Pulmonary Surfactant: A Mighty Thin Film.

Pulmonary surfactant is a critical component of lung function in healthy individuals. It functions in part by lowering surface tension in the alveoli, thereby allowing for breathing with minimal effort. The prevailing thinking is that low surface tension is attained by a compression-driven squeeze-out of unsaturated phospholipids during exhalation, forming a film enriched in saturated phospholipids that achieves surface tensions close to zero. A thorough review of past and recent literature suggests that the compression-driven squeeze-out mechanism may be erroneous. Here, we posit that a surfactant film enriched in saturated lipids is formed shortly after birth by an adsorption-driven sorting process and that its composition does not change during normal breathing. We provide biophysical evidence for the rapid formation of an enriched film at high surfactant concentrations, facilitated by adsorption structures containing hydrophobic surfactant proteins. We examine biophysical evidence for and against the compression-driven squeeze-out mechanism and propose a new model for surfactant function. The proposed model is tested against existing physiological and pathophysiological evidence in neonatal and adult lungs, leading to ideas for biophysical research, that should be addressed to establish the physiological relevance of this new perspective on the function of the mighty thin film that surfactant provides.

Bronchopneumonia with interstitial pneumonia in beef feedlot cattle: Characterization and laboratory investigation.

Bronchopneumonia with interstitial pneumonia (BIP) has been considered a variant of acute interstitial pneumonia (AIP) rather than a distinct disease. This study compared 18 BIP, 24 bronchopneumonia (BP), and 13 AIP cases in feedlot beef cattle. Grossly, BIP cases typically had cranioventral lung lesions of similar morphology and extent as BP cases, but the caudodorsal lung appeared overinflated, bulged on section, and had interlobular edema and emphysema. Gross diagnosis of BIP had 83% sensitivity and 73% specificity relative to histopathology. Histologic lesions of BIP in cranioventral areas were of chronic BP, while caudodorsal lesions included alveolar and bronchiolar damage and inflammation, interstitial hypercellularity, and multifocal hemorrhages. In BIP cases, cranioventral lung lesions were more chronic than caudodorsal lesions. Histologic scores and microbiology data were comparable in cranioventral lung of BIP versus BP cases and caudodorsal lung of BIP versus AIP cases, with differences reflecting a more chronic disease involving less virulent bacteria in BIP versus BP. Mycoplasma bovis infection was similarly frequent among groups, and a viral cause of BIP was not identified. Lesion morphology and similar blood cytokine concentrations among groups argued against sepsis as a cause of lung injury. Surfactant dysfunction was identified in BIP and BP, and was only partially the result of protein exudation. These and other findings establish BIP as a distinct condition in which chronic cranioventral BP precedes acute caudodorsal interstitial lung disease, supporting a role of chronic inflammation in heightened sensitivity to 3-methylindole or another lung toxicant.

PepBiotics, novel cathelicidin-inspired antimicrobials to fight pulmonary bacterial infections.

BACKGROUND: Antimicrobial peptides are considered potential alternatives to antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. METHODS: Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of PepBiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. RESULTS: A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using ITC and 31P solid state NMR. CONCLUSIONS: PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. GENERAL SIGNIFICANCE: These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

National Preclinical Sepsis Platform: developing a framework for accelerating innovation in Canadian sepsis research.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies-defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis-are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical  research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.

The COVID-19 pandemic: a target for surfactant therapy?

INTRODUCTION: The dramatic impact of COVID-19 on humans worldwide has initiated an extraordinary search for effective treatment approaches. One of these is the administration of exogenous surfactant, which is being tested in ongoing clinical trials. AREAS COVERED: Exogenous surfactant is a life-saving treatment for premature infants with neonatal respiratory distress syndrome. This treatment has also been tested for acute respiratory distress syndrome (ARDS) with limited success possibly due to the complexity of that syndrome. The 60-year history of successes and failures associated with surfactant therapy distinguishes it from many other treatments currently being tested for COVID-19 and provides the opportunity to discuss the factors that may influence the success of this therapy. EXPERT OPINION: Clinical data provide a strong rationale for using exogenous surfactant in COVID-19 patients. Success of this therapy may be influenced by the mechanical ventilation strategy, the timing of treatment, the doses delivered, the method of delivery and the preparations utilized. In addition, future development of enhanced preparations may improve this treatment approach. Overall, results from ongoing trials may not only provide data to indicate if this therapy is effective for COVID-19 patients, but also lead to further scientific understanding and improved treatment strategies.

