DODAB vesicles containing lysophosphatidylcholines: The relevance of acyl chain saturation on the membrane structure and thermal properties.

The saturated LPC18:0 and unsaturated LPC18:1 lysophosphatidylcholines have important roles in inflammation and immunity and are interesting targets for immunotherapy. The synthetic cationic lipid DODAB has been successfully employed in delivery systems, and would be a suitable carrier for those lysophosphatidylcholines. Here, assemblies of DODAB and LPC18:0 or LPC18:1 were characterized by Differential Scanning Calorimetry (DSC) and Electron Paramagnetic Resonance (EPR) spectroscopy. LPC18:0 increased the DODAB gel-fluid transition enthalpy and rigidified both phases. In contrast, LPC18:1 caused a decrease in the DODAB gel-fluid transition temperature and cooperativity, associated with two populations with distinct rigidities in the gel phase. In the fluid phase, LPC18:1 increased the surface order but, differently from LPC18:0, did not affect viscosity at the membrane core. The impact of the different acyl chains of LPC18:0 and 18:1 on structure and thermotropic behavior should be considered when developing applications using mixed DODAB membranes.

Modes of action of lysophospholipids as endogenous activators of the TRPV4 ion channel.

The Transient Receptor Potential Vanilloid 4 (TRPV4) channel has been shown to function in many physiological and pathophysiological processes. Despite abundant information on its importance in physiology, very few endogenous agonists for this channel have been described, and very few underlying mechanisms for its activation have been clarified. TRPV4 is expressed by several types of cells, such as vascular endothelial, and skin and lung epithelial cells, where it plays pivotal roles in their function. In the present study, we show that TRPV4 is activated by lysophosphatidic acid (LPA) in both endogenous and heterologous expression systems, pinpointing this molecule as one of the few known endogenous agonists for TRPV4. Importantly, LPA is a bioactive glycerophospholipid, relevant in several physiological conditions, including inflammation and vascular function, where TRPV4 has also been found to be essential. Here we also provide mechanistic details of the activation of TRPV4 by LPA and another glycerophospholipid, lysophosphatidylcholine (LPC), and show that LPA directly interacts with both the N- and C-terminal regions of TRPV4 to activate this channel. Moreover, we show that LPC activates TRPV4 by producing an open state with a different single-channel conductance to that observed with LPA. Our data suggest that the activation of TRPV4 can be finely tuned in response to different endogenous lipids, highlighting this phenomenon as a regulator of cell and organismal physiology. KEY POINTS: The Transient Receptor Potential Vaniloid (TRPV) 4 ion channel is a widely distributed protein with important roles in normal and disease physiology for which few endogenous ligands are known. TRPV4 is activated by a bioactive lipid, lysophosphatidic acid (LPA) 18:1, in a dose-dependent manner, in both a primary and a heterologous expression system. Activation of TRPV4 by LPA18:1 requires residues in the N- and C-termini of the ion channel. Single-channel recordings show that TRPV4 is activated with a decreased current amplitude (conductance) in the presence of lysophosphatidylcholine (LPC) 18:1, while LPA18:1 and GSK101 activate the channel with a larger single-channel amplitude. Distinct single-channel amplitudes produced by LPA18:1 and LPC18:1 could differentially modulate the responses of the cells expressing TRPV4 under different physiological conditions.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

A novel receptor for platelet-activating factor and lysophosphatidylcholine in Trypanosoma cruzi.

The lipid mediators, platelet-activating factor (PAF) and lysophosphatidylcholine (LPC), play relevant pathophysiological roles in Trypanosoma cruzi infection. Several species of LPC, including C18:1 LPC, which mimics the effects of PAF, are synthesized by T. cruzi. The present study identified a receptor in T. cruzi, which was predicted to bind to PAF, and found it to be homologous to members of the progestin and adiponectin family of receptors (PAQRs). We constructed a three-dimensional model of the T. cruzi PAQR (TcPAQR) and performed molecular docking to predict the interactions of the TcPAQR model with C16:0 PAF and C18:1 LPC. We knocked out T. cruzi PAQR (TcPAQR) gene and confirmed the identity of the expressed protein through immunoblotting and immunofluorescence assays using an anti-human PAQR antibody. Wild-type and knockout (KO) parasites were also used to investigate the in vitro cell differentiation and interactions with peritoneal mouse macrophages; TcPAQR KO parasites were unable to react to C16:0 PAF or C18:1 LPC. Our data are highly suggestive that PAF and LPC act through TcPAQR in T. cruzi, triggering its cellular differentiation and ability to infect macrophages.

Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

BACKGROUND: Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. METHODS: In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. RESULTS: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. CONCLUSIONS: Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

Interplay Between EGFR and the Platelet-Activating Factor/PAF Receptor Signaling Axis Mediates Aggressive Behavior of Cervical Cancer.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase widely expressed in cervical tumors, being correlated with adverse clinical outcomes. EGFR may be activated by a diversity of mechanisms, including transactivation by G-protein coupled receptors (GPCRs). Studies have also shown that platelet-activating factor (PAF), a pro-inflammatory phospholipid mediator, plays an important role in the cancer progression either by modulating the cancer cells or the tumor microenvironment. Most of the PAF effects seem to be mediated by the interaction with its receptor (PAFR), a member of the GPCRs family. PAFR- and EGFR-evoked signaling pathways contribute to tumor biology; however, the interplay between them remains uninvestigated in cervical cancer. In this study, we employed The Cancer Genome Atlas (TCGA) and cancer cell lines to evaluate possible cooperation between EGFR, PAFR, and lysophosphatidylcholine acyltransferases (LPCATs), enzymes involved in the PAF biosynthesis, in the context of cervical cancer. It was observed a strong positive correlation between the expression of EGFR × PAFR and EGFR × LPCAT2 in 306 cervical cancer samples. The increased expression of LPCAT2 was significantly correlated with poor overall survival. Activation of EGFR upregulated the expression of PAFR and LPCAT2 in a MAPK-dependent fashion. At the same time, PAF showed the ability to transactivate EGFR leading to ERK/MAPK activation, cyclooxygenase-2 (COX-2) induction, and cell migration. The positive crosstalk between the PAF-PAFR axis and EGFR demonstrates a relevant linkage between inflammatory and growth factor signaling in cervical cancer cells. Finally, combined PAFR and EGFR targeting treatment impaired clonogenic capacity and viability of aggressive cervical cancer cells more strongly than each treatment separately. Collectively, we proposed that EGFR, LPCAT2, and PAFR emerge as novel targets for cervical cancer therapy.

Bioactive lipids regulate Trypanosoma cruzi development.

Trypanosoma cruzi is the etiological agent of Chagas disease. These parasites undergo dramatic morphological and physiological changes during their life cycle. The human-infective metacyclic trypomastigotes differentiate from epimastigotes inside the midgut of the Triatominae insect vector. Our group has shown that the saliva and feces of Rhodnius prolixus contains a lysophospholipid, lysophosphatidylcholine (LPC), which modulates several aspects of T. cruzi infection in macrophages. LPC hydrolysis by a specific lysophospholipase D, autotaxin (ATX), generates lysophosphatidic acid (LPA). These bioactive lysophospholipids are multisignaling molecules and are found in human plasma ingested by the insect during blood feeding. Here, we show the role of LPC and LPA in T. cruzi proliferation and differentiation. Both lysophospholipids are able to induce parasite proliferation. We observed an increase in parasite growth with different fatty acyl chains, such as C18:0, C16:0, or C18:1 LPC. The dynamics of LPC and LPA effect on parasite proliferation was evaluated in vivo through a time- and space-dependent strategy in the vector gut. LPC but not LPA was also able to affect parasite metacyclogenesis. Finally, we determined LPA and LPC distribution in the parasite itself. Such bioactive lipids are associated with reservosomes of T. cruzi. To the best of our knowledge, this is the first study to suggest the role of surrounding bioactive lipids ingested during blood feeding in the control of parasite transmission.

Lysophosphatidylcholine Induces NLRP3 Inflammasome-Mediated Foam Cell Formation and Pyroptosis in Human Monocytes and Endothelial Cells.

