Michael V. Rector

Loss of Anion Transport without Increased Sodium Absorption Characterizes Newborn Porcine Cystic Fibrosis Airway Epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR⁻(/)⁻ pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR⁻(/)⁻ epithelia showed markedly reduced Cl⁻ and HCO₃⁻ transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR⁻(/)⁻ pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl⁻ conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl⁻ and HCO₃⁻ in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Produces Abnormalities in Tracheal Development in Neonatal Pigs and Young Children

RATIONALE Although airway abnormalities are common in patients with cystic fibrosis (CF), it is unknown whether they are all secondary to postnatal infection and inflammation, which characterize the disease. OBJECTIVES To learn whether loss of the cystic fibrosis transmembrane conductance regulator (CFTR) might affect major airways early in life, before the onset of inflammation and infection. METHODS We studied newborn CFTR⁻(/)⁻ pig trachea, using computed tomography (CT) scans, pathology, and morphometry. We retrospectively analyzed trachea CT scans in young children with CF and also previously published data of infants with CF. MEASUREMENTS AND MAIN RESULTS We discovered three abnormalities in the porcine CF trachea. First, the trachea and mainstem bronchi had a uniformly small caliber and cross-sections of trachea were less circular than in controls. Second, trachealis smooth muscle had an altered bundle orientation and increased transcripts in a smooth muscle gene set. Third, submucosal gland units occurred with similar frequency in the mucosa of CF and control airways, but CF submucosal glands were hypoplastic and had global reductions in tissue-specific transcripts. To learn whether any of these changes occurred in young patients with CF, we examined CT scans from children 2 years of age and younger, and found that CF tracheas were less circular in cross-section, but lacked differences in lumen area. However, analysis of previously published morphometric data showed reduced tracheal lumen area in neonates with CF. CONCLUSIONS Our findings in newborn CF pigs and young patients with CF suggest that airway changes begin during fetal life and may contribute to CF pathogenesis and clinical disease during postnatal life.

The ΔF508 Mutation Causes CFTR Misprocessing and Cystic Fibrosis–Like Disease in Pigs

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs. Four Legs Good, Two Legs Bad In Animal Farm, George Orwell describes a pasture in which the pigs lead an animal revolt, resulting eventually in the porcine dwellers becoming indistinguishable from the human ones against whom they revolted. Scientists similarly wish for pigs to model humans, although as large animal models of human disease, not despotic rulers. Ostedgaard et al. extended this idea to cystic fibrosis (CF), generating pigs that carry the most common human CF mutation, Δ508. CF is a devastating genetic disease characterized by difficulty breathing, progressive disability, persistent infections, and, often, early death. CF is caused by a mutation in the gene that encodes the CF transmembrane conductance regulator (CFTR), which is an anion channel that modulates the components of sweat, digestive juices, and mucus. The most common mutation in CF patients results in an altered version of CFTR, CFTR-Δ508, which is found in 1 of 25 people of Caucasian descent. CF is difficult to study in human patients, and mouse models do not accurately reflect the human disease. Pigs may provide a better model of CF because they have more similar anatomy, biochemistry, physiology, size, and genetics to humans than mice. Thus, the authors generated a pig model of CF with the CFTR-Δ508 mutation. Similar to pigs that completely lack expression of CFTR, the CFTR-Δ508 pigs developed CF symptoms that mimicked those in human patients. In these animals, much of the CFTR-Δ508 protein was misprocessed; specifically, it was retained in the endoplasmic reticulum and rapidly degraded. However, pigs with CFTR-Δ508 retained small amounts of CFTR conductance (~6%), although this level of function was not sufficient to prevent disease. This new model may help to determine which levels of CFTR are sufficient for function and, therefore, guide future therapeutic strategies. After all, all animal models are equal, but some are more equal than others. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTRΔF508/ΔF508 airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3′,5′-monophosphate (cAMP) agonists were less potent at stimulating current in CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/ΔF508 pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass

Diabetes is a common and significant co-morbidity in cystic fibrosis (CF). The pathogenesis of cystic fibrosis related diabetes (CFRD) is incompletely understood. Because exocrine pancreatic disease is similar between humans and pigs with CF, the CF pig model has the potential to contribute significantly to the understanding of CFRD pathogenesis. We determined the structure of the endocrine pancreas in fetal, newborn and older CF and non-CF pigs and assessed endocrine pancreas function by intravenous glucose tolerance test (IV-GTT). In fetal pigs, pancreatic insulin and glucagon density was similar between CF and non-CF. In newborn and older pigs, the insulin and glucagon density was unchanged between CF and non-CF per total pancreatic area, but increased per remnant lobular tissue in CF reflecting exocrine pancreatic loss. Although fasting glucose levels were not different between CF and non-CF newborns, CF newborns demonstrated impaired glucose tolerance and increased glucose area under the curve during IV-GTT. Second phase insulin secretion responsiveness was impaired in CF newborn pigs and significantly lower than that observed in non-CF newborns. Older CF pigs had elevated random blood glucose levels compared with non-CF. In summary, glycaemic abnormalities and insulin secretion defects were present in newborn CF pigs and spontaneous hyperglycaemia developed over time. Functional changes in CF pig pancreas were not associated with a decline in islet cell mass. Our results suggest that functional islet abnormalities, independent of structural islet loss, contribute to the early pathogenesis of CFRD.

Cystic fibrosis transmembrane conductance regulator in sarcoplasmic reticulum of airway smooth muscle: Implications for airway contractility

RATIONALE An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

CFTR is required for maximal transepithelial liquid transport in pig alveolar epithelia

A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR(-/-) pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR(+/-) epithelia was similar to that in CFTR(+/+) epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR(-/-) T2AECs but had no effect on CFTR(+/+) epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR(-/-) alveolar epithelia but similar between CFTR(+/+) and CFTR(+/-) epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR(+/+) but not in CFTR(-/-) T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.

Expression of Human Paraoxonase 1 Decreases Superoxide Levels and Alters Bacterial Colonization in the Gut of Drosophila melanogaster

Paraoxonases (PON) are a family of proteins (PON1, 2 and 3) with multiple enzymatic activities. PON1 interferes with homoserine lactone-mediated quorum sensing in bacteria and with reactive oxygen species (ROS) in humans and mice. PON1 gene mutations have been linked to multiple traits, including aging, and diseases of the cardiovascular, nervous and gastrointestinal system. The overlapping enzymatic activities in the PON family members and high linkage disequilibrium rates within their polymorphisms confound animal and human studies of PON1 function. In contrast, arthropods such as Drosophila melanogaster have no PON homologs, resulting in an ideal model to study interactions between PON genotype and host phenotypes. We hypothesized that expression of PON1 in D. melanogaster would alter ROS. We found that PON1 alters expression of multiple oxidative stress genes and decreases superoxide anion levels in normal and germ-free D. melanogaster. We also found differences in the composition of the gut microbiota, with a remarkable increase in levels of Lactobacillus plantarum and associated changes in expression of antimicrobial and cuticle-related genes. PON1 expression directly decreased superoxide anion levels and altered bacterial colonization of the gut and its gene expression profile, highlighting the complex nature of the interaction between host genotype and gut microbiota. We speculate that the interaction between some genotypes and human diseases may be mediated by the presence of certain gut bacteria that can induce specific immune responses in the gut and other host tissues.

Role of PON in Anoxia-Reoxygenation Injury: A Drosophila Melanogaster Transgenic Model

Background Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury. Methods Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded. Results Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection. Conclusion Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection.