The effects of mechanical forces on lung functions

Respiration Physiology 119 (2000) 1 – 17 www.elsevier.com/locate/resphysiol

Frontiers review

The effects of mechanical forces on lung functions Hubert R. Wirtz a, Leland G. Dobbs b,* b

a Department of Medicine (I), Uni6ersity of Leipzig, 04103 Leipzig, Germany Departments of Medicine and Pediatrics, Cardio6ascular Research Institute, Uni6ersity of California, San Francisco, Laurel Heights Campus, Suite 150, 3333 California Street, Box 1245, San Francisco, CA 94118, USA

Accepted 18 October 1999

Abstract The lung is a dynamic organ that is subjected to mechanical forces throughout development and adult life. This review article addresses the types of mechanical forces in the lung and their effects on development and normal lung functions. The effects of mechanical forces on the various different cell types of the lung are discussed, as are the mechanisms underlying mechanotransduction. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Forces, mechanical, lung, cell types; Lung function, mechanical lung forces; Mechanics of breathing, lung function

1. Introduction The lung is a dynamic organ subjected to varying mechanical forces throughout life. During fetal development, the lung is subject to both tonic distention and fetal breathing movements. After birth, cyclical expansion and relaxation is brought about by changes in transpulmonary pressure produced by the muscles of the chest wall and diaphragm. Changes in lung volume not only result in airflow but also affect many aspects of lung metabolism, function and growth. The focus of this review is the influence that mechanical forces play in lung development and * Corresponding author. Tel.: +1-415-4765995; fax: +1415-4763586. E-mail address: [email protected] (L.G. Dobbs)

metabolism. Mechanical forces may be secondary to gradients in gravity, motion, osmotic forces and interactions between cells and/or matrix components. Physical forces are important even with unicellular organisms such as Paramecium and Stylonychia. With normal movement, following collision of the organism with an object, the ciliary beat pattern reverses and the ciliary beat frequency increases, providing an immediate correction in the directionality of movement. Where appropriate, the effects that mechanical forces have on nonpulmonary systems will be referred to for comparison purposes. For more general reviews of mechanotransduction, the reader is referred to several excellent recent publications (e.g. Banes et al., 1995; Chien et al., 1998).

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2. The nature of physical forces in the lung Although physical forces are often referred to by imprecise terms such as ‘stretch’ or ‘distention’, they are more accurately defined as follows: ‘stress’ (force per unit area), ‘strain’ (the change in length in relation to the initial length), and ‘shear stress’ (force per unit surface area in the direction of flow exerted at the fluid/surface interface). The term ‘stretch’ has been used by many investigators synonymously with ‘strain’. In this review we will use the term ‘strain’ more broadly to describe forces leading to cellular deformations that are not necessarily confined to one-dimension and that are not caused by fluid shear stress or an increase in ambient hydrostatic pressure. Osmotic forces, which modulate signal transduction in some cell types may also prove to be important in cells that transport ions and water, such as pulmonary endothelial and epithelial cells. Cells within a complex three-dimensional structure such as the lung are likely to be subjected to a variety of different physical forces. One might speculate that most lung cells would be subjected to some degree of strain. Strain might be more prominent in cells of the alveolar wall and epithelium during breathing, while shear forces may have differential effects on various portions of the vascular bed, with a gradient from the pulmonary arterial endothelium to the capillaries. Shear stress forces may play a role in cells other than vascular endothelium, such as the effects of pleural fluid on pleural mesothelial cells and the fluid hypophase on airway and alveolar epithelial cells. Vascular endothelium might also be subjected to strain and hydrostatic pressure. Finally, physical forces can be generated within cells. Strain can be generated by cytoskletal rearrangements (such as actin contraction) that caused tension to be transmitted throughout the cell, including the nucleus. The term ‘autobaric effects’ has been proposed by Banes et al. (1995) for these types of physical forces that are generated intracellularly, in contrast to ‘parabaric effects’ generated by intercellular or cell – matrix interactions. These authors have proposed a model in which the basal equilibrium stress state of cells can be altered by extracellular perturba-

tions. As a consequence of perturbations and in order to minimize intracellular strain, the interactions between cytoskeletal components and integrins may be strengthened, actin cables may polymerize, and tension on focal adhesions may increase, resulting in stimulation of matrix protein synthesis and secretion and an increase in focal adhesion complexes. These complex interactions ultimately result in a realignment of cell structure. The opposite may occur when strain is reduced. Ingber and coworkers have postulated that there is an equilibrium between tensile forces within the cell and independent but interconnected compression-resistant intracellular structures (reviewed in Chicurel et al., 1998); they have called this ‘tensegrity’, based on architectural principles of tensional integrity. Evidence supporting this hypothesis has been garnered from studies of cells transfected with microtubule-associated proteins labeled with fluorescent probes. Individual microtubules buckle under compression as they extend and push against cytoskeletal elements being compressed by microfilaments (Kaech et al., 1996). Based on structural analogies, Ingber et al. (1993a) have proposed that tensegrity theory may apply both at the cellular level and also in tissue organization and biological architecture. Furthermore, the three-dimensional architecture of cells within a tissue as well as the interactions among cells and matrix components may determine the manner in which strain affects cellular functions. For example, fetal lung cells exhibit increased DNA synthesis when strained in a gel, but respond differently when strained as a monolayer (Liu et al., 1995a). Changes in cellular shape (modulated by matrix components) may result in changes in differentiation and/or proliferation. Conversely, physical forces have been shown to stimulate cells to modulate the synthesis of matrix components (e.g. Leung et al., 1976) and to strengthen the integrin–cytoskeleton linkages of focal adhesion complexes (FAC) (Choquet et al., 1997). These observations demonstrate that physical forces, the extracellular matrix, and the tensile status of the cell are interdependent, in accordance with assumptions of the tensegrity model. Some components of the extracellular matrix have the

