Pressure Points: Endothelial Responses to Hemodynamic Stress in Health and Pulmonary Arterial Hypertension

Background: Hemodynamic forces exert a profound influence on endothelial cell signaling and, when abnormal, contribute centrally to human vascular disease. Pulmonary arterial hypertension (PAH) is characterized by both hemodynamic derangement and pulmonary arterial endothelial cell (PAEC) dysfunction. Despite importance in disease initiation and progression, the combined effects of shear and pressure forces on PAEC biology remain incompletely understood, particularly in the context of PAH. Methods: PAECs obtained at explant from controls and from patients with idiopathic or congenital heart disease-associated PAH (CHD-PAH) were cultured in a custom resistor-coupled microfluidic platform and exposed to static, low (3 dyne/cm²), or high (20 dyne/cm²) shear stress under either low or elevated (60 mmHg) pressure. After 24 hours, we assessed cellular morphology and performed systems-level transcriptomic analysis via bulk RNA sequencing, incorporating analyses of PAH subtype and donor sex. Results: PAECs (n=18 donors) aligned with flow under high, but not low, shear, and alignment was not significantly altered by disease state or pressure. Shear stress fundamentally reorganized the PAEC transcriptome and the “dose-response” to increasing shear differed across biological pathways in six statistically significant patterns. Increasing shear led to divergence in transcription between control and PAH cells, particularly in pathways involved in immune activation, stress signaling, and vascular remodeling, with subtype differences also observed. Pressure alone had modest effects on transcription, though CHD-PAH PAECs especially displayed pressure-induced stress and inflammatory signaling. We identified sexual dimorphism in the endothelial shear response, noting male cells under shear enriched for pathways involved in proliferation and inflammation and female cells enriched for lipid metabolism and stress responses. Conclusions: Shear and pressure forces profoundly influence PAEC transcription, with responses shaped by disease state, PAH subtype, and sex. These findings highlight the need for further investigation into mechanosensitive pathways in PAH as potential targets for novel therapies. Pulmonary arterial endothelial cells (PAECs) from control subjects and individuals with idiopathic pulmonary arterial hypertension (IPAH) or congenital heart disease-associated PAH (CHD-PAH) were seeded into microfluidic culture chambers, grown to confluence overnight under gentle, intermittent flow, and then exposed to defined combinations of pressure and shear stress for 24 hours. Pressure and shear stress were set by varying the length of a resistor channel and adjusting the flow rate of perfused culture media. After 24 hours RNA was collected and sequenced. Sequencing was performed in two batches: batch 1 contained pressure and shear stress as variables, batch 2 contained shear stress alone as a variable, with no manipulation of pressure