Nonvisualization of fetal gallbladder increases the risk of cystic fibrosis.

OBJECTIVE: The aim of our study is to evaluate the prevalence of cystic fibrosis (CF) in fetuses referred for genetic testing because of ultrasonographic sign (nonvisualized fetal gallbladder--NVFGB). METHOD: We reviewed the results of CFTR gene analysis over the period 2002 to 2009 in all consecutive cases referred because of NVFGB in Western France. We correlated these data with the presence of a more classical ultrasonographic finding (fetal echogenic bowel - FEB). RESULTS: Cystic fibrosis was diagnosed in 5 of the 37 fetuses with NVFGB (13.5%, 95% confidence interval (CI): [2.5%; 24.5%]) and in only 9 of the 229 other cases referred because of FEB (3.9%, 95% CI: [3.2%; 14.7%]). In our series, all CF-affected fetuses with NVFGB also had FEB. The risk of CF was 11.6-fold higher in fetuses with both indications (NVFGB + FEB) than in fetuses with isolated FEB (45.5% vs 3.9%, RR = 11.6, 95% CI: [4.7%; 28.8%], p = 0.0001). We also estimated that the residual risk of CF was less than 1 in 68 (1.5%) when a single mutation was identified in the fetus by our molecular protocol. CONCLUSION: Ultrasonographic evidence of NVFGB is an additional risk factor for CF in cases with FEB.

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

beta-adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent beta2 component in pulmonary hypertension.

AIMS: beta-adrenoceptor (beta-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects. METHODS: Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS-/- mice were used for pharmacological characterization of beta-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-beta-AR antibodies. RESULTS: In pulmonary arteries from normoxic mice, isoproterenol (beta-AR agonist) and procaterol (selective beta2-AR agonist) elicited relaxation, while cyanopindolol and CL316243 (beta3-AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (beta1- or beta2-AR antagonists, respectively) and also partially inhibited by N omega-nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS-/- mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for beta2-AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal. CONCLUSION: beta1- and beta2-AR, but not beta3-AR, mediate relaxation of mice pulmonary arteries. The beta2-AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the beta2-AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.

Role of reactive oxygen species and gp91phox in endothelial dysfunction of pulmonary arteries induced by chronic hypoxia.

1. This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia-induced pulmonary hypertension. 2. In pulmonary arteries from control mice, the NO-synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) potentiated contraction to prostaglandin F2alpha (PGF2alpha) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine-induced relaxation was slightly inhibited by polyethyleneglycol-superoxide dismutase (PEG-SOD) or catalase. 3. In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L-NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2-induced contraction. PEG-SOD or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L-NAME-insensitive) relaxations in extra- and intrapulmonary arteries from hypoxic mice. 4. Exposure of gp91phox(-/-) mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox(-/-) mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5. Chronic hypoxia increases ROS levels and impairs endothelial NO-dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia-induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium-dependent relaxation appears to be mediated by ROS, in a gp91phox-independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO-dependent relaxation

Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

Orphan missense mutations in the cystic fibrosis transmembrane conductance regulator: A three-step biological approach to establishing a correlation between genotype and phenotype.

More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a three-step in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling.