The Role of Serotonin Transporter in Human Lung ...downloads.hindawi.com/journals/crj/2017/9064046.pdf · The Role of Serotonin Transporter in Human Lung ... hydronephrosis ... allele

Research ArticleThe Role of Serotonin Transporter in Human LungDevelopment and in Neonatal Lung Disorders

E C C Castro1 P Sen2 W T Parks3 C Langston1 and C Galambos4

1Department of Pathology amp Immunology Texas Childrenrsquos Hospital Baylor College of Medicine6621 Fannin St MC-1195 Houston TX 77030 USA2Department of Pediatrics Baylor College of Medicine 6621 Fannin St MC-1195 Houston TX 77030 USA3Department of Pathology Magee Womenrsquos Hospital UPMC Pittsburgh PA 15213 USA4Department of Pathology and Laboratory Medicine Childrenrsquos Hospital Colorado University School of Medicine of Colorado13123 East 16th Avenue PO Box 120 Aurora CO 80045 USA

Correspondence should be addressed to C Galambos csabagalamboschildrenscoloradoorg

Received 6 October 2016 Revised 8 December 2016 Accepted 10 January 2017 Published 20 February 2017

Academic Editor Elie El Agha

Copyright copy 2017 E C C Castro et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Introduction Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT) and isassociated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillarydysplasia with misalignment of pulmonary veins (ACDMPV) Serum serotonin regulation has been linked to pulmonary vascularfunction and disease and serotonin transporter (SERT) is thought to be one of the key regulators in these processes We sought tofind evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACDMPVMethodsWe used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal humanfetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology In addition we tested for a SERT genepromoter defect in ACDMPV patients ResultsWe found that SERT protein expression begins at 30 weeks of gestation increasesto term and stays high postnatally ACDMPV patients had diminished SERT expression without SERT promoter alterationConclusion We concluded that SERTserotonin pathway is crucial in the process of pulmonary vascular remodelingadaptationat birth and plays a key role in the pathobiology of ACDMPV

1 Introduction

There is a remarkable change in the structure and function ofthe pulmonary vasculature during the transition from fetalto extrauterine life In the fetus the pulmonary vasculature isa high resistance low flow system and thus right ventricularoutput is shunted to the placenta for gas exchange At birtheven with the first breath pulmonary vascular resistance fallsdramatically and pulmonary blood flow increases rapidlyFailure of this transition can lead to serious neonatal lungdisorders characterized by the clinical syndrome of PersistentPulmonary Hypertension of the Newborn (PPHN) [1 2]A lethal form of PPHN alveolar capillary dysplasia withmisalignment of pulmonary veins (ACDMPV) is char-acterized by vascular abnormalities including a dysplasticpulmonary microvascular network with drastically reduced

numbers of capillaries that are often markedly expandedand malpositioned misalignment of pulmonary veins andabnormally thickened small arteriesarterioles as well as lob-ular underdevelopment Clinically infants with ACDMPVcommonly present with respiratory failure and treatment-resistant PHT at or a few days after birth Associated morbidconditions are well-known A significantly high number(approximately 80) of the babies with ACDMPV have gas-trointestinal cardiac or genitourinary anomalies in additionto the lung disorder [3 4] Recently FOXF1 abnormalitieswere detected in up to 70 of ACDMPV cases however theexact mechanism through which these genetic abnormalitieslead to ACDMPV is yet to be elucidated [5]

The role of vasoactive mediators in achieving successfulpulmonary vascular remodeling at birth has begun to emerge[6] Bioavailability of the potent vasoconstrictor serotonin

HindawiCanadian Respiratory JournalVolume 2017 Article ID 9064046 10 pageshttpsdoiorg10115520179064046

2 Canadian Respiratory Journal

has been shown to be predominantly regulated by theserotonin transporter (SERT) in the lung endothelium [7ndash15] Abnormal SERT expression can lead to dysfunction ofa variety of organs including the lung [16ndash21] Importantlymaternal use of pharmacologic SERT inhibitors (SSRIs) inthe third trimester of pregnancy has been linked to thedevelopment of PPHN and altered pulmonary vasculaturehas been proposed as a possible mechanism [22 23] Thisassociation indicates a likely critical role for SERT in thenormal adaptation of the fetal pulmonary vasculature toextrauterine lifeThis role of SERT is further substantiated byan animal study in which SSRI exposure in utero induced PHand pulmonary arterial smooth muscle hyperplasia resem-bling that seen in PPHN [24] A recent study has shownthat serotonin plays a key role in maintaining high levelsof pulmonary vascular resistance and that SSRI exposureincreases serotonin activity causing pulmonary hypertensionin the chronically prepared newborn lamb [25]

Current data suggests that disorders associated withPPHN have an overall mortality rate of 10ndash20 althoughACDMPV is uniformly lethal despite the use of inhalednitric oxide therapy often combined with extracorporealmembrane oxygenation [11 26] As the bioavailability ofserotonin in the body is primarily regulated by pulmonarySERT [7ndash15] there is likely a fundamental role for SERT inthe regulation of pulmonary vascular tone particularly forthe major changes that occur related to birth

Thus we hypothesize that human pulmonary SERT mayplay a role in the adaptation of fetal pulmonary vasculature toextrauterine life and that abnormalities of SERT activity leadto neonatal pulmonary hypertensionHerewe present a studyof SERT expression in pre- and postnatal human lungs andin cases of newborn and childhood PHT of varied etiologyincluding ACDMPV

2 Material and Methods

21 Tissue Samples Approval for this study was obtainedthrough review by the University of Pittsburgh and BaylorCollege of Medicine Institutional Review Boards

