INTRODUCTION

Hydrogen sulfide (H2S) has recently emerged as a biologicallyactive gas with multiple physiological effects (1-6). H2S issynthesized endogenously from l-cysteine through the actions ofcystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE).Alternatively, l-cysteine can be converted into 3-mercaptopyruvateby glutamate oxaloacetate transaminase (GOT), and then to H2Sthrough 3-mercaptopyruvate sulphurtransferase (MPST) (1-4, 6).GOT is a pyridoxal phosphate-dependent enzyme which exists incytoplasmic (GOT1) and inner-membrane mitochondrial (GOT2)forms (7).

Collectively, the involvement of H2S on heart and bloodvessel physiology has received more attention than any otherorgan system and the effects of H2S have been investigated innumerous mammalian and non-mammalian blood vessels (1, 3,6, 8-11). Although CSE is considered the predominant H2S-generating enzyme in the cardiovascular system (1, 10), recentevidences point to the relevant role of MPSTin some vascularbeds, including the human coronary artery (12). H2S may induceeither vascular relaxation or contraction and these disparate

responses may occur in different vascular beds within a singlespecies or even in the same vessel at different levels of H2Sexposure (8, 11). Several studies have investigated the effects ofH2S in human vessels. H2S-induced relaxation has beendemonstrated in internal mammary (13), pulmonary (14),mesenteric (15), and intrarenal (16) arteries, as well as inperfused human placentas (17). However, low concentrations ofH2S (5 × 10–5M) evoked a contraction in the human mammaryartery (13). Furthermore, administration of H2S to healthyvolunteers did not provoke any acute changes in heart rate, bloodpressure, respiration, or temperature (18).

The umbilical circulation is not only the pathway forgaseous and nutritional/metabolic exchange, but it is also thesingle most important determinant of systemic vascularresistance within the fetal circulation (19-21). The early view ofthe human placenta as a passive organ in which blood flowdepends only on the arteriovenous pressure difference has beenmodified by recent evidence that the regulation of vasomotortone in the vessels of the fetoplacental circulation is important tomaintain an adequate blood supply to the fetus (22, 23). Recentinvestigations suggest that endogenous H2S is required for

JOURNALOF PHYSIOLOGYAND PHARMACOLOGY 2017, 68, 5, 737-747

www.jpp.krakow.pl

R. MOHAMMED1, L. PROVITERA2, G. CAVALLARO 2, D. LATTUADA 2,3, G. ERCOLI4, F. MOSCA2, E. VILLAMOR 1

VASOMOTOR EFFECTS OF HYDROGEN SULFIDE IN HUMAN UMBILICALVESSELS

1Department of Pediatrics, Maastricht University Medical Center (MUMC+), School for Oncology and Developmental Biology(GROW), University of Maastricht, Maastricht, the Netherlands; 2Neonatal Intensive Care Unit, Department of Clinical Sciences

and Community Health, Foundation IRCCS CA'Granda Ospedale Maggiore Polyclinic, University of Milan, Milan, Italy; 3Lino Rossi Research Center for the Study and Prevention of Unexpected Perinatal Death and SIDS; Department of Biomedical,Surgical and Dental Sciences, University of Milan, Milan, Italy; 4Division of Pathology, Foundation IRCCS CA'Granda Ospedale

Maggiore Polyclinic, University of Milan, Milan, Italy

Hydrogen sulfide (H2S) has recently emerged as a biologically active gas with multiple effects on the cardiovascularsystem. We aimed to investigate the vasomotor actions of sodium sulfide (Na2S), which forms H2S and HS– in solution, inhuman umbilical artery (HUA) and vein (HUV) rings. In addition, we examined by immunocytochemistry the expressionand localization of cystathionine β-synthase (CBS), cystathionine lyase (CSE), and 3-mercaptopyruvate sulphurtransferase(MPST), the enzymes responsible for endogenous H2S production. Human umbilical vessels were compared with chickenembryo umbilical vessels. HUAand HUVexpressed a robust signal for CSE, CBS, and 3-MPSTin both endothelial andsmooth muscle cells. However, HUA rings did not respond to Na2S (10–6M-10–3M) either at resting tone or duringcontraction evoked by serotonin or KCl. Similarly, the extraembryonic part of chicken allantoic artery did not respond toNa2S. In contrast, Na2S induced a concentration-dependent contraction in HUVrings under resting tone and aconcentration-dependent relaxation when the HUVrings were contracted with serotonin (42 ± 5% relaxation) or KCl (12± 5% relaxation). Na2S-induced contraction of HUVwas impaired following removal of extracellular Ca2+, endothelialdenudation, NO synthase inhibition (L-NAME), or soluble guanylate cyclase (sGC) inhibition (ODQ). Na2S-inducedrelaxation of HUVwas impaired by the KATP channel inhibitor glibenclamide. In conclusion, H2S does not have vasomotoreffects on HUAbut induced contraction (mediated through inactivation of the NO/sGC axis) and relaxation (mediatedthrough KATP channels) in HUV. Our data suggest a role for H2S in the venous side of human umbilical circulation.

