Oxidized phospholipids as novel mediators of neurodegeneration.

Neurodegeneration drives the progression of many neurological diseases. Inflammation and oxidative stress occurring in the CNS promote lipid peroxidation, leading to the generation of oxidized phospholipids such as oxidized phosphatidylcholines (OxPCs). OxPCs have been proposed as biomarkers of oxidative stress, where their detection in lesions in multiple sclerosis (MS), frontotemporal lobe dementia, spinal cord injury, and amyotrophic lateral sclerosis (ALS) implies that oxidative insult had occurred. However, recent findings highlight OxPCs as potent neurotoxic species requiring neutralization by microglia. Here, we summarize the science of OxPCs, including lessons from non-CNS diseases. We discuss the potential of OxPCs as common drivers of injury across neurological conditions and encourage investigations of OxPCs as novel neurotoxins.

Oxidized phosphatidylcholines found in multiple sclerosis lesions mediate neurodegeneration and are neutralized by microglia.

Neurodegeneration occurring in multiple sclerosis (MS) contributes to the progression of disability. It is therefore important to identify and neutralize the mechanisms that promote neurodegeneration in MS. Here, we report that oxidized phosphatidylcholines (OxPCs) found in MS lesions, previously identified as end-product markers of oxidative stress, are potent drivers of neurodegeneration. Cultured neurons and oligodendrocytes were killed by OxPCs, and this was ameliorated by microglia. After OxPC injection, mouse spinal cords developed focal demyelinating lesions with prominent axonal loss. The depletion of microglia that accumulated in OxPC lesions exacerbated neurodegeneration. Single-cell RNA sequencing of lesioned spinal cords identified unique subsets of TREM2high mouse microglia responding to OxPC deposition. TREM2 was detected in human MS lesions, and TREM2-/- mice exhibited worsened OxPC lesions. These results identify OxPCs as potent neurotoxins and suggest that enhancing microglia-mediated OxPC clearance via TREM2 could help prevent neurodegeneration in MS.

Oxidized phosphatidylcholines trigger ferroptosis in cardiomyocytes during ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner (P < 0.05). POVPC and PONPC also caused a significant decrease in Ca2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells (P < 0.05). PONPC depressed maximal respiration rate (P < 0.01; 54%) and spare respiratory capacity (P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease.NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli.

AIMS: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs. METHODS AND RESULTS: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease. CONCLUSION: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.

Toxicological assessment of arsenic-containing phosphatidylcholines in HepG2 cells.

Arsenolipids include a wide range of organic arsenic species that occur naturally in seafood and thereby contribute to human arsenic exposure. Recently arsenic-containing phosphatidylcholines (AsPCs) were identified in caviar, fish, and algae. In this first toxicological assessment of AsPCs, we investigated the stability of both the oxo- and thioxo-form of an AsPC under experimental conditions, and analyzed cell viability, indicators of genotoxicity and biotransformation in human liver cancer cells (HepG2). Precise toxicity data could not be obtained owing to the low solubility in the cell culture medium of the thioxo-form, and the ease of hydrolysis of the oxo-form, and to a lesser degree the thioxo-form. Hydrolysis resulted amongst others in the respective constituent arsenic-containing fatty acid (AsFA). Incubation of the cells with oxo-AsPC resulted in a toxicity similar to that determined for the hydrolysis product oxo-AsFA alone, and there were no indices for genotoxicity. Furthermore, the oxo-AsPC was readily taken up by the cells resulting in high cellular arsenic concentrations (50 µM incubation: 1112 ± 146 µM As cellular), whereas the thioxo-AsPC was substantially less bioavailable (50 µM incubation: 293 ± 115 µM As cellular). Speciation analysis revealed biotransformation of the AsPCs to a series of AsFAs in the culture medium, and, in the case of the oxo-AsPC, to as yet unidentified arsenic species in cell pellets. The results reveal the difficulty of toxicity studies of AsPCs in vitro, indicate that their toxicity might be largely governed by their arsenic fatty acid content and suggest a multifaceted human metabolism of food derived complex arsenolipids.

Fibrin-Targeted Polymerized Shell Microbubbles as Potential Theranostic Agents for Surgical Adhesions.

