title: Resveratrol ameliorates abnormalities of fluid and electrolyte secretion in a hypoxia-Induced model of acquired CFTR deficiency.
abstract: OBJECTIVE
  Ineffective mucociliary clearance (MCC) is a common pathophysiologic process that underlies airway inflammation and infection. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed by the cystic fibrosis transmembrane conductance regulator (CFTR). Decreased transepithelial Cl(-) transport secondary to an acquired CFTR deficiency may exacerbate respiratory epithelial dysfunction by diminishing MCC and increasing mucus viscosity. The objectives of the present study are to 1) develop a model of acquired CFTR deficiency in sinonasal epithelium using hypoxia, 2) investigate whether the polyphenol resveratrol promotes CFTR-mediated anion transport, 3) explore resveratrol mechanism of action and determine therapeutic suitability for overcoming acquired CFTR defects, and 4) test the drug in the hypoxic model of acquired CFTR deficiency in preparation for a clinical trial in human sinus disease. We hypothesize that hypoxia will induce depletion of airway surface liquid (ASL) secondary to acquired CFTR deficiency and that resveratrol will restore transepithelial Cl(-) secretion and recover ASL hydration.
  
  
  METHODS
  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 current = ΔISC ) evaluated in Ussing chambers. Resveratrol was tested using primary cells and HEK293 cells expressing human CFTR by Ussing chamber and patch clamp techniques under both phosphorylating and nonphosphorylating conditions. CFTR activation was evaluated in human 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. Effects of hypoxia and resveratrol on ASL were tested using confocal laser scanning microscopy (CLSM) and micro-optical coherence tomography (µOCT).
  
  
  RESULTS
  Hypoxia significantly decreased ΔISC (in µA/cm(2) ) attributable to CFTR at 12 and 24 hours of exposure in both MNSE (13.55 ± 0.46 [12 hours]; 12.75 ± 0.07 [24 hours] vs. 19.23 ± 0.18 [control]; P < 0.05) and HSNE (19.55 ± 0.56 [12 hours]; 17.67 ± 1.13 [24 hours] vs. 25.49 ± 1.48 [control]; P < 0.05). We have shown that resveratrol (100 μM) enhanced CFTR-dependent Cl(-) secretion in HSNE to an extent comparable to the recently Food and Drug Administration-approved CFTR potentiator, ivacaftor. Cl(-) transport across human sinonasal explants (78.42 ± 1.75 vs. 1.75 ± 1.5 [control]; P < 0.05) and in vivo murine nasal epithelium (-4 ± 1.8 vs. -0.8 ± 1.7 mV [control]; P < 0.05) were also significantly increased by the drug. No increase in cAMP or CFTR R-D phosphorylation was detected. Inside-out patches showed increased CFTR open probability (NPo/N (N = channel number]) compared to controls in both MNSE (0.329 ± 0.116 vs. 0.119 ± 0.059 [control]; P < 0.05) and HEK293 cells (0.22 ± 0.048 vs. 0.125 ± 0.07 [control]; P < 0.05). ASL thickness was decreased under hypoxic conditions when measured by CLSM (4.19 ± 0.44 vs. 6.88 ± 0.67 [control]; P < 0.05). A 30-minute apical application of resveratrol increased ASL depth in normal epithelium (8.08 ± 1.68 vs. 6.11 ± 0.47 [control]; P < 0.05). Furthermore, hypoxia-induced abnormalities of fluid and electrolyte secretion in sinonasal epithelium were restored with resveratrol treatment (5.55 ± 0.74 vs. 3.13 ± 0.17 [control]; P < 0.05).
  
  
  CONCLUSIONS
  CFTR activation with a leading edge Cl(-) secretagogue such as resveratrol represents an innovative approach to overcoming acquired CFTR defects in sinus and nasal airway disease. This exciting new strategy bears further testing in non-CF individuals with chronic rhinosinusitis.
  
  
  METHODS
  N/A. Laryngoscope, 125:S1-S13, 2015.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
authors.forename: Bradford A
authors.initials: BA
authors.lastname: Woodworth
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000627:therapeutic use*; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects*; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000860:Hypoxia / Q000150:complications*; D017136:Ion Transport / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008954:Models, Biological; D009079:Mucociliary Clearance / Q000187:drug effects*; D009297:Nasal Mucosa / Q000187:drug effects / Q000378:metabolism; D010256:Paranasal Sinuses / Q000166:cytology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D000077185:Resveratrol; D013267:Stilbenes / Q000627:therapeutic use*; D013552:Swine
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D018118:Chloride Channels; D013267:Stilbenes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D000077185:Resveratrol
keywords: CFTR; Resveratrol; Ussing chamber; acquired CFTR deficiency; airway surface liquid; chronic rhinosinusitis; cystic fibrosis; electrophysiology; hypoxia; ion transport; patch clamp; sinusitis