A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.     

A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells

Lipopolysaccharide (LPS)-induced vascular endothelial cell (VEC) dysfunction is an important contributing factor in vascular diseases. Recently, we found that LPS impaired VEC by inducing autophagy. Our previous researches showed that a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) selectively protected VEC function. The objective of the present study is to investigate whether and how 3BDO inhibits LPS-induced VEC autophagic injury. Our results showed that LPS induced autophagy and led to increase of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential (MMP) in Human umbilical vein vascular endothelial cells (HUVECs). Furthermore, LPS significantly increased p8 and p53 protein levels and the nuclear translocation of p53. All of these effects of LPS on HUVECs were strongly inhibited by 3BDO. Importantly, the ROS scavenger N-acetylcysteine (NAC) could inhibited LPS-induced autophagy and knockdown of p8 by RNA interference inhibited the autophagy, p53 protein level increase, the translocation of p53 into nuclei and the ROS level increase induced by LPS in HUVECs. The data suggested that 3BDO inhibited LPS-induced autophagy in HUVECs through inhibiting the ROS overproduction, the increase of p8 and p53 expression and the nuclear translocation of p53. Our findings provide a potential tool for understanding the mechanism underlying LPS-induced autophagy in HUVECs and open the door to a novel therapeutic drug for LPS-induced vascular diseases.     

Treponema denticola induces interleukin-8 and macrophage chemoattractant protein 1 production in human umbilical vein epithelial cells

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Phosphatidylcholine-specific phospholipase C, p53 and ROS in the association of apoptosis and senescence in vascular endothelial cells

Previously, we found that phosphatidylcholine-specific phospholipase C (PC-PLC) participated in apoptosis signaling of vascular endothelial cells (VECs). Here, to explore whether PC-PLC is involved in the association of apoptosis and senescence in VECs, we analyzed p53 expression and intracellular reactive oxygen species (ROS) levels in young and senescent VECs before and after inhibiting PC-PLC activity. The results showed that suppressing PC-PLC inhibited apoptosis and the elevation of p53 expression induced by apoptosis in young cells, but not in senescent cells, and that inhibiting PC-PLC depressed intracellular ROS levels both in young and senescent cells. The data suggested that PC-PLC was involved in the association of apoptosis and senescence. Its function might be closely related to the level of p53 in VECs.     

胰高血糖素样肽1对脂多糖诱导的血管内皮细胞炎性反应的影响

目的：探讨胰高血糖素样肽1（glucagon like peptide 1,GLP-1）对脂多糖（1ipopolysaccharide,LPS）诱导的血管内皮细胞（VEC）炎性反应的影响。方法：以体外培养的人动脉VEC为研究模型,将细胞分为四组（对照组、LPS刺激组、LPS±GLP-1组、GLP-1组）,Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红（HE）染色观察细胞间连接的形态特征,用示踪剂Rhodamine Bisothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素（IL）-6和IL-8的变化。结果：GLP-1（100nM）可减少LPS（1μg／mL）刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示：GLP-1可明显降低LPS刺激引起的VEC通透性增加[由（2．57±0．19）×10^-5cm／s降至（2．10±0．18）×10^-5cm／s,P〈0．05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由（42130±6522）pg／ml降至（27478±5096）pg／ml和（18376±1561）pg／ml降至（14414±927）pg／ml,均P〈0．05]。结论：GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加．改善LPS诱导的内皮细胞炎性损伤。     

胰高血糖素样肽l对脂多糖诱导的血管内皮细胞炎性反应的影响

目的:探讨胰高血糖素样肽l(glucagon like peptide 1,GLP-1)对脂多糖(1ipopolysaccharide,LPS)诱导的血管内皮细胞(VEC)炎性反应的影响。方法:以体外培养的人动脉VEC为研究模型,将细胞分为四组(对照组、LPS刺激组、LPS+GLP-1组、GLP-1组),Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红(HE)染色观察细胞间连接的形态特征,用示踪剂Rhodamine B isothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素(IL)-6和IL-8的变化。结果:GLP-1(100 nM)可减少LPS(1μg/mL)刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示:GLP-1可明显降低LPS刺激引起的VEC通透性增加[由(2.57±0.19)×10-5cm/s降至(2.10±0.18)×10-5cm/s,P0.05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由(42130±6522)pg/ml降至(27478±5096)pg/ml和(18376±1561)pg/ml降至(14414±927)pg/ml,均P0.05]。结论:GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加,改善LPS诱导的内皮细胞炎性损伤。     

Targeting annexin A7 by a small molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells and inhibited atherosclerosis in apolipoprotein E?/? mice

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E^−/− (apoE^−/−) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE^−/− mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE^−/− mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE^−/− mice.     

长链非编码RNA KCNQ1OT1影响脂多糖诱导的血管内皮细胞凋亡及其炎性因子表达的机制

该研究探讨了长链非编码RNA KCNQ1OT1对脂多糖(LPS)诱导的血管内皮细胞(VEC)凋亡和炎性因子表达的影响以及其可能机制.通过体外培养VEC,分别转染KCNQ1OT1过表达载体、miR-223抑制剂或共转染KCNQ1OT1过表达载体与miR-223模拟物后,用1.0mg/mLLPS干预24h,然后采用RT-q...     

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions,adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1β, tumor necrosis factor-α (TNF-α) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts

Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.     

Cilostazol attenuates MCP-1 and MMP-9 expression in vivo in LPS-administrated balloon-injured rabbit aorta and in vitro in LPS-treated monocytic THP-1 cells

Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) are involved in vascular inflammation. We tested the hypothesis, and explored the underlining mechanisms that cilostazol, a phosphodiesterase 3 inhibitor with antiplatelet and antithrombotic properties, inhibits lipopolysaccharide (LPS)-induced MCP-1 and MMP-9 expression. In a rabbit aorta balloon-injury model, administration of LPS increased macrophage infiltration and MCP-1 and MMP-9 expression; cilostazol supplementation prevented this phenomenon and reduced intimal hyperplasia. In contrast, the reverse zymography showed that cilostazol did not affect TIMP-1 expression in serum. In monocytic THP-1 cells, cilostazol and N6,O2'-dibutyryl-cAMP (dioctanoyl-cAMP, a cAMP analog) dose-dependently inhibited LPS-induced MCP-1 protein expression and MMP-9 activation, but did not affect the tissue inhibitor of metalloproteinase-1. Quantitative real-time polymerase chain reaction (PCR) showed that cilostazol inhibited MCP-1 and MMP-9 mRNA expression. Cilostazol significantly inhibited LPS-induced activation of p38, JNK, and nuclear factor-kappaB, and the respective inhibitors of p38 and JNK greatly reduced the level of LPS-induced MCP-1 and MMP-9, suggesting the involvement of the p38 and JNK pathways. In conclusion, cilostazol administered with LPS in vivo reduced neointimal hyperplasia and macrophage infiltration in the balloon-injured rabbit aorta; in vitro, cilostazol inhibits LPS-induced MCP-1 and MMP-9 expression. These data suggest that cilostazol may play an important role in preventing endotoxin- and injured-mediated vascular inflammation.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

Rhamnocitrin extracted from Nervilia fordii inhibited vascular endothelial activation via miR-185/STIM-1/SOCE/NFATc3

BackgroundVascular endothelial activation is pivotal for the pathological development of various infectious and inflammatory diseases. Therapeutic interventions to prevent endothelial activation are of great clinical significance to achieve anti-inflammatory strategy. Previous studies indicate that the total flavonoids from the endemic herbal medicine Nervilia fordii (Hance) Schltr exerts potent anti-inflammatory effect and protective effect against endotoxin lipopolysaccharide (LPS)-induced acute lung injury, and shows clinical benefit in severe acute respiratory syndromes (SARS). However, the exact effective component of Nervilia fordii and its potential mechanism remain unknown.PurposeThe aim of this study was to investigate the effect and mechanism of rhamnocitrin (RH), a flavonoid extracted from Nervilia fordii, on LPS-induced endothelial activation.MethodsThe in vitro endothelial cell activation model was induced by LPS in human umbilical vein endothelial cells (HUVECs). Cell viability was measured to determine the cytotoxicity of RH. RT-PCR, Western blot, fluorescent probe and immunofluorescence were conducted to evaluate the effect and mechanism of RH against endothelial activation.ResultsRH was extracted and isolated from Nervilia fordii. RH at the concentration from 10⁻⁷ M-10⁻⁵ M inhibited the expressions of interlukin-6 (IL-6) and -8 (IL-8), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell-adhesion molecule-1 (VCAM-1), and plasminogen activator inhibitor-1 (PAI-1) in response to LPS challenge. Mechanistically, RH repressed calcium store-operated Ca²⁺ entry (SOCE) induced by LPS, which is due to downregulation of stromal interaction molecule-1 (STIM-1) following upregulating microRNA-185 (miR-185). Ultimately, RH abrogated LPS-induced activation of SOCE-mediated calcineurin/NFATc3 (nuclear factor of activated T cells, cytoplasmic 3) signaling pathway.ConclusionThe present study identifies RH as a potent inhibitor of endothelial activation. Since vascular endothelial activation is a pivotal cause of excessive cytokine production, leading to cytokine storm and severe pathology in infectious diseases such as SARS and the ongoing COVID-19 pneumonia disease, RH might suggest promising therapeutic potential in the management of cytokine storm in these diseases.     

Lysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.     

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.     

Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3'methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Effects of CXCL4/CXCR3 on the lipopolysaccharide-induced injury in human umbilical vein endothelial cells

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well-known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)-treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory-induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose- and time-dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS-induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin-4 and Zonula occluden-1[ZO-1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS-treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4-stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway.