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The prevalence of dilated cardiomyopathy (DCM) is increasing globally, highlighting the need for innovative therapeutic approaches to prevent its onset. In this study, we examined the energetic and epigenetic distinctions between dilated and non-dilated human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and assessed the effects of class I and II HDAC inhibitors (HDACi) on these cells and their cardiomyogenic differentiation. Cells were isolated from myocardium biopsies using explant outgrowth methods. Mitochondrial and histone deacetylase activities, ATP levels, cardiac transcription factors, and structural proteins were assessed using flow cytometry, PCR, chemiluminescence, Western blotting, and immunohistochemistry. The data suggest that the tested HDAC inhibitors improved acetylation and enhanced the energetic status of both types of cells, with significant effects observed in dilated myocardium-derived hmMSCs. Additionally, the HDAC inhibitors activated the cardiac transcription factors Nkx2-5, HOPX, GATA4, and Mef2C, and upregulated structural proteins such as cardiac troponin T and alpha cardiac actin at both the protein and gene levels. In conclusion, our findings suggest that HDACi may serve as potential modulators of the energetic status and cardiomyogenic differentiation of human heart hmMSCs. This avenue of exploration could broaden the search for novel therapeutic interventions for dilated cardiomyopathy, ultimately leading to improvements in heart function.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa 2+ ) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa 2+ was highest in chondrocytes, whereas iCa 2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa 2+ , Cav1.2 and the proliferation of all cells and affected iCa 2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs ( SOX9 and COL2A1 expression); however, purposeful iCa 2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

The degradation of cartilage, due to trauma, mechanical load or diseases, results in abundant loss of extracellular matrix (ECM) integrity and development of osteoarthritis (OA). Chondroitin sulfate (CS) is a member of the highly sulfated glycosaminoglycans (GAGs) and a primary component of cartilage tissue ECM. In this study, we aimed to investigate the effect of mechanical load on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MCSs) encapsulated into CS-tyramine-gelatin (CS-Tyr/Gel) hydrogel in order to evaluate the suitability of this composite for OA cartilage regeneration studies in vitro. The CS-Tyr/Gel/BM-MSCs composite showed excellent biointegration on cartilage explants. The applied mild mechanical load stimulated the chondrogenic differentiation of BM-MSCs in CS-Tyr/Gel hydrogel (immunohistochemical collagen II staining). However, the stronger mechanical load had a negative effect on the human OA cartilage explants evaluated by the higher release of ECM components, such as the cartilage oligomeric matrix protein (COMP) and GAGs, compared to the not-compressed explants. Finally, the application of the CS-Tyr/Gel/BM-MSCs composite on the top of the OA cartilage explants decreased the release of COMP and GAGs from the cartilage explants. Data suggest that the CS-Tyr/Gel/BM-MSCs composite can protect the OA cartilage explants from the damaging effects of external mechanical stimuli. Therefore, it can be used for investigation of OA cartilage regenerative potential and mechanisms under the mechanical load in vitro with further perspectives of therapeutic application in vivo.

Articular cartilage is vulnerable to mechanical overload and has limited ability to restore lesions, which leads to the development of chronic diseases such as osteoarthritis (OA). In this study, the chondrogenic responses of human bone marrow mesenchymal stem cells (BMMSCs) and OA cartilage-derived chondrocytes in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr)/Gel) hydrogels with or without experimental mechanical load have been investigated. Chondrocytes were smaller in size, had slower proliferation rate and higher level of intracellular calcium (iCa 2+ ) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-β3, chondrocytes more intensively secreted cartilage oligomeric matrix protein (COMP) and expressed collagen type II ( COL2A1 ) and aggrecan ( ACAN ) genes but were more susceptible to mechanical load compared to BMMSCs. ICa 2+ was more stably controlled in CS-Tyr/Gel/BMMSCs than in CS-Tyr/Gel/chondrocytes ones, through the expression of L-type channel subunit CaV1.2 ( CACNA1C ) and Serca2 pump ( ATP2A2 ) genes, and their balance was kept more stable. Due to the lower susceptibility to mechanical load, BMMSCs in CS-Tyr/Gel hydrogel may have an advantage over chondrocytes in application for cartilage regeneration purposes. The mechanical overload related cartilage damage in vivo and the vague regenerative processes of OA chondrocytes might be associated to the inefficient control of iCa 2+ regulating channels.

