Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC and PLC pathways

Abstract

1. The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2. Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-phosphorylation mediated by VEGF, but did not alter immediate (0-10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5. In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.

Figure 1

VEGF effect on cGMP production. BAEC were pretreated with (IBMX, 500 μM; 10 min) prior to VEGF stimulation in a DMEM/5 mM CaCl₂ solution. Cells were scraped, proteins lyophilized and cGMP production detected by a RIA kit. (A) VEGF (1 nM) induced a time-dependent increase of cGMP production. (B) Effect of VEGF at various concentrations (0.01–1 nM) on cGMP production for a 10-min stimulation. ***P<0.001 as compared to control buffer (PBS).

Figure 2

Effect of several pathway inhibitors on VEGF-induced cGMP production. BAEC were pretreated with IBMX (500 μM; 10 min), and with inhibitors or antagonists (up to 60 min) prior to VEGF stimulation in a DMEM/5 mM CaCl₂ solution. cGMP production was detected by a RIA kit. Effect of VEGF (1 nM) on cGMP production upon a pretreatment with (A) eNOS inhibitor (L-NAME, 100 μM) or PLC pathway inhibitors (U73122, 10 μM; U73343, 10 μM; GF109203X, 5 μM; Ro318220, 1 μM or W-7, 250 μM). (B) cGMP synthesis upon stimulation with VEGF (1 nM) or A23187 (10 μM) on cGMP in a 5 mM CaCl₂. Effect of VEGF (1 nM) on cGMP production upon a pretreatment with two chelators of calcium (EGTA/AM 100 μM or BAPTA/AM 10 μM) in a CaCl₂-free solution. Effect of VEGF (1 nM) on cGMP production upon a pretreatment with (C) two PI3K inhibitors (Wortmannin, 500 nM or LY294002, 50 μM), (D) PAF mediator inhibitors (SB203580, 10 μM; PD98059, 10 μM or SB203347, 10 μM) or PAF receptor antagonists (BN52021 10 μM; LAU8080, 100 nM or CV3988, 1 μM). Effect of PAF (100 nM or 1 μM) on cGMP production. ***P<0.001 as compared to control buffer (PBS), †††P<0.001 as compared to VEGF (1 nM), *P<0.05 as compared to control buffer (PBS) in a DMEM CaCl₂ free solution and †P<0.05 as compared to VEGF (1 nM) in a DMEM CaCl₂ free solution.

Figure 3

VEGF effect on eNOS phosphorylation at Serine 1177. Confluent BAEC were stimulated with agonists, cells were lysed and proteins were separated on a 7.5% SDS–PAGE. Western blot analysis was performed with rabbit polyclonal anti-phospho-eNOS (Ser¹¹⁷⁷) antibodies. (A) Time-dependent eNOS activation mediated by VEGF, the maximal phosphorylation was considered as control (100%). Studies (B–E) were performed with the same inhibitors described in Figure 2, and VEGF-mediated phosphorylation was considered as control (100%).

Figure 4

VEGF effect on Akt phosphorylation at Serine 473. Confluent BAEC were stimulated with agonists, cells were lysed and proteins separated on a 7.5% SDS–PAGE. Western blot analysis was performed with rabbit polyclonal anti-phospho-Akt (Ser⁴⁷³) antibodies. Studies (A–E) were performed as detailed in Figure 3.

Figure 5

Effect of IBMX pretreatment on VEGF-induced immediate cGMP synthesis. Confluent BAEC were incubated 10 min in a DMEM 5 mM CaCl₂ solution. IBMX (500 μM) was added for different periods of time (0, 2.5, 5, 10 min) prior to a 10-min stimulation with VEGF (1 nM). Cells were scraped, proteins lyophilized and cGMP production detected by a RIA kit. ***P<0.001 as compared to control buffer (PBS).

Figure 6

Effect of PI3K and PKC inhibitors on VEGF-induced immediate and delayed cGMP synthesis. Immediate cGMP synthesis was measured as described in Figure 2. Delayed cGMP synthesis was performed using confluent BAEC incubated 10 min with PKC inhibitors (GF109203X, 5 μM or Ro318220, 1 μM) or PI3K inhibitors (Wortmannin, 500 nM or LY294002, 50 μM) in a DMEM 5 mM CaCl₂ solution prior to VEGF (1 nM) stimulation. IBMX (500 μM) was added at 5 min after VEGF stimulation and the experiment was stopped 30 min after VEGF stimulation. In another set of experiments and under the same conditions (IBMX added 5 min after VEGF stimulation), we quantified cGMP synthesis produced between 5 and 10 min (data not shown) and subtracted this amount from total delayed cGMP synthesis. Therefore, in Figure 6 (black columns) we show delayed cGMP synthesis observed between 10 and 30 min after VEGF stimulation. ***P<0.001 as compared to control buffer (PBS) for immediate cGMP synthesis; *P<0.05 as compared to control buffer (PBS) for delayed cGMP synthesis and †P<0.05 as compared to VEGF (1 nM) for delayed cGMP.

Figure 7

Effect of VEGF-induced nitric oxide on PAF synthesis. BAEC were grown to confluence. The media was replaced by 1 ml of HBSS-HEPES (10 mM)+CaCl₂ (5 mM). Inhibitors were added 15 min prior to stimulation. VEGF (1 nM) was added to stimulate BAEC: alone as a control, with L-NAME (100 μM) pretreatment or in addition with the exogenous NO donor SNP (500 μM). L-NAME (100 μM) and SNP (500 μM) were added without VEGF to evaluate their effect on basal PAF synthesis. ***P<0.001 as compared to control buffer (PBS).

Figure 8

Proposed intracellular pathways for the induction of NO and PAF synthesis by VEGF in endothelial cells. VEGF induces immediate NO synthesis through the PLC-Ca²⁺/CaM pathway and the induction of delayed NO synthesis implies Akt and PKC activition. In addition, NO and PAF synthesis mediated by VEGF involves two independent pathways.