Lack of matrix metalloproteinase 3 in mouse models of lung injury ameliorates the pulmonary inflammatory response in female but not in male mice.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a complex pulmonary disorder in which the local release of cytokines and chemokines appears central to the pathophysiology. OBJECTIVE: Based on the known role of matrix metalloproteinase-3 (MMP3) in inflammatory processes, the objective was to examine the role of MMP3 in the pathogenesis of ARDS through the modulation of pulmonary inflammation. MATERIALS AND METHODS: Female and male, wild type (MMP3+/+) and knock out (MMP3-/-) mice were exposed to two, clinically relevant models of ARDS including (i) lipopolysaccharide (LPS)-induced lung injury, and (ii) hydrochloric acid-induced lung injury. Parameters of lung injury and inflammation were assessed through measurements in lung lavage including total protein content, inflammatory cell influx, and concentrations of mediators such as TNF-α, IL-6, G-CSF, CXCL1, CXCL2, and CCL2. Lung histology and compliance were also evaluated in the LPS model of injury. RESULTS: Following intra-tracheal LPS instillation, all mice developed lung injury, as measured by an increase in lavage neutrophils, and decrease in lung compliance, with no overall effect of genotype observed. Increased concentrations of lavage inflammatory cytokines and chemokines were also observed following LPS injury, however, LPS-instilled female MMP3-/- mice had lower levels of inflammatory mediators compared to LPS-instilled female MMP3+/+ mice. This effect of the genotype was not observed in male mice. Similar findings, including the MMP3-related sex differences, were also observed after acid-induced lung injury. CONCLUSION: MMP3 contributes to the pathogenesis of ARDS, by affecting the pulmonary inflammatory response in female mice in relevant models of lung injury.

Apolipoprotein E-deficient mice are susceptible to the development of acute lung injury.

BACKGROUND: Apolipoprotein E (apoE) has been shown to play a pivotal role in the development of cardiovascular disease, attributable to its function in lipid trafficking and immune modulating properties; however, its role in modulating inflammation in the setting of acute lung injury (ALI) is unknown. OBJECTIVE: To determine whether apoE-deficient mice (apoE-/-) are more susceptible to ALI compared to wild-type (WT) animals. METHODS: Two independent models of ALI were employed. Firstly, WT and apoE-/- mice were randomized to acid aspiration (50 µl of 0.1 N hydrochloric acid) followed by 4 h of mechanical ventilation. Secondly, WT and apoE-/- mice were randomized to 72 h of hyperoxia exposure or room air. Thereafter, the intrinsic responses of WT and apoE-/- mice were assessed using the isolated perfused mouse lung (IPML) setup. Finally, based on elevated levels of oxidized low-density lipoprotein (oxLDL) in apoE-/-, the effect of oxLDL on lung endothelial permeability and inflammation was assessed. RESULTS: In both in vivo models, apoE-/- mice demonstrated greater increases in lung lavage protein levels, neutrophil counts, and cytokine expression (p < 0.05) compared to WT mice. Experiments utilizing the IPML setup demonstrated no differences in intrinsic lung responses to injury between apoE-/- and WT mice, suggesting the presence of a circulating factor as being responsible for the in vivo observations. Finally, the exposure of lung endothelial cells to oxLDL resulted in increased monolayer permeability and IL-6 release compared to native (nonoxidized) LDL. CONCLUSIONS: Our findings demonstrate a susceptibility of apoE-/- animals to ALI that may occur, in part, due to elevated levels of oxLDL.

The effects of exogenous surfactant administration on ventilation-induced inflammation in mouse models of lung injury.

