The Patatin-like Phospholipase Domain Containing Protein 7 (PNPLA7) Facilitates M2 Polarization of Macrophages Through a PPARγ-Dependent Mechanism / 中国生物化学与分子生物学报

Lysophosphatidylcholine (LPC) modulates the dynamic and integral process of macrophage polarization in immune responses, tissue inflammation and remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) was identified as an LPC-preferring lysophospholipase recently. However, the expression and role of PNPLA7 in macrophage polarization remained unknown. In the present study, PNPLA7 was found to be upregulated in the process of macrophage polarization toward an alternatively activated (M2) phenotype stimulated with interleukin 4 (IL-4) (P<0.05). We found that knockdown and overexpression of PNPLA1 decreased and increased the expression of M2 marker genes, including arginase 1 (Argl) and chitinase-like 3 (Ym\ ), respectively (P<0.05). Further studies showed that PNPLA7 regulated the expression of peroxisome proliferator activated receptor-γ (P P A R γ) at the mRNA and protein levels during M2 polarization (P < 0.05). However, the phosphorylation of signal transducer and activator of transcription 6 (STAT6) was not influenced by PNPLA7. These findings suggest that PNPLA7 favors macrophage anti-inflammatory M2 polarization through a PPAR

Effects of high glucose and lysophosphatidylcholine (LPC) on RAW264.7 macrophages during No-cardia farcinica infection / 中华微生物学和免疫学杂志

Objective To investigate the effects of high glucose and lysophosphatidylcholine (LPC) on the immune function of in vitro cultured macrophages during Nocardia farcinica infection. Meth-ods RAW264.7 macrophages were cultured in vitro under different conditions as follows: routine culture (control group),50 mmol/L glucose (high glucose group),10 mg/L LPC(LPC groupⅠ),25 mg/L LPC (LPC groupⅡ) and 50 mmol/L glucose+25 mg/L LPC(high glucose and LPC group). The activity of mac-rophages in each group was tested after 6,12,24 and 36 h of culture. After 24 h of culture, macrophages were collected from every group and co-cultured with Nocardia farcinica. Dynamic phagocytosis rates were detected at 1,2,3,4,5 and 6 h after co-culture. Toxic effects of Nocardia farcinica on macrophages and concentrations of IL-10 and TNF-α were measured at 1,3 and 6 h after co-culture. Results Macrophages in all four experimental groups showed decreased activity as compared with those in the control group (P<0.01). Phagocytosis of Nocardia farcinica by macrophages was also reduced by high glucose and LPC. Phagocytosis rates of high glucose group and LPC groupⅡ at 1 and 2 h,LPC groupⅠat 1,2 and 3 h,and high glucose and LPC group at 1,2,3 and 4 h after co-culture were significantly lower than that of the con-trol group (P<0.05 or P<0.01). Compared with the control group, significantly reduced toxic effects on macrophages caused by Nocardia farcinica was observed in the experimental groups (P<0.05 or P<0.01). Compared with the control group,LPC groupsⅠand Ⅱ and high glucose and LPC group had decreased se-cretion of IL-10 at 3 h,and high glucose group and LPC groupⅠhad decreased secretion of TNF-α at 1 h(P<0.05). Conclusion Culture macrophages under the conditions of high glucose and LPC would reduce their activity and impair their ability to phagocytose Nocardia farcinica. Moreover, high glucose and LPC might have impacts on the toxic effects of Nocardia farcinica on macrophages and the secretion of IL-10 and TNF-α.

Bioactive Lysophosphatidylcholine 16:0 and 18:0 Are Elevated in Lungs of Asthmatic Subjects

PURPOSE: Asthma is a chronic inflammatory disease of the airways, and is associated with upregulation of phospholipase A2 (PLA2), the enzyme that hydrolyzes phosphatidylcholine, producing lysophosphatidylcholine (LPC) and free fatty acids. LPC is a lipid mediator with known pro-inflammatory and pro-atherogenic properties, and is believed to be a critical factor in cardiovascular diseases. We postulate that asthmatic subjects have an elevated content of LPC in the lung lining fluids. METHODS: Eight non-asthmatic controls and seven asthmatic subjects were recruited for broncho-alveolar lavage fluids (BALF) collection for analysis of LPC by high performance liquid chromatography-tandem mass spectrometry. RESULTS: LPC16:0 and LPC18:0 were significantly elevated in the BALF of asthmatics with impaired lung function characteristic of moderate asthma, but not mild asthma. The increased LPC content in BALF was accompanied by increased PLA2 activity. Furthermore, qRT-PCR analysis of the BALF cell fraction indicated increased secretory PLA2-X (sPLA2-X). CONCLUSIONS: The increased LPC content in the lung lining fluids is a potential critical lipid mediator in the initiation and/or progression of airway epithelial injury in asthma.

