Mesenchymal stem cells protect from hypoxia-induced alveolar epithelial-mesenchymal transition.

Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-ß1 mRNA expression and the secretion of active TGF-ß1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-ß1 expression and in TGF-ß1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-ß1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxia-induced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF.

Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury.

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1ß, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

Hypoxia-induced inhibition of epithelial Na(+) channels in the lung. Role of Nedd4-2 and the ubiquitin-proteasome pathway.

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the ß-Liddle mouse strain mice carrying a truncation of ß-ENaC C-terminus abolishing the interaction between ß-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated ß-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

Neural mechanisms underlying breathing complexity.

Breathing is maintained and controlled by a network of automatic neurons in the brainstem that generate respiratory rhythm and receive regulatory inputs. Breathing complexity therefore arises from respiratory central pattern generators modulated by peripheral and supra-spinal inputs. Very little is known on the brainstem neural substrates underlying breathing complexity in humans. We used both experimental and theoretical approaches to decipher these mechanisms in healthy humans and patients with chronic obstructive pulmonary disease (COPD). COPD is the most frequent chronic lung disease in the general population mainly due to tobacco smoke. In patients, airflow obstruction associated with hyperinflation and respiratory muscles weakness are key factors contributing to load-capacity imbalance and hence increased respiratory drive. Unexpectedly, we found that the patients breathed with a higher level of complexity during inspiration and expiration than controls. Using functional magnetic resonance imaging (fMRI), we scanned the brain of the participants to analyze the activity of two small regions involved in respiratory rhythmogenesis, the rostral ventro-lateral (VL) medulla (pre-Bötzinger complex) and the caudal VL pons (parafacial group). fMRI revealed in controls higher activity of the VL medulla suggesting active inspiration, while in patients higher activity of the VL pons suggesting active expiration. COPD patients reactivate the parafacial to sustain ventilation. These findings may be involved in the onset of respiratory failure when the neural network becomes overwhelmed by respiratory overload We show that central neural activity correlates with airflow complexity in healthy subjects and COPD patients, at rest and during inspiratory loading. We finally used a theoretical approach of respiratory rhythmogenesis that reproduces the kernel activity of neurons involved in the automatic breathing. The model reveals how a chaotic activity in neurons can contribute to chaos in airflow and reproduces key experimental fMRI findings.

Idiopathic interstitial lung disease with anti-SSA antibody.

OBJECTIVES: Consensus is lacking on the immunological tests to perform for diagnosis of interstitial lung diseases (ILDs). In particular, the value of detecting anti-SSA antibody in this context is unknown. We aimed to determine whether the detection of anti-SSA antibody in patients with ILD can identify a subgroup of patients with CTD. METHODS: We compared the characteristics of patients with newly diagnosed apparently idiopathic ILD with anti-SSA antibody [anti-SSA(+) group] and without anti-SSA antibody (control group). RESULTS; Anti-SSA(+) patients (n = 15) more often had extra-respiratory signs (xerostomia and ocular dryness), auto-immune features, a CT scan pattern of non-specific interstitial pneumonia and more severe lung function alteration than controls (n = 30). Of patients who were anti-SSA(+), 2 met the criteria for SS and 13 (86%) of 15 met the criteria for the diagnosis of undifferentiated CTD. CONCLUSIONS: Our results suggest that identification of anti-SSA antibody in patients with early ILD can reveal a specific subgroup of patients with more ground glass opacity and more severe lung function impairment than those without anti-SSA antibody.

Ventilatory chaos is impaired in carotid atherosclerosis.

Ventilatory chaos is strongly linked to the activity of central pattern generators, alone or influenced by respiratory or cardiovascular afferents. We hypothesized that carotid atherosclerosis should alter ventilatory chaos through baroreflex and autonomic nervous system dysfunctions. Chaotic dynamics of inspiratory flow was prospectively evaluated in 75 subjects undergoing carotid ultrasonography: 27 with severe carotid stenosis (>70%), 23 with moderate stenosis (<70%), and 25 controls. Chaos was characterized by the noise titration method, the correlation dimension and the largest Lyapunov exponent. Baroreflex sensitivity was estimated in the frequency domain. In the control group, 92% of the time series exhibit nonlinear deterministic chaos with positive noise limit, whereas only 68% had a positive noise limit value in the stenoses groups. Ventilatory chaos was impaired in the groups with carotid stenoses, with significant parallel decrease in the noise limit value, correlation dimension and largest Lyapunov exponent, as compared to controls. In multiple regression models, the percentage of carotid stenosis was the best in predicting the correlation dimension (p<0.001, adjusted R(2): 0.35) and largest Lyapunov exponent (p<0.001, adjusted R(2): 0.6). Baroreflex sensitivity also predicted the correlation dimension values (p = 0.05), and the LLE (p = 0.08). Plaque removal after carotid surgery reversed the loss of ventilatory complexity. To conclude, ventilatory chaos is impaired in carotid atherosclerosis. These findings depend on the severity of the stenosis, its localization, plaque surface and morphology features, and is independently associated with baroreflex sensitivity reduction. These findings should help to understand the determinants of ventilatory complexity and breathing control in pathological conditions.

