Abstract-Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF 165 . In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF 165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF 165 -mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF 121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF 165 -mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF. Key Words: vascular endothelial growth factor receptor-2 Ⅲ transendothelial electrical resistance Ⅲ chemotaxis Ⅲ mitogen-activated protein kinase Ⅲ acute lung injury V ascular endothelial growth factor (VEGF) was first isolated and purified from tumor cells because of its ability to markedly increase vascular permeability to plasma proteins. 1 VEGF is expressed normally in most tissues, including lung, 2,3 with abundant expression of VEGF in alveolar cells of normal rats. 3,4 Published data support a significant role for VEGF in the development of acute lung injury and acute respiratory distress syndrome (ARDS) after various insults. [5][6][7][8][9][10] In the adult lung, VEGF may promote vascular injury 6 -9,11 via effects on increased endothelial permeability. On the other hand, VEGF stimulates endothelial cell proliferation 12 and chemotaxis, 13 potential reparative responses to pulmonary vascular injury. Because cellular responses to VEGF may be either injurious or reparative, understanding how the endothelium transduces specific VEGF-mediated signals is critical.Vascular endothelial cells express 2 tyrosine kinase receptors for VEGF, termed VEGF receptor 1 (VEGFR1) 14 and VEGF receptor 2 (VEGFR2). 15 Intermolecular cross-talk between VEGFR1 and VEGFR2 has been reported, 16 -18 and represents one possible pathway for differential regulation of VEGF signals. Endothelial cells also express neuropilin-1 (Npn-1), a class 3 s...