NHLBI Workshop Summary Effect of Physical Forces on Lung Structure, Function, and Metabolism 1 , 2
DAVID J. RILEY, Chairman, D. EUGENE RANNELS and ROBERT B. LOW, Co-Chairmen; LEEANN JENSEN, and THOMAS P. JACOBS
Introduction
The living organism continually is subjected to a variety of internal and external physical forces at the cell, tissue, and organ levels. The lung, for example, normally is exposed to transmural pressure gradients required for inflation and to shear stress produced by circulation of blood and by airflow during ventilation. In addition, internal forces are exerted on lung tissue and cells from smooth muscle contraction, from tension generated by the cytoskeleton of lung cells,and from surface forces at the air-tissue interface. The biologic effects of physical forces and their mechanisms have been studied in a number of cell types and tissues such as bone and the cardiovascular and skeletal muscle systems. This has led to an appreciation of their role in cellular and tissue growth, differentiation, and metabolism as well as integrated function and in repair after injury. The effects of physical forces on lung structure, function, and metabolism have not been evaluated systematically. Little information is available regarding their role in lung development or in the maintenance of normal function. The role of physical forces in repair of parenchymal injury, in vascular remodeling with pulmonary hypertension, and in interstitial fibrosis or emphysema remains largely unexplored. A workshop sponsored by the Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, was held in April 1989to summarize the state of knowledge regarding the effects of physical forces on the lung. New developments in celland organ physiology related to mechanical effects on biologic systems were discussed and evaluated, particularly as they might relate to the respiratory system. Current efforts in this area were assessed to identify 910
promising directions for future research. Investigators from various disciplines met to discuss five major topics: biomechanics, cell physiology and biology, mechanical signal transduction, effects of physical forces on the lung, and manpower and training. Biomechanics
Application of engineering principles and measurements to biologic systems is required to define the role of physical forces in the regulation of biologic function. It is imperative that measurements of physical force in all experimental conditions be described in units and in terms derived from physics and engineering. Static and dynamic forces on biomaterials should be quantitated in specific physical terms such as stress, strain, and shear stress. These are defined as follows:stress, force per unit area; strain, change in length divided by initial length; shear stress, force per unit surface area in the direction of flow exerted at the fluid/surface interface. Other terms such as stretch and deformation have been used, but these are not classically defined physical parameters. Although the application of physical principles to the study of simple biomaterials is technically straightforward, data analysis and interpretation are far more complex when dealing with complicated structures such as the lung. The anatomic arrangements of tissues make it difficult to attribute the effects of mechanical perturbations to individual cells, matrix, or other structural components. This necessitated development of a number of model systems that utilize isolated tissues and cells in which the response to physical force can be explored at various levels of complexity. Examples of experimental approaches that have been used in an effort to un-
derstand the role of physical force in biologic regulation are listed in table 1. Forces can be applied to whole organs, tissue strips, or cells cultured on deformable surfaces or in collagen gels; cells in culture can be subjected to hydrostatic or shear stress, and events generated by stress or strain can be measured in cell membranes deformed by application of suction to membrane "patches" or byosmotic swelling.Numerous end points can be investigated, including morphology, cell shape and cytoskeletal properties, protein synthesis and secretion, enzyme activity, and cell division. Signal transduction can be explored in terms of plasma membrane ion channels and activation of second messenger systems such as the cyclic AMP cascade. (Received in original form December 15, 1989 and in revised form February 5, 1990) 1 List ofparticipants: Judith Aggeler, Davie, CA; Albert Banes, Chapel Hill, Nt; Thomas Borg, Columbia, SC; Hazel M. Coleridge, San Francisco, CA; Edward D. Crandall, NeW York, NY;Leland G. Dobbs, San Francisco, CA;. Randall O. Dull, Chicago, IL; John N. Evans, Burlington, vn Yuang-Cheng B. Fung, La Jolla, CA; Albert K. Harris, Jr., Chapel Hill, NC; Donald Ingber, Boston, MA; Thomas P. Jacobs, Bethesda, MD; LeeAnn Jensen, Bethesda, MD; Brian L. Langille, Toronto, ONl; Robert D: Low, Burlington, V'I; Edward J. Macarak, Philadelphia, P!\.; Robert R. Mercer, Durham, NC; Howard E. Morgan, Danville, PA; Catherine E. Morris, Ottawa, ONl; Maurizio Pacifici, Philadelphia, PA; Marlene Rabinovitch, Toronto, ON'!; D. Eugene Rannels, Jr., Hershey, PA; David J. Riley, New Brunswick, NJ; Una S. Ryan, Miami, FL; Frederick Sachs, Buffalo, NY; Stephen Schwartz, Seattle, WA; Frederick Silver, Piscataway, NJ; Gene Sprague, San Antonio, TX; Robert L. Trelstad, Piscataway, NJ; Herman Vandenburgh, Providence, RI; David M. Warshaw, Burlington, VI; and Peter Watson, Danville, PA. 2 Correspondence and requests for reprints should be addressed to Thomas P. Jacobs, Ph.D., Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, 5333 Westbard Ave., Bethesda, MD 20892.
AM REV RESPIR DIS 1990; 142:910-914
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NHLBI WORKSHOP SUMMARY
TABLE 1 EXAMPLES OF PHYSICAL FORCES APPLIED TO BIOMATERIALS* Model Tissues Stress on tissue strips Tendon Muscle Skin Myometrium Blood vessel Cells Growth on deformable membranes Smooth muscle cells Epithelial cells Chondrocytes Endothelial cells Skeletal muscle cells Cardiac muscle cells Growth in collagen gels Hydrostatic pressure Chondrocytes Endothelial cells Shear stress Endothelial cells
Cell membranes Patch clamp Osmotic swelling
End Points
Glycosaminoglycan synthesis Protein synthesis and degradation DNA synthesis Protein synthesis, cell growth Protein and DNA synthesis
Protein synthesis, cell division DNA synthesis Glycosaminoglycan synthesis Fibronectin synthesis Protein synthesis, intermediary metabolism Protein synthesis Myotube formation Glycosaminoglycan synthesis Morphology Morphology Shape LDL uptake Release of vasoactive substances Ion channel activation Ion channel activation Accumulation of cyclic AMP
* This table is not intended to provide a complete summary of the subject.
