Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Background and purpose:

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Experimental approach:

We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.

Key results:

We showed that exposure of EA.hy926 cells to Deta-NO (125–1000 µM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 µM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.

Conclusions and implications:

These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.     

油酰乙醇胺对人脐静脉内皮细胞血管细胞黏附分子-1表达的影响

目的探讨油酰乙醇胺(OEA)对肿瘤坏死因子α(TNF-α)诱导的人脐静脉内皮细胞血管细胞黏附分子-1(VCAM-1)表达的影响。方法体外培养人脐静脉内皮细胞,分别加入3种不同浓度的OEA(10,50,100μmol/L)或非诺贝特(10,50,100μmol/L)共同孵育10 h,再加入TNF-α共同孵育6 h,采用实时定量逆转录聚合酶链式反应和酶联免疫吸附剂检测测定VCAM-1以及mRNA和蛋白的表达,并采用Western blot方法检测过氧化物酶体增殖物激活受体α(PPAR-α)的蛋白表达。结果与非诺贝特相比,不同浓度的OEA更加显著地抑制人脐静脉内皮细胞VCAM-1mRNA和蛋白的表达,随着浓度的增大,抑制作用逐渐增强。Western bolt结果显示OAE能明显增强PPAR-α蛋白的表达。结论OEA对TNF-α引起的内皮细胞受损起到保护作用,其机理可能与上调过PPAR-α有关。     

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

Aim:

To investigate the protective effects of prostaglandin E₁ (PGE₁) against H₂O₂-induced oxidative damage on human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with PGE₁ (0.25, 0.50, and 1.00 μmol/L) for 24 h and exposed to H₂O₂ (200 μmol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR.

Results:

PGE₁ (0.25−1.00 μmol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H₂O₂. PGE₁ also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE₁ significantly increased NO content, eNOS protein, and mRNA expression.

Conclusion:

PGE₁ effectively protected endothelial cells against oxidative stress induced by H₂O₂, an activity that might depend on the up-regulation of NO expression.     

Ginsenoside Rg3 alleviates the migration,invasion, and angiogenesis of lung cancer cells by inhibiting the expressions of cyclooxygenase-2 and vascular endothelial growth factor

Lung cancer (LC) is a common cancer with high incidence and mortality rates. In recent years, ginsenoside Rg3 (Rg3), a traditional medicine, is widely used for the treatment of LC. Herein, we concentrate on assessing the effect of Rg3 on LC cell migration and invasion. The effects of Rg3 (0, 25, 50, and 100 μg/ml) on the viability, migration, invasion, angiogenesis, and expressions of epithelial–mesenchymal transition (EMT)-related proteins, cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) of LC cell lines were evaluated by cell counting kit-8 (CCK-8), scratch, transwell, tube formation, and western blot assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess transfection efficiency. COX2 overexpression plasmid and short hairpin RNA for VEGF (shVEGF) were applied to evaluate whether the effect of Rg3 is related to COX2 and VEGF through rescue assay. In this study, Rg3 significantly dose-dependently suppressed the viability, migration, invasion, angiogenesis, and protein expressions of N-cadherin, vimentin, COX2, and VEGF in H1299 and A549 cells, while promoting the expression of E-cadherin protein. COX2 overexpression markedly reversed the effects of Rg3 on the viability, migration, invasion, angiogenesis, and EMT-related protein expression levels in LC cells; however, such effects of COX2 overexpression were offset by VEGF knockdown. In sum, Rg3 alleviates the migration, invasion, and angiogenesis of LC cells by inhibiting the expressions of COX2 and VEGF.     

Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway

Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.     

