Large Surfactant Aggregates Research Articles

Stabilization of CO2-foams in brine by reducing drainage and coarsening using alkyldimethylamine oxides as surfactants

Producing CO2-foams in saline environments can be a great challenge in many industrial applications, since fast gas diffusion and reduction of electrical double layer promote fast coarsening and coalescence of bubbles. Amine oxides are zwitterionic surfactants that have been shown to improve foam stability by intermolecular hydrogen bonding, but have not been investigated for CO2-foams yet. This paper reports a study on the use of commercial alkyldimethylamine oxides (CxDAO) as foaming agents in brine for CO2-foams, based on the interactions present at the low pH imposed by the dissolution of the CO2 and the presence of salts. Despite the presence of salt and CO2 increased the protonation degree of amine oxide molecules, favoring hydrogen bonding, a significant improvement in foam stability was only observed when using the long chain surfactant (C14DAO). This behavior was attributed to the synergy between increased attractive hydrophobic interactions of alkyl chains and reduced repulsion among ionic surfactant heads, which led to formation of large surfactant aggregates in bulk, as demonstrated by the significant increase in viscosity of the aqueous phase. There was also a decrease in foam drainage and coarsening rates for the foams using C14DAO in brine, compared to results in deionized water, suggesting that formation of hydrogen bonds is not enough to arrest the destabilization in CO2-foams. Moreover, the linear rate of bubble growth obtained for these foams (∝t) was higher than that reported for N2– or air-foams (∝t1/2), likely due to the high solubility of CO2 in the aqueous phase, which accelerated the coarsening phenomena. The results of this work offer insight on the future design of foaming formulations for stabilizing CO2-foams in applications that require the use of high salinity brines, such as carbon sequestration and oil recovery.

Methane Inhalation Protects Against Lung Ischemia-Reperfusion Injury in Rats by Regulating Pulmonary Surfactant via the Nrf2 Pathway.

Methane (CH4) exerted protective effects against lung ischemia-reperfusion (I/R) injury, but the mechanism remains unclear, especially the role of pulmonary surfactant. Therefore, this study aimed to explore the effects of CH4 inhalation on pulmonary surfactant in rat lung I/R injury and to elucidate the mechanism. Rats were randomly divided into three groups (n = 6): the sham, I/R control, and I/R CH4 groups. In the sham group, only thoracotomy was performed on the rats. In the I/R control and I/R CH4 groups, the rats underwent left hilum occlusion for 90 min, followed by reperfusion for 180 min and ventilation with O2 or 2.5% CH4, respectively. Compared with those of the sham group, the levels of large surfactant aggregates (LAs) in pulmonary surfactant, lung compliance, oxygenation decreased, the small surfactant aggregates (SAs), inflammatory response, oxidative stress injury, and cell apoptosis increased in the control group (P < 0.05). Compared to the control treatment, CH4 increased LA (0.42 ± 0.06 vs. 0.31 ± 0.09 mg/kg), oxygenation (201 ± 11 vs. 151 ± 14 mmHg), and lung compliance (16.8 ± 1.0 vs. 11.5 ± 1.3 ml/kg), as well as total antioxidant capacity and Nrf2 protein expression and decreased the inflammatory response and number of apoptotic cells (P < 0.05). In conclusion, CH4 inhalation decreased oxidative stress injury, inflammatory response, and cell apoptosis, and improved lung function through Nrf2-mediated pulmonary surfactant regulation in rat lung I/R injury.