Stimulated Bronchial Epithelial Cells Release Bioactive Lysophosphatidylcholine 16:0, 18:0, and 18:1

PURPOSE: In human subjects and animal models with acute and chronic lung injury, the bioactive lysophosphatidylcholine (LPC) is elevated in lung lining fluids. The increased LPC can promote an inflammatory microenvironment resulting in lung injury. Furthermore, pathological lung conditions are associated with upregulated phospholipase A2 (PLA2), the predominant enzyme producing LPC in tissues by hydrolysis of phosphatidylcholine. However, the lung cell populations responsible for increases of LPC have yet to be systematically characterized. The goal was to investigate the LPC generation by bronchial epithelial cells in response to pathological mediators and determine the major LPC species produced. METHODS: Primary human bronchial epithelial cells (NHBE) were challenged by vascular endothelial growth factor (VEGF) for 1 or 6 h, and condition medium and cells collected for quantification of predominant LPC species by high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The cells were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for PLA2. The direct effects of LPC in inducing inflammatory activities on NHBE were assessed by transepithelial resistance as well as expression of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1). RESULTS: VEGF stimulation of NHBE for 1 or 6 h, significantly increased concentrations of LPC16:0, LPC18:0, and LPC18:1 in condition medium compared to control. The sPLA2-selective inhibitor (oleyloxyethyl phosphorylcholine) inhibited the VEGF-induced release of LPC16:0 and LPC18:1 and PLA2 activity. In contrast, NHBE stimulated with TNF did not induce LPC release. VEGF did not increase mRNA of PLA2 subtypes sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Exogenous LPC treatment increased expression of IL-8 and MMP-1, and reduced the transepithelial resistance in NHBE. CONCLUSIONS: Our findings indicate that VEGF-stimulated bronchial epithelial cells are a key source of extracellular LPCs, which can function as an autocrine mediator with potential to induce airway epithelial inflammatory injury.

Research on Protective Effects and Mechanism of Resveratrol on Endothelial Cells / 世界科学技术-中医药现代化

This article was aimed to study the protective effects and mechanism of resveratrol (Res) on endothelial injury induced by atherosclerosis ( AS ) . Lysophosphatidylcholine ( LPC ) was applied to induce the model of injured endothelial cells. Flow cytometer was used to detect the rate of cell apoptosis. TUNEL staining was used to detect the cell apoptosis. MTT colorimetric test was used to detect the cell viability. Automatic biochemistry analyzer was used to measure the LDH level . ELISA was used to detect TNF-α level . RT-PCR was used to determine the mRNA levels of calcitonin gene-related peptide (CGRP). The results showed that Res with the concentration of 20 μmol/L can effectively slow down the degree of endothelial cell injury induced by LPC which caused the cell disability, high LDH level and high TNF-α level (P < 0.01). Res showed effects on mRNA expression of CGRP. It was concluded that Res can protect against endothelial injury induced by AS, the mechanism of which may be associated with accelerating CGRP synthesis and release by activating VR1.

Lysophosphatidylcholine Increases Ca2+ Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A(2), has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca2+ influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca2+ influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca2+ current (ICa(L)) activity and to elucidate the mechanism of LPC-induced change of ICa(L) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased ICa(L) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of ICa(L) was not significantly changed by LPC. Staurosporine (100 nanometer) or chelerythrine (3 micrometer, which is a potent inhibitor of PKC, significantly decreased basal ICa(L), and LPC-induced increase of ICa(L) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal ICa(L) significantly, and LPC-induced enhancement of ICa(L) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased ICa(L) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of ICa(L) might be, at least in part, responsible for increased Ca2+ influx in atherosclerotic artery.