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capability of modulating growth factor activities. Aspects of cellular differentiation, apoptosis, motility, signal transduction, gene expression, chromosome movement and remodeling of the extracellular matrix (ECM) are influenced by cellshape changes brought about by exogenous or by cell-generated mechanical forces (Chicurel et al., 1998). Although many aspects of cellular responses to mechanical perturbation appear to be present in all anchorage-dependent cells, some celluar responses have evolved to serve organ-specific functions, such as surfactant metabolism in the lung

2.1. Quantitation of physical forces There have been several different approaches to quantifying the extent to which cells undergo mechanical deformation with changes in the extent of lung inflation. The interpretation of these studies is complicated because the degree of unfolding or stretching of the alveolar epithelium is dependent on the lung volume history prior to fixation (Oldmixon and Hoppin, 1991) and because investigators have measured different endpoints, such as the surface area of the alveolar tissue–air interface (Mercer et al., 1987), collagen fiber length (Mercer and Crapo, 1990), or epithelial basement membrane surface area (Tschumperlin and Margulies, 1999). Because changes in either collagen fibril length or basement membrane surface area may correlate with the extent of distention/contraction of the basal portion of the epithelial cell, these measurements may reflect how inflation and deflation affect basal cell surface area. These studies suggest that, when lung volume increases from 40 to 100% TLC, epithelial cell basal surface area increases between 16% (Bachofen et al., 1987) and 34 – 35% (Mercer and Crapo, 1990; Tschumperlin and Margulies, 1999). There have been few attempts to measure the magnitude of the physical forces applied to cells in in vitro models. The force induced by gravity on a cell has been estimated to be less than 1 mdyne/cell (Albrecht-Buehler, 1990). Tensile forces generated within the cytoskeleton and exerted on adhesion structures may be 50 – 10 000 times greater than this (Dennerll et al., 1988).

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Many different experimental designs have been used to study the biologic effects of physical forces on lung cells. In most in vitro model systems, cells or organ explants are cultured on distensible surfaces such as membranes or gels. In these models, the amount of pressure or suction applied to the distensible substratum may not be an accurate guide to estimating the forces applied to the biologic material, because the compliance of the substratum will vary with the material used, the thickness, the surface area. The increase in cellular surface area during strain may be a better parameter to compare the effects of various different models systems. In most of the devices, cells are cultured on a coated surface of a distensible membrane, which is then distended by downward, upward, or lateral application of force. In the initial commercially-available Flexercell™ device, the membrane is subjected to a vacuum applied to the underside of the membrane. Changes in membrane surface area are lower towards the center of the membrane (and negative in the exact center) in comparison to the more peripheral areas. Cellular surface area increases have been reported to range between 6.5 and 8.1% halfway between the center of the membrane and the perimeter of the membrane in the initial depending on whether bone, muscle or lung cells were stretched in a downward deflection by applying 20 kPa of suction (Anderson et al., 1993). In a differently-designed newer device (Flexrcell FX 3000), cells cultured on a membrane surface are distended by circumferential stretch of the membrane. We have developed a device in our laboratory in which cells cultured on a membrane are distended by a hydrostatic force applied to the bottom of the membrane. In this device, the cellular surface area of alveolar epithelial cells could be increased by 25% (Wirtz and Dobbs, 1990). The change in membrane surface area was greater than the change in cellular surface area, as has been reported in a similar but differently-designed device (Winston et al., 1989). Taken together, these observations demonstrate that the responses of cells to mechanical deformation may not be linear, perhaps because of alterations of cell–matrix adhesions, and underlines the importance of measuring the extent of cell deformation in different experimental models.

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3. Fetal lung growth During the last third of gestation, the lung grows proportionately with increasing body weight. Lung growth occurs as an increase in cell number, with DNA/g lung being constant during this period (because of space considerations, we refer to the review by Kitterman (1996) rather than cite individual references). Fetal lungs are kept inflated to a volume similar to postnatal functional residual capacity by the active secretion of fluid (Kitterman, 1996). Pulmonary hypoplasia, a major cause of death in the neonate (Liggins, 1984), results from various conditions such as congenital diaphragmatic hernia, oligohydramnios, renal agenesis and prolonged rupture of fetal membranes (Kitterman, 1996). In contrast, chronic tracheal occlusion during the fetal period, which may result from the rare clinical condition of laryngeal atresia or from experimental manipulation, leads to overdistension and hyperplasia of the lung. It has therefore been suggested that mechanical distension is the major factor influencing lung growth during fetal development. During the last trimester, intermittent breathing movements generate pressure changes similar to those with postnatal breathing. It is believed that fetal breathing movements are important for normal fetal lung growth because absence of fetal breathing movements results in lung hypoplasia (reviewed in Kitterman, 1996). Because oligohydramnios and abolition of fetal breathing movements are additive regarding the extent of lung hypoplasia, they may act by different pathways. Conceivably, fetal breathing movements might result in strain-induced fine tuning of lung growth because fluid is shifted back and forth between apical and basal lobes.

