Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse primary aortic endothelial cells during expression of dominant negative PPAR gamma (V290M).

ABSTRACT: Ligand-mediated activation of the nuclear hormone receptor PPAR gamma lowers blood pressure and improves glucose tolerance in humans. Two naturally occurring mutations (P467L, V290M) in the ligand binding domain of PPAR gamma have been described in humans that lead to severe insulin resistance and hypertension. Experimental evidence suggests that these mutant versions of PPAR gamma act in a dominant negative fashion. To better understand the molecular mechanisms underlying PPAR gamma action in the vasculature, we determined the global gene expression profile in primary aortic endothelial cells in response to endothelial cell specific expression of a dominant negative isoform of PPAR gamma (V290M). Experiment Overall Design: We generated transgenic mice specifically targeting expression of dominant negative human PPAR gamma to the endothelium using an endothelial-specific promoter (vascular endothelial cadherin or CDH5). Primary aortic endothelial cells were isolated from 10 non-transgenic and 10 EC-DN mice by Dominion Pharmakine (http://www.pharmakine.com/). For each group, 5 cultures of cells were established. Each culture was derived from 2 mice and remained separate from the other cultures. Cellular RNA was prepared using conventional methods and quality was assessed using the Bioanalyzer 2100 (Agilent Technologies). For the microarray hybridizations, RNA from 3 of the cultures in each group was used. All the microarray procedures were conducted at the University of Iowa DNA Core facility using standard Affymetrix protocols. In brief, approximately 50 ng of total RNA was used as input to a two-step amplification procedure (NuGen, http://www.nugeninc.com/) to generate biotin-labeled RNA fragments for hybridization to the Affymetrix GeneChip Mouse Genome 430 2.0 array.

ORGANISM(S): Mus musculus

SUBMITTER: Henry Keen

PROVIDER: E-GEOD-11870 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pharmacological activation of the transcription factor PPAR gamma lowers blood pressure and improves glucose tolerance in humans. In contrast, naturally occurring mutations (e.g., P467L, V290M) in the ligand binding domain of PPAR gamma in humans leads to severe insulin resistance and early-onset hypertension. Experimental evidence, including whole genome expression profiling, suggests that these mutant versions of PPAR gamma act in a dominant negative manner. Because PPAR gamma is expressed in a variety of cell types and tissues, we generated a transgenic mouse model (SP467L) specifically targeting dominant negative PPAR gamma to the vascular smooth muscle cells in order to determine the action of PPAR gamma in the blood vessel independent of its systemic metabolic actions. In the data set provided herein, we examined the gene expression profile in thoracic aorta from SP467L mice and their control littermates using the Affymetrix Mouse Genome 430 2.0 array. We generated transgenic mice specifically targeting expression of mutant dominant negative human PPAR gamma (P467L) to vascular smooth muscle using a smooth muscle-specific promoter (smooth muscle myosin heavy chain or SMMHC). Thoracic aortas were isolated from male transgenic mice and corresponding non-transgenic littermate controls. Total RNA was prepared using conventional methods and quality was assessed using the Bioanalyzer 2100 (Agilent Technologies). For the microarray hybridizations, 2 samples from control mice and 3 samples from transgenic mice were used. Each sample was an independent biological replicate generated by pooling total RNA from 6-8 separate mouse aortas. All procedures were conducted at the University of Iowa DNA Core facility using standard Affymetrix protocols. In brief, approximately 3-5 ug of total RNA was used as input to a one-step amplification procedure to generate biotin-labeled RNA fragments for hybridization to the Affymetrix Mouse Genome 430 2.0 array.

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Transcription profiling of mouse primary aortic endothelial cells during expression of dominant negative PPAR gamma (V290M).

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