Endothelial disruptive proinflammatory effects of nicotine and e-cigarettevapor exposures

Kelly S. Schweitzer,1 Steven X. Chen,1 Sarah Law,1 Mary Van Demark,1 Christophe Poirier,1

Matthew J. Justice,1 Walter C. Hubbard,2 Elena S. Kim,1 Xianyin Lai,3 Mu Wang,3 William D. Kranz,4

Clinton J. Carroll,4 Bruce D. Ray,5 Robert Bittman,6† John Goodpaster,4 and Irina Petrache1,3,7

1Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of ClinicalPharmacology, The Johns Hopkins University, Baltimore, Maryland; 3Department of Biochemistry and Molecular Biology,Indiana University School of Medicine, Indianapolis, Indiana; 4Department of Chemistry and Chemical Biology; IndianaUniversity-Purdue University, Indianapolis, Indiana; 5Department of Physics, Indiana University-Purdue University,Indianapolis, Indiana; 6Queens College, City University of New York, Flushing, New York; and 7Richard L. RoudebushVeterans Affairs Medical Center, Indianapolis, Indiana

Submitted 2 January 2015; accepted in final form 4 May 2015

Schweitzer KS, Chen SX, Law S, Van Demark M, Poirier C,Justice MJ, Hubbard WC, Kim ES, Lai X, Wang M, KranzWD, Carroll CJ, Ray BD, Bittman R, Goodpaster J, PetracheI. Endothelial disruptive proinflammatory effects of nicotine ande-cigarette vapor exposures. Am J Physiol Lung Cell Mol Physiol309: L175–L187, 2015. First published May 15, 2015;doi:10.1152/ajplung.00411.2014.—The increased use of inhaled nic-otine via e-cigarettes has unknown risks to lung health. Havingpreviously shown that cigarette smoke (CS) extract disrupts the lungmicrovasculature barrier function by endothelial cell activation andcytoskeletal rearrangement, we investigated the contribution of nico-tine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primarylung microvascular endothelial cells were exposed to nicotine, e-Cigsolution, or condensed e-Cig vapor (1–20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containingextract, nicotine free-CS extract (10–20%) caused significantly lessendothelial permeability as measured with electric cell-substrate im-pedance sensing. Nicotine exposures triggered dose-dependent loss ofendothelial barrier in cultured cell monolayers and rapidly increasedlung inflammation and oxidative stress in mice. The endothelialbarrier disruptive effects were associated with increased intracellularceramides, p38 MAPK activation, and myosin light chain (MLC)phosphorylation, and was critically mediated by Rho-activated kinasevia inhibition of MLC-phosphatase unit MYPT1. Although nicotine atsufficient concentrations to cause endothelial barrier loss did nottrigger cell necrosis, it markedly inhibited cell proliferation. Augmen-tation of sphingosine-1-phosphate (S1P) signaling via S1P1 improvedboth endothelial cell proliferation and barrier function during nicotineexposures. Nicotine-independent effects of e-Cig solutions werenoted, which may be attributable to acrolein, detected along withpropylene glycol, glycerol, and nicotine by NMR, mass spectrometry,and gas chromatography, in both e-Cig solutions and vapor. These resultssuggest that soluble components of e-Cig, including nicotine, causedose-dependent loss of lung endothelial barrier function, which is asso-ciated with oxidative stress and brisk inflammation.

tobacco; permeability; cell proliferation; sphingosine-1-phosphate;inflammation

CIGARETTE SMOKING IS THE primary causative factor for chronicobstructive pulmonary disease (COPD), the third leading cause

of death worldwide. We have shown that in addition to injuringthe lung epithelium, soluble components of cigarette smoke(CS) can be directly injurious to lung endothelial cells bydisrupting the lung endothelial barrier function (22). It is notknown whether nicotine, the main component of CS, could beresponsible for this effect. Furthermore, it is not knownwhether inhalation of the vapor released by electronic ciga-rettes (e-Cig) has similar effects as CS on lung endothelium.Given the increasing use of e-Cig, which results in inhalationof vapor produced by heating nicotine-containing liquid, it isimportant to define their biological effects on the lung endo-thelial barrier function.

Previous studies of nicotine in endothelial cells have gener-ated diverse results, showing inhibition of human umbilicalvein endothelial cell (HUVEC) proliferation (1), but enhancedproliferation of endothelial progenitor cells (35). Furthermore,high doses of nicotine were shown to inhibit cytokines requiredfor neovascularization during bone healing (14, 26) and to affectthe vasoreactivity of various vascular networks (8, 9, 16, 17).However, the effects of nicotine on lung barrier function are notknown and, given that the loss of endothelial integrity contributesto lung inflammation and injury, they are important to define.

The mechanisms by which nicotine triggers systemic endo-thelial cell responses have been shown to involve increases inNO signaling molecules (18) and reactive oxygen species(ROS) (16), as well as generation of proapoptotic metabolites(28), events that would be expected to also impair lung endo-thelial barrier function. However, nicotine has been shown tohave discrepant effects, either decreasing or increasing theexpression of intracellular adhesion molecules in HUVEC (24,25) via signaling pathways involving PKC, p38, and ERK1/2MAPK (29, 33). Little is known about the direct effect ofnicotine on the lung cell endothelial barrier and the mecha-nisms by which nicotine would exert such effects.

Endothelial cellular barrier is tightly regulated by the acto-myosin cytoskeleton, whose contraction is governed by myosinlight chain kinase (MLCK) and Rho kinase enzymatic activi-ties. We have previously shown that CS extracts caused endo-thelial barrier dysfunction via oxidative stress, p38 MAPKactivation, and ceramide release, which are generated upstreamof Rho kinase activation and cellular contraction (22). Inaddition, CS-induced barrier dysfunction may be attributed toloss of intercellular tethering forces (22), which are typically

Address for reprint requests and other correspondence: I. Petrache, IndianaUniv., Division of Pulmonary, Allergy, Critical Care and Occupational Med-icine, Walther Hall-R3 C400, 980 W. Walnut St., Indianapolis, IN 46202-5120(e-mail: ipetrach@iu.edu).

† Deceased.

Am J Physiol Lung Cell Mol Physiol 309: L175–L187, 2015.First published May 15, 2015; doi:10.1152/ajplung.00411.2014.

http://www.ajplung.org L175

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

reinforced by sphingosine-1-phosphate (S1P) signaling (10). Weinvestigated whether nicotine, one of the hundreds of moleculespresent in CS extracts, is sufficient to alter lung endothelial barrierfunction by affecting cytoskeletal regulation.

Using measurements of endothelial monolayer barrier func-tion in cultured primary cells via transcellular electrical cellularimpedance sensing (ECIS) (27) and in vivo assessment ofoxidative stress and extravasated inflammatory cells in thebronchoalveolar lavage, we show that nicotine and e-Cigsolutions or vapor condensates cause dose-dependent cell in-jury manifested by decreased barrier function and decreasedcell proliferation, via specific signaling pathways.