It is now possible to investigate the physical properties and responses of individual cellsto external force. Recent research, for example, has described the mechanical properties of isolated single smooth muscle cells (1). An elegant preparation in which individual cells are mounted in a force-measuring device permits correlation of cellular mechanics with cytoskeletal and contractile protein function. As a second example, hypotonic media are used to produce mechanical signals at the cellular level (2), offering a potentially important model for investigation of the biologic implications of physical perturbation of the plasma membrane (2).
through specific matrix-receptor interactions. These effects, in turn, influence the secretion and organization of the extracellular matrix itself, and they result in a "dynamic reciprocity" between cells and the three-dimensionalmatrices with which they are associated. The initial deposition of extracellular matrix is critical giventhis reciprocal relationship. Matrix deposition is a highly organized, multistep process involving coordinated intracellular and extracellular events. In tendon, for example, matrix-synthesizing cells exert control of fibril formation by regulating the amount and kinds of matrix elements produced, their orientation upon secretion, and the secretion of enzymes needed for final modification and assembly of mature Cell Physiology and Biology collagen type I fibrils. The requirement Workshop participants discussedat length for increased tensile strength of tendon the mechanisms by which physical force during embryonic development in reis transmitted through biologic systems, sponse to increased mechanical forces particularlywith regardto cell-matrixinter- may account for the tight coupling to inactions. Biologic responses generated by creased collagen content, fibril, and fimechanical perturbations in cultured ber diameter and fibril length, as well as cells were presented and discussed with to the differentiated phenotype of tenregardto potentialunderlyingmechanisms. don fibroblasts. An important future Studies in which cells are exposed to goal is to establish the mechanism of this extracellular matrix molecules show that apparently causal relationship. specific matrix components affect celluThe ability of cells to exert physical lar movement, shape, polarity, metabo- forces on neighboring cells and the malism, and differentiation (3), presumably trix is not limited to muscle, as nearly
all differentiated cell types have contractile capability (4). Contractile forces appear to be involved in cell migration during embryonic growth, wound closure, locomotion, and matrix alignment (4). The consequence of the interplay between internal forces and those imposed externally is illustrated by the observation that cellsin culture orient in response to force (5). Alignment perpendicular or parallel to the axis of force has been described for a number of cell types and appears to depend on the pattern of stress imposed (6, 7). Physical force also influences differentiated function by affecting cell shape. This is clearly evident from model systems that show the influence of reconstituted basement membrane components on gene expression and protein secretion by cultured mammary epithelium (8). Another example is induction of the expression of differentiationspecific collagen genes by the manipulation of the shape of cultured chondrocytes (9). Attendant changes in the regulation of protein synthesis may reflect a functional association of polyribosomes with the cytoskeleton. The endothelial cell, by its strategic location, is subjected to both stress and shear stress. Endothelial cells in vitro change shape in response to unidirectional fluid shear stress in a reversible timeand shear-dependent manner (10). Exposure to shear stress causes reorganization of the cytoskeletonand significantchanges in cell surface properties. Additionally, it also stimulates the synthesis and release of substances involved in vascular homeostasis such as prostacyclin (PGI 2) , tissue plasminogen activator, and endothelin. Shear stress also may increase low density lipoprotein uptake, fluid endocytotic rate, and histamine-forming capacity. Flow-induced dilatation of large arteries has been attributed to the release of endothelium-derived relaxing factor. Evidence for an influence of shear stress on endothelial cell function in vivo comes from studies which demonstrate that decreasing flow rates result in reorganization of the arterial architecture and narrowing of the vascular lumen only in the presence of intact endothelium. Thus, the endothelium may mediate flow-induced structural changes in the vesselwall, consistent with the known ability of endothelial cellsto regulate smooth muscle cell proliferation and protein turnover (11). Characterization of the mechanoreceptors responsible for the multiple effects of shear stress on the endothelium remains an important subject for future in-
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NHLBI WORKSHOP SUMMARY
vestigation. Smooth muscle within the vessel wall may also respond to mechanical forces. For example, several studies show realignment of smooth muscle cells in response to cyclic tensional deformation in culture, whereas others show that cyclic deformation increases extracellular matrix production. Mechanical Signal Transduction
The molecular mechanisms by which mechanical stimuli are transduced into a variety of biologic responses remain poorly defined, although significant progress is being made toward understanding these events. Twobroad types of specific mechanoreceptors have been described: specialized nerve endings, which are part of an integrated neural response to movement of organs (12), and stretch-activated ion channels, which operate at the cellular level(13). Mechanoreceptors of the former type are important in the respiratory system and will be discussed in the section on the lung. Ion channels are transmembrane proteins that permit signaling of cellsby control of the selective movement of ions. Mechanically activated ion channels have been identified that are distinct from channels that respond to voltage changes or various ligands (13). Their identification will help facilitate analysis of the cellular and molecular events through which cells respond to mechanical forces in terms of altered gene transcription, cell shape, or cell division. A direct coupling of stretch-activated ion channels to these complex biologic responses, however,has not been demonstrated. Mechanically activated ion channels have been demonstrated directly in a number of cell types (13).A method that allows exploration of their properties involves application of pressure or suction by a patch clamp pipette, which results in channel opening or closing. These channels have variable ion selectivities, although most are cation-selective and prefer K+ or Na", The energy required to open such channels is provided by membrane strain rather than by metabolism of high energy phosphates. The channels are highly sensitive and require a force in the range of 0.5 to 3.0 dyn/cm' for halfmaximal activation (13). Stretch-inactivated channels also have been described that open at low force and close with increasing force (14). The co-localization of stretch-activated and inactivated channels suggests that distinct populations of channels with reciprocal responses can coexist within a small area of the membrane. Isolation of these channels in pure
form for chemical, structural, and functional analysis is a critical area for future investigation. Although mechanically activated ion channels have been found in a variety of cell types, their presence in endothelial cells may be of particular importance in mediating hemodynamic effects on vascular function (see above). Twotypes of ion channels have been identified in cultured endothelial cells subjected to shear stress. The first is a K+sensitive channel in which K+ current increases as a function of shear stress. It has been suggested that flow-mediated hyperpolarization may alter underlying smooth muscle contractility via direct coupling through gap-junctions or indirectly by release of smooth muscle cell constricting or relaxing factors (10). Nonselective ion channels also exist in endothelial cells; these could also contribute to additional responses to hemodynamic force (15). Mechanical tension also may affect ion channels in smooth muscle cells. Tension applied to blood vessels promotes the influx of Ca 2 + via a pathway with unique pharmacologic properties. This pathway may mediate myogenic tone in response to increased tension on the blood vessel wall. Other molecular mechanisms by which mechanical stimuli may be transduced into morphologic and biochemical responses are under investigation. For example, the effects of mechanical stress on intermediary metabolism and protein turnover in muscle have been studied (6). These effects may be mediated via activation of a plasma membrane sodium pump (6). Newly synthesized prostaglandins PGE2 and PGF2 may also be important. Finally, in studies of isolated hearts, increased protein synthesis caused by elevated perfusion pressure is associated with activation of the cyclic-AMP-dependent protein kinase. Forces applied to cells may be redistributed by tension-bearing cytoskeletal and contractile elements. Physical forces that act through the cytoskeleton may function as an alternative signal transduction mechanism to the more commonly considered plasma-membrane-derived second messengers by conveying information directly to the nucleus (16). Effects of Physical Forces on the Lung
The lung is continuously subjected to physical forces by pressure distending the parenchyma, by airflow and gradients of pressure in the tracheobronchial tree, and
