The long pentraxin PTX3 in vascular pathology

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor acting as a non-redundant component of the humoral arm of innate immunity and involved in tuning inflammation, in matrix deposition and female fertility.     

Ginkgolide B reduces inflammatory protein expression in oxidized low-density lipoprotein-stimulated human vascular endothelial cells

Ginkgolide B is a herbal constituent extracted from leaves of the ginkgo biloba tree. Previous studies have shown that ginkgolide B is a specific platelet activating factor (PAF) receptor antagonist, and it suppresses PAF-mediated platelet activation via competitive binding. In this study, the effect of ginkgolide B on nicotinamide adenine dinucleotide phosphate oxidase and other inflammatory proteins in ox-LDL (low-density lipoprotein)-stimulated human vascular endothelial cells was investigated. Another PAF receptor antagonist CV3988 was employed to compare with ginkgolide B in this study. Our results show that the enhancement of Nox4 expression and reactive oxygen species generation was attenuated by ginkgolide B in cells treated with ox-LDL but not with CV3988. Increases in monocyte chemoattractant protein-1 and intercellular adhesion molecule 1 expression induced by ox-LDL, however, were inhibited by both ginkgolide B and CV3988. The translocation of NF-kappaB p65 (NF-κB) into the nucleus was inhibited by both ginkgolide B and CV3988. In conclusion, both ginkgolide B and CV3988 can inhibit the expression of inflammatory proteins by blocking NF-κB translocation. It seems that ginkgolide B possesses some pharmacological action on intracellular oxidative stress in association with the downregulation of Nox4 expression.     

Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential sensitivity to statin-induced toxicity.     

Lovastatin reduces nuclear factor kappaB activation induced by C-reactive protein in human vascular endothelial cells

The role of C-reactive protein (CRP) in atherogenesis has been supported by more recent data. Some studies have demonstrated marked up-regulation inflammatory responses in endothelial cells subjected to CRP. The nuclear factor-kappaB (NF-kappaB) signal transduction is known to play a key role in the expression of these proatherogenic entities. Statins have anti-inflammatory properties independent of their cholesterol-lowering effects. Therefore, we studied the effects of CRP and lovastatin on NF-kappaB activation in human umbilical vein endothelial cells (HUVECs). By using an electrophoretic mobility shift assays (EMSA), we found that CRP (50 microg/ml) increased activation of NF-kappaB and degradation of inhibitory kappa B (IkappaB) in HUVECs, reaching a maximal effect after the incubation with CRP for 1 h. Lovastatin (10(-5) mol/l) diminished NF-kappaB activation induced by CRP. Furthermore, lovastatin may block NF-kappaB activation by causing a stabilization of the IkappaB-alpha in cellular cytoplasm with western blotting analysis. Preincubation of HUVECs with pyrrolidinethiocarbamate (PDTC, NF-kappaB inhibitor) diminished CD40 expression induced by CRP with flow cytometry. Our results suggest that CRP increases activation of NF-kappaB and induces CD40 expression in HUVECs partly via activation of NF-kappaB. Lovastatin, through the inhibition of NF-kappaB activation, reduces the inflammation involved in the pathogenesis of atherosclerosis.     

Aidi injection reduces doxorubicin-induced cardiotoxicity by inhibiting carbonyl reductase 1 expression

ContextAidi injection (ADI), a traditional Chinese medicine antitumor injection, is usually combined with doxorubicin (DOX) for the treatment of malignant tumours. The cardiotoxicity of DOX is ameliorated by ADI in the clinic. However, the relevant mechanism is unknown.ObjectiveTo investigate the effects of ADI on DOX-induced cardiotoxicity and its mechanism.Materials and methodsICR mice were randomly divided into six groups: control, ADI-L, ADI-H, DOX, DOX + ADI-L and DOX + ADI-H. DOX (i.p., 0.03 mg/10 g) was administered in the presence or absence of ADI (i.p., 0.1 or 0.2 mL/10 g) for two weeks. Heart pathology and levels of AST, LDH, CK, CK-MB and BNP were assessed. H9c2 cells were treated with DOX in the presence or absence of ADI (1, 4, 10%). Cell viability, caspase-3 activity, nuclear morphology, and CBR1 expression were then evaluated. DOX and doxorubicinol (DOXol) concentrations in heart, liver, kidneys, serum, and cells were analysed by UPLC-MS/MS.ResultsHigh-dose ADI significantly reduced DOX-induced pathological changes and the levels of AST, LDH, CK, CK-MB and BNP to normal. Combined treatment with ADI (1, 4, 10%) improved the cell viability, and IC50 increased from 68.51 μM (DOX alone) to 83.47, 176.9, and 310.8 μM, reduced caspase-3 activity by 39.17, 43.96, and 61.82%, respectively. High-dose ADI inhibited the expression of CBR1 protein by 32.3%, reduced DOXol levels in heart, serum and H9c2 cells by 59.8, 72.5 and 48.99%, respectively.Discussion and ConclusionsADI reduces DOX-induced cardiotoxicity by inhibiting CBR1 expression, which provides a scientific basis for the rational use of ADI.     

