Alveolar epithelial glycocalyx degradation mediates surfactant dysfunction and contributes to acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in patients with ARDS. Using mass spectrometry of airspace fluid noninvasively collected from mechanically ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding, which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

The impact of maternal protein restriction during perinatal life on the response to a septic insult in adult rats.

Although abundant evidence exists that adverse events during pregnancy lead to chronic conditions, there is limited information on the impact of acute insults such as sepsis. This study tested the hypothesis that impaired fetal development leads to altered organ responses to a septic insult in both male and female adult offspring. Fetal growth restricted (FGR) rats were generated using a maternal protein-restricted diet. Male and female FGR and control diet rats were housed until 150-160 d of age when they were exposed either a saline (control) or a fecal slurry intraperitoneal (Sepsis) injection. After 6 h, livers and lungs were analyzed for inflammation and, additionally, the amounts and function of pulmonary surfactant were measured. The results showed increases in the steady-state mRNA levels of inflammatory cytokines in the liver in response to the septic insult in both males and females; these responses were not different between FGR and control diet groups. In the lungs, cytokines were not detectable in any of the experimental groups. A significant decrease in the relative amount of surfactant was observed in male FGR offspring, but this was not observed in control males or in female animals. Overall, it is concluded that FGR induced by maternal protein restriction does not impact liver and lung inflammatory response to sepsis in either male or female adult rats. An altered septic response in male FGR offspring with respect to surfactant may imply a contribution to lung dysfunction.

The effect of maternal protein restriction during perinatal life on the inflammatory response in pediatric rats.

Fetal growth restriction can affect health outcomes in postnatal life. This study tested the hypothesis that the response to an inflammatory pulmonary insult is altered in pediatric fetal growth restricted rats. Using a low-protein diet during gestation and postnatal life, growth-restricted male and female rats and healthy control rats were exposed to an inflammatory insult via the intratracheal instillation of heat-killed bacteria. After 6 h, animal lungs were examined for lung inflammation and status of the surfactant system. The results showed that in response to an inflammatory insult, neutrophil infiltration was decreased in both male and female rats in the growth-restricted animals compared with the control rats. The amount of surfactant was increased in the growth-restricted animals compared with the control rats, regardless of the inflammatory insult. It is concluded that fetal growth restriction results in increased surfactant and altered neutrophil responses following pulmonary insult.

The effects of aging and exercise on lung mechanics, surfactant and alveolar macrophages.

Purpose: Advancing age leads to changes to the respiratory system associated with increased susceptibility to lung diseases, and exercise may counteract this effect. To explore the underlying processes, we investigated the effects of aging and exercise on lung mechanics, alveolar macrophage function, and surfactant pools and activity, in mice. It was hypothesized that aging would impact lung mechanics, macrophage polarization, and the status of the surfactant system, and that these changes would be mitigated by exercise. Methods: Male C57BL/6 mice were housed from 2-3 to 22 months, for the aged group, or until 4 months of age for young mice. Mice in both groups were randomized to voluntarily running exercise or to non-exercise, for a 2-month period. Mice were euthanized and lung mechanics were analyzed using a flexiVent ventilator. Subsequently, the lungs were lavaged to obtain pulmonary surfactant and alveolar macrophages. Pulmonary surfactant was analyzed for pool sizes and activity whereas alveolar macrophages were examined for response to pro and anti-inflammatory stimuli. Results: Changes in lung mechanics, such as increased compliance and decreased airway resistance, were associated with aging but were not affected by exercise. The quantity as well as the biophysical activity of the pulmonary surfactant system was unaffected by either aging or exercise. More alveolar macrophages were recovered from exercising aged mice compared to both the young and non-exercising groups. Macrophages in this aged exercise group were more responsive to an anti-inflammatory stimulus. Conclusions: Our data supports previous literature that suggest the development of emphysema-like alterations to lung mechanics with aging. This effect was independent of exercise. Our data also indicates that surfactant is unaffected by aging and exercise. Alveolar macrophage properties and numbers were affected by exercise in the aging lung and may represent the main, potentially beneficial, effect of exercise on the pulmonary system.

