Effects of varying shear stress profiles on the regulation of pulmonary artery endothelial cell gene expression: a focus on selected mediators of vascular tone, inflammation and angiogenesis

Abstract

Abstract Background Pulmonary arterial hypertension (PAH) is a complex pathology characterized by obliterative vascular remodeling that leads to right heart failure and death. Predisposition to PAH is associated with mutations in the BMPR2 gene in approximately 70–80% of familial cases and around 30% for that of sporadic PAH. The study of the pathogenetic basis of PAH is often performed in static endothelial cultures. Such two-dimensional, isolated cell microenvironments fail to consider the heterogeneity in mechanical stress acting on endothelial cells in various regions of the pulmonary vascular tree. In the remodeled pulmonary vasculature, low and oscillatory shear stress is observed in the proximal pulmonary artery with high shear stress in distal pre-capillary pulmonary arterioles. Therefore, the impact of varied shear profiles (including both laminar and oscillatory flow) on pulmonary artery endothelial cell (and that of BMPR2-deplete) gene expression of common vasoactive (EDN1, ENOS), proinflammatory (IL6, IL8) and angiogenic mediators (ANG2, VEGFA), are poorly described. Purpose To evaluate the effects of shear stress magnitude, including unidirectional and oscillatory flow on BMPR2-knockdown human pulmonary artery endothelial cell (HPAEC) gene expression of EDN1, ENOS, IL6, IL8, ANG2 and VEGFA. Methods HPAECs were transfected with siRNA directed against BMPR2 (siB2) or with a non-targeting control (siCon). Cells were exposed to 10 hours of laminar or oscillatory flow (1Hz; 1.5 dyn/cm2, 15 dyn/cm2 or 90 dyn/cm2) using a parallel-plate fluid flow chamber system. Measurement of mRNA expression was performed using qPCR. Results Shear stress intensity and flow type (unidirectional and oscillatory) mediated diverse effects on HPAEC gene expression across the markers studied. Changes in gene expression were calculated relative to that of static siCon-transfected HPAECs and in such a manner are summarized as fold changes in the table below. Asterisks are shown where significant fold differences are reported. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. aP≤0.05, bP≤0.05, cP≤0.05, denote comparisons between groups. Of note, no significant differences in gene expression were observed between static siCon and static siB2. Conclusions For the markers studied, different magnitudes of shear stress and flow profiles (together with BMPR2 loss) exhibit varied patterns of gene expression in the pulmonary vascular endothelium. As such, this illustrates the need for wider study of in vitro endothelial-shear stress interactions in understanding mechanisms of remodeling in PAH. Funding Acknowledgement Type of funding sources: None. Table 1