Intracellular Surfactant Research Articles

ErbB deletion effect on pulmonary intracellular surfactant distribution

Background: Alveolar epithelial type II cells (AEII) synthesize, store, and recycle surfactant. Lipids and primarily hydrophobic surfactant proteins (SPs) are stored in lamellar bodies (Lbs) while the hydrophilic SPs and the precursors of hydrophobic SPs are stored in multivesicular bodies (mvb). ErbB4-receptor and its ligand neuregulin (NRG) are important regulators of fetal lung development and fetal surfactant synthesis. ErbB4 deletion leads predominantly to an alveolar simplification. We hypothesized that ErbB4 deletion affects the ultrastructure of AEII, specifically Lb and the intracellular distribution of the immunomodulating hydrophilic SP-A and the hydrophobic SP-B. Material and Methods: Using a HER4 transgenic cardiac rescue mouse model, AEII were characterized stereologically in lungs of juvenile transgenic HER4heart+/− and HER4heart−/− mice. The ultrastructure of Lb and the intracellular distribution of SPs were evaluated by immune electron microscopy. A preferential nonrandom labeling of a compartment for SP is present, if the relative labeling index (RLI) > 1and the chi-quadrat test is significant. Results: HER4 deletion had no significant effects on size of AEII and volume fractions of subcellular organelles as well as on the volume weighted mean volume of Lb in HER4heart−/− when compared to HER4heart+/− mice. The cytoplasm was preferentially labeled for SP-A in the AEII of both genotypes. Lbs were preferential labeled for SP-A in the AEII of HER4heart−/−, but not in the AEII of HER4heart+/− mice. SP-B was preferentially distributed over Lbs in AEII independent of the genotype, however, the evaluated RLI was significantly higher in HER4heart−/− mice. Conclusion: HER4 deletion does not affect the ultrastructure of AEII and influence the distribution of SP-A and SP-B only moderately. Responsible for this could be compensatory mechanisms caused by the redundancy of ErbB receptors.

Novel opportunities from bioimaging to understand the trafficking and maturation of intracellular pulmonary surfactant and its role in lung diseases.

Pulmonary surfactant (PS), a complex mixture of lipids and proteins, is essential for maintaining proper lung function. It reduces surface tension in the alveoli, preventing collapse during expiration and facilitating re-expansion during inspiration. Additionally, PS has crucial roles in the respiratory system's innate defense and immune regulation. Dysfunction of PS contributes to various respiratory diseases, including neonatal respiratory distress syndrome (NRDS), adult respiratory distress syndrome (ARDS), COVID-19-associated ARDS, and ventilator-induced lung injury (VILI), among others. Furthermore, PS alterations play a significant role in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The intracellular stage involves storing and releasing a specialized subcellular organelle known as lamellar bodies (LB). The maturation of these organelles requires coordinated signaling to organize their intracellular organization in time and space. LB's intracellular maturation involves the lipid composition and critical processing of surfactant proteins to achieve proper functionality. Over a decade ago, the supramolecular organization of lamellar bodies was studied using electron microscopy. In recent years, novel bioimaging tools combining spectroscopy and microscopy have been utilized to investigate the in cellulo intracellular organization of lamellar bodies temporally and spatially. This short review provides an up-to-date understanding of intracellular LBs. Hyperspectral imaging and phasor analysis have allowed identifying specific transitions in LB's hydration, providing insights into their membrane dynamics and structure. A discussion and overview of the latest approaches that have contributed to a new comprehension of the trafficking and structure of lamellar bodies is presented.

