An antihypertensive lactoferrin hydrolysate inhibits angiotensin I-converting enzyme, modifies expression of hypertension-related genes and enhances nitric oxide production in cultured human endothelial cells

1 An antihypertensive lactoferrin hydrolysate inhibits angiotensin Iconverting enzyme, modifies expression of hypertension-related genes and enhances nitric oxide production in cultured human endothelial cells. Aurora García-Tejedora,#, José V. Gimeno-Alcañíza,#, Sandra Tavárezb, Eulalia Alonsob, Juan B. Salomc,d, Paloma Manzanaresa,* aDepartamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas (IATACSIC), Ave Agustín Escardino 7, 46980 Paterna, Valencia, Spain bDepartamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Ave Blasco Ibáñez 17, 46010 Valencia, Spain cUnidad Mixta de Investigación Cerebrovascular, Instituto de Investigación Sanitaria Hospital La Fe, Ave Campanar 21, 46009 Valencia, Spain dDepartamento de Fisiología, Universidad de Valencia, Ave Blasco Ibáñez 17, 46010 Valencia, Spain #Both authors contributed equally to this work. *Corresponding author: Tel.: 34-96-3900022; Fax: 34-96-3636301; e-mail address: [email protected] 2 Abstract This study was aimed to explore whether an antihypertensive lactoferrin hydrolysate (LFH) can inhibit angiotensin I-converting enzyme (ACE) activity and modify the expression of genes related to hypertension in human umbilical vein endothelial cells (HUVEC). LFH induced significant inhibition of ACE activity but it did not affect ACE mRNA levels after 24 h of exposure. LFH treatment significantly affected the expression of genes encoding for proteins involved in nitric oxide pathway such as soluble guanylate cyclase 1 3 subunit (GUCY1A3; 4.42-fold increase) and nitric oxide synthase trafficking (NOSTRIN; 2.45-fold decrease). Furthermore, expression of the PTGS2/COX-2 gene encoding prostaglandin-endoperoxide synthase 2 a key component of prostaglandin synthesis was significantly increased (2.23-fold). Moreover, NOSTRIN mRNA downregulation was consistent with reduced NOSTRIN protein expression and increased NO production observed in HUVEC. The present study reveals the complexity of the effects exerted by LFH opening avenues for the better understanding of its antihypertensive effects. Keywords: lactoferrin hydrolysate, ACE inhibition, endothelial cells, transcriptomic analysis, NOSTRIN, nitric oxide. 3 Abbreviations ACE, angiotensin I-converting enzyme; ACTB, -actin; ARG2, arginase type II; AT1, angiotensin type 1 receptor; B2, kinin type 2 receptor; BCA, bicinchoninic acid method; ECE, endothelin converting enzyme; eNOS, endothelial nitric oxide synthase; Fmoc, N-(9-fluorenyl) methoxycarbonyl; GUCY1A3, guanylate cyclase 1, soluble, alpha 3; HUVEC, human umbilical vein endothelial cells; LF, bovine lactoferrin; LFH, lactoferrin pepsin hydrolysate; NO, nitric oxide; NOSTRIN; nitric oxide synthase trafficking; PBS, phosphate buffered saline solution; PTGS2, prostaglandin-endoperoxide synthase 2; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RASS, renin-angiotensin-aldosterone system; RPL13A, ribosomal protein L13a; SBP, systolic blood pressure; SHRs, spontaneously hypertensive rats. 4 1. Introduction Hypertension is one of the most prevalent risk factors associated with cardiovascular diseases, the leading cause of death in Western countries. During the last two decades many studies have focused on the dietary prevention of hypertension development, with particular interest in food-derived bioactive peptides with inhibitory effects on angiotensin I converting enzyme (ACE). Some of these food-derived peptides can control arterial blood pressure in hypertensive animals after a single oral dose and also during chronic administration. Moreover, some of them may reduce systolic and diastolic blood pressure in hypertensive patients (Martínez-Maqueda, Miralles, Recio, & Hernández-Ledesma, 2012). ACE, as part of the renin-angiotensin-aldosterone system (RAAS), hydrolyzes both the inactive angiotensin I into vasoconstrictor angiotensin II and the vasodilator bradykinin into inactive peptide fragments leading to increased blood pressure (Carey & Siragy, 2003). Most well-known effects of angiotensin II, including vasoconstriction and release of vasopressin and aldosterone, are mediated by angiotensin type 1 receptors (AT1) (Brewster & Perazella, 2004) whereas bradykinin acts mainly via kinin type 2 receptors (B2) which lead to the production of nitric oxide (NO), a vasorelaxing factor (Hillmeister & Persson, 2012). Despite numerous efforts, the in vivo mechanism underlying vasoactive and blood pressure lowering effects of antihypertensive food-derived peptides has not yet been fully established. Beyond in vivo ACE inhibition reported for some peptides and hydrolysates (Yang, Yang, Chen, Tzeng, & Han, 2004; Lu et al., 2011; Wang et al., 2012; García-Tejedor et al., 2014), antihypertensive effects can be mediated via other components of the RAAS system, such as renin which 5 catalyzes the cleavage of the N-terminal region of angiotensinogen to release angiotensin I (Udenigwe, Lin, Hou, & Aluko, 2009; Fitzgerald et al., 2012; Ajibola, Fashakin, Fagbemi, & Aluko, 2013). Moreover, emerging evidence points to the arginine–nitric oxide pathway, the endothelin system or opioid receptors as molecular targets for food-derived antihypertensive peptides (Udenigwe & Mohan, 2014). In previous studies, antihypertensive properties of peptides derived from bovine lactoferrin (LF), a well-characterized component of milk whey, were extensively characterized (Centeno et al., 2006; Ruiz-Giménez et al., 2010). Focusing on the RAAS system, we have shown vasoactive effects of a LF pepsin hydrolysate (LFH) through ACE inhibition (Ruiz-Giménez et al., 2007) and its orally antihypertensive effect in spontaneously hypertensive rats (SHR) after acute administration (Ruiz-Giménez et al., 2012). Moreover, chronic administration of LFH resulted in reductions of circulating ACE activity, angiotensin II and aldosterone levels, as well as a compensatory increase of renin activity (Fernández-Musoles, Manzanares, Burguete, Alborch, & Salom, 2013a). Recently we have suggested that inhibition of angiotensin II-induced vasoconstriction by blocking angiotensin AT1 receptors is a potential mechanism also contributing along with ACE inhibition to the antihypertensive effect of LFH (Fernández-Musoles et al., 2014). Beyond its effect on the RAAS system, LFH might act as dual vasopeptidase inhibitor since it also inhibits endothelinconverting enzyme (ECE), a key enzyme of the endothelin system involved in vascular tone and blood pressure regulation (Fernández-Musoles et al., 2010; Fernández-Musoles et al., 2013b). 6 Endothelial cells line the internal surface of blood vessels, play critical roles in vascular biology and represent a good model to evaluate the molecular mechanisms involved in blood pressure regulation. Since the effects of antihypertensive peptides might result from synergic interactions with several targets, global approaches represent a feasible strategy for revealing the action of these peptides through distinct pathways. With the aim of characterizing the effect of LFH at the cellular level, its inhibitory effect on ACE activity in cultured human endothelial cells was determined. Moreover, the effects of LFH on the expression of a panel of genes related to hypertension were evaluated in these cells. Finally, in the light of mRNA expression results, the effects of LFH on the NO pathway, including NO production, have been specifically assessed. 