High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension
Computational modeling indicated that a pathological level of high shear stress (HSS, 100 dyn/cm2) is generated in distal pulmonary arteries (PA) (100-500 um) in a congenital heart defect with increased PA blood flow causing PA hypertension (PAH), and in idiopathic PAH with occlusive vascular remodeling.  The response of human PA endothelial cells (EC) to HSS compared to physiologic laminar shear stress (LSS, 15 dyn/cm2), was therefore assessed.  Endothelial-mesenchymal transition (EndMT), a feature of PAH not previously attributed to HSS was observed. HSS did not alter induction of the transcription Krüppel-like factors (KLF) 2/4, but H3K27ac peaks containing motifs for an ETS-family transcription factor (ERG) were reduced, as was the interaction between ERG and KLF2/4 and ERG expression.  In PAEC under LSS, reducing ERG by siRNA caused EndMT related to decreased bone morphogenetic protein receptor 2 (BMPR2), cadherin 5 (CDH5) and platelet and endothelial cell adhesion molecule 1 (PECAM1), and increased Snail/Slug (SNAI1/2) and smooth muscle alpha a-2 actin (ACTA2).  In PAEC under HSS, transfection of ERG prevented EndMT.  We induced HSS in mice by an aorto-caval shunt that causes a progressive increase in PAH over eight weeks and used an adeno-associated viral vector (AAV2-ESGHGYF) to replenish ERG selectively in PAEC.  Elevated PA pressure and resistance, EndMT and vascular remodeling assessed by muscularization of peripheral arteries were markedly reduced by ERG delivery in the aorto-caval shunt mice. Thus, agents that restore ERG in the pulmonary vasculature will be of therapeutic benefit in overcoming the adverse effect of HSS on progressive PAH. Overall design: Pulmonary artery endothelial cells were pre-conditioned by exposure to 15 dyn/cm2 of unidirectional uniform LSS for 24 h. Cells were then exposed to an additional 24 h of LSS, or 24 h of 100 dyn/cm2 unidirectional uniform HSS. Shear stress was generated using the Ibidi Perfusion System (Ibidi). Static (ST) controls were performed simultaneously with the shear stress experiments, cultured on 0.4 mm chamber slides using the same EC media. CUT&RUN was performed using the CUTANA CUT&RUN kit (#14-1048, EpiCypher, USA) following manufacturer's instructions.