Effect of Astragalus Membranaceus on the Interaction of Mesangial Cells and Endothelial Cells / 中国中医药信息杂志

Objective To study the effect of astragalus membranaceus on the interaction of mesangial cells and endothelial cells in the media of high glucose and lysophosphatidylcholine. Methods The model of intercellular interaction between endothelial cells and mesangial cells of diabetic nephropathy was established and divided into 4 groups:control, mannitol, high glucose and lysophosphatidylcholine, intervented with astragalus membranaceus. Endothelial cells and mesangial cells were co-cultured in DMEN with or without astragalus membranaceus in high glucose and lysophosphatidylcholine media up to 24 hours. The level of collagen Ⅳ and fibrocentin were determined by enzyme linked immunosorbent assay in the culture media. Results The level of collagen Ⅳ and fibrocentin of high glucose and lysophosphatidylcholine group was markedly higher than that of control group in co-culture and the ordinary monolayer cell culture (P

Effects of pravastatin on the proliferation of vascular smooth muscle cells and cell adhesion induced by lysophosphatidylcholine / 中国药理学通报

Aim This study was designed to investigate the effects of pravastatin,a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor,on vascular smooth muscle cells (VSMCs) proliferation and leukocyte-endothelial cell adhesion induced by lysophosphatidylcholine (LPC).Methods Cultured VSMCs from rabbit thoracic aorta were incubated with various concentrations of LPC in the absence or presence of different concentrations of pravastatin. MTT was used to evaluate the proliferation of VSMCs. We determined the effects of LPC and pravastatin on neutrophil K562 adhesion to endothelial cells ECV304 by directly counting non-adhesive K562 cells.Results Incubation of VSMCs with LPC (1～10 ?mol?L -1) stimulated proliferation of VSMCs in a time- and dose-dependent manner,while pravastatin (0.3～1 mmol?L -1) treatment prevented the proliferation of VSMCs caused by LPC. Moreover, incubation of ECV304 with 3 ?mol ? L -1 LPC for 12 h significantly enhanced K562 cells adhesion to endothelial cells, whereas pretreatment with pravastatin reduced leukocyte-endothelial cell adhesion. Conclusion These results suggest that pravastatin can antagonize the effects of VSMCs proliferation and leukocyte-endothelial cell adhesion induced by LPC.

The inhibitive effect of Cariporide in LPC-induced adhesion of monocytes and endothelial cells by suppressing expression of ICAM-1 and P-selectin in vitro / 中国药理学通报

Aim To investigate the effects of Cariporide on expression of intercellular adhesion molecule-1(ICAM-1) and P-selectin and adhesion of monocytes and endothelial cells induced by lysophophatidylcholine(LPC).Methods The ratio of adhesion of monocytes and endothelial cells was assessed by measuring protein content.The expression of ICAM-1 and P-selectin in endothelial cells was examined by enzyme-linked immunosorbent assay(ELISA).Intracellular pH of endothelial cells was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF).Results The results showed that LPC enhanced the adhesion of monocytes and endothelial cells in a concentration-dependant and time-related manner.The dosage of 5,10 and 20 ?mol?L-1 Cariporide reduced the adhesive ratio of endothelial cells induced by LPC(5 mg?L-1)from 36% to 23%,18% and 16%(P

Increase of Intracellular Ca2+ Concentration Induced by Lysophosphatidylcholine in Murine Aortic Endothelial Cells

Effects of oxidized low-density lipoprotein (ox-LDL), l-alpha-stearoyl-lysophosphatidylcholine (LPC), on intracellular Ca2+ concentration were examined in mouse endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i) with fura 2-AM and reverse transcription-polymerase chain reaction (RT-PCR). LPC increased [Ca2+]i under the condition of 1.5 mM [Ca2+]o but did not show any effect under the nominally Ca2+-free condition. Even after the store depletion with 30microM 2,5-di-tert- butylhydroquinone (BHQ) or 30microM ATP, LPC could still increase the [Ca2+]i under the condition of 1.5 mM [Ca2+]o. The time required to increase [Ca2+]i (about 1 minute) was longer than that for ATP-induced [Ca2+]i increase (10-30 seconds). LPC-induced [Ca2+]i increase was completely blocked by 1microM La3+. Transient receptor potential channel(trpc) 4 mRNA was detected with RT-PCR. From these results, we suggest that LPC increased [Ca2+]i via the increase of Ca2+ influx through the Ca2+ routes which exist in the plasma membrane.