Annexin Peptide Ac2-26 Suppresses TNFα-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells

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.

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

“…[15,35,36] In addition, it was reported that ANXA1 peptide Ac2-26 suppressed TNF-α-induced inflammatory response via inhibition of VCAM-1 and ICAM-1 in endothelial cells. [37] In the present study, HDL-induced ANXA1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin in TNF-α-activated endothelial cells. Our novel results indicated that ANXA1 was a crucial mediator for anti-inflammation, which enriched protective mechanism of HDL.…”

Differential effects of superoxide dismutase and superoxide dismutase/catalase mimetics on human breast cancer cells

“…HMECs and MDA-MB-231 were seeded in 12-well plates (BD Falcon TM , New South Wales, Australia) and transfected using Lipofectamine 2000 (Life Technologies, Victoria, Australia) 400 ng of pGL3-NF-jB and 100 ng of a reference plasmid (pRL-SV40; Promega, Wisconsin, USA) simultaneously. Luciferase assay was performed using dual luciferase kit from Promega (Promega, Wisconsin, USA) as described previously [22].…”

Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

“…Additional evidence in dermal microvascular ECs indicated that activation of NF-kB, a downstream effector of TNF-aemediated inflammatory signaling, was involved in TNF-aeinduced ROS generation and involved NADPH oxidase activation. 15 In aortic vascular ECs, cross talk between oxidative and inflammatory pathways was proposed to affect endothelial dysfunction and cardiovascular disease. 16 We previously found that VEGF promoted CEC migration by activating the small GTPase Rac1.…”