Foam cells are specialized lipid-loaded macrophages derived from monocytes and are a key pathological feature of atherosclerotic lesions. Lysophosphatidylcholine (LPC) is a major lipid component of the plasma membrane with a broad spectrum of proinflammatory activities and plays a key role in atherosclerosis. However, the role of LPC in lipid droplet (LD) biogenesis and the modulation of inflammasome activation is still poorly understood. In the present study, we investigated whether LPC can induce foam cell formation through an analysis of LD biogenesis and determined whether the cell signaling involved in this process is mediated by the inflammasome activation pathway in human endothelial cells and monocytes. Our results showed that LPC induced foam cell formation in both types of cells by increasing LD biogenesis via a NLRP3 inflammasome-dependent pathway. Furthermore, LPC induced pyroptosis in both cells and the activation of the inflammasome with IL-1ß secretion, which was dependent on potassium efflux and lysosomal damage in human monocytes. The present study described the IL-1ß secretion and foam cell formation triggered by LPC via an inflammasome-mediated pathway in human monocytes and endothelial cells. Our results will help improve our understanding of the relationships among LPC, LD biogenesis, and NLRP3 inflammasome activation in the pathogenesis of atherosclerosis.

Lipidomic Profiling of Plasma and Erythrocytes From Septic Patients Reveals Potential Biomarker Candidates.

BACKGROUND: Sepsis remains the primary cause of death from infection, despite advances in modern medicine. The identification of reliable diagnostic biomarkers for the early detection of this disease is critical and may reduce the mortality rate as it could allow early treatment. The purpose of this study was to describe the changes in the plasma and red cells blood lipidome profiling of patients diagnosed with sepsis and septic shock with the aim to identify potentially useful metabolic markers. METHODS: Lipids from plasma and erythrocytes from septic patients (n = 20) and healthy controls (n = 20) were evaluated by electrospray ionization quadrupole time-of-flight mass spectrometry, and the fatty acid composition of the phospholipids fraction of erythrocytes was determined by gas chromatography. The data were treated with multivariate data analysis, including principal component analysis and (orthogonal) partial least squares discriminant analysis. RESULTS: Potential biomarkers including lysophosphatidylcholines (lyso-PCs) and sphingomyelin (SMs) with specific fatty acid chains were identified. Both Lyso-PCs and SMs were downregulated, whereas the saturated and unsaturated phosphatidylcholines (PCs) were upregulated in the plasma and erythrocytes of septic patients. An increase in oleic acid (C18:1 n-9) accompanied by a decrease in the unsaturation index as well as in the levels on n-3 polyunsaturated fatty acids was observed in erythrocytes phospholipids patients as compared with healthy controls. CONCLUSIONS: These results suggest that lipidome profiling has great potential in discovering potential clinical biomarkers for sepsis and helping to understand its underlying mechanisms.

Triatoma infestans relies on salivary lysophosphatidylcholine to enhance Trypanosoma cruzi transmission.

Triatoma infestans is a mandatory haematophagous vector of Chagas disease in Brazil. Despite a large number of studies on the anti-haemostatic molecules present in its saliva, the role of its salivary components on parasite transmission is poorly understood. Here, we show that the bioactive lipid molecule, lysophosphatidylcholine (LPC), is present in the salivary gland of T. infestans. We characterized the lipid profiles of each unit of the T. infestans salivary gland. We noticed that LPC is present in the three units of the salivary gland and that the insect feeding state does not influence its proportion. T. infestans saliva and LPC can enhance T. cruzi transmission to mice by dramatically altering the profile of inflammatory cells at the site of inoculation on mouse skin, facilitating the transmission of T. cruzi to the vertebrate host. Consequently, the mortality curves of either saliva- or LPC-injected mice display significant higher mortality rates than the control. Altogether, these results implicate LPC as one of key salivary molecule involved in Chagas disease transmission.

Regulation of thermoTRPs by lipids.

The family of Transient Receptor Potential (TRP) ion channels is constituted by 7 subfamilies among which are those that respond to temperature, the thermoTRPs. These channels are versatile molecules of a polymodal nature that have been shown to be modulated in various fashions by molecules of a lipidic nature. Some of these molecules interact directly with the channels on specific regions of their structures and some of these promote changes in membrane fluidity or modify their gating properties in response to their agonists. Here, we have discussed how some of these lipids regulate the activity of thermoTRPs and included some of the available evidence for the molecular mechanisms underlying their effects on these channels.

Role of ERK1/2 activation and nNOS uncoupling on endothelial dysfunction induced by lysophosphatidylcholine.