Viscosity-driven stabilization of CO2-in-brine foams using mixtures of cocamidopropyl hydroxysultaine and sodium dodecyl sulfate

Foam stability is one of the key factors determining the success of any foam-field application, and for that, it is imperative to identify surfactant formulations able to reduce the extensive foam drainage and coarsening occurring in harsh environments, such as in high salinity brines. In this work, mixtures of cocamidopropyl hydroxysultaine (CAHS) and sodium dodecyl sulfate (SDS) at 1 wt% were evaluated as foaming agents to obtain CO2-in-brine foams with improved stability, compared to the single surfactant foams. The results showed that the mixture with excess of the zwitterionic surfactant (2:1, CAHS:SDS) produced a CO2-foam with a half-life four times higher than that of the foam formed using single components, at the same surfactant concentration. These foams exhibited a drastic reduction in both drainage and coarsening mechanisms, which was attributed to the significant increase (four orders of magnitude) in bulk phase viscosity due to the formation of large surfactant aggregates in the brine, as confirmed by dynamic light scattering (DLS) and rheological measurements. The analysis of the gas fraction in the foams formulated with the surfactant mixtures revealed that they were also able to comprise large amounts of CO2 (83–88% per volume of aqueous phase), as a result of retarding the diffusion of CO2 through the viscous aqueous phase of the foam. The results obtained in this work demonstrated that the synergy exhibited by CAHS and SDS in brine had a direct impact not only in the viscosity-driven stabilization of the CO2-foams, but also for the capture and retention of large amounts of CO2 inside the foam, which can have a direct impact in the sweeping efficiency, in the case of EOR, and in the CO2 storage, in the case of carbon capture and storage (CCS) technologies.

Mild Hypothermia Enhances Lung Surfactant Activity: Delving into the Molecular Mechanisms

Pulmonary surfactant (PS) is a lipid-protein complex that reduces the surface tension at the respiratory air-liquid interface of alveoli, minimizing the work of breathing. The composition and structure of PS are directly responsible for its mechanical properties in a healthy lung or under pathological conditions. We previously demonstrated that PS activity improves after 72h of Whole Body Hypothermia (WBH, 33 °C) in neonates with and without lung injury. PS performance is significantly better when samples are tested under cooling condition. Moreover, decreasing neonatal lung temperature, SP-C and poli-unsaturated phospholipids are significantly reduced in surfactant large aggregates, promoting the simultaneous increase in DPPC percentage. To understand the molecular mechanisms under this temperature-dependent improvement, we designed a surfactant in vitro model. We combined surfactant lipids at different percentages (DPPC, POPG, POPC and DOPC) with purified porcine surfactant proteins SP-B and SP-C. We tested the biophysical activity of the resulting synthetic models upon breathing-like dynamics in a Captive Bubble Surfactometer, at both 37 °C and 33 °C. We also analyzed the thermotropic profiles of these protein-lipid mixtures by Differential Scanning Calorimetry. Finally, to further confirm our results, we studied both the lateral structure and the lipid composition of porcine PS once subjected to compression at the air-liquid interface. To do so, we coupled Langmuir-Blodgett Balance experiments with Epifluorescence Microscopy and Lipidomic Analysis. Our in vitro data suggests that the improvement in PS activity at 33 °C depends on a better and preferential exclusion from the air-liquid interface of less active phospholipids. This process occurs during compression (at expiration) and SP-C may play a crucial role.

Surfactant status and respiratory outcome in premature infants receiving late surfactant treatment.

BackgroundMany premature infants with respiratory failure are deficient in surfactant, but the relationship to occurrence of bronchopulmonary dysplasia (BPD) is uncertain.MethodsTracheal aspirates were collected from 209 treated and control infants enrolled at 7–14 days in the Trial of Late Surfactant. The content of phospholipid, surfactant protein-B, and total protein were determined in large aggregate (active) surfactant.Results.At 24 h, surfactant treatment transiently increased surfactant protein-B content (70%, p<0.01) but did not affect recovered airway surfactant or total protein/phospholipid. The level of recovered surfactant during dosing was directly associated with content of surfactant protein-B (r=0.50, p<0.00001) and inversely related to total protein (r=0.39, p<0.0001). For all infants, occurrence of BPD was associated with lower levels of recovered large aggregate surfactant, higher protein content and lower SP-B levels. Tracheal aspirates with lower amounts of recovered surfactant had an increased proportion of small vesicle (inactive) surfactant.Conclusions.We conclude that many intubated premature infants are deficient in active surfactant, in part due to increased intra-alveolar metabolism, low SP-B content and protein inhibition, and that the severity of this deficit is predictive of BPD.Late surfactant treatment at the frequency used did not provide a sustained increase in airway surfactant.

Structural organization of surfactant aggregates in vacuo: a molecular dynamics and well-tempered metadynamics study.

Experimental investigations using mass spectrometry have established that surfactant molecules are able to form aggregates in the gas phase. However, there is no general consensus on the organization of these aggregates and how it depends on the aggregation number and surfactant molecular structure. In the present paper we investigate the structural organization of some surfactants in vacuo by molecular dynamics and well-tempered metadynamics simulations to widely explore the space of their possible conformations in vacuo. To study how the specific molecular features of such compounds affect their organization, we have considered as paradigmatic surfactants, the anionic single-chain sodium dodecyl sulfate (SDS), the anionic double-chain sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and the zwitterionic single-chain dodecyl phosphatidyl choline (DPC) within a wide aggregation number range (from 5 to 100). We observe that for low aggregation numbers the aggregates show in vacuo the typical structure of reverse micelles, while for large aggregation numbers a variety of globular aggregates occur that are characterized by the coexistence of interlaced domains formed by the polar or ionic heads and by the alkyl chains of the surfactants. Well-tempered metadynamics simulations allows us to confirm that the structural organizations obtained after 50 ns of molecular dynamics simulations are practically the equilibrium ones. Similarities and differences of surfactant aggregates in vacuo and in apolar media are also discussed.

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

Biophysical inhibition of synthetic vs. naturally-derived pulmonary surfactant preparations by polymeric nanoparticles

Reasonable suspicion has accumulated that inhaled nano-scale particulate matter influences the biophysical function of the pulmonary surfactant system. Hence, it is evident to provide novel insights into the extent and mechanisms of nanoparticle–surfactant interactions in order to facilitate the fabrication of safe nanomedicines suitable for pulmonary applications.Negatively- and positively-charged poly(styrene) nanoparticles (diameters of ~100nm) served as model carriers. Nanoparticles were incubated with several synthetic and naturally-derived pulmonary surfactants to characterize the sensitivity of each preparation to biophysical inactivation. Changes in surface properties (i.e. adsorption and dynamic surface tension behavior) were monitored in a pulsating bubble surfactometer.Both nanoparticle formulations revealed a dose-dependent influence on the biophysical behavior of all investigated pulmonary surfactants. However, the surfactant sensitivity towards inhibition depended on both the carrier type, where negatively-charged nanoparticles showed increased inactivation potency compared to their positively-charged counterparts, and surfactant composition. Among the surfactants tested, synthetic mixtures (i.e. phospholipids, phospholipids supplemented with surfactant protein B, and Venticute®) were more susceptible to surface-activity inhibition as the more complex naturally-derived preparations (i.e. Alveofact® and large surfactant aggregates isolated from rabbit bronchoalveolar lavage fluid).Overall, nanoparticle characteristics and surfactant constitution both influence the extent of biophysical inhibition of pulmonary surfactants.

Late administration of surfactant replacement therapy increases surfactant protein-B content: a randomized pilot study

Background:Surfactant dysfunction may contribute to the development of bronchopulmonary dysplasia (BPD) in persistently ventilated preterm infants. We conducted a multicenter randomized, blinded, pilot study to assess the safety and efficacy of late administration of doses of a surfactant protein-B (SP-B)-containing surfactant (calfactant) in combination with prolonged inhaled nitric oxide (iNO) in infants ≤1,000 g birth weight (BW).Methods:We randomized 85 preterm infants ventilated at 7–14 d after birth to receive either late administration of surfactant (up to 5 doses) plus prolonged iNO or iNO alone. Large aggregate surfactant was isolated from daily tracheal aspirates (TAs) for measurement of SP-B content, total protein, and phospholipid (PL).Results:Late administration of surfactant had minimal acute adverse effects. Clinical status as well as surfactant recovery and SP-B content in tracheal aspirate were transiently improved as compared to the controls; these effects waned after 1 d. The change in SP-B content with surfactant dosing was positively correlated with SP-B levels during treatment (r = 0.50, P = 0.02).Conclusion:Low SP-B values increased with calfactant administration, but the relationship of this response to SP-B levels suggests that degradation is a contributing mechanism for SP-B deficiency and surfactant dysfunction. We conclude that late therapy with surfactant in combination with iNO is safe and transiently increases surfactant SP-B content, possibly leading to improved short- and long-term respiratory outcomes.

Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice

Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A2. Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls. These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition.

Secretory Phospholipase A2-Mediated Depletion of Phosphatidylglycerol in Early Acute Respiratory Distress Syndrome

IntroductionSecretory phospholipases A2 (sPLA2) hydrolyze phospholipids in cell membranes and extracellular structures such as pulmonary surfactant. This study tests the hypothesis that sPLA2 are elevated in human lungs during acute respiratory distress syndrome (ARDS) and that sPLA2 levels are associated with surfactant injury by hydrolysis of surfactant phospholipids. MethodsBronchoalveolar lavage (BAL) fluid was obtained from 18 patients with early ARDS (<72 hours) and compared with samples from 10 healthy volunteers. Secreted phospholipase A2 levels were measured (enzyme activity and enzyme immunoassay) in conjunction with ARDS subjects’ surfactant abnormalities including surfactant phospholipid composition, large and small aggregates distribution and surface tension function. ResultsBAL sPLA2 enzyme activity was markedly elevated in ARDS samples relative to healthy subjects when measured by ex vivo hydrolysis of both phosphatidylglycerol (PG) and phosphatidylcholine (PC). Enzyme immunoassay identified increased PLA2G2A protein in the ARDS BAL fluid, which was strongly correlated with the sPLA2 enzyme activity against PG. Of particular interest, the authors demonstrated an average depletion of 69% of the PG in the ARDS sample large aggregates relative to the normal controls. Furthermore, the sPLA2 enzyme activity against PG and PC ex vivo correlated with the BAL recovery of in vivo PG and PC, respectively, and also correlated with the altered distribution of the large and small surfactant aggregates. ConclusionsThese results support the hypothesis that sPLA2-mediated hydrolysis of surfactant phospholipid, especially PG by PLA2G2A, contributes to surfactant injury during early ARDS.

Adaptation to low body temperature influences pulmonary surfactant composition thereby increasing fluidity while maintaining appropriately ordered membrane structure and surface activity

The interfacial surface tension of the lung is regulated by phospholipid-rich pulmonary surfactant films. Small changes in temperature affect surfactant structure and function in vitro. We compared the compositional, thermodynamic and functional properties of surfactant from hibernating and summer-active 13-lined ground squirrels (Ictidomys tridecemlineatus) with porcine surfactant to understand structure-function relationships in surfactant membranes and films. Hibernating squirrels had more surfactant large aggregates with more fluid monounsaturated molecular species than summer-active animals. The latter had more unsaturated species than porcine surfactant. Cold-adapted surfactant membranes displayed gel-to-fluid transitions at lower phase transition temperatures with reduced enthalpy. Both hibernating and summer-active squirrel surfactants exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and DPH anisotropy revealed that surfactant bilayers from both groups of squirrels possessed similar ordered phase characteristics at low temperatures. While ground squirrel surfactants functioned well during dynamic cycling at 3, 25, and 37°C, porcine surfactant demonstrated poorer activity at 3°C but was superior at 37°C. Consequently the surfactant composition of ground squirrels confers a greater thermal flexibility relative to homeothermic mammals, while retaining tight lipid packing at low body temperatures. This may represent the most critical feature contributing to sustained stability of the respiratory interface at low lung volumes. Thus, while less effective than porcine surfactant at 37°C, summer-active surfactant functions adequately at both 37°C and 3°C allowing these animals to enter hibernation. Here further compositional alterations occur which improve function at low temperatures by maintaining adequate stability at low lung volumes and when temperature increases during arousal from hibernation.

