Accumulation Of Lysophosphatidylcholine Research Articles

ACSL1 Aggravates Thromboinflammation by LPC/LPA Metabolic Axis in Hyperlipidemia Associated Myocardial Ischemia-Reperfusion Injury.

Acute myocardial infarction (AMI)is associated with well-established metabolic risk factors, especially hyperlipidemia and obesity. Myocardial ischemia-reperfusion injury (mIRI) significantly offsets the therapeutic efficacy of revascularization. Previous studies indicated that disrupted lipid homeostasis can lead to lipid peroxidation damage and inflammation, yet the underlying mechanisms remain unclear. Here, the study demonstrates that hyperlipidemia is a key driver of mIRI. Long-chain fatty acyl-CoA synthetase 1 (ACSL1) is upregulated in both hyperlipidemia and AMI patients. ACSL1 expression is induced by a high-fat microenvironment (oxLDL and palmitic acid) in a concentration-dependent manner. Interestingly, the protein level is positively correlated with total cholesterol level and thromboinflammatory biomarkers. Furthermore, ACSL1 reprogrammed lipid metabolism in monocytes, leading to the accumulation of lysophosphatidylcholine (LPC)/lysophosphatidic acid (LPA). The monocytic LPC/LPA axis accelerated lipid peroxidation and neutrophil extracellular traps (NETs)-induced thromboinflammation via the paracrine effect. The main LPA producer Autotaxinis is also induced under high-fat conditions and then exerts thromboinflammation response through converted LPC to LPA. Finally, ACSL1 knockdown or NETs release inhibitor (DNase I or GSK484) significantly alleviated mIRI in mice. These findings highlight ACSL1 and NETosis as potential key targets for preventing mIRI and underscore the lipid peroxidation in the mechanisms of ACSL1-mediated thromboinflammation.

Vesicle budding caused by lysolipid-induced asymmetry stress

Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20–30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a “budding power” of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20–30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively.

Spatiotemporal Variation of Residual Cortisol Obstructs Nutrient Acquisition in Animal-Derived Foods by Perturbing Glycerophospholipid Metabolism.

Cortisol is inevitably secreted by pigs due to the physical and psychological stressors produced by mixed group transportation and preslaughter handling. Accumulated cortisol in animal tissues enters the human body through the food chain and entails potential risks to human health. An integrated lipidome and proteome analysis was conducted to investigate the effect of the spatiotemporal variation of residual cortisol on nutrient acquisition in pork. A total of 55 crucial lipid molecules associated with cortisol residue were identified based on debiased sparse partial correlation analysis. Label-free proteomics was applied to screen 58 differentially abundant proteins (including phospholipase A2, lysophosphatidylcholine acyltransferase, and CTP:phosphocholine cytidylyltransferase), indicating that cortisol residue perturbed the glycerophospholipid biosynthetic process and glycerophospholipid metabolic process. Cortisol induced downregulations of cPLA2 encoding genes and decreased phospholipase A2 activity, resulting in the bioaccumulation of phosphatidylethanolamine (from 36.86 to 43.18 mg kg-1). Cortisol increased the activity of CTP:phosphocholine cytidylyltransferase by improving the availability of fatty acids and aggregating the inactive L-form (lipid-independent form) to the active H-form (lipid-associated form). The metabolic pathways perturbed by cortisol resulted in phosphatidylcholine degradation (from 93.73 to 58.28 mg kg-1) and lysophosphatidylcholine accumulation (from 3.39 to 5.16 mg kg-1). These findings indicated that cortisol residue deteriorated meat quality and obstructed nutrient acquisition in animal-origin foods.

An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.

