a dose-dependent manner; however, a high dosage of both NICD and Jag1 decreased the efficiency of osteogenic differentiation. NICD dose-dependently increased activity of the CSL luciferase reporter but a high dosage of NICD caused a decrease in the activity of the reporter. A high dosage of both Notch components NICD and Jag1 induced apoptosis. In co-culture experiments where only half of the cells were transduced with either NICD or Jag1, only NICD increased osteogenic differentiation according to the dosage, while Jag1-transduced cells differentiated almost equally independently on dosage. In conclusion, activation of Notch promotes osteogenic differentiation in a tissue-specific dose-dependent manner; both NICD and Jag1 are able to increase osteogenic potential but at moderate doses only and a high dosage of Notch activation is detrimental to osteogenic differentiation. This result might be especially important when considering possibilities of using Notch activation to promote osteogenesis in clinical applications to bone repair.

Abstract Background Calcific aortic valve disease is the 2nd most frequent cause of open heart surgery. The valve interstitial cells (VIC) are crucial for calcification. SNF472 (a derivative of phytic acid) is a calcification inhibitor currently in clinical development for the treatment of cardiovascular calcification (Phase 2 CaLIPSO trial, EudraCT 2016–002834–59). SNF472 has been shown to inhibit vascular calcification in several preclinical models. Purpose 1. Establish a new model of calcification in cultured human VIC; 2. Investigate whether SNF472 would inhibit calcification in this model, and 3. Study if SNF472 might inhibit ongoing calcification processes. Methods Healthy and calcified aortic valves were obtained from heart transplant recipients and patients undergoing aortic valve replacement due to calcific valve disease, respectively. VIC were isolated and seeded in basic growth medium, osteogenic differentiation medium (Osteodiff) alone, and with addition of different concentrations of SNF472. The following series of studies were performed: 1. VIC from healthy and calcified valves were cultured for three weeks with Osteodiff; 2. VIC from calcified valves were cultured for 3 weeks in Osteodiff media with 0, 1, 3, 10, 30, or 100 μM SNF472; 3. VIC from calcified valves were cultured for 3 weeks in Osteodiff media in total, but after 1 or 2 weeks 30 or 100 μM SNF472 was added to the cultures (n=8). Calcification was visualized by Alzarin Red staining and quantified by spectrophotometry. Statistics analysis was performed nonparametric One-Way ANOVA (Friedman and Kruskal–Wallis tests) with Dunn's post-test. Results Calcification was found to be 30% stronger in cultures of VIC from calcified valves as compared to cultured VIC from healthy valves (p=0.03). SNF472 successfully inhibited VIC calcification in a dose-dependent manner. SNF472 concentrations of 1, and 3 μM inhibited calcification by 7% (not significant) and 66% (p=0.08) respectively. Concentrations of 10, 30, and 100 μM completely inhibited calcification. 30 and 100 μM of SNF472 added after 1 week reduced ongoing calcification by 84% (p<0.01) and 100% (p<0.01) respectively. When given after 2 weeks of ongoing calcification non-significant inhibition was still observed (21 and 30%, respectively). Conclusions VIC from calcified valves have a more pro-calcification phenotype than VIC from healthy valves. SNF472 is able to inhibit the development VIC calcification in vitro. By early intervention SNF472 is also able to stop the progression of ongoing calcification. SNF472 shows to be a promising therapy to treat heart valve calcification. Acknowledgement/Funding EC FP7 (GA 609020), Balearic Islands Government grant (ES01/TCAI/41_2017), FEDER 2014-2020, Laboratoris Sanifit, Palma, Spain; University of Oslo

Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-q PCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

The purpose of the present study was to investigate whether SNF472, the hexasodium salt of myo-inositol hexaphosphate (IP6 or phytate): 1. Inhibits induced calcification in cultured aortic valve interstitial cells (VIC) as an in vitro model of aortic valve stenosis and 2. Whether inhibition is different in VIC obtained from healthy and calcified aortic valves. VIC from healthy (n = 5) and calcified (n = 7) human aortic valves were seeded in basic growth medium, osteogenic differentiation medium alone, or in osteogenic medium with SNF472 (3, 10, and 30 mu M) and cultivated for 3 weeks. Calcification was quantified spectrophotometrically after Alizarin Red staining. In VIC from calcified valves, a complete inhibition of calcification was observed with SNF472 concentrations of 10 and 30 0.4 (p < .01), significantly stronger than in VIC from healthy valves. When SNF472 was added to VIC after 1 week in osteogenic medium, 30 and 100 mu M SNF472 inhibited the progression of ongoing calcification by 81 and 100% (p < .01), respectively. The same concentrations of SNF472 given after 2 weeks reduced calcification by 35 and 40% respectively (not significant). SNF472 inhibited both the formation and the progression of calcification with the strongest effect in VIC from calcified valves.

Objective: Valve interstitial cells (VICs) are crucial for the development of calcific aortic valve disease. We aimed to compare the phenotype and functions of VICs from calcified and healthy aortic valves. Methods: VICs were isolated from human calcified and healthy aortic valves sampled during aortic valve replacement or transplantation. To induce calcification, VICs were cultured 21 days in osteogenic medium. Expression of osteogenic marker bone morphogenetic protein 2 (BMP2) was analyzed by RT-qPCR, calcification was quantified using Alizarin Red staining. To differentiate VICs into myofibroblasts, we stimulated them with TGFβ1. The myofibroblastic markers alpha-smooth muscle actin (αSMA) and calponin was measured by FACS and immunostaining. For adipogenic differentiation, cells were stimulated with adipogenic medium and the expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG) and lipoprotein lipase (LPL) was analyzed by RT-qPCR. The expression of stem cell-associated marker CD106 and aldehyde dehydrogenase activity were measured by FACS. Cell proliferation was manually counted in Bürker chamber. Results: Unlike VICs from healthy valves, VICs from calcified valves cultured for 21 days without osteogenic stimulation stained positively with Alizarin Red confirming their calcific phenotype. Stimulation with osteogenic medium increased calcification and expression of BMP2 in both cells from calcified and healthy valves. Upon stimulation with myofibroblastic or adipogenic medium, VICs from calcified valves had lower expression of myofibroblastic (p=0.0001 for αSMA, p=0.03 for calponin) and adipogenic (p=0.007 for PPARG, p=0.03 for LPL) markers than VICs from healthy valves. VICs from calcified valves had lower expression of stem-cell associated markers CD106 (p=0.04) and aldehyde dehydrogenase (p=0.04) suggesting their lower “stemness” degree. Cells from healthy valves proliferated faster than cells from calcified valves. Conclusions: VICs from calcified aortic valves have a pro-osteogenic phenotype with less ability for proliferation and myofibroblastic differentiation as well as reduced multipotency compared to VICs from healthy valves.

Background: Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. Methods: Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 μg/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. Results: Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 (BMP2) (5-fold increase from control; p = 0.02) and decreased expression of mRNA of myofibroblastic markers: α-smooth muscle actin (ACTA2) (50% reduction from control; p = 0.0006) and calponin (CNN1) (80% reduction from control; p = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin (POSTN) was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, p = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. Conclusion: Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.

PB, with better control of hyperphosphatemia in prevalent hemodialysis patients.

AIMS:Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups.METHODS AND RESULTS:We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves.CONCLUSION:Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

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

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.