Published ahead of print on July 25, 2002, doi:10.1164/rccm.200203-207OC
© 2002 American Thoracic Society
Role of Deformation-induced Lipid Trafficking in the Prevention of Plasma Membrane Stress FailurePulmonary and Critical Care Medicine Division, Mayo Clinic and Foundation, Rochester, Minnesota Correspondence and requests for reprints should be addressed to Nicholas E. Vlahakis, M.D., Stabile 8-62, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: vlahakis.nicholas{at}mayo.edu
Cells experience plasma membrane stress failure when the matrix to which they adhere undergoes large deformations. In the lung, such a mechanism might explain mechanical ventilation–associated cell injury. We have previously shown that in alveolar epithelial cells, deformation induces lipid trafficking to the plasma membrane, thereby accommodating the required increase in the cell surface area. We now show that cell wounding is strain amplitude and rate dependent and that under conditions of impaired exocytosis strain-induced cell wounding is significantly increased. In addition, the susceptibility of cells to mechanical injury was not correlated with changes in cell stiffness. Using a dual-labeling technique, we differentiated between cell populations that were reversibly and irreversibly injured and showed that interventions that impair deformation-induced lipid trafficking also reduce the likelihood of plasma membrane resealing. Our findings suggest that cell plasticity and remodeling responses such as deformation-induced lipid trafficking are more important for cytoprotection from strain injury than are the innate mechanical properties of the cell. We also conclude that in deformation experiments, tests of cell membrane integrity cannot be interpreted as tests of cell viability because an intact plasma membrane after deformation does not mean that no injury had occurred.
Key Words: exocytosis • plasma membrane wounding • plasma membrane resealing • ventilator-induced lung injury
Cells experience plasma membrane stress failure when the matrix to which they adhere undergoes large deformations (1–3). In the lung, this mechanism might explain epithelial and endothelial cell disruptions associated with high tidal volume mechanical ventilation (4, 5). To simulate alveolar volume change during mechanical ventilation, alveolar epithelial cells are grown on malleable substrata, which when stretched deform the overlying cells, resulting in a large surface-to-volume ratio (3, 6, 7). In fibroblasts and neurons, unfolding of excess plasma membrane and exocytosis allow cells to increase their surface area while protecting the lipid bilayer from lytic tension (8, 9). Recently, we reported that in alveolar epithelial cells, intracellular lipids are trafficked to the plasma membrane after substratum stretch (7). This deformation-induced lipid trafficking (DILT) is vesicular in nature and energy and strain amplitude dependent and accommodates deformation-induced changes in cell surface area. These prior studies led us to postulate that DILT is important for protection against deformation-related cell injury. To test this hypothesis, we measured the susceptibility of alveolar epithelial cells to stretch amplitude and rate-induced plasma membrane wounding. Cell wounding is commonly assessed using fluorescently labeled, membrane-impermeable macromolecules such as fluorescein-labeled dextran (FDx) (2, 3, 10–12). FDx enters the cell through plasma membrane breaks and is retained in the cytoplasm when the membrane defect reseals (11, 13, 14). However, if a wounded cell fails to reseal a plasma membrane defect, FDx cannot be retained and the cell cannot be distinguished from an uninjured cell. We present here a novel dual-labeling technique using first FDx and, after stretching, propidium iodide (PI), which labels the nuclei of injured and nonresealed cells. Using this approach, we were able to identify three cell populations: (1) wounded and resealed cells, (2) wounded cells that had not resealed, and (3) uninjured cells with intact plasma membranes. Our findings highlight that live–dead exclusion assays performed at the end of a deformation experiment might be both false positive (lethal wounding is not distinguished from wounding and resealing) and false negative (wounded and nonresealed cells might not retain exclusion dyes). Additional experiments were performed under conditions of impaired exocytosis, induced by cold temperature or cholesterol depletion. These interventions, which impaired DILT, also made cells more prone to injury and altered the cells' resistance to shape change. Because changes in cell mechanical properties might determine a cell's susceptibility to injury, we measured the effects of cytoskeleton active agents on DILT along with their effects on apparent cell stiffness. Based on these measurements, we conclude that remodeling responses such as DILT are more important for protecting the plasma membrane against stress failure than are the inherent strength and organization of stress-bearing elements such as the subcortical cytoskeleton.
Experimental Design for Determination of Plasma Membrane Breaks When deformation-induced breaks occur, fluorescently labeled molecules—typically membrane impermeable—enter the cell along a concentration gradient. Cells were incubated with FDx (70 kD; Fluka, Milwaukee, WI) and then strained as described later here. After cell deformation, cells were incubated with PI (668 D; Sigma, St. Louis, MO) or in a few experiments with unesterified calcein (622 D; Molecular Probes, Eugene, OR). Cells with FDx or nuclear PI labeling were counted in five low-power (x40) microscope fields in each culture well and were tabulated as a percentage of the total cells counted. FDx-positive/PI-negative cells were considered wounded and resealed, and PI-positive/FDx-negative cells were considered wounded and nonresealed.
Alveolar Epithelial Cell Culture
Cell Strain Devices
Labeling of Lipid Membranes
Cell Imaging
Magnetic Twisting Cytometry
Statistical Analysis
Substratum Strain Induces Plasma Membrane Wounds in Adherent Alveolar Epithelial Cells The fractions of injured cells and their resealing responses in different alveolar epithelial culture systems after exposure to a single large deforming stress are shown in Figure 1 . A549 (n = 5, 27 wells), L2 (n = 2, eight wells), and rat alveolar epithelial cells (n = 2, 12 wells) were rapidly stretched by 25%. The susceptibility to stretch-induced wounding and the probability of subsequent resealing varied across cell types. The fraction of wounded A549, L2, and primary rat alveolar epithelial cells was 6%, 21%, and 46%, respectively. The rates of resealing were 38%, 78%, and 21%, respectively.
Plasma Membrane Resealing after Cell Wounding Maintains Cell Viability Cell viability was determined for both FDx- and PI-positive A549 cells and Day 5 primary rat alveolar epithelial cells (n = 3, eight wells). After deformation, the adherent cells were reincubated with fresh nutrient media. Twelve and 24 hours later, the number of FDx- and PI-positive cells was recounted. There was no difference (p > 0.05) in the number of FDx-positive cells when compared immediately after stretch and 12 and 24 hours after stretch; however, no PI-positive cells were seen. Because PI is itself cytotoxic, we repeated these experiments using a nontoxic second label, namely unesterified calcein. No calcein-positive cells could be identified after 12 or 24 hours, suggesting that failure to reseal plasma membrane wounds rapidly is a lethal event.
Plasma Membrane Wounding Is Strain Amplitude and Strain Rate Dependent
Figure 3 (n = 4, 12 wells) shows that in A549 and L2 cells, the susceptibility to injury varies not only with strain amplitude but also with strain rate. In A549 cells (Figure 3A), at the highest strain rate of 140% per second, a single stretch of 25% produced two times (4.9% versus 2.6%, p < 0.0001) and three times (4.9% versus 1.3%, p < 0.0001) more wounding than at rates of 40% per second and 12% per second, respectively. When stretched at a rate of 3% per second, a 25% stretch produced no irreversible cell injury. Similarly, in L2 cells (Figure 3B), a single stretch of 25% with a strain rate of 140% per second resulted in 0.7 times (23.1% versus 15.9%, p < 0.0001) and 5.5 times (23.1% versus 4.2%, p < 0.0001) more wounding than at rates of 40% per second and 12% per second, respectively.
Cold Temperature and Cholesterol Depletion Inhibits DILT To test whether vesicular lipid trafficking is important for protecting cells from mechanical injury, we first had to demonstrate that exposure to cold temperature and cholesterol depletion indeed inhibited DILT. The effects of cooling on the stretch-related decrease in FM1-43 fluorescence (a marker of exocytosis) in A549 cells (two experiments, six cells) and in primary rat alveolar epithelial cells (two experiments, six cells) are shown in Figure 4A . At room temperature, a single stretch of 25% caused an eightfold and a threefold decrease in FM1-43 fluorescence, compatible with a significant exocytic response. Also note that at 4°C, the same stretch produced no significant change in FM1-43 fluorescence, consistent with inhibition of DILT. Figure 4B shows that treatment of A549 cells (n = 3, 18 cells) and alveolar epithelial cells (n = 3, 11 cells) with ß-methyl cyclodextrin also results in inhibition of DILT compared with untreated and stretched epithelial cells. ß-Methyl-cyclodextrin was found to reduce the total cholesterol content of A549 cells by up to 45% (data not shown).
