Role of NADPH Oxidase-4 in human endothelial progenitor cells

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization

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

<p class="Para">Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂) have been implicated in development and progression of breast cancer. In the present study, we have evaluated the effects of the superoxide dismutase (SOD) mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 on superoxide and H₂O₂ formation as well as proliferation, adhesion, and migration of MCF-7 and MDA-MB-231 cells. Superoxide and H₂O₂ production was examined using dihydroethidium and Amplex red assays, respectively. Cell viability and adhesion were measured using a tetrazolium-based MTT assay. Cell proliferation was determined using trypan blue assay. Cell cycle progression was analyzed using flow cytometry. Clonal expansion of a single cell was performed using a colony formation assay. Cell migration was measured using transwell migration assay. Dual luciferase assay was used to determine NF-κB reporter activity. EUK 134 effectively reduced both superoxide and H₂O₂, whereas MnTmPyP removed superoxide but enhanced H₂O₂ formation. EUK 134 effectively attenuated viability, proliferation, clonal expansion, adhesion, and migration of MCF-7 and MDA-MB-231 cells. In contrast, MnTmPyP only reduced clonal expansion of MCF-7 and MDA-MB-231 cells but had no effect on adhesion and cell cycle progression. Tumor necrosis factor-alpha-induced NF-κB activity was reduced by EUK 134, whereas MnTmPyP enhanced this activity. These data indicate that the SOD mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 exert differential effects on breast cancer cell growth. Inhibition of H₂O₂ signaling using EUK 134-like compound might be a promising approach to breast cancer therapy.</p

Hypoxic Conditioning Enhances the Angiogenic Paracrine Activity of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O(2)) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC(CM)) was assessed by implanting ASC(CM)-treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC(CM) treatment compared to normoxic control ASC(CM) treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC(CM) vs. 4.1%±0.7% in the normoxic ASC(CM), P<0.05). Reduction of VEGF-A and ANG levels in the ASC(CM) with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC(CM) were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC(CM) for in vivo angiogenesis-related applications as an effective cell-free technology

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

<div><p>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.</p></div

Annexin-1 peptide Ac2-26 inhibited tumour necrosis factor (TNFα)-mediated response in HMECs.

<p>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, <i>n</i> = 3 to 5. * <i>P</i><0.05 <i>vs</i> control without TNFα stimulation; ^†<i>P</i><0.05 <i>vs</i> control with TNFα.</p

The stimulatory effect of tumour necrosis factor (TNFα) is NADPH oxidase dependent in HMECs.

<p>(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<i>; n</i> = 3. * <i>P</i><0.05 <i>vs</i> control without TNFα stimulation; † <i>P</i><0.05 <i>vs</i> control with TNFα stimulation.</p

Tumour necrosis factor (TNFα) stimulated NADPH-dependent superoxide release and cell adhesion molecule expression in HMECs.

<p>(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, <i>n</i> = 5 to 8. * <i>P</i><0.05 <i>vs</i> control.</p

The effect of annexin-1 peptide Ac2-26 on TNFα-induced NF-κB promoter activity in HMECs.

<p>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, <i>n</i> = 4. *<i>P</i><0.05 vs control without TNFα, ^†P<0.05 vs control with TNFα.</p