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The actin cytoskeleton is involved in the regulation of the endothelial barrier. Arginine bioavailability is an important factor that determines actin cytoskeleton dynamics. Pathogenic microorganisms can use arginine-hydrolyzing enzymes to disrupt the confluence of vascular endothelium for subsequent dissemination. In this study, the effect of streptococcal arginine deiminase on human umbilical vein endothelial cell (HUVEC) monolayer confluence and actin cytoskeleton structure in vitro was studied. The study was carried out on sonicated streptococcal cell supernatants (SSCSs) of the parental strain (Streptococcus pyogenes M49-16) and its isogenic mutant with the inactivated arginine deiminase gene (S. pyogenes M49-16delArcA). L-arginine concentration was evaluated by modified colorimetric Sakaguchi method. Actin cytoskeleton structure was analyzed by cell staining with Alexa Fluor 488-labeled phalloidin. HUVEC monolayer confluence was evaluated morphologically after crystal violet staining. It was found that in the presence of the parental strain-derived SSCS, a significant decrease in arginine concentration in the endothelial cell culture medium caused dynamic changes in actin cytoskeleton structure. After a 48-h incubation under this conditions, lamella and stress fiber formation was observed. After 72 h, the content of F-actin decreased, and confluence of the endothelial cell monolayer was disrupted. Similar changes were detected under neither standard conditions nor in the presence of mutant strain-derived SSCS. The results obtained show that pathogenic microbes are capable of using arginine depletion to downregulate endothelial barrier function for their dissemination in the host organism. [ABSTRACT FROM AUTHOR]

Fracture healing is a complex process in which the periosteum and endosteum become the main sources of osteoblast progenitor cells. However, cellular mechanisms and signaling cascades underlying the early stages of osteoblast progenitors differentiation in adult bone are still not well understood. Therefore, we performed shotgun proteomics analysis of primary culture of isolated human osteoblasts from femur of adult donors in undifferentiated conditions and on the fifth day of osteogenic differentiation in vitro. This is an early timepoint in which we have observed no extracellular matrix mineralization yet. 1612 proteins identified with at least two unique peptides were included in proteomics analysis. Data are available via ProteomeXchange with identifier PXD033697. Despite the fact, that matrix mineralization starts only after induction of osteogenic differentiation, we revealed unexpectedly weak physiological shift associated with a decrease of cells proliferative activity and changes in proteins involved in extracellular matrix secretion and organization. We demonstrated that osteoblasts were positive for markers of later osteogenic differentiation stages during standard cultivation: osteopontin, osteocalcin, BMP-2/4 and RUNX2. Therefore, further differentiation required for matrix mineralization needs minimal physiological changes. [ABSTRACT FROM AUTHOR]

This review considers the main modern methods and approaches of fluorescence microscopy used in the study of intracellular signaling cascades. The methods for the analysis of fixed samples and live-cell imaging are discussed, and special attention is paid to the methods of super-resolution microscopy. The limits of application and limitations of the methods are analyzed, scientific advances of recent years obtained using the described methods are presented. [ABSTRACT FROM AUTHOR]

Endoglin, a co-receptor of TGF-β family growth factors, is an endothelial cells marker. In our previous work we have demonstrated that monoclonal antibodies (MABs) against endoglin can change the functional properties of endothelial cells EA.hy926. The aim of the present work was to study the effects of these antibodies on HUVEC endothelial cells, as well as to correlate the data obtained for HUVEC and EA.hy926 cells. Comparison of in vitro models based on the permanent EA.hy926 endothelial cell line and primary HUVECs revealed, along with common morpho-functional properties, a number of dissimilarities in the le-vels of adhesion molecules and the expression of endothelial differentiation genes. Anti-endoglin MABs 2C8 and 4E4 inhibited HUVEC cells migration, reduced their adhesion to solid substrate, influenced the arrangement of actin microfilaments, and impeded the formation of caspillary-like structures. These effects were revealed either in presence of TGF-β1 in culture medium or under hypoxia. Supplementation of the growth medium with MABs 2C8 promoted endoglin shedding from HUVEC membrane both in hypoxia and normoxia. Several impacts of anti-endoglin MABs on HUVEC cultures were similar to those detected on EA.hy926 cells. However the effect of MABs 2C8 on endoglin shedding by HUVEC and EA.hy926 cells was opposite. [ABSTRACT FROM AUTHOR]