The evolving field of the microbiome and microbiota has become a popular research topic. The human microbiome is defined as a new organ and is considered a living community of commensal, symbiotic and pathogenic microorganisms within a certain body space. The term 'microbiome' is used to define the entire genome of the microbiota. Bacteria, archaea, fungi, algae and small protists are all members of the microbiota, followed by phages, viruses, plasmids and mobile genetic elements. The composition, heterogeneity and dynamics of microbiomes in time and space, their stability and resistance, essential characteristics and key participants, as well as interactions within the microbiome and with the host, are crucial lines of investigation for the development of successful future diagnostics and therapies. Standardization of microbiome studies and harmonized comparable methodologies are required for the transfer of knowledge from fundamental science into the clinic. Human health is dependent on microbiomes and achieved by nurturing beneficial resident microorganisms and their interplay with the host. The present study reviewed scientific knowledge on the major components of the human respiratory microbiome, i.e. bacteria, viruses and fungi, their symbiotic and parasitic roles, and, also, major diseases of the human respiratory tract and their microbial etiology. Bidirectional relationships regulate microbial ecosystems and host susceptibility. Moreover, environmental insults render host tissues and microbiota disease-prone. The human respiratory microbiome reflects the ambient air microbiome. By understanding the human respiratory microbiome, potential therapeutic strategies may be proposed., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Raudoniute et al.)

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1 ) and cardiac troponin T (TNNT2 ). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase ( PTK2 ) and activated ACTC1 and TNNT2 . Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Osteoarthritis (OA) and cardiovascular diseases (CVD) share many similar features, including similar risk factors and molecular mechanisms. A great number of cardiovascular drugs act via different ion channels and change ion balance, thus modulating cell metabolism, osmotic responses, turnover of cartilage extracellular matrix and inflammation. These drugs are consumed by patients with CVD for many years; however, information about their effects on the joint tissues has not been fully clarified. Nevertheless, it is becoming increasingly likely that different cardiovascular drugs may have an impact on articular tissues in OA. Here, we discuss the potential effects of direct and indirect ion channel modulating drugs, including inhibitors of voltage gated calcium and sodium channels, hyperpolarization-activated cyclic nucleotide-gated channels, β-adrenoreceptor inhibitors and angiotensin-aldosterone system affecting drugs. The aim of this review was to summarize the information about activities of cardiovascular drugs on cartilage and subchondral bone and to discuss their possible consequences on the progression of OA, focusing on the modulation of ion channels in chondrocytes and other joint cells, pain control and regulation of inflammation. The implication of cardiovascular drug consumption in aetiopathogenesis of OA should be considered when prescribing ion channel modulators, particularly in long-term therapy protocols.

Osteoarthritis (OA) is a long-term chronic joint disease characterized by the deterioration of bones and cartilage, which results in rubbing of bones which causes joint stiffness, pain, and restriction of movement. Tissue engineering strategies for repairing damaged and diseased cartilage tissue have been widely studied with various types of stem cells, chondrocytes, and extracellular matrices being on the lead of new discoveries. The application of natural or synthetic compound-based scaffolds for the improvement of chondrogenic differentiation efficiency and cartilage tissue engineering is of great interest in regenerative medicine. However, the properties of such constructs under conditions of mechanical load, which is one of the most important factors for the successful cartilage regeneration and functioning in vivo is poorly understood. In this review, we have primarily focused on natural compounds, particularly extracellular matrix macromolecule-based scaffolds and their combinations for the chondrogenic differentiation of stem cells and chondrocytes. We also discuss different mechanical forces and compression models that are used for In Vitro studies to improve chondrogenic differentiation. Summary of provided mechanical stimulation models In Vitro reviews the current state of the cartilage tissue regeneration technologies and to the potential for more efficient application of cell- and scaffold-based technologies for osteoarthritis or other cartilage disorders.

