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TRIOLOGICAL SOCIETYCANDIDATE THESIS

Resveratrol Ameliorates Abnormalities of Fluid and Electrolyte

Secretion in a Hypoxia-Induced Model of Acquired CFTR Deficiency

Bradford A. Woodworth, MD, FACS

Objective/Hypothesis: Ineffective mucociliary clearance (MCC) is a common pathophysiologic process that underliesairway inflammation and infection. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed bythe cystic fibrosis transmembrane conductance regulator (CFTR). Decreased transepithelial Cl2 transport secondary to anacquired CFTR deficiency may exacerbate respiratory epithelial dysfunction by diminishing MCC and increasing mucus viscos-ity. The objectives of the present study are to 1) develop a model of acquired CFTR deficiency in sinonasal epithelium usinghypoxia, 2) investigate whether the polyphenol resveratrol promotes CFTR-mediated anion transport, 3) explore resveratrolmechanism of action and determine therapeutic suitability for overcoming acquired CFTR defects, and 4) test the drug in thehypoxic model of acquired CFTR deficiency in preparation for a clinical trial in human sinus disease. We hypothesize thathypoxia will induce depletion of airway surface liquid (ASL) secondary to acquired CFTR deficiency and that resveratrol willrestore transepithelial Cl2 secretion and recover ASL hydration.

Study Design: Basic science.Methods: Murine nasal septal (MNSE) and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic

conditions (1% O2, 5% CO2) and transepithelial ion transport (change in short-circuit current5 DISC) evaluated in Ussingchambers. Resveratrol was tested using primary cells and HEK293 cells expressing human CFTR by Ussing chamber andpatch clamp techniques under both phosphorylating and nonphosphorylating conditions. CFTR activation was evaluated inhuman explants and by murine in vivo (nasal potential difference) assessment. Cellular cyclic adenosine monophosphate(cAMP) (ELISA) and subsequent CFTR regulatory domain (R-D) phosphorylation (gel-shift assay) were also evaluated. Effectsof hypoxia and resveratrol on ASL were tested using confocal laser scanning microscopy (CLSM) and micro-optical coherencetomography (mOCT).

Results: Hypoxia significantly decreased DISC (in mA/cm2) attributable to CFTR at 12 and 24 hours of exposure in bothMNSE (13.556 0.46 [12 hours]; 12.756 0.07 [24 hours] vs. 19.236 0.18 [control]; P< 0.05) and HSNE (19.556 0.56 [12hours]; 17.676 1.13 [24 hours] vs. 25.496 1.48 [control]; P<0.05). We have shown that resveratrol (100 lM) enhancedCFTR-dependent Cl2 secretion in HSNE to an extent comparable to the recently Food and Drug Administration-approvedCFTR potentiator, ivacaftor. Cl2 transport across human sinonasal explants (78.426 1.75 vs. 1.7561.5 [control]; P<0.05)and in vivo murine nasal epithelium (2461.8 vs. 20.86 1.7 mV [control]; P<0.05) were also significantly increased by thedrug. No increase in cAMP or CFTR R-D phosphorylation was detected. Inside-out patches showed increased CFTR open prob-ability (NPo/N (N5 channel number]) compared to controls in both MNSE (0.32960.116 vs. 0.11960.059 [control];P< 0.05) and HEK293 cells (0.226 0.048 vs. 0.1256 0.07 [control]; P< 0.05). ASL thickness was decreased under hypoxicconditions when measured by CLSM (4.196 0.44 vs. 6.886 0.67 [control]; P< 0.05). A 30-minute apical application of resver-atrol increased ASL depth in normal epithelium (8.0861.68 vs. 6.1160.47 [control]; P< 0.05). Furthermore, hypoxia-induced abnormalities of fluid and electrolyte secretion in sinonasal epithelium were restored with resveratrol treatment(5.556 0.74 vs. 3.136 0.17 [control]; P<0.05).

Conclusions: CFTR activation with a leading edge Cl2 secretagogue such as resveratrol represents an innovativeapproach to overcoming acquired CFTR defects in sinus and nasal airway disease. This exciting new strategy bears furthertesting in non-CF individuals with chronic rhinosinusitis.

Key Words: Resveratrol, hypoxia, CFTR, cystic fibrosis, chronic rhinosinusitis, sinusitis, Ussing chamber, airway surfaceliquid, electrophysiology, patch clamp, ion transport, acquired CFTR deficiency.

Level of Evidence: N/A.Laryngoscope, 125:S1–S13, 2015

From the Department of Otolaryngology–Head and Neck Surgery, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

Editor’s Note: This Manuscript was accepted for publication March 24, 2015.

This article received the Edmund Prince Fowler Award from the Triological Society and was presented at the Combined Otolaryngology SpringMeetings, Boston, MA April 24, 2015.

This work was supported by the National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1K08HL107142-05) and NIH/National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-04). The author has no other funding, financial relationships, or con-flicts of interest to disclose.

*Send correspondence to Bradford A. Woodworth, MD, BDB 563, 1720 2nd Ave S, Birmingham, AL 35294. E-mail: [email protected]

DOI: 10.1002/lary.25335

Laryngoscope 125: October 2015 Woodworth: Resveratrol Ameliorates Abnormalities in Acquired CFTR Deficiency

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INTRODUCTIONChronic rhinosinusitis (CRS) causes significant

morbidity and detriments to quality of life for 16% of theU.S. population and accrues an estimated aggregatedcost of 8.6 billion dollars annually in health care expen-ditures.1–3 Patients with CRS mark worse scores forphysical pain and social functioning on quality-of-lifequestionnaires than those suffering from chronicobstructive pulmonary disease, congestive heart failure,or angina.2 Conventional CRS treatments are comprisedof antibiotics, steroids, and surgical intervention. Newtherapies using safe but effective compounds that eitheraugment the benefit of current treatments or targetinherent properties of sinonasal mucosa are clearlyneeded.

