**2. The airway epithelium**

The succession of events leading from the defective CFTR to the clinical symptoms is not completely understood. However, it is obvious that the abnormal ion transport with hyper‐

cholangiocytes leads to a disturbance of the fluid lining the airways and the bile ducts [6-10].

The *CFTR* gene was identified in 1989 and this has sharply accelerated the research on CF. The gene, which is situated on the long arm of human chromosome 7 (7q31.2), spans approximately 250 kilobases (kb) of nucleotide sequences together with its promoter and regulatory regions. The 27 exons form a 6.5 kb long coding sequence, which is capable of encoding a protein of

The *CFTR* gene product is not limited to the cells of epithelial origin. In fact*, CFTR* mRNA transcripts and/or CFTR protein have been demonstrated in lung fibroblasts, blood cells, hematopoietic stem/progenitor stem cells (HSPC), alveolar macrophages, and smooth muscle cells [12-14]. In addition to its typical plasma membrane location, CFTR was also found in membranous organelles such as lysosomes of alveolar macrophages [15] and in both apical

Although over 1,900 different mutations in the *CFTR* gene are known (Cystic Fibrosis Mutation Database, http://www.genet.sickkids.on.ca/cftr/Home.html), approximately 66% of the patients worldwide carry the F508del mutation (a deletion of three nucleotides that results in a loss of phenylalanine at position 508 of the CFTR protein) with somewhat higher prevalence in Western Europe and USA [17]. This type of mutation causes an incorrectly assembled CFTR protein resulting in endoplasmatic reticulum (ER) retention and degradation of the protein [18] as well as defective regulation [19]. Patients homozygous for F508del usually have more pronounced clinical manifestations compared to heterozygotes and genotypes without

Based on the amino-acid sequence and its structure, CFTR is identified as a member of the superfamily of ATP-binding cassette (ABC) transporters. However, among the thousands of ABC family members, only CFTR is an ion channel [24, 25]. ABC transporters are ubiquitous in the entire animal kingdom due to their role in coupling transport to ATP hydrolysis. They also are involved in many genetic diseases [26]. Like other ABC transporters CFTR contains two membrane-spanning domains (MSDs), two hydrophilic nucleotide-binding domains (NBDs) located at the cytoplasmic site of the protein, and, as a unique feature among ABC transporters, a regulatory domain (R domain) located between NBD1 and MSD2. The R domain contains several consensus phosphorylation sites for protein kinases A (PKA) and C

of kinase and phosphatase activity within the cell and by cellular ATP levels [28]. Activation of PKA causes the phosphorylation of multiple serine residues within the R domain leading to conformational changes in this domain [29] relieving its inhibitory functions on CFTR

secretion in airway epithelial cells and

channel is tightly controlled by the balance

and HCO3-

absorption of Na+

4 Regenerative Medicine and Tissue Engineering

*1.1.1. The CFTR gene*

1480 amino acids [11].

*1.1.2. The CFTR protein*

and impaired Cl-

and basolateral membrane of the sweat duct [16].

(PKC) [27]. The opening and closing of the CFTR Cl-

F508del [20-22] although these differences are highly variable [23].

The airway epithelium is a target for potentially noxious substances and pathogens. It plays a critical role in maintaining a sterile undamaged airway and also separates the connective tissue as well as the smooth muscle from the airway luminal contents. In addition to its barrier function, the airway epithelium has a regulated fluid and ion transport together with a secretory function, although its function is mainly absorptive [46]. It can produce mucus, and can release mediators of the immune system such as lysozyme, lactoferrin, mucous glycopro‐ tein, immunoglobulins, chemokines, cytokines, lectins and β-defensin (cationic antimicrobial peptides) [47, 48].

Furthermore, the airway epithelium produces antioxidants such as glutathione and ascorbic acid [49]. Aside from these protective functions it also regulates the airway physiology via production of smooth muscle relaxant factors such as prostaglandin E2, nitric oxide and enzymes, which catabolize smooth muscle contractile agonists [50, 51].

In normal human airways the surface epithelium is on average 50 μm thick and rests on a basement membrane. The epithelium in the major bronchi and proximal bronchioles is ciliated pseudostratified with the main cell types: ciliated and secretory columnar cells, and underlying basal cells. In addition, immune cells, inflammatory cells and phagocytic cells migrate to and remain within the epithelium [52].

More distally, in the terminal bronchioles, the epithelium changes towards a simple ciliated columnar and, finally, to simple cuboidal epithelium with ciliated and non-ciliated cells (Clara cells) [53]. In addition brush cells (columnar with microvilli only) have been identified in the respiratory tract from nose to alveoli [54]. Scattered along the respiratory tree, various progenitor niches are present in the airway epithelium [55].

