Simvastatin attenuates ventilator-induced lung injury in mice

In acute respiratory failure, mechanical ventilation (MV) is a life saving treatment without alternatives, and MV is also employed following surgery or trauma. One third of all patients in intensive care units worldwide receive MV [ 1]. However, particularly in preinjured lungs even minimal MV-associated physical stress may evoke ventilator-induced lung injury (VILI), an important undesirable effect of respirator therapy [ 2, 3]. VILI is characterized by a pulmonary inflammatory response with the liberation of cytokines, recruitment of leukocytes to the lung and increased lung permeability, consecutively resulting in lung edema, surfactant dysfunction, impaired lung compliance and deterioration of pulmonary gas exchange [ 4]. Clinical studies of Amato et al. and the ARDS Network revealed that minimization of MV-induced physical stress by reduction of tidal volumes to 6 ml/kg significantly improved the clinical outcome of mechanically ventilated patients [ 5, 6]. However, even low tidal volume ventilation of healthy lungs causes lung injury [ 7], and particularly preinjured lungs are sensitive to the development of VILI even in the setting of lung-protective ventilation [ 2, 3]. As the necessity to guarantee sufficient gas exchange limits a further substantial reduction of tidal volumes, new adjuvant pharmacological therapies in addition to lung-protective ventilation are needed to prevent VILI.

Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor belonging to the group of statins may be a promising drug candidate for adjuvant pharmacotherapy in MV. Besides well-known lipid lowering properties, simvastatin exhibits pleiotropic effects that attenuated acute lung injury (ALI), including reduction of pulmonary microvascular leakage, limitation of pulmonary leukocyte infiltration, and attenuation of pulmonary and systemic hyperinflammation in different experimental settings [ 8– 11]. Moreover, statins may alter inflammatory responses in humans. Healthy volunteers subjected to lipopolysacharide (LPS) inhalation developed lung inflammation, which was attenuated by simvastatin treatment [ 12]. Further, statin treatment was associated with improved survival in sepsis and severe community acquired pneumonia [ 13– 16].

Pulmonary and systemic hyperinflammation, leukocyte recruitment to the lungs, and the development of pulmonary microvascular leakage are crucial components of VILI [ 4, 17]. We thus hypothesized that simvastatin may reduce VILI and may be a promising adjuvant pharmacologic strategy to limit VILI in addition to lung protective ventilation.

In the current study, anesthetized mice were subjected to mechanical ventilation for six hours. Simvastatin treatment markedly attenuated ventilator-induced pulmonary microvascular permeability and endothelial injury, recruitment of neutrophils and Gr-1 ^high monocytes, as well as proinflammatory cytokine levels in the lung, and improved oxygenation considerably.

Mouse models have been successfully used to investigate pathomechanisms of VILI [ 18– 20]. The currently employed mouse model allowed us to analyze key features of VILI while avoiding detrimental lung injury due to high airway pressures, tidal volumes or respiration rates. Although a V _T of 6 ml/kg is recommended for lung protective ventilation, we employed a V _T of 12 ml/kg which allowed for limitation of respiratory rates in our model, an important independent trigger of VILI in mice [ 21]. Further lung stress and lung strain, generated by a V _T of 12 ml/kg affecting healthy lungs in the current model may apply in ventilated areas of inhomogeneously injured lungs even under lung protective ventilation according to the baby lung concept of the inhomogeneous ARDS lung [ 22, 23]. To further enhance clinical relevance, we prevented hemodynamic instability by fluid support and metabolic acidosis by adequate infusion of sodium bicarbonate. In summary, a mouse model was established for the current study, which evoked moderate lung injury by ventilation for a six-hour period.

Microvascular leakage, a hallmark of VILI evokes lung edema, reduction of lung compliance, surfactant dysfunction, and finally deterioration of pulmonary gas exchange [ 4]. Statins prevented pulmonary hyperpermeability in ALI evoked by different stimuli, including endotoxin and ischemia/reperfusion [ 8– 10]. Of note, simvastatin treatment also reduced VILI-associated pulmonary hyperpermeability and improved pulmonary gas exchange in the current study.

