Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

Levosimendan attenuates pulmonary vascular remodeling.

PURPOSE: The calcium-sensitizing drug levosimendan increases myocardial contractility and, by activating K(+)-channels, dilates pulmonary vessels. In the acute setting, levosimendan is clinically used to treat right heart failure in pulmonary hypertension. As K(+)-channel activation elicits several beneficial effects in the vascular system, we hypothesized that levosimendan also attenuates the remodeling process in the monocrotaline model of rat pulmonary hypertension. METHODS AND RESULTS: Animal subgroups received levosimendan, the K(+)-channel opener nicorandil, or levosimendan together with the K(+)-adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) blocker glibenclamide. Morphometric analyses revealed that levosimendan and nicorandil attenuated the increased pulmonary vascular medial wall thickness after monocrotaline challenge. Accordingly, in vivo BrdU assays revealed that levosimendan significantly diminished proliferation of pulmonary arterial smooth muscle cells (PASMCs), and this effect was attenuated by glibenclamide. Levosimendan also reduced right ventricular hypertrophy, but this effect was not glibenclamide sensitive and not recapitulated by nicorandil. In cell culture, levosimendan had a direct inhibitory effect on the platelet-derived growth factor (PDGF)-induced proliferation of PASMCs, which however required high concentrations of the compound, pointing towards an endothelial effect. Indeed, levosimendan increased cyclic guanosine monophosphate (cGMP) in human umbilical vein endothelial cells (HUVECs) and impaired the tumor necrosis factor-α (TNF-α)-induced inflammatory expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), and monocyte chemotactic protein-1 (MCP-1). In luciferase reporter gene assays in HUVECs, levosimendan dose-dependently attenuated the TNF-α-stimulated increase of proinflammatory transcription factors activator protein 1 (AP1), hypoxia-inducible factor-1α (HIF-1α), and nuclear factor-κB (NF-κB). CONCLUSIONS: Levosimendan attenuates pulmonary vascular remodeling, presumably by an antiproliferative and anti-inflammatory effect which is mediated by cellular hyperpolarization. The compound also has a direct inhibitory effect on cardiac hypertrophy, which is however K(+)-channel independent.

Decorin deficiency in diabetic mice: aggravation of nephropathy due to overexpression of profibrotic factors, enhanced apoptosis and mononuclear cell infiltration.

Although deficiency of the small leucine-rich proteoglycan decorin aggravates diabetic nephropathy in mice, the precise mechanisms of action are not fully understood. In the present study we used decorin-deficient mice (Dcn(-/-)) to further elucidate the molecular mechanisms involved in the protective action of decorin in diabetes. We discovered that streptozotocin-induced diabetes in Dcn(-/-) mice led to increased proteinuria associated with enhanced cyclin-dependent kinase inhibitor p27Kip1 in podocytes and tubular epithelial cells. Furthermore, lack of decorin increased the rate of apoptosis and caused overexpression of the IGF-IR in tubular epithelial cells of diabetic kidneys. In vitro experiments using human proximal renal epithelial cells showed that recombinant decorin was bound to the IGF-IR and protected against high glucose-mediated apoptosis. Furthermore, overexpression of TGFbeta1 and CTGF triggered by decorin deficiency resulted in enhanced accumulation of extracellular matrix in diabetic kidneys. Notably, diabetic Dcn(-/-) kidneys revealed marked upregulation of the proinflammatory proteoglycan biglycan and enhanced infiltration of mononuclear cells. Collectively, our results indicate that decorin is a protective agent during the development of diabetic nephropathy. Future therapeutic approaches that would either enhance the endogenous production of decorin or deliver recombinant decorin to the diseased kidney might improve the outcome of patients with diabetic nephropathy.

Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli.

AIMS/HYPOTHESIS: We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-beta1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. METHODS: THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/-THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. RESULTS: In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. CONCLUSIONS/INTERPRETATION: Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.