BACKGROUND AND AIMS: Lysophosphatidylcholine (LPC) - a main component of oxidized LDL - is involved in endothelial dysfunction that precedes atherosclerosis, with an increased superoxide anions and a reduced NO production via endothelial NO synthase (eNOS) uncoupling. However, there is no evidence about the mechanisms involved in neuronal NOS (nNOS) uncoupling. Extracellular signal-regulated kinase (ERK) is related to the control of NO production and inflammatory gene transcription activation in atherosclerosis. Our aim was to investigate the role of nNOS/ERK1/2 pathway on endothelial dysfunction induced by LPC, in mouse aorta and human endothelial cells. METHODS: Thoracic aorta from wild type mice was used to perform vascular reactivity studies in the presence or absence of LPC. Human endothelial cells were used to investigate the effect of LPC on expression of nNOS and his products NO and H2O2. RESULTS: LPC reduced acetylcholine (ACh)-induced vasodilation in mouse aorta (EmaxCT/LPC = âˆ¼95 ± 2/62 ± 3%, p = 0.0004) and increased phenylephrine-induced vasoconstriction (EmaxCT/LPC = âˆ¼4 ± 0,1/6 ± 0,1 mN/mm, p = 0.0002), with a reduction in NO (fluorescence intensityCT/LPC = 91 ± 3/62±2 × 103, p = 0.0002) and H2O2 (fluorescence intensityCT/LPC = âˆ¼16 ± 0,8/10 ± 0,7 × 103, p = 0.0041) production evocated by ACh. An inhibition of nNOS by TRIM (EmaxCT/CT+TRIM = âˆ¼93 ± 1/43 ± 3%, p = 0,0048; EmaxLPC/LPC+TRIM = âˆ¼62 ± 3/65 ± 3%) or H2O2 degradation by catalase (EmaxCT/CT+cat = âˆ¼93 ± 1/46 ± 2%, p < 0,001; EmaxLPC/LPC+cat = âˆ¼62,8 ± 3,2/60,5 ± 4,7%) reduced the relaxation in the control but not in LPC group. PD98059, an ERK1/2 inhibitor, abolished the increase in vasoconstriction in LPC-treated vessels (EmaxLPC/LPC+PD = âˆ¼6 ± 0,1/3 ± 0,1 mN/mm, p = 0.0001). LPC also reduced the dimer/monomer proportion and increased nNOSser852 phosphorylation. CONCLUSIONS: LPC induced nNOS uncoupling and nNOSSer852 phosphorylation, reduced NO and H2O2 production and improved superoxide production by modulating ERK1/2 activity in human and murine endothelial cells.

Schistosomal-derived lysophosphatidylcholine triggers M2 polarization of macrophages through PPARγ dependent mechanisms.

Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFß and production of IL-10 and PGE2 24h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis.

Here, There, and Everywhere: The Ubiquitous Distribution of the Immunosignaling Molecule Lysophosphatidylcholine and Its Role on Chagas Disease.

Chagas disease is a severe illness, which can lead to death if the patients are not promptly treated. The disease is caused by the protozoan parasite Trypanosoma cruzi, which is mostly transmitted by a triatomine insect vector. There are 8-10 million people infected with T. cruzi in the world, but the transmission of such disease by bugs occurs only in the Americas, especially Latin America. Chronically infected patients will develop cardiac diseases (30%) and up digestive, neurological, or mixed disorders (10%). Lysophosphatidylcholine (LPC) is the major phospholipid component of oxidized low-density lipoproteins associated with atherosclerosis-related tissue damage. Insect-derived LPC powerfully attracts inflammatory cells to the site of the insect bite, enhances parasite invasion, and inhibits the production of nitric oxide by T. cruzi-stimulated macrophages. The recognition of the ubiquitous presence of LPC on the vector saliva, its production by the parasite itself and its presence both on patient plasma and its role on diverse host × parasite interaction systems lead us to compare its distribution in nature with the title of the famous Beatles song "Here, There and Everywhere" recorded exactly 50 years ago in 1966. Here, we review the major findings pointing out the role of such molecule as an immunosignaling modulator of Chagas disease transmission. Also, we believe that future investigation of the connection of this ubiquity and the immune role of such molecule may lead in the future to novel methods to control parasite transmission, infection, and pathogenesis.