Endoplasmic reticulum stress produced by Thapsigargin affects the occurrence of spike-wave discharge by modulating unfolded protein response pathways and activating immune responses in a dose-dependent manner.

The Endoplasmic Reticulum (ER) is associated with many cellular functions, from post-transcriptional modifications to the proper folding of proteins, and disruption of these functions causes ER stress. Although the relationship between epileptic seizures and ER stress has been reported, the contribution of ER stress pathways to epileptogenesis is still unclear. This study aimed to investigate the possible effects of ER stress-related molecular pathways modulated by mild- and high-dose Thapsigargin (Tg) on absence epileptic activity, CACNA1H and immune responses in WAG/Rij rats. For this purpose, rats were divided into four groups; mild-dose (20 ng) Tg, high-dose (200 ng) Tg, saline, and DMSO and drugs administered intracerebroventriculary. EEG activity was recorded for 1 h and 24 h after drug administration following the baseline recording. In cortex and thalamus tissues, GRP78, ERp57, GAD153 protein changes (Western Blot), Eif2ak3, XBP-1, ATF6, CACNA1H mRNA expressions (RT-PCR), NF-κB and TNF-α levels (ELISA) were measured. Mild-dose-Tg administration resulted in increased spike-wave discharge (SWD) activity at the 24th hour compared to administration of saline, and high-dose-Tg and it also significantly increased the amount of GRP78 protein, the expression of Eif2ak3, XBP-1, and CACNA1H mRNA in the thalamus tissue. In contrast, high-dose-Tg administration suppressed SWD activity and significantly increased XBP-1 and ATF6 mRNA expression in the thalamus, and increased NF-κB and TNF-α levels. In conclusion, our findings indicate that Tg affects SWD occurrence by modulating the unfolded protein response pathway and activating inflammatory processes in a dose-dependent manner.

Investigation of the differentiation potential of pericyte cells as an alternative source of mesenchymal stem cells.

BACKGROUND: The mesenchymal stem cells (MSCs) with characterized by their multipotency and capacity to differentiate into various tissue cell types, have led to their incorporation in regenerative medicine research. However, the limited numbers of MSCs in the human body and their diverse differentiation capabilities in tissues highlight the need for exploring alternative regenerative cell sources. In this study, therefore, we conducted molecular level examinations to determine whether pericytes, specialized cell communities situated near blood vessels, could serve as a substitute for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this context, the potential application of pericytes surrounds the vessels when MSCs are insufficient for functional purposes. METHODS: The pericytes utilized in this investigation were derived from the placenta and characterized at the third passage. Similarly, the hBM-MSCs were also characterized at the third passage. The pluripotent properties of the two cell types were assessed at the gene expression level. Thereafter, both pericytes and hBM-MSCs were directed towards adipogenic, osteogenic and chondrogenic differentiation. The cells in both groups were examined on days 7, 14, and, 21 and their differentiation status was compared both immunohistochemically and through gene expression analysis. RESULTS: Upon comparing the pluripotency characteristics of placental pericytes and hBM-MSCs, it was discovered that there was a substantial upregulation of the pluripotency genes FoxD3, Sox2, ZPF42, UTF1, and, Lin28 in both cell types. However, no significant expression of the genes Msx1, Nr6a1, Pdx1, and, GATA6 was observed in either cell type. It was also noted that pericytes differentiate into adipogenic, osteogenic and, chondrogenic lineages similar to hBM-MSCs. DISCUSSION: As a result, it has been determined that pericytes exhibit high differentiation and proliferation properties similar to those of MSCs, and therefore can be considered a suitable alternative cell source for regenerative medicine and tissue engineering research, in cases where MSCs are not available or insufficient. It is notable that pericytes have been suggested as a potential substitute in studies where MSCs are lacking.

Production of alginate macrocapsule device for long-term normoglycaemia in the treatment of type 1 diabetes mellitus with pancreatic cell sheet engineering.

Type 1 diabetes-mellitus (T1DM) is characterized by damage of beta cells in pancreatic islets. Cell-sheet engineering, one of the newest therapeutic approaches, has also been used to create functional islet systems by creating islet/beta cell-sheets and transferring these systems to areas that require minimally invasive intervention, such as extrahepatic areas. Since islets, beta cells, and pancreas transplants are allogeneic, immune problems such as tissue rejection occur after treatment, and patients become insulin dependent again. In this study, we aimed to design the most suitable cell-sheet treatment method and macrocapsule-device that could provide long-term normoglycemia in rats. Firstly, mesenchymal stem cells (MSCs) and beta cells were co-cultured in a temperature-responsive culture dish to obtain a cell-sheet and then the cell-sheets macroencapsulated using different concentrations of alginate. The mechanical properties and pore sizes of the macrocapsule-device were characterized. The viability and activity of cell-sheets in the macrocapsule were evaluatedin vitroandin vivo. Fasting blood glucose levels, body weight, and serum insulin & C-peptide levels were evaluated after transplantation in diabetic-rats. After the transplantation, the blood glucose level at 225 mg dl-1on the 10th day dropped to 168 mg dl-1on the 15th day, and remained at the normoglycemic level for 210 days. In this study, an alginate macrocapsule-device was successfully developed to protect cell-sheets from immune attacks after transplantation. The results of our study provide the basis for future animal and human studies in which this method can be used to provide long-term cellular therapy in T1DM patients.

