Abstract
The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5–1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.
Highlights
Annexin-1 is the first member of the annexin superfamily which, in humans, consists of at least 12 members each of which has a unique N-terminal sequence [1]
We explored whether interfering with the assembly of a functional NADPH oxidase affects tumour necrosis factor alpha (TNFa) mediated ICAM-1 and VCAM-1expression by transfecting Human dermal microvascular endothelial cells (HMECs) cells with a plasmid carrying dominant negative Rac1 (N17Rac1)
We examined the effect of the annexin peptide Ac2-26 on both basal and TNFa stimulated superoxide release, ICAM-1and VCAM-1 gene expression since Ac2-26 has previously been shown to block the response to TNFa
Summary
Annexin-1 ( termed lipocortin-1) is the first member of the annexin superfamily which, in humans, consists of at least 12 members each of which has a unique N-terminal sequence [1]. The annexin-1 peptide and its N-terminal derivative Ac2-26 have been shown to have protective effects in both brain and cardiac tissue following ischemia/reperfusion injury [2,3,4]. These effects have been attributed to the anti-inflammatory actions of annexin-1 and Ac2-26. Annexin-1 is found intracellularly in gelatinase granules of neutrophils [5] and in human serum, in inflammatory conditions such as myocardial infarction [6] and colitis [7] These findings, together with observations from in vitro and in vivo models of inflammation strongly suggest an anti-inflammatory role for annexin-1. Data suggest that these effects are mediated through the specific interaction of annexin-1 with the formyl peptide receptor (FPR) [10] and FPR like receptor (FPRL-1) [11]
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