Whey protein hydrolysate induced modulation of endothelial cell gene expression

Journal of Functional Foods 40 (2018) 102–109

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Whey protein hydrolysate induced modulation of endothelial cell gene expression Martina B. O'Keeffe, Richard J. FitzGerald

MARK

⁎

Department of Biological Sciences, University of Limerick, Castletroy, Limerick, Ireland

A R T I C L E I N F O

A B S T R A C T

Keywords: Whey protein hydrolysate ACE inhibition Endothelial cells Endothelial nitric oxide synthase (eNOS) Endothelin-1 Vasodilation

Whey protein concentrate (WPC) hydrolysates generated using Neutrase®, Alcalase® and Flavourzyme® and their associated ultrafiltration fractions inhibited angiotensin-1-converting enzyme activity (71.14 ± 1.05, 73.22 ± 0.99 and 51.52 ± 5.80% inhibition when assayed at 14.3 µg/mL). Incubation of human umbilical vein endothelial cells with 5 kDa permeates of Neutrase®- and Alcalase®-hydrolysed WPC for 48 h resulted in the beneficial differential expression of genes relevant to blood pressure control, as measured by microarray. Furthermore, real-time reverse transcriptase polymerase chain reaction demonstrated an upregulation of endothelial nitric oxide synthase (+2.14 ± 0.45 and 2.36 ± 0.27-fold) and down-regulation of endothelin-1 (−0.58 ± 0.09 and −0.82 ± 0.11-fold) following incubation with the 5 kDa permeates of Alcalase®- and Neutrase®-hydrolysates, respectively. Peptide sequences within the 5 kDa permeate of the Alcalase®-hydrolysed WPC were identified, many of which have previously been demonstrated as having ACE-inhibitory and/or other bioactivities. These WPC hydrolysates potentially represent sources of bioactive peptides for the beneficial regulation of endothelial cell function.

1. Introduction Cardiovascular disease (CVD) was associated with 31% of all global deaths in 2015 (WHO, 2017). Hypertension is one of the major risk factors for CVD. Diet plays a major role in the control of hypertension and CVD. Therefore, there is interest in developing foods having antihypertensive properties. Food proteins contain peptides that, when released by, e.g., enzymatic hydrolysis, fermentation, processing etc., have bioactive effects. Milk protein-derived bioactive peptides have been associated with a range of bioactivities including anti-microbial, anti-oxidative, anti-thrombotic, anti-hypertensive and immunomodulatory effects (Korhonen & Pihlanto, 2003; Nongonierma, O’Keeffe, & FitzGerald, 2016). The renin-angiotensin system (RAS) is an important regulator of hypertension. Inhibition of angiotensin 1 converting enzyme (ACE), an important component of the RAS, has a number of effects in lowering blood pressure, including the reduced production of the vasoconstrictor, angiotensin 1 and the decreased breakdown of the potent vasodilator, bradykinin. ACE inhibition has been extensively investigated in terms of the potential blood pressure lowering effects of food protein derived peptides (Norris & FitzGerald, 2013). Two of the best known milk protein-derived ACE inhibitory peptides (Val-Pro-Pro and Ile-Pro-Pro) have been identified in a sour milk drink (Ameal S) fermented with Lactobacillus helveticus and

⁎

Saccharomyces cerevisiae strains (Nakamura, Yamamoto, Sakai, & Takano, 1995). Numerous studies have shown Val-Pro-Pro and Ile-ProPro, or products containing these peptides, to have beneficial effects on blood pressure in spontaneously hypertensive rats and hypertensive humans (Cicero, Aubin, Azais-Braesco, & Borghi, 2013; Norris & FitzGerald, 2013, for review). Much attention has also focused on antihypertensive effects of food protein-derived peptides through pathways other than ACE inhibition. These include inhibition of renin and endothelin, blocking of calcium channels and angiotensin receptors, as well as modulation of the nitric oxide pathway (Udenigwe & Mohan, 2014). The nitric oxide (NO) system is a major controller of vasodilation. NO regulates vascular tone at rest, mediates changes in blood flow to meet metabolic demand of tissue, and dilates vessel diameter in response to increased blood flow (Kelm, 2003). NO is synthesised from Larginine and oxygen by nitric oxide synthase (NOS). There are three forms of NOS; neuronal (nNOS), inducible (iNOS) and that produced by endothelial cells (eNOS). Elevations in eNOS activity may result in increased NO secretion by endothelial cells in turn leading to vasorelaxation. Consumption of a novel whey protein-derived extract (NOP47) which increases endothelial NO was reported to improve brachial artery flow mediated dilation in healthy (Ballard et al., 2009) and overweight (Ballard et al., 2013) adults. However, the peptide sequence

Corresponding author. E-mail addresses: martina.okeeff[email protected] (M.B. O'Keeffe), dick.fi[email protected] (R.J. FitzGerald).

https://doi.org/10.1016/j.jff.2017.11.001 Received 12 July 2017; Received in revised form 20 October 2017; Accepted 2 November 2017 1756-4646/ © 2017 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 40 (2018) 102–109

M.B. O'Keeffe, R.J. FitzGerald

Sentandreu & Toldra, 2006) with some modifications. The amount of ACE enzyme employed was 3 mU/mL. UF permeates of hydrolysates were investigated for ACE inhibitory activity at a final concentration in the assay of 14.3 µg/mL.

