Transport of a Prolyl Endopeptidase Inhibitory Peptide across the

Article pubs.acs.org/JAFC

Transport of a Prolyl Endopeptidase Inhibitory Peptide across the Blood−Brain Barrier Demonstrated Using the hCMEC/D3 Cell Line Transcytosis Assay Maria Hayes,*,† Lars Fredrik Moen,§ Mark A. E. Auty,# and Tor Erling Lea§ †

Food BioSciences Department, Teagasc, The Irish Agricultural and Food Development Authority, Ashtown, Dublin 15, Ireland Department of Chemistry, Biotechnology and Food Science, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences (NMBU), 1432 Ås, Oslo area, Norway # Food Chemistry and Technology Department, National Food Imaging Centre, Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland §

ABSTRACT: The blood−brain barrier (BBB) remains a significant hurdle for treatment of central nervous system (CNS) and mental health disorders. A prolyl endopeptidase (PEP) inhibitory peptide with the amino acid sequence proline−proline−leucine (PPL) was chemically synthesized labeled with 5-FAM and assessed using a transcytosis assay for its ability to cross the BBB. Transport of this peptide across the BBB was determined using an in vitro model of the human BBB, which utilizes the human cerebral microvascular endothelial cell line (hCMEC/D3). Uptake and transport of 5-FAM-PPL across the hCMEC/D3 cell model was determined using confocal microscopy and mass spectrometry. This is an important parameter in determining whether peptides may reach the target organ (i.e., the brain and central nervous system).This work assessed, for the first time, the ability of a food-derived PEP inhibitory peptide to cross the BBB without the use of animal models. KEYWORDS: blood−brain barrier, endothelium, hCMEC/D3 cell line, transcytosis, prolyl endopeptidase, inhibitory peptides

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been demonstrated. For example, an αs1-casein hydrolysate previously showed anxiolytic activity in animal models as well as in healthy subjects by oral digestion compared to a placebo.5 In addition, a PEP inhibitory hydrolysate was generated recently from barbel fish skin gelatin.3 Many promising therapeutic candidates fail due to the presence of barriers between the blood and brain in cerebral capillariesthe blood−brain barrier (BBB). The BBB impedes several therapeutic and indeed pathological agents from eliciting a desired or negative health effect at an attainable dose. The hCMEC/D3 cell line, derived from a primary cell culture through coexpression of hTERT and the SV40 T large T antigen via an efficient lentiviral vector system, retains most of its morphological and functional characteristics of brain endothelial cells, even without coculture with glial cells, and constitutes a reliable in vitro model of the human BBB.6,7 This is an important parameter in determining whether peptides may reach the target organ (i.e., the brain) and have a positive health effect. This work assessed the ability of a PEP inhibitory peptide with the amino acid sequence PPL to cross the hCMEC/D3 cell line, which was used as a model of the BBB. This is the first work to detail transport of PEP inhibitory peptides across hCMEC/D3 and is useful as animal models were not used.

INTRODUCTION Mental health may be defined as cognitive and emotional wellbeing and the absence of a mental health disorder. Cognitive decline and depression are serious problems in many societies currently. Globally, it is estimated that as many as 450 million people suffer from a mental or behavioral disorder, and one in four families have at least one member with a mental health problem. Determinants of mental health are complex; however, emerging evidence for nutrition as a crucial factor in the high prevalence and incidence of mental health disorders suggests that diet is as important to psychiatry as it is to cardiology, endocrinology, and gastroenterology.1 Amyloidosis is associated with the development of several diseases including Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease, adult-onset diabetes, endocrine tumors, and macular degradation.2 Indeed, significant epidemiological evidence has emerged which suggests that mental health diseases including AD belong to the “diseases of civilisation” caused by modern Western diets. Several enzymes are important in the control and regulation of mental health including prolyl endopeptidase (PEP) (EC 3.4.21.26), acetylcholinesterase (AChE) (EC 3.1.1.7), and β-site APP cleaving enzyme (BACE1). PEP, also known as prolyl oligopeptidase (POP), is a highly conserved serine protease, and it is thought that abnormal PEP levels are related to neuropathological disorders including depression, mania, schizophrenia, and senile dementia of the AD type.3 Inhibitors of PEP may improve memory by blocking the metabolism of endogenous neuropeptides.4 In recent times, the anxiolytic and neuroprotective activities of food-derived peptides and enzymatic digests of proteins have © 2015 American Chemical Society

