title: Gene Expression Profiling In Rat Smooth Muscle Cells Modulated by Rapamycin and Paclitaxel.

status: Public on Dec 31 2006

city: Chapel Hill

first: Monte

last: Willis

address: 103 Mason Farm Road

department: Pathology & Laboratory Medicine

institute: UNC

summary:
Background: Drug-eluting stents (DES) have rapidly become a standard therapy for treatment of obstructive coronary artery disease. Differences exist in therapeutic responses to different DES formulations, and mechanisms to predict drug responses in the preclinical setting have not been standardized. Results: We have used gene expression profiling to characterize the patterns of gene regulation within cultures of rat aortic smooth muscle cells (RASMC) treated with rapamycin or paclitaxel, the two drugs widely used in commercially available DES. These studies further validate the use of the in vitro RASMC culture system as a model of insulin resistance. Gene expression profiles easily distinguish RASMC grown under normal or high glucose conditions, and we therefore followed this approach in order to understand the activity of these drugs under conditions that approximate those seen in type 2 diabetics. Remarkably, although both drugs are used to arrest smooth muscle proliferation when delivered by DES, there were major differences in gene regulatory responses. Paclitaxel caused marked changes in expression of tubulin-related genes, and also caused striking changes in the transcription of VEGF, PDGF, JAG-1 and their respective receptors, suggesting an important effect on paracrine and autocrine response to mitogens. However, the gene expression signatures elicited by paclitaxel showed little variation under different cell culture conditions. In contrast, these gene expression responses to rapamycin varied considerably depending on the glycemic conditions of culture, and rapamycin had a dramatic dose- and metabolic status- dependent effect on the transcription of key members of the AKT signaling axis, providing a transcriptional explanation for the paradoxical proliferative effect of rapamycin at low dose in the setting of high glucose concentrations and insulin resistance. Conclusions: Gene expression signatures for drugs eluted from coronary stents vary dramatically in ways that correlate with known differences in biological activities of these drugs. Gene expression profiling may provide a useful preclinical method to characterize the activities of candidate drugs for stent impregnation and to understand their biological activities.
Keywords: Cardiovascular disease, drug eluting stent, vascular smooth muscle, rapamycin, paclitaxel

overall_design:
Reagents: Paclitaxel, rapamycin, insulin, dimethyl sulfoxide and glucose were obtained from Sigma-Aldrich (St. Loius, MO, USA). Dulbecco’s Minimum Essential Medium (DMEM), Trypsin/ EDTA, antibiotics/antimycotics, tissue culture grade phosphate-buffered saline (PBS) and trypan blue were obtained from GibCO (Grand Island, NY, USA). Filtered, mycoplasma-/endotoxin-free fetal bovine serum (FBS) was purchased from Gemini Bioproducts (Woodland, CA, USA). RNeasy Mini Kits and Rnase-free DNase I were obtained from Qiagen Sciences (Maryland, USA). Microarray slides, hybridization chambers, hybridization buffer, Low Input Linear RNA Amplification/Labeling kits and free-radical scavenging wash buffer were obtained from Agilent Technologies (Palo Alto, CA, USA). Cyanine-5 and cyanine-3 CTP was purchased from Perkin Elmer/NEN (Wellesley, MA, USA). 20X standard saline citrate (SSC) buffer was obtained from the Promega Corporation (Madison, WI, USA). All other salts, buffers and detergents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture: RASMC were explanted from aortas of 1 day old rat pups, purified and cultured essentially as previously described (10) Patterson (9). RASMC were maintained in DMEM containing 5 mM glucose, 10% FBS and an antibiotic/antimycotic cocktail (penicillin 100 IU/ml; streptomycin, 100 μg/ml; and amphotericin-B, 2 μg/ml) at 37°C in a 5% CO2 /100% humidity atmosphere. RASMC were used between passage level 8 and 11. Cells were plated into 60mm polystyrene dishes (Falcon cat num 35100) and grown to 85% confluence. Dishes of RASMC were quiessed for 72 hrs prior to stimulation or drug treatment by reduction of the FBS concentration to 0.1%. Normo- or hyper-glycemic conditions (5 or 25 mM glucose, respectively) were established in the final 24 hrs of quiessesence by re-feeding with medium containing 5mM or 25mM glucose (respectively) supplemented with 0.1% FBS and antibiotic/antimycotic cocktail. RASMC were stimulated with 100nM insulin (or PBS vehicle) for 30 min at 37°C prior to drug treatment. Un-stimulated and insulin stimulated cells grown under normal and high glucose conditions were treated with paclitaxel (0.01ng/mL, 1ng/mL, or 1000ng/mL final concentration), rapamycin (0.01ng/mL, 1ng/mL or 100ng/mL final concentration), or vehicle alone (dimethylsulfoxide, DMSO) for either 6 or 24 hours at 37°C prior to harvesting RNA.