Ultrastructural analysis of the intracellular surfactant in lungs of healthy and ovalbumin sensitized and challenged Brown Norway rats

Introduction: In human and experimentally induced asthma, a dysfunction of the intra-alveolar-surface active agent (surfactant) has been demonstrated. Type II alveolar epithelial cells (AEII) synthesize, secrete and recycle surfactant. Prior to secretion, intracellular surfactant is stored in specific secretory organelles of AEII. The lamellar bodies (Lb) represent its ultrastructural correlate. The aim of this study was to investigate whether disturbances of the intra-alveolar surfactant are accompanied by alterations in the intracellular surfactant.Material and Methods: Brown-Norway rats were sensitized twice with ovalbumin (OVA) and heat killed Bordetella pertussis bacilli. During airway challenge, an aerosol of 5% ovalbumin/saline solution (0.25 l/min) was nebulized. 24 h after airway challenge, lungs were fixed by vascular perfusion. AEII and their Lb were characterized stereologically by light and electron microscopy.Results: In both groups, AEII were structurally intact. The number of AEII per lung and their number-weighted mean volume did not differ (controls: 49 × 106, 393 µm3; asthmatics: 44 × 106, 390 µm3). A mean of 90 Lb in AEII of asthmatics and of 93 Lb in AEII of controls were evaluated. The Lb mean total volume was 59 µm in asthmatics and 68 µm in controls. Values of both parameters did not reach significance. Also, the size distribution and mean volume of Lb was not influenced by asthma induction, because the volume weighted mean volume of Lb (2.18 µm in asthmatics compared to 1.87 µm in controls) and the numerical weighted mean volume (0.96 µm in asthmatics and 0.75 µm in controls) were comparable in both groups.Conclusion: The obtained results suggest that asthma-induced surfactant dysfunction is not related to disturbances in the intracellular surfactant´s ultrastructural correlates.

Amphiphilic Proteins Coat Membraneless Organelles and Act as Biological Surfactants

The intracellular milieu is emulsion-like, with multiple membraneless compartments that assemble due to liquid-liquid phase separation. Recent reports indicate that membraneless organelles may be surrounded by a surface layer of distinct composition, forming a core-shell condensate in which both components are necessary for biological function, such as Atg19 in Ape1 autophagy. Here, we took a bio-inspired approach to understand how amphiphilic, surfactant-like proteins may alter the structure and properties of membrane-less organelles. We designed and examined a family of amphiphilic proteins, for example, GST-mCherry-RGG, MBP-RGG-GFP, and variations thereof. These amphiphilic fusion proteins contain an intrinsically disordered RGG domain rich in Arginine and Glycine, and structured domains such as Glutathione S-transferase (GST), Maltose Binding Protein (MBP), and fluorescent proteins. The RGG domain has a high propensity to phase separate, whereas GST and MBP are water-soluble. When one of these amphiphilic proteins is mixed with RGG-RGG (tandem RGG) protein in vitro, the two proteins assemble into an emulsion-like core-shell structure, with phase-separating tandem RGG protein forming the core droplet, and the amphiphilic surfactant protein forming the surface layer. Importantly, we found that the MBP-RGG-GFP surfactant concentration controls droplet size. At low surfactant concentrations, the protein droplets were relatively large, while the droplet size decreased at higher surfactant concentrations. Moreover, we demonstrated enzymatic control of surfactant protein adsorption at the interface using TEV protease. Interestingly, mixing GST-mCherry-RGG and MBP-RGG-GFP with tandem RGG led to competitive adsorption favoring GST-mCherry-RGG. Based on these experiments, we propose a model and mechanism of intracellular surfactant proteins that adsorb to membraneless organelles. This work advances our understanding of the relationship between multi-component structure and function of biomolecular condensates, pointing towards the significant role that surfactant proteins may play in regulating the biochemistry of biomolecular condensates.

Mechanical ventilation-induced alterations of intracellular surfactant pool and blood\u2013gas barrier in healthy and pre-injured lungs