Background: In this study the effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) on the energetic status and cardiomyogenic differentiation of human healthy and dilated myocardium-derived mesenchymal stromal cells (hmMSC) have been investigated., Methods: The hmMSC were isolated from the healthy and dilated post-operation heart biopsies by explant outgrowth method. Cell proliferation, HDAC activity, mitochondrial membrane potential, and level of adenosine triphosphate (ATP) were evaluated. The effect of SAHA on mitochondrial parameters has been investigated also by Seahorse XF analyzer and cardiomyogenic differentiation was confirmed by the expression of transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T and alpha cardiac actin at gene and protein levels., Results: Dilated myocardium-derived hmMSC had almost 1.5 folds higher HDAC activity compared to the healthy cells and significantly lower mitochondrial membrane potential and ATP level. HDAC class I and II inhibitor SAHA improved energetic status of mitochondria in dilated myocardium-isolated hmMSC and increased expression of cardiac specific proteins during 14 days of exposure of cells to SAHA., Conclusions: HDAC inhibitor SAHA can be a promising therapeutic for dilated cardiomyopathy (DCM). Dilated hmMSC exposed to SAHA improved energetic status and, subsequently, cardiomyogenic differentiation. Data suggest that human dilated myocardium-derived MSC still have cardio tissue regenerative potential, which might be stimulated by HDAC inhibitors.

Cardiovascular diseases are the most common cause of human death in the developing world. Extensive evidence indicates that various toxic environmental factors and unhealthy lifestyle choices contribute to the risk, incidence and severity of cardiovascular diseases. Alterations in the genetic level of myocardium affects normal heart development and initiates pathological processes leading to various types of cardiac diseases. Homeobox genes are a large and highly specialized family of closely related genes that direct the formation of body structure, including cardiac development. Homeobox genes encode homeodomain proteins that function as transcription factors with characteristic structures that allow them to bind to DNA, regulate gene expression and subsequently control the proper physiological function of cells, tissues and organs. Mutations in homeobox genes are rare and usually lethal with evident alterations in cardiac function at or soon after the birth. Our understanding of homeobox gene family expression and function has expanded significantly during the recent years. However, the involvement of homeobox genes in the development of human and animal cardiac tissue requires further investigation. The phenotype of human congenital heart defects unveils only some aspects of human heart development. Therefore, mouse models are often used to gain a better understanding of human heart function, pathology and regeneration. In this review, we have focused on the role of homeobox genes in the development and pathology of human heart as potential tools for the future development of targeted regenerative strategies for various heart malfunctions.

Cardiac muscle is the hardest working muscle in the body, pumping approximately 70 g of blood with every heartbeat, circulating 9500 l of blood daily and contracting over 3 billion times during the average human's life. Heart failure - a heterogeneous syndrome - is a major and increasing health care problem worldwide and a leading cause of hospitalization and morbidity in elderly. Adequate heart tissue regeneration in human is lacking. Challenges to engineer heart tissue and employ it in vitro or in regenerative medicine remain to be solved. First of all, cardiac tissue bioengineering requires robust and powerful cells capable of differentiating into cardiomyogenic lineages in combination with effective, safe and highly specialized biomaterials, hydrogels and/or scaffolds for recreating the native extracellular microenvironment. Advances in stem cell and biomaterial science already provided an increasing array of cell resources, their cultivation technologies and biomatrices for efficient and safe cardiac tissue reconstruction. In order to develop new cardiac tissue mimicking technologies in vitro, it is necessary to analyze the advantages and drawbacks of already established biosystems. Therefore, in this paper, we provide a comprehensive overview of recently employed cells, 2D and 3D biomatrices for cardiac tissue engineering and review the current state-of-the-art in this field as well as future directions.