Cholesterol Depletion Increases the Susceptibility of Alveolar Epithelial Cells to Stretch-related Injury Having demonstrated inhibition of DILT, we next sought to measure the effect of this inhibition on the cells' susceptibility to deformation-related wounding. The incidence of stretch-related plasma membrane wounding was significantly increased in cholesterol-depleted cells compared with placebo-treated controls. In A549 cells and primary alveolar epithelial cells, cholesterol depletion raised the fraction of wounded cells from 1.4 ± 1.5% to 4.0 ± 1.3% (p < 0.0001; Figure 5A) and from 49 ± 3.2% to 59 ± 2% (p < 0.0003; Figure 5B), respectively. Furthermore, the fraction of wounded cells that healed was substantially reduced after cholesterol depletion. The resealing rate of wounded and cholesterol depleted A549 cells was 46 ± 7.7% compared with 73 ± 30.3% in normal controls (p < 0.006; Figure 5A). Similar effects were observed in alveolar epithelial cells in which cholesterol depletion lowered the resealing rate from 24 ± 3.4% to 15 ± 2% (p < 0.0004; Figure 5B).
Cooling Increases the Susceptibility of A549 Cells to Stretch-related Injury We have previously demonstrated that cooling changes the mechanical properties of cells and increases their susceptibility to deformation-related injury (1); however, in prior experiments, we had assessed injury solely on the basis of FDx retention and thereby implicitly assumed a lack of temperature effect on the resealing rate. Figure 6 shows that this assumption is incorrect. Compared with 37°C, low temperature increased the number of cells with stretch-induced wounding. At the 3% per second, 12% per second, 40% per second, and 140% per second strain rate, low temperature increased total wounding by 2.5-fold, 4-fold, 4-fold, and 3.5-fold, respectively (p < 0.001). In addition, low temperature also decreased the number of cells that resealed stretch-induced plasma membrane breaks. At the 3% per second, 12% per second, 40% per second, and 140% per second strain rate, low temperature increased the proportion of nonresealed plasma membrane breaks by 40-fold, 9.5-fold, 5-fold, and 2-fold, respectively (p < 0.001). Therefore, analyses that are entirely based on FDx retention can substantially underestimate the effects of an intervention such as cooling on stretch injury.
Inhibition of DILT Increases the Susceptibility to Stretch Injury Independent of the Cells' Resistance to Shape Change We had previously speculated that the inherent mechanical properties of cells determine their susceptibility to deformation injury (1, 3). To test this hypothesis, we measured the effects of cholesterol depletion, cooling, and cytoskeletal active agents on apparent A549 cell stiffness as shown in Figure 7 . These interventions have diverse effects on the cells' resistance to a shape change; however, these same cytoskeleton active agents all raise the cells' susceptibility to stretch injury. Figure 8 shows that after microfilament depolymerization with cytochalasin D (n = 3, nine wells), the percentage of wounded A549 cells increased from 3.8 ± 0.8% to 6.0 ± 1.4% (p < 0.0001), whereas there was an increase in the proportion of nonresealed cells by 3-fold (p < 0.0001). Changes in microtubular assembly (colchicine; four experiments, 12 wells) and disassembly (paclitaxel; four experiments, 15 wells) resulted in a 34% increase (5.1 ± 1.9% to 8.6 ± 1.7%, p < 0.0001) and a 29% increase (4.4 ± 0.7% to 5.8 ± 1.1%, p < 0.001), respectively, in the number of wounded cells. Both colchicine and paclitaxel inhibited resealing, increasing the proportion of cells that did not reseal by 1.5-fold (p < 0.001).
Strikingly, this apparent discordance in findings is explained by the fact that these agents all impair DILT (Figure 9) . In the presence of cytochalasin D, colchicine, and paclitaxel, DILT was inhibited 6-fold, 12-fold, and 11-fold, respectively (p < 0.0001, 10 cells) as compared with cells stretched without cytoskeletal agents present.
In this study, we show that inhibition of DILT by low temperature and cholesterol depletion results in an increased probability of cell wounding. This finding highlights the importance of lipid exocytosis as a means to accommodate deformation-induced changes in cell shape and in turn to prevent plasma membrane breaks. We also show that despite the opposite effects of low temperature and cholesterol depletion on apparent cell stiffness, the probability of wounding increased. Similarly, the probability of cell wounding increased after both increases and decreases in cytoskeletal stiffness. These findings are explained, at least in part, by their inhibition of vesicular lipid trafficking to the plasma membrane. We therefore conclude that a dynamic remodeling process such as DILT is a more important determinant of cell wounding than the mechanical properties of the cell's stress-bearing elements before deformation. By using a dual-labeling technique for identifying plasma membrane wounding, we also show that substratum deformation results in plasma membrane breaks that either reseal (maintaining cell viability) or do not reseal (resulting in cell death). We demonstrate that inhibition of DILT not only increases the probability of cell wounding but also decreases the probability of wound resealing, suggesting that DILT plays a role not only in wound prevention but also in wound resealing.