Maternal protein restriction during perinatal life affects lung mechanics and the surfactant system during early postnatal life in female rats.

Limited information is available on how fetal growth retardation (FGR) affects the lung in the neonatal period in males and females. This led us to test the hypothesis that FGR alters lung mechanics and the surfactant system during the neonatal period. To test this hypothesis a model of FGR was utilized in which pregnant rat dams were fed a low protein diet during both the gestation and lactation period. We subsequently analyzed lung mechanics using a FlexiVent ventilator in male and female pups at postnatal day 7 and 21. Lung lavage material was obtained at postnatal day 1, 7 and 21, and was used for analysis of the surfactant system which included measurement of the pool size of surfactant and its subfraction as well as the surface tension reducing ability of the surfactant. The main result of the study was a significantly lower lung compliance and higher tissue elastance which was observed in FGR female offspring at day 21 compared to control offspring. In addition, female LP offspring exhibited lower surfactant pool sizes at postnatal day 1compared to controls. These changes were not observed in the male offspring. It is concluded that FGR has a different impact on pulmonary function and on surfactant in female, as compared to male, offspring.

Impact of ventilation-induced lung injury on the structure and function of lamellar bodies.

Alterations to the pulmonary surfactant system have been observed consistently in ventilation-induced lung injury (VILI) including composition changes and impairments in the surface tension reducing ability of the isolated extracellular surfactant. However, there is limited information about the effects of VILI on the intracellular form of surfactant, the lamellar body. It is hypothesized that VILI leads to alterations of lamellar body numbers and function. To test this hypothesis, rats were randomized to one of three groups, nonventilated controls, control ventilation, and high tidal volume ventilation (VILI). Following physiological assessment to confirm lung injury, isolated lamellar bodies were tested for surfactant function on a constrained sessile drop surfactometer. A separate cohort of animals was used to fix the lungs followed by examination of lamellar body numbers and morphology using transmission electron microscopy. The results showed an impaired ability of reducing surface tension for the lamellar bodies isolated from the VILI group as compared with the two other groups. The morphological assessment revealed that the number, and the relative area covered by, lamellar bodies were significantly decreased in animals with VILI animals as compared with the other groups. It is concluded that VILI causes significant alterations to lamellar bodies. It is speculated that increased secretion causes a depletion of lamellar bodies that cannot be compensated by de novo synthesis of surfactant in these injured lungs.

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.

The effect of matrix metalloproteinase-3 deficiency on pulmonary surfactant in a mouse model of acute lung injury.

The acute respiratory distress syndrome (ARDS) is characterized by arterial hypoxemia accompanied by severe inflammation and alterations to the pulmonary surfactant system. Published data has demonstrated a protective effect of matrix metalloproteinase-3 (Mmp3) deficiency against the inflammatory response associated with ARDS; however, the effect of Mmp3 on physiologic parameters and alterations to surfactant have not been previously studied. It was hypothesized that Mmp3 deficient (Mmp3(-/-)) mice would be protected against lung dysfunction associated with ARDS and maintain a functional pulmonary surfactant system. Wild type (WT) and Mmp3(-/-) mice were subjected to acid-aspiration followed by mechanical ventilation. Mmp3(-/-) mice maintained higher arterial oxygenation compared with WT mice at the completion of ventilation. Significant increase in functional large aggregate surfactant forms were observed in Mmp3(-/-) mice compared with WT mice. These findings further support a role of Mmp3 as an attractive therapeutic target for drug development in the setting of ARDS.

The effect of diet-induced serum hypercholesterolemia on the surfactant system and the development of lung injury.