2. Materials and methods 2.1. Materials Bovine LF was provided by FrieslandCampina Domo (Zwolle, The Netherlands). Bicinchoninic acid protein assay kit, porcine pepsin, gelatin, monoclonal anti-- Actin antibody and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). ACE substrate o-aminobenzoyl-Gly-p-nitro-Phe-Pro was provided by Bachem Feinchemikalien (Bubendorf, Switzerland). Primary human umbilical vein endothelial cells (HUVEC) pooled from two to four different umbilical cords (Advancell, Barcelona, Spain) were a kind gift of Dr. Isabel Fariñas (Department of Cellular Biology, Universidad de Valencia, Spain). EndoGRO-VEGF complete medium kit was provided by Millipore (Darmstadt, Germany). Phosphate buffered saline solution (PBS) and trypsin-EDTA solution were obtained from HyClone (Logan, UT, USA). RNeasy Mini Kit, RT2 First Strand Kit and Human 7 Hypertension RT² Profiler™ PCR Array from SABiosciences were purchased from Qiagen (Valencia, CA, USA). LightCycler® 480 SYBR Green I Master and Complete Mini protease-inhibitor cocktail were provided by Roche Diagnostics (Basel, Switzerland). Protran BA 85 nitrocellulose, horseradish peroxidase-linked species-specific secondary antibodies and ECL Select™ Western Blotting Detection Reagent were purchased from GE Healthcare (Buckinghamshire, United Kingdom). Polyclonal anti-mNOSTRIN antibody was a kind gift of Dr. Stephanie Oess (Goethe Universität, Frankfurt am Main, Germany). 2.2. Bovine Lactoferrin Enzymatic Hydrolysate (LFH) and Synthetic Peptides Bovine lactoferrin was hydrolyzed using porcine pepsin and the product was subjected to ultrafiltration through a polyethersulfone membrane with a 3 kDa cutoff (Vivascience, Sartorius Stedim Biotech, Aubagne, France) as previously described (Ruiz-Giménez et al., 2012). The permeate (LFH) was kept at -20oC until use. Peptides of sequences LIWKL and RPYL were purchased at >95% purity from GenScript Corporation (Piscataway, NJ, USA), wherein they were synthesized by solid phase methods using N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry. Stock solutions (10 mM) of each peptide were prepared in water, sterilized by filtration and stored at -20oC. Protein content of LFH was estimated by the bicinchoninic acid method (BCA) using bovine serum albumin as standard (Ruiz-Giménez et al., 2012). Synthetic peptide concentration was based on the dry weight of the peptides. 2.3. Cell Culture 8 HUVEC were maintained in culture using EndoGRO-VEGF complete medium kit and seeded onto plasticware previously coated with a sterile solution containing 1% (w/v) gelatin in distilled water, for at least 90 minutes at 37ºC. Cell integrity was checked daily using an inverted phase-contrast microscope, and they were subcultured before reaching 90% confluence. Cells from passages 3 to 5 were washed with PBS solution, detached with trypsin-EDTA solution, resuspended in complete medium and evaluated for viability by microscopic observation of cells stained with 0.2 % trypan blue. HUVEC were seeded onto gelatin-coated 6-well plates at a cellular density of 25.000 viable cells/cm2. Twenty four hours after seeding, the corresponding treatment was added directly to the cell culture supernatant, and maintained in contact with the cells for additional 24 hours. The treatments were used at the following final concentrations: LFH 700 µg/mL; RPYL and LIWKL, 100 µM. At the end of the experiments cell viability was determined by the trypan blue exclusion method. No loss of viability was observed after any of the treatments. 2.4. ACE Activity Measurements Three different biological replicates of either the control (non-treated cells), or LFH-treated cells, were evaluated for ACE activity present in cell culture supernatants. Activity was measured in all the replicates just before treatment (zero time) and at 1, 3, 6 and 24h after addition of LFH. ACE activity was measured using the fluorescent method described by Sentandreu & Toldrá (2006) with some modifications. Fifty µL samples were taken at every time point along the experiment, and transferred to a 96-well black plate. Two hundred µL of the internally quenched fluorescent substrate o-aminobenzoyl-Gly-p-nitro-Phe- 9 Pro (0.45 mM) were added and the production of the fluorescent product by ACE activity was recorded (excitation 320 nm, emission 405 nm) every 30 minutes for at least 4 hours using a Fluoroskan Ascent FL (ThemoFisher, Waltham, MA ). Relative ACE activity in treated cells was referred to controls at the same time points. 2.5. RNA Isolation and Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) Analysis After the 24h treatment period, cell culture supernatants were aspired and stored at -20oC for nitric oxide measurements. The cells were washed with PBS and lysed for obtaining total RNA and total protein extracts using RNeasy Mini Kit. Total RNA was DNase-digested on column and was finally eluted in RNase-free water. The RNA concentration and purity was measured using a Nanodrop ND1000 spectrophotometer (ThermoFisher). All the PCR reactions were carried out in 96-well plate format in a LightCycler 480 instrument (Roche). For the transcriptomic analysis of HUVEC, 2 µg of total RNA were retro-transcribed using RT2 First Strand Kit, which includes an additional step for genomic DNA elimination. The cDNA obtained was used as template for the quantitation of the mRNA levels of 84 genes related to hypertension, by the use of the Human Hypertension RT² Profiler™ PCR Array. The complete gene list, including housekeeping genes and PCR controls is accessible online (www.SABiosciences.com). The qPCR reactions were carried out using the RT²SYBR green qPCR mastermix. The CT data were obtained by the LighCycler 480 software (release 1.5.0) and were analysed by the CT method using web-based software specific for the analysis of PCR array data 16 Inhibition of ACE activity by LFH at a concentration of 700 µg/mL was shown in cultured HUVEC supernatants. LFH-inhibitory action on ACE was comparable to that reported for two casein trypsin hydrolyates which showed IC50 values in the µg/mL level (Rousseau-Ralliard et al., 2010). Despite inhibition of ACE activity at 24 h, LFH did not affect ACE mRNA levels at the same time point. Although transcriptional regulation of ACE at earlier time points cannot be discarded, our results are in agreement with previous works that describe no significant changes in expression of genes associated with RAAS in aorta of SHR after repeated administration of the well-known ACE-inhibitory peptides VPP and IPP were described (Yamaguchi, Kawaguchi, & Yamamoto, 2009). In HUVEC, VPP and IPP had no effect on ACE protein expression, although ACE mRNA levels were not evaluated (Hirota et al., 2011). By contrast, downregulation of the mRNA levels of renin, ACE and AT1 was reported in kidney from SHRs after repeated treatment with the ACE-inhibitory sequences IQP and RVPSL (Lu et al., 2011; Yu, Yin, Zhao, Chen, & Liu, 2014). Despite ECE has also been described as a target for LFH (Fernández-Musoles et al., 2013b), and similarly to what we describe here for ACE gene expression, we did not find significant changes in ECE relative mRNA level or in expression of other genes associated with the endothelin system (EDN1, EDN2, EDNRA, EDNRB, see Table 2). Time-course effect of LFH in HUVEC on mRNA levels of genes associated with RAAS and endothelin system are necessary to discard any regulation at the mRNA level at shorter exposure times. Regulation via post-transcriptional or post-translational mechanisms such as mRNA stability and translation and protein-protein interactions cannot be ruled out (Chattopadhyay et al., 2005; Kohlstedt et al., 2013). 