Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (IleArg-Trp) in vascular smooth muscle cells against angiotensin II stimulation Wang Liao, Subhadeep Chakrabarti, Sandra T. Davidge, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03513 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Journal of Agricultural and Food Chemistry

Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp)

1

on vascular smooth muscle cells against angiotensin II stimulation

2 3

Wang Liao, † Subhadeep Chakrabarti, †,∥,⊥ Sandra T. Davidge, ‡,§,∥,⊥ Jianping Wu *,†,∥

4 5 6 7

†

Department of Agricultural, Food & Nutritional Science, ‡ Department of Obstetrics &

Gynecology,

§

Department of Physiology,

∥

Cardiovascular Research Centre, ⊥ Women and

8

Children’s Health Research Institute

9

University of Alberta, Edmonton, Alberta, T6G 2P5, Canada

10 11 12

Corresponding author

13

*E-mail: [email protected]. 4-10 Ag/For Centre, University of Alberta, Edmonton, AB T6G

14

2P5, Canada. Phone: (780) 492-6885. Fax: (780) 492-4265.

15 16 17 18 19 20 21 22

ACS Paragon Plus Environment


23

ABSTRACT

24

25

The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin

26

II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal

27

proliferation, oxidative stress and inflammation in vascular smooth muscle cells (VSMCs) that

28

contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-

29

Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well

30

as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on

31

VSMCs are still unclear. In the present study, we evaluated the anti-proliferative, anti-oxidant,

32

and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was

33

found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production

34

and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-

35

κB pathway. These findings could further our understanding on the antihypertensive mechanism

36

of IRW beyond vascular endothelium.

37 38 39 40

KEYWORDS: IRW, VSMC, proliferation, oxidative stress, inflammation

41 42


Page 2 of 26

Page 3 of 26

43


INTRODUCTION

44

Hypertension is a global health challenge, accounting for about 6% of the total deaths

45

worldwide.1 Hypertension is also a major risk factor for renal and cardiovascular diseases.2 The

46

renin angiotensin system (RAS) is one of the major regulators of blood pressure and fluid and

47

electrolyte homeostasis,3 in which, angiotensin II (Ang II) is the main effector peptide of

48

vasoconstriction.4 Hence, angiotensin converting enzyme (ACE) inhibitor (to reduce the

49

production of Ang II), or angiotensin II type I receptor (AT1R) blocker (to inhibit the binding of

50

Ang II to its target tissues) are the first-line drugs for hypertension therapy.5

51

Ang II is more than a potent vasoconstrictor. It also plays multiple roles in controlling the

52

functional and structural integrity of the vascular wall.6 In vascular smooth muscle cells

53

(VSMCs), over stimulation of Ang II could lead to aberrant proliferation,7 excessive superoxide

54

production8 and inflammation,6 which would further promote vascular remodelling and

55

inflammation, playing pivotal roles in atherosclerotic diseases.4 The anti-proliferative, anti-

56

oxidant and anti-inflammatory actions of ACE-inhibitory agents have been documented.

57

However, as pharmacological drugs require long-term (often life long) therapy with considerable

58

side-effects, there is an unmet need for safer alternatives from naturally based sources.12,13

9-11

59

In the past three decades, food protein-derived antihypertensive peptides have received

60

substantial interest as natural alternatives to clinical drugs.14 Antihypertensive peptides derived

61

from egg white, milk and soy proteins have been reported and the in vivo effects of some of these

62

peptides have been validated on spontaneously hypertensive rats (SHRs).15-19 Functional food

63

and nutraceuticals developed from these peptides may provide new opportunities for treatment

64

and prevention of hypertension. IRW (Ile-Arg-Trp), an egg white ovotransferrin-derived

65

tripeptide, was identified and characterized as a potent ACE inhibitor.20 The anti-hypertensive



66

activity of IRW is associated with amelioration of endothelial inflammation and oxidative

67

stress,21 enhancement of nitric oxide-mediated vasodilation17 and upregulation of angiotensin

68

converting enzyme 2 (ACE2) expression.22 However, beneficial effects of IRW on VSMCs, if

69

any, have not been investigated previously.

70

Given this background, we aimed to investigate effects of IRW on proliferation, oxidative

71

stress and inflammation in VSMCs under Ang II stimulation in this study.

72

MATERIALS and METHODS

73

Reagents. Dulbecco’s phosphate buffered saline (PBS), Ang II and dithiothreitol (DTT) were

74

from Sigma Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and

75

fetal bovine serum (FBS) were purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Type 1

76

Collagenase used for cell splitting was from Worthington Biochemical Corporation (Lakewood,

77

NJ, USA). Penicillin-Streptomycin as well as Gentamicin was from Life Technologies (Carlsbad,

78

CA, USA). Triton-X-100 was from VWR International (West Chester, PA, USA). The tripeptide

79

IRW was synthesized by Genscript (Piscataway, NJ, USA). Peptide sequence and purity (99.8

80

%) were validated by HPLC-MS/MS. The peptide was dissolved in PBS at a stock concentration

81

of 10 mM, aliquoted and stored at -20οC until use.

