Introduction Vascular endothelial growth factor (VEGF) is a potent mitogenic, angiogenic and permeability factor that has been implicated in the development of lung injury and repair in a number of respiratory diseases such as ARDS and IPF VEGF₁₆₅a functions via VEGF receptors in particular VEGFR-2, leading to a diverse and complex network of signalling pathways including activation of both the MAPK pathway and eNOS This results in changes to cell permeability, migration and proliferation. We have investigated the downstream signalling mechanisms regulated by VEGF₁₆₅a in pulmonary and systemic endothelial cells. Understanding the signalling pathway used by VEGF₁₆₅a to regulate lung biology is critical to preferentially induce specific beneficial effects.

Methods Human Umbilical Vein Endothelial Cells (HUVEC) and Human Lung Microvascular Endothelial Cells (HUMVEC-L) were treated with 20ng/ml of VEGF165a lysed and studied using phosphospecific antibodies which measure the phosphorylation/activation of key signalling molecules. Phosphorylation of VEGFR-2 was measured using phosphotyrosine-specific antibody to tyr1175 and tyr1214. Phosphorylation and hence activation of MEK, MAPK and eNOS were also measured. The effects of VEGF isoforms on cell permeability in a time and dose dependent manner were measured by using a transwell system and “Electrical Cell-Substrate Impedance Sensor” (ECIS). Changes in the cellular distribution of VE-cadherin a protein known to be involved in the regulation of cell permeability was assessed by immunofluorescent labelling and confocal microscopy.

Results Phosphorylation of VEGFR-2 at tyr1175 and tyr1214was induced between 5 and 10min (n=4; >5 fold increase). Activation of MEK and p44/42 MAPK (members of the MAPK pathway which regulates cell proliferation) were seen over a similar time course to that of VEGFR-2 (n=4; >5 fold increase) (Figs 1A, B). Phosphorylation of eNOS which regulates cell permeability was also observed (n=3; >2 fold) and indeed VEGF165a increased permeability in both HUVEC and HUMVEC-L (Huvec p<0.001); (Humvec-l p<0.01) (Fig 1). Finally we showed that in both cell types VEGF induced changes in the cellular distribution of VE-cadherin.

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Abstract S56 Figure 1

Conclusion These results demonstrate that signalling pathways, previously suggested to induce mitogenesis or permeability are activated by VEGF 165a in HUVEC and HMVEC-l cells, identifying potential future therapeutic targets.