Yifan Yuan

BACKGROUND: Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases. Paracrine signaling is highly enriched in the lung and plays a crucial role in regulating vascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. METHODS: In this study, we used single-cell RNA-sequencing–based computational pipelines to systematically profile ligand-receptor interactions within the lung microvascular niche and identified VEGF-D (vascular endothelial growth factor-D) as a key local factor with previously unrecognized barrier-protective properties in models of acute lung injury. RESULTS: Our single-cell RNA-sequencing data revealed that, under physiological conditions, soluble ligand-receptor interactions between mesenchymal cells, in particular alveolar fibroblasts, and microvascular endothelial cells are predominantly associated with pathways involved in maintaining vascular integrity as compared with all other cells. On treatment with top identified ligands, we found that VEGF-D significantly enhanced endothelial barrier function and conferred protection against inflammatory challenges induced by TNF-α (tumor necrosis factor-α), IL (interleukin)-1β, and thrombin. This barrier-protective effect of VEGF-D was significantly attenuated by inhibition of VEGFR2 (vascular endothelial growth factor receptor 2), either through small interfering RNA (siRNA) knockdown or pharmacological blockade using specific VEGFR2 inhibitors. Intravenous administration of recombinant VEGF-D in lipopolysaccharide-induced acute lung injury models significantly reduced vascular permeability (7339±2510 arbitrary unit [a.u.] [lipopolysaccharides] versus 5350±1821 a.u. [lipopolysaccharides+VEGF-D]; P 6 whole blood cells/mL [lipopolysaccharides] versus 0.540±0.190×10 ⁶ whole blood cells/mL [lipopolysaccharide+VEGF-D]; P VEGFR2 ^iECKO (VEGFR2 inducible endothelial cells knockout) mice, confirming that VEGF-D mediates its effects via VEGFR2-dependent signaling. CONCLUSIONS: This study demonstrates an unexpected protective role for VEGF-D in promoting lung endothelial barrier integrity and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis.

Analysis of pulmonary vascular dysfunction in various lung pathologies remains challenging due to the lack of functional ex vivo models. Paracrine signaling in the lung plays a critical role in regulating endothelial maturation and vascular homeostasis. Previously, we employed single-cell RNA-sequencing (scRNAseq) to systematically map ligand–receptor (L/R) interactions within the lung vascular niche. However, the functional impact of these ligands on endothelial biology remained unknown. Here, we systematically evaluated selected ligands in vitro to assess their effects on endothelial barrier integrity, anti-inflammatory responses, and phenotypic maturation. Among the top soluble ligands, we found that adrenomedulin (ADM) exhibited superior barrier enhancing effect on human pulmonary endothelial cell monolayers, 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. Perfusion is commonly used in bioengineered vascular model systems. Shear stress (15 dynes/cm²) alone increased endothelial characteristics, including homeostatic markers such as CDH5, NOS3, TEK, and S1PR1. ADM treatment maintained the enhanced level of these markers under shear stress and further improved anti-coagulation by increasing THBD and decreasing F3 expression and synergistically enhanced the expression of the native lung aerocyte capillary endothelial marker EDNRB. This effect was completely attenuated by a blockade of ADM receptor, RAMP2. Together, these findings identify ADM/RAMP2 signaling as a key paracrine pathway that enhances vascular barrier integrity, anti-inflammatory phenotype, and endothelial homeostasis, providing a framework for improving the physiological relevance of engineered vascular models.

Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases. Paracrine signaling is highly enriched in the lung and plays a crucial role in regulating vascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. In this study, we employed single-cell RNA-sequencing (scRNAseq)-based computational pipelines to systematically profile ligand-receptor (L/R) interactions within the lung microvascular niche and identified vascular endothelial growth factor-D (VEGF-D) as a key local factor with previously unrecognized barrier-protective properties in models of acute lung injury. Our scRNAseq data revealed that, under physiological conditions, soluble L/R interactions between mesenchymal cells, in particular alveolar fibroblast, and microvascular endothelial cells are predominantly associated with pathways involved in maintaining vascular integrity as compared to all other cells. Upon treatment with top identified ligands, we found that VEGF-D significantly enhanced endothelial barrier function and conferred protection against inflammatory challenges induced by tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and thrombin. This barrier-protective effect of VEGF-D was significantly attenuated by inhibition of VEGFR2, either through siRNA knockdown or pharmacological blockade using specific VEGFR2 inhibitors. Intravenous administration of recombinant VEGF-D in lipopolysaccharides (LPS)-induced acute lung injury models significantly reduced vascular permeability (7339 ± 2510 (LPS) v.s. 5350 ± 1821 (LPS + VEGF-D), p < 0.05), immune cell infiltration (0.791 ± 0.199 x 10 ⁶ WBC/mL (LPS) v.s. 0.540 ± 0.190 x 10 ⁶ WBC/mL (LPS + VEGF-D), p < 0.01), and the expression of pro-inflammatory markers TNF-α and IL-6 in the lung tissue. This effect was abolished in VEGFR2 ^iECKO mice, confirming that VEGF-D mediates its effects via VEGFR2-dependent signaling. This study demonstrates an unexpected protective role for VEGF-D in promoting lung endothelial barrier integrity and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis.

Therapeutic lung regeneration is predicated upon successful reconstitution of lung alveoli, the functional units of gas exchange. Here, we identify requisite multimodal cues that are critical to reconstructing the alveolar epithelium and alveoli in lung scaffolds. Alveolar reconstruction in vitro is divided into several distinct phases. In the first phase, endothelial cells coordinate with fibroblasts and select exogenous factors to promote alveolar scaffold population with surfactant-secreting alveolar epithelial type 2 cells (AEC2s). After formation of organized epithelial alveolar-like structures, subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-level mechanical strain to induce differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ . These results outline a rational strategy to engineer an alveolus of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial units, and reveal the critical interplay amongst biochemical, cellular, and mechanical niche cues within the reconstituting alveolus.