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Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β2 adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o− cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o− cells. High concentrations inhibited Cl− conductance. In CFBE41o− airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β2 adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr+ mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr−/− mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference.
This article describes the use of the flavonoid quercetin as an important adjunct for detecting surface-localized ΔF508 CFTR by nasal potential difference, a topic of considerable interest in the field of cystic fibrosis (CF) therapeutics, and relevant to emerging therapies in clinical testing. Our findings elucidate important mechanistic features of quercetin-mediated CFTR activation in vitro, and provide data in mice and humans that support the use of the agent in subjects with CF homozygous for ΔF508 CFTR, to identify individuals with residual surface protein, and in the evaluation of correctors of ΔF508 CFTR processing.
The most common cause of cystic fibrosis (CF) is attributable to the deletion of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), which causes misfolding of the protein product in the endoplasmic reticulum (ER) and accelerated degradation in the proteosome. At least one copy of the ΔF508 mutation is found in approximately 90% of patients with CF, and is therefore a logical target for treating the disease. The absence of CFTR at the cell plasma membrane results in defective ion transport and subsequent clinical manifestations of CF (1). Several maneuvers were identified that correct ΔF508 CFTR misprocessing, including cell growth at low temperature (2), treatment with chemical chaperones that promote ER escape (3–5), and exposure to small molecules (identified by high-throughput screening programs and other means) that promote protein localization to the plasma cell membrane (6–9).
Numerous laboratories showed that the ΔF508 CFTR demonstrates defective channel gating (10–12). These findings are evident in airway cell monolayers, because ΔF508 CFTR rescue using low temperatures or small molecule correctors of misprocessing do not consistently restore the cAMP-dependent activation of Cl− transport, a finding that is also germane to measures of ion transport across the human nasal membrane (13, 14). Thus, proof-of-concept studies testing agents that correct ΔF508 CFTR processing may be hampered if assays of CFTR activity, such as nasal potential difference (NPD), fail to stimulate membrane-localized ΔF508 CFTR adequately. Recently, an evaluation of the ΔF508 CFTR processing corrector (VX-809) has entered Phase 2 testing in human subjects, and includes assessment via NPD, further emphasizing the importance of an adequate stimulus within the measurement of NPD (NCT00865904).
The standard stimulus of CFTR-dependent Cl− transport is perfusion with β2 adrenergic receptor (β2 AR) agonists (e.g., isoproterenol) in the presence of a Cl− secretory gradient. If cAMP-dependent activation is defective in human airway cells expressing surface-localized ΔF508 CFTR, the identification of a molecule capable of activating the mutant channel that is also suitable for use in NPD protocols may improve the sensitivity of the assay to detect ΔF508 CFTR at the cell surface. Because flavonoids are among the best-described activators of CFTR activity (15–17), we sought to determine the most suitable agent that could be applied to the measurement of NPD. Some results of these studies were previously reported in abstracts (18, 19).
The CFBE41o− and Fisher rat thyroid (FRT) cells stably transduced with ΔF508 or WT CFTR cDNA were grown on Costar permeable inserts and mounted in Ussing chambers, and the short-circuit current (Isc) was measured under voltage clamp conditions, as previously described (13, 20). Briefly, cells were seeded on 0.4-μm permeable supports (5 × 105 cells/filter, 6.5-mm diameter; Costar, Bethesda, MD) after coating with fibronectin. Cells were grown to confluence, and then switched to at an air–liquid interface 48 hours before mounting in modified Ussing chambers (Jim's Instruments, Iowa City, IA), and initially bathed on both sides with identical Ringer solution. Measurements of Isc were obtained by using an epithelial voltage clamp (University of Iowa Bioengineering, Iowa City, IA). A 3-mV pulse of 1-second duration was imposed every 100 seconds to monitor resistance, which was calculated using Ohm's law. To measure stimulated Isc, the mucosal bathing solution was changed to a low Cl− sodium gluconate solution plus 100 μM amiloride. Agonists (flavonoids, isoproterenol, and forskolin) were added to the bathing solutions as indicated (for a minimum 5 min of observation under each condition). Glibenclamide (200 μM) or CFTRInh-172 (10 μM) was added to the mucosal bathing solution at the end of the experiments to block CFTR-dependent Isc. All chambers were maintained at 37°C, vigorously stirred, and gassed with 95%O2/5% CO2.