Glycogen synthase kinase 3 activity mediates neuronal pentraxin 1 expression and cell death induced by potassium deprivation in cerebellar granule cells

Expression of neuronal pentraxin 1 (NP1) is part of the apoptotic cell death program activated in mature cerebellar granule neurons when potassium concentrations drop below depolarizing levels. NP1 is a glycoprotein homologous to the pentraxins of the acute phase immune response, and it is involved in both synaptogenesis and synaptic remodeling. However, how it participates in the process of apoptotic neuronal death remains unclear. We have studied whether the signaling pathways known to control neuronal cell death and survival influence NP1 expression. Both activation of the phosphatidylinositol 3-kinase/Akt (PI-3-K/AKT) pathway by insulin-like growth factor I and pharmacological blockage of the stress activated c-Jun NH(2)-terminal kinase (JNK) offer transitory neuroprotection from the cell death evoked by nondepolarizing concentrations of potassium. However, neither of these neuroprotective treatments prevents the overexpression of NP1 upon potassium depletion, indicating that nondepolarizing conditions activate additional cell death signaling pathways. Inhibiting the phosphorylation of the p38 mitogen-activated protein kinase without modifying JNK, neither diminishes cell death nor inhibits NP1 overexpression in nondepolarizing conditions. In contrast, impairing the activity of glycogen synthase kinase 3 (GSK3) completely blocks NP1 overexpression induced by potassium depletion and provides transient protection against cell death. Moreover, simultaneous pharmacological blockage of both JNK and GSK3 activities provides long-term protection against the cell death evoked by potassium depletion. These results show that both the JNK and GSK3 signaling pathways are the main routes by which potassium deprivation activates apoptotic cell death, and that NP1 overexpression is regulated by GSK3 activity independently of the PI-3-K/AKT or JNK pathway.     

哇巴因抑制钠泵阻β1亚单位和VE-cadherin减弱血管内皮细胞连接功能

目的 探讨钠泵抑制剂哇巴因对血管内皮细胞连接的影响及机制.方法 以人脐静脉内皮细胞(HUVECs)为靶细胞,Hoechst33342/PI双荧光染色法观察哇巴因作用后细胞凋亡或坏死特征,透射电镜和光镜观察细胞形态结构变化.应用半定量聚合酶链反应检测钠泵α1亚单位、β1亚单位、VE-cadherin和Snail mRNA的表达.结果 0.1μmol·L-1哇巴因作用HuVECs 24～48 h,细胞死亡以凋亡为主,10μmol·L-1哇巴因作用24 h,引起细胞坏死;对照组细胞间的细胞连接数量多,结构清晰,而经哇巴因作用后,细胞连接丧失,细胞脱落.哇巴因作用HuVEcs后,钠泵α1亚单位和Snail表达上调,β1亚单位和VE-cadherin表达下降,其改变均呈剂量和时间依赖性.结论 哇巴因通过下调血管内皮细胞钠泵β1亚单位和VE-cadherin的表达使细胞连接功能减弱.     