Different mechanisms of endothelial barrier protection by HMG-CoA reductase inhibitors have been reported, including inhibition of the RhoA/Rho kinase pathway with consecutive reduction of endothelial myosin light chain phosphorylation [ 24– 26], stabilization of endothelial junctions by polymerization of cortical actin [ 25], as well as downregulation of endothelial caldesmon and upregulation of integrin β4 expression in endothelial cells [ 25]. Although these mechanisms were not evaluated in detail in the current study, they may have been contributing to the observed improvement of barrier function in murine VILI. Notably, an additional way of endothelial cell protection by simvastatin has now been observed by electron microscopy. Simvastatin attenuated VILI-evoked cell swelling and loss of intracellular vesicle structures in lung endothelium, which are indicators of energy depletion and impaired cell metabolism. Previous in vitro and in vivo studies linked cyclic stretch with apoptosis and necrosis of pulmonary epithelial cells [ 27, 28]. In line with the works of Vaneker et al. this study provides ultrastructural in vivo evidence for lung endothelial cell injury following ventilation with moderate tidal volumes [ 29]. The observed morphologic findings resemble alterations observed in capillary stress failure previously described by West et al. To the best of our knowledge this is the first study showing that a pharmacologic treatment attenuated endothelial injury VILI. This previously undescribed effect of simvastatin treatment suggests a so far unknown beneficial effect of HMG-CoA reductase inhibitors, which may be further examined in future studies.

In VILI, PMN and Gr-1 ^high monocytes infiltrate the lungs and have been identified as major effector cells for the development of tissue damage [ 30– 32]. Reportedly, simvastatin inhibited tissue leukocyte infiltration in ALI both in animal experiments and in humans [ 8, 9, 12]. Leukocyte rolling, adhesion and transmigration were attenuated by simvastatin, at least partly by reduction of adhesion molecules including CEACAM-1, VCAM-1 and PCAM-1 [ 33– 36]. In line, the significant recruitment of PMN and Gr-1 ^high monocytes in murine VILI was diminished by simvastatin in the current study. Moreover, an MV-induced increase of circulating PMN and Gr-1 ^high monocytes in the blood was even more pronounced in simvastatin-treated mice. This observation may suggest that simvastatin-evoked inhibition of endothelial leukocyte recruitment contributed to reduced pulmonary and concomitantly increased blood counts of PMN and Gr-1 ^high monocytes.

Simvastatin reduced production and liberation of various cytokines in animal models of ALI, sepsis and asthma as well as in humans following LPS-inhalation [ 9, 11, 12, 37– 40]. In the current study, VILI-associated pulmonary production of IL-1β, MIP-1α and IL-12p40 was reduced by simvastatin treatment. Thus, alteration of chemotaxis may have been contributing to the limitation of PMN and Gr-1 ^high monocyte influx into the lungs in this study. Particularly IL-1β may be a key mediator in VILI, as IL-1β blockade as well as IL-1β deficiency resulted in reduced pulmonary PMN recruitment and hyperpermeability in animal models of VILI [ 41]. Therefore, dampening of pulmonary IL-1β production by simvastatin may have been adding to the observed attenuation of microvascular leakage, pulmonary leukocyte recruitment and endothelial cell injury.

Although increasing evidence derived from experimental and observational studies suggests beneficial effects of simvastatin in ALI as well as in pneumonia [ 8– 11, 14, 16, 42], a retrospective study analyzing an ALI patient cohort did not find an outcome improvement by conventional statin treatment [ 43]. Of note, statin doses of 5 mg/kg/d did not improve experimental ALI [ 8], whereas higher doses of 10 to 20 mg/kg/d evoked protective effects. Further, previous studies suggested a delay of at least 6 h for the development of barrier-protective effects by simvastatin [ 24, 25]. Thus, mice were pretreated with 20 mg/kg/d simvastatin commencing 24 h before the onset of ventilation in the current study. Although mandatory for this experimental approach, simvastatin pre-treatment does not match the clinical scenario. However, animal studies are limited to hours while ARDS patients often are ventilated for days or even weeks. Taking this long time course in account we believe that simvastatin may deliver its beneficial effects over time when it is given with the initation of MV. Notably, an upcoming randomized controlled NHLBI sponsored trial is going to investigate statin therapy in ALI (NCT00979121). As patients included in this trial will presumably receive respirator therapy, the effects of statins on VILI observed in the current experimental study may possibly contribute to the outcome of the treatment arm.

This study shows, for the first time, that high-dose simvastatin markedly reduced VILI-associated microvascular leakage and improved pulmonary gas exchange in mechanically ventilated mice. Simvastatin prevented recruitment of PMN and Gr-1 ^high monocytes to the lung, limited pulmonary cytokine production and attenuated endothelial injury in VILI. The data suggest that high-dose simvastatin offers a promising perspective to prevent VILI in addition to lung protective ventilation.