High Levels of Avenanthramides in Oat-Based Diet Further Suppress

Jan 3, 2018 - The Ldlr–/– mice were fed a low fat, high fat, high fat containing regular oat brans with low levels of Avns (HFLA), or high fat con...
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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

High Levels of Avenanthramides in Oat-Based Diet Further Suppress High Fat Diet-Induced Atherosclerosis in Ldlr−/− Mice Michael Thomas,† Sharon Kim,† Weimin Guo,† William F. Collins,‡ Mitchell L. Wise,§ and Mohsen Meydani*,† †

Vascular Biology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, Massachusetts 02111, United States ‡ Bioproducts and Bioprocesses, Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Central Experimental Farm, Ottawa, Ontario K1A 0C6, Canada § USDA-Cereal Crops Research Unit, 502 Walnut Street, Madison, Wisconsin 53726, United States ABSTRACT: Oats, in addition to cholesterol-lowering properties, contain unique antioxidants called avenanthramides (Avns), which inhibit both inflammatory cytokines and adhesion molecules in endothelial cells in culture. This study evaluated the effects of Avns of oats on atherosclerosis in Ldlr−/− mice, one of the most commonly used atherosclerosis mouse models with their similar cholesterol distributions to humans. The Ldlr−/− mice were fed a low fat, high fat, high fat containing regular oat brans with low levels of Avns (HFLA), or high fat containing regular oat brans with high levels of Avns (HFHA) diet. After 16 weeks of intervention, blood cholesterol and extent of aortic lesions were evaluated. We found that both oat-based diets reduced high fat diet-induced atheroma lesions in the aortic valve (p < 0.01). Furthermore, the effects of oat-based diets are more profound in HFHA mice than mice fed HFLA. Total plasma cholesterol levels were similarly reduced in both oat-supplemented mice. We concluded that oat bran diets reduce atheroma lesions and higher levels of Avns further reduce aortic lesions compared to regular oat bran. These preliminary in vivo data indicate that consumption of oats bran, with high Avns, has demonstrable beneficial effects on prevention of cardiovascular disease. KEYWORDS: oats, avenanthramide, atherosclerosis, Ldlr−/−mice, VCAM-1

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INTRODUCTION Studies have clearly shown that the consumption of oats lowers total plasma as well as LDL cholesterol, and it reduces the risk of coronary heart disease (CHD).1 β-Glucan is the active component of soluble fiber in oats. It interferes with the reabsorption of bile acid in the gut and reduces cholesterol levels.2 In addition to cholesterol-lowering effects, oat consumption has recently been shown to improve endothelial function and reduce blood pressure,3,4 potentially through modulation of vascular endothelium production of nitric oxide (NO). Oats, in addition to containing β-glucan, are a good source of protein and lipids as well as several antioxidants including vitamin E, phytic acid, phenolics, and Avenanthramides (Avns), a group of unique soluble bioactive compounds that are not present in other food crops.5 Avns from oats exhibit potent antioxidant activity in vitro and in vivo.6−11 Combined with vitamin C, Avns synergistically inhibited LDL oxidation in vitro.12 In addition, we and others have reported that Avns, through interactions with molecular signaling pathways, can reduce inflammation in endothelial cells through modulation of nuclear transcription factor (NF-κB),13 which regulates expression of several inflammatory cytokines.14 Moreover, Avns were recently shown to induce heme oxygenase-1 expression via the Nrf-2/ARE (antioxidant response element) pathway in HK-2 cells.15 We have reported that Avns suppressed endothelial expression of chemokines such as monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, pro-inflammatory cytokines such as IL-1 and IL-6, and © XXXX American Chemical Society

