PHYSICAL FORCES AND FETAL LUNGS

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Based on clinical observations and physiological studies, it has been realized for a long time that physical factors play important roles in regulating fetal lung growth and maturation (12, 13, 18, 19). Physical force-related lung disorders during the perinatal period could be very severe or lethal. Pulmonary hypoplasia is the most common single autopsy finding in the first week after birth (20, 35). It is associated with a variety of congenital disorders and malformations, which have in common a reduction in lung volume and/or fetal breathing movements (20, 35). For example, lesions such as congenital diaphragmatic hernia (8) and thoracic abnormalities (6) limit the amplitude of fetal breathing movements and reduce the liquid volume and pressure in the potential airway and alveolar space. In utero tracheal occlusion is one of the most potent stimuli of fetal lung growth (4). Clinical trials have been conducted to test whether this approach could be used to treat severe pulmonary hypoplasia (7, 15). However, it has been found from animals that prolonged tracheal occlusion causes qualitative and quantitative deficiencies of the lung alveolar type II epithelial cells, resulting in significant reduction of lung surfactant and surfactant proteins (SPs) (17). To properly overcome pulmonary hypoplasia, mechanisms regulating mechanical stretch-induced cell proliferation and differentiation need to be elucidated.

Bronchopulmonary dysplasia is the most common cause of chronic lung disease in infants (36). Mechanical ventilation-induced lung injury, as well as pulmonary immaturity, oxygen toxicity, and secondary infection, are the major etiologic factors for this disease. Many clinical studies and animal models have been developed to investigate the effects of physical forces on fetal and neonatal lung growth, maturation, and injury (3). However, it is difficult to ascribe the effects of mechanical perturbation to individual components, because of the cellular and spatial complexity of lung tissue. Models at the cellular and molecular levels have been developed over the last decade. The effects of physical forces on the functions of lung cells have drawn increasing attention (27). In this paper, we will focus on mechanical stretch-induced fetal lung cell proliferation, differentiation, and cellular injury, and related signal transduction events.

	PHYSICAL FORCES AND FETAL LUNG CELL PROLIFERATION

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In a fetal sheep model, it was found that, after tracheal obstruction, fetal lung DNA synthesis rates increased to a maximum at day 2, were reduced but still elevated at day 4, and returned to control levels by day 10 (16). However, the relative contributions of fetal breathing movements vs. lung volume cannot be easily resolved by using experimental animal models, because these two mechanisms are interdependent, i.e., breathing movements influence regional lung volume and the effectiveness of breathing movements depends on an adequate lung volume. Most experimental approaches in animal models cannot discriminate between the effects of the static stretch of lung liquid in the potential airways and the effects of cyclic stretch derived from fetal breathing movements (13). For example, spinal cord transection has been used to abolish fetal breathing movements, but it also reduces fluid production from the potential airway (14). In vitro studies, therefore, are very helpful in determining the role of stretch in fetal lung cell proliferation.

Different from regular breathing, the bursts of fetal breathing movements are separated by resting periods (12). Using a three-dimensional (3D) cell culture model (24, 45), it has been shown that an intermittent mechanical stretch regimen (5% elongation, 60 cycles/min, 15 min/h) stimulated DNA synthesis and cell division of mixed fetal rat lung cells (26). A continuous cyclic stretch (1 cycle/min) has been shown to increase DNA synthesis of primary cultured fetal rabbit pneumocytes cultured as a monolayer (43). Continuous stretch (60 cycles/min) also stimulated proliferation of a human embryonic fibroblast cell line (IMR-90) (1). In addition, the 3D cell-stretch model increased proliferation of primary cultured fetal rat lung fibroblasts as well as epithelial cells (29, 54) in a gestational-dependent manner (54). Therefore, cyclic mechanical stretch appears to stimulate fetal lung cell growth directly.

Mechanical stretch-induced fetal lung cell proliferation is mediated by growth factors. It has been found that conditioned medium collected from stretched fetal rat lung cells contained proliferating activities for static cultured fetal rat lung epithelial cells (31). Similarly, conditioned medium from the human embryonic fibroblasts (IMR-90 cell line) enhanced proliferation of static cultured cells (1). Further studies with fetal rat lung cells demonstrated that mechanical stretch stimulated gene expression and protein synthesis of platelet-derived growth factor B (PDGF-B) and its -receptor (23). These in vitro results are in agreement with observations from animal models. Either lung liquid drainage (16) or spinal cord transection (14) decreased mRNA levels of insulin-like growth factor II, whereas tracheal occlusion increased insulin-like growth factor II mRNA expression (16). Therefore, mechanical stretch could effectively and inversely regulate the production of growth factors and enhance the responsiveness of cells to growth factors by upregulating the expression of related receptors, which can further mediate cell proliferation through autocrine and/or paracrine mechanism(s) (27).

Mechanical stretch-induced cell-proliferating activities are mediated through intracellular signal transduction pathways (27). It appears that mechanical stretch-induced activation of protein kinase C (PKC) is critical for the mediation of stretch-induced proliferation of fetal rat lung cells (28). PKC activation requires increased diacylglycerol (DAG) and Ca²⁺ mobilization from its intracellular storage with inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] as the second messenger (28). Mechanical stretch activates protein tyrosine kinases (PTK), such as pp60^src, which consequently activates phospholipase C (PLC) by increasing its tyrosine phosphorylation (25). Activated PLC resulted in hydrolysis of phosphatidylinositol 4,5-diphosphate to generate DAG and Ins(1,4,5)P₃ (28). In addition, phospholipase D is also activated by mechanical stretch, which maintains the prolonged elevation of DAG levels after the onset of mechanical stretch (28). The entry of extracellular Ca²⁺ through stretch-activated ion channels is also involved in stretch-induced PKC activation and cell proliferation. Blocking these steps on the signal pathway inhibited or attenuated stretch-induced fetal lung cell growth (23, 25, 28-30). Therefore, the PTK-PLC-PKC axis appears to be a major pathway for stretch-induced proliferation of fetal rat lung cells (Fig. 1).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Mechanical stretch-activated intracellular signal transduction pathways. Putative mechanisms of mechanical stretch-induced fetal lung cell proliferation are used as an example to demonstrate the complexity of mechanotransduction in fetal lung cells. Mechanical stretch activates protein tyrosine kinases (PTK) and consequently activates phospholipase C (PLC) via its tyrosine phosphorylation. PLC mediates the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC), which requires Ca2+ mobilized by IP3 from intracellular storage and the entry of extracellular Ca2+ via stretch-activated ion channels. Phosphotidylcholine (PC) is another source of DAG through stretch-induced activation of phospholipase D (PLD). PKC and other signals may activate transcriptional factors that bind to special response elements, such as stretch response element (SRE) and shear stress response element (SSRE). Increased gene expression and production of growth factors and their receptors may further regulate cell proliferation via related signal pathways.

How PKC mediates stretch-induced fetal cell proliferation is still unknown. One possible mechanism is to activate transcriptional factors, which can bind to the regulatory elements of the promoter region of the target gene. A shear stress-response element has been characterized for the PDGF-B chain promoter (39) and has also been found in several other genes that could be induced by shear stress (39). A stretch response element (42) and several other similar elements, which could mediate physical force-initiated gene expression in various cell types, have also been found from different genes (summarized in Ref. 27). Whether similar mechanisms are present in fetal lung cells needs to be elucidated. Once the expression of growth factors is upregulated, newly synthesized growth factors may activate their specific receptors and further stimulate cell proliferation (Fig. 1). It is likely that the interaction between mechanotransduction pathways and growth factor-related signal transduction pathways is overlapping, which requires further investigation.

In addition to the PTK-PLC-PKC axis, several other signal transduction pathways are also activated by mechanical stretch in fetal or adult lung cells (27). For example, cAMP is increased by stretch in fetal lung cells (43, 46). The pp125^FAK pathway is activated by stretch in airway smooth muscle cells (47) and so is mitogen-activating protein kinase in type II pneumocytes (38). However, the downstream cellular functions mediated by these pathways still need to be elucidated.

	PHYSICAL FORCES AND LUNG CELL DIFFERENTIATION

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Maturation of the fetal lung is a complicated process that requires proper coordination of cell growth and differentiation at each stage of lung development. Mechanical force-initiated signals have been found to affect the gene expression, protein synthesis, and secretion of many differentiation-related molecules. The effects and signals vary, depending on the molecules of interest, gestational stages, experimental conditions, and many confounding factors. Thus in contrast to the stretch-induced cell proliferation, mechanical forces likely affect these molecules through many different mechanisms and signaling pathways.

Effects of mechanical forces on extracellular matrix molecule production from fetal lung cells. Synthesis and secretion of extracellular matrix (ECM) molecules is one of the important signs of lung maturation. ECM plays important roles in holding different cell types together and mediating cell proliferation and differentiation via cell-ECM interactions. The components of ECM can be divided into four broad categories: collagens, noncollagenous glycoproteins (such as fibronectin and lamimin), glycosaminoglycans (GAGs) and proteoglycans, and elastic fibers (11).

Effects of mechanical stretch on fetal lung epithelial cells. It has long been thought that hormonal factors control fetal lung differentiation, whereas mechanical factors control growth (9, 17). However, mechanical stretch also affects fetal lung epithelial cell differentiation. The pulmonary alveolar epithelium of the mature lung is composed of two types of cells. Type I cells, which cover most of the alveolar wall for gas exchange, are extremely large and very thin. Type II cells are cuboidal-shaped cells containing morphologically distinct lamellar bodies, the storage organelles of pulmonary surfactant. Type II cells are likely progenitors of type I cells. The morphology, function, and phenotype of alveolar epithelial cells continuously change during development. During early lung development, undifferentiated alveolar epithelial cells have a cuboidal appearance but contain few lamellar bodies. They further differentiate into type II cells, and some of them then turn to type I cells.

