Olive oil and herbs, two key components of the Mediterranean diet, are known for their beneficial effects on humans. In our study, we incorporated aromatic and medicinal herbs into local monovarietal olive oils via maceration procedures for enrichment. We identified the herbal-derived ingredients that migrate to olive oils and contribute positively to their total phenolic content and functional properties, such as radical scavenging activity. Thus, we characterized the essential oil composition of the aromatic herbs (GC-MS), and we determined the phenolic content and antioxidant capacity of the additives and the virgin olive oils before and after enrichment. The herbal phenolic compounds were analyzed by LC-LTQ/Orbitrap HRMS. We found that olive oils infused with Origanum vulgare ssp. hirtum, Rosmarinus officinalis and Salvia triloba obtained an increased phenolic content, by approximately 1.3 to 3.4 times, in comparison with the untreated ones. Infusion with S. triloba led to a significantly higher antioxidant capacity. Rosmarinic acid, as well as phenolic glucosides, identified in the aromatic herbs, were not incorporated into olive oils due to their high polarity. In contrast, phenolic aglycones and diterpenes from R. officinalis and S. triloba migrated to the enriched olive oils, leading to a significant increase in their phenolic content and to an improvement in their free radical scavenging capacity.
Antioxidants , Plants, Medicinal , Humans , Antioxidants/chemistry , Olive Oil/chemistry , Phytochemicals , Rosmarinic Acid , Plant Oils/chemistry
Antimicrobial peptides (AMPs) are a chemically and structurally heterogeneous family of molecules produced by a large variety of living organisms, whose expression is predominant in the sites most exposed to microbial invasion. One of the richest natural sources of AMPs is insects which, over the course of their very long evolutionary history, have adapted to numerous and different habitats by developing a powerful innate immune system that has allowed them to survive but also to assert themselves in the new environment. Recently, due to the increase in antibiotic-resistant bacterial strains, interest in AMPs has risen. In this work, we detected AMPs in the hemolymph of Hermetia illucens (Diptera, Stratiomyidae) larvae, following infection with Escherichia coli (Gram negative) or Micrococcus flavus (Gram positive) and from uninfected larvae. Peptide component, isolated via organic solvent precipitation, was analyzed by microbiological techniques. Subsequent mass spectrometry analysis allowed us to specifically identify peptides expressed in basal condition and peptides differentially expressed after bacterial challenge. We identified 33 AMPs in all the analyzed samples, of which 13 are specifically stimulated by Gram negative and/or Gram positive bacterial challenge. AMPs mostly expressed after bacterial challenge could be responsible for a more specific activity.
Red blood cells (RBCs) release hemoglobin (Hb)-containing extracellular vesicles (EVs) throughout their lifespan in the circulation, and especially during senescence, by spleen-facilitated vesiculation of their membrane. During ex vivo aging under blood bank conditions, the RBCs lose Hb, both in soluble form and inside EVs that accumulate as a part of storage lesion in the supernatant of the unit. Spontaneous hemolysis and vesiculation are increasingly promoted by the storage duration, but little is known about any physiological linkage between them. In the present study, we measured the levels of total extracellular and EV-enclosed Hb (EV-Hb) in units of whole blood (n = 36) or packed RBCs stored in either CPDA-1 (n = 99) or in CPD-SAGM additive solution (n = 46), in early, middle, and late storage. The spectrophotometry data were subjected to statistical analysis to detect possible correlation(s) between storage hemolysis and EV-Hb, as well as the threshold (if any) that determines the area of this dynamic association. It seems that the percentage of EV-Hb is negatively associated with hemolysis levels from middle storage onward by showing low to moderate correlation profiles in all strategies under investigation. Moreover, 0.17% storage hemolysis was determined as the potential cut-off, above which this inverse correlation is evident in non-leukoreduced CPDA units. Notably, RBC units with hemolysis levels > 0.17% are characterized by higher percentage of nanovesicles (<100 nm) over typical microvesicles (100-400 nm) compared with the lower hemolysis counterparts. Our results suggest an ordered loss of Hb during RBC accelerated aging that might fuel targeted research to elucidate its mechanistic basis.
Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor's characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (ßThal+). For this purpose, RBC units from fourteen ßThal+ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the ßThal+ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the ßThal+ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in ßThal+ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that ßThal+ erythrocytes maintain a considerable "proteo-vigilance" during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.
Acute respiratory distress syndrome (ARDS) is a multifactorial life-threatening lung injury, characterized by diffuse lung inflammation and increased alveolocapillary barrier permeability. The different stages of ARDS have distinctive biochemical and clinical profiles. Despite the progress of our understanding on ARDS pathobiology, the mechanisms underlying its pathogenesis are still obscure. Herein, we review the existing literature about the implications of phospholipases 2 (PLA2s), a large family of enzymes that catalyze the hydrolysis of fatty acids at the sn-2 position of glycerophospholipids, in ARDS-related pathology. We emphasize on the versatile way of participation of different PLA2s isoforms in the distinct ARDS subgroup phenotypes by either potentiating lung inflammation and damage or by preserving the normal lung. Current research supports that PLA2s are associated with the progression and the outcome of ARDS. We herein discuss the transcellular communication of PLA2s through secreted extracellular vesicles and suggest it as a new mechanism of PLA2s involvement in ARDS. Thus, the elucidation of the spatiotemporal features of PLA2s expression may give new insights and provide valuable information about the risk of an individual to develop ARDS or advance to more severe stages, and potentially identify PLA2 isoforms as biomarkers and target for pharmacological intervention.
Extracellular Vesicles , Respiratory Distress Syndrome , Biomarkers , Extracellular Vesicles/metabolism , Humans , Inflammation , Phospholipases A2/metabolism , Respiratory Distress Syndrome/diagnosis
Secretory phospholipase-IIA A2 (sPLA2-IIA) is expressed in a variety of cell types under inflammatory conditions. Its presence in the bronchoalveolar lavage (BAL) fluid of patients with acute respiratory distress syndrome (ARDS) is associated with the severity of the injury. Exosomal type extracellular vesicles, (EVs), are recognized to perform intercellular communication. They may alter the immune status of recipient target cells through cargo shuttling. In this work, we characterized the exosomal type EVs isolated from BAL fluid of patients with early and late ARDS as compared to control/non-ARDS patients, through morphological (confocal and electron microscopy) and biochemical (dynamic light scattering, qRT-PCR, immunoblotting) approaches. We provide evidence for the presence of an sPLA2-IIA-carrying EV pool that coprecipitates with exosomes in the BAL fluid of patients with ARDS. PLA2G2A mRNA was present in all the samples, although more prominently expressed in early ARDS. However, the protein was found only in EVs from early phase ARDS. Under both forms, sPLA2-IIA might be involved in inflammatory responses of recipient lung cells during ARDS. The perception of the association of sPLA2-IIA to the early diagnosis of ARDS or even with a mechanism of development and propagation of lung inflammation can help in the adoption of appropriate and innovative therapeutic strategies.
Helleborus cyclophyllus Boiss is a rhizomatous plant species, with strong allelochemical properties, that has been used since ancient times for its therapeutic properties. In the present study we investigated the ability of an aqueous-soluble fraction of the methanol extract of H. cyclophyllus Boiss leaves, to induce apoptotic cell death on A549 human bronchial epithelial adenocarcinoma cells. A primary human lung fibroblasts' cell line was used as a model of normal-healthy cells for comparison. Cell morphology was examined after appropriate staining, cytotoxic activity of the extract was determined by the MTT assay, the type of cell death was analyzed by flow cytometry, confirmation of apoptosis was evaluated with the analysis of caspase-3, PARP1 by western blotting, while the chemical composition was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). H. cyclophyllus Boiss extract was selectively active on A549 cells inducing significant morphological changes, even at low concentrations. Characteristic morphological alterations included the release of vesicular formations from A549 cell membranes (ectosomes), detachment of cells from their substrate, generation of a large vesicle into the cytoplasm (thanatosome) and the formation of apoptotic bodies. The selective apoptotic action on treated cells was also confirmed by biochemical criteria. Low concentrations, however, did not affect normal cells. The phytochemical analysis of the extract revealed the presence of cardiac glucosides, bufadienolides and phytoecdysteroids. To the best of our knowledge, the above-mentioned sequences of events leading selectively cancer cells to apoptosis, has not been reported before.
Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F17 KRIV21 QR23 IK25 DF27 LR-NH2 were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R23 and K25 in the hydrophilic side, V21 and F27 in the hydrophobic side of the interphase, and F17 that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL25 DFLR-NH2 resulted in significant cytotoxicity against A549 cancer cells with an IC50 value 3.90 µg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQI23 IKK26 FLR-NH2 , which incorporates the IIKK motif and the peptides FKRIVQL23 L24 KK26 L27 LR-NH2 and Ac-FKRIVQL23 L24 KK26 L27 LR-NH2 , which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3-7.5 µg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC50 12.9-9.8 µg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Antineoplastic Agents/pharmacology , Candida parapsilosis/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , A549 Cells , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Microbial Sensitivity Tests
Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P ⺠hypereosinophilic syndrome.
Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Docosahexaenoic Acids/pharmacology , Leukemia/metabolism , Cell Line, Tumor , Gene Expression/drug effects , Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , Humans , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism
Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.
Extracellular Vesicles/physiology , Animals , Cell Communication , Cell-Derived Microparticles/physiology , Clinical Trials as Topic , Drug Delivery Systems , Exosomes/physiology , Humans , Nanomedicine , Theranostic Nanomedicine
The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A(2) (PLA(2)) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C(12)-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA(2). Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA(2). Mass spectrometry showed that only C(12)-NBD-FA, the PLA(2 )hydrolysis product, was detected in the reaction mixture, indicating that PLA(2) recognizes PVA-SbQ/C(12)-NBD-PtdCho as a surface to perform catalysis.
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Phosphatidylcholines/metabolism , Phospholipases A2/metabolism , Pulmonary Alveoli/enzymology , Respiratory Mucosa/enzymology , 4-Chloro-7-nitrobenzofurazan/chemistry , 4-Chloro-7-nitrobenzofurazan/metabolism , Animals , Chromatography, High Pressure Liquid , Computational Biology , Feasibility Studies , Fluorescent Dyes/chemistry , Humans , Hydrogen-Ion Concentration , Limit of Detection , Membranes, Artificial , Microscopy, Atomic Force , Phosphatidylcholines/chemistry , Phospholipases A2/blood , Pilot Projects , Polyvinyl Alcohol/analogs & derivatives , Polyvinyl Alcohol/chemistry , Pyridinium Compounds/chemistry , Reproducibility of Results , Spectrometry, Fluorescence , Stereoisomerism , Substrate Specificity , Sus scrofa
Azithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages.
Azithromycin/pharmacology , Lung/drug effects , Macrophages, Alveolar/drug effects , Phospholipases A2/metabolism , Respiratory Distress Syndrome/metabolism , Respiratory Mucosa/drug effects , Case-Control Studies , Cell Line, Tumor , Humans , Lipopolysaccharides/pharmacology , Lung/cytology , Lung/metabolism , Macrophages, Alveolar/metabolism , Organ Specificity , Phospholipases A2/genetics , Respiratory Mucosa/metabolism
Dipalmitoylphosphatidylcholine, (DP-PtdCho), the major phospholipid component of lung surfactant is biosynthesized via a de novo pathway, the last step of which is catalyzed by CDP-choline:cholinephosphotransferase (CPT) and two remodeling steps: a deacylation and a reacylation one, catalyzed by an acidic, Ca²âº-independent phospholipase A2 (aiPLA2) and a lyso-phosphatidylcholine acyltransferase (LPCAT), respectively. The aim of our study was to investigate whether a low magnitude, non-injurious static mode of mechanical stretch can induce phosphatidylcholine (PtdCho) biosynthesis and its remodeling to DP-PtdCho in the A549 cell-line, a model of alveolar type II cells. The deformation of A549 cells did not cause any release of lactate dehydrogenase, or phospholipids into the cell culture supernatants. An increase in PtdCho levels was observed after 1 h of static stretching, especially among the DP-PtdCho molecular species, as indicated by targeted lipidomics approach and site-directed fatty acyl-chain analysis. Moreover, although sphingomyelin (CerPCho) levels were unaffected, the DP-PtdCho/CerPCho ratio increased. Induction was observed in CPT, LPCAT and aiPLA2 enzymatic activities and gene expression. Finally, incubation of the cells with MJ33 suppressed aiPLA2 activity and DP-PtdCho production. Our data suggest that mild static mechanical stretch can promote the biosynthesis of PtdCho and its remodeling to DP-PtdCho in lung epithelial cells. Thus, low magnitude stretch could contribute to protective mechanisms rather than to injurious ones.