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder associated with alterations to the pulmonary surfactant system. Recent studies showed that supra-physiological levels of cholesterol in surfactant contribute to impaired function. Since cholesterol is incorporated into surfactant within the alveolar type II cells which derives its cholesterol from serum, it was hypothesized that serum hypercholesterolemia would predispose the host to the development of lung injury due to alterations of cholesterol content in the surfactant system. Wistar rats were randomized to a standard lab diet or a high cholesterol diet for 17-20 days. Animals were then exposed to one of three models of lung injury: i) acid aspiration ii) ventilation induced lung injury, and iii) surfactant depletion. Following physiological monitoring, lungs were lavaged to obtain and analyze the surfactant system. The physiological results showed there was no effect of the high cholesterol diet on the severity of lung injury in any of the three models of injury. There was also no effect of the diet on surfactant cholesterol composition. Rats fed a high cholesterol diet had a significant impairment in surface tension reducing capabilities of isolated surfactant compared to those fed a standard diet exposed to the surfactant depletion injury. In addition, only rats that were exposed to ventilation induced lung injury had elevated levels of surfactant associated cholesterol compared to non-injured rats. It is concluded that serum hypercholesterolemia does not predispose rats to altered surfactant cholesterol composition or to lung injury. Elevated cholesterol within surfactant may be a marker for ventilation induced lung damage.

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.

The biological effects of lung-derived mediators on the liver.

Despite the use of lung-protective mechanical ventilation (MV), the mortality of patients with acute lung injury remains at 30 to 40%, predominantly due to multiorgan failure. The objective of this study was to determine the biological significance of lung-derived mediators on peripheral organ inflammation. The authors utilized an isolated perfused mouse lung model of lipopolysaccharide (LPS)-induced lung inflammation and protective MV to collect lung-derived mediators. Aliquots of perfusate from these animals (or appropriate controls) were then injected intravenously into a cohort of normal animals whose livers were subsequently assessed in vivo using intravital video microscopy. Perfusate from LPS-inflamed lungs contained significantly higher concentrations of inflammatory mediators than perfusate from saline-instilled lungs. Assessment of livers in the second cohort of animals 120 minutes after perfusate injection revealed decreased sinusoidal blood flow, leukocytosis, and increased cell death in those receiving perfusate from LPS-inflamed lungs compared to perfusate from saline controls. There were no differences between control animals that received pure perfusate or pure LPS mixed with perfusate. These results showed that lung-derived mediators had a significant biological effect on nonpulmonary organs within a short period of time after administration. Therapies targeting these mediators may prevent multiorgan failure and death in patients with acute lung injury.

Surfactant protein-A reduces translocation of mediators from the lung into the circulation.

The objective of this study was to characterize a mouse model of lung inflammation and determine the effect of surfactant protein A (SP-A, or sftpa) on the transfer of inflammatory mediators from these injured lungs into the systemic circulation. Lung inflammation was induced in either sftpa-deficient (-/-) or wild-type (+/+) spontaneously breathing, adult mice via intranasal lipopolysaccharide (LPS). Four hours later, lungs were isolated, perfused, and mechanically ventilated for 2 hours. Perfusate was collected for analysis over the duration of ventilation and lung lavage was obtained in groups of animals immediately before and after mechanical ventilation (MV). Lavage analysis showed an increase in interleukin-6 (IL6) and tumor necrosis factor-alpha (TNFalpha) 4 hours after LPS, with a further increase in IL6 following MV. LPS and MV also caused an increase in total cell and neutrophil numbers as well as total protein in the lavage compared to controls. Perfusate analysis revealed a significant increase in IL6 and TNFalpha after LPS and MV, with significantly greater levels of these mediators in sftpa (-/-) versus (+/+) mice. The authors conclude that LPS followed by MV resulted in lung inflammation and injury, and that SP-A significantly influenced inflammatory mediator release from these inflamed lungs into the perfusate.

The effects of long-term conventional mechanical ventilation on the lungs of adult rats.

