claudin-5 as a novel estrogen target in vascular...

Claudin-5 as a Novel Estrogen Target inVascular Endothelium

Malgorzata Burek; Paula A. Arias-Loza; Norbert Roewer; Carola Y. Forster

Objective—Estrogens have multiple effects on vascular physiology and function. In the present study, we look for directestrogen target genes within junctional proteins.

Methods and Results—We use murine endothelial cell lines of brain and heart origin, which express both subtypes ofestrogen receptor, ER and ER. Treatment of these cells with 17-estradiol (E2) led to an increase in transendothelialelectric resistance and a most prominent upregulation of the tight junction protein claudin-5 expression. A significantincrease of claudin-5 promoter activity, mRNA, and protein levels was detected in cells from both vascular beds. Inprotein lysates and in immunoreactions on brain sections from ovariectomized E2-treated mice, we noticed an increasein claudin-5 protein and mRNA content. Treatment of cells with a specific ER agonist, diarylpropionitrile, revealedthe same effect as E2 stimulation. Moreover, we detected significantly lower claudin-5 mRNA and protein content inER knockout mice.

Conclusions—We describe claudin-5 as a novel estrogen target in vascular endothelium and show in vivo (brainendothelium) and in vitro (brain and heart endothelium) effects of estrogen on claudin-5 levels. The estrogen-inducedincrease in junctional protein levels may lead to an improvement in vascular structural integrity and barrier function ofvascular endothelium. (Arterioscler Thromb Vasc Biol. 2010;30:298-304.)

Key Words: claudin-5 endothelium estrogen receptor tight junction vascular biology

Experimental and population-based studies indicate thatwomen are protected from stroke and cardiovascular

disease; however, this advantage is lost with menopause.1

The protective effects of female sex hormones, particularlyestrogens, such as the natural endogenous estrogen 17-estradiol (E2), were confirmed in in vitro experiments and invivo in animal models. Studies with estrogen receptor (ER)knockout mice confirmed that the vasoprotective effects ofestrogen are ER-dependent.2,3 Cellular signaling of estrogensis mediated through 2 ER subtypes, ER and ER, which areexpressed at differing levels in many tissues.4,5 The ERactivate the transcription of genes directly by binding toestrogen response elements (ERE) in the promoter region ofgenes. ER-induced transcription may also be indirectly me-diated by interaction with Sp1 and AP-1 transcription fac-tors.6,7 Although both ER subtypes are expressed in endothe-lial and smooth muscle cells of blood vessels, most of E2effects on the vasculature, such re-endothelialization8 andendothelial nitric oxide production,9 are ER-mediated butnot ER-mediated.

Intercellular junctions between endothelial cells are impor-tant for vascular integrity and function. Claudin-5 is anintegral membrane tight junction protein that belongs to afamily of transmembrane proteins with 4 transmembrane

domains, of which, currently, 24 members have been de-scribed. Claudins play an essential role in the control ofparacellular ion flux and in the maintenance of cell polarity.10

Claudin-5 protein was initially considered to be an endothe-lium-specific member of the claudin protein family.11 How-ever, in recent years claudin-5 protein expression was dem-onstrated in numerous other cells and tissues, such asSchwann cells, olfactory epithelium, and in the lateral mem-branes of cardiomyocytes.12–14 Claudin-5 knockout mice areborn alive but die within 1 day after birth without anymorphological abnormalities. The integrity of the blood–brain barrier of these mice is severely affected, being selec-tively permeable to small molecules (800-Da).15

After transient focal ischemia in animal models, tightjunction proteins, such as claudin-5 and occludin, are de-graded, leading to the opening of the blood–brain barrier,brain edema, and neuronal cell death.16 Thus, enhancedstability of these structural elements of the vessel lining mayunderlay the vasoprotective effects observed for estrogens.17

Therefore, in the present study, we searched for novelestrogen targets among proteins of the junctional complex.Herein, we describe for the first time to our knowledge thetight junction protein claudin-5 as a novel estrogen target invascular endothelium.

Received May 12, 2009; revision accepted October 26, 2009.From Institute of Anatomy and Cell Biology (M.B., C.Y.F.), Department of Anaesthesia and Critical Care (M.B., N.R., C.Y.F.), and Department of

Medicine (P.A.A.-L.), University of Wurzburg, Wurzburg, Germany.Correspondence to Malgorzata Burek, University of Wurzburg, Department of Anaesthesia and Critical Care, Oberdurrbacherstr. 6, 97080 Wurzburg,

Germany. E-mail [email protected]© 2010 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.109.197582

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Materials and MethodsDetailed explanation of experimental procedures is provided as supple-mentary material (available online at http://atvb.ahajournals.org).

ChemicalsThe 17-estradiol (E2) and propylpyrazole (PPT) were purchasedfrom Sigma. ICI 182.780 and diarylpropionitrile (DPN) were pur-chased from Biotrend Chemicals AG.

PlasmidsCloning of the murine claudin-5 promoter (GenBank Accessionnumber EU623469) fragment 500/111 in pGL3-basic (Promega)was described previously.18

Cell CulturesMurine microvascular cerebral endothelial cell lines (cEND) andcerebellar endothelial cell lines (cerebEND), as well as the micro-vascular myocardial endothelial cell line (MyEND), were isolatedand immortalized as described previously.19–21

Animal TreatmentThe current study was approved by the institutional animal researchcommittee. Twelve-week-old female C57BL/6 mice (Harlan, Ey-strup, Germany) were sham-operated or ovariectomized and micro-osmotic pumps were implanted subcutaneously (model 1004; Alzet).Animals treated with estradiol received mini-pumps filled withcyclodextrin encapsulated estradiol (Sigma) at a dose of 2 g/kg perday. Placebo-treated animals were implanted with micro-osmoticpumps filled with vehicle only (cyclodextrin 23 mg/mL watersolution). Five animals per group were analyzed.

Transient Transfection and Reporter Gene AssayTransient transfection of endothelial cell lines with claudin-5 pro-moter construct and reporter gene assay were performed as previ-ously described.18

Western Blot AnalysisProtein extracts from cells or tissue were analyzed by Western blot.Antibodies against claudin-5 (Invitrogen), -actin (Sigma), ER,and anti-ER (Santa Cruz Biotechnology) were used. Densitometricanalysis using Scion Image Beta 4.02 (Scion Corp) was performedfor quantification.

Real-Time Polymerase Chain ReactionRNA was isolated from tissue or cells and reverse-transcribed. Real-time polymerase chain reaction was performed using the TaqMan PCRMaster Mix (Applied Biosystems). Assays for claudin-5, occludin,vascular endothelium-cadherin, and GAPDH were obtained from Ap-plied Biosystems.

Transendothelial Electric ResistanceTransendothelial electric resistance of cell monolayer grown on trans-well (0.4 m pores; Falkon) was measured using an assembly contain-ing current-passing and voltage-measuring electrodes (World PrecisionInstruments Inc). Resistances of blank filters were subtracted from thoseof filters with cells before final resistances ( cm2) were calculated.All experiments were repeated at least 3 times.

ImmunostainingFrozen brains from wild-type and ER knockout mice were a kindgift from T. Pelzer (University Wurzburg, Department of Medicine,with the permission of Dr K.S. Korach, Receptor Biology Section,NIEHS, NIH, Research Triangle Park, NC).22 Brain sections werestained with antibodies against platelet/endothelial cell adhesionmolecule-1 (1:100; BD Biosciences) and claudin-5 (1:100; Invitro-gen). FITC-conjugated/carbocyarin 3-conjugated secondary antibod-ies were used. The slides were analyzed using a Leica TCSSPconfocal microscope. Optical density measurements were performed

on 10 randomly selected vessels from 5 sections each using LeicaConfocal Software (LCS Lite; Leica Microsystems) toquantify fluorescence.

Analysis and StatisticsThroughout the experiments, averaged values were reported asmeansSD. Mann-Whitney U-test was performed and P0.05 wasconsidered statistically significant.

ResultsClaudin-5 Is a Target Gene for Estrogen Action inMicrovascular EndotheliumIn a first step, estrogen effects on endothelial barrier proper-ties were tested on microvascular endothelial cells frommurine cerebellum (cerebEND) by measuring changes intransendothelial electric resistance in response to treatmentwith 108 M E2 for 4, 8, and 24 hours. The cerebEND forma tight monolayer in vitro.20 Transendothelial electric resis-tance was increased by 150.620.8% after 4 hours, by122.2618.1% after 8 hours, and by 143.9819.9% after 24hours of treatment (Figure 1A). Next, we explored whetherand which junctional proteins could be the molecular targetsof estrogen action. We analyzed the expression of occludin,vascular endothelium-cadherin, and claudin-5 by real-timepolymerase chain reaction (Figure 1B). We demonstrated thatclaudin-5 mRNA was increased up to 2.80.15-fold after 24hours of treatment with E2. We also observed a smallincrease in occludin and vascular endothelium-cadherinmRNA expression.

The 2 ER subtypes, ER and ER, are expressed differ-entially in diverse tissues.23 To control their expression levelsin the 3 tested endothelial cell lines, we performed Westernblot analysis (Figure 1C). We were able to detect strongprotein expression of both ER subtypes in all endothelial celllines tested.

The Claudin-5 Promoter Is Regulated by E2To examine transcriptional regulation of claudin-5, wepreviously cloned the murine claudin-5 promoter.18 Analysisof the promoter sequence with a Genomatix MatInspector(http://www.genomatix.de) and TFSEARCH (http://www.cbrc.jp)revealed several potential transcription factor binding sites.Selected potential transcription factor binding sites are shown ina schematic diagram in Figure 2A. For the present investigation,we failed to identify a conventional ERE, 5-AGGTCANN-NTGACCT-3.24 However, at positions 449 to 463, themurine claudin-5 promoter contains the sequence 5-GGG-TCATGCTGCTAA-3 (consensus nucleotides are written initalics). This sequence has the highest homology of the publishedERE consensus sequence. We could also detect a putative Sp1binding site (5-GGGGCAGAGT-3) at positions 45 to 55.

The claudin-5 promoter fragment 500/111 was clonedinto the pGL3-basic vector upstream of the luciferase geneand was then transfected into cEND, cerebEND, and MyENDcell lines. After transfection, the cells were stimulated with108 M E2 for 24 hours. Afterward, lysates were prepared toevaluate induction of the luciferase enzyme activity thatserved as an indicator of promoter transactivation (Figure 2B).All cell lines showed an increased promoter activity in responseto E2. In MyEND, promoter activity increased 1.360.03 fold,

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in cerebEND promoter activity increased 1.50.11-fold, and incEND promoter activity increased 3.70.34-fold.

Claudin-5 mRNA and Protein Induction by E2To examine whether E2-dependent claudin-5 upregulationcould be detected also on the protein and mRNA level, wefirst performed a dose–response curve in cerebEND cells(Figure 3A, B). We treated the cells with 106, 108, 1010,and 1012 M of E2 for 24 hours and performed Western blotsfor claudin-5 and -actin. Claudin-5 protein level was in-creased in comparison to the untreated sample 3.30.35-fold,3.00.18-fold, 2.40.17-fold, and 2.20.16-fold in 106,108, 1010, and 1012 M samples, respectively (Figure 3A),as determined by densitometric analyses of Western blots.Expression of claudin-5 mRNA increased in comparison tothe untreated sample 1.30.16-fold, 1.50.27-fold, 1.30.11-fold,and 1.20.04 fold in 106, 108, 1010, and 1012 M samples,

respectively (Figure 3B). Next, we performed a time–responsecurve in cerebEND at constant physiological ligand concentra-tion (108 M). We treated the cerebEND for 6, 12, 24, 48, and72 hours with E2 (Figure 3C). The cells were then lysed andWestern blots for claudin-5 and -actin were performed.Claudin-5 protein levels were increased by 3.90.75-fold,4.070.86-fold, and 5.231.28-fold after 6,12, and 24 hours ofE2 treatment, respectively, as determined by densitometricanalyses of Western blots. The amount of protein in lysates after48 hours and 72 hours of stimulation was similar to that of theuntreated control. Accordingly, for further experiments we chosethe 24-hour stimulation time.

Next, we evaluated whether other endothelial cell linesfrom different vascular beds, cEND and MyEND, display asimilar claudin-5 protein expression pattern as cerebEND inresponse to E2 (Figure 3D). After 24 hours of E2 treatment,claudin-5 protein levels were increased by 2.80.16-fold incEND and by 2.60.2-fold in MyEND in comparison tountreated controls. We also tested the mRNA expression ofclaudin-5 after 24 hours of treatment with E2 in all cell lines(Figure 3E). Similar to the protein levels, we observedsignificant increases in claudin-5 mRNA expression, with a1.810.31-fold increase in cEND, a 1.460.13-fold increasein cerebEND, and a 1.610.3-fold increase in MyEND. Thecomparatively delayed induction of claudin-5 expression inresponse to E2 treatment indicated that the effect was causedby genomic action of estrogen. To examine this hypothesis,we included in the treatment solution the nonselective estro-

Figure 1. Claudin-5 is a target for estrogen action in microvas-cular endothelium. A, E2 administration increases transendothe-lial electric resistance. CerebEND were seeded on collagen IVcoated transwells, grown to confluence in differentiationmedium, followed by stimulation with 108 M E2. Transendothe-lial electric resistance was measured after 4, 8, and 24 hours.Values are expressed as perecentage over the untreated con-trol, which was set as 100%. B, Effects of E2 on mRNA expres-sion of cell contact proteins. CerebEND were stimulated for 24hours with or without 108 M E2, followed by total RNA extrac-tion and quantitative real-time polymerase chain reaction.Occludin, vascular endothelium-cadherin, and claudin-5 expres-sion were normalized to GAPDH and expressed as fold overuntreated cells, which was set as 1. C, Estrogen receptors, ERand ER, are expressed in endothelial cell lines. Cellular lysatesof cEND, cerebEND, MyEND were subjected to Western blotanalysis with anti-ER and anti-ER antibodies. *P0.05.

Figure 2. Murine claudin-5 promoter activation by E2. A, Sche-matic diagram of the putative transcription factor binding sitesin murine claudin-5 promoter fragment 500/111 (GenBankAccession number EU623469). The “1” indicates the transcrip-tional initiation site as determined by MatInspector (Genomatix).GRE, glucocorticoid response element; Sp1, stimulating protein1; ERE, estrogen response element. B, Transactivation of theclaudin-5 promoter in cEND, cerebEND, and MyEND inresponse to E2. Endothelial cell lines were transfected with the500/111 claudin-5 promoter construct and stimulated with108 M E2 24 hours later. After 24 hours of stimulation, the cellswere lysed and luciferase activity was measured. Each transfec-tion reaction in an assay was performed in triplicate and assayswere repeated 3 times. Values are normalized for protein con-tent and expression of cotransfected Renilla luciferase reportergene. Data (meansSD) are expressed as fold over untreatedcontrol, which was arbitrarily set as 1. *P0.05.

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gen receptor antagonist ICI 182.780 and examined claudin-5mRNA levels (Figure 3E). The levels of claudin-5 mRNA inthe samples treated with E2 /ICI 182.780 were comparable tothe untreated control (Figure 3E), indicating that estrogenaction was blocked when ICI 182.780 was present.

E2 Regulation of Claudin-5 Expression In Vivo inthe MouseTo investigate whether upregulation of claudin-5 expressionalso takes place in vivo, we sham-operated or ovariectomizedmice and randomized them to receive either placebo orestradiol-filled osmotic mini-pumps. After 2 weeks of treat-ment after the operation, there were significant differences incontrol and E2-treated animals in weight and size of theuterus (sham-operated: 26.91 mg; ovariectomized:15.20.3 mg; ovariectomized E2: 106.915 mg; P0.05),which is an estrogen target organ, indicating applicability ofthe experimental set-up for studying generalized estrogeneffects. We analyzed claudin-5 expression in the brains ofsham-operated, E2-treated (ovariectomized E2), and placebo-treated ovariectomized (control) mice (Figure 4). The Westernblot analysis showed in sham-operated animals a 0.840.05-fold decrease and in ovariectomized E2 brains a 1.530.11-foldincrease of claudin-5 protein levels as compared to controlovariectomized brains (Figure 4A). Similarly, the mRNA levelincreased up to 1.60.1-fold in ovariectomized E2 brains, and insham-operated animals the mRNA level was lowered to0.920.13-fold as compared to ovariectomized mRNA sample,which was set as 1 (Figure 4B).

To further examine claudin-5 regulation in vivo, we per-formed immunofluorescence stainings with anti-claudin-5 andwith anti- platelet/endothelial cell adhesion molecule-1 antibody,the latter serving as an endothelial marker protein, on brainsections from sham-operated, ovariectomized, and ovariecto-mized E2 mice (Figure 4C). Quantification of fluorescencesignals revealed that in brains from E2-treated ovariecto-

mized mice, claudin-5 staining was enhanced 2.30.55-fold(P0.05) and in sham-operated mice claudin-5 staining wasenhanced 1.50.18-fold (P0.05) in comparison to ovariec-tomized controls, which confirmed claudin-5 upregulation byE2 treatment.

Figure 3. E2 regulation of claudin-5 mRNAand protein expression in endothelial celllines. A, Dose–response of cerebENDuntreated or treated with E2. CerebENDwere left untreated or were treated with106, 108, 1010, and 1012 M of E2 for24 hours, followed by lysis and Westernblot with anti-claudin-5 and anti--actinantibody. B, Claudin-5 mRNA dose–re-sponse to E2. CerebEND were leftuntreated or were treated with 106, 108,1010, and 1012 M of E2 for 24 hours,followed by total RNA isolation, reversetranscription, and real-time polymerasechain reaction analysis. Data (meansSD)are expressed as fold over untreated con-trol, which was set as 1. C, Time–re-sponse of cerebEND untreated or treatedwith E2. CerebEND were left untreated orwere treated for 6, 12, 24, 48, and 72hours with 108 M E2, followed by lysis

and Western blot with anti-claudin-5 and anti--actin antibody. D, Expression of claudin-5 protein in endothelial cell lines. ThecEND and MyEND were treated with or without 108 M E2 for 24 hours and subjected to Western blot analysis with anti-claudin-5 andanti--actin antibody. E, Claudin-5 mRNA expression in endothelial cell lines after treatment with E2 or E2/ICI 182.780. The cEND,cerebEND, and MyEND were left untreated or were either treated with 108 M E2 or 108 M E2/1 mol/L ICI 182.780 for 24 hours, fol-lowed by real-time polymerase chain reaction analysis. Data (meansSD) are expressed as fold over untreated control, which was setas 1. *P0.05.

Figure 4. In vivo regulation of claudin-5 in brain of ovariecto-mized mice. A, Western blot analysis of lysates from brain ofsham-operated or ovariectomized mice. Protein lysates frombrain of sham-operated or ovariectomized mice untreated ortreated with 2 g/kg per day of E2 (ovariectomized E2) wereprepared and subjected to Western blot analysis using anti-claudin-5 and anti--actin antibody. B, Claudin-5 mRNA expres-sion in brain from sham-operated, ovariectomized, and ovariec-tomized E2 mice. Total RNA was isolated from brain tissue ofsham-operated, ovariectomized, and ovariectomized E2 mice,reverse-transcribed, and analyzed in real-time polymerase chainreaction. Data (meansSD) are expressed as fold overuntreated control, which was set as 1. *P0.05. C, Confocalfluorescence microscopy depicting localization of claudin-5 andplatelet/endothelial cell adhesion molecule-1 in blood vesselsfrom brain of sham-operated and ovariectomized mice with(ovariectomized E2) and without (ovariectomized) estrogen treat-ment. Scale bar, 50 m.



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E2 Effects on Claudin-5 Expression ArePotentially Mediated Via ERWestern blot analysis had revealed the expression of both ERsubtypes, ER and ER, in cEND, cerebEND, and MyEND(Figure 1C). To test which of the ER subtypes could bemediating claudin-5 regulation, we transfected the claudin-5promoter construct 500/111 into cEND and treated thecells with different selective synthetic ER agonists, such asPPT (ER agonist) and DPN (ER agonist), at a concentra-tion of 108 M (Figure 5A). After stimulation, the cells werelysed and promoter activity assay was performed. We ob-served 1.690.27-fold promoter transactivation in the pres-ence of E2 and 1.70.17-fold promoter transactivation at-tributable to DPN treatment. There was no transactivation ofthe claudin-5 promoter in cells stimulated with PPT. Next, weexamined the level of claudin-5 expression in brains fromER knockout mice (Figure 5B–D). Western blot analysis ofbrains from wild-type and ER/ mice showed a 0.770.07-fold decrease in the level of claudin-5 protein (Figure 5B).Similarly, the claudin-5 mRNA expression in ER/ mice was0.60.12-fold lower as compared to the samples from wild-typeanimals (Figure 5C). Next, we performed immunofluorescencestainings with anti-claudin-5 and with anti-platelet/endothelialcell adhesion molecule-1 antibody on brain sections from wild-type and ER/ mice (Figure 5D). Quantification of fluores-cence signals revealed that in brains from ER/ mice,

claudin-5 staining was 0.60.04-fold (P0.05) lower in com-parison to wild-type animals.

DiscussionProtective effects of estrogens on the vasculature have beenpreviously reported.17 However, the molecular mechanismsof these effects are not completely understood. Intercellularjunctions are important structural elements that contribute tothe structural and functional integrity of the vessel wall.Therefore, we hypothesized that influence on endothelialjunction properties might underlay protective effects of es-trogens on the vasculature. So, we designed experimentsaiming at providing functional proof of this hypothesis bystudying estrogen effects on endothelial permeability and,subsequently, identifying possible estrogen targets amongendothelial intercellular junction proteins. To enable compar-isons between endothelial cells derived from central nervoussystem and from non-neural vasculature, we included in theanalyses immortalized endothelial cell lines of brain and heartorigin, which have been previously proven to posses thetypical endothelial properties.19,20,25 Estrogen effects on en-dothelial barrier functions were initially confirmed by thedetection of an E2-mediated increase in transendothelialelectric resistance of endothelial monolayers. Additionally,we detected increased expression of claudin-5, occludin, andvascular endothelium-cadherin after treatment with E2. Be-cause E2-effects on claudin-5 expression were the mostprominent, we proceeded to analyze in detail the E2-mediatedregulation of claudin-5.

The analysis of the murine claudin-5 promoter18 revealedno canonical ERE in the promoter sequence.24 However, weidentified an imperfect ERE and potential Sp1 transcriptionfactor binding site. The regulation of the claudin-5 promotercould take place either directly by interaction of ER with theDNA via an imperfect ERE or indirectly by interaction withSp1 protein. In the present study, we report for the first timeto our knowledge an increased claudin-5 promoter activity byestradiol treatment of cEND, cerebEND, and MyEND celllines. The exact molecular mechanism and promoter elementsresponsible for E2 regulation remain to be determined.

Claudin-5 is expressed ubiquitously in the vascular endo-thelium.11 Overexpression of claudin-5 improved blood–brain barrier function,26 reduced paracellular cation perme-ability in Madin-Darby canine kidney II cells,27 and narrowedparacellular cleft.28 Here, we demonstrate that with treatmentwith estrogen, claudin-5 protein levels significantly increasedin cEND, cerebEND, and MyEND. The increase was time-dependent and dose-dependent, being strongest after 24 hoursof 108 M E2 stimulation. E2 also mediated the induction ofclaudin-5 expression at the mRNA level, and this effect wasblocked by co-administration of the nonselective ER antag-onist ICI 182.780.

In humans, ER and ER are expressed in vascularendothelial cells.29 We also detected expression of both ERsubtypes in the murine endothelial cell lines used for thisstudy. The presence of both ER subtypes raised the questionof which of the ER could be responsible for the observedclaudin-5 regulation. We demonstrate that in the presence ofDPN, but not after the stimulation with PPT, claudin-5

Figure 5. ER is a potential claudin-5 promoter regulator. A,Transactivation of the claudin-5 promoter in response to E2,DPN, and PPT. The cEND were transfected with the 500/111claudin-5 promoter construct. Cells were stimulated with 108

M E2, 108 M PPT, or 108 M DPN for 24 hours, lysed, andsubjected to luciferase reporter gene assay. Data (meansSD)are expressed as fold over untreated control, which was arbi-trarily set as 1. B, Western blot analysis of lysates from brain ofER/ and wild-type mice. Protein lysates from brain ofER/ and wild-type mice were prepared and subjected toWestern blot analysis using anti-claudin-5 and anti--actin anti-body. C, Claudin-5 mRNA expression in brain from ER/ andwild-type mice. Total RNA was isolated from brain tissue ofER/ and wild-type mice, reverse-transcribed, and analyzedin real-time polymerase chain reaction. Data (meansSD) areexpressed as fold over sample from wild-type animals, whichwas set as 1. *P0.05 D, Confocal fluorescence microscopydepicting localization of claudin-5 and platelet/endothelial celladhesion molecule-1 in blood vessels from brain of ER/ andwild-type mice. Scale bar, 50 m.



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promoter activation was increased, indicating that ER mightbe responsible for claudin-5 regulation. Both ER subtypeshave been described to play a role in vascular biology andfunction.30 The ER and ER knockout mice differ fromeach other in several ways. The ER/ mice present uterusatrophy and polycystic ovaries and both male and femalemice are sterile, whereas the ER/ mice exhibit differentdegrees of subfertility. However, this first ER knockoutmice study31 reported residual estradiol binding capacity inuterine tissue of 5% to 10%32; a clear-cut leakage of E2 effectwas attributed incorrectly to ER. These mice were subse-quently shown to present a transcriptional leakage attribut-able to a non-natural alternative splicing of the ER mRNA,resulting in the expression of a truncated 55-kDa isoform.33,34

Such an ER isoform, lacking a major part of the B domainand thus the AF-1 transactivation function, however, wassufficient to mediate the estradiol effect on the endothelialnitric oxide production.33 In ER/ mice, morphologicalchanges have been demonstrated in prostatic epithelium andin the brain.22,35 These abnormalities were not found in 2other ER/ models.36,37 Further abnormalities demon-strated in ER/ were development of hypertension onaging, abnormal vascular and myocardial function, and re-duced expression of cell adhesion molecules in various celltypes3,38,39; however, the present work is the first demonstra-tion, to our knowledge, of a direct endothelial action of E2through ER. Our experiments with the brains from ER/

mice22 and in an ovariectomized mouse model showed thatclaudin-5 expression is E2-regulated not only in vitro but alsoin vivo. We showed a significant increase of claudin-5mRNA and protein in brain tissue from ovariectomized micetreated with E2 and a significant decrease of claudin-5mRNA and protein in brains from ER/ mice.

In summary, this study describes a new target gene ofestrogen action in vascular endothelium. Using a murineclaudin-5 promoter construct and immortalized microvascu-lar endothelial cell lines of brain and heart origin, we showE2-mediated claudin-5 upregulation on promoter, mRNA,and protein levels. Moreover, our observations could beconfirmed for brain endothelium in experiments in ovariec-tomized E2-treated mice and in ER/ mice. Whether asimilar regulation of claudin-5 also occurs in other endotheliashould be determined in future work.

AcknowledgmentsThe authors are grateful to Katharina Mattenheimer for excellenttechnical assistance and to Esther Asan (University of Wurzburg,Institute of Anatomy and Cell Biology), Kinga Blecharz (Universityof Wurzburg, Department of Anesthesia and Critical Care), and TheoPelzer (University of Wurzburg, Department of Medicine) forhelpful discussions and critical reading of the manuscript.

Sources of FundingThis research was supported by SFB 688 grant from the DeutscheForschungsgemeinschaft to C.Y.F.

DisclosureNone.

References1. Barton M, Meyer MR, Haas E. Hormone replacement therapy and ath-

erosclerosis in postmenopausal women: does aging limit therapeuticbenefits? Arterioscler Thromb Vasc Biol. 2007;27:1669–1672.

2. Pare G, Krust A, Karas RH, Dupont S, Aronovitz M, Chambon P,Mendelsohn ME. Estrogen receptor-alpha mediates the protective effectsof estrogen against vascular injury. Circ Res. 2002;90:1087–1092.

3. Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW,Thoren P, Smithies O, Gustafsson JA, Mendelsohn ME. Abnormalvascular function and hypertension in mice deficient in estrogen receptorbeta. Science. 2002;295:505–508.

4. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A,Jensen E, Scrace G, Waterfield M, Chambon P. Cloning of the humanestrogen receptor cDNA. Proc Natl Acad Sci U S A. 1985;82:7889–7893.

5. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA.Cloning of a novel receptor expressed in rat prostate and ovary. Proc NatlAcad Sci U S A. 1996;93:5925–5930.

6. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM,Webb P. Estrogen receptor pathways to AP-1. J Steroid Biochem MolBiol. 2000;74:311–317.

7. Safe S, Kim K. Nuclear receptor-mediated transactivation through inter-action with Sp proteins. Prog Nucleic Acid Res Mol Biol. 2004;77:1–36.

8. Brouchet L, Krust A, Dupont S, Chambon P, Bayard F, Arnal JF.Estradiol accelerates reendothelialization in mouse carotid artery throughestrogen receptor-alpha but not estrogen receptor-beta. Circulation. 2001;103:423–428.

9. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J,Chambon P, Bayard F, Arnal JF. Estradiol alters nitric oxide productionin the mouse aorta through the alpha-, but not beta-, estrogen receptor.Circ Res. 2002;90:413–419.

10. Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE.Structure and function of claudins. Biochim Biophys Acta. 2008;1778:631–645.

11. Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J CellBiol. 1999;147:185–194.

12. Poliak S, Matlis S, Ullmer C, Scherer SS, Peles E. Distinct claudins andassociated PDZ proteins form different autotypic tight junctions in myeli-nating Schwann cells. J Cell Biol. 2002;159:361–372.

13. Sanford JL, Edwards JD, Mays TA, Gong B, Merriam AP, Rafael-Fortney JA. Claudin-5 localizes to the lateral membranes of cardiomyo-cytes and is altered in utrophin/dystrophin-deficient cardiomyopathicmice. J Mol Cell Cardiol. 2005;38:323–332.

14. Steinke A, Meier-Stiegen S, Drenckhahn D, Asan E. Molecular compo-sition of tight and adherens junctions in the rat olfactory epithelium andfila. Histochem Cell Biol. 2008;130:339–361.

15. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M,Tsukita S. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol. 2003;161:653–660.

16. Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA. Matrixmetalloproteinase-mediated disruption of tight junction proteins incerebral vessels is reversed by synthetic matrix metalloproteinase inhib-itor in focal ischemia in rat. J Cereb Blood Flow Metab. 2007;27:697–709.

17. Miller VM, Duckles SP. Vascular actions of estrogens: functional impli-cations. Pharmacol Rev. 2008;60:210–241.

18. Burek M, Forster CY. Cloning and characterization of the murineclaudin-5 promoter. Mol Cell Endocrinol. 2009;298:19–24.

19. Forster C, Silwedel C, Golenhofen N, Burek M, Kietz S, Mankertz J,Drenckhahn D. Occludin as direct target for glucocorticoid-inducedimprovement of blood-brain barrier properties in a murine in vitro system.J Physiol. 2005;565:475–486.

20. Silwedel C, Forster C. Differential susceptibility of cerebral and cere-bellar murine brain microvascular endothelial cells to loss of barrierproperties in response to inflammatory stimuli. J Neuroimmunol. 2006;179:37–45.

21. Golenhofen N, Ness W, Wawrousek EF, Drenckhahn D. Expression andinduction of the stress protein alpha-B-crystallin in vascular endothelialcells. Histochem Cell Biol. 2002;117:203–209.

22. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M,Korach KS, Gustafsson JA, Smithies O. Generation and reproductivephenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci U S A.1998;95:15677–15682.

23. Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J,Tujague M, Strom A, Treuter E, Warner M, Gustafsson JA. Estrogen



Dow

nloaded from


receptors: how do they signal and what are their targets. Physiol Rev.2007;87:905–931.

24. Klock G, Strahle U, Schutz G. Oestrogen and glucocorticoid responsiveelements are closely related but distinct. Nature. 1987;329:734–736.

25. Forster C, Waschke J, Burek M, Leers J, Drenckhahn D. Glucocorticoideffects on mouse microvascular endothelial barrier permeability are brainspecific. J Physiol. 2006;573:413–425.

26. Honda M, Nakagawa S, Hayashi K, Kitagawa N, Tsutsumi K, Nagata I,Niwa M. Adrenomedullin improves the blood-brain barrier functionthrough the expression of claudin-5. Cell Mol Neurobiol. 2006;26:109–118.

27. Wen H, Watry DD, Marcondes MC, Fox HS. Selective decrease inparacellular conductance of tight junctions: role of the first extracellulardomain of claudin-5. Mol Cell Biol. 2004;24:8408–8417.

28. Piontek J, Winkler L, Wolburg H, Muller SL, Zuleger N, Piehl C,Wiesner B, Krause G, Blasig IE. Formation of tight junction: deter-minants of homophilic interaction between classic claudins. FASEB J.2008;22:146–158.

29. Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovas-cular gender differences. Science. 2005;308:1583–1587.

30. Deroo BJ, Korach KS. Estrogen receptors and human disease. J ClinInvest. 2006;116:561–570.

31. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O.Alteration of reproductive function but not prenatal sexual developmentafter insertional disruption of the mouse estrogen receptor gene. Proc NatlAcad Sci U S A. 1993;90:11162–11166.

32. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB,Smithies O, Korach KS. Analysis of transcription and estrogen insensi-

tivity in the female mouse after targeted disruption of the estrogenreceptor gene. Mol Endocrinol. 1995;9:1441–1454.

33. Pendaries C, Darblade B, Rochaix P, Krust A, Chambon P, Korach KS,Bayard F, Arnal JF. The AF-1 activation-function of ERalpha may bedispensable to mediate the effect of estradiol on endothelial NO produc-tion in mice. Proc Natl Acad Sci U S A. 2002;99:2205–2210.

34. Kos M, Denger S, Reid G, Korach KS, Gannon F. Down but not out? Anovel protein isoform of the estrogen receptor alpha is expressed in theestrogen receptor alpha knockout mouse. J Mol Endocrinol. 2002;29:281–286.

35. Wang L, Andersson S, Warner M, Gustafsson JA. Morphological abnor-malities in the brains of estrogen receptor beta knockout mice. Proc NatlAcad Sci U S A. 2001;98:2792–2796.

36. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M.Effect of single and compound knockouts of estrogen receptors alpha(ERalpha) and beta (ERbeta) on mouse reproductive phenotypes.Development. 2000;127:4277–4291.

37. Antal MC, Krust A, Chambon P, Mark M. Sterility and absence ofhistopathological defects in nonreproductive organs of a mouseERbeta-null mutant. Proc Natl Acad Sci U S A. 2008;105:2433–2438.

38. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, Warner M,Gustafsson JA. Involvement of estrogen receptor beta in terminal differ-entiation of mammary gland epithelium. Proc Natl Acad Sci U S A.2002;99:15578–15583.

39. Forster C, Kietz S, Hultenby K, Warner M, Gustafsson JA. Character-ization of the ERbeta/mouse heart. Proc Natl Acad Sci U S A. 2004;101:14234–14239.



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Malgorzata Burek, Paula A. Arias-Loza, Norbert Roewer and Carola Y. FörsterClaudin-5 as a Novel Estrogen Target in Vascular Endothelium

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2009 American Heart Association, Inc. All rights reserved.

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Supplement

Materials and Methods

Chemicals

17β-estradiol (E2) and propylpyrazole (PPT) were purchased from Sigma (Sigma,

Taufkirchen, Germany), ICI 182.780 (fulvestrant) and diarylpropionitrile (DPN) were

purchased from Biotrend Chemicals AG (Zürich, Switzerland). Stock solution were prepared

in dimethylsulphoxide (DMSO) and kept at -20 °C. Desired dilution was made before the

experiment in cell culture medium. The final concentration of DMSO in the treatment

medium was lower than 0.01% (v/v). To obtain the best receptor specificity, PPT and DPN

were used at a concentration of 10-8

M 1.

Plasmids

Cloning of the murine claudin-5 promoter (GenBank Accession No.: EU623469) fragment -

500/+111 in pGL3-basic (Promega, Mannheim, Germany) was described previously 2.

Cell Cultures

Murine microvascular cerebral (cEND) and cerebellar (cerebEND) endothelial cell lines as

well as the microvascular myocardial endothelial cell line MyEND were isolated and

immortalized as described previously 3-5

. Endothelial cell lines were cultivated in Dulbecco's

modified Eagle's medium (DMEM) with 10% Fetal Calf Serum (FCS). All cultures were

supplemented with 100 i.u. ml–1

penicillin and 100 mg ml

–1 streptomycin (1% PEST) (Sigma).

Cells were maintained in an atmosphere of 5.0% CO2–95% O2 and at 37°C. Endothelial cell

lines were grown on plates coated with collagen IV (Fluka, Taufkirchen, Germany).

Animal Treatment

The current study was approved by the institutional animal research committee

(Tierschutzbeauftragter) and the animals were kept under conventional conditions in the

animal facility. Twelve weeks old female C57BL/6 mice (Harlan, Germany), under an

adequate depth anesthesia (tribromoethanol / amylene hydrate - “Avertin”; 2,5% WT/vol, 6

µl/g BW ip.), were sham operated or ovariectomized and micro-osmotic pumps were

implanted subcutaneously (Alzet, model 1004). Animals treated with estradiol received

minipumps filled with cyclodextrin encapsulated estradiol (Sigma) at a dosis of 2 µg/kg/day.

This dosis was chosen to ensure normal physiological response to estradiol 6. Placebo treated

animals were implanted micro-osmotic pumps filled with vehicle only (cyclodextrin 23mg/ml

water solution). Five animals per group were analyzed. The animals used for the study were

also employed to study the cardiovascular metabolic effects of estradiol (Arias-Loza P.A.,

unpublished data) therefore after 2 weeks of treatment the animals were fasted overnight, and

were killed by decapitation under glucose stimulus (1,5 mg/g body weight).

Transient Transfection and Reporter Gene Assay

Endothelial cell lines were seeded on a collagen IV coated 6-well cell culture plate at a

density of 3 x 105 cells per well. The cells were grown in DMEM medium with 10% FCS to

confluence prior to transfection. Transient transfection experiments using the Effectene

reagent (Qiagen, Hilden, Germany) were performed as suggested by the manufacturer in

DMEM medium supplemented with 1% FCS, using 2 µg of claudin-5 promoter construct

DNA and 1 µg of the internal control reporter pTRL-TK 7. After 24 hours the cells were

washed and 2 ml fresh medium with or without 10-8

M E2 was added. After 24 hours

stimulation the cells were lysed and the cellular extracts were analyzed for luciferase activity.

Measurement of both firefly and Renilla luciferase activity was performed with the Dual-

Luciferase assay kit (Promega) according to the manufacturers’ instructions. Protein

concentration was estimated by standard Bradford protein assay. Cell lysate (20µl) was used

for assaying the enzymatic activities, using a LB9507 luminometer with dual injector

(Berthold, Bad Wildbad, Germany). Each lysate was measured twice. Promoter activities

were expressed as relative light units (RLU) normalized for the protein content and the

activity of Renilla luciferase in each extract. The data were calculated as the mean of three

identical set-ups and expressed as fold over the untreated control, which was arbitrarily set

as 1.

Western Blot Analysis

Cells were plated on collagen IV coated dishes at a density of 1.1 x 105

cells per 3.5 cm2 and

grown in DMEM medium with 10% FCS to confluence. At confluence, cells were maintained

at 1% FCS for 48 hours followed by treatment with E2 as indicated in the figure legends. For

Western blot analyses, cells or tissue samples were extracted in Laemmli sample buffer.

Protein contents were quantified by protein estimation directly from SDS-PAGE loading

buffer using 0.1% (w/v) Amidoschwarz (AppliChem, Darmstadt, Germany) in 25% (v/v)

methanol–5% (v/v) acetic

acid, and equal protein amounts were loaded on SDS-

polyacrylamide gels for Western blot analysis. For immunoblotting, proteins were transferred

in Kyhse-Andersen

transfer buffer 8 to Hybond nitrocellulose

membranes (Amersham,

Braunschweig, Germany) which were blocked with 10% (w/v) low fat milk in PBS and

incubated overnight at 4°C with the respective primary antibody (in PBS plus 10% low fat

milk). Mouse monoclonal anti-mouse claudin-5 antibody was purchased from

Invitrogen (CA,

USA), anti-β-actin antibody was purchased from Sigma, anti-ERα and anti-ERβ antibodies

were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). As secondary

antibody,

horseradish peroxidase-labelled goat anti-mouse IgG or goat anti-rabbit IgG

(Jackson Immuno Res. Laboratory, West Grove, PA, USA) was used diluted

1: 3000 with

PBS/ 10% low fat milk. Bound immunoglobulins were visualized by the

enhanced

chemiluminescence technique (ECL, Amersham). Densitometric analysis using Scion Image

Beta 4.02 (Scion Corp., MD, USA) was performed for quantification.