1,2-Dipalmitoylphosphatidylcholine/biosynthesis , Pulmonary Alveoli/cytology , 1-Acylglycerophosphocholine O-Acyltransferase/genetics , 1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Cell Line/drug effects , Cell Survival , Diacylglycerol Cholinephosphotransferase/metabolism , Gene Expression Regulation , Glycerophosphates/pharmacology , Humans , L-Lactate Dehydrogenase/metabolism , Peroxiredoxin VI/genetics , Phospholipases A2/metabolism , Phospholipids/metabolism , Pulmonary Alveoli/metabolism , Sphingomyelins/metabolism , Stress, Mechanical
Alveolar epithelial type II cells (AT II) in which lung surfactant synthesis and secretion take place, are subjected to low magnitude stretch during normal breathing. The aim of the study was to explore the effect of mild stretch on phospholipase A(2) (PLA(2)) activation, an enzyme known to be involved in surfactant secretion. In A549 cells (a model of AT II cells), we showed, using a fluorometric assay, that stretch triggers an increase of total PLA(2) activity. Western blot experiments revealed that the cytosolic isoform cPLA(2) is rapidly phosphorylated under stretch, in addition to a modest increase in cPLA(2) mRNA levels. Treatment of A549 cells with selective inhibitors of the MEK/ERK pathway significantly attenuated the stretch-induced cPLA(2) phosphorylation. A strong interaction of cPLA(2) and pERK enzymes was demonstrated by immunoprecipitation. We also found that inhibition of PI3K pathway attenuated cPLA(2) activation after stretch, without affecting pERK levels. Our results suggest that low magnitude stretch can induce cPLA(2) phosphorylation through the MEK/ERK and PI3K-Akt pathways, independently.
Epithelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipases A2, Cytosolic/metabolism , Androstadienes/pharmacology , Butadienes/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cell Survival , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Flavonoids/pharmacology , Fluorometry , Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , Humans , Immunoblotting , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Nitriles/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phospholipases A2, Cytosolic/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Stress, Mechanical , Time Factors , Wortmannin
The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AMΦ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A2 (PLA2) activity and TLR4 expression were evaluated as markers of cell response. AMΦ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA2 production. On the contrary, upon stimulation, in control patients the intracellular PLA2, (mainly cPLA2) levels were increased, but secretion of PLA2 (mainly sPLA2-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA2 production in cells from both groups of patients. Increased relative changes of total PLA2 activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA2 induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA2, its activated form, p-cPLA2, and sPLA2-IIA were the predominant PLA2 types within the cells, while extracellularly only sPLA2-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA2 isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.