Importance of Defining Cell Injury as Wounding or Resealing First, this distinction facilitates a more accurate assessment of factors determining either wounding or resealing. This is illustrated by our results regarding the effect of strain amplitude and strain rate. If total cell wounding is not accounted for, one might conclude that resealing increases proportionally with both interventions; however, by measuring total wounding, it is appreciated that they have opposite effects on the probability of resealing. The biologic reason for this is unclear and warrants further study but does suggest that wounding per se might have determinants that are separate from resealing. Second, the determinations of cell death by the use of cellular markers depending on plasma membrane integrity potentially are unsuitable for deformation experiments. Many of the cells considered to be "dead" might merely be injured with resealed plasma membrane breaks. Numerous deformation experiments to date have used such an approach and now must be reassessed in light of this new data (2, 11, 12, 18). This in turn has "downstream" implications regarding interpretation of deformation-induced cellular responses. For example, protein measurement will be "confounded" by differing contributions from the irreversibly wounded and resealed cell populations (19). Thus, the degree of wounding and resealing must be established for all experimental conditions and deformation parameters (including strain rate). Third, although the study of alveolar epithelial biology using cell lines has limitations (20), our data support the notion that important biologic phenomena are shared by alveolar epithelial cell lines and primary cell cultures, particularly as it pertains to studies of cell injury and repair mechanisms in vitro. We studied human A549 cells, rat L2 cells, and rat primary alveolar epithelial cells and found that the determinants of DILT, wounding, and resealing were similar in primary cells and cell lines. It is of note that injured primary cells were less likely to reseal than wounded A549 and L2 cells. The reasons underlying this remain to be determined, but such factors as differences in state of polarization and interconnectedness, rate constant for cytoskeletal remodeling and vesicular trafficking, or more global differences in mechanosensing and mechanotransduction might all play a role.
Cell Plasticity Is More Important for Determination of Wounding Than the Mechanical Properties of the Stress-Bearing Elements We have previously postulated on the adaptive responses of the lipid surface to deforming stress in alveolar epithelial cells (3). First, the cell might unfold plasma membrane "ruffles," thus preventing any increase in plasma membrane tension. Second, the plasma membrane, once unfolded, might be able to accommodate a 2–3% lateral strain before yielding (8, 9, 25). Third, if these responses are overwhelmed, the plasma membrane might either undergo stress failure or further adapt its stress-bearing elements to prevent plasma membrane breaks. For example, after deformation, the protein components of actin-extracellular matrix adhesions remodel (26), forming larger and stronger focal adhesion complexes (27). Lipid trafficking to the cell surface is another such adaptive mechanism and has been demonstrated in numerous in vitro cell systems using various deformation profiles (8, 9, 13, 25, 28). We have shown that this phenomenon also occurs in alveolar epithelial cells (7). We now demonstrate that after inhibition of DILT by cholesterol depletion and cold temperature, the probability of wounding is increased. These interventions have opposite effects on cell stiffness: decreased by plasma membrane cholesterol depletion and increased by cold. One might predict that opposite effects on cell rigidity should have opposite effects on the probability of wounding (8, 24, 29); however, our findings show that both interventions resulted in an increase in cell wounding. Cholesterol depletion, low temperature, and cytoskeletal alteration all had one effect in common: inhibition of DILT. Thus, together, these results suggest that the mechanical properties of the cell's stress-bearing elements (namely the plasma membrane and the cytoskeleton) are less of a determinant of plasma membrane wounding than is DILT, a process that actively remodels the stress-bearing capability of the cell. Of note, cellular tensegrity does not account for such active remodeling of cellular stress-bearing elements (30). These results also point out that the cytoskeleton is not solely a structural force-bearing unit of the cell but rather is integrally involved in facilitating signal transduction and vesicle trafficking between organelles and the plasma membrane (31–33). Also, it would seem that the rate at which the cell is able to remodel its stress-bearing elements will determine the probability of wounding. This supposition is indeed supported by our findings that wounding is increased with increasing strain rate. Not only was wounding found to be increased with inhibition of DILT, but concomitantly, the rate of wound resealing