Abstract
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.
Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP-43919) and from the Heart and Stroke Foundation of Québec to Dr Sirois. Mr. Neagoe is recipient of PhD studentship from the CIHR, Dr Sirois is recipient of a senior scientist scholarship from the Fonds de la recherche en santé du Québec (FRSQ).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.