Egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) inhibits

Data were analyzed by one way analysis of variance (ANOVA) coupled with Tukey post-hoc. 126 test by the ... but up-regulate MasR expression in Ang II-...
0 downloads 4 Views 942KB Size
Subscriber access provided by JAMES COOK UNIVERSITY LIBRARY

Bioactive Constituents, Metabolites, and Functions

Egg white-derived antihypertensive peptide IRW (IleArg-Trp) inhibits Ang II-stimulated migration of vascular smooth muscle cells via angiotensin type I receptor Wang Liao, Hongbing Fan, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00483 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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 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 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.

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 24

Journal of Agricultural and Food Chemistry

1

Egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) inhibits Ang II-stimulated

2

migration of vascular smooth muscle cells via angiotensin type I receptor

3

Wang Liao†, Hongbing Fan† and Jianping Wu †‡*

4

5

6 †

7

Department of Agricultural, Food and Nutritional Science; ‡ Cardiovascular Research Centre, University of Alberta, Edmonton, AB, Canada

8

9

10

11

12

13

14

15

*

16

Alberta, Canada, T6G 2P5 (telephone 1-780-492-6885; fax 1-780-492-4265; e-mail:

17

[email protected])

Corresponding author: Dr. Jianping Wu, 4-10 Ag/For Centre, University of Alberta, Edmonton,

18

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

20

Abstract

21

Excessive proliferation, inflammation, oxidative stress and migration induced by angiotensin II

22

(Ang II), occurring in vascular smooth muscle cells (VSMCs) during vascular remodelling, are a

23

major pathogenesis of hypertension. Antihypertensive peptides derived from food proteins are

24

promising alternatives in preventing/treating hypertension and associated complications. In

25

addition to reducing high blood pressure in spontaneously hypertensive rats, egg white

26

ovotransferrin-derived antihypertensive IRW (Ile-Arg-Trp) was shown to exert anti-proliferative,

27

anti-oxidant and anti-inflammatory effects in A7r5 cells (a vascular smooth muscle cell line)

28

against Ang II stimulation, further indicating its potential in retarding vascular remodelling.

29

Since its regulatory role on migration of VSMC is unclear, the objective of this study was to

30

evaluate the anti-migrant activity of IRW in Ang II-stimulated A7r5 cells. It was found that IRW

31

could down-regulate matrix metallopeptidase 9 (MMP9) expression and inhibit migration of Ang

32

II-stimulated A7r5 cells, which was associated with inactivation of p38/MAPK signalling. More

33

importantly, the anti-migrant activity of IRW in Ang II-stimulated A7r5 cells was dependent on

34

angiotensin type I receptor (AT1R). Our study provided the first evidence that egg

35

ovotransferrin-derived antihypertensive peptide IRW inhibited migration of VSMCs.

36

37

Key words: Ovotransferrin; Antihypertensive peptides; IRW; Vascular smooth muscle cell;

38

Migration

39

40

41

ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

Journal of Agricultural and Food Chemistry

42

Introduction

43

Vascular smooth muscle cell (VSMC) is a highly specialized cell in vasculature. By contraction

44

and relaxation, VSMC can alter the luminal diameter, enabling blood vessels to maintain an

45

appropriate blood pressure.1 Physiologically, synthetic VSMC and quiescent VSMC represent

46

the two ends of a spectrum of VSMCs with intermediate phenotypes. In normal conditions,

47

quiescent VSMC is the predominant phenotype. While, in response to stimulus, VSMC could

48

transform from quiescent phenotype to synthetic phenotype, leading to vascular remodelling,2

49

which is a common feature among major cases of hypertension.3 Excessive proliferation,

50

inflammation, oxidative stress and migration are typical intracellular events in VSMCs during

51

vascular remodelling.4 Specifically for migration, it occurs after over proliferation, during which,

52

VSMCs migrate from media to intima and subsequently proliferate in intima, ultimately leading

53

to the formation of vascular lesions.5

54

The renin angiotensin system (RAS) plays an essential role in blood pressure regulation.

55

Angiotensin II (Ang II) is a potent vasoconstrictor in the RAS.

56

participates in vascular remodelling mainly through angiotensin type I receptor (AT1R). In

57

return, vascular remodelling could further increase peripheral resistance and enhance

58

hypertension.6 The effects of some AT1R antagonists in mitigating vascular remodelling have

59

been reported.7,8 However, as pharmaceutical antihypertensive drugs are always associated with

60

side-effects, development safer alternatives from natural sources is highly suggested.9,10

61

Food protein-derived antihypertensive peptides have attracted substantial attentions during the

62

past decades. Numerous antihypertensive peptides have been characterized from various food

63

proteins.11 IRW (Ile-Arg-Trp), characterized from egg white ovotransferrin by our lab is one of

ACS Paragon Plus Environment

However, Ang II also

Journal of Agricultural and Food Chemistry

64

the most potent antihypertensive peptides.12 Followed by the validation of its blood pressure-

65

lowering effect in spontaneously hypertensive rats (SHRs), mechanisms underlying its

66

antihypertensive activity have been addressed mainly including amelioration of endothelial

67

dysfunction, enhancement of nitric-oxide-mediated vasodilation13 and upregulation of

68

angiotensin converting enzyme 2 (ACE2) at mRNA level.14 Furthermore, we found that IRW

69

treatment could ameliorate proliferation, oxidative stress and inflammation in VSMCs against

70

Ang II stimulation,15 indicating the potential of IRW in retarding vascular remodelling. However,

71

the role of IRW in inhibiting migration of VSMCs is ambiguous.

