Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells.

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Mechanical stimuli to airway remodeling.

The airway is exposed to a variety of mechanical stimuli, the most prominent of which is the acute compressive stress caused by bronchoconstriction. The folding of the airway wall into a rosette pattern during bronchoconstriction creates a complex stress field, with the highest stresses compressing the epithelial layer at the inner surface of the airway wall. The epithelial cells lining the airway possess the capacity to modulate the inflammatory environment of the airway wall, and produce factors that influence the recruitment, proliferation, and activity of fibroblasts and smooth muscle cells. A variety of in vitro studies have demonstrated that airway epithelial cells, along with lung fibroblasts and smooth muscle cells, are responsive to mechanical stimuli. Airway epithelial cells exposed to compressive stresses matched to those occurring in the constricted airway increase expression of genes relevant to airway remodeling, and increase the collagen synthesis of cocultured fibroblasts. These findings demonstrate that mechanical stress may contribute to the remodeling of the asthmatic airway.

Mechanical stress is communicated between different cell types to elicit matrix remodeling.

Tissue remodeling often reflects alterations in local mechanical conditions and manifests as an integrated response among the different cell types that share, and thus cooperatively manage, an extracellular matrix. Here we examine how two different cell types, one that undergoes the stress and the other that primarily remodels the matrix, might communicate a mechanical stress by using airway cells as a representative in vitro system. Normal stress is imposed on bronchial epithelial cells in the presence of unstimulated lung fibroblasts. We show that (i) mechanical stress can be communicated from stressed to unstressed cells to elicit a remodeling response, and (ii) the integrated response of two cell types to mechanical stress mimics key features of airway remodeling seen in asthma: namely, an increase in production of fibronectin, collagen types III and V, and matrix metalloproteinase type 9 (MMP-9) (relative to tissue inhibitor of metalloproteinase-1, TIMP-1). These observations provide a paradigm to use in understanding the management of mechanical forces on the tissue level.

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being approximately 0. 35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Deformation-induced injury of alveolar epithelial cells. Effect of frequency, duration, and amplitude.

The onset of ventilator-induced lung injury (VILI) is linked to a number of possible mechanisms. To isolate the possible role of alveolar epithelial deformation in the development of VILI, we have developed an in vitro system in which changes in alveolar epithelial cell viability can be measured after exposure to tightly controlled and physiologically relevant deformations. We report here a study of the relative effect of deformation frequency, duration, and amplitude on cell viability. We exposed rat primary alveolar epithelial cells to a variety of biaxial stretch protocols, and assessed deformation-induced cell injury quantitatively, using a fluorescent cell viability assay. Deformation-induced injury was found to depend on repetitive stretching, with cyclic deformations significantly more damaging than tonically held deformations. In cyclically deformed cells, injury occurred rapidly, with the majority of cell death occurring during the first 5 min of deformation. Deformation-induced injury was increased with the frequency of sustained cyclic deformations, but was not dependent on the deformation rate during a single stretch. Reducing the amplitude of cell deformations by superimposing small cyclic deformations on a tonic deformation significantly reduced cell death as compared with large-amplitude deformations with the same peak deformation.

Alveolar epithelial surface area-volume relationship in isolated rat lungs.

In vitro studies of the alveolar epithelial response to deformation require knowledge of the in situ mechanical environment of these cells. Because of the presence of tissue folding and crumpling, previous measurements of the alveolar surface area available for gas exchange are not equivalent to the epithelial surface area. To identify epithelial deformations in uniformly inflated lungs representative of the in vivo condition, we studied isolated Sprague-Dawley rat lungs (n = 31) fixed by perfusion with glutaraldehyde on deflation after cycling three times at high lung volume (10-25 cmH2O). The epithelial basement membrane in 45 electron micrographs (x12,000)/rat was traced, digitally scanned, and analyzed. Epithelial basement membrane surface area (EBMSA) was computed from a morphometric relationship. EBMSA was found to increase 5, 16, 12, and 40% relative to EBMSA at 24% total lung capacity at lung volumes of 42, 60, 82, and 100% total lung capacity, respectively. The increases in EBMSA suggest that epithelial cells undergo significant deformations with large inflations and that alveolar basement membrane deformation may contribute to lung recoil at high lung pressures.

Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro.

Deformation of the alveolar epithelial basement membrane with lung inflation has been implicated in blood-gas barrier breakdown during the development of ventilator-induced lung injury. To determine the vulnerability of alveolar epithelial cells to deformation-induced injury, we developed a cell-stretching device that subjects cells to cyclic, equibiaxial strains. Alveolar epithelial type II cells from primary culture were tested 1 and 5 days after seeding, during which time the cells underwent major morphological and phenotypic changes. Cells were subjected to changes in surface area of 12, 24, 37, and 50%, which corresponded to lung inflation of approximately 60, 80, 100, and >100% of total lung capacity. Deformation-induced injury of alveolar epithelial cells, assessed with a fluorescent cell viability assay, increased with deformation magnitude and decreased with time elapsed after seeding. In cells stretched after 1 day in culture, the percentage of dead cells after a single deformation ranged from 0.5 to 72% over the range of deformations used. In cells stretched at 5 days, the percentage of dead cells ranged from 0 to 9% when exposed to identical deformation protocols. These results suggest that morphological and phenotypic changes with time in culture fundamentally change the vulnerability of alveolar epithelial cells to deformation.