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Effects of Saskatoon berry powder on monocyte adhesion to vascular wall of leptin receptor-deficient diabetic mice
Effects of Saskatoon Berry Powder on Monocyte Adhesion to Vascular Wall of Leptin Receptor-Deficient Diabetic Mice Ruozhi Zhao, Khuong Le, Wende Li, Song Ren, Mohammed H. Moghadasian, Trust Beta, Garry X. Shen PII: DOI: Reference:
S0955-2863(14)00079-5 doi: 10.1016/j.jnutbio.2014.03.016 JNB 7190
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
3 August 2013 19 March 2014 20 March 2014
Please cite this article as: Zhao Ruozhi, Le Khuong, Li Wende, Ren Song, Moghadasian Mohammed H., Beta Trust, Shen Garry X., Effects of Saskatoon Berry Powder on Monocyte Adhesion to Vascular Wall of Leptin Receptor-Deficient Diabetic Mice, The Journal of Nutritional Biochemistry (2014), doi: 10.1016/j.jnutbio.2014.03.016
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ACCEPTED MANUSCRIPT JNB-13-481 R3
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Effects of Saskatoon Berry Powder on Monocyte Adhesion to Vascular
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Wall of Leptin Receptor-Deficient Diabetic Mice
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Ruozhi Zhao1, Khuong Le2, Wende Li3, Song Ren1,
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Mohammed H. Moghadasian2, Trust Beta3, Garry X. Shen1 Departments of Internal Medicine and Physiology, 2Department of Human Nutritional
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Garry X. Shen MD PhD
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Corresponding author:
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Sciences, 3Department of Food Sciences, University of Manitoba
Diabetes Research Group
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University of Manitoba
835-715 McDermot Ave Winnipeg, MB R3E 3P4 Canada Tel: 204-789-3816 Fax: 204-789-3987
Email: [email protected] Running title: Saskatoon berry on monocyte adhesion in diabetic mice Word counts for abstract: 249; text: 3,500
Key words: Saskatoon berry, db/db mice, monocyte adhesion, inflammatory mediators. 1
ACCEPTED MANUSCRIPT Abstract Hypothesis: Atherosclerotic cardiovascular complications are the leading cause of death in
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diabetic patients. Monocyte adhesion is an early event for atherogenesis. Previous studies demonstrated that dark-skin berries had cardiovascular protective effects. We hypothesize that SB
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powder may reduce monocyte adhesion in leptin receptor-deficient (db/db) diabetic mice. Methods: Wild-type and db/db mice were fed with chow or supplemented with SB powder.
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Anthocyanins in SB powder were identified using mass spectrometry. Mouse monocytes were
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incubated with mouse aorta. Monocyte adhesion was counted under microscopy. Inflammatory or metabolic markers in blood or tissue were analyzed using immunological or biochemical methods.
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Results: SB powder significantly reduced monocyte adhesion to aorta from diabetic db/db mice
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compared to regular chow. The increased monocyte adhesion to aorta was normalized in db/db mice treated with ≥5% of SB powder for 4 weeks. Increased contents of NAPDH oxidase-4, heat
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shock factor-1, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM)-1,
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P-selectin, tumor necrosis factor-α, plasminogen activator inhibitor (PAI)-1 and urokinase plasminogen activator in aorta or heart apex, elevated plasma PAI-1 and MCP-1 were detected in db/db mice on chow compared to wild-type mice on the same diet. 5% SB powder inhibited the increases of inflammatory, fibrinolytic or stress regulators in aorta or heart apex of db/db mice. Monocyte adhesion positively correlated with blood glucose, cholesterol, body weight, heart MCP1, PAI-1 or ICAM-1. Conclusion: The findings suggest that SB powder attenuated monocyte adhesion to aorta of db/db mice, which was potentially mediated through inhibiting the inflammatory, stress and/or fibrinolyic regulators. 2
ACCEPTED MANUSCRIPT Introduction
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The prevalence of diabetes mellitus (DM) rapidly increased in most countries during last 30 years, and the trend is expected to continue. Nine out of ten diabetic patients are type 2 DM
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(T2DM) (1). Atherosclerotic cardiovascular complications are the predominant cause of death in diabetic patients. Vascular inflammation plays a critical role in the etiology of atherosclerosis
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(2). Monocyte adhesion on endothelium has been considered as an early event in vascular
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inflammation (3). Previous studies demonstrated that monocyte adhesion may be regulated by cytokines, adhesion molecules, metabolic factors and oxidative stress (4, 5). Elevations of
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inflammatory mediators, including interleukins, tumor necrosis factor (TNF)-α, monocyte
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chemotactic protein (MCP)-1 and intracellular adhesion molecule (ICAM)-1, were detected in diabetic patients (6, 7). Hyperglycemia and hyperketonemia are associated with increased
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monocyte adhesion to endothelial cells (EC) (8, 9). Previous studies in our laboratory
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demonstrated that glycated low density lipoprotein (LDL) may increase the production of reactive oxygen species, activate NADPH oxidase (NOX) and impair mitochondrial respiration in cultured EC or fibroblasts (10-12). The effect of diabetes on monocyte adhesion and relevant regulators in animal models remains to be investigated.
Previous studies demonstrated that products of a number of dark-skin fruits had cardiovascular protective effects in animal models or in small trials of human study (13, 14). Anthocyanins are common flavonoids in the dark-skin phyto-products. A recent study found that purple sweet potato extracts and cyanidin inhibited TNF-α-induced monocyte adhesion 3
ACCEPTED MANUSCRIPT and the production multiple cytokines or adhesion molecules in cultured EC (15). Previous studies by our group demonstrated that cyanidin-3-glucoside or delphindin-3-glucoside
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inhibited glycated or oxidized LDL-induced oxidative stress and cell injury in vascular EC (16, 17). Saskatoon berries (SB) naturally grow or are planted in Canada prairie areas and Northern
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states of USA. Previous studies by Hosseinian and Beta demonstrated that SB powder had relatively higher contents of anthocyanins compared to blue berries, strawberries, raspberries,
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inflammation has not been documented.
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chokecherry or seabuckthorn (18). The effect of SB on monocyte adhesion or vascular
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The present study examined the effect of SB powder on the adhesion of monocytes to aorta and relevant inflammatory, stress and fibrinolytic regulators in leptin receptor-knockout
SB powder
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(db/db) mice, a common rodent model of T2DM
Materials and Methods
Smoky SB was purchased from Prairie Lane Saskatoons (Portage, Manitoba). The berries were cleaned and frozen at -70°C, and then lyophilized using a freeze-drier without an addition of supplement. Dried whole berries were powered and stored in vacuum-sealed bags at -20°C. All processes were conducted in sterile stainless steel containers.
Animals 4
ACCEPTED MANUSCRIPT Male db/db mice and C57BL/J counterparts (6 weeks of age) were obtained from the Jackson Laboratory (Bar Harbour, ME). Mice were housed in an air-conditioned room and
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received regular chow for 1 week for stabilization. Animals were randomized into indicated groups (n=5-8/group). Mice in control group received regular mouse chow, and animals in the
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SB treatment groups received chow supplemented with 0.2-20% (w/w) SB for up to 5 weeks. Body weights were measured at weekly basis. Heart, aortae, and blood were harvested from
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db/db and wild-type mice after euthanasia. The protocol of animal experiments was approved
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Monocyte adhesion to intima of aorta
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by the Animal Protocol Management and Review Committee in the University of Manitoba.
Aortae were freshly harvested at the level of aortic arch to the level of abdominal aorta
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beyond renal arteries and submersed in ice-cold Hank’s balance salt solution (HBSS).
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Perivascular adipose tissue was removed from freshly isolated aortae. The aortae were
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longitudinally opened in fresh ice-cold HBSS and fixed with 27-gauge needles on the bottom of 35-mm culture dish within 1 h after harvesting. Mouse WEHI-274.1 monocytes were labelled with 3 µg/mL of TRITC (Molecular Probes, Burlington, ON) for 15 min. Free dye was removed from cells through overlaid on fetal calf serum and a centrifugation at 400xg as previously described (19). The abundance of labeled monocytes represents >95% of total cells assessed using flow cytometer. Fluorescently labelled or unlabeled monocytes (1 x 105) were added to each dish containing aorta. Mouse monocytes were incubated with aortic strips at room temperature for 30 min on a rotator mixer. Un-adhered monocytes were removed using 2 washes with HBSS. Adhered monocytes were fixed using 2% glutaraldehyde in HBSS. Five fields 5
ACCEPTED MANUSCRIPT (2 mm in diameter/field) were observed for each aortic strip for monocyte counting under fluorescent microscopy using 10X magnification. One field was at center and 4 fields at each
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corner of aortic strip. The combined examining areas represent approximately 1/4 of total area (typically 10 mm x 6 mm) of each aortic strip (Fig.1B). The averages of adhered monocytes to
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the fields in each aorta were used for data analysis.