Received: Revised: Accepted: Published: 146

September 25, 2015 December 17, 2015 December 21, 2015 December 21, 2015 DOI: 10.1021/acs.jafc.5b04696 J. Agric. Food Chem. 2016, 64, 146−150

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Journal of Agricultural and Food Chemistry

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PET membrane filter inserts (0.4 μm, 0.7 mm diameter, Falcon, Corning BV, Amsterdam, The Netherlands) were used in 24-well cell culture plates (Falcon, Corning BV). Prior to cell barrier coating, the transparent PET membrane filter inserts were coated with rat tail collagen type I (0.35 mL) at a concentration of 0.1 mg/mL and incubated at 37 °C overnight. Inserts were subsequently washed with PBS and incubated for 1 h, after which time PBS was removed and replaced with the assay medium. The inserts were incubated with assay medium at 37 °C for at least 1 h. Optimum medium volumes were calculated to be 0.5 and 1.5 mL for apical and basolateral chambers, respectively, on the basis of previous works.9 After the transwell inserts were incubated with assay media for 1 h, medium was removed and hCMEC/D3 cells seeded onto the apical side of the inserts at a density of 50000 cells in 0.5 mL of assay medium, and 1.5 mL of fresh medium was added to the basolateral chamber. Following 6 h of cell culture, the assay medium in each apical chamber was removed and replaced with prewarmed fresh medium to avoid nonattached cells forming multilayers as previously suggested.9 The assay medium was changed every 3 days following transwell apical insert seeding with hCMEC/D3 cells, and cells were grown to confluence for 7 days. Uptake of 5-FAM-PPL by hCMEC/D3 Human Brain Endothelial Cells. Following culture of the hCMEC/D3 cells, uptake of 5-FAM-PPL was assessed as previously described with minor modifications.10 Sixty-five thousand cells/cm2 were cultured for 2 days on rat type I collagen-coated coverslips (diameter = 2.2 cm) positioned in 6-well culture dishes and then incubated with 50 μg/ mL fluorescent 5-FAM-PPL at 37 °C for 2 h. After that, cells were rinsed three times with PBS and fixed with a 10% formalin solution. Cells were permeabilized with 0.2% Triton-X100 in PBS for 15 min then rinsed twice and incubated with a solution of 1% phalloidin (staining actin filaments) in PBS for 1 h and then with 20 μM 4′,6diamidino-2-phenylindole (DAPI) nuclear staining in PBS for 10 min. Samples were imaged using a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) fitted with a ×63 oil immersion objective (N.A. = 1.4). Dual-channel imaging was used to sequentially image cells labeled with (a) DAPI (excitation, 405 nm; emission bandwidth, 420−460 nm) and (b) Alexa488 (excitation, 488 nm; emission bandwidth, 510−550 nm). Eight-bit images 1024 × 1024 pixels in size were acquired at a zoom factor of 3.0, and channels were pseudocolored blue (DAPI-stained) and green (Alexa488stained). Transendothelial Electrical Resistance (TEER). Cells were incubated with 5-FAM-PPL at a peptide concentration of 50 μg/mL, respectively. The peptide was applied to the cells when the TEER value was at its highest. The TEER value was measured using a EVOM2 epithelial voltohmmeter, Endohm-6 STX2 electrode (World Precision Instruments, Sarasota, FL, USA). TEER values were recorded as ohms·cm2 (Ω·cm2) with every assay medium change.11 In addition to TEER measurements, the paracellular permeability of fluorescein (Na-F) was assessed from the literature. Peptide Transcytosis Assay. This assay was performed in EndGro complete medium, which was reconstituted as previously stated. The method used is shown in Figure 2. On the day of the assay, cell assay medium was changed. After incubation at 37 °C for 1 h, medium was removed. Subsequently, filter inserts with or without cells were individually incubated apically with the fluorescent-labeled peptide 5-FAM PPL at a concentration of 50 μg/mL for 1 h at 37 °C, following which the entire apical and basolateral volumes (referred to as the values of stock post loading) were collected (Figure 2). The monolayers were washed at room temperature three times for 3−5 min each time, and all washes were collected, stored, and frozen at −20 °C until future use in MS analysis. Samples were cleaned up for further analysis using Pierce graphite spin columns available as part of the Pierce titanium dioxide phosphopeptide enrichment and cleanup kit (Thermo Fischer Scientific, Waltham, MA, USA). The graphite spin columns were used according to the manufacturer’s instructions. Briefly, the columns were prepared by placing them in 1.5 mL Eppendorf tubes followed by centrifugation at 2000g for 1 min to remove storage buffer. The columns were then rinsed twice with 100 μL of NH4OH and subsequently centrifuged at 2000g. The graphite