Humanity faces an increasing impact of air pollution worldwide, including threats to human health. Air pollutants prompt and promote chronic inflammation, tumourigenesis, autoimmune and other destructive processes in the human body. Post-translational modification of proteins, for example citrullination, results from damaging attacks of pollutants, including smoking, air pollution and others, rendering host tissues immunogenic. Citrullinated proteins and citrullinating enzymes, deiminases, are more prevalent in patients with COPD and correlate with ongoing inflammation and oxidative stress. In this study, we installed an in-house-designed diesel exhaust delivery and cannabidiol vaporization system where mice were exposed to relevant, urban traffic-related levels of diesel exhaust for 14 days and assessed integrity of alveolar tissue, gene expression shifts and changes in protein content in the lungs and other tissues of exposed mice. Systemic presence of modified proteins was also tested. The protective effect of phytocannabinoids was investigated as well. Data obtained in our study show subacute effects of diesel exhaust on mouse lung integrity and protein content. Emphysematous changes are documented in exposed mouse lungs. In parallel, increased levels of citrulline were detected in the alveolar lung tissue and peripheral blood of exposed mice. Pre-treatment with vaporized cannabidiol ameliorated some damaging effects. Results reported hereby provide new insights into subacute lung tissue changes that follow diesel exhaust exposure and suggest possible dietary and/or other therapeutic interventions for maintaining lung health and healthy ageing., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Atmospheric particulate matter (PM) constitutes the major part of urban air pollution and is a heterogeneous mixture of solid and liquid particles of different origin, size, and chemistry. Human exposure to PM in urban areas poses considerable and significant adverse effects on the respiratory system and human health in general. Major contributors to PM content are combustion-related sources such as diesel vehicles, household, and industrial heating. PM is composed of thousands of different high molecular weight organic compounds, including poly-aromatic hydrocarbons (PAHs). The aim of this study was to clarify the cytotoxic effects of the extract of actual urban PM1 with high benzo[a]pyrene (BaP) content collected in Eastern European mid-sized city during winter heating season on human bronchial epithelial cells (BEAS-2B). Decreased cell viability, alteration of cell layer integrity, increased apoptosis, and oxidative stress were observed during the 3-day exposure to the PM extract. In addition, following PM exposure pro-inflammatory cytokine expression was upregulated at gene and protein levels. Morphology and motility changes, i.e., decreased cells' ability to cover scratch area, were also documented. We report here that the extract of urban PM1 may induce bronchial epithelium changes and render it pro-inflammatory and compromised within 3 days.

Studies of lung diseases in vitro often rely on flat, plastic-based monocultures, due to short lifespan of primary cells, complicated anatomy, lack of explants, etc. We hereby present a native 3D model with cues for repopulating epithelial cells. Abilities of mesenchymal stem cells (MSC) to modulate bacterial lipopolysaccharide (LPS) and cigarette smoke-induced injury to pulmonary epithelium were tested in our model. Post-mortem human lung tissue was sliced, cut and decellularized. Resulting matrix pads were reseeded with pulmonary epithelium (A549 line). Markers of the layer integrity and certain secreted proteins in the presence of cigarette smoke extract (CSE) and LPS were assessed via Western blot, ELISA and RT-PCR assays. In parallel, the effects of MSC paracrine factors on exposed epithelial cells were also investigated at gene and protein levels. When cultured on native 3D matrix, A549 cells obtain dual, type I- and II-like morphology. Exposure to CSE and LPS leads to downregulation of several epithelial proteins and suppressed proliferation rate. MSC medium added to the model restores proliferation rate and some of the epithelial proteins, i.e. e-cadherin and beta-catenin. CSE also increases secretion of pro-inflammatory cytokines by epithelial cells and upregulates transcription factor NFκB. Some of these effects might be counteracted by MSC in our model. We introduce repopulated decellularized lung matrix that highly resembles in vivo situation and is convenient for studies of disease pathogenesis, cytotoxicology and for exploring therapeutic strategies in the human lung context in vitro. MSC paracrine products have produced protecting effects in our model., Competing Interests: The authors declare that they have no conflict of interest.

Background: The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs., Methods: hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone., Results: Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores., Conclusions: mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.

