溶血磷脂酰胆碱诱导内皮细胞表达血管内皮生长因子及丹酚酸B的抑制作用

研究溶血磷脂酰胆碱对内皮细胞中血管内皮生长因子表达的影响以及丹酚酸B的保护作用。在人脐静脉内皮细胞株ECV30 4培养基中加入溶血磷脂酰胆碱或溶血磷脂酰胆碱 +丹酚酸B ,用酶联免疫吸附试验检测各组内皮细胞培养上清液中血管内皮生长因子蛋白含量 ;用原位杂交检测血管内皮生长因子mRNA的表达。结果显示 ,培养的ECV30 4中未见血管内皮生长因子mRNA的表达 ,溶血磷脂酰胆碱刺激后可见血管内皮生长因子mRNA的高表达 ,加入丹酚酸B后阳性反应明显低于溶血磷脂酰胆碱组。酶联免疫吸附试验结果显示 ,溶血磷脂酰胆碱可使ECV30 4细胞条件培养基中血管内皮生长因子蛋白表达明显增加 ,丹酚酸B可明显降低其含量。以上结果提示 ,溶血磷脂酰胆碱能诱导ECV30 4表达高水平的血管内皮生长因子 ,丹酚酸B可明显降低其含量     

丹酚酸B对溶血磷脂酰胆碱刺激内皮细胞产生MMP-2的抑制作用

目的　研究抗氧化剂丹酚酸B对溶血磷脂酰胆碱 (LPC)诱导牛主动脉内皮细胞 (BAEC)产生MMP 2的影响。方法　采用体外培养牛主动脉内皮细胞的方法 ,用LPC单独或与丹酚酸B共同作用于BAEC ,明胶酶谱法测定细胞培养上清中基质金属蛋白酶 2 (MMP 2 )活性。结果　LPC显著促进BAEC产生MMP 2 ,当浓度为 0 .0 2 5 μg·L-1时有明显的刺激作用 (P <0 .0 5 ) ,0 .2 5 μg·L-1时作用增强 (P <0 .0 1)。丹酚酸B能抑制LPC刺激MMP 2产生的作用 ,抑制作用具有剂量依赖性 ,在浓度为 0 .0 1μmol·L-1抑制作用达到显著水平 (P <0 .0 5 ) ,在 0 .1μmol·L-1时抑制作用达到极显著性水平 (P <0 .0 1)。结论　LPC能刺激内皮细胞产生MMP 2 ,抗氧化剂丹酚酸B能抑制LPC的刺激作用。     

钙离子信使通路在溶血卵磷脂促进巨噬泡沫细胞胆固醇外流中的作用

探讨溶血卵磷脂对巨噬泡沫细胞胆固醇外流影响及第二信使细胞内钙在这一效应中所起的作用 ,为动脉粥样硬化的有效防治提供理论依据。分离培养小鼠腹腔巨噬细胞 ,用乙酰化低密度脂蛋白负载使之形成巨噬泡沫细胞 ;酶学荧光法检测细胞胆固醇外流 ,荧光分光光度法检测细胞内钙 ,分别螯合细胞外钙和抑制蛋白激酶C活性后检测溶血卵磷脂对巨噬泡沫细胞胆固醇外流的影响。结果发现 ,在 10～ 80 μmol/L浓度范围内 ,各剂量溶血卵磷脂组培养基中游离胆固醇浓度明显高于对照组。各剂量溶血卵磷脂引起胞内钙离子浓度升高 ,当用EGTA螯合细胞外钙后 ,溶血卵磷脂不引起胞内钙离子浓度升高 ,以及不再促进巨噬泡沫细胞胆固醇外流。当蛋白激酶C活性受抑制后 ,溶血卵磷脂不再促进细胞胆固醇外流。溶血卵磷脂在 10～ 80 μmol/L浓度范围内能够促进巨噬泡沫细胞胆固醇外流 ,这一效应与胞内第二信使细胞内钙浓度升高有关 ,而且钙离子 -蛋白激酶C这一信使通路介导了溶血卵磷脂促进巨噬泡沫细胞胆固醇外流效应。     