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells.

BACKGROUND: Hyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown. METHODS: We evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients. RESULTS: Neither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4. CONCLUSIONS: These results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.

The impact of bariatric surgery on comorbidities and medication use among obese patients.

BACKGROUND: The risks and benefits of bariatric surgery have rarely been evaluated in large multiyear patient samples. This study identifies the short- and long-term impact of bariatric surgery on comorbidities and medication use among obese patients. METHODS: A comprehensive analysis of 5,502 obese patients who underwent bariatric surgery was performed. Submissions of reimbursement claims, including diagnostics and medication use, were compared in the 90 days preceding the surgery and 30 up to 1,110 days following the surgery. Presurgery and postsurgery frequency counts were performed on diagnostics and medication use to identify trends. RESULTS: Among 5,502 patients, significant decreases in the prevalence of reported comorbidities were observed during the short-term postsurgery period and sustained for up to 3 years of follow-up. Compared to the presurgery period, significant decreases (p < 0.05) were observed after 3 years for total cardiovascular disorders (43.6% vs. 14.2%), diabetes mellitus (19.9% vs. 7.7%), chronic obstructive pulmonary disease and other respiratory conditions (57.7% vs. 16.2%), diseases of the musculoskeletal system and connective tissue (32.6% vs. 27.7%), and mental disorders (30.7% vs. 14.8%). Over the same period, the frequency of medication use decreased significantly for a number of conditions including infections, pain, respiratory, cardiovascular, gastroenterologic, lipidemic, and diabetic conditions. Anemia, however, increased from 3.8% to 9.9%, and use of nutritional supplements increased significantly. CONCLUSION: Bariatric surgery was associated with significant reductions in reported claims for short- and long-term health outcomes and reduced medication use for major disease categories.

Predictive factors of weight loss 1 year after laparoscopic gastric bypass in obese patients.

BACKGROUND: Substantial weight loss is achieved in majority of severely obese subjects undergoing laparoscopic gastric bypass (LGBP) but some fail to obtain expected results. Our aim was to identify preoperative factors that could influence weight loss (WL) 1 year after LGBP. METHODS: We studied the predictive value of clinical, biological, and dietary preoperative factors on weight loss in obese patients referred for LGBP. WL was assessed according to mean absolute weight loss (AWL) and mean percent excess weight loss (%EWL) 1 year after LGBP. RESULTS: One hundred twenty-three subjects were included (112 women, age 42 ± 10 years; weight 127 ± 23 kg; BMI 47 ± 8 kg/m(2)). Mean AWL was 39.4 ± 10.5 kg at 1 year, corresponding to a mean %EWL of 70.5 ± 21.2%. AWL was positively correlated with initial weight, BMI, and energy intake and negatively with age, female sex, and treatment for hypertension and diabetes. %EWL was negatively correlated with initial weight, BMI, and positively correlated with triglycerides and ferritinemia. In multivariate analysis, %EWL was negatively correlated only with initial BMI (p < 0.001). AWL was positively correlated with initial BMI and male sex (both p < 0.001), and negatively correlated with protein intake (p = 0.039) and treatment for diabetes (p = 0.021), but not with biomarkers of diabetes and insulin resistance. CONCLUSION: Initial BMI appears to be a strong determinant of individual WL, but predictive factors differ when WL was expressed as %EWL or AWL. The treatment of diabetes rather than diabetes itself appears to affect WL.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

Traditional anthropometric parameters still predict metabolic disorders in women with severe obesity.

It is well established that fat distribution rather than the total quantity of fat is the major determinant of cardiovascular risk in overweight subjects. However, it is not known whether the concept of fat distribution still makes sense in severely obese subjects. Particularly, the role of visceral fat accumulation and/or of adipocyte hypertrophy in insulin resistance (IR) has not been studied in this population. Therefore, the aim of this study was to clarify the determinants of metabolic disorders in severely obese women. We performed a cross-sectional study in 237 severely obese women (BMI >35 kg/m(2)). We assessed total body fat mass and fat distribution by anthropometric measurements (BMI and waist-to-hip ratio (WHR)) and by dual-energy X-ray absorptiometry (DXA). In 22 women, we measured subcutaneous and visceral adipocyte size on surgical biopsies. Mean BMI was 44 +/- 7 kg/m(2) (range 35-77), mean age 37 +/- 11 years (range 18-61). Lipid parameters (triglycerides, high-density lipoprotein cholesterol) and IR markers (fasting insulin and homeostasis model assessment (HOMA) index) correlated with fat distribution, whereas inflammatory parameters (C-reactive protein, fibrinogen) correlated only with total fat mass. An association was observed between android fat distribution and adipocyte hypertrophy. Visceral adipocyte hypertrophy was associated with both IR and hypertension, whereas subcutaneous fat-cell size was linked only to hypertension. Our results obtained in a large cohort of women showed that fat distribution still predicts metabolic abnormalities in severe obesity. Furthermore, we found a cluster of associations among fat distribution, metabolic syndrome (MS), and adipocyte hypertrophy.

Chaotic dynamics of cardioventilatory coupling in humans: effects of ventilatory modes.

Cardioventilatory coupling (CVC), a transient temporal alignment between the heartbeat and inspiratory activity, has been studied in animals and humans mainly during anesthesia. The origin of the coupling remains uncertain, whether or not ventilation is a main determinant in the CVC process and whether the coupling exhibits chaotic behavior. In this frame, we studied sedative-free, mechanically ventilated patients experiencing rapid sequential changes in breathing control during ventilator weaning during a switch from a machine-controlled assistance mode [assist-controlled ventilation (ACV)] to a patient-driven mode [inspiratory pressure support (IPS) and unsupported spontaneous breathing (USB)]. Time series were computed as R to start inspiration (RI) and R to the start of expiration (RE). Chaos was characterized with the noise titration method (noise limit), largest Lyapunov exponent (LLE) and correlation dimension (CD). All the RI and RE time series exhibit chaotic behavior. Specific coupling patterns were displayed in each ventilatory mode, and these patterns exhibited different linear and chaotic dynamics. When switching from ACV to IPS, partial inspiratory loading decreases the noise limit value, the LLE, and the correlation dimension of the RI and RE time series in parallel, whereas decreasing intrathoracic pressure from IPS to USB has the opposite effect. Coupling with expiration exhibits higher complexity than coupling with inspiration during mechanical ventilation either during ACV or IPS, probably due to active expiration. Only 33% of the cardiac time series (RR interval) exhibit complexity either during ACV, IPS, or USB making the contribution of the cardiac signal to the chaotic feature of the coupling minimal. We conclude that 1) CVC in unsedated humans exhibits a complex dynamic that can be chaotic, and 2) ventilatory mode has major effects on the linear and chaotic features of the coupling. Taken together these findings reinforce the role of ventilation in the CVC process.

Gene regulation in the adaptive process to hypoxia in lung epithelial cells.

Lung alveolar epithelial cells are normally very well oxygenated but may be exposed to hypoxia in many pathological conditions such as pulmonary edema, acute respiratory distress syndrome, chronic obstructive pulmonary diseases, or in some environmental conditions such ascent to high altitude. The ability of alveolar epithelial cells to cope with low oxygen tensions is crucial to maintain the structural and functional integrity of the alveolar epithelium. Alveolar epithelial cells appear to be remarkably tolerant to oxygen deprivation as they are able to maintain adequate cellular ATP content during prolonged hypoxic exposure when mitochondrial oxidative phosphorylation is limited. This property mostly relies on the ability of the cells to rapidly modify their gene expression program, stimulating the expression of genes involved in anaerobic energy supply and repressing expression of genes involved in some ATP-consuming cellular processes. This adaptive strategy of the cells is mostly, but not entirely, dependent on the expression of hypoxia-inducible factors (HIFs), known to be responsible for orchestrating a large number of hypoxia-sensitive genes. This review focuses on the role of HIF isoforms expressed in alveolar epithelial cells exposed to hypoxia and on the specific hypoxic gene regulation that takes place in alveolar epithelial cells either through HIF-dependent or -independent pathways.

Nutritional consequences of adjustable gastric banding and gastric bypass: a 1-year prospective study.

BACKGROUND: Gastric bypass (GBP) is more efficient than adjustable gastric banding (AGB) on weight loss and comorbidities, but potentially induces more nutritional deficits. However, no study has compared the prevalence of nutritional deficiencies after these two bariatric procedures. WE PROSPECTIVELY COMPARED: To prospectively compare the prevalence of nutritional deficiencies after AGB and GBP. METHODS: We have performed a 1-year prospective study of nutritional parameters in 70 consecutive severe obese patients, who had undergone bariatric surgery, 21 AGB and 49 GBP. After GBP, multivitamin supplements were systematically prescribed and vitamin B12 supplementation was introduced if a deficiency was observed. RESULTS: Patients lost more weight after GBP than after AGB (40 +/- 13 vs 16 +/- 8 kg, p < 0.001). Vitamins B1 and C and iron deficiencies were frequent before surgery but were not worsened by GBP. AGB only induced a slight decrease of vitamin B1 at 1 year, whereas GBP induced significant decreases of vitamins B12 and E, serum prealbumin, and creatinine concentrations, with only minor clinical consequences. Anemia was observed in 10% of the patients after bariatric surgery. Hemoglobin concentration was not correlated to vitamin B12 or folate concentrations but was related to iron status. Risk of iron deficiency anemia was better assessed by transferrin saturation than by serum ferritin concentration in this obese population. CONCLUSION: Severe nutritional deficits can be avoided after bariatric surgery if patients are systematically supplemented with multivitamin and carefully monitored. However, specific care is required to avoid iron and vitamin B12 deficiencies, anemia, and protein malnutrition.

Multispecies breath analysis faster than a single respiratory cycle by optical-feedback cavity-enhanced absorption spectroscopy.

We demonstrate a first application, of optical-feedback cavity-enhanced absorption spectroscopy (OF-CEAS) to breath analysis in a medical environment. Noninvasive monitoring of trace species in exhaled air was performed simultaneous to spirometric measurements on patients at Bichat Hospital (Paris). The high selectivity of the OF-CEAS spectrometer and a time response of 0.3 s (limited by sample flow rate) allowed following the evolution of carbon monoxide and methane concentrations during individual respiratory cycles, and resolving variations among different ventilatory patterns. The minimum detectable absorption on this time scale is about 3 x 10(-10) cm(-1). At the working wavelength of the instrument (2.326 microm), this translates to concentration detection limits of approximately 1 ppbv (45 picomolar, or approximately 1.25 microg/m(3)) for CO and 25 ppbv for CH(4), well below concentration values found in exhaled air. This same instrument is also able to provide measurement of NH(3) concentrations with a detection limit of approximately 10 ppbv; however, at present, memory effects do not allow its measurement on fast time scales.

Source of human ventilatory chaos: lessons from switching controlled mechanical ventilation to inspiratory pressure support in critically ill patients.

Ventilatory flow measured at the airway opening in humans exhibits a complex dynamics that has the features of chaos. Currently available data point to a neural origin of this feature, but the role of respiratory mechanics has not been specifically assessed. In this aim, we studied 17 critically ill mechanically ventilated patients during a switch form an entirely machine-controlled assistance mode (assist-controlled ventilation ACV) to a patient-driven mode (inspiratory pressure support IPS). Breath-by-breath respiratory variability was assessed with the coefficient of variation of tidal volume, total cycle time, inspiratory time, expiratory time, mean inspiratory flow, duty cycle. The detection of chaos was performed with the noise titration technique. When present, chaos was characterized with numerical indexes (correlation dimension, irregularity; largest Lyapunov exponent, sensitivity to initial conditions). Expectedly, the coefficients of variations of the respiratory variables were higher during IPS than during ACV. During ACV, noise titration failed to detect nonlinearities in 12 patients who did not exhibit signs of spontaneous respiratory activity. This indicates that the mechanical properties of the respiratory system were not sufficient to produce ventilatory chaos in the presence of a nonlinear command (ventilator clock). A positive noise limit was found in the remaining 5 cases, but these patients exhibited signs of active expiratory control (highly variable expiratory time, respiratory frequency higher than the set frequency). A positive noise limit was also observed in 16/17 patients during IPS (p<0.001). These observations suggest that ventilatory chaos predominantly has a neural origin (intrinsic to the respiratory central pattern generators, resulting from their perturbation by respiratory afferents, or both), with little contribution of respiratory mechanics, if any.

Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse.

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the beta-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the beta-ENaC gene locus resulting in low levels of beta-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that beta-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. beta-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but alpha- and gamma-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The beta2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in alpha- and gamma-ENaC protein expression observed in mutant mouse lung, low expression of beta-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate beta-ENaC expression appears to be required for AFC stimulation by beta2-agonists.