17 In this study, expression of the PTGS2/COX-2 gene was significantly increased by LFH treatment. COX-1 and COX-2 are the first and rate-limiting enzymes involved in the conversion of arachidonic acid to prostaglandins, thromboxane A2 and prostacyclin. It is generally assumed that COX-2 plays a detrimental role in cardiovascular homeostasis since COX-2-derived thromboxanes might induce vasoconstriction and potentiate an inflammatory state. However, in healthy humans, COX-2 generates mainly prostacyclin, a potent vasodilator and platelet inhibitor (McAdam et al., 1999). Moreover, it has been described that ACE inhibitors increase expression of COX-2 and prostacyclin levels in different experimental models (Kohlstedt, Busse, & Fleming, 2005) thus suggesting that COX-2 induction, like that provoked by LFH, may potentiate vasodilator activity. VPP and IPP treatment in SHR significantly increased the expression of COX-1 gene in aorta, but not that of COX-2 (Yamaguchi, Kawaguchi, & Yamamoto, 2009). In this work, although a slight increase in the expression of COX-1 gene was detected (1.28-fold), it did not reach statistical significance (P = 0.0609). Involvement of COX in the antihypertensive effect of milk-derived peptides deserves further research. ACE inhibitors are considered to act protectively on endothelium by inhibiting production of angiotensin II and degradation of bradykinin, which facilitates NO production. NO is an endogenous vasodilatory gaseous molecule that continuously regulates the diameter of blood vessels and maintains an antiproliferative and anti-apoptopic environment in the vessel wall, thus contributing to protect the endothelium (Sessa, 2004). The action of NO as a vasodilator is mediated by the activation of vascular smooth muscle soluble guanylate cyclase (sGC), the physiological receptor for NO (Arnold, Mittal, Katsuki, & Murad, 1977) 18 and presumably the most relevant molecular target for NO-releasing drugs in human cardiovascular therapy (Zabel, Weeger, La, & Schmidt, 1998). sGC is a signal transduction enzyme that forms the second messenger molecule cyclic GMP which in turn modulates the activity of several effector proteins that lead to vasorelaxation (Schmidt, Lohmann, & Walter, 1993). Our results showed increased NO production and upregulated expression of sGC (GUCY1) following LFH treatment in HUVEC, suggesting an elevation of cyclic GMP in HUVEC, as described for vasodilator compounds which in vivo activate sGC in vascular tissue (Galle et al., 1999). Endothelial NO is produced by eNOS which converts the amino acid Larginine into L-citrulline and NO (Geller & Billiar, 1998). eNOS is tightly controlled by coand post-translational lipid modifications, phosphorylation on multiple residues and regulated protein-protein interactions (Qian & Fulton, 2013). Significant increases of eNOS at mRNA or protein levels in aorta of SHR after administration of VPP and IPP (Yamaguchi, Kawaguchi, & Yamamoto, 2009) or a casein hydrolysate (Sánchez et al., 2011) have been reported, as well as an increase in eNOS phosphorylation in endothelial cells cultured with ACEinhibitory peptides (Shimizu et al., 2010; Ko et al., 2012). In our study, LFH treatment showed no effect on eNOS mRNA (Table 2) or protein levels (results not shown). By contrast, data reported here reveal eNOS-interacting protein NOSTRIN as a target for LFH in endothelial cells. In HUVEC, NOSTRIN expression was significantly downregulated after LFH treatment for 24 h. Moreover, this result was consistent with the reduced protein expression detected by immunoblot analysis (60%) and the increased NO production (45%) observed in HUVEC after 24h LFH treatment. NOSTRIN is a protein which modulates 19 subcellular distribution of eNOS and thus NO release. Overexpression of NOSTRIN can promote the translocation of eNOS from the plasma membrane to intracellular vesicles, with a concomitant reduction in eNOS enzyme activity and inhibition of NO synthesis (Zimmermann et al., 2002). Conversely, decreased NOSTRIN expression also