Western blotting
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Targeted proteins in protein extracts of heart apex and ascending aorta were analysed using Western blotting analysis as previously described (20). Monoclonal or polyclonal
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antibodies against mouse PAI-1, urokinase plasminogen activator (uPA), heat shock factor-1
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(HSF1), ICAM-1, P-selectin, TNF-α, MCP-1 or β-actin were obtained from Santa Cruz (Santa Cruz, CA), Cedarlane (Burlington, ON) or Abcam (Cambrige, MA). Corresponding secondary antibody
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conjugated with horse radish peroxidase (Santa Cruz) and enhanced chemilluminence reagents
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(GE Healthcare, Buckinghamshire, UK) were applied to visualize targeted antigens on nitrocellulose membranes. The abundances of the antigens was assessed using Chemi-Doc system and Quantity One software (BioRad, Hercules, CA), and normalized with the abundance of β-actin in corresponding samples.
Identification of anthocyanins in SB powder Anthocyanins in SB powder were extracted and analyzed using Waters high performance liquid chromatography-mass spectrometry (HPLC-MS) as previously described (18). Purified anthocyanins were loaded on a solid-phase extraction C18 cartridge of a Water 6
ACCEPTED MANUSCRIPT high performance liquid chromatography (HPLC) system. Anthocyanins were eluted with acidified methanol and the solution was dried at ambient temperature under nitrogen gas. The
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residue was re-dissolved in HPLC mobile-phase solution for the determination of anthocyanins using Waters Quatro Micro API mass spectrometer (Waters Corp, Milford, Massachusetts, USA)
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equipped with an ESI Multi-Mode Ionization probe as described (18). Standards of anthocyanins
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were obtained by Polyphenols Laboratories (Sandnes, Norway).
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Analyses of variables in blood
The levels of glucose in mouse blood were measured using AlphaTRAK glucose
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monitoring system from Abbott. Serum cholesterol was measured using Sekisui Diagnostics SL reagents. Plasma PAI-1 and MCP-1 were measured using ELISA kits (Cedarlane for mouse PAI-1,
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Thermo Scientific for mouse MCP-1).
Statistical analysis
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Continuous data were presented as means of values ± standard deviation. Data from multiple groups were analyzed using the one-way variance assay followed with post-hoc tests using Microsoft Excel software. Data from two groups using Students’ t-test. Correlations between 2 groups on continuous data were analyzed using liner regression analysis using Sigma Window software. Differences at p<0.05 were considered as significant.
RESULTS
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ACCEPTED MANUSCRIPT Anthocyanin composition of SB powder SB powder contained 11.5% moisture. The total anthocyanins in freeze-dried powder of
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Smoky SB were 5,011.0 mg/kg (dry weight basis or dwb). Four types of anthocyanins, cyanidin3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside and cyanidin-3-xyloside, were
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identified in the Smoky SB powder. The contents of individual anthocyanin in the freeze-dried powder were 3,708.2 mg/kg (dwb) for cyanidin-3-galactoside, 903.4 mg/kg (dwb) for cyanidin-
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3-glucoside, 345.0 mg/kg (dwb) as equivalent of cyanidin-3-glucoside for cyanidin-3-arabinoside
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and 314.5 mg/kg (dwb) as equivalent of cyanidin-3-glucoside for cyanidin-3-xyloside. The predominant anthocyanins were cyanidin-3-galactoside (74.00 %), and cyanidin-3-glucoside
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(18.03 %) in the Smoky SB powder.
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Effect of SB powder on monocyte adhesion in db/db mice The adhesion of mouse WEHI 274.1 monocytes to the intima of aorta from db/db mice
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was increased by 137% compared to wild-type mice (p<0.01). Monocyte adhesion to aorta of db/db mice was significantly decreased by 0.2-20% SB powder compared to db/db mice without exposure to SB powder (p<0.01). The monocyte adhesion was normalized in db/db mice fed with ≥5% SB powder (Fig. 1A). Fig. 1B demonstrated a scheme for field selection for monocyte accounting on aorta under microscopy. In a separate experiment, treatment with 5% SB powder for 4 weeks significantly reduced the adhesion of monocytes to aorta db/db mice compared to the diabetic mice without an exposure to SB powder (p<0.01, Fig. 2).
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ACCEPTED MANUSCRIPT Effects of SB powder on inflammatory mediators in plasma or heart apex of db/db mice The levels of MCP-1 in plasma were increased in db/db diabetic mice by 2-folds
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compared to wild-type mice (p<0.01). Treatment with 5% SB powder for 4 weeks reduced MCP1 in C57 mice by 30% compared to controls (p<0.05). The SB powder treatment reduced the
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levels of MCP-1 in the db/db mice by 32% compared to db/db mice fed with regular chow
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(p<0.01). The level of MCP-1 in heart apex was significantly increased in db/db mice assessed using Western blotting. SB treatment did not significantly affect the levels of MCP-1 in heart
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apex of wild-type mice. The abundance of MCP-1 in heart apex of db/db mice was significantly
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decreased after 4 weeks feeding of SB powder (Fig 3).
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The abundances of TNF-α, P-selectin and ICAM-1 in heart apex of db/db mice were
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increased compared to wild-type mice (p<0.05 or 0.01). 5% SB powder significantly increased
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the level of ICAM-1 (p<0.05), but not that of TNF-α or P-selectin, in heart apex of C57 mice. The SB powder treatment significantly inhibited the abundances of TNF α, P-selectin and ICAM-1 in heart apex of db/db mice compared to those on regular chow (p<0.05 or 0.01, Fig. 4).
Effects of SB powder on NOX4 and HSF1 in heart apex of db/db mice The results of the present study demonstrate that the levels of NOX4 and HSF1 were increased in heart apex of db/db mice. Treatment with 5% SB powder for 4 weeks significantly reduced the abundance of NOX4, but not HSF1, in wild-type mice. In db/db mice fed with 5% SB 9
ACCEPTED MANUSCRIPT powder for 4 weeks, the levels of abundance of NOX4 and HSF1 were significantly decreased in
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heart apex of compared to that in db/db mice on regular diet (p<0.05, Fig. 5).
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Impact of SB powder on fibrinolysis regulators in heart apex
SB powder (5% for 4 weeks) reduced the abundance of PAI-1 in heart apex (p<0.05), but
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did not significantly affect that of uPA, in wild-type mice on regular chow. The levels of PAI-1
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and uPA in heart apex of db/db mice fed with regular chow were significantly higher than wildtype mice on regular chow (p<0.01). The abundances of PAI-1 and uPA in the heart apex of
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db/db mice were significantly reduced by 5% SB powder compared to db/db mice without an
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exposure to SB powder (p<0.05 or 0.01, Fig 5).
Elevated levels of PAI-1 were detected in plasma of db/db mice compared to wild-type
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mice (p<0.01). PAI-1 plasma in wild-type mice was significantly lowered by treatment with 5% SB powder for 4 weeks (p<0.01). The increase of PAI-1 in plasma of db/db mice was reduced by the SB powder treatment compared to db/db mice without exposure to SB powder (p<0.01, Fig.6).
Effects of SB powder on the contents of fibrinolytic, stress and inflammatory regulators in aorta of db/db mice
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ACCEPTED MANUSCRIPT In order to determine the effect of SB powder on the contents of stress, inflammatory and fibrinolytic regulators in aortic wall, we extracted proteins from pooled ascending aorta
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from each group of wild-type and db/db mice fed with chow or 5% SB powder described in Fig.2 and conducted Western blotting for PAI-1, uPA, NOX4, HSF1, ICAM-1, P-selectin, TNF-α and
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MCP-1. The results indicated that the effect of diabetes and SB powder on the contents of the fibrinolytic, stress and inflammatory regulators in aortae were similar to that in heart apex
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(Fig.7).
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Effects of SB powder on body weight, glucose, cholesterol and relationship with monocyte adhesion or inflammatory mediators
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Body weights of db/db mice at baseline (7 weeks of age) were not significantly different
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from wild-type mice at the same age. Significantly higher body weights were detected in db/db
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mice fed with SB powder compared to that in wild-type mice on the same diet (p<0.01), but the levels of body weight between mice on various dosages of SB powder were not significantly different. At baseline, relatively higher levels of blood glucose were detected in each groups of db/db mice compared to wild-type mice, but significant increase in blood glucose between db/db and wild-type mice was only detected in those assigned for 20% SB powder (p<0.05). At 5 weeks after, the levels of blood glucose in db/db mice receiving chow or 0.2% SB powder were higher than wild-type mice receiving the same diet (p<0.05). In db/db mice fed with regular chow for 5 weeks, the mean level of glucose was 90% higher than that at baseline. In db/db mice received 0.2%, 1%, 5% and 20% SB powder, the mean levels of blood glucose were 11
ACCEPTED MANUSCRIPT 21%, 29%, 41% and 17% lower than that in db/db mice without an exposure to SB powder. The levels in blood glucose between the groups of db/db and wild-type mice fed with or without SB
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powder were not statistically significant. At baseline, significant difference in serum total cholesterol between wild-type and db/db mice was only detected in mice assigned for 5% SB
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powder (p<0.05). At the end of 5 weeks of experimental feeding, the levels of serum cholesterol in db/db mice fed with 0-20% of SB powder were significantly higher than that in
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wild-type mice fed with corresponding diet (p<0.05 or 0.01). No significant difference in serum
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cholesterol was detected between baseline and 5 weeks later in wild-type or db/db mice fed
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with regular chow or various dosages of SB powder (Table 1).
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The intensities of monocyte adhesion positively correlated with the levels of blood
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glucose, cholesterol, body weight, and the abundance of MCP-1, PAI-1 or ICAM-1 in the heart
Table 2).