was found to be decreased. The mechanisms of cell wound repair have been studied extensively and reviewed recently (11, 12, 21). It has been postulated that resealing can occur either spontaneously by passive lateral diffusion of lipid molecules within the injured plasma membrane edges or by "patch" resealing via rapid fusion of lipid vesicles into a large "lipid patch" in close proximity to the wound (12) or exocytosis of intracellular lipid resulting in decreased plasma membrane tension (8, 25). Our results show that wound resealing in alveolar epithelial cells involves a vesicular process of exocytosis that is independent of the mechanical properties of either the plasma membrane or the cytoskeleton. They also highlight the conflicting literature regarding the importance of an intact actin cytoskeleton for facilitation of wound resealing (34). The motivation for these studies is the clinical entity of ventilator-induced lung injury. Numerous studies have focused on the manifestations and physiologic consequences of ventilator-induced lung injury in animal models and humans. The cytopathologic lesions of ventilator-induced lung injury, namely plasma membrane blebbing, intercellular and intracellular epithelial and endothelial gap formation, and denudation of the basement membrane have all been carefully documented, yet research on the pathophysiology and molecular mechanisms of mechanical lung injury has focused largely on downstream events such as edema formation and inflammation, which are consequences of cellular mechanotransduction and stress failure. Comparatively little is known about the actual deformation experienced by lung cells during mechanical ventilation, nor have the adaptive responses of lung cells to deforming stresses been rigorously defined. In this study, we chose to elucidate alveolar epithelial cell injury and repair mechanisms in an in vitro setting. Although repeated deformation has obvious direct relevance to mechanical ventilation, we chose a single, held stretch because it was simple and addressed basic and broad unanswered questions about alveolar epithelial cell mechanics and deformation-related remodeling. The cytoskeleton is the principal stress-bearing element of the plasma membrane, a source of mechanosensing, an avenue for transport of proteins and lipid cargo, and actively remodels in response to deformation; however, the quasi-static mechanical properties of the cytoskeleton did not predict the susceptibility of cells to deformation injury. Rather, our observations are consistent with the hypothesis that a deformed cell unable to actively regulate its surrounding lipid bilayer cannot keep bilayer tension below lytic levels. This remodeling and cell plasticity, as opposed to the inherent static mechanical properties of the cytoskeleton, determine the fate of a cell that is forced to assume a certain shape. The concept is akin to the volutrauma idea. Dreyfuss and Saumon (4) and Hernandez and colleagues (35) showed that pressure applied to the airway is damaging to the lung only if this pressure causes a large lung volume excursion. Scaled down to the individual cell, this means that for a given external force, a "rigid" cytoskeleton simply prevents a large shape change from occurring; however, when the deformation of the surrounding matrix, for example, the basement membrane, imposes a large cell strain then having a rigid cytoskeleton simply means that a large deforming stress will be generated. Such a stress could become lytic to a "weak" structure, causing it to fail. Remodeling of stress bearing elements (the cytoskeleton) or of potentially stress bearing elements (the lipid bilayer of the plasma membrane) is the principal mechanism by which the cell can protect itself from such a catastrophic event. Because remodeling is a rate-dependent process, one might speculate that the dependence of wounding on strain rate reflects limits in the rate at which lipid vesicles can be transported to and fuse with the plasma membrane. It could also reflect limits in the rate at which the subcortical cytoskeleton can remodel. More work will need to be done to define the relevant rate constants. The observation that strain rate, not just strain amplitude, is an important determinant of cell injury might also have clinical relevance on the appropriate choice of flow and rate settings during mechanical ventilation. The most important message of our work, however, is that mechanisms of deformation-related cell remodeling such as DILT are appropriate and underappreciated treatment targets in the search for pharmacoprotective agents against ventilator-induced lung injury.
The authors thank Dr Gary C. Sieck for use and help with confocal microscopy and Dr. Vishwajeet Puri for help with cholesterol quantification.
Funded by a Glaxo-Wellcome Research Fellowship grant, a grant from the Brewer Foundation, and National Institutes of Health grants GM 22942 and HL-63178. This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form March 14, 2002; accepted in final form July 22, 2002
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
INTRODUCTION