Polyclonal NIH-3T3 cells expressing the hemagglutinin (HA)-tagged R-domain were treated with quercetin for 20 minutes, and compared with forskolin (20 μM) × 5 minutes as a positive control. After lysis, equal amounts (50 μg) of total cell lysate were electrophoresed through a 12% SDS-PAGE gel, and immunoblotted with antibody to the HA tag (Covance, Cumberland, VA). Phosphorylation of the R-domain was visualized as a 2- to 4-kD shift in migration, as previously described (21). Cellular cAMP was measured using an ELISA-based detection kit (Cayman Chemicals, Ann Arbor, MI) according to published methods (13).
Mice were anesthetized with ketamine, and each mouse's tail was gently abraded, placed in lactated Ringer solution, and connected through a calomel cell to a high-impedance voltmeter (VF-1; World Precision Instruments, Sarasota, FL), as previously described (22, 23). Perfusion solutions included Ringer lactate plus amiloride (100 μM), a low Cl− concentration solution with amiloride (100 μM), and an agonist in low Cl− solution. Each condition was studied for 5 to 10 minutes until a stable signal was achieved before changing to the next solution (23). Additional details are described in the online supplement.
All subjects provided written, informed consent, and the study was approved by the Institutional Review Board of the University of Alabama at Birmingham. The NPD studies were performed in accordance with the report by Knowles and colleagues (24) and the standard operating procedure of the CF-Therapeutics Development Network (perfusion method) using electronic data capture (Biopac, San Francisco, CA), KCl calomel electrodes (Fischer Scientific, Pittsburgh, PA), and 3% agar bridges. Additional details are described in the online supplement.
Forskolin was obtained from Calbiochem (San Diego, CA), and genistein from Sigma-Aldrich (St. Louis, MO). Quercetin (Jarrow Formulas, Los Angeles, CA) was freshly reconstituted for each experiment in DMSO at a 20 mg/ml stock concentration, and passed through a 0.2-μM filter to remove particulates. Quercetin from an alternative source (Sigma-Aldrich) was used for confirmation.
For Isc, cAMP, and Western blot measurements, descriptive statistics (mean, SD, and SEM) were compared using the Student t test or ANOVA, as appropriate. All statistical tests were two-sided, and were performed at a 5% significance level (i.e., α = 0.05), using SPSS software (Chicago, IL) and Microsoft Excel (Seattle, WA).
To identify compounds that potentiate ΔF508 CFTR activity, 20 flavonoid agents were screened in ΔF508 CFTR-transduced CFBE41o− cells after temperature correction of aberrant protein processing (growth at 27°C × 48 h). As shown in Figure 1, cell monolayers were evaluated in modified Ussing chambers after prestimulation with forskolin (20 μM) and exposure to incrementally increasing doses (1, 10, and 50–100 μM) of the respective flavonoid agents. The concentrations of flavonoids tested were based on previous experience with genistein and other flavonoids on CF processing and maturation (13, 17, 25, 26). Several agents stimulated Cl− conductance above that produced by forskolin and other cAMP agonists, which are poor stimuli of ΔF508 CFTR activity in this airway cell type (13). Genistein, a well-characterized flavonoid activator of ΔF508 CFTR, was found to be the most potent stimulus in our screen, but was not considered further because of potential human safety concerns with repeated nasal administration and off-target effects (27). Equol and apigenin were avoided because of their observed effects on ΔF508 CFTR processing. Of the agents that activated ΔF508 CFTR residing at the cell surface, quercetin was selected for additional evaluation because of its well-established safety profile for human use, including clinical trials in a variety of disorders encompassing cancer and heart disease, and because of its usefulness as an anti-inflammatory agent (28–32).