MATERIALS AND METHODS

Materials and Reagents. The hCMEC/D3 cell line (CLU512 hCMEC/D3 101614. 3A P25, October 16, 2014) was obtained under license from Nordic Biosite (Nordic BioSite Ås, Oslo, Norway) and used for experiments between passages 25 and 35. The growth medium and assay medium used for hCMEC/D3 cell propagation and barrier forming, respectively, was EndoGRO-MV Complete Culture Media supplemented as previously described with human basic fibroblast growth factor (Sigma-Aldrich, Dublin, Ireland; no. F0291) and penicillin 10000 units−streptomycin, 10000 μg mL−1 (Life Technologies, Dublin, Ireland). Dimethyl sulfoxide (DMSO) was supplied by Sigma-Aldrich (Dublin, Ireland). All other chemicals used were of molecular biology grade. Peptide Synthesis. The peptide PPL, identified previously from barbel fish skin gelatin3 and generated previously from meat proteins using in silico analysis,8 was tagged with a fluorescent label (5-FAM), custom synthesized and quality assured using ultraperformance liquid chromatography (UPLC) and mass spectrometry (MS) analysis (Selleck Chemicals LLC, Houston, TX, USA). Cultivation of hCMEC/D3 Cell Monolayer Grown on Permeable Supports. The hCMEC/D3 cells were cultured according to previously reported methods with slight modifications.9,10 A cryovial containing approximately 1 × 106 hCMEC/D3 cells was removed from liquid nitrogen and thawed quickly in a water bath at 37 °C. Once defrosted, cells were gently suspended with prewarmed culture medium and centrifuged for 10 min at 1300 rpm. The cell pellet was suspended in fresh growth medium and cultured at a concentration of 144,000 cells per milliliter in 25 cm2 rat tail collagen type-I coated culture flasks. Cells were cultured at 37 °C, 5% CO2. EndGro-MV complete culture medium supplemented with the antibiotics streptomycin and penicillin was changed every 3 days, and cells were grown until they were 90% confluent as observed using a Lecia cell microscope (Figure 1). Cells were passaged at least twice