Beneficial effects of synthetic KL4surfactant in experimental lung transplantation

The aim of this study was to investigate whether intratracheal administration of a new synthetic surfactant that includes the cationic, hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL₄), might be effective in reducing ischaemia-reperfusion injury after lung transplantation. Single left lung transplantation was performed in Landrace pigs 22 h post-harvest. KL₄ surfactant at a dose of 25 mg total phospholipid·kg body weight⁻¹ (2.5 mL·kg body weight⁻¹) was instilled at 37°C to the donor left lung (n = 8) prior to explantation. Saline (2.5 mL·kg body weight⁻¹; 37°C) was instilled into the donor left lung of the untreated group (n = 6). Lung function in recipients was measured during 2 h of reperfusion. Recipient left lung bronchoalveolar lavage (BAL) provided native cytometric, inflammatory marker and surfactant data. KL(4) surfactant treatment recovered oxygen levels in the recipient blood (mean ± sd arterial oxygen tension/inspiratory oxygen fraction 424 ± 60 versus 263 ± 101 mmHg in untreated group; p=0.01) and normalised alveolar-arterial oxygen tension difference. Surfactant biophysical function was also recovered in KL₄ surfactant-treated lungs. This was associated with decreased C-reactive protein levels in BAL, and recovery of surfactant protein A content, normalised protein/phospholipid ratios, and lower levels of both lipid peroxides and protein carbonyls in large surfactant aggregates. These findings suggest an important protective role for KL₄ surfactant treatment in lung transplantation.

Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60-80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60-100% oxygen, whereas levels of T1alpha, a protein expressed by type I cells, were comparably increased in mice exposed to 40-100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to > or = 60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

A Specific Phospholipase C Activity Regulates Phosphatidylinositol Levels in Lung Surfactant of Patients with Acute Respiratory Distress Syndrome

Lung surfactant (LS) is a lipid-rich material lining the inside of the lungs. It reduces surface tension at the liquid/air interface and thus, it confers protection of the alveoli from collapsing. The surface-active component of LS is dipalmitoyl-phosphatidylcholine, while anionic phospholipids such as phosphatidylinositol (PtdIns) and primarily phosphatidylglycerol are involved in the stabilization of the LS monolayer. The exact role of PtdIns in this system is not well-understood; however, PtdIns levels change dramatically during the acute respiratory distress syndrome (ARDS) evolution. In this report we present evidence of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid, which may regulate PtdIns levels. Characterization of this extracellular activity showed specificity for PtdIns and phosphatidylinositol 4,5-bisphosphate, sharing the typical substrate concentration-, pH-, and calcium-dependencies with mammalian PI-PLCs. Fractionation of BAL fluid showed that PI-PLC did not co-fractionate with large surfactant aggregates, but it was found mainly in the soluble fraction. Importantly, analysis of BAL samples from control subjects and from patients with ARDS showed that the PI-PLC specific activity was decreased by 4-fold in ARDS samples concurrently with the increase in BAL PtdIns levels. Thus, we have identified for the first time an extracellular PI-PLC enzyme activity that may be acutely involved in the regulation of PtdIns levels in LS.

Transient in utero disruption of Cystic Fibrosis Transmembrane Conductance Regulator causes phenotypic changes in Alveolar Type II cells in adult rats