It has been widely accepted that acinar gland serous cells are the predominant site for CFTR expression in the human large airways, arguing for a dominant role of submucosal glands in the volume regulation of airway surface liquid (ASL) and CF [56-59]. However, these findings have later been debated. It has been demonstrated that normal (but not the F508del) surface airway epithelia express CFTR in every ciliated cell, also in glandular ducts, with decreased expression towards the distal airways. This suggests a key role for the superficial epithelium in the initiation of ASL volume depletion and as the site for early disease [60]. It also supports a role for CFTR in regulating glandular secretion homeostasis, but predominantly in the submucosal ducts rather than in the serous acini as was earlier proposed.

#### **2.1. Ion and water transport in airway epithelium**

Net vectorial fluid transport depends critically on ENaC and CFTR operating in concert with the paracellular and transcellular pathways [61].

*Fluid absorption* is mainly controlled by the transport of Na+ through apical ENaC, which is also the dominant basal ion transport process. *Fluid secretion* is regulated by cell-to-lumen move‐ ment of Cl- , via CFTR, CaCC and volume regulated chloride channel, and/or HCO3 via the interactions between CFTR and the SLC26 channel. In both cases the transport occurs along the electrochemical gradient and the movement of counterions likely takes place predomi‐ nantly through leaky tight junctions [61].

Over the basolateral membrane a Na+ gradient is maintained by the Na+ -K+ -ATPase, which pumps 3 Na+ ions out of the cell for every 2 K+ ions coming in. As a result the intracellular concentration of Na+ is low (20 mM), whereas the K+ concentration is high (150 mM) [62]. In addition, the Na+ -K+ -2Cl co-transporter moves Cl against its electrochemical gradient and accumulates Cl- inside the cell to be released via apical channels. Secretion of Cl is electrically coupled to efflux of K+ through basolateral K+ conductance channels [63]. Through the paracellular pathway, Cl is absorbed or Na+ secreted and the water-flow is regulated by diffusion following osmotic gradients.

The maintenance of the electro-osmotic gradients is dependent on limiting back diffusion. The tightness of the paracellular barrier and the molecular selectivity together contribute to the overall epithelial transport characteristics [64]. In many epithelia the transport of different ions is performed by different cell types, however, in airway epithelia the ciliated cell is responsible for both secreting Cl and absorbing Na+ [65].

#### **2.2. The airway surface liquid**

production of smooth muscle relaxant factors such as prostaglandin E2, nitric oxide and

In normal human airways the surface epithelium is on average 50 μm thick and rests on a basement membrane. The epithelium in the major bronchi and proximal bronchioles is ciliated pseudostratified with the main cell types: ciliated and secretory columnar cells, and underlying basal cells. In addition, immune cells, inflammatory cells and phagocytic cells migrate to and

More distally, in the terminal bronchioles, the epithelium changes towards a simple ciliated columnar and, finally, to simple cuboidal epithelium with ciliated and non-ciliated cells (Clara cells) [53]. In addition brush cells (columnar with microvilli only) have been identified in the respiratory tract from nose to alveoli [54]. Scattered along the respiratory tree, various

It has been widely accepted that acinar gland serous cells are the predominant site for CFTR expression in the human large airways, arguing for a dominant role of submucosal glands in the volume regulation of airway surface liquid (ASL) and CF [56-59]. However, these findings have later been debated. It has been demonstrated that normal (but not the F508del) surface airway epithelia express CFTR in every ciliated cell, also in glandular ducts, with decreased expression towards the distal airways. This suggests a key role for the superficial epithelium in the initiation of ASL volume depletion and as the site for early disease [60]. It also supports a role for CFTR in regulating glandular secretion homeostasis, but predominantly in the

Net vectorial fluid transport depends critically on ENaC and CFTR operating in concert with

the dominant basal ion transport process. *Fluid secretion* is regulated by cell-to-lumen move‐

interactions between CFTR and the SLC26 channel. In both cases the transport occurs along the electrochemical gradient and the movement of counterions likely takes place predomi‐

, via CFTR, CaCC and volume regulated chloride channel, and/or HCO3-

gradient is maintained by the Na+

through apical ENaC, which is also


ions coming in. As a result the intracellular

concentration is high (150 mM) [62]. In

against its electrochemical gradient and

conductance channels [63]. Through the

secreted and the water-flow is regulated by

via the


is electrically

enzymes, which catabolize smooth muscle contractile agonists [50, 51].

progenitor niches are present in the airway epithelium [55].

**2.1. Ion and water transport in airway epithelium**

the paracellular and transcellular pathways [61].

nantly through leaky tight junctions [61].

Over the basolateral membrane a Na+


diffusion following osmotic gradients.

ment of Cl-

pumps 3 Na+

concentration of Na+

coupled to efflux of K+

paracellular pathway, Cl-

addition, the Na+

*Fluid absorption* is mainly controlled by the transport of Na+

ions out of the cell for every 2 K+

is low (20 mM), whereas the K+

through basolateral K+

is absorbed or Na+

co-transporter moves Cl-

accumulates Cl- inside the cell to be released via apical channels. Secretion of Cl-

submucosal ducts rather than in the serous acini as was earlier proposed.

remain within the epithelium [52].

6 Regenerative Medicine and Tissue Engineering

ASL, the fluid covering the airway epithelium, consists of a periciliary layer (PCL), which is a watery layer surrounding the cilia, and of mucus on top of the cilia. Mucus is produced mainly by the submucosal glands, while a small amount is produced by the goblet cells. In normal airways PCL height is defined as the length of an outstretched cilium (~6 μm) [66], whereas the ASL layer (mucus plus PCL) varies in thickness of 20-150 μm for different species (20-58 μm in humans) [67]. ASL is the first line of defense against inhaled pathogens and is important for mucociliary clearance. It contains *e.g.*, mucins, phospholipids, albumin, lactoferrin, lysozyme, proteases, defensins and other peptides, ions and water [68], see also paragraph 2.1. The composition, volume and physical properties of the ASL depend manly on secretions of the airway submucosal glands and the absorptive properties of the surface epithelial cells. Regulation of the balance between absorption and secretion determines the net transport of ions across the epithelium through transcellular and paracellular pathways and, thus the mass of salt on an epithelial surface [69].

#### **2.3. Pathogenesis of CF lung disease**

The lung of CF patients is normal at birth, but soon after birth an endobronchiolitis ensues with surprisingly few pathogenic bacterial species (*Pseudomonas aeruginosa* in most cases), and which is associated with an intense neutrophilic response localized to the peribronchial and endobronchial spaces [70-72]. The neutrophil-dominated inflammatory response is harmful for the host by causing exaggerated production of inflammatory cytokines and proteases which may sustain infection [73]. CF primarily affects the airways and submucosal glands with sparing of the interstitium and alveolar spaces until late in the disease [74, 75]. The CF lung disease is characterized by a picture of airway epithelial injury [76] and remodeling, such as squamous metaplasia [77], cell hyperproliferation [78], basal and goblet cell hyperplasia, and hypersecretion of mucus due to the inflammatory profile [79-81]. The epithelial regeneration characterized by successive steps of cell adhesion and migration, proliferation, pseudostrati‐ fication, and terminal differentiation is disturbed and characterized by delayed differentiation, increased proliferation, and altered pro-inflammatory responses [82].

There are several hypotheses about the early pathogenetic steps in the CF lung disease and how defective CFTR leads to the airway disease:

**•** *The low ASL volume hypothesis* claims that the ASL is isotonic both normally and in CF. CFTR functions both as a Cl channel and as an inhibitor of the ENaC. In CF airway epithelia, with an absence of either molecular or functional CFTR, there will be unregulated Na+ absorption and a decreased capacity to secrete Cl- . This leads to dehydration of the airway surface, with a collapsed PCL, concentration of mucins within the mucus layer, and adhesion of mucus to the airway surface [83].


An interesting question is what the role of aquaporins (AQP) is in the production of ASL, compared to paracellular water flow and CFTR. In the epididymis, CFTR appears to regulate AQP-mediated water permeability [93]. In this tissue, CFTR is co-localized with AQP9 in the apical membrane, and this association promotes the activation of AQP9 by cAMP [94]. In a heavily debated study, concerning the clinical benefit of nebulized hypertonic saline in cystic fibrosis, an important role of amiloride-inhibitable AQP water channels in the generation of ASL was proposed [95]. However, although the positive effect of hypertonic saline as such is not disputed, the question whether this effect is mediated by AQP has received conflicting answers [96, 97] and is still open. Recently, it has been found that interleukin (IL)-13 enhances the expression of CFTR but abolishes the expression of AQP in airway epithelial cells [98]. In conclusion, the relation between CFTR and AQP needs further study.

The differences in the proposed hypotheses are due to difficulties in determining the accurate composition of the ASL because of the very small depth of the layer. Among the problems encountered there are difficulties to collect an adequate amount of ASL without disturbing the epithelium and inducing secretion from submucosal glands or leakage of interstitial fluid into the lumen, which may modify the composition of the ASL [99].

Furthermore, fluid secretion by submucosal glands differs markedly between mammalian species. For example, in transgenic mice that serve as animal models for CF, the fluid transport in the airways is much less affected than in CF patients [100]. It is also possible that variant forms of ENaC or different regulatory components operate in different systems [101].
