Vascular Endothelial Growth Factor-D Improves Lung Vascular Integrity During Acute Lung Injury

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<0.05), immune cell infiltration (0.791±0.199×106 whole blood cells/mL [lipopolysaccharides] versus 0.540±0.190×106 whole blood cells/mL [lipopolysaccharide+VEGF-D]; P<0.01), and the expression of proinflammatory markers TNF-α, IL-6, and keratinocyte chemoattractant in the lung tissue. This effect was abolished in VEGFR2iECKO (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.