K e y w o r d s :hydrogen sulfide, sodium sulfide, human, placenta, umbilical artery, umbilical vein, chicken embryo, nitric oxide,soluble guanylate cyclase

healthy placental vasculature and that a decrease in CSE/H2Sactivity may contribute to the pathogenesis of preeclampsia (17,24). Moreover, an important part of the experimental evidenceon the role of H2S in endothelial (patho)physiology has beenobtained using human umbilical vein (HUV) endothelial cellcultures (25-27). HUVendothelial cells are the most simple andavailable human endothelial cell type and are one of the he mostcommonly used models in vascular disease research (28, 29).There are, however, no functional studies that have investigatedthe vasomotor effects of H2S on human umbilical vessels.Therefore, our study was designed to investigate the response ofhuman umbilical artery (HUA) and HUVto Na2S, which formsH2S and HS– in solution (30, 31), and the mechanismsresponsible for this response. Reactivity in human vessels wascompared with reactivity in umbilical arteries from chickenembryos. In addition, we examined by immunocytochemistrythe expression and localization of the enzymes responsible forendogenous H2S production.

MATERIALS AND METHODS

Human umbilical vessels

Fresh umbilical cords (n = 24) were obtained followingdelivery of healthy babies from healthy normotensive mothers,either by vaginal delivery or by elective caesarean section.Collection and processing of the umbilical cords was performedanonymously. Approval was granted by the Research EthicsCommittee of Maastricht University Medical Center (METCprotocol 16-4-047).

HUAs and HUVs were dissected from the middle portion ofthe umbilical cord and cut into 2 - 3 mm rings. After dissection,two L-shaped stainless steel wires were inserted into the arteriallumen and the rings were introduced in Allhin organ chambersfilled with Krebs-Ringer bicarbonate (KRB) buffer at 37°C,gassed with 21% O2/5% CO2. One wire was attached to thechamber and the other to an isometric force-displacementtransducer (model PRE 206-4, Cibertec, Madrid, Spain). Theisometric force signal was amplified, A/D converted (PowerLab,ADInstruments Pty., Castle Hill, Australia), and recorded (Chartv3.4, ADInstruments Pty.) (32). Preliminary length-tensioncurves suggested that a resting tension of 19.6 mN (2 g force)gave optimal contractile responses to KCl 62.5 mM, thereforethis level of resting tension was used in all experiments.

Na2S (10–6M-10–3M), which forms H2S and HS- in solution,was used to analyze the response to H2S because of itsavailability with a reduced amount of elemental sulfurimpurities (28, 29). Na2S was dissolved in deoxygenatedHEPES buffer under 100% N2 and titration to pH 7.4 with HCl.The response to Na2S was tested in vessels at resting tone orcontracted with KCl (62.5 mM), or serotonin (5-HT, 10–6M).Pilot experiments showed that these concentrations produced asubmaximal (75 – 80%) contraction to KCl and 5-HT.Concentration increments of Na2S were made once theresponse had reached a plateau, or after 5 – 10 min if noresponse had occurred or a clear plateau was not reached.Experiments were performed under three different oxygenationconditions: 95% O2/5% CO2 (hyperoxia), 21% O2/5%CO2/74% N2 (normoxia) or 95% N2/5% CO2 (hypoxia). Inorder to assess the mechanisms involved in the response of theumbilical vessels to Na2S, some experiments were performedin the presence of the following pharmacological tools: the NOsynthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester(L-NAME; 10–4M), the soluble guanylate cyclase (sGC)inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ,10-5M), the cyclooxygenase inhibitor indomethacin (10–5M),