The development of new therapies for surgical adhesions has proven to be difficult as there is no consistently effective way to assess treatment efficacy in clinical trials without performing a second surgery, which can result in additional adhesions. We have developed lipid microbubble formulations that use a short peptide sequence, CREKA, to target fibrin, the molecule that forms nascent adhesions. These targeted polymerized shell microbubbles (PSMs) are designed to allow ultrasound imaging of early adhesions for diagnostic purposes and for evaluating the success of potential treatments in clinical trials while acting as a possible treatment. In this study, we show that CREKA-targeted microbubbles preferentially bind fibrin over fibrinogen and are stable for long periods of time (∼48 h), that these bound microbubbles can be visualized by ultrasound, and that neither these lipid-based bubbles nor their diagnostic-ultrasound-induced vibrations damage mesothelial cells in vitro. Moreover, these bubbles show the potential to identify adhesionlike fibrin formations and may hold promise in blocking or breaking up fibrin formations in vivo.

Syntheses and cytotoxicity of phosphatidylcholines containing ibuprofen or naproxen moieties.

In this study, novel phosphatidylcholines containing ibuprofen or naproxen moieties were synthesized in good yields and high purities. Under the given synthesis conditions, the attached drug moieties racemized, which resulted in the formation of phospholipid diastereomers. The comperative studies of the cytotoxicity of ibuprofen, naproxen and their phosphatidylcholine derivatives against human promyelocytic leukemia HL-60, human colon carcinoma Caco-2, and porcine epithelial intestinal IPEC-J2 cells were carried out. The results of these studies indicated that phospholipids with NSAIDs at both sn-1 and sn-2 positions (15 and 16) were more toxic than ibuprofen or naproxen themselves, whereas 2-lysophosphatidylcholines (7 and 8) were less toxic against all tested cell lines. Phospholipids with NSAIDs at sn-1 and palmitic acid at sn-2 (9 and 10) were also less toxic against Caco-2 and normal cells (IPEC-J2).

High selective and sensitive detection of Zn(II) using tetrapeptide-based dansyl fluorescent chemosensor and its application in cell imaging.

The zinc ions (Zn2+) play extremely irreplaceable role in the organism and the environment, the design and synthesis of a biomolecule-based fluorescence chemosensor for the detection of Zn2+ with high sensitivity is very important. Herein, a novel tetrapeptide-based dansyl fluorescent "turn-on" response chemosensor (L) has been designed and synthesized by solid phase peptide synthesis (SPPS). As designed, L can detect Zn2+ ions with specifically and sensitively based on photo-induced electron transfer (PET) mechanism in 100% aqueous solutions, and other metal ions do not interfere with Zn2+ ions recognition. The stoichiometric ratio of L with Zn2+ ions was 2:1, which matches with fluorescence titration and Job-plot assay. In addition, the reversibility and circularly process of the detection of L was confirmed by adding bonding agent Na2EDTA. Moreover, L exhibits excellent biocompatibility and low biotoxicity with the limit of detection (LOD) for Zn2+ about 18 nM, and has been successfully utilized for fluorescence imaging of Zn2+ ions in living HeLa cells under physiological conditions.

Synthesis of lipid-black phosphorus quantum dot bilayer vesicles for near-infrared-controlled drug release.

Black phosphorus quantum dots are incorporated into liposomal bilayers to produce a drug delivery system with excellent near-infrared (NIR) photothermal properties and drug release capability controlled by light. In vitro experiments demonstrate its good biocompatibility and NIR-light-induced chemo-photothermal antitumor efficiency.

Dibucaine in Ionic-Gradient Liposomes: Biophysical, Toxicological, and Activity Characterization.

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 µM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.

Nano-formulation for topical treatment of precancerous lesions: skin penetration, in vitro, and in vivo toxicological evaluation.

With the aim of improving the topical delivery of the antineoplastic drug 5-fluorouracil (5FU), it was loaded into ultradeformable liposomes composed of soy phosphatidylcholine and sodium cholate (UDL-5FU). The liposome populations had a mean size of 70 nm without significant changes in 56 days, and the ultradeformable formulations were up to 324-fold more elastic than conventional liposomes. The interaction between 5FU and the liposomal membrane was studied by three methods, and also release profile was obtained. UDL-5FU did penetrate the stratum corneum of human skin. At in vitro experiments, the formulation was more toxic on a human melanoma-derived than on a human keratinocyte-derived cell line. Cells captured liposomes by metabolically active processes. In vivo toxicity experiments were carried out in zebrafish (Danio rerio) larvae by studying the swimming activity, morphological changes, and alterations in the heart rate after incubation. UDL-5FU was more toxic than free 5FU. Therefore, this nano-formulation could be useful for topical application in deep skin precancerous lesions with advantages over current treatments. This is the first work that assessed the induction of apoptosis, skin penetration in a Saarbrücken penetration model, and the toxicological effects in vivo of an ultradeformable 5FU-loaded formulation.