The protein kinase A (PKA)/cAMP-dependent phosphorylation of the CFTR R-domain is a critical step in channel activation, but this stimulus was shown to be insufficient to activate ΔF508 CFTR fully at the plasma membrane of airway epithelial cells (13, 33). To examine the mechanistic basis of CFTR activation by quercetin, we measured the effects of quercetin on cAMP levels and on R-domain phosphorylation. Quercetin had small but detectable effects on cell cAMP concentrations (126 pmol over vehicle control) that were less than 10% of that observed with forskolin treatment but greater than those seen with genistein (Figure 2A). As opposed to the potent cAMP agonist forskolin, quercetin had no detectable effect on the phosphorylation-dependent mobility shift of the recombinant CFTR R-domain (Figure 2B), suggesting that its effects were independent of CFTR phosphorylation, despite reports of quercetin as a phosphatase inhibitor and/or PKA activator in other cell types (34, 35). Together, the results of Figure 2 provide evidence that the quercetin-induced activation of ΔF508 CFTR differs from classic cAMP-signaling agonists, and is consistent with a mechanism of action that imparts direct effects on CFTR that are independent of R-domain phosphorylation, as reported with other flavonoid agents including genistein (36–38).
To define the dose–response relationship between quercetin and ΔF508 CFTR activation, and to confirm its effects in a distinct ΔF508 CFTR-expressing cell model, additional studies were performed in ΔF508 CFTR-transduced FRT monolayers after the temperature correction of ΔF508 misprocessing (27°C × 48 h). This cell type is frequently used to examine ΔF508 CFTR function and processing, including high-throughput screens of CFTR potentiators and ΔF508 CFTR correctors (7, 39). Quercetin activated CFTR-dependent Cl− conductance at 1 and 10 μg/ml, but inhibited Isc after the addition of higher concentrations (>20 μg/ml; Figures 3A and 3B). The Cl− transport stimulated by quercetin (10 μg/ml) was not affected by forskolin (20 μM) pretreatment, insofar as quercetin stimulated Isc by 57.6 ± 2.7 μA/cm2 in the presence of forskolin, compared with 53.4 ± 3.8 μA/cm2 produced by quercetin alone (P = NS, n = 4, data not shown). In monolayers without temperature correction of ΔF508 CFTR processing (i.e., grown at 37°C), the effect of quercetin on Cl− conductance was minimal, establishing the specificity of its effect on ΔF508 CFTR residing at the cell surface. Experiments in wild-type CFTR-expressing FRT monolayers suggested a peak effect at 10 μg/ml (Figure 3C). Confirmatory experiments performed in ΔF508 CFTR-transduced CFBE41o− cells after low temperature treatment showed a similar dose–response relationship (Figure 3D) and specificity for surface-residing ΔF508 CFTR (no activation seen with growth at 37°C). As seen in FRT cells, the quercetin-dependent activation of Cl− transport did not require forskolin preactivation in temperature-corrected ΔF508 CFBE41o− cells. The dose–response curve in CFBE41o− cells transduced with wild-type CFTR was similar to that of ΔF508 CFTR-transduced cells (Figure 3E). Experiments using parental CFBE41o− cells (without ΔF508 or wild-type CFTR transduction and undetectable expression of endogenous ΔF508 CFTR) (13) showed no activation by quercetin (0 ± 0 μA/cm2, data not shown), confirming the specificity of the agonist for CFTR.
We next examined whether quercetin increased ΔF508 CFTR Cl− conductance above that produced by the β2 AR agonist isoproterenol, a well-established regulatory pathway to activate CFTR in vivo and a standard agonist for NPD studies. These experiments were designed to mirror CFTR detection in humans using NPD protocols (40), and used isoproterenol (10 μM) and a Cl− secretory gradient in the presence of amiloride. These studies were limited to ΔF508 CFTR-transduced CFBE41o− cells after low temperature growth (Figure 4), because FRT cells do not express β2 ARs. Quercetin (20 μg/ml) significantly increased the Cl− conductance above isoproterenol stimulation, increasing Isc 12.3 μA/cm2 above that produced by isoproterenol alone. To confirm that pre-exposure to quercetin did not induce tachyphylaxis (an effect that would impede its use in human NPD protocols requiring repeated measurements), monolayers were treated with 20 μg/ml quercetin for two 30-minute intervals, 24 and 48 hours before evaluation in Ussing chambers. Pre-exposure to quercetin had no measurable effect on the subsequent activation of corrected ΔF508 CFTR by the compound (Figure 4D). The activation of CFTR-dependent ion transport was confirmed in primary human airway cells from a normal (non-CF) donor (Figures 4E and 4F).