the voltage-gated K+ channel (KV) inhibitor 4-aminopyridine(4-AP, 10–2M), the KV7 inhibitors linopirdine (10–5M) and XE-991 (10–5M), the ATP-sensitive K+ channel (KATP) inhibitorglibenclamide (10–5M), the large-conductance Ca2+-activatedK+ channel (BKCa) inhibitor iberiotoxin (10–7M), theintermediate-conductance KCa (IK Ca) channel inhibitor TRAM-34 (10–6M), the small-conductance KCa (SKCa) channelinhibitor apamin (5 × 10–8M), the TP receptor antagonistSQ29548 (10–6M), and the dual endothelin (ET) receptorantagonist bosentan (10–5M). In some experiments, theendothelium was removed by gentle rubbing of the vessellumen with a stainless steel wire. The dependency onextracellular Ca2+ of the response to Na2S was examined byomission of Ca2+ from the KRB solution and addition of EGTA(10–3M).

Chicken embryo vessels

All experimental procedures were carried out in accordancewith the Dutch Law on Animal Experimentation and theEuropean Directive for the Protection of Vertebrate Animals Usedfor Experimental and Other Scientific Purposes (86/609/EU) andapproved by the Committee on Animal Experimentation of theUniversity of Maastricht. Fertilized eggs from White Leghornchickens ('t Anker, Ochten, The Netherlands) were incubated at37.8°C, 45% humidity and rotated once per hour over an angle of90° (Incubator model 25HS, Masalles Comercial, Spain).

Umbilical vessels were sampled at 19 days of incubation(hatching at 21 days). The extent of the egg air space wasdetermined by candling, and the overlying shell was removed. Acut about 2 cm long was then made in the shell membrane andthe underlying chorioallantoic membrane (CAM), the line ofincision being chosen so as to avoid major blood vessels. Bygripping the embryo's beak it was possible to draw it out throughthe incision in the CAM and lay it on its back in a Petri-dishcoated with silicon. The yolk sac was tipped out and the embryowas sacrificed by decapitation. The extraembryonic allantoicvessels were separated from the CAM close to their point ofinsertion into it. The preparation was rinsed with cold KRBbuffer and a midline laparotomie and sternotomy wereperformed. With the aid of a dissecting microscope, the ischiaticand the cranial mesenteric arteries were located and the intra-and extraembryonic trajectories of the allantoic and yolk sacarteries were followed. Vascular rings were obtained from theischiatic artery (distal to the origin of the allantoic artery), theproximal intraembryonic part of the allantoic artery(immediately after its origin in the ischiatic artery), and theextraembryonic part of the allantoic artery (in the middle part ofits trajectory between the embryo and the chorioallantoicmembrane), and the extraembryonic part of the yolk sac artery(33). Vascular rings were mounted as ring segments between anisometric force transducer and a displacement device in amyograph (model 610M; Danish Myo Technology, Aarhus,Denmark). The myograph organ bath (5 mLvolume) was filledwith KRB buffer maintained at 39°C and bubbled with 21%O2/74% N2/5% CO2 (Po2~18 kPa). After an equilibration period,the vessels were distended to a resting tension corresponding toa transmural pressure of 2.66 kPa. This pressure correspond tothe mean arterial blood pressure reported in 19d chickenembryos and elicit the highest contractile response to KCl, asdetermined in previous experiments. After 30 min of incubationat basal tone, a control reference contraction was elicited byraising the K+ concentration of the buffer to 62.5 mM. Thepreparations were washed three times and allowed to recoverbefore a new stimulation. Then concentration-response curves toNa2S were performed in vessels under resting tone or contractedwith KCl (62.5 mM) or 5-HT(10–6M).

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Immunohistochemistry

Formalin-fixed, paraffin-embedded human umbilical cordswere cut into 4 µm sections and placed on electrostaticallypolarized slides. Immunohistochemistry was performed usingthe automated system BenchMark XT(Ventana MedicalSystems Inc., Tucson, Arizona, USA). Reactions were revealedusing UltraView™ Universal DAB, a biotin-free, multimer-based detection system, according to the manufacturer'sinstructions. The immunostaining procedure was performedusing CBS (clone 3E1, WH0000875M1), CSE (HPA023300),GOT2 (HPA018139), MPST(HPA001240) (Sigma- Aldrich,USA), and GOT1 (NBP1-54778) (Novus Biological, USA) -specific antibodies. Primary antibody solutions were diluted at

1:100 (CBS and GOT1) or 1:200 (CSE, GOT2, and MPST) inVentana Diluent. For GOT1 and GOT2, the signal was enhancedwith the Ventana amplification kit. Negative controls wereperformed by replacement of primary antibody with equalconcentrations of nonimmune serum. Images were scannedusing an Aperio CS2 Scanscope (Leica, Germany).

Drugs and solutions

KRB buffer contained (in mmol L–1): NaCl (118.5), KCl(4.75), MgSO4·7H2O (1.2), KH2PO4 (1.2), NaHCO3 (25.0),CaCl2 (2.5), glucose (5.5). HEPES buffer contained (in mmol L-1): NaCl (142.9), KCl (4.75), MgSO4·7H2O (1.2), KH2PO4 (1.2),CaCl2 (2.5), glucose (5.5), HEPES (15.0). Solutions containing

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Fig. 1. Representative traces showing the effect ofNa2S (1 µM–1 mM) on human umbilical artery (A, B)and vein (A, C, D) rings at resting tone (A) orcontracted with serotonin (5-HT, 1 µM; B, C) or KCl(62.5 mM; D). Vertical bars indicate tension scale(mN/mm). Horizontal bars show time (min). Valuesindicate log M [Na2S]. Arrows without a valueindicate half-log increments in concentration. Therelaxant effects of sodium nitroprusside (SNP, 0.1mM) and forskolin (0.1 µM) are shown in panel B.Experiments were performed under 21% O2.

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Fig. 3. Mechanisms involved in the contraction(A) and the relaxation (B) induced by Na2S(10–3M) in human umbilical vein rings. Vesselswere at resting tone (A) or contracted withserotonin (10-6M, B). In control experiments,vessels were exposed to Na2S under basal tone(A) or following precontraction with serotonin(B) but the different inhibitors of thecontraction/relaxation pathways were notpresent. All the experiments were performedunder 21% O2. *P < 0.05, ***P < 0.001 versuscontrol.

Fig. 2. Mean (± S.E.M.) cumulative dose-response curves for Na2Sin human umbilical vein rings contracted with KCl (62.5 mM, n =6) or serotonin (5-HT, 1 µM, n = 12). Experiments were performedunder 21% O2. Mean contraction induced by KCl was 9.6 ± 1.2mN/mm. Mean contraction induced by 5-HTwas 8.3 ± 1.1 mN/mm.*P < 0.001 for difference in maximal relaxation when comparedwith KCl-contracted veins.

different concentrations of K+ were prepared by replacing NaClby an equimolar amount of KCl. Glibenclamide was obtainedfrom Alexis Biochemicals (Lausen, Switzerland/ Farmingdale,NY, USA). All other drugs were obtained from Sigma Chemical(St. Louis, MO, USA). All drugs were initially dissolved indistilled deionized water, except ODQ which was dissolved indimethylsulphoxide (DMSO), indomethacin which wasdissolved in ethanol and 4-APwhich was directly dissolved inKRB buffer.

Analysis of data

Results are expressed as mean ± S.E.M. and n reflects thenumber of umbilical cords or embryos from which the ringswere obtained. Contractions are expressed in terms of activewall tension (mN/mm, calculated as the force divided by twicethe length of the segment) or as a percentage of the referencecontraction to KCl (62.5 mM) performed for each individualring at the beginning of the experiment. The relaxant responsesare expressed as the percentage of reduction of the contractioninduced by KCl or 5-HT. Sensitivity/potency (expressed aspEC50 = –logEC50) and efficacy (expressed as Emax) were

calculated by nonlinear regression analysis of the concentration-response curves. Differences between mean values wereassessed by Student's t-test or one-way ANOVA followed byBonferroni's post hoc t-test. Differences were consideredsignificant at a P< 0.05. All analyses were performed usingGraphPad Prism (version 5.00 for Windows, GraphPadSoftware, San Diego California USA, www.graphpad.com).

RESULTS

Human umbilical vessels

HUA rings did not respond to Na2S either under resting tone(Fig. 1A) or during contraction evoked by 5-HT(Fig. 1B) or KCl(not shown). In contrast, the NO donor sodium nitroprusside(10–4M) and the adenylate cyclase stimulator forskolin (10–5M)relaxed 5-HT-contracted HUArings (Fig. 1B). Bubbling theorgan chamber with 0%, 21%, or 95% oxygen did not affect thelack of responsiveness of HUAs to Na2S (data not shown).

