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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
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Egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) inhibits Ang II-stimulated
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migration of vascular smooth muscle cells via angiotensin type I receptor
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Wang Liao†, Hongbing Fan† and Jianping Wu †‡*
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Department of Agricultural, Food and Nutritional Science; ‡ Cardiovascular Research Centre, University of Alberta, Edmonton, AB, Canada
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*
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Alberta, Canada, T6G 2P5 (telephone 1-780-492-6885; fax 1-780-492-4265; e-mail:
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[email protected])
Corresponding author: Dr. Jianping Wu, 4-10 Ag/For Centre, University of Alberta, Edmonton,
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Abstract
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Excessive proliferation, inflammation, oxidative stress and migration induced by angiotensin II
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(Ang II), occurring in vascular smooth muscle cells (VSMCs) during vascular remodelling, are a
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major pathogenesis of hypertension. Antihypertensive peptides derived from food proteins are
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promising alternatives in preventing/treating hypertension and associated complications. In
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addition to reducing high blood pressure in spontaneously hypertensive rats, egg white
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ovotransferrin-derived antihypertensive IRW (Ile-Arg-Trp) was shown to exert anti-proliferative,
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anti-oxidant and anti-inflammatory effects in A7r5 cells (a vascular smooth muscle cell line)
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against Ang II stimulation, further indicating its potential in retarding vascular remodelling.
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Since its regulatory role on migration of VSMC is unclear, the objective of this study was to
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evaluate the anti-migrant activity of IRW in Ang II-stimulated A7r5 cells. It was found that IRW
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could down-regulate matrix metallopeptidase 9 (MMP9) expression and inhibit migration of Ang
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II-stimulated A7r5 cells, which was associated with inactivation of p38/MAPK signalling. More
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importantly, the anti-migrant activity of IRW in Ang II-stimulated A7r5 cells was dependent on
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angiotensin type I receptor (AT1R). Our study provided the first evidence that egg
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ovotransferrin-derived antihypertensive peptide IRW inhibited migration of VSMCs.
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Key words: Ovotransferrin; Antihypertensive peptides; IRW; Vascular smooth muscle cell;
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Migration
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Introduction
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Vascular smooth muscle cell (VSMC) is a highly specialized cell in vasculature. By contraction
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and relaxation, VSMC can alter the luminal diameter, enabling blood vessels to maintain an
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appropriate blood pressure.1 Physiologically, synthetic VSMC and quiescent VSMC represent
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the two ends of a spectrum of VSMCs with intermediate phenotypes. In normal conditions,
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quiescent VSMC is the predominant phenotype. While, in response to stimulus, VSMC could
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transform from quiescent phenotype to synthetic phenotype, leading to vascular remodelling,2
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which is a common feature among major cases of hypertension.3 Excessive proliferation,
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inflammation, oxidative stress and migration are typical intracellular events in VSMCs during
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vascular remodelling.4 Specifically for migration, it occurs after over proliferation, during which,
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VSMCs migrate from media to intima and subsequently proliferate in intima, ultimately leading
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to the formation of vascular lesions.5
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The renin angiotensin system (RAS) plays an essential role in blood pressure regulation.
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Angiotensin II (Ang II) is a potent vasoconstrictor in the RAS.
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participates in vascular remodelling mainly through angiotensin type I receptor (AT1R). In
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return, vascular remodelling could further increase peripheral resistance and enhance
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hypertension.6 The effects of some AT1R antagonists in mitigating vascular remodelling have
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been reported.7,8 However, as pharmaceutical antihypertensive drugs are always associated with
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side-effects, development safer alternatives from natural sources is highly suggested.9,10
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Food protein-derived antihypertensive peptides have attracted substantial attentions during the
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past decades. Numerous antihypertensive peptides have been characterized from various food
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proteins.11 IRW (Ile-Arg-Trp), characterized from egg white ovotransferrin by our lab is one of
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the most potent antihypertensive peptides.12 Followed by the validation of its blood pressure-
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lowering effect in spontaneously hypertensive rats (SHRs), mechanisms underlying its
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antihypertensive activity have been addressed mainly including amelioration of endothelial
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dysfunction, enhancement of nitric-oxide-mediated vasodilation13 and upregulation of
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angiotensin converting enzyme 2 (ACE2) at mRNA level.14 Furthermore, we found that IRW
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treatment could ameliorate proliferation, oxidative stress and inflammation in VSMCs against
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Ang II stimulation,15 indicating the potential of IRW in retarding vascular remodelling. However,
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the role of IRW in inhibiting migration of VSMCs is ambiguous.
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In order to have a further insight on regulatory roles of IRW in cellular events of VSMCs during
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vascular remodelling, the objectives of this study were to investigate effects of IRW on Ang II-
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stimulated migration of VSMCs and characterize the involved signaling pathways.
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Materials and Methods
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Reagents
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Dulbecco’s modified Eagle medium (DMEM), Opti-MEM and fetal bovine serum (FBS) were
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purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Penicillin-Streptomycin as well as
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Gentamicin was from Life Technologies (Carlsbad, CA, USA). Dulbecco’s phosphate buffered
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saline (PBS) and dithiothreitol (DTT) were from Sigma Aldrich (St Louis, MO, USA). Ang II
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was purchased from Sigma Aldrich. IRW was synthesized by Genscript (Piscataway, NJ, USA)
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with a purity > 98% and the sequence was validated by HPLC-MS/MS.
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Cell culture
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Similar to our previous study,15,16 A7r5 cells (cat# ATCC CRL-1444, Manassas, VA, USA),
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which is a VSMC cell-line from rat aorta, were used in this study between passages 4 and 11.
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The cells were received were grown in DMEM supplemented with 10% FBS and antibiotics
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(Penicillin-Streptomycin and Gentamicin) at a CO2 level of 5%. Cell culture media was replaced
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with DMEM supplemented with 1% FBS and antibiotics prior to the treatment. Cells were
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treated with 50 µM of IRW combined with 1 µM of mas receptor (MasR) antagonist A77917
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(Abcam) or 50 µM of IRW combined with 100 µM of AT1R antagonist losartan potassium18
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(Tocris, Oakville, ON, Canada) 1 h prior to adding 1 µM of Ang II for different time periods.
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Western blot
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After 23-hour co-treatment of IRW+specific receptor antagonist+Ang II, cell lysates were
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prepared as we described in our previous study.15 Total proteins of the cell lysates were separated
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by 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
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nitrocellulose membranes and immunoblotted with antibody against matrix metallopeptidase 9
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(MMP9, NOVUS Biologicals, Littleton, CO, USA). Goat anti-rabbit IRDye 680RD or Donkey
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anti-mouse 800CW from Licor Biosciences (Lincoln, NE, USA) was used as the secondary
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antibody. Protein bands were detected by Licor Odyssey BioImager (Licor Biosciences).
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Quantification of protein bands were performed using Image Studio Lite 5.2 (Licor Biosciences).
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Each band was normalized to the loading control α-tubulin (rabbit polyclonal antibody from
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Abcam, Cambridge, MA, cat# ab15246).
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For intracellular signalling study, the co-treatment time of IRW and Ang II was 15 min. Protein
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samples were harvested and run in SDS-PAGE, followed by membrane transfer as indicated
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above. Protein bands for phospho-extracellular signal-regulated kinases 1/2 (p-ERK1/2, Cell
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Signaling Technology, Danvers, MA, USA), phspho-p38 (p-p38, NOVUS Biologicals) and
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phospho-c-Jun N-terminal Kinase (p-JNK, NOVUS Biologicals) were normalized to the
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corresponding “total” form (ERK1/2, Cell Signaling Technology; p38, Santa Cruz
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Biotechnology, Dallas, TX, USA; JNK, NOVUS Biologicals).
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Cell migration assay
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Migration of A7r5 cells was evaluated by CytoSelectTM wound healing assay kit (Cell Biolabs,
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Inc., San Diego, CA, USA) according to the manual. Briefly, cells were seeded into a 24-well
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plate with a wound healing insert in the middle of each well. Cells were cultured for overnight
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until the monolayer forms. The wound healing area was created by removing the insert. The
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wound healing insert was removed to create a 0.9 mm-wound healing area. Cells then were
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treated with 50 µM of IRW combined with 100 µM of AT1R antagonist losartan potassium
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(Tocris) 1 h before the addition of 1 µM Ang II. The co-treatment period of IRW, AT1R
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antagonist and Ang II was 4 h.19 Afterwards, cells were fixed by fixation solution and cell
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nucleus were stained by DAPI fluorescence stain. Cells were visualized by Olympus IX83
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fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). Distance of the wound area was
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measured by ImgeJ software (https://imagej.net/Welcome). Less distance indicated a higher
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migration ability of the cells. Results of each treatment group were normalized to the
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corresponding untreated.
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Statistics
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Data were analyzed by one way analysis of variance (ANOVA) coupled with Tukey post-hoc
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test by the PRISM 5 statistical software (GraphPad Software, Inc., La Jolla, CA, USA) with a
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significance level of P