by hemodynamic forcesin the pulmonary vasculature. Normal ventilatory fluctuations are enhanced by periodic "sighs" and by exercise, which increases both the rate and depth of ventilatory excursions. Ventilatory movements exert stress on the tissue, and it has been shown that ventilation modulates several metabolic pathways in the lung. Few studies, however, have demonstrated that physical force directly affects individual components of the lung. Little is known of the effects of mechanical forces on respiratory muscles. One study found increased protein and collagen synthesis in isolated rat diaphragm muscle after mechanical stimulation (17). Because the position of the diaphragm is altered in diseases associated with hyperinflation of the lungs, abnormal passive tension on diaphragmatic musclesmay influencetheir metabolism. Immobilization of skeletal muscle results in increased protein degradation and muscle atrophy (18). These effects may contribute to respiratory muscle atrophy in patients requiring long-term mechanical ventilation. Mechanoreceptors within the respiratory system have been studied as part of the integrated neural response involved in control of breathing (19). Afferent nerve fibers from muscle spindles .in the chest wall provide information to the respiratory neurons about forces exerted by respiratory muscles and about thoracic movements (19). Stretch receptors, classified as rapidly and slowly adapting based on their electrical activities and the distribution of their terminals, are located within the smooth muscle layer of the extrapulmonaryairways (20). They are activated by distortion of the airway walls during inflation rather than by changes in lung volume (20). Their putative function is to minimize ventilatory work output for a given minute volume. However, few studies have explored the degree to which bronchial transmural pressure or longitudinal stretching contribute to stimulation of . the sensory terminals. Studies are needed to determine how activity of the two types of mechanoreceptors correlates with changes in wall tension. Future studies also should define the biophysical properties of respiratory stretch receptors and should explore the molecular basis of force transduction by these mechanoreceptors. Cellular and extracellular components of the pulmonary vasculature also may respond to physical signals. High intravascular pressure is an important factor in stimulating growth of smooth muscle
NHLBI WORKSHOP SUMMARY
cells in large arteries. Cellular proliferation in this case may be a response to metabolic changes caused by vascular damage or to growth factors. Recent studies of mechanical tension applied to pulmonary artery segments indicate that collagen and elastin synthesis are increased by short-term tension, but DNA synthesis is not affected (21). These changes are not observed in vessels denuded of endothelium, indicating a role for the endothelium in mediating the early response. An endothelial cell-derived growth factor may be involved since only vesselswith intact endothelium express the proto-oncogene v-sis after application of tension (21). When cultured newborn lamb endothelial cells are subjected to pulsatile stress, soluble factors active on smooth muscle cells are released, but, in initial studies, they appeared to be growth-inhibitory. These and other results suggest that cell-cell interactions and soluble factors that act in an autocrine or paracrine fashion, in addition to direct mechanical stimulation, may playa physiologically significant role in mediating the effects of physical forces on pulmonary vascular remodeling. Definition of the manner in which changes in transpulmonary pressure deform the peripheral lung is critical to determining the nature of parenchymal stress-strain relationships. The issue of whether normal lung inflation leads to unfolding of the alveolar septal wall or to deformation of the alveoli, coupled with changes in their dimensions, has not been clearly resolved. Recent three-dimensional, reconstructions of individual alveoli suggest that, under physiologic conditions of lung inflation, both alveolar volume and surface area increase as a function of lung volume, and alveoli exhibit hysteresis (22). The positioning of connective tissue fibers at the alveolar septal edges that form the boundaries of alveolar ducts suggests they function as loadbearing elements within the lung parenchyma. The latter tissue components are more important as determinants of parenchymal stress-strain relationships at higher lung volumes. Despite these descriptive advances, the micro mechanics of the parenchymal region remain poorly understood. Normal levels of stress and strain in the alveolar epithelial, interstitial, and endothelial layers, or within the cell types present in each, are not known. The coupling between forces borne by connective tissue elements and the cells that produce these elements may represent a critical site of
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transduction between physical forces and employ different approaches to this probcellular responses in maintenance and lem. Becauseof this diversity, communicagrowth of the lung, as well as in repara- tion among investigators with common tive processes. interests has been difficult. Development It is clear from severalexperiments that of a comprehensive understanding of the stresses associated with lung inflation role of mechanical forces in biology will produce a variety of biochemical respons- require more effective exchange of scies. For example, increased ventilation ele- entific information to enhance the revates surfactant levels in bronchoalveo- search effort. lar lavage fluid and modulates producAll participants in the workshop ention of surfactant components, glucose, couraged integrated interdisciplinary inand fatty acids in isolated perfused lungs. vestigation, with particular emphasis on Rapid induction of metabolic changes in bioengineering. Many biologists have a ornithine decarboxylase, transport of poor understanding of engineering conpolyamines, cyclicAMP content, and ac- cepts involved in the study and quantitativation of the cyclic-AMP-dependent tion of mechanical forces; bioengineers protein kinase occur with overinflation often lack broad training in experimenof the remaining lung after partial pneu- tal biology. Thus, there is a need to fosmonectomy and with increased inflation ter closer collaboration between biologists and bioengineers to develop new of isolated perfused lungs (23). A number of experiments have demon- theoretical and methodologic approaches strated that mechanical strain affects for studying the biologic effects of memetabolic changes in isolated cells (see chanical force, particularly at the celluabove), but few studies of lung cells have lar and subcellular levels. Development been reported. Recent data suggest that of knowledge in the area of cellular biothe type II pulmonary epithelial cell mechanics is critical to advancing reresponds to distortion of the culture sur- search in this field. A major reason for insufficient inface with increased surfactant secretion (24). Stress-activated secretion is inhibit- tegration of engineering into biologic ed by surfactant apoprotein-A, but does studies is the structure of training for biolnot appear to be mediated by a cyclic- ogists and engineers. There is little forAMP-dependent mechanism. In what mal teaching of bioengineering princimay be a related phenomenon, changes ples in biology curricula, and bioenin cell shape by interaction with extracel- gineers generally are not sufficiently lular matrix components clearly modu- trained in applying biologic approaches late type II cell differentiation and me- to research. Restructured training in both tabolism. The effects of physical forces disciplines at the graduate and poston other cell types from the lung paren- graduate levels is needed to improve the chyma have not been reported. Finally, integration of professional efforts in there has been very little investigation of these fields. the effect of physical forces on repair of lung injury. Hyperventilation worsens Recommendations elastase-induced emphysema in hamsters 1. Encourage biologists, biophysicists, as determined by lung elastic recoil, but, in other studies, no adverse effects of andengineers to worktogether in thearea hyperventilation are evident based on of the biologic effects of mechanical morphologic or physiologic criteria. Al- forcesand theirtransduction through the though viscoelastic properties of lung useofmoderncellular andmolecularapstrips have been described, mechanical proaches. The importance of studying tension has not been applied directly to the effects of mechanical forces on biolung tissue to determine metabolic ef- logic systems is well established. Discovfects. Thus, there remains a need to de- ery of potential transduction mechanisms fine the influence of physical forces on such as stretch-activated ion channels has the outcome of the repair process after added new impetus to this research. New concepts of integrated cell function also lung injury and disease. have stimulated interest in this area and Manpower and Training provide opportunities for fruitful invesStudy of the effects of mechanical forces tigation. More effort is required to deon biologic systems is a relatively under- velop innovative strategies to overcome developed field of scientificinvestigation. the relative lack of techniques to meaScientists of diversebackgrounds, includ- sure cellular biomechanics and to improve existing technologies at the celluing biophysicists, cell biologists, bioengineers, and cell and organ physiologists lar and the molecular levels.
