Abstract Body: Background
Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling driven by smooth muscle cell (SMC) phenotypic modulation in response to hemodynamic stress. Although elevated pulmonary arterial pressure is central to disease progression, mechanistic insight is limited by the lack of human-relevant models capable of sustaining defined pressure states. We hypothesize that increasing pathologic pressure elicits graded SMC remodeling mediated by endoplasmic reticulum (ER) stress, and that a bioengineered 3D vessel platform enables interrogation of pressure- and hypoxia-dependent mechanisms in PAH.

Methods
We bioengineered a pulmonary artery construct composed of human pulmonary artery SMCs embedded within a hydrogel and maintained under long-term perfusion in a custom bioreactor. Pathologic pressure states were established using downstream resistance, producing a linear relationship between flow rate and inlet pressure. Structural stability, stiffness, and cell viability were assessed at baseline. Constructs were perfused for two weeks under mildly elevated (>20 mmHg) or severely pathologic (>50 mmHg) mean pressures combined with normoxic (21% O2) or hypoxic (5% O2) conditions. Optimized protocols enabled recovery of SMCs from pressurized 3D constructs for transcriptomic analysis. Parallel 2D hypoxia studies were performed to define hypoxia-induced ER stress and remodeling pathways.

Results
Engineered vessels demonstrated high structural fidelity (4-mm lumen: 91.3%, n=3), consistent stiffness (8-14 kPa, n=3), and preserved SMC viability prior to perfusion. Inlet pressure was linearly proportional to flow rate in both small (r²=0.9978, n=3) and large (r²=0.9948, n=2) constructs, enabling reproducible control of clinically relevant pulmonary arterial pressure states. Post-culture SMCs were successfully recovered from perfused 3D constructs and submitted for bulk RNA sequencing. In complementary 2D experiments, hypoxia robustly induced ER stress and remodeling-associated gene expression, establishing candidate pathways relevant to PAH-associated SMC dysfunction.

Conclusion
We establish a human-relevant 3D pulmonary artery model capable of long-term perfusion under defined pressure and oxygen conditions and enabling molecular interrogation of pressure-driven SMC remodeling. This platform provides a novel approach to study pulmonary vascular mechanobiology and supports future integration of hypoxia severity and inflammatory cues.