MATERIALS AND METHODS

Reagents and Pharmacological Inhibitors

Unless otherwise stated, all chemical reagents were purchased fromSigma-Aldrich (St. Louis, MO). Free radical scavenger and precursorto glutathione N-acetylcysteine (NAC; 0.5 M) and p38 MAPK inhib-itor SB203580 (5 �M) were from Santa Cruz Biotechnology (Dallas,TX). ERK1/2 inhibitor PD98059 (50 �M) and JNK inhibitorSP600125 (50 �M) were from Calbiochem (San Diego, CA). The S1Panalogs (S)-FTY720 phosphonate (1S), (S)-FTY720 enephosphonate(2S), (R)-FTY720 phosphonate (1R), and (R)-FTY720 enephospho-nate (2R) (all solubilized in MeOH) were synthesized as previouslydescribed (13). Ceramide synthase inhibitor, fumonisin (FB1, 10�M), was from Cayman Chemical (Ann Arbor, MI); and the serinepalmitoyl transferase inhibitor, myriocin (Myr; 50 nM), was fromBiomol International (Plymouth Meeting, PA). The neutral sphingo-myelinase (nSMase) inhibitor GW4869 (GW; 20 �M) and acidsphingomyelinase (aSMase) inhibitor imipramine (Imi; 50 �M) werefrom Calbiochem. Nicotine solutions [Vanilla Dream (e-Cig 1), Ken-tucky Prime (e-Cig 2), and nicotine-free Kentucky Prime] wereobtained from Sigma. E-Cig solutions for vaporization were pur-chased from World of Vapor (Indianapolis, IN). E-Cig used togenerate vapor (iClear 16) was from Innokin (Shenzhen, China).

Cells

Primary rat lung endothelial cells (RLEC; from Dr. Troy Stevens,University of Southern Alabama) and human bronchial epithelial cellline Beas-2B (American Type Culture Collection, Manassas, VA)were cultured in DMEM with high glucose (DMEM-HG) fromInvitrogen (Hercules, CA) containing 10% FBS and 1% penicillin/streptomycin. Primary mouse lung endothelial cells (MLEC; from Dr.Patty Lee, Yale University) were grown as RLEC but with 20% FBS.Primary human microvascular cells-lung derived (HMVEC-LBl;Lonza) were grown in Endothelium Cell Growth Basal Medium(EBM-2) with supplements (EGM-2MV SingleQuot Kit supple-mented with growth factors). All cultures were maintained at 37°Cwith 5% CO2. Before treatments (2 h), the culture medium wasreplaced with basal media containing 2% FBS.

Transcellular Electrical Resistance Measurements

Electrical resistance across cell monolayers was measured usingECIS (Applied Biophysics, Troy, NY), as previously described (20).Cells were cultured on gold microelectrodes and the total resistancewas measured in real time across monolayers and recorded continu-ously every 2–4 min over 24 h. Shown in the figures are select (e.g.,5-h and 20-h) time points as representative of changes induced byexposures studied. Experiments were conducted after cells wereconfluent [i.e., transendothelial electrical resistance (TER) achieved asteady state]. TER values (in ohms) for each time point were normal-ized to the initial resistance value (at the beginning of the recording)and plotted as normalized TER.

Animal Experiments

All experiments were performed according to Indiana UniversitySchool of Medicine Institutional Animal Care and Use Committeeguidelines and approved protocols. C57Bl/6 mice (4-mo-old females)were nebulized (nebulizer unit 2.5–4.0 VMD, Aerogen; Galway,Ireland) using either one dose of nicotine (2 �g) and harvestedimmediately, or two doses of e-Cig solution (1 �g each) and harvestedafter either 30 min or 24 h. Controls were nebulized with saline andharvested at similar time points.

Oxidative Stress Measurements

Mouse plasma and bronchoalveolar lavage fluid. As a marker ofoxidative damage, 8-hydroxydeoxyguanosine (8-OHdG) was quanti-fied in plasma (1:10 dilution) or bronchoalveolar lavage fluid (BALF)using an OxiSelect Oxidative DNA Damage ELISA kit (Cell Biolabs;San Diego, CA). Total nitrotyrosine was determined in plasma (1:10)using competitive ELISA (Hycult; Uden, Netherlands) following themanufacturer’s specifications.

Rat lung endothelial cells. Cells were grown on gelatin-coatedcoverslips pretreated with nicotine (10 mM, 30 min) with or withoutNAC (0.5 M). ROS were detected using an Image-iT LIVE GreenReactive Oxygen Species Detection Kit (Invitrogen) following themanufacturer’s instructions. Nuclei were stained using DAPI (Invit-rogen). Photographs were captured using a Nikon 80i fluorescentmicroscope.

CS Extract and e-Cig Vapor Condensate

Aqueous CS extract was obtained from filtered research-gradecigarettes (2R4F) or nicotine-free cigarettes (1R5F) from the Ken-tucky Tobacco Research and Development Center (University ofKentucky, Lexington, KY) as previously described (22). A stock CSextract (100%) was prepared by bubbling smoke from two cigarettesinto 20 ml of PBS at a rate of 1 cigarette/min to 0.5 cm above the filter,followed by pH adjustment to 7.4 and 0.2-�m filtration. A similarprocedure was followed for air control (AC) extract preparation bybubbling ambient air. Treatments were performed with CS or ACextract concentrations ranging from 1% to 20% (vol:vol). Condensede-Cig vapor was collected in a 25-ml side-armed Erlenmeyer flaskplaced under vacuum while connected to the e-Cig via Tygon tubing.The temperature of the heating coil inside the e-Cig was not measured.A vacuum trap was created to collect the postvaporized condensate ofe-Cig solutions using a gel-loading tip as a constriction point. A totalof 125 �l of condensate was collected from vaporization of 600 �l ofe-Cig solution over �30 min and applied to cell cultures in indicatedconcentrations (vol:vol).

Ceramide Determination

Following treatments, the culture medium was washed with PBSand cells were collected in methanol, followed by lipid extractionutilizing a modified Bligh and Dyer method, and sphingolipid analy-ses were performed via combined liquid chromatography-tandemmass spectrometry using an AB-Sciex API4000 triple quadrupolemass spectrometer (Foster City, CA) interfaced with an Agilent 1100series liquid chromatograph (Agilent Technologies, Wilmington, DE)as previously described (19). Ceramide analytes were ionized viapositive ion electrospray ionization. Elution of the ceramides wasdetected by multiple reaction monitoring characteristic for 14:0, 16:0,18:0, 18:1, 20:0, 24:0, and 24:1 ceramides. C17:0-ceramide wasemployed as an internal standard. All ceramide measurements werenormalized by lipid phosphorus (Pi) (19).

Cell Proliferation and Toxicity Assays

MTT assay (Invitrogen) was used to determine cell proliferation/metabolic activity following the manufacturer’s instructions. RLEC

L176 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

were plated in triplicate at 2,500 cells/ml for 18 h and the medium wasthen replaced with 2% FBS-containing medium with inhibitors ortheir vehicle controls for 2 h, followed by addition of nicotine andovernight incubation before assay. An assay using the Cell CountingKit-8 (CCK-8; Dojindo, Rockville, MD) was performed on 1.6 � 104

cells/well plated on 96-well tissue culture plates and treated the nextday as indicated. The kit’s CCK-8 solution was added and the platewas read using a spectrophotometer at 450 nm. Cytotoxicity/LDHassay (Roche, Indianapolis, IN) was performed in HLMVEC plated intriplicate onto 96-well dishes at 20 � 105 or 50 � 105 cells/well.

Immunoblotting

Cells were grown in six-well dishes, washed with ice-cold PBSimmediately after treatment, and collected by centrifugation. Cellswere lysed in standard RIPA buffer with protease and phosphataseinhibitors (Complete and Phostop, respectively; Roche). Samplescontaining equal protein amounts as determined by bicinchoninic acidprotein analysis (Pierce; Rockford, IL) were resolved using SDS-PAGE, transferred onto polyvinylidene difluoride using semidrytransfer (Bio-Rad; Hercules, CA), and probed with the followingprimary antibodies (all from Cell Signaling, Beverly, MA, unlessotherwise stated): phospho-p38 (1:500); total p38 (1:1,000); phospho-MLC (1:500); and phospho-myosin phosphatase target subunit 1(phos-MYPT1, 1:500). Horseradish peroxidase-conjugated secondaryantibodies to rabbit, rat, or mouse were from Amersham (Piscataway,NJ). Protein expression was detected using enhanced chemilumines-cence (ECL-plus; Amersham), quantified by densitometry, and nor-malized by a housekeeping protein, vinculin (1:10,000; Calbiochem).

Nuclear Magnetic Resonance

Samples for NMR spectroscopy were prepared by dissolving 25 �l ofe-Cig solution (World of Vapor) in 675 �l of [2H4] methanol fromCambridge Isotope Laboratories (Andover, MA). NMR spectra at 1Hwere acquired at 25°C on a Varian Inova 500 equipped with a 5-mmtriple-resonance pulsed-field gradient probe. All spectra were acquiredwith 90° pulse with 8,192 complex points, 64 transients, and 3-s recycledelay.

Mass Spectrometry

One microliter of each sample was diluted with 199 �l of 50% acetonenitrile in water with 0.1% formic acid. The samples were analyzed usinga Thermo Scientific Orbitrap Velos Pro hybrid ion trap-Orbitrap massspectrometer through direct infusion using a syringe pump. The flow ratewas 3 �l/min and the resolution was 60,000.

Gas Chromatography-Mass Spectrometry

All experiments used an Agilent 6890N gas chromatograph cou-pled with an Agilent 5975 mass spectrometer. The method utilized anoven program with an initial temperature of 40°C held for 1 min, aramp of 20°C/min, and a final temperature of 300°C held for 1 min.The carrier gas was hydrogen, with a flow rate of 2.5 ml/min and asplit ratio of 20:1. The inlet was set at 250°C. The mass spectrometeroperated in electron ionization mode, with a scan range of m/z50–550, and a solvent delay of 2 min. In an initial experiment todetermine the ingredients of each sample, 25 mg of nicotine, nicotine-containing, and nicotine-free e-Cig solutions, and e-Cig condensedvapor were placed in a 25-ml volumetric flask and diluted to the markwith dichloromethane. The samples were filtered with a polytetrafluo-roethylene syringe filter and analyzed. In a separate quantitationexperiment, nicotine and quinoline were diluted with dichloromethaneto produce four standard solutions: a 100 mg/ml nicotine, 1 mg/mlquinoline solution; a 10 mg/ml nicotine, 1 mg/ml quinoline solution;a 1 mg/ml nicotine, 1 mg/ml quinoline solution; and a 0.1 mg/mlnicotine, 1 mg/ml quinoline solution. The ratio of nicotine to quino-line in each standard was determined by peak integration, and this

information was used to create a calibration curve. Approximately 50mg of three condensed vapor samples was transferred into a 2-mlvolumetric flask, spiked with 2 mg of quinolone, and diluted to themark with dichloromethane. These samples were also analyzed viagas chromatography-mass spectrometry using the same method andcompared against the calibration curve to determine the amount ofnicotine in each sample.

Statistical Analysis

SigmaStat 3.5 (San Jose, CA) or Prism 6 (San Diego, CA) softwarewas utilized for comparisons among groups by ANOVA as indicated,followed by intergroup comparisons with Tukey’s post hoc testing.For experiments in which two conditions were being compared, atwo-tailed Student’s t-test was used. All data are expressed asmeans � SE and statistically significant differences were consideredif P � 0.05.

RESULTS

To investigate the contribution of nicotine in CS extract tothe loss of lung endothelial barrier function, we compared theeffect of soluble extract from nicotine-containing and nicotine-free cigarettes. Primary RLEC exposed to nicotine-containingCS extract (10% vol:vol) exhibited increased monolayer per-meability as measured by ECIS in a time-dependent manner,with �40% decrease in TER at 5 h and �50% at 20 h (Fig.1A), consistent with our previous report (22). In contrast,similar concentrations of nicotine-free CS extract had a mark-edly diminished effect, with no loss of barrier function at 5 hand only � 30% loss of TER at 20 h (Fig. 1A). These resultssuggest that nicotine directly contributes to the damaging effectof soluble CS extract on lung endothelial barrier. We nexttested whether exposure to nicotine itself decreases the endo-thelial barrier function. Upon incubation of primary RLECwith increasing concentrations of nicotine (up to 50 mM for upto 15 h), we noted significant time- and dose-dependent de-creases in TER (Fig. 1B). Using a similar setup, monolayers ofprimary mouse lung endothelial cells or human lung microvas-cular endothelial cells challenged with nicotine exhibited sim-ilar time and dose-dependent decreases in TER (Fig. 1, C andD), indicating that nicotine effects are not species-specific.

Similar to pure, analytical-grade nicotine solutions testedabove, two separate nicotine-containing solutions (e-Cig 1 ande-Cig 2) used for vaporization in commercially available e-Cigs also triggered barrier dysfunction in RLEC, consistentwith their purported nicotine concentration (Fig. 2A). Unex-pectedly, barrier dysfunction was also induced by exposures tosimilar volumes of an e-Cig solution (e-Cig 2) that lackednicotine (Fig. 2A). Of note, the nicotine-free e-Cig 2 wasmarketed as having the same flavor as nicotine-containinge-Cig 2 and shared the same manufacturer. Since vaporizationof e-Cig solutions may generate different metabolites than theoriginal solution due to heating, we investigated whether thecondensed vapor isolated from an e-Cig affected the endothe-lial barrier. For similar volumes as nicotine solutions, the e-Cigvapor condensate was less potent on altering RLEC barrierfunction (Fig. 2A). The barrier disruptive effect of e-Cigsolutions on human lung endothelial cells was nicotine doserelated (Fig. 2B) and of similar magnitude to that caused byexposures to 3% CS extract, a relatively low concentration wehave previously shown to not cause cell death (22). The vaporcondensate of e-Cig also caused a dose-related loss of endo-

L177NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

thelial barrier that required a higher volume compared withnonvaporized solutions of e-Cig (Fig. 2C).

