Assessment of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CFTR-null mice after bone marrow transplantation

Several studies have demonstrated that bone marrow (BM)-derived cells give rise to rare epithelial cells in the gastrointestinal (GI) and respiratory tracts after BM transplantation into myeloablated recipients. We investigate whether, after transplantation of cystic fibrosis transmembrane conductance regulator (CFTR)-positive BM-derived cells, BM-derived GI and airway epithelial cells can provide CFTR activity in the GI tract and nasal epithelium of recipient cystic fibrosis mice. CFTR-/- mice were transplanted with wild-type BM after receiving different doses of irradiation, and CFTR activity was assessed in vivo in individual mice over time by using rectal and nasal potential difference analyses and in vitro by Ussing chamber analysis. The data suggest that rare BM-derived epithelial cells in the GI and nasal epithelium detected in CFTR-/- transplanted mice provide a modest level of CFTR-dependent chloride secretion. Detection of CFTR mRNA and protein in tissues of transplanted CFTR-/- mice supports these data.     

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.     

Stem cells and cell therapies for cystic fibrosis and other lung diseases

This review will critically evaluate recent findings suggesting that embryonic stem cells and stem cells derived from adult tissues, including bone marrow and umbilical cord blood, may be utilized in repair and regeneration of injured or diseased lungs. This is an exciting and rapidly moving field that holds promise as a novel therapeutic approach for cystic fibrosis and other lung diseases. However, while early studies suggested substantial lung remodeling, particularly with bone marrow-derived cells, more recent findings suggest that engraftment of adult marrow-derived cells in lung is a rare event of uncertain significance. Most recently, it has been suggested that a more relevant role of adult marrow-derived stem cells in lung is modulation of local inflammatory and immune responses. This review will also describe recent advances in understanding of local stem and progenitor cells in lung and their roles in lung development and repair.     

Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice.

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance. This alternative conductance (Cl-a) was detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). We conclude that (i) there is an intracellular Ca(2+)-regulated Cl- conductance that is molecularly distinct from CFTR; and (ii) the level of expression of this alternative Cl- conductance in the epithelium is an important determinant of the severity of organ-level disease in CF.     

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na⁺ absorption, a defective cAMP-dependent Cl⁻ conductance, higher concentrations of Na⁺, Cl⁻, K⁺, and Ca²⁺ in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca²⁺ entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.     

供体骨髓来源干细胞在骨髓嵌合小鼠肠上皮中定植和分化研究

目的 观察骨髓移植后供体骨髓来源干细胞（bone marrow derived stem cells，BMDSCs）是否能够在受体肠上皮中定植并分化为肠上皮细胞。方法 使用密度为1．073g／mL的Percoll分离GFP转基因小鼠的骨髓有核细胞。利用^60Co放射源对C57BL／6小鼠进行10Gy致死剂量照射，照射后4h经尾静脉移植绿色荧光蛋白（green fluorescence protein，GFP）转基因小鼠骨髓细胞建立嵌合小鼠模型，移植后1月和6月取小肠标本石蜡包埋。使用GFP抗体鉴定GFP阳性供体细胞在小肠上皮的表达情况，并使用GFP抗体和细胞角蛋白（PCK）抗体进行双标以判断BMDSCs是否分化上皮细胞，同时结合绒毛蛋白（Villin）和GFP的免疫荧光双标及连续切片染色肠道特异转录因子CDX2和GFP来分析骨髓来源细胞是否分化为肠道功能性上皮细胞。结果供体骨髓来源细胞在嵌合鼠的骨髓和小肠上皮层中均有定植，且小肠上皮中GFP阳性细胞能够同时表达PCK、Villin和CDX2。结论 供体骨髓来源干细胞可以在骨髓嵌合小鼠的肠上皮定植，并分化为具有正常功能的小肠上皮细胞。     

Bone marrow stem cells do not repopulate the healthy upper respiratory tract

Recent studies reported differentiation of both bone marrow and tissue-specific stem cells into cells of other organs. The demonstration that bone marrow stem cells differentiate into human hepatocytes in vivo has raised the possibility of new therapeutic approaches for liver disease. For diseases such as cystic fibrosis (CF), correction of the respiratory epithelium is being attempted by gene therapy. Differentiation of bone marrow stem cells into epithelium of the lung and airway was recently reported in an animal model, and would provide an alternative approach. We examined the nasal epithelium of female patients up to 15 years after gender-mismatched bone marrow transplantation. Donor-derived epithelial cells were sought with a combination of Y-chromosome fluorescence in situ hybridization and anti-cytokeratin antibody.In nasal brushing samples from 6 transplant-recipients, a median of 2.5% (range, 0.7-18.1%) of nuclei was male and identified as being of donor-origin. However, a complete absence of staining with anti-cytokeratin antibodies confirmed that these were not epithelial cells, but were likely to be either intraepithelial lymphocytes or mesenchymal cells.Following whole bone marrow transplantation, bone marrow progenitor cells do not differentiate into respiratory epithelium of the healthy upper airway. The differences between this and other studies could relate to the cells transplanted, to differential rates of turnover, or to the requirement for specific triggers to stimulate migration and differentiation. In the absence of such conditions, whole bone marrow transplantation is unlikely to provide a route for correction of the CF airway.     

The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond

Although intrapulmonary progenitor cells are traditionally believed to be the source for regenerating cells in response to lung injury, recent mounting evidence indicates that a significant proportion of the mesenchymal cells involved in this repair/remodeling process may be derived from extrapulmonary sources, such as the recently described circulating fibrocyte as well as other bone marrow-derived progenitor cells. Studies tracking CD34 and/or CD45 markers of fibrocytes show their presence in injured murine lung tissue. Moreover, bone marrow chimeric mice with green fluorescence protein (GFP)-expressing marrow cells show abundant GFP-expressing fibroblasts in their lungs in response to lung injury. However, although fibrocytes express CD34 and CD45, and appear to have the capacity to differentiate to myofibroblasts, these properties are not evident in the bone marrow-derived fibroblasts. Induction of CCL21 (SLC) and CXCR12 (SDF1alpha) in injured lung tissue, and their respective cognate receptors, CCR7 and CXCR4, in fibroblasts from injured lungs, suggests recruitment of these extrapulmonary progenitor cells via these chemokines. This is supported by evidence that antibody neutralization of CXCR12 reduces recruitment of fibrocytes and pulmonary fibrosis. In contrast, other studies suggest a protective effect for bone marrow-derived cells. Thus, although suggesting that influx of extrapulmonary fibroblast progenitor cells occurs in response to lung injury, these recent studies do not yet provide clear insight as to the actual phenotype and fate of the recruited cells, the identity of the progenitor cell population in bone marrow, and most important, the function or role of these cells in pathogenesis of the idiopathic interstitial pneumonias.     

Circulating vascular progenitor cells do not contribute to compensatory lung growth

The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow-derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene-transgenic strains. flk-1+/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell-specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow-derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow-derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow-derived progenitor cell contribution to the concomitant vascular growth and remodeling processes.     

Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.     

Allergen-induced peribronchial fibrosis and mucus production mediated by IkappaB kinase beta-dependent genes in airway epithelium

In response to inflammation or injury, airway epithelial cells express inducible genes that may contribute to allergen-induced airway remodeling. To determine the contribution of epithelial cell NF-kappaB activation to the remodeling response, we generated CC10-Cre(tg)/Ikkbeta(delta/delta) mice in which NF-kappaB signaling through IkappaB kinase beta (IKKbeta) is selectively ablated in the airway epithelium by conditional Cre-recombinase expression from the Clara cell (CC10) promoter. Repetitive ovalbumin challenge of mice deficient in airway epithelial IKKbeta prevented nuclear translocation of the RelA NF-kappaB subunit only in airway epithelial cells, resulting in significantly lower peribronchial fibrosis in CC10-Cre(tg)/Ikkbeta(delta/delta) mice compared with littermate controls as assessed by peribronchial trichrome staining and total lung collagen content. Levels of airway mucus, airway eosinophils, and peribronchial CD4+ cells in ovalbumin-challenged mice were also reduced significantly upon airway epithelial Ikkbeta ablation. The diminished inflammatory response was associated with reduced expression of NF-kappaB-regulated chemokines, including eotaxin-1 and thymus- and activation-regulated chemokine, which attract eosinophils and Th2 cells, respectively, into the airway. The number of peribronchial cells expressing TGF-beta1, as well as TGF-beta1 amounts in bronchoalveolar lavage, were also significantly reduced in mice deficient in airway epithelium IKKbeta. Overall, these studies show an important role for NF-kappaB regulated genes in airway epithelium in allergen-induced airway remodeling, including peribronchial fibrosis and mucus production.     

Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.     

Lung disease associated with the IVS8 5T allele of the CFTR gene

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The 5T allele in intron 8 (IVS8) causes abnormal splicing in the CFTR gene, and is associated with lung disease when it occurs in cis with a missense mutation in the CFTR gene, R117H. However, the 5T variant alone has not been reported to cause lung disease. We describe two adult female patients with CF-like lung disease associated with the 5T allele. One patient's genotype is 5T-TG12-M470V/5T-TG12-M470V, and the other is DeltaF508/5T-TG12-M470V; full sequencing of the CFTR gene revealed no other mutation on the same allele as the 5T variant. The levels of full-length CFTR mRNA in respiratory epithelia were very low in these patients (11 and 6%, respectively, of total CFTR mRNA expression). Both patients had defective CFTR-mediated chloride conductance in the sweat ductal and/or acinar epithelia (sweat chloride, mmol/L, mean +/- SEM: 40.0 +/- 5.0 [n = 8 samples] and 80. 0 +/- 3.5 [n = 6 samples]) and airway epithelia (mV, mean +/- SEM CFTR-mediated Cl(-) conductance of 1.2 +/- 2.2 [n = 5 studies] and -6.75 +/- 8.1 [n = 4 studies]). These data suggest that the 5T polythymidine tract sequence on specific haplotype backgrounds (TG12 and M470V) may cause a low level of full-length functional CFTR protein and CF-like lung disease.     

Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis

Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.     

Potential for antioxidant therapy of cystic fibrosis

PURPOSE OF REVIEW: Changes in redox state clearly play a role in airway inflammation and mucus rheology. Furthermore CFTR (cystic fibrosis transmembrane conductance regulator), the defective protein in cystic fibrosis (CF), not only is regulated by redox state but also directly modulates the epithelial redox environment through transepithelial flux of glutathione. The purpose of this review is to explore the potential therapeutic interest of antioxidant molecules in CF. RECENT FINDINGS: Several antioxidants have been shown to have mucolytic and anti-inflammatory properties. Some antioxidants such as zinc and vitamin C may also help increase epithelial chloride secretion through CFTR-dependent and independent pathways. Other antioxidants are showing promise in helping CFTR mobilization to plasma membranes. SUMMARY: The many levels of potential application offered by antioxidants make this class of molecules one of the promising areas of therapeutic development for CF. Several redox-modulating agents have a high likelihood of providing useful approaches for the treatment of many aspects of CF airway disease.