3.1. Al6eolar epithelial de6elopment in the fetal lung Until recently, fetal lung differentiation was believed to be regulated principally by hormonal factors. However, several lines of experimental evidence suggest that mechanical factors modulate differentiation as well as lung growth. Tracheal ligation, compared to drainage of lung liquid, not

only caused changes in lung growth, but appeared to modulate alveolar differentiation in that there was a qualitative impression that the ratio of type II cells/type I cells was higher in the drained, hypoplastic lungs (Alcorn et al., 1977). Tracheal ligation in sheep with surgically-produced diaphragmatic hernia caused a decrease in both the alveolar phospholipid content and the incorporation of choline into phospholipids by isolated type II cells (O’Toole et al., 1996, but not Hedrick et al., 1994). In recent studies, experimental over- or underdistention resulted in changes in alveolar epithelial development. For example, tracheal ligation in fetal sheep caused an increase in lung growth but a decrease in the content of saturated phosphatidylcholine, SP-A, SP-B, and the relative percent of type II cells; in contrast, spinal cord transection, which abolished fetal breathing movements, resulted in opposite effects (Joe et al., 1997). Taken together, these in vitro studies suggest that overdistention favors expression of the type I cell phenotype at the expense of the type II cell phenotype; underdistention has the opposite effects. This hypothesis has been directly tested in vitro. Studies of mechanical effects in vitro support the hypothesis that mechanical distention promotes expression of the alveolar epithelial type I cell phenotype and suppresses expression of the type II cell phenotype. In contrast, contraction (or lack of distention) has the opposite effects, favoring expression of the type II cell phenotype at the expense of the type I cell phenotype. Gutierrez et al. (1999) have studied the effects of 18 h of mechanical distention on both fetal lung explants and type II cells isolated from adult rat lungs (Gutierrez et al., 1998). Although the amount of distention was unknown in the fetal lung explants, the type II cells were distended to increase the surface area by approximately 22%. In both cases, distention increased expression of RTI40, an apical integral membrane protein specific within the lung for type I cells. With fetal lung explants, this effect was large, approximately ten times greater than undistended controls. In contrast, distention inhibited expression of surfactant proteins B and C. The inhibitory effects were also approximately 10-fold. Mechanical distention affected transcrip-

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tion of each of these marker genes. Taken together, the findings in vivo and in vitro suggest that both continuous distention due to physiologic liquid filling of the lung together with some degree of tracheal/pharyngeal resistance and intermittent strain during fetal breathing movements may all be important for proper lung growth and maturation. These findings are summarized in Table 1.

3.2. Fetal lung growth: mechanisms on a cellular le6el Liu et al. (1992) have developed a model for examination of the influence of mechanical strain on mixed fetal rat lung cells. In this model, mixed lung cells obtained from rats at 19 days of gestation were seeded into gelfoam sponges; with time in culture, a lining cell layer develops within cavities in the sponge. The gelfoam sponges were elongated; presumably the cells were subjected to mechanical forces, although the extent of cellular deformation was not quantified. Gelfoams were stretched 60 min − 1 for 15 min/h for a total duration of 48 h. After four days of culture, [3H]thymidine incorporation increased by 60% and cell number by 12%. These increases were inhibited by a high seeding density (4 × 105/cm2 and higher), decreased strain (1 versus 5 or 10% foam elongation), increased concentration of fetal bovine serum, lower stretching frequency (6 min − 1 versus 30 or 60 min − 1) and decreased total stretch duration (6 h versus 12, 24, 48 h). DNA synthesis in both control and stretched cells was inhibited equivalently by incubation with actinomycin or cycloheximide. This model demonstrates a direct effect of cyclical intermittent strain on

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DNA synthesis in fetal lung cells, and a dependency of this effect on the amplitude, frequency, periodicity and total duration of strain. With an in vivo sheep model, continuous lung distension also caused an increase in DNA synthesis. Tracheal obstruction resulted in increased lung to body weight ratio and increased DNA synthesis determined by [3H]thymidine incorporation, while continuous drainage of pulmonary liquid with a catheter reduced both parameters when compared to control animals. Another study demonstrating that obstruction of one mainstem bronchus leads to hyperplasia of that lung and hypoplasia of the contralateral lung suggested that the mechanically induced growth of one lung does not result in concomitant growth of the other lung, e.g. induced by humoral factors (Moessinger et al., 1990). Although the mechanisms responsible for these effects remain unknown, mechanical factors clearly modulate expression of mRNA of some growth factors. Tracheal obstruction causes an increase in mRNA for insulin-like growth factor II (IGF-II), while draining of pulmonary fluid has the opposite effect (Hooper et al., 1993). IGF-II expression has been localized to lung fibroblasts in the mesenchyme and interlobar septa (Han et al., 1988). PDGF-AA, PDGF-BB and the corresponding receptors PDGF-a-R and PDGF-b-R have all been found in fetal rat lung cells (Caniggia et al., 1993), and an involvement of PDGF in fetal rat lung growth has been suggested by experiments in which antisense PDGF-B causes a reduction in DNA synthesis in embryonic rat lung explants (Souza et al., 1994) and fetal lung epithelial cells (Buch et al., 1994). PDGF-B and PDGFb-R gene and protein expression were increased in

Table 1 Mechanical factors and fetal lung growth and differentiation Factor effect (example)

Lung growth

Surfactant system maturation

Alveolar differentiation

Continuous distention (tracheal ligation)

+

Possible − (?)

Intermittent distention (fetal breathing movements) Underdistention (diaphragmatic hernia, lung liquid drainage)

+ −

Possible + (?) None (?)

Type I cell favored; type II cell inhibited Possibly type II cell favored (?) Type II cell favored

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the intermittent but prolonged strain model of Liu et al. (1995b) described in the previous paragraph. The PDGF-B gene promotor in bovine aortic endothelial cells has been shown to contain a cis-acting fluid shear-stress-responsive element that does not encode binding sites for any previously known transcription factor (Resnick et al., 1993). A core binding sequence (GAGACC) was determined; and this sequence was also found in other endothelial genes induced by shear stress. Experimental evidence from several sources suggest that calcium may be an important second messenger in mechano-sensitive fetal lung growth (e.g. Liu et al., 1994). Calcium fluxes were not altered by L-type calcium channel inhibitors or amiloride, which inhibits Na+/Ca2 + exchange, but were inhibited by gadolinium, a known inhibitor of stretch-activated channels (Yang and Sachs, 1989). Not surprisingly, protein kinase C phospholipase C-g (PLC-g) and D (PLD) activation, and protein-tyrosine kinase (PTK) activity (Liu et al., 1996), and pp60src kinase and cortactin appear to be involved. These events may be similar to the welldescribed signaling cascade in endothelial cells stimulated by shear-stress. In this model system, early events following mechanical stimuli involve K+ and Ca2 + fluxes, followed by MAP kinase signaling, NFkB activation and binding to appropriate shear-stress response elements in DNA, followed by growth factor upregulation, and cytoskeletal rearrangements (Davies et al., 1997).

