Inhaled Activated Protein C Protects Mice from Ventilator-Induced Lung Injury.

Abstract

Introduction: Activated Protein C (APC), an endogenous anticoagulant, improves tissue microperfusion and endothelial cell survival in systemic inflammatory states such as sepsis, but intravenous administration may cause severe bleeding. We have thus addressed the role of APC delivered locally by inhalation in preventing acute lung injury from alveolar overdistention and the subsequent ventilator-induced lung injury (VILI). We also assessed the effects of APC on the activation status of Extracellular-Regulated Kinase 1/2 (ERK) pathway, which has been shown to be involved in regulating pulmonary responses to mechanical stretch. Methods:Inhaled APC (12.5 μg drotrecogin-α × 4 doses) or saline was given to tracheotomized C57/Bl6 mice starting 20 min prior to initiation of injurious mechanical ventilation with tidal volume 25 mL/Kg for 4 hours and then hourly thereafter; control groups receiving inhaled saline were ventilated with 8 mL/Kg for 30 min or 4 hr. We measured lung function (respiratory system elastance H), arterial blood gases, surrogates of vascular leak (broncho-alveolar lavage (BAL) total protein and angiotensin-converting enzyme (ACE)-activity), and parameters of inflammation (BAL neutrophils and lung tissue myeloperoxidase (MPO) activity). Morphological alterations induced by mechanical ventilation were examined in hematoxylin-eosin lung tissue sections. The activation status of ERK was probed in lung tissue homogenates by immunoblotting and in paraffin sections by immunohistochemistry. The effect of APC on ERK signaling downstream of the thrombin receptor was tested on A549 human lung epithelial cells by immunoblotting. Statistical analyses were performed using ANOVA with appropriate post-hoc testing. Results:In mice subjected to VILI without APC, we observed hypoxemia, increased respiratory system elastance and inflammation, assessed by BAL neutrophil counts and tissue MPO activity. BAL total protein levels and ACE activity were also elevated by VILI, indicating compromise of the alveolo-capillary barrier. In addition to preserving lung function, inhaled APC prevented endothelial barrier disruption and attenuated hypoxemia and the inflammatory response. Mechanistically, we found a strong activation of ERK in lung tissues by VILI, which was prevented by APC, suggestive of pathogenetic involvement of the Mitogen-Activated Kinase pathway. In cultured human lung epithelial cells challenged by thrombin, APC abrogated the activation of ERK and its downstream effector, cytosolic Phospholipase A 2 .Conclusions: Topical application of APC by inhalation may effectively reduce lung injury induced by mechanical ventilation in mice.

Plasma‐derived human antithrombin attenuates ventilator‐induced coagulopathy but not inflammation in a Streptococcus pneumoniae pneumonia model in rats

“…We found no effect of AT on ventilator-induced coagulopathy and inflammation in non-infected rats. This might be surprising, as APC have been shown to be protective in healthy lungs subjected to ventilation [45,49]. In the present study, coagulation was only mildly activated by the ventilator, mechanical ventilation in combination with pneumonia, however, strongly increased TATc production.…”

“…There is an ongoing discussion on the exact mechanism by which APC prevents morbidity and mortality. In humans challenged with LPS, APC limits pulmonary coagulopathy [43] and in experimental acute lung injury, APC limits coagulation, neutrophil influx [44] and the prevention of endothelial barrier disruption, thereby improving gas exchange and reducing cytokine production [45]. However, in a recent randomized clinical trial, APC treatment did not reduce ventilator‐free days or 60‐day mortality in patients without severe sepsis possibly as a result of a lack of statistical power [46].…”

Overexpression of Activated Protein C is Detrimental During Severe Experimental Gram-Negative Sepsis (Melioidosis)*

“…Before their use in the experiments, all mice were checked for transgene expression by PCR. Although one could argue that it would have been better to use mice overexpressing murine APC instead of human APC, it is less likely that such mice would yield significantly different results since both rhAPC (28,29,31) and recombinant murine APC (rmAPC [32,33]) have shown (protective) effects in murine models of LPS-induced endotoxemia (31), LPS-induced lung injury (32), ventilator-induced lung injury (33), and pneumococcal pneumonia (28,29). Of note, although APC high mice respond to thrombin with increased APC production, our results cannot be extrapolated to mice with enhanced levels of the zymogen protein C. Our results are in contrast with previous data in murine models of pneumosepsis and endotoxin-induced lung inflammation.…”

Bronchoalveolar hemostasis in lung injury and acute respiratory distress syndrome

“…In addition, inhaled activated protein C significantly diminishes pulmonary inflammation in a murine model of intranasal LPS‐induced ARDS [75,76]. In line with these findings, nebulized activated protein C effectively reduced histological alterations and inflammation in a murine model of ventilator‐induced lung injury [77]. In endotoxin‐challenged sheep, activated protein C inhalation improved gas exchange and prevented a decline in effective lung volume [78].…”