COX2 expression is downregulated in a shear stress magnitude-dependent manner in BMPR2-silenced HPAECs: implications for prostacyclin deficiency in PAH

Introduction: Mutations in the BMPR2 gene predispose to pulmonary arterial hypertension (PAH). In addition, reduced bioavailability of prostacyclin and nitric oxide (NO) with increased expression of endothelin-1 (EDN1) disrupts vascular tone and contributes to the pathogenesis. Shear-regulated genes, COX2 and NO synthase (ENOS), are involved in the production of prostacyclin and NO, respectively, whilst EDN1 expression is also regulated by flow. Emerging data suggests that BMPR2 deficiency may instigate a defective molecular and morphological response of endothelial cells to flow. As such the integrity of COX2, EDN1 and ENOS in human pulmonary artery endothelial cells (HPAECs), under concomitant dynamic fluid flow and reduced BMPR2 expression is unclear, hence the aim of this study. Methods: HPAECs were transfected with siRNA to BMPR2 (siB) or with control sequences (siC). Cells were cultured under static conditions or exposed to 10 hours of laminar or oscillatory flow (1.5, 15 or 90 dyn/cm2) using a parallel-plate fluid flow chamber system. Quantification of COX2, ENOS and EDN1 mRNA was performed using qPCR. Results: Under laminar flow at 15 dyne/cm2 only, there was significantly decreased (28%) flow-induced (relative to static siC) COX2 mRNA in siB HPAECs versus siC. No changes in EDN1 or ENOS mRNA levels were observed between siC and siB under flow, although oscillatory flow at 15 dyne/cm2 (for both siC and siB), inhibited ENOS mRNA synthesis and induced EDN1. Conclusion: BMPR2 loss may disrupt the flow-driven production of COX2 in HPAECs, which may be an underlying mechanism of endothelial dysfunction in PAH.