These results suggested that although nicotine in CS extractsis sufficient to trigger endothelial barrier dysfunction, theeffects of e-Cig solutions and vapors are only in part nicotine-dependent. Since commercially available e-Cig extracts andvapors are not well regulated or biochemically defined, wedetermined the composition of these solutions compared withthat of CS extract and analytical-grade nicotine solutions.NMR confirmed the presence of nicotine in e-Cig solutionsmarked as nicotine-containing and confirmed the lack of nic-otine in nicotine-free e-Cig solutions tested (Fig. 3A). Inaddition, NMR detected the propylene glycol and glycerol ine-Cig solutions, and acrolein in e-Cig vapor condensate. Thesecompounds, in particular nicotine, acrolein, and glycerol, wereconfirmed using high-resolution mass spectrometry (MS) (Fig.3B) by comparing the monoisotopic mass of each compoundwith its theoretical monoisotopic mass (Table 1). MS could notdetect propylene glycol, likely because of its poor ionization,but confirmed the lack of nicotine in nicotine-free e-Cig solu-tions and, demonstrating increased sensitivity compared withNMR, detected acrolein not only in condensed e-Cig vapor, butalso in all e-Cig solutions tested. This finding suggested thatheating e-Cig solutions produced vapor was not a necessary

step to produce acrolein. We confirmed these results using athird complementary method, gas chromatography (GC) (Fig.3C). Quantitative GC analysis determined that the condensede-Cig vapor generated for our experiments lost up to four timesthe nicotine compared with the e-Cig (stock) solutions used forvaporization. These measurements suggested that the concen-trations of nicotine used in these experiments are within therange of nicotine that is inhaled by the average smoker percigarette (1–2 mg total nicotine).

Given the relatively high concentrations of nicotine appliedto cells in cultures, we ensured that the nicotine effect on theendothelium was not due to cell toxicity/necrosis, as deter-mined by lactate dehydrogenase (LDH) release (data notshown). However, nicotine exposure of RLEC significantlyand dose-dependently decreased MTT activity, a test thatreflects cellular metabolic activity and decreased CCK8 activ-ity, a marker of cellular proliferation (Fig. 4, A and B).Nicotine-containing e-Cig solutions had similar inhibitory ef-fect on endothelial proliferation as measured by CCK8 activity(Fig. 4C). These results indicated that nicotine and e-Cig evenat nontoxic (nonlethal) exposure levels significantly impact thebehavior of relevant primary cell cultures.

To investigate whether inhalation of nicotine and e-Cigsolutions also trigger short-term (immediate, 30 min, or 24 h)

Fig. 1. Effect of nicotine on lung endothelial and epithelial barrier function. A: transcellular electrical resistance (TER) measured at the indicated time pointnormalized to TER at baseline (at the beginning of the measurement, before any treatment) in cells exposed to ambient air control extract (AC),nicotine-containing cigarette smoke extract (CS), or nicotine-free CS extract (all solutions were 10% vol:vol) measured by electrical cellular impedance sensing(ECIS) in primary lung microvascular endothelial cells. Values are means � SE, n � 4–10, one-way ANOVA (with Tukey’s post hoc testing for intergroupcomparisons). B–D: normalized TER measured at the indicated time (hours) in primary lung rat microvascular endothelial cells (RLEC, B), primary mouse lungendothelial cells (C), and primary human lung microvascular endothelial cells (D) exposed to the indicated concentrations of nicotine. Values are means � SE,n � 5–56, one-way ANOVA with Tukey’s post hoc testing.

L178 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

pulmonary responses in vivo, we administered 1 �g of nicotineor 2 �g of e-Cig to mice via nebulization. These doses areequivalent to smoking one or two cigarettes, respectively.There was a trend toward a rapid increase in polymorphonu-clear cells in the BALF at 24 h (Table 2) indirectly reflectinga permissive endothelial barrier for inflammatory cell extrav-asation. In addition, there was evidence of systemic oxidativeand nitroxidative stress, indicated by increased 8-OHdG andnitrotyrosine levels in plasma in response to inhalation analyt-ical-grade nicotine (Fig. 5, A and B). These changes wereparalleled by increases in the oxidative stress marker 8-OHdGlevels in the BALF (Fig. 5C). Oxidative stress tended to

increase by �15% and �10% compared with saline vehicle inmice exposed to e-Cig solutions, as measured by 8-OHdGlevels in plasma and BALF, respectively (data not shown).Overall, these studies indicate that even brief exposures oflungs to nicotine via inhalation are associated with pulmonaryresponses such as inflammation and oxidative stress, whichmay cause or be the result of altered lung endothelial barrierfunction. A direct oxidative stress-inducing effect of nicotineexposure was confirmed in cell cultures using a fluorescently-labeled ROS indicator and the ROS scavenger NAC (Fig. 5D).

To define the signaling pathways by which nicotine impairslung endothelial barrier function, we focused on the mecha-

Fig. 2. Effect of commercial electronic cigarette (e-Cig) solutions on lung endothelial barrier. A and B: normalized TER measured in cells (RLEC in A and humanlung microvascular endothelial cells in B) exposed to nicotine (15 mM, 5 h), to CS extract (CSE with similar nicotine content), or to e-Cig extracts or condensedvapor (commercial preparation with the indicated nicotine content; 5 h). Values are means � SE, n � 4–10, one-way ANOVA with Tukey’s post hoc testing.C: normalized TER measured in human lung microvascular endothelial cells exposed to the indicated volume (microliters, �l) of e-Cig or condensed e-Cig vapor.Values are means � SE n � 4–10, one-way ANOVA with Tukey’s post hoc testing.

L179NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

Fig. 3. Composition of e-Cig and condensed e-Cig vapor. A: spectra from indicated solutions analyzed with NMR [resonances are � 0.05 parts per million (ppm)],which detected methanol solvent OH 4.87, s; 3.30, quintet; nicotine, H2 8.50, d; H6 8.44, dd; H4 7.85, dt; H5 7.42, dd; H9a 3.24, t; H7 3.20, dd; H9b 2.37, dd;H11a 2.26, m; HN-methyl 2.17, s; H10a 1.98, m; H10b 1.88, m; H11b 1.77, m; propylene glycol H2, 3.78, m; H1 3.42, d; H3 1.15, d; glycerol, H2 3.66, tt; and H1,3

3.57, dm. In some spectra, a small aldehyde singlet (presumed acrolein) is visible at 9.77 ppm. Spectra from noted molecules were obtained from high-resolutionelectrospray ionization-mass spectrometry (ESI-MS, B) or gas chromatography (C) analyses of indicated solutions.

L180 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

nisms previously shown to be important in CS extract-inducedendothelial permeability such as ROS, MAPK, and sphingo-lipid pathways, as well as cytoskeletal/cellular contractilityeffectors (22). ROS played a critical role in the upstreamactivation of signaling pathways induced by CS extract, whichdecreased the lung endothelial barrier in cell culture models(22). Despite increased ROS induced by nicotine in vivo and invitro, treatment of RLEC with NAC, a potent ROS scavengerthat attenuates CS extract-induced barrier dysfunction (Fig.

6A), failed to reduce the nicotine-induced loss of barrier inthese cells. This result suggested that the mechanism by whichhigh concentrations of nicotine-induced barrier dysfunctionmay be distinct from those engaged by whole CS extract(containing lower nicotine concentrations).

At concentrations shown to cause barrier dysfunction, nic-otine significantly activated p38 MAPK in RLEC, similar toCS extract, whereas nicotine-free CS extract (in similar con-

Table 1. Theoretical and detected monoisotopic mass ofcompounds identified by mass spectrometry in e-Cig

Compound FormulaTheoretical Monoisotopic

Mass*Detected Monoisotopic

Mass

Nicotine C10H14N2 163.12352 163.12157Acrolein C3H4O 57.03403 57.03297Glycerol C3H8O3 93.05516 93.05386

e-Cig, e-cigarette extract.

Fig. 4. Effect of nicotine on proliferation of lung endothelial cells. Cell proliferation was determined with the metabolic activity indicator, MTT (A), or the celldivision marker, CCK-8 (B), in primary rat lung microvascular endothelial cells exposed to increasing concentrations of nicotine or e-Cig (C) solutions. Valuesare means � SE, n � 3, one-way ANOVA with Tukey’s post hoc testing.

Table 2. Cells detected in bronchoalveolar fluid of miceexposed to inhaled e-Cig or saline control and collected atthe indicated time

Treatment Time Macrophages Lymphocytes PMN Number

Saline 30 min 33,228 � 9,264 106 � 82 0 � 0 3e-Cig 1 30 min 28,278 � 6,664 56 � 34 0 � 0 3Saline 24 h 87,128 � 21,520 1,205 � 399 0 � 0 3e-Cig 1 24 h 62,317 � 13,064 2,122 � 1,862 561 � 427 3

Values are means � SE. PMN, polymorphonuclear cells.

L181NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

centrations) failed to induce phospho-p38 (Fig. 6B). Despitenicotine-dependent activation of p38 MAPK, treatment ofRLEC with p38 inhibitor SB203580 (5 �M) did not reducenicotine-induced endothelial permeability (Fig. 6C). Becauseneither the ERK inhibitor PD98059 (at 50 �M) nor the JNKinhibitor SP600125 (at 50 �M) attenuated nicotine-inducedendothelial cell permeability (data not shown), we concludethat nicotine induces MAPK-independent alterations in thelung endothelial barrier.

Nicotine activated the actin-myosin apparatus, as measuredby increased phosphorylation of MLC (Fig. 6D). Nicotine-induced MLC phosphorylation was partially inhibited by theROS scavenger NAC and abolished by p38 inhibitor (Fig. 6D).MLC phosphorylation occurs by activation of the MLC kinase(MLCK) or by inhibition of MLC phosphatase. We investi-gated whether the myosin phosphatase target subunit 1,MYPT1, is inhibited (via phosphorylation) during nicotineexposure. Nicotine increased MYPT1 phosphorylation within15 min of application, and for up to 60 min (Fig. 6E).

Nicotine-induced MYPT1 phosphorylation was prevented bytreatment with the Rho kinase inhibitor Y27632 (Fig. 6E).Unlike NAC or p38 inhibition, Rho kinase inhibition signifi-cantly attenuated nicotine-induced barrier dysfunction inRLEC (Fig. 6F), implicating a critical role for Rho kinase-induced MYPT1 inhibition for nicotine’s effect on endothelialpermeability.

We have previously shown that Rho kinase was also a keymediator of endothelial permeability induced by CS extract,and that ceramides were involved in the upstream signalingleading to Rho kinase activation. We therefore interrogated therole of the sphingolipid pathway in nicotine-induced endothe-lial effects. First, nicotine exposure significantly increased Cer16:0, total ceramides, and total dihydroceramides (precursorsof ceramides in the de novo sphingolipid pathway) in RLEC(Fig. 7A). This effect was not cell type- or host species-specific, because incubation of cells in the human bronchialcell line Beas-2B with varying concentrations of nicotine alsocaused a significant and dose-dependent increase in total cer-

Fig. 5. Oxidative stress induced by nicotine. A: nitrotyrosine levels from the plasma of C57Bl/6 mice nebulized with one dose of nicotine and harvestedimmediately. Levels of 8-OHdG in plasma (B) or bronchoalveolar lavage fluid (BALF, C) of C57Bl/6 mice nebulized with one dose of nicotine and collectedimmediately. Values are means � SE, n � 3 per group, Student’s t-test. D: detection of reactive oxygen species (ROS, green) in rat lung microvascularendothelial cells exposed to nicotine (10 mM for 30 min) with or without N-acetylcysteine (NAC, 0.5 M) using Image-iT LIVE Green Reactive Oxygen SpeciesDetection Kit and DAPI staining of nuclei (blue).

L182 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

amides (Fig. 7B) and dihydroceramides (data not shown).Pharmacological inhibition of neutral sphingomyelinase, orany of the other enzymes involved in ceramide production suchas acid sphingomyelinase, serine palmitoyltransferase, andceramide synthase (with imipramine, myriocin, and fumonisinB1, respectively) did not attenuate the nicotine-induced de-crease in TER (Fig. 7C). In contrast, treatment with analogs ofS1P, a barrier-enhancing, downstream metabolite of ceramide,significantly attenuated nicotine-induced endothelial permea-bility in RLEC (Fig. 7D). We tested S1P analogs because theS1P molecule has a short duration of action and it is imprac-tical to use with exposures that lead to a relatively slow onsetof increased permeability. Indeed, concomitant treatment ofS1P (5 �M) with nicotine did not attenuate permeabilityresponses to nicotine (data not shown). Nonetheless, FTY720mono-(1) or bi-(2) phosphonate enantiomers (R or S) of theS1P analog FTY720, significantly inhibited the decrease inlung endothelial permeability triggered by nicotine exposure(Fig. 7E). This effect was associated with decreased nicotine-

induced MYPT1 and MLC phosphorylation (Fig. 7F), suggest-ing that FTY720 analogs affected, at least in part, actin cyto-skeletal contraction. Because S1P is also a pro-proliferativesignaling molecule, we investigated whether increased endo-thelial cell proliferation could explain improvement in theendothelial barrier induced by the S1P agonists. Interestingly,only the FTY-2S agonist significantly increased cell prolifer-ation, as measured by the MTT assay (Fig. 7G), indirectlysuggesting that cell proliferation is not the main mechanism bywhich S1P agonists exert barrier protective effects in responseto nicotine. Because it is not known whether S1P could alsoameliorate CS extract-induced permeability, we interrogatedthe effect of S1P agonists in primary human lung microvascu-lar endothelial cells, along with its dependence on S1P receptor1 (S1P1) signaling. All FTY agonists, with the exception ofFTY-1S, significantly improved CS extract-induced endothe-lial permeability; this effect was abolished by knockdown ofS1P1 with specific siRNA (Fig. 7H). These studies revealedthat nicotine triggers selective signaling pathways that partially

Fig. 6. Signaling in nicotine-induced endothelial barrier dysfunction. A: normalized TER measured in rat lung endothelial cells exposed to CS (10%) or to nicotine(15 mM) for the indicated time (hours), and effect of the antioxidant N-acetylcysteine (NAC, 0.5 M, means � SE, n � 2–12). B: p38 MAPK activation by nicotinein lung endothelium detected by Western blot analysis for phospho- and total p38 (�, , , and � isoforms) in rat lung microvascular endothelial cells (RLEC)exposed to ambient air control extract (AC), CS extract (CS), or nicotine solution at the indicated concentrations and time points. The blot is representative ofn � 3. C: normalized TER measured in RLEC exposed to nicotine (15 mM) for the indicated time (hours), and effect of a p38 inhibitor (SB203580, 30 �M,means � SE, n � 6–50), one-way ANOVA with Tukey’s post hoc test. D: myosin light chain kinase activation detected by immunoblotting for phospho-myosinlight chain (Ser19) of RLEC following exposure to nicotine solution (15 mM for 1 h) in the absence or presence of the antioxidant NAC (0.5 M), the ERK-MAPKinhibitor PD98059 (PD 50 �M), or the p38 inhibitor SB203580 (SB, 30 �M). E: myosin phosphatase inhibition detected by phosphorylation of myosinphosphatase target subunit 1 (MYPT1) in RLEC exposed to nicotine (15 mM) for the indicated time points in the presence of 3 �M Rho kinase inhibitor Y29632(RhoKinh). F: normalized TER measured in RLEC exposed to nicotine (10 mM) for the indicated time, and effect of a Rho kinase inhibitor (Y29632, 3 �M).Values are means � SE, n � 10, one-way ANOVA with Tukey’s post hoc test.

