Critical Role of the Small GTPase RhoA in the Development of Pulmonary Edema Induced by Pseudomonas aeruginosa  in Mice

Abstract

Background:Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia in critically ill patients. We have reported previously that P. aeruginosa exotoxins S and T mediate in vitro the increase in protein permeability across lung endothelial cell monolayers via a RhoA-dependent mechanism. However, whether inhibition of RhoA would significantly attenuate P. aeruginosa-mediated lung injury in mice is unknown. Methods: P. aeruginosa-induced paracellular protein permeability was measured across bovine lung endothelial and rat alveolar epithelial type II cell monolayers with 125 I-albumin. Some cell monolayers were pretreated with RhoA inhibitor CGX0287 1 h before P. aeruginosa exposure. At 4 h after exposure, lung endothelial and epithelial permeability, bacterial counts, bronchoalveolar lavage fluid levels of keratinocyte-derived chemokine, myeloperoxidase activity, and alveolar fluid clearance were measured. Some mice were treated intraperitoneally with CGX0287 1 h before or after airspace instillation of P. aeruginosa. Results: RhoA inhibition attenuated in vitro P. aeruginosamediated increase in lung endothelial and epithelial permeability to protein and in vivo the development of pulmonary edema and inhibition of alveolar fluid clearance associated with P. aeruginosa pneumonia. Furthermore, RhoA inhibition decreased the systemic dissemination of P. aeruginosa and neutrophil activity in the lung tissue observed after airspace instillation of these bacteria. Conclusions: The small GTPase RhoA plays a critical role in mediating lung injury associated with P. aeruginosa pneumonia in mice. Thus, transient blockade of RhoA could attenuate lung damage caused by P. aeruginosa in critically ill patients.

Cytoprotective-Selective Activated Protein C Attenuates Pseudomonas aeruginosa–Induced Lung Injury in Mice

“…Our laboratory demonstrated that ExoS and ExoT, two cytotoxins from the Type III secretion system, are responsible for the P. aeruginosa-mediated increase in protein permeability across lung endothelial cell monolayers via the inhibition of Rac1, combined with the activation of RhoA (13). In a subsequent in vivo study, we found that the inhibition of RhoA attenuated the increase in lung endothelial and alveolar epithelial permeability to protein and the development of pulmonary edema, and further, that this restored positive alveolar fluid clearance in a murine model of P. aeruginosa pneumonia (14). Thus, we hypothesized that the mediators decreasing the activity of RhoA in the lung cells constituting the alveolar-capillary barrier may attenuate the lung damage caused by P. aeruginosa.…”

“…Accordingly, ExoS and ExoT, two cytotoxins from the Type III secretion system, are responsible for the P. aeruginosa-mediated increase in protein permeability across lung endothelial cell monolayers via an inhibition of Rac1 and an activation of RhoA (13). In an in vivo study, we found that the inhibition of RhoA attenuates the increase in lung endothelial and alveolar epithelial permeability to protein, the development of pulmonary edema, and the inhibition of alveolar fluid clearance in a murine model of P. aeruginosa pneumonia (14). aPC favorably elevates the ratio of Rac1/RhoA activity in the lung endothelium, apparently by activating the S1P pathway through EPCR/PAR-1-dependent mechanisms, and indirectly by preventing the formation of thrombin and its detrimental effects on the barrier properties of the lung endothelium and alveolar epithelium (4).…”

“…A Kaplan-Meier analysis was used to compare survival between the two experimental groups of mice. We previously reported that the increase in transendothelial albumin flux induced by P. aeruginosa was blocked by the inhibition of the small GTPase RhoA (14) or of RhoA activated kinase (ROCK), the immediate downstream effector of RhoA (13). Other investigators showed that aPC inhibits the activation and activity of RhoA in lung endothelial cells, and increases the activity of Rac1 (6).…”

“…We previously reported that the inhibition of the small GTPase RhoA attenuated the development of pulmonary edema, and restored positive alveolar fluid clearance in a murine model of P. aeruginosa pneumonia (14). aPC can restore a physiologically favorable ratio of elevated Rac1/RhoA activity in the lung endothelium exposed to inflammatory mediators by activating the S1P pathway through an EPCR/PAR-1-dependent mechanism (6,7).…”

“…Mice were anesthetized and instilled with P. aeruginosa (50 ml of phosphate-buffered saline containing 1 3 10 7 colony-forming units of P. aeruginosa), as reported previously (14).…”