Self-induced mechanical stress can trigger biofilm formation in uropathogenic Escherichia coli.

Bacterial biofilms represent an important medical problem; however, the mechanisms of the onset of biofilm formation are poorly understood. Here, using new controlled methods allowing high-throughput and reproducible biofilm growth, we show that biofilm formation is linked to self-imposed mechanical stress. In growing uropathogenic Escherichia coli colonies, we report that mechanical stress can initially emerge from the physical stress accompanying colony confinement within micro-cavities or hydrogel environments reminiscent of the cytosol of host cells. Biofilm formation can then be enhanced by a nutrient access-modulated feedback loop, in which biofilm matrix deposition can be particularly high in areas of increased mechanical and biological stress, with the deposited matrix further enhancing the stress levels. This feedback regulation can lead to adaptive and diverse biofilm formation guided by the environmental stresses. Our results suggest previously unappreciated mechanisms of the onset and progression of biofilm growth.

Induction of the plasminogen activator system by mechanical stimulation of human bronchial epithelial cells.

Mechanical stimulation of the airway epithelium, as would occur during bronchoconstriction, is a potent stimulus and can activate profibrotic pathways. We used DNA microarray technology to examine gene expression in compressed normal human bronchial epithelial cells (NHBE). Compressive stress applied continuously over an 8-h period to NHBE cells led to the upregulation of several families of genes, including a family of plasminogen-related genes that were previously not known to be regulated in this system. Real-time PCR demonstrated a peak increase in gene expression of 8.0-fold for urokinase plasminogen activator (uPA), 16.2-fold for urokinase plasminogen activator receptor (uPAR), 4.2-fold for plasminogen activator inhibitor-1 (PAI-1), and 3.9-fold for tissue plasminogen activator (tPA). Compressive stress also increased uPA protein levels in the cell lysates (112.0 versus 82.0 ng/ml, P = 0.0004), and increased uPA (4.7 versus 3.3 ng/ml, P = 0.02), uPAR (1.3 versus 0.86 ng/ml, P = 0.007), and PAI-1 (50 versus 36 ng/ml, P = 0.006) protein levels in cell culture media. Functional studies demonstrated increased urokinase-dependent plasmin generation in compression-stimulated cells (0.0090 versus 0.0033 OD/min, P = 0.03). In addition, compression led to increased activation of matrix metalloproteinase (MMP)-9 and MMP-2 in a urokinase-dependent manner. In postmortem human lung tissue, we observed an increase in epithelial uPA and uPAR immunostaining in the airways of two patients who died in status asthmaticus compared with minimal immunoreactivity noted in airways from seven lung donors without asthma. Together these observations suggest an integrated response of airway epithelial cells to mechanical stimulation, acting through the plasminogen-activating system to modify the airway microenvironment.

Asthma: one hundred years of treatment and onward.

There have been four types of drug treatment of asthma that have been used over the past 100 years. Belladonna alkaloids, derived from the thorn-apple plant were used in 1905, and chemically synthesized entities in this class are still in use today. Western medicine began to use adrenergic stimulants approximately 100 years ago, but they were likely used in Asian medicine long before that. Systemic treatment with corticosteroids was introduced into the treatment of asthma in the mid-20th century; inhaled corticosteroids have been in use for over 35 years. The last 40 years have also seen the development of the first targeted asthma treatments: cromones, antileukotrienes, and anti-IgE. As we learn more of the biology of asthma, we anticipate that more effective targeted asthma treatments will be developed.

Bronchial epithelial compression regulates epidermal growth factor receptor family ligand expression in an autocrine manner.

The epidermal growth factor receptor (EGFR), an important signaling pathway in airway biology, is stimulated by compressive stress applied to human airway epithelial cells. Although the EGFR ligand, heparin-binding epidermal growth factor-like growth factor (HB-EGF), is known to be released as a result of this stimulation, whether compressive stress enhances expression of other EGFR ligands, and the duration of mechanical compression required to initiate this response, is not known. Human airway epithelial cells were exposed to compressive stress, and expression of four EGFR ligands was examined by quantitative PCR. Cells were exposed to: (1) continuous compressive stress over 8 h, (2) compression with and without EGFR inhibitor (AG1478), or (3) time-limited compression (3.75, 7.5, 15, 30, and 60 min). Compressive stress produced a sustained upregulation of the EGFR ligands HB-EGF, epiregulin, and amphiregulin, but not transforming growth factor-alpha. Inhibition with AG1478 demonstrated that expression of HB-EGF, epiregulin, and amphiregulin is dependent on the signaling via the EGFR. Immunostaining for epiregulin protein demonstrated increased expression with compression and attenuation with EGFR inhibition. The response of all three EGFR ligands persisted long after the mechanical stimulus was removed. Taken together, these data suggest the possibility of a mechanically activated EGFR autocrine feedback loop involving selected EGFR ligands.

Effects of cyclic opening and closing at low- and high-volume ventilation on bronchoalveolar lavage cytokines.

OBJECTIVE: To examine the mechanisms of ventilator-induced lung injury at low and high lung volumes. DESIGN: Prospective, randomized, laboratory study. SETTING: University research laboratory. SUBJECTS: Eighty-eight adult male Sprague-Dawley rats. INTERVENTIONS: Mechanical ventilation using low and high lung volumes. MEASUREMENTS AND MAIN RESULTS: An ex vivo rat lung model was used. In study I (ventilation at low lung volumes), rat lungs (n = 40) were randomly assigned to various modes of ventilation: a) opening and closing with positive end-expiratory pressure (PEEP; control): tidal volume 7 mL/kg and PEEP 5 cm H2O; b) opening and closing from zero end-expiratory pressure (ZEEP): tidal volume 7 mL/kg and PEEP 0; or c) atelectasis. Peak inspiratory pressure was monitored at the beginning and end of 3 hrs of ventilation. At the end of 3 hrs of ventilation, the lungs were lavaged, and the concentrations of tumor necrosis factor-alpha, macrophage inflammatory protein-2, and interleukin-6 cytokines were measured in the lavage. In study II (ventilation at high volumes), rat lungs (n = 45) were randomly assigned to a) cyclic lung stretch: pressure-controlled ventilation, peak inspiratory pressure 50 cm H2O, and PEEP 8 cm H2O; b) continuous positive airway pressure at 50 cm H2O (CPAP50); or c) CPAP at the mean airway pressure of the cyclic stretch group (CPAP 31 cm H2O). Bronchoalveolar lavage cytokine concentrations (tumor necrosis factor-alpha, macrophage inflammatory protein-2, and interleukin-6) were measured at the end of 3 hrs of ventilation. In the low volume study, there was no difference in bronchoalveolar lavage cytokine concentrations between the PEEP group and the atelectatic group. All cytokines were significantly higher in the ZEEP group compared with the atelectasis group. Macrophage inflammatory protein-2 was significantly higher in the ZEEP group compared with the PEEP group. Lung compliance, as reflected by change in peak inspiratory pressure, was also significantly worse in the ZEEP compared with the PEEP group. In the high-volume study, tumor necrosis factor-alpha and interleukin-6 were significantly higher in the cyclic stretch group compared with the CPAP 31 group. There was no significant difference between the cytokine concentrations in the cyclic stretch group compared with the CPAP 50 group. CONCLUSION: We conclude that at low lung volumes, cyclic opening and closing from ZEEP leads to greater increases in bronchoalveolar lavage cytokines than atelectasis. With high-volume ventilation, over time, the degree of overdistension is more associated with increases in bronchoalveolar lavage cytokines than cyclic opening and closing alone.