L183NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

overlap those engaged by CS extract to disrupt the barrier andproliferative functions of endothelial cells (Fig. 8).

DISCUSSION

The results presented indicate that nicotine has dose-relateddeleterious pulmonary effects that result in loss of lung endothelial

barrier function, acute lung inflammation, and decreased lungendothelial cell proliferation. These findings enhance our under-standing of how CS exposure causes inflammation and furtherelucidate the pulmonary effects of nicotine inhalation.

The preservation of an intact endothelial barrier is deter-mined by a balance of contracting cytoskeletal forces and the

Fig. 7. Role of sphingolipids in cellular responses to nicotine. Ceramide (A) and dihydroceramide (B) levels in RLEC following exposure to nicotine (15 mMfor 4 h) and in human lung epithelial cells Beas-2B (C) following exposure to the indicated nicotine concentrations (mM) for the indicated time. Values aremeans � SE, n � 3, Student’s t-test. D: effect of ceramide synthesis inhibitors including that of ASMase with imipramine (Imi, 50 �M), of nSMase with GW4869(GW, 15 �M), of the de novo pathway with myriocin (Myr, 50 nM); or of ceramide synthases in the recycling pathway with fumonisin B1 (FB1, 5 �M); ortheir respective vehicle controls dH2O (for FB1, Imi, and Myr) or DMSO (for GW). E: normalized TER of RLEC exposed to nicotine (15 mM for 5 h) and impactof S1P receptor agonists (FTY phosphonate analogs 1S, 1R, 2S, 2R, 10 �M) or vehicle (methanol). Values are means � SE, n � 15–45, Student’s t-test. F:myosin phosphatase inhibition MLCK activation and MLCK activation detected by phospho-Mypt1 (Thr 696) and phospho-MLC (Ser19) immunoblottingfollowed by densitometry in RLEC exposed to nicotine (15 mM for 20 min) and vehicle (methanol) or the indicated FTY720 analog (10 �M). Valuesare means � SE, n � 3, one-way ANOVA with Tukey’s post hoc test. G: cell proliferation measured with MTT in RLEC exposed to nicotine (15 mM)in the presence or absence of S1P receptor agonists (FTY phosphonate analogs 1S, 1R, 2S, 2R, 10 �M) or vehicle (methanol). Values are means � SE,n � 3. H: TER of primary human lung microvascular cells exposed to CS (3%) or air extract (3%) and attenuated with S1P receptor agonists (FTYphosphonate analogs 1S, 1R, 2S, 2R, 10 �M) in the presence or absence of S1PR1-specific siRNA. Values are means � SE, n � 4 –14, ANOVA withTukey’s post hoc test.

L184 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

integrity of cell-cell contacts, both of which can be affected byexposure to soluble components of CS extract (22). In thiswork we identified that nicotine, which can be absorbed in thecirculation as a component of CS or e-Cig, disrupts endothelialbarrier by increasing actomyosin contractile signaling, primar-ily by Rho kinase-dependent phosphorylation and thereforeinhibition of endothelial myosin phosphatase, causing in-creased MLC phosphorylation. Interestingly, although nicotinecaused oxidative stress and activated p38 MAPK similarly toCS extract (22), neither p38 MAPK inhibition nor the ROSscavenger NAC were sufficient to restore barrier functionfollowing nicotine exposure, in contrast to their remarkableeffect on CS-induced barrier dysfunction. These results suggestseveral possible explanations that include a threshold of MLCphosphorylation that is needed for barrier dysfunction, which isachievable by Rho kinase activation but not by p38 MAPKalone. Such a concept is supported by a recent report in whichRho kinase was found, under certain conditions, to activatep38, but not vice versa (34). Alternatively, nicotine-activatedRho kinase may have additional targets that cause barrierdysfunction besides MLC phosphorylation, as supported by therecent finding of a critical role for Rho kinase isoform 2 inregulating cellular junctional tension (2). Finally, at least the-oretically, nicotine-activated p38 MAPK may have additionalunexpected barrier-enhancing activities that counteract itsMLC phosphorylation effects. Either inhibition of Rho kinaseor enhancement of S1P to S1P1 signaling significantly coun-teracted the barrier-disruptive effects of nicotine (Fig. 8) andCS extract. The novel finding of a protective effect of S1P1agonists on the CS/nicotine-disrupted endothelial barrier is notsurprising given reports of similar S1P1-dependent protectiveeffects of FTY phosphonates against lung endothelial perme-ability during sepsis or acute lung injury (23, 31, 32), or duringsynergistic conditions of cystic fibrosis transmembrane con-ductance regulator (CFTR) inhibition and CS extract exposure(3). FTY phosphonates acted at least in part by activatingMYPT1 and inhibiting MLC phosphorylation, although anadditional effect on intercellular tethering cannot be ruled out.That S1P augmentation did not recapitulate the effects of FTY

phosphonates may be due to the short half-life of the molecule,or to complementary, S1P-independent mechanisms of actionof FTY phsosphonates (7).

Using various pharmacological inhibitors of nicotinic recep-tors to test their involvement in nicotine’s effects on thepulmonary endothelium, we could not identify a protectiveeffect against barrier dysfunction (data not shown). However,it is possible that untested receptors and other mediators mayregulate nicotine-altered endothelial barrier function. This maybe true especially in response to high, cytotoxic nicotineexposure levels shown to inhibit prostaglandin and endothelinexpression in bovine pulmonary endothelial cells (25), butthese were not studied here. The concentrations of nicotineused in our cell culture studies were derived from detaileddose-response testing, were noncytotoxic, and induced signif-icant effects at levels higher than those absorbed in the circu-lation by individuals who smoke, but which may be achievedin tissue levels with high nicotine concentrations, such as thelung (6, 30). The effect of nicotine on barrier function may beorgan dependent because other studies have shown an im-provement in the gut barrier function by cholinergic actions ofnicotine on enteric glial cells (4).

Although many of the nicotine effects on the lung endothe-lium were dose-dependent, nicotine-independent deleteriouseffects of e-Cig solutions were also noted. We have identifiedacrolein as putative mediator for nicotine-independent toxicityon the basis of its presence in both e-Cig solution and vaporand on a large body of literature showing adverse pulmonaryeffects of acrolein, including on endothelial intercellular teth-ering molecules (12). The signaling effects on nicotine-freee-Cig vapors on the lung endothelial barrier remain to beinvestigated.

