VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells

BACKGROUND: Alternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF(165)a and VEGF(165)b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways. METHOD: To test this hypothesis we investigated the physiological effect of VEGF(165)a and VEGF(165)b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors. RESULTS: VEGF(165)a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF(165)b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF(165)a and VEGF(165)b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF(165)a and VEGF(165)b on paracellular permeability and the effect of VEGF(165)a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s). CONCLUSION: This study demonstrates that the novel isoform VEGF(165)a and VEGF(165)b induce differing effects on permeability in pulmonary microvascular endothelial cells.