Microarray of pulmonary tissue obtained from multipleautopsy cases was employed to establish the specificity andsensitivity of SERT immunostaining of lung endotheliumAdditionally SERT immunostaining of the vascular endothe-lial of different organs was similarly assessed

Physiologic temporal-spatial expression of SERT wasstudied using lung sections from 30 autopsy cases encompa-ssing a range of ages including 27 cases of 12 13 16 18 34 39and 40 weeks of gestational age (WGA) and 3 postnatal lungsof ages 1 5 and 10 years Cases with significant pulmonarypathology were excluded from this group

SERT expression in a variety pulmonary hypertensivedisorders was studied employing lung sections obtained atautopsy from 29 children with PHT due to ACDMPV(119899 = 14) congenital alveolar dysplasia (119899 = 1) congenitaldiaphragmatic hernia (119899 = 5) bronchopulmonary dysplasia(119899 = 3) secondary PHT in congenital heart disease (119899 = 1)and idiopathic primary PHT (119899 = 5) For these 29 casespatient histories and histologic sections of the lung were

reviewed to confirm the initial diagnosis All ACDMPVpatients were tested for mutation in FOXF1 and 4 weretested for rearrangements at 16q24 locus Among the 14ACDMPV patients three had mutations in FOXF1 Of the11 without identified mutation 4 were further identified withrearrangements at 16q24 locus with deletions identified inthree (Table 1)

22 Immunohistochemistry SERT (mousemonoclonal cloneMAB5618 Millipore Temecula CA USA) and CD31 (mousemonoclonal clone JC70A Dako Carpinteria CA USA)antibodies were used as follows after the routine stepsof deparaffinization and dehydration SERT antigen wasretrieved by steam-heat treatment in 1mM EDTA + 10mMTris buffer at pH 90 After incubation of primary SERT anti-body for 30 minutes in room temperature at a dilution of14000 diaminobenzidine (DAB) staining was carried outwith utilization of the avidin-biotin-peroxidase system Allsections were counterstained with hematoxylin Diffusebrown membranous endothelial staining was interpretedas positive while absence of brown staining was deemednegative For positive controls brain sections including theRaphe nucleus (rich in serotoninergic neurons and recom-mended by manufacturer) were used adrenal tissue wasused as negative control (Figure 1) The ACD cases werealways stained in batches containing the positive and negativecontrol as well as the pan-endothelial marker CD31 to assesspulmonary endothelial cell preservation Diffuse and intenseendothelial cell staining was required otherwise the case wasrejected

23 SERT Staining Assessment SERT immunohistochem-istry was assessed and recorded as positive and negative Inaddition the terms diffuse and patchy were added for thedistribution pattern of the stain

24 PCR and Sequencing The PCR was carried out in aTC-512 thermocycler (Techne Minneapolis MN) utilizing100 ng template DNA using the following protocol 5min at95∘C followed by 35 cycles of 1min at 95∘C 1min at 60∘C2min at 72∘C and final extension for 10min at 72∘C ThePCR products were purified using a Sephadex G-50 col-umn and sequenced on a ABI 3130XL DNA Genetic Analy-zer (Life Technologies Grand Island NY) following theman-ufacturerrsquos protocol The primers used were as follows for-ward-GCCAGCACCTAACCCCTAATGand reverse-GAA-TACTGGTAGGGTGCAAGGAG

3 Results

31 Specificity and Sensitivity of SERT to Human EndotheliumThe autopsy tissue microarray showed that in normal lungsSERT is expressed specifically in pulmonary endothelial cellswith a diffuse crisp brown staining after birth (Figure 1) Wealso found positive staining in the epithelial cells of gastricglands (44 cases) hepatocytes (34 cases) and renal tubularepithelium (34 cases) and in axons of the brain (34 cases)and Schwann cells in the nerve (34 cases) One case of 4showed minimal staining of myocardium and 14 showed

Canadian Respiratory Journal 3

Table 1 ACDMVP patient population with testing for mutation andor deletions in FOXF1 Cases not tested for deletions typically hadfragmented andor poorly preserved DNA making testing problematic

Serialnumber Gender Survival in days Gestation Weight Diagnosis FOXF1 mutation FOXF1

deletion Other associated anomalies

1 F 5 39-57 3850 Autopsy NM NT

Coarctation of aorta ventricularseptal defect mitral valve

malformation tricuspid valvemalformation malformed atrial

septum

2 M 22 39 3240 Autopsy NM NTBilateral hydronephrosis

secondary to posterior urethralvalves

3 F 33 36-12 3120 Autopsy NM NT None4 M 12 37 NA Autopsy NM NT Right lung with two lobes

5 M 4 38 3295 Autopsy NM NTMarked hypertrophy of urinarybladder wall bilaterally dilated

ureters and renal pelves

6 F 9 40 4180 Autopsy NM NTButterfly vertebra imperforateanus abnormal pulmonary lobar

configuration

7 F 30 41-27 3595 Autopsy NM NT Small thymus with adrenalglands single umbilical artery

8 M 47 40 3025 Autopsy NM ND Bilateral ureteropelvic junctionobstruction hydronephrosis

9 M 10 32 2470 Autopsy c225CgtApTyr75lowast

Bilateral bilobed lungsmalrotation of the intestinebilateral hydronephrosis andhydroureters annular pancreasand gastroduodenal stenosis