72

In order to have a further insight on regulatory roles of IRW in cellular events of VSMCs during

73

vascular remodelling, the objectives of this study were to investigate effects of IRW on Ang II-

74

stimulated migration of VSMCs and characterize the involved signaling pathways.

75

Materials and Methods

76

Reagents

77

Dulbecco’s modified Eagle medium (DMEM), Opti-MEM and fetal bovine serum (FBS) were

78

purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Penicillin-Streptomycin as well as

79

Gentamicin was from Life Technologies (Carlsbad, CA, USA). Dulbecco’s phosphate buffered

80

saline (PBS) and dithiothreitol (DTT) were from Sigma Aldrich (St Louis, MO, USA). Ang II

81

was purchased from Sigma Aldrich. IRW was synthesized by Genscript (Piscataway, NJ, USA)

82

with a purity > 98% and the sequence was validated by HPLC-MS/MS.

83

Cell culture

ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

Journal of Agricultural and Food Chemistry

84

Similar to our previous study,15,16 A7r5 cells (cat# ATCC CRL-1444, Manassas, VA, USA),

85

which is a VSMC cell-line from rat aorta, were used in this study between passages 4 and 11.

86

The cells were received were grown in DMEM supplemented with 10% FBS and antibiotics

87

(Penicillin-Streptomycin and Gentamicin) at a CO2 level of 5%. Cell culture media was replaced

88

with DMEM supplemented with 1% FBS and antibiotics prior to the treatment. Cells were

89

treated with 50 µM of IRW combined with 1 µM of mas receptor (MasR) antagonist A77917

90

(Abcam) or 50 µM of IRW combined with 100 µM of AT1R antagonist losartan potassium18

91

(Tocris, Oakville, ON, Canada) 1 h prior to adding 1 µM of Ang II for different time periods.

92

Western blot

93

After 23-hour co-treatment of IRW+specific receptor antagonist+Ang II, cell lysates were

94

prepared as we described in our previous study.15 Total proteins of the cell lysates were separated

95

by 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to

96

nitrocellulose membranes and immunoblotted with antibody against matrix metallopeptidase 9

97

(MMP9, NOVUS Biologicals, Littleton, CO, USA). Goat anti-rabbit IRDye 680RD or Donkey

98

anti-mouse 800CW from Licor Biosciences (Lincoln, NE, USA) was used as the secondary

99

antibody. Protein bands were detected by Licor Odyssey BioImager (Licor Biosciences).

100

Quantification of protein bands were performed using Image Studio Lite 5.2 (Licor Biosciences).

101

Each band was normalized to the loading control α-tubulin (rabbit polyclonal antibody from

102

Abcam, Cambridge, MA, cat# ab15246).

103

For intracellular signalling study, the co-treatment time of IRW and Ang II was 15 min. Protein

104

samples were harvested and run in SDS-PAGE, followed by membrane transfer as indicated

105

above. Protein bands for phospho-extracellular signal-regulated kinases 1/2 (p-ERK1/2, Cell

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

106

Signaling Technology, Danvers, MA, USA), phspho-p38 (p-p38, NOVUS Biologicals) and

107

phospho-c-Jun N-terminal Kinase (p-JNK, NOVUS Biologicals) were normalized to the

108

corresponding “total” form (ERK1/2, Cell Signaling Technology; p38, Santa Cruz

109

Biotechnology, Dallas, TX, USA; JNK, NOVUS Biologicals).

110 111

Cell migration assay

112

Migration of A7r5 cells was evaluated by CytoSelectTM wound healing assay kit (Cell Biolabs,

113

Inc., San Diego, CA, USA) according to the manual. Briefly, cells were seeded into a 24-well

114

plate with a wound healing insert in the middle of each well. Cells were cultured for overnight

115

until the monolayer forms. The wound healing area was created by removing the insert. The

116

wound healing insert was removed to create a 0.9 mm-wound healing area. Cells then were

117

treated with 50 µM of IRW combined with 100 µM of AT1R antagonist losartan potassium

118

(Tocris) 1 h before the addition of 1 µM Ang II. The co-treatment period of IRW, AT1R

119

antagonist and Ang II was 4 h.19 Afterwards, cells were fixed by fixation solution and cell

120

nucleus were stained by DAPI fluorescence stain. Cells were visualized by Olympus IX83

121

fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). Distance of the wound area was

122

measured by ImgeJ software (https://imagej.net/Welcome). Less distance indicated a higher

123

migration ability of the cells. Results of each treatment group were normalized to the

124

corresponding untreated.

125

Statistics

126

Data were analyzed by one way analysis of variance (ANOVA) coupled with Tukey post-hoc

127

test by the PRISM 5 statistical software (GraphPad Software, Inc., La Jolla, CA, USA) with a

ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

Journal of Agricultural and Food Chemistry

128

significance level of P