The Role of Zinc on Liver Fibrosis by Modulating ZIP14 Expression Throughout Epigenetic Regulatory Mechanisms.

Zinc plays a pivotal role in tissue regeneration and maintenance being as a central cofactor in a plethora of enzymatic activities. Hypozincemia is commonly seen with chronic liver disease and is associated with an increased risk of liver fibrosis development and hepatocellular carcinoma. Previously favorable effects of zinc supplementation on liver fibrosis have been shown. However, the underlying mechanism of this effect is not elucidated. Liver fibrosis was induced in mice by using CCl4 injection, followed by treatment with zinc chloride (ZnCl2) both at fibrotic and sham groups, and their hepatocytes were isolated. Our results showed that the administration of ZnCl2 restored the depleted cytosolic zinc levels in the hepatocytes isolated from the fibrotic group. Also, alpha-smooth muscle actin (αSMA) expression in hepatocytes was decreased, indicating a reversal of the fibrotic process. Notably, ZIP14 expression significantly increased in the fibrotic group following ZnCl2 treatment, whereas in the sham group ZIP14 expression decreased. Chromatin immunoprecipitation (ChIP) experiments revealed an increased binding percentage of Metal-regulatory transcription factor 1 (MTF1) on ZIP14 promoter in the hepatocytes isolated from fibrotic mice compared to the sham group after ZnCl2 administration. In the same group, the binding percentage of the histone deacetylase HDAC4 on ZIP14 promoter decreased. Our results suggest that the ZnCl2 treatment ameliorates liver fibrosis by elevating intracellular zinc levels through MTF1-mediated regulation of ZIP14 expression and the reduction of ZIP14 deacetylation via HDAC4. The restoration of intracellular zinc concentrations and the modulation of ZIP14 expression by zinc orchestrated through MTF1 and HDAC4, appear to be essential determinants of the therapeutic response in hepatic fibrosis. These findings pave the way for potential novel interventions targeting zinc-related pathways for the treatment of liver fibrosis and associated conditions.

Cardioprotective role of a magnolol and honokiol complex in the prevention of doxorubicin-mediated cardiotoxicity in adult rats.

Doxorubicin (DOXO) induces marked cardiotoxicity, though increased oxidative stress while there are some documents related with cardioprotective effects of some antioxidants against organ-toxicity during cancer treatment. Although magnolia bark has some antioxidant-like effects, its action in DOXO-induced heart dysfunction has not be shown clearly. Therefore, here, we aimed to investigate the cardioprotective action of a magnolia bark extract with active component magnolol and honokiol complex (MAHOC; 100 mg/kg) in DOXO-treated rat hearts. One group of adult male Wistar rats was injected with DOXO (DOXO-group; a cumulative dose of 15 mg/kg in 2-week) or saline (CON-group). One group of DOXO-treated rats was administered with MAHOC before DOXO (Pre-MAHOC group; 2-week) while another group was administered with MAHOC following the 2-week DOXO (Post-MAHOC group). MAHOC administration, before or after DOXO, provided full survival of animals during 12-14 weeks, and significant recoveries in the systemic parameters of animals such as plasma levels of manganese and zinc, total oxidant and antioxidant statuses, and also systolic and diastolic blood pressures. This treatment also significantly improved heart function including recoveries in end-diastolic volume, left ventricular end-systolic volume, heart rate, cardiac output, and prolonged P-wave duration. Furthermore, the MAHOC administrations improved the structure of left ventricles such as recoveries in loss of myofibrils, degenerative nuclear changes, fragmentation of cardiomyocytes, and interstitial edema. Biochemical analysis in the heart tissues provided the important cardioprotective effect of MAHOC on the redox regulation of the heart, such as improvements in activities of glutathione peroxidase and glutathione reductase, and oxygen radical-absorbing capacity of the heart together with recoveries in other systemic parameters of animals, while all of these benefits were observed in the Pre-MAHOC treatment group, more prominently. Overall, one can point out the beneficial antioxidant effects of MAHOC in chronic heart diseases as a supporting and complementing agent to the conventional therapies.