influences eNOS subcellular localization and contributes to increase NO levels in endothelial cells (McCormick et al., 2011). To the best of our knowledge this is the first time that NOSTRIN is pointed out as a target for antihypertensive peptides. Other enzymes involved in the NO pathway have been proposed as molecular targets for antihypertensive peptides. Treatment of endothelial cells with a snake venom antihypertensive peptide was related to the activation of argininosuccinate synthetase, a key enzyme of the arginine-citrulline cycle which provides arginine to eNOS (Guerreiro et al., 2009). In this context, our results show a modest but significant 30% increase in relative abundance of ARG2 mRNA in LFH-treated HUVEC. ARG2 encodes for one of the two isoforms of mammalian arginase, the enzyme which shares and competes for the substrate L-arginine with eNOS in endothelial cells. Increased expression of arginase II encoded by ARG2 reduces basal NO synthesis (Li et al., 2001), and this has been proposed as a protective mechanism against the toxicity generated by NO overproduction and NO-derived radicals (Dawson, Dawson, London, Bredt, & Snyder, 1991; Gotoh & Mori, 1999). Further research is in progress to investigate the participation of eNOS and enzymes involved in arginine metabolism in the antihypertensive effect of LFH. In contrast to that found for LFH, the sequences LIWKL and RPYL did not elicit any significant change in the level of expression of PTGS2/COX-2, 20 GUCY1A3 or NOSTRIN genes. These results might reveal a negligible contribution of both peptides to the effects on mRNA levels provoked by LFH treatment, at least at the time point tested, and suggest that other peptides of the hydrolysate could contribute to such effect on gene expression in HUVEC. Synergistic effects of individual peptides cannot be discarded as suggested for the stimulatory effect on mucin gene expression in human intestinal cells of a casein hydrolysate and derived peptides (Martínez-Maqueda, Miralles, CruzHuerta, & Recio, 2013). Finally, stability of LFH and peptides in HUVEC during treatment has not been addressed here, and potential degradation by the action of cellular peptidases including ACE should be considered. Undoubtedly the possible effects of individual sequences contained in LFH as well as bioavailability issues merit further research. 5. Conclusions This in vitro study showed that LFH was able to inhibit ACE, modify the expression of hypertension-related genes and increase NO production in human endothelial cells. The HUVEC in vitro model used in this study is a widely validated model and has allowed us the detailed analysis of human gene expression, although data on the mechanism of action of LFH and derived peptides in human subjects need to be obtained. Work is underway to fulfill this issue. Data from the human in vitro model suggest NOSTRIN as a target for LFH. NOSTRIN downregulation at both mRNA and protein levels could be partly responsible for the enhanced NO production in HUVEC at the concentration tested. Other concentrations which would be closer to a physiological situation need to be tested in order to confirm the role of NOSTRIN as a functional target 21 of LFH. Results from the PCR array serve as a ground point for future studies on the complex mechanism of action underlying the antihypertensive effects of ACEinhibitory peptides derived from food proteins, including lactoferrin-derived peptides. Acknowledgements This work was supported by grant AGL2010-21009 from Ministerio de Educación y Ciencia - FEDER, Consolider Ingenio 2010, Fun-C-Food, CSD2007-00063 and RETICS INVICTUS RD12/0014/0004 from Instituto de Salud Carlos III. A. García-Tejedor is recipient of a predoctoral fellowship from Ministerio de Educación y Ciencia (BES-2011-044424). Authors thank Dr I. Fariñas for kind gift of HUVEC and Dr. S. Oess for kindly providing polyclonal anti-mNOSTRIN antibody. References Ajibola, C. F., Fashakin, J. B., Fagbemi, T. N., & Aluko, R. E. (2013). Renin and angiotensin converting enzyme inhibition with antioxidant properties of African yam bean protein hydrolysate and reverse-phase HPLC-separated peptide fractions. Food Research International, 52, 437-444. Arnold, W. P., Mittal, C. K., Katsuki, S., & Murad, F. (1977). Nitric oxide activates guanylate cyclase and increase guanosine 3,5-monophosphate levels in various tissue preaparations. 