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

Abstract

We recently demonstrated that Tie2 receptor activation on human neutrophils by both angiopoietins (Ang1 and Ang2) promoted platelet-activating factor synthesis, beta(2)-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 beta-granules. Treatment of neutrophils with phorbol myristate acetate (PMA), N-Formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor-alpha (TNF-alpha) induced VEGF release. Maximal effect was observed with PMA (80 nM) stimulation inducing a complete release of VEGF content (565 +/- 100 pg/ml; 6 x 10(6) 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 x 10(6) 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.

Prognostic Significance of Neutrophil to Lymphocyte Ratio, Lymphocyte to Monocyte Ratio, and Platelet to Lymphocyte Ratio in Patients with Nasopharyngeal Carcinoma

“…Neutrophils, as a type of inflammatory cells, are considered to be involved in different steps of tumor development through the production of a variety of cytokines, such as oncostatin M, hepatocyte growth factor, and transforming growth factor- (TGF-) β [12]. In addition, neutrophils promote tumor angiogenesis through the release of angiogenic factors, such as vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor-2 [13, 14]. On the other hand, lymphocytes are also responsible for immune surveillance to remove tumor cells.…”

Group V Secreted Phospholipase A2 Induces the Release of Proangiogenic and Antiangiogenic Factors by Human Neutrophils

“…The signals responsible for the constitutive expression of angiogenic mediators, in particular VEGF-A 165b , in PMNs are not known. VEGF-A is stored almost exclusively in the specific (β) granules (49), by contrast, Ang1 is predominantly located in the cytosolic fraction (50). Further studies are needed to understand the molecular details of VEGF-A 165b expression in resting and hGV-stimulated PMNs.…”

“…In particular, PMNs release a variety of proangiogenic and antiangiogenic factors and play important roles in several models of inflammatory and tumor angiogenesis (36, 49–51). Indeed, PMNs release VEGF-A in response to fMLF, LPS, and phorbolmyristate acetate (PMA), while Ang1 is secreted only in response to PMA (50). It is unknown whether sPLA 2 s can modulate the production of proangiogenic and antiangiogenic factors from PMNs.…”

Characterization of FGF receptor expression in human neutrophils and their contribution to chemotaxis

“…The study has been approved by the human ethical committee of the Montreal Heart Institute, and all subjects provided written informed consent. Neutrophils were isolated using Ficoll-Hypaque gradient, as described previously (32,36,44), and resuspended in RPMI medium (Lonza, Allendale, NJ) supplemented with 25 mM HEPES (N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid) and 1% penicillin-streptomycin. Ninety-eight (98) percent of the isolated cells were polymorphonuclear cells, as determined with a Coulter counter, and viability was found to be Ͼ98%, as assessed by Trypan blue dye exclusion assay.…”

“…Interestingly, many of the mediators that neutrophils respond to are also extensively involved in angiogenesis, a phenomenon that leads to the formation of new blood vessels from preexisting vasculature. Factors such as vascular endothelial growth factor (VEGF), angiopoietins (Ang1, Ang2), and interleukins (IL)-1, -6, and -8, all of which are extensively involved at different stages of angiogenesis, have been shown to modulate neutrophil survival, degranulation, respiratory burst, adhesion, and chemotaxis (5,11,28,32,36).…”