Cytosolic and calcium-independent phospholipase A2 mediate glioma-enhanced proangiogenic activity of brain endothelial cells

Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs. The glioma CM effect was significantly reduced by cytosolic (cPLA2) and Ca++-independent (iPLA2) phospholipase A2, cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA2 and iPLA2 enzyme activities and phosphorylation of cPLA2, significantly stimulated after 24h CM co-incubation, were attenuated by PLA2, PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA2, phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA2 was in parallel observed. Electroporation of anti-iPLA2 and cPLA2 antibodies and siRNAs directed against iPLA2 and cPLA2 significantly inhibited cell proliferation and migration. Incubation of CM- or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA2 and iPLA2 protein levels. The findings suggest that PLA2 activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA2 is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis. © 2010 Elsevier Inc.