Examining the effect of activated cytotoxic (CD8+) T-cell exosomes to the lung cancer.

Lung cancer continues to be a major health problem worldwide owing to its incidence, and causes physical, psychological, social, and economic problems. Activated cytotoxic T cells (ACTC) are positively correlated with the tumor microenvironment (TME), improving the prognosis of cancer patients. Recently, ACTC-derived exosomes (ACTC-dExo) were implicated in this effect by inhibiting mesenchymal stem cells, which may promote metastasis in the TME. Exosomes are thought to be advantageous for the specific delivery of drugs to cancer cells because they have the characteristics of natural liposomes, are nanosized, and remain largely stable in the blood due to the protein and lipid content they carry on their membranes. In this study, we aimed to determine the cytotoxic and metastatic inhibitory effects of ACTC-dExo in A549 cells in vitro. Cytotoxic CD8+ T cells were isolated from whole blood obtained from healthy individuals and cultured for 5-7 days after stimulation. The ACTC-dExo serum-free culture medium was collected by ultracentrifugation. Characterization and quantification of the isolated exosomes were performed using flow cytometry, electron microscopy, zeta-sizer measurements, and bicinchoninic acid (BCA) assays. We co-cultured ACTC and ACTC-dExo with A549 cells for 48 h. The viability of A549 cells was evaluated using a WST-1 assay. The metastasis-related genes MMP2, MMP9, TWIST, SNAI1, and CDH1 were detected by qRT-PCR, and MMP2 and MMP9 proteins were evaluated by confocal microscopy. In addition, changes in cell migration were investigated using a scratch assay. ACTC-dExo were found to have anti-proliferative and anti-metastatic effects and reduced cancer cell proliferation and metastatic properties.

Investigation of impacts of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of mesenchymal stem cell through Notch/Hedgehog signaling pathways.

OBJECTIVE: Decellularization is the process to obtain natural scaffolds with tissue integrity and extracellular matrix components, and recellularization is used to produce tissue-like constructs with specific cell types. In this study, rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) were cultured on decellularized heart extracellular matrix. These cells were then induced to differentiate into cardiomyogenic cells under the stimulatory effect of vascular endothelial growth factor (VEGF) and other chemicals. This study aimed to investigate the effect of the cardiac extracellular matrix and VEGF on cardiomyogenic differentiation in the context of the Notch and Hedgehog signaling pathways. METHODS: Heart samples extracted from rats were decellularized by serial application of detergent to remove cells from the tissue, and then recellularized with rBM-MSCs. The recellularized tissue matrices were then analyzed for cardiomyogenesis. Cardiomyogenic differentiation was performed on decellularized heart extracellular matrix (ECM; three-dimensional scaffolds) and culture plates (two-dimensional cell culture system) for 28 days to understand the effects of the heart extracellular matrix. In addition, differentiation was induced with and without the stimulatory effect of VEGF to understand the effect of VEGF on cardiomyogenic differentiation of rBM-MSCs. RESULTS: Immunofluorescence staining showed that decellularization of the heart was performed effectively and successfully. After decellularization process, the heart extracellular matrix was completely free of cells. It was observed that rBM-MSCs transplanted onto the heart extracellular matrix remained viable and proliferated for 21 days after recellularization. The rBM-MSCs promoted cardiomyogenic differentiation in the conventional differentiation medium but were inversely affected by both VEGF and heart extracellular matrix proteins. Lower expression of connexin43 and cardiac troponin I genes was observed in cells induced by either matrix proteins or VEGF, compared to cells differentiated by chemical agents alone. CONCLUSION: In this study, we investigated the effect of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of rBM-MSCs. On the decellularized cardiac extracellular matrix, rBM-MSCs maintained their viability by adhering to the matrix and proliferating further. The adhesion of the cells to the matrix also produced a physical stimulus that led to the formation of histological structures resembling myocardial layers. Chemical stimulation of the decellularized heart extracellular matrix and cardiomyogenic differentiation supplements resulted in increased expression of cardiomyogenic biomarkers through modulation of the Notch and Hedgehog signaling pathways.

Letrozole Decreased Testosterone-Induced Cell Proliferation and Prolactin Secretion also Increased Apoptosis in MMQ and GH3 Rat Prolactinoma Cell Lines.

