Vascular endothelial growth factor (VEGF) undergoes alternate splicing producing isoforms with differing functional effects. The most biologically active and extensively studied isoform is VEGF165. An isoform that causes inhibition of endothelial proliferation, migration and permeability, VEGF165b has also been identified (Bates et al. 2002). We have previously investigated the downstream signalling mechanisms in response to VEGF165 in pulmonary and systemic endothelial cells and have now compared these to the effects of VEGF165b.
HUVEC and Human Lung Microvascular Endothelial Cells (HMVEC-L) were treated with both VEGF isoforms. Phosphorylation of VEGFR-2 (tyr1175 and tyr1214) was measured along with phosphorylation/activation of pMEK1/2, p44/42MAPK (regulating cell proliferation) and eNOS (involved in cell permeability). We have previously shown the functional effects of VEGF165/VEGF165b on HMVEC-L proliferation (Varet et al. 2010). We have now explored the functional effects of VEGF165/ VEGF165b on cell permeability parameters by Endohm and Electrical Cell-Substrate Impedance Sensor (ECIS) measurements and modification in VE-cadherin cell distribution. The effects of the eNOS inhibitor L-NIO were also investigated.
VEGF165 induced maximal phosphorylation of VEGFR-2 at tyr1175 and tyr1214 between 5 and 10min (>10 fold increase), VEGF165b induced less than 5 fold increase compared to control. Comparable results for both isoforms were seen for activation of pMEK1/2, p42/44MAPK and eNOS. The two permeability assessments showed an increase in cells permeability due to VEGF165 (HUVEC p < 0.001); (HMVEC-l p < 0.01) in contrast to VEGF165b. This may be reflected by the differential changes in the cellular distribution of VE-cadherin induced in both cell types by VEGF isofroms. Treatment with L-NIO inhibited the effect of VEGF165b suggesting a potential regulatory mechanism..
VEGF165b induces differential responses to VEGF165 in HUVEC and HMVEC-L. These observations suggest separate pathways for the regulation of mitogenesis and permeability which may be targeted.
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