Na2S induced a concentration-dependent contraction inHUV rings under resting tone (Fig. 1A) and a concentration-

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Fig. 4. Immunostaining forcystathionine β-synthase (CBS),cystathionine lyase (CSE), glutamateoxaloacetate transaminase (GOT), and3-mercaptopyruvate sulphurtransferase(MPST) in human umbilical arteries.Scale bars: left panels, 200 µm; rightpanels, 60 µm.

dependent relaxation when the HUVrings were contractedwith 5-HT (Figs. 1C and 2, pEC50 = 4.11 ± 0.3) or KCl (Figs.1D and 2). As shown in Fig. 2, Na2S-induced relaxation wasmarkedly reduced when the HUVrings were contracted withKCl. When HUVrings were precontracted with KCl, a plateaurelaxant effect could not be observed and, therefore, the pEC50

could not be calculated. Neither the contraction nor therelaxation evoked by Na2S in HUV rings was affected bybubbling the organ chamber with 0%, 21%, or 95% oxygen(data not shown).

In order to characterize the mechanisms involved in Na2S-induced contraction and relaxation in HUVrings, we performedadditional concentration-response curves in the presence ofseveral inhibitors of common relaxant or contractile pathways orunder different experimental conditions (i.e., endothelialdenudation or elimination of extracellular Ca2+). Incubation ofHUV rings with some of the inhibitors evoked a contractileresponse (L-NAME: 0.31 ± 0.02 mN/mm, n = 6; ODQ: 0.34 ±0.02 mN/mm, n = 6; indomethacin: 0.21 ± 0.01 mN/mm, n = 6).As shown in Fig. 3A, Na2S-induced contraction of HUVwasimpaired following endothelial denudation or in the presence ofthe NOS inhibitor L-NAME, or the sGC inhibitor ODQ.

Removal of extracellular Ca2+ abolished Na2S-inducedcontraction of HUV rings. In contrast, incubation with thecyclooxygenase inhibitor indomethacin, the TP receptorantagonist SQ29548, or the dual ETreceptor antagonistbosentan did not significantly affect Na2S-induced contraction ofHUV rings (Fig. 3A). Na2S-induced relaxation of 5-HT-contracted HUVrings was impaired by the presence of the KATP

channel inhibitor glibenclamide but unaffected by endothelialdenudation or by the presence of L-NAME, ODQ,indomethacin, the KV inhibitor 4-AP, the KV7 inhibitorslinopirdine and XE-991, the BKCa inhibitor iberiotoxin, the IKCa

inhibitor TRAM-34, or the SKCa inhibitor apamin (Fig. 3B).The immunohistochemistry study showed that HUA(Fig. 4)

and HUV(Fig. 5) expressed a robust signal for CSE, CBS, and3-MPSTin both endothelial and smooth muscle cells. Althoughwith a lower intensity, GOT1 was also expressed by HUAendothelial and smooth muscle cells (Fig. 4). In contrast, a veryfaint, almost undetectable, expression of GOT1 was observed inHUV cells (Fig. 5). GOT2 was expressed in endothelial cellsfrom both HUA(Fig. 4) and HUV(Fig. 5). Controls processedin the absence of primary antibody did not show any staining(Fig. 6).

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Fig. 5. Immunostaining forcystathionine β-synthase (CBS),cystathionine lyase (CSE), glutamateoxaloacetate transaminase (GOT), and3-mercaptopyruvate sulphurtransferase(MPST) in human umbilical veins.Scale bars: left panels, 200 µm; rightpanels, 60 µm.

Chicken embryo vessels

None of the chicken vessels studied (i.e., ischiatic artery,intraembryonic allantoic artery, extraembryonic allantoic artery,and yolk sac artery) responded to Na2S under resting tone. Whenthe vessels were contracted with 5-HT(10–6M), Na2S induced a

concentration-dependent relaxation in all the vessels with theexception of the extraembryonic allantoic artery. As shown inFig. 7, the relaxation induced Na2S in intraembryonic allantoicarteries (pEC50 = 3.81 ± 0.3) was significantly less marked thanthe relaxation observed in ischiatic (pEC50 = 4.02 ± 0.4) and yolksac arteries (pEC50 = 3.92 ± 0.4).

