Regulation of VEGF-induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways.

1. Vascular endothelial growth factor (VEGF) is a potent angiogenic and inflammatory mediator. We have recently shown that this latter effect requires the activation of Flk-1 receptor and subsequent endothelial cell (EC) PAF synthesis. However, the intracellular events that regulate EC PAF synthesis upon Flk-1 stimulation by VEGF remain to be elucidated. 2. Using specific inhibitors and Western blot analysis, we herein report that in bovine aortic endothelial cells (BAEC), VEGF induces the synthesis of PAF through the cascade activation of Flk-1 receptor, phospholipase Cgamma (PLCgamma), protein kinase C (PKC) and p42/44 mitogen-activated protein kinases (MAPK). 3. Moreover, we demonstrate that VEGF-mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso-PAF to PAF. 4. Interestingly, we observed that VEGF also promoted the activation of the phosphatidyl inositol-3-phosphate kinase (PI3K) pathway, and that its blockade potentiated PAF synthesis following a VEGF treatment. Consequently, it appears that the PI3K pathway acts as a negative regulator of EC PAF synthesis. 5. Taken together, these results allow a better understanding of the intracellular events activated upon EC stimulation by VEGF, and shed a new light on the mechanisms by which VEGF induces PAF synthesis.

Vascular endothelial growth factor production by isolated rat hepatocytes after cold ischemia-warm reoxygenation.

Inflammatory disturbances in the liver microcirculation have been associated with preservation injury of hepatic grafts. Vascular endothelial growth factor (VEGF), a proinflammatory growth factor released by hepatocytes, acts on sinusoidal endothelial cells, but its implication in graft injury is still unclear. We studied VEGF production by rat hepatocytes after cold ischemia and warm reoxygenation and compared the capacity of University of Wisconsin (UW) and sodium-lactobionate-sucrose (SLS) preservation solutions to maintain this hepatocellular function. Isolated hepatocytes were kept for 0, 24, and 48 hours at 4 degrees C in either solution (cold ischemia), then incubated for 1 to 24 hours at 37 degrees C (warm reoxygenation). We assessed cell viability and production of VEGF messenger RNA (mRNA) and protein. Cell viability decreased in a linear time-dependent fashion by 10% after 48 hours of cold preservation and by an additional 40% after 24 hours of warm culture. Very little VEGF mRNA could be detected after up to 48 hours of simple cold preservation in either solution. However, subsequent warm culture led to a robust and rapid increase in VEGF mRNA expression within the first hour, which declined to close to background levels within 8 to 12 hours in culture. This effect was more important in cells preserved in SLS than UW solution. Similarly, cold preservation alone did not trigger VEGF secretion. VEGF secretion was detected after culturing hepatocytes at 37 degrees C and reached a maximal secretion rate within 12 to 15 hours. However, VEGF production by preserved cells was reduced compared with unstored cells. In conclusion, cold ischemia and warm reoxygenation triggers VEGF mRNA expression by hepatocytes, but subsequent VEGF secretion is partially impaired, suggesting posttranslational defects.

VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2.

1. Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA-independent transacylase, CoA-IT; phospholipase A2, PLA2; and lyso-PAF acetyltransferase, lyso-PAF-AT) may participate in PAF synthesis, we assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC). 2. VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 3. A pretreatment with a CoA-IT and lyso-PAF-AT inhibitor (Sanguinarin; 500 nM) blocked VEGF-induced PAF synthesis by 95%, a specific CoA-IT inhibitor (SKF45905; 10 - 50 microM) was without effect, confirming the crucial role of the PLA2 and lyso-PAF-AT. 4. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 microM and LY311727; 100 microM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 microM) had no significant effect. 5. RT - PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. 6. Treatment with cytosolic and calcium-independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 microM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. 7. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent.

Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell (EC) proliferation and migration in vitro as well as inflammation in vivo. We showed recently that VEGF effect on vascular permeability was dependent on the synthesis of platelet-activating factor (PAF) by EC. Consequently, we sought to evaluate by antisense knockdown of gene expression the contribution of VEGF receptors (Flt-1 and Flk-1) on these events. VEGF (10(-11) to 10(-8) M) elicited a dose-dependent increase of bovine aortic EC proliferation, migration, and PAF synthesis by up to 2.05-, 1.31- and 35.9-fold above basal levels, respectively. A treatment with two modified antisense oligomers (1-5 x 10(-7) M) directed against Flk-1 mRNA blocked by 100, 91, and 85% the proliferation, migration, and PAF synthesis mediated by VEGF, respectively. A treatment with two antisense oligomers directed against Flt-1 mRNA failed to modulate these activities. The use of placenta growth factor (up to 10(-8) M), an Flt-1-specific agonist, induced only a slight increase (0.6-fold) of PAF synthesis. These data illustrate the crucial role of Flk-1 in EC stimulation by VEGF. The capacity to inhibit the protein synthesis of Flt-1 and Flk-1 by antisense oligonucleotides provides a new approach to block VEGF pathological effects in vivo.