Nutrients Turned into Toxins: Microbiota Modulation of Nutrient Properties in Chronic Kidney Disease.

In chronic kidney disease (CKD), accumulation of uremic toxins is associated with an increased risk of death. Some uremic toxins are ingested with the diet, such as phosphate and star fruit-derived caramboxin. Others result from nutrient processing by gut microbiota, yielding precursors of uremic toxins or uremic toxins themselves. These nutrients include l-carnitine, choline/phosphatidylcholine, tryptophan and tyrosine, which are also sold over-the-counter as nutritional supplements. Physicians and patients alike should be aware that, in CKD patients, the use of these supplements may lead to potentially toxic effects. Unfortunately, most patients with CKD are not aware of their condition. Some of the dietary components may modify the gut microbiota, increasing the number of bacteria that process them to yield uremic toxins, such as trimethylamine N-Oxide (TMAO), p-cresyl sulfate, indoxyl sulfate and indole-3 acetic acid. Circulating levels of nutrient-derived uremic toxins are associated to increased risk of death and cardiovascular disease and there is evidence that this association may be causal. Future developments may include maneuvers to modify gut processing or absorption of these nutrients or derivatives to improve CKD patient outcomes.

Cationic DOPC-Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals.

Histopathological and ultra-structural characterization of local neuromuscular damage induced by repeated phosphatidylcholine/deoxycholate injection.

Phosphatidylcholine/deoxycholate (PC/DC) combination is frequently used for injection lipolysis in body contouring and size reduction of subcutaneous lipomas. Nonetheless, studies that assess possible injurious effects of PC/DC combination on tissues at injection sites are inadequate. The current work attempts to evaluate the effects of repeated PC/DC injection on skeletal muscles and neural tissues at the injection site. For this purpose, female Wistar rats were randomly assigned into 2 groups, 10 rats each, and injected percutaneously via either normal saline (control group) or PC/DC (treated group) in the groin area for 4 consecutive days. Biopsies were harvested on the 4(th) day for histopathological studies. The results of the present work demonstrated that repeated injection of PC/DC caused neural damage and intense inflammation at the injection site leading to skeletal muscle degeneration, necrosis and fibrosis. Electron microscopic examination of the neural tissues in the injected area showed intra-neural fibroblasts, deposition of intra-neural collagen fibers and marked myelin degeneration. In addition, PC/DC injection caused thickening of intra-neural blood vessel walls and evident endo-neural mast cells. The current data highlight the attendant risk of neuromuscular injury associated with repeated PC/DC injection during the treatment of undesirable fat deposits and lipomas.

Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells.

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.

A pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis.

Lipid-laden macrophages, or "foam cells," are observed in the lungs of patients with fibrotic lung disease, but their contribution to disease pathogenesis remains unexplored. Here, we demonstrate that fibrosis induced by bleomycin, silica dust, or thoracic radiation promotes early and sustained accumulation of foam cells in the lung. In the bleomycin model, we show that foam cells arise from neighboring alveolar epithelial type II cells, which respond to injury by dumping lipids into the distal airspaces of the lungs. We demonstrate that oxidized phospholipids accumulate within alveolar macrophages (AMs) after bleomycin injury and that murine and human AMs treated with oxidized phosphatidylcholine (oxPc) become polarized along an M2 phenotype and display enhanced production of transforming growth factor-ß1. The direct instillation of oxPc into the mouse lung induces foam cell formation and triggers a severe fibrotic reaction. Further, we show that reducing pulmonary lipid clearance by targeted deletion of the lipid efflux transporter ATP-binding cassette subfamily G member 1 increases foam cell formation and worsens lung fibrosis after bleomycin. Conversely, we found that treatment with granulocyte-macrophage colony-stimulating factor attenuates fibrotic responses, at least in part through its ability to decrease AM lipid accumulation. In summary, this work describes a novel mechanism leading to foam cell formation in the mouse lung and suggests that strategies aimed at blocking foam cell formation might be effective for treating fibrotic lung disorders.

Design and characterization of an ocular topical liposomal preparation to replenish the lipids of the tear film.

