Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using in vitro and ex vivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

Impact of Truncated Oxidized Phosphatidylcholines on Phospholipase A2 Activity in Mono- and Polyunsaturated Biomimetic Vesicles.

The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A2 (sPLA2) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA2 modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA2 activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA2 activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA2 activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA2 activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA2 activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Chronic and Binge Alcohol Ingestion Increases Truncated Oxidized Phosphatidylcholines in Mice Lungs Due to Increased Oxidative Stress.

Heavy alcohol drinking has negative health effects in multiple organs. It predisposes lungs to inflammatory conditions associated with acute lung injury and increased incidence of pneumonia and sepsis, which may lead to death due to acute respiratory distress syndrome in some individuals with alcohol use disorder (AUD). In general, rodent models of alcohol exposure either do not recapitulate multiple organ injuries as seen in humans or require longer duration to establish tissue injury and inflammation. The recently introduced NIAAA model of alcohol-induced liver injury, characterized by a marked increase in steatosis and liver damage with 10 days of a liquid diet containing 5% ethanol followed by a single ethanol binge (5 g/kg). Therefore, we employed this model to explore the status of surfactant phospholipids, oxidative stress, tissue injury markers and inflammatory cytokines in lungs. In lungs of C57BL/6J mice, the alcohol feeding significantly increased levels of the surfactant phospholipid dipalmitoyl phosphatidylcholine (DPPC) as well as the truncated oxidized phosphatidylcholines palmitoyl oxovaleryl phosphatidyl-choline (POVPC), palmitoyl glutaryl phosphatidyl-choline (PGPC), palmitoyl oxo-nonanoyl phosphatidyl-choline (ALDO-PC), and palmitoyl azelaoyl phosphatidyl-choline (PAzePC) at 9 h post-binge. Additionally, gene expression of the enzymes catalyzing lipid oxidation, such as arachidonate 15-lipoxygenase (Alox15), prostaglandin synthase 2 (Ptgs2), Cytochrome P450 2E1 (Cyp2E1) and NADPH oxidase 1 (Nox1) were significantly increased. Furthermore, ethanol increased levels of the inflammatory cytokine Interleukin-17 in bronchoalveolar lavage fluid. In conclusion, the NIAAA alcohol feeding model might be suitable to study alcohol-induced lung injury and inflammation.

The Levels of Oxidized Phospholipids in High-Density Lipoprotein During the Course of Sepsis and Their Prognostic Value.

Purpose: To examine the levels of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero phosphatidylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphatidylcholine (PGPC) (the oxidized phosphatidylcholines) in HDL during the course of sepsis and to evaluate their prognostic value. Materials and Methods: This prospective cohort pilot study enrolled 25 septic patients and 10 healthy subjects from 2020 to 2021. The HDLs were extracted from patient plasmas at day 1, 3 and 7 after sepsis onset and from healthy plasmas (total 81 plasma samples). These HDLs were then subjected to examining POVPC and PGPC by using an ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) system. We further measured the levels of 38 plasma cytokines by Luminex and evaluated the correlation of HDL-POVPC level with these cytokines. Patients were further stratified into survivors and non-survivors to analyze the association of HDL-POVPC level with 28-day mortality. Results: Septic patients exhibited significant increase of HDL-POVPC at day 1, 3 and 7 after sepsis onset (POVPC-D1, p=0.0004; POVPC-D3, p=0.033; POVPC-D7, p=0.004, versus controls). HDL-PGPC was detected only in some septic patients (10 of 25) but not in healthy controls. Septic patients showed a significant change of the plasma cytokines profile. The correlation assay showed that IL-15 and IL-18 levels were positively correlated with HDL-POVPC level, while the macrophage-derived chemokine (MDC) level was negatively correlated with HDL-POVPC level. Furthermore, HDL-POVPC level in non-survivors was significantly increased versus survivors at day 1 and 3 (POVPC-D1, p=0.002; POVPC-D3, p=0.003). Area under ROC curves of POVPC-D1 and POVPC-D3 in predicting 28-day mortality were 0.828 and 0.851. POVPC-D1and POVPC-D3 were the independent risk factors for the death of septic patients (p=0.046 and 0.035). Conclusions: HDL-POVPC was persistently increased in the course of sepsis. POVPC-D1 and POVPC-D3 were significantly correlated with 28-mortality and might be valuable to predict poor prognosis.

Partial Mitigation of Oxidized Phospholipid-Mediated Mitochondrial Dysfunction in Neuronal Cells by Oxocarotenoids.