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2. Encourage quantitation ofphysical forces in experimental studiesalongwith the use of standard terms in reporting results. Application of bioengineering concepts and principles to complex tissues is not straightforward. It is nevertheless important to quantitate both the forces applied and the resulting responses at the tissue and the cell levels. Quantitation of mechanical forces in biologic work often is inadequate. In all studies on the effects of mechanical force on biomaterials, for example, relevant parameters should be measured quantitatively and expressed in conventional physical terms. Sound bioengineering principles should be used in experimental design. Development of new techniques to apply micromechanical measurements must be encouraged.
3. Improve research and training opportunitiesthat integrate bioengineering with biologicapproaches and teaching. Engineering principles are fundamental to all work on mechanical forces, but often the integration of engineering concepts into biologic experiments is inadequate. Interdisciplinary training programs and research activities that involvebioengineers, biophysicists, and biologists should be encouraged to enhance such integration.
4. Promote research on the effects of physicalforces on the lung and its components. Although the lung is subjected to a variety of mechanical forces, it is not known how these forces affect normal lung function. The effects of physical force in injury and disease also can be expectedto be important, but they remain unexplored. Fewpublished studies of the effects of mechanical force on various tissues and cells have been carried out that are physiologically relevant to the lung. New models for exploration of the effects of mechanical forces on the lung
NHLBI WORKSHOP SUMMARY
should be developed and existing models should be refined in order to enlarge the existing base of knowledge. It is particularly important to encourage investigations of the mechanisms by which mechanical force is transduced to alter tissue or cellular function. It is recommended that research on these problems be promoted but not targeted to specific areas. References 1. Warshaw DM, Fay FS. Cross-bridge elasticity in single smooth muscle cells. J Gen Physiol1983; 82:157-99. 2. Christensen O. Mediation of cell volume regulation by Ca 2 + influx through stretch-activated channels. Nature 1987; 330:66-8. 3. Ekblom P, VestweberD, Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Bioi 1986; 2:27-47. 4. Harris AK. Traction and its relationship to contraction in tissue cell formation. In: Bellairs R, Curtis A, Dunn G, eds. Cellbehavior. Cambridge: Cambridge University Press, 1982; 109-34. 5. Banes AJ, Gilbert J, Taylor D, Monbureau O. A newvacuum-operated stress-providinginstrument that applies static or variable duration cycle tension or compression to cells in vitro. J Cell Sci 1985; 75:35-42. 6. Vandenburgh HH. Motion into mass: how does tension stimulate muscle growth? Med Sci Sports Exerc 1987; 19:5142-9. 7. Harris AK. Tissue culture cells on deformable substrate: biomechanical implications. J Biomech Eng 1984; 106:19-24. 8. Li EY-H, Aggeler J, Farson DA, Hatier C, Hassess J, BissellMJ. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc Nat! Acad Sci USA 1987; 84:136-40. 9. Schmid TM, Linsenmayer TF. Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Bioi 1985; 107:373-81. 10. Davies PF. Endothelial cells, hemodynamic forces, and the localization of atherosclerosis. In: Ryan US, ed. Endothelial cells. Boco Raton, FL: CRC Press, 1988; 123-39. 11. Bowen-Pope DF, Majesky MW, Ross R. Orowth factors for vascular smooth muscle cells. In: Campbell JH, Campbell OR, eds. Vascular smooth muscle cells in culture. Vol I. Boca Raton, FL: CRC Press, 1987; 71-92.
12. Munger BL, Ide C. The enigma of sensitivity in Pacini an corpuscles: a critical review and hypothesis of mechano-electric transduction. Neurosci Res 1987; 5:1-15. 13. Sachs F. Mechanical transduction in biological systems. CRC Crit Rev Biomed Eng 1988; 16:141-69. 14. Morris CE, Sigurdson WJ. Stretch-inactivated ion channels coexist with stretch-activated ion channels. Science 1989; 243:807-9. 15. Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature 1987; 325:811-3. 16. Ingber DE, Folkman J. Tension and compression as basic determinates of cell form and function: utilization of a cellular tensegrity mechanism. In: Stein W, Bronner F, eds. Cell shape: determinates, regulation and regulatory role. Orlando, FL: Academic Press, 1989; 1-31. 17. Reeds PJ, Palmer RM, Smith RH. Protein and collagen synthesis in rat diaphragm muscle incubated in vitro: the effect of alterations in tension produced by electrical or mechanical means. Int J Biochem 1980; 11:7-14. 18. Ooldspink DF. The influence of immobilization and stretch on protein turnover of rat skeletal muscle. J Physiol (Lond) 1977; 264:267-82. 19. Sant'Ambrogio G. Nervous receptors of the tracheobronchial tree. Annu Rev Physiol 1987; 49:611-27. 20. Coleridge HM, Coleridge J CG. Reflexes evoked from tracheobronchial tree and lungs. In: Fishman AP, section ed.; Cherniack NS, Widdicombe JO, volume eds. Handbook of physiology,the respiratory system. VolII. Control of breathing. Part 1. Bethesda: American Physiological Society, 1986; 395-429. 21. Tozzi CA, Poiani GJ, Harangozo AM, Boyd CD, Riley DJ. Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium. J Clin Invest 1989; 84:1005-12. 22. Mercer RR, Laco JM, Crapo JD. Threedimensional reconstruction of alveoli in the rat lung for determination of pressure-volumerelationships. J Appl Physiol 1987; 62:1480-7. 23. Rannels DE. Role of physical forces in compensatory growth of the lung. Am J Physiol1989; 257:LI79-89. 24. Wirtz H, Dobbs LG. Phosphatidylcholine secretion is stimulatd by a single mechanical stretch of rat alveolar type II cellscultured on silastic membranes. FASEB J 1988; 2:A708.
