Comment[ArrayExpressAccession]	E-GEOD-37127
MAGE-TAB Version	1.1						
Public Release Date	2013-05-01
Investigation Title	Positive and Negative Spatial Gradients of High Wall Shear Stress Have Different Effects on Endothelial Gene Expression						
Comment[Submitted Name]	Positive and Negative Spatial Gradients of High Wall Shear Stress Have Different Effects on Endothelial Gene Expression
Experiment Description	Intracranial aneurysms tend to form at bifurcation apices, where flow impingement causes high frictional force (or wall shear stress, WSS) and flow acceleration and deceleration that create positive and negative streamwise gradients in WSS (WSSG), respectively. In vivo, intracranial aneurysms initiate under high WSS and positive WSSG. Little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile EC gene expression exposed to positive WSSG vs. negative WSSG for 24 hours in a flow chamber with converging and diverging channels, respectively.  WSS varied between 3.5 and 28.4 Pa in each gradient channel. GO and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. A subset of characteristic genes was validated using qPCR: Genes for ADAMTS1, CKAP2 and NCEH1 had higher expression under positive WSSG compared to negative WSSG while TAGLN, THBS1, VCAM1, CCL2, and CSF2 had lower expression. To determine if these patterns of expression are also exhibited in vivo, we tested whether the extracellular matrix related protein ADAMTS1 and proliferation were modulated by positive WSSG during intracranial aneurysm initiation. An aneurysm was induced at the basiliar terminus in rabbits by bilateral carotid ligation. WSSG at the bifurcation was determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for ADAMTS1 and the proliferation marker, Ki-67. Endothelial ADAMTS1 protein and Ki-67 were significantly higher in regions with positive WSSG compared to adjacent sites where WSSG was negative. Our results indicate that WSSG can elicit distinct gene expression profiles in ECs.  Increased matrix processing and high levels of proliferation under positive WSSG could contribute to intracranial aneurysm initiation by causing transient gaps in the endothelium or disrupting EC signals to smooth muscle cells.   Time-matched bovine aortic endothelial cells were exposed to positive wall shear stress gradient, negative wall shear stress gradient, and two no gradient samples: uniform WSS of 3.5 Pa and high WSS of 28.4 Pa for 24 hrs in an in vitro flow loop system. RNA was extracted and hybridized on Affymetrix microarrays. There were 12 samples in total, four flow conditions with three replicates each.						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Dolan	Dolan	Meng	Sim	Kolega		
Person First Name	Jennifer	Jennifer	Hui	Fraser	John		
Person Mid Initials		M		J			
Person Email	jdolan@buffalo.edu						
Person Affiliation	University at Buffalo						
Person Address	Neuroscience, University at Buffalo, 3435 Main Street, Buffalo, NY, USA						
Person Roles	submitter						
Protocol Name	P-GSE37127-1	P-GSE37127-5	P-GSE37127-6	P-GSE37127-2	P-GSE37127-3	P-GSE37127-4	P-GSE37127-7
Protocol Description	All analyses were performed in R/Bioconductor. Affymetrix bovine CEL files were preprocessed and normalized using RMA [see supplementary file on Series record]. Informative, valid probe sets were determined using FARMS (Talloen et al., 2007, Bioinformatics 23(21):2897-2902). FARMS represents an unbiased approach to remove non-informative probe sets based on their intra-probe set correlation. Sample to sample data exploration was performed using unsupervised hierarchial clustering and principle component analysis. Differential gene expression analysis was performed using a linear model approach and employing an empirical Bayes method for calculation of statistical significance (Bioconductor,limma package). We identified those probe sets whose expression were significantly enriched or depleted by at least two fold change and statistically significant following 5% FDR adjustment of p-values. ID_REF =  VALUE = Log base 2 RMA signal, FARMS-filtered	Conducted using Affymetrix standard protocols.	Standard protocol described in Affymetrix GeneChip 3' IVT Express Kit.	24 hrs before the flow experiments started culture media was changed and cells were allowed to equilibrate in flow media containing DMEM, 5% FBS, 1% pen/strep, and 8% Dextran 70. For treatment, cells were subjected to either positive wall shear stress gradient or a negative wall shear stress gradient by placement in a flow loop system in which media was circulated over the cells to produce the mentioned flow conditions. After 24 hours the experiment was ended and coverslips of cells removed from the chamber.	Bovine aortic endothelial cells were seeded on glass coverslips and allowed to grow to confluence for 5 days. Cells were maintained in DMEM with 10% FBS and 1% penicilin/streptomycin.	Total RNA was extracted following Qiagen RNeasy Mini Kit protocol using spin column technology. Purity and integrity were confirmed.	Affymetrix GeneChip Scanner and software.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	STRESS						
Experimental Factor Type	stress						
Comment[SecondaryAccession]	GSE37127						
Comment[GEOReleaseDate]	2013-05-01						
Comment[ArrayExpressSubmissionDate]	2012-04-09						
Comment[GEOLastUpdateDate]	2013-05-02						
Comment[AEExperimentType]	transcription profiling by array						
Comment[AdditionalFile:Data1]	GSE37127_WSSG_Matrix_following_RMA.txt						
SDRF File	E-GEOD-37127.sdrf.txt