6-Benzylaminopurine causes lipid dyshomeostasis via disruption of glycerophospholipid metabolism in zebrafish

6-Benzylaminopurine (6-BA) is ubiquitous in agricultural production and is accessible to humans through diets. The modulation of lipid metabolism by 6-BA has been previously demonstrated in plants and oleaginous microorganisms. Therefore, whether it alters lipid homeostasis in other living organisms requires further investigation. In this study, doses ≥10 mg 6-BA/L caused malformation of the yolk sac, steatosis, and other hepatopathies in zebrafish larvae. Exposure to 25 mg 6-BA/L resulted in increased levels of triglyceride and total cholesterol. Results of transcriptomic analysis indicated that 6-BA alters genes associated with fatty acid and glycerophospholipid metabolism. Among them, the expression levels of hmgcra, elovl7b, and apobb.2 were downregulated, whereas those of lpcat3, bco1l, cyp7al, fabp1b.1, elp6, pde6ha, apoa4b.2_2, sgk1, dgkaa, and mogat2 were upregulated. Correspondingly, a study of the metabolome identified lysophosphatidylcholine (LPC) as the major differentially expressed metabolite in response to 6-BA treatment. Therefore, abnormal accumulation of LPCs and dyshomeostasis of glycerophospholipid metabolism were identified as potential mechanisms causing the toxicity of 6-BA, which should be assessed to understand the risks of 6-BA and the products contaminated by it. Environmental implication6-Benzylaminopurine (6-BA), an important residue in “toxic bean sprouts,” is ubiquitous in agricultural production and is common in typical diets. Its regulation of lipid metabolism has been demonstrated in plants and oleaginous microorganisms. Whether it alters lipid homeostasis in other organisms and the underlying mechanisms remain largely unknown. The worldwide use of 6-BA and the potential exposure of humans have aroused public attention owing to its hazardous effects; thus, its hazardous effects, particularly those on lipid homeostasis, deserve careful clarification.

Spns1 is a lysophospholipid transporter mediating lysosomal phospholipid salvage

The lysosome is central to the degradation of proteins, carbohydrates, and lipids and their salvage back to the cytosol for reutilization. Lysosomal transporters for amino acids, sugars, and cholesterol have been identified, and the metabolic fates of these molecules in the cytoplasm have been elucidated. Remarkably, it is not known whether lysosomal salvage exists for glycerophospholipids, the major constituents of cellular membranes. By using a transport assay screen against orphan lysosomal transporters, we identified the major facilitator superfamily protein Spns1 that is ubiquitously expressed in all tissues as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, with LPC and LPE being the lysosomal breakdown products of the most abundant eukaryotic phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively. Spns1 deficiency in cells, zebrafish embryos, and mouse liver resulted in lysosomal accumulation of LPC and LPE species with pathological consequences on lysosomal function. Flux analysis using stable isotope-labeled phospholipid apolipoprotein E nanodiscs targeted to lysosomes showed that LPC was transported out of lysosomes in an Spns1-dependent manner and re-esterified back into the cytoplasmic pools of phosphatidylcholine. Our findings identify a phospholipid salvage pathway from lysosomes to the cytosol that is dependent on Spns1 and critical for maintaining normal lysosomal function.

Radiation-induced lipoprotein-associated phospholipase A2 increases lysophosphatidylcholine and induces endothelial cell damage

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1β, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.

CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion

Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.

Insufficient Lymph Drainage Causes Abnormal Lipid Accumulation and Vein Wall Degeneration.