Real-Time PCR

For real-time PCR, RNA was isolated from tissue or cells treated or untreated with 10-6

, 10-8

,

10-10

and 10-12

M E2 or 10-8

M E2/ 1 µM ICI 182.780 using RNeasyMini kit (Qiagen). One

microgram of RNA was used for the cDNA synthesis using iSCRIPT cDNA synthesis kit

(Bio-Rad, München, Germany). Real-time PCR was performed using the TaqMan PCR

Master Mix (Applied Biosystems, Foster City, CA). Assays for claudin-5 (Mm00727012_s1),

occludin (Mm00500912_m1), VE-cadherin (Mm00486938_ m1) and

GAPDH

(Mm99999915_g1) were obtained from Applied Biosystems.

All PCR reactions were

performed in triplicate for each target. Data were acquired with the ABI PRISM 7300 system

(Applied Biosystems). The ABI PRISM 7300 SDS software (relative quantification study)

was used to determine the threshold cycle (Ct) for each reaction and gene expression was

normalized to the expression of the endogenous housekeeping gene GAPDH based on the 2-

∆∆Ct method.

Transendothelial Electrical Resistance

Cells were plated in DMEM medium with 10% FCS on top of collagen IV-coated transwell

chambers for six-well plates (0.4 µm pores; Falcon, Heidelberg,

Germany) at densities of 1 x

104 cells per well. When they had

reached confluence, the different experimental sets

of cells

were transferred to 1% FCS or were treated with 10-8

M E2 at 1% FCS, respectively. The

control set was maintained in basal medium (DMEM, 1% FCS) for an additional 8 hours, at

which point

the permeability measurement was performed. Transendothelial

electrical

resistance (TER) was measured using an assembly containing current-passing and voltage-

measuring electrodes (World Precision Instruments Inc., New Haven, CT, USA). Resistances

of blank filters were subtracted from those of filters with cells before

final resistances (in

Ω x cm2) were calculated.

All experiments were repeated at least three times.

Immunostaining

Frozen brains from wild type and ERβ knockout mice were a kind gift from T. Pelzer

(University Würzburg, Department of Medicine, with the permission of Dr. K.S. Korach,

Receptor Biology Section, NIEHS, NIH, Research Triangle Park, North Carolina)9. Brains

from sham operated, OVX and OVX E2-treated mice were frozen in liquid nitrogen-cooled

isopentane. Ten-µm-thick sections were cut in a cryostat and

mounted on glass slides

(Superfrost Plus, Menzel). Frozen sections were air dried for 30 min. After washing with ice-

cold methanol

and acetone, each for 3 min, sections were fixed for 10 min

at room

temperature in 4% formaldehyde prepared freshly from paraformaldehyde. All sections were

treated with 0.1% Triton X-100 in PBS for 5 min, incubated with 10% normal goat serum

in

PBS for 30 min at 25°C to reduce unspecific binding of secondary antibodies, and exposed in

sequence to primary antibodies: rat monoclonal antibody to mouse PECAM-1

(platelet/endothelial cell adhesion molecule 1 10

) (BD Biosciences, Heidelberg, Germany;

1:100), rabbit polyclonal anti-claudin-5 (Invitrogen, Camarillo, CA; 1:100) and FITC-

/Carbocyarin 3 (Cy3)-conjugated goat anti-rat and goat anti-rabbit secondary

antibodies,

respectively. Incubation without primary antibody served as a background control. After

incubation, coverslips were mounted onto slides using 60% (w/v) glycerol and 1.5% (w/v)

n-

propylgallate (Fluka) as antifading component. The slides were analysed using a Leica TCSSP

confocal microscope. Optical density measurements were carried out on 10 randomly selected

vessels from five sections each using Leica Confocal Software (LCS Lite) (Leica

Microsystems, Heidelberg, Germany) to quantify fluorescence.

Analysis and Statistics

Throughout the experiments, averaged values were reported as means ± standard deviation

(s.d.). Mann–Whitney U-test was performed and P<0.05 (*) was considered statistically

significant.

Supplemental References:

1. Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA,

Katzenellenbogen BS. Activities of estrogen receptor alpha- and beta-selective ligands

at diverse estrogen responsive gene sites mediating transactivation or transrepression.

Mol Cell Endocrinol. 2003;206:13-22.

2. Burek M, Forster CY. Cloning and characterization of the murine claudin-5 promoter.

Mol Cell Endocrinol. 2009;298:19-24.

3. Forster C, Silwedel C, Golenhofen N, Burek M, Kietz S, Mankertz J, Drenckhahn D.

Occludin as direct target for glucocorticoid-induced improvement of blood-brain

barrier properties in a murine in vitro system. J Physiol. 2005;565:475-486.

4. Silwedel C, Forster C. Differential susceptibility of cerebral and cerebellar murine

brain microvascular endothelial cells to loss of barrier properties in response to

inflammatory stimuli. J Neuroimmunol. 2006;179:37-45.

5. Golenhofen N, Ness W, Wawrousek EF, Drenckhahn D. Expression and induction of

the stress protein alpha-B-crystallin in vascular endothelial cells. Histochem Cell Biol.

2002;117:203-209.

6. Andersson N, Islander U, Egecioglu E, Lof E, Swanson C, Moverare-Skrtic S, Sjogren

K, Lindberg MK, Carlsten H, Ohlsson C. Investigation of central versus peripheral

effects of estradiol in ovariectomized mice. J Endocrinol. 2005;187:303-309.

7. Lorenz WW, McCann RO, Longiaru M, Cormier MJ. Isolation and expression of a

cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A.

1991;88:4438-4442.

8. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer

tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem

Biophys Methods. 1984;10:203-209.

9. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS,

Gustafsson JA, Smithies O. Generation and reproductive phenotypes of mice lacking

estrogen receptor beta. Proc Natl Acad Sci U S A. 1998;95:15677-15682.

10. Newman PJ, Berndt MC, Gorski J, White GC, 2nd, Lyman S, Paddock C, Muller WA.

PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin

gene superfamily. Science. 1990;247:1219-1222.

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