Adrenomedullin-RAMP2 enhances endothelial cell maturation synergically with shear stress

Diseases in pulmonary vasculature remain challenging to study due to the lack of functional ex vivo models. Paracrine signals are enriched in whole lung tissue and are critical in regulating endothelial maturation and vascular homeostasis. Previously, we employed single-cell RNA-sequencing (scRNAseq) to systematically profile ligand-receptor (L/R) interactions within the lung vascular niche. Here, we leveraged in vitro cell culture models to evaluate different paracrine signals identified from scRNAseq data and determined that adrenomedullin (ADM)-RAMP2 could improve the endothelial maturation under dynamic flow conditions. Among the top 20 soluble ligands, we found ADM has a superior effect on enhancing vascular barrier function, as evidenced by electrical cell impedance sensing (ECIS) and XperT assays. ADM also exhibited anti-inflammatory properties, decreasing ICAM1 and increasing IkBa expression in a dose-dependent manner. Interestingly, under physiological shear stress (15 dyn/cm ² ), ADM synergistically enhanced the expression of the native lung aerocyte capillary (aCap) endothelial marker EDNRB, increased anti-coagulation by increasing THBD and decreasing F3 expression. This effect was completely attenuated by blocking RAMP2, a receptor of ADM. This study highlights the potential of native paracrine cues, such as ADM/RAMP2 signaling, to enhance cellular and physiological maturation in engineered vascular models.