The noted dose-dependent antiproliferative effects of nico-tine on lung endothelial cells may have implications in angio-genesis and in lung injury repair. Our results on primary lungendothelial cells are in contrast to pro-proliferative effects ofnicotine on human umbilical vascular endothelial cells (11),systemic vasculature, or on lung cancer cells (15), suggestingcell type-specific effects of low-dose nicotine.

Fig. 8. Schematic of signaling events detected in lung endothelial cells exposed to nicotine. Arrows indicate activation and blocked lines indicate inhibition.Nicotine activates Rho kinase, which in turn inhibits the myosin phosphatase target subunit 1, MYPT1, enhancing phosphorylation of myosin light chains(MLC-P) to increase endothelial permeability. Rho kinase may have other targets in the cell to increase endothelial permeability because nicotine-inducedoxidative stress (ROS)-dependent p38 MAPK activation also contributed to myosin light chain phosphorylation (MLC-P), but not sufficiently to alone increasepermeability. Nicotine also increases the ceramide/sphingosine-1-phosphate (S1P) ratios, which may inhibit lung endothelial cell proliferation. Enhancing S1Psignaling opposes the decreased cell proliferation and the increase in permeability induced by nicotine in part by inhibiting MLC phosphorylation and restoringthe lung endothelial barrier function.

L185NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

Intravital lung microcopy in animals with intact circulation(no pump-perfusion) demonstrated that the in vitro finding ofCS extracts causing decreased endothelial barrier function wasparalleled by increased lung inflammation in vivo, as measuredby increased adherence of circulating leukocytes to the lungmicrovasculature within 20 min of CS inhalation, withoutpulmonary edema (21). This previous work led us to comple-ment our investigations of nicotine in cell culture models within vivo studies of acute lung and systemic effects of nebulizednicotine and e-Cig extracts, mimicking the inhalation of e-Cigvapors by humans. We found that nicotine and e-Cig extractscaused rapid oxidative and nitroxidative stress observed in theBALF and plasma as well as a trend toward greater neutrophillung inflammation at 24 h following inhalation as measured bythe relatively less sensitive method of BALF cytospins, ratherthan intravital microscopy. Although future studies will deter-mine how these acute inflammatory lung responses translateinto long-term effects of recurrent e-Cig exposures, we antic-ipate these will include dose-dependent sustained oxidative-stress and inflammatory lung damage with limitation of endo-thelial repair. In this context, ceramide/S1P balance may serveas an important rheostat of alveolar integrity, as was observedin experimental models of COPD (5). By augmenting barrier-enhancing and angiogenic S1P signaling via S1P1, such as wasshown here with pharmacological agonists, one may be able toimprove barrier function in vivo and potentially attenuate thechronic damage caused by e-Cig inhalation.

The clinical implications of this work related to the potentialdetrimental lung effects of exposure to e-Cig. Although furtherstudies are needed to determine the usual levels of absorbede-Cig vapor that are harmful to human lung health, our resultsin primary human and murine lung endothelial cells and inanimal models indicate they are dose related.

ACKNOWLEDGMENTS

We thank Margie Albrecht, Alayna Hutchinson, and Yuan Gu for experttechnical assistance. NMR spectra were acquired at the IUPUI School ofScience NMR Center.

GRANTS

Support for this study was provided by National Institutes of Health GrantsRO1 HL-077328 and R21 DA-029249 to I. Petrache. The American ChemicalSociety Project SEED Program sponsored S. Law.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.S.S., C.P., B.D.R., R.B., J.G., and I.P. conception and design of research;K.S.S., S.X.C., S.L., M.J.V.D., C.P., M.J.J., W.C.H., E.S.K., X.L., M.W.,W.D.K., C.J.C., B.D.R., R.B., and J.G. performed experiments; K.S.S., S.X.C.,S.L., M.J.V.D., C.P., M.J.J., W.C.H., E.S.K., X.L., M.W., W.D.K., C.J.C.,B.D.R., J.G., and I.P. analyzed data; K.S.S., C.P., W.C.H., M.W., W.D.K.,B.D.R., J.G., and I.P. interpreted results of experiments; K.S.S., S.X.C., S.L.,M.J.V.D., X.L., M.W., W.D.K., C.J.C., B.D.R., J.G., and I.P. prepared figures;K.S.S., B.D.R., J.G., and I.P. drafted manuscript; K.S.S., W.C.H., M.W.,B.D.R., J.G., and I.P. edited and revised manuscript; K.S.S., S.X.C., S.L.,M.J.V.D., C.P., M.J.J., W.C.H., X.L., M.W., W.D.K., C.J.C., B.D.R., J.G., andI.P. approved final version of manuscript.

REFERENCES

1. An N, Andrukhov O, Tang Y, Falkensammer F, Bantleon HP, OuyangX, Rausch-Fan X. Effect of nicotine and porphyromonas gingivalislipopolysaccharide on endothelial cells in vitro. PloS One 9: e96942, 2014.

2. Beckers CM, Knezevic N, Valent ET, Tauseef M, Krishnan R, Rajen-dran K, Corey Hardin C, Aman J, van Bezu J, Sweetnam P, vanHinsbergh VW, Mehta D, van Nieuw Amerongen GP. ROCK2 primesthe endothelium for vascular hyperpermeability responses by raisingbaseline junctional tension. Vascul Pharmacol. First published April 11,2015; doi:10.1016/j.vph.2015..03.017.

3. Brown MB, Hunt WR, Noe JE, Rush NI, Schweitzer KS, Leece TC,Moldobaeva A, Wagner EM, Dudek SM, Poirier C, Presson RG Jr,Gulbins E, Petrache I. Loss of cystic fibrosis transmembrane conduc-tance regulator impairs lung endothelial cell barrier function and increasessusceptibility to microvascular damage from cigarette smoke. Pulm Circ4: 260–268, 2014.

4. Cheadle GA, Costantini TW, Bansal V, Eliceiri BP, Coimbra R.Cholinergic signaling in the gut: a novel mechanism of barrier protectionthrough activation of enteric glia cells. Surg Infect (Larchmt) 15: 387–393,2014.

5. Diab KJ, Adamowicz JJ, Kamocki K, Rush NI, Garrison J, Gu Y,Schweitzer KS, Skobeleva A, Rajashekhar G, Hubbard WC, Berdy-shev EV, Petrache I. Stimulation of sphingosine 1-phosphate signaling asan alveolar cell survival strategy in emphysema. Am J Respir Crit CareMed 181: 344–352, 2010.

6. Dobrinas M, Choong E, Noetzli M, Cornuz J, Ansermot N, Eap CB.Quantification of nicotine, cotinine, trans-3=-hydroxycotinine and vareni-cline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation. JChromatogr B Analyt Technol Biomed Life Sci 879: 3574–3582, 2011.

7. Dudek SM, Camp SM, Chiang ET, Singleton PA, Usatyuk PV, ZhaoY, Natarajan V, Garcia JG. Pulmonary endothelial cell barrier enhance-ment by FTY720 does not require the S1P1 receptor. Cell Signal 19:1754–1764, 2007.

8. El-Mas MM, El-Gowilly SM, Gohar EY, Ghazal AR. Pharmacologicalcharacterization of cellular mechanisms of the renal vasodilatory effect ofnicotine in rats. Eur J Pharmacol 588: 294–300, 2008.