Mechanical ventilation triggers the manifestation of lung injury and pre-injured lungs are more susceptible. Ventilation-induced abnormalities of alveolar surfactant are involved in injury progression. The effects of mechanical ventilation on the surfactant system might be different in healthy compared to pre-injured lungs. In the present study, we investigated the effects of different positive end-expiratory pressure (PEEP) ventilations on the structure of the blood–gas barrier, the ultrastructure of alveolar epithelial type II (AE2) cells and the intracellular surfactant pool (= lamellar bodies, LB). Rats were randomized into bleomycin-pre-injured or healthy control groups. One day later, rats were either not ventilated, or ventilated with PEEP = 1 or 5 cmH2O and a tidal volume of 10 ml/kg bodyweight for 3 h. Left lungs were subjected to design-based stereology, right lungs to measurements of surfactant proteins (SP−) B and C expression. In pre-injured lungs without ventilation, the expression of SP-C was reduced by bleomycin; while, there were fewer and larger LB compared to healthy lungs. PEEP = 1 cmH2O ventilation of bleomycin-injured lungs was linked with the thickest blood–gas barrier due to increased septal interstitial volumes. In healthy lungs, increasing PEEP levels reduced mean AE2 cell size and volume of LB per AE2 cell; while in pre-injured lungs, volumes of AE2 cells and LB per cell remained stable across PEEPs. Instead, in pre-injured lungs, increasing PEEP levels increased the number and decreased the mean size of LB. In conclusion, mechanical ventilation-induced alterations in LB ultrastructure differ between healthy and pre-injured lungs. PEEP = 1 cmH2O but not PEEP = 5 cmH2O ventilation aggravated septal interstitial abnormalities after bleomycin challenge.

Aging impairs alveolar epithelial type II cell function in acute lung injury.

Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 μg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.

Cell fate analysis in fetal mouse lung reveals distinct pathways for TI and TII cell development

Alveolar type I (TI) cells are large squamous cells that cover >95% of the internal surface area of the lung; type II (TII) cells are small cuboidal cells with distinctive intracellular surfactant storage organelles. Based on autoradiographic studies in the 1970s, the long-held paradigm of alveolar epithelial development has been a linear progression from undifferentiated progenitor cells through TII cells to TI cells. Subsequent data support the existence of more complex pathways. Recently, a bipotent TI/TII progenitor cell at embryonic day E18 has been inferred both from marker expression in developing airways and from statistical analyses of gene expression data obtained from single-lung embryonic cells. To study cell lineage directly by fate mapping, we developed new transgenic mouse models in which rtTA is driven either by the rat podoplanin or the mouse Sftpc gene to mark cells irreversibly in development. Using these models, we found two distinct lineage pathways. One pathway, evident as early as E12-15, is devoted almost exclusively to TI cell development; a second pathway gives rise predominantly to TII cells but also a subpopulation of TI cells. We have defined the molecular phenotypes of these distinct progenitor populations and have identified potential regulatory factors in TI and TII cell differentiation. By analyzing gene pathways in mature TI and TII cells, we identified potential novel functions of each cell type. These results provide novel insights into lung development and suggest a basis for testing strategies to promote alveolar differentiation and repair, including potential transplantation of lineage-specific progenitor cells.

Air-blood barrier thickening and alterations of alveolar epithelial type 2 cells in mouse lungs with disrupted hepcidin/ferroportin regulatory system.

Iron accumulates in the lungs of patients with common respiratory diseases or transfusion-dependent beta-thalassemia. Based on our previous work, we hypothesized that systemic iron overload affects the alveolar region of the lung and in particular the surfactant producing alveolar epithelial type II (AE2) cells. Mice with a point mutation in the iron exporter ferroportin, a model for human hemochromatosis type 4 were compared to wildtype mice (n = 5 each). Lungs were fixed and prepared for light and electron microscopy (EM) according to state-of-the-art protocols to detect subcellular iron localization by scanning EM/EDX and to perform design-based stereology. Iron was detected as electron dense particles in membrane-bound organelles, likely lysosomes, in AE1 cells. AE2 cells were higher in number but had a lower mean volume in mutated mice. Lamellar body volume per AE2 cell was lower but total volume of lamellar bodies in the lung was comparable to wildtype mice. While the volume of alveoli was lower in mutated mice, the volume of alveolar ducts as well as the surface area, volume and the mean thickness and composition of the septa was similar in both genotypes. The thickness of the air-blood barrier was greater in the mutated than in the WT mice. In conclusion, disruption of systemic iron homeostasis affects the ultrastructure of interalveolar septa which is characterized by membrane-bound iron storage in AE1 cells, thickening of the air-blood barrier and hyperplasia and hypotrophy of AE2 cells despite normal total intracellular surfactant pools. The functional relevance of these findings requires further analysis to better understand the impact of iron on intra-alveolar surfactant function.