Macrophages, Alveolar/enzymology , Monocytes/enzymology , Phospholipases A2/metabolism , Respiratory Distress Syndrome/enzymology , Adult , Aged , Biomarkers/metabolism , Blotting, Western , Cells, Cultured , Female , Flow Cytometry , Humans , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Alveolar/drug effects , Male , Middle Aged , Monocytes/drug effects , Respiratory Distress Syndrome/immunology , Toll-Like Receptor 4/metabolism , Young Adult
INTRODUCTION: Activated Protein C (APC), an endogenous anticoagulant, improves tissue microperfusion and endothelial cell survival in systemic inflammatory states such as sepsis, but intravenous administration may cause severe bleeding. We have thus addressed the role of APC delivered locally by inhalation in preventing acute lung injury from alveolar overdistention and the subsequent ventilator-induced lung injury (VILI). We also assessed the effects of APC on the activation status of Extracellular- Regulated Kinase 1/2 (ERK) pathway, which has been shown to be involved in regulating pulmonary responses to mechanical stretch. METHODS: Inhaled APC (12.5 microg drotrecogin-alpha x 4 doses) or saline was given to tracheotomized C57/Bl6 mice starting 20 min prior to initiation of injurious mechanical ventilation with tidal volume 25 mL/Kg for 4 hours and then hourly thereafter; control groups receiving inhaled saline were ventilated with 8 mL/Kg for 30 min or 4 hr. We measured lung function (respiratory system elastance H), arterial blood gases, surrogates of vascular leak (broncho-alveolar lavage (BAL) total protein and angiotensin-converting enzyme (ACE)-activity), and parameters of inflammation (BAL neutrophils and lung tissue myeloperoxidase (MPO) activity). Morphological alterations induced by mechanical ventilation were examined in hematoxylin-eosin lung tissue sections. The activation status of ERK was probed in lung tissue homogenates by immunoblotting and in paraffin sections by immunohistochemistry. The effect of APC on ERK signaling downstream of the thrombin receptor was tested on A549 human lung epithelial cells by immunoblotting. Statistical analyses were performed using ANOVA with appropriate post-hoc testing. RESULTS: In mice subjected to VILI without APC, we observed hypoxemia, increased respiratory system elastance and inflammation, assessed by BAL neutrophil counts and tissue MPO activity. BAL total protein levels and ACE activity were also elevated by VILI, indicating compromise of the alveolo-capillary barrier. In addition to preserving lung function, inhaled APC prevented endothelial barrier disruption and attenuated hypoxemia and the inflammatory response. Mechanistically, we found a strong activation of ERK in lung tissues by VILI, which was prevented by APC, suggestive of pathogenetic involvement of the Mitogen-Activated Kinase pathway. In cultured human lung epithelial cells challenged by thrombin, APC abrogated the activation of ERK and its downstream effector, cytosolic Phospholipase A2. CONCLUSIONS: Topical application of APC by inhalation may effectively reduce lung injury induced by mechanical ventilation in mice.
Anticoagulants/administration & dosage , Anticoagulants/pharmacology , Protein C/administration & dosage , Protein C/pharmacology , Ventilator-Induced Lung Injury/prevention & control , Administration, Inhalation , Animals , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred C57BL
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.
Phosphatidylinositol Phosphates/metabolism , Phosphoinositide Phospholipase C/metabolism , Pulmonary Surfactants/metabolism , Respiratory Distress Syndrome/enzymology , Adult , Aged , Bronchoalveolar Lavage Fluid , Calcium/metabolism , Demography , Extracellular Space/enzymology , Female , Humans , Hydrolysis , Intracellular Space/enzymology , Male , Middle Aged , Phosphatidylinositol 4,5-Diphosphate , Respiratory Distress Syndrome/pathology , Substrate Specificity
Phospholipases A2 (PLA2) catalyse the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids. In acute lung injury-acute respiratory distress syndrome (ALI-ARDS) several distinct isoenzymes appear in lung cells and fluid. Some are capable to trigger molecular events leading to enhanced inflammation and lung damage and others have a role in lung surfactant recycling preserving lung function: Secreted forms (groups sPLA2-IIA, -V, -X) can directly hydrolyze surfactant phospholipids. Cytosolic PLA2 (cPLA2-IVA) requiring Ca2+ has a preference for arachidonate, the precursor of eicosanoids which participate in the inflammatory response in the lung. Ca(2+)-independent intracellular PLA2s (iPLA2) take part in surfactant phospholipids turnover within alveolar cells. Acidic Ca(2+)-independent PLA2 (aiPLA2), of lysosomal origin, has additionally antioxidant properties, (peroxiredoxin VI activity), and participates in the formation of dipalmitoyl-phosphatidylcholine in lung surfactant. PAF-AH degrades PAF, a potent mediator of inflammation, and oxidatively fragmented phospholipids but also leads to toxic metabolites. Therefore, the regulation of PLA2 isoforms could be a valuable approach for ARDS treatment.
