Restoring Perivascular Adipose Tissue Function in Obesity Using Exercise.

PURPOSE: Perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating vascular tone. This effect is mediated via sympathetic nervous stimulation of PVAT by a mechanism which involves noradrenaline uptake through organic cation transporter 3 (OCT3) and ß3-adrenoceptor-mediated adiponectin release. In obesity, autonomic dysfunction occurs, which may result in a loss of PVAT function and subsequent vascular disease. Accordingly, we have investigated abnormalities in obese PVAT, and the potential for exercise in restoring function. METHODS: Vascular contractility to electrical field stimulation (EFS) was assessed ex vivo in the presence of pharmacological tools in ±PVAT vessels from obese and exercised obese mice. Immunohistochemistry was used to detect changes in expression of ß3-adrenoceptors, OCT3 and tumour necrosis factor-α (TNFα) in PVAT. RESULTS: High fat feeding induced hypertension, hyperglycaemia, and hyperinsulinaemia, which was reversed using exercise, independent of weight loss. Obesity induced a loss of the PVAT anti-contractile effect, which could not be restored via ß3-adrenoceptor activation. Moreover, adiponectin no longer exerts vasodilation. Additionally, exercise reversed PVAT dysfunction in obesity by reducing inflammation of PVAT and increasing ß3-adrenoceptor and OCT3 expression, which were downregulated in obesity. Furthermore, the vasodilator effects of adiponectin were restored. CONCLUSION: Loss of neutrally mediated PVAT anti-contractile function in obesity will contribute to the development of hypertension and type II diabetes. Exercise training will restore function and treat the vascular complications of obesity.

Effects of Apatinib on the "Stemness" of Non-Small-Cell Lung Cancer Cells In Vivo and Its Related Mechanisms.

Objective: To investigate the effects of Apatinib on the "stemness" of lung cancer cells in vivo and to explore its related mechanisms. Methods: A xenograft model of lung cancer cells A549 was established in nude mice and randomized into a control group (n = 4) and an Apatinib group (n = 4). Tumor tissues were harvested after 2 weeks, and mRNA was extracted to detect changes in stemness-related genes (CD133, EPCAM, CD13, CD90, ALDH1, CD44, CD45, SOX2, NANOG, and OCT4) and Wnt/ß-catenin, Hedgehog, and Hippo signal pathways. Results: Compared with the control group, the volume and weight of nude mice treated with Apatinib were different and had statistical significance. Apatinib inhibited the expressions of ABCG2, CD24, ICAM-1, OCT4, and SOX2 and upregulated the expressions of CD44, CD13, and FOXD3. Apatinib treatment also inhibited the Wnt/ß-catenin, Hedgehog, and Hippo signaling pathways. Conclusion: Apatinib suppressed the growth of non-small-cell lung cancer cells by repressing the stemness of lung cancer through the inhibition of the Hedgehog, Hippo, and Wnt signaling pathways.

Inhibition of DNA Methyltransferase by RG108 Promotes Pluripotency-Related Character of Porcine Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) have been identified in almost all adult human tissues and been used in numerous clinical trials for a variety of diseases. Studies have shown that MSCs would undergo cellular senescence when cultured over a long term, which is brought on by increased epigenetic modifications, including DNA methylation. However, the mechanism of MSCs senescence is not well studied. In this study, the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on senescence, apoptosis, and pluripotency gene expressions in porcine bone marrow (pBM)-MSCs were investigated. First, we determined the optimized dose and time of RG108 treatment in pBM-MSCs to be 10 µM for 48 hours, respectively. Under these conditions, the pluripotency genes (NANOG, POU5F1), the anti-senescence genes (TERT, bFGF), and the anti-apoptosis gene (BCL2) were increased, whereas the apoptotic gene (BAX) was decreased. RG108 protected against apoptosis when pBM-MSC induces apoptosis with H2O2 for 1.5 hours. We also found that RG108 significantly induced the expression of NANOG and POU5F1 by decreasing DNA methylation in gene promoter regions. These results indicate that an optimized dose of RG108 may promote the pluripotency-related character of pBM-MSCs through improving cellular anti-senescence, anti-apoptosis, and pluripotency, which provide a better cell origin for stem cell therapy.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

OBJECTIVE: Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. DESIGN: We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. RESULTS: HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40µM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44+ cells, CD105+ cells, and STRO-1+ cells was significantly abolished by apigenin. CONCLUSION: Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia.

Comparison of colony formation of human spermatogonial stem cells (SSCs) with and without collagen.

OBJECTIVE: To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia. METHODS: The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks. RESULTS: Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups. CONCLUSIONS: According to positive effects of collagen and growth factors on the colonisation of spermatogonial stem cells, it seems that using the cells may lead to better colonisation of this type of stem cells.

Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer worldwide. Cancer stem cells (CSC) model theoretically contribute to tumor growth, metastasis, and chemo-radioresistance. Cisplatin is a widely used chemotherapeutic agent for OSCC treatment. The aim of this study was to compare stemness genes expression in chemo-sensitive and chemo-resistant specimens and further explore the potential markers that may lead to induce chemo-resistance in OSCC. METHODS: The study method is the treatment of OC2 cells with cisplatin select cisplatin-resistant OC2 cells. Self-renewal ability was evaluated by cultivating parental and cisplatin-resistant OC2 cells within sphere-forming assay after serial passages. Differential expression profile of stemness markers between parental and cisplatin-resistant OC2 cells was elucidated. The parental and cisplatin-resistant OC2 cells were assessed for migration/invasion/clonogenicity tumorigenic properties in vitro. Expression of stemness markers in chemo-sensitive and chemo-resistant patients with OSCC was performed by immunohistochemistry staining in vivo. RESULTS: Sphere-forming/self-renewal capability was increased in cisplatin-resistant OC2 cells. Cisplatin-resistant OC2 cells highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2). Furthermore, cisplatin-resistant OC2 cells increased migration/invasion/clonogenicity ability. Notably, up-regulation of Oct4 and Nanog expression was significantly observed in cisplatin-resistant patients with OSCC (**P < 0.01). CONCLUSIONS: These data indicate that cancer stem-like properties were expanded during the acquisition of cisplatin resistance in OSCC. Clinically, oral cancer stemness markers (Oct4 and Nanog) overexpression may promote the OSCC's recurrence to resist cisplatin.

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation.

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.