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apex of wild-type or db/db mice fed with or without SB powder treatment (p<0.05 or 0.01,
Discussion The present study demonstrated following major novel findings: a) administration of SB powder decreased monocyte adhesion to aorta of db/db diabetic mice; b) treatment with 5% SB powder (w/w) significantly inhibited the increases of MCP-1 in blood, and the upregulations of TNF-α, P-selectin, ICAM-1 and MCP-1 in the aorta or heart apex of db/db mice; c) 5% SB powder significantly reduced the abundance of PAI-1 and uPA in the aorta or heart apex, and 12
ACCEPTED MANUSCRIPT the plasma levels of PAI-1 in db/db mice; d) 5% SB powder inhibited the increases of both
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NOX4 and HSF1 in the aorta or heart apex of diabetic mice.
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Monocytes in blood are the precursors of macrophages in vascular wall, and macrophages are major cellular components of atherosclerotic lesions. The adhesion of
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monocytes to vascular endothelium initializes transmembrane migration of monocytes to
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vascular wall and the development of lipid-rich foam cells in atherogenetic lesions. A number of biological factors or conditions affect monocyte adhesion to cultured EC or vascular intima in
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animal models (21). Previous studies indicated that monocyte adhesion was enhanced under diabetic conditions (22). The present study demonstrated an increased normal monocyte
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adhesion to aorta from db/db diabetic mice, which suggests diabetic condition induced pro-
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monocyte adhesion in aortic intima or endothelium. The adhesion of monocytes was significantly reduced in diabetic mice fed with all tested dosages (0.2-20%) of SB powder
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compared to db/db mice without a SB exposure, which implies that monocyte adhesion to aorta from diabetic db/db mice by dietary treatment with SB powder.
It has been widely accepted that MCP-1 plays a key role in monocyte adhesion to vascular endothelium through chemotaxis and in diabetic vascular complications (23). The present study indicated that the levels of MCP-1 in blood circulation and heart apex or aorta were elevated in db/db mice. SB treatment inhibited the increase of MCP-1 in blood or the heart apex or aorta of the diabetic mice. The findings suggest that MCP-1 may contribute to 13
ACCEPTED MANUSCRIPT diabetes-induced monocyte adhesion, and the decrease in monocyte adhesion in SB powdertreated diabetic mice may be partially due to the inhibitory effect of SB powder on MCP-1. The
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increased contents MCP-1 and other inflammatory or oxidative stress related mediators in mouse aorta or heart apex were altered by diabetes or SB powder in a similar pattern. This
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suggests that SB powder altered the protein expression of the inflammatory or oxidative stress meditors not only in aortic wall, but also in heart apex (mainly myocardium) in db/db or wild-
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type mice.
Monocyte adhesion may be affected by adhesion molecules on cell surface of EC or
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monocytes. Previous studies suggest that P-selectin and ICAM-1 mediate the attachment and
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rolling over of monocytes respectively on endothelium (24, 25). The present study demonstrated that the abundances of P-selectin and ICAM-1 in the heart apex or aorta of
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db/db mice compared to that in control mice. SB power attenuated the expression of P-selectin
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and ICAM-1 in the heart apex or aorta of db/db mice, which may contribute to the reduction of monocyte adhesion in diabetic mice fed with SB powder. The cellular source of the adhesion molecules responsible to SB powder or its components-induced decrease of monocyte adhesion may be further investigated in subsequent studies.
Previous studies indicated that uPA and PAI-1 may affect monocyte adhesion (26). Glycated LDL increased the expression of PAI-1 in EC (27). Our recent studies demonstrated that PAI-1 is required for glycated LDL-induced monocyte adhesion to EC (28). The results of the present study indicated that the levels of PAI-1 in blood circulation and in the heart apex or 14
ACCEPTED MANUSCRIPT aorta of db/db mice were significantly higher than that in wild-type mice. SB powder prevented the increase in PAI-1 in blood circulation, and the abundances of PAI-1 in the heart apex or
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aorta of db/db mice. The abundances of PAI-1 in hearts positively correlated with monocyte adhesion to aorta of the mice. The results of the in vivo experiments in the present study
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supported the findings from in vitro results (28) that PAI-1 plays an important role in diabetesassociated metabolic disorder-induced monocyte adhesion. Relationship between uPA and PAI-
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1 in diabetes-induced monocyte adhesion, and the role of uPA in SB powder-induced inhibition
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of monocyte adhesion remains to be clarified in subsequent studies.
Previous studies in our laboratory demonstrated that NOX and HSF1 are involved in the
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upstream regulation of PAI-1 in glycated LDL-treated EC or fibroblasts (29). NOX components
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are required for the activation of HSF1, and HSF1 mediates the transcription of PAI-1 induced by glycated LDL (10, 20). The abundances of NOX-4 and HSF1 were increased in streptozotocin-
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induced or db/db genetic diabetic mice (12). Cyanidin-3-glucoside reduced glycated LDLinduced increased in NOX4 in EC (17). Anthocyanins containing in SB powder may contribute to the decreases of NOX4, HSF1 and PAI-1 in the heart apex or aorta of db/db mice.
Anthocyanin contents of four SB cultivars, Smoky, Thiessen, Honeywood and Northline SB, were reported recently (30). Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3arabinoside and cyanidin-3-xyloside were 1.77, 0.40, 0.27 and 0.15 mg/g (fresh weight, FW) for Smoky SB cultivar, 3.31, 1.02, 0.52 and 0.33 mg/g (FW) for Thiessen SB cultivar, 3.08, 0.80, 0.45 and 0.29 mg/g (FW) for Honeywood SB cultivar, and 3.28, 0.45, 0.33 and 0.19 mg/g (FW) for 15
ACCEPTED MANUSCRIPT Northline cultivar, respectively. Although SB cultivar and growing environment affect the level of individual anthocyanins, their order as cyanidin-3-galactoside (63-77 %) cyanidin-3-
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glucoside (10-20 %) cyanidin-3-arabinoside (7-10 %) cyanidin-3-xyloside (4-6 %), which is consistent with the anthocyanin components detected in the Smoky SB powder used in the
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present study.
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The results of correlation analysis demonstrated the monocyte adhesion positively
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correlates with inflammatory mediators, fibrinolytic regulators in heart apex, body weights, blood glucose and cholesterol in db/db and control mice, which suggests that diabetes-
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associated metabolic factors play important roles in the regulation of vascular inflammation under diabetic condition. The results of the present study demonstrated that the levels of blood
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glucose in db/db mice fed with 0.2-20% of SB powder were relatively lower than db/db mice
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without an exposure to SB powder, but the differences were not statistically significant or dose-
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dependent. The potential relationship between the inhibitory effects of SB powder on monocyte adhesion and blood glucose remains to further be investigated.
In conclusion, the results of the present study demonstrated that monocyte adhesion was evidently increased in db/db mice compared to that wild-type mice on regular diet, that was associated with the upregulations of multiple inflammatory mediators (MCP-1, ICAM-1 and P-selectin), stress regulators (NOX4 and HSF1) and fibrinolytic regulators (PAI-1 and uPA) in cardiovascular tissue. Oral administration of SB powder, a traditional fruit for residents in Canadian Prairie and Northern staties in USA, effectively reduced monocyte adhesion and 16
ACCEPTED MANUSCRIPT attenuated the disorders of the inflammatory, stress and fibrinolytic regulators in
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cardiovascular tissue of db/db mice.
Author contributions: RZ contributed to cell biological, immunological data and initial
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manuscript preparation; KL contributed to animal experiments and biochemical data; WL
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contributed to HPLC-MS analysis and SB powder preparation; SR contributed to cell biological data; MHM was responsible for animal experimental planning; TB was responsible to the
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interpretation of HPLC-MS results on berry components and consultant on berry cultivars; GXS
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was responsible to general experimental design and manuscript writing.
Acknowledgements for funding:
The authors thank for the grant supports from Canadian
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Diabetes Association, Canadian Institutes of Health Research, and Manitoba Health Research
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Council for G. X. S., and National Scientific and Engineer Research Council for M. H. M.
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ACCEPTED MANUSCRIPT 18. Hosseinian FS, Beta T (2007) Saskatoon and wild blueberries have higher anthocyanin
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contents than other Manitoba berries. J Agric Food Chem 55:10832-108388
19. Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in
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hypercholesterolemia is attenuated by L-arginine. Circulation. 1994 May;89(5):2176-82.