adhesion molecules such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and Eselectin.14,16 We have also reported Avns inhibited proliferation of vascular smooth muscle cells (VSMC) and increased NO production by endothelial cells in cell culture systems,17,18 all of which suggests potential anti-inflammatory and antiatherogenic properties of Avns.16 Taking an oat bath provides soothing effects on irritated skin of a person with poison ivy; this effect is likely due to Avns’ anti-inflammatory and antihistamine properties, as the anti-irritation effect of Avns has been demonstrated in animal models of skin irritation.19 It is worth mentioning that Avns possess a similar chemical structure to that of the drug called Tranilast [N-(3′,4′dimethoxycinnamoyl)-anthranilic acid], which was originally developed as an antihistamine drug (see Figure 1) but discovered to inhibit cytokine-induced NF-κB activation in vascular endothelial cells as well as prevent VSMC proliferation and restenosis in preclinical trials.20−22 Our studies in cell culture systems were the first to report and highlight the presence of anti-inflammatory properties in oats with potential additional benefit to cardiovascular health.16 In oats, Avns are not distributed evenly throughout the kernel but are localized primarily in the bran layers. These observations suggest that the Received: October 24, 2017 Revised: December 15, 2017 Accepted: December 15, 2017

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DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Composition of Study Diets for Ldlr−/− Mice for 16 Weeks: Low Fat Control (LFC), High Fat Control (HFC), High Fat Low Avns Oat Bran (HFLA), and High Fat High Avns Oat Bran (HFHA) ingredient caseina b DL-methionine corn starch maltodextrin sucrose cellulose anhydrous milkfatc corn oil mineral mix, AIN93G-MX calcium phosphate, dibasic potassium phosphate, monobasic vitamin mix, Teklad cholesterol oat bran (low Avns)d oat bran (high Avns)e Avenanthramides (mg) calories/1000 g macronutrient contents

Figure 1. Chemical structure of the different forms of Avenanthramides alongside the chemical structure of Tranilast.

consumption of oat bran and oatmeal, with their high Avns content, would provide additional health benefits through their anti-inflammatory activities on CVD risk factors, apart from their capacity to lower cholesterol levels. Notwithstanding the aforementioned properties of Avns, there have been few in vivo studies to determine Avns pharmacokinetics from a food matrix such as oatmeal and oat bran to establish bioavailability and potential dose−response parameters. However, preliminary results from human trials suggest that the levels of Avns in regular oats are probably too low to elicit these effects in vivo. In this study, oats with considerably higher concentrations of Avns were produced using a new patented process involving “false malting” in which the oats are maintained dormant during malting, thus do not germinate, but actively increase Avns levels.21

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protein carbohydrate fat

MATERIALS AND METHODS

Animals and Diets. A total of 30 male, 5-week-old, Ldlr−/− mice (Jackson Laboratory, Bar Harbor, ME) were used in this study due to their predisposition to developing atherosclerosis. The Ldlr−/− mouse has a lipoprotein-cholesterol distribution similar to that of normolipidemic humans, so it responds to a high fat, high cholesterol diet, making it a useful model for studying atherosclerosis in relation to a variety of dietary interventions.31 Mice were housed individually in polycarbonate shoebox-type cages under a 12 h light/dark cycle with ad libitum access to food and distilled water. Body weight was measured twice weekly for the 16 weeks of the dietary treatment period. Diets for this study are listed in Table 1. Oats used in this study were certified Canadian hull-less, hairless spring oat variety from the Agriculture and Agri-Food Canada (AAFC, Ottawa, ON Canada), grown in 2009. Both regular unmalted oats and malted oats were processed specifically for this study. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts University. Preparation of Oat Bran with High Avns. To prepare oats with high levels of Avns, a false-malted method was used. This methodology is patented by our collaborator Dr. Collins and currently is licensed to CEAPRO Inc., of Edmonton, Alberta, Canada.28 Briefly, clean whole oats were dried to a moisture content of approximately 3% in a forced air oven at 35 °C for 72 h. The oats were then heat-treated in a forced air oven at 70 °C for 144 h to mitigate microbial growth during subsequent malting steps and to induce secondary dormancy in the kernels. After this treatment the oats were divided into two lots and stored at −20 °C until utilized. It should be pointed out that by using this pretreatment, the oats will not germinate but will retain the ability to greatly accumulate avenanthramides upon subsequent “false malting” even with prolonged storage at −20 °C.23 Subsequently, they can be traditionally abrasion-milled to produce a bran fraction. The first lot of heat-treated oats was steeped in tap water for 18 h at 30 °C in a closed vessel to provide a partial anaerobic environment.