(s) of NOP-47 are not reported in the literature. Some milk proteinderived peptides may exert their antihypertensive effects through inhibition of the potent vasoconstrictor, endothelin-1 (ET-1). Incubation of the whey protein-derived lactokinin peptide, Ala-Leu-Pro-Met-HisIle-Arg (ALPMHIR), with endothelial cells resulted in a 29% reduction in basal ET-1 (Maes et al., 2004). However, in general, there is a lack of information on the mechanism of action of many antihypertensive hydrolysates/peptides. The main aims of this study therefore were (i) to assess the ACEinhibitory properties of UF fractions of WPC hydrolysates (produced using Alcalase®, Neutrase® and Flavourzyme®) and (ii) to investigate the potential antihypertensive effects of WPC-derived peptides though pathways other than ACE inhibition, by assessing differential expression of vasomodulatory genes in HUVECs exposed to 5 kDa permeates of Alcalase® and Neutrase® WPC hydrolysates.

2.4. Cell culture and microarray analysis Routine cell maintenance and microarray analysis was carried out as described by O'Keeffe and FitzGerald (2014). In short, HUVECs were maintained in MCDB-131 with FBS (10%, v/v), VEGF (1 ng/mL), FGF (2 ng/mL), IGF (2 ng/mL) and EGF (10 ng/mL). For microarray analysis HUVECs between passages 6 and 10 were grown on 10 cm2 dishes and were incubated with vehicle control (media)/unhydrolysed WPC/5 kDa permeate of Alcalase®- or Neutrase®-hydrolysed WPC. RNA was isolated using Trizol® following the manufacturer’s instructions (Invitrogen, CA, USA). Microarray analysis was carried out by Almac Diagnostics (Craigavon, Northern Ireland) using an Affeymetrix® Human Genome U133 Plus 2.0 array (Affeymetrix, Santa Clara, CA, USA) as described by Knudsen et al. (2012). All RNA samples passed spectrophotometric (A260/280 and A260/230 ratios of ∼2.0) and Bioanalyser quality control analyses at Almac Diagnostics. The Human Genome U133 Plus 2.0 Array analyses the expression levels of over 47,000 transcripts and variants. This includes 38,500 well-characterised human genes. A number of controls are included on the array, e.g., hybridisation controls (bioB, bioC, bioD, cre), Poly-A controls (dap, lys, phe, thr) and housekeeping/control genes (GAPDH, beta-actin, STAT-1). The full list of target genes is available at https://www.thermofisher.com/order/ catalog/product/900466. Gene regulation in the three treatment groups (unhydrolysed WPC, 5 kDa permeate of Alcalase® WPH and 5 kDa permeate of Neutrase® WPH) was expressed as a function of gene expression in vehicle-treated HUVECs. The gene expression ratios were generated using an Affymetrix® default ratio builder error model. The data was processed using the Rosetta Error Model with further stringency being applied through the use of specific filters, i.e., subtraction of three times the average standard deviation of the background intensity and a fold-change filter whereby > 1.5-fold change was required for stringent lists. Pathway analysis was carried out using MetaCore™ data mining software (GeneGo, CA, USA) in order to determine the specific molecular pathways that were most regulated.

2. Materials and methods 2.1. Materials Whey protein concentrate (WPC80, 75.8% protein) manufactured from sweet whey was purchased from a commercial supplier (Carberry Milk Products, Ballineen, Ireland). Alcalase® 2.4L, Neutrase® and Flavourzyme® 500L were generously donated by Novozymes A/S (Bagsvaerd, Denmark). MCDB-131, L-glutamine, foetal bovine serum (FBS), trypsin and trizol® were all from Invitrogen (CA, USA). Epidermal growth factor (EGF), insulin growth factor (IGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) were from Peprotech (London, UK). O-aminobenzoylglycyl-p-nitro-Lphenylalanyl-L-proline (Abz-Gly-p-nitro-Pro-OH) and o-aminobenzoylglycine (Abz-Gly) were from Bachem (79576 Weil am Rhein, Germany). Rabbit-lung acetone powder and sodium tetraborate decahydrate (borax) were from Sigma (Wicklow, Ireland). Human umbilical vein endothelial cells (HUVECs) were from Lonza Biologics (Slough, UK). The QuantiTect reverse transcription kit was from Qiagen (Crawley, West Sussex, UK). The LightCycler® FastStart DNA master plus sybr green I kit and the LightCycler® capillaries were from Roche Diagnostics (West Sussex, UK). 2.2. Enzymatic hydrolysis of whey protein concentrate, membrane processing and spray-drying of hydrolysates

2.5. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated from HUVECs that had been incubated with unhydrolysed WPC/5 kDa permeate of the Alcalase® WPH/5 kDa permeate of the Neutrase WPH using the Trizol® procedure according to manufacturer’s instructions (Invitrogen, CA, USA). Traces of genomic DNA were eliminated and first strand cDNA was synthesised from 0.8 μg RNA using the QuantiTect® reverse transcription kit as per manufacturer’s instructions (Qiagen, Crawley, West Sussex, UK). Thereafter, aliquots (5 μL of 1:10 diluted) of first strand cDNA were used as templates in each real-time PCR reaction (20 μL) using the LightCycler® FastStart DNA master plus sybr green I kit following the manufacturer’s instructions (Roche Diagnostics, West Sussex, UK). Oligonucleotide primers were designed using the Roche Universal ProbeLibrary version 2.43 software and included the forward and reverse primers for endothelial nitric oxide synthase (eNOS); 5′-GACCC TCACCGCTACAACAT-3′ and 5′-CCGGGTATCCAGGTCCAT-3′, respectively, forward and reverse primers for endothelin-1 (ET-1); 5′-TCTCT GCTGTTTGTTTGTGGCTTG-3′ and 5′-GAGCTCAGCGCCTAAGACTG-3′, respectively, and forward and reverse primers for hypoxanthine-phosphoribosyl-transferase (HPRT); 5′-CGTGATTAGTGATGATGAACCAG-3′ and 5′-CGAGCAAGACGTTCAGTCCT-3′, respectively. Real-time RT-PCR reactions were carried out on the LightCycler® Carousel system (Roche Diagnostics, West Sussex, UK) and were analysed using the relative quantification function of LightCycler® version 4.0 software. Fold change was determined using the delta Ct model and as a function of

WPC hydrolysates (WPHs) were generated, membrane processed and spray dried as previously described by O'Keeffe and FitzGerald (2014). In short, WPC (10% w/v, protein) was allowed to hydrate at room temperature for 1 h. Hydrolysis was carried out using Neutrase®, Alcalase® and Flavourzyme® at an enzyme: substrate ratio of 0.3% (v/ w) at 50 °C for 4 h and using a pH Stat (842 Stat Titrino, Methrom, Herisau, Switzerland) to maintain the pH at 7.0. Hydrolysates were subsequently heated to 80 °C for 20 min to inactivate the enzyme. Cooled hydrolysates were processed through UF membranes having nominal molecular mass cut-offs of 0.2 µm, 5 kDa and 1 kDa using a bench-scale ultrafiltarion system (Sartoflow Alpha, Sartorius AG, Goettingen, Germany). A 650 Da permeate fraction was generated by passing the 1 kDa permeate through a 650 Da membrane using a tangential flow filtration (TFF) in a Minimate™ TFF Capsule with an Omega™ 65D membrane (Pall Life Sciences, Ann Arbor, MI, USA). Hydrolysates and UF fractions were spray-dried using a bench-top B290 mini spray dryer (Buchi Labortechnik AG, Switzerland). The inlet and outlet temperatures were 140–160 and ≤70 °C, respectively. 2.3. Angiotensin converting enzyme (ACE) inhibition assay The ACE activity was from rabbit lung acetone powder and ACE inhibition of UF permeates of hydrolysates was determined as previously described (Norris, Casey, FitzGerald, Shields, & Mooney, 2012; 103

Journal of Functional Foods 40 (2018) 102–109

M.B. O'Keeffe, R.J. FitzGerald

to the production of peptides with significantly higher (p < .05) ACE inhibitory properties than those generated by Flavourzyme®. While further ultrafiltration of the Neutrase® and Alcalase® 5 kDa permeates though a 1 kDa membrane resulted in no significant enhancement of ACE inhibitory activity. Concentration of low molecular mass peptides in the 1 kDa permeate of the Flavourzyme® hydrolysate led to an increase in ACE inhibition from 51% in the 5 kDa permeate to 65% inhibition in the 1 kDa permeate. This enhancement of ACE inhibitory activity on concentration of low molecular mass peptides has previously been found in peptides derived from whey protein (Pan, Cao, Guo, & Zhao, 2012), casein (Miguel, Contreras, Recio, & Aleixandre, 2009), soy protein (Wu & Ding, 2002), and Atlantic salmon skin proteins (Gu, Li, Liu, Yi, & Cai, 2011). In contrast, no enhancement of ACE inhibitory activity was observed on ultrafiltration of a 5 kDa permeate of a Corolase PP WPH through 1 and 0.65 kDa membranes (O'Keeffe, Conesa, & FitzGerald, 2017).

the house-keeping gene, HPRT. 2.6. Ultra high performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ESI-MS/MS) The 5 kDa UF permeate of the Alcalase™ WPH was reconstituted at a concentration of 0.1 mg/mL in mobile phase A (0.1% formic acid, FA, in MS-grade H2O) and passed through a 0.22 µm filter (Restek Ireland, Belfast, N. Ireland). Mobile phase B was 0.1% FA in acetonitrile (ACN). The sample was separated on a Dionex Ultimate 3000 UHPLC system (Thermo Scientific, MA, USA) connected to an Impact HD Ultra high resolution (UHR) Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using an Aeris™ 1.7 µm PEPTIDE XB-C18 150 × 2.1 mm column (Phenomenex, Cheshire, UK). MS/MS methods specific for (i) a wide range of peptide masses and (ii) short peptides with low mass range were as previously described (O'Keeffe and FitzGerald, 2015; O'Keeffe, Norris, Alashi, Aluko, & FitzGerald, 2017). Mass spectra were searched against the SwissProt database limited to Bos taurus using PEAKS Studio 7.5 software (Bioinformatics Solutions Inc., Waterloo, Canada) as well as the de novo function of the PEAKS software. A false discovery rate (FDR) of 1% and an average local confidence (ALC, average of the confidence in the amino acid assignment in each position of the peptide sequence, Ma et al., 2003) cut-off of 70% were applied to results. Peptide sequences from the main milk proteins are reported.