Stereological assessment of the blood-air barrier and the surfactant system after mesenchymal stem cell pretreatment in a porcine non-heart-beating donor model for lung transplantation.

More frequent utilization of non-heart-beating donor (NHBD) organs for lung transplantation has the potential to relieve the shortage of donor organs. In particular with respect to uncontrolled NHBD, concerns exist regarding the risk of ischaemia/reperfusion (IR) injury-related graft damage or dysfunction. Due to their immunomodulating and tissue-remodelling properties, bone-marrow-derived mesenchymal stem cells (MSCs) have been suspected of playing a beneficial role regarding short- and long-term survival and function of the allograft. Thus, MSC administration might represent a promising pretreatment strategy for NHBD organs. To study the initial effects of warm ischaemia and MSC application, a large animal lung transplantation model was generated, and the structural organ composition of the transplanted lungs was analysed stereologically with particular respect to the blood-gas barrier and the surfactant system. In this study, porcine lungs (n = 5/group) were analysed. Group 1 was the sham-operated control group. In pigs of groups 2-4, cardiac arrest was induced, followed by a period of 3 h of ventilated ischaemia at room temperature. In groups 3 and 4, 50 × 106 MSCs were administered intravascularly via the pulmonary artery and endobronchially, respectively, during the last 10 min of ischaemia. The left lungs were transplanted, followed by a reperfusion period of 4 h. Then, lungs were perfusion-fixed and processed for light and electron microscopy. Samples were analysed stereologically for IR injury-related structural parameters, including volume densities and absolute volumes of parenchyma components, alveolar septum components, intra-alveolar oedema, and the intracellular and intra-alveolar surfactant pool. Additionally, the volume-weighted mean volume of lamellar bodies (lbs) and their profile size distribution were determined. Three hours of ventilated warm ischaemia was tolerated without eliciting histological or ultrastructural signs of IR injury, as revealed by qualitative and quantitative assessment. However, warm ischaemia influenced the surfactant system. The volume-weighted mean volume of lbs was reduced significantly (P = 0.024) in groups subjected to ischaemia (group medians of groups 2-4: 0.180-0.373 μm³) compared with the sham control group (median 0.814 μm³). This was due to a lower number of large lb profiles (size classes 5-15). In contrast, the intra-alveolar surfactant system was not altered significantly. No significant differences were encountered comparing ischaemia alone (group 2) or ischaemia plus application of MSCs (groups 3 and 4) in this short-term model.

Stereological and biophysical characteristics of the ovine surfactant system and its changes caused by ovine pulmonary adenocarcinoma

Surfactant covers the inner surface of lung alveoli and lowers the surface tension to prevent alveoli from collapsing. A lack of surfactant or its dysfunction causes dyspnea. The Jaagsiekte Sheep Retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA), whose typical clinical appearance is fluid running from nostrils. This fluid might contain surfactant as alveolar type II pneumocytes (AEII) are target cells for JSRV. Therefore, the progressive dyspnea during OPA might be caused partially by surfactant alterations.Bronchoalveolar and intracellular surfactant as well as the biophysical function of surfactant were analyzed in OPA sheep and controls. Transmission electron microscopy and stereological methods were used to characterize ultrastructure and distribution of surfactant subtypes in AEII and bronchoalveolar lavage fluid (BALF). Pulsating Bubble Surfactometry enabled studying the surface activity of the surfactant, while lung volumes were detected by computed tomography.The methods used are suitable to determine intraalveolar and intracellular surfactant subtypes in OPA sheep and controls. OPA sheep showed more lamellar body-like forms, multivesicular vesicles and tubular myelin in BALF compared to controls. These higher amounts of active surfactant subtypes might be a consequence of a higher surfactant production and release. Surfactant subtypes in AEII of OPA sheep showed smaller and more immature lamellar bodies compared to controls. The surfactant surface activity of OPA sheep does not show obvious defects. In conclusion, the general quality of surfactant in OPA appears to be equivalent to surfactant produced in controls, however, dyspnea of OPA might be triggered by quantity of fluid production.