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Fig. 6. Negative immunostainingcontrols in human umbilical arteries (Aand C) and veins (B and D). Sectionswere developed without primaryantibodies to control for the secondaryantibodies and the label (UltraviewUniversal Dab Kit, RocheDiagnostics). A and B were performedwith standard protocol, C and D wereperformed with the Ventanaamplification kit. Scale bars: leftpanels, 500 µm; right panels, 200 µm.

Fig. 7. Mean (± S.E.M.) cumulative dose-response curves for Na2S inchicken embryo artery rings contracted with serotonin (1 µM).Experiments were performed under 21% O2. Curves without acommon letter (a, b, c) are significantly different (P< 0.05).

DISCUSSION

To the best of our knowledge, this is the first studyevaluating the effects of H2S in human umbilical vessels. Weobserved that the main enzymes responsible for endogenous H2Sproduction were expressed in HUAand HUVvascular smoothmuscle and/or endothelial cells. However, the response to Na2S,which forms H2S and HS– in solution, was different in arteriesand veins. Thus, Na2S did not elicit a vasomotor response inHUA rings but evoked a concentration-dependent contraction inHUV rings under basal tone and a concentration-dependentrelaxation when the HUVs were pre-contracted with 5-HTorKCl. Na2S-induced contraction of HUVrings was almostabolished in the absence of extracellular Ca2+ and impaired byendothelial denudation or by inhibition of NO synthase or sGC.On the other hand, Na2S-mediated relaxation of HUVs wasendothelium-independent and impaired by the presence of theKATP inhibitor glibenclamide. Altogether, our data suggest a rolefor H2S in the venous side of human umbilical circulation.

CSE is expressed in endothelial and/or smooth muscle cellsin many vascular beds from several species and, for years, it hasbeen considered as the main enzymatic source of H2S in thevascular system (34, 35). Nevertheless, all three H2S-producingenzymes (i.e., CSE, CBS, 3-MPST) have been reported to beexpressed in vascular cells (17, 35-38). Cindrova-Daviesshowed that, in human placentas, CBS was immunolocalizedonly to the syncytiotrophoblast, whereas CBS was localized tosmooth muscle cells encircling arteries in stem villi (17). In thepresent study, we observed that umbilical vessels expressedCSE, CBS, and 3-MPSTin both endothelial and smooth musclecells. Moreover, GOT, the enzyme responsible for theconversion of l-cysteine into 3-mercaptopyruvate (anintermediate step for the production of H2S by 3-MPST) wasalso expressed in umbilical vessels. Therefore, human umbilicalvessels are equipped with the enzymatic machinery responsiblefor the endogenous synthesis of H2S. Interestingly, only thesmooth muscle of the veins was responsive to exogenous H2S.

With our data, we can only speculate on the mechanismsunderlying the differences between HUAs and HUVs on theresponse to Na2S. Previous studies described differencesbetween HUAs and HUVs on the response to physiological andpharmacological stimuli, such as oxygen (39), local anesthetics(40), calcium channel blockers (41), or arginase inhibitors (42).This suggests the presence of important differences betweenHUAs and HUVs affecting both vascular smooth muscle andendothelial cells. It has been suggested that part of thesedifferences could be related to the fact that, under physiologicalconditions, pO2 levels are higher in the umbilical vein (4 - 6 kPa)than in the umbilical arteries (2 - 4.7 kPa) (42, 43). Moreover,the reactivity of human umbilical and placental chorionic platevessels is modified by the level of oxygenation (43-45). Of note,in the majority of the studies examining ex vivo reactivity ofumbilical vessels, organ baths were bubbled with 95% (40, 41,46, 47) or 21% O2 (42, 48, 49) that is in great excess comparedto the O2 that reaches tissues (50). In our study, we bubbled theorgan baths with three different gas mixtures containing 0, 21,and 95% O2. Our aim was not to reproduce (patho)physiologicalconditions, but rather to examine the response to Na2S under awide range of oxygenation. The lack of response of HUAringsto Na2S was observed under the three O2 levels of and thecontraction/relaxation observed in HUVrings was unaffected byoxygenation. Therefore, the differences between arteries andveins were not mediated by O2.