BACKGROUND: Ventilation-induced lung injury is often studied in animal models by using ventilation strategies with high-tidal volumes and high-oxygen concentration over a relatively short period of time. The injury induced by these ventilation strategies includes alterations to the surfactant system and up-regulation of inflammatory markers. Whether these responses to ventilation occur with more clinically relevant ventilation strategies is not known. OBJECTIVE: To assess how healthy adult rats respond to 24 hrs of conventional mechanical ventilation with respect to lung physiology, markers of inflammation, and alterations to pulmonary surfactant, and how this is affected by the oxygen concentration. INTERVENTIONS: Adult rats were mechanically ventilated for 24 hrs with a tidal volume of 8 mL/kg, 5 cm H2O positive end-expiratory pressure, at 60 breaths/min with either 21% or 100% oxygen. Animals were monitored for blood oxygenation and other physiologic parameters. After ventilation, lungs were lavaged and analyzed for inflammatory markers and pulmonary surfactant. These outcomes were compared with measurements obtained from spontaneously breathing rats exposed to either 21% or 100% oxygen for 24 hrs. MAIN RESULTS: Twenty-four hours of ventilation did not result in significant changes in blood oxygenation. Inflammatory markers, such as interleukin-6 concentration and the number of neutrophils in the lavage, were increased in ventilated animals compared with the nonventilated controls, regardless of the level of inspired oxygen. The amount of active surfactant was increased after ventilation; however, the surface activity of this material was impaired as compared with controls. CONCLUSION: Prolonged mechanical ventilation of health lungs with a physiologically benign strategy can contribute to the inflammatory response and cause alterations to pulmonary surfactant.

Lung mechanics in the TIMP3 null mouse and its response to mechanical ventilation.

Tissue inhibitor of metalloproteinase-3 (TIMP3) null mice develop emphysema-like airspace enlargement due to an enzymatic imbalance. This study investigates how these abnormalities alter lung mechanics and the response to 2 different mechanical ventilation strategies. Phenotypically, TIMP3 null mice had increased compliance, and decreased resistance, tissue damping, and tissue elastance over wild-type controls. Decreased compliance and increased resistance were observed following the injurious ventilation strategy; however, the TIMP3 null response to both ventilation strategies was similar to wild-type mice. In conclusion, TIMP3 null mice have significant alterations in lung mechanics; however, this does not affect their response to ventilation.

Protein inhibition of surfactant during mechanical ventilation of isolated rat lungs.

This study tested the hypothesis that material leaking into the airspace from the vasculature during ventilation interferes with surfactant function and contributes to decreases in lung compliance. Rats were euthanized and the lungs were isolated either with or without flushing of the vasculature, followed by mechanical ventilation and analysis of lung compliance and lung lavage analysis. Flushed lungs had higher lung compliance compared to the non-flushed lungs. This was associated with lower protein concentrations and improved surfactant activity. It is concluded that during mechanical ventilation, leakage of proteins results in surfactant inhibition and thereby contribute to decreased lung compliance.

Differential response of TIMP-3 null mice to the lung insults of sepsis, mechanical ventilation, and hyperoxia.

An imbalance in matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) leads to excessive or insufficient tissue breakdown, which is associated with many disease processes. The TIMP-3 null mouse is a model of MMP/TIMP imbalance, which develops air space enlargement and decreased lung function. These mice responded differently to cecal ligation and perforation (CLP)-induced septic lung injury than wild-type controls. The current study addresses whether the TIMP-3 knockout lung is susceptible to different types of insults or only those involving sepsis, by examining its response to lipopolysaccharide (LPS)-induced sepsis, mechanical ventilation (MV), and hyperoxia. TIMP-3 null noninjured controls of each insult consistently demonstrated significantly higher compliance vs. wild-type mice. Null mice treated with LPS had a further significantly increased compliance compared with untreated controls. Conversely, MV and hyperoxia did not alter compliance in the null lung. MMP abundance and activity increased in response to LPS but were generally unaltered following MV or hyperoxia, correlating with compliance alterations. All three insults produced inflammatory cytokines; however, the response of the null vs. wild-type lung was dependent on the type of insult. Overall, this study demonstrated that 1) LPS-induced sepsis produced a similar response in null mice to CLP-induced sepsis, 2) the null lung responded differently to various insults, and 3) the null susceptibility to compliance changes correlated with increased MMPs. In conclusion, this study provides insight into the role of TIMP-3 in response to various lung insults, specifically its importance in regulating MMPs to maintain compliance during a sepsis.