Objective: Previously, we analyzed human varicose veins (VV) using imaging mass spectrometry (IMS) and detected the abnormal accumulation of lipid molecules in the walls of VV, possibly due to insufficient lipid drainage by the lymphatic vessels. In this study, we created an animal model of lymphatic insufficiency to investigate the effects of insufficient lymph drainage on vein walls. Methods: In rats, the lymphatic collecting vessels surrounding the femoral vein were ligated on one side (the model tissue), which caused the local retention of lymphatic fluid in the perivascular tissue. The equivalent contralateral tissue was used as a control. A histological study of the femoral vein and the surrounding perivascular tissue was conducted. IMS was used to analyze the distribution of lipid molecules in the perivascular tissue. Results: Fourteen days after the procedure, the lymphatic vessels in the model tissue were significantly dilated. Furthermore, IMS revealed that the composition of the lipid molecules in the perivascular regions of the model tissue had altered. Compared with the control tissue, the model tissue exhibited marked perivascular accumulation of lysophosphatidylcholine (1-acyl 16:0), phosphatidylcholine (16:0/20:4), and triglycerides (52:2). Interestingly, the walls of the femoral veins running through the model tissue were 3.4-fold thicker than those of the femoral veins running through the control tissue. The number of tumor necrosis factor α-positive adipocytes was increased in the perivascular regions of the model tissue. Conclusion: The findings of this study indicated that the accumulation of lymphatic fluid due to insufficient lymph drainage changes the structure of vein walls, and such changes might be associated with chronic venous insufficiency. (This is a translation of Jpn J Phlebol 2015; 26: 227-235.).

Abstract B06: TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism

Abstract Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by multi-organ proliferation of TSC2-deficient cells with aberrant activation of the mechanistic/mammalian Target of Rapamycin Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Up to 80% of women with TSC develop lymphangioleiomyomatosis (LAM), which consists of diffuse proliferation of TSC2-deficient smooth muscle-like cells with progressive cystic destruction of the lung. LAM can also occur as a sporadic disorder of women. A downstream effector of mTORC1, the Sterol Regulatory Element-Binding Protein (SREBP), regulates the transcription of de novo fatty acid synthesis enzymes in TSC2-deficient cells. The impact of loss of the TSC proteins on the lipidome and how complex lipid species are affected by rapamycin and its analogs are unknown. Here, we report the first systematic study of the TSC lipidome, revealing unexpected findings. Using mass spectrometry, we profiled 131 complex lipid species discovered elevated levels (p<0.05) of 4 lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, C20:4) in the plasma of LAM patients compared with healthy control women. LPC are a class of bioactive lipids generated by phospholipase A (PLA) activity. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro pre-clinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid, including the LPC precursors phosphatidylcholines (PC) and sphingolipids, and neutral lipid species, including triacylglycerols and cholesterol esters. To determine whether TSC2-deficient cells generate LPC using de novo synthesized PC, TSC2-deficient and TSC2-expressing cells were labeled with deuterated choline (choline chloride 1,1,2,2-d4) for 6 hours, and lipids isolated to measure choline incorporation into two saturated LPC (C16:0 and C18:0) and two parent PC (C32:0 and C34:0) species. Incorporation of deuterated choline into PC and LPC was greater than 2 fold higher in TSC2-deficient cells compared to TSC2-expressing controls (p<0.05). Surprisingly, treatment with rapamycin, the mTOR catalytic inhibitor Torin1, or downregulation of the mTORC1-regulated lipogenic transcription factor SREBP (Sterol Regulatory Element-Binding Protein) did not suppress LPC in TSC2-deficient cells, while they suppressed other phospholipid species. Finally, pharmacologic and genetic approaches revealed that inhibition of distinct isoforms of PLA2 may decrease the proliferation of TSC2-deficient but not TSC2-expressing cells. Collectively, these results demonstrate for the first time that TSC2-deficient cells have enhanced choline phospholipid metabolism, revealing a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies. Citation Format: Carmen Priolo, Stéphane J. H. Ricoult, Damir Khabibullin, Harilaos Filippakis, Jane Yu, Brendan Manning, Clary Clish, Elizabeth P. Henske. TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B06.

Tuberous sclerosis complex 2 loss increases lysophosphatidylcholine synthesis in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease affecting women. LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. The TSC protein complex inhibits the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), which is a master regulator of cellular metabolism. Using mass spectrometry-based lipid profiling, we analyzed plasma from patients with LAM and discovered elevated levels of four lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, and C20:4) compared with those in healthy control women. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro preclinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid and neutral lipid species. Treatment with rapamycin or torin1 or down-regulation of sterol regulatory element-binding protein (SREBP), a lipogenic transcription factor, did not suppress LPC in TSC2-deficient cells. Inhibition of distinct isoforms of phospholipase A2 decreased the proliferation of TSC2-deficient cells. Collectively, these results demonstrate that TSC2-deficient cells have enhanced choline phospholipid metabolism and reveal a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.