group 1: LFC (g/kg ) diet

group 2: HFC (g/kg ) diet

group 3: HFLA ( g/kg) diet

group 4: HFHA ( g/kg) diet

200 3 435.5 100 100 65 35 10 35

200 3 270 100 100 65 200 10 35

120 5 0 100 100 54 166 10 35

111 5 6.9 100 100 53 169 10 35

0.7

0.7

0

0

5.8

5.8

0

0

10 0 0

10 0.1 0

10 0.1 400

10 0.1 0

0

0

0

400

0

0

8.8

480

3500 % kcal from

4400 % kcal from

4400 % kcal from

20.1 67.9 12

16 40.8 43.2

16 40.8 43.2

4400 % kcal from 16 41 43

a

Casein is 88% protein. bExtra methionine added to compensate for oat’s deficiency of methionine. cAnhydrous milkfat, 230 mg of cholesterol/100 g. Extra cholesterol was added to increase to 0.055%. d Data listed in table is experimental. Expected values were as follows: 400 g of oat bran (low Avns) provides ≈16 mg of Avns, 68.8 g of protein, 248 g of carbohydrate, 35.2 g of fat, 48 g of fiber of which 17.6 g is soluble fiber (β-glucan), and 7.2 g of ash. eData listed in table is experimental. Expected values were as follows: 400 g of oat bran (high Avns) provides ≈480 mg of Avns, 76.8 g of protein, 247.2 g of carbohydrate, 32.4 g of fat, 43.2 g of fiber of which 15.2 g is soluble fiber (β-glucan), and 7.2 g of ash. These conditions tend to promote secondary dormancy and to raise the moisture content from about 3% to 35−40% by weight. The oats were then surface-sanitized by emersion in an aqueous solution of 0.25% sodium hypochlorite for 1 h at room temperature, rinsed with tap water, and subjected to specific “malting” conditions.23 Briefly, the oats were spread evenly on a stainless steel screen in a malting tray and covered with a clear plastic lid. The tray was then placed in a forced air drying oven for 96 h and maintained at 35 °C. Every 24 h during this “malting” period, the tray was removed and the oats rinsed thoroughly with tap water before being returned to the malting oven. After 96 h, the oats were air-dried to about 14% moisture in a forced air drying oven at 35 °C and then dried to about 3% moisture at 70 °C for 2 days. After drying, the malted oats were vacuum-packed and stored at −20 °C until processed for bran preparation. Oat bran was prepared from the dried oats using an abrasion mill Satake TM05 test mill (Satake USA, Stafford, TX). On the basis of weight, approximately 10% of the starting kernel was abraded and the resulting bran fraction vacuum-packed and stored at −20 °C until mixed into the mouse diet. Samples were retained for quantitative analyses of Avns. B

DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Preparation of Oat Bran with Low Avns. The second lot of heat-treated oats was used as the “un-malted oats”. Oat bran was prepared as described above using abrasion milling and the resulting bran fraction vacuum-packed and stored at −20 °C until it was mixed into a mouse diet. Determination of Avn Concentration in the Prepared Diet. Three 1.5 g pelleted samples from each final mixed diet preparations were collected and analyzed for Avns content. Briefly, food samples were extracted three times with 10 mL of 80% ethanol in 10 mM sodium phosphate buffer (pH 2). Extracts were pooled and rotaryevaporated under vacuum at 50 °C until dry. The residue was resuspended in 1.5 mL of methanol and injected in 2.0 μL onto a Shimadzu NexEra UHPLC system (Kyoto, Japan) using a Kinetex 2.1 × 50 mm, 100 Å pore, 1.7 μm diameter, C-18 column (Phenomenex, Torrance, CA) at 40 °C with a Shimadzu SPD-M20A photodiode array detector. The mobile phase consisted of buffer A (H2O with 5% acetonitrile and 0.1% formic acid) and buffer B (acetonitrile with 0.1% formic acid). A gradient of 10−17% B over 2.1 min, then 17−33% B from 2.3 to 3.2 min, and then to 60% B at 3.4 min returning to 10% B at 4.0 min at a flow rate of 1.0 mL/min. Authentic Avns standards Avn C, A, and B were synthesized, according to methods previously described15,24 and used to generate standard curves for the corresponding Avns. Avns AA and R were quantified using the curve for A, QQ, and Q based on the curve for C and P on the curve for B. Identification of the Avns was based on mass spectral data obtained by LC-MS, as previously described,15,24 as well as their retention times relative to the authentic standards and to each other, as determined previously23 (Figure 2).