O2 has an antagonistic effect on H2S, leading to its oxidationand consequently attenuating its biological actions (51).However, it should be noted that all previous studies examiningthe response of human tissues to exogenous H2S were performed

under 95% O2 (13-17, 52). Our results suggest that O2

concentration does not affect the response of human vessels toexogenous H2S. In contrast, studies in other species have yieldedcontradictory results, ranging from more sensitivity to H2S underhypoxia (53) to vascular effects of H2S that were independent ofthe experimental O2 conditions (9).

Both exogenous and endogenous H2S can activate multiplesecond messenger systems leading to relaxation (in some casescontraction) of vascular smooth muscle. The pathways utilizedand the effect seem to depend on the vascular bed studied, thespecies examined, and on the H2S source used (35). Of note, theconcentrations of H2S gas solution, or NaSH and Na2S, requiredto induce vascular relaxation are often in the high micromolarrange. These concentrations are several fold higher than thereported levels of H2S gas in blood (1, 54, 55).

In our study, H2S relaxed HUVs in a range of concentrationssimilar to those reported to produce effects in vessels such as rataorta (11, 56), rat mesenteric artery (57), or human internalmammary artery (13). Interestingly, Cindrova-Davies et al.reported that nanomolar concentrations of the H2S donor NaHSinduced a significant relaxation in human perfused placentas(17). This suggests that small resistance vessels of thefetoplacental circulation are more sensitive to H2S-inducedrelaxation than the large vessels of umbilical cord.

It has been suggested that the major physiological role ofH2S in the vasculature may not be to act as a direct vasodilator,but rather to regulate local concentrations and activity of NO(10). In our experiments, incubation of HUVrings with the NOSinhibitor L-NAME or the sGC inhibitor ODQ induced a toniccontraction, suggesting that there is a basal production of NO byHUVs. Moreover, Na2S induced a lower contraction in the HUVrings in which this basal production of NO was impaired (i.e.,HUV rings without endothelium or incubated with L-NAME orODQ). Altogether, this suggests that Na2S-induced contractionof HUV rings was mediated through an inhibitory effect of H2Son the endogenous production of NO. Accordingly, Geng et al.,demonstrated that exogenous H2S reduced endothelial NOSactivity and transcript abundance in HUVendothelial cells (58).Ali et al. have also shown that constriction of rat aortic rings byNaHS was attenuated after endothelial denudation (59).However, in the same preparation, concentrations of NaHSgreater than 100 mM resulted in vascular relaxation. A similarbiphasic effect (contraction at low concentrations and relaxationat high concentrations) has been demonstrated by Cancanviovaet al. using Na2S instead of NaHS (11). Moreover, the dualvasodilator and vasoconstrictor effect of H2S has also beenobserved in human internal mammary artery (13). In accordancewith our results, contraction of human internal mammary arterywas related to the inactivation of NO, whereas relaxation wasmediated through KATP channels.

Na2S-induced relaxation in HUVrings was markedlyreduced when the vessels were contracted by increasing theextracellular concentration of potassium ions, providing strongindication that potassium channel activation played a major rolein the relaxation. Moreover, glibenclamide, a KATP channelblocker, partially impaired the relaxation of HUVrings causedby Na2S, suggesting that part of the relaxation is due to increasedopen time of the KATP channels. The currently available dataindicate that H2S relaxes mammalian blood vessels mostly byopening KATP channels in smooth muscle cells (1, 2, 60).Accordingly, glibenclamide attenuated H2S-induced relaxationin human internal mammary artery (13) human mesenteric artery(15) and human perfused placenta (17). Unfortunately, themechanisms involved in H2S-induced relaxation in humanpulmonary arteries have not been characterized (14).Glibenclamide also impaired H2S-induced relaxation in othermammalian vessels such as rat thoracic aorta (61-63), or rat

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mesenteric arteries (57). In addition, patch-clamp studies havedemonstrated that H2S increases KATP-dependent current andinduces hyperpolarization in mammalian vascular smoothmuscle cells (62, 63). However, glibenclamide failed to affectthe relaxing effect of H2S in mouse aorta (64) and rat coronaryartery (65). In the latter vessel, H2S-induced relaxation wasimpaired by the presence of the KV channel blocker 4-AP(65).Moreover, the Kv7-blockers linopirdine and XE-991 impairedH2S-induced relaxation in rat aorta. However, in the HUVneither 4-AP nor the Kv7 blockers affected H2s-inducedrelaxation suggesting lack of involvement of Kv channels in therelaxant response.