Loss of lymphatic vessels and regional lipid accumulation is associated with great saphenous vein incompetence

Recent studies suggest that biologic changes in the vein wall associated with varicose veins (VVs) occur not only in valvular tissue but also in nonvalvular regions. We previously used imaging mass spectrometry (IMS) to determine the distribution of lipid molecules in incompetent valve tissue. In this study, we used IMS to analyze incompetent great saphenous veins (GSVs) in patients with varicose vein (VV) to assess the distribution of lipid molecules. We obtained GSV tissue from 38 VV patients (50 limbs) who underwent GSV stripping. For the control veins (CV), we obtained GSV samples from 10 patients undergoing infrainguinal bypass with reversed GSV grafting for peripheral artery occlusive disease (10 limbs). Conventional and immunofluorescence staining were performed for histopathologic examination. The total lipid content in the homogenized vein tissue was determined. The localization of each lipid molecule in the vein wall was assessed by IMS. The histologic examination showed the VV walls were significantly thicker than the CV walls, and only the VV adventitia was positive for lipid staining. The VV wall had higher concentrations of phospholipids and triglycerides than the CV wall. IMS revealed an abnormal accumulation of lysophosphatidylcholine (LPC; 1-acyl 16:0) and phosphatidylcholine (diacyl 16:0/20:4) in the VV intima and media. Triglyceride was found only in VV adventitia. The number of lymphatic vessels, as measured by staining with D2-40, a lymphatic vessel-specific marker, was significantly lower in the VV adventitia than in the CV adventitia. Lymphatic vessel reduction may be associated with insufficient lymphatic drainage in the VV adventitia causing histologic changes in VV tissue. The accumulation of LPC (1-acyl 16:0) and PC (diacyl 16:0/20:4) in the VV intima and media may be associated with chronic inflammation, leading to VV tissue degeneration. Furthermore, insufficient lipid drainage by lymphatic vessel may be responsible for accumulation of lipid molecules and subsequent vein wall degeneration.

E0036 Regulation of T-type Ca ion channel in Lysophosphatidylcholine-stimulated cardiomyocytes

ObjectivesTo study the effect of lysophosphatidylcholine (LPC) in myocardial cells in T-type calcium channel currents (ICaT), and the hypothesis that LPC accumulation in intracellular and/or interstitial space in cardiomyocytes may...

Supernatant of Aged Erythrocytes Causes Lung Inflammation and Coagulopathy in a “Two-Hit”In Vivo Syngeneic Transfusion Model

Transfusion of erythrocytes is associated with increased morbidity in certain patient groups. Storage time of erythrocytes may contribute to respiratory complications. Using a syngeneic in vivo transfusion model, we investigated whether transfusion of stored rat erythrocytes causes lung injury in healthy and in lipopolysaccharide-primed rats in a "two-hit" model of lung injury. Rats were infused with aged rat erythrocytes (14 days of storage) and washed aged erythrocytes or supernatant of aged erythrocytes. Controls received fresh rat erythrocytes (0 days of storage) or saline. In the "two-hit" model of lung injury, lipopolysaccharide was used as a "first hit" before transfusion. Rat and control human erythrocyte products were analyzed for lysophosphatidylcholine accumulation. In healthy rats, transfusion of aged erythrocytes caused mild pulmonary inflammation but no coagulopathy. In lipopolysaccharide-pretreated rats, transfusion of aged erythrocytes augmented lung injury by inducing coagulopathy, both in the pulmonary and systemic compartment, when compared with transfusion with fresh erythrocytes. When transfused separately, supernatant of aged erythrocytes, but not washed aged erythrocytes, mediated coagulopathy in the "two-hit" model. Analysis of the supernatant of aged erythrocytes (rat and human) showed no lysophosphatidylcholine accumulation. Transfusion of aged erythrocytes induces lung injury in healthy rats. In a "two-hit" model, injury induced by aged erythrocytes was characterized by coagulopathy and was abrogated by washing. Washing of aged erythrocytes may decrease pulmonary complications in patients with an inflammatory condition who are exposed to a blood transfusion.