Normal sinonasal mucociliary function is a criticalhost defense mechanism that clears the upper airwaysof inhaled pathogens such as bacteria, dust, and aero-sols. Sinonasal respiratory epithelium is a highly regu-lated barrier that is central to function of themucociliary apparatus. Mucociliary clearance (MCC) isdependent on inherent biological properties of intactrespiratory epithelium, including proper ciliary beatingand the airway surface liquid (ASL). The ASL is com-posed of a periciliary fluid (sol) layer and a mucus (gel)layer.4 Respiratory epithelium modifies the volume ofthe periciliary fluid layer5 and the viscoelastic propertiesof the mucus in a fashion that depends on vectorialtransepithelial ion transport.6,7 The active and passivetransport of electrolytes (primarily sodium [Na1], chlo-ride [Cl2], and bicarbonate [HCO3

2]) modulate ASL, andsignificant alterations in ion transport can be detrimen-tal to the airway, as clearly exemplified by the diseasecystic fibrosis (CF), which fibrosis affects approximately30,000 persons in the United States (70,000 worldwide)and is the most common lethal inherited disease amongCaucasians.8 In CF, mutations in the gene encodingCFTR result in decreased apical membrane anion con-ductance and cause dysregulated Na1 absorption.8

These abnormalities of fluid and electrolyte transportpromote thick mucus formation and immobile exocrinesecretions in multiple organ systems. In the sinuses ofCF individuals, chronic stasis of inspissated mucus incombination with bacterial infection result in nearly uni-versal inflammatory paranasal sinus disease.9–12

The CFTR is a member of the ATP-binding cassetteprotein family and is comprised of multiple domains,including two transmembrane domains (TMs) and twonucleotide binding domains (NBDs). In addition, CFTRhas a single regulatory domain (R-D) that functions inpart to control CFTR activity.13 Activation of CFTR isthought to occur by a two-step process that involves 1)phosphorylation of the R-D, and 2) dimerization of thetwo NBDs, facilitating ATP binding and activation of theCl2 channel by inducing a conformational change in theTMs. Clinically important mutations in the CFTR genecan affect transcription, translation, posttranslationalmodification, channel gating and function, and proteinstability and turnover at the plasma membrane. Themost common cause of CF is due to the deletion of phe-nylalanine at CFTR position 508 (F508del CFTR), which

results in misfolding of the protein product in the endo-plasmic reticulum and accelerated degradation by theproteosome. The absence of CFTR at the plasma mem-brane results in defective ion transport and the clinicalmanifestations of CF.13 Other mutations, such asG551D, result in adequate levels of CFTR protein at theapical cell surface, but the channels exhibit defectivegating.14 The genetic mutations in individuals afflictedwith CF contribute to phenotypic expression of the dis-ease. However, recent evidence indicates that wild-type(WT) CFTR processing, endocytic recycling, and functioncan also be markedly repressed by various environmen-tal insults, including cigarette smoke exposure, high alti-tude/hypoxemia, inflammation, and infectious agents,and may be a contributing factor in CRS and other dis-ease of MCC such as chronic obstructive pulmonary dis-ease (COPD).15–24 Hypoxia has been suggested to haveconsiderable influence in the pathophysiology of CRSamong non-CF individuals.25–27 Blockage of sinus ostiamay lead to hypoxia, resulting in decreased MCC, elabo-ration of inflammatory mediators, and bacterial coloniza-tion.27 In addition, accumulated secretions and impactedmucus lead to poor oxygen (O2) delivery, resulting innear anaerobic tissue conditions that accelerate progres-sion of disease.28 Aaneas et al.29 used an oxygen probeto cannulate the maxillary sinuses of CF patients withCRS and found severely repressed pO2, including someindividuals with complete anoxia. Hypoxia in the sinusesof CRS patients could contribute to propagation of sinusdisease by creating a localized CFTR-deficient environ-ment where abnormalities of fluid and electrolyte trans-port persist. In either case, a strategy aimed atstimulating electrolyte secretion with drugs that targetCFTR, therefore promoting mucus clearance, would fur-nish a valuable addition to the pharmacologic armamen-tarium available for treatment of sinusitis.

Several small organic molecules that activate CFTRhave been identified through high-throughput screeningof compound libraries containing millions of discreteagents. Pharmaceutical companies have pursued CFTRpotentiators for therapy of diseases of mucus clearance,including both CF and COPD. One such agent, ivacaftor(Vertex Phamaceuticals, Boston MA), represents a novelCF therapeutic recently approved for treatment of CFpatients with at least one copy of the G551D mutation.30

In this CF population, ivacaftor improves CFTR channelopening and thereby augments endpoints predictive ofbenefit, including MCC, sweat Cl2, and lung function.31

Importantly, the same strategy being applied to activateCl2 secretion in the lower airways of CF patients is alsoapplicable to impaired sinus and nasal mucusclearance.31

Mechanistically, ivacaftor increases channel openprobability (Po) in both G511D mutated and WT CFTRin the presence of R-D phosphorylation. This is bestdemonstrated in vitro following activation of cyclic aden-osine monophosphate (cAMP)/protein kinase A (PKA)-dependent pathways by drugs such as forskolin that con-fer phosphorylation of the R-D.32 Other compounds suchas the flavonoid, genistein, increase CFTR channel Poby enhancing channel-open time and decreasing closed


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time, a phenomenon also observed as decreased run-down of CFTR channels studied by membrane patchexcision.33,34 Flavonoid compounds related to genisteinalso exhibit the capacity to activate CFTR Po,35–37 par-ticularly after forskolin-mediated phosphorylation.38

Based on these and other functional studies, several lab-oratories have concluded that flavonoids may directlybind to CFTR, possibly at the interface between the twoNBDs.39,40 Other molecules identified by high through-put compound library screening, such as the isoxazoleUCCF2152 (3-[2-bezyloxy-phenyl]-5-chloromethyl-isoxa-zole), induce PKA-dependent phosphorylation of the Rdomain.41 In summary, multiple agents are known toactivate CFTR Cl2 transport by a variety of distinctmechanisms and are therefore candidates for targetingbasal CFTR activity and acquired ion transport defectsthat underlie pathogenesis in CRS.

Resveratrol is an organic polyphenol found in manyplants and vegetables, including the skins of red fruits,trees, some flowering plants, and peanuts.42,43 Flavo-noids are also plant-based compounds that possess thecommon flavone ring structure (2-phenyl-c-benzopyr-one).38 Resveratrol lacks the central ring of the commonthree-ring flavone backbone but is otherwise structurallysimilar to flavonoids as a polyphenolic molecule. Broadlyrecognized for its immune-modulator activity attribut-able to inhibition of NF-jB signaling,44 resveratrolmarkedly reduces inflammatory pathways mediated bygranulocyte–macrophage colony-stimulating factor (GM-CSF), IL-8 (or the murine homologue KC), induciblenitric oxide synthase, and COX-2.44 However, utility as atherapeutic agent for acquired CFTR dysfunction insinus disease has not been previously investigated.