Mitochondria are important (patho)physiological sources of reactive oxygen species (ROS) that mediate mitochondrial dysfunction and phospholipid oxidation; an increase in mitochondrial content of oxidized phospholipid (OxPL) associates with cell death. Previously we showed that the circulating OxPL 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) increases in patients with Alzheimer's disease (AD), and associates with lower plasma antioxidant oxocarotenoids, zeaxanthin, and lutein. Since oxocarotenoids are metabolized in mitochondria, we propose that during AD, lower concentrations of mitochondrial zeaxanthin and lutein may result in greater phospholipid oxidation and predispose to neurodegeneration. Here, we have investigated whether non-toxic POVPC concentrations impair mitochondrial metabolism in differentiated (d)SH-SY5Y neuronal cells and whether there is any protective role for oxocarotenoids against mitochondrial dysfunction. After 24 hours, glutathione (GSH) concentration was lower in neuronal cells exposed to POVPC (1-20 µM) compared with vehicle control without loss of viability compared to control. However, mitochondrial ROS production (determined by MitoSOX oxidation) was increased by 50% only after 20 µM POVPC. Following delivery of lutein (0.1-1 µM) and zeaxanthin (0.5-5 µM) over 24 hours in vitro, oxocarotenoid recovery from dSH-SY5Y cells was > 50%. Co-incubation with oxocarotenoids prevented loss of GSH after 1 µM but not 20 µM POVPC, whereas the increase in ROS production induced by 20 µM POVPC was prevented by lutein and zeaxanthin. Mitochondrial uncoupling increases and ATP production is inhibited by 20 µM but not 1 µM POVPC; carotenoids protected against uncoupling although did not restore ATP production. In summary, 20 µM POVPC induced loss of GSH and a mitochondrial bioenergetic deficit in neuronal cells that was not mitigated by oxocarotenoids.

c-Kit modifies the inflammatory status of smooth muscle cells.

BACKGROUND: c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. METHODS: High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W-v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. RESULTS: The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFß-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. DISCUSSION: Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

Phospholipid oxidation and carotenoid supplementation in Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease, characterised by decline of memory, cognitive function and changes in behaviour. Generic markers of lipid peroxidation are increased in AD and reactive oxygen species have been suggested to be involved in the aetiology of cognitive decline. Carotenoids are depleted in AD serum, therefore we have compared serum lipid oxidation between AD and age-matched control subjects before and after carotenoid supplementation. The novel oxidised phospholipid biomarker 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) was analysed using electrospray ionisation tandem mass spectrometry (MS) with multiple reaction monitoring (MRM), 8-isoprostane (IsoP) was measured by ELISA and ferric reducing antioxidant potential (FRAP) was measured by a colorimetric assay. AD patients (n=21) and healthy age-matched control subjects (n=16) were supplemented with either Macushield™ (10mg meso-zeaxanthin, 10mg lutein, 2mg zeaxanthin) or placebo (sunflower oil) for six months. The MRM-MS method determined serum POVPC sensitively (from 10µl serum) and reproducibly (CV=7.9%). At baseline, AD subjects had higher serum POVPC compared to age-matched controls, (p=0.017) and cognitive function was correlated inversely with POVPC (r=-0.37; p=0.04). After six months of carotenoid intervention, serum POVPC was not different in AD patients compared to healthy controls. However, POVPC was significantly higher in control subjects after six months of carotenoid intervention compared to their baseline (p=0.03). Serum IsoP concentration was unrelated to disease or supplementation. Serum FRAP was significantly lower in AD than healthy controls but was unchanged by carotenoid intervention (p=0.003). In conclusion, serum POVPC is higher in AD patients compared to control subjects, is not reduced by carotenoid supplementation and correlates with cognitive function.

Toxicity of oxidized phosphatidylcholines in cultured human melanoma cells.

The oxidized phospholipids (oxPL) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are generated from 1-palmitoyl-2-arachidonoyl-phosphatidylcholine under conditions of oxidative stress. These oxPL are components of oxidized low density lipoprotein. They are cytotoxic in cells of the arterial wall thus playing an important role in the development and progression of atherosclerosis. The toxic lipid effects include inflammation and under sustained exposure apoptosis. The aim of this study was to find out whether such toxic effects, especially apoptosis, are also elicited by oxPL in melanocytic cells in order to assess their potential for therapeutic intervention. FACS analysis after staining with fluorescent markers was performed to identify the mode of lipid-induced cell death. Activation of sphingomyelinase which generates apoptotic ceramide was measured using an established fluorescence assay. Ceramide profiles were determined by mass spectrometry. We found that 50µM POVPC induce cell death in human melanoma cells isolated from different stages of tumor progression but affect primary human melanocytes to a much lesser extent. In contrast, 50µM PGPC was only apoptotic in two out of four cell lines used in this study. The toxicity of both compounds was associated with efficient lipid uptake into the tumor cells and activation of acid sphingomyelinase. In several but not all melanoma cell lines used in this study, activation of the sphingomyelin degrading enzyme correlated with an increase in the concentration of the apoptotic mediator ceramide. The individual patterns of the newly formed ceramide species were also cell line-specific. PGPC and POVPC may be considered potential drug candidates for topical skin cancer treatment. They are toxic in malignant cells. The respective oxidized phospholipids are naturally formed in the body and resistance to these compounds is not likely to occur.

Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,ß-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.