10 M 05 32 2435 Autopsy c850dupTpTyr284Leufslowast11

Lymphangiectasia renal corticalcysts pneumothorax renal

obstructive dysplasia

11 M 46 40 5057 Autopsy c1138TgtCp380Argextlowast73

Malrotation of the colon withMeckelrsquos diverticulum

12 F 13 40 3600 Biopsy NM Upstream None13 F 25 NA 3676 Biopsy NM Genic Abnormal lobation

14 M 40 38 2900 Autopsy NM Genic

Ventricular septal defectduodenal atresia annularpancreas imperforate anushydronephrosis tetralogy of

FallotNM no mutation NT not tested ND tested no deletion Genic deletions involving FOXF1 sequences Upstream deletions involving upstream sequences ofFOXF1 not involving the gene itself

limited staining in the epithelium of tracheal mucous glandsIn no organs other than the lung was there positive SERTstaining of vascular endothelium

32 Expression of SERT in Pre- and Postnatal Human LungsLungs from the antenatal period included all stages of lungdevelopment including pseudoglandular (119899 = 3 Figure 2(a))canalicular (119899 = 13 Figure 2(b)) saccular (119899 = 5 Fig-ure 2(c)) and alveolar (119899 = 6 Figure 2(d)) At very young age(below 29WGA) there was no SERT staining Focal capillaryendothelial staining (arrows) of low intensity was first notedat 30ndash34 WGA (Figure 2(c)) By 37 WGA SERT staining

became more diffuse (arrows) and intense and at 40 WGAalmost all the capillaries were SERT positive when comparedto CD31 staining Postnatally SERT stain remained diffuselypositive (arrows) within the pulmonary microvasculature(Figures 2(e) and 2(f))

33 Expression of SERT in Lungs with Maladaptive PHTSERT expression was absent or only very minimal in thepulmonary microvasculature in every ACDMPV case (Fig-ures 3(a)ndash3(e)) The ACDMPV cases were always stained inbatches containing the positive control stained with the pan-endothelialmarker CD31 to assess pulmonary endothelial cell


(a) (b) (c)

Figure 1 SERT expression is specific and sensitive to the lung microvasculature (331015840-diaminobenzidine SERT antibody expression in brown)Autopsy tissue microarrays from newborns to 10 years of age showed crisp and diffuse SERT expression (arrow) in lung endothelium ((a)200x inset is highmagnification 600x) with positive controls ((b) 200x) highlighting numerous serotoninergic neurons (arrows) in the Raphenucleus (note negative endothelial staining of the adjacent small artery on the right arrow head) all other organs had negative endothelialcell SERT staining as seen here in the adrenal ((c) 20x no brown stain noted)

preservation (Figure 3(f))The pulmonary capillaries from allother cases with PHT of other etiologies showed diffuse andstrong SERT staining (Figure 4)

34 Length Polymorphism (LPR) and Sequence Analysis ofSERT (5-HHT) Promoter in the DNA of ACDMVP PatientsSeveral SERT promoter defects have been linked to humandisorders [27ndash36] Thus we were interested in knowing ifsuch genetic abnormalitymight play a role in the regulation ofSERT expression in ACDMVP patientsThe PCR result withACDMPV DNA samples showed an amplicon of uniformlength corresponding to the long (L normal) allele of thelength polymorphic region (LPR) of the SERT promoterThe presence of only one band indicates that these patientswere all homozygous for that allele (Figure 5) Sequenceanalysis of all the amplicons showed perfect homology withthe ref seq (NM 0010454) in the GenBank (data not shown)The ACDMPV cases included patients with and withoutidentified FOXF1 abnormalities (Table 1)

4 Discussion

We found that SERT is specifically expressed in themicrovas-culature of the developing human lung beginning at about 30WGA and that SERT expression increases near birth SERTexpression in the pulmonary microvasculature was absent inall 14 ACDMPV patients tested

We did not identify any decrease in SERT expression inthe lungs of neonates and young children with PHT due toother causes In addition SERT promoter length polymor-phism in all ACDMPV patients was without abnormality

Our study is the first direct evidence of a functionalabnormality of the maldeveloped pulmonary microvascu-lature of ACDMPV In this uniformly lethal conditiondeath is associated with treatment-resistant PHT suggest-ing functionally altered pulmonary vasculature [3] In theadult population decreased SERT activity in the pulmonaryvasculature either drug-induced genotypical or idiopathichas been implicated in the development of pulmonary

arterial hypertension [37ndash39] The pulmonary microvascu-lature of ACDMPV patients is markedly and uniformlydepleted of SERT expression suggesting that disruption ofSERTserotonin pathway is a key component of the pathome-chanism of ACDMPV Because of the rarity of this disorderwe were unable tomeasure serotonin levels in the ACDMPVpatient group Based on recent evidence that serotonin isexclusively regulated by lung SERT activity and that increasedserotonin levels lead to increased pulmonary vascular resis-tance and PHT [25] it seems likely that decreased SERTactivity leads to increased serotonin levels in ACDMPVpatients Age-matched control studies of serum serotoninmeasurements might provide direct evidence for diminishedSERT function in patients with ACDMPV

The finding that the expression of SERT peaks at or nearbirth points to a regulatory role for SERT in governing normalpulmonary vascular growth and remodeling particularly atthe critical period near birth SERT expression begins in thelate saccular stage (30WGA) while the morphologic changesof ACDMPV begin earlier likely before the canalicular stage(starting at sim20WGA) [40] Therefore the SERT expressiondefectmay be a consequence of an earlier as yet unknown pri-mary event The regular association of FOXF1 abnormalitieswith ACDMPV suggests that detailed studies targeting therelationship between FOXF1 and SERT may provide valuabledata regarding the pathomechanism of this uniformly fatalneonatal lung disorder

Due to the rarity of ACDMPV and because it has largelybeen defined histologically with most diagnoses made atautopsy examination it has been very challenging to identifycases in which tissue appropriate for complex molecularstudies can be obtained [41ndash43] There have been only rarecases in which fresh tissues have been obtained for geneticor complex molecular studies involving RNA analyses Withincreasing awareness of this disorder there have been rarecases in which antemortem diagnosis and sample acquisitionhave become possible but for the most part formalin fixedand paraffin embedded tissues have been those availableto study this disease With this in mind we designed our


16 weeks

(a)

28 weeks

(b)

30 weeks

(c)

37 weeks

(d)

1 year

(e)

10 years

(f)

Figure 2Expression of SERT in the pulmonary endotheliumduring development (331015840-diaminobenzidine SERTantibody expression in brown)(a) At 16 WGA (pseudoglandular stage) no SERT expression is detected (400x) (b) At 28 WGA (canalicular stage) no SERT expression isdetected (400x) (c) At 30 WGA (saccular stage) patchy capillary SERT staining (arrows) is detected (400x) (d) At 37 WGA (alveolar stage)diffuse and intense microvascular staining of SERT (arrows) is evident (400x) (e) At one year of age SERT staining (arrows) is diffuse andintense (400x) (f) At 10 years of age SERT staining (arrows) remains diffusely present (400x)

immunohistochemistry study very cautiously to ensure acc-uracy and reproducibility Positive and negative SERT con-trols (Figure 1) with positive pan-endothelial marker (CD31)controls (Figure 3(f)) were stained in batch with the patientsrsquoslides and blinded for the investigatorsrsquo assessment Becausewe found that among 26 patients with PH only the ACDMPV patients had negative or very minimal SERT stainingwe are confident that our findings are reliable even withoutadditional RNA studies RNA studies perhaps using rare andfortuitously obtained tissue from diagnostic lung biopsieswould be needed to confirm this data

The genetics of SERT promoter regulation is complex andseveral abnormalities have been described that can lead to a

decrease in SERT protein expression [27ndash36] We attemptedto link decreased SERT expression to SERT promoter lengthpolymorphism (SERT-LP) which has been shown to play arole in the pathogenesis of inflammatory bowel disease [29]and autism [41] A 43-bp deletion or insertion located withinthe SERT promoter results in a short or long allele and shortallele has been proposed to reduce SERT expression [31 32]Because all our tested subjects were homozygous for the Lallele we concluded that SERT-LP was not responsible forthe decreased SERT activity in ACDMPV patients Whilewe have no data to suggest that the SERT gene is abnormal inconcert with the FOXF1 in ACDMPV patients the identifi-cation of genetic abnormalities in the SERTgene (either in the


(a) (b)

(c) (d)

(e) (f)

Figure 3 ACDMPV pulmonary microvasculature lacks SERT expression (331015840-diaminobenzidine SERT) Representative sections from 5different ACD cases ((a)ndash(e)) show absence (no brown stain noted) of SERT staining (200x) (f) Positive control stained with the pan-endothelial marker CD31 (brown stain) to assess pulmonary endothelial cell preservation)


BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)

Figure 4 SERT staining is diffusely present in various pulmonary hypertension disorders other than ACDMPV (331015840-diaminobenzidine arrowsshowing SERT antibody expression in brown) BPD bronchopulmonary dysplasia (200x arrows) CAD congenital alveolar dysplasia (100x)IPHN idiopathic (primary) pulmonary hypertension (200x) and 2nd PHT secondary PH due to congenital heart disease (200x)

1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L

Figure 5 PCR study of SERT promoter length polymorphism showsthe uniform presence of the long allele in 13 ACDMVP patients (1ndash13)L is the DNA ladder and C is the amplicon from a control template

coding or in the regulatory regions) could provide a potentialexplanation for the diminished SERT protein expression

Most pediatric pathologists radiologists and neonatol-ogists consider ACDMPV to be a difficult to diagnoseneonatal lung disease Our study suggests that absence ofSERT immunostaining in the pulmonary vasculature ininfants and beyond is an excellent surrogate marker for thisdisorder currently diagnosed only by microscopic examina-tion of the lung with hematoxylin and eosin staining [3]Theuniformabsence of SERT immunostaining inACDMPVcould become a powerful diagnostic tool for ACDMPV inpediatric pathology practice Additionally imaging of alteredSERT expression in patients with CNS disease has been anactive area of medical diagnostic practice and research and

thus several radiotracers that specifically bind SERT havebeen developed Proton Emission Tomography (PET) andSingle Proton Emission Computer Tomography (SPECT)imaging studies have shown that SERT-radiotracers bind notonly to SERT in the CNS but also to lung endothelial cellsapparently with high affinity [44ndash50] SERT-radiotracershave been tested in children and found to be safe [47] Ininfants with ACDMPV without SERT expression as ourdata suggest an imaging study such as PETSPECT utilizingSERT-radiotracer could be a novel and powerful diagnos-tic method for ACDMPV Significantly SERT-radiotracersare currently available and with our data showing thatACDMPV lungs lack SERT expression this imagingmethodcould theoretically be clinically implemented immediatelyThe availability of a new diagnostic imaging study forACDMPV couldmake invasive neonatal lung biopsy unnec-essary

Because diminished SERT expression was unique toACDMPV patients with all other patients with neonatalor childhood PH diseases having normal SERT expressionour study provides evidence of a role for SERT-serotoninregulation in the pathogenesis of ACDMPV Since theprecise mechanism leading to ACDMPV is unknown ourfindings could lead to focused studies to test SERT-relevantpathways [51] in ACDMPV patients and possibly animalmodels [52 53] Serotonin has been shown to upregulateRho-kinase signaling which leads to pulmonary vascular


smooth muscle proliferation and pulmonary hypertension[54] SERT-mediated serotonin uptake is regulated by a num-ber of signaling molecules including p38-MAPK [55] andSyn-1A [56] In the future identification of SERTserotoninpathway defects could also direct the development of newtherapeutic strategies targeting their related functions innewborns with ACDMPV Controllingmodulating SERTexpression andor serotonin levels could potentially slow theprogression of pulmonary hypertension in such infants

We conclude that abnormalities in pulmonary SERTprotein expression contribute to the pathomechanism ofACDMPV We speculate that the absence of SERT activityleading to high levels of serotonin results in the maintenanceof high pulmonary vascular resistance and low pulmonaryblood flow seen during fetal life and contributes to the charac-teristic vascular abnormalities and pulmonary hypertensionof ACDMPV

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthorswould like to thankDr StevenHAbmanDepart-ment of Pediatrics University of Colorado and Dr RonJaffeDivision of Pediatric PathologyUniversity of PittsburghMedical Center for their advice and help with this manu-script

References

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[2] D L Levin M A Heymann J A Kitterman et al ldquoPersistentpulmonary hypertension of the newbornrdquo Journal of Pediatricsvol 89 pp 626ndash633 1976

[3] N B Bishop P Stankiewicz and R H Steinhorn ldquoAlveolarcapillary dysplasiardquoAmerican Journal of Respiratory andCriticalCare Medicine vol 184 no 2 pp 172ndash179 2011

[4] P Sen N Thakur D W Stockton C Langston and B A Bej-jani ldquoExpanding the phenotype of alveolar capillary dysplasia(ACD)rdquo Journal of Pediatrics vol 145 no 5 pp 646ndash651 2004

[5] P Stankiewicz P Sen S S Bhatt et al ldquoGenomic and GenicDeletions of the FOX Gene Cluster on 16q241 and InactivatingMutations of FOXF1 Cause Alveolar Capillary Dysplasia andOther Malformationsrdquo American Journal of Human Geneticsvol 84 no 6 pp 780ndash791 2009

[6] J Gien G J Seedorf V Balasubramaniam N Markham andS H Abman ldquoIntrauterine pulmonary hypertension impairsangiogenesis in vitro role of vascular endothelial growth factor-nitric oxide signalingrdquo American Journal of Respiratory andCritical Care Medicine vol 176 no 11 pp 1146ndash1153 2007

[7] A F Junod ldquoMetabolism production and release of hormonesand mediators in the lungrdquoThe American Review of RespiratoryDisease vol 112 no 1 pp 93ndash108 1975

[8] B R Pitt ldquoMetabolic functions of the lung and systemic vaso-regulationrdquo Federation Proceedings vol 43 no 11 pp 2574ndash2577 1984

[9] J M Strum and A F Junod ldquoRadioautographic demonstrationof 5-hydroxytryptamine- 3 H uptake by pulmonary endothelialcellsrdquo The Journal of Cell Biology vol 54 no 3 pp 456ndash4671972

[10] S I Said ldquoMetabolic functions of the pulmonary circulationrdquoCirculation Research vol 50 no 3 pp 325ndash333 1982

[11] B Wojciak-Stothard and S G Haworth ldquoPerinatal changes inpulmonary vascular endothelial functionrdquo Pharmacology andTherapeutics vol 109 no 1-2 pp 78ndash91 2006

[12] C N Gillis and J A Roth ldquoPulmonary disposition of circulat-ing vasoactive hormonesrdquo Biochemical Pharmacology vol 25no 23 pp 2547ndash2553 1976

[13] F Al-Ubaidi and Y S Bakhle ldquoMetabolism of vasoactivehormones in human isolated lungrdquo Clinical Science vol 58 no1 pp 45ndash51 1980

[14] CNGillis LHCronau SMandel andG LHammond ldquoInd-icator dilution measurement of 5-hydroxytryptamine clearanceby human lungrdquo Journal of Applied Physiology vol 46 pp 1178ndash1183 1979

[15] S Ramamoorthy A L Bauman K RMoore et al ldquoAntidepres-sant- and cocaine-sensitive human serotonin transportermolecular cloning expression and chromosomal localizationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 6 pp 2542ndash2546 1993

[16] A Nocito F Dahm W Jochum et al ldquoSerotonin mediates oxi-dative stress and mitochondrial toxicity in a murine model ofnonalcoholic steatohepatitisrdquo Gastroenterology vol 133 no 2pp 608ndash618 2007

[17] M Marzioni S Glaser H Francis et al ldquoAutocrineparacrineregulation of the growth of the biliary tree by the neuroen-docrine hormone serotoninrdquo Gastroenterology vol 128 no 1pp 121ndash137 2005

[18] A Nocito P Georgiev F Dahm et al ldquoPlatelets and platelet-derived serotonin promote tissue repair after normothermichepatic ischemia in micerdquo Hepatology vol 45 no 2 pp 369ndash376 2007

[19] A Nocito F DahmW Jochum et al ldquoSerotonin regulates mac-rophage-mediated angiogenesis in amousemodel of colon can-cer allograftsrdquo Cancer Research vol 68 no 13 pp 5152ndash51582008

[20] G M Mawe M D Coates and P L Moses ldquoReview articleintestinal serotonin signalling in irritable bowel syndromerdquoAlimentary Pharmacology and Therapeutics vol 23 no 8 pp1067ndash1076 2006

