LPS-mediated neutrophil VEGF-A release is modulated by cannabinoid receptor activation

Abstract

Neutrophils (PMNs) are innate immune cells with primary roles in inflammation and in host defense against infections. Both inflammatory and tumor angiogenesis are modulated by a sequential, coordinated production of angiogenic factors such as vascular endothelial growth factors (VEGFs), angiopoietins, hepatocyte growth factor (HGF), and chemokines. These factors are produced by several immune cells, including PMNs. Activation of cannabinoid receptor type-1 (CB₁ ) and -2 (CB₂ ) has been suggested as a new strategy to modulate in vitro and in vivo angiogenesis. We sought to investigate whether activation of CB₁ and CB₂ by CB agonists modulate LPS-mediated angiogenic activity of human PMNs. Highly purified PMNs were isolated from buffy coats of healthy donors. Cells were stimulated with CB₁ and CB₂ agonists/antagonists alone and/or in combination with LPS. Angiogenic factors in cell-free supernatants were measured by ELISA. The modulation of activation markers of PMNs by CB agonists was evaluated by flow cytometry. Angiogenesis in vitro was measured as tube formation by optical microscopy. Endothelial cell permeability was assessed by an in vitro vascular permeability assay. LPS-activated PMNs released VEGF-A, CXCL8, and HGF. Preincubation of PMNs with low concentrations of CB₁ and CB₂ agonists inhibited VEGF-A release induced by LPS, but did not affect CXCL8 and HGF production. The effects of CB agonists on VEGF-A release induced by LPS were reversed by preincubation with CB antagonists. CB agonists modulated in vitro angiogenesis and endothelial permeability induced by supernatants of LPS-activated PMNs through the reduction of VEGF-A. Neutrophils play a central role in the control of bacterial infections and in the outcome of sepsis. The latter condition is associated with an increase in circulating levels of VEGF-A. We demonstrated that low concentrations of CB agonists inhibit VEGF-A release from LPS-activated PMNs. These results suggest that CB agonists might represent a novel therapeutic strategy in patients with sepsis.

Keywords: angiogenesis; angiopoietins; cannabinoid receptors; endocannabinoids; hepatocyte growth factor; neutrophils; vascular endothelial growth factor.