Alteration of the Systemic and Microcirculation by a Single Oral Dose of Flavan-3-Ols

Several systematic reviews have reported that flow mediated dilatation (FMD) was significantly increased in subjects after ingestion of chocolate that contains flavan-3-ols; however, the mechanisms responsible for this effect are not clear. In this study, we evaluated the effects of a single oral dose of flavan-3-ols on the systemic circulation and microcirculation in the cremaster muscle using intravital video microscopy in vivo. The cremaster muscle in rats was spread over a plastic chamber and a gastric tube was placed into the stomach. Blood flow in the cremasteric artery was determined using a laser Doppler flowmeter, while blood pressure and heart rate were measured by the tail-cuff method. Red blood cell velocity in arterioles and blood flow in the artery were significantly increased 5 min after the administration of 10 mg/kg flavan-3-ols compared with distilled water treatment. The number of capillaries recruited in the cremaster muscle was also significantly increased 15 min after treatment. Microscopic observation confirmed that increased shear stress on endothelial cells was maintained during the measurement period. The mean arterial blood pressure and heart rate were also significantly elevated soon after administration and returned to baseline before the end of the observation period. Plasma nitrate and nitrite levels, and NO phosphorylation of aortic tissue were significantly increased at 60 min after administration of flavan-3-ols. According to these results, a single oral dose of flavan-3-ols elevates blood pressure and flow transiently, and these effects induce NO production through increased shear stress on endothelial cells.


Introduction
Cocoa bean, the seed of Theobroma cocoa, is one of the ingredients in chocolate and cocoa. It is known to be rich in polyphenols, such as the flavan 3-ol monomers, (+)-catechin and (-)-epicatechin; and oligomers, such as the B-type flavan 3-ols linked by C4-C8 bonds [1][2][3]. Recent epidemiological meta-analyses have suggested that ingestion of chocolate reduces the risk of cardiovascular disease and stroke [4,5]. Numerous studies support the idea that flavan-3ols in cocoa reduce the risk of cardiovascular disease by improving hypertension, dyslipidemia and glucose intolerance [6][7][8][9][10][11][12]. In addition, flow-meditated dilation (FMD), which is a non-invasive method to assess endothelial function, was shown to be significantly increased shortly after ingestion of dark chocolate in healthy or mild hypertensive subjects [13][14][15]. It is well known that endothelial function is affected by cardiovascular risk factors, and can also be influenced by exercise [16,17] or behavioral factors [18]. It was reported that the blood NO metabolite level was increased after ingestion of chocolate; however, there is little information about the mechanisms responsible for this effect.
In this study, we evaluated the acute effects of flavan-3-ols on the systemic circulation and microcirculation in skeletal muscle using intravital video microscopy under physiological conditions. We also examined changes in eNOS phosphorylation in aortic tissue and nitrate/nitrite levels in blood to elucidate the mechanisms of the acute response to flavan-3-ols.

Materials
Urethane and Krebs-Ringer bicarbonate buffer were purchased from Sigma Chemicals (St. Louis, MO, USA). The flavan-3-ol fraction was provided by Meiji Co., Ltd (Tokyo, Japan), and the concentrations of polyphenols are shown in Table 1. The total polyphenol content was determined by the Prussian blue method [19], and each polyphenol was measured by high performance liquid chromatography (HPLC) [20].

Animals and diets
This study was approved by the Animal Care and Use Committee of the Shibaura Institute of Technology (Permit Number: 27-2956). All animals received humane care under the guidelines of this institution. Male Wistar rats weighting 200-250 g were obtained from Saitama Experimental Animal Supply (Tokyo, Japan). The rats were kept in a room with controlled lighting (12 h light and dark cycles at a regulated temperature of 23-25uC). The diet was certified diet obtained from the Oriental Yeast Co., Ltd., Tokyo, Japan.