Parkinson's disease is a widespread neurodegenerative disease, which is characterized by the death of dopaminergic neurons in the substantia nigra of the midbrain. Clinically, the disease is manifested by tremor, bradykinesia, muscle rigidity, and other motor and non-motor symptoms that ultimately lead to disability. To date, there are only symptomatic treatment options for Parkinson's disease; therefore, the search for new approaches is one of the most important directions of therapy for this disease. In the 1970's the idea of using cell replacement therapy based on the local nature and specificity of damage to a particular type of neuron in Parkinson's disease originated. The selection of the source of cells, the method and place of introduction, indications for this operation, and peculiarities of patient management have been in development for a long time. The efficiency of cell replacement therapy has been confirmed by a number of studies on animal models. Clinical trials have already begun and several more are planned soon. This review describes the main prerequisites for the use of cell replacement therapy in Parkinson's disease, the stages of development of this method, and clinical trials that have started in the last few years. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

The rigid skeleton formed by networks of A- and B-type lamins is responsible for maintaining the nucleus shape. Moreover, the change in the ratio of lamin proteins in its composition, apparently, is a key factor determining mechanical properties of the cell nucleus, in particular plasticity. In this paper, the effect of the component composition of the nuclear lamina on the resistance of cells to mechanical stress and on the cell motility was considered. Expression of mutant forms of lamin A is accompanied by changes in the distance between microdomains of lamins within the nuclear membrane, and its increased bubbling relative to wild-type cells and cells overexpressing lamin A is observed. An increased number of deformed nuclei are noted when exposed to osmotic shock. The assessment of the effect of a change in the molecular composition of the nuclear lamina due to an increase (decrease) in expression of lamin A, as well as the introduction of progerin in an experimental wound model, showed that there are no differences under conditions of unlimited space. [ABSTRACT FROM AUTHOR]

The mechanism of valve calcification, representing the main cause of aortic stenosis (AS) formation and progression still remains unclear. Currently, the role of the OPG/RANKL/RANK system is considered as one of important variants of valve calcification pathogenesis. In this study we have investigated the differences in the levels of OPG and sRANKL involved in the calcification processes in tissues of patients with severe AS. The study was performed on AS patients, on patients with aortic dilatation, and also on AS patients with aortic dilatation. Increased level of RANKL was found in AS patients and it was higher in valve than in aortic tissue. In patients with aortic dilatation the RANKL level negatively correlated with aortic dilatation. The results of this study confirm importance of the OPG/RANKL/RANK system for calcification in AS patients. Although patients of all groups had comparable values of OPG (including patients with aortic dilatation), increased RANKL level was found only in AS patients. This suggests involvement of some additional mechanisms influencing the increase of RANKL expression. [ABSTRACT FROM AUTHOR]

Differential diagnosis of arrhythmogenic cardiomyopathy (ACM) during the disease latent phase is a challenging clinical problem that requires identification of early ACM biomarkers. Because extracellular nucleic acids are stable, specific, and can be easily detected, they can be used as reliable biomarkers of various diseases. In this study, we analyzed the levels of extracellular microRNAs and mitochondrial DNA in the conditioned medium collected from cardiomyocytes differentiated from induced pluripotent stem cells of ACM patients and healthy donor. Several microRNAs were expressed differently by the affected and healthy cardiomyocytes; therefore, they could be considered as potential ACM biomarkers. [ABSTRACT FROM AUTHOR]

