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

Barley (1–3)β-D-Glucan (BBG) enhances angiogenesis. Since pasta is very effective in providing a BBG-enriched diet, we hypothesized that the intake of pasta containing 3% BBG (P-BBG) induces neovascularization-mediated cardioprotection. Healthy adult male C57BL/6 mice fed P-BBG (n = 15) or wheat pasta (Control, n = 15) for five-weeks showed normal glucose tolerance and cardiac function. With a food intake similar to the Control, P-BBG mice showed a 109% survival rate (P < 0.01 vs. Control) after cardiac ischemia (30 min)/reperfusion (60 min) injury. Left ventricular (LV) anion superoxide production and infarct size in P-BBG mice were reduced by 62 and 35% (P < 0.0001 vs. Control), respectively. The capillary and arteriolar density of P-BBG hearts were respectively increased by 12 and 18% (P < 0.05 vs. Control). Compared to the Control group, the VEGF expression in P-BBG hearts was increased by 87.7% (P < 0.05); while, the p53 and Parkin expression was significantly increased by 125% and cleaved caspase-3 levels were reduced by 33% in P-BBG mice. In vitro, BBG was required to induce VEGF, p53 and Parkin expression in human umbelical vascular endothelial cells. Moreover, the BBG-induced Parkin expression was not affected by pifithrin-α (10 uM/7days), a p53 inhibitor. In conclusion, long-term dietary supplementation with P-BBG confers post-ischemic cardioprotection through endothelial upregulation of VEGF and Parkin.

. 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.

Results
Effects of P-BBG diet on body weight, food intake and blood glucose levels. As shown in Fig. 1A, starting from the baseline, mice fed with the P-BBG diet did not gain weight compared to the Control group. The difference in body weight gain between groups was significant after three, four and five weeks, yet the daily food intake was similar between the two groups ( Fig. 1B). At the fifth week of the diet, the basal blood glucose levels (T0) were similar to corresponding values before the diet (baseline) in each experimental group. During the intraperitoneal glucose tolerance test (IPGTT), the profile of blood glucose levels was similar between the two groups before (Fig. 1C) and after (Fig. 1D) the experimental feeding.
Effects of P-BBG diet on heart rate and cardiac function. As shown in Fig. 2, after five weeks of regular feeding, the P-BBG diet did not adversely affect the heart rate, the left ventricular (LV) end-diastolic wall thickness, and the LV systolic function of mice. Fig. 3A, 44% of the mice fed with the P-BBG diet survived after one hour of post-ischemic reperfusion, whereas only 21% of the Control animals survived after a similar injury at the same time. Hearts harvested from Control mice that had undergone I/R, showed a larger LV necrotic infarct area than those collected from infarcted P-BBG mice (Fig. 3B). The Masson and hematoxylin-eosin staining of LV tissue revealed contraction band necrosis in the hearts of Control mice (Fig. 3C), but not in treated mice (Fig. 3D).

Effects of P-BBG diet on survival, infarct scar size and myocardial structure after left ventricular ischemia/reperfusion injury. As shown in
Effects of P-BBG diet on capillary and arteriolar density, VEGF and dectin-1 protein levels. The myocardial capillary (Fig. 4A-B) and arteriolar ( Fig. 4C-D) density of the P-BBG mice was significantly higher than that of the Control hearts. As shown in panels E and F of Fig. 4, cardiac VEGF was considerably more evident in P-BBG than in the Control mice. Similarly, the VEGF protein levels of P-BBG hearts were significantly higher than those of the Control hearts (Fig. 4G). Higher levels of VEGF did not affect the myocardial expression of dectin-1, whose protein levels were similar in both groups (Fig. 4H).
Effects of P-BBG diet on levels of myocardial oxidative stress, cleaved caspase-3, HIF1-α, pAkt/ Akt, pSTAT3/STAT3 and pNF-kB/NF-kB. As shown in panels A and B of Fig. 5, the myocardial production of anion superoxide in response to I/R insult was significantly reduced in P-BBG compared to the Control hearts. At one hour of post-ischemic reperfusion, the myocardial cleavage of caspase-3 in the P-BBG hearts was markedly attenuated compared to the Control hearts (Fig. 5C) in the presence of similar protein levels of Effects of P-BBG diet on myocardial levels of p53 and Parkin. As shown in Fig. 6A, the myocardial protein expression of p53 was higher in P-BBG than the Control mice. Conversely, Parkin protein levels were significantly upregulated in P-BBG compared with the Control hearts (Fig. 6B). Although Parkin was weakly detectable in the Control hearts (Fig. 6C), its expression was highly focused in the coronary endothelial cells of the P-BBG hearts (Fig. 6D).
