Endothelins Mediate Neutrophil Activation, ProMMP-9 Release and Endothelial Cell Detachment

Abstract

Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml(-1)) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B(4) (LTB(4)); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-alpha (TNF-alpha 10 ng ml(-1)) or interleukin-1 (IL-1; 10 ng ml(-1)) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19-37) (10(-9) M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10(-6) M; a selective endothelin A (ET(A)) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50-70% inhibition) as well as the levels of LTB(4) (70-100% inhibition) and proMMP-9 (40-50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60-75% inhibition). Moreover, the exogenous addition of ET-1 (10(-9) M) to neutrophil suspensions induced neutrophil spreading and release of LTB(4) and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.

Effect of Gedunin on Acute Articular Inflammation and Hypernociception in Mice

“…Interestingly, in addition to gedunin effect on neutrophils, it is noteworthy that such limonoid also modulates tEnd.1 cell functions, as observed by diminished adhesion of neutrophils to tEnd.1 monolayers. The mechanisms by which gedunin reduces neutrophil attachment to endothelial cells remains to be investigated; however, it might modulates the expression of adhesion molecules or even the secretion of inflammatory mediators, including ET-1, as previously described by Toffoli and colleagues [ 58 ]. Reduced numbers of lipid bodies observed in gedunin-treated ET-1 stimulated leukocytes corroborates the direct capability of gedunin to modulate leukocyte functions.…”

Lipoxin A4 attenuates zymosan‐induced arthritis by modulating endothelin‐1 and its effects

“…LXA 4 decreased in vitro neutrophil chemotaxis, adherence and transmigration across the vascular endothelium and epithelium (McMahon and Godson, 2004; Filep et al ., 2005), CD11b/CD18 expression (Filep et al ., 1999) and blocked superoxide generation (Levy et al ., 1999) and TNF‐α production and actions (Hachicha et al ., 1999). Previous in vitro data demonstrate that ETs cause direct neutrophil activation (Toffoli et al ., 2007), adhesion (Zouki et al ., 1999) and migration in vitro (Elferink and de Koster, 1994). Interestingly, in the present study, we showed that LXA 4 seemed to directly impair ET‐1‐induced neutrophil activation and migration through a mechanism that needs further elucidation.…”

Peri-interventional endothelin-A receptor blockade improves long-term outcome in patients with ST-elevation acute myocardial infarction

“…Furthermore the reduction of myeloperoxidase (MPO), a mediator of neutrophil activation (34) and an independent predictor of cardiovascular events (35,36), was more pronounced in patients of the treatment group (▶ Figure 1 A), suggesting that ET-A receptor blockade decreases neutrophil activity which may in turn translate into lower MMP-9 levels. Indeed, it has previously been observed in vitro that pre-incubating neutrophils with BQ-123 leads to a significant reduction of activation and to a decrease in MMP-9 release (17).…”

“…ET receptors are found on a broad variety of cells, most importantly on neutrophils which play a crucial role in coronary atherothrombosis (15) and are locally recruited and activated (16). Pre-incubation of neutrophils with an ET-A antagonist decrease subsequent activation (17). Neutrophils mediate the breakdown of extracellular matrix (ECM) mainly by release of matrix metallopeptidase-9 (MMP-9) particularly in the early hours of AMI (18).…”