Lung remodeling in aging surfactant protein D deficient mice

Pulmonary surfactant, a mixture of lipids and proteins at the air–liquid interface of alveoli, prevents the lungs from collapsing due to surface tension. One constituent is surfactant-associated protein-D (SP-D), a protein involved in surfactant homeostasis and innate immunity. Mice deficient in SP-D (SP-D (−/−)) has been described as developing a characteristic phenotype which affects the surfactant system (including changes in the intra-cellular and intra-alveolar surfactant pool, alveolar epithelial type II cells and alveolar macrophages), lung architecture and its inflammatory state (development of an emphysema-like pathology, inflammatory cell infiltration). Furthermore, it has been described that these mice develop sub-pleural fibrosis and a thickening of alveolar septal walls. The aim of the present study was to systematically investigate the long term progression of this phenotype with special focus on parenchymal remodeling, whether there are progressive emphysematous changes and whether there is progressive septal wall thickening which might indicate the development of pulmonary fibrosis. By means of design-based stereology and light microscopy, lungs of wild type (wt) and SP-D (−/−) mice of four age groups (3, 6, 12 and ∼18 months) were investigated. The data do not suggest a relevant spontaneous pro-fibrotic remodeling or a destructive process in the aging SP-D (−/−) mice. We demonstrated neither a significant destructive emphysema nor significant thickening of alveolar septal walls, but the data suggest an increase in the number weighted mean alveolar volume in aging SP-D (−/−) mice without loss of alveoli or alveolar epithelial surface area per lung. This increase may reflect over-distension due to altered mechanical properties of alveoli. In the light of our findings and data from the literature, the question arises as to whether a lack of SP-D promotes structural changes in the lung which have been described as being associated with aging lungs. Furthermore, data from wt mice point to an ongoing alveolarization during adult life in C57Bl/6NCrl mice. Our data may promote further studies on the morphological development of the aging lung and the role of SP-D in this process.

ABCA3, a key player in neonatal respiratory transition and genetic disorders of the surfactant system.

Genetic disorders of the surfactant system are rare diseases with a broad range of clinical manifestations, from fatal respiratory distress syndrome (RDS) in neonates to chronic interstitial lung disease (ILD) in children and adults. ABCA3 [ATP-binding cassette (ABC), subfamily A, member 3] is a lung-specific phospholipid transporter critical for intracellular surfactant synthesis and storage in lamellar bodies (LBs). Its expression is developmentally regulated, peaking prior to birth under the influence of steroids and transcription factors. Bi-allelic mutations of the ABCA3 gene represent the most frequent cause of congenital surfactant deficiency, indicating its critical role in lung function. Mutations affect surfactant lipid and protein processing and LBs' morphology, leading to partial or total surfactant deficiency. Approximately 200 mutations have been reported, most of which are unique to individuals and families, which makes diagnosis and prognosis challenging. Various typesof mutations, affecting different domains of the protein, account in part for phenotype diversity. Disease-causing mutations have been reported in most coding and some non-coding regions of the gene, but tend to cluster in the first extracellular loop and the second nucleotide-binding domain (NBD), leading to defective glycosylation and trafficking defects and interfering with ATP binding and hydrolysis respectively. Mono-allelic damaging and benign variants are often subclinical but may act as disease modifiers in lung diseases such as RDS of prematurity or associate with mutations in other surfactant-related genes. Diagnosis is complex but essential and should combine pathology and ultrastructure studies on lung biopsy with broad-spectrum genetic testing of surfactant-related genes, made possible by recent technology advances in the massive parallel sequencing technology.

Linking progression of fibrotic lung remodeling and ultrastructural alterations of alveolar epithelial type II cells in the amiodarone mouse model.