DAVID J. RILEY, Chairman, D. EUGENE RANNELS and ROBERT B. LOW, Co-Chairmen; LEEANN JENSEN, and THOMAS P. JACOBS
Introduction
The living organism continually is subjected to a variety of internal and external physical forces at the cell, tissue, and organ levels. The lung, for example, normally is exposed to transmural pressure gradients required for inflation and to shear stress produced by circulation of blood and by airflow during ventilation. In addition, internal forces are exerted on lung tissue and cells from smooth muscle contraction, from tension generated by the cytoskeleton of lung cells,and from surface forces at the air-tissue interface. The biologic effects of physical forces and their mechanisms have been studied in a number of cell types and tissues such as bone and the cardiovascular and skeletal muscle systems. This has led to an appreciation of their role in cellular and tissue growth, differentiation, and metabolism as well as integrated function and in repair after injury. The effects of physical forces on lung structure, function, and metabolism have not been evaluated systematically. Little information is available regarding their role in lung development or in the maintenance of normal function. The role of physical forces in repair of parenchymal injury, in vascular remodeling with pulmonary hypertension, and in interstitial fibrosis or emphysema remains largely unexplored. A workshop sponsored by the Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, was held in April 1989to summarize the state of knowledge regarding the effects of physical forces on the lung. New developments in celland organ physiology related to mechanical effects on biologic systems were discussed and evaluated, particularly as they might relate to the respiratory system. Current efforts in this area were assessed to identify 910
promising directions for future research. Investigators from various disciplines met to discuss five major topics: biomechanics, cell physiology and biology, mechanical signal transduction, effects of physical forces on the lung, and manpower and training. Biomechanics
Application of engineering principles and measurements to biologic systems is required to define the role of physical forces in the regulation of biologic function. It is imperative that measurements of physical force in all experimental conditions be described in units and in terms derived from physics and engineering. Static and dynamic forces on biomaterials should be quantitated in specific physical terms such as stress, strain, and shear stress. These are defined as follows:stress, force per unit area; strain, change in length divided by initial length; shear stress, force per unit surface area in the direction of flow exerted at the fluid/surface interface. Other terms such as stretch and deformation have been used, but these are not classically defined physical parameters. Although the application of physical principles to the study of simple biomaterials is technically straightforward, data analysis and interpretation are far more complex when dealing with complicated structures such as the lung. The anatomic arrangements of tissues make it difficult to attribute the effects of mechanical perturbations to individual cells, matrix, or other structural components. This necessitated development of a number of model systems that utilize isolated tissues and cells in which the response to physical force can be explored at various levels of complexity. Examples of experimental approaches that have been used in an effort to un-
derstand the role of physical force in biologic regulation are listed in table 1. Forces can be applied to whole organs, tissue strips, or cells cultured on deformable surfaces or in collagen gels; cells in culture can be subjected to hydrostatic or shear stress, and events generated by stress or strain can be measured in cell membranes deformed by application of suction to membrane "patches" or byosmotic swelling.Numerous end points can be investigated, including morphology, cell shape and cytoskeletal properties, protein synthesis and secretion, enzyme activity, and cell division. Signal transduction can be explored in terms of plasma membrane ion channels and activation of second messenger systems such as the cyclic AMP cascade. (Received in original form December 15, 1989 and in revised form February 5, 1990) 1 List ofparticipants: Judith Aggeler, Davie, CA; Albert Banes, Chapel Hill, Nt; Thomas Borg, Columbia, SC; Hazel M. Coleridge, San Francisco, CA; Edward D. Crandall, NeW York, NY;Leland G. Dobbs, San Francisco, CA;. Randall O. Dull, Chicago, IL; John N. Evans, Burlington, vn Yuang-Cheng B. Fung, La Jolla, CA; Albert K. Harris, Jr., Chapel Hill, NC; Donald Ingber, Boston, MA; Thomas P. Jacobs, Bethesda, MD; LeeAnn Jensen, Bethesda, MD; Brian L. Langille, Toronto, ONl; Robert D: Low, Burlington, V'I; Edward J. Macarak, Philadelphia, P!\.; Robert R. Mercer, Durham, NC; Howard E. Morgan, Danville, PA; Catherine E. Morris, Ottawa, ONl; Maurizio Pacifici, Philadelphia, PA; Marlene Rabinovitch, Toronto, ON'!; D. Eugene Rannels, Jr., Hershey, PA; David J. Riley, New Brunswick, NJ; Una S. Ryan, Miami, FL; Frederick Sachs, Buffalo, NY; Stephen Schwartz, Seattle, WA; Frederick Silver, Piscataway, NJ; Gene Sprague, San Antonio, TX; Robert L. Trelstad, Piscataway, NJ; Herman Vandenburgh, Providence, RI; David M. Warshaw, Burlington, VI; and Peter Watson, Danville, PA. 2 Correspondence and requests for reprints should be addressed to Thomas P. Jacobs, Ph.D., Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, 5333 Westbard Ave., Bethesda, MD 20892.
AM REV RESPIR DIS 1990; 142:910-914
911
NHLBI WORKSHOP SUMMARY
TABLE 1 EXAMPLES OF PHYSICAL FORCES APPLIED TO BIOMATERIALS* Model Tissues Stress on tissue strips Tendon Muscle Skin Myometrium Blood vessel Cells Growth on deformable membranes Smooth muscle cells Epithelial cells Chondrocytes Endothelial cells Skeletal muscle cells Cardiac muscle cells Growth in collagen gels Hydrostatic pressure Chondrocytes Endothelial cells Shear stress Endothelial cells
Cell membranes Patch clamp Osmotic swelling
End Points
Glycosaminoglycan synthesis Protein synthesis and degradation DNA synthesis Protein synthesis, cell growth Protein and DNA synthesis
Protein synthesis, cell division DNA synthesis Glycosaminoglycan synthesis Fibronectin synthesis Protein synthesis, intermediary metabolism Protein synthesis Myotube formation Glycosaminoglycan synthesis Morphology Morphology Shape LDL uptake Release of vasoactive substances Ion channel activation Ion channel activation Accumulation of cyclic AMP
* This table is not intended to provide a complete summary of the subject.