厄贝沙坦对溶血磷脂酰胆碱所致人静脉内皮细胞损害的影响

目的 观察厄贝沙坦（Irbesartan,Irb）对由溶血磷脂酰胆碱（Lysophosphatidyl choline,LPC）所致人脐静脉内皮细胞（Human umbilical vein endothelial cells,HUVECs）损害的影响。方法 体外培养HUVECs,分为（1）正常对照组;（2）低浓度LPC组（10μg/L）;（3）中浓度LPC组（20μg/L）;（4）高浓度LPC组（40μg/L）;（5）Irb对照组（含LPC20μg/L）;（6）低浓度Irb组(10-7μmol/L+LPC20μg/L);（7）中浓度Irb组(10-6μmol/L+LPC20μg/L);（8）高浓度Irb组(10-5μmol/L+LPC20μg/L)。采用放免及RT-PCR法分别观测LPC对HUVECs血管紧张素II(Angiotensin II,ATII)蛋白及AT1型受体（Angontensin II 1 receptor,AT1R）、AT2型受体（Angontensin II2 receptor,AT2R）mRNA（messenger Ribonucleic Acid）的表达的影响;采用化学比色法测定一氧化氮（NO）含量,一氧化氮合酶（NOS）及超氧化物歧化酶（SOD）活性;并观测使用Irb干预后的效果。结果 与正常对照组相比,LPC使HUVECs ATII蛋白及AT1RmRNA的表达显著增加,使NO含量、NOS及SOD活性显著下降;经Ibr干预后显著增加了HUVECs NO含量、NOS及SOD的活性。结论 Irb可以对LPC所致的内皮细胞损害发挥部分保护作用。     

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 A₂, 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 (I_Ca(L)) activity and to elucidate the mechanism of LPC-induced change of I_Ca(L) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased I_Ca(L) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of I_Ca(L) was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine (3 µM), which is a potent inhibitor of PKC, significantly decreased basal I_Ca(L), and LPC-induced increase of I_Ca(L) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal I_Ca(L) significantly, and LPC-induced enhancement of I_Ca(L) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased I_Ca(L) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of I_Ca(L) might be, at least in part, responsible for increased Ca²⁺ influx in atherosclerotic artery.     

PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30 μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKC? inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKC? in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKC? in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKC?-mediated serine phosphorylation of Cx43. PKC? inhibitors might therefore be effective in prevention of LPC-related diseases.     

Metabolic basis for asthma and rhinitis: An integrated approach

Observations that suggest a metabolic basis for the association between asthma and rhinitis and their pathogenesis are reviewed. We have observed increased plasma lysophosphatidylcholine (LPC) levels and decreased (Na⁺-K⁺)-ATPase activity in the leukocytes of patients with asthma and rhinitis. In asthmatic patients leukocytes also showed significantly lower Ca⁺⁺-ATPase activity. LPC increases cell membrane permeability to Na⁺ and Ca⁺⁺, causes membrane depolarization, potentiates IgE response, promotes phagocytic activity, inhibits adenylate cyclase, and stimulates phosphodiesterase, thus decreasing cAMP levels in the cells. These changes, when accompanied by failure of Na/K and Ca pumps, would lead to increased cytosolic Ca⁺⁺ and enhanced release of mediators from the mast cells in the airway lumen, producing a state of airway inflammation. We have also observed abnormal fall in specific airway conductance at residual volume in these patients, thereby showing small airway obstruction in asthma as well as in rhinitis. This could be due to airway inflammation caused by mechanisms described above. Large airway obstruction seen in asthma could be due to failure of Ca pump, normal activity of which seems essential for proper efflux of Ca⁺⁺ from airway smooth muscle cells. We suspect that increased plasma LPC levels and failure of the homeostatic ionic pumps occur due to some defect in oxidative metabolism and suggest that asthma and rhinitis could be seen as metabolic disorders.     

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.