BackgroundMechanicosensory mechanisms regulate cell differentiation during lung organogenesis. We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) was integral to stretch-induced growth and development and that transient expression of antisense-CFTR (ASCFTR) had negative effects on lung structure and function. In this study, we examined adult alveolar type II (ATII) cell phenotype after transient knock down of CFTR by adenovirus-directed in utero expression of ASCFTR in the fetal lung.ResultsIn comparison to (reporter gene-treated) Controls, ASCFTR-treated adult rat lungs showed elevated phosphatidylcholine (PC) levels in the large but not in the small aggregates of alveolar surfactant. The lung mRNA levels for SP-A and SP-B were lower in the ASCFTR rats. The basal PC secretion in ATII cells was similar in the two groups. However, compared to Control ATII cells, the cells in ASCFTR group showed higher PC secretion with ATP or phorbol myristate acetate. The cell PC pool was also larger in the ASCFTR group. Thus, the increased surfactant secretion in ATII cells could cause higher PC levels in large aggregates of surfactant. In freshly isolated ATII cells, the expression of surfactant proteins was unchanged, suggesting that the lungs of ASCFTR rats contained fewer ATII cells. Gene array analysis of RNA of freshly isolated ATII cells from these lungs showed altered expression of several genes including elevated expression of two calcium-related genes, Ca2+-ATPase and calcium-calmodulin kinase kinase1 (CaMkk1), which was confirmed by real-time PCR. Western blot analysis showed increased expression of calmodulin kinase I, which is activated following phosphorylation by CaMkk1. Although increased expression of calcium regulating genes would argue in favor of Ca2+-dependent mechanisms increasing surfactant secretion, we cannot exclude contribution of alternate mechanisms because of other phenotypic changes in ATII cells of the ASCFTR group.ConclusionDevelopmental changes due to transient disruption of CFTR in fetal lung reflect in altered ATII cell phenotype in the adult life.

THE EFFECTS OF HYPEROXIA EXPOSURE ON LUNG FUNCTION AND PULMONARY SURFACTANT IN A RAT MODEL OF ACUTE LUNG INJURY

The objective of this study was to determine if prolonged hyperoxia exposure would deplete antioxidants, resulting in excessive oxidative stress that would lead to oxidation of pulmonary surfactant and contribute to lung dysfunction. Rats were exposed to either hyperoxic (> 95% O2) or normoxic (21% O2) oxygen concentrations for 48 or 60 hours. Pulmonary compliance, inflammatory cells, and total protein levels were measured as indicators of lung injury. Bronchoalveolar lavage (BAL) samples were analyzed for surfactant composition, antioxidant content, and markers of oxidative stress. Antioxidants were also measured in lung tissue and plasma samples. Hyperoxia exposure for 60 hours resulted in increased protein and inflammatory cells in BAL, and lower pulmonary compliance, compared to all other groups. Total surfactant and surfactant large aggregates were increased following 48 hours of hyperoxia exposure, with a further increase following 60 hours. Animals exposed to 60 hours of hyperoxia also demonstrated lower ascorbate levels in lung tissue, increased lipid peroxides in BAL, and increased oxidation of phosphatidylglycerol species in surfactant. This study demonstrates that the balance of oxidant/antioxidant components is disrupted within the lung during periods of hyperoxia, and that although surfactant lipids may be susceptible to oxidative damage, they do not likely represent a major mechanism for the lung dysfunction observed.

SURFACTANT DYSFUNCTION IN LUNG CONTUSION WITH AND WITHOUT SUPERIMPOSED GASTRIC ASPIRATION IN A RAT MODEL

This study investigates surfactant dysfunction in rats with lung contusion (LC) induced by blunt chest trauma. Rats at 24 h postcontusion had a decreased percent content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL) and altered large-aggregate composition with decreased phosphatidylcholine (PC), increased lyso-PC, and increased protein compared with uninjured controls. The surface activity of large aggregates on a pulsating bubble surfactometer was also severely impaired at 24 h postcontusion. Decreases in large surfactant aggregate content and surface activity were improved, but still apparent, at 48 and 72 h postcontusion compared with uninjured control rats and returned to normal by 96 h postcontusion. The functional importance of surfactant abnormalities in LC injury was documented in pilot studies showing that exogenous surfactant replacement at 24 h postcontusion improved inflation/deflation lung volumes. Additional experiments investigated a clinically relevant combination of LC plus gastric aspiration (combined acid and small gastric food particles) and found reductions in large surfactant aggregates in BAL similar to those for LC. However, rats given LC + combined acid and small gastric food particles versus LC had more severe surfactant dysfunction based on decreases in surface activity and alterations in large aggregate composition. Combined data for all animal groups had strong statistical correlations between surfactant dysfunction (increased minimum surface tension, decreased large aggregates in BAL, decreased aggregate PC, and increased aggregate lyso-PC) and the severity of inflammatory lung injury (increased total protein, albumin, protein/phospholipid ratio, neutrophils, and erythrocytes in BAL plus increased whole lung myeloperoxidase activity). These results show that surfactant dysfunction is important in the pathophysiology of LC with or without concurrent gastric aspiration and provides a rationale for surfactant replacement therapy in these prevalent clinical conditions.