The objectives of the current study were to 1) useoxygen restriction to create a model of acquired CFTRdeficiency in sinonasal epithelium; 2) investigate whetherthe polyphenol resveratrol stimulates CFTR-mediatedanion transport in vitro, ex vivo, and in vivo; 3) explorethe mechanistic action of resveratrol on CFTR functionand activity in sinonasal epithelium to determine thera-peutic suitability for acquired CFTR defects in humansinus disease; and 4) test the drug in the hypoxic model ofacquired CFTR deficiency in preparation for a clinicaltrial of mucociliary activators in human sinus disease. Wehypothesize that hypoxia induces the depletion of ASLsecondary to alterations in transepithelial ion transportin a similar fashion to CF disease, and that the impact ofthe environmental hypoxic insult can be amelioratedthrough the application of resveratrol.

MATERIALS AND METHODSUniversity of Alabama at Birmingham Institutional Ani-

mal Care and Use Committee and Institutional Review Board(IRB) approval were obtained prior to initiation of the study.Written informed consent was obtained from each participanton a document approved by the IRB.

Tissue CulturePrimary sinonasal epithelial cells (from humans, mice,

and pigs) were cultured at an air–liquid interface according to

previously established protocols.45–53 All of the murine nasalseptal epithelial (MNSE) cells were obtained from congenic C57/BL6 WT and CFTR2/2 mice. Primary human sinonasal epithe-lial (HSNE) cells were prepared and cultured on collagen-coatedCostar 6.5-mm-diameter permeable filter supports (Corning,Lowell, MA) submerged in culture media. Differentiation andciliogenesis occurred in all cultures within 10 to 14 days. Allexperiments were performed only when cell monolayers werefully differentiated with widespread ciliogenesis and transepi-thelial resistances (Rt)>300 X. cm2. A recombinant humanCFTR-expressing HEK293 cell line (D060) was also employedfor patch clamp analysis.54

For hypoxia experiments, cells were incubated in a sealedmodular incubator chamber at 378C, with an environment com-prised of a 1% O2, 5% CO2 (the remainder consisting of nitro-gen), or a physiologic environment (21% O2, 5% CO2).

ElectrophysiologyUssing Chamber Short Circuit (ISC) Measurements.

To investigate pharmacologic manipulation of vectorial ion trans-port, a large array of electrophysiology assays were employed.Human sinonasal epithelial explants were removed from patientsundergoing endoscopic surgery for pituitary tumor, benign sino-nasal tumor, or cerebrospinal fluid leak repair. Specimens wereimmediately stored in ice-cold DMEM/F-12 media (Invitrogen,Grand Island, NY, USA) and transferred to the laboratory within15 minutes. A thin layer of epithelial tissue was dissected fromthe surgical specimen, placed on a slider with an open area of0.031- to 0.71-cm2, and mounted between Ussing-type hemicham-bers (Easy Mount, Physiologic Instruments Inc., San Diego, CA)to evaluate resveratrol activity ex vivo. Transwell inserts con-taining monolayers as well as tissues were arranged in Ussingchambers (VCC 600; Physiologic Instruments Inc.) and continu-ously analyzed under short-circuit conditions following fluidresistance compensation using automatic voltage clamps. Bathsolutions for the transwell filters were warmed to 378C, and eachsample continuously gas lifted with 95% O2 to 5% CO2 or 94%N2/1%O2/5% CO2 mixture for hypoxia experiments. The bathsolution contained (mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4,0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose with pH 7.3 to7.4. Chemicals were acquired from Sigma (St. Louis, MO). Eachsolution was formulated as 10003 stock and used at 13 inthe Ussing chamber. All experiments were performed with lowCl2 (6 mM) in the mucosal bath. Pharmacologic preparationswere the following: amiloride (100 mM), ivacaftor (10 mM), forsko-lin (20 mM), and INH-172 (10 mM). Resveratrol (trans isoform)was utilized at a range of doses to establish a working concentra-tion. Amiloride blocks epithelial sodium (Na1) channels andsupports the notion that variation in short-circuit current (DISC)from subsequent manipulation is due to effects on Cl2 channelactivity. Forskolin stimulates the cystic fibrosis transmembraneconductance regulator via cAMP-mediated/PKA-dependent phos-phorylation of the R-D, as described above. INH-172 is a selectiveinhibitor of CFTR and aids in determining the degree of contribu-tion of CFTR to the ISC under experimental conditions. Corre-sponding DMSO (vehicle) control solutions for resveratrol andivacaftor were studied in parallel. The ISC was assessed at onecurrent measurement per second. By convention, a positivedeflection in ISC was defined as the net movement of anions inthe serosal to mucosal direction.

Single-Channel Studies. Conventional patch clamp tech-niques were adapted to record single-channel currents by cell-attached and inside-out configuration using MNSE andHEK293 cells expressing WT human CFTR. Recording pipetteswere constructed from borosilicate glass capillaries (WarnerInstruments, Hamden CT) using a Narishige PP83


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microelectrode puller and fire polished with a PP90 microforge(Narishige, Tokyo, Japan). The pipettes were partially filledwith a solution containing (in mmol/l) 150 CsCl, 1 CaCl2, 1MgCl2 and 10 HEPES (pH 7.2), with tip resistances of 6 to 8MX. All experiments were performed at room temperature (20–228C). Single-channel currents were obtained using an Axopatch200B patch clamp amplifier (Axon Instruments, Molecular Devi-ces, Sunnyvale, CA), with voltage commands and data acquisi-tion controlled by Clampex software (pClamp 10, AxonInstruments) and digitized (Digidata 1440A interface, AxonInstruments) at a sampling frequency of 1 kHz. MNSE orHEK293 cells seeded on glass coverslips were mounted in aflow-through chamber. To acquire seals, bath solutions con-tained (in mmol/l) 140 NaCl, 4.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 10glucose, and 10 HEPES pH 7.4. For inside-out patches, bathsolution was comprised of (in mmol/l) 150 CsCl, 5 EDTA, and 10HEPES pH 7.4 designed to minimize channel rundown. Resver-atrol was used at a working concentration of 100 mM. Vehiclecontrols were included in all studies (DMSO 0.02%). Confirma-tion that stimulated currents were due to CFTR activity wasachieved using the inhibitor INH-172 at 10 mM. Single-channelrecordings were analyzed using pClamp 10 software whereVcom 5 command potential (Axon Instruments). Tracings werefiltered postacquisition at 500 Hz.

Nasal Potential Difference Assay. A 3-step protocol wasused, as described previously.55 Animals were anesthetized withketamine (200 mg/kg), acepromazine (0.6 mg/kg), and xylazine(30 mg/kg) by intraperitoneal injection prior to testing. First,nasal cavities of anesthetized mice (C57/BL6) were perfusedsequentially with 1) Ringer’s solution containing 140 mM NaCl,5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, andamiloride 50 lM (pH 7.3); 2) amiloride 1 a low-Cl2-containingsolution (NMDG, 6 mM Cl2, pH 7.3); and 3) amiloride 1 low-Cl2-containing solution 1 resveratrol, forskolin 20 mM, orDMSO control. Because of the continuous presence of amiloride(50 mM) and the complete replacement of Na1 with themembrane-impermeant cation NMDG (140 mM) in the perfu-sion solution, hyperpolarization in this setting reflects Cl2

secretion rather than Na1 absorption. Each condition was stud-ied for 5 to 10 minutes until a steady signal was achieved.Activity of the test solution was measured from the stable base-line to the highest point of hyperpolarization. All traces wereinterpreted in a blinded fashion.

CFTR R-D Phosphorylation and cAMP LevelsBecause activation of CFTR-mediated anion transport

requires PKA-dependent phosphorylation of the R-D, an ELISA-based detection kit (Cayman Chemicals, Ann Arbor, MI) wasused to measure stimulation of cellular cAMP by resveratrol inMNSE cultures, as previously described.56,57 To confirm absenceof a cAMP/PKA dependent mechanism, polyclonal NIH-3T3cells expressing HA-tagged R-D were treated with resveratrolfor 20 minutes and compared to forskolin (20 mM) 3 5 minutesas a positive control and to DMSO as a negative control. Follow-ing lysis, equal amounts (50 mg) of total cell lysate were electro-phoresed through a 12% sodium dodecyl sulfate-polyacrylamidegel (SDS-PAGE) and immunoblotted with antibody to the HAtag (Covance, Cumberland, VA). Phosphorylation of the R-Dwas visualized as a 2 to 4 kD shift in migration, as previouslydescribed.56

Airway Surface Liquid DepthConfocal Laser Scanning Microscopy. Cells were

washed three times with PBS followed by administration of 100lM CellTracker Green BODIPY (Invitrogen, cat. no.: C2102) to

the basolateral medium and 10 mg/ml Texas Red (Invitrogen,cat. no.: D-1863) in FC-70 (Flourinert FC-70, Fisher, cat. no.:NC 9062226) to the apical side 2 hours before confocal visual-ization. Cells incubated at 1% O2/5% CO2 were compared tophysiologic O2 (air control). Resveratrol, resveratrol 1

INH2172 (10 mM), or vehicle (DMSO) control were added toapical (in 30-ml volume) chambers, and imaging was performedwith a Zeiss LSM 710 Confocal Microscope (Oberkochen, Ger-many) using Zeiss Plan Apo 203 .8 na dry objective in 1-micronsteps. Airway surface liquid (ASL) depth was measured in theorthogonal (head on X–Z) image view. Two regions of interestwere analyzed for each monolayer, and average ASL depth wasmeasured for five equally distributed locations in each region.

Statistical AnalysisStatistical analyses were conducted using Excel 2010

(Microsoft Corp., Redmond, WA) and GraphPad Prism 6.0 soft-ware (GraphPad Prism, La Jolla, Ca) with significance set atP<0.05. Statistical evaluation utilized paired and unpairedStudent t tests, the Mann-Whitney rank sum test, or the analy-sis of variance followed by Tukey-Kramer multiple comparisontest, as appropriate.

RESULTS

Hypoxic Primary Sinonasal Epithelial CulturesAre a Valid In Vitro Model of Acquired CFTRDeficiency

In order to establish and confirm the validity ofhypoxia-induced acquired CFTR deficiency, MNSE andHSNE cultures were incubated for 12 and 24 hours at1% O2 and evaluated in Ussing chambers using pharma-cologic manipulation. The change in short-circuit current(DISC 5mA/cm2) attributable to CFTR (forskolin-stimu-lated transport) was significantly decreased at 12 and 24hours in MNSE (Fig. 1A, 1B), incubated in an oxygen-restricted environment (13.55 6 0.46 [12 hours];12.75 6 0.07 [24 hours] vs. 19.23 6 0.18 [control];P< 0.05). Inhibition with the specific CFTR inhibitor,INH-172, was also markedly decreased (210.79 6 0.10[12 hours]; 29.57 6 0.28 [24 hours] vs. 217.19 6 1.80[control]; P<0.05) indicating overall deficiency of CFTR-dependent anion transport after hypoxia. Of note, ISC

attributable to epithelial Na1 channel (ENaC) transport,as measured by amiloride blockade, was nearly absentby 12 hours (20.16 6 0.01 vs. 27.76 6 0.80; P< 0.05).

Similar to MNSE, forskolin-stimulated DISC inHSNE (Fig. 2A, 2B) was sensitive to hypoxic stress anddemonstrated significant reductions in CFTR-mediatedCl2 transport (19.55 6 0.56 [12 hours]; 17.67 6 1.13 [24hours] vs. 25.49 6 1.48 [control]; P< 0.05). DecreasedDISC following INH-172 pharmacologic blockade verifieda decreased contribution of CFTR to the ISC

(223.67 6 0.05 [12 hours]; 223.21 6 1.86 [24 hours] vs.232.66 6 1.15 [control]; P< 0.05). Sodium absorption inHSNE (amiloride blockade) appeared to be resistant tohypoxia, and in fact was significantly increased at 12hours (221.13 6 0.27 [12 hours] vs. 210.45 6 1.05 [24hours]; P< 0.05) and returned to baseline by 24 hours(212.89 6 0.37). HSNE also demonstrated recovery ofCl2 transport following 24 hours in physiologic O2 (21%)(25.12 6 1.24). However, Na1 absorption was


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significantly inhibited following return to atmosphericO2 environment (26.05 6 0.33).