4. Compensatory lung growth Mechanical factors play a role in the compensatory lung growth that occurs following pneumonectomy. Compensatory lung growth is believed to result in normal lung architecture (i.e. with normal cellular proportions, alveolar and capillary volumes, alveolar wall thickness with normal amounts of collagen and elastin). To develop normal lung artchitecture, a four

step process has been proposed (Berger and Burri, 1985): air space distention, cell proliferation, tissue remodeling and eventual restoration of normal tissue architecture. Among various factors that have been proposed to modulate compensatory lung growth, including alveolar hypoxia, changes in pH or PCO2, or mechanical distention of the lung, mechanical induction is the factor best supported by experimental data. For example, the instillation of space-occupying material into the thorax blunted compensatory growth in a dose dependent manner (Brody et al., 1978). In patients, morphometric and radiologic data confirm that the residual lung tissue is overinflated following surgical resection. Various biochemical factors have been implicated in the process of compensatory lung growth. Rannels et al. (1986) reported that as early as 3 h post pneumonectomy, there is an increased uptake of polyamines which persisted during compensatory lung growth. By day 3 following pneumonectomy, there is an accumulation of protein and RNA, followed by an increase in DNA synthesis. Adrenal steroids inhibit compensatory lung growth (Rannels and Rannels, 1988). In rats following pneumonectomy, there was an increase in both cAMP content and in cAMP-dependent protein kinase activity in the remaining lung beginning at 24 h, peaking in 3 days, and returning to baseline at 7 days. Sham-operated controls exhibited no change in cAMP content (Rannels and Rannels, 1988). Similar results were obtained in a model of isolated perfused lungs distended by CPAP, in which an increase in cAMP was seen as early as 20 min, continuing throughout the 2-h observation period (Russo et al., 1989). The difference in response times was attributed to a differing extent of tissue distention, the difference of dynamic overdistention in vivo to the static distention in vitro, or overriding factors in vivo possibly associated with the surgical procedure. These studies demonstrate that in vivo and in vitro overdistention of the lung results in a significant rise of an important cellular second messenger.

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5. Lung cells

5.1. Lung fibroblasts Surprisingly little information is available on the effects of physical forces on fibroblasts of pulmonary origin. In a recent study, Bishop et al. (1993) investigated the effect of mechanical deformation on fibroblast proliferation and autocrine growth factor activity. IMR-90, a passaged cell line derived from human fetal lung fibroblasts, were cultured on collagen-coated silastic membranes and stretched so that the resulting increase in membrane surface area was approximately 10%. Cyclical distention at 1 Hz for up to 5 days resulted in realigning of fibroblasts perpendicular to the stretch stimulus; there was also an increase in cell number in stretched cultures (+160% of control on day 4). The tissue culture medium of distended cultures was mitogenic for static cultures, suggesting that unknown autocrine growth factors were involved.

5.2. Pleural mesothelial cells Pleural mesothelial cells (PMC) are subjected to mechanical stimuli from distention of the lung during inflation and to shear forces exerted by a fluid film between the visceral and parietal pleura. It has been reported that the average diameter of visceral PMC increased from 27 to 45 mm during lung expansion in rabbits (Wang, 1985). Shear force has been estimated to be as high as 20 dyn/cm2 at a breathing frequency of 20 min − 1. Waters et al. (1996, 1997) have developed a model for testing the effect of mechanical forces on the production of growth factors by mesothelial cells. PMC were attached to microcarrier beads and exposed to shear stress (5 – 16 dyn/cm2) by perfusing a column of cell covered beads; in separate experiments, PMC were subjected to cyclical deformation (Flexcell model, 30 cycles/min, average of 10% cellular area increase). Fluid shear stress and, to a lesser extent, mechanical strain increased endothelin-1 (ET-1) release by PMC. In contrast, PDGF secretion, which occurs in static PMC, was not increased by either shear stress or mechanical deformation. The authors hypothesized a role of

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pleural mesenchymal cells in stimulation of compensatory lung growth. Because ET-1 is a potent vaso- and bronchoconstrictor, the possible role of ET-1 release with abnormally high breathing frequency or increased tidal volume during mechanical ventilation may warrant further investigation.

5.3. Cells of the pulmonary artery wall Remodeling of the capillary wall follows an increase in vascular wall stress, the classical example being thickening of the capillary basement membrane in mitral stenosis. Experimental models of increasing vessel wall stress are complex, involving stress to structures other than capillary walls and (in the case of hypoxia), potential stimuli in addition to mechanical factors. Several groups (Mecham et al., 1987; Poiani et al., 1990) demonstrated an increase in elastin and and collagen proteins and mRNAs in animals under hypoxic conditions in vivo. West and coworkers studied two methods of increasing capillary wall stress, hyperinflation or increasing capillary pressure by cyclically raising venous pressure in isolated perfused rat lungs (Berg et al., 1997; Parker et al., 1997). Both model systems caused an increase in the mRNA content of various ECM components and growth factors, although the effects on mRNAs for certain procollagens varied in the two systems. Cells of the vascular wall may be subjected to various different mechanical forces. Endothelial cells are subjected to shear stress and cells of the media (smooth muscle cells) and adventitia (fibroblasts) are subjected to mechanical deformation during vessel distention and to changes in hydrostatic (blood) pressure. Chronic pulmonary hypertension is also associated with marked remodeling of the pulmonary vascular bed (Wagenvoort and Wagenvoort, 1979). Although this effect is widely accepted, experimental data on the effects of strain on pulmonary vascular cells are limited. Both constant strain and hydrostatic pressure, two different ways of mechanically perturbing cells, have been reported by (Kolpakov et al., 1995) to stimulate different effects on rabbit pulmonary artery cells in vitro. In this model, isolated pulmonary artery strips were placed in tissue