9. Galanzha EL, Chowhury P, Tuchim VV, Zharov VP. Monitoring ofnicotine impact in microlymphatics of rat mesentery with time-resolvedmicroscopy. Lymphology 38: 181–192, 2005.

10. Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, GerthofferWT, Bamberg JR, English D. Sphingosine 1-phosphate promotes endo-thelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.J Clin Invest 108: 689–701, 2001.

11. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS,Johnson FL, Cooke JP. Nicotine stimulates angiogenesis and promotestumor growth and atherosclerosis. Nat Med 7: 833–839, 2001.

12. Jang AS, Concel VJ, Bein K, Brant KA, Liu S, Pope-Varsalona H,Dopico RA Jr, Di YP, Knoell DL, Barchowsky A, Leikauf GD.Endothelial dysfunction and claudin 5 regulation during acrolein-inducedlung injury. Am J Respir Cell Mol Biol 44: 483–490, 2011.

13. Lu X, Sun C, Valentine WJ, Shuyu E, Liu J, Tigyi G, Bittman R.Chiral vinylphosphonate and phosphonate analogues of the immunosup-pressive agent FTY720. J Org Chem 74: 3192–3195, 2009.

14. Ma L, Zheng LW, Sham MH, Cheung LK. Effect of nicotine on geneexpression of angiogenic and osteogenic factors in a rabbit model of boneregeneration. J Oral Maxillofac Surg 68: 777–781, 2010.

15. Ma X, Jia Y, Zu S, Li R, Jia Y, Zhao Y, Xiao D, Dang N, Wang Y. �5Nicotinic acetylcholine receptor mediates nicotine-induced HIF-1alphaand VEGF expression in non-small cell lung cancer. Toxicol Appl Phar-macol 278: 172–179, 2014.

16. Mayhan WG, Sharpe GM. Chronic exposure to nicotine alters endothe-lium-dependent arteriolar dilatation: effect of superoxide dismutase. JAppl Physiol 86: 1126–1134, 1999.

17. Mimura K, Tomimatsu T, Sharentuya N, Tskitishvili E, Kinugasa-Taniguchi Y, Kanagawa T, Kimura T. Nicotine restores endothelialdysfunction caused by excess sFlt1 and sEng in an in vitro model ofpreeclamptic vascular endothelium: a possible therapeutic role of nicotinicacetylcholine receptor (nAChR) agonists for preeclampsia. Am J ObstetGynecol 202: 464 e461–e466, 2010.

18. Park HS, Cho K, Park YJ, Lee T. Chronic nicotine exposure attenuatesproangiogenic activity on human umbilical vein endothelial cells. J Car-diovasc Pharmacol 57: 287–293, 2011.

19. Petrache I, Natarajan V, Zhen L, Medler TR, Richter AT, Cho C,Hubbard WC, Berdyshev EV, Tuder RM. Ceramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease in mice. NatMed 11: 491–498, 2005.

L186 NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from

20. Petrache I, Verin AD, Crow MT, Birukova A, Liu F, Garcia JG.Differential effect of MLC kinase in TNF-alpha-induced endothelial cellapoptosis and barrier dysfunction. Am J Physiol Lung Cell Mol Physiol280: L1168–L1178, 2001.

21. Presson RG Jr, Brown MB, Fisher AJ, Sandoval RM, Dunn KW,Lorenz KS, Delp EJ, Salama P, Molitoris BA, Petrache I. Two-photonimaging within the murine thorax without respiratory and cardiac motionartifact. Am J Pathol 179: 75–82, 2011.

22. Schweitzer KS, Hatoum H, Brown MB, Gupta M, Justice MJ, BeteckB, Van Demark M, Gu Y, Presson RG Jr, Hubbard WC, Petrache I.Mechanisms of lung endothelial barrier disruption induced by cigarettesmoke: role of oxidative stress and ceramides. Am J Physiol Lung Cell MolPhysiol 301: L836–L846, 2011.

23. Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation ofsphingosine 1-phosphate-induced endothelial cytoskeletal rearrangementand barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, andalpha-actinin. FASEB J 19: 1646–1656, 2005.

24. Speer P, Zhang Y, Gu Y, Lucas MJ, Wang Y. Effects of nicotine onintercellular adhesion molecule expression in endothelial cells and integrinexpression in neutrophils in vitro. Am J Obstet Gynecol 186: 551–556,2002.

25. Suzuki N, Ishii Y, Kitamura S. Effects of nicotine on production ofendothelin and eicosanoid by bovine pulmonary artery endothelial cells.Prostaglandins Leukot Essent Fatty Acids 50: 193–197, 1994.

26. Theiss SM, Boden SD, Hair G, Titus L, Morone MA, Ugbo J. Theeffect of nicotine on gene expression during spine fusion. Spine 25:2588–2594, 2000.

27. Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electricalmethod for detection of endothelial cell shape change in real time: assessment ofendothelial barrier function. Proc Natl Acad Sci USA 89: 7919–7923, 1992.

28. Tithof PK, Elgayyar M, Schuller HM, Barnhill M, Andrews R.4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, in-duces apoptosis of endothelial cells. Am J Physiol Heart Circ Physiol 281:H1946–H1954, 2001.

29. Ueno H, Pradhan S, Schlessel D, Hirasawa H, Sumpio BE. Nicotineenhances human vascular endothelial cell expression of ICAM-1 andVCAM-1 via protein kinase C, p38 mitogen-activated protein kinase,NF-kappaB, and AP-1. Cardiovasc Toxicol 6: 39–50, 2006.

30. Urakawa N, Nagata T, Kudo K, Kimura K, Imamura T. Simultaneousdetermination of nicotine and cotinine in various human tissues using capillary gaschromatography/mass spectrometry. Int J Legal Med 106: 232–236, 1994.

31. Wang L, Dudek SM. Regulation of vascular permeability by sphingosine1-phosphate. Microvasc Res 77: 39–45, 2009.

32. Wang L, Sammani S, Moreno-Vinasco L, Letsiou E, Wang T, CampSM, Bittman R, Garcia JG, Dudek SM. FTY720 (s)-phosphonatepreserves sphingosine 1-phosphate receptor 1 expression and exhibitssuperior barrier protection to FTY720 in acute lung injury. Crit Care Med42: e189–e199, 2014.

33. Wang Y, Wang Z, Zhou Y, Liu L, Zhao Y, Yao C, Wang L, Qiao Z.Nicotine stimulates adhesion molecular expression via calcium influx andmitogen-activated protein kinases in human endothelial cells. Int JBiochem Cell Biol 38: 170–182, 2006.

34. Wu T, Xing J, Birukova AA. Cell-type-specific crosstalk between p38MAPK and Rho signaling in lung micro- and macrovascular barrierdysfunction induced by Staphylococcus aureus-derived pathogens. TranslRes 162: 45–55, 2013.

35. Yu M, Liu Q, Sun J, Yi K, Wu L, Tan X. Nicotine improves thefunctional activity of late endothelial progenitor cells via nicotinic acetyl-choline receptors. Biochem Cell Biol 89: 405–410, 2011.

L187NICOTINE-INDUCED SIGNALING IN LUNG ENDOTHELIAL CELLS

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00411.2014 • www.ajplung.org

by 10.220.33.6 on April 18, 2017

http://ajplung.physiology.org/D

ownloaded from