Figure 1. hCMEC/D3 cells grown in EndGRO-MV medium in 25 cm2 culture flasks and photographed using the Leica EM UC7 ultramicrotome (Leica Microsystems, Mannheim, Germany) showing elongated, contact-inhibited morphology typical of hCMEC/D3 cells at 90% confluence. before use. Assay medium was screened following cell culture for mycoplasma contamination using the MycoAlert PLUS Mycoplasma detection kit (Lonza, USA). Trypsinization of hCMEC/D3 Cells. Confluent hCMEC/D3 cells were split by trypsinization. First, cell medium was removed using an aseptic technique, and cells were rinsed with phosphate-buffered saline−CaCl2−MgCl2 (PBS) (5 mL per 25 cm2 flask). A trypsin− EDTA solution was added (2 mL,) and cells were incubated for 3 min at 37 °C to enable detachment of cells. Detached cells were observed under the microscope and trypsin inactivated by addition of 3 mL of assay medium containing fetal bovine serum (FBS, Gibco, UK). hCMEC/D3 Cell Culture for Transcytosis Assay. For all transcytosis assays, high density pore (1 × 108 pores/cm2) transparent 147

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line.12 Intact endothelium in adult blood vessels has a low turnover rate. If junctions are disrupted, the endothelium is able to proliferate, a process that slows again if cell−cell contact is re-formed.13,14 Stable endothelial cell contacts create signals to counteract cell proliferation.14 In this study, the transwell barrier composed of hCMEC/D3 cells, which was representative of the BBB, exhibited confluent and contact-inhibited morphology (Figure 2) similar to that reported previously by Ye and colleagues.14 Contact-inhibited cells, including the endothelium, have a reduced response to specific growth factors. The establishment of intercellular contact transfers negative intracellular signals between hCMEC/D3 cells, which restrains the capacity of the cells to respond to proliferative signals. Adhesive transmembrane proteins known as the vascular endothelium cadherins (VE cadherins) have been implicated in this process.15 TEER Values. Prior to transport studies, it is standard practice to perform well-established tests to confirm that the barrier functionality has been attained.14 TEER values of ∼56 Ω·cm2 were obtained during the course of this work. This value correlates well with previously reported TEER values for the endothelial cell line hCMEC/D3 of 30−60 Ω·cm2. The literature also reports that this cell line always gives a low TEER value compared to the in vivo values.12,15 TEER values in vivo are usually ≥1000 Ω·cm2.15 Transport of N-fluorescein, was not observed, providing further evidence of the integrity of the hCMEC/D3 barrier. Transcytosis Assay. To characterize transport of the peptide 5-FAM-PPL across the hCMEC/D3 cell monolayer, a 50 μg/mL solution was applied to the cells. After incubation for 1 h in a 5% carbon dioxide (CO2) incubator at 37 °C, fluid was recovered from both the apical and basolateral layers and transferred to 1.5 mL eppendorfs, which were subsequently frozen and the contents freeze-dried. The concentration of the peptide applied to the cell line is in line with previous studies in which the transcytosis assay was used. For example, Fernández et al.16 applied 13.5 nM palytoxin to human intestinal cells (Caco-2) previously. MS Analysis and Microscopy Work. The permeability of the hCMEC/D3 cell monolayers to the peptide 5-FAM-PPL was evaluated after incubation for 1 h, and the presence of the peptide in both the apical and basolateral layer was identified using mass spectrometry (MS). Dried samples were resuspended in water and processed as described earlier prior to MS analysis. Following MS analysis, the peptide 5-FAM-PPL was identified in fluid collected from both the apical and basolateral layer as well as in the cell. The peptide PPL 5-FAM-PPL has a mass of 685.26 g/mol. Figure 4 shows the presence of 5-FAMPPL in samples taken from the basolateral (well) side, as described under Materials and Methods. This presence of 5FAM-PPL is indicated by the mass peaks of 342.63 (monoisotopic charge of 1) and 685.26 (charge of 2). 5FAM-PPL is lipid soluble and is hydrophobic. Peptide transport across the BBB presents conflicting results. Work reviewed by Zlokovic15 presents strong evidence that peptides can be differentially transported at the BBB. Furthermore, confocal microscopy of resin-embedded hCMEC/D3 cells shows uptake of 5-FAM-PPL into the cells as shown in Figure 3A. The nucleus was imaged using DAPI staining (blue), whereas the 5FAM-PPL peptide is shown in green. The control (Figure 3B) sample, where no peptide was applied to the cells, shows only the stained nuclei (blue). In addition, Figure 4 demonstrates that the peptide 5-FAM-PPL is capable of passing through the

