Role of Gata5 in vascular endothelial cells. Homo sapiens

Despite its high prevalence and economic burden, the etiology of human hypertension remains incompletely understood. Here we identify the transcription factor Gata5, as a new gene involved in regulation of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells (mEC) and its genetic inactivation in mice leads to hypertension, vascular endothelial dysfunction and renal inflammation. Aged Gata5-Null mice develop salt-sensitivity and target-organ damage reminiscent of the progression of human hypertension. Endothelial-specific inactivation of Gata5 increases BP and leads to vascular endothelial dysfunction, confirming the endothelial component of Gata5 inactivation-related hypertension. To directly assess the effect of loss of GATA5 on endothelial cells, we generated a stable GATA5 knockdown cell line (HDMEC-Gata5KO) by infecting human dermal microvascular endothelial cells with a lentiviral vector containing an anti-Gata5 shRNA followed by a transcriptomic analysis. The control cells were infected with a lentivirus containing an empty vector pLKO2. Overall design: GATA5 MISSION® TRC2 pLKO.shRNA (TRCN0000431556) and MISSION® TRC2 pLKO-puro Non-Mammalian shRNA Control (SHC202) plasmids were purchased from Sigma-Aldrich. Viral particles were generated by co-transfection of phoenix cells with the lentiviral vector (GATA5 TRC2 shRNA or the shRNA control plasmids), the packaging vector psPAX2 and the envelope vector pMD2G. Co-transfection was performed using the Qiagen’s Effectene transfection reagent (301425). Twenty-four hours after transfection, medium was discarded and replaced by Endothelial Growth Medium 2 (Promocell, C-22021). Forty-eight hours later, the medium (EGM2) was collected, filtered, and used for infection of Human Dermal Microvascular Endothelial Cells (HDMEC; Promocell, C-14016). Infected cells were selected with addition of puromycin (Sigma-Aldrich, P8833) to culture medium at the concentration of 2.5 μg/ml for 15 days. Control (HDMEC-pLKO-Ctrl) and HDMEC-GATA5-KD cells were then grown and maintained in EGM2 containing puromycin at the concentration of 0.25 μg/ml. RNA was extracted and hybridized on Affymetrix microarray

尽管人类高血压具有高患病率与沉重的经济负担，但其病因学仍未完全阐明。本研究鉴定出转录因子Gata5作为参与血压（blood pressure, BP）调控的新基因。Gata5在微血管内皮细胞（microvascular endothelial cells, mEC）中表达，小鼠体内该基因的遗传失活会引发高血压、血管内皮功能障碍与肾脏炎症。老年Gata5敲除小鼠会出现盐敏感性与靶器官损伤，这与人类高血压的疾病进展特征相似。内皮细胞特异性的Gata5失活会升高血压并引发血管内皮功能障碍，证实了Gata5失活相关高血压的内皮细胞致病机制。为直接评估Gata5缺失对内皮细胞的影响，本研究通过携带抗Gata5短发夹RNA（short hairpin RNA, shRNA）的慢病毒载体感染人真皮微血管内皮细胞（human dermal microvascular endothelial cells, HDMEC），构建了稳定的Gata5敲低细胞系HDMEC-Gata5KO，并开展了转录组学分析。对照组细胞则用携带空载体pLKO2的慢病毒进行感染。实验整体设计如下：从Sigma-Aldrich公司购得GATA5 MISSION® TRC2 pLKO.shRNA（TRCN0000431556）与MISSION® TRC2 pLKO-puro 非哺乳动物shRNA对照（SHC202）质粒。通过将慢病毒载体（GATA5 TRC2 shRNA或shRNA对照质粒）、包装载体psPAX2与包膜载体pMD2G共转染Phoenix细胞，以制备病毒颗粒。转染操作使用Qiagen公司的Effectene转染试剂（货号301425）完成。转染24小时后，弃去原培养基，更换为内皮细胞生长培养基2（Endothelial Growth Medium 2, EGM2；Promocell，货号C-22021）。转染48小时后，收集培养基（EGM2）并过滤，用于感染人真皮微血管内皮细胞（HDMEC；Promocell，货号C-14016）。将感染后的细胞置于添加了嘌呤霉素（Sigma-Aldrich，货号P8833，浓度2.5 μg/ml）的培养基中筛选15天。随后将对照组细胞（HDMEC-pLKO-Ctrl）与HDMEC-GATA5-KD细胞在添加了0.25 μg/ml嘌呤霉素的EGM2培养基中培养并维持。提取RNA后，在Affymetrix基因芯片上进行杂交实验。