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culture and subjected to varying magnitudes of constant strain or hydrostatic pressure for 4 days. Protein synthesis, the percentage of procollagen type I-positive cells, relative rates of elastin and collagen synthesis, and total actin content in pulmonary artery strips were measured. Strain was applied to pulmonary artery strips in form of a load corresponding to a wall stress of 12, 25 or 45 mmHg of intravascular pressure. A total of 45 mmHg compared to 12 mmHg caused a significant increase in the rates of synthesis of collagen (62%) and elastin (50%) and an overall increase in actin (60%). Fibroblasts of the adventitia did not show similar changes. Wall strain, but not hydrostatic pressure alone led to an increase in cellular replication in the media and adventitia. These results indicate, that pressure stimuli may initiate a hypertrophic stimulus with increased deposition of matrix proteins as well as a hyperplastic stimulus. The presence or absence of endothelium in these preparations did not alter the effects of mechanical perturbation except for a reduction in the cellular replication in smooth muscle cells of the media. Conversely, in pulmonary artery preparations subjected to constant strain, procollagen and m-RNA for 6-sis (a protooncogene encoding a peptide highly homologous to the B chain of PDGF) were increased (Tozzi et al., 1989), these effects were dependent on the presence of an intact endothelium. A possible explanation of the conflicting results of the role of the endothelium may be the large difference in the duration of strain in these two models: 4 h versus 4 days. Endothelial factors appear to be produced rapidly in response to constant strain, whereas other cells of the pulmonary vascular wall require longer periods of constant strain in order to express, synthesize and secrete matrix proteins. Different patterns seems to exist for the systemic and the pulmonary vasculatures, with cyclic strain in systemic vasculature resulting in cellular proliferation, whereas pulmonary vasculature subjected to constant increases in pressure responds with an increased deposition of matrix components. This is demonstrated by the fact, that the increase in procollagen and 6-sis mRNA in pulmonary artery segments following increased pres-

sure have not been observed in jugular vein and aortic vessels (Tozzi et al., 1989). Further evidence that the pulmonary vasculature exhibits inherently different responses compared to the systemic vasculature is that cyclical stretch of pulmonary artery smooth muscle cells (PAC1 cell line and early passage cells from lambs) does not cause the increases in total protein synthesis, RNA synthesis, cell number or mRNA for a1(I) and a1(III) collagen (Kulik and Alvarado, 1993) seen in studies with systemic vasculature. Hydrostatic pressure (i.e. continuous transmural pressure) in the study using rabbit pulmonary artery strips (Kolpakov et al., 1995) appeared to have less effect compared to strain with respect to protein synthesis. However, bovine pulmonary artery endothelial cells (BPAEC) subjected to hydrostatic pressure responded with cellular proliferation (Acevedo et al., 1993). Conditioned medium from cells under increased hydrostatic pressure was mitogenic; the mitogenic factor(s) could be inhibited by either suramin, a growth factor receptor inhibitor, or by a neutralizing monoclonal antibody against bovine bFGF. It was therefore postulated that bFGF release, present in small amounts even under control conditions (3 mmHg) in BPAEC cells, is enhanced when hydrostatic pressure is elevated.

5.4. Airway smooth muscle cells Hyperplasia of airway smooth muscle cells (ASMC) occurs in obstructive lung disease. The mechanisms responsible for this are unknown, but there are likely to be multiple etiologies, including inflammation. One possible contributing factor is that is that elevated airway pressure may cause distention in distal airways because they contain fewer structural supports (cartilage, collagen). Experimentally, elevated transpulmonary pressure causes ASMC hyperplasia (Takizawa and Thurlbeck, 1971). Based on these observations, Smith et al. (1994) studied the effects of cyclic strain on proliferation of ASMC, using canine ASMC in the Flexcell device (Flexcell Corp., McKeesport, PA), with a pattern of a 2-sec distention followed by a 2-sec relaxation, for 14 days. These investigators used two different controls, cells on identical

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plates but not subjected to distention/relaxation and cells cultured on a rocking platform. The stretched cells assumed a more elongated, spindle shape oriented perpendicular to the direction of strain and exhibited an increase in cell number, with a peak in incorporation of 3[H]thymidine at day 5 but not by day 14. These studies demonstrate that cyclical strain induces changes in airway cell morphology, orientation, and proliferation.

5.5. Airway epithelial cells In studies of rabbit tracheal mucosal cells cultured from explant tissue, Sanderson and Dirksen (1986) showed that mechanical stimulation with a glass microprobe resulted in a transient increase in ciliary beat frequency of ]20%. Interestingly, the duration but not the frequency of the response depended on the strength of the stimulation. The response was calcium-dependent. Both the increases in intracellular calcium and the increase in ciliary beat were transmitted to neighboring cells. The initial increase in calcium as well as the propagation of the response to neighboring cells was mediated by IP3 (Boitano et al., 1992). Because stretching and hypoosmotic swelling of tracheal epithelial cells also causes an increase in intracellular IP3, Felix et al. (1996), suggested that a deformation of the cell (e.g. dimpling of the cellular surface by increasing the load on cilia) rather than a manipulation of cilia is the trigger of the reported cellular reactions. Thus ciliary movement would not perturb the mechanical sensor. One might infer the physiologic implication that mechanical sensors mediate an autoregulatory mechanism of increasing mucociliary clearance efficiency by responding to an increased particle or mucous load. The propagation of the ciliary beat response, with a increasing lag time between stimulus and response in the cells more distal to the initial stimulus, might result in coordinated responses as particles approach and move away from a given tracheal location. Inhaled particles are in fact drawn into the phoshoplipidaqueous layer above airway epithelial cells (Gehr et al., 1996) with an inward force strong enough to result in a dimpling of airway epithelial cells.