Acute oxidative stress does not affect the levels of H4 histone acetylation, VEGF and MnSOD in viable endothelial cells treated with barley β-D-glucan. As shown in Fig. 7, the BBG-induced increase in endothelial levels of H4 histone acetylation (panel A), VEGF (panel B) and MnSOD (panel C) were not altered during the transient exposure of HUVECs to exogenous H 2 O 2 . The decay of cell viability following the acute exposure to hydrogen peroxide was significantly preserved by long-term treatment with BBG (panel D).
BBG induces the protein expression of Parkin independently of p53 activity. The interplay between p53, VEGF and Parkin governs tissue homeostasis through the modulation of angiogenesis 31 and cellular tolerance to stress 32 . BBG increases the expression of p53 (Fig. 8A) without affecting cell viability, but preserving the cell viability under H 2 O 2 (Fig. 8B). Although pifithrin-alpha (PFT-α), a well-known inhibitor of p53 activity, does not affect cell viability in resting conditions, it is unable to protect the cells against acute oxidative stress (Fig. 8B). Interestingly, the BBG-induced increase in protein levels of Parkin is not affected by PFT-α. In line with the BBG effects on the viability of H 2 O 2 -stressed cells during co-exposure to PFT-α (Fig. 8B), BBG attenuates the decay of Parkin protein levels in HUVECs exposed to acute oxidative stress independently of p53 activity   Fig. 8C). Conversely, the inhibition of p53 activity by PFT-α counteracts the BBG-induced VEGF expression in HUVECs at rest and during stress (Fig. 8D).
BBG prevents the rise of anion superoxide levels in a p53-dependent manner. BBG precludes the rise of anion superoxide levels in H 2 O 2 -stressed endothelial cells, as previously reported by us 22 . HUVECs co-treated for seven days with BBG and non-toxic concentration of PFT-α do not show anion superoxide levels higher than Control cells (Fig. 9A). Interestingly, the inhibition of p53 activity by PFT-α counteracts the BBG-induced reduction of anion superoxide levels in HUVECs exposed to acute oxidative stress (Fig. 9B).

Discussion
Our study demonstrates that the long-term supplementation of a low fat diet with pasta enriched with BBG increases the survival rate of mice undergoing LV I/R injury. The magnitude of the LV infarct scar size and of the contraction necrotic bands was safely attenuated in mice fed with P-BBG for five weeks. The dietary intake of P-BBG also prevented an increase in body weight without altering the systemic glucose tolerance, cardiac function, and the well-being of mice. Compared to the Control group, the histological analysis revealed a higher density of coronary capillaries and arterioles in the explanted hearts of P-BBG mice, which support higher tissue viability downstream of the obstructed coronary tree. Regarding the cardioprotective pathways, our data revealed, for the first time, the relationship between myocardial VEGF, p53 and Parkin protein upregulation, vasculogenesis enhancement and reduction of caspase-3 cleavage at the early stages of post-ischemic reperfusion conferred by the sustained dietary intake of P-BBG.
It is largely recognised that cardiac ischemic preconditioning enhances the myocardial protein expression of VEGF 33 , a growth factor that induces myocardial angiogenesis and cell survival 34 . In line with the cardioprotective effects of VEGF, other researchers have perfused the heart with VEGF 35 or have upregulated the myocardial VEGF expression using gene therapy 36,37 in order to enhance the local adaptive response naturally occurring under oxidative microenvironment. However, none of these invasive approaches may be ethically permissible in healthy subjects. Hence, the urgent need to develop a noninvasive strategy to enhance collateral artery growth in the adult myocardium and to promote the survival of cardiac cells following I/R injury.
In a previous study, we demonstrated that chronic exposure of endothelial cells to water-soluble BBG enhances the pro-angiogenic and pro-survival expression of manganese superoxide dismutase (MnSOD) 22 , which is a mediator of ischemic preconditioning 38 .
In the current study, we thus hypothesized that the daily dietary intake of water-soluble BBG might promote the formation of new coronary vessels in healthy subjects and prevent myocardial I/R injury.
For this purpose, healthy mice were fed with a low fat diet supplemented with pasta made with a mixture of barley flour and wheat flour, which guarantees a high content of viscous polysaccharides and a regular BBG intake. Our hypothesis is supported by the evidence that pasta safely provides a BBG-enriched diet 27,39 . To the best of our knowledge, no previous study has assessed the effects of diet supplemented with BBG-enriched pasta to counteract the onset of myocardial I/R injury.
