Post-ischemic Intravenous Administration of Allogeneic Dental Pulp-Derived Neurosphere Cells Ameliorated Outcomes of Severe Forebrain Ischemia in Rats.

BACKGROUND: Transplantation of bone marrow or adipose-derived mesenchymal stem cells (MSCs) for various neurological disorders has yielded promising results in models of focal cerebral ischemia. Dental pulp stem cells (DPSCs) are a type of MSC. In serum-free culture, they can form neurospheres that contain nestin-positive neuronal progenitor cells. We hypothesized that transplantation of dental pulp-derived neurosphere cells would ameliorate outcomes of global cerebral ischemia, the pathophysiology of which is known to resist conventional treatments. We also hypothesized that transplantation of dental pulp-derived cells would provide some neuroprotection in this pathology due to the presence of DPSCs. METHODS: Using adult rats, ischemia was induced by two-vessel occlusion of both carotid arteries in combination with systemic hypotension. Allogeneic dental pulp cells from juvenile rats were cultured in advance in serum-free medium to obtain neurospheres. Dental pulp-derived neurosphere cells or dental pulp-derived cells were intravenously administered at 3 h after ischemic insult, with normal saline as a control. Animals were observed for 14 days after ischemia. Neurological outcome was assessed using the water-maze test and neuromotor test. Histological outcome was measured by counting the percentage of dead neurons in the hippocampal CA1 and CA3 regions. RESULTS: Transplantation of both dental pulp-derived neurosphere cells and dental pulp-derived cells significantly improved survival rate and water-maze test results. Neurosphere cell transplantation was related to significantly better neuromotor test and histological outcomes, as indicated by the reduced percentage of dead neurons in CA1. CONCLUSIONS: Transplantation of dental pulp-derived neurosphere cells ameliorated outcomes of global cerebral ischemia. It was also demonstrated that dental pulp-derived cell administration provided some neuroprotection.

Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma.

Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ERbeta in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ERbeta isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription-polymerase chain reaction were used to evaluate methylation status and expression of ERbeta isoforms. The relative abundance of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ERbeta5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ERbeta gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5-aza-2' deoxycytidine resulted in reexpression of the ERbeta gene. The results of our present study suggest that ERbeta is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of epithelial ovarian cancer.

Decreased expression of RIZ1 and its clinicopathological significance in epithelial ovarian carcinoma: correlation with epigenetic inactivation by aberrant DNA methylation.

The retinoblastoma protein-interacting zinc finger gene (RIZ1) is considered a tumor suppressor gene. The purpose of the present study was to examine the expression of RIZ1 and evaluate its clinicopathological significance in ovarian carcinoma. Immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for RIZ1 and its clinicopathological significance was examined. DNA methylation status of RIZ1 was also studied. All (6/6) of the normal, 5/9 of benign, and 4/9 of borderline tissues were positive for RIZ1 protein. In ovarian cancer tissues 32.9% (54/164) were positive for RIZ1. Decreased expression of RIZ1 was significantly correlated with histological subtypes (P < 0.0001), high tumor grade (P = 0.0153) and advanced clinical stage (P = 0.0345), and high Ki67 index (P = 0.0117) but was not associated with the overall prognoses of the patients (P = 0.519). The presence of methylated band was detected in 2/9 cell lines, and 5/69 ovarian cancer tissues. Median values of relative RIZ1 expression in cell lines with methylation were significantly lower than those without methylation (P = 0.0404), and treatment of 5-aza-2'deoxycitidine resulted in demethylation and re-expression of RIZ1. Reduced expression of RIZ1 may play an important role in the pathogenesis and/or development of epithelial ovarian carcinoma, and is considered to be caused in part by aberrant DNA methylation.

MDM2: a novel mineralocorticoid-responsive gene involved in aldosterone-induced human vascular structural remodeling.

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.