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5.6. Al6eolar type II cells, surfactant Mead and Collier (1959) made the important observation that the diminished lung compliance caused by mechanical ventilation with low and invariant tidal volumes could be normalized by a single large inflation of the lungs. The authors hypothesized that the large inflation restored normal compliance by stimulating surfactant secretion. Experimental models with excised lungs demonstrated the prolonged inflation at room temperature caused an increase in dipalmitoylphosphatidylcholine (DPPC) recovery, but that there was no increase in DPPC if inflation was performed either at 6°C or when lungs were inflated without oxygen. In ventilated animals, the use of larger tidal volumes resulted in increased phospholipid (PL) recovery by broncho-alveolar lavage (Wyszogrodski et al., 1975). The increased PL fraction was less surface active than expected, but surface activity could be restored by the use of positive end-expiratory pressure (PEEP). The authors concluded that increased ventilation led to increased usage of surfactant, and that this process was reversed when ventilation was interrupted under conditions that allow normal metabolic acitivity. Oyarzun and Clements (1978) reported an increase in the amount of phospholipid in BAL from spontaneously-breathing rabbits following hyperventilation. This increase was inhibited by propranolol, atropine, and indomethacin but was not affected by prostaglandins. Increased ventilation also led to an increased uptake of labeled DPPC (Oyarzun et al., 1980). Further evidence of hyperventilation-induced surfactant release was provided by the studies of Nicholas and Barr (Nicholas and Barr, 1981), showing that even brief periods of hyperventilation (or even one deep breath (Nicholas et al., 1982)) led to increased BAL phospholipid content; the increase was related to the amount of lung inflation. Massaro and Massaro (Massaro and Massaro, 1983) showed morphologic evidence that deep inflation induced surfactant release by showing a decrease in the number of lamellar bodies (the surfactantcontaining secretory organelle of alveolar type II cells) in type II cells following deep inflations.

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From these studies, it had been hypothesized that mechanical forces during breathing stimulated surfactant secretion and that these forces could be triggered by one deep inflation. In mammals, breathing at tidal volume is punctuated by periodic deep inflations (sighs) (Bartlett, 1971). We tested the hypothesis that direct mechanical distention of type II cells stimulates surfactant secretion. Type II cells cultured on silicone membranes were distended and then relaxed once in a device designed and constructed in our laboratory; cells were distended by an increase of 5 – 25% of the cellular surface area. Following a single distention/relaxation, there was an increase in surfactant secretion lasting 30 – 60 min. The increase in surfactant secretion was dependent on the extent of stretch, with a threshold of approximately 15% increase in cellular surface area below which secretion was not stimulated. Secretion was preceded by a fast transient increase of [Ca2 + ]i, presumably from intracellular stores (Wirtz and Dobbs, 1990). Taken together, the experiments performed with intact animals, excised lungs, and cultured type II cells support the hypothesis that mechanical distention is a potent stimulus for surfactant secretion both in vitro and in vivo. Periodic deep breaths (sighs) may serve to continually maintain surfactant stores by providing newly secreted surfactant material to replace surfactant, which has a rapid turnover rate (reviewed in Wright and Clements, 1987). Increased utilization of surfactant by expansion and recompression of the surface film during hyperventilation (e.g. in exercise) would therefore be counteracted by increased secretion. As described above (see Section 3.1), mechanical factors modulate surfactant protein gene expression at the level of transcription; distention inhibits expression of SP-B and SP-C. Contraction has the opposite effects (Gutierrez and Dobbs, personal communication). Distention favors expression of the type I cell phenotype and inhibits expression of the type II cell phenotype, whereas contraction has the opposite effects. These results may have bearing on the design of future studies of mechanical ventilation, suggesting that breathing patterns may affect the state of

alveolar epithelial cell differentiation and surfactant synthesis and metabolism.

6. Mechanotransduction: how are mechanical forces sensed and how is the mechanical signal transduced to cause changes in gene expression and cell metabolism? The question of how mechanical forces are transduced is under active study in many laboratories, although the areas of most active investigation are in non-pulmonary systems. We will briefly review some of these findings because the mechanisms are likely to be applicable to mechanotransduction in the lung.

6.1. Mechanoreception at focal adhesions Cells adhere to neighboring cells and to the extracellular matrix (ECM) via transmembrane receptors of the cadherin and integrin families, respectively. These receptors are bound in vivo to one other (cadherins: cell-to-cell contact) or to ECM proteins such as fibronectin (integrins: ECM adhesion). On the cytoplasmic face of the cell membrane, these receptors are coupled, either directly or indirectly, to a multitude of proteins whose functions are not completely understood (Ben-Ze’ev et al., 1994). These proteins have been called cytoskeletal plaque proteins and, as a group, form the focal adhesion complex (FAC), which serves as a macromolecular scaffold which mechanically couples the cytoplasmic portion of integrins to the actin cytoskeleton. The FAC contains various types of molecules, including those associated with actin (e.g. vinculin, talin, and a-actinin), focal adhesion kinase (pp125 FAK), a number of oncogene products, signaling molecules (e.g. tyrosine (pp60src) and serine protein kinases, and inositol lipid kinases), and some growth factor receptors. These proteins, bound indirectly to both structural molecules of both the ECM and the cytoskeleton, are key candidates for transforming physical (mechanical) to biochemical signals (Chicurel et al., 1998). Integrin receptors have been shown to be involved in mechanotransduction in experiments using mag-

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netic drag forces on integrin receptors, inducing physical anchoring of tyrosine-phosphorylated proteins to the cytoskeleton (Schmidt et al., 1998). In a model of shear-stress stimulated endothelial cells, Chien, Shyy and coworkers have performed elegant experiments to describe a signaling cascade, with a specific mechano-senstive integrin causing activation of focal adhesion kinase (Li et al., 1997) and subsequent activation of c-Src, the Ras signaling pathway, the MAPkinase pathway, and downstream effects on transcription factors and gene expression (Jalali et al., 1998). These experiments with vascular endothelium describe a model system in which mechanical forces are transduced via cell surface receptors and downstream signaling cascades to modulate gene expression and various other cellular functions. It is likely that specific components of this type of pathway [i.e. specific portions of the MAPkinase system or other cascades, specific ‘second messengers’ (Ca2 + , cAMP, etc), and specific transcription factors] will vary, depending on the stimulus, tissues, cell types, and endpoints, but the overall schema may prove to be a general model for mechanotransduction. Strain-stimulated growth of fetal lung cells has been shown to involve an increase in protein tyrosine phosphorylation, especially substrates of pp60src, and activation as well as translocation of this nonreceptor tyrosine kinase from cytosolic to cytoskeletal compartment (Liu et al., 1996). Although the mechanisms underlying pp60src activation remained unknown, it has been speculated that mechanical strain may caused physical approximation of pp60src and AFAP-110, a cytoskeleton associated protein with four SH2 binding sites. AFAP-110 may thus compete with pp60src’s C-terminal Tyr(P)-527 sequence, which, by binding to the SH2 domain, renders the kinase inactive. Phosphorylation of pp60src is a rapid event, as demonstrated in tendon cells subjected to a 1 Hz strain regimen; in this system, an increase in pp60src phosphorylation was observed after one load cycle (1 sec) (Banes et al., 1995). The product of the c-Src gene, pp60src, is widespread in distribution but is particularly abundant in nervous tissue, where it is associated

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with motile structures of the neural growth cone. Overexpression of p50csk, which results in phosphorylation and down regulation of Src tyrosine kinase in HeLa cells, caused rounding of the cells and less firm attachment (Bergman et al., 1995). Csk in these experiments formed a complex with focal adhesion protein paxillin in cells and the SH2 domain of the csk molecule interacted with pp125FAK and paxillin in vitro. Taken together, these findings support a role for adhesion molecules and non-receptor tyrosine kinases in linking adhesion via the FAC to a variety of cellular functions, including differentiation. Considering that physical perturbation may stimulate pathways similar to adhesion, it is conceivable that mechanical stimulation may itself be linked directly to regulating the differentiated state in certain tissues.

6.2. Mechanically acti6ated/inacti6ated ion channels Stretch-activated calcium (Sachs, 1986) and potassium (Olesen et al., 1988) channels have been recognized for almost 15 years. Although stretch-inactivated ion channels have been described in invertebrates, they have not been identified in endothelial cells. In a recent review, it was suggested that the net effect of activating stretch-sensitive channels would be an influx of ions resulting in depolarization of the cell (Gudi and Frangos, 1994). The physical force required for channel opening is :1 dyn/cm (Sachs, 1991). It is unclear whether stretch-sensitive channels are activated/inactivated directly by a conformational change or indirectly via an intermediate mechano-sensitive protein which then opens the channel by a signaling pathway. Although excised patch experiments suggest a direct effect (Sachs, 1991), G proteins have been implicated in the process because K+ channel activation is commonly mediated by G proteins (Gudi and Frangos, 1994). Because disruption of actin filaments in endothelial cells does not render stretch-activated ion channels inactive, it has been postulated that F-actin stress fibers do not mediate these events.

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6.3. Possible in6ol6ement of G proteins in shear stress-induced effects

6.4. Mechanically-induced alterations of nuclear structures

There is substantial evidence supporting the involvement at multiple levels of both heteromeric and ‘small’ GTP-binding proteins in shear stress. Once again, the role of GTP-binding proteins has been most completely described in endothelial cells. Gudi and Frangos (1994) suggested direct G protein activation to be a very early, if not the first event, in mechanotransduction of shear stress. Flow-induced stimulation of PGI2 release (both the initial burst and the sustained activation) is mediated by a pertussis toxin (PTX)-sensitive G protein (Berthiaume and Frangos, 1992). Shear stress-induced PDGF expression involves generation of IP3 and DAG, followed by activation of PKC (Hsieh et al., 1992). Flow-stimulated NO production is increased acutely following an increase in flow, but is also upregulated independently by a sustained exposure to steady flow. Flow increase-induced NO release is calcium/calmodulin dependent and requires an increase in [Ca2 + ]i and stimulation of calcium-dependent potassium channels. Taken together, these types of experiments suggest that phospholipase C is involved is shear-stress processes. However PTX-sensitive G proteins do not appear to be involved in shear stress-induced PDGF expression (Kuchan et al., 1994). More than one class of heteromeric G proteins, such as PTX-insenstive Gq proteins, may be involved in mechanotransduction. In endothelial cells, small GTPases also appear to be involved in mediating shear stress-induced gene expression. Li et al. (1996) have shown that p21ras is transiently and rapidly activated by shear stress, resulting in preferential activation of c-Jun NH2 terminal kinases JNK1 and JNK2 and subsequent stimulation of transcription of a shearstress sensitive gene. In summary, there is now good evidence that various types of GTP-binding proteins, both heterotrimeric and small GTPases, are involved with early steps of mechanotransduction.