20. Zhao R, Shen G.X (2007) Involvement of heat shock factor-1 in glycated low density
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lipoprotein-induced upregulation of plasminogen activator inhibitor-1 in vascular endothelial
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cells. Diabetes 56:1436-1444
21. Pamukcu B, Lip GY, Devitt A, Griffiths H, Shantsila E (2010) The role of monocytes in
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atherosclerotic coronary artery disease. Ann Med 42:394-403
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22. Burlet E, Jain SK (2013) Manganese supplementation reduces high glucose-induced
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monocyte adhesion to endothelial cells and endothelial dysfunction in Zucker diabetic fatty rats. J Biol Chem 288:6409-6416
23. Dragomir E, Simionescu M (2006) Monocyte chemoattractant protein-1--a major contributor to the inflammatory process associated with diabetes. Arch Physiol Biochem 112:239-244
24. Zakynthinos E, Pappa N (2009) Inflammatory biomarkers in coronary artery disease. J Cardiol 53:317-333 21
ACCEPTED MANUSCRIPT 25. Galkina E, Ley K (2007) Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb
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Vasc Biol 27:2292-2301
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26. Viswanathan K, Richardson J, Togonu-Bickersteth B, Dai E, Liu L, Vatsya P, Sun YM, Yu J, Munuswamy-Ramanujam G, Baker H, Lucas AR (2009) Myxoma viral serpin, Serp-1, inhibits
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human monocyte adhesion through regulation of actin-binding protein filamin B. J Leukoc Biol
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85:418-426
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27. Zhang, J.Y., Ren S., Sun D.F., Shen, G.X (1998) Influence of Glycation on LDL-induced Generation of Fibrinolytic Regulators in Vascular Endothelial Cells. Arterioscler Thromb Vasc
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Biol 18:1140-1148
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28. Zhao R, Ren S, Moghadasian M, Shen GX (2012) Role of plasminogen activator inhibitor-1 in
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(abstr)
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glycated LDL-induced monocyte adhesion on vascular endothelial cells. Can J Diabetes 36:S50
29. Zhao R, Moghadasian M, Shen GX (2011) Involvement of NADPH oxidase in upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice. Free Radical Res 45:1013-1023
30. Lavola A, Karjalainen R, Julkunen-Tiitto R (2012) Bioactive Polyphenols in Leaves, Stems, and Berries of Saskatoon (Amelanchier alnifolia Nutt.) Cultivars. J Agric Food Chem 60:1020−1027
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Fig.1 Dose responses of Saskatoon berry (SB) powder on monocyte adhesion in leptin
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receptor-knockout (db/db). A: C57 control mice and db/db mice (male, 7 weeks of age) were treated with 0, 0.2, 1.0, 5.0 and 20.0% of SB powder (w/w) for 5 weeks. Freshly isolate aorta
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were incubated with 1x105 of TRITC labelled WEHI.274.1 mouse monocytes for 30 min at room
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temperature at a rotating plate. Unattached monocytes were washed away from aorta by 2 washes. Monocytes remaining or the surface of aorta were fixed and counted under
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microscope with 10x magnification. Values were expressed in mean ± SD (n = 5 mice/group, averages of monocytes/field from 5 fields/aorta). B: a scheme for the size and location of
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selected fields on aortic strip selected counting monocyte adhesion on aorta under microscopy.
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**: p<0.01 versus wild-type mice on regular chow; ++: p<0.01 versus db/db mice without SB
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powder treatment.
Fig.2 Effect of SB powder on monocyte adhesion to aorta of db/db (db) mice. C57 and db mice (male, 7 weeks) were treated with regular chow or 5% SB power for 4 weeks. Fresh aortae were incubated with labelled or unlabeled monocytes (1x105 cells/aorta) for 30 min and then processed as described in the legend of Fig.1. A. typical images: C57 (regular chow) + unlabeled monocytes; C57 (regular chow) + labelled monocytes; C57 fed with SB power + labelled monocytes; db mice (regular chow) + labelled monocytes; db mice fed with SB powder + labelled monocytes. B. Quantitative changes in monocyte adhesion (adhesion of unlabeled monocytes to aortae were not counted). Values were expressed in mean ± SD (n = 8 23
ACCEPTED MANUSCRIPT animals/group, averages of monocytes/field from 5 fields/aorta). **: p<0.01 versus wild-type
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mice on regular chow; +: p<0.05 versus db mice without SB treatment.
Fig.3 Effect of SB powder on monocyte chemotactic protein-1 (MCP-1) in plasma or heart
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apex of db/db (db) mice. C57 or db mice were fed with regular chow or 5% SB powder for 4
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weeks (n = 8/group). A: MCP-1 in plasma of mice was measured using ELISA. Values were expressed in mean ± SD (pg/ml, n = 8/group). B: MCP-1 in heart apex was assessed using
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Western blotting. Values were expressed in mean ± SD after normalization with the abundance
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of β-actin (fold of control, n = 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-
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type mice or regular chow; ++: p<0.01 versus db mice without SB powder administration.
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Fig.4 Effect of SB powder on tumor necrosis factor-α (TNF-α), P-selectin and intracellular
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adhesion molecule-1 (ICAM-1) in heart apex of db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). TNF-α, P-selectin and ICAM-1 in heart apex were assessed using Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n= 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-type mice on regular chow; +, ++: p<0.05 or 0.01 versus db mice without SB powder administration.
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ACCEPTED MANUSCRIPT Fig.5 Effect of SB powder on plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPA), NADPH oxidase 4 (NOX4) and heat shock factor-1 (HSF-1) in heart apex of
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db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). Abundances of PAI-1, uPA, NOX4 and HSF1 in heart apex were assessed using
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Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n = 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-
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type mice on regular chow; +, ++: p<0.05 or 0.01 versus db/db mice without exposure to SB
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powder.
Fig.6 Effect of SB powder on PAI-1 in plasma of db/db (db) mice. C57 or db mice were fed with
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chow or 5% SB powder for 4 weeks. PAI-1 in plasma of mice was measured using ELISA. Values
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were expressed in mean ± SD µg/mL (n = 8/group). **: p<0.01 versus wild-type mice on regular;
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++: p<0.01 versus db mice without SB powder administration.
Fig.7 Effects of SB powder on fibrinolytic, stress and inflammatory regulators in ascending aorta of db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). Abundances of PAI-1, uPA, NOX4, HSF1, ICAM-1, P-selectin, TNF-α and MCP-1 in ascending aorta of the mice were assessed using Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n = 3 assays). x: p<0.05 versus wild-type mice; *: p<0.01 versus wild-type mice on regular chow; +: p<0.01 versus db/db mice without exposure to SB powder. 25
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Figure 7
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ACCEPTED MANUSCRIPT Table 1 Body weights, blood glucose and cholesterol in db/db and control mice with and without
5.0
20.0
0
5 weeks
5 weeks
17.8 ± 0.3 21.9 ± 1.5
db/db
22.9 ± 3.7 39.8 ± 3.1** 12.1 ± 7.3
C57
16.4 ± 3.1
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23.5 ± 3.5
8.5 ± 0.9
7.8 ± 0.8
18.4 ± 2.9
42.6 ± 4.6** 13.1 ± 4.7
C57
16.6 ± 3.0
23.5 ± 1.1
db/db
20.2 ± 1.9
40.4 ± 5.7 ** 9.9 ± 8.6
C57
17.1 ± 0.7
db/db C57 db/db
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db/db
23.4 ± 1.1
8.3 ± 1.1
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C57
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1.0
0
8.5 ± 1.8
Cholesterol (mg/dl)
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5 weeks
82.9 ± 38.6
74.2 ± 28.9
23.0 ± 13.0* 164.9 ± 106.2 125.5 ± 23.1* 7.9 ± 0.7
84.7 ± 33.3
76.5 ± 14.6
18.1 ± 8.2*
168.3 ± 67.7
147.2 ± 52.1*
10.0 ± 2.8
94.2 ± 30.5
81.7 ± 26.0
16.3 ± 14.6 170.4 ± 69.0 8.6 ± 2.1
22.3 ± 4.2
46.0 ± 3.4** 9.9 ± 8.6
13.5 ± 6.7
17.1 ± 0.7
24.1 ± 2.3
6.5 ± 0.7
10.4 ± 3.0
80.0 ± 25.9
43.8 ± 4.0** 14.6 ± 4.0
19.0 ± 8.1
15.9 ± 29.9 169.6 ± 36.3**
25.3 ± 6.3
84.0 ± 12.1
159.6 ± 30.7**
7.7 ± 2.1
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Glucose (mM0
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Body weight (g)
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SBE (%) Mice
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Saskatoon berry (SB) powder administration
83.7 ± 10.9
131.6 ± 36.6* 170.8 ± 30.7** 87.6 ± 7.6
Mean ± SD (n = 5/group). *, **: p<0.05 or 0.01 versus C57 control mice with same treatment at identical time point.
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ACCEPTED MANUSCRIPT Table 2 Correlation between monocyte adhesion, body weight, glucose, cholesterol, PAI-1, MCP-1 and
Weight
Glucose
Glucose
0.47**
Chol.
0.90**
0.46**
PAI-1
0.50**
0.73**
MCP-1
0.71**
0.60**
ICAM-1
0.53**
0.63**
Monocyte adhesion
0.37*
Chol.
PAI-1
MCP-1
ICAM-1
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Variables
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ICAM-1 in diabetic and control mice with and without SB administration
0.52**
0.81**
0.40*
0.95**
0.80**
0.39*
0.79**
0.73**
0.84**
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0.54**
0.64**
Chol.: total serum cholesterol, Glucose: fasting blood glucose. Values in the table are r values. Values in the table are r. *, **: p<0.05 or 0.01 (n = 32 animals, n =3-4/group (randomly selected) for Western blotting from experiments described in Table 1). Body weights and blood tests for glucose and cholesterol were obtained just prior to tissue collection.