3.2. Effect of WPHs on the expression of genes related to blood pressure control To date, many antihypertensive food protein derived peptides have been reported to exert their antihypertensive effects, at least in part, through inhibition of ACE (Norris & FitzGerald, 2013; Norris, Harnedy, & FitzGerald, 2013). Recent attention has focused on other antihypertensive pathways that may be activated by the ingestion of food protein-derived peptides (Udenigwe & Mohan, 2014). A microarray approach was taken herein to assess the regulation of genes associated with hypertension and endothelial function following incubation of HUVECs with unhydrolysed WPC or the 5 kDa permeates of Alcalase®and Neutrase®-hydrolysed WPC. As the ACE inhibitory activities of the Flavourzyme® WPH UF fractions were lower than those of the Alcalase® and Neutrase® WPH UF fractions (Table 1) it was decided not to continue with this sample. The 5 kDa permeates were chosen for further study as there was no enhancement of ACE inhibitory activity observed following further UF through 1 or 0.65 kDa membranes (Table 1). Furthermore, the Neutrase® and Alcalase® 5 kDa permeate fractions were previously shown to have beneficial effects on ORAC activity, on the regulation of antioxidant genes as well as having the ability to increase the level of glutathione and catalase activity in HUVECs (O'Keeffe and FitzGerald, 2014). A number of genes involved in the maintenance of vascular homeostasis were beneficially regulated by all three of the treatments (unhydrolysed WPC and the 5 kDa permeates of Alcalase®- and Neutrase®-hydrolysed WPC) compared to vehicle (media alone) treated cells (Table 2). These include genes from the RAS pathway; angiotensin I converting enzyme (ACE), angiotensinogen and membrane metallo-endopeptidase, all of which were down-regulated. Down-regulation of ACE may result in a reduction in the vasoconstrictor, angiotensin II, and inhibition of the breakdown of the potent vasodilator, bradykinin, leading to vasorelaxation. Bradykinin acts to stimulate the production of NO by endothelial cells (Furchgott, 1983). It has been shown that inhibition of the RAS improves endothelial function by either increasing NO production or by activating eNOSrelated NO production (Unger, 2002). Moreover, inhibition of the RAS has been shown to lead to a reduction of periadventitial inflammation and atherosclerotic lesion formation (Fukuda, Enomoto, Nagai, & Sata, 2009). Expression of the gene encoding for ACE was downregulated by all three treatments herein, with the greatest reduction being achieved by incubation of HUVECs with the 5 kDa permeate of Neutrase® hydrolysed WPC (−4.34-fold). Physicochemical analysis of the unhydrolysed WPC and the Neutrase® and Alcalase® WPHs by reverse phase (RP) HPLC and gel permeation chromatography (GPC) HPLC has previously shown the presence of different profiles in these samples (O'Keeffe and FitzGerald, 2014), thereby potentially explaining the differences in gene regulation observed herein. It has previously been reported, in a study of male subjects with mild hypertension, that administration of a casein hydrolysate containing the ACE-inhibitory

2.7. Statistical analysis Data were analysed for statistical significance (p < .05) by one way analysis of variance (ANOVA) and, when significantly different, by Bonferroni post hoc analysis using GraphPad® Prism 4.0 software (GraphPad Software, San Diego, CA, USA). 3. Results and discussion 3.1. ACE inhibitory activity of the WPC hydrolysates WPH 5 kDa UF permeates produced with the food-grade enzyme preparations, Neutrase®, Alcalase® and Flavourzyme® have previously been shown to have in vitro antioxidant activity (measured by the oxygen radical absorbance capacity, ORAC, assay). In addition, they had the ability to beneficially regulate antioxidant genes and increase glutathione and catalase activity in HUVECs (O'Keeffe and FitzGerald, 2014). Analysis of the ACE-inhibitory properties of these hydrolysates herein revealed that all three enzyme preparations yielded hydrolysates with the ability to inhibit ACE (Table 1). ACE inhibition was significantly (P < .0001) increased upon hydrolysis. ACE inhibition by the unhydrolysed WPC was 10.2 ± 3.2%, while the greatest inhibition overall (73.74 ± 1.05%) was found in the < 1 kDa fraction of the Alcalase® hydrolysate. Neutrase® and Alcalase® hydrolysis of WPC led Table 1 Angiotensin converting enzyme (ACE) inhibitory activity (expressed as % inhibition of total activity when tested at 14.3 µg/mL) of 5, 1 or 0.65 kDa ultrafiltration permeates of Neutrase®-, Alcalase®- or Flavourzyme®-hydrolysed whey protein concentrate. ACE inhibition (%)a

Alcalase® WPH Neutrase® WPH Flavourzyme® WPH

5 kDa UF permeate

1 kDa UF permeate

0.65 kDa UF permeate

71.14 ± 1.27a 71.59 ± 0.99a 51.52 ± 5.8b

73.74 ± 1.05a 73.22 ± 3.3a 65.18 ± 0.54a

70.55 ± 5.96a 70.61 ± 4.74a 56.75 ± 1.25a

a ACE inhibition was assayed at 14.3 µg/mL of the ultrafiltration fraction of the hydrolysates. Data represent the mean ± SEM for at least 3 independent determinations. Different superscript letters represent significant difference within columns (P < .05). ACE: angiotensin converting enzyme; UF: ultrafiltration; WPH: whey protein hydrolysate.