First, we have observed that P-BBG-enriched diet precludes gain weight in mice. Our data are consistent with recent clinical study showing that daily intake of barley beta-D-Glucan prevents visceral fat obesity and body weight gain in humans 40 . In fact, the ability of soluble BBG to form highly viscous solutions in the human gut slows gastric emptying, digestion and absorption of dietary fat 41 .
In our study, the survival rate at an early stage of post-ischemic reperfusion was significantly higher in mice fed with P-BBG than with the wheat pasta. Compared to the Control group, the IS/AAR ratio revealed that the left ventricles of P-BBG mice were less injured and the contraction band necrosis, a hallmark of immediate necrotic cell death during the first minutes of reperfusion 42 , was undetectable in treated hearts. Since myocardial injury is induced after an overnight fasting period, it is conceivable that cardioprotection depends on increased myocardial angiogenesis due to the sustained intake of P-BBG. Our hypothesis is well supported by previous clinical evidence showing that individual tolerance to cardiac post-ischemic reperfusion injury is due to a higher density of coronary collaterals 4,5 .
In line with our hypothesis, the myocardial density of coronary capillaries and arterioles was higher in mice fed with a diet supplemented with P-BBG than with regular pasta. Although the myocardial protein levels of dectin-1, a receptor that mediates endothelial β-D-Glucan effects 43 , were similar in both experimental groups, the marked increase in VEGF protein levels in the P-BBG hearts is noteworthy and is closely linked to the regular intake of BBG and to the weight loss. Indeed, we cannot exclude that BBG-based diet may induce VEGF expression in other tissues. It is known that an increase in VEGF expression in adipose tissue can result in increasing vascular density 44 , in reducing adipocyte size and in precluding gain weight in mice 45,46 .
In addition, the myocardial levels of anion superoxide decreased significantly in the reperfused hearts of mice fed with P-BBG rather than in the Control mice. are shown. The full-length blots/gels are presented in Supplementary Figure 1B. The myocardial protein levels of dectin-1 are similar in both Control (n = 7) and P-BBG (n = 7) hearts. Levels of dectin-1 are expressed as arbitrary units of dectin-1 (28 kDa, MW)/GAPDH (37 kDa, MW) ratio. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight); α-SMA: α-smooth muscle actin; CD31: cluster of differentiation 31; VEGF: vascular endothelial growth factor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. All measurements are mean ± SD. *p < 0.05 vs. Control.
SCIENTIFIC REPORTs | 7: 13424 | DOI:10.1038/s41598-017-13949-1 These new in vivo findings strongly support our previous study 22 . In fact, VEGF likely enhances the local expression of MnSOD 23 , a key anti-oxidant enzyme, which contributes to the angiogenesis 22 and higher cardiac tolerance against ischemia/reperfusion injury 47 in P-BBG mice.
Since natural compounds may promote VEGF/antioxidant signaling and inhibit the onset of apoptosis in hearts undergoing I/R 48 , we assessed the myocardial levels of cleaved caspase-3, an established pro-apoptotic factor. The levels of cleaved caspase-3 were significantly lower in P-BBG hearts, even though the myocardial protein expression of HIF-1α, a transcriptional factor involved in the induction of VEGF expression 49 as well as in the inhibition of apoptosis 50 , was similar in both experimental groups. These results are in agreement with our previous in vitro study 22 and suggest the activation of different signaling pathways by BBG.
Although several studies have found a key role for phosphorylated Akt to mediate the cardioprotective phosphatidylinositide 3-kinase (PI3K) signaling pathway 51 during preconditioning, the myocardial levels of phospho-Akt/total-Akt ratio were the same in treated and untreated mice.
We then examined the role of the P-BBG diet on myocardial STAT3 signaling pathways since STAT3 activation has been implicated in the inhibition of caspase-3 cleavage 52 and also in the upregulation of MnSOD 53 and VEGF 54 genes. Surprisingly, cardiac STAT3 was also similarly expressed and phosphorylated in both experimental groups.
Lastly, although active NF-kB promotes the VEGF-dependent modulation of MnSOD expression 55 , the myocardial levels of phospho-NF-kB/total-NF-kB ratio were similar in both the P-BBG and Control hearts.
Although the activation of the pathways so far investigated plays a role in regulating VEGF levels during preconditioning, it seems that they are not activated by BBG in our model. Since p53 has been recently considered an important regulator of cardiac gene transcription 56 , we have hypothesized that it may play a key role in mediating the BBG-induced cardioprotection.
Farhang Ghahremani et al. 57 demonstrated that p53 induces the expression of VEGF. In our study, the expression of p53 in P-BBG hearts was higher than in the Control hearts. It is conceivable that p53 leads to VEGF promoter activation by acting synergistically with HIF-1α, as previously demonstrated by others 57 .
We have demonstrated, for the first time, that dietary intake of P-BBG induces myocardial p53 upregulation without promoting systemic toxicity or cardiac cell death. Our findings are not in agreement with a previous study that found that dietary anti-oxidants increase p53 levels to the point where they induce apoptosis 58 . This can be explained by the fact that P-BBG diet also increased the myocardial protein expression of Parkin, an E3 ubiquitin ligase, which plays a key role in promoting mitophagy 59 and in delaying caspase-3 activation 60 . Since healthy mice were safely fed with the P-BBG-enriched diet for five weeks, our results would seem to suggest that the physiological interplay between higher levels of p53 and Parkin may contribute to the non-invasive regulation of the VEGF expression and myocardial viability status.
As β-Glucan receptors are expressed on the surface of endothelial cells 61 , the upregulation of Parkin protein was localized in the coronary endothelial cells of P-BBG hearts. The binding affinity of dectin-1 to β-D-Glucan and its biological activity are similar in humans and mice 22,62 , it would thus appear that we have revealed BBG-dependent mechanisms in cultured HUVECs at rest and under acute oxidative stress. On day 7 after the exposure of HUVECs to 3% BBG, the levels of acetylated H4 histone, VEGF and MnSOD increased and were not affected by acute oxidative stress. Our in vitro results suggest that the acute oxidative burst induced by I/R injury in vivo did not counteract the BBG-induced regulatory effects on the abovementioned proteins. Finally, we have demonstrated that the 7-day treatment of HUVECs with BBG similarly increased the protein expression of p53 and Parkin in the presence of increased histone acetylation. Our data agree with previous studies showing that class I HDAC inhibitors, such as BBG, upregulate the protein expression of p53 63 and Parkin 64 . As a function of expression levels, p53 can either prevent or promote apoptosis 65 . In vitro, however, BBG-induced p53 upregulation did not affect the viability of endothelial cells.
Although the inhibition of p53 activity is essential to enhancing the mitophagic activity of Parkin 66 , it is still unknown whether the VEGF and Parkin protein expression induced by BBG depends on p53 activity. Consequently we performed additional experiments by treating HUVECs with BBG in the presence of PFT-α, an established inhibitor of p53 activity 66 . Although PFT-α did not alter the expression of Parkin, the BBG-induced upregulation of Parkin was not affected by inhibition of p53 activity.
At least, BBG mitigates the H 2 O 2 -induced reduction of Parkin protein expression independently of p53 activity. Hence, our results suggest that the Parkin expression induced by BBG is independent of p53 activity. Conversely, the BBG-induced increase in VEGF protein levels was hampered by PFT-α, which confirms that BBG increased the VEGF expression in a p53-dependent manner. Similarly, PFT-α hindered the BBG-induced decrease in anion superoxide levels in stressed cells, which confirms that BBG decreased the oxidative stress in a p53-dependent manner. Our findings reveal for the first time the impact of BBG on the simultaneous endothelial expression of VEGF and Parkin interacting differently with p53 activity. Moreover, we have demonstrated that the anti-oxidant effects of BBG depends on the p53 activation.    Therefore, by enhancing class I HDAC inhibition, the long-term dietary intake of P-BBG may simultaneously promote the neoangiogenesis and higher tolerance to oxidative stress due to enhanced endothelial expression of VEGF/MnSOD signaling and Parkin (the proposed mechanism is summarized in Fig. 10).

Conclusions
To the best of our knowledge, this is the first study to show in vivo data demonstrating that a sustained dietary intake of pasta enriched with BBG safely increases coronary collaterals, limits the infarct size, and reduces mortality. This is achieved through the simultaneous myocardial upregulation of p53, VEGF and Parkin without interfering with the ability of cardiac cells to phosphorylate Akt, STAT3 and NF-kB during the post-ischemic reperfusion. Higher p53, VEGF and Parkin protein levels in endothelial cells chronically treated with BBG are not affected by exposure to acute oxidative stress. Finally, Parkin responsiveness to BBG is not driven by the inhibition of p53 activity, which in turn may contribute to VEGF expression 31 and anion superoxide decay.
We believe our findings will help in the design a novel nutraceutical approach in the clinical noninvasive prevention of cardiac I/R injury.

