Urotensin II (U-II; cyclo [5][6][7][8][9][10] [H-Glu-Thr-Pro-Asp-Cys-Phe-TrpLys-Tyr-Cys-Val-OH]) is a potent vasoconstrictor in mammals, and it is postulated that it plays a central role in cardiovascular homeostasis. Thus, we initiated a structure-to-function analysis of this peptide characterized by a N-terminal tail and a cyclic core formed through a disulfide bridging. A total of 41 analogs focusing on these characteristics were developed and evaluated using a binding assay on membranes from a stable HEK-293 cell line containing the human or rat U-II receptor, a functional assay for Ca 2ϩ mobilization on transiently transfected CHO-K1 cells with the human or rat U-II receptor, and a rat thoracic aorta bioassay. At first, the focus was applied on peptide compounds containing exocyclic modifications. From this series, it appeared that only valine-11 played a significant role although it is not an essential amino acid. Similarly, endocyclic and ring transformations of hU-II were also studied. In most cases, a detrimental effect on affinity and biological activity was observed. However, two compounds, [Tyr 6 ]hU-II and [Phe 9 ]hU-II, retained affinity and activity. So far, our binding, functional, and pharmacological data clearly demonstrated the minor contribution of the N-terminal segment and the essential role of the cyclic structure. More particularly, three residues within the loop, i.e., Trp-7, Lys-8, and Tyr-9, are required for receptor recognition and activation. This three-pole feature, kept by the disulfide bond in a correct spatial arrangement, appears as the key pharmacophore for the U-II receptor.

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 beta2-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.

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-A(165), VEGF-A(121), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased vascular permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) 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-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) 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-A(165)-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-A(165) proinflammatory activities.