Long-term Intake of Pasta Containing Barley (1–3)Beta-D-Glucan Increases Neovascularization-mediated Cardioprotection through Endothelial Upregulation of Vascular Endothelial Growth Factor and Parkin

Figures

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  Figure 1. Long-term intake of P-BBG diet prevents body weight gain without altering glucose tolerance. (A) Time-dependent effects of diet supplemented with P-BBG (n = 15) or regular pasta (Control; n = 15) on murine body weight. (B) Time-dependent changes in food intake in both experimental groups. (C) Levels of blood glicemia before (T0) and after intraperitoneal bolus of glucose solution (1 mg/g body weight) at baseline in P-BBG (n = 15) and Control group (n = 15). (D) Levels of blood glicemia before (T0) and after intraperitoneal bolus of glucose solution (1 mg/g body weight) after 5 weeks of experimental feeding in both experimental groups. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight). All measurements are mean ± SD. *p < 0.05 vs. 0 weeks; § p < 0.05 vs. Control.

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  Figure 2. Long-term intake of P-BBG diet does not alter cardiac function. (A) The heart rate at baseline and after five weeks of diet is similar in P-BBG (n = 15) and Control (n = 15) mice. (B) The sustained intake of diet supplemented with P-BBG does not alter both the end-systolic and end-diastolic left ventricular (LV) wall thickness, which are similar to Control mice. (C) The indexes of global LV function are not altered by diet in each experimental group. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight); EST: end-systolic thickness; EDT: end-diastolic thickness; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening. All measurements are mean ± SD.

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  Figure 3. Effects of long-term intake of P-BBG diet on left ventricular ischemia/reperfusion injury. (A) The survival rate of mice chronically fed with P-BBG diet (n = 15) exposed to cardiac ischemia/reperfusion injury is significantly higher than Control (n = 15) mice. (B) P-BBG diet reduced the left ventricular (LV) infarct size compared to Control mice. The LV infarct size was quantified as a percentage of the ischemic area at risk (IS/AAR (%)) (see methods section). (C) Effects of Control diet on the occurrence of contraction band necrosis in Masson (black arrows) and in hematoxylin-eosin (transparent arrows) staining of LV tissue. (D) Effects of P-BBG diet on the occurrence of contraction band necrosis in Masson and in hematoxylin-eosin staining of LV tissue. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight); IS/AAR: infarct size/area at risk; HE: hematoxylin-eosin. All measurements are mean ± SD. *p < 0.05 vs Control.

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  Figure 4. Effects of long-term intake of P-BBG diet on left ventricular capillary and arteriolar density, VEGF and dectin-1 protein levels. (A) Representative images of left ventricular (LV) coronary arterioles stained with α–smooth muscle actin in Control and P-BBG hearts. (B) The P-BBG diet (n = 15) increased the number of LV coronary arterioles compared to Control group (n = 15); the results are shown as number of arterioles per mm2. (C) Representative images of LV coronary capillaries stained with CD31 in Control and P-BBG hearts. (D) The P-BBG diet (n = 15) increased the number of LV coronary capillaries compared to Control diet (n = 15); the results are shown as number of capillaries per mm2. (E) Representative images of myocardial VEGF detected in Control and P-BBG left ventricles. (F) The P-BBG diet (n = 15) determined a detectable elevation of myocardial VEGF compared to Control diet (n = 15); the results are shown as arbitrary units of intense VEGF staining. (G) P-BBG diet (n = 7) increased the myocardial protein expression of VEGF compared to Control diet (n = 7). Levels of VEGF are expressed as arbitrary units of VEGF (24 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images…

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  Figure 5. Effects of P-BBG diet on levels of myocardial oxidative stress, cleaved caspase-3, HIF-1α, pAkt/Akt, pSTAT3/STAT3 and pNF-kB/NF-kB. (A) Representative images of DHE (dihydroethidium) staining of Control and P-BBG left ventricles. (B) P-BBG diet (n = 15) prevented an increase in anion superoxide generation following cardiac ischemia/reperfusion injury compared to Control diet (n = 15); the results are shown as arbitrary units of fluorescence intensity (see Methods). (C) The myocardial levels of cleaved caspase-3 were lower in P-BBG (n = 7) than Control (n = 7) hearts. Levels of cleaved caspase-3 are expressed as arbitrary units of cleaved caspase-3 (19 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of cleaved caspase 3 AND GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1C. (D) The myocardial levels of HIF-1α are similar in P-BBG (n = 7) and Control (n = 7) hearts. Levels of HIF-1α are expressed as arbitrary units of HIF-1α (110 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of HIF-1α and GAPDH are shown. The full-length blots/gels are presented in Supplementary…