阿托伐他汀对内皮细胞微粒诱导人脐静脉内皮细胞VCAM-1和ICAM-1表达的影响

目的:探讨阿托伐他汀对内皮细胞微粒(EMPs)诱导的人脐静脉内皮细胞(HUVECs)表达血管细胞粘附分子(VCAM)-1和细胞间粘附分子(ICAM)-1的影响。方法:取生长良好的第4,5代人脐静脉内皮细胞,将细胞分为3大组:对照组、EMPs组、EMPs+阿托伐他汀组。对照组加入培养基,EMPs组以不同浓度的EMPs(0/mL,1×102/mL,1×103/mL,1×104/mL,1×105/mL)与HUVECs共同孵育24 h,EMPs+阿托伐他汀组以不同浓度的阿托伐他汀(0.05,0.1,1.0,10μmol.L-1)与HUVECs作用1 h后,加入105/mL EMPs共同孵育24 h。分别采用实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测VCAM-1和ICAM-1 mRNA和蛋白的表达。结果:HUVECs受EMPs刺激后,VCAM-1和ICAM-1 mRNA及蛋白表达呈浓度依赖性增加,阿托伐他汀可不同程度上抑制EMPs的作用。结论:阿托伐他汀抗动脉粥样硬化作用可能部分与抑制EMPs诱导的内皮细胞VCAM-1和ICAM-1的表达有关。     

川芎嗪治疗非增殖期糖尿病视网膜病变的临床疗效及其对血清 HIF－1、VEGF的影响

目的：观察川芎嗪治疗非增殖期糖尿病视网膜病变（ NPDR）的临床疗效及其对血清低氧诱导因子－1（HIF－1）、血管内皮生长因子（VEGF）表达水平的影响。方法将60例NPDR患者（120只眼）随机分为对照组30例（60只眼）和试验组30例（60只眼）。对照组予以调节血糖、饮食控制、运动疗法等常规治疗;试验组在对照组基础上,加用川芎嗪0．24 g＋0．9％氯化钠250 mL静脉滴注qd。2组疗程均为2周。比较2组的临床疗效和血清HIF－1、VEGF的表达以及不良反应发生率。结果治疗后,试验组的临床总有效率96．67％显著优于对照组83．33％（P＜0．05）。试验组患者血清HIF－1、VEGF的表达（25．26±10．14）,（88．36±16．54） ng? L－1明显低于对照组（37．59±10．26）,（102．40±21．35） ng? L－1（ P＜0．05）。2组患者在治疗期间均未出现明显不良反应。结论川芎嗪对NPDR有较好的临床疗效,其作用机制可能与下调HIF－1、VEGF的表达有关。     

Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

Aim:

To investigate whether sphingosine-1-phosphate (S1P), a potent angiogenic factor, induced vascular endothelial growth factor-C (VEGF-C) expression in endothelial cells in vitro and to examine its underlying mechanisms.

Methods:

Human umbilical vein endothelial cells (HUVECs) were examined. VEGF-C mRNA expression in the cells was assessed using real-time PCR. VEGF-C protein and FGFR-1 phosphorylation in the cells were measured with ELISA. RNA interference was used to downregulate the expression of matrix metalloproteinase-2 (MMP-2), fibroblast growth factor-1 (FGF-1) and FGF receptor-1 (FGFR-1).

Results:

Incubation of HUVECs with S1P (1, 5, and 10 μmol/L) significantly increased VEGF-C expression. The effect was blocked by pretreatment with the MMP inhibitor GM6001 or the FGFR inhibitor SU5402, but not the EGFR inhibitor AG1478. The effect was also blocked in HUVECs that were transfected with FGFR-1 or MMP-2 siRNA. Furthermore, incubation of HUVECs with S1P (5 μmol/L) significantly increased FGFR-1 phosphorylation, which was blocked by GM6001. Moreover, knockdown of FGF-1, not FGF-2, in HUVECs with siRNAs, blocked S1P-induced VEGF-C expression.

Conclusion:

S1P induces VEGF-C expression through a MMP-2/ FGF-1/FGFR-1-dependent pathway in HUVECs.     

1H-1,2,4-triazol-3-yl-anilines: novel potent inhibitors of vascular endothelial growth factor receptors 1 and 2

Novel derivatives of 1,2,4-triazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity comparable to that of Vatalanib and Vandetanib in both homogenous time-resolved fluorescence enzymatic and cellular assays. Several active molecules feature high intrinsic permeability (>30 x 10(-5) cm/min) across Caco-2 cell monolayer.     

Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.     

Effects of hesperidin on cyclic strain-induced endothelin-1 release in human umbilical vein endothelial cells

1. Hesperidin, a member of the flavanone group of flavonoids, can be isolated in large amounts from the rinds of some citrus species and has been reported to have antihypotensive and vasodilator properties. However, the mechanism of action of hesperidin in the prevention and treatment of vascular diseases remains unclear. 2. The vascular endothelium can produce potent contracting factors, such as endothelin (ET)-1, and endothelium-derived relaxing factors, such as nitric oxide (NO). The aims of the present study were to test the hypothesis that hesperidin may alter strain-induced ET-1 secretion and NO production and to identify the putative underlying signalling pathways in human umbilical vein endothelial cells (HUVEC). 3. Hesperidin (10 and 100 micromol/L) inhibited strain-induced ET-1 secretion. Hesperidin also inhibited strain-induced increases in the formation of reactive oxygen species and extracellular signal-regulated kinase (ERK) phosphorylation. 4. Hesperidin treatment of HUVEC enhanced NO production, endothelial NO synthase (eNOS) activity and the phosphorylation of eNOS and Akt. Furthermore, hesperidin modulated strain-induced ET-1 release and suppressed ERK phosphorylation in part via the NO/protein kinase G pathway. 5. In summary, we have demonstrated that hesperidin inhibits strain-induced ET-1 secretion and enhances NO production in HUVEC.     

HIF,hypoxia and the role of angiogenesis in non-small cell lung cancer

Importance of the field: The role of angiogenesis in the initiation and progression of NSCLC and the molecular alterations leading to the growth of tumor vasculature are areas of great interest and recent therapeutic success.

Areas covered in this review: VEGF and its receptors play critical roles in the development of tumor vasculature and can be targeted by agents such as bevacizumab in the treatment of NSCLC. Furthermore, tumor hypoxia and the expression of the hypoxia-inducible factor (HIF) family of proteins are also linked to poorer survival in these patients. Recent studies using genetically engineered mouse models expressing stabilized HIF validate the importance of HIF in the evolution of NSCLC and demonstrate genetically that HIF is involved in NSCLC.

What the reader will gain: An overview of the key pathways and mediators of tumor angiogenesis, their relevance to the pathogenesis of NSCLC, and an update on the current status of angiogenesis inhibitors in NSCLC.

Take home message: Angiogenesis is a key mediator of NSCLC progression. Several antiangiogenic strategies are in clinical use and under development. While candidate predictive biomarkers of response to antiangiogenic therapy exist, they await independent and prospective validation.     

Effects of the selective COX-2 inhibitors celecoxib and rofecoxib on human vascular cells

Rheumatoid arthritis (RA) is associated with a reduced life expectancy considered to be partly caused by cardiovascular events. A growing concern is that accelerated atherosclerosis is driven by inflammatory mechanisms similar to those responsible for RA. Therefore, selective COX-2 inhibitors, which are widely used for the symptomatic treatment of pain and inflammation in RA, may have an impact on atherosclerotic processes. Their anti-inflammatory properties might provoke anti-atherogenic effects but on the other hand, selective inhibition of anti-thrombotic prostacyclin and COX-2 independent effects might promote the risk of increased prothrombotic activity. In the current study, the effects of the presently marketed selective COX-2 inhibitors celecoxib and rofecoxib on vascular cells have been investigated. Celecoxib inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. At high concentrations, it induced apoptosis and the modulation of inhibitory cell cycle proteins. In contrast rofecoxib-even at high concentrations-had no effect on cell proliferation, apoptosis or cell cycle distribution indicating that celecoxib and rofecoxib do not affect the same signal transduction pathways in endothelial cells. Both drugs did not affect apoptosis induction or cell cycle proliferation in human vascular smooth muscle cells. The observed effects on endothelial cells appear to be COX-independent since both drugs selectively inhibited COX-2-activity and the applied concentrations lay beyond the IC(50) for inhibition of prostacyclin production. Regarding endothelial apoptosis as a relevant event in the initiation and progression of atherosclerosis the present data put forward the hypothesis that the presently marketed COX-2 inhibitors have a different impact on atherosclerotic processes.     

