Pascal N. Bernatchez

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 migrationin 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 × 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.

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

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. 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 Ser1177‐phosphorylation and NO synthesis measured by cGMP production. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser1177‐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. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser1177‐phosphorylation nor cGMP synthesis mediated by VEGF. 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.

Estrogen Regulation of Endothelial and Smooth Muscle Cell Migration and Proliferation: Role of p38 and p42/44 Mitogen-Activated Protein Kinase

Objective—Restenosis is a major limitation of percutaneous coronary intervention. Migration and proliferation of vascular cells remain a cornerstone in neointimal formation. The cardioprotection of estrogen is well recognized, but the intracellular mechanisms related to these beneficial effects are not completely understood. Methods and Results—We investigated the effects of 17&bgr;-estradiol (17&bgr;E) on mitogen-activated protein kinase (MAPK) activity and the migration and proliferation of porcine aortic endothelial cells (PAECs) and porcine smooth muscle cells (PSMCs). Treatment with 17&bgr;E (10−8 mol/L) abrogated p38 and p42/44 MAPK phosphorylation mediated by platelet-derived growth factor-BB as well as the migration and proliferation of PSMCs. In contrast, treatment with 17&bgr;E (10−8 mol/L) induced the phosphorylation of p38 and p42/44 MAPK and the migration and proliferation of PAECs. Interestingly, the effects of 17&bgr;E on PSMCs and PAECs were reversed by selective estrogen receptor antagonists (tamoxifen, 4-OH-tamoxifen, and raloxifen). These results suggest that in PSMCs, 17&bgr;E inhibits chemotactic and mitogenic effects of platelet-derived growth factor-BB as well as p38 and p42/44 MAPK phosphorylation. In contrast, 17&bgr;E promotes in PAECs the phosphorylation of p42/44 and p38 MAPK as well as the migration and proliferation of these cells. Conclusions—Treatment with 17&bgr;E has a dual beneficial effect: the improvement of vascular healing and the prevention of restenosis after angioplasty.

Relative effects of VEGF‐A and VEGF‐C on endothelial cell proliferation, migration and PAF synthesis: Role of neuropilin‐1

Vascular endothelial growth factor (VEGF‐A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet‐activating factor (PAF). Recently, neuropilin‐1 (NRP‐1) has been described as a coreceptor of KDR which potentiates VEGF‐A activity. However, the role of NRP‐1 in numerous VEGF‐A activities remains unclear. To assess the contribution of NRP‐1 to VEGF‐A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt‐1, NRP‐1, KDR or KDR and NRP‐1. Cells were stimulated with VEGF‐A, which binds to Flt‐1, KDR and NRP‐1, and VEGF‐C, which binds to KDR only. VEGF‐A was 12.4‐fold more potent than VEGF‐C in inducing KDR phosphorylation in PAEC‐KDR. VEGF‐A and VEGF‐C showed similar potency to mediate PAEC‐KDR proliferation, migration, and PAF synthesis. On PAEC‐KDR/NRP‐1, VEGF‐A was 28.6‐fold more potent than VEGF‐C in inducing KDR phosphorylation and PAEC‐KDR/NRP‐1 proliferation (1.3‐fold), migration (1.7‐fold), and PAF synthesis (4.6‐fold). These results suggest that cooperative binding of VEGF‐A to KDR and NRP‐1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP‐1 receptors. In contrast, stimulation of PAEC‐Flt‐1 and PAEC‐NRP‐1 with VEGF‐A or VEGF‐C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP‐1 on EC preferentially increases KDR activation by VEGF‐A as well as KDR‐mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF‐A and VEGF‐C have equipotent biological activities on EC in absence of NRP‐1. J. Cell. Biochem. 85: 629–639, 2002.

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

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). VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 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 μM) was without effect, confirming the crucial role of the PLA2 and lyso‐PAF‐AT. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 μM and LY311727; 100 μM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 μM) had no significant effect. RT – PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. Treatment with cytosolic and calcium‐independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 μM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

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

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. 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 Cγ (PLCγ), protein kinase C (PKC) and p42/44 mitogen‐activated protein kinases (MAPK). Moreover, we demonstrate that VEGF‐mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso‐PAF to PAF. 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. 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.

A New Role for the Muscle Repair Protein Dysferlin in Endothelial Cell Adhesion and Angiogenesis

Objective—Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC. Methods and Results—EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing. Conclusion—Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.

Sphingosine 1‐phosphate effect on endothelial cell PAF synthesis: Role in cellular migration

Sphingosine 1‐phosphate (S1P) and vascular endothelial growth factor (VEGF) are two inflammatory mediators capable of promoting endothelial cell (EC) migration and angiogenesis. As VEGF inflammatory effect is mediated by the synthesis of endothelial platelet‐activating factor (PAF) which is also contributing to VEGF chemotactic activity, we wanted to assess if S1P can trigger PAF synthesis in EC and if S1P‐induced migration is PAF‐dependent. Treatment of bovine aortic EC (BAEC) with S1P (10−10–10−6 M) increased dose‐ and time‐dependently the synthesis of PAF by up to 3.3‐fold above the basal level, with a maximal amount of PAF detected at 20 min post‐stimulation. This biological response was attenuated by inhibiting p38 mitogen‐activated protein kinase (MAPK), cytosolic or secreted phospholipase A2 (cPLA2, sPLA2) activity, suggesting that p38 MAPK activation by S1P promotes the conversion of membrane phospholipids into PAF through the combined activation of cPLA2 and sPLA2. Interestingly, pretreatment of BAEC with extracellular PAF receptor antagonists (BN52021, 10−5 M and CV3988, 10−6 M) reduced by up to 42% the cellular migration induced by S1P (10−6 M). These data demonstrate the capacity of S1P to induce PAF synthesis, which contributes in part to S1P chemotactic activity.