Gene expression changes in primary aortic endothelial cells during expression of dominant negative PPAR gamma (V290M).

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). 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.

配体介导的核激素受体PPARγ（PPAR gamma）激活可降低人类血压并改善糖耐量。人类中已报道过PPARγ配体结合域内的两种自然发生突变（P467L、V290M），可导致严重胰岛素抵抗与高血压。实验证据表明，这些突变型PPARγ以显性负调控方式发挥作用。为深入阐明PPARγ在血管系统中的作用分子机制，本研究针对原代主动脉内皮细胞过表达显性负调控型PPARγ（V290M）的响应情况，测定了其全基因表达谱。本研究利用内皮细胞特异性启动子（血管内皮钙粘蛋白，即CDH5），构建了将显性负调控型人PPARγ特异性表达于内皮细胞的转基因小鼠模型。本研究委托Dominion Pharmakine公司（http://www.pharmakine.com/）从10只非转基因小鼠与10只EC-DN转基因小鼠中分离原代主动脉内皮细胞。每组均建立5个细胞培养株，每株细胞来源于2只小鼠，且各培养株之间相互独立。采用常规方法提取细胞总RNA，并通过Agilent 2100生物分析仪（Agilent Technologies）评估RNA质量。针对芯片杂交实验，每组选取3个细胞培养株的RNA进行实验。所有芯片实验均在爱荷华大学DNA核心实验室完成，采用Affymetrix标准实验流程。简言之，实验取约50 ng总RNA作为起始模板，通过两步扩增流程（NuGen公司，http://www.nugeninc.com/）制备生物素标记的RNA片段，用于与Affymetrix GeneChip Mouse Genome 430 2.0芯片进行杂交。