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.

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Abstract T2 Figure 1.

HMVEC-L and HUVEC cells stimulated with 20 ng/ml of VEGF₁₆₅a, VEGF₁₆₅b, VEGF₁₆₅ a+b or without any stimulation (control) A, immunoblotting of primary HUMVECL treated for 5 to 10min and immunoblotted for the phosphorylation of p-VEGFR-2, p-MEK, p-MAPK and p-eNOS using phosphospecific antibodies (comparable results for HUVEC cells, data not shown). B C, Measurement of TEER by Endohm (inserts 0.4mm pore size). VEGF₁₆₅a reduces resistance (increased permeability) ***p < 0.001 (45min onwards) and VEGF₁₆₅b increases resistance (decrease permeability) **p < 0.01 (15 to 45min) compared with control. In combination VEGF₁₆₅b ameliorates VEGF₁₆₅a effect in HMVEC-L. D E, Electrical Cell-Substrate Impedance Sensor (ECIS) measurement (B well assay 8W10E+); VEGF₁₆₅a reduces the resistance (increased permeability) VEGF₁₆₅b increases resistance (decreases permeability) in comparison to control. Data were analysed using one-way ANOVA and Bonferroni post test analysis.

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 VEGF_165/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.