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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 ExtracellularRegulated 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 A2. Conclusions: Topical application of APC by inhalation may effectively reduce lung injury induced by mechanical ventilation in mice. Introduction Acute lung injury (ALI), a form of pulmonary edema due to increased microvascular permeability, is a major cause of respiratory failure, morbidity and mortality in the ICU. Commonly seen as a complication of sepsis, ALI can be exacerbated by the use of mechanical ventilation, which is the main life-support modality for these patients [1,2]. Overdistention of diseased alveoli by positive pressure promotes inflammation and further disrupts the alveolocapillary membrane [3-5]. This occurs by mechanisms including capillary stress failure, plasma membrane microrupture and lung cell death [6-9]. In addition, * Correspondence: sorfanos@med.uoa. gr , akotanid@med.uoa. gr 1 2nd Dept. of Critical Care, "Attikon" Hospital, National and Kapodistrian University of Athens Medical School, Rimini 1, Haidari, 12462, Greece 5 1st Dept of Critical Care, "Evangelismos" Hospital, National and Kapodistrian University of Athens Medical School, Ipsilandou 45-47, 10675 Athens, Greece † Contributed equally Full list of author information is available at the end of the article BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maniatis et al. Critical Care 2010, 14:R70 http://ccforum.com/content/14/2/R70 Page 2 of 14 stretch can elicit biochemical signaling events through activation of mechanical sensors present in lung cells, including stretch-activated ion channels, the cytoskeleton, and integrins [5]. The mitogen-activated protein kinase (MAPK) enzyme family transduces many of these signals and mediates cellular responses to stretch. The importance of these enzymes is underscored by the beneficial effects observed by MAPK antagonism in various models of ventilator-induced lung injury (VILI) [10-12]. Limiting tidal volume (Vt) can reduce mortality by preventing ventilator injury but this complication may still occur with substantial frequency [4,13-16]. Therefore, additional therapeutic options are clearly needed and intervening in the underlying biochemical pathways with drugs could be a rational approach. A recent development in the management of sepsis has been the launch of activated protein C (APC), an endogenous anticoagulant with additional cytoprotective, immunomodulatory and endothelial barrier-enhancing properties, which constitute important defence mechanisms in sepsis [17-21]. These attributes make APC an attractive consideration for clinical conditions associated with increased microvascular permeability in general, including ALI, irrespective of the presence of sepsis. In fact, as recently reported, intravenous APC could be useful at reversing experimental ALI caused by mechanical ventilation [22]. In clinical studies, low plasma levels of protein C have been linked to increased risk of death in patients with ALI [23] and APC supplementation improved tissue perfusion in septic patients [24]. Moreover, infusion of recombinant human APC has been shown to reduce mortality of patients with severe sepsis and at high risk of death [25-27] presumably by reversing the sepsis-associated proinflammatory and procoagulant state and thus preserving organ function. However, no benefit could be demonstrated in pediatric patients or in adults with less severe sepsis [28] or non-septic ALI [29]. These results, coupled with the drug's potential to cause bleeding and its substantial cost, have recently dampened the enthusiasm of the critical care community toward this agent and more studies have been ordered by international authorities. In this context, exploring alternate dosing schemes and routes of administration may be helpful in elucidating how to optimally use the agent. The issue of bleeding could be addressed in the lung by local delivery, which would allow build-up of high concentrations of APC in the alveolar space, thus maximizing local cyto-protection, while minimizing the risk of systemic anticoagulation-related side effects. We and others have previously demonstrated the feasibility and efficacy of this approach in various experimental systems [30-32]. In this work we tested the utility of inhaled APC in a mouse model of ALI induced by high tidal volume (HVt) positive-pressure mechanical ventilation. As VILI arises from excessive stretch primarily of pulmonary epithelial and endothelial cells, we hypothesized that airspace delivery of APC would protect mouse lungs against this type of insult by targeting the affected cell populations directly. In this respect we measured lung function, indices of microvascular permeability and inflammation in order to quantify the extent of lung injury and the effect of APC. We then specifically explored the activation status of the MAPK pathway in order to gain mechanistic insight into the APC action in our model. We found that inhaled APC was associated with improved lung function and oxygenation, in addition to a reduction in lung inflammation and vascular leak. Extracellular-regulated kinase 1/2 (ERK) activation was attenuated by APC in lung homogenates, in addition to A549 human lung epithelial cells challenged with thrombin. Materials and methods Mice Ten to fourteen week-old C57/Black6 male mice, with a mean ± standard deviation weight at the time of experiments of 22.7 ± 1.5 g, were purchased from the Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece. Mice were housed at 20 to 22°C, 55 ± 5% humidity, and a 12 hour light-dark cycle; food and water were given ad libitum. All experimentation was approved by an internal Institutional Review Board, as well as by the veterinary service of the local governmental prefecture. Reagents All reagents were purchased from Sigma (Ilioupoli, Greece) or as specified.