Hypoxia Depletes Airway Surface LiquidTo assess whether the ion transport deficiencies

observed in the Ussing chamber would confer abnormal-ities of ASL, hypoxic MNSE were evaluated at 24 hoursby CLSM and the mOCT techniques. ASL depth wasdecreased under hypoxic conditions when measured byCLSM (in mm: 4.19 6 0.44, hypoxia; 6.88 6 0.67, n�5 percondition, control; P< 0.05) providing direct evidencethat altered ion transport in this culture model leads toASL depletion (Fig. 3).

Resveratrol Is a Robust CFTR-Dependent Cl2

SecretagogueFollowing confirmation of our model of hypoxia-

induced CFTR deficiency, a comprehensive evaluation ofresveratrol was implemented to assess its utility as aCl2 secretagogue in sinonasal epithelium. Dose-dependent CFTR-mediated anion transport (DISC) wasanalyzed using MNSE cultures in Ussing chambers.Resveratrol activated CFTR-mediated Cl2 conductancein a dose-dependent fashion up to 500 mM, but it demon-strated modest inhibitory effects on total CFTR activa-tion with posttreatment forskolin (100 nM) atconcentrations> 100 mM (Fig. 4A and 4B). Thus, 100 mMresveratrol (DISC, 13.51 6 0.77 vs. 4.4 6 0.66 [control];

Fig. 2. HSNE exhibits a CF-like ion transport phenotype. Hypoxic(1%) HSNE cells were studied in Ussing chambers under identicalconditions to MNSE, plus the addition of a 24-hour recoveryperiod under physiologic O2 (21%). Representative tracings at 24hours (A) show stable amiloride-sensitive ISC but diminishedCFTR-mediated ISC (forskolin). Summary data (B) reveal a relativeincrease in DISC attributable to sodium blockade with amiloride at12 hours that returns to baseline at 24 hours. Forskolin-stimulatedDISC was sensitive to hypoxic stress and demonstrated significantreduction in CFTR-mediated Cl2 transport (n�6 per condition).Decreased DISC from INH-172 blockade verified the contributionof CFTR to the ISC. HSNE demonstrated significant recovery ofCl2 transport following 24 hours in a physiologic O2 (21%) envi-ronment. (Adapted from Blount et al.63). CFTR 5 cystic fibrosistransmembrane conductance regulator; HSNE 5 human sinonasalepithelial; MNSE 5 murine nasal septal.

Fig. 1. Hypoxia induces acquired defects in transepithelial iontransport in murine nasal epithelia. Murine nasal septal cellsgrown on transwell permeable supports and incubated in 1% O2

or physiologic O2 (control, 21%) for 12 or 24 hours were mountedin Ussing chambers under short-circuit conditions and sequen-tially exposed to amiloride (100 mM), forskolin (20 mM), and INH-172 (10 mM). Representative tracings (at 24 hours) are shown in(A). By convention, a positive deflection in the tracing representsmovement of anion in the serosal to mucosal direction. Summarydata is represented in (B). Under these conditions, change inshort-circuit current (DISC) was significantly decreased at all timepoints with amiloride (used as a means to monitor epithelialsodium channels) and forskolin (a pharmacologic activator ofCFTR). CFTR blockade by INH-72 was also significantlydecreased compared to control (n�6 per condition). (Adaptedfrom Blount et al.63). CFTR 5 cystic fibrosis transmembrane con-ductance regulator.


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P< 0.05) was considered an optimal dose for subsequentstudies.

CFTR2/2 knockout MNSE on the same geneticbackground were used to confirm that resveratrol-stimulated DISC is mediated through a CFTR-dependentmechanism (Fig. 4C). Following application of amiloride,resveratrol (100 mM) resulted in no measurable transepi-thelial secretion; therefore, no contribution from alterna-tive Cl2 transport pathways (i.e., calcium-activated Cl2

channels) was implicated.Activities in culture derived from other mammalian

species (including human) were next evaluated. Murine([DISC, 14.2 6 1.5 vs. 0.8 6 0.2 (control)], human[17.4 6 0.7 vs. 1.0 6 0.2 (control)], and porcine [6.8 6 0.3vs. 1.1 6 0.3 (control]) sinonasal airway epithelial cellsexhibited significant CFTR-mediated anion secretion fol-lowing application of the drug (P< 0.05) (Fig. 5A and5B). Resveratrol activated between 50% and 70% of totalCFTR-dependent ISC, as judged by addition of saturating(20 mM) forskolin (murine 22.0 6 1.8, human 29.4 6 1.5,and porcine 14.2 6 0.2). The drug produced equivalentlevels of CFTR-mediated anion transport when com-pared to ivacaftor (16.5 6 1.9 at an optimal concentrationof 10 mM) in human sinonasal epithelial cultures(Fig. 5C).

Resveratrol Activates Human Sinonasal CFTR-Mediated Cl2 Transport Ex Vivo

Once in vitro evaluation was confirmed, activity infreshly excised sinus tissue from individuals undergoingendoscopic sinus surgery was performed (n 5 5; pairedsamples) (Fig. 6A and 6B). Resveratrol briskly stimu-lated CFTR-mediated Cl2 transport in human sinusexplants (78.42 6 1.75 vs. 1.75 6 1.5 [control], P< 0.05).Amiloride-sensitive ISC (2193.5 6 23.5 vs. 179.6 6 5.7[control]), representing epithelial sodium channel activ-ity, total stimulated ISC with forskolin (resvera-trol 1 forskolin 163.0 6 67.9 vs. control 1 forskolin152.4 6 30.0), and CFTR inhibition with INH-172(2165.7 6 40.9 vs. 145.2 6 46.8) were equivalent betweengroups, indicating that physiologic ion transport wasintact among resveratrol treated and untreated mucosalsamples.