[21] M D Gershon and J Tack ldquoThe serotonin signaling systemfrom basic understanding to drug development for functionalGI disordersrdquoGastroenterology vol 132 no 1 pp 397ndash414 2007

[22] C D Chambers S Hernandez-Diaz L J Van Marter et alldquoSelective serotonin-reuptake inhibitors and risk of persistentpulmonary hypertension of the newbornrdquo The New EnglandJournal of Medicine vol 354 no 6 pp 579ndash587 2006

[23] C D Chambers K A Johnson L M Dick R J Felix and K LJones ldquoBirth outcomes in pregnant women taking fluoxetinerdquoNew England Journal of Medicine vol 335 no 14 pp 1010ndash10151996

[24] E Fornaro D Li J Pan and J Belik ldquoPrenatal exposure tofluoxetine induces fetal pulmonary hypertension in the ratrdquoAmerican Journal of Respiratory and Critical Care Medicine vol176 no 10 pp 1035ndash1040 2007

[25] C Delaney J Gien T R Grover G Roe and S H Abman ldquoPul-monary vascular effects of serotonin and selective serotonin


reuptake inhibitors in the late-gestation ovine fetusrdquo AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 301 no 6 pp L937ndashL944 2011

[26] M C Walsh-Sukys J E Tyson L L Wright et al ldquoPersistentpulmonary hypertension of the newborn in the era before nitricoxide practice variation and outcomesrdquo Pediatrics vol 105 no1 pp 14ndash20 2000

[27] D L Murphy A Lerner G Rudnick and K-P Lesch ldquoSero-tonin transporter gene genetic disorders and pharmacogenet-icsrdquoMolecular Interventions vol 4 no 2 pp 109ndash123 2004

[28] D L Murphy and K-P Lesch ldquoTargeting the murine serotonintransporter insights into human neurobiologyrdquoNature ReviewsNeuroscience vol 9 no 2 pp 85ndash96 2008

[29] R Colucci C Blandizzi M Bellini N Ghisu M Tonini andM Del Tacca ldquoThe genetics of the serotonin transporter andirritable bowel syndromerdquo Trends in Molecular Medicine vol14 no 7 pp 295ndash304 2008

[30] G Carrasco M A Cruz A Dominguez V Gallardo P Migueland C Gonzalez ldquoThe expression and activity of monoamineoxidase A but not of the serotonin transporter is decreased inhuman placenta from pre-eclamptic pregnanciesrdquo Life Sciencesvol 67 no 24 pp 2961ndash2969 2000

[31] W Ni and S W Watts ldquo5-Hydroxytryptamine in the cardio-vascular system focus on the serotonin transporter (SERT)rdquoClinical and Experimental Pharmacology and Physiology vol 33no 7 pp 575ndash583 2006

[32] S Eddahibi N Hanoun L Lanfumey et al ldquoAttenuated hypo-xic pulmonary hypertension in mice lacking the 5-hydroxytry-ptamine transporter generdquoThe Journal of Clinical Investigationvol 105 no 11 pp 1555ndash1562 2000

[33] S Eddahibi M Humbert E Fadel et al ldquoSerotonin transporteroverexpression is responsible for pulmonary artery smoothmuscle hyperplasia in primary pulmonary hypertensionrdquo TheJournal of Clinical Investigation vol 108 no 8 pp 1141ndash11502001

[34] S Eddahibi and S Adnot ldquoAnorexigen-induced pulmonaryhypertension and the serotonin (5-HT) hypothesis lessons forthe future in pathogenesisrdquo Respiratory Research vol 3 articleno 9 2001

[35] S Eddahibi A Chaouat N Morrell et al ldquoPolymorphism ofthe serotonin transporter gene and pulmonary hypertension inchronic obstructive pulmonary diseaserdquo Circulation vol 108no 15 pp 1839ndash1844 2003

[36] C H Huang and S L Santangelo ldquoAutism and serotonintransporter gene polymorphisms a systematic review andmeta-analysisrdquo American Journal of Medical Genetics Part BNeuropsychiatric Genetics vol 147 no 6 pp 903ndash913 2008

[37] H Zhang M Xu J Xia and R-Y Qin ldquoAssociation betweenserotonin transporter (SERT) gene polymorphism and idio-pathic pulmonary arterial hypertension a meta-analysis andreview of the literaturerdquoMetabolism Clinical and Experimentalvol 62 no 12 pp 1867ndash1875 2013

[38] M R MacLean ldquoPulmonary hypertension and the serotoninhypothesis where are we nowrdquo International Journal of ClinicalPractice Supplement vol 156 pp 27ndash31 2007

[39] M R MacLean and Y Dempsie ldquoThe serotonin hypothesis ofpulmonary hypertension revisitedrdquo Advances in ExperimentalMedicine and Biology vol 661 pp 309ndash322 2010

[40] J Kimura and G H Deutsch ldquoKey mechanisms of early lungdevelopmentrdquo Pediatric and Developmental Pathology vol 10no 5 pp 335ndash347 2007

[41] B Antao M Samuel E Kiely L Spitz and M Malone ldquoCon-genital alveolar capillary dysplasia and associated gastrointesti-nal anomaliesrdquo Fetal and Pediatric Pathology vol 25 no 3 pp137ndash145 2006

[42] B P Chelliah D Brown M Cohen A J Talleyrand and SShen-Schwarz ldquoAlveolar capillary dysplasiamdasha cause of persis-tent pulmonary hypertension unresponsive to a second courseof extracorporeal membrane oxygenationrdquo Pediatrics vol 96no 6 pp 1159ndash1161 1995