BACKGROUND: Mechanical ventilation (MV) is an essential supportive therapy for acute lung injury (ALI); however it can also contribute to systemic inflammation. Since pulmonary surfactant has anti-inflammatory properties, the aim of the study was to investigate the effect of exogenous surfactant administration on ventilation-induced systemic inflammation. METHODS: Mice were randomized to receive an intra-tracheal instillation of a natural exogenous surfactant preparation (bLES, 50 mg/kg) or no treatment as a control. MV was then performed using the isolated and perfused mouse lung (IPML) set up. This model allowed for lung perfusion during MV. In experiment 1, mice were exposed to mechanical ventilation only (tidal volume =20 mL/kg, 2 hours). In experiment 2, hydrochloric acid or air was instilled intra-tracheally four hours before applying exogenous surfactant and ventilation (tidal volume =5 mL/kg, 2 hours). RESULTS: For both experiments, exogenous surfactant administration led to increased total and functional surfactant in the treated groups compared to the controls. Exogenous surfactant administration in mice exposed to MV only did not affect peak inspiratory pressure (PIP), lung IL-6 levels and the development of perfusate inflammation compared to non-treated controls. Acid injured mice exposed to conventional MV showed elevated PIP, lung IL-6 and protein levels and greater perfusate inflammation compared to air instilled controls. Instillation of exogenous surfactant did not influence the development of lung injury. Moreover, exogenous surfactant was not effective in reducing the concentration of inflammatory cytokines in the perfusate. CONCLUSIONS: The data indicates that exogenous surfactant did not mitigate ventilation-induced systemic inflammation in our models. Future studies will focus on altering surfactant composition to improve its immuno-modulating activity.

Pulmonary-derived phosphoinositide 3-kinase gamma (PI3Kγ) contributes to ventilator-induced lung injury and edema.

BACKGROUND: Ventilator-induced lung injury (VILI) occurs in part by increased vascular permeability and impaired alveolar fluid clearance. Phosphoinositide 3-kinase gamma (PI3Kγ) is activated by mechanical stress, induces nitric oxide (NO) production, and participates in cyclic adenosine monophosphate (cAMP) hydrolysis, each of which contributes to alveolar edema. We hypothesized that lungs lacking PI3Kγ or treated with PI3Kγ inhibitors would be protected from ventilation-induced alveolar edema and lung injury. METHODS: Using an isolated and perfused lung model, wild-type (WT) and PI3Kγ-knockout (KO) mice underwent negative-pressure cycled ventilation at either -25 cmH2O and 0 cmH2O positive end-expiratory pressure (PEEP) (HIGH STRESS) or -10 cmH2O and -3 cmH2O PEEP (LOW STRESS). RESULTS: Compared with WT, PI3Kγ-knockout mice lungs were partially protected from VILI-induced derangement of respiratory mechanics (lung elastance) and edema formation [bronchoalveolar lavage (BAL) protein concentration, wet/dry ratio, and lung histology]. In PI3Kγ-knockout mice, VILI induced significantly less phosphorylation of protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), production of nitrate and nitrotyrosine, as well as hydrolysis of cAMP, compared with wild-type animals. PI3Kγ wild-type lungs treated with AS605240, an inhibitor of PI3Kγ kinase activity, in combination with enoximone, an inhibitor of phosphodiesterase-3 (PDE3)-induced cAMP hydrolysis, were protected from VILI at levels comparable to knockout lungs. CONCLUSIONS: Phosphoinositide 3-kinase gamma in resident lung cells mediates part of the alveolar edema induced by high-stress ventilation. This injury is mediated via altered Akt, eNOS, NO, and/or cAMP signaling. Anti-PI3Kγ therapy aimed at resident lung cells represents a potential pharmacologic target to mitigate VILI.

Phosphoinositide-3 kinase gamma activity contributes to sepsis and organ damage by altering neutrophil recruitment.

RATIONALE: Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase γ (PI3Kγ) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. OBJECTIVES: This study aimed to determine the specific role of the kinase activity of PI3Kγ in the pathogenesis of sepsis and multiorgan damage. METHODS: PI3Kγ wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation-induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3Kγ inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. MEASUREMENTS AND MAIN RESULTS: Both genetic and pharmaceutical PI3Kγ kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3Kγ pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. CONCLUSIONS: This study establishes PI3Kγ as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Pulmonary atelectasis during low stretch ventilation: "open lung" versus "lung rest" strategy.