Tissue Staining. After euthanasia, the chest cavity was opened and the heart and aorta were perfused via injection of cold buffer solution through the apex of the left ventricle. The heart and descending aorta were dissected out. The descending aorta was removed and stored in a 10% buffered formaldehyde solution for en face staining of the luminal side of the aorta with Oil Red O to identify fatty streaks and atheroma lesions. The portion of the heart distal to the aortic root was removed and the remaining portion containing the aortic tricuspid valve was immersed in OCT compound and then frozen in liquid nitrogen for cryostat sectioning. Frozen sections were collected at intervals of 20 μm beginning at the heart’s apex and until the aortic root was observed. The 20th section from the aortic ring and subsequent two sections were collected. These sections contained a significant portion of aortic valves; thus, they were used for qualitative and quantitative measurement of lesions. For general visualization of the valves and plaque, H&E staining was performed. Additionally, the state of inflammation within the valves was assessed by fluorescent staining with VCAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Images of H&E and fluorescently stained valves were captured on an Olympus FSX100 microscope. The descending aorta was cut longitudinally and pinned down on a black wax platform for en face evaluation of fatty streaks and atheroma lesions. Lesions were visualized through staining with freshly prepared Oil Red O in isopropanol. Resulting images were captured under a dissection microscope. To calculate the extent of atheroma lesions, we used the NIH Image-J software program. In H&E stained sections, the total area of lesions was measured, and a ratio of lesion area to the perimeter of the vessel was established to account for differently sized vessels. Intensity of fluorescent VCAM-1 stain was compared to total intensity of fluorescent nucleic acid stain to account for differences in tissue thickness, and then it was compared to vessel perimeter. En face measurements were calculated by comparing total area of the aorta to lesion area. Analysis of Blood Cholesterol. Total cholesterol in plasma was assessed using a fluorometric assay performed according to the manufacturer’s instructions (Cholesterol Fluorometric Assay Kit, Cayman Chemical Company, Ann Arbor, Michigan, USA). Briefly, mouse plasma was diluted with assay buffer. Esters were hydrolyzed, and all cholesterol was then oxidized to produce ketones and hydrogen peroxide. Products were reacted with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), and fluorescence was measured using a CytoFluor Multi-Well Plate Reader (Applied Biosystems, Beverly, MA, USA) against standard solutions. Statistical Analysis. Normality was determined by Shapiro-Wilk test. Homogeneity of variance was evaluated with Bartlett’s test. Data were analyzed using two-tailed unpaired t test with Welch’s correction or one-way ANOVA with Turkey’s or Bonferroni’s posthoc test when comparing the means of all samples.

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RESULTS Among the multiple forms of Avns present in the false malted formulation, Avns C, A, B, Q, O, and P were the major components of total Avns in the false-malted diet mix (Figure 2); thus, the total content of Avns was based on the concentrations of these six components. As expected, analysis of diets showed a substantially higher concentration of Avns in the false-malted oat bran formulation compared to the formulation with regular oat bran (Figure 3) (450.9 ± 18.6 mg/kg diet versus 10.08 ± 0.60 mg/kg diet). Treatment of Ldlr−/− mice with HFC diet, as well as diets containing either regular oat bran (HFLA) or high Avns oat bran (HFHA), significantly increased body weight compared to mice fed LFC diet. Inclusion of either bran in HF diet formulation had no differential effect on the weight gain of mice compared to HFC (Figure 4). The plasma concentration of total cholesterol in Ldlr−/− mice is higher than those of C57/BL mice, the background strain to Ldlr−/− mice. Therefore, mice fed LFC