[43] R Rabah and J M Poulik ldquoCongenital alveolar capillary dys-plasia with misalignment of pulmonary veins associated withhypoplastic left heart syndromerdquo Pediatric and DevelopmentalPathology vol 4 no 2 pp 167ndash174 2001

[44] J Booij J De Jong K De Bruin R Knol M M L De Winand B L F Van Eck-Smit ldquoQuantification of striatal dopa-mine transporters with123I-FP-CIT SPECT is influenced bythe selective serotonin reuptake inhibitor paroxetine a double-blind placebo-controlled crossover study in healthy controlsubjectsrdquo Journal of Nuclear Medicine vol 48 no 3 pp 359ndash366 2007

[45] K-J Lin C-Y Liu S-P Wey et al ldquoBrain SPECT imaging andwhole-body biodistribution with [123I]ADAMmdasha serotonintransporter radiotracer in healthy human subjectsrdquo NuclearMedicine and Biology vol 33 no 2 pp 193ndash202 2006

[46] A B Newberg K Plossl P D Mozley et al ldquoBiodistributionand imaging with 123I-ADAM a serotonin transporter imagingagentrdquo Journal of Nuclear Medicine vol 45 no 5 pp 834ndash8412004

[47] I Makkonen R Riikonen H Kokki M M Airaksinen andJ T Kuikka ldquoSerotonin and dopamine transporter binding inchildren with autism determined by SPECTrdquo DevelopmentalMedicine and Child Neurology vol 50 no 8 pp 593ndash597 2008

[48] S Hesse P Brust P Mading et al ldquoImaging of the brainserotonin transporters (SERT) with 18F-labelled fluoromethyl-McN5652 and PET in humansrdquo European Journal of NuclearMedicine and Molecular Imaging vol 39 no 6 pp 1001ndash10112012

[49] P Brust J Zessin H Kuwabara et al ldquoPositron emissiontomography imaging of the serotonin transporter in the pigbrain using [11C](+)-McN5652 and S-([18F]fluoromethyl)-(+)-McN5652rdquo Synapse vol 47 no 2 pp 143ndash151 2003

[50] P Brust R Hinz H Kuwabara et al ldquoIn vivo measurementof the serotonin transporter with (S)-([18F]fluoromethyl)-(+)-McN5652rdquo Neuropsychopharmacology vol 28 no 11 pp 2010ndash2019 2003

[51] J Haase A-M Killian F Magnani and C Williams ldquoReg-ulation of the serotonin transporter by interacting proteinsrdquoBiochemical Society Transactions vol 29 no 6 pp 722ndash7282001

[52] CGalambos Y-SNgAAli et al ldquoDefective pulmonary devel-opment in the absence of heparin-binding vascular endothelialgrowth factor isoformsrdquo American Journal of Respiratory Celland Molecular Biology vol 27 no 2 pp 194ndash203 2002

[53] R N N Han and D J Stewart ldquoDefective lung vascular devel-opment in endothelial nitric oxide synthase-deficient micerdquoTrends in Cardiovascular Medicine vol 16 no 1 pp 29ndash342006

[54] M J Connolly and P I Aaronson ldquoKey role of the RhoARhokinase system in pulmonary hypertensionrdquo Pulmonary Phar-macology andTherapeutics vol 24 no 1 pp 1ndash14 2011


[55] D J Samuvel LD JayanthiN R Bhat and S Ramamoorthy ldquoArole for p38 mitogen-activated protein kinase in the regulationof the serotonin transporter evidence for distinct cellularmechanisms involved in transporter surface expressionrdquo Jour-nal of Neuroscience vol 25 no 1 pp 29ndash41 2005

[56] S Eddahibi N Morrell M-P drsquoOrtho R Naeije and S AdnotldquoPathobiology of pulmonary arterial hypertensionrdquo EuropeanRespiratory Journal vol 20 no 6 pp 1559ndash1572 2002

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


has been shown to be predominantly regulated by theserotonin transporter (SERT) in the lung endothelium [7ndash15] Abnormal SERT expression can lead to dysfunction ofa variety of organs including the lung [16ndash21] Importantlymaternal use of pharmacologic SERT inhibitors (SSRIs) inthe third trimester of pregnancy has been linked to thedevelopment of PPHN and altered pulmonary vasculaturehas been proposed as a possible mechanism [22 23] Thisassociation indicates a likely critical role for SERT in thenormal adaptation of the fetal pulmonary vasculature toextrauterine lifeThis role of SERT is further substantiated byan animal study in which SSRI exposure in utero induced PHand pulmonary arterial smooth muscle hyperplasia resem-bling that seen in PPHN [24] A recent study has shownthat serotonin plays a key role in maintaining high levelsof pulmonary vascular resistance and that SSRI exposureincreases serotonin activity causing pulmonary hypertensionin the chronically prepared newborn lamb [25]

Current data suggests that disorders associated withPPHN have an overall mortality rate of 10ndash20 althoughACDMPV is uniformly lethal despite the use of inhalednitric oxide therapy often combined with extracorporealmembrane oxygenation [11 26] As the bioavailability ofserotonin in the body is primarily regulated by pulmonarySERT [7ndash15] there is likely a fundamental role for SERT inthe regulation of pulmonary vascular tone particularly forthe major changes that occur related to birth

Thus we hypothesize that human pulmonary SERT mayplay a role in the adaptation of fetal pulmonary vasculature toextrauterine life and that abnormalities of SERT activity leadto neonatal pulmonary hypertensionHerewe present a studyof SERT expression in pre- and postnatal human lungs andin cases of newborn and childhood PHT of varied etiologyincluding ACDMPV

