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

Abstract

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.

Figure 1

PAF biosynthesis induced by VEGF is Ca²⁺-dependent. Confluent BAEC (6-well tissue culture plate) were incubated with ³H-acetate and stimulated with VEGF (1 nM) for 15 min with various concentrations of CaCl₂ (0 – 10 mM). The radioactive polar lipids samples were extracted by the Bligh and Dyer procedure. The samples were injected into a 4.6×250 mm Varian Si-5 column and eluted with a mobile phase (H₂O:CHCl₃:MeOH; 5 : 40 : 55; 0.5 ml min⁻¹). Fractions were collected every min after injection and radioactivity was determined with a β-counter. The values are means of at least six experiments. ^*P<0.05 and ^***P<0.001 as compared to control buffer (PBS) as determined by analysis of variance followed by an unpaired Student's t-test.

Figure 2

Biosynthesis of lyso-PAF and PAF via the remodelling pathway. The initial hydrolysis of the acyl moiety of alkylacylglycerophosphocholine to form lyso-PAF and arachidonic acid (AA) can be catalyzed by the action of a direct phospholipase A₂ (PLA₂) or a CoA-independent transacylase (CoA-IT). Lyso-plasmalogen and other lyso-glycerophospholipids can act as the acyl acceptor in the CoA-independent transacylase type of reaction. The lyso-PAF is then converted to PAF by the acetyl-CoA:lyso-PAF acetyltransferase. Above, are the names of selective inhibitors used to identify the enzymes involved in VEGF-mediated PAF synthesis upon remodelling pathway activation. R=(CH₂)_n-CH₃ where n=15 to 17 and R′=(CH₂)₂N⁺(CH₃)₃.

Figure 3

Effect of the remodelling pathway inhibitors on VEGF-induced PAF synthesis. (A) Confluent BAEC (6-well tissue culture plate) were pretreated 5 or 30 min with the remodelling pathway inhibitors Sanguinarine (Sang; 500 μM), SKF45905 (SKF; up to 50 μM), AACOCF₃ (AACOCF₃; 50 μM), MAFP (MAFP; 10 μM), BEL (BEL; 10 μM), Scalaradial (Scal; 10 μM), SB203347 (SB; 10 μM) and LY311727 (LY; up to 100 μM) stimulated with VEGF (1 nM)+³H-acetate+10 mM CaCl₂, and the lipids were extracted and purified as described in Figure 1. The values are means of at least four experiments. (B) Confluent HUVEC were treated as described for BAEC. ^*P<0.05 and ^***P<0.001 as compared to control buffer (PBS), and †P<0.05, ††P<0.01, †††P<0.001 as compared to VEGF (1 nM) as determined by analysis of variance followed by an unpaired Student's t-test.

Figure 4

Expression of group IIA and V sPLA₂ by BAEC and HUVEC. Confluent BAEC and HUVEC (100 mm tissue culture plate) were rinsed and stimulated with VEGF (1 nM) for 15 min. Cells were scraped and total proteins were isolated. Forty μg of crude proteins were separated by a 10% SDS – PAGE and transblotted onto a PVDF membrane. Proteins were detected by a sPLA₂ (human synovial) polyclonal antiserum which recognizes both group IIA and group V sPLA₂. Lane 1: P388D1 macrophage cell lysate (group V sPLA₂ positive control). Lane 2: Human synovial fluid sPLA₂ (group IIA sPLA₂ positive control). Lane 3: BAEC lysate. Lane 4: HUVEC lysate.

Figure 5

Expression of group IIA and V sPLA₂ mRNA by BAEC and HUVEC. Confluent BAEC and HUVEC were lysed, total RNA was extracted and used for RT – PCR. The sizes of the expected amplified fragments are 450 base-pairs for group IIA sPLA₂ (A) and 225 base-pairs for group V sPLA₂ (B). Human brain and intestine mRNA were used as negative and positive controls respectively, for group IIA sPLA₂ RT – PCR (A). Human brain and human heart mRNA were used as positive controls for group V sPLA₂ RT – PCR (B).