	IMPORTANT ISSUES FOR MECHANOTRANSDUCTION STUDIES

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Above, we described that mechanical stretch has different effects on gene expression and other cellular functions when applied under different experimental models (in vivo vs. in vitro), on different cells (primary culture vs. cell line), and with different cell culture conditions (static vs. cyclic stretch, 2D vs. 3D culture, single cell types vs. mixed cells, etc.). These discrepancies of experimental results from mechanotransduction studies raise many questions that need to be addressed in future investigations.

In vivo vs. in vitro. The complexity and chronic nature of in vivo experimental systems make mechanical force-induced intracellular or intercellular signal transduction studies difficult or impossible. Cell stretch and lung explant stretch models in vitro could provide invaluable information if they are used properly. In tissue culture studies, lung tissues or cells are subjected to mechanical stretch in standard culture media. These simplified conditions provide a relatively "clean" background; thus confounding factors could be selected as specific aims for studies or "add back" to the study system. For example, fetal lung cells or tissues collected from different gestational stages have been subjected to mechanical stretch with various combination ratios (54), and the combined effects of hormonal factors such as glucocorticoids have been studied, together with mechanical stretch on fetal rat lung cells (34).

Cell-cell interaction. Interactions between different cell types or the interactions among the same type of cells via autocrine and paracrine mechanisms are likely important for mechanical force-initiated signaling. It has been found that mechanical stretch in monolayer culture stimulated the expression and production of a differentiation factor, parathyroid hormone-related peptide (PTHRP), from fetal lung epithelial cells and increased the responsiveness of fetal lung fibroblasts to PTHRP (49). PTHRP released by type II cells can specifically bind to its receptor on contiguous fibroblasts, stimulating cAMP as the second messenger, to induce specific functions of fetal lung fibroblasts (49). It has been postulated that this paracrine mechanism is involved in augmenting glucocorticoid binding, increasing metabolic activities (such as lipoprotein lipase elaboration and triglyceride uptake), and stimulating production of cytokines (such as interleukin-6 and -11). These cytokine molecules may act as intercellular mediators and, thereby, increase the synthesis of surfactant phospholipids and SPs from alveolar epithelial cells (49). Mechanical stretch-induced upregulation of PDGF-B and its -receptor is another example of autocrine and/or paracrine communication (23). Thus, on one hand, stretch increases the production of growth factors, and, on the other hand, stretch enhances the responsiveness of target cells toward these proliferating factors.

3D vs. 2D culture: maintaining cell phenotypes. An important issue in studying lung-specific functions and gene regulations is to maintain the phenotypes of lung cells. For this reason, primary cultured cells are commonly used, even though it is very expensive, time consuming, and technically demanding with greater interexperimental variations. To prevent the loss of cell phenotype, rat alveolar epithelial cells, for example, were stretched 1 day after being inoculated on fibronectin-coated membranes (10). Fetal lung explants have been used to maintain the 3D property of lung tissues (10). Mixed fetal rat lung cells in organotypic culture is another method to create miniature "alveolar-like structures" in vitro (26, 29, 44). Culturing lung cells in a 3D environment has been considered an important issue (22), specifically for mechanotransduction studies (27). However, data from mixed cell cultures are usually difficult to interpret, and the 3D structure of tissues makes the observation of cell morphology and other microscopic studies less possible. Therefore, mechanotransduction studies using monolayer cell culture are needed, but some important cellular responses have only been observed when different cell types were coexisting (22, 29).

Cofactors of mechanotransduction pathways. The presence of costimuli is also very important to simulate in vivo conditions for mechanotransduction studies. At different gestational stages, the endocrine system provides a different hormoral environment for the fetal lung cells. These environmental conditions need to be considered carefully when planning related studies in vitro. Mechanical stretch and oxygen toxicity are the two major components of ventilation-induced acute lung injury, especially in infants (36). The combined effects of stretch and oxygen need to be studied. The transmission of signals initiated by mechanical stretch could depend on the activation of other pathways. For example, PGI₂ and its stable chemical analog iloprost enhanced mechanical stretch-induced surfactant secretion in alveolar epithelial type II cells by increasing cAMP content, whereas the increase in cGMP content, induced by a nitric oxide donor, spermine NONOate, has no such effect (41). Prostacyclin could bind to its cellular membrane receptor through G protein to activate the cAMP-protein kinase A pathway and to facilitate mechanical stretch-activated signals for surfactant secretion (41). Similar mechanisms may exist for other factors when they are applied to cells together with mechanical forces.

Static vs. cyclic stretch. A major debate over the past three decades has been the importance of fluid-derived airway expansion vs. fetal breathing movements in fetal lung development (17, 18), because both are interdependent in animal models. With the use of cell or tissue culture models, it has been shown that both static (tonic) (9, 10, 51) and cyclic stretches (1, 2, 26, 45) have profound effects on cellular functions. Mechanical stretch is applied to the substratum on which cells or lung explants are cultured. Detachment and reattachment of cells through cell-ECM interaction continuously change the adhesion of cells to the surface of culture substratum. Thus, when prolonged static stretch is applied to the substratum, the tension applied to the cells could be released by the rearrangement of cells. Similarly, it has been found that the alignment of cells can be changed by cyclic stretch. Randomly distributed fetal lung cells on culture surface could be rearranged perpendicularly to the direction of the stretch (1, 2).