Mutations in the LMNA gene resulting in the substitution of the highly conserved arginine 482 residue in the globular C-terminal domain of lamin A/C are associated with the Dunnigan-type familial partial lipodystrophy (FPLD2) often accompanied by impairments in the muscle tissue development. The mechanisms underlying these impairments remain unknown. The purpose of our work was to investigate the effects of the LMNA gene mutation R482L on the muscle differentiation and intramuscular fat accumulation using C2C12 mouse myoblasts transduced with the lentiviral constructs carrying the wild-type human LMNA gene (LMNA-WT) or the LMNA-R482L mutant gene. After stimulation of myogenesis and adipogenesis in the transduced cell, expression of muscle and adipose tissue differentiation markers, morphology of differentiated myotubes, and formation of intramuscular lipid droplets were analyzed. C2C12 cells carrying the LMNA-R482L construct exhibited upregulated desmin expression at all stages of muscle differentiation and transformed into hypertrophied myotubules (in comparison with C2C12 myoblasts transduced with LMNA-WT). Reduced expression levels of the myogenic transcription factor Myf6 in the cells with the LMNA-R482L mutant indicated delayed maturation of muscle fibers. These cells more actively accumulated fat in response to the stimulation of adipose differentiation than myoblasts modified with the wild-type LMNA; they also expressed the markers of lipid droplets, such as FABP4 (fatty acid-binding protein 4), ATGL (adipose triglyceride lipase), and PLIN2 (perilipin 2). Therefore, the R482L mutation of the LMNA gene affects the dynamics of C2C12 myoblast differentiation into myotubules and stimulates formation of fat deposits in the myoblasts and myotubules in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Nitrogen-doped thin titanium dioxide films formed by the reactive magnetron sputtering method on the surface of PLLA electrospun microfibers scaffold were investigated. It was shown that the chemical composition of the films is shifting from titanium dioxide (TiO2) composites saturated with C-NH, C=N, N-C=N and HN-C=O compounds to solid solutions of titanium oxides (TixOy) and titanium oxynitrides (TiOxNy) with the increased time of the treatment. An empirical model describing changes in the chemical composition of the surface due to the treatment was proposed. It was shown that the modification of the PLLA microfibers scaffolds surface improves cell-scaffold and cell-cell interactions with the highest number of viable adherent cells observed on the scaffold treated for 4 min. [ABSTRACT FROM AUTHOR]

The development of heart failure caused by acute myocardial infarction is accompanied by massive necrotic death of cardiomyocytes in lesion areas and subsequent pathological myocardial remodeling. Traditionally, the possibility of heart reparation has been considered to be severely limited or absent in the postnatal period. Endogenous cardiac stem cells with a regenerative potential have recently been described, but the mechanisms of activation of these cells remain poorly understood. The aim of our work was to obtain cardiac stem cells from the ischemic area of the myocardium and compare their functional properties with stem cells isolated from the healthy area of the myocardium. RT-PCR was used to quantify the gene expression in cardiac stem cells. In addition, differentiated cells were stained for specific markers using immunocytochemical method. Cardiac stem cells originating from the infarction area had a higher proliferative potential and a greater propensity to migrate in comparison to the cells originated from a healthy myocardial area. The expression level of several specific markers of cardiogenic, osteogenic and adipogenic differentiation upon induction of corresponding differentiation was higher in the cells from the infarction area than in cells from the healthy myocardium. We conclude that myocardial ischemia activates the internal regenerative potential of cardiac stem cells. [ABSTRACT FROM AUTHOR]

Comparative in vitro study examined the osteogenic potential of interstitial cells of aortic valve obtained from the patients with aortic stenosis and from control recipients of orthotopic heart transplantation with intact aortic valve. The osteogenic inductors augmented mineralization of aortic valve interstitial cells (AVIC) in patients with aortic stenosis in comparison with the control level. Native AVIC culture of aortic stenosis patients demonstrated overexpression of osteopontin gene ( OPN) and underexpression of osteoprotegerin gene ( OPG) in comparison with control levels. In both groups, AVIC differentiation was associated with overexpression of RUNX2 and SPRY1 genes. In AVIC of aortic stenosis patients, expression of BMP2 gene was significantly greater than the control level. The study revealed an enhanced sensitivity of AVIC to osteogenic inductors in aortic stenosis patients, which indicates probable implication of OPN, OPG, and BMP2 genes in pathogenesis of aortic valve calcification. [ABSTRACT FROM AUTHOR]

