Alexandre Brkovic

Vascular permeability induced by VEGF family members in vivo: Role of endogenous PAF and NO synthesis

We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet‐activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF‐induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF‐A165, VEGF‐A121, and VEGF‐C (1 µM) which activate VEGFR‐2 (Flk‐1) receptor increased vascular permeability, whereas a treatment with VEGFR‐1 (Flt‐1) analogs; PlGF and VEGF‐B (1 µM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L‐NAME) abrogated protein extravasation mediated by VEGF‐A165. As opposed to PAF (0.01‐1 µM), treatment with acetylcholine (ACh; up to 100 µM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 µM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF‐A165 vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR‐2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF‐A165‐mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF‐A165 proinflammatory activities. J. Cell. Biochem. 100: 727–737, 2007.

Angiopoietin chemotactic activities on neutrophils are regulated by PI-3K activation.

Angiopoietins (Ang1 and Ang2) modulate blood vessel integrity during the angiogenic process through the activation of tyrosine kinase receptor (Tie2). We recently detected Tie2 expression on neutrophils and reported that angiopoietins induce acute proinflammatory events including neutrophil β2‐integrin activation and their adhesion onto endothelial cells. Herein, we investigated the effect of angiopoietins on neutrophil migration and their capacity to modulate CXCL8/IL‐8 chemotactic properties. Using a Boyden chamber assay, we observed that Ang1 and Ang2 (up to 10 nM; 60 min) increased the migration of neutrophils, and the maximal effect was achieved at 1 nM (72% and 114% increase, respectively) as compared with untreated cells. Angiopoietins induce a rapid and transient Akt phosphorylation, and pretreatment of neutrophils with PI‐3K inhibitors, wortmannin (100 nM) and LY294002 (500 nM), reduced Ang1‐mediated neutrophil migration by 100% and 78% and Ang2 chemotactic activity by 100% and 71%, respectively. Treatment of neutrophils with CXCL8/IL‐8 (up to 50 nM; 60 min) increased basal neutrophil migration by 257% at its optimal concentration (10 nM), and pretreatment of neutrophils with corresponding PI‐3K inhibitors reduced CXCL8/IL‐8 (1 nM) chemotactic effect. Pretreatment of neutrophils with Ang1 or Ang2 (10 nM; 15 min) potentiated neutrophil migration induced by CXCL8/IL‐8 (1 or 10 nM; 60 min) by 263% and 238% and by 177% and 164%, respectively. Finally, both angiopoietins showed a synergistic effect on the induction of Akt phosphorylation mediated by CXCL8/IL‐8. In summary, our data demonstrate that angiopoietins increase neutrophil migration through PI‐3K activation and can enhance proinflammatory activities of other cytokines.

Expression and release of angiopoietin-1 from human neutrophils: Intracellular mechanisms

We recently demonstrated that Tie2 receptor activation on human neutrophils by both angiopoietins (Ang1 and Ang2) promoted platelet-activating factor synthesis, β2-integrin activation, and cell migration. Herein, we wanted to assess if human neutrophils express angiopoietins and further delineate their mechanisms of release. Employing Reverse transcriptase-polymerase chain reaction, Real time quantitative transcriptase-polymerase chain reaction, FACScan analysis and ELISA approaches, we observed that neutrophils express Ang1 but not Ang2. For each condition, vascular endothelial growth factor (VEGF) detection was performed as positive control. Using nitrogen cavitation, we observed that Ang1 is localized in the cytosolic fraction whereas VEGF is found in β-granules. Treatment of neutrophils with phorbol myristate acetate (PMA), N-Formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α) induced VEGF release. Maximal effect was observed with PMA (80 nM) stimulation inducing a complete release of VEGF content (565 ± 100 pg/ml; 6 × 106 neutrophils), corresponding to a 18.9-fold increase as compared to phosphate buffer saline (PBS) treated neutrophils. By contrast, only a treatment with PMA (80 nM) induced Ang1 release. PMA treatment induced also a complete release of Ang1 (661 ± 148 pg/ml; 6 × 106 neutrophils), corresponding to 2.8-fold increase as compared to PBS-treated neutrophils. In both cases, PMA-mediated release of VEGF and Ang1 was nearly maximal by 15 min. Finally, we observed that the induction of Ang1 release was calcium-independent whereas VEGF release was not. These data demonstrate the capacity of human neutrophils to synthesize Ang1, which is stored and released differently as compared to VEGF. These data suggest a different cascade of events regarding the distribution of selected growth factors during inflammation and angiogenesis.

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms

Recently identified, angiopoietin‐1 (Ang1) and ‐2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities. As inflammation exists in a mutually dependent association with angiogenesis, we sought to determine if Ang1 and/or Ang2 could modulate proinflammatory activities, namely P‐selectin translocation, in bovine aortic endothelial cells (EC) and dissect the mechanisms implicated. P‐selectin, an adhesion molecule found in the Weibel‐Palade bodies of EC, is translocated rapidly to the cell surface upon EC activation during inflammatory processes. Herein, we report that Ang1 and Ang2 (1 nM) are capable of mediating a rapid Tie2 phosphorylation as well as a rapid and transient endothelial P‐selectin translocation maximal within 7.5 min (125% and 100% increase, respectively, over control values). In addition, we demonstrate for the first time that angiopoietin‐mediated endothelial P‐selectin translocation is calcium‐dependent and regulated through phospholipase C‐γ activation.

Priming effect of homocysteine on inducible vascular cell adhesion molecule-1 expression in endothelial cells

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis, as well as for arterial and venous thrombosis. However, the mechanisms through which elevated circulating levels of homocysteine cause vascular injury and promote thrombosis remain unclear. Here, we tested the hypothesis that homocysteine (Hcy) sensitizes endothelial cells to the effect of inflammatory mediators. Human umbilical vein endothelial cells (HUVEC) were incubated with Hcy 1.0 mM for varying time points, and then treated in the absence or presence of 1.5 U/ml thrombin or 10 mg/ml lipopolysaccharide (LPS). Hcy alone had no effect on the expression of vascular cell adhesion molecule (VCAM)-1. However, Hcy enhanced thrombin- and LPS-mediated induction of VCAM-1 mRNA and protein levels. Consistent with these results, pretreatment of HUVEC with Hcy resulted in a two-fold increase in LSP-mediated induction of leukocyte adhesion. The latter effect was significantly inhibited by anti-VCAM-1 antibodies. Together, these findings suggest that Hcy sensitizes HUVEC to the effect of inflammatory mediators thrombin and LPS, at least in part through VCAM-1 expression and function.