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.

Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH.

Although the GraS sensor kinase of Staphylococcus aureus is known for the sensing of and resistance to cationic antimicrobial peptides (CAMPs), we recently established that it also signals in response to acidic pH, which is encountered on human skin concurrently with CAMPs, antimicrobial unsaturated free fatty acids (uFFA), and calcium. We therefore evaluated how these environmental signals would affect GraS function and resistance to antimicrobial uFFA. Growth at pH 5.5 promoted increased resistance of S. aureus USA300 to linoleic and arachidonic acids but not palmitoleic or sapienic acid. However, enhanced resistance to these C16:1 uFFA was achieved by supplementing acidic medium with 0.5 mM calcium or subinhibitory CAMPs. Enhanced resistance to uFFA at acidic pH was dependent on GraS and GraS-dependent expression of the lysyl-phosphatidylglycerol synthase enzyme MprF, through a mechanism that did not require the lysyl-transferase function of MprF. In addition to enhanced resistance to antimicrobial uFFA, acidic pH also promoted increased production of secreted proteases in a GraS-dependent manner. During growth at pH 5.5, downstream phenotypes of signaling through GraS, including resistance to uFFA, MprF-dependent addition of positive charge to the cell surface, and increased production of secreted proteases, all occurred independently of acidic amino acids in the extracytoplasmic sensor loop of GraS that were previously found to be required for sensing of CAMPs. Cumulatively, our data indicate that signaling through GraS at acidic pH occurs through a mechanism that is distinct from that described for CAMPs, leading to increased resistance to antimicrobial uFFA and production of secreted proteases.IMPORTANCEStaphylococcus aureus asymptomatically colonizes 30% of humans but is also a leading cause of infectious morbidity and mortality. Since infections are typically initiated by the same strain associated with asymptomatic colonization of the nose or skin, it is important to understand how the microbe can endure exposure to harsh conditions that successfully restrict the growth of other bacteria, including a combination of acidic pH, antimicrobial peptides, and antimicrobial fatty acids. The significance of our research is in showing that acidic pH combined with antimicrobial peptide or environmental calcium can signal through a single membrane sensor protein to promote traits that may aid in survival, including modification of cell surface properties, increased resistance to antimicrobial fatty acids, and enhanced production of secreted proteases.

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.

New insights into exogenous surfactant as a carrier of pulmonary therapeutics.

As an organ system, the lung has unique advantages and disadvantages for localized drug delivery. Its direct contact with the external environment allows for the upper airways to be easily accessible to intrapulmonary delivery. However, its complex branching structure makes direct delivery to the peripheral airways challenging. This review will discus the utility of exogenous surfactant, a lipoprotein complex currently used to treat neonatal respiratory distress syndrome, as a carrier for pulmonary therapeutics to enhance the delivery of these drugs to the deeper regions of the lung. The focus is to provide an update on the many tools available to develop new surfactant-based therapeutics using computer modeling, in vitro approaches, and in vivo testing, which may ultimately lead to clinical trials. Two clinical conditions, Acute Respiratory Distress Syndrome and Bacterial Pneumonia are utilized throughout as prototypical examples of pulmonary conditions in which surfactant drug combination may be beneficial. Consequently, the pharmaceuticals discussed are primarily those with antimicrobial or anti-inflammatory activities.

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.

The Antibacterial and Anti-inflammatory Activity of Chicken Cathelicidin-2 combined with Exogenous Surfactant for the Treatment of Cystic Fibrosis-Associated Pathogens.