Chronic injury of alveolar epithelial type II cells (AE2 cells) represents a key event in the development of lung fibrosis in animal models and in humans, such as idiopathic pulmonary fibrosis (IPF). Intratracheal delivery of amiodarone to mice results in a profound injury and macroautophagy-dependent apoptosis of AE2 cells. Increased autophagy manifested in AE2 cells by disturbances of the intracellular surfactant. Hence, we hypothesized that ultrastructural alterations of the intracellular surfactant pool are signs of epithelial stress correlating with the severity of fibrotic remodeling. With the use of design-based stereology, the amiodarone model of pulmonary fibrosis in mice was characterized at the light and ultrastructural level during progression. Mean volume of AE2 cells, volume of lamellar bodies per AE2 cell, and mean size of lamellar bodies were correlated to structural parameters reflecting severity of fibrosis like collagen content. Within 2 wk amiodarone leads to an increase in septal wall thickness and a decrease in alveolar numbers due to irreversible alveolar collapse associated with alveolar surfactant dysfunction. Progressive hypertrophy of AE2 cells and increase in mean individual size and total volume of lamellar bodies per AE2 cell were observed. A high positive correlation of these AE2 cell-related ultrastructural changes and the deposition of collagen fibrils within septal walls were established. Qualitatively, similar alterations could be found in IPF samples with mild to moderate fibrosis. We conclude that ultrastructural alterations of AE2 cells including the surfactant system are tightly correlated with the progression of fibrotic remodeling.

NOS2 Is Critical to the Development of Emphysema in Sftpd Deficient Mice but Does Not Affect Surfactant Homeostasis

RationaleSurfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.ObjectiveIn order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).MethodsStructural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd−/−) and iNOS single knockout mice (NOS2−/−) as well as wild type (WT) littermates.Measurements and ResultsDiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd−/− demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.ConclusioniNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.

Profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement

BackgroundWe previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen’s protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment. MethodsDonor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation. ResultsPretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes. ConclusionDonor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen’s protective effects against I/R injury during transplantation.

NOS2 mediates lung structure and function changes in the SP-D model of emphysema without improving surfactant homeostasis

The collectin Surfactant protein D (SP-D) is an innate host defense protein with well-established immuno-modulatory properties. Mice deficient in SP-D (SP-D−/−) spontaneously develop typical emphysematous and inflammatory alterations: a significant decrease in alveolar number, accumulation of enlarged alveolar macrophages overproduction of the inducible nitric oxide synthase (iNOS), hypertrophy and hyperplasia of alveolar type II cells and disturbances of surfactant homoeostasis. Using design-based stereology, wild type, iNOS(−/−), SP-D(−/−) and double knockout (DiNOS) mice at 12 weeks of age were measured for a series of structural parameters. Lung resistance and elastance as functions of frequency at positive end-expiratory pressures in the range of 0–9 cm H2O were measured using Flexivent. The additional ablation of the iNOS gene improved emphysematous alterations in the SP-D(−/−) mouse resulting in a significant increase in alveolar number and a reduction in mean alveolar volume. DiNOS mice exhibited reduced amount of enlarged alveolar macrophages and altered their inflammatory phenotype relative to SP-D(−/−) mice. Neither disturbances of intracellular surfactant homeostasis nor the hyperplasia or hypertrophy of alveolar type II cells were corrected by iNOS ablation. However, despite a lack of effect on surfactant homeostasis, iNOS ablation did normalize lung function. The increased iNOS-activity in SP-D(−/−) mice is critical to the development of inflammation and consequent emphysema but is not important in altered surfactant homeostasis, implying that emphysema and alterations of type II cells are mediated via different pathways. The structural remodeling appears to dominate in terms of lung function.

Lung preservation in experimental ischemia/reperfusion injury and lung transplantation: A comparison of natural and synthetic surfactants