Background: Inflammatory dilated cardiomyopathy (iDCM) is a common debilitating disease with poor prognosis that often leads to heart failure and may require heart transplantation. The aim of this study was to evaluate sera and biopsy samples from chronic iDCM patients, and to investigate molecular mechanism associated with left ventricular remodeling and disease progression in order to improve therapeutic intervention., Methods: Patients were divided into inflammatory and non-inflammatory DCM groups according to the immunohistochemical expression of inflammatory infiltrates markers: T-lymphocytes (CD3), active-memory T lymphocyte (CD45Ro) and macrophages (CD68). The inflammation, apoptosis, necrosis and fibrosis were investigated by ELISA, chemiluminescent, immunohistochemical and histological assays., Results: The pro-inflammatory cytokine IL-6 was significantly elevated in iDCM sera (3.3 vs. 10.98 μg/ml; P < 0.05). Sera levels of caspase-9, -8 and -3 had increased 6.24-, 3.1- and 3.62-fold, (P < 0.05) and only slightly (1.3-, 1.22- and 1.03-fold) in biopsies. Significant release of Hsp60 in sera (0.0419 vs. 0.36 ng/mg protein; P < 0.05) suggested a mechanistic involvement of mitochondria in cardiomyocyte apoptosis. The significant MMP9/TIMP1 upregulation in biopsies (0.1931 - 0.476, P < 0.05) and correlation with apoptosis markers show its involvement in initiation of cell death and ECM degradation. A slight activation of the extrinsic apoptotic pathway and the release of hsTnT might support the progression of chronic iDCM., Conclusions: Data of this study show that significant increase of IL-6, MMP9/TIMP1 and caspases-9, -8, -3 in sera corresponds to molecular mechanisms dominating in chronic iDCM myocardium. The initial apoptotic pathway was more activated by the intramyocardial inflammation and might be associated with extrinsic apoptotic pathway through the pro-apoptotic Bax. The activated intrinsic form of myocardial apoptosis, absence of necrosis and decreased fibrosis are most typical characteristics of chronic iDCM. Clinical use of anti-inflammatory drugs together with specific anti-apoptotic treatment might improve the efficiency of therapies against chronic iDCM before heart failure occurs.

Background: Cardiac fibrosis disrupts the normal myocardial structure and has a direct impact on heart function and survival. Despite already available digital methods, the pathologist's visual score is still widely considered as ground truth and used as a primary method in histomorphometric evaluations. The aim of this study was to compare the accuracy of digital image analysis tools and the pathologist's visual scoring for evaluating fibrosis in human myocardial biopsies, based on reference data obtained by point counting performed on the same images., Methods: Endomyocardial biopsy material from 38 patients diagnosed with inflammatory dilated cardiomyopathy was used. The extent of total cardiac fibrosis was assessed by image analysis on Masson's trichrome-stained tissue specimens using automated Colocalization and Genie software, by Stereology grid count and manually by Pathologist's visual score., Results: A total of 116 slides were analyzed. The mean results obtained by the Colocalization software (13.72 ± 12.24%) were closest to the reference value of stereology (RVS), while the Genie software and Pathologist score gave a slight underestimation. RVS values correlated strongly with values obtained using the Colocalization and Genie (r>0.9, p<0.001) software as well as the pathologist visual score. Differences in fibrosis quantification by Colocalization and RVS were statistically insignificant. However, significant bias was found in the results obtained by using Genie versus RVS and pathologist score versus RVS with mean difference values of: -1.61% and 2.24%. Bland-Altman plots showed a bidirectional bias dependent on the magnitude of the measurement: Colocalization software overestimated the area fraction of fibrosis in the lower end, and underestimated in the higher end of the RVS values. Meanwhile, Genie software as well as the pathologist score showed more uniform results throughout the values, with a slight underestimation in the mid-range for both., Conclusion: Both applied digital image analysis methods revealed almost perfect correlation with the criterion standard obtained by stereology grid count and, in terms of accuracy, outperformed the pathologist's visual score. Genie algorithm proved to be the method of choice with the only drawback of a slight underestimation bias, which is considered acceptable for both clinical and research evaluations., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9857909611227193.