糖网明目颗粒对缺氧/高糖状态下血管内皮细胞相关因子表达的影响

目的观察糖网明目颗粒(TWK)对缺氧/高糖状态下血管内皮细胞EA.hy926相关因子的影响。方法用Co Cl250μmol·L-1和葡萄糖40 mmol·L-1干预人脐静脉内皮细胞EA.hy926复制缺氧和高糖模型;分别给予TWK 1.0和2.5 g·L-1干预24 h,MTT法检测细胞存活率;半定量逆转录(RT)-PCR法检测缺氧诱导因子1α(HIF-1α)、血管内皮细胞生长因子受体2(VEGFR-2)和细胞间黏附分子1(ICAM-1)基因表达;ELISA法检测HIF-1α,VEGFR-2和ICAM-1蛋白含量。结果缺氧和高糖均能使EA.hy926细胞存活率升高,显著上调HIF-1α,VEGFR-2和ICAM-1 mRNA表达和蛋白含量(P<0.05)。TWK干预后,与模型组比较,细胞存活率显著下降,HIF-1α,VEGFR-2和ICAM-1 mRNA水平和蛋白含量显著降低(P<0.05),并且随着浓度的增加,药效有增强的趋势,但差异无统计学意义。TWK 2.5 g·L-1使缺氧细胞存活率由(109.9±6.9)%降至(69.6±2.2)%(P<0.05);HIF-1α,VEGFR-2和ICAM-1基因表达分别由0.292±0.017,0.210±0.013和0.724±0.027降低至0.204±0.014,0.061±0.010和0.476±0.018(P<0.05);HIF-1α,VEGFR-2和ICAM-1蛋白含量分别由56.2±4.9,1756±145和(339±33)ng·L-1降低至38.4±1.5,1057±118和(226±15)ng·L-1(P<0.05)。TWK 2.5 g·L-1使高糖细胞存活率由(111.4±7.3)%下降至(70.5±2.9)%(n=5,P<0.05);HIF-1α,VEGFR-2和ICAM-1 mRNA表达分别由0.397±0.021,0.289±0.034和0.755±0.031降低至0.247±0.015,0.079±0.005和0.531±0.025(P<0.05);HIF-1α,VEGFR-2和ICAM-1蛋白含量分别由56±4,1824±179和(339±31)ng·L-1降低至37±1,1170±110和(260±19)ng·L-1(P<0.05)。TWK 1.0 g·L-1也具有同样的作用效果。结论 TWK可能是通过调控内皮细胞HIF-1α,VEGFR-2和ICAM-1等新生血管相关因子的mRNA表达和蛋白分泌从而发挥防治糖尿病视网膜病变的作用。     

链霉素及H7对机械牵张离体心肌HIF-1α、VEGF的影响及机制探讨

目的研究链霉素及H7对机械牵张大鼠心肌组织低氧诱导分子-1α和血管内皮细胞生长因子表达的影响,并探讨二者在其中的作用机制。方法采用大鼠离体灌流心脏模型,膨胀左心室30min,RT-PCR法检测左室心肌细胞HIF-1α、VEGF mRNA的表达,免疫组化观察二者在心肌细胞中定位,Western blot检测HIF-1α蛋白的表达,利用链霉素作为牵张敏感离子通道(SACs)阻断剂研究SACs和PKC抑制剂H7在其中的可能作用。结果与不牵张组HIF-1α和VEGF mRNA无表达的比较,牵张可以明显增加HIF-1α和VEGF mRNA的表达(P<0·05或P<0·01);而链霉素、H7可以明显减少HIF-1α和VEGF mRNA的表达(P<0·05);但是二者不能完全抑制急性牵张刺激激活的HIF-1α和VEGF mRNA水平升高(P<0·05),HIF-1α和VEGF在胞质和胞核中均有表达,并检测到HIF-1α蛋白表达。结论链霉素、H7对膨胀左室致HIF-1α、VEGF表达有明显抑制作用,提示心室膨胀经SACs-PKC-激活胞内信号诱导HIF-1α、VEGF表达。同时链霉素并不能完全抑制HIF-1α、VEGF表达,提示膨胀心室致HIF-1α、VEGF表达进而引起心室肥厚尚有其他传导途径,仍需进一步研究。     

Differential effects of ABT-510 and a CD36-binding peptide derived from the type 1 repeats of