Resveratrol Stimulates CFTR-Dependent Cl2

Secretion Across In Vivo Murine NasalEpithelium

To establish resveratrol’s capabilities to activate Cl2

transport in nasal epithelium in vivo, the murine nasalpotential difference assay was utilized. Mice perfused

Fig. 3. ASL depth is decreased in hypoxic MNSE. BecauseCFTR secretes Cl2 ions to regulate ASL depth, an importantdeterminant of normal mucociliary clearance, we evaluatedwhether hypoxic incubation for 24 hours would induce ASLdepletion in MNSE by confocal microscopy (panel A representsconfocal images). Incubation in 1% O2 decreased ASL depthcompared with control monolayers (4.19 6 0.44 (hypoxia) versus6.88 6 0.67 lm (21% O2 control); P<0.05) (B). ASL 5 airway sur-face liquid; CFTR 5 cystic fibrosis transmembrane conductanceregulator; MNSE 5 murine nasal septal.


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Fig. 4. Resveratrol stimulates CFTR-mediated Cl2 transport in vitro.Representative Ussing chamber tracings reveals resveratrol activationof Cl2 transport in a dose-dependent fashion (A). Note the negativedeflection after a peak effect with subsequent inhibition of forskolin(100 nM) response at higher concentrations of resveratrol. Summarydata (B) of resveratrol stimulation vs. total stimulation (resvera-trol 1 forskolin [20 mM]) of transepithelial Cl2 secretion (n�3 per con-dition). Resveratrol concentrations>100 uM result in modestlydecreased total stimulation. (C) Ussing chamber tracings of CFTR2/2

MNSE monolayers exposed to resveratrol (100 mM) or DMSO controlfollowing amiloride (100 nM) blockade shows no measurableresponse, indicating Cl2 secretagogue activity is dependent on thepresence of CFTR. (Adapted from Alexander et al.64). CFTR 5 cysticfibrosis transmembrane conductance regulator; MNSE 5 murinenasal septal; Dimethyl sulfoxide. [Color figure can be viewed in theonline issue, which is available at www.laryngoscope.com.]

Fig. 5. Resveratrol activates CFTR-mediated Cl2 secretion in pri-mary sinonasal epithelial cell cultures from several mammalianspecies. (A) Representative Ussing chamber tracings show resver-atrol (100 mM) activation of Cl2 transport in murine, porcine, andhuman sinonasal epithelial cell cultures. (B) Graphic representationof resveratrol stimulation versus total stimulation (resveratrol [100mM] 1 forskolin [20 uM]) for transepithelial Cl2 transport in thesecells. Significant stimulation (P<0.05) of Cl2 transport was dem-onstrated in primary sinonasal cultures as compared to untreatedvehicle controls (DMSO) in all species. Total stimulation (additionof 20- mM forskolin) was also significantly greater than resveratrolalone (P<0.05). Resveratrol (as a percentage of total stimulation)consistently activated 50% to 70% of total CFTR-mediated aniontransport. (C) Resveratrol activates wild-type CFTR-mediatedanion transport to the same magnitude as ivacaftor (10 mM) inhuman sinonasal epithelial cultures. (Adapted from Zhang et al.54).CFTR 5 cystic fibrosis transmembrane conductance regulator;DMSO 5 Dimethyl sulfoxide. [Color figure can be viewed in theonline issue, which is available at www.laryngoscope.com.]


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with resveratrol (100 uM) in the presence of amiloride(100 mM) and a Cl2 secretory gradient demonstrated sig-nificant hyperpolarization (24 6 1.87 mV) comparedwith continued depolarization seen in mice perfusedwith vehicle and identical conditions [20.93 6 1.69 mV,P< 0.01). Activity was increased compared to the posi-tive control (forskolin, 21.65 6 1.78 mV), although thiswas not statistically significant (Fig. 7A and 7B).

CFTR Is Activated in the Absence of R-DPhosphorylation

PKA/cAMP-dependent phosphorylation of the CFTRR-D is a critical step in channel activation, but it hasbeen previously demonstrated that flavonoid compounds(e.g., genistein58) activate CFTR through a mechanismindependent of PKA/cAMP-dependent phosphorylation.To examine whether resveratrol might activate CFTRvia PKA-dependent pathways, cAMP levels and R-Dphosphorylation were evaluated. Unlike the cAMP ago-nist forskolin, resveratrol had no measurable effect onthe phosphorylation-dependent mobility shift of recombi-

nant CFTR R-D and did not elevate cellular cAMP(Fig. 8).

Resveratrol Increases CFTR Open ProbabilityOnce we recognized that resveratrol did not activate

CFTR via PKA-dependent pathways, patch clamp tech-niques were required to evaluate impact on CFTR chan-nel open time. Segments of the apical plasma membranesealed within the tip of a patch pipette were evaluatedafter application of test compounds to the intracellularsurface in the excised, inside-out patch clamp configura-tion. In patches from apical membranes of MNSE cells,resveratrol stimulated an ;8 pS chloride channel con-sistent with CFTR potentiation in the absence of ATPand PKA. These experiments are the first to character-ize single-channel CFTR in freshly isolated/low passagenumber sinonasal epithelia. Patches were excised and

Fig. 6. Resveratrol activates CFTR-mediated Cl2 secretion inhuman sinonasal mucosa ex vivo. Representative Ussing chambertracings of full-thickness human sinonasal tissue explants demon-strating robust activation of CFTR-dependent Cl2 secretion with100-mM resveratrol compared to DMSO controls (A). Ussingchamber summary data evaluating paired sinus mucosal samplesfrom five individuals and demonstrating significant activation(P<0.05) of ex vivo CFTR-dependent Cl2 transport (B). (Adaptedfrom Zhang et al.54). CFTR 5 cystic fibrosis transmembrane con-ductance regulator; DMSO 5 Dimethyl sulfoxide.

Fig. 7. Murine nasal potential difference measurements demon-strate activation of Cl2 transport in vivo. (A) Mice underwent astandardized NPD protocol with the addition of 100 uM resveratrol(or vehicle control) in the final perfusion solution (20 mg/mL in Cl2

free gluconate solution and amiloride [100 mM]). A representativetracing is shown from C57 mice stimulated with resveratrol in thefinal perfusate. (B) Resveratrol perfusion resulted in a 24 mVmean NPD polarization that was significantly different than inmice-receiving vehicle alone (P<0.05). (Adapted from Alexanderet al. 64). NPD 5 nasal potential difference. [Color figure can beviewed in the online issue, which is available at www.laryngoscope.com.]