Increased PAFAH and Oxidized Lipids Are Associated With Inflammation and Atherosclerosis in Hypercholesterolemic Pigs

To study the association of PAF-acetyl hydrolase (PAFAH) activity with inflammation, oxidative stress, and atherosclerosis in hypercholesterolemic swine. Cholesterol-rich diet feeding of miniature pigs was associated with an increase in PAFAH activity and an increase of the PAFAH to PON1 ratio. PLA2G7 RNA (coding for PAFAH) expression was increased in blood monocytes and plaque macrophages. Increased PAFAH activity was associated with higher plasma lysophosphatidylcholine and correlated with oxidized LDL. In THP1 monocytes and macrophages and in human blood-derived macrophages, oxidized LDL induced PLA2G7 RNA expression. Atherogenic diet feeding induced the accumulation of macrophages and oxidized LDL in the arterial wall leading to atherosclerosis. PAFAH activity correlated positively with plaque size and TNFalpha expression in plaque macrophages. We demonstrated that an increase in PAFAH activity was associated with increased levels of lysophosphatidylcholine, oxidized LDL, and inflammation, resulting in accelerated atherosclerosis in hypercholesterolemic minipigs. The significant correlation between PLA2G7 RNA expression in plaque macrophages and plasma PAFAH activity suggests that the latter is a consequence, rather than a cause of macrophage accumulation. Our cell experiments suggest that oxidized LDL can induce PAFAH, resulting in accumulation of lysophosphatidylcholine that increases the inflammatory action of oxidized LDL.

Lysophosphatidylcholine molecular species in low density lipoprotein and high density lipoprotein in alloxan-induced diabetic rats: effect of probucol.

To elucidate the contribution of diabetic state toward the accumulation of lysophosphatidylcholine (LPC) in low density lipoprotein (LDL), the LPC molecular species in LDL and high density lipoprotein (HDL) obtained from alloxan-induced diabetic rats were determined by high performance liquid chromatography. The palmitoyl LPC (PLPC) per protein was increased in the LDL of diabetic rats (p < 0.05), whereas the stearoyl LPC (SLPC) decreased in both the LDL and HDL of diabetic rats (p < 0.001) in comparison to nondiabetic rats. After the dietary administration of probucol for 4 weeks, the concentrations of SLPC and PLPC in the LDL of diabetic rats and of SLPC in the LDL of nondiabetic rats all significantly decreased by probucol (all; p < 0.01), with a concomitant decrease of the plasma concentrations of total and HDL cholesterol, and phospholipid, compared with those without probucol. The level of TBARS per protein in LDL increased in diabetic rats, and decreased by probucol (p < 0.01). In conclusion, the oxidative stress is thought to be an important factor to accumulate LPC in LDL, although the paradoxical decrease of SLPC in diabetic LDL suggests a non-oxidative metabolism with a preference for the LPC molecular species. The reducing effect of probucol on LPC may not be solely attributed to its antioxidative effect.

The ischemic metabolite lysophosphatidylcholine increases rat coronary arterial tone by endothelium-dependent mechanisms