We next examined the effects of quercetin on anion transport in vivo via standardized murine NPD measurements. As shown in Figure 5, mice expressing wild-type CFTR (Cftr+/− and Cftr+/+ mice of a Cftrtm1Kth C57BL6/J genetic background) demonstrated significant hyperpolarization after perfusion with quercetin (20 μg/ml) in the presence of amiloride (100 μM) and a Cl− secretory gradient (−2.9 ± 0.7 mV), compared with the continued depolarization seen in mice perfused with vehicle and otherwise identical conditions (+0.9 ± 0.5 mV). The Cftr−/− mice did not demonstrate repolarization after quercetin perfusion, confirming the specificity of quercetin's effects in CFTR-dependent anion transport in vivo (Figure 5C). Consistent with the effects of quercetin on ΔF508 CFTR cells without temperature correction, quercetin perfusion did not hyperpolarize the nasal epithelial membrane of mice homozygous for murine ΔF508 CFTR, compared with isoproterenol or vehicle control (Figure 5D). In normal (non-CF) human subjects, sequential perfusion with quercetin at 5, 10, and 20 μg/ml was well tolerated, and demonstrated a sequential dose-dependent polarization of the nasal membrane, indicative of CFTR-dependent anion transport (Figure 6). No adverse events were reported, and no changes were seen in the nasal examination rating or total nasal symptom score before and after the NPD procedure. The results confirm findings observed in polarized epithelial model systems, and demonstrate that quercetin retains bioactivity to stimulate CFTR Cl− transport when topically applied to the nasal mucosa in an NPD protocol.
The detection of plasma membrane-localized ΔF508 CFTR is a vital step in the development of therapies designed to treat the underlying cause of CF in the majority of patients with CF. In this study, we evaluated several flavonoid compounds for the activation of surface ΔF508 CFTR, and identified a number of agents that robustly activate ΔF508 CFTR-dependent Cl− transport in airway monolayers after temperature correction of aberrant processing. One agent in particular, quercetin, was selected for further investigation because it demonstrated efficient activation of ΔF508 CFTR-dependent Isc, and has a well-established safety profile in human subjects. Two other flavonoids, equol and apigenin, also demonstrated potent ΔF508 CFTR activation, but were not studied further because of their inferior safety profile in the literature. Furthermore, additional studies indicated that these agents affect ΔF508 CFTR misprocessing, a characteristic that could confound studies intended to detected ΔF508 CFTR correction. Using two polarized epithelial models, we showed that quercetin produces a dose-dependent activation of ΔF508 CFTR after cell surface localization (low temperature rescue) (Figure 3). In both ΔF508 CFTR expressing FRT and CFBE41o− cells, the effect was specific for CFTR at concentrations below 100 μg/mL, as indicated by the lack of effect on anion transport in ΔF508 CFTR-transduced cells without temperature correction (or in cells lacking the ΔF508 CFTR transgene). When repeated in a sequence analogous to a standard NPD protocol, quercetin stimulated CFTR-dependent Isc without evidence of tachyphylaxis, indicating that the compound may be suitable for clinical protocols requiring repeated exposure (Figure 4D). Together with results demonstrating the stimulation of CFTR-dependent anion transport across the nasal epithelium in vivo (Figures 5 and and6),6), the findings provide support for further studies of this agent in human NPD protocols as a means to enhance the detection of ΔF508 CFTR at the plasma membrane. Based on these findings, a multicenter study is being conducted to examine the response of ΔF508 homozygotes to quercetin perfusion compared with isoproterenol, and is intended to determine whether the response to quercetin is associated with a more mild pulmonary phenotype.
In the Calu-3 serous glandular cell line, Illek and Fischer showed that quercetin (at concentrations >40 μM, equivalent to 12.1 μg/ml) stimulated Cl− secretion (half-maximal doses were 22.1 ± 4.5 μM) in vitro, whereas higher doses were inhibitory (26). Our findings indicate that quercetin produced maximal Isc at approximately 10 μg/ml (33 μM) in vitro, and hyperpolarized the NPD response between 5 to 20 μg/ml. These results concerning the effects of quercetin in the murine airway extend earlier studies, and firmly establish the CFTR dependence of observations at the concentrations tested. Interestingly, Illek and Fischer (26) suggested that pretreatment of Calu-3 cells with forskolin may sensitize to subsequent quercetin stimulation. Our results in ΔF508 CFTR-expressing FRT cells did not show this effect, insofar as quercetin stimulated Isc to a similar degree, regardless of forskolin pretreatment. This could indicate differences in the response of wild-type CFTR versus the ΔF508 protein, or other variables attributable to the cell types being tested (26). The inhibition of anion conductance at higher concentrations observed in our studies was reported with other flavonoids (26) and in studies of colonic epithelia (41). These results emphasize the importance of using the appropriate concentration for studies intended to activate CFTR. Moreover, tissue-specific differences may mediate anion conductance in response to quercetin. These concerns may be abrogated through the use of the NPD assay, which requires topical administration directly to the nasal epithelia, as opposed to systemic treatment in which pharmacokinetics are more difficult to control, and unintended consequences of CFTR inhibition may be more likely in off-target tissues.