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel's wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells' response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained. [ABSTRACT FROM AUTHOR]

Nuclear lamins form nuclear lamina located under the inner nuclear membrane. It was believed that the nuclear lamina plays predominantly a structural role. Recently, its involvement in regulatory processes have been described, e.g., chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to development of laminopathies, primarily affecting tissues of mesenchymal origin. Today, the mechanisms of the lamina regulation of cell differentiation are largely unknown. In the present work, we studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and in С2С12 cell line. The genome of satellite and С2С12 cells was modified by cell transduction via lentiviral constructs encoding LMNA G232E associated with the development of Emery-Dreyfus muscular dystrophy and LMNA R571S associated with the development of dilated cardiomyopathy. Cell morphology was assessed with immunofluorescence, and expression of myogenic genes was analyzed by qPCR. We showed that the analyzed mutations reduced the cell ability to differentiate (to fuse and to form myotubes). We proposed that these mutations enhanced expression of early and reduced expression of late markers of myogenesis. Thus, mutations in nuclear lamins can modify the process of muscle differentiation. [ABSTRACT FROM AUTHOR]

Plakophilin-2 is a desmosomal protein encoded by PKP2 gene. Desmosomal proteins are usually considered as structural proteins with the main function of maintaining intercellular interactions. Genetic studies revealed that mutations in desmosomal genes could lead to arrhythmogenic right ventricular cardiomyopathy, a heart disease characterized by substitution of cardiomyocytes by adipose and fibrotic tissue predominantly in the right ventricle. Wnt signaling is a signal transduction pathway presumably involved in the development of pathology. The purpose of this study was to investigate Wnt activity in cells with wild-type and mutant PKP2 gene during adipogenic and cardiomyogenic differentiation. We used multipotent mesenchymal stromal cells and iPS cells generated from a patient carrying PKP2 gene mutation. We found that cells with mutant PKP2 exhibited lower transcriptional activity of β-catenin, a key player of Wnt signaling, as well as attenuated expression of its targets SOX2 and SOX9. These findings suggest a signaling role of plakophilin2 by regulating Wnt activity [ABSTRACT FROM AUTHOR]

Nuclear 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. [ABSTRACT FROM AUTHOR]

The study of pathogenesis of muscle disorders needs an appropriate cell model. In the field of muscle research, there is no general cell line considered standard for studying muscular and neuromuscular diseases. Most cell lines are incapable of differentiation into a muscle lineage exhibiting morphological and physiological properties of mature muscle cells and can hardly be genetically modified. The goal of our study was to find an informative cell model of muscle differentiation suitable for examination of muscular pathogenesis in vitro. We assayed cultured human mesenchymal stem cells (MSCs), mature murine muscle fibers, and primary murine satellite cells. It was shown that MSCs had very low capacity for myogenic differentiation; they were able to differentiate only in the presence of C2C12 cells. Lentiviral transduction had a toxic effect in primary myofiber cultures, and positively transduced cells were unable to respond to electrical stimulation, i.e., were functionally inactive. Satellite cells proved to be the most appropriate cell model, since they were easily transduced with lentiviruses and rapidly formed myotubes in the differentiation medima. Functional analysis of induced myotubes showed their ability to react to electrical and chemical stimulation. The patch-clump technique showed the presence of potassium and calcium channels. Collectively, the results show that cultured satellite cells are the most promising cell line for further experiments to explore molecular pathways in muscle pathologies. [ABSTRACT FROM AUTHOR]