Figure 2. Chromatogram of oat bran containing high avenathramides (Avns) overlaid with chromatogram of oat bran containing low Avns. C, Avenanthramide (Avn)-C; AA, Avn-AA; B, Avn-B; QQ, Avn-QQ; A, Avn-A; Q, Avn-Q; O, Avn-O; P, Avn-P. Dietary Intervention. All mouse diets were prepared by Teklad (Harlan Laboratories, Madison, WI) according to the formulation shown in Table 1. During the 16-week dietary intervention period, mice were divided into the following four dietary groups: low fat control (LFC, n = 7), high fat control (HFC, n = 7) intended to mimic a Western-style diet, high fat diet supplemented at a ratio of 400 g/kg feed with low Avns oat bran (HFLA, n = 8), and high fat diet supplemented (400 g/kg) with high Avns oat bran (HFHA, n = 8). After the 16-week dietary treatment, mice were asphyxiated with CO2 followed by cervical dislocation and blood samples were collected by heart puncture. C

DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Concentration of plasma cholesterol after 16 weeks of dietary intervention in Ldlr−/− mice. Cholesterol esters were hydrolyzed and cholesterol was oxidized to produce hydrogen peroxide. Hydrogen peroxide was then detected by reaction with 10acetyl-3,7-dihydroxyphenoxazine (ADHP) in the presence of horseradish peroxidase. Fluorescence was detected using excitation wavelength of 530 nm and emission wavelength of 585 nm. Low fat control (LFC) n = 5, high fat control (HFC) n = 6, high fat low Avns (HFLA) n = 4, and high fat high Avns (HFHA) n = 4. Data are mean ± SEM. Compared with LFC and HFC groups, HFLA and HFHA treatment tends to have decreased plasma cholesterol levels, but no significant statistical differences were found.

Figure 3. Concentration of total Avenanthramides (Avns) in high fat low Avns (HFLA) diet compared to high fat high Avns (HFHA) diet. Avns determined by UHPLC. Pelleted HFLA and HFHA diet was crushed and extracted three times. Pooled extracts were used to determine Avns concentration. Data represents mean ± SEM of three samples.

Figure 4. Effect of including a high amount of fat in the diet on body weights within 16 weeks of study. All animal weights were taken upon starting the 16-week dietary intervention and after completing 16 weeks. Week 0 weight was subtracted from week 16 weight to yield total weight gained during intervention period. n = 7 for low fat control (LFC) and high fat control (HFC), n = 8 for high fat low Avns (HFLA) and high fat high Avns (HFHA). Data are mean ± SEM. *, LFC compared with HFC, P < 0.05. #, LFC compared with HFLA and HFHA, P < 0.05; no significant statistical differences among HFC, HFLA, and HFHA groups were found.

Figure 6. Lesions of the descending aorta from Ldlr−/− mice visualized with Oil Red O stain. Dietary groups as follows: (a) low fat control (LFC), (b) high fat control (HFC), (c) high fat low Avns (HFLA), and (d) high fat high Avns (HFHA). (e) Lesion and aorta areas measured using ImageJ software to generate a value of percentage of luminal surface of aorta containing lesions. HFHA group of Ldlr−/− mice had significantly less lesions in the descending aorta compared to HFLA, HFC, and LFC groups. Data are mean ± SEM n = 7 for LFC and HFC, n = 7 for HFLA, and n = 8 for HFHA.

LFC mice (Figure 7), but the HFC mice had robust lesions accounting for more than a 50% increase in lesion per area of the tricuspid valve. Inclusion of oat brans containing low Avns significantly reduced high fat diet-induced formation of advanced fatty lesions (Figure 7). However, there was no difference in the suppression of fatty lesions between mice fed diets containing low or high Avns oat bran (Figure 7). Immunostaining with VCAM-1 antibody of frozen sections of aortic valves (Figure 8) tended to show a reduction of this inflammatory marker in aortic valves from mice fed either high or low Avns oat bran (Figure 8).