RNA extraction and microarray analysis: Total RNA was extracted from RASMC using the Qiagen RNeasy Minikit in accordance with the manufacturer’s instructions. RNA integrity was verified by assay on an Agilent BioAnalyzer 2100. Five hundred nanograms of RASMC total RNA was labeled with Cyanine-5 CTP in a T-7 transcription reaction using the Agilent Low Input Linear RNA Amplification/Labeling System. Labeled cRNA from test samples was hybridized to Agilent G4130A Rat 22K microarray slides in the presence of equimolar concentrations of Cyanine-3 CTP labeled rat reference RNA prepared from pools of 1 day old rat pups(11).

Statistical Methods: Microarray data (N=224 arrays) were loess normalized and genes were filtered for features having a normalized intensity of < 30 aFU in either channel and for <70% good data across the entire dataset. Missing data points were imputed using the k nearest-neighbors algorithm (three nearest neighbors). Principal component analysis (PCA) was used to identify eigenvectors in the data matrices that contributed to differences between the four replicate runs that were unrelated to the biological conditions under study (13), and were removed from the dataset using scripts written in the R Statistical Language and Environment (“R”; Version 2.2.1, build r36812, release date 2005-12-20. See supplemental materials for R scripts). The dataset was standardized with a custom Perl script (ActiveState Perl 5.8.1, build 807, release date 2003-11-6. See supplemental materials for Perl scripts).

Lists of differentially expressed genes were identified using the statistical analysis of microarray algorithm (14-16) (“SAM”, Version 2.21, release date 2005-8-24. Typical false discovery rate of < 5%), two-tailed heteroscedastic T-Test (significance threshold of p<0.05), and custom R scripts written in our laboratory (see supplemental materials). Unsupervised, semi-supervised and supervised clustering analysis was performed on genelists essentially as described (17, 18) using Cluster (Version 2.11, http://rana.lbl.gov/EisenSoftware.htm). Heatmaps of cluster analyses were visualized with JavaTreeView (Version 1.0.12, release date 2005-3-14; http://sourceforge.net/projects/jtreeview/).

High-level pathway analysis and mapping to gene ontology (“GO”, http://www.geneontology.org/) categories were performed on genelists using the Expression Analysis Systematic Explorer (“EASE” Version 1.21, released date 2003-6-9; http://david.niaid.nih.gov/david/ease.htm) and PathArtTM (Jubilant Biosys, Version 2005 R3) analysis environments. The significance analysis of function and expression (“SAFE”(4)) algorithm was used for ab initio statistical analysis of the GO categories present within our dataset (Wilcoxon statistic significance threshold of p<0.05). The SAFE technique was extended to allow exporting of genelists and ontologies for heatmap visualizations of the expression patterns of genes present within the GO categories using customizations to the SAFE R scripting and Perl scripts (see supplemental materials).

