Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from human umbilical vein endothelial cells (HUVEC) treated with DMSO, telmisartan, or telmisartan and amlodipine

ABSTRACT: Objective M-bM-^@M-^S Telmisartan, an angiotensin II type 1 (AT1) receptor blocker, and amlodipine, a calcium channel blocker, are antihypertensive agents clinically used as monotherapy or in combination. They exert beneficial cardiovascular effects independently of blood pressure lowering and classic mechanisms of action. In this study, we investigate molecular mechanisms responsible for the off-target effects of telmisartan and telmisartan-amlodipine in endothelial cells (EC), using an unbiased approach. Approach and Results M-bM-^@M-^S In human umbilical vein endothelial cells, microarray analysis of gene expression followed by pathway enrichment analysis and qRT-PCR validation revealed that telmisartan modulates the expression of key genes responsible for cell cycle progression and apoptosis. AmlodipineM-bM-^@M-^Ys effect was similar to control. EC exposed to telmisartan, but not amlodipine, losartan or valsartan, exhibited a dose-dependent impairment of cell growth and failed to enter the S-phase of the cell cycle. Similarly, telmisartan inhibited proliferation in COS-7 cells lacking the AT1 receptor. In telmisartan-treated EC, phosphorylation and activation of Akt as well as MDM2 was reduced, leading to accumulation of p53 in the nucleus, where it represses the transcription of cell cycle promoting genes. Phosphorylation of GSK3M-NM-2 was also reduced, resulting in rapid proteolytic turnover of CyclinD1. Telmisartan induced downregulation of proapoptotic genes and protected EC from serum starvation- and 7-ketocholesterol-induced apoptosis. Conclusions M-bM-^@M-^S Telmisartan exerts antiproliferative and antiapoptotic effects in EC. This may account for the improved endothelial dysfunction observed in the clinical setting. HUVEC were treated with 100 M-BM-5mol/L telmisartan or 5 M-BM-5mol/L amlodipine or both in dimethyl sulfoxide (DMSO) or an equivalent volume of DMSO alone, as control, for 24 hours. Microarray analysis was carried out on two biological replicates per group (total of six samples). See PROTOCOL section for experimental details.

ORGANISM(S): Homo sapiens

SUBMITTER: Mauro Siragusa

PROVIDER: E-GEOD-42808 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Studies of human embryonic stem cells (hESCs) commonly describe the non-functional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of microRNAs, including hESC-specific microRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC microRNA cluster. Most importantly, we show that the hESC-enriched microRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and thus demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of microRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. Karyotypically normal hESC lines CCTL-14 (46, XX, at passages 20M-bM-^@M-^S30 and 242M-bM-^@M-^S250, respectively) was used in this study (for detailed characterization of hESC lines see International Stem Cell Registry: http://www.iscr-admin). For UVC irradiation, culture media were aspirated and cells were washed once with phosphate-buffered saline (PBS) without Mg2+ and Ca2+ (pH 7.4). For each time point, nine culture dishes were independently irradiated with a dose of 3 J/m2. Cells were then further incubated in culture media (37M-BM-0C/5% CO2) until they were harvested at 4, 8, and 16 hrs, respectively, post-UVC treatment. Differentiation and culture of NPCs derived from hESCs To induce neural differentiation, we utilized a protocol based on embryoid body (EB) formation with simultaneous all-trans-retinoic acid (RA) treatment. Colonies of hESCs were transferred to a non-adherent cell culture dish in the presence of differentiation media [DMEM/F12:Neurobasal 1:1, N2, B27 (Invitrogen), 2 mmol/l L-Glutamine (all media components from Invitrogen, Carlsbad, CA, USA), 1M-CM-^W MEM non-essential amino acids, 0.5% penicillin-streptomycin, 100 M-BM-5mol/l M-NM-2-2 mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml FGF-2 (PeproTech, Rocky Hill, NJ, USA)]. RA was added (Sigma-Aldrich, concentration 0.5 M-BM-5mol/l) into fresh differentiation media on days 6, 10, and 14 after the beginning of EB development. On day 16, cells were dissociated using Accutase (Sigma-Aldrich) and plated on a gelatin-coated cell culture dish in maintenance media (DMEM/F12, N2, B27, L-Glutamine, MEM non-essential amino acids, 0.5% penicillin-streptomycin, 100 M-BM-5mol/l M-NM-2-2 mercaptoethanol, and 20 ng/ml FGF-2). Cells were passaged twice and subsequently harvested for RNA isolation.

Project description:Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor antagonist and a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, reportedly has beneficial effects on insulin sensitivity. We studied the effects of telmisartan on the adipose tissue macrophage phenotype in high fat-fed mice. Telmisartan was administered for 5 weeks to high fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in epididymal fat tissues were examined. Insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight and adipocyte size without affecting the amount of food intake. Telmisartan treatment reduced the number of CD11c-positive cells and crown-like structures. Telmisartan reduced the mRNA expressions of M1 macrophage markers, such as TNF-alpha and IL-6, and increased the expression of M2 markers, such as IL-10 and Mgl2. The reduction of M1 macrophage markers, as well as the increased gene expression of M2 markers especially IL-10, is a possible mechanism for the improvement of insulin sensitivity by telmisartan. Six-week-old male C57BL/6J mice were purchased from CLEA Japan. The mice were fed a chow that contained 10% of its calories from fat (control) or a high-fat diet (HFD) that contained 30% of its calories from fat for 24 weeks. The high fat-fed mice were randomized to 3 groups. Either telmisartan (~3 mg/kg/day) in drinking water (HFD+Tel), candesartan (~3 mg/kg/day) in drinking water (HFD+Can), or a HFD without any drugs (HFD) was administered for the next 5 weeks. Two mice were treated per group. Epididymal adipose tissues were rapidly removed from each mouse. Gene expression in epididymal fat tissue was analyzed using a GeneChip® system with the Mouse Genome 430 2.0 Array, which was spotted with 45,101 probe sets (Affymetrix, Santa Clara, CA, USA). Sample preparation for the array hybridization was performed according to the manufacturer’s instructions. In short, 5 μg of total RNA was used to synthesize double-stranded cDNA using the GeneChip® Expression 3′-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix). Biotin-labeled cRNA was then synthesized from the cDNA using GeneChip® Expression 3′-Amplification Reagents for IVT Labeling (Affymetrix). After fragmentation, the biotinylated cRNA was hybridized to arrays at 45 °C for 16 h. The arrays were washed, stained with streptavidin-phycoerythrin, and scanned using a probe array scanner. The scanned chip was analyzed using the GeneChip Analysis Suite software (Affymetrix). Hybridization intensity data were converted into a presence/absence call for each gene, and changes in gene expression between experiments were detected by a comparison analysis. Data was shown as the fold change relative to the expression level of normal chow-fed mice.

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Expression data from human umbilical vein endothelial cells (HUVEC) treated with DMSO, telmisartan, or telmisartan and amlodipine

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