Silencing KRIT1 Partially Reverses the Effects of Disturbed Flow on the Endothelial Cell Transcriptome

Endothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow are primarily mediated by the elevation of transcription factors Krüppel-like factors 2 and 4 (KLF2 and KLF4). Notably, turbulent flow patterns, which predispose to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4 and reulting changes in the transcriptome that exacerbate inflammation and thrombosis. The CCM complex, comprising KRIT1, CCM2, and CCM3 suppress expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the effects of turbulent flow on the human endothelial transcriptome. We conducted bulk RNA sequencing (RNA-seq) on human umbilical vein endothelial cells (HUVECs) after silencing KRIT1 expression using specific siRNAs. The endothelial cells were exposed to pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), or no flow for 24 hours. Overall design: HUVECs transfected with siRNA for 48 hours were replated using 0.05% Trypsin/EDTA (Thermo Fisher, 25300054) on 38 mm × 76 mm glass plates at 95% confluence. A subset of the glass plates were then assembled into a parallel-plate flow channel as previously described22 for each condition: siScr+PS, siKrit1+PS, siSCr+OS, and siKrit1+OS. The flow system was maintained at 37°C and ventilated with 95% humidified air with 5% CO2. HUVECs were exposed to pulsatile flow (PS) (12 ± 4 dyn/cm2, 1 Hz) or oscillatory flow (OS) (1 ± 4 dyn/cm2, 1 Hz) for 24 hours as described previously23. Additional control plates of siScr- and siKrit1-transfected HUVECs were subject to static conditions (no flow) and maintained in the same culture medium and conditions described above in an adjacent incubator.