first: Peter

last: Charles

first: Sabeen

last: Mapara

first: Joel

last: Parker

first: Robert

last: Herrmann

first: Cam

last: Patterson


title: Expression data from human fibroblasts 6 and 12 hours after 8-oxoguanine treatment

status: Public on Jan 27 2011

city: Galveston

first: Istvan

last: Boldogh

state: TX

address: 301 University Blvd.

country: USA

institute: University of Texas Medical Branch at Galveston

summary:
8-oxoguanine is excised from DNA by 8-oxoguanine DNA glycosylase-1 during DNA base excision repair.
We used microarrays to gain insight into the potential biological function of the excised, free 8-oxoguanine base.

overall_design:
MRC5, human diploid fibroblasts were exposed to 8-oxoguanine and changes in gene expression compared to mock control were analyzed 6 hours after exposure.
MRC5, human diploid fibroblasts were exposed to 8-oxoguanine and changes in gene expression compared to mock control were analyzed 12 hours after exposure.

last: German

first: Gyorgy

last: Hajas

last: Szaniszlo


title: Campylobacter jejuni gene expression in germ-free mice

status: Public on Apr 14 2011

city: East Lansing

first: Linda

middle: S.

last: Mansfield

state: MI

address: 181 Food Safety Bldg.

department: Microbiology and Molecular Genetics

institute: Michigan State University

summary: Expression arrays comparing Campylobacter jejuni NCTC11168 during growth in the cecum of germ-free C57 BL/6 IL-10 knockout mice to C. jejuni NCTC11168 during growth in Bolton broth.

sample_organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819

overall_design: Four biological replicates comparing C. jejuni NCTC11168 growth in vivo to in vitro. Two biological replicates were dye swaps.

first: John

middle: P

last: Jerome

first: Anne

middle: E

last: Plovanich-Jones

first: Julia

middle: A

last: Bell

middle: S


title: Campylobacter jejuni comparative genome: control strain C. jejuni subsp. jejuni ATCC 700819 (NCTC 11168) vs. 12 other strains

status: Public on Oct 21 2010

summary:
Gene content comparison of control C. jejuni subsp. jejuni strain 11168 which colonizes and causes disease in C57BL/6 IL-10-/- mice versus C. jejuni strains D6844, D6845, D6846, D6847, D6848, D6849, D0121, D0835, D2586, D2600,33560 and NW in the C57BL/6 IL-10-/- mice.

Keywords: DNA/DNA comparison

overall_design: Two genome comparison of disease strain versus non disease strain of C.j., 4 Biological replicates - 2 of which were dye swaps


title: Muscle gene expression patterns in pigs with divergent phenotypes for fatness traits

status: Public on Mar 01 2010

city: Lleida

first: Romi

middle: N

last: Pena

address: 191 Av Alcalde Rovira Roure

department: Genètica i Millora Animal

country: Spain

institute: IRTA

summary: Background: Marketing products with added-value characteristics is a current trend in livestock production systems. Regarding meat, selection for intramuscular fat and muscular fatty acid composition is a way to improve the palatability and juiciness of meat while assuring a healthy fat content. This represents selecting animal with a different muscular metabolic profile with respect to the extended selection of lean animals.   Results: The present study has analysed the muscular gene expression profiles of 68 commercial Duroc pigs belonging to two groups with extreme phenotypes for traits strongly related with lipid deposition and composition. This has allowed us to compare the physiological and metabolic implications of selecting for each of these extreme groups. Rather than upregulation of a single pathway, the main differences lied on the transcriptional levels of genes related with lipogenesis and lipolysis, revealing the existence of a cycle where triacylglycerols are continuously synthesized and degraded. Most strikingly, several genes which enhanced fatty acid β-oxidation and favoured insulin signalling and glucose uptake were upregulated in the fattest animals, indicating that the events leading to peripheral insulin resistance in humans with increased levels of intramuscular fat and obesity do not take place in these pigs. Moreover, neither was detected the well-characterised low-grade inflammatory state observed in overweighed humans.   Conclusion: As a whole, our data suggest that selection for increasing intramuscular fat content in pigs would lead to a shift but not a disruption of the metabolic homeostasis of muscle cells. Future studies on the post-translational changes affecting protein activity or expression as well as information about protein location within the cell would be needed to fully understand how lipid deposition affects muscle physiology in pigs.