Methods
Experimental protocol and animal model of left ventricular I/R injury. Thirty adult male C57BL/6 J mice (body weight 22-25 g; 4-8 weeks old) were housed with a constant light and dark phase of 12 hours at 20-23 °C. They were fed for five weeks with a low fat diet (LFD; 3.085 Kcal/g metabolizable energy; 3.5% fat; 17.6% proteins; A. Rieper SpA, BZ, Italy) supplemented with BBG-enriched pasta (3 g/100 g of dry weight) (P-BBG, n = 15) or regular wheat pasta (Control, n = 15), kindly provided by Granoro srl (Corato, Italy). The body weight and food intake of mice were assessed once a week. The composition of the diets is summarized in Table 1.
At the beginning and the end of the experimental feeding, transthoracic echocardiography 67 and an intraperitoneal glucose tolerance test 68 were performed in 6 h fasted mice. At the end of the fifth week of the diet, the mice underwent acute myocardial I/R, as previously described 69 . Briefly, the chest was opened and the left anterior descending coronary artery was transiently ligated at 2 mm lower than the tip of the left auricle in animals anesthetized with isoflurane (1.5-2%) in 100% oxygen with a flow rate of 0.4 L/min until loss of righting reflex, ventilated via a tracheal intubation at a tidal volume of 0.30 ml and respiratory rate of 100 breaths/min (Ugo Basile Srl, Varese, Italy). Body temperature was maintained constant between 36.8 °C and 37 °C by a thermoregulated surgical table connected to a rectal probe (Ugo Basile Srl, Varese, Italy). Thirty minutes of coronary occlusion was followed by reperfusion for 60 min under monitoring of three-lead electrocardiography (ECG). At the end of myocardial reperfusion, the LAD was reoccluded and the phtalocyanine blue dye was injected into the LV cavity to perfuse the nonischemic portions of the myocardium. Then, the heart was rapidly removed for infarct size, histological and molecular assessment. All animal procedures were approved by the Italian Ministry of Health and conducted in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.
Each parameter was measured using B-mode guided M-mode imaging in the parasternal short axis-view at the level of the papillary muscles. Images were analysed offline in a blinded fashion. The global cardiac function was calculated as the percentage ejection fraction of the left ventricle (%LVFE). Heart rate was simultaneously obtained with a three-lead ECG monitor.
Intraperitoneal glucose tolerance test. The glucose tolerance test was performed as previously described 68 . Briefly, glucose sterile solution was intraperitoneally injected (1 mg/g body weight) in each awake 6 h fasting mouse after baseline blood glucose measurement. The blood glucose levels were measured at 10, 20, 30, 60, and 120 min after bolus glucose.
Infarct size, histological and immunohistochemical assays. The LV infarct size was assessed as previously described 28 . Each one-millimeter-thick left ventricular transverse section was photographed with a light microscopy (Olympus BX43) at 10X original magnification and digitized by a video system (Olympus DP20  Table 1. Composition of normal diet supplemented with regular or functional pasta. P-BBG, low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight). camera) interfaced with a computer with dedicated software (CellSens Dimension, Olympus) for morphometric and/or color analysis. The infarct size (IS; non stained by 1% solution of 2,3,5-triphenyltetrazolium chloride, Sigma-Aldrich) was measured with Image J software, and expressed as percentage of the ischemic area at risk (AAR; non stained by phtalocyanine blue dye, Sigma-Aldrich Chemical Co, MO, USA). Serial 5-μm frozen heart sections were stained with H&E for overall morphology assessment. The density of the left ventricular coronary arterioles and capillaries stained by immunohistochemistry was quantified as the number of smooth muscle α-actin -(α-SMA, 1:100, Santa Cruz Biotechnology Inc, USA) and/or CD31-positive structures (1:100, Abcam Inc., Cambridge, UK) per mm 2 using Image J. Finally, to show the cell source of VEGF, additional LV sections were further stained with an anti-VEGF antibody (1:100, Santa Cruz Biotechnology Inc, USA).
Detection of myocardial anion superoxide generation. We used a dihydroethidium assay (DHE; Sigma-Aldrich Chemical Co, MO, USA) to examine the anion superoxide formation in the frozen heart tissue sections, as previously described 71 . In vitro, endothelial superoxide anion generation was determined by staining of HUVECs in each experimental condition as previously described by us 22 . Fluorescence microscopy was performed with a Leica TCS DMIRE 2 (LCS Lite Software; Leica, Wetzlar, Germany).