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S0955-2863(14)00079-5 doi: 10.1016/j.jnutbio.2014.03.016 JNB 7190
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
3 August 2013 19 March 2014 20 March 2014
Please cite this article as: Zhao Ruozhi, Le Khuong, Li Wende, Ren Song, Moghadasian Mohammed H., Beta Trust, Shen Garry X., Effects of Saskatoon Berry Powder on Monocyte Adhesion to Vascular Wall of Leptin Receptor-Deficient Diabetic Mice, The Journal of Nutritional Biochemistry (2014), doi: 10.1016/j.jnutbio.2014.03.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT JNB-13-481 R3
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Effects of Saskatoon Berry Powder on Monocyte Adhesion to Vascular
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Wall of Leptin Receptor-Deficient Diabetic Mice
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Ruozhi Zhao1, Khuong Le2, Wende Li3, Song Ren1,
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Mohammed H. Moghadasian2, Trust Beta3, Garry X. Shen1 Departments of Internal Medicine and Physiology, 2Department of Human Nutritional
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Garry X. Shen MD PhD
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Corresponding author:
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Sciences, 3Department of Food Sciences, University of Manitoba
Diabetes Research Group
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University of Manitoba
835-715 McDermot Ave Winnipeg, MB R3E 3P4 Canada Tel: 204-789-3816 Fax: 204-789-3987
Email: [email protected] Running title: Saskatoon berry on monocyte adhesion in diabetic mice Word counts for abstract: 249; text: 3,500
Key words: Saskatoon berry, db/db mice, monocyte adhesion, inflammatory mediators. 1
ACCEPTED MANUSCRIPT Abstract Hypothesis: Atherosclerotic cardiovascular complications are the leading cause of death in
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diabetic patients. Monocyte adhesion is an early event for atherogenesis. Previous studies demonstrated that dark-skin berries had cardiovascular protective effects. We hypothesize that SB
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powder may reduce monocyte adhesion in leptin receptor-deficient (db/db) diabetic mice. Methods: Wild-type and db/db mice were fed with chow or supplemented with SB powder.
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Anthocyanins in SB powder were identified using mass spectrometry. Mouse monocytes were
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incubated with mouse aorta. Monocyte adhesion was counted under microscopy. Inflammatory or metabolic markers in blood or tissue were analyzed using immunological or biochemical methods.
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Results: SB powder significantly reduced monocyte adhesion to aorta from diabetic db/db mice
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compared to regular chow. The increased monocyte adhesion to aorta was normalized in db/db mice treated with ≥5% of SB powder for 4 weeks. Increased contents of NAPDH oxidase-4, heat
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shock factor-1, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM)-1,
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P-selectin, tumor necrosis factor-α, plasminogen activator inhibitor (PAI)-1 and urokinase plasminogen activator in aorta or heart apex, elevated plasma PAI-1 and MCP-1 were detected in db/db mice on chow compared to wild-type mice on the same diet. 5% SB powder inhibited the increases of inflammatory, fibrinolytic or stress regulators in aorta or heart apex of db/db mice. Monocyte adhesion positively correlated with blood glucose, cholesterol, body weight, heart MCP1, PAI-1 or ICAM-1. Conclusion: The findings suggest that SB powder attenuated monocyte adhesion to aorta of db/db mice, which was potentially mediated through inhibiting the inflammatory, stress and/or fibrinolyic regulators. 2
ACCEPTED MANUSCRIPT Introduction
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The prevalence of diabetes mellitus (DM) rapidly increased in most countries during last 30 years, and the trend is expected to continue. Nine out of ten diabetic patients are type 2 DM
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(T2DM) (1). Atherosclerotic cardiovascular complications are the predominant cause of death in diabetic patients. Vascular inflammation plays a critical role in the etiology of atherosclerosis
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(2). Monocyte adhesion on endothelium has been considered as an early event in vascular
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inflammation (3). Previous studies demonstrated that monocyte adhesion may be regulated by cytokines, adhesion molecules, metabolic factors and oxidative stress (4, 5). Elevations of
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inflammatory mediators, including interleukins, tumor necrosis factor (TNF)-α, monocyte
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chemotactic protein (MCP)-1 and intracellular adhesion molecule (ICAM)-1, were detected in diabetic patients (6, 7). Hyperglycemia and hyperketonemia are associated with increased
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monocyte adhesion to endothelial cells (EC) (8, 9). Previous studies in our laboratory
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demonstrated that glycated low density lipoprotein (LDL) may increase the production of reactive oxygen species, activate NADPH oxidase (NOX) and impair mitochondrial respiration in cultured EC or fibroblasts (10-12). The effect of diabetes on monocyte adhesion and relevant regulators in animal models remains to be investigated.
Previous studies demonstrated that products of a number of dark-skin fruits had cardiovascular protective effects in animal models or in small trials of human study (13, 14). Anthocyanins are common flavonoids in the dark-skin phyto-products. A recent study found that purple sweet potato extracts and cyanidin inhibited TNF-α-induced monocyte adhesion 3
ACCEPTED MANUSCRIPT and the production multiple cytokines or adhesion molecules in cultured EC (15). Previous studies by our group demonstrated that cyanidin-3-glucoside or delphindin-3-glucoside
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inhibited glycated or oxidized LDL-induced oxidative stress and cell injury in vascular EC (16, 17). Saskatoon berries (SB) naturally grow or are planted in Canada prairie areas and Northern
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states of USA. Previous studies by Hosseinian and Beta demonstrated that SB powder had relatively higher contents of anthocyanins compared to blue berries, strawberries, raspberries,
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inflammation has not been documented.
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chokecherry or seabuckthorn (18). The effect of SB on monocyte adhesion or vascular
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The present study examined the effect of SB powder on the adhesion of monocytes to aorta and relevant inflammatory, stress and fibrinolytic regulators in leptin receptor-knockout
SB powder
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(db/db) mice, a common rodent model of T2DM
Materials and Methods
Smoky SB was purchased from Prairie Lane Saskatoons (Portage, Manitoba). The berries were cleaned and frozen at -70°C, and then lyophilized using a freeze-drier without an addition of supplement. Dried whole berries were powered and stored in vacuum-sealed bags at -20°C. All processes were conducted in sterile stainless steel containers.
Animals 4
ACCEPTED MANUSCRIPT Male db/db mice and C57BL/J counterparts (6 weeks of age) were obtained from the Jackson Laboratory (Bar Harbour, ME). Mice were housed in an air-conditioned room and
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received regular chow for 1 week for stabilization. Animals were randomized into indicated groups (n=5-8/group). Mice in control group received regular mouse chow, and animals in the
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SB treatment groups received chow supplemented with 0.2-20% (w/w) SB for up to 5 weeks. Body weights were measured at weekly basis. Heart, aortae, and blood were harvested from
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db/db and wild-type mice after euthanasia. The protocol of animal experiments was approved
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Monocyte adhesion to intima of aorta
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by the Animal Protocol Management and Review Committee in the University of Manitoba.
Aortae were freshly harvested at the level of aortic arch to the level of abdominal aorta
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beyond renal arteries and submersed in ice-cold Hank’s balance salt solution (HBSS).
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Perivascular adipose tissue was removed from freshly isolated aortae. The aortae were
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longitudinally opened in fresh ice-cold HBSS and fixed with 27-gauge needles on the bottom of 35-mm culture dish within 1 h after harvesting. Mouse WEHI-274.1 monocytes were labelled with 3 µg/mL of TRITC (Molecular Probes, Burlington, ON) for 15 min. Free dye was removed from cells through overlaid on fetal calf serum and a centrifugation at 400xg as previously described (19). The abundance of labeled monocytes represents >95% of total cells assessed using flow cytometer. Fluorescently labelled or unlabeled monocytes (1 x 105) were added to each dish containing aorta. Mouse monocytes were incubated with aortic strips at room temperature for 30 min on a rotator mixer. Un-adhered monocytes were removed using 2 washes with HBSS. Adhered monocytes were fixed using 2% glutaraldehyde in HBSS. Five fields 5
ACCEPTED MANUSCRIPT (2 mm in diameter/field) were observed for each aortic strip for monocyte counting under fluorescent microscopy using 10X magnification. One field was at center and 4 fields at each
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corner of aortic strip. The combined examining areas represent approximately 1/4 of total area (typically 10 mm x 6 mm) of each aortic strip (Fig.1B). The averages of adhered monocytes to
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the fields in each aorta were used for data analysis.
Western blotting
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Targeted proteins in protein extracts of heart apex and ascending aorta were analysed using Western blotting analysis as previously described (20). Monoclonal or polyclonal
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antibodies against mouse PAI-1, urokinase plasminogen activator (uPA), heat shock factor-1
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(HSF1), ICAM-1, P-selectin, TNF-α, MCP-1 or β-actin were obtained from Santa Cruz (Santa Cruz, CA), Cedarlane (Burlington, ON) or Abcam (Cambrige, MA). Corresponding secondary antibody
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conjugated with horse radish peroxidase (Santa Cruz) and enhanced chemilluminence reagents
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(GE Healthcare, Buckinghamshire, UK) were applied to visualize targeted antigens on nitrocellulose membranes. The abundances of the antigens was assessed using Chemi-Doc system and Quantity One software (BioRad, Hercules, CA), and normalized with the abundance of β-actin in corresponding samples.
Identification of anthocyanins in SB powder Anthocyanins in SB powder were extracted and analyzed using Waters high performance liquid chromatography-mass spectrometry (HPLC-MS) as previously described (18). Purified anthocyanins were loaded on a solid-phase extraction C18 cartridge of a Water 6
ACCEPTED MANUSCRIPT high performance liquid chromatography (HPLC) system. Anthocyanins were eluted with acidified methanol and the solution was dried at ambient temperature under nitrogen gas. The
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residue was re-dissolved in HPLC mobile-phase solution for the determination of anthocyanins using Waters Quatro Micro API mass spectrometer (Waters Corp, Milford, Massachusetts, USA)
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equipped with an ESI Multi-Mode Ionization probe as described (18). Standards of anthocyanins
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were obtained by Polyphenols Laboratories (Sandnes, Norway).
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Analyses of variables in blood
The levels of glucose in mouse blood were measured using AlphaTRAK glucose
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monitoring system from Abbott. Serum cholesterol was measured using Sekisui Diagnostics SL reagents. Plasma PAI-1 and MCP-1 were measured using ELISA kits (Cedarlane for mouse PAI-1,
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Thermo Scientific for mouse MCP-1).
Statistical analysis
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Continuous data were presented as means of values ± standard deviation. Data from multiple groups were analyzed using the one-way variance assay followed with post-hoc tests using Microsoft Excel software. Data from two groups using Students’ t-test. Correlations between 2 groups on continuous data were analyzed using liner regression analysis using Sigma Window software. Differences at p<0.05 were considered as significant.
RESULTS
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ACCEPTED MANUSCRIPT Anthocyanin composition of SB powder SB powder contained 11.5% moisture. The total anthocyanins in freeze-dried powder of
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Smoky SB were 5,011.0 mg/kg (dry weight basis or dwb). Four types of anthocyanins, cyanidin3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside and cyanidin-3-xyloside, were
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identified in the Smoky SB powder. The contents of individual anthocyanin in the freeze-dried powder were 3,708.2 mg/kg (dwb) for cyanidin-3-galactoside, 903.4 mg/kg (dwb) for cyanidin-
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3-glucoside, 345.0 mg/kg (dwb) as equivalent of cyanidin-3-glucoside for cyanidin-3-arabinoside
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and 314.5 mg/kg (dwb) as equivalent of cyanidin-3-glucoside for cyanidin-3-xyloside. The predominant anthocyanins were cyanidin-3-galactoside (74.00 %), and cyanidin-3-glucoside
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(18.03 %) in the Smoky SB powder.
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Effect of SB powder on monocyte adhesion in db/db mice The adhesion of mouse WEHI 274.1 monocytes to the intima of aorta from db/db mice
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was increased by 137% compared to wild-type mice (p<0.01). Monocyte adhesion to aorta of db/db mice was significantly decreased by 0.2-20% SB powder compared to db/db mice without exposure to SB powder (p<0.01). The monocyte adhesion was normalized in db/db mice fed with ≥5% SB powder (Fig. 1A). Fig. 1B demonstrated a scheme for field selection for monocyte accounting on aorta under microscopy. In a separate experiment, treatment with 5% SB powder for 4 weeks significantly reduced the adhesion of monocytes to aorta db/db mice compared to the diabetic mice without an exposure to SB powder (p<0.01, Fig. 2).
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ACCEPTED MANUSCRIPT Effects of SB powder on inflammatory mediators in plasma or heart apex of db/db mice The levels of MCP-1 in plasma were increased in db/db diabetic mice by 2-folds
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compared to wild-type mice (p<0.01). Treatment with 5% SB powder for 4 weeks reduced MCP1 in C57 mice by 30% compared to controls (p<0.05). The SB powder treatment reduced the
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levels of MCP-1 in the db/db mice by 32% compared to db/db mice fed with regular chow
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(p<0.01). The level of MCP-1 in heart apex was significantly increased in db/db mice assessed using Western blotting. SB treatment did not significantly affect the levels of MCP-1 in heart
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apex of wild-type mice. The abundance of MCP-1 in heart apex of db/db mice was significantly
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decreased after 4 weeks feeding of SB powder (Fig 3).
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The abundances of TNF-α, P-selectin and ICAM-1 in heart apex of db/db mice were
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increased compared to wild-type mice (p<0.05 or 0.01). 5% SB powder significantly increased
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the level of ICAM-1 (p<0.05), but not that of TNF-α or P-selectin, in heart apex of C57 mice. The SB powder treatment significantly inhibited the abundances of TNF α, P-selectin and ICAM-1 in heart apex of db/db mice compared to those on regular chow (p<0.05 or 0.01, Fig. 4).
Effects of SB powder on NOX4 and HSF1 in heart apex of db/db mice The results of the present study demonstrate that the levels of NOX4 and HSF1 were increased in heart apex of db/db mice. Treatment with 5% SB powder for 4 weeks significantly reduced the abundance of NOX4, but not HSF1, in wild-type mice. In db/db mice fed with 5% SB 9
ACCEPTED MANUSCRIPT powder for 4 weeks, the levels of abundance of NOX4 and HSF1 were significantly decreased in
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heart apex of compared to that in db/db mice on regular diet (p<0.05, Fig. 5).
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Impact of SB powder on fibrinolysis regulators in heart apex
SB powder (5% for 4 weeks) reduced the abundance of PAI-1 in heart apex (p<0.05), but
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did not significantly affect that of uPA, in wild-type mice on regular chow. The levels of PAI-1
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and uPA in heart apex of db/db mice fed with regular chow were significantly higher than wildtype mice on regular chow (p<0.01). The abundances of PAI-1 and uPA in the heart apex of
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db/db mice were significantly reduced by 5% SB powder compared to db/db mice without an
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exposure to SB powder (p<0.05 or 0.01, Fig 5).
Elevated levels of PAI-1 were detected in plasma of db/db mice compared to wild-type
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mice (p<0.01). PAI-1 plasma in wild-type mice was significantly lowered by treatment with 5% SB powder for 4 weeks (p<0.01). The increase of PAI-1 in plasma of db/db mice was reduced by the SB powder treatment compared to db/db mice without exposure to SB powder (p<0.01, Fig.6).
Effects of SB powder on the contents of fibrinolytic, stress and inflammatory regulators in aorta of db/db mice
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ACCEPTED MANUSCRIPT In order to determine the effect of SB powder on the contents of stress, inflammatory and fibrinolytic regulators in aortic wall, we extracted proteins from pooled ascending aorta
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from each group of wild-type and db/db mice fed with chow or 5% SB powder described in Fig.2 and conducted Western blotting for PAI-1, uPA, NOX4, HSF1, ICAM-1, P-selectin, TNF-α and
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MCP-1. The results indicated that the effect of diabetes and SB powder on the contents of the fibrinolytic, stress and inflammatory regulators in aortae were similar to that in heart apex
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(Fig.7).
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Effects of SB powder on body weight, glucose, cholesterol and relationship with monocyte adhesion or inflammatory mediators
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Body weights of db/db mice at baseline (7 weeks of age) were not significantly different
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from wild-type mice at the same age. Significantly higher body weights were detected in db/db
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mice fed with SB powder compared to that in wild-type mice on the same diet (p<0.01), but the levels of body weight between mice on various dosages of SB powder were not significantly different. At baseline, relatively higher levels of blood glucose were detected in each groups of db/db mice compared to wild-type mice, but significant increase in blood glucose between db/db and wild-type mice was only detected in those assigned for 20% SB powder (p<0.05). At 5 weeks after, the levels of blood glucose in db/db mice receiving chow or 0.2% SB powder were higher than wild-type mice receiving the same diet (p<0.05). In db/db mice fed with regular chow for 5 weeks, the mean level of glucose was 90% higher than that at baseline. In db/db mice received 0.2%, 1%, 5% and 20% SB powder, the mean levels of blood glucose were 11
ACCEPTED MANUSCRIPT 21%, 29%, 41% and 17% lower than that in db/db mice without an exposure to SB powder. The levels in blood glucose between the groups of db/db and wild-type mice fed with or without SB
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powder were not statistically significant. At baseline, significant difference in serum total cholesterol between wild-type and db/db mice was only detected in mice assigned for 5% SB
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powder (p<0.05). At the end of 5 weeks of experimental feeding, the levels of serum cholesterol in db/db mice fed with 0-20% of SB powder were significantly higher than that in
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wild-type mice fed with corresponding diet (p<0.05 or 0.01). No significant difference in serum
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cholesterol was detected between baseline and 5 weeks later in wild-type or db/db mice fed
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with regular chow or various dosages of SB powder (Table 1).
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The intensities of monocyte adhesion positively correlated with the levels of blood
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glucose, cholesterol, body weight, and the abundance of MCP-1, PAI-1 or ICAM-1 in the heart
Table 2).
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apex of wild-type or db/db mice fed with or without SB powder treatment (p<0.05 or 0.01,
Discussion The present study demonstrated following major novel findings: a) administration of SB powder decreased monocyte adhesion to aorta of db/db diabetic mice; b) treatment with 5% SB powder (w/w) significantly inhibited the increases of MCP-1 in blood, and the upregulations of TNF-α, P-selectin, ICAM-1 and MCP-1 in the aorta or heart apex of db/db mice; c) 5% SB powder significantly reduced the abundance of PAI-1 and uPA in the aorta or heart apex, and 12
ACCEPTED MANUSCRIPT the plasma levels of PAI-1 in db/db mice; d) 5% SB powder inhibited the increases of both
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NOX4 and HSF1 in the aorta or heart apex of diabetic mice.