One limitation of our study is that we combined data fromumbilical cords obtained after vaginal deliveries and electivecesarean sections. In general, elective cesarean sections areconsidered less stressful for the fetus as compared to vaginaldeliveries (66, 67). There is also evidence that vaginal deliveriesare associated with an increased oxidative stress (68). The birthmodes also differ in the amount of mechanical strain put on theumbilical cord. Hoenicka et al., investigated the differences inreactivity of HUVs obtained after vaginal delivery and electivecesarean section (67). HUVrings from vaginal deliveriesshowed a significantly lower contraction in response to KCl and5-HT. However, none of the vessel sections contained any signsof endothelial damage or other histological alterations,regardless of the mode of delivery (67). In our study, collectionof the umbilical cord was performed anonymously. Therefore,we cannot analyze the effects of H2S on vessels depending ondelivery mode. Nevertheless, the differences between HUVrings from cesarean sections and vaginal deliveries might notaffected the validity of our results since vessels from both groupswere randomly distributed throughout the study and were likelypresent in a similar proportion in each experimental protocol. Inaddition, rings from the same cord were used as control in theexperiments aimed to analyze the mechanisms ofcontraction/relaxation.

Although the enzymes responsible for endogenous H2Sproduction were expressed in HUAs, Na2S did not evoke anyvasomotor response in this preparation. In order to investigatewhether this lack of responsiveness to exogenous H2S was aparticular characteristic of the umbilical arteries, we performedadditional experiments in chicken umbilical vessels. Thechicken umbilical arterial system comprises two allantoicarteries and the yolk sac artery (33). The allantoic arteries bringthe deoxygenated blood into contact with the CAM whereexchange of gases through the shell occurs and the allantoic veinreturns oxygenated blood to embryo (69-71). The yolk sacmembrane is the nutritional organ of the late chicken embryoand is supplied by the yolk sac artery, a branch of the cranialmesenteric or omphalomesenteric artery (33, 70, 71). In aprevious study we characterized the reactivity of allantoic andyolk sac arteries. We observed that the allantoic artery, did notshow, in its extraembryonic portion, α-adrenergic-mediatedcontraction or acetylcholine (ACh)-induced relaxation, whereasboth phenomena were present in the yolk sac artery (33). Lackof relaxation to ACh, lack of innervation, and lowresponsiveness to α-adrenoceptor agonists are characteristicsalso present in HUAs. Therefore, from the two types of arterieswhich form the chicken embryo umbilical system, theextraembryonic allantoic arteries are the ones with more incommon with HUAs (33). Interestingly, both α-adrenergic-mediated contraction and ACh-mediated relaxation were presentin the intraembryonic segment of the allantoic artery anddecreased up to disappear along the trajectory of the artery (33).In the present study, we noted a similar finding for Na2S-inducedrelaxation which was present in the intraembryonic but absent inthe extraembryonic part of the allantoic artery. In contrast, Na2S

relaxed the yolk sac artery with similar potency and efficacythan the observed in pulmonary and systemic chicken embryovessels (9). Taken together, the results in HUAand theextraembryonic part of chicken allantoic artery suggest that thelack of responsiveness to H2S is an intrinsic characteristic of theextra-fetal umbilical arterial circulation.

In conclusion, this first functional characterization of theeffects of H2S in HUVs suggests a role for the so-named thirdgasotransmitter, besides NO and CO (73), in the umbilicalcirculation. The blood vessels of the umbilical cord arenecessary for the communication between the growing fetus andthe placenta, so a thorough knowledge of the mechanisms andstructures responsible for their contractile state is fundamentalfor the study of the physiology and the pathophysiology offetoplacental blood flow. Our results warrant further researchaimed at determining the activity of H2S-generating enzymes inthe umbilical vessels as well in small fetoplacental arteries andveins. These investigation would lead to a better understandingof the role of H2S in the fetomaternal circulation underphysiological and pathological conditions (25).

Conflict of interests: None declared.

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R e c e i v e d :June 13, 2017A c c e p t e d :September 25, 2017

Author's address: Prof. Eduardo Villamor, Department ofPediatrics, Maastricht University Medical Center (MUMC+). P.Debyelaan 25. P.O. Box 5800. 6202 AZ Maastricht. TheNetherlands.E-mail: e.villamor@mumc.nl

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