The involvement of extracellular signal-regulated kinases 1 and 2 (ERK1,2), stress kinase p38 and c-Jun NH2 -terminal kinases 1 and 2 (JNK1,2) on Hsp70-upregulation following mild heat shock, and resulting cell protection, was studied on rabbit primary myoblasts. Cells subjected to heat stress (42°C; 60 min) showed a significantly enhanced amount of heat-shock-induced protein 70 (Hsp70), correlating with sustained phosphorylation of MAP kinases ERK1,2, inhibition of p38 and JNK1,2 activation. Induced Hsp70 did not autocrinally suppress activation of transcription factor c-Jun, suggesting involvement of the latter in the protection of myoblasts following heat shock. The inhibition of stress kinases p38, JNK1,2, and MEK1,2 by SP600125, SB203580, and UO126, respectively, established the involvement of JNK1,2 and p38 as upstream, and ERK1,2 as downstream targets of Hsp70 induction. Moreover, the effect of the MEK1,2 inhibitor UO126 revealed a new pathway of c-Jun activation by ERK1,2 in myogenic heat-stressed stem cells. The presented data show that transient activation of JNK1, JNK2, and p38 is necessary for Hsp70 induction and ensuing cell protection. In conclusion, affecting myogenic stem cell protective mechanisms might be a useful strategy in improving stem cell survival and their expanded application in therapy., (Copyright © 2013 Wiley Periodicals, Inc.)

We induced upregulation of stanniocalcin-1 (STC-1) by various mild and long lasting stresses and assayed its influence on mitochondrial membrane potential (MMP) in the neural crest-derived cell line Paju. The obtained data showed that starvation (24-96 h), exposure to 10nM TPA, and low concentrations (0.05-1 μM) of As2O3 significantly (3-5 times) upregulated Paju cell STC-1 RNA and stabilized the mitochondrial membrane potential (MMP). However, high concentrations of As2O3 (2.5-5.0 μM) increased intracellular ROS and free calcium levels and, consequently, suppressed STC-1 and MMP. The results show that cells preconditioned by various mild stresses expressed more STC-1 and their MMP were more resistant to a secondary exposure to As2O3 (2.5-5 μM, 96 h) demonstrating mitohormesis. We suggest that MMP deviation from control levels, to an extent innocuous to cell viability, is a general signal for STC-1-induction and MMP-protection. Our findings of Paju cell MMP-regulation may be of great importance for inventing new ways to prevent neurodegenerative diseases and unravel the mechanisms behind drug resistance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Improvement in the yield of adult organism stem cells, and the ability to manage their differentiation and survival potential are the major goals in their application in regenerative medicine and in the adult stem cell research. We have demonstrated that adult rabbit muscle-derived cell lines with an unlimited proliferative potential in vitro can differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. Studies of cell survival in vitro showed that differentiated cells, except neurogenic ones, are more resistant to apoptosis inducers compared to proliferating cells. Resistance to death signals correlated with the level of protein kinase AKT phosphorylation. Skeletal muscle-derived cell lines can be multipurpose tools in therapy. Enhanced resistance of differentiated cells to certain types of damage shows their potential for long-term survival and maintenance in an organism. This article was published online on 29 January 2013. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 6 March 2013., (© 2013 International Federation for Cell Biology.)

iHsp70 [inducible Hsp70 (heat-shock protein 70)] family members (iHsp70, Hsp72 and Hsp70) are highly conserved proteins that act as molecular chaperones and promote cell survival during various forms of stress. Our data indicate that cultured adult rabbit myoblasts do not express iHsp70 under normal growth conditions, although increased expression was detectable 0.5-72 h following a 42°C heat shock for 15-60 min. The intracellular iHsp70 level reached a maximum 8 h after onset of the heat shock, which correlated with its increased accumulation in nuclei. Inhibition of iHsp70 expression by quercetin showed that sustained activation of JNK (c-Jun N-terminal kinase) 2 and suppression of c-Jun phosphorylation were responsible for myoblast death after heat shock. The data also demonstrate that activation of transcription factor c-Jun depends mostly on JNK1, whereas JNK2 had higher affinity and was translocated to nuclei together with c-Jun. We have also shown that the JNK signalling pathway is an upstream effect of iHsp70 expression. These findings provide further in-depth understanding of the implication of the pro-survival signalling kinases JNK1 and JNK2 and their target, c-Jun, in expression of iHsp70 and regulation of myogenic stem cell survival and death mechanisms after heat shock. Mild heat shock before transplantation might be a way of improving myogenic stem cell survival.