Coffee-Derived Exosome-Like Nanoparticles: Are They the Secret Heroes?

BACKGROUND: Regular coffee consumption has beneficial and preventative effects on liver and chronic neurodegenerative diseases. However, the studies performed with the ingredients found in coffee beverages have not clarified the responsible mechanisms. Exosomes are small, membrane-coated cargo packages secreted by prokaryote and eukaryote cells. Exosomes regulate intercellular communication and affect cellular metabolic activities even among different species. In this study, we aimed to isolate and characterize the edible plant-derived exosome-like nanoparticles from roasted hot coffee beverages, hypothesizing that the edible plant-derived exosome-like nanoparticles were responsible for the beneficial effects of coffee. METHODS: Size exclusion chromatography and commercial kits were used for the isolation process. Efficient coffee edible plant-derived exosome-like nanoparticle fractions were determined by an ultraviolet-visible spectrophotometer. Harvested coffee edible plant-derived exosome-like nanoparticles were characterized by transmission electron microscopy. The quantification procedure was performed using a commercial kit. Coffee edible plant-derived exosome-like nanoparticles' proliferative effects on human hepatic stellate cells and human hepatocellular carcinoma cells were studied using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Whole-exosome RNA sequencing was performed. RESULTS: Transmission electron microscopy scanning analysis indicated round-shaped nanoparticles with sizes ranging from 40 to 100 nm. Both size exclusion chromatography and kit-isolated edible plant-derived exosome-like nanoparticle samples showed maximum absorbance at 227.5 nm in ultraviolet-visible spectrophotometer analysis. Regarding the quantitation results, kit isolation was more efficient than the size exclusion chromatography method when the harvested particle numbers were compared. An important MTT assay finding confirmed the observed beneficial effects of coffee beverages: coffee edible plant-derived exosome-like nanoparticles significantly suppressed hepatocellular carcinoma cell proliferation. As a result of sequencing, we identified 15 mature miRNAs. A MapReduce-based MicroRNA Target Prediction Method (The DIANA tools' MR-microT algorithm) highlighted 2 genes specifically associated with the miRNAs that we obtained: KMT2C and ZNF773. CONCLUSION: For the first time in the literature, coffee edible plant-derived exosome-like nanoparticles were identified. These nanoparticles may have therapeutic effects on chronic liver diseases. Experimental studies, therefore, should be performed on disease models to demonstrate their efficacy.

Cardioprotective effect of extracellular vesicles derived from ticagrelor-pretreated cardiomyocyte on hyperglycemic cardiomyocytes through alleviation of oxidative and endoplasmic reticulum stress.

Extracellular vesicles (EVs) play important roles in diabetes mellitus (DM) via connecting the immune cell response to tissue injury, besides stimulation to muscle insulin resistance, while DM is associated with increased risks for major cardiovascular complications. Under DM, chronic hyperglycemia, and subsequent increase in the production of reactive oxygen species (ROS) further lead to cardiac growth remodeling and dysfunction. The purinergic drug ticagrelor is a P2Y12 receptor antagonist. Although it is widely used in cardioprotection, the underlying molecular mechanism of its inhibitory effect on diabetic cardiomyopathy is poorly elucidated. Here, we aimed to understand how ticagrelor exerts its cardio-regulatory effects. For this purpose, we investigated the anti-oxidative and cardioprotective effect of EVs derived from ticagrelor-pretreated cardiomyocytes under DM conditions. To mimic DM in cardiomyocytes, we used high glucose incubated H9c2-cells (HG). HG cells were treated with EVs, which were derived from either ticagrelor-pretreated or untreated H9c2-cells. Our results demonstrated that ticagrelor-pretreated H9c2-derived EVs significantly decreased the hyperglycemia-induced aberrant ROS production, prevented the development of apoptosis and ER stress, and alleviated oxidative stress associated miRNA-expression profile. Importantly, EVs derived from ticagrelor-pretreated H9c2-cells enhanced endothelial cell migration and tube formation, suggesting a modulation of the EV profile in cardiomyocytes. Our data, for the first time, indicate that ticagrelor can exert an important regulatory effect on diabetic cardiomyopathy through extracellular vesicular modulation behind its receptor-inhibition-related effects.

Generation and characterization of human induced pluripotent stem cell line METUi001-A from a 25-year-old male patient with relapsing-remitting multiple sclerosis.

Multiple sclerosis is a chronic disease characterized by inflammation, demyelination, and axonal damage in the central nervous system. Here, we established an induced pluripotent stem cell (iPSC) line METUi001-A from the peripheral blood mononuclear cells of a 25-year-old male individual with clinically diagnosed Relapsing-Remitting Multiple Sclerosis (RRMS) using the integration-free Sendai reprogramming method. We demonstrated that the iPSCs are free of exogenous Sendai reprogramming vectors, have a normal male karyotype, express pluripotency markers, and differentiate into the three germ layers. The iPSC line can serve as a valuable resource to generate cellular model systems to investigate molecular mechanisms underlying RRMS.

Graphene Oxide Nanosheets Interact and Interfere with SARS-CoV-2 Surface Proteins and Cell Receptors to Inhibit Infectivity.

Nanotechnology can offer a number of options against coronavirus disease 2019 (COVID-19) acting both extracellularly and intracellularly to the host cells. Here, the aim is to explore graphene oxide (GO), the most studied 2D nanomaterial in biomedical applications, as a nanoscale platform for interaction with SARS-CoV-2. Molecular docking analyses of GO sheets on interaction with three different structures: SARS-CoV-2 viral spike (open state - 6VYB or closed state - 6VXX), ACE2 (1R42), and the ACE2-bound spike complex (6M0J) are performed. GO shows high affinity for the surface of all three structures (6M0J, 6VYB and 6VXX). When binding affinities and involved bonding types are compared, GO interacts more strongly with the spike or ACE2, compared to 6M0J. Infection experiments using infectious viral particles from four different clades as classified by Global Initiative on Sharing all Influenza Data (GISAID), are performed for validation purposes. Thin, biological-grade GO nanoscale (few hundred nanometers in lateral dimension) sheets are able to significantly reduce copies for three different viral clades. This data has demonstrated that GO sheets have the capacity to interact with SARS-CoV-2 surface components and disrupt infectivity even in the presence of any mutations on the viral spike. GO nanosheets are proposed to be further explored as a nanoscale platform for development of antiviral strategies against COVID-19.

2D MXenes with antiviral and immunomodulatory properties: A pilot study against SARS-CoV-2.

Two-dimensional transition metal carbides/carbonitrides known as MXenes are rapidly growing as multimodal nanoplatforms in biomedicine. Here, taking SARS-CoV-2 as a model, we explored the antiviral properties and immune-profile of a large panel of four highly stable and well-characterized MXenes - Ti3C2Tx, Ta4C3T x , Mo2Ti2C3T x and Nb4C3T x . To start with antiviral assessment, we first selected and deeply analyzed four different SARS-CoV-2 genotypes, common in most countries and carrying the wild type or mutated spike protein. When inhibition of the viral infection was tested in vitro with four viral clades, Ti3C2T x in particular, was able to significantly reduce infection only in SARS-CoV-2/clade GR infected Vero E6 cells. This difference in the antiviral activity, among the four viral particles tested, highlights the importance of considering the viral genotypes and mutations while testing antiviral activity of potential drugs and nanomaterials. Among the other MXenes tested, Mo2Ti2C3T x also showed antiviral properties. Proteomic, functional annotation analysis and comparison to the already published SARS-CoV-2 protein interaction map revealed that MXene-treatment exerts specific inhibitory mechanisms. Envisaging future antiviral MXene-based drug nano-formulations and considering the central importance of the immune response to viral infections, the immune impact of MXenes was evaluated on human primary immune cells by flow cytometry and single-cell mass cytometry on 17 distinct immune subpopulations. Moreover, 40 secreted cytokines were analyzed by Luminex technology. MXene immune profiling revealed i) the excellent bio and immune compatibility of the material, as well as the ability of MXene ii) to inhibit monocytes and iii) to reduce the release of pro-inflammatory cytokines, suggesting an anti-inflammatory effect elicited by MXene. We here report a selection of MXenes and viral SARS-CoV-2 genotypes/mutations, a series of the computational, structural and molecular data depicting deeply the SARS-CoV-2 mechanism of inhibition, as well as high dimensional single-cell immune-MXene profiling. Taken together, our results provide a compendium of knowledge for new developments of MXene-based multi-functioning nanosystems as antivirals and immune-modulators.

Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 in vitro.

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico, and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48 h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression.

Human Gut Commensal Membrane Vesicles Modulate Inflammation by Generating M2-like Macrophages and Myeloid-Derived Suppressor Cells.

Immunomodulatory commensal bacteria modify host immunity through delivery of regulatory microbial-derived products to host cells. Extracellular membrane vesicles (MVs) secreted from symbiont commensals represent one such transport mechanism. How MVs exert their anti-inflammatory effects or whether their tolerance-inducing potential can be used for therapeutic purposes remains poorly defined. In this study, we show that MVs isolated from the human lactic acid commensal bacteria Pediococcus pentosaceus suppressed Ag-specific humoral and cellular responses. MV treatment of bone marrow-derived macrophages and bone marrow progenitors promoted M2-like macrophage polarization and myeloid-derived suppressor cell differentiation, respectively, most likely in a TLR2-dependent manner. Consistent with their immunomodulatory activity, MV-differentiated cells upregulated expression of IL-10, arginase-1, and PD-L1 and suppressed the proliferation of activated T cells. MVs' anti-inflammatory effects were further tested in acute inflammation models in mice. In carbon tetrachloride-induced fibrosis and zymosan-induced peritonitis models, MVs ameliorated inflammation. In the dextran sodium sulfate-induced acute colitis model, systemic treatment with MVs prevented colon shortening and loss of crypt architecture. In an excisional wound healing model, i.p. MV administration accelerated wound closure through recruitment of PD-L1-expressing myeloid cells to the wound site. Collectively, these results indicate that P. pentosaceus-derived MVs hold promise as therapeutic agents in management/treatment of inflammatory conditions.

Differential expression of genes participating in cardiomyocyte electrophysiological remodeling via membrane ionic mechanisms and Ca2+-handling in human heart failure.

Excitation-contraction coupling in normal cardiac function is performed with well balanced and coordinated functioning but with complex dynamic interactions between functionally connected membrane ionic currents. However, their genomic investigations provide essential information on the regulation of diseases by their transcripts. Therefore, we examined the gene expression levels of the most important voltage-gated ionic channels such as Na+-channels (SCN5A), Ca2+-channels (CACNA1C and CACNA1H), and K+-channels, including transient outward (KCND2, KCNA2, KCNA5, KCNA8), inward rectifier (KCNJ2, KCNJ12, KCNJ4), and delayed rectifier (KCNB1) in left ventricular tissues from either ischemic or dilated cardiomyopathy (ICM or DCM). We also examined the mRNA levels of ATP-dependent K+-channels (KCNJ11, ABCC9) and ERG-family channels (KCNH2). We further determined the mRNA levels of ryanodine receptors (RyR2; ARVC2), phospholamban (PLB or PLN), SR Ca2+-pump (SERCA2; ATP2A1), an accessory protein FKBP12 (PPIASE), protein kinase A (PPNAD4), and Ca2+/calmodulin-dependent protein kinase II (CAMK2G). The mRNA levels of SCN5A, CACNA1C, and CACNA1H in both groups decreased markedly in the heart samples with similar significance, while KvLQT1 genes were high with depressed Kv4.2. The KCNJ11 and KCNJ12 in both groups were depressed, while the KCNJ4 level was significantly high. More importantly, the KCNA5 gene was downregulated only in the ICM, while the KCNJ2 was upregulated only in the DCM. Besides, mRNA levels of ARVC2 and PLB were significantly high compared to the controls, whereas others (ATP2A1, PPIASE, PPNAD4, and CAMK2G) were decreased. Importantly, the increases of KCNB1 and KCNJ11 were more prominent in the ICM than DCM, while the decreases in ATP2A1 and FKBP1A were more prominent in DCM compared to ICM. Overall, this study was the first to demonstrate that the different levels of changes in gene profiles via different types of cardiomyopathy are prominent particularly in some K+-channels, which provide further information about our knowledge of how remodeling processes can be differentiated in HF originated from different pathological conditions.

A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System.

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Liver fibrosis.

Liver fibrosis is a wound-healing response generated against an insult to the liver that causes liver injury. It has the potential to progress into cirrhosis, and if not prevented, it may lead to liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. In addition to HSCs, numerous studies have supported the potential contribution of bone marrow-derived cells and myofibroblasts to liver fibrosis. The liver is a heterogeneous organ; thus, molecular and cellular events that underlie liver fibrogenesis are complex. This review aims to focus on major events that occur during liver fibrogenesis. In addition, important antifibrotic therapeutic approaches and experimental liver fibrosis models will be discussed.

Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+ -transporters in heart failure.

Zn2+ -homoeostasis including free Zn2+ ([Zn2+ ]i ) is regulated through Zn2+ -transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn2+ -transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn2+ ]i was significantly high in doxorubicin-treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα-phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn2+ ]i using zinc-ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn2+ transporters. Additionally, increased [Zn2+ ]i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn2+ ]i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn2+ transporters on an intersection pathway with increased [Zn2+ ]i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well-controlled [Zn2+ ]i via Zn2+ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF.

Role of FLT3 in the proliferation and aggressiveness of hepatocellular carcinoma.

BACKGROUND/AIM: Previously we showed that Fms-like tyrosine kinase (FLT3) changes its cellular localization upon partial hepatectomy, suggesting a role in liver regeneration. FLT3 was also shown to play an important function in cellular proliferation and activation of PI3K and Ras. Thus, we aimed to investigate the role of FLT3 in hepatocellular tumorigenesis utilizing in vitro and in vivo models. MATERIALS AND METHODS: We used Snu398 cells that express FLT3. We investigated these cells' in vitro proliferation and invasion abilities by treatment with the FLT3 inhibitor K-252a or by knocking-down with FLT3 shRNA,. Furthermore, the effect of blocking FLT3 activity and expression during in vivo tumorigenesis was assessed with xenograft models. RESULTS: After K-252a treatment or stable knock-down, these cells' proliferation and migration abilities were highly diminished in vitro. In addition, significant diminution in tumorigenicity of Snu398 cells was also obtained in vivo. When FLT3 knocked-down Snu398 cells were injected into nude mice, we did not detect αSMA expression in these tumors, suggesting a role for FLT3 in in vivo invasiveness. CONCLUSION: Our data provided evidence that FLT3 has a crucial role both in hepatocarcinogenesis and its invasiveness. Therefore, targeting FLT3 and/or its activity may be a promising tool for combating hepatocellular carcinomas.

Determination of the presence of diphtheria toxin in the myocardial tissue of rabbits and a female subject by using an immunofluorescent antibody method.

BACKGROUND: Clinical diagnosis of diphtheria is often difficult, in particular in countries where the disease is rarely observed, such as Turkey. In 2011, after 12 years of no recorded diphtheria cases in Turkey, a 34-year-old woman was diagnosed with diphtheria; she later died of myocarditis. In this study, we aimed to demonstrate the diagnostic potential of an immunofluorescent antibody method to determine the presence of diphtheria toxin (DT) in the myocardial cells of DT-injected rabbits and the female subject. METHODS: We randomly divided rabbits into two groups: a control group and a DT-injected group. Diphtheria intoxication was simulated in the rabbits by intravenous injection of DT. The myocardium of the rabbits and the female subject were harvested for histopathologic and immunofluorescence examination. A mouse monoclonal anti-DT antibody was used for the immunofluorescent antibody method. RESULTS: The presence of DT in the myocardial cells of both the rabbits and the female subject was visualized using the immunofluorescent method. CONCLUSIONS: Laboratory diagnosis of diphtheria is challenging because of non-toxigenic C. diphtheriae strains and/or the dysfunction of DT. However, visualizing the presence of DT in the myocardial tissue may act as an indicator of biologically active DT. We validated that an immunofluorescent method, which utilizes a monoclonal anti-DT (A-subunit specific) antibody, is a useful diagnostic tool to determine the presence of DT in the myocardium of rabbits and human.

Resveratrol affects histone 3 lysine 27 methylation of vessels and blood biomarkers in DOCA salt-induced hypertension.

Hypertension is a risk factor for the cardiovascular diseases. Although, several drugs are used to treat hypertension, the success of the antihypertensive therapy is limited. Resveratrol decreases blood pressure in animal models of hypertension. This study researched the mechanisms behind the effects of resveratrol on hypertension. Hypertension was induced by using the deoxycorticosterone acetate (DOCA)-induced (15 mg/kg twice per week, subcutaneously) salt-sensitive hypertension model of Wistar rats. Hypertension caused a decrease in endothelium-dependent relaxations of the isolated thoracic aorta. Resveratrol treatment (50 mg/l in drinking water) prevented DOCA salt-induced hypertension, but did not improve endothelial dysfunction. Plasma nitric oxide (NO), asymmetric dimethylarginine (ADMA), total antioxidant capacity (TAC) and hydrogen sulfide (H2S) levels were not changed by DOCA salt application. However, treatment of resveratrol significantly decreased ADMA and increased TAC and H2S levels. NO level in circulation was not significantly changed by resveratrol. DOCA salt application and resveratrol treatment also caused an alteration in the epigenetic modification of vessels. Staining pattern of histone 3 lysine 27 methylation (H3K27me3) in the aorta and renal artery sections was changed. These results show that preventive effect of resveratrol on DOCA salt-induced hypertension might due to its action on the production of some blood biomarkers and the epigenetic modification of vessels that would focus upon new aspect of hypertension prevention and treatment.

Di-peptidyl peptidase-4 inhibitor sitagliptin protects vascular function in metabolic syndrome: possible role of epigenetic regulation.

Metabolic syndrome (MetS) is a complex medical disorder characterized by insulin resistance, hypertension, and high risk of coronary disease and stroke. Microvascular rarefaction and endothelial dysfunction have also been linked with MetS, and recent evidence from clinical studies supports the efficacy of incretin-based antidiabetic therapies for vascular protection in diabetes. Previous studies pointed out the importance of dipeptidyl peptidase-4 (DPP-4) inhibition in endothelial cells due to getting protection against metabolic pathologies. We therefore aimed to investigate the acute effects of a DPP-4 inhibitor, sitagliptin, on vascular function in rats with high-sucrose diet-induced MetS. In order to elucidate the mechanisms implicated in the effects of DPP-4 inhibition, we tested the involvement of NO pathway and epigenetic regulation in the MetS. Acute use of sitagliptin protects the vascular function in the rats with MetS in part due to NO pathway via restoring the depressed aortic relaxation responses mediated by receptors. Application of sitagliptin enhanced the depressed phosphorylation levels of both the endothelial NO synthase and the apoptotic status of protein kinase B, known as Akt, in endothelium-intact thoracic aorta from rats with MetS. One-hour application of sitagliptin on aortic rings from rats with MetS also induced remarkable histon posttranslational modifications such as increased expression of H3K27Me3, but not of H3K27Me2, resulting in an accumulation of the H3K27Me3. Our findings suggest that, in addition to its well-known hypoglycemic action, sitagliptin may also have beneficial effects on hyperglycemia-induced vascular changes in an endotheium-dependent manner. These present results with sitagliptin aside from the glycaemic control, may demonstrate its important role in the treatment of patients with MetS.

Patterned carbon nanotubes as a new three-dimensional scaffold for mesenchymal stem cells.

We investigated the cellular adhesive features of mesenchymal stem cells (MSC) on non-coated and collagen coated patterned and vertically aligned carbon nanotube (CNT) structures mimicking the natural extra cellular matrix (ECM). Patterning was achieved using the elasto-capillary induced by water treatment on the CNT arrays. After confirmation with specific markers both at transcript and protein levels, MSCs from different passages were seeded on either collagen coated or non-coated patterned CNTs. Adhesion and growth of MSCs on the patterned CNT arrays were examined using scanning electron microscopy image analysis and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. The highest MSC count was observed on the non-coated patterned CNTs at passage zero, while decreasing numbers of MSCs were found at the later passages. Similarly, MTT assay results also revealed a decrease in the viability of the MSCs for the later passages. Overall, the cell count and viability experiments indicated that MSCs were able to better attach to non-coated patterned CNTs compared to those coated with collagen. Therefore, the patterned CNT surfaces can be potentially used as a scaffold mimicking the ECM environment for MSC growth which presents an alternative approach to MSC-based transplantation therapy applications.

Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: In vitro interactions with platelets and rat stem cells.

Copolymerization of 2-hydroxyethylmethacrylate (HEMA) with glycidylmethacrylate (GMA) in the presence of α-α'-azoisobisbutyronitrile (AIBN) resulted in the formation of hydrogel films carrying reactive epoxy groups. Thirteen kinds of different molecules with pendant NH2 group were used for modifications of the p(HEMA-GMA) films. The NH2 group served as anchor binding site for immobilization of functional groups on the hydrogel film via direct epoxy ring opening reaction. The modified hydrogel films were characterized by FTIR, and contact angle studies. In addition, mechanical properties of the hydrogel films were studied, and modified hydrogel films showed improved mechanical properties compared with the non-modified film, but they are less elastic than the non-modified film. The biological activities of these films such as platelet adhesion, red blood cells hemolysis, and swelling behavior were studied. The effect of modified hydrogel films, including NH2, (using different aliphatic CH2 chain lengths) CH3, SO3H, aromatic groups with substituted OH and COOH groups, and amino acids were also investigated on the adhesion, morphology and survival of rat mesenchymal stem cells (MSCs). The MTT colorimetric assay reveals that the p(HEMA-GMA)-GA-AB, p(HEMA-GMA)-GA-Phe, p(HEMA-GMA)-GA-Trp, p(HEMA-GMA)-GA-Glu formulations have an excellent biocompatibility to promote the cell adhesion and growth. We anticipate that the fabricated p(HEMA-GMA) based hydrogel films with controllable surface chemistry and good stable swelling ratio may find extensive applications in future development of tissue engineering scaffold materials, and in various biotechnological areas.

The effect of estrogen on bone marrow-derived rat mesenchymal stem cell maintenance: inhibiting apoptosis through the expression of Bcl-xL and Bcl-2.

Mesenchymal Stem Cells (MSCs) have high therapeutic value for regenerative medicine and tissue engineering due to their differentiation potential and non-immunogenic characteristics. They are also considered as an effective in vivo delivery agent because of their ability to migrate to the site of injury. A major roadblock in their use for cell-based therapies is their rareness in vivo. Therefore, it is important to obtain increased number of functional MSCs in vitro in order to have adequate numbers for therapeutic regiments. We aimed to investigate the role of estrogen and its mechanism in obtaining more MSCs. MSCs were isolated from female and ovariectomized rats and cultured in the presence and absence of 10(-7) M estrogen. In the presence of estrogen, not only their CFU-F activity increased but also apoptotic rate decreased as shown by TUNEL staining leading to obtain more MSCs. Also the number of the cells in the colonies increased upon estrogen treatment. To reveal the mechanism of this effect, we focused on Bcl-2 family of proteins. Our immunoblotting experiments combined with knockdown studies suggested a critical role for anti-apoptotic Bcl-x(L) and Bcl-2. Estrogen treatment up regulated the expression Bcl-x(L) and Bcl-2. When we knocked down the expression of bcl-x ( L ) and bcl-2, MSCs lacking these genes showed an increase in the apoptotic rate in contrast to normal MSCs following estrogen treatment. Therefore, estrogen treatment will be of great advantage for cell-based therapies in order to get more functional MSCs and may provide opportunities to develop new strategies for debilitating diseases.