and HFC both had a high level of plasma cholesterol levels (Figure 5). Inclusion of either form of oats (low or high Avns) in the high fat diets (both HFLA and HFHA) lowered total cholesterol levels in plasma by 21% compared to HFC mice (p = 0.05). En face examination of the whole aortic luminal surface showed the presence of fatty streaks in Ldlr−/− mice, which are genetically prone to atherogenesis (Figure 6). When the diet of these mice contains a high amount of carbohydrate and fat, they develop atherosclerosis at a faster rate. Therefore, mice in the HFC group developed robust fatty lesions, with lesions accounting for 58% more of the total luminal surface than those of LFC group (Figure 6). Interestingly, inclusion of oat bran with low Avns into the HF diet at a concentration of 400 g/kg significantly reduced development of fatty lesions to a level comparable to that of LFC (p < 0.05). What is more intriguing is that inclusion of oat bran with high Avns into the HF diet remarkably reduced fatty lesion formation by 64% (p < 0.05) compared to HFC and by 46.4% compared to LFC (Figure 6). Analysis of H&E staining of aortic valves for fatty lesions showed a presence of some fatty deposits in aortic valves of

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DISCUSSION Atherosclerosis is an inflammatory disease of the arteries in which monocytes, macrophages, T cells, oxidized/modified lipoprotein, platelets, inflammatory cytokines, and chemokines participate under oxidative conditions. Our earlier cell culture studies have revealed that oats, in addition to having cholesterol-lowering properties through their β-glucan content, also possess antioxidant and anti-inflammatory properties due D

DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 7. Sections of aortic tricuspid valves lesions in Ldlr−/− mice after 16 weeks stained with H&E: (a) low fat control (LFC), (b) high fat control (HFC), (c) high fat low Avns (HFLA), and (d) high fat high Avns (HFHA); lesions are identified by arrows. (e) Lesion and luminal perimeter measured using ImageJ software to generate a ratio of lesion to perimeter of vessel. Ldlr−/− mice in groups (c) and (d) who were fed a diet containing high fat and oat brans (HFLA and HFHA) had a significantly (p < 0.01) lower lesion area at the aortic tricuspid valve than those consuming HF diet alone. Data are mean ± SE n = 6 for LFC and HFC and n = 6 for HFLA and HFHA.

Figure 8. Sections of aortic tricuspid valve, fluorescently labeled to visualize VCAM-1 expression (red) with nuclear counterstain (green). Dietary groups shown in top panel: (a) low fat control (LFC), (b) high fat control (HFC), (c) high fat low Avns (HFLA), and (d) high fat high Avns (HFHA); lesion areas are identified with arrows. (e) ImageJ software was used to determine the ratio of red-labeled to green-labeled tissue in the section. This value was then adjusted as a ratio per vessel perimeter and multiplied by 1000. A nonsignificant trend of reduced VCAM-1 expression with increasing Avns content is shown. Data are mean ± SE n = 6 for LFC and HFC, n = 5 for HFLA, and n = 7 HFHA. Compared with HFC groups, HFHA treatment trends to have decreased VCAM-1 expression levels, but no significant statistical difference was found.

to the presence of Avns.16 Cell culture studies in our lab, as well as others, have demonstrated that these unique alkaloids, present only in oats, possess anti-inflammatory and potentially antiatherogenic properties, which complement the cholesterollowering property of oats.13,16,19 Therefore, we aimed to investigate the potential antiatherogenic properties of Avns in vivo using Ldlr−/− mice, an animal model of human atherosclerosis. We report here that the inclusion of oat bran with high levels of Avns in the diet significantly suppresses high fat diet-induced atherosclerosis in this mouse model of atherosclerosis. These findings, particularly the data collected from the descending aorta (Figure 6), provide significant histopathological evidence in support of earlier observations that Avns of oat bran suppress several markers of inflammation in cell culture systems.16,25 At present, we do not know the mechanism of action of Avns on the modulation of atherosclerosis. However, Avns have been shown to possess strong antioxidative properties,7 which may allow the regulation of redox status and suppress lesion development through modulation of monocyte and macrophage activity. Our earlier cell culture studies provide evidence that Avns modulate NF-κB, a transcription factor with a key role in regulating inflammation.13,26,27 While inclusion of either formulation of oats reduced total plasma cholesterol almost equally, it is important to note that the expression of VCAM-1 within the aortic tricuspid valve lesions showed a trend of VCAM-1 reduction with increasing Avns concentration in the diet. This indicates that reductions in