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Monocytes in blood are the precursors of macrophages in vascular wall, and macrophages are major cellular components of atherosclerotic lesions. The adhesion of
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monocytes to vascular endothelium initializes transmembrane migration of monocytes to
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vascular wall and the development of lipid-rich foam cells in atherogenetic lesions. A number of biological factors or conditions affect monocyte adhesion to cultured EC or vascular intima in
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animal models (21). Previous studies indicated that monocyte adhesion was enhanced under diabetic conditions (22). The present study demonstrated an increased normal monocyte
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adhesion to aorta from db/db diabetic mice, which suggests diabetic condition induced pro-
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monocyte adhesion in aortic intima or endothelium. The adhesion of monocytes was significantly reduced in diabetic mice fed with all tested dosages (0.2-20%) of SB powder
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compared to db/db mice without a SB exposure, which implies that monocyte adhesion to aorta from diabetic db/db mice by dietary treatment with SB powder.
It has been widely accepted that MCP-1 plays a key role in monocyte adhesion to vascular endothelium through chemotaxis and in diabetic vascular complications (23). The present study indicated that the levels of MCP-1 in blood circulation and heart apex or aorta were elevated in db/db mice. SB treatment inhibited the increase of MCP-1 in blood or the heart apex or aorta of the diabetic mice. The findings suggest that MCP-1 may contribute to 13
ACCEPTED MANUSCRIPT diabetes-induced monocyte adhesion, and the decrease in monocyte adhesion in SB powdertreated diabetic mice may be partially due to the inhibitory effect of SB powder on MCP-1. The
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increased contents MCP-1 and other inflammatory or oxidative stress related mediators in mouse aorta or heart apex were altered by diabetes or SB powder in a similar pattern. This
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suggests that SB powder altered the protein expression of the inflammatory or oxidative stress meditors not only in aortic wall, but also in heart apex (mainly myocardium) in db/db or wild-
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type mice.
Monocyte adhesion may be affected by adhesion molecules on cell surface of EC or
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monocytes. Previous studies suggest that P-selectin and ICAM-1 mediate the attachment and
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rolling over of monocytes respectively on endothelium (24, 25). The present study demonstrated that the abundances of P-selectin and ICAM-1 in the heart apex or aorta of
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db/db mice compared to that in control mice. SB power attenuated the expression of P-selectin
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and ICAM-1 in the heart apex or aorta of db/db mice, which may contribute to the reduction of monocyte adhesion in diabetic mice fed with SB powder. The cellular source of the adhesion molecules responsible to SB powder or its components-induced decrease of monocyte adhesion may be further investigated in subsequent studies.
Previous studies indicated that uPA and PAI-1 may affect monocyte adhesion (26). Glycated LDL increased the expression of PAI-1 in EC (27). Our recent studies demonstrated that PAI-1 is required for glycated LDL-induced monocyte adhesion to EC (28). The results of the present study indicated that the levels of PAI-1 in blood circulation and in the heart apex or 14
ACCEPTED MANUSCRIPT aorta of db/db mice were significantly higher than that in wild-type mice. SB powder prevented the increase in PAI-1 in blood circulation, and the abundances of PAI-1 in the heart apex or
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aorta of db/db mice. The abundances of PAI-1 in hearts positively correlated with monocyte adhesion to aorta of the mice. The results of the in vivo experiments in the present study
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supported the findings from in vitro results (28) that PAI-1 plays an important role in diabetesassociated metabolic disorder-induced monocyte adhesion. Relationship between uPA and PAI-
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1 in diabetes-induced monocyte adhesion, and the role of uPA in SB powder-induced inhibition
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of monocyte adhesion remains to be clarified in subsequent studies.
Previous studies in our laboratory demonstrated that NOX and HSF1 are involved in the
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upstream regulation of PAI-1 in glycated LDL-treated EC or fibroblasts (29). NOX components
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are required for the activation of HSF1, and HSF1 mediates the transcription of PAI-1 induced by glycated LDL (10, 20). The abundances of NOX-4 and HSF1 were increased in streptozotocin-
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induced or db/db genetic diabetic mice (12). Cyanidin-3-glucoside reduced glycated LDLinduced increased in NOX4 in EC (17). Anthocyanins containing in SB powder may contribute to the decreases of NOX4, HSF1 and PAI-1 in the heart apex or aorta of db/db mice.
Anthocyanin contents of four SB cultivars, Smoky, Thiessen, Honeywood and Northline SB, were reported recently (30). Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3arabinoside and cyanidin-3-xyloside were 1.77, 0.40, 0.27 and 0.15 mg/g (fresh weight, FW) for Smoky SB cultivar, 3.31, 1.02, 0.52 and 0.33 mg/g (FW) for Thiessen SB cultivar, 3.08, 0.80, 0.45 and 0.29 mg/g (FW) for Honeywood SB cultivar, and 3.28, 0.45, 0.33 and 0.19 mg/g (FW) for 15
ACCEPTED MANUSCRIPT Northline cultivar, respectively. Although SB cultivar and growing environment affect the level of individual anthocyanins, their order as cyanidin-3-galactoside (63-77 %) cyanidin-3-
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glucoside (10-20 %) cyanidin-3-arabinoside (7-10 %) cyanidin-3-xyloside (4-6 %), which is consistent with the anthocyanin components detected in the Smoky SB powder used in the
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present study.
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The results of correlation analysis demonstrated the monocyte adhesion positively
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correlates with inflammatory mediators, fibrinolytic regulators in heart apex, body weights, blood glucose and cholesterol in db/db and control mice, which suggests that diabetes-
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associated metabolic factors play important roles in the regulation of vascular inflammation under diabetic condition. The results of the present study demonstrated that the levels of blood
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glucose in db/db mice fed with 0.2-20% of SB powder were relatively lower than db/db mice
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without an exposure to SB powder, but the differences were not statistically significant or dose-
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dependent. The potential relationship between the inhibitory effects of SB powder on monocyte adhesion and blood glucose remains to further be investigated.
In conclusion, the results of the present study demonstrated that monocyte adhesion was evidently increased in db/db mice compared to that wild-type mice on regular diet, that was associated with the upregulations of multiple inflammatory mediators (MCP-1, ICAM-1 and P-selectin), stress regulators (NOX4 and HSF1) and fibrinolytic regulators (PAI-1 and uPA) in cardiovascular tissue. Oral administration of SB powder, a traditional fruit for residents in Canadian Prairie and Northern staties in USA, effectively reduced monocyte adhesion and 16
ACCEPTED MANUSCRIPT attenuated the disorders of the inflammatory, stress and fibrinolytic regulators in
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cardiovascular tissue of db/db mice.
Author contributions: RZ contributed to cell biological, immunological data and initial
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manuscript preparation; KL contributed to animal experiments and biochemical data; WL
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contributed to HPLC-MS analysis and SB powder preparation; SR contributed to cell biological data; MHM was responsible for animal experimental planning; TB was responsible to the
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interpretation of HPLC-MS results on berry components and consultant on berry cultivars; GXS
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was responsible to general experimental design and manuscript writing.
Acknowledgements for funding:
The authors thank for the grant supports from Canadian
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Diabetes Association, Canadian Institutes of Health Research, and Manitoba Health Research
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Council for G. X. S., and National Scientific and Engineer Research Council for M. H. M.
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1. Sherwin R, Jastreboff AM (2012) Year in diabetes 2012: The diabetes tsunami. J Clin
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3. Wrigley BJ, Lip GY, Shantsila E (2011) The role of monocytes and inflammation in the
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11. Sangle GV, Roy Chowdhury SK, Xie X, Halayko AJ, Stelmark G, Shen GX (2010) Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated LDL. Free Radical Biol Med 48:781-790
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ACCEPTED MANUSCRIPT 12. Zhao R, Le K, Moghadasian MH, Shen GX (2013) Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1
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13. Wallace TC (2011) Anthocyanins in cardiovascular disease. Adv Nutr 2:1-7
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14. Stoclet JC, Schini-Kerth V (2011) Dietary flavonoids and human health. Ann Pharm Fr 69:78-
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15. Chao PY, Huang YP, Hsieh WB (2013) Inhibitive effect of purple sweet potato leaf extract
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ACCEPTED MANUSCRIPT 18. Hosseinian FS, Beta T (2007) Saskatoon and wild blueberries have higher anthocyanin
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19. Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in
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hypercholesterolemia is attenuated by L-arginine. Circulation. 1994 May;89(5):2176-82.