In other studies, Lim and colleagues reported no activation of chloride efflux with the addition of quercetin (1 or 5 μM) to forskolin (13 μM) in IB3–1 bronchial epithelial cells after the correction of endogenous ΔF508 CFTR with 4-phenylbuturate (42). This disparity may be attributable to the low concentrations of quercetin tested, because our experiments also showed that no ΔF508 CFTR activation occurred with 3.3 μM quercetin. Plasma membrane ΔF508 CFTR levels can also be markedly influenced by cell polarity and by growth at an air–liquid interface (13, 43, 44). Because the studies with IB3–1 cells were conducted under nonpolarizing conditions, this fundamental difference may have contributed to the previously reported negative results. These effects may also represent additional cell model-specific responses to flavonoid exposure, as reported by Hansen and colleagues regarding the quercetin modulation of heat shock protein-70 synthesis (45).
Although previous work with quercetin in Calu-3 cells demonstrated a cAMP/PKA-dependent activation of CFTR (26), our results provide evidence that the quercetin signaling that promotes corrected ΔF508 CFTR activation may differ from that produced by classic PKA-dependent stimuli, and this effect may be unique to flavonoids. Quercetin produced a small increase in cAMP relative to forskolin, despite potent stimulation of ion transport compared with cAMP agonist (Figure 2). The cAMP levels resulting from quercetin treatment, however, were greater than those produced by genistein. Previous work from our laboratory has shown that ΔF508 CFTR channel activation (by genistein) can be partially blocked by inhibiting PKA in CFBE41o− cells (13). This implies that ΔF508 CFTR requires some level of PKA activity (and thus PKA dependent RD phosphorylation) for genistein to confer Cl− transport. While no RD phosphorylation was detected following quercetin treatment (further distinguishing the mechanistic basis of flavonoid versus PKA activation pathways; see Figure 2B), we speculate that quercetin may provide two stimuli that together optimize ΔF508-CFTR activation (including both cAMP-dependent and -independent effects), particularly in cells where endogenous cAMP levels could be limiting.
Several laboratories showed that ΔF508 CFTR, in addition to a well-described cellular processing defect, also exhibits significant abnormalities in channel gating at the cell surface. For example, ΔF508 CFTR exhibits reduced sensitivity to phosphorylation dependent activation in membrane patch and whole-cell studies (46). More recent findings by Wang and colleagues using a series of molecules (including genistein, NPPB-AM, and curcumin), indicate that ΔF508 CFTR activation required stimuli independent of cAMP and PKA to rescue mutant channel function fully (47). Studies from our laboratory and from others using intact airway monolayers established that low temperature or pharmacologically corrected ΔF508 CFTR is not fully activated by cAMP stimuli, including the β2 agonists albuterol and isoproterenol or the A2B adenosine receptor agonists adenosine or 5′-N-ethylcarboxamide adenosine. This defect appears to be more pronounced in pulmonary epithelia, including human alveolar and airway cells (13, 33). These findings provide a compelling rationale to investigate more sensitive biomarkers of ΔF508 CFTR activity after processing correction, including agents such as quercetin. For example, current methods may not detect effective biochemical corrections of ΔF508 CFTR, insofar as chemically corrected ΔF508 CFTR may not be activated by the cAMP pathway because of persistent gating defects. As such, improved surface activation of CFTR by an agent such as quercetin would be expected to decrease the number of subjects required to observe a relevant improvement in CFTR-dependent NPD response, simplifying the designs of clinical trials.