lesion development are not only due to reductions in total cholesterol levels brought about by β-glucan but also due to properties specific to the Avns. Our earlier findings on the suppressive effect of Avns on IL-1β-induced in vitro adhesion of monocytes to the endothelial monolayer and inflammatory cytokines and chemokines support our present in vivo findings. It is also worth mentioning that the metabolism of Avns by mouse and human gut microbiota28 may contribute to the biological activity of Avns in vivo. Avns have been found to be bioavailable in both humans and rodents. A human study discovered a dose-dependent response of Avn metabolites AvnA, Avn-B, and Avn-C in plasma concentrations after the consumption of 0.5 or 1 g of Avn mixture extracted from oats to be roughly 13−375 nmol/L.30 In another study, rats were administered a high dose of total Avns and were able to successfully measure Avn metabolites Avn-A, Avn-B, Avn-C in the plasma, and accumulation in hepatic, cardiac, and skeletal tissue.29 The present preliminary results provide histological evidence that an oat-based diet is capable of suppressing aortic fatty lesions induced by consuming a high amount of saturated E

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Journal of Agricultural and Food Chemistry fat in Ldlr−/− mice. We believe that the presence of β-glucan, phytosterols, vitamin E, and other phenolic compounds found within oats contribute to the suppression of high fat dietinduced atherosclerosis. However, incorporating bran from false-malted oats with very high levels of Avns in the diet of Ldlr−/− mice significantly increased the effectiveness of oats in the reduction of atheroma lesion development. In conclusion, these findings suggest that oat bran containing high levels of Avns may reduce the development of aortic lesions. These preliminary in vivo data indicate that consumption of oats, especially oat bran from false-malted oats, may have more beneficial effects on the prevention of cardiovascular disease.