Figure 2. Transcytosis assay method used to assess transport of the peptide 5-FAM-PPL across the blood−brain barrier. hCMEC/D3 cells grown to confluence on rat tail collagen type I coated PET membrane filter inserts (and serum starved for 1 h prior to assay experiments) were incubated apically with the peptide for 60 min at 37 °C. After 1 h, media from the apical and basolateral chambers were collected to assess for the presence of the peptide using MS. was activated by adding 100 μL of acetonitrile followed by centrifugation at 2000g for 1 min before the addition of 100 μL of 1% trifluoroacetic acid (TFA) in water (1:99 v/v) followed again by centrifugation of the columns at 2000g for 1 min. After column preparation, 50 μL of the recovered basolateral insert fractions, thought to contain the peptide, and 50 μL of the apical fraction were added separately to individual columns and allowed to bind to the columns for 10 min with periodical vortexing. The columns were then centrifuged at 1000g for 3 min, and the flow-through was discarded. The columns were subsequently placed in new tubes and washed with 200 μL of 1% TFA followed by centrifugation at 20009g for 1 min. The columns were then placed in new tubes and spun at 2000g for 1 min following the addition of 100 μL of 0.1% formic acid (FA) in a 50:50 acetonitrile/water (v/v) ratio. This step was repeated three times, and the resulting elutions were freeze-dried. The samples were run on a Thermo Scientific Q Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system. Peptides were resuspended in 0.1% formic acid. Each sample was loaded onto a fused silica emitter (75 μm i.d., pulled using a laser puller (Sutter Instruments P2000)), packed with Reprocil Pur C18 (1.9 μm) reverse phase medium, and was separated by an increasing acetonitrile gradient over 31 min at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 320 °C and with a potential of 2300 V applied to the frit. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. A highresolution (70000) MS scan (m/z 100−1500) was performed using the Q Exactive to select the 12 most intense ions prior to MS/MS analysis using HCD. Data were analyzed using Xcalibur Data Acquisition and Processing Software version 3.0.

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RESULTS AND DISCUSSION The peptide PPL was previously identified from both barbel fish skin gelatin3 and meat proteins8 and is known to have a PEP inhibitory IC50 value of 2.86 mM.8 The aim of this work was to determine if this peptide could pass through a representative model of the BBB, which was developed using the hCMEC/D3 cell line. Selection and Cultivation of hCMEC/D3 Cell Monolayer. hCMEC/D3 cells were cultured to confluent monolayers on rat tail collagen type-I coated filter inserts and were used to investigate the apical-to-basolateral transport of PPL. The hCMEC/D3 cell line was chosen as a model for the BBB as recent work by Eigenmann et al.,12 which assessed four cell lines that are often used as blood−brain barrier models, found that the cell type-specific adherens junction protein vascular endothelium (VE)-cadherin as well as the tight junction (TJ) protein ZO-1 were expressed in hCMEC/D3.12 In addition, Claudin-5 expression was detected when using the hCMEC/D3 148

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hCMEC/D3 cells, which represent the BBB, to reach the basolateral layer, and this finding is in agreement with Zlokovic’s work and other works.15,17 Previously, β-casomorphin was assessed for its potential to be transported across the BBB, but this was carried out using rat models in vivo.18 The quantity of peptide found in the basolateral layer was not determined; however, minimal transport of peptides could have an important impact on central nervous system (CNS) functions because only small amounts are needed for physiologic, pharmacologic and/or pathologic effects. Size, surface charge, and molecular signaling influence the ability of substances to cross the blood−brain barrier through these various pathways.19 In recent years, research concerning bioactive peptides from foods has focused on the ability of peptides to positively affect mental health.20,21 However, the bioavailabilities of these compounds and their ability to cross the BBB have not been assessed. Here, we provide strong evidence for the transport of the peptide 5-FAM-PPL across hCMEC/D3 cells. Further work will involve deciphering the mechanism of transport across the BBB and in vivo animal model studies.