Lysophosphatidylcholine (LPC), a hydrolysis product of phospholipid degradation, accumulates in the ischemic myocardium. Using isolated hearts or rat coronary septal arteries, we tested the impact of LPC in modulating basal function or the responses to vasoactive agents. Sustained perfusion of hearts with LPC augmented coronary perfusion pressure (CPP) and reduced left ventricular developed pressure (LVDP). By mechanisms that have yet to be identified, these effects on CPP and LVDP were exaggerated when LPC was removed from the perfusate. Although LPC (or its washout) had no direct effect on vascular tone in the isolated coronary artery, it selectively potentiated the receptor-coupled vasoconstrictor response to U-46619, a thromboxane A2 mimetic. Interestingly, when LPC was washed out, the potentiation to U-46619 was even more pronounced. Both the immediate and residual effects of LPC were endothelium-dependent. EDHF was likely the sole mediator responsible for the direct effects of LPC on U-46619-vasoconstriction, whereas the augmented vasoconstrictor responses following LPC washout may in part be related to an increase in ET-1, and a striking reduction in the bioavailability of NO. Our data suggest that in addition to reducing the accumulation of LPC to prevent ischemia-reperfusion (I/R) damage, efforts targeting an improved endothelium-dependent regulation of vascular tone could be an attractive approach to limit the cardiac damage induced by I/R.

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography–mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

Phospholipase A2 and lysophospholipid roles in receptor activation of TRPC5 channel

TRPC5 is a widely expressed calcium‐permeable channel that is stimulated by several agonists acting at various G‐protein coupled receptors. The mechanisms coupling the receptors to TRPC5 are, however, unknown. Because we have shown previously that TRPC5 is activated by intracellular lysophosphatidylcholine (LPC) (Flemming et al 2006, J Biol Chem 281, 4977–82) we investigated the hypothesis that receptor coupling to TRPC5 results, at least in part, from accumulation of LPC as a consequence of receptor‐activation of a calcium‐independent phospholipase A2 (iPLA2). Tetracycline inducible TRPC5‐expressing HEK 293 cells were studied using fluo‐4 to measure calcium‐entry in real‐time 96‐well plate assays. The agonists employed were carbachol (CCh) and sphingosine‐1‐phosphate (S1P), which act at different receptors. Pre‐incubation of cells with LPC, but not arachidonic acid, abolished TRPC5 activation by CCh, reduced stimulation by S1P and left unaffected activation by gadolinium. Bromoenol lactone, an iPLA2 inhibitor, suppressed TRPC5 activation by CCh or S1P but not activation by LPC or gadolinium; a similar effect occurred following knock‐down of iPLA2‐VIA using RNAi. The data suggest receptor‐coupling to TRPC5 involves iPLA2‐VIA and LPC.The research is supported by the Wellcome Trust.

The catalytically active secretory phospholipase A2 type IIA is involved in restenosis development after PTCA in human coronary arteries and generation of atherogenic LDL

Secretory phospholipase A2 type IIA (sPLA2) may actively contribute to atherogenesis, acting either within the arterial wall or in plasma. Proinflammatory eicosanoids and lysophospholipids, generated through hydrolysis of cell membrane phospho-lipids by sPLA2, initiate and prolong the inflammatory process. In the present study we examined the possible involvement of sPLA2 in development of restenosis in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). We also investigated whether serum sPLA2 could catalyze accumulation of lysophosphatidylcholine (LPC) in LDL. Concentrations and catalytic activities of sPLA2 were measured in blood serum of 49 consenting patients immediately before, 1-7 and 180 days after PTCA. All patients had repeat angiograms at 180-day follow-up. Restenosis was registered in 19 patients. Accumulation of LPC in LDL was evaluated by thin-layer chromatography after incubation of blood serum with LDL. Serum sPLA2 concentrations increased in all study patients by day 1 post-PTCA, but the increase was significantly greater and more protracted in patients who developed restenosis. Catalytic activities increased significantly 6 days post-PTCA in patients who developed restenosis, whereas for patients without restenosis there was no change in serum sPLA2 activity throughout the study period in spite of the sPLA2 presence in blood. Incubation of blood serum (6 days post-PTCA) with LDL resulted in accumulation of LPC only for those patients who subsequently developed restenosis. Manoalide, a specific inhibitor of sPLA2, completely blocked the LPC accumulation. The data indicate that elevated serum sPLA2 activity after PTCA is associated with restenosis development and may be involved in atherogenic modification of LDL in blood serum.