Cell development and differentiation in the fetal lung are regulated by mechanical, physiologic, and biochemical factors . The markers for peripheral lung development include maturation of alveolar type II (ATII) cells as evidenced by the appearance of lamellar bodies and increased expression of surfactant phospholipids and proteins, and transformation of ATII cells into type I cells [2–4]. Previously studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein are expressed in adult ATII cells and that the CFTR-like chloride channel is functional [5–7]. However, its role in development of fetal ATII cells is unclear. One possible mechanism is that CFTR-mediated chloride secretion in the fetal lung epithelium can concomitantly increase fluid secretion and lung fluid volume [7, 8]. Lung distention due to increased fluid secretion and fluid volume can accelerate peripheral lung development and ATII cell maturation as demonstrated in tracheal occlusion studies [9, 10]. Conversely, decreased lung volume in congenital diaphragmatic hernia can result in impaired lung growth and differentiation [9, 11]. Multiple studies utilizing in utero gene transfer and transient over-expression of CFTR have shown increased fetal lung volume, and accelerated maturation of ATII cells [11–14].
Mutations of CFTR gene have been associated with Cystic Fibrosis (CF); however, the mechanism for its direct participation in the disease pathology remains unclear . CFTR is found in the epithelial cells of many organs including the lung. The crucial role of CFTR in the cellular development and cell differentiation in the lung has become somewhat clear with studies involving in utero gene transfer technology developed by Larson and Cohen [13, 15, 16]. This technique circumvents the early developmental role of CFTR and allows investigations into the role of CFTR (or any other gene) in a stage-specific manner in accessible organs. Using this technique, recent studies have shown a role of CFTR in fetal lung development because its over-expression increases mechanical stretch in the lung .
Pulmonary surfactant is essential for the biophysical and immunologic integrity of the lungs and for maintenance of the patency of small airways and alveoli [17–19]. Phosphatidylcholine (PC) is the major phospholipid and principal surface-active constituent in pulmonary surfactant. Four surfactant proteins – SP-A, SP-B, SP-C, and SP-D – are present, each of which plays a role in lowering of the surface tension or in the innate host-defense mechanisms in the lung. Several investigations have used differential centrifugation of the bronchoalveolar lavage (BAL) fluid for further fractionation into large aggregates (LA) and small aggregates (SA) of lung surfactant . The LA fraction contains multilamellar structures, the tubular myelin, surfactant phospholipids and most of the SP-A, SP-B and SP-C proteins. It represents the newly secreted surface-active form of alveolar surfactant, which is capable of reducing the surface tension and is the precursor for the lighter SA fraction of lung surfactant and contains mostly small vesicles, surfactant phospholipids and small amounts of SP-A, SP-B and SP-C proteins [20–22].
The secretion of surfactant phospholipids in ATII cells occurs by exocytosis of lamellar bodies [23, 24]. Surfactant homeostasis is under feedback regulation by surfactant proteins which increase surfactant clearance and uptake by ATII cells [23, 25–27]. Different physiological, biochemical and pharmacological factors also regulate surfactant secretion through specific cell surface receptors like β-adrenergic, purinergic and endothelin receptors or by directly affecting second messenger like calcium, cAMP, and diacylglycerol [23, 28, 29]. Although the expression of surfactant phospholipids and proteins increases with lung and ATII cell maturation, temporal expression of each constituent may be independently regulated [30–32].
Adenovirus-mediated transfer of genes into cells has been used in several cell types including some hard to transfect cells like ATII cells. Our group has previously used this technique for in utero transfer of CFTR constructs and demonstrated gene-specific effects on lung development in various species [4, 11–13, 16, 33–37]. The expression of adenovirus-mediated transferred gene was transient and lasted only 48–72 h post-transfer [13, 34]. In our previous studies, we have repeatedly shown lack of inflammation in control animals that were treated with adenovirus-reporter gene constructs confirming our interpretations that the effects were due to the target gene transfer into presumably pluripotent cells in the developing lung. Our previous study employing in utero treatment with antisense-CFTR (ASCFTR) construct showed airway thickening with collagen deposits in the small and large airways and increased airway reactivity to acetylcholine in 100 days old rats .
This study was undertaken to determine if transient disruption of lung organogenesis can result in altered phenotype of ATII cells in the adult lung. Because our previous studies suggested accelerated maturation of ATII cells with transient over expression of CFTR in fetal lung , we postulated that a transient knock down of CFTR in fetal lung would cause delayed or erratic development and cause persistent effects in ATII cell phenotype. We now show that a similar approach utilizing transient in utero disruption of CFTR caused changes in surfactant protein expression and in the alveolar surfactant phospholipid pool in lungs of adult ASCFTR rats. Studies with isolated adult lung ATII cells from these animals also showed changes in surfactant secretion suggesting altered cell phenotype. The latter was also confirmed by gene array analysis of RNA from freshly isolated ATII cells, which showed altered expression of several genes including calcium regulating genes, the Ca2+-transporting ATPase (Atp2c2, also known as Spca2) and calcium-dependent calmodulin kinase kinase1 (CaMkk1). The latter is known to cause phosphorylation and stimulation of calcium-calmodulin kinase I (CaMKI) [38, 39]. The expression levels of CaMKI, which also regulates surfactant secretion in ATII cells , were elevated in ATII cells from ASCFTR rats. Thus, transient in utero knock down of CFTR causes changes in lung development which is reflected in altered ATII cell phenotype in the adult lung.