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. 2013 Apr 24;8(4):e60790.
doi: 10.1371/journal.pone.0060790. Print 2013.

Annexin peptide Ac2-26 suppresses TNFα-induced inflammatory responses via inhibition of Rac1-dependent NADPH oxidase in human endothelial cells

Hitesh M Peshavariya  1 , Caroline J Taylor, Celeste Goh, Guei-Sheung Liu, Fan Jiang, Elsa C Chan, Gregory J Dusting
Affiliations

Annexin peptide Ac2-26 suppresses TNFα-induced inflammatory responses via inhibition of Rac1-dependent NADPH oxidase in human endothelial cells

Hitesh M Peshavariya et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tumour necrosis factor (TNFα) stimulated NADPH-dependent superoxide release and cell adhesion molecule expression in HMECs.
(A) Superoxide production detected by lucigenin enhanced chemiluminescence (normalized to control without TNFα stimulation) following 6 and 24 h TNFα treatment (20 ng/ml). (B) Intracellular cell adhesion molecule-1 (ICAM-1) mRNA (expressed as fold change) following 6 and 24 h TNFα stimulation (C) ICAM-1 protein expression following 6 and 24 h TNFα stimulation. (D) Vascular cell adhesion molecule-1 (VCAM-1) mRNA (expressed as fold change) following 6 and 24 h TNFα stimulation (E) VCAM-1 protein expression following 6 and 24 h TNFα stimulation. Data are mean ± SEM, n = 5 to 8. * P<0.05 vs control.
Figure 2
Figure 2. The stimulatory effect of tumour necrosis factor (TNFα) is NADPH oxidase dependent in HMECs.
(A) Inhibitors of other enzymatic sources of ROS, allopurinol (Allo, 100 µM), indomethacin (Indo, 3 µM), L-NAME (100 µM) and rotenone (Rote, 1 µM) did not affect TNFα-induced superoxide generation, but it was inhibited by the Nox inhibitor diphenyleneiodonium (DPI, 1 µM). Cells were treated with TNFα (20 ng/ml) for 24 h prior to incubation with inhibitors, which were incubated with HMECs for 30 min prior to measurement of superoxide. (B, C) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 mRNA upregulation. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. (D) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 protein expression. GAPDH was used as a reference. (E,F) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on mRNA expression of ICAM-1 and VCAM-1 respectively. mRNA expression was expressed as fold change and normalized to control (Ctrl) with TNFα stimulation. (G) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on protein expression of ICAM-1 and VCAM-1. GAPDH was used to confirm equal loading. Data are mean ± SEM; n = 3. * P<0.05 vs control without TNFα stimulation; † P<0.05 vs control with TNFα stimulation.
Figure 3
Figure 3. Annexin-1 peptide Ac2-26 inhibited tumour necrosis factor (TNFα)-mediated response in HMECs.
Ac2-26 (0.5 and 1.5 µM) suppressed TNFα (20 ng/ml) stimulated (A) NADPH dependent superoxide production measured by lucigenin-enhanced chemiluminescence and (B) total intracellcular ROS production detected by DCFH2-DA. (C) Ac2-26 (0.5 µM) inhibited the stimulatory effect of TNFα on ICAM-1 mRNA expression, without affecting its basal level. (D) Ac2-26 (0.5 µM) inhibited the stimulatory effect of TNFα on VCAM-1 mRNA expression, without affecting its basal level. (E) FPRL-1 antagonist WRW4 prevented the inhibitory effect of Ac2-26 on TNFα (20 ng/ml) stimulated superoxide generation. mRNA expression data was normalized to control with TNFα stimulation. Data are mean ± SEM, n = 3 to 5. * P<0.05 vs control without TNFα stimulation; † P<0.05 vs control with TNFα.
Figure 4
Figure 4. The stimulatory effect of tumour necrosis factor (TNFα) on adhesion molecule gene upregulation requires Rac1 in HMECs.
(A) TNFα (2–50 ng/ml) reduced Nox4 gene expression (B) and pretreatment of Ac2-26 (0.5 µM) did not affect the TNFα mediated mRNA downregulation. TNFα (2–50 ng/ml) alone (C) and in combination of Ac2-26 (0.5 µM) (D) did not affect Nox2 mRNA expression. TNFα (20 ng/ml) stimulated ICAM-1 mRNA (E) and protein (G) and VCAM-1 mRNA (F) and protein (G) upregulation was blunted by Ac2-26 (0.5 µM) and diphenyleneiodonium (DPI, 1 µM) in cells trasnfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1 and VCAM-1 upregulation and this inhibition was not potentiated by pretreatment of Ac2-26 and DPI. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. GAPDH was used to confirm equal loading. Data are mean ± SEM, n = 5 to 8. * P<0.05 vs control without TNFα stimulation; † P<0.05 vs control with TNFα stimulation. There was no difference (not significant, NS) between control Ac2-26 and DPI in the presence of TNFα in cells transfected with N17Rac1.
Figure 5
Figure 5. The effect of annexin-1 peptide Ac2-26 on TNFα-induced NF-κB promoter activity in HMECs.
TNFα (20 ng/ml) induced NF-κB promoter activity is decreased by pretreatment of Ac2-26 (0.5 µM) and diphenyleneiodonium (DPI, 1 µM). Luciferase activity is expressed as relative luminescence units (RLU) and is normalised to control without TNFα stimulation. Data are mean ± SEM, n = 4. *P<0.05 vs control without TNFα, †P<0.05 vs control with TNFα.
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Grants and funding

This work was supported by Project Grants from the National Health and Medical Research Council (NHMRC) of Australia and Grants-in-Aid from the National Heart Foundation of Australia. GJD also receives a Principal Research Fellowship from NHMRC. The O'Brien Institute and Center for Eye Research Australia acknowledges the Victorian State Government's Department of Innovation, Industry and Regional Development's Operational Infrastructure Support Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.