In addition to indirect effects on transcriptional events, there may be direct effects on nuclear structures from mechanically perturbing cells. The tensegrity model predicts that application of mechanical force to the cell will be transduced to the nucleus (Ingber, 1993b). The nuclear protein matrix is involved in chromosome organization and contains fixed sites for regulation of DNA replication and transcription (Nelson et al., 1986). Mechanical forces may therefore directly alter gene expression and DNA synthesis, either by rearranging DNA regulatory nuclear membrane-associated proteins by allowing previously restricted DNA molecules to unfold (Roberts and D’Urso, 1988) or by modulating nuclear pore size and nucleo-cytosplasmic transport of mRNA (Feldherr and Akin, 1990).

6.5. Signal transduction (second messenger systems) Following mechanical perturbations, several signaling cascades appear to be activated. A list of second messengers involved downstream from mechanical stimulation of cells has been reviewed and detailed schematically by Banes et al. (1995). Although one can find elements of almost every known signalling pathway in these and other reports, there appear to be three predominant pathways. One involves the phospholipase C-mediated breakdown of phosphatidylinositol 4,5 bisphosphate (PIP2) and possibly activation of other membrane phospholipases. As a result, IP3 and DAG are elevated, causing a rise in [Ca2 + ]i and activation of PKC. The elevation of [Ca2 + ]i may result in relatively rapid processes, such as exocytosis, while PKC activation modulates gene expression (see below) and may stimulate slower, longer lasting cellular responses. These pathways are involved in shear stress-induced effects in endothelial cells (Ishida et al., 1997) and cyclic stretch-induced effects in fetal lung cells, as detailed above. A second signaling pathway involves activation of adenylyl cyclase, resulting in an increase in

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cellular cAMP and activation of protein kinase A (PKA). Activation of adenylyl cyclase subtypes I and VII is sensitive to cytochalasin treatment and osmotic volume changes. This mechanism has been demonstrated to be involved in several different systems: the adaptive changes of cardiac pressure overload (e.g. Watson et al., 1989); compensatory lung growth (Rannels and Rannels, 1988); and extracellular osmotic changes (Watson and Krupinski, 1994). A third important signaling pathway is the MAP kinase cascade, which is important in the regulation of shear stress-stimulated gene transcription in endothelial cells (see above, in Section 6.3).

6.6. Links to gene expression Second messengers may, by coupling to transcription factors, activate these factors and thereby transfer information rapidly to the nucleus. In this manner, a signal cascade is triggered in which early response genes (IERG) regulate the expression of genes responsible for adaptation to the altered situation which led to the initial signalling event. For these reasons, transcription factors have been sometimes called ‘third messengers’. At least four cis-acting elements functional in mechanosensitive gene transcription have been identified. The best characterized are the shear stress-responsive element (SSRE) initially described by Resnick et al. (1993) and the shear TRE element described by Shyy et al. (1995). The SSRE ‘core’ element is a 6 bp element that is known to be sensitive not only to shearstress, but also to other types of mechanical forces (Resnick and Gimbrone, 1995; Chien et al., 1998). The ‘shear TRE element’ is a divergent TRE (phorbol ester tissue responsive element) that also can transduce mechanical signals to transcriptional events. More recent data suggest that mechanotransduction may also occur via binding and/or displacement of Sp1 (Lin et al., 1997) and Egr-1 (Silverman et al., 1997). As various mechano-sensitive systems are developed, it seems likely that the number of regulatory cis-acting elements and their transcription factors will increase.

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7. Physical forces and the lung, a perspective In recent years, there has been a growing recognition that physical forces are important in pathophysiologic processes in human disease states. Three areas in which physical forces clearly are important are fetal lung development, surfactant metabolism, and mechanical ventilation. Early in development, the correct interplay of lung distention by liquid filling and fetal breathing movements regulates both lung growth and maturation; an incorrect pattern during the fetal period results in insufficient lung growth and disordered maturation. The individual contributions of constant and repetitive distention to these processes are under current investigation in various in vitro model systems (Gutierrez et al., 1998) and the results of these investigations may lead to therapeutic approaches designed to prevent lung hypoplasia and possibly to accelerate pulmonary maturation. It has been recognized for some time that intermittent deep breaths are an important stimulus for surfactant secretion. It is unclear under what circumstances this stimulus can be compensated for by other regulatory processes or whether it will result in low surface activity and concomitant atelectatic lung (Wirtz and Schmidt, 1992). There is a renewed interest in the effects of mechanical forces (both pressure and volume) on the lung functions in patients requiring mechanical ventilation. High alveolar pressures and/or volumes that cause direct strain or increased tissue shear are the presumed cause of mechanical ventilation-induced lung injury; recent findings demonstrate increased amounts of inflammatory cytokines such as TNFa and IL-6 with mechanical ventilation (e.g. von Bethmann et al., 1998). These three areas are part of an emerging realization that physical forces, in addition to well-recognized humoral factors, affect multiple aspects of lung growth and function. A matter of some controversy remains the manner and extent to which individual lung cells are mechanically deformed during ventilatory movements. The difficulties in determining this experimentally were discussed above (Section 2.1). However it is conceivable that the spectrum of distention in vivo is quite large and dependent on

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various unknown factors, such as location within the lung or the anatomic state of the lung. For example, there may be more distention/contraction in lungs that are ‘small’ due to fluid overload, leakage and impaired surface activity as in acute lung injury and ARDS. Other pathologic processes such as interstitial fibrosis or regional differences in lung compliance caused by local disease may also affect how various cells experience physical forces. Recent experimental findings that lung cells contain sensing and signaling mechanisms for mechanical forces suggest possible specific areas of research to establish the mechanisms underlying mechanotransduction. Elucidating these roles will be important for both basic scientists and pulmonary physicians.

Acknowledgements This work was supported, in part, by grants R01 57426 and PPG HL 24075 from the USA National Institute of Heart, Lung, and Blood, (Dobbs) and a grant from the VERUM foundation, Munich (Wirtz).

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