It is now possible to investigate the physical properties and responses of individual cellsto external force. Recent research, for example, has described the mechanical properties of isolated single smooth muscle cells (1). An elegant preparation in which individual cells are mounted in a force-measuring device permits correlation of cellular mechanics with cytoskeletal and contractile protein function. As a second example, hypotonic media are used to produce mechanical signals at the cellular level (2), offering a potentially important model for investigation of the biologic implications of physical perturbation of the plasma membrane (2).
through specific matrix-receptor interactions. These effects, in turn, influence the secretion and organization of the extracellular matrix itself, and they result in a "dynamic reciprocity" between cells and the three-dimensionalmatrices with which they are associated. The initial deposition of extracellular matrix is critical giventhis reciprocal relationship. Matrix deposition is a highly organized, multistep process involving coordinated intracellular and extracellular events. In tendon, for example, matrix-synthesizing cells exert control of fibril formation by regulating the amount and kinds of matrix elements produced, their orientation upon secretion, and the secretion of enzymes needed for final modification and assembly of mature Cell Physiology and Biology collagen type I fibrils. The requirement Workshop participants discussedat length for increased tensile strength of tendon the mechanisms by which physical force during embryonic development in reis transmitted through biologic systems, sponse to increased mechanical forces particularlywith regardto cell-matrixinter- may account for the tight coupling to inactions. Biologic responses generated by creased collagen content, fibril, and fimechanical perturbations in cultured ber diameter and fibril length, as well as cells were presented and discussed with to the differentiated phenotype of tenregardto potentialunderlyingmechanisms. don fibroblasts. An important future Studies in which cells are exposed to goal is to establish the mechanism of this extracellular matrix molecules show that apparently causal relationship. specific matrix components affect celluThe ability of cells to exert physical lar movement, shape, polarity, metabo- forces on neighboring cells and the malism, and differentiation (3), presumably trix is not limited to muscle, as nearly
all differentiated cell types have contractile capability (4). Contractile forces appear to be involved in cell migration during embryonic growth, wound closure, locomotion, and matrix alignment (4). The consequence of the interplay between internal forces and those imposed externally is illustrated by the observation that cellsin culture orient in response to force (5). Alignment perpendicular or parallel to the axis of force has been described for a number of cell types and appears to depend on the pattern of stress imposed (6, 7). Physical force also influences differentiated function by affecting cell shape. This is clearly evident from model systems that show the influence of reconstituted basement membrane components on gene expression and protein secretion by cultured mammary epithelium (8). Another example is induction of the expression of differentiationspecific collagen genes by the manipulation of the shape of cultured chondrocytes (9). Attendant changes in the regulation of protein synthesis may reflect a functional association of polyribosomes with the cytoskeleton. The endothelial cell, by its strategic location, is subjected to both stress and shear stress. Endothelial cells in vitro change shape in response to unidirectional fluid shear stress in a reversible timeand shear-dependent manner (10). Exposure to shear stress causes reorganization of the cytoskeletonand significantchanges in cell surface properties. Additionally, it also stimulates the synthesis and release of substances involved in vascular homeostasis such as prostacyclin (PGI 2) , tissue plasminogen activator, and endothelin. Shear stress also may increase low density lipoprotein uptake, fluid endocytotic rate, and histamine-forming capacity. Flow-induced dilatation of large arteries has been attributed to the release of endothelium-derived relaxing factor. Evidence for an influence of shear stress on endothelial cell function in vivo comes from studies which demonstrate that decreasing flow rates result in reorganization of the arterial architecture and narrowing of the vascular lumen only in the presence of intact endothelium. Thus, the endothelium may mediate flow-induced structural changes in the vesselwall, consistent with the known ability of endothelial cellsto regulate smooth muscle cell proliferation and protein turnover (11). Characterization of the mechanoreceptors responsible for the multiple effects of shear stress on the endothelium remains an important subject for future in-
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NHLBI WORKSHOP SUMMARY
vestigation. Smooth muscle within the vessel wall may also respond to mechanical forces. For example, several studies show realignment of smooth muscle cells in response to cyclic tensional deformation in culture, whereas others show that cyclic deformation increases extracellular matrix production. Mechanical Signal Transduction
The molecular mechanisms by which mechanical stimuli are transduced into a variety of biologic responses remain poorly defined, although significant progress is being made toward understanding these events. Twobroad types of specific mechanoreceptors have been described: specialized nerve endings, which are part of an integrated neural response to movement of organs (12), and stretch-activated ion channels, which operate at the cellular level(13). Mechanoreceptors of the former type are important in the respiratory system and will be discussed in the section on the lung. Ion channels are transmembrane proteins that permit signaling of cellsby control of the selective movement of ions. Mechanically activated ion channels have been identified that are distinct from channels that respond to voltage changes or various ligands (13). Their identification will help facilitate analysis of the cellular and molecular events through which cells respond to mechanical forces in terms of altered gene transcription, cell shape, or cell division. A direct coupling of stretch-activated ion channels to these complex biologic responses, however,has not been demonstrated. Mechanically activated ion channels have been demonstrated directly in a number of cell types (13).A method that allows exploration of their properties involves application of pressure or suction by a patch clamp pipette, which results in channel opening or closing. These channels have variable ion selectivities, although most are cation-selective and prefer K+ or Na", The energy required to open such channels is provided by membrane strain rather than by metabolism of high energy phosphates. The channels are highly sensitive and require a force in the range of 0.5 to 3.0 dyn/cm' for halfmaximal activation (13). Stretch-inactivated channels also have been described that open at low force and close with increasing force (14). The co-localization of stretch-activated and inactivated channels suggests that distinct populations of channels with reciprocal responses can coexist within a small area of the membrane. Isolation of these channels in pure
form for chemical, structural, and functional analysis is a critical area for future investigation. Although mechanically activated ion channels have been found in a variety of cell types, their presence in endothelial cells may be of particular importance in mediating hemodynamic effects on vascular function (see above). Twotypes of ion channels have been identified in cultured endothelial cells subjected to shear stress. The first is a K+sensitive channel in which K+ current increases as a function of shear stress. It has been suggested that flow-mediated hyperpolarization may alter underlying smooth muscle contractility via direct coupling through gap-junctions or indirectly by release of smooth muscle cell constricting or relaxing factors (10). Nonselective ion channels also exist in endothelial cells; these could also contribute to additional responses to hemodynamic force (15). Mechanical tension also may affect ion channels in smooth muscle cells. Tension applied to blood vessels promotes the influx of Ca 2 + via a pathway with unique pharmacologic properties. This pathway may mediate myogenic tone in response to increased tension on the blood vessel wall. Other molecular mechanisms by which mechanical stimuli may be transduced into morphologic and biochemical responses are under investigation. For example, the effects of mechanical stress on intermediary metabolism and protein turnover in muscle have been studied (6). These effects may be mediated via activation of a plasma membrane sodium pump (6). Newly synthesized prostaglandins PGE2 and PGF2 may also be important. Finally, in studies of isolated hearts, increased protein synthesis caused by elevated perfusion pressure is associated with activation of the cyclic-AMP-dependent protein kinase. Forces applied to cells may be redistributed by tension-bearing cytoskeletal and contractile elements. Physical forces that act through the cytoskeleton may function as an alternative signal transduction mechanism to the more commonly considered plasma-membrane-derived second messengers by conveying information directly to the nucleus (16). Effects of Physical Forces on the Lung
The lung is continuously subjected to physical forces by pressure distending the parenchyma, by airflow and gradients of pressure in the tracheobronchial tree, and
by hemodynamic forcesin the pulmonary vasculature. Normal ventilatory fluctuations are enhanced by periodic "sighs" and by exercise, which increases both the rate and depth of ventilatory excursions. Ventilatory movements exert stress on the tissue, and it has been shown that ventilation modulates several metabolic pathways in the lung. Few studies, however, have demonstrated that physical force directly affects individual components of the lung. Little is known of the effects of mechanical forces on respiratory muscles. One study found increased protein and collagen synthesis in isolated rat diaphragm muscle after mechanical stimulation (17). Because the position of the diaphragm is altered in diseases associated with hyperinflation of the lungs, abnormal passive tension on diaphragmatic musclesmay influencetheir metabolism. Immobilization of skeletal muscle results in increased protein degradation and muscle atrophy (18). These effects may contribute to respiratory muscle atrophy in patients requiring long-term mechanical ventilation. Mechanoreceptors within the respiratory system have been studied as part of the integrated neural response involved in control of breathing (19). Afferent nerve fibers from muscle spindles .in the chest wall provide information to the respiratory neurons about forces exerted by respiratory muscles and about thoracic movements (19). Stretch receptors, classified as rapidly and slowly adapting based on their electrical activities and the distribution of their terminals, are located within the smooth muscle layer of the extrapulmonaryairways (20). They are activated by distortion of the airway walls during inflation rather than by changes in lung volume (20). Their putative function is to minimize ventilatory work output for a given minute volume. However, few studies have explored the degree to which bronchial transmural pressure or longitudinal stretching contribute to stimulation of . the sensory terminals. Studies are needed to determine how activity of the two types of mechanoreceptors correlates with changes in wall tension. Future studies also should define the biophysical properties of respiratory stretch receptors and should explore the molecular basis of force transduction by these mechanoreceptors. Cellular and extracellular components of the pulmonary vasculature also may respond to physical signals. High intravascular pressure is an important factor in stimulating growth of smooth muscle
NHLBI WORKSHOP SUMMARY
cells in large arteries. Cellular proliferation in this case may be a response to metabolic changes caused by vascular damage or to growth factors. Recent studies of mechanical tension applied to pulmonary artery segments indicate that collagen and elastin synthesis are increased by short-term tension, but DNA synthesis is not affected (21). These changes are not observed in vessels denuded of endothelium, indicating a role for the endothelium in mediating the early response. An endothelial cell-derived growth factor may be involved since only vesselswith intact endothelium express the proto-oncogene v-sis after application of tension (21). When cultured newborn lamb endothelial cells are subjected to pulsatile stress, soluble factors active on smooth muscle cells are released, but, in initial studies, they appeared to be growth-inhibitory. These and other results suggest that cell-cell interactions and soluble factors that act in an autocrine or paracrine fashion, in addition to direct mechanical stimulation, may playa physiologically significant role in mediating the effects of physical forces on pulmonary vascular remodeling. Definition of the manner in which changes in transpulmonary pressure deform the peripheral lung is critical to determining the nature of parenchymal stress-strain relationships. The issue of whether normal lung inflation leads to unfolding of the alveolar septal wall or to deformation of the alveoli, coupled with changes in their dimensions, has not been clearly resolved. Recent three-dimensional, reconstructions of individual alveoli suggest that, under physiologic conditions of lung inflation, both alveolar volume and surface area increase as a function of lung volume, and alveoli exhibit hysteresis (22). The positioning of connective tissue fibers at the alveolar septal edges that form the boundaries of alveolar ducts suggests they function as loadbearing elements within the lung parenchyma. The latter tissue components are more important as determinants of parenchymal stress-strain relationships at higher lung volumes. Despite these descriptive advances, the micro mechanics of the parenchymal region remain poorly understood. Normal levels of stress and strain in the alveolar epithelial, interstitial, and endothelial layers, or within the cell types present in each, are not known. The coupling between forces borne by connective tissue elements and the cells that produce these elements may represent a critical site of
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transduction between physical forces and employ different approaches to this probcellular responses in maintenance and lem. Becauseof this diversity, communicagrowth of the lung, as well as in repara- tion among investigators with common tive processes. interests has been difficult. Development It is clear from severalexperiments that of a comprehensive understanding of the stresses associated with lung inflation role of mechanical forces in biology will produce a variety of biochemical respons- require more effective exchange of scies. For example, increased ventilation ele- entific information to enhance the revates surfactant levels in bronchoalveo- search effort. lar lavage fluid and modulates producAll participants in the workshop ention of surfactant components, glucose, couraged integrated interdisciplinary inand fatty acids in isolated perfused lungs. vestigation, with particular emphasis on Rapid induction of metabolic changes in bioengineering. Many biologists have a ornithine decarboxylase, transport of poor understanding of engineering conpolyamines, cyclicAMP content, and ac- cepts involved in the study and quantitativation of the cyclic-AMP-dependent tion of mechanical forces; bioengineers protein kinase occur with overinflation often lack broad training in experimenof the remaining lung after partial pneu- tal biology. Thus, there is a need to fosmonectomy and with increased inflation ter closer collaboration between biologists and bioengineers to develop new of isolated perfused lungs (23). A number of experiments have demon- theoretical and methodologic approaches strated that mechanical strain affects for studying the biologic effects of memetabolic changes in isolated cells (see chanical force, particularly at the celluabove), but few studies of lung cells have lar and subcellular levels. Development been reported. Recent data suggest that of knowledge in the area of cellular biothe type II pulmonary epithelial cell mechanics is critical to advancing reresponds to distortion of the culture sur- search in this field. A major reason for insufficient inface with increased surfactant secretion (24). Stress-activated secretion is inhibit- tegration of engineering into biologic ed by surfactant apoprotein-A, but does studies is the structure of training for biolnot appear to be mediated by a cyclic- ogists and engineers. There is little forAMP-dependent mechanism. In what mal teaching of bioengineering princimay be a related phenomenon, changes ples in biology curricula, and bioenin cell shape by interaction with extracel- gineers generally are not sufficiently lular matrix components clearly modu- trained in applying biologic approaches late type II cell differentiation and me- to research. Restructured training in both tabolism. The effects of physical forces disciplines at the graduate and poston other cell types from the lung paren- graduate levels is needed to improve the chyma have not been reported. Finally, integration of professional efforts in there has been very little investigation of these fields. the effect of physical forces on repair of lung injury. Hyperventilation worsens Recommendations elastase-induced emphysema in hamsters 1. Encourage biologists, biophysicists, as determined by lung elastic recoil, but, in other studies, no adverse effects of andengineers to worktogether in thearea hyperventilation are evident based on of the biologic effects of mechanical morphologic or physiologic criteria. Al- forcesand theirtransduction through the though viscoelastic properties of lung useofmoderncellular andmolecularapstrips have been described, mechanical proaches. The importance of studying tension has not been applied directly to the effects of mechanical forces on biolung tissue to determine metabolic ef- logic systems is well established. Discovfects. Thus, there remains a need to de- ery of potential transduction mechanisms fine the influence of physical forces on such as stretch-activated ion channels has the outcome of the repair process after added new impetus to this research. New concepts of integrated cell function also lung injury and disease. have stimulated interest in this area and Manpower and Training provide opportunities for fruitful invesStudy of the effects of mechanical forces tigation. More effort is required to deon biologic systems is a relatively under- velop innovative strategies to overcome developed field of scientificinvestigation. the relative lack of techniques to meaScientists of diversebackgrounds, includ- sure cellular biomechanics and to improve existing technologies at the celluing biophysicists, cell biologists, bioengineers, and cell and organ physiologists lar and the molecular levels.