Experimental procedures
Thirty two animals were fed a basal diet for 4 days and then allocated to two groups, with each group treated 4 ml/kg distilled water (vehicle group; n = 16) or 10 mg/kg flavan-3-ol fraction (flavan-3-ols group; n = 16), flavan-3-ols was dissolved in distilled water. Microscopic observation was carried out using eight rats then sacrificed under anesthesia, laser Doppler and tail cuff measurement was performed the other eight rats in each group. A gastric tube was inserted under urethan anesthesia (1 g/kg, SC). The cremaster muscle was exteriorized and carefully spread out on a dedicated plastic chamber with an optical port for transillumination; the surface was superfused with Krebs-Ringer bicarbonate buffer (pH 7.3-7.4) in an environment with 95% N 2 and 5% CO 2 at 37uC. Thirty min after post-surgical equilibration period, a single unbranched arteriole with a resting inner diameter of 15-20 mm was selected from the microscopic images for measurement of red blood cell velocity and diameter of the vessels. After 15 min of baseline observation, 10 mg/kg of flavan-3-ols or distilled water was administered orally to animals through the fixed gastric tube. The dosage of flavan-3-ols was determined according to a preliminary experiment (data not shown). The microcirculation was visualized by placing the chamber on a three-way movable stage, and the cremaster was transilluminated with a 150-W halogen light. The microcirculation was observed using an intravital microscope (M5A, Olympus) equipped with a chargecoupled video camera (DXC-107S, Sony, Tokyo). The images were displayed on a high-resolution television monitor at a final magnification of 14506 and stored for off-line analysis. Red blood cell (RBC) velocity and the number of newly-recruited capillaries were measured using a video image with 8-bit gray levels at a resolution of 5126512 pixels. RBC velocity was measured by monitoring the change in the position of a RBC over time in successive frames, and each velocity measurement was repeated 3 times. The number of newly-recruited capillaries was counted over an 8466307 mm area of the cremaster muscle throughout the observation period.
A schematic diagram of the experimental apparatus is shown in Fig.1 (a). The cremasteric arterial blood flow was determined using a laser Doppler blood flowmeter (Periscan PIM-2, Perimed Co. Ltd.), and blood pressure and heart rate were determined simultaneously by the tail-cuff method (BP-98A Softron, Tokyo Japan) every 6 min. The cremaster muscle was exteriorized under anesthesia as described above. After all the measurements were completed, 4 to 5 ml of blood sample was collected from the abdominal vein using a heparinized syringe. The aorta was removed by dissection, snap frozen in liquid nitrogen and stored at -80uC until analysis.

Western blotting analysis
Aortic tissue was homogenized in a microtube with lysis buffer (CelLytic TM MT cell lysis reagent; Sigma Aldrich, Japan) containing a protease inhibitor (Sigma Aldrich, Japan) and 0.2% SDS. Protein concentration was measured by the Bradford method. Protein (50 mg) was separated by SDS-PAGE using a 4-12% Bis-Tris gel and transferred onto a polyvinylidene difluoride membrane (Life Technology). The membrane was blocked with membrane-blocking reagent (GE Healthcare) for 1 hour. After blocking, the membrane was probed with a rabbit polyclonal primary antibody against phosphorylated eNOS (Ser 1177, 1:800; sc-12972 Santa Cruz Biotech, Dallas, TX, USA) and an a-tublin antibody (1:2000; ab4074, Abcam) for 2 hours. After the primary antibody reaction, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:100000) for 1 hour. Immunoreactivity was detected by chemiluminescence using the ECL Select TM Western Blotting Reagent (GE Healthcare). Fluorescence band images were analyzed using Just TLC (SWEDAY) analysis software. Values were normalized to those for a-tubulin.

Measurement of nitrate and nitrate in plasma
The plasma nitrate and nitrite concentrations were determined by using the Griess reaction [21]. Briefly, plasma was incubated with the same volume of nitrate reductase in 0.1 M potassium phosphate buffer containing 1 mM bnicotinamide adenine dinucleotide phosphate (NADPH) and 2 units of nitrate reductase/mL. Samples were allowed to incubate overnight at 37uC. Griess reagent (1% sulphanilamide, 0.1% naphthyl-ethylenediamine dihydrochloride in 5% phosphoric acid) was added, and the samples were incubated for an additional 115 min at room temperature. The total amount of nitrite was measured at 540 nm.

Data analysis and statistical methods
Data are expressed as the means and standard deviations. Statistical analyses were performed using Student's t-test or Dunnett's test. P,0.05 was considered significant.

Changes in microcirculation in the cremaster muscle after treatment with flavan-3-ols
Changes in the microcirculation of the cremaster muscle before and after oral administration of flavan-3-ols are shown in supplemental videos 1 (before treatment) and 2 (5 min after flavan-3-ols treatment). The marked elevation of RBC velocity in the arterioles can be seen in the video image, and this confirms that endothelial cells were exposed to severe shear stress. The average RBC velocity in the arterioles shortly after treatment with flavan-3-ols is shown in Fig.2 (a), and a significant difference was observed compared with vehicle from 5 min after treatment until the end of the observation period. At the end of observation period, RBC velocity in flavan-3-ols group was increased about 1.7-fold compared with vehicle group. The number of capillaries recruited in the rat cremaster muscle is shown in Fig. 2(b). A significant increase in capillary recruitment was observed after flavan-3-ols treatment compared with vehicle treatment at 15 and 20 min (1.25 and 1.35-fold increase) after administration. Blood flow in the cremasteric artery is shown in Fig 2 (C). There was a significant difference between vehicle-treated and flavan-3-olstreated rats 5 min after treatment, and the elevated blood flow in the flavan-3-ols group was maintained during the measurement period. At the end of measurement period, blood flow of cremaster artery in flavan 3-ols group was increased 3.5-fold compared with vehicle group. Changes in heart rate and blood pressure Changes of blood pressure and heart rate measured by the tail cuff method are shown in Fig. 3 (a,b). Mean blood pressure was significantly increased soon after treatment with flavan-3-ols;the elevation from 4 to 7 mmHg was maintained until 42 min and then returned to baseline (Fig. 3a). A similar result was observed for heart rate: there was a significant increase (from 18 to 25 beat/ min) until 18 min after administration of flavan-3-ols and then a return to baseline. eNOS phosphorylation in aorta and nitrate and nitrite levels in blood Fig.4 shows eNOS phosphorylation in the aorta (a) and blood nitrate and nitrite levels (b) 60 min after administration of flavan-3-ols. Aortic phosphorylated eNOS was nearly doubled compared with vehicle group, and blood nitrite and nitrate concentrations were also significantly elevated (1.7 fold) by treatment with flavan-3-ols.