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  Figure 6. Effects of P-BBG diet on levels of myocardial p53 and Parkin. (A) P-BBG diet (n = 7) increased the myocardial protein levels of p53 compared to Control (n = 7) diet. Levels of p53 are expressed as arbitrary units of p53 (53 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of p53 and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1F. (B) P-BBG diet (n = 7) increased the myocardial protein levels of Parkin compared to Control diet (n = 7). Levels of Parkin are expressed as arbitrary units of Parkin (52 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of Parkin and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1G. (C) Representative images of Parkin localization in coronary endothelium of Control left ventricles. (D) Representative images of Parkin localization in coronary endothelium of P-BBG left ventricles. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight); GAPDH: glyceraldehyde 3-phosphate dehydrogenase. All measurements are mean ± SD. *p < 0.05 vs. Control.

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  Figure 7. Acute oxidative stress does not affect levels of H4 histone acetylation, VEGF and MnSOD in viable endothelial cells chronically treated with barley β-D-glucan. (A) 7-day treatment with 3% barley β-D-glucan (BBG) increases the endothelial levels of acetyl H4 histone compared to untreated HUVECs, which are not affected by acute oxidative stress. Levels of acetyl H4 (14 kDa, MW)/H4 (14 kDa, MW), normalized on GAPDH levels, are expressed as arbitrary units. Representative images of cropped densitometric bands of acetyl H4, H4 and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 2A. (B) The protein levels of VEGF in HUVECs increase after long-term exposure to 3% BBG compared to untreated cells at rest and during acute oxidative stress. Levels of VEGF are expressed as arbitrary units of VEGF (24 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of VEGF and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 2B. (C) The protein levels of MnSOD in HUVECs are increased after long-term exposure to 3% BBG compared to untreated cells, yet are not altered after 1 h exposure to hydrogen…

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  Figure 8. BBG induces the protein expression of Parkin independently of p53 activity. (A) 7-day treatment with 3% barley β-D-glucan (BBG) increases the endothelial levels of p53 compared to untreated HUVECs. Levels of p53 are expressed as arbitrary units of p53 (53 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of p53 and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 2D. (B) The sustained co-treatment of HUVECs with 3% BBG and PFT-α (Inhibitor) does not impair cell viability. BBG preserves the viability of H2O2-stressed cells, but not Inhibitor. (C) The sustained treatment of HUVECs with 3% BBG increases the protein levels of Parkin compared to untreated cells (Control). The co-treatment with PFT-α does not affect the BBG-induced protein expression of Parkin. In addition, the decay of Parkin protein expression in stressed BBG-treated cells is smaller than in stressed untreated cells. (D) HUVECs co-treated with 3% BBG and PFT-α show lower protein levels of VEGF. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; PFT-α: pifithrin-alpha; H2O2: hydrogen peroxide; VEGF: vascular endothelial growth factor. All…

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  Figure 9. BBG prevents the rise of anion superoxide levels in a p53-dependent manner. (A) Representative images of DHE (dihydroethidium) staining of HUVECs at rest and after acute oxidative stress (+H2O2). (B) At rest, the 7-day treatment with 3% BBG (β-D-Glucan), PTF-α (Inhibitor) and β-D-Glucan + Inhibitor did not increase the anion superoxide levels in untreated HUVECs (Control). At stress (+H2O2), β-D-Glucan prevented an increase in anion superoxide generation compared to untreated cells (Control). Conversely, the 7-day co-treatment with β-D-Glucan + Inhibitor precluded the anti-oxidant effects of BBG in cells exposed to acute oxidative stress. The results are shown as arbitrary units of fluorescence intensity (see Methods). PFT-α: pifithrin-alpha; H2O2: hydrogen peroxide. All measurements are mean ± SD. *p < 0.05 vs. corresponding resting condition; #p < 0.05 vs. Control + H2O2; §p < 0.05 vs. Inhibitor + H2O2; ^p < 0.05 vs. β-D-Glucan + Inhibitor + H2O2.

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  Figure 10. Proposed mechanism of BBG-mediated regulation of endothelial tolerance to ischemia-reperfusion injury The proposed mechanism of BBG-mediate regulation of endothelial tolerance to oxidative stress following ischemia-reperfusion injury through upregulation of VEGF and Parkin. BBG: barley-derived (1.3) β-D-Glucan; HDAC: class I histone deacetylase; Ac: acetyl; VEGF: vascular endothelial growth factor; VEGFR1: vascular endothelial growth factor receptor type 1; HIF-1α: hypoxia-inducible factor 1-alpha; MnSOD: manganese superoxide dismutase; O2−: anion superoxide.