PURPOSE: Dry eye (DE) includes a group of diseases related to tear film disorders. Current trends for DE therapy focus on providing lipid components to replace the damaged lipid layer. Formulations that contain aqueous and mucin-like compounds may have additional therapeutic benefits for DE patients. The aim of this work was to design and evaluate novel formulations having the potential to become topical treatment for DE. METHODS: Unpreserved liposomal formulations composed of phosphatidylcholine (PC), cholesterol, and α-tocopherol (vit E) were prepared by the thin-film hydration technique. Formulations were characterized in terms of liposome size, pH, surface tension, osmolarity, and viscosity. In vitro tolerance assays were performed on macrophage, human corneal, and conjunctival cell lines at short- and long-term exposures. In vivo ocular tolerance was studied after instillation of the formulation. RESULTS: The mean liposome size was less than 1 µm and surface tension < 30 mN/m for all formulations. The final liposomal formulation (PC-cholesterol-vit E in a ratio of 8:1:0.8) had physiological values of pH (6.45 ± 0.09), osmolarity (289.43 ± 3.28 mOsm), and viscosity (1.82 ± 0.02 mPa · s). Cell viability was greater than 80% in the corneal and conjunctival cells. This formulation was well tolerated by experimental animals. CONCLUSIONS: The unpreserved liposomal formulation has suitable properties to be administered by a topical ophthalmic route. The liposome-based artificial tear had good in vitro and in vivo tolerance responses. This formulation, composed of a combination of liposomes and bioadhesive polymers, may be used successfully as a tear film substitute in DE therapy.

The role of phosphatidylcholine and deoxycholic acid in inflammation.

AIMS: Phosphatidylcholine with deoxycholic acid (PC/DA) is widely used to reduce localized fat deposits with mild adverse effects. We previously demonstrated that PC induces lipolysis with mild PMN infiltration, while DA induces adipose tissue damage. Therefore, the aim of this study was to extend our understanding of the pro-inflammatory responses of PC, DA, and PC/DA. MAIN METHODS: We evaluated the level of edema and polymononuclear (PMN) infiltration by histopathological examination. Myeloperoxidase (MPO) activity was analyzed using an MPO activity assay kit. Levels of inflammatory cytokines (IL-1ß and IL-6) and PGE2 were measured by ELISA. KEY FINDINGS: A low and high dose of PC failed to induce an inflammatory response, whereas DA led to an intense inflammatory response in a dose dependent manner. Combined PC/DA treatment resulted in a mild inflammatory response that was notably less severe than higher DA. Together, these results demonstrated that DA plays a role in inflammation caused by combined PC/DA. Histopathological examination and measurement of MPO activity indicated that DA was the primary cause of edema and PMN infiltration. Further, increased levels of cytokines (IL-1ß and IL-6) and PGE2 demonstrated that DA might directly induce inflammation, whereas PC alone has no effect on inflammation. SIGNIFICANCE: These results indicate that DA rather than PC is responsible for inflammation, and that PC may not aggravate inflammatory responses induced by DA. Thus, the results of this study suggest that the adverse effects of PC/DA during localized fat treatment may be solely due to DA.

Effects of fullerene C60 on proteomic profile of Danio rerio fish embryos.

The effects of phosphatidylcholine-based phospholipid nanoparticles containing fullerene C60 on Danio rerio fish embryos were studied. Exposure of the embryos with the nanoparticles for 48 h did not lead to appreciable changes in the number of protein bands in SDS-PAGE in comparison with the control (exposure in medium with phosphatidylcholine). Mass spectrometric identification of proteins showed differences in the proteomic profiles of the samples. The content of vitellogenins changed after exposure with phosphatidylcholine-based nanoparticles with C60 fullerenes. This could indicate low toxicity of the nanoparticles towards D. rerio embryos under experimental conditions.

Nimodipine-loaded mixed micelles: formulation, compatibility, pharmacokinetics, and vascular irritability study.

BACKGROUND: The clinical application of nimodipine (NIM) is limited by several unfavorable properties, which are induced by its low aqueous solubility. In the present study, nimodipine-loaded egg phosphatidylcholine-sodium glycocholate mixed micelles (NIM-EPC-SGC-MMs) were prepared to improve the water solubility of NIM, thus allowing it to be more applicable for clinical use. METHODS: NIM-EPC-SGC-MMs were prepared using the coprecipitation method and the factors influencing formulation quality were optimized. After formulation, water solubility, solubilizing efficiency, drug loading, particle size, physical compatibility, pharmacokinetics, and vascular irritability were determined. RESULTS: The mean size of the NIM-EPC-SGC-MMs was 6.099 ± 0.048 nm under optimized conditions. The water solubility of NIM in EPC-SGC-MMs was enhanced 250-fold compared with free NIM. The physical compatibility, pharmacokinetic, and vascular irritability studies showed that, in comparison to the commercially available NIM injections, NIM-EPC-SGC-MMs presented better physical compatibility, the same pharmacokinetic profile, and less risk of local vascular irritation and phlebitis. CONCLUSION: EPC-SGC-MMs represent a promising new formulation suitable for the intravenous delivery of NIM.