Role of Gata5 in vascular endothelial cells

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. 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