哇巴因抑制钠泵β1亚单位和VE-cadherin减弱血管内皮细胞连接功能

目的探讨钠泵抑制剂哇巴因对血管内皮细胞连接的影响及机制。方法以人脐静脉内皮细胞(HUVECs)为靶细胞,Hoechst33342/PI双荧光染色法观察哇巴因作用后细胞凋亡或坏死特征,透射电镜和光镜观察细胞形态结构变化。应用半定量聚合酶链反应检测钠泵α1亚单位、β1亚单位、VE-cadherin和SnailmRNA的表达。结果0.1μmol.L-1哇巴因作用HUVECs24～48h,细胞死亡以凋亡为主,10μmol·L-1哇巴因作用24h,引起细胞坏死;对照组细胞间的细胞连接数量多,结构清晰,而经哇巴因作用后,细胞连接丧失,细胞脱落。哇巴因作用HUVECs后,钠泵α1亚单位和Snail表达上调,β1亚单位和VE-cadherin表达下降,其改变均呈剂量和时间依赖性。结论哇巴因通过下调血管内皮细胞钠泵β1亚单位和VE-cadherin的表达使细胞连接功能减弱。     

Celecoxib modulates adhesion of HT29 colon cancer cells to vascular endothelial cells by inhibiting ICAM-1 and VCAM-1 expression

BACKGROUND AND PURPOSE: Cyclooxygenase-2 (COX-2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX-2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM-1 and VCAM-1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS-coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM-1 and VCAM-1, to assess further the potential protective effects of celecoxib on cancer development. EXPERIMENTAL APPROACH: Celecoxib was incubated for 4 h with HT29 cells and HUVEC and adhesion was quantified by a computerized micro-imaging system. Expression analysis of ICAM-1 and VCAM-1 cell adhesion molecules was performed by western blot. KEY RESULTS: Celecoxib (1 nM-10 microM) inhibited, with the same potency, adhesion of HT29 cells to resting HUVEC or to HUVEC stimulated by tumour necrosis factor-alpha (TNF-alpha), mimicking inflammatory conditions. Analysis of ICAM-1 and VCAM-1 expression showed that celecoxib inhibited expression of both molecules in TNF-alpha-stimulated HUVEC, but not in resting HUVEC; inhibition was concentration-dependent and maximal (about 50%) at 10 microM celecoxib. CONCLUSIONS AND IMPLICATIONS: In conclusion, our data show that celecoxib inhibits HT29 cell adhesion to HUVEC and expression of ICAM-1 and VCAM-1, in stimulated endothelial cells. These effects may contribute to the chemopreventive activity of celecoxib in the development of colorectal cancer.     

牛磺酸对高糖诱导的大鼠血管内皮细胞氧化应激与凋亡的保护作用

目的观察牛磺酸对高糖培养诱导大鼠血管内皮细胞氧化应激与凋亡的影响。方法组织块法原代培养大鼠胸主动脉内皮细胞,取对数生长期的第4代内皮细胞进行试验。培养体系中的葡萄糖浓度保持30 mmol/L,加入终浓度为0,2.5,5,10,20 mmol/L的牛磺酸共培养24 h,流式细胞仪检测细胞内活性氧(ROS)水平及细胞凋亡率,比色法测定细胞培养基中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果与0 mmol/L比较,2.5~20 mmol/L组细胞内ROS水平依次降低,组间比较差异均有统计学意义(P<0.05);5和10 mmol/L牛磺酸抑制细胞凋亡作用明显。5~20 mmol/L组MDA含量逐渐降低,而SOD活性逐渐增强。结论牛磺酸能削弱高糖诱导大鼠血管内皮细胞的氧化应激,抑制细胞凋亡。     

Reduction of GTP cyclohydrolase I feedback regulating protein expression by hydrogen peroxide in vascular endothelial cells

We examined the effect of H(2)O(2) on the expression of GTP cyclohydrolase I (GTPCH) feedback regulating protein (GFRP). Addition of H(2)O(2) to endothelial cells decreased GFRP mRNA levels, in contrast to the increase of tetrahydrobiopterin (BH(4)) content and GTPCH mRNA levels. The inhibitors of nitric oxide (NO) synthase and GTPCH had no influence on the decrease of GFRP mRNA levels in H(2)O(2)-treated cells. It is suggested that H(2)O(2) induces BH(4) synthesis through not only induction of GTPCH but also reduction of GFRP. The decrease of GFRP mRNA level appears to be independent of the produced NO and BH(4).     