Aromatase enzyme plays an essential role in estrogen-induced tumorigenesis. It is expressed in the normal pituitary and more significantly in prolactinoma tissues. The aim of this study was to investigate the effects of an aromatase inhibitor, letrozole, on MMQ and GH3 rat prolactinoma cell lines and evaluate the possible mechanism of action. MMQ and GH3 cells were characterized with demonstrating aromatase enzyme and estrogen receptor alpha expression by PCR and immunofluorescence staining. After dose optimization for testosterone (T) and letrozole (L), four groups were established: only the testosteron-treated group (T) to detect cell proliferation; only letrozole-treated group (L) to investigate apoptotic effects; testosterone and letrozole concomitant-treated group to demonstrate inhibition of testosterone induced cell proliferation with letrozole treatment s(T + L) and control group (C) with no treatment. The proliferation rate of cells was determined by WST-1. For the detection of apoptotic and necrotic cells, Annexin V and caspase-3 labeling was used. Prolactin and estrogen levels were measured with ELISA, and the mRNA expression of aromatase and Esr1 was also determined. Testosterone induced the proliferation of MMQ and GH3 cells and further increased prolactin and estradiol levels. Adding letrozole to testosterone resulted in decreased cellular proliferation and even induced apoptosis. Also, letrozole administration significantly decreased prolactin and estradiol levels. However, letrozole alone had no effects on proliferation and apoptosis. Gene expression of aromatase and Esr1 was also significantly decreased by letrozole treatment. This in vitro study demonstrated that treatment of testosterone proliferating cells with letrozole resulted in decreased prolactin levels and cell proliferation and induced apoptosis, and further loss of aromatase and Esr1 mRNA expression were observed. Although this is an in vivo study, the results showed unique and novel findings which may easily be adapted to clinical use for further verification.

Resveratrol improves vascular endothelial dysfunction in the unpredictable chronic mild stress model of depression in rats by reducing inflammation.

Chronic psychological stress may cause depression and it is a risk factor for vascular endothelial dysfunction. Inflammation may contribute to endothelial dysfunction. Resveratrol, which has antiinflammatory and vasculoprotective properties, has been reported its beneficial effects on endothelial dysfunction induced by hypertension, diabetes and, aging. The effects of resveratrol on stress-induced endothelial dysfunction is not investigated yet. This study aimed to investigate the efficacy of resveratrol on vascular function in the unpredictable chronic moderate stress (UCMS) model of rats and to examine the possible mechanisms of resveratrol by assessment of proinflammatory markers. Male rats were assigned to 4 groups (n = 8 for each group): Control, Control+Resveratrol, UCMS, UCMS+Resveratrol. UCMS and UCMS+Resveratrol groups were exposed to the UCMS procedure for 12 weeks. Resveratrol (20 mg/kg/day, i.p., during 12 weeks) was given to the Control+Resveratrol and UCMS+Resveratrol groups.Then depressive-like behaviors were evaluated by forced swimming test. After behavioral tests, systolic blood pressure was recorded. Endothelial function of the thoracic aorta was evaluated by isolated organ bath system. Vascular eNOS expression and inflammatory markers such as TNF-α, IL-1ß, IL-6, CRP, ICAM1, MCP in serum and vascular tissue were analyzed to explore the mechanisms of resveratrol. UCMS resulted in depressive-like behavior, endothelial dysfunction and increased inflammatory cytokines in both serum and tissue samples. Resveratrol treatment improved depressive-like behavior, ameliorated vascular dysfunction, and reversed stress-induced inflammation. Our findings suggest that resveratrol exerted antidepressant-like effect and prevented vascular endothelial dysfunction by reducing systemic and peripheral inflammation in UCMS-induced depression in rats. Therefore, resveratrol may be a therapeutic option with a vasculoprotective effect in depression.

The effect of mesenchymal stromal cells in the microenvironment on cancer development.

Inflammatory signals secreted from the tumor microenvironment are thought to promote tumor growth and survival. It has been reported that stromal cells in the tumor microenvironment have similar characteristics to tumor-associated cells. In addition miRNAs play critical roles in various diseases, including cancer. In this study, we aimed to investigate the effects of co-culture of cancer cells and stromal cells isolated from normal and malignant breast tissue on each other and the possible effects of miRNAs on these interactions. The characterized stromal cells were co-cultured with an MDA-MB-231 cancer cell line. The proliferation capacity of the experimental groups was evaluated using the WST-1 assay. The expression of breast cancer-specific miRNAs and related genes were assessed by real-time PCR. ELISA assay was performed to determine the concentration of some cytokines and chemokines. We found that the microenvironment plays an important role in the development of cancer, confirming the changes in the expression of oncogenic and tumor suppressor miRNA and their target genes after co-culture with malignant stromal cells. As a result of the studies, specific gene expressions of related signaling pathways were detected in correlation with miRNA changes and the effects of tumor microenvironment on tumorigenesis were revealed in detail. miRNAs have been shown to play an important role in cancer development in recent studies. The idea that these small molecules can be used in diagnosis and treatment is becoming stronger day by day. We believe that new treatment approaches involving the tumor microenvironment and using miRNAs as markers are promising.