Stem cells (SCs) vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. Comparative analysis of their biological properties is essential for making an optimal SC choice for regenerative therapy. Using immunocytochemistry, flow cytometry, histochemistry, and RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissues and cultured under similar conditions without any differentiation-promoting factors. The cultures were similar in having a high proportion of proliferating cells positive for nuclear antigen (PCNA). In both cultures, immunophenotyping has revealed high expression of mesenchymal stem-cell surface markers CD29, CD44, CD73, and CD105; low expression of CD31, CD34, and CD45; and variability in CD117, CD146, and CD309 expression. The only difference in the CD marker profile was the significantly lower expression of CD90 in the culture of SCs from SC-AT than from SEC-AT. Histochemical analysis showed a lack of Oil Red O-positive cells in both cultures and an about ten times higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In both cultures, immunocytochemistry detected low expression of the slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Expression of the gap junction protein connexin-43 was markedly higher in cells from SC-AT cultures. Only the cells of these cultures expressed the epithelial cell marker cytokeratin-19. GATA4 mRNA expression detected with RT-PCR was identified in SEC-AT rather than in SC-AT cells. Our results suggest that SC-AT is enriched compared to SEC-AT with epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs and can be considered an alternative source for cell cardiotherapy. [ABSTRACT FROM AUTHOR]

Thoracic aortic aneurysm (TAA) develops as a result of complex sequential events that dynamically alter the structure and composition of the aortic vascular extracellular matrix (ECM). The main cellular elements that alter the composition of aortic wall are smooth muscle cells (SMCs). The purpose of the present work was to study alterations of smooth muscle cell functions derived from the patients with TAA and from healthy donors. Since it is believed that TAA associates with bicuspid aortic valve (BAV) and with tricuspid aortic valve (TAV) differed in their pathogenesis, we have compared SMCs and tissue samples from BAV and TAV patients and healthy donors. The comparison was done by several parameters: SMC growth, migration and apoptotic dynamics, metalloproteinase MMP2 and MMP9 activity (zymography), and elastin, collagen, and fibrillin content (Western blot) in both tissue samples and cultured SMCs. Proliferation of BAV and TAV SMCs was decreased and migration ability in scratch tests was increased in TAV-derived SMCs compared to donor cells. BAV-cells migration ability was not changed compared to donor SMCs. Elastin content was decreased in TAA SMCs, whereas the content of fibrillin and collagen was not altered. At the same time, the elastin and collagen protein level was significantly higher in tissue samples of TAA patients than in donorderived samples. SMC proliferation and migration is differently affected in TAV and BAV-associated TAA that supports the idea on different nature of these two TAA groups. Our data also show that SMC functional properties are altered in TAA patients and these alterations could play a significant role in the disease pathogenesis. [ABSTRACT FROM AUTHOR]

Mutations in the lamin A/C gene (LMNA) lead to severe disorders collectively called laminopathies. The mechanisms by which lamin mutations cause the diseases are not clear. Since the mesenchymal lineages, adipose tissue in particular, are mostly affected in laminopathies, the aim of the study was to estimate the effect of LMNA mutations on differentiation of mesenchymal stem cells, adipose tissue stromal cells (ATSCs), into adipose lineages. ATSCs transduced with lentiviral vectors carrying LMNA gene mutations associated with various syndromes (myodystrophy, cardiomyopathy, lipodystrophy, progeroid syndrome) were induced to adipose differentiate. It was found that introduction of genetic constructions with LMNA gene point mutations G465D, R482L, and R527C promote adipogenic differentiation compared to wild-type lamin gene; mutation R471C reduced the differentiation. Introduction of R471C or R527C lamin mutations profoundly increased the expression of adipogenesis markers PPARG, SREBP1, and adipsin. Mutations in A/C lamin gene strongly and variously affect the differentiation of mesenchymal stem cells that probably underlie the pathogenic changes in patients with laminopathies. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a fundamental biological process that ensures the transition of a gene from one ganeration to another via male gametes. This process relies on a rare population of testicular cells called spermatogonial stem cells (SSCs), which self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding study of SSCs, their biological properties remain largely unknown, which is partly due to the very limited availability of these cells. Here, we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs that can be successfully used to enrich them. [ABSTRACT FROM AUTHOR]

We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after γ-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after γ-irradiation, however, this was followed not by a G1/S arrest but by a short and reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells, we investigated the expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest thatp21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after γ-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functionalp21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following γ-irradiation. Afterγ-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population. Oncogene (2000) 19, 3858–3865 [ABSTRACT FROM AUTHOR]

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-qPCR, 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. [ABSTRACT FROM AUTHOR]