Surfactant inactivation results from ischemia/reperfusion injury and plays a major role in the pathogenesis of primary graft dysfunction after clinical lung transplantation. Thus, prophylactic administration of exogenous surfactant preparations before the onset of ischemia/reperfusion has proven to be effective in preserving pulmonary structure and function. Various natural and synthetic surfactant preparations exhibit differences regarding the biochemical composition and biophysical properties. In this study we compared the efficacy of preservation of pulmonary structure and function of the natural surfactant preparations Curosurf and Survanta to that of a synthetic surfactant containing an analog of surfactant protein C (SPC-33) in a rat model of ischemia/reperfusion injury. The oxygenation capacity and peak inspiratory pressure during the reperfusion period were recorded. By applying design-based stereology at the light- and electron-microscopic level, pathologic alterations, including alveolar edema, injury of the blood-air barrier and the intra-alveolar as well as intracellular surfactant pools, were quantified. The best oxygenation and preservation of lung structure was achieved with Curosurf. Survanta treatment was associated with the most severe injury of the blood-air barrier, and SPC-33 demonstrated signs of microatelectasis. The intra-alveolar surfactant pool after Curosurf and SPC-33 was dominated by active surfactant subtypes, whereas Survanta was associated with the highest fraction of inactive surfactant. The intracellular surfactant pool did not show any differences between the treatment groups. Taken together, Curosurf achieved the best structural and functional lung preservation, whereas Survanta was inferior to both Curosurf and SPC-33.

Ultrastructural changes of the intracellular surfactant pool in a rat model of lung transplantation-related events

BackgroundIschemia/reperfusion (I/R) injury, involved in primary graft dysfunction following lung transplantation, leads to inactivation of intra-alveolar surfactant which facilitates injury of the blood-air barrier. The alveolar epithelial type II cells (AE2 cells) synthesize, store and secrete surfactant; thus, an intracellular surfactant pool stored in lamellar bodies (Lb) can be distinguished from the intra-alveolar surfactant pool. The aim of this study was to investigate ultrastructural alterations of the intracellular surfactant pool in a model, mimicking transplantation-related procedures including flush perfusion, cold ischemia and reperfusion combined with mechanical ventilation.MethodsUsing design-based stereology at the light and electron microscopic level, number, surface area and mean volume of AE2 cells as well as number, size and total volume of Lb were determined in a group subjected to transplantation-related procedures including both I/R injury and mechanical ventilation (I/R group) and a control group.ResultsAfter I/R injury, the mean number of Lb per AE2 cell was significantly reduced compared to the control group, accompanied by a significant increase in the luminal surface area per AE2 cell in the I/R group. This increase in the luminal surface area correlated with the decrease in surface area of Lb per AE2. The number-weighted mean volume of Lb in the I/R group showed a tendency to increase.ConclusionWe suggest that in this animal model the reduction of the number of Lb per AE2 cell is most likely due to stimulated exocytosis of Lb into the alveolar space. The loss of Lb is partly compensated by an increased size of Lb thus maintaining total volume of Lb per AE2 cell and lung. This mechanism counteracts at least in part the inactivation of the intra-alveolar surfactant.

Fine structure of tubular myelin from mammalian lungs

Tubular myelin and intracellular surfactant images are presented from human, rabbit, rat, and dog lung. The samples represent a long‐term study of normal as well as cystic fibrosis specimens. Tubular myelin is seen in its typical lattice‐like form as it is found on the extracellular surface. Surfactant within lamellar bodies is also shown with a unique relationship to mitochondria. Images from lung parenchyma and rabbit lung washes reveal the complex architecture of tubular myelin. Specimens are fixed with glutaraldehyde and secondary fix common to electron microscopy protocols. Comparative study of cystic fibrosis samples and normal lung did not reveal any differences in tubular myelin pattern. It is hoped that an analysis of these specimens will yield further information regarding cellular surfactant production, its arrangement, and its appearance on the cell surface in both the human normal and disease condition.

Effect of Perfluorohexane on the Expression of Cellular Adhesion Molecules and Surfactant Protein A in Human Mesothelial Cells In Vitro

The intraperitoneal instillation of perfluorocarbons augmented systemic oxygenation and was protective in mesenteric ischemia-reperfusion and experimental lung injury. To study biocompatibility and potential anti-inflammatory effects of intraperitoneal perfluorocarbons, we evaluated the influence of perfluorohexane and/or inflammatory stimuli on human mesothelial cells in vitro. Perfluorohexane exposure neither impaired cell viability nor induced cellular activation. TNFα enhanced ICAM-1 expression, which was not attenuated by simultaneous perfluorohexane treatment. Concentration of intracellular surfactant protein A tended to be higher in perfluorohexane treated cells compared to controls. Our in vitro data add further evidence that intraperitoneal perfluorocarbon application is feasible without adverse local effects.