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Gene Description Fold change ACE Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 -1.65 ACTA2 Actin, alpha 2, smooth muscle, aorta -1.20 ADM Adrenomedullin 1.58 ADRA1D Adrenergic, alpha-1D-, receptor 1.64 ADRB1 Adrenergic, beta-1-, receptor 1.05 ARG2 Arginase, type II 1.30* ATP2C1 ATPase, Ca++ transporting, type 2C, member 1 1.05 ATP6AP2 ATPase, H+ transporting, lysosomal accessory protein 2 - 1.35* AVPR1B Arginine vasopressin receptor 1B 1.35 BMPR2 Bone morphogenetic protein receptor, type II (serine/threonine kinase) -1.27 CAV1 Caveolin 1, caveolae protein, 22kDa 1.04 CHRNA1 Cholinergic receptor, nicotinic, alpha 1 (muscle) 1.08 CHRNB1 Cholinergic receptor, nicotinic, beta 1 (muscle) 1.08 CLIC1 Chloride intracellular channel 1 -1.33 CLIC4 Chloride intracellular channel 4 -1.15 CLIC5 Chloride intracellular channel 5 -2.70 ECE1 Endothelin converting enzyme 1 -1.18 EDN1 Endothelin 1 1.57 EDN2 Endothelin 2 -2.56 EDNRA Endothelin receptor type A -1.23 EDNRB Endothelin receptor type B -1.00 EPHX2 Epoxide hydrolase 2, cytoplasmic -1.52 GCH1 GTP cyclohydrolase 1 -1.07 GCHFR GTP cyclohydrolase I feedback regulator -1.43 GUCY1A3 Guanylate cyclase 1, soluble, alpha 3 4.42** 33 GUCY1B3 Guanylate cyclase 1, soluble, beta 3 1.81* HIF1A Hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) -1.13 ITPR1 Inositol 1,4,5-trisphosphate receptor, type 1 -1.09 ITPR2 Inositol 1,4,5-trisphosphate receptor, type 2 -1.10 KCNMA1 Potassium large conductance calcium-activated channel, subfamily M, alpha member 1 -1.17 MYLK Myosin light chain kinase 1.21 MYLK2 Myosin light chain kinase 2 -1.19 NOS3 Nitric oxide synthase 3 (endothelial cell) 1.17 NOSIP Nitric oxide synthase interacting protein 1.20 NOSTRIN Nitric oxide synthase trafficking - 2.45** NPR1 Natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) -1.56 P2RX4 Purinergic receptor P2X, ligand-gated ion channel, 4 1.22 PDE3A Phosphodiesterase 3A, cGMP-inhibited -1.20 PDE3B Phosphodiesterase 3B, cGMP-inhibited -1.27 PDE5A Phosphodiesterase 5A, cGMP-specific 1.11 PLCG1 Phospholipase C, gamma 1 -1.31 PLCG2 Phospholipase C, gamma 2 (phosphatidylinositol-specific) -1.13 PRKG1 Protein kinase, cGMP-dependent, type I 1.03 PTGIR Prostaglandin I2 (prostacyclin) receptor (IP) 1.22 PTGS1 Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) 1.28 PTGS2 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) 2.23* S1PR1 Sphingosine-1-phosphate receptor 1 -1.19 SCNN1A Sodium channel, nonvoltage-gated 1 alpha -1.79 SLC7A1 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 1.04 SPHK1 Sphingosine kinase 1 -1.39 SPHK2 Sphingosine kinase 2 -1.03 aFinal concentration in the assay 700 µg/mL. bFold change was calculated by the CT method . *significantly different from control P < 0.05; ** significantly different from control P < 0.01 (Student’s t-test; n=4). Fold change Nostrin mRNA 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 Fold change GUCY1A3 mRNA 0 1 2 3 4 5 6 Control LFH LIWKL RPYL Fold change PTGS2 mRNA 0,0 0,5 1,0 1,5 2,0 A B C * * * Figure 1 García-Tejedor et al., 2014 control LFH Control LFH Relative NOSTRIN protein level 0 20 40 60 80 100 b-Actin NOSTRIN Figure 2 García-Tejedor et al 2014 * Time (h) 1,5 3 6 24 Nitrite (M) 0,0 0,2 0,4 0,6 0,8 1,0 1,2 * Figure 3 García-Tejedor et al., 2014