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of measuring the rate at which cells acidify their environment in response to ligand-receptor binding. By measuring the extracellular acidification response (ECAR) we characterized some aspects of ligand-B2 receptor interaction in SHP-77 cell line. SHP-77 cells maximally acidified their environment within 30 s after the exposure to bradykinin (BK) or the BK agonist, B9972, with the maximum effect seen at a ligands concentration of 1 microM. Fetal bovine serum (FBS) modulated the binding of BK or B9972, showing that B9972 is a partial agonist. In addition, the binding of BK agonist or antagonist to the B2 receptor showed different ECAR and different interaction with other intracellular and plasma membrane proteins. Our microphysiometrical results showed that two parameters, antagonist binding affinity (pD2) and antagonist potency (pIC50) are required to characterize BK antagonist activity for the B2 receptor in the SHP-77 cell line. The previously used parameter of B2 antagonist activity, pA2, had high variation and poor correlation with the inhibition of SHP-77 cell growth in vitro and suppression of tumor growth when SHP-77 cells were injected to mice. Our results permit us to conclude that BK agonists and antagonists differ in their interactions with the B2 receptor and consequently elicit different cell responses. Based on our results, we have developed a new microphysiometrical assay for analyzing the activity of BK agonists and antagonist in SHP-77 cells, which may facilitate the discovery of new potent anticancer drugs.

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (elL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70-fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR I thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant elL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCRI did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of elL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.

Bradykinin (BK) is an autocrine growth factor for lung and prostate cancers. BK also facilitates tumor extension by increasing tissue permeability and stimulating angiogenesis. Peptide BK antagonists are in development as potential new drugs for lung cancer. Newer nonpeptide BK antagonists have even higher potency against lung cancer, in vitro and in vivo. These compounds have now been applied to the study of prostate cancers, and have been found to be effective. Prostate cancer cell line PC3 is derived from a late-stage, hormone-independent, metastatic tumor; its growth is difficult to inhibit. Our established BK antagonists, while less effective against this line of prostate cancer in xenografts in nude mice than against lung cancer, are active and have led the way to development of new peptide and nonpeptide agents for prostate cancer. In addition to inhibiting cancer cell growth directly, they inhibit angiogenesis mediated by vascular endothelial growth factor, and inhibit increased tissue permeability mediated by membrane metalloproteases in these tumors. This class of compounds offers hope for development of new drugs for refractory prostate cancer.

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50 microM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N'-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion. Cultures exposed to rhein for 15 min and subsequently rinsed and incubated for 16 h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis. No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca2+ concentration. We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.

In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied. The steady-state reactions of A. thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E. coli enzyme (B.W. Lennon, C.H. Williams, Jr., Biochemistry, vol. 35 (1996), pp. 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced. Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16%. Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential. E(1)7. In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX. Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged. In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns. These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide. Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction. The relatively high rate of quinone reduction by A. thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme. These factors make plant TR an attractive target for redox active and alkylating pesticide action.

The rotenone-insensitive reduction of quinones and aromatic nitrocompounds by mitochondrial NADH: ubiquinone reductase (complex I, EC 1.6.99.3) has been studied. It was found that these reactions proceed via a mixed one- and two-electron transfer. The logarithms of the bimolecular rate constants of oxidation (TN/Km) are proportional to the one-electron-reduction potentials of oxidizers. The reactivities of nitrocompounds are close to those of quinones. Unlike the reduction of ferricyanide, these reactions are not inhibited by NADH. However, they are inhibited by NAD+ and ADP-ribose, which also act as the mixed-type inhibitors for ferricyanide. TN/Km of quinones and nitrocompounds depend on the NAD+/NADH ratio, but not on NAD+ concentration. They are diminished by the limiting factors of 2.5-3.5 at NAD+/NADH greater than 200. It seems that rotenone-insensitive reduction of quinones and nitrocompounds takes place near the NAD+/NADH and ferricyanide binding site, and the inhibition is caused by induced conformational changes after the binding of NAD+ or ADP-ribose.