Evaluation of the Effect of Diclofenac Sodium and 5-Fluourasil in a 3D Cholesteatoma Cell Culture Model.

INTRODUCTION: Middle ear cholesteatoma is a benign disease with invasive and destructive clinical behaviors. It increases the rate of both chronic otitis media complications and revision surgeries. The most effective treatment of middle ear cholesteatoma is surgical excision, and there is no medical treatment for this disease. Exploring new medical treatment options may help to create treatment alternatives instead of surgery. MATERIALS AND METHODS: Required cholesteatoma tissues for cell culture were excised from 4 different participants who underwent surgery in our clinic and agreed to give tissue for the study. Cholesteatoma-derived keratinocytes and fibroblasts were cocultured in temperature-sensitive culture dishes to make a three-dimensional (3D) cholesteatoma model. Then, the effects of 1% and 2% diclofenac sodium on viability and cell proliferation rates were examined using WST-1 and annexin-V tests. RESULTS: Cell viability and proliferation rates were found to be lower and apoptosis rates were higher in the diclofenac sodium group versus the negative and positive control groups. CONCLUSION: In this present study, we described a new 3D cholesteatoma cell culture model developed using cell sheet technology and demonstrated the efficacy of diclofenac sodium on cholesteatoma for the first time in the literature. It may be used in patients with chronic otitis media with cholesteatoma, but further studies investigating ototoxic and neurotoxic effects of this molecule are needed.

Restorative effect of resveratrol on expression of endothelial and neuronal nitric oxide synthase in cavernous tissues of chronic unpredictable mild stress-exposed rats: an impact of inflammation.

We investigated the effect of resveratrol on endothelial and neuronal nitric oxide synthase (eNOS and nNOS) expression in the corpus cavernosum from chronic unpredictable mild stress (CUMS)-exposed rats in order to examine possible role of proinflammatory cytokines, which might play a role on erectile dysfunction (ED). Rats were randomly and equally divided into four groups such as control, control+resveratrol, CUMS and CUMS + resveratrol (20 mg/kg/day, i.p/8 weeks). Sucrose intake and forced swimming tests were used to evaluate depressive-like behaviors. nNOS, eNOS expressions, inflammatory markers, corticosterone and testosterone levels were analyzed either in blood samples and/or penile tissues. CUMS-exposed rats displayed depressive-like behaviors, reduced penile nNOS and eNOS expressions, and serum testosterone levels and enhanced serum and penile tissue levels of proinflammatory markers compared to controls. Resveratrol reversed depressive-like behaviors and suppressed serum and penile levels of proinflammatory markers, increased nNOS and eNOS expressions and testosterone levels in CUMS-exposed rats. Resveratrol exerted antidepressant-like effects and protected the development of CUMS-induced impairment of cavernosal eNOS and nNOS expressions associated with ED, which might be related to its anti-inflammatory action.

Depression induced by chronic stress leads to penile cavernosal dysfunction: protective effect of anti-TNF-α treatment.

Psychological stress may lead to erectile dysfunction (ED), and inflammation has been evaluated as a major contributing factor. The goal of this study was to investigate the effects of etanercept (ETN), an anti-tumor necrosis factor α (TNF-α) protein, on cavernosal function in the unpredictable chronic mild stress (UCMS) rat model of depression. Animals were divided into 4 groups: animals not exposed to UCMS, animals not exposed to UCMS and treated with ETN, animals exposed to UCMS, and animals treated with ETN while exposed to UCMS. UCMS significantly impaired the neurogenic and endothelium-dependent relaxation responses; reduced cavernosal endothelial nitric oxide (NO) synthase (eNOS) and neuronal NO synthase (nNOS) expressions; decreased testosterone levels; enhanced systemic levels of corticosterone, TNF-α, interleukin 1ß (IL-1ß), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1); and also increased cavernosal levels of TNF-α, IL-1ß, and IL-6 in rats. ETN administration restored NO-mediated neurogenic and endothelium-dependent relaxation responses of the corpus cavernosum, increased cavernosal eNOS and nNOS expressions, enhanced testosterone levels, and decreased corticosterone levels in UCMS-exposed rats. Also, systemic inflammatory markers and cavernosal proinflammatory cytokine levels were reduced by ETN. Our results demonstrate the role of TNF-α-mediated inflammation in the development of depression and ED in rats exposed to chronic stress.