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spontaneous activity recorded at 2Vcom 5 150 mV.Channel function was enhanced within seconds ofadministration of 100 mM resveratrol (Fig. 9A, upper

tracing). This observation was validated in D060/HEK293 cells expressing exogenous human WT-CFTR(Fig. 9A, lower tracing). Under cell-free conditions, anincrease in channel Po (rather than new channel inser-tion) was determined to account for CFTR activation.Complete inhibition of stimulated current by INH-172further supports the finding that CFTR activity is ampli-fied by resveratrol.

Open probability (NPo/N, where N represents chan-nel number) was calculated from single-channel record-ings following perfusion with 100-mM resveratrol andunder control conditions. Open probability was signifi-cantly augmented following the application of resvera-trol in MNSE cells (0.329 6 0.116 vs. 0.119 6 0.059 forcontrol, P< 0.05). This result was confirmed in D060/HEK293 cells expressing human WT-CFTR, where openprobability was significantly increased compared to con-trol (0.22 6 0.048 vs. 0.125 6 0.07, P< 0.05) (Fig. 9B).

Resveratrol Activates Transepithelial Cl2

Secretion in Hypoxic Cells and ReversesHypoxia-Induced Reduction of ASL Depth

Following comprehensive evaluation of the Cl2

secretagogue activity of resveratrol and its underlyingmechanism of action, we next investigated the ability ofthe agent to compensate for the acquired defects inCFTR-mediated anion secretion demonstrated in hypoxicsinonasal epithelium. After 24 hours incubation in ahypoxic environment, MNSE and HSNE filters were

Fig. 8. Resveratrol does not increase cellular cAMP or phosphoryl-ate the CFTR R-D. Cellular cAMP was minimal in the presence ofresveratrol and not significantly changed when compared toDMSO control. The adenylate cyclase agonist forskolin was usedas a positive control. Polyclonal NIH-3T3 cells expressing HA-tagged R-domain were treated with resveratrol for 20 minutes,and compared to forskolin (20 mM) or DMSO control in an assayof R-D phosphorylation (inset). Phosphorylation results in a 2–4kD shift in migration (white arrow). (Adapted from Alexanderet al.64). cAMP 5 cyclic adenosine monophosphate; CFTR 5 cysticfibrosis transmembrane conductance regulator; R-D 5 regulatorydomain; DMSO 5 Dimethyl sulfoxide. [Color figure can be viewedin the online issue, which is available at www.laryngoscope.com.]

Fig. 9. Resveratrol stimulates single CFTR channel activity. (A)Under nonphosphorylating conditions, application of resveratrolenhanced-channel activity in both MNSE (upper tracing) andD060/HEK293 cells expressing exogenous WT human CFTR(bottom tracing). Patches were excised and spontaneous channelfunction recorded at –Vcom 5 150 mV. Activity was stimulatedwithin seconds of application of 100 mM resveratrol. Similarenhancement was observed in human CFTR-expressing D060cells and abrogated by addition of INH-172. Dotted lines indicatezero current level. (B) Open probability (NPo/N where N repre-sents channel number) was calculated from single-channelrecordings under control conditions and after perfusion with 100-mM resveratrol obtained in similar experiments to those displayedin (A). Data are presented as mean 6 SEM. Data points werecompared with control using paired Student t test (*P<0.05).(Adapted from Zhang et al.54). ASL 5 airway surface liquid;CFTR 5 cystic fibrosis transmembrane conductance regulator;MNSE 5 murine nasal septal; DMSO 5 Dimethyl sulfoxide.


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evaluated in the Ussing chamber for the ability ofresveratrol to stimulate CFTR-mediated anion transport.The drug significantly increased CFTR-dependent Cl2

transport in MNSE (11.51 6 0.23 vs. 0.2 6 0.05 [control];P< 0.05) and HSNE (10.8 6 0.7 vs. 0.3 6 0.05 [control];P< 0.05) when compared to control vehicle (Fig. 10). Asa percentage of the maximal CFTR stimulus induced byforskolin (20 mM) in murine (21.2 6 0.89) and human(22.3 6 1.4) monolayers, resveratrol activated 54.3% and48.4% of total ISC, respectively.

With evidence that resveratrol activates CFTR-mediated anion transport in cells that are deficient inthe ability to secrete transepithelial Cl2 due to acquiredCFTR deficiency, we next assessed whether resveratrolmitigated the depleted ASL demonstrated in oxygen-restricted cultures. First, we established that Cl2 secre-tagogue activity attributable to resveratrol would trans-late to hydration of ASL in epithelium compared tocorresponding DMSO vehicle control in a normal oxygenenvironment. Resveratrol increased ASL depth in MNSEcultures after a 30-minute apical application (in lm:8.08 6 1.68 vs. 6.11 6 0.47, DMSO control, P< 0.05, n� 5per condition) (Fig. 11), indicating a robust treatmenteffect resulting from stimulation of apical Cl2 secretionand enhanced CFTR channel Po. ASL hydration was sig-nificantly diminished by addition of the INH-172(3.54 6 0.34, P< 0.05). INH-172 also suppresses constitu-tively activated CFTR, accounting for the overalldecrease in ASL depth. The effects of drug were thenmeasured on hypoxic MNSE and compared to the controlsolution. Hypoxia-induced abnormalities of fluid andelectrolyte secretion in sinonasal epithelium wereimproved by a 30-minute application of resveratrol fol-lowing 24 hours of hypoxia (5.55 6 0.74 vs. 3.13 6 0.17,

n�5 per condition, P<0.05), providing confirmationthat the compound mitigates effects of acquired CFTRdeficiency on depleted ASL (Fig. 12).