overall_design: 68 gluteus medius samples form 68 animals belonging to two groups of 34 animals each: HIGH group had higher carcass, plasma and muscle fat content; LOW group had lower carcass, plasma and muscle fat content

first: A

last: Cánovas

first: R

last: Quintanilla

first: M

last: Amills


title: Transcriptional profile of Sacchromyces cerevisiae in different levels of oxygen provision

status: Public on Oct 05 2009

city: VTT

first: Eija

last: Rintala

country: Finland

institute: VTT Technical Research Centre of Finland

summary: Saccharomyces cerevisiae CEN.PK113-1A was grown in glucose-limited chemostat culture with 0%, 0.5%, 1.0%, 2.8% or 20.9% O2 in the inlet gas (D= 0.10 /h, pH5, 30C).

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: Four (0,0.5,2.8 and 20.9% oxygen) or seven samples (1% oxygen) were analysed. Samples were either from four (0,1,20.9% oxygen) or two independent cultivations (0.5, 2.8% oxygen). One sample (VTT_Ys23_13_1%) was found to be an outlier and was removed from the analysis.


title: Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease

status: Public on Nov 30 2009

city: Bari

first: Paola

last: Pontrelli

address: Piazza G.Cesare 11

department: Dept. of Precision and Regenerative Medicine and Ionian Area (DIMEPRE-J)

country: Italy

institute: University of Bari

summary: Several reports have focused on the identification of biological elements involved in the development of abnormal systemic biochemical alterations in chronic kidney disease, but this abundant literature results most of the time fragmented. To better define the cellular machinery associated to this condition, we employed an innovative high-throughput approach based on a whole transcriptomic analysis and classical biomolecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both chronic kidney disease patients in conservative treatment (CKD, n=9) and hemodialysis (HD, n=17) compared to healthy subjects (NORM) (p<0.001, FDR=1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system (OXPHOS). Western blotting for COXI and COXIV, key constituents of the complex IV of OXPHOS, performed on an independent testing-group (12 NORM, 10 CKD and 14 HD) confirmed the elevated synthesis of these subunits in CKD/HD patients. However, complex IV activity was significantly reduced in CKD/HD patients compared to NORM (p<0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to NORM. Taken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress.

overall_design: For microarray analysis, we studied subjects included in the training group. This population included 8 healthy subjects (NORM), 9 patients with Chronic kidney disease (CKD) on stage II-III (CKD II-III) (mean±SD of estimated GFR by MDRD formula: 41.4±4.3 ml/min) and 17 patients undergoing hemodialysis treatment (HD).All HD patients were stably treated, for at least 1 year, three times a week (4-5 hours per session), using synthetic membrane dialyzers. During the study period, no CKD patients received dialysis treatment. In addition, all patients suffering from infectious diseases, diabetes, chronic lung diseases, neoplasm, or inflammatory diseases and patients receiving antibiotics, corticosteroids, or nonsteroidal anti-inflammatory agents were excluded. No patients had symptomatic coronary artery diseases or a family history of premature cardiovascular diseases. For all subjects, 20 ml of whole blood were collected. For HD patient the biological material was obtained at the beginning of the second HD session of the week. PBMC were isolated by density separation over a Ficoll-PaqueTM (GE healthcare, Sweden) gradient (460g for 30 min). Cells were then counted and their viability was assessed by trypan blue exclusion (>90% PBMC were viable). Total RNA was isolated by RNeasy mini kit Qiagen (QIAGEN AG, Basel, Switzerland) from a minimum of 5 milion cryopreserved PBMC. RNA was, then, processed and hybridized to the GeneChip Human Genome Plus 2.0 or U133A oligonucleotide microarray (Affymetrix, Santa Clara, CA, USA)