Phospholipases A2/classification , Phospholipases A2/metabolism , Respiratory Distress Syndrome/enzymology , Animals , Humans , Respiratory Distress Syndrome/therapy
INTRODUCTION: Use of the prone position in patients with acute lung injury improves their oxygenation. Most of these patients die from multisystem organ failure and not from hypoxia, however. Moreover, there is some evidence that the organ failure is caused by increased cell apoptosis. In the present study we therefore examined whether the position of the patients affects histological changes and apoptosis in the lung and 'end organs', including the brain, heart, diaphragm, liver, kidneys and small intestine. METHODS: Ten mechanically ventilated sheep with a tidal volume of 15 ml/kg body weight were studied for 90 minutes. Five sheep were placed in the supine position and five sheep were placed in the prone position during the experiment. Lung changes were analyzed histologically using a semiquantitative scoring system and the extent of apoptosis was investigated with the TUNEL method. RESULTS: In the supine position intra-alveolar hemorrhage appeared predominantly in the dorsal areas, while the other histopathologic lesions were homogeneously distributed throughout the lungs. In the prone position, all histological changes were homogeneously distributed. A significantly higher score of lung injury was found in the supine position than in the prone position (4.63 +/- 0.58 and 2.17 +/- 0.19, respectively) (P < 0.0001). The histopathologic changes were accompanied by increased apoptosis (TUNEL method). In the supine position, the apoptotic index in the lung and in most of the 'end organs' was significantly higher compared with the prone position (all P < 0.005). Interestingly, the apoptotic index was higher in dorsal areas compared with ventral areas in both the prone and supine positions (P < 0.003 and P < 0.02, respectively). CONCLUSION: Our results suggest that the prone position appears to reduce the severity and the extent of lung injury, and is associated with decreased apoptosis in the lung and 'end organs'.
Lung/pathology , Prone Position/physiology , Respiration, Artificial/adverse effects , Respiratory Distress Syndrome/pathology , Supine Position/physiology , Animals , Lung/physiology , Multiple Organ Failure/etiology , Multiple Organ Failure/pathology , Multiple Organ Failure/therapy , Respiration, Artificial/methods , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/therapy , Sheep , Tidal Volume/physiology
OBJECTIVE: To identify diagnostic markers distinguishing between acute lung injury/acute respiratory distress syndrome (ALI/ARDS) due to fat embolism syndrome (FES) and that due to other causes, and to investigate whether phospholipase A2 and platelet-activating factor (PAF) play a role in the pathogenesis of ALI due to FES. DESIGN AND SETTING: A prospective study in a 14-bed ICU. PATIENTS: We studied 13 patients with FES, 11 with ALI/ARDS from other causes (6 without trauma, ALI/ARDS group 1; 7 with trauma, ALI/ARDS group 2) and 5 without cardiopulmonary disease. MEASUREMENTS AND RESULTS: We compared broncholveolar lavage (BAL) fluid alterations in the respective groups. Total BAL protein in FES group was significantly higher compared to in ALI/ARDS group 1 and controls but ALI/ARDS group 2. Higher total phospholipids were found than in other groups. The alterations in individual phospholipid classes were similar to those in ALI/ARDS patients. However, total cholesterol, lipid esters, and monoglycerides were significantly higher in FES than in other groups. The level of PAF in FES was significantly higher and there was an inverse correlation between PAF and PAF-acetylhydrolase. Phospholipase A2 activity was significantly higher in both FES and ALI/ARDS groups than in control. CONCLUSIONS: The levels of neutral lipids and especially cholesterol and cholesterol esters in BAL can be used to distinguish patients with FES from ALI/ARDS due to other predisposing factors. Phospholipase A(2) may be involved in the development, and PAF-acetylhydrolase in the downregulation of inflammation in FES.
Embolism, Fat/complications , Embolism, Fat/diagnosis , Phospholipases A/metabolism , Platelet Activating Factor/metabolism , Respiratory Distress Syndrome/etiology , Adolescent , Adult , Biomarkers/metabolism , Bronchoalveolar Lavage Fluid/immunology , Case-Control Studies , Causality , Diagnosis, Differential , Embolism, Fat/metabolism , Female , Humans , Male , Middle Aged , Phospholipases A2 , Prospective Studies , Respiratory Distress Syndrome/metabolism , Wounds and Injuries/complications , Wounds and Injuries/metabolism