OBJECTIVE: Limiting tidal volume (VT) may minimize ventilator-induced lung injury (VILI). However, atelectasis induced by low VT ventilation may cause ultrastructural evidence of cell disruption. Apoptosis seems to be involved as protective mechanisms from VILI through the involvement of mitogen-activated protein kinases (MAPKs). We examined the hypothesis that atelectasis may influence the response to protective ventilation through MAPKs. DESIGN: Prospective randomized study. SETTING: University animal laboratory. SUBJECTS: Adult male 129/Sv mice. INTERVENTIONS: Isolated, nonperfused lungs were randomized to VILI: VT of 20 mL/kg and positive end-expiratory pressure (PEEP) zero; low stretch/lung rest: VT of 6 mL/kg and 8-10 cm H2O of PEEP; low stretch/open lung: VT of 6 mL/kg, two recruitment maneuvers and 14-16 cm H2O of PEEP. Ventilator settings were adjusted using the stress index. MEASUREMENT AND MAIN RESULT: Both low stretch strategies equally blunted the VILI-induced derangement of respiratory mechanics (static volume-pressure curve), lung histology (hematoxylin and eosin), and inflammatory mediators (interleukin-6, macrophage inflammatory protein-2 [enzyme-linked immunosorbent assay], and inhibitor of nuclear factor-kB[Western blot]). VILI caused nuclear swelling and membrane disruption of pulmonary cells (electron microscopy). Few pulmonary cells with chromatin condensation and fragmentation were seen during both low stretch strategies. However, although cell thickness during low stretch/open lung was uniform, low stretch/lung rest demonstrated thickening of epithelial cells and plasma membrane bleb formation. Compared with the low stretch/open lung, low stretch/lung rest caused a significant decrease in apoptotic cells (terminal deoxynucleotidyl transferase mediated deoxyuridine-triphosphatase nick end-labeling) and tissue expression of caspase-3 (Western blot). Both low stretch strategies attenuated the activation of MAPKs. Such reduction was larger during low stretch/open lung than during low stretch/lung rest (p < 0.001). CONCLUSION: Low stretch strategies provide similar attenuation of VILI. However, low stretch/lung rest strategy is associated to less apoptosis and more ultrastructural evidence of cell damage possibly through MAPKs-mediated pathway.

Polymyxin-B hemoperfusion inactivates circulating proapoptotic factors.

OBJECTIVE: To test the hypothesis that extracorporeal therapy with polymyxin B (PMX-B) may prevent Gram-negative sepsis-induced acute renal failure (ARF) by reducing the activity of proapoptotic circulating factors. SETTING: Medical-Surgical Intensive Care Units. PATIENTS AND INTERVENTIONS: Sixteen patients with Gram-negative sepsis were randomized to receive standard care (Surviving Sepsis Campaign guidelines) or standard care plus extracorporeal therapy with PMX-B. MEASUREMENTS AND RESULTS: Cell viability, apoptosis, polarity, morphogenesis, and epithelial integrity were evaluated in cultured tubular cells and glomerular podocytes incubated with plasma from patients of both groups. Renal function was evaluated as SOFA and RIFLE scores, proteinuria, and tubular enzymes. A significant decrease of plasma-induced proapoptotic activity was observed after PMX-B treatment on cultured renal cells. SOFA and RIFLE scores, proteinuria, and urine tubular enzymes were all significantly reduced after PMX-B treatment. Loss of plasma-induced polarity and permeability of cell cultures was abrogated with the plasma of patients treated with PMX-B. These results were associated to a preserved expression of molecules crucial for tubular and glomerular functional integrity. CONCLUSIONS: Extracorporeal therapy with PMX-B reduces the proapoptotic activity of the plasma of septic patients on cultured renal cells. These data confirm the role of apoptosis in the development of sepsis-related ARF.

Expression of Pax2 in human renal tumor-derived endothelial cells sustains apoptosis resistance and angiogenesis.

The transcription factor Pax2 is known to play a key role during renal development and to act as an oncogene favoring renal tumor growth. We recently showed that endothelial cells derived from human renal carcinomas display abnormal characteristics of survival and angiogenic properties. In the present study we found that renal tumor-derived endothelial cells, but not normal endothelial cells, expressed Pax2 protein and mRNA. To down-regulate Pax2 expression, we transfected tumor-derived endothelial cells with an anti-sense PAX2 vector whereas we transfected normal human microvascular endothelial cells with a sense PAX2 vector to induce Pax2 expression. The inhibition of Pax2 expression in tumor-derived endothelial cells induced an increase in tumor suppressor PTEN expression and a decrease in Akt phosphorylation. In addition, decreased apoptosis resistance, adhesion, invasion, and in vitro and in vivo angiogenesis were observed. Conversely, Pax2 induction in normal endothelial cells conferred to these cells a proinvasive, proangiogenic phenotype similar to that of tumor-derived endothelial cells. These results indicate that Pax2 is involved in renal tumor angiogenesis and its expression may antagonize that of the PTEN tumor suppressor gene, affecting the Akt-survival pathway and promoting angiogenesis.