82

Cell culture. Rat aortic VSMC cell line A7r5 was purchased from ATCC (cat# ATCC CRL-

83

1444, Manassas, VA, USA). The cells were grown in DMEM supplemented with 10% FBS and

84

antibiotics (Penicillin-Streptomycin and Gentamicin) until they reached 80% confluence. For

85

actual experiments, the confluent cells were placed in a quiescing medium (DMEM + 1% FBS +

86

antibiotics) and then treated with 50 µM of IRW 1 h prior to the addition of 1 µM of Ang II for


Page 4 of 26

Page 5 of 26


87

different time periods. Cells between passages 4 and 11 (since receiving) were used for all

88

experiments.

89

Cell proliferation assay. Cell proliferation was evaluated by bromodeoxyuridine (BrDU)

90

incorporation assay. A7r5 cells were seeded into a 48 well plate and grown in DMEM with 10%

91

of FBS for overnight. After the medium was replaced by quiescing medium, 50 µM of IRW was

92

added 1 h prior to stimulation of Ang II (1 µM). The co-treatment period with IRW and Ang II

93

was 23 h. Afterwards, BrDU reagent (Invitrogen) diluted in quiescing medium at a final

94

concentration of 1% was added for 1 h incubation at 37 ℃. Then the cells were fixed by 70%

95

ethanol (20 min, room temperature). The

96

hydrochloric acid (20 min, room temperature) followed by permeabilization by 0.1% Triton-X-

97

100 in PBS (5 min, room temperature) and finally, blocking was done in 1% bovine serum

98

albumin (BSA) in PBS (60 min, room temperature). Mouse monoclonal antibody against BrDU

99

(1:1000; Cell Signaling, Beverly, MA, USA) was diluted in PBS with 0.1% BSA and incubated

100

at 4 °C overnight. Upon washing, cell was treated with anti-mouse secondary antibody

101

(Molecular Probes, Eugene, OR, USA) for 30 minutes in the dark. Nuclei were stained with the

102

Hoechst33342 nuclear dye (Molecular Probes). Visualization was performed by Olympus IX81

103

fluorescent microscope (Carson Scientific Imaging Group; Markham, ON, Canada). For each

104

data point, 3 fields were randomly selected and the number of nuclei counted. The percentage of

105

nuclei positive for BrDU stain was noted in each field and the mean value was calculated.

106

antigen was exposed by incubation with 1M

Superoxide detection. Cellular superoxide generation was detected by dihydroethidium (DHE)

107

staining similar to our previous work.21 Reactive oxygen species (ROS) could react with DHE to

108

form ethidium, which then binds to nuclear DNA and release nuclear fluorescence.23 A7r5 cells

109

were seeded into 48 well plate and grown in DMEM with 10% of FBS until confluent. After the



Page 6 of 26

110

medium was replaced by quiescing medium, 50 µM of IRW was added 1 h prior to stimulation

111

of Ang II (1 µM). The co-treatment period of IRW and Ang II was 30 min. Then cells were

112

treated with 20 µM of DHE and incubated in dark for 30 min. Afterwards, the cells were washed

113

3 times. Fluorescence signal was detected by Olympus IX81 fluorescent microscope (Carson

114

Scientific Imaging Group). For each data point, images from 3 random fields were taken. Mean

115

fluorescence intensity (MFI)

116

(http://imagej.net/Welcome). MFI/cell was calculated based on the cell number in each field.

117

Results were presented as % of the untreated group.

of

each

image was

quantified

by ImageJ

software

118

Western blot. At the end of experiment, the A7r5 cells were lysed in boiling hot Laemmle’s

119

buffer containing 50 mM DTT (a reducing agent) and 0.2% Triton-X-100 to prepare samples for

120

western blotting. These cell lysates were then run in 9% sodium dodecyl sulfate polyacrylamide

121

gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and immunoblotted

122

with antibodies. Protein bands for cyclooxygenase 2 (COX2) (rabbit monoclonal antibody from

123

Abcam, cat# ab52237), inducible nitric oxide synthase (iNOS) (mouse monoclonal antibody

124

from BD Transduction Laboratories, cat# 610432) and inhibitory κBα (IκBα) (Rabbit polyclonal

125

antibody from Cell Signaling, cat# 9242) and AT1R (rabbit polyclonal antibody from Santa Cruz

126

Biotechnology, cat# sc-1173) were normalized to the loading control α-tubulin (rabbit polyclonal

127

antibody from Abcam, cat# ab15246). Phospho-p65 (p-p65) (rabbit polyclonal antibody from

128

Cell Signling, cat# 3033) were normalized to the total p65 (mouse monoclonal antibody from

129

Santa Cruz Biotechnology, cat# sc-8008). Anti-tubulin was used at 0.4 µg/ml, while all other

130

antibodies were used at 0.5-1 µg/ml. Goat anti-rabbit IRDye 680RD or Donkey anti-mouse

131

800CW from Licor Biosciences (Lincoln, NE, USA) was used as the secondary antibody. Protein

132

bands were detected by Licor Odyssey BioImager (Licor Biosciences) and quantified by


Page 7 of 26


133

densitometry using Image Studio Lite 5.2 (Licor Biosciences). Cell lysates from untreated cells

134

were loaded on every gel and all data were expressed as % of the corresponding untreated.

135

Statistics. All data have been presented as mean ± SEM (standard error of mean) of 4 to 6

136

independent experiments. Data were analyzed by one way analysis of variance (ANOVA) with

137

Tukey’s post-hoc test. The PRISM 5 statistical software (Graph Pad Software, San Diego, CA)

138

was used for all analyses. P

Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide

Recommend Documents