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AUTHOR INFORMATION

Corresponding Author

*(M.H.) Phone: +353 (0) 1 8059957. Fax: +353 (0) 1 8059500. E-mail: [email protected]. Funding

This work was supported by short-term scientific mission (STSM) funding from the French National Institute for Agricultural Research INRA leaders of the EU COST Action INFOGEST FA1005 2011−2015. Notes

Figure 3. (A) Uptake of 5-FAM PPL (50 μg/mL) by the hCMEC/D3 endothelial cell monolayer after incubation for 2 h at 37 °C, 5% CO2, versus untreated cells (B). The peptide is colored green in the cytoplasm, and cell nuclei are stained blue with DAPI.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Stine Indrelid and Katarzyna Kuczkowska (NMBU) for assistance with cell culture.

Figure 4. MS spectra obtained for determination of the peptide 5-FAM-PPL in the basolateral layer of the hCMEC/D3 cell model. This peptide was identified using a Thermo Scientific Q-Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system and with the software package Xcalibur Data Acquisition and Processing Software version 3.0 following MS/MS analysis. 149

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assessment of nanoparticle transport mechanisms across barriers. Analyst 2015, 140, 83−97. (15) Zlokovic, B. V. Cerebrovascular permeability of peptides: manipulations of transport systems at the blood brain barrier. Pharm. Res. 1995, 12, 1395−1406. (16) Fernandéz, D. A.; Louzao, M. C.; Vilarino, N.; Espina, B.; Fraga, M.; Vieytes, M. R.; Roman, A.; Poli, M.; Botana, L. M. The kinetics, mechanistic and cytomorphological effects of palytoxin in human intestinal cells (Caco-2) explain its lower than parenteral oral toxicity. FEBS J. 2013, 280, 3906−3918. (17) Gavard, J. Endothelial permeability and VE-cadherin, a wacky comradeship. Cell Adhesion Migration 2013, 7, 465−471. (18) Ermisch, A.; Ruhle, H. J.; Neubert, K.; Hartrodt, B.; Landgauf, R. On the blood-brain barrier to peptides: [3H]β- Casomorphin-5 uptake by eighteen brain regions in vivo. J. Neurochem. 1983, 41, 1229−1233. (19) Abbott, N. J.; Ronnback, L.; Hansson, E. Astrocyte-endothelial interactions at the blood−brain barrier. Nat. Rev. Neurosci. 2006, 7, 41−53. (20) Selhub, E.; Logan, A. C.; Bested, A. C. Fermented foods, microbiota and mental health: ancient practice meets nutritional psychiatry. J. Physiol. Anthropol. 2014, 33 (2), 1−12. (21) Lister, J.; Fletcher, P. J.; Nobrega, J. N.; Remington, G. Behavioral effects of food derived opioid-like peptides in rodents: implications for schizophrenia? Pharmacol., Biochem. Behav. 2015, 7, 70−78.

ABBREVIATIONS USED AD, Alzheimer’s disease; BACE1, β-site APP cleaving enzyme; BBB, blood−brain barrier; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; hCMEC/D3, human cerebral microvascular endothelial cell line; hTERT, human telomerase reverse transcriptase; FA, formic acid; FAM, 6-carboxyfluorescein; MALDI-TOF, matrixassisted laser desorption/ionization−time-of-flight mass spectrometry; MW-SPPS, microwave-assisted solid phase peptide synthesis; PEP, prolyl endopeptidase; PET, polyester; RPHPLC, reverse phase high-performance liquid chromatography; TEER, transendothelial electrical resistance; TFA, trifluoroacetic acid; VE-cadherins, vascular endothelium cadherins

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DOI: 10.1021/acs.jafc.5b04696 J. Agric. Food Chem. 2016, 64, 146−150