Cystic fibrosis (CF) is characterized by recurrent airway infections with antibiotic-resistant bacteria and chronic inflammation. Chicken cathelicin-2 (CATH-2) has been shown to exhibit antimicrobial activity against antibiotic-resistant bacteria and to reduce inflammation. In addition, exogenous pulmonary surfactant has been suggested to enhance pulmonary drug delivery. It was hypothesized that CATH-2 when combined with an exogenous surfactant delivery vehicle, bovine lipid extract surfactant (BLES), would exhibit antimicrobial activity against CF-derived bacteria and downregulate inflammation. Twelve strains of CF-pathogens were exposed to BLES+CATH-2 in vitro and killing curves were obtained to determine bactericidal activity. Secondly, heat-killed bacteria were administered in vivo to elicit a pro-inflammatory response with either a co-administration or delayed administration of BLES+CATH-2 to assess the antimicrobial-independent, anti-inflammatory properties of BLES+CATH-2. CATH-2 alone exhibited potent antimicrobial activity against all clinical strains of antibiotic-resistant bacteria, while BLES+CATH-2 demonstrated a reduction, but significant antimicrobial activity against bacterial isolates. Furthermore, BLES+CATH-2 reduced inflammation in vivo when either co-administered with killed bacteria or after delayed administration. The use of a host-defense peptide combined with an exogenous surfactant compound, BLES+CATH-2, is shown to exhibit antimicrobial activity against antibiotic-resistant CF bacterial isolates and reduce inflammation.

Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo.

The development of antibiotic resistance by Pseudomonas aeruginosa is a major concern in the treatment of bacterial pneumonia. In the search for novel anti-infective therapies, the chicken-derived peptide cathelicidin-2 (CATH-2) has emerged as a potential candidate, with strong broad-spectrum antimicrobial activity and the ability to limit inflammation by inhibiting Toll-like receptor 2 (TLR2) and TLR4 activation. However, as it is unknown how CATH-2 affects inflammation in vivo, we investigated how CATH-2-mediated killing of P. aeruginosa affects lung inflammation in a murine model. First, murine macrophages were used to determine whether CATH-2-mediated killing of P. aeruginosa reduced proinflammatory cytokine production in vitro Next, a murine lung model was used to analyze how CATH-2-mediated killing of P. aeruginosa affects neutrophil and macrophage recruitment as well as cytokine/chemokine production in the lung. Our results show that CATH-2 kills P. aeruginosa in an immunogenically silent manner both in vitro and in vivo Treatment with CATH-2-killed P. aeruginosa showed reduced neutrophil recruitment to the lung as well as inhibition of cytokine and chemokine production, compared to treatment with heat- or gentamicin-killed bacteria. Together, these results show the potential for CATH-2 as a dual-activity antibiotic in bacterial pneumonia, which can both kill P. aeruginosa and prevent excessive inflammation.

Voluntary running exercise protects against sepsis-induced early inflammatory and pro-coagulant responses in aged mice.

BACKGROUND: Despite many animal studies and clinical trials, mortality in sepsis remains high. This may be due to the fact that most experimental studies of sepsis employ young animals, whereas the majority of septic patients are elderly (60 - 70 years). The objective of the present study was to examine the sepsis-induced inflammatory and pro-coagulant responses in aged mice. Since running exercise protects against a variety of diseases, we also examined the effect of voluntary running on septic responses in aged mice. METHODS: Male C57BL/6 mice were housed in our institute from 2-3 to 22 months (an age mimicking that of the elderly). Mice were prevented from becoming obese by food restriction (given 70-90% of ad libitum consumption amount). Between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating 1-3 days of running with 1-2 days of rest. At 22 months, mice were intraperitoneally injected with sterile saline (control) or 3.75 g/kg fecal slurry (septic). At 7 h post injection, we examined (1) neutrophil influx in the lung and liver by measuring myeloperoxidase and/or neutrophil elastase in the tissue homogenates by spectrophotometry, (2) interleukin 6 (IL6) and KC in the lung lavage by ELISA, (3) pulmonary surfactant function by measuring percentage of large aggregates, (4) capillary plugging (pro-coagulant response) in skeletal muscle by intravital microscopy, (5) endothelial nitric oxide synthase (eNOS) protein in skeletal muscle (eNOS-derived NO is putative inhibitor of capillary plugging) by immunoblotting, and (6) systemic blood platelet counts by hemocytometry. RESULTS: Sepsis caused high levels of pulmonary myeloperoxidase, elastase, IL6, KC, liver myeloperoxidase, and capillary plugging. Sepsis also caused low levels of surfactant function and platelet counts. Running exercise increased eNOS protein and attenuated the septic responses. CONCLUSIONS: Voluntary running protects against exacerbated sepsis-induced inflammatory and pro-coagulant responses in aged mice. Protection against pro-coagulant responses may involve eNOS upregulation. The present discovery in aged mice calls for clinical investigation into potential beneficial effects of exercise on septic outcomes in the elderly.