104

Journal of Functional Foods 40 (2018) 102–109

tripeptides, VPP and IPP, for one week resulted in an improvement of vascular endothelial function (Hirota et al., 2007). Prostaglandin I2 synthase was upregulated 3.65-fold in HUVECs incubated with the 5 kDa permeate of the Alcalase® hydrolysate while remaining unchanged on incubation of HUVECs with WPC or the 5 kDa permeate of the Neutrase® hydrolysate. Prostaglandin I2 synthase activity results in the catalysis of prostaglandin H to the vasodilator, prostacyclin. An impairment of endothelium-dependent vasodilation has been found in patients with hypertension (Lind, Granstam, & Millgard, 2000). Peptides and hydrolysate fractions from casein and soy protein have previously been shown to influence the modulation of endothelial cell proliferation and to cause alterations in the release of NO and prostaglandin I2 (Ringseis et al., 2005). Vascular endothelial growth factor (VEGF) C was up-regulated herein by treatment with the 5 kDa permeate of the Neutrase® hydrolysate (+1.96), whilst remaining unchanged following WPC or 5 kDa permeate of the Alcalase® hydrolysate treatment. Another form of VEGF (VEGF A) was upregulated by treatment with WPC (+1.87) and by the 5 kDa permeate of the Alcalase® hydrolysate (+2.05). VEGF leads to the production of the vasodilator, NO (Papapetropoulos, Garcia-Cardena, Madri, & Sessa, 1997) and the maintenance of vascular homeostasis. To our knowledge, none of these genes have previously been reported to be beneficially regulated by hydrolysates of WPC. A study on the effects of a peptic hydrolysate of bovine lactoferrin reported that the prostaglandin-endoperoxide synthase trafficking (PTGS2) gene was upregulated 2.23fold (García-Tejedor et al., 2015). Apolipoprotein E (Apo E) was upregulated following treatment of HUVECs with intact WPC and the 5 kDa permeate of Neutrase® WPH by +2.20- and +3.08-fold, respectively. ApoE plays a protective role against atherosclerosis (Curtiss & Boisvert, 2000) while ApoE deficiency has been shown to lead to the accumulation of cholesterol in the blood and is linked with CVD (Ang, Cruz, Hendel, & Granville, 2008). To our knowledge, no other studies report on the regulation of the gene expression of Apo E following incubation of HUVECs with a WPH. Incubation of HUVECs with the 5 kDa permeate of the Neutrase® WPH for 48 h resulted in a down-regulation (−5.75-fold) of the urotensin (UT) 2 gene. The UT2 gene encodes for a highly potent endogenously expressed vasoconstrictor peptide (Watanabe, Kanome, Miyazaki, & Katagiri, 2006). Expression of UT2 is increased in human cardiovascular disease states (Zhu, Zhu, & Moore, 2006). Therefore, the down-regulation of the UT2 gene observed in this microarray may lead to vasodilation and improvement in endothelial function and may result in the beneficial regulation of the atherosclerosis pathway. To our knowledge, this is the first report of the regulation of UT2 in HUVECs by a WPH. MetaCore™ pathway analysis was carried out on the three gene lists (i.e. the list of genes regulated following incubation of HUVECs with (i) intact WPC, (ii) the 5 kDa permeates of the Neutrase®- and (iii) Alcalase®-WPHs) in order to rank the molecular pathways that were differentially regulated due to each of the three treatments (Table 3). Interestingly, no pathway was regulated to a significant level (p > .05) by intact WPC, while all of the top 20 pathways were significantly (p < .05) regulated by the 5 kDa permeates of Alcalase® and Neutrase® WPHs. Pathways associated with VEGF signalling were highly ranked by all three treatments but to a greater extent by the hydrolysates than the intact WPC. In both the 5 kDa permeate of the Neutrase®- and Alcalase®-WPH treated cells the VEGF-family signalling pathway was ranked number 3 (i.e., this pathway was the third most regulated pathway by this particular treatment with 9 genes out of the 32 in the pathway regulated) while in the WPC-treated cells a similar pathway (VEGF signalling and activation) was ranked 7th (8 genes out of 32 were regulated). VEGF signalling via the VEGFR2 pathway was ranked 11th in the list of regulated pathways from HUVECs treated with the 5 kDa permeate of the Neutrase® WPH (7 out of 29 genes were regulated) and 17th in the list from HUVECs treated with the 5 kDa permeate of the Alcalase® WPH. The cross-talk between VEGF and

+1.96 +1.77

+3.08 +1.76 +2.54 – – –

–

+2.20 – –

–

Apolipoprotein E (AI358867) Plasminogen activator, urokinase (K03226) Plasminogen activator, tissue (NM_000930)

Urotensin 2 (NM_021995)

–

+3.65 +2.05 – – – +1.87 – – Prostaglandin I2 (prostacyclin, D38145) synthase Vascular endothelial growth factor A (M27281) Vascular endothelial growth factor C (U58111) Thrombomodulin (AW119113)

– −1.52 –

– −1.60 –

−5.75

Involved in maintaining blood pressure and in the pathogenesis of essential hypertension and preeclampsia Cleaves peptides at the amino side of hydrophobic residues and inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin and bradykinin Catalysis of prostaglandin H to prostacyclin (vasodilator) Active in angiogenesis, vasculogenesis and endothelial cell growth Active in angiogenesis, vasculogenesis and endothelial cell growth Binds thrombin resulting in activation of protein C, degradation of clotting factors and a reduction in thrombin production Essential for the normal catabolism of triglyceride-rich lipoprotein constituents Converts plasminogen to the active enzyme plasmin Converts plasminogen to the active enzyme plasmin. Involved in cell migration and tissue remodelling Highly potent vasoconstrictor

Converts angiotensin I into the vasopressor angiotensin II −2.18 −2.30

Angiotensin 1 Converting enzyme (peptidyl dipeptidase A) (AW057540) Angiotensinogen (serpin peptidase inhibitor, Clade A, member 8) (NM_000029) Membrane metallo-endopeptidase (NM_007287)

−4.34

Gene function (Weizmann Institute of Science, 2017) Δ-fold Neutrase® 5 kDa Permeate Δ-fold Alcalase® 5 kDa permeate Δ-fold unhydrolysed WPC Gene name (Accession Number)

Table 2 Genes involved in blood pressure control pathways that were differentially expressed, as determined by microarray analyses, following incubation of human umbilical vein endothelial cells (HUVECs) with unhydrolysed whey protein concentrate (WPC), or the 5 kDa permeates of Alcalase®- or Neutrase®-hydrolysed WPC.

M.B. O'Keeffe, R.J. FitzGerald

105

Catecholamine metabolism Immune response_IL4 signalling pathway Blood coagulation, GPVI-dependent platelet activation

Cell adhesion, Chemokines and adhesion

Regulation of lipid metabolism, Regulation of acetyl-CoA carboxylase 1 activity in keratinocytes Development, Transcription regulation of granulocyte development wtCFTR and delta508 traffic/Clathrin coated vesicles formation (norm and CF) Serotonin – melatonin biosynthesis and metabolism Plasmalogen biosynthesis Immune response _T cell receptor signalling pathway Signal transduction, Activation of PKC via G-Protein coupled receptor DNA damage, ATM/ATR regulation of G1/S checkpoint

Development, VEGF-family signalling Cell cycle, Spindle assembly and chromosome separation Retinol metabolism

Development_Alpha-1 adrenergic receptors activation of ERK

Glycine, serine, cysteine and threonine metabolism Immune response, Oncostatin M signalling via JAK-Stat in human cells

3 4 5

6

7

10 11 12 13 14

15 16 17

18

19 20

106 .4594 .4624

.4513

.4513 .4513 .4513

.4357 .4357 .4418 .4418 .4513

.4304 .4357

.4292

.4249

.4083 .4093 .4241

.4083

.4083

Development_A3 receptor signalling Apoptosis and survival, Regulation of Apoptosis by Mitochondrial Proteins Normal wtCFTR traffic/ER-to-Golgi Delta508-CFTR traffic/ER-to-Golgi in CF Immune response _T cell receptor signalling pathway Apoptosis and survival_HTR1A signalling Development, Delta-, type opioid receptor signalling via G-protein alpha-14 Immune response_IL4 anti-apoptotic action G-protein signalling, N-RAS regulation pathway Development, VEGF signalling via VEGFR2 – generic cascades Transcription, Ligand-dependent activation of the ESR1/ SP pathway Development, CNTF receptor signalling Immune response _IL1 signalling pathway

Development, VEGF signalling and activation Development, EPO-induced MAPK pathway Translation _Non-genomic (rapid) action of Androgen Receptor Development, Leptin signalling via PI3K-dependent pathway Development, ERBB-family signalling

G-protein signalling, Gαq signalling cascades

Immune response_CXCR4 signalling via second messenger

.0146 .0147

.0121

.0080 .0107 .0121

.0040 .0040 .0046 .0053 .0065

.0036 .0039

.0029

.0023

.0015 .0017 .001856

.0011

.0011

P value

Development, Angiotensin activation of Akt Development, VEGF signalling and activation

G-protein signalling, Proinsulin C-peptide signalling

Histamine metabolism Development, VEGF signalling via VEGFR2 – generic cascades Blood coagulation, GPIb-IX-V-dependent platelet activation Proteolysis, Role of Parkin in the Ubiquitin-Proteasomal Pathway Regulation of lipid metabolism, Regulation of lipid metabolism via LXR, NF-Y and SREBP Cell adhesion_Alpha-4 integrins in cell migration and adhesion Transcription, PPAR Pathway Development_A1 receptor signalling

Apoptosis and survival_Beta-2 adrenergic receptor anti-apoptotic action Cell adhesion, Chemokines and adhesion Selenoaminoacids and taurine, hypotaurine metabolism

.0014

Immune response, MIF – the neuroendocrine-macrophage connector Development, VEGF-family signalling Cell adhesion, Integrin inside-out signalling Development, Delta- and kappa-type opioid receptors signalling via beta-arrestin Nitrogen metabolism

.0232 .0240

.0203

.0203 .0203 .0203

.0140 .0141 .0186 .0191 .0203

.0100 .0124

.0090

.0057

.0018 .0056 .0057

.0008

P value

Cell adhesion, Cell-matrix glycoconjugates

Pathway

Neutrase® 5 kDa permeate

GPCR: G-protein coupled receptor; Gαq: G-Protein alpha-q; IL: interleukin; VEGF: vascular endothelial growth factor; EPO: erythropoietin; MAPK: mitogen activated protein kinase.