地塞米松通过抑制p53蛋白降解诱导成骨细胞凋亡

目的 观察地塞米松对成骨细胞凋亡的影响并探讨其作用机制。方法 分别采CellTiter-Glo?荧光细胞活力检测试剂盒和Annexin V-FITC/PI双染凋亡检测试剂盒测定地塞米松作用后MC3T3-E1细胞的增殖和凋亡。蛋白质印迹法(Western blotting)检测地塞米松作用后MC3T3-E1细胞和hFOB1.19细胞胱天蛋白酶3(caspase-3)/裂解caspase-3和PARP/裂解PARP的表达。用Western blotting和PCR分析地塞米松作用后MC3T3-E1细胞中p53和检控点激酶2(Chk2)的表达。探讨p53敲除和Chk2敲除在地塞米松诱导MC3T3-E1细胞凋亡中的作用。结果 地塞米松能显著抑制MC3T3-E1细胞的生长并诱导其凋亡。地塞米松能通过促进p53的表达诱导成骨细胞凋亡。地塞米松对P53蛋白表达的调节作用是通过上调Chk2来实现的,Chk2与P53相互作用,抑制P53蛋白的降解。P53基因敲除可通过降低裂解caspase-3和裂解PARP的表达来抑制地塞米松诱导的MC3T3-E1细胞凋亡,经Dex处理的MC3T3-E1和hFOB1.1...     

Paeonol inhibits oxidized low density lipoprotein-induced monocyte adhesion to vascular endothelial cells by inhibiting the mitogen activated protein kinase pathway

Atherosclerosis is a chronic inflammatory disease characterized by increased expression of adhesion molecules, which contribute to monocytes adhesion to vascular endothelial cells (VECs). Paeonol, an active compound isolated from cortex Moutan, has been shown to have therapeutic effects on atherosclerotic animals. The present study aims to investigate whether paeonol can inhibit monocyte adhesion to vascular endothelial cells induced by oxidized Low-Density Lipoprotein (ox-LDL) and its possible therapeutic molecular mechanism. Exposure to ox-LDL (50, 100 μg/mL) induced damaged to VECs leading to decreased survival rates (p<0.01). Paeonol (7.2-18.0 μM) partially restored survival and reduced lactate dehydrogenase (LDH) release in VECs in a concentration-dependent manner (p<0.01). Adhesion of monocytes to VECs was dramatically prevented by paeonol at 21.6 and 25.2 μM (p<0.01). In addition, paeonol (14.4-21.6 μM) repressed the expression of vascular cell adhesion molecule-1 (VCAM-1) and lowered the levels of phosphor-c-Jun N-terminal kinase (P-JNK)1/2, phosphor-extracellular signal-regulated kinase (P-ERK)1/2 and P-p38 in a dose-dependent manner. The molecular effects of paeonol were more pronouced when companied with mitogen activated protein kinases (MAPKs) inhibitors. These data suggest that paeonol (10.8-25.2 μM), at certain concentrations, prevents monocyte adhesion to VEC induced by ox-LDL, probably by means of blocking one or more target proteins on MAPKs signaling pathway. These results indicate that paeonol has potential protective effects on the development of atherosclerosis.     

Particulate matter-mediated release of long pentraxin 3 (PTX3) and vascular endothelial growth factor (VEGF) in vitro: Limited importance of endotoxin and organic content

Exposure to particulate matter (PM) is associated with adverse health effects, but it is still relatively unknown which role PM sources and physicochemical properties play in the observed effects. It was postulated that PM in vitro induces release of long pentraxin 3 (PTX3) and vascular endothelial growth factor (VEGF) and that endotoxin and organic compounds present in the PM regulate this release. A contact coculture of THP-1 human leukemia monocytes and A549 human adenocarcinoma alveolar pneumocytes was exposed to PM from Traffic, Wood, Diesel, and Quartz (10–40 µg/cm²) for 12–64 h to determine release of PTX3 and VEGF. The role of endotoxin and the organic fraction in the mediator release was assessed using polymyxin B sulfate and organic extracts, respectively. Finally, antagonists were used to investigate whether the early proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α affected the PTX3 and VEGF release. All PM samples induced a time-dependent release of both PTX3 and VEGF. Traffic mediated the greatest release of PTX3, whereas Wood and Diesel were more potent inducers of VEGF. The endotoxin content did not markedly affect release of either mediator, while the organic fraction exerted no significant effect on VEGF release and limited influence on PTX3 release. In addition, the IL-1 and TNF-α agonists affected PTX3 release more strongly than VEGF release. In conclusion, the current data show a limited impact of endotoxin and organic compounds on PTX3 and VEGF release. Further, the observed differences in response patterns may point toward differential regulation of PM-mediated release of PTX3 and VEGF.     

Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation

Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated.In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1–10 μM) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47^phox translocation from cytosol to membranes.In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20 mg). We observed that as early as 2 h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients.This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug.