Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants.

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR

New era of cystic fibrosis: full mutational analysis and personalized therapy

Despite its apparently simple genetics, cystic fibrosis (CF) is a rather complex genetic disease. A lot of variability in the steps of the path from the cystic fibrosis transmembrane conductance regulator (CFTR ) gene to the clinical manifestations originates an uncertain genotype - phenotype relationship. A major determinant of this uncertainty is the incomplete knowledge of the CFTR mutated genotypes, due to the high number of CFTR mutations and to the higher number of their combinations in trans and in cis. Also the very limited knowledge of functional effects of CFTR mutated alleles severely impairs our diagnostic and prognostic ability. The final phenotypic modulation exerted by CFTR modifier genes and interactome further complicates the framework. The next generation sequencing approach is a rapid, lowcost and high-throughput tool that allows a near complete structural characterization of CFTR mutated genotypes, as well as of genotypes of several other genes cooperating to the final CF clinical manifestations. This powerful method perfectly complements the new personalized therapeutic approach for CF. Drugs active on specific CFTR mutational classes are already available for CF patients or are in phase 3 trials. A complete genetic characterization has been becoming crucial for a correct personalized therapy. However, the need of a functional classification of each CFTR mutation potently arises. Future big efforts towards an ever more detailed knowledge of both structural and functional CFTR defects, coupled to parallel personalized therapeutic interventions decisive for CF cure can be foreseen

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations

Targeted therapies to improve CFTR function in cystic fibrosis

Cystic fibrosis is the most common genetically determined, life-limiting disorder in populations of European ancestry. The genetic basis of cystic fibrosis is well established to be mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that codes for an apical membrane chloride channel principally expressed by epithelial cells. Conventional approaches to cystic fibrosis care involve a heavy daily burden of supportive treatments to combat lung infection, help clear airway secretions and maintain nutritional status. In 2012, a new era of precision medicine in cystic fibrosis therapeutics began with the licensing of a small molecule, ivacaftor, which successfully targets the underlying defect and improves CFTR function in a subgroup of patients in a genotype-specific manner. Here, we review the three main targeted approaches that have been adopted to improve CFTR function: potentiators, which recover the function of CFTR at the apical surface of epithelial cells that is disrupted in class III and IV genetic mutations; correctors, which improve intracellular processing of CFTR, increasing surface expression, in class II mutations; and production correctors or read-through agents, which promote transcription of CFTR in class I mutations. The further development of such approaches offers great promise for future therapeutic strategies in cystic fibrosis

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

<div><p>Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in <i>Cftr^F508del</i> homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the <i>F508del-CFTR</i> mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from <i>F508del</i> homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both <i>in vivo</i>, in mice, and <i>in vitro</i>, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 <i>F508del-CFTR</i> homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells <i>in vivo</i>, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of <i>TNF/TNF-alpha (tumor necrosis factor)</i> and <i>CXCL8</i> (<i>chemokine [C-X-C motif] ligand 8</i>) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the <i>F508del-CFTR</i> mutation.</p></div

A Genotypic-oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macro-categories of Cystic Fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, 125 different mutated alleles (11 of which with novel mutations and 10 of which complex) and 225 genotypes were found. A strong correlation between mutational patterns at the genotypic level and phenotypic macro-categories emerged. This specificity appears to be largely dependent on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macro-categories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype - phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype - phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway

Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.This work was supported by a center grant UID/MULTI/04046/2019 to BioISI and project PTDC/BIA-CEL/28408/2017 and IF2012 to PM, both from FCT, Portugal. AMM was recipient of fellowship SFRH/BD/52490/2014 from BioSYS PhD programme PD65-2012, and PB of fellowship SFRH/BPD/94322/2013.N/