Class I and II Histone Deacetylase Inhibitors as Therapeutic Modulators of Dilated Cardiac Tissue-Derived Mesenchymal Stem/Stromal Cells.

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.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

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 (iCa2+) 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 iCa2+ was highest in chondrocytes, whereas iCa2+ 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 iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ 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 iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

The Effects of Mechanical Load on Chondrogenic Responses of Bone Marrow Mesenchymal Stem Cells and Chondrocytes Encapsulated in Chondroitin Sulfate-Based Hydrogel.

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 (iCa2+) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-ß3, 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. ICa2+ 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 iCa2+ regulating channels.

The Effect of Heat Shock on Myogenic Differentiation of Human Skeletal-Muscle-Derived Mesenchymal Stem/Stromal Cells.

Muscle injuries, degenerative diseases and other lesions negatively affect functioning of human skeletomuscular system and thus quality of life. Therefore, the investigation of molecular mechanisms, stimulating myogenic differentiation of primary skeletal-muscle-derived mesenchymal stem/stromal cells (SM-MSCs), is actual and needed. The aim of the present study was to investigate the myogenic differentiation of CD56 (neural cell adhesion molecule, NCAM)-positive and -negative SM-MSCs and their response to the non-cytotoxic heat stimulus. The SM-MSCs were isolated from the post operation muscle tissue, sorted by flow cytometer according to the CD56 biomarker and morphology, surface profile, proliferation and myogenic differentiation has been investigated. Data show that CD56(+) cells were smaller in size, better proliferated and had significantly higher levels of CD146 (MCAM) and CD318 (CDCP1) compared with the CD56(-) cells. At control level, CD56(+) cells significantly more expressed myogenic differentiation markers MYOD1 and myogenin (MYOG) and better differentiated to the myogenic direction. The non-cytotoxic heat stimulus significantly stronger stimulated expression of myogenic markers in CD56(+) than in CD56(-) cells that correlated with the multinucleated cell formation. Data show that regenerative properties of CD56(+) SM-MSCs can be stimulated by an extracellular stimulus and be used as a promising skeletal muscle regenerating tool in vivo.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

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.

Mechanotransducive Biomimetic Systems for Chondrogenic Differentiation In Vitro.

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.

The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats' pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Improves Energetic Status and Cardiomyogenic Differentiation of Human Dilated Myocardium-Derived Primary Mesenchymal Cells.

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.

The Role of Cardiac T-Cadherin in the Indicating Heart Failure Severity of Patients with Non-Ischemic Dilated Cardiomyopathy.

Background and objectives: T-cadherin (T-cad) is one of the adiponectin receptors abundantly expressed in the heart and blood vessels. Experimental studies show that T-cad sequesters adiponectin in cardiovascular tissues and is critical for adiponectin-mediated cardio-protection. However, there are no data connecting cardiac T-cad levels with human chronic heart failure (HF). The aim of this study was to assess whether myocardial T-cad concentration is associated with chronic HF severity and whether the T-cad levels in human heart tissue might predict outcomes in patients with non-ischemic dilated cardiomyopathy (NI-DCM). Materials and Methods: 29 patients with chronic NI-DCM and advanced HF were enrolled. Patients underwent regular laboratory investigations, echocardiography, coronary angiography, and right heart catheterization. TNF-α and IL6 in serum were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, endomyocardial biopsies were obtained, and the levels of T-cad were assessed by ELISA and CD3, CD45Ro, CD68, and CD4- immunohistochemically. Mean pulmonary capillary wedge pressure (PCWP) was used as a marker of HF severity, subdividing patients into two groups: mean PCWP > 19 mmHg vs. mean PCWP < 19 mmHg. Patients were followed-up for 5 years. The study outcome was composite: left ventricular assist device implantation, heart transplantation, or death from cardiovascular causes. Results: T-cad shows an inverse correlation with the mean PCWP (rho = -0.397, p = 0.037). There is a tendency towards a lower T-cad concentration in patients with more severe HF, as indicated by the mean PCWP > 19 mmHg compared to those with mean PCWP ≤ 19 mmHg (p = 0.058). Cardiac T-cad levels correlate negatively with myocardial CD3 cell count (rho = -0.423, p = 0.028). Conclusions: Univariate Cox regression analysis did not prove T-cad to be an outcome predictor (HR = 1, p = 0.349). However, decreased T-cad levels in human myocardium can be an additional indicator of HF severity. T-cad in human myocardium has an anti-inflammatory role. More studies are needed to extend the role of T-cad in the outcome prediction of patients with NI-DCM.

Lung alveolar tissue destruction and protein citrullination in diesel exhaust-exposed mouse lungs.

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.

Cytoprotective Effects of Mangiferin and Z-Ligustilide in PAH-Exposed Human Airway Epithelium in Vitro.

According to World Health Organisation (WHO) air pollution increases the risk of cardiovascular disorders, respiratory diseases, including COPD, lung cancer and acute respiratory infections, neuro-degenerative and other diseases. It is also known that various phytochemicals may mitigate such risks. This study tested if phytochemicals mangiferin (MNG) and Z-ligustilide (Z-LG) may protect PAH-exposed human lung bronchial epithelial cells (BEAS-2B). Organic PAH extract was obtained from the urban fine PM with high benzo(a)pyrene content collected in Eastern European mid-sized city during winter heating season. Cell proliferation traits and levels of intracellular oxidative stress were examined. Effect of MNG (0.5 µg/mL) alone or in combination with PAH on bronchial epithelium wound healing was evaluated. Both phytochemicals were also evaluated for their antioxidant properties in acellular system. Treatment with MNG produced strong cytoprotective effect on PAH-exposed cells (p < 0.01) while Z-LG (0.5 µg/mL) exhibited strong negative effect on cell proliferation in untreated and PAH-exposed cells (p < 0.001). MNG, being many times stronger antioxidant than Z-LG in chemical in vitro assays (p < 0.0001), was also able to decrease PAH-induced oxidative stress in the cell cultures (p < 0.05). Consequently MNG ameliorates oxidative stress, speeds up wound healing process and restores proliferation rate in PAH-exposed bronchial epithelium. Such protective effects of MNG in air pollution affected airway epithelium stimulate further research on this promising phytochemical.

Pro-inflammatory effects of extracted urban fine particulate matter on human bronchial epithelial cells BEAS-2B.

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.

The Role of Serum Adiponectin for Outcome Prediction in Patients with Dilated Cardiomyopathy and Advanced Heart Failure.

Clinical interpretation of patients' plasma adiponectin (APN) remains challenging; its value as biomarker in dilated cardiomyopathy (DCM) is equivocal. We evaluated whether circulating APN level is an independent predictor of composite outcome: death, left ventricle assist device (LVAD) implantation, and heart transplantation (HT) in patients with nonischemic DCM. 57 patients with nonischemic DCM (average LV diastolic diameter 6.85 cm, LV ejection fraction 26.63%, and pulmonary capillary wedge pressure 22.06 mmHg) were enrolled. Patients underwent echocardiography, right heart catheterization, and endomyocardial biopsy. During a mean follow-up of 33.42 months, 15 (26%) patients died, 12 (21%) patients underwent HT, and 8 (14%) patients were implanted with LVAD. APN level was significantly higher in patients who experienced study endpoints (23.4 versus 10.9 ug/ml, p = 0.01). APN was associated with worse outcome in univariate Cox proportional hazards model (HR 1.04, CI 1.02-1.07, p = 0.001) but lost significance adjusting for other covariates. Average global strain (AGS) is an independent outcome predictor (HR 1.42, CI 1.081-1.866, p = 0.012). Increased circulating APN level was associated with higher mortality and may be an additive prognostic marker in DCM with advanced HF. Combination of serum (APN, BNP, TNF-α) and echocardiographic (AGS) markers may increase the HF predicting power for the nonischemic DCM patients.

Native matrix-based human lung alveolar tissue model in vitro: studies of the reparatory actions of mesenchymal stem cells.

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.

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19.

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.

Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

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.

Scaffolds and cells for tissue regeneration: different scaffold pore sizes-different cell effects.

During the last decade biomaterial sciences and tissue engineering have become new scientific fields supplying rising demand of regenerative therapy. Tissue engineering requires consolidation of a broad knowledge of cell biology and modern biotechnology investigating biocompatibility of materials and their application for the reconstruction of damaged organs and tissues. Stem cell-based tissue regeneration started from the direct cell transplantation into damaged tissues or blood vessels. However, it is difficult to track transplanted cells and keep them in one particular place of diseased organ. Recently, new technologies such as cultivation of stem cell on the scaffolds and subsequently their implantation into injured tissue have been extensively developed. Successful tissue regeneration requires scaffolds with particular mechanical stability or biodegradability, appropriate size, surface roughness and porosity to provide a suitable microenvironment for the sufficient cell-cell interaction, cell migration, proliferation and differentiation. Further functioning of implanted cells highly depends on the scaffold pore sizes that play an essential role in nutrient and oxygen diffusion and waste removal. In addition, pore sizes strongly influence cell adhesion, cell-cell interaction and cell transmigration across the membrane depending on the various purposes of tissue regeneration. Therefore, this review will highlight contemporary tendencies in application of non-degradable scaffolds and stem cells in regenerative medicine with a particular focus on the pore sizes significantly affecting final recover of diseased organs.

Molecular mechanisms behind progressing chronic inflammatory dilated cardiomyopathy.

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.

Quantification of myocardial fibrosis by digital image analysis and interactive stereology.

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.

A variety of mild stresses upregulate stanniocalcin-1 (STC-1) and induce mitohormesis in neural crest-derived cells.

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.