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oats (Avena sativa L.) and structure-antioxidant activity relationships. J. Agric. Food Chem. 2003, 51 (3), 594−600. (9) Chen, C.-Y.; Milbury, P. E.; Li, T.; O’leary, J.; Blumberg, J. B. Antioxidant capacity and bioavailibility of oat avenanthramides. FASEB J. 2005, 19, A1477. (10) Ren, Y.; Yang, X.; Niu, X.; Liu, S.; Ren, G. Chemical characterization of the avenanthramide-rich extract from oat and its effect on D-galactose-induced oxidative stress in mice. J. Agric. Food Chem. 2011, 59 (1), 206−11. (11) Koenig, R.; Dickman, J. R.; Kang, C.; Zhang, T.; Chu, Y. F.; Ji, L. L. Avenanthramide supplementation attenuates exercise-induced inflammation in postmenopausal women. Nutr. J. 2014, 13, 21. (12) Chen, C. Y.; Milbury, P. E.; Kwak, H. K.; Collins, F. W.; Samuel, P.; Blumberg, J. B. Avenanthramides and phenolic acids from oats are bioavailable and act synergistically with vitamin C to enhance hamster and human LDL resistance to oxidation. J. Nutr. 2004, 134 (6), 1459− 1466. (13) Guo, W.; Wise, M. L.; Collins, F. W.; Meydani, M. Avenanthramides, polyphenols from oats, inhibit IL-1beta-induced NF-kappaB activation in endothelial cells. Free Radical Biol. Med. 2008, 44 (3), 415−29. (14) Collins, T.; Cybulsky, M. I. NF-kB: pivotal mediator or innocent bystander in atherosclerosis? J. Clin. Invest. 2001, 107, 255−264. (15) Fu, J.; Zhu, Y.; Yerke, A.; Wise, M. L.; Johnson, J.; Chu, Y.; Sang, S. Oat avenanthramides induce heme oxygenase-1 expression via Nrf2-mediated signaling in HK-2 cells. Mol. Nutr. Food Res. 2015, 59 (12), 2471−9. (16) Liu, L.; Zubik, L.; Collins, F. W.; Marko, M.; Meydani, M. The antiatherogenic potential of oat phenolic compounds. Atherosclerosis 2004, 175 (1), 39−49. (17) Nie, L.; Wise, M.; Peterson, D.; Meydani, M. Mechanism by which avenanthramide-c, a polyphenol of oats, blocks cell cycle progression in vascular smooth muscle cells. Free Radical Biol. Med. 2006, 41 (5), 702−8. (18) Nie, L.; Wise, M. L.; Peterson, D. M.; Meydani, M. Avenanthramide, a polyphenol from oats, inhibits vascular smooth muscle cell proliferation and enhances nitric oxide production. Atherosclerosis 2006, 186 (2), 260−6. (19) Sur, R.; Nigam, A.; Grote, D.; Liebel, F.; Southall, M. D. Avenanthramides, polyphenols from oats, exhibit anti-inflammatory and anti-itch activity. Arch. Dermatol. Res. 2008, 300 (10), 569−74. (20) Isaji, M.; Miyata, H.; Ajisawa, Y. Tranilast: A new application in the cardiovascular field as an antiproliferative drug. Cardiovasc. Drug Rev. 1998, 16 (3), 288−299. (21) Azuma, H.; Banno, K.; Yoshimura, T. Pharmacological properties of N-(3′,4′-dimethoxycinnamoyl) anthranilic acid (N-5′), a new anti-atopic agent. Br. J. Pharmacol. 1976, 58 (4), 483−8. (22) Spiecker, M.; Lorenz, I.; Darius, H. Tranilast Inhibits CytokineInduced Nuclear Factor κB Activation in Vascular Endothelial Cells. Molecul Pharmacol 2002, 62 (4), 856−863. (23) Collins, F. W.; Burrows, V. D.;, Canadian Patent #2,756,554 and US Patent Application #20120082740) 2011. (24) Wise, M. L. Effect of chemical systemic acquired resistance elicitors on avenanthramide biosynthesis in oat (Avena sativa). J. Agric. Food Chem. 2011, 59 (13), 7028−38. (25) Liu, L.; Zubik, L.; Marko, M. G.; Collins, F. W.; Meydani, M. The antiatherogenic and anti-inflammatory potential of oat phenolics. FASEB J. 2002, 16, A223. (26) Guo, W.; Nie, L.; Wu, D.; Wise, M. L.; Collins, F. W.; Meydani, S. N.; Meydani, M. Avenanthramides inhibit proliferation of human colon cancer cell lines in vitro. Nutr. Cancer 2010, 62 (8), 1007−16. (27) Guo, W.; Nie, L.; Wu, D.; Wise, M. L.; Collins, F. w.; Meydani, S. N.; Meydani, M. Avenanthramides inhibit proliferation of human colon cancer cell lines in vitro. Nutr. Cancer 2010, 62, 1007. (28) Wang, P.; Chen, H.; Zhu, Y.; McBride, J.; Fu, J.; Sang, S. Oat avenanthramide-C (2c) is biotransformed by mice and the human microbiota into bioactive metabolites. J. Nutr. 2015, 145 (2), 239−45. (29) Koenig, R. T.; Dickman, J.; Wise, M. L.; Ji, L. Avenanthramides are bioavailable and accumulate in hepatic, cardiac, and skeletal muscle

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Cell: 617-780-7040. Office: 617-556-3126. ORCID

Sharon Kim: 0000-0002-8996-9752 Author Contributions

W.F.C., M.L.W., and M.M. designed research; M.T. and S.K. conducted research; W.F.C. provided essential reagents; M.T. and W.G. analyzed data; M.T. and M.M. wrote the paper; M.M. had primary responsibility for final content. All authors read and approved this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the U.S. Department of Agriculture − Agricultural Research Service (ARS), under Agreement #58-1950-0-014, and an HNRCA at Tufts University Pilot Award. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. We thank Stephanie Marco for her assistance in the preparation of the manuscript. We also thank Dr. Donald Smith for animal assistance.

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DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.7b04860 J. Agric. Food Chem. XXXX, XXX, XXX−XXX