20. Zhao R, Shen G.X (2007) Involvement of heat shock factor-1 in glycated low density
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lipoprotein-induced upregulation of plasminogen activator inhibitor-1 in vascular endothelial
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cells. Diabetes 56:1436-1444
21. Pamukcu B, Lip GY, Devitt A, Griffiths H, Shantsila E (2010) The role of monocytes in
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22. Burlet E, Jain SK (2013) Manganese supplementation reduces high glucose-induced
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monocyte adhesion to endothelial cells and endothelial dysfunction in Zucker diabetic fatty rats. J Biol Chem 288:6409-6416
23. Dragomir E, Simionescu M (2006) Monocyte chemoattractant protein-1--a major contributor to the inflammatory process associated with diabetes. Arch Physiol Biochem 112:239-244
24. Zakynthinos E, Pappa N (2009) Inflammatory biomarkers in coronary artery disease. J Cardiol 53:317-333 21
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Vasc Biol 27:2292-2301
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26. Viswanathan K, Richardson J, Togonu-Bickersteth B, Dai E, Liu L, Vatsya P, Sun YM, Yu J, Munuswamy-Ramanujam G, Baker H, Lucas AR (2009) Myxoma viral serpin, Serp-1, inhibits
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human monocyte adhesion through regulation of actin-binding protein filamin B. J Leukoc Biol
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85:418-426
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27. Zhang, J.Y., Ren S., Sun D.F., Shen, G.X (1998) Influence of Glycation on LDL-induced Generation of Fibrinolytic Regulators in Vascular Endothelial Cells. Arterioscler Thromb Vasc
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Biol 18:1140-1148
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28. Zhao R, Ren S, Moghadasian M, Shen GX (2012) Role of plasminogen activator inhibitor-1 in
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glycated LDL-induced monocyte adhesion on vascular endothelial cells. Can J Diabetes 36:S50
29. Zhao R, Moghadasian M, Shen GX (2011) Involvement of NADPH oxidase in upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice. Free Radical Res 45:1013-1023
30. Lavola A, Karjalainen R, Julkunen-Tiitto R (2012) Bioactive Polyphenols in Leaves, Stems, and Berries of Saskatoon (Amelanchier alnifolia Nutt.) Cultivars. J Agric Food Chem 60:1020−1027
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ACCEPTED MANUSCRIPT Legends of figures
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Fig.1 Dose responses of Saskatoon berry (SB) powder on monocyte adhesion in leptin
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receptor-knockout (db/db). A: C57 control mice and db/db mice (male, 7 weeks of age) were treated with 0, 0.2, 1.0, 5.0 and 20.0% of SB powder (w/w) for 5 weeks. Freshly isolate aorta
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were incubated with 1x105 of TRITC labelled WEHI.274.1 mouse monocytes for 30 min at room
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temperature at a rotating plate. Unattached monocytes were washed away from aorta by 2 washes. Monocytes remaining or the surface of aorta were fixed and counted under
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microscope with 10x magnification. Values were expressed in mean ± SD (n = 5 mice/group, averages of monocytes/field from 5 fields/aorta). B: a scheme for the size and location of
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selected fields on aortic strip selected counting monocyte adhesion on aorta under microscopy.
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**: p<0.01 versus wild-type mice on regular chow; ++: p<0.01 versus db/db mice without SB
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powder treatment.
Fig.2 Effect of SB powder on monocyte adhesion to aorta of db/db (db) mice. C57 and db mice (male, 7 weeks) were treated with regular chow or 5% SB power for 4 weeks. Fresh aortae were incubated with labelled or unlabeled monocytes (1x105 cells/aorta) for 30 min and then processed as described in the legend of Fig.1. A. typical images: C57 (regular chow) + unlabeled monocytes; C57 (regular chow) + labelled monocytes; C57 fed with SB power + labelled monocytes; db mice (regular chow) + labelled monocytes; db mice fed with SB powder + labelled monocytes. B. Quantitative changes in monocyte adhesion (adhesion of unlabeled monocytes to aortae were not counted). Values were expressed in mean ± SD (n = 8 23
ACCEPTED MANUSCRIPT animals/group, averages of monocytes/field from 5 fields/aorta). **: p<0.01 versus wild-type
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mice on regular chow; +: p<0.05 versus db mice without SB treatment.
Fig.3 Effect of SB powder on monocyte chemotactic protein-1 (MCP-1) in plasma or heart
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apex of db/db (db) mice. C57 or db mice were fed with regular chow or 5% SB powder for 4
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weeks (n = 8/group). A: MCP-1 in plasma of mice was measured using ELISA. Values were expressed in mean ± SD (pg/ml, n = 8/group). B: MCP-1 in heart apex was assessed using
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Western blotting. Values were expressed in mean ± SD after normalization with the abundance
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of β-actin (fold of control, n = 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-
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type mice or regular chow; ++: p<0.01 versus db mice without SB powder administration.
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Fig.4 Effect of SB powder on tumor necrosis factor-α (TNF-α), P-selectin and intracellular
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adhesion molecule-1 (ICAM-1) in heart apex of db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). TNF-α, P-selectin and ICAM-1 in heart apex were assessed using Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n= 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-type mice on regular chow; +, ++: p<0.05 or 0.01 versus db mice without SB powder administration.
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ACCEPTED MANUSCRIPT Fig.5 Effect of SB powder on plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPA), NADPH oxidase 4 (NOX4) and heat shock factor-1 (HSF-1) in heart apex of
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db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). Abundances of PAI-1, uPA, NOX4 and HSF1 in heart apex were assessed using
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Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n = 3/group, randomly selected). *, **: p<0.05 or 0.01 versus wild-
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type mice on regular chow; +, ++: p<0.05 or 0.01 versus db/db mice without exposure to SB
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powder.
Fig.6 Effect of SB powder on PAI-1 in plasma of db/db (db) mice. C57 or db mice were fed with
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chow or 5% SB powder for 4 weeks. PAI-1 in plasma of mice was measured using ELISA. Values
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were expressed in mean ± SD µg/mL (n = 8/group). **: p<0.01 versus wild-type mice on regular;
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++: p<0.01 versus db mice without SB powder administration.
Fig.7 Effects of SB powder on fibrinolytic, stress and inflammatory regulators in ascending aorta of db/db (db) mice. C57 or db mice were fed with chow or 5% SB powder for 4 weeks (n = 8/group). Abundances of PAI-1, uPA, NOX4, HSF1, ICAM-1, P-selectin, TNF-α and MCP-1 in ascending aorta of the mice were assessed using Western blotting. Values were expressed in mean ± SD after normalization with the abundance of β-actin (fold of control, n = 3 assays). x: p<0.05 versus wild-type mice; *: p<0.01 versus wild-type mice on regular chow; +: p<0.01 versus db/db mice without exposure to SB powder. 25
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ACCEPTED MANUSCRIPT Table 1 Body weights, blood glucose and cholesterol in db/db and control mice with and without
5.0
20.0
0
5 weeks
5 weeks
17.8 ± 0.3 21.9 ± 1.5
db/db
22.9 ± 3.7 39.8 ± 3.1** 12.1 ± 7.3
C57
16.4 ± 3.1
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23.5 ± 3.5
8.5 ± 0.9
7.8 ± 0.8
18.4 ± 2.9
42.6 ± 4.6** 13.1 ± 4.7
C57
16.6 ± 3.0
23.5 ± 1.1
db/db
20.2 ± 1.9
40.4 ± 5.7 ** 9.9 ± 8.6
C57
17.1 ± 0.7
db/db C57 db/db
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db/db
23.4 ± 1.1
8.3 ± 1.1
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C57
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1.0
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8.5 ± 1.8
Cholesterol (mg/dl)
0
5 weeks
82.9 ± 38.6
74.2 ± 28.9
23.0 ± 13.0* 164.9 ± 106.2 125.5 ± 23.1* 7.9 ± 0.7
84.7 ± 33.3
76.5 ± 14.6
18.1 ± 8.2*
168.3 ± 67.7
147.2 ± 52.1*
10.0 ± 2.8
94.2 ± 30.5
81.7 ± 26.0
16.3 ± 14.6 170.4 ± 69.0 8.6 ± 2.1
22.3 ± 4.2
46.0 ± 3.4** 9.9 ± 8.6
13.5 ± 6.7
17.1 ± 0.7
24.1 ± 2.3
6.5 ± 0.7
10.4 ± 3.0
80.0 ± 25.9
43.8 ± 4.0** 14.6 ± 4.0
19.0 ± 8.1
15.9 ± 29.9 169.6 ± 36.3**
25.3 ± 6.3
84.0 ± 12.1
159.6 ± 30.7**
7.7 ± 2.1
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Glucose (mM0
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Body weight (g)
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SBE (%) Mice
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Saskatoon berry (SB) powder administration
83.7 ± 10.9
131.6 ± 36.6* 170.8 ± 30.7** 87.6 ± 7.6
Mean ± SD (n = 5/group). *, **: p<0.05 or 0.01 versus C57 control mice with same treatment at identical time point.
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ACCEPTED MANUSCRIPT Table 2 Correlation between monocyte adhesion, body weight, glucose, cholesterol, PAI-1, MCP-1 and
Weight
Glucose
Glucose
0.47**
Chol.
0.90**
0.46**
PAI-1
0.50**
0.73**
MCP-1
0.71**
0.60**
ICAM-1
0.53**
0.63**
Monocyte adhesion
0.37*
Chol.
PAI-1
MCP-1
ICAM-1
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ICAM-1 in diabetic and control mice with and without SB administration
0.52**
0.81**
0.40*
0.95**
0.80**
0.39*
0.79**
0.73**
0.84**
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0.54**
0.64**
Chol.: total serum cholesterol, Glucose: fasting blood glucose. Values in the table are r values. Values in the table are r. *, **: p<0.05 or 0.01 (n = 32 animals, n =3-4/group (randomly selected) for Western blotting from experiments described in Table 1). Body weights and blood tests for glucose and cholesterol were obtained just prior to tissue collection.
34