Discussion
In the present study, we observed increased RBC velocity in the cremasteric arterioles (Fig.2a) and blood flow in the cremasteric artery (Fig.2b) soon after oral administration of flavan-3-ols, and these observations indicate severe shear stress on endothelial cells (video 1 and 2). We also found that mean blood pressure and heart rate were significantly increased immediately by the ingestion of flavan-3-ols. The significant elevation of blood pressure was maintained 42 min after treatment with flavan-3-ols and then returned to baseline (Fig.3). The heart rate was also significantly increased during the 18-min measurement period and then also returned to baseline. As the changes of heart rate and blood pressure were shown in Fig.2, there was slight reduction of heart rate and blood pressure in vehicle group during the early part of measurements. Though there were no significant changes in absolute value in vehicle group both heart rate and blood pressure during experimental period. The current study shows acute oral supplementation affected blood pressure or flow by gastric reflux, however elevation induced by flavan-3-ols lasted a number of minutes, and there were significant differences between distilled water and flavan-3-ols treatment rats. Further studies required to   Alteration of Circulation by Flavan-3-Ols PLOS ONE | www.plosone.org elucidate the mechanisms of initial rise in blood pressure and heart rate induced by flavan-3-ols. It is well known that eNOS phosphorylation can be induced via shear stress sensors in the plasma membrane of endothelial cells [22,23]. Increased shear stress was reported to induce nitric oxide synthase (eNOS) dissociation from caveolin and binding of calmodulin and hsp90 complex. Caveolin-free eNOS undergoes various protein kinasemediated phosphorylation and changes to an active form, finally catalyzing the conversion of molecular oxygen to nitric oxide (NO) using a terminal guanidino group from L-alginine [24]. In the present study, aortic eNOS was significantly phosphorylated 60 min after treatment with flavan-3-ols, resulting in an elevation of nitrate and nitrite concentrations in blood (Fig.4). In previous reports, acute physiological stress caused a rise in blood pressure and local blood flow that caused vasodilatation through induction of endothelial-derived relaxing factors [16,17]. In addition, interventional trials have indicated a significant elevation of FMD several hours after chocolate ingestion. A chocolate-induced FMD has been estimated by the rise in NO production, since there was elevation of nitrate and nitrite levels in blood [13][14][15]. The results of the present study suggested that the elevation of blood flow in skeletal muscle by treatment with flavan-3-ols was mediated by increased NO production in endothelial cells. These hemodynamic changes resulted in increased shear stress, which led to more NO release and vasodilatation. The NO-induced vasodilatation probably acted to reduce the elevated blood pressure [25,26].
Several previous studies was indicated that (-)-epicatechin in flavan-3-ols activated eNOS directly [27][28][29]. Although it was well investigated that (-)-epicatechin was distributed in blood as metabolites as glucuronide and/or sulfate forms [30,31]. In addition, there was few information regarding the effect epicatechin oligomers on eNOS activities [32]. It was also known that epicatechin oligomers were poorly absorbed in blood [33,34]. The contribution of the component in flavan-3-ols to eNOS activation and its mechanisms may be required additional discussion.
In this study, we found an increase in newly recruited capillaries in the cremaster muscle (Fig. 2b), and the elevation of blood flow in cremasteric artery was continued after blood pressure was returned to normal level. It is well established that an increased number of functioning capillaries is detected in tissues with a decreased oxygen partial pressure [35]. For example, the number of newly recruited capillaries is increased by contraction of skeletal muscle, which requires O 2 for ATP production during exercise [36]. Further investigation is needed to elucidate the mechanism for increased capillary recruitment in the cremaster muscle by the ingestion of flavan-3-ols.

Conclusions
In conclusion, we found an increase in RBC velocity in arterioles and blood flow in artery in the cremaster muscle, along with transient elevation of blood pressure and heart rate after a single oral administration of flavan-3-ols. In this condition, the elevation of shear stress on endothelial cells was confirmed by microscopic observation. These results suggest that the mechanism of FMD elevation after acute chocolate administration was partly due to enhanced NO release from endothelial cells due to increased shear stress.

Supporting Information
Video S1 Microcirculation in cremaster muscle of before administration of flavan-3-ols.