8 9

2

Transcription Assembly of RNA Polymerase II preinitiation complex on TATA-less promoters Tyrosine metabolism p.1 (dopamine)

Pathway

Pathway

P value

Alcalase® 5 kDa permeate

Intact WPC

1

Rank

Table 3 MetaCore™ pathway analysis of the regulation of molecular pathways in human umbilical vein endothelial cells incubated with unhydrolysed whey protein concentrate (WPC), or the 5 kDa permeates of Alcalase®- or Neutrase®-hydrolysed WPC.

M.B. O'Keeffe, R.J. FitzGerald

Journal of Functional Foods 40 (2018) 102–109

Journal of Functional Foods 40 (2018) 102–109

M.B. O'Keeffe, R.J. FitzGerald

angiopoietin 1 signalling pathway was ranked equally by all three treatments. VEGF stimulates the release of biologically active NO (Papapetropoulos et al., 1997) which may lead to improved endothelial function. VEGF signalling also leads to activation of the calcium signalling pathway and the resultant production of the vasodilator, prostacyclin, from arachidonic acid through the action of cytosolic phospholipase A2 (cPLA2) and cyclo-oxygenase 2 (COX2). Both of these actions may lead to improved endothelial cell function. The G-protein alpha-q signalling cascade pathway was ranked number 2 in the list of regulated genes from HUVECs incubated with the 5 kDa permeate of Alcalase® WPH with 8 out of 25 genes being regulated (p = .0011). The G alpha-q/11 signalling pathway results in the activation of phospholipase C beta (PLCB), which catalyses hydrolysis of phosphoinositide 4,5-biphosphate to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Neves, Ram, & Iyengar, 2002). IP3 leads to Ca2+ mobilisation from internal stores, which in turn leads to vasoconstriction. DAG activates protein kinase C epsilon. On examining the genes in this pathway that were regulated in HUVECs incubated with the 5 kDa permeate of Alcalase® WPH it was seen that both PLCB1 and PLCG1 were down-regulated (-1.72 and –2.03 respectively) which may result in an overall reduction in vasoconstriction. Protein kinase C epsilon type (PRKCE) was up-regulated, +2.23fold. PRKCE has been shown to mediate cardio-protection to the ischemic heart (Cross, Murphy, Bolli, Ping, & Steenbergen, 2002). Both protein kinase B (Akt) 2 and Akt3 were down-regulated (−2.63-fold and −1.98-fold, respectively). The expression of the Akt1 gene was unregulated; loss of expression of this gene has been shown to result in severe atherosclerosis by increased inflammatory mediators and reduced eNOS phosphorylation (Fernandez-Hernando et al., 2007). Moreover, the NF-kappa-B inhibitor alpha was upregulated 2.03-fold. NF-kB activation has been shown to play an important role by transactivating several genes involved in the inflammatory process (Tak & Firestein, 2001), thus the negative regulation of NF-kB transcription observed herein may result in an anti-inflammatory response which may, for example, combat the early signs of atherosclerosis. This microarray analysis suggests that incubation of HUVECs with the 5 kDa permeates of both the Neutrase®- and Alcalase®- WPHs results in potentially beneficial modulation of a number of different genes involved in the maintenance of vascular homeostasis, which may have a beneficial effect on, e.g., hypertension as well as atherosclerosis. To our knowledge this appears to be the first study demonstrating the major pathways regulated by incubation of HUVECs with hydrolysates of WPC.

Table 4 Regulation of expression of endothelial nitric oxide synthase (eNOS) or endothelin-1 (ET1) genes in human umbilical vein endothelial cells (HUVECs) treated with 5 or 1 kDa permeates of Neutrase® or Alcalase® hydrolysates of whey protein concentrate (WPC) in growth media for 48 h by real time reverse transcriptase polymerase chain reaction (RTPCR). Sample Vehicle Intact WPC Alcalase 5 kDa permeate Neutrase 5 kDa permeate

eNOS (fold change) 1.00 1.09 2.14 2.36

± ± ± ±

a

0.00 0.23a 0.45a,b 0.27b

ET-1 (fold change) 1.00 0.87 0.58 0.82

± ± ± ±

0.00a 0.14a 0.09b 0.11a

Data represent the mean ± SEM for at least 3 independent determinations. Different superscript letters represent significant difference within columns (P < .05).

expression was upregulated in spontaneously hypertensive rats consuming a daily dose of 800 mg/kg body weight of a casein hydrolysate for 6 weeks (Sánchez et al., 2011) while there was an increase in NO production in HUVECs following incubation with an antihypertensive bovine lactoferrin hydrolysate (García-Tejedor et al., 2015). VPP and IPP, two peptides isolated from a casein fermentate have been previously shown to result in an increase in the production of NO in cultured endothelial cells (Hirota et al., 2011), while the egg-derived peptide, IRW, resulted in an upregulation of eNOS in spontaneously hypertensive rats (Majumder et al., 2013). However, to our knowledge, with the exception of NOP-47 (the WPC-derived peptide/extract whose sequence(s) and means of production are not reported in the literature, Ballard et al., 2009, 2013), this is the first report of a WPH which regulates the expression of eNOS and ET-1 in HUVECs.

3.4. Peptide identification in UF fractions of Alcalase® WPH The 5 kDa permeate of the Alcalase® WPH was found to have the greatest effect on the down-regulation of ET-1, significantly greater upregulation of eNOS gene expression as well as having a beneficial effect on the regulation of many genes related to blood pressure control. It was therefore decided to analyse the 5 kDa permeate of the Alcalase® WPH using UPLC-ESI-MS/MS in order to identify the peptides therein. A list of the peptide sequences identified can be seen in Supplementary Table 1 (identified from database search) and Supplementary Table 2 (identified by de novo sequencing). Furthermore, the peptide sequences were searched against a milk bioactive peptide database (MBPDB, available at: http://mbpdb.nws.oregonstate.edu; Nielsen, Beverly, Qu, & Dallas, 2017) and a general bioactive peptide database, BIOPEP (available at: http://www.uwm.edu.pl/biochemia/index.php/en/ biopep; Minkiewicz, Dziuba, Iwaniak, Dziuba, & Darewicz, 2008). Many of the identified peptide sequences have previously been reported to have various bioactivities including ACE-inhibitory, antioxidant, DPP-IV inhibitory activity etc., (Supplementary Tables 1 and 2). For example, the ACE inhibitory peptides Val-Leu-Asp-Thr-Asp-Tyr-Lys from β-lactoglobulin and Tyr-Gly-Leu-Phe from α-lactalbumin were identified. Furthermore, there were many examples of peptides identified within the 5 kDa permeate of the Alcalase® WPH whose sequences were truncated versions of bioactive peptides. These include the ACEinhibitory peptides Leu-Asp-Ala-Gln-Ser-Ala-Pro-Leu-Arg and Leu-AspIle-Gln-Lys-Val-Ala-Gly-Thr-Trp from β-lactoglobulin (the truncated versions of these peptides, Asp-Ala-Gln-Ser-Ala-Pro-Leu and Asp-IleGln-Lys-Val-Ala-Gly-Thr, respectively, were identified herein). These ACE-inhibitory peptides may account for the ACE-inhibitory properties of the 5 kDa permeate of the Alcalase® WPH. Many other peptides were identified that have not been reported as bioactive in MBPDM or BIOPEP. Some of these peptide sequences may be responsible for the beneficial modulation of vasomodulatory genes in HUVECs via various pathways. In conclusion, enzymatic hydrolysis of WPC using Alcalase®, Neutrase® or Flavourzyme® herein resulted in the generation of

3.3. Real-time reverse-transcriptase (RT)-PCR Real-time RT-PCR was carried out in order to confirm the results of some genes of interest from the less stringent list of genes (< 1.5-fold regulation and less rigorous background elimination) from the microarray analysis. This analysis was carried out on total RNA isolated from HUVEC-treated (unhydrolysed WPC and the 5 kDa permeates of Alcalase® and Neutrase® WPHs) cells. Real-time RT-PCR experiments revealed that expression of the eNOS gene in HUVECs was upregulated following 48 h incubation with the 5 kDa permeates of the Neutrase®(2.36-fold, significantly greater than vehicle and intact WPC-treated cells, p < .05) and Alcalase®- (2.14-fold) WPHs (Table 4). Furthermore, expression of the ET-1 gene was seen to be down-regulated following incubation with the 5 kDa permeate of the Neutrase® WPH (0.825-fold) and significantly down-regulated (P < .05) by the 5 kDa permeate of Alcalase® WPH (0.5825-fold, Table 4). This indicates that incubation with 5 kDa permeate of the Neutrase®- and Alcalase®- WPHs result in significant beneficial effects on human endothelial cells in culture. NO produced by eNOS regulates systemic blood pressure, angiogenesis and vascular remodelling (Moncada & Higgs, 1993; Murohara et al., 1998; Rudic et al., 1998), all of which play a crucial role in the maintenance of vascular homeostasis. Aortic eNOS 107

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peptides that (i) inhibit ACE activity in vitro and (ii) beneficially regulate vasomodulatory genes, including, but not limited to those of the RAS system. These UF permeates of WPH, which have previously been shown to beneficially modulate oxidative stress in HUVECs, were shown herein to regulate genes which may evoke a vasodilatory response in HUVECs which appears to be mediated by a number of different pathways; both of these responses are recognised as being beneficial in counteracting hypertension. It will be necessary, however, to investigate if this response at the gene expression level is translated to differential expression of the corresponding proteins in HUVECs. Identification of the many peptides within the 5 kDa permeate of the Alcalase® WPH has been achieved, however, the peptide sequences leading to the specific effects demonstrated remains to be elucidated. Furthermore, in vivo studies are required to confirm if the vasomodulatory effects observed in cells translate to antihypertensive effects in humans.

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