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2. Encourage quantitation ofphysical forces in experimental studiesalongwith the use of standard terms in reporting results. Application of bioengineering concepts and principles to complex tissues is not straightforward. It is nevertheless important to quantitate both the forces applied and the resulting responses at the tissue and the cell levels. Quantitation of mechanical forces in biologic work often is inadequate. In all studies on the effects of mechanical force on biomaterials, for example, relevant parameters should be measured quantitatively and expressed in conventional physical terms. Sound bioengineering principles should be used in experimental design. Development of new techniques to apply micromechanical measurements must be encouraged.
3. Improve research and training opportunitiesthat integrate bioengineering with biologicapproaches and teaching. Engineering principles are fundamental to all work on mechanical forces, but often the integration of engineering concepts into biologic experiments is inadequate. Interdisciplinary training programs and research activities that involvebioengineers, biophysicists, and biologists should be encouraged to enhance such integration.
4. Promote research on the effects of physicalforces on the lung and its components. Although the lung is subjected to a variety of mechanical forces, it is not known how these forces affect normal lung function. The effects of physical force in injury and disease also can be expectedto be important, but they remain unexplored. Fewpublished studies of the effects of mechanical force on various tissues and cells have been carried out that are physiologically relevant to the lung. New models for exploration of the effects of mechanical forces on the lung
NHLBI WORKSHOP SUMMARY
should be developed and existing models should be refined in order to enlarge the existing base of knowledge. It is particularly important to encourage investigations of the mechanisms by which mechanical force is transduced to alter tissue or cellular function. It is recommended that research on these problems be promoted but not targeted to specific areas. References 1. Warshaw DM, Fay FS. Cross-bridge elasticity in single smooth muscle cells. J Gen Physiol1983; 82:157-99. 2. Christensen O. Mediation of cell volume regulation by Ca 2 + influx through stretch-activated channels. Nature 1987; 330:66-8. 3. Ekblom P, VestweberD, Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Bioi 1986; 2:27-47. 4. Harris AK. Traction and its relationship to contraction in tissue cell formation. In: Bellairs R, Curtis A, Dunn G, eds. Cellbehavior. Cambridge: Cambridge University Press, 1982; 109-34. 5. Banes AJ, Gilbert J, Taylor D, Monbureau O. A newvacuum-operated stress-providinginstrument that applies static or variable duration cycle tension or compression to cells in vitro. J Cell Sci 1985; 75:35-42. 6. Vandenburgh HH. Motion into mass: how does tension stimulate muscle growth? Med Sci Sports Exerc 1987; 19:5142-9. 7. Harris AK. Tissue culture cells on deformable substrate: biomechanical implications. J Biomech Eng 1984; 106:19-24. 8. Li EY-H, Aggeler J, Farson DA, Hatier C, Hassess J, BissellMJ. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc Nat! Acad Sci USA 1987; 84:136-40. 9. Schmid TM, Linsenmayer TF. Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Bioi 1985; 107:373-81. 10. Davies PF. Endothelial cells, hemodynamic forces, and the localization of atherosclerosis. In: Ryan US, ed. Endothelial cells. Boco Raton, FL: CRC Press, 1988; 123-39. 11. Bowen-Pope DF, Majesky MW, Ross R. Orowth factors for vascular smooth muscle cells. In: Campbell JH, Campbell OR, eds. Vascular smooth muscle cells in culture. Vol I. Boca Raton, FL: CRC Press, 1987; 71-92.
12. Munger BL, Ide C. The enigma of sensitivity in Pacini an corpuscles: a critical review and hypothesis of mechano-electric transduction. Neurosci Res 1987; 5:1-15. 13. Sachs F. Mechanical transduction in biological systems. CRC Crit Rev Biomed Eng 1988; 16:141-69. 14. Morris CE, Sigurdson WJ. Stretch-inactivated ion channels coexist with stretch-activated ion channels. Science 1989; 243:807-9. 15. Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature 1987; 325:811-3. 16. Ingber DE, Folkman J. Tension and compression as basic determinates of cell form and function: utilization of a cellular tensegrity mechanism. In: Stein W, Bronner F, eds. Cell shape: determinates, regulation and regulatory role. Orlando, FL: Academic Press, 1989; 1-31. 17. Reeds PJ, Palmer RM, Smith RH. Protein and collagen synthesis in rat diaphragm muscle incubated in vitro: the effect of alterations in tension produced by electrical or mechanical means. Int J Biochem 1980; 11:7-14. 18. Ooldspink DF. The influence of immobilization and stretch on protein turnover of rat skeletal muscle. J Physiol (Lond) 1977; 264:267-82. 19. Sant'Ambrogio G. Nervous receptors of the tracheobronchial tree. Annu Rev Physiol 1987; 49:611-27. 20. Coleridge HM, Coleridge J CG. Reflexes evoked from tracheobronchial tree and lungs. In: Fishman AP, section ed.; Cherniack NS, Widdicombe JO, volume eds. Handbook of physiology,the respiratory system. VolII. Control of breathing. Part 1. Bethesda: American Physiological Society, 1986; 395-429. 21. Tozzi CA, Poiani GJ, Harangozo AM, Boyd CD, Riley DJ. Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium. J Clin Invest 1989; 84:1005-12. 22. Mercer RR, Laco JM, Crapo JD. Threedimensional reconstruction of alveoli in the rat lung for determination of pressure-volumerelationships. J Appl Physiol 1987; 62:1480-7. 23. Rannels DE. Role of physical forces in compensatory growth of the lung. Am J Physiol1989; 257:LI79-89. 24. Wirtz H, Dobbs LG. Phosphatidylcholine secretion is stimulatd by a single mechanical stretch of rat alveolar type II cellscultured on silastic membranes. FASEB J 1988; 2:A708.