In addition, agents such as quercetin could help detect and discriminate the ΔF508 CFTR that resides in the plasma membrane, but that is unresponsive to endogenous activation because of a persistent gating defect, thus helping define the mechanistic basis underlying a ΔF508 CFTR processing corrector under study. Although the mechanism by which quercetin and other flavonoids activate CFTR is unknown, our findings support a direct effect on the CFTR Cl− channel that can overcome the defective gating of cell surface-localized ΔF508 CFTR. These results are further supported by observations from whole-cell studies with quercetin (26) and single-channel studies with other flavonoids (47).
Because the endoplasmic reticulum-associated degradation of ΔF508 CFTR was postulated to be less than 100% efficient in vitro (45), and because small amounts of ΔF508 CFTR at the cell membrane were speculated to account for phenotypic differences among ΔF508 CFTR homozygous individuals (48), the evaluation of CFTR-dependent Cl− conductance by NPD using quercetin (or other potentiating stimuli) could be used as an approach to detect individuals with small but relevant amounts of ΔF508 CFTR at the plasma membrane. Such an approach could identify ΔF508 CFTR homozygotes who are potential candidates for treatment with other CFTR potentiators, including the agent VX-770, currently under investigation in patients with CF harboring the G551D CFTR mutation (49). The relative safety of the approach, as confirmed during the human NPD studies described here, makes this assessment feasible.
Flavonoids are ubiquitous in the human diet, are highly bioavailable, and have been extensively studied with regard to their pharmacology and toxicology (50). Multiple laboratory clinical trials point to an excellent safety profile of quercetin, including studies using systemic, oral, and intravenous administration at levels far exceeding those found to activate ΔF508 CFTR (51–53). For example, Shoskes and colleagues reported that oral quercetin (500 mg, twice daily) was well tolerated and significantly improved symptoms in men with chronic prostatitis (54). The intravenous administration of high doses of quercetin was well tolerated over a 6- to 7-month period in a study examining quercetin as a tyrosine kinase inhibitor (51). More recently, oral quercetin (730 mg daily) for 28 days produced no significant adverse events in a hypertension trial (55). Carcinogenic testing in hamsters by Morino and colleagues (56), and additional studies involving systemic exposure in rats (57, 58), further indicate the safety of the agent in animal models. This extensive preclinical and clinical experience, coupled with our results in vitro and in vivo, provide strong support for further studies to examine quercetin in subjects with CF as a means to activate ΔF508 CFTR.
The authors thank Dr. Eric J. Sorscher for reviewing the manuscript and for infrastructural support, and Dr. Melissa Ashlock for critical appraisal and support of the work. The authors are also grateful to Ms. Heather Hathorne and Ms. Ginger Reeves for assistance in performing NPD procedures, Ms. Meenakshi Sthanam for technical expertise, and Ms. Cheryl Owens for assistance preparing the manuscript.
This research was funded by National Institutes of Health grants 1K23DK075788–01 (S.M.R.), 1R03DK084110-01 (S.M.R.), and 1P30DK072482–01A1 (to Eric J Sorscher; see Acknowledgments), and Cystic Fibrosis Foundation grants ROWE08XX (S.M.R.), R464-CR07 (to Eric J Sorscher), and CLANCY02YO (J.P.C.). This study also benefited from National Institutes of Health funding to the Purdue University-University of Alabama at Birmingham Botanicals Center for Age-Related Disease from the National Center for Complementary and Alternative Medicine, and b funding from the National Institutes of Health Office of Dietary Supplements (P50 AT00477, C. Weaver, principal investigator).
The contents of this article are solely the responsibility of the authors, and do not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases, the National Heart, Lung, and Blood Institute, or the National Institutes of Health.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10. 1165/rcmb.2009-0281OC on December 30, 2009
Author Disclosure: L.C.P. has received sponsored grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (in excess of $100,000). S.M.R. has served on the advisory boards for Vertex Pharmaceuticals (up to $1,000) and PTC Therapeutics ($1,001–$5,000), has received industry-sponsored grants from Novartis Pharmaceuticals (in excess of $100,000), Vertex Pharmaceuticals (in excess of $100,000), and PTC Therapeutics (in excess of $100,000), and has received sponsored grants from the Cystic Fibrosis Foundation ($50,000–$100,000) and the National Institutes of Health (in excess of $100,000). None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.