Sphingosine-1-phosphate promotes the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase (eNOS) expressing endothelial-like cells.

BACKGROUND: Adipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability. RESULTS: ASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K. CONCLUSIONS: S1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks

GCM2 Silencing in Parathyroid Adenoma is associated with Promoter Hypermethylation and Gain of Methylation on Histone 3

PURPOSE: Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of parathyroid glands. We sought to identify if the epigenetic alterations in the GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. EXPERIMENTAL DESIGN: mRNA and protein expression of GCM2 were analyzed by RT-qPCR and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of GCM2 promoter were measured by bisulfite sequencing and ChIP-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in PTH-C1 cells by treating with 5-aza 2\u27deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. RESULTS: mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P\u3c0.001) and higher H3K9me3 levels in GCM2 promoter (P\u3c0.04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a non-significant change in GCM2 transcription. CONCLUSION: These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be promising avenues of research for parathyroid adenoma therapeutics

Heterogeneity in the radiosensitizing effects of the DNA ligand hoechst-33342 in human tumor cell lines.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I & II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 & U-87) and squamous carcinoma (4197 & 4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc).Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&7 fold increase in the cell death (loss of clonogenic cell survival) in U-87& BMG-1 and a 3 fold increase in 4197 &4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy

Abstract Background Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. Results Volume of spheroids, fed daily with fresh medium, increased exponentially during 7–28 days of growth through three population doublings. Proportion of G1-phase cells was higher (~60%) than exponentially growing monolayer cells (~48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2–3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1α was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (~2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. Conclusion Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.</p

A Rare Case of Necrotizing Myopathy and Fibrinous and Organizing Pneumonia with Anti-EJ Antisynthetase Syndrome and SSA Antibodies.

BACKGROUND Idiopathic inflammatory myopathies are autoimmune disorders that can involve the skin, joints, muscles, and lungs. The most common of these disorders are dermatomyositis, polymyositis, overlap syndrome, and inclusion body myositis. Necrotizing autoimmune myopathy is an idiopathic inflammatory myopathy that is rarely associated with Sjögren\u27s syndrome. The most common lung findings associated with anti-EJ antisynthetase syndrome are nonspecific interstitial pneumonia and usual interstitial pneumonia; this condition is rarely associated with fibrinous and organizing pneumonia. CASE REPORT Here, we present a rare case of necrotizing myopathy and fibrinous and organizing pneumonia in a 34-year-old African American man with Sjögren\u27s syndrome and anti-EJ antibodies. The patient\u27s presenting symptoms were cough and proximal muscle weakness of the extremities. He had elevated serum creatine kinase level, aldolase level, and erythrocyte sedimentation rate. Myositis panel was positive for anti-EJ antibodies. Chest radiography was consistent with bilateral interstitial infiltrates. CT chest showed patchy bilateral infiltrates. Quadriceps muscle biopsy revealed widespread necrotic fibers and lung biopsy showed fibrinous and organizing pneumonia. The patient responded well to immunoglobulin therapy, mycophenolate, and prednisone, which resulted in complete resolution of bilateral infiltrates and improved muscle pain and weakness. CONCLUSIONS Myopathies are characterized by myalgia and muscle weakness due to muscle fiber dysfunction and are associated with autoimmune diseases. Histopathological features may differ in idiopathic inflammatory myopathies. It is important to recognize the rare association of anti-EJ autoantibodies with necrotizing myopathy and interstitial lung disease, which responds well to methylprednisolone and intravenous immunoglobulin