Oxygen Administration Improves Survival but Worsens Cardiopulmonary Functions in Chlorine-exposed Rats.

Chlorine is a highly reactive gas that can cause significant injury when inhaled. Unfortunately, its use as a chemical weapon has increased in recent years. Massive chlorine inhalation can cause death within 4 hours of exposure. Survivors usually require hospitalization after massive exposure. No countermeasures are available for massive chlorine exposure and supportive-care measures lack controlled trials. In this work, adult rats were exposed to chlorine gas (LD58-67) in a whole-body exposure chamber, and given oxygen (0.8 FiO2) or air (0.21 FiO2) for 6 hours after baseline measurements were obtained. Oxygen saturation, vital signs, respiratory distress and neuromuscular scores, arterial blood gases, and hemodynamic measurements were obtained hourly. Massive chlorine inhalation caused severe acute respiratory failure, hypoxemia, decreased cardiac output, neuromuscular abnormalities (ataxia and hypotonia), and seizures resulting in early death. Oxygen improved survival to 6 hours (87% versus 42%) and prevented observed seizure-related deaths. However, oxygen administration worsened the severity of acute respiratory failure in chlorine-exposed rats compared with controls, with increased respiratory acidosis (pH 6.91 ± 0.04 versus 7.06 ± 0.01 at 2 h) and increased hypercapnia (180.0 ± 19.8 versus 103.2 ± 3.9 mm Hg at 2 h). In addition, oxygen did not improve neuromuscular abnormalities, cardiac output, or respiratory distress associated with chlorine exposure. Massive chlorine inhalation causes severe acute respiratory failure and multiorgan damage. Oxygen administration can improve short-term survival but appears to worsen respiratory failure, with no improvement in cardiac output or neuromuscular dysfunction. Oxygen should be used with caution after massive chlorine inhalation, and the need for early assisted ventilation should be assessed in victims.

Bronchiolitis Obliterans and Pulmonary Fibrosis after Sulfur Mustard Inhalation in Rats.

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-ß, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-ß, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-ß, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Integrin α3ß1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat.

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbß3 and α5ß1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3ß1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3ß1 to the Fn fragments. Integrin α3ß1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3ß1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3ß1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3ß1 binding to Fn.

Synergistic effects of particulate matter and substrate stiffness on epithelial-to-mesenchymal transition.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-ß) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-ß. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is dose- and time-dependent. In response to low levels of TGF-ß on soft surfaces, either added exogenously or produced through contraction induced by the stiffness agonist thrombin, cells initiate EMT; on removal of the TGF-ß, they revert to an epithelial phenotype. Overall, the results from this first part of our study identified matrix stiffness or cell contractility as critical targets for the control of EMT in fibrotic diseases. For the second part of our study, we wanted to investigate whether exposure to PM2.5, which might have higher toxicity than coarser PM because of its small size and large surface-to-mass ratio, altered the observed stiffness-mediated EMT. Again, we cultured ATII cells on increasingly stiff substrates with or without the addition of three concentrations of PM2.5. We found that exposure to PM2.5 was involved in increased stiffness-mediated EMT, as shown by increases in mesenchymal markers, cell contractility, and TGF-ß activation. Most notably, on substrates with an elastic modulus (E) of 8 kilopascals (kPa), a physiologically relevant range for pulmonary fibrosis, the addition of PM2.5 resulted in increased mesenchymal cells and EMT; these were not seen in the absence of the PM2.5. Overall, this study showed that there is a delicate balance between substrate stiffness, TGF-ß, and EMT. Furthermore, we showed that exposure to PM2.5 is able to further mediate this interaction. The higher levels of EMT seen with exposure to PM2.5 might have been a result of a positive feedback loop, in which enhanced exposure to PM2.5 through the loss of cell-cell junctions during the initial stages of EMT led to the cells being more susceptible to the effects of surrounding immune cells and inflammatory signals that can further activate TGF-ß and drive additional EMT progression. Overall, our work--showing increased cell contractility, TGF-ß activation, and EMT in response to substrate stiffness and PM2.5 exposure--highlights the importance of both the micromechanical and biochemical environments in lung disease. These findings suggest that already-fibrotic tissue might be more susceptible to further damage than healthy tissue when exposed to PM2.5.

Environmental particulate (PM2.5) augments stiffness-induced alveolar epithelial cell mechanoactivation of transforming growth factor beta.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor--beta (TGFß) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFß. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFß. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFß activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFß, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFß that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFß signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.