The role of BMPR2 and SOX17 deficiency in endothelial dysfunction: implications for pulmonary arterial hypertension
File(s)
Author(s)
Mahomed, Abdul Sattar Fakee
Type
Thesis
Abstract
Background: Pulmonary arterial hypertension (PAH) is characterized by endothelial cell dysfunction and obliterative lung vascular remodelling, culminating in right-sided heart failure and death. Bone morphogenetic protein receptor type 2 (BMPR2) is firmly established as a PAH-risk gene, whereby deficiency of functional BMPR2 is known to disrupt endothelial homeostasis. In addition, mutations in the SRY-box transcription factor 17 (SOX17) gene have recently been associated with development of the disease. However, given its only recent implication in PAH, the understanding of SOX17 deficiency in endothelial dysfunction is in its infancy. Blood flow-induced shear stress is well documented as a regulator of endothelial function and the transcriptome. Emerging, albeit limited evidence, suggests that the dysfunctional endothelium in PAH, including due to loss of BMPR2, exhibit defective shear stress responsiveness. Therefore, this project sought to investigate the effects BMPR2 and SOX17 deficiency on the regulation of shear-induced gene expression (selected PAH/endothelial function markers).
Approach and results: siRNA-mediated BMPR2-silenced (siBMPR2) human pulmonary artery endothelial cells (HPAECs) or that of non-targeting control treated cells (siControl) were exposed to PAH-relevant shear stress profiles; laminar or bidirectional oscillatory flow (1Hz) at 1.5, 15 or 90 dyne/cm2 or cultured under static conditions. Under laminar flow (15 dyne/cm2) BMPR2 knockdown led to decreased shear-induced (relative to static siControl) production of several genes including, cyclooxygenase 2 (COX2), interleukin (IL)6 and IL8, compared with siControl cells. Notably these observations selectively occurred under shear magnitudes of 15 dyne/cm2 (and not 1.5 or 90 dyne/cm2) and almost exclusively under laminar flow. SOX17 knockdown in HPAECs (static) resulted in the identification of marked overexpression of angiostatic chemokines, C-X-C motif chemokine ligand 10 (CXCL10) and CXCL11, driven by excessive nuclear factor kappa B (NF-κB) p65 activity, compared with siControl HPAECs. In addition, SOX17 silencing upregulated mRNA of a diverse array of markers, most of which have previously been described in the pathobiology of PAH, for example, EDN1 (endothelin-1). Monolayer integrity of SOX17-silenced HPAECs was also reduced, as determined by real-time impedance measurements.
Conclusion: The application of specific shear stress profiles disrupts pulmonary endothelial gene regulation under conditions of reduced BMPR2. SOX17 loss in HPAECs induces mediators and characteristics of endothelial dysfunction previously established in PAH, in particular the upregulation of angiostatic factors CXCL10 and CXCL11.
Approach and results: siRNA-mediated BMPR2-silenced (siBMPR2) human pulmonary artery endothelial cells (HPAECs) or that of non-targeting control treated cells (siControl) were exposed to PAH-relevant shear stress profiles; laminar or bidirectional oscillatory flow (1Hz) at 1.5, 15 or 90 dyne/cm2 or cultured under static conditions. Under laminar flow (15 dyne/cm2) BMPR2 knockdown led to decreased shear-induced (relative to static siControl) production of several genes including, cyclooxygenase 2 (COX2), interleukin (IL)6 and IL8, compared with siControl cells. Notably these observations selectively occurred under shear magnitudes of 15 dyne/cm2 (and not 1.5 or 90 dyne/cm2) and almost exclusively under laminar flow. SOX17 knockdown in HPAECs (static) resulted in the identification of marked overexpression of angiostatic chemokines, C-X-C motif chemokine ligand 10 (CXCL10) and CXCL11, driven by excessive nuclear factor kappa B (NF-κB) p65 activity, compared with siControl HPAECs. In addition, SOX17 silencing upregulated mRNA of a diverse array of markers, most of which have previously been described in the pathobiology of PAH, for example, EDN1 (endothelin-1). Monolayer integrity of SOX17-silenced HPAECs was also reduced, as determined by real-time impedance measurements.
Conclusion: The application of specific shear stress profiles disrupts pulmonary endothelial gene regulation under conditions of reduced BMPR2. SOX17 loss in HPAECs induces mediators and characteristics of endothelial dysfunction previously established in PAH, in particular the upregulation of angiostatic factors CXCL10 and CXCL11.
Version
Open Access
Date Issued
2022-06
Date Awarded
2022-10
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Wort, Stephen John
Burke-Gaffney, Anne
Sponsor
Imperial College London
Publisher Department
National Heart and Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)