A respiratory-gated micro-CT comparison of respiratory patterns in free-breathing and mechanically ventilated rats.

In this study, we aim to quantify the differences in lung metrics measured in free-breathing and mechanically ventilated rodents using respiratory-gated micro-computed tomography. Healthy male Sprague-Dawley rats were anesthetized with ketamine/xylazine and scanned with a retrospective respiratory gating protocol on a GE Locus Ultra micro-CT scanner. Each animal was scanned while free-breathing, then intubated and mechanically ventilated (MV) and rescanned with a standard ventilation protocol (56 bpm, 8 mL/kg and PEEP of 5 cm H2O) and again with a ventilation protocol that approximates the free-breathing parameters (88 bpm, 2.14 mL/kg and PEEP of 2.5 cm H2O). Images were reconstructed representing inspiration and end expiration with 0.15 mm voxel spacing. Image-based measurements of the lung lengths, airway diameters, lung volume, and air content were compared and used to calculate the functional residual capacity (FRC) and tidal volume. Images acquired during MV appeared darker in the airspaces and the airways appeared larger. Image-based measurements showed an increase in lung volume and air content during standard MV, for both respiratory phases, compared with matched MV and free-breathing. Comparisons of the functional metrics showed an increase in FRC for mechanically ventilated rats, but only the standard MV exhibited a significantly higher tidal volume than free-breathing or matched MV Although standard mechanical ventilation protocols may be useful in promoting consistent respiratory patterns, the amount of air in the lungs is higher than in free-breathing animals. Matching the respiratory patterns with the free-breathing case allowed similar lung morphology and physiology measurements while reducing the variability in the measurements.

Lung Lavage and Surfactant Replacement During Ex Vivo Lung Perfusion for Treatment of Gastric Acid Aspiration-Induced Donor Lung Injury.

BACKGROUND: Ex vivo lung perfusion (EVLP) provides opportunities to treat injured donor lungs before transplantation. We investigated whether lung lavage, to eliminate inflammatory inhibitory components, followed by exogenous surfactant replacement, could aid lung recovery and improve post-transplant lung function after gastric aspiration injury. METHODS: Gastric acid aspiration was induced in donor pigs, which were ventilated for 6 hours to develop lung injury. After retrieval and 10 hours of cold preservation, EVLP was performed for 6 hours. The lungs were randomly divided into 4 groups (n = 5, each): (1) no treatment (control), (2) lung lavage, (3) surfactant administration, and (4) lung lavage, followed by surfactant administration. After another 2-hour period of cold preservation, the left lung was transplanted and reperfused for 4 hours. RESULTS: Physiologic lung function significantly improved after surfactant administration during EVLP. The EVLP perfusate from the lavage + surfactant group showed significantly lower levels of interleukin (IL)-1ß, IL-6, IL-8, and secretory phospholipase A2. Total phosphatidylcholine was increased, and minimum surface tension was recovered to normal levels (≤5 mN/m) in the bronchioalveolar fluid after surfactant administration. Lysophosphatidylcholine in bronchioalveolar fluid was significantly lower in the lavage + surfactant group than in the surfactant group. Post-transplant lung function was significantly better in the lavage + surfactant group compared with all other groups. CONCLUSIONS: Lung lavage, followed by surfactant replacement during EVLP, reduced inflammatory mediators and prevented hydrolysis of phosphatidylcholine, which contributed to the superior post-transplant function in donor lungs with aspiration injury.

Lung remodeling associated with recovery from acute lung injury.

Acute respiratory distress syndrome (ARDS) is a disease with a variety of causes and is defined by severe hypoxemia. Whereas ARDS carries a mortality of approximately 30 %, patients that survive may ultimately regain near normal pulmonary physiology. The critical pathophysiological processes in ARDS are alveolar barrier dysfunction and overwhelming inflammation. This encompasses damage to the epithelial and endothelial layers, thickening of the interstitial matrix, edema with inactivation of pulmonary surfactant at the alveolar surface and marked inflammation mediated by infiltrating neutrophils and pro-inflammatory macrophages. For patients that survive the disease, these are the critical processes that require repair and remodeling to allow for the recovery of ARDS. As such, the current review focuses on the experimental studies that have begun to elucidate the mechanisms involved in restoring the alveolar barrier following injury.

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.