Surfactant Function and Composition in Premature Infants Treated With Inhaled Nitric Oxide

We hypothesized that inhaled nitric oxide treatment of premature infants at risk for bronchopulmonary dysplasia would not adversely affect endogenous surfactant function or composition. As part of the Nitric Oxide Chronic Lung Disease Trial of inhaled nitric oxide, we examined surfactant in a subpopulation of enrolled infants. Tracheal aspirate fluid was collected at specified intervals from 99 infants with birth weights <1250 g who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Large-aggregate surfactant was analyzed for surface activity with a pulsating bubble surfactometer and for surfactant protein contents with an immunoassay. At baseline, before administration of study gas, surfactant function and composition were comparable in the 2 groups, and there was a positive correlation between minimum surface tension and severity of lung disease for all infants. Over the first 4 days of treatment, minimum surface tension increased in placebo-treated infants and decreased in inhaled nitric oxide-treated infants. There were no significant differences between groups in recovery of large-aggregate surfactant or contents of surfactant protein A, surfactant protein B, surfactant protein C, or total protein, normalized to phospholipid. We conclude that inhaled nitric oxide treatment for premature infants at risk of bronchopulmonary dysplasia does not alter surfactant recovery or protein composition and may improve surfactant function transiently.

Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration

BackgroundAlterations to pulmonary surfactant composition have been encountered in the Acute Respiratory Distress Syndrome (ARDS). However, only few data are available regarding the time-course and duration of surfactant changes in ARDS patients, although this information may largely influence the optimum design of clinical trials addressing surfactant replacement therapy. We therefore examined the time-course of surfactant changes in 15 patients with direct ARDS (pneumonia, aspiration) over the first 8 days after onset of mechanical ventilation.MethodsThree consecutive bronchoalveolar lavages (BAL) were performed shortly after intubation (T0), and four days (T1) and eight days (T2) after intubation. Fifteen healthy volunteers served as controls. Phospholipid-to-protein ratio in BAL fluids, phospholipid class profiles, phosphatidylcholine (PC) molecular species, surfactant proteins (SP)-A, -B, -C, -D, and relative content and surface tension properties of large surfactant aggregates (LA) were assessed.ResultsAt T0, a severe and highly significant reduction in SP-A, SP-B and SP-C, the LA fraction, PC and phosphatidylglycerol (PG) percentages, and dipalmitoylation of PC (DPPC) was encountered. Surface activity of the LA fraction was greatly impaired. Over time, significant improvements were encountered especially in view of LA content, DPPC, PG and SP-A, but minimum surface tension of LA was not fully restored (15 mN/m at T2). A highly significant correlation was observed between PaO2/FiO2 and minimum surface tension (r = -0.83; p < 0.001), SP-C (r = 0.64; p < 0.001), and DPPC (r = 0.59; p = 0.003). Outcome analysis revealed that non-survivors had even more unfavourable surfactant properties as compared to survivors.ConclusionWe concluded that a profound impairment of pulmonary surfactant composition and function occurs in the very early stage of the disease and only gradually resolves over time. These observations may explain why former surfactant replacement studies with a short treatment duration failed to improve outcome and may help to establish optimal composition and duration of surfactant administration in future surfactant replacement studies in acute lung injury.