VEGF-D, unlike VEGF-A, improves vascular integrity in the lung and exerts a protective effect during acute lung injury

Background: Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases, such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and COVID-19. Paracrine signals are enriched throughout the lung tissue and are critical in maintaining functional microvascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. Our objective in this study is to determine the local factors in lung microvascular niche exhibiting barrier protective and anti-inflammatory properties as potential therapeutic targets for improving vascular integrity during ALI. Hypothesis: Paracrine factors expressed under physiological conditions within the lung microvascular niche facilitate barrier protection and exhibit anti-inflammatory properties and can serve as potential therapeutic targets to enhance vascular integrity during ALI. Methods: 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. The impact of identified ligands on vascular barrier function and anti-inflammatory response was further assessed in vitro cell culture assays and lipopolysaccharide (LPS)-induced lung injury models. Results: Our scRNAseq data revealed that, under physiological conditions, soluble L/R interactions between mesenchymal cells, in particular alveolar fibroblast, and capillary 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 observed that VEGF-D, unlike the effect of VEGF-A and VEGF-C, 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 and anti-inflammatory effect of VEGF-D was significantly attenuated by inhibition of VEGFR2, either through siRNA knockdown or pharmacological blockade using specific VEGFR2 inhibitors (SU5416, XL184, SU5614). Intravenous administration of recombinant VEGF-D in LPS-induced ALI models significantly reduced vascular permeability (7339 ± 2510 (LPS) v.s. 5350 ± 1821 (LPS + VEGF-D), p < 0.05) and 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) into the lung tissue. Levels of pro-inflammatory markers, including TNF-α and IL-6, in bronchoalveolar lavage fluid were also significantly decreased. Importantly, in VEGFR2 iECKO mice, the impact of VEGF-D on immune cell infiltration and lung injury index was abolished, 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, distinguishing it from VEGF-A, and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis. K99/R00HL159261 (Y.Y.), UL1TR003098 (Y.Y.) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.