A Biomimetic Miniaturized <i>In Vitro</i> Model to Target Early Markers of Neonatal Pulmonary Vascular Injury

Pulmonary vascular disease (PVD) is a major contributor to morbidity in preterm infants as it is associated with a significant risk to devevlop pulmonary hypertension, especially in infants diagnosed with prematurity-associated lung disease (PLD), also known as bronchopulmonary dysplasia (BPD). However, the earliest events of vascular injury triggered by postnatal mechanical and oxygen-related stress remain poorly understood, largely due to the limitations of existing in vitro models. We therefore developed a biomimetic, miniaturized Pulmonary In Vitro Perfusion (PIPE) system that integrates pathophysiologically relevant shear stress with controlled oxygen exposure for the exposure of a human co-culture of pulmonary microvascular endothelial cells and pulmonary artery smooth muscle cells. Advancing the system to a triple co-culture, circulating THP-1 monocytes capture early endothelial-smooth muscle-immune cell interactions. Shear stress alone induced early proliferative and extracellular matrix-related responses in endothelial cells and resulted in enhanced monocyte recruitment without disrupting barrier integrity. When combined with oxygen exposure, the model revealed a dose-dependent injury pattern: moderate hyperoxia (FiO 2 0.40) had minimal acute effects, whereas severe hyperoxia (FiO 2 0.85) impaired endothelial barrier function, increased ROS production, promoted monocyte transmigration, activated apoptosis (Caspase 3), and elevated soluble collagen synthesis. This dynamic in vitro system reveals early drivers in vascular injury and recapitulates key characteristics of PVD, thereby introducing a translational platform for the dissection of disease mechanisms and evaluation of therapeutic strategies targeting vascular injury in the developing lung.