DISCUSSIONThe data presented in the current study suggests

that a low oxygen environment profoundly affects nor-mal ion transport physiology in both MNSE and HSNEand leads to acquired defects in CFTR-mediated trans-port. Although stimulation of transepithelial Cl2 trans-port would be expected to hydrate the ASL, increasedNa1 absorption (as demonstrated in CF) reduces watercontent, leading to decreased clearance of dehydratedand eventually impacted mucus.13 Notably, ENaC chan-nels exhibited a time-dependent blockade, as detected byamiloride-dependent ISC in MNSE. Thus, both Na1

absorption and Cl2 secretion are reduced in the hypoxicmurine model; an indication that the overall influence ofhypoxia on the ASL could be mitigated by opposingeffects on epithelial Na1 and Cl2 transport (with a net

Fig. 10. Resveratrol restores transepithelial Cl2 transport inhypoxic cells. Murine nasal septal and human sinonasal epithelialmonolayers were subjected to 24 hours of hypoxia, and the effectof resveratrol was evaluated in Ussing chambers. Resveratrol sig-nificantly increased ISC compared to vehicle controls in hypoxicmurine (11.51 6 0.23 vs. 0.2 6 0.05 (control); P<0.05) and human(10.8 6 0.7 vs. 0.3 6 0.05(control); P<0.05) monolayers; maximalcystic fibrosis transmembrane conductance regulator stimulusinduced by forskolin indicates resveratrol activated ISC to 54.3%and 48.4% in murine (21.2 6 0.89) and human (22.3 6 1.4) mono-layers, respectively (DMSO 5 Dimethyl Sulfoxide).

Fig. 11. Resveratrol hydrates ASL. Confocal images demonstratetreatment effect form stimulation of apical Cl2 secretion andenhanced CFTR channel Po (red-ASL; green-cell marker) (A).Resveratrol significantly increased ASL depth (in lm) in murinenasal septal epithelial cultures (8.08 6 1.68 vs. 6.11 6 0.47, control,*P<0.05). Addition of INH2172 inhibited resveratrol-dependentASL accumulation (3.54 6 0.34, *P<0.05) (B). (Adapted from Zhanget al.54). ASL 5 airway surface liquid; DMSO 5 Dimethyl Sulfoxide.


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effect of diminished ASL depth, as measured in ourexperiments). HSNE exhibited a marked reduction inforskolin-stimulated ISC over 24 hours of low-oxygenexposure. In contrast to the murine situation, however,Na1 absorption increased during the first 12 hours (asmeasured by amiloride blockade of ENaC). The findingthat HSNE develops a globally decreased transepithelialCl2 secretion and increased Na1 absorption in responseto hypoxic conditions indicates that in human sinonasalmucosa, the detrimental effects on ion transport thatimpact ASL hydration (Cl2 and HCO3

2 secretionthrough CFTR; Na1 absorption through ENaC) are com-pounded by tissue hypoxia. Thus, the ion transport prop-erties are consistent with CF airway pathophysiology(decreased Cl2 secretion and enhanced Na1 absorption),resulting in dehydration of the ASL, and ultimately dis-rupted mucociliary transport.

CFTR activation has been shown to confer excellenttherapeutic benefit in CF, a disease characterized bydefective MCC. The drug ivacaftor has recently beenFood and Drug Administration-approved for CF patientswith at least one copy of the G551D mutation, but theagent is very expensive (approximately $300,000/year inthe U.S.).59 Recent studies have also shown that ivacaf-tor augments ASL depth, accelerates MCC, and pharma-cologically reverses acquired CFTR dysfunction due tocigarette smoke exposure.20 Given this precedent,resveratrol represents an excellent candidate for treat-

ment of acquired Cl2 transport defects based on anestablished safety profile,60 activity equivalent to ivacaf-tor with regard to WT human CFTR stimulation, andwell described antiinflammatory benefits. In addition,the working concentration of resveratrol was 100 mM, adrug concentration routinely achievable in topicallyapplied, superperfused, or aerosolized solutions inhuman subjects in vivo.61,62 Single-channel patch clampanalysis indicates that the mechanism underlyingresveratrol activity is CFTR channel potentiation; chan-nel open probability increases with drug application, asdemonstrated in both WT MNSE cells and a recombi-nant human CFTR expressing cell line. Resveratrol’sconserved activity across several mammalian speciesalso provides a means to study MCC activation in ani-mal models (unlike ivacaftor, which has no effect onmurine or porcine CFTR in primary nasal airway epithe-lium [data not shown]).31

Because resveratrol is a robust CFTR channel poten-tiator in sinonasal epithelia, we hypothesized that thedrug could ameliorate acquired CFTR dysfunction con-ferred by hypoxic incubation. The compound effectivelystimulated transepithelial anion secretion in hypoxic epi-thelial cells and increased ASL thickness in both controland hypoxic monolayers, indicating that 1) the increase inhydration is most likely attributable to potentiatingeffects on CFTR channels; and 2) acquired CFTR dysfunc-tion represents a therapeutic target that can be addressed

Fig. 12. Resveratrol restores hypoxia-depleted ASL. Because api-cal fluid is applied with resveratrol treatment, hypoxia controlsrequire the corresponding control (DMSO) vehicle. Confocalimages are demonstrated in panel A. Resveratrol treatment signif-icantly mitigated hypoxia-induced reductions in ASL depth(3.13 6 0.17 lm [DMSO 1 hypoxia] versus 5.55 6 0.74 lm [resver-atrol 1 hypoxia] P<0.05), reaching 91% of the ASL depth foundin normal murine nasal septal monolayers incubated with DMSO(6.11 6 0.47, from Fig. 11) (B). ASL 5 airway surface liquid;DMSO 5 Dimethyl sulfoxide.


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with resveratrol and/or other Cl2 secretagogues. Activa-tion that is independent of PKA provides additional evi-dence of direct CFTR binding of the compound, and thepotentiating effects of the drug represent an optimalapproach to directly stimulate CFTR, fluid and electrolytetransport, and ASL hydration. Because acquired defectsof CFTR in chronic diseases such as sinusitis can result inboth decreased channel expression and increased turn-over in nasal airway epithelium,63 potentiating CFTRchannels already situated in the plasma membrane couldserve as an effective strategy for reversing ASL dehydra-tion in individuals with CRS.

CONCLUSIONHypoxia-induced–acquired CFTR dysfunction can be

ameliorated by resveratrol potentiation of CFTR chan-nels, resulting in improved epithelial function. CFTR acti-vation with Cl2 secretagogues such as resveratrolrepresents an innovative approach to overcomingacquired CFTR defects in sinus and nasal airway disease.Additional studies to define the role of CFTR activation byresveratrol are indicated in CRS and could result in noveland improved therapeutic strategies for the disease.

AcknowledgmentsShaoyan Zhang, Ph.D., procurement of data; Daniel Skin-ner, B.S., procurement of data; Carmel McNicholas, Ph.D.,expertise in patch clamp technique; John C. Kappes, M.D.,generous gift of D060 cells; Eric J. Sorscher, M.D.,mentorship.

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