2 Material and Methods

21 Tissue Samples Approval for this study was obtainedthrough review by the University of Pittsburgh and BaylorCollege of Medicine Institutional Review Boards

Microarray of pulmonary tissue obtained from multipleautopsy cases was employed to establish the specificity andsensitivity of SERT immunostaining of lung endotheliumAdditionally SERT immunostaining of the vascular endothe-lial of different organs was similarly assessed

Physiologic temporal-spatial expression of SERT wasstudied using lung sections from 30 autopsy cases encompa-ssing a range of ages including 27 cases of 12 13 16 18 34 39and 40 weeks of gestational age (WGA) and 3 postnatal lungsof ages 1 5 and 10 years Cases with significant pulmonarypathology were excluded from this group

SERT expression in a variety pulmonary hypertensivedisorders was studied employing lung sections obtained atautopsy from 29 children with PHT due to ACDMPV(119899 = 14) congenital alveolar dysplasia (119899 = 1) congenitaldiaphragmatic hernia (119899 = 5) bronchopulmonary dysplasia(119899 = 3) secondary PHT in congenital heart disease (119899 = 1)and idiopathic primary PHT (119899 = 5) For these 29 casespatient histories and histologic sections of the lung were

reviewed to confirm the initial diagnosis All ACDMPVpatients were tested for mutation in FOXF1 and 4 weretested for rearrangements at 16q24 locus Among the 14ACDMPV patients three had mutations in FOXF1 Of the11 without identified mutation 4 were further identified withrearrangements at 16q24 locus with deletions identified inthree (Table 1)

22 Immunohistochemistry SERT (mousemonoclonal cloneMAB5618 Millipore Temecula CA USA) and CD31 (mousemonoclonal clone JC70A Dako Carpinteria CA USA)antibodies were used as follows after the routine stepsof deparaffinization and dehydration SERT antigen wasretrieved by steam-heat treatment in 1mM EDTA + 10mMTris buffer at pH 90 After incubation of primary SERT anti-body for 30 minutes in room temperature at a dilution of14000 diaminobenzidine (DAB) staining was carried outwith utilization of the avidin-biotin-peroxidase system Allsections were counterstained with hematoxylin Diffusebrown membranous endothelial staining was interpretedas positive while absence of brown staining was deemednegative For positive controls brain sections including theRaphe nucleus (rich in serotoninergic neurons and recom-mended by manufacturer) were used adrenal tissue wasused as negative control (Figure 1) The ACD cases werealways stained in batches containing the positive and negativecontrol as well as the pan-endothelial marker CD31 to assesspulmonary endothelial cell preservation Diffuse and intenseendothelial cell staining was required otherwise the case wasrejected

23 SERT Staining Assessment SERT immunohistochem-istry was assessed and recorded as positive and negative Inaddition the terms diffuse and patchy were added for thedistribution pattern of the stain

24 PCR and Sequencing The PCR was carried out in aTC-512 thermocycler (Techne Minneapolis MN) utilizing100 ng template DNA using the following protocol 5min at95∘C followed by 35 cycles of 1min at 95∘C 1min at 60∘C2min at 72∘C and final extension for 10min at 72∘C ThePCR products were purified using a Sephadex G-50 col-umn and sequenced on a ABI 3130XL DNA Genetic Analy-zer (Life Technologies Grand Island NY) following theman-ufacturerrsquos protocol The primers used were as follows for-ward-GCCAGCACCTAACCCCTAATGand reverse-GAA-TACTGGTAGGGTGCAAGGAG

3 Results

31 Specificity and Sensitivity of SERT to Human EndotheliumThe autopsy tissue microarray showed that in normal lungsSERT is expressed specifically in pulmonary endothelial cellswith a diffuse crisp brown staining after birth (Figure 1) Wealso found positive staining in the epithelial cells of gastricglands (44 cases) hepatocytes (34 cases) and renal tubularepithelium (34 cases) and in axons of the brain (34 cases)and Schwann cells in the nerve (34 cases) One case of 4showed minimal staining of myocardium and 14 showed








septum







configuration



















(a) (b) (c)




4 Discussion








16 weeks

(a)

28 weeks

(b)

30 weeks

(c)

37 weeks

(d)

1 year

(e)

10 years

(f)






(a) (b)

(c) (d)

(e) (f)



BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























septum







configuration



















(a) (b) (c)




4 Discussion








16 weeks

(a)

28 weeks

(b)

30 weeks

(c)

37 weeks

(d)

1 year

(e)

10 years

(f)






(a) (b)

(c) (d)

(e) (f)



BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)




4 Discussion








16 weeks

(a)

28 weeks

(b)

30 weeks

(c)

37 weeks

(d)

1 year

(e)

10 years

(f)






(a) (b)

(c) (d)

(e) (f)



BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















16 weeks

(a)

28 weeks

(b)

30 weeks

(c)

37 weeks

(d)

1 year

(e)

10 years

(f)






(a) (b)

(c) (d)

(e) (f)



BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)



BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BPD

(a)

CAD

(b)

IPHT

(c)

2nd PHT

(d)


1 2 3 4 5 6 7 8 9 10 11 12 13 C

400bp

300bp

L









Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Competing Interests


Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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The Role of Serotonin Transporter in Human Lung ...downloads.hindawi.com/journals/crj/2017/9064046.pdf · The Role of Serotonin Transporter in Human Lung ... hydronephrosis ... allele