Host response to intratracheally instilled bacteria in ventilated and nonventilated rats.

OBJECTIVE: Pneumonia occurs in approximately 7% of hospitalized patients. Susceptibility to certain bacteria such as Pseudomonas aeruginosa increases in critically ill patients, particularly those requiring mechanical ventilation. Previous studies investigating this susceptibility have used injurious modes of ventilation. The objective of this study was to evaluate the host's response to intratracheal instillation of P. aeruginosa in the setting of noninjurious mechanical ventilation and compare this with normal, spontaneously breathing animals receiving bacteria. DESIGN: Randomized, controlled in vivo animal study. SETTING: Research laboratory at a university-affiliated institution. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized into four groups: spontaneously breathing given saline, spontaneously breathing given bacteria, mechanically ventilated given saline, and mechanically ventilated given bacteria. The ventilation strategy used involved low stretch (tidal volume of 8 mL/kg) with a positive end-expiratory pressure of 5 cm H2O. MEASUREMENTS AND MAIN RESULTS: Lung compliance, bacterial recovery, surfactant, total cells, and cytokine concentrations in the lung lavage were analyzed after 4 hrs. Results showed that neither ventilation nor bacteria alone altered lung function, although the combination of ventilation and Pseudomonas significantly decreased arterial oxygenation and lung compliance. Increases in lavage cell counts, cytokines, and surfactant were observed in both groups administered bacteria compared with animals given saline. However, there were no significant differences in bacterial recovery, cell counts, cytokines, and surfactant measurements in the groups given bacteria. CONCLUSIONS: These data suggest that bacterial instillation with low-stretch ventilation had a significant effect on lung function but did not alter the inflammatory response to a bacterial challenge over this time course compared with spontaneously breathing animals.

High-frequency oscillation and exogenous surfactant administration in lung-injured adult sheep.

OBJECTIVE: To evaluate the effects of high-frequency oscillation on the response to exogenous surfactant in lung-injured adult sheep. DESIGN: A prospective, controlled, in vivo, animal laboratory study. SETTING: Animal research facility of a health sciences university. SUBJECTS: Twenty-eight adult sheep. INTERVENTIONS: Animals were anesthetized and instrumented with a tracheostomy and vascular catheters. Following whole lung saline lavage, animals were randomized to one of four groups: Group S-CMV received surfactant and was ventilated for 4 hrs using a conventional mechanical ventilation strategy, group S-HFOV/CMV received surfactant and was ventilated with a high-frequency oscillation technique for 2 hrs and a conventional mechanical strategy for 2 hrs, group HFOV/CMV underwent the latter ventilatory strategies without receiving surfactant, and group HFOV was ventilated with high-frequency oscillation only for 4 hrs. At the end of the ventilatory period, the distributions of ventilation and surfactant were evaluated in animals that received surfactant. MEASUREMENTS AND MAIN RESULTS: Animals in the S-CMV group had a significantly greater mean PaO2 value at the end of the experimental period than animals in the S-HFOV/CMV or HFOV/CMV groups. Evaluation of the distribution of ventilation relative to surfactant demonstrated that animals ventilated with high-frequency oscillation followed by conventional mechanical ventilation had a significantly greater disproportionate distribution of ventilation relative to surfactant compared with the CMV-only group. CONCLUSIONS: A period of high-frequency oscillation, as used in this study, immediately following exogenous surfactant administration mitigates the host's response to surfactant when subsequently switched to conventional mechanical ventilation.