Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Abstract

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2 O2 -stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2 (-) ) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2 (-) , cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.

Keywords: angiogenesis; antioxidants; beta-glucan; endothelial cells; histone deacetylases.

Fig. 1

β-d-glucan treatment promotes endothelial cell survival and MnSOD expression under chronic oxidative stress. (A) HUVECs survival after 24 hrs treatment with 50 μM H₂O₂, alone or in combination with 3% β-d-glucan. Untreated and unstressed cells were used as control. (B) Quantification of the relative intensity of fluorescence in DHE-positive cells, compared to control condition. (C) Quantification of the relative intensity of luminescence in cells producing superoxide anion, compared with control condition. (D) Representative western blot bands for MnSOD and GAPDH. (E) Measurement of the level of MnSOD expression normalized over loading control (GAPDH). (mean ± SD; n = 4) *P < 0.05 versus control; §P < 0.05 versus H₂O₂.

Fig. 2

β-d-glucan treatment increases eNOS phosphorylation and histone H4 acetylation in viable endothelial cells. (A) Representative western blot bands for HSP70, HIF1-α, p-AKT, AKT, p-eNOS, eNOS and GAPDH in each experimental condition. (B) Measurement of the level of HSP70, HIF1-α, AKT, eNOS expression normalized over loading control (GAPDH). Phosphorylation level of eNOS and AKT was quantified normalizing the amount of phosphorylated protein over total protein: p-eNOS/eNOS and p-AKT/AKT. (mean ± SD; n = 4) *P < 0.05 versus control.

Fig. 3

β-d-glucan treatment increases nitric oxide generation in stressed endothelial cells. (A) Representative images of DAF staining of HUVECs at rest or with H₂O₂, in the presence of β-d-glucan or vehicle. (B) Quantification of the relative intensity of fluorescence in DAF-FM diacetate positive cells at rest or during oxidative stress (H₂O₂), in the presence of β-d-glucan or vehicle. (mean ± SD; n = 3) *P < 0.05 versus control.

Fig. 4

β-d-glucan treatment increases histone H4 acetylation and promotes human capillary formation in vitro. (A) Representative western blot bands for histone H4 (H4) and acetylated histone H4 (Acetyl-H4) in each experimental condition. Measurement of the level of H4 expression normalized over loading control (GAPDH). Acetylation level of H4 was quantified normalizing the amount of Acetyl-H4 protein over total protein. (B and C) Representative images of tube formation from HUVECs without exogenous growth factors. (D and E) Representative images of tube formation from HUVECs with exogenous growth factors. (F) Measure of total length of tubes from HUVECs without exogenous growth factors. (G) Measure of total length of tubes from HUVECs with exogenous growth factors. Intrinsic tube formation ability was tested alone (control) or with 3% β-d-glucan; at rest (left side) or during oxidative stress (stress, right side) (mean ± SD; n = 4) *P < 0.05 versus control; **P < 0.01 versus control; §P < 0.05 versus H₂O₂.

Fig. 5

β-d-glucan treatment rescues the vasculogenic activity under chronic oxidative stress in vivo. (A) Representative images of Zebrafish transgenic Tg (kdrl: EGFP)^s843Tg embryos at 24 hpf (hours post-fertilization), alone (control) or treated at 70% epiboly stage with 3% β-d-glucan, PMA, and PMA+3% β-d-glucan. Brackets: normal caudal plexus; stars: injured caudal plexus; scale bar 100 μm. (B) At each experimental condition, representative images of caudal view of embryos, at 24 hpf. DLAV: dorsal longitudinal anastomotic vessel (CVP, caudal vein plexus). White arrow: end of DLAV; scale bar 150 μm. (C) Measurement of the distance between the end of DLAV and the tip of CPV at each experimental condition. *P < 0.05 versus control.

Fig. 6

NaBu does not increase Endothelial MnSOD expression. (A) HUVECs survival after 24 hrs treatment with 50 μM H₂O₂, alone or with increasing dose of sodium butyrate (NaBu) ranging from 5 to 500 μM. (B) Representative western blot bands for MnSOD, histone H4 (H4), acetylated histone H4 (Ac-H4) and GAPDH in each experimental condition. (C) Measurement of the level of H4 expression normalized over loading control (GAPDH). Acetylation level of H4 was quantified normalizing the amount of Acetyl-H4 protein over total protein. (D) Measurement of the level of MnSOD expression normalized over loading control (GAPDH). nt: control (mean ± SD; n = 4) *P < 0.05 versus control; **P < 0.01 versus control: ***P < 0.001 versus control; §P < 0.05 versus H₂O₂.

Fig. 7

NaBu does not affect β-d-glucan-induced MnSOD up-regulation under chronic oxidative stress. (A) Representative western blot bands for MnSOD, histone H4 (H4), acetylated histone H4 (Ac-H4) and GAPDH in each experimental condition. (B) Measurement of the level of MnSOD and H4 expression normalized over loading control (GAPDH). Acetylation level of H4 was quantified normalizing the amount of Acetyl-H4 protein over total protein. Cells were exposed to H₂O₂ for 24 hrs, alone or in combination with 3% β-d-glucan, NaBu (5 μM), or both. Unstressed and untreated cells were used as control (mean ± SD; n = 4). *P < 0.05 versus control; §P < 0.05 versus H₂O₂.

Fig. 8

NaBu does not affect β-d-glucan-induced superoxide anion down-regulation under chronic oxidative stress. (A) Quantification of the relative intensity of fluorescence in DHE positive cells in each experimental condition at rest or during oxidative stress (H₂O₂). (B) Representative images of DHE staining of HUVECs at rest or with H₂O₂, in the presence of either β-d-glucan, NaBu (5 μM), or both (mean ± SD; n = 4). *P < 0.05 versus control; ***P < 0.001 versus control; §P < 0.05 versus H₂O₂.

Fig. 9

NaBu does not affect β-d-glucan-induced capillary formation under chronic oxidative stress. (A) In vitro angiogenesis assay without exogenous growth factors. Intrinsic tube formation ability was tested, alone (untreated) or with 3% β-d-glucan, NaBu (5 μM,) or both; at rest (left side) or during oxidative stress (right side). (C) In vitro angiogenesis assay with exogenous growth factors. Tube formation ability was tested, alone (untreated) or in combination with 3% β-d-glucan, NaBu (5 μM), or both; at rest (left side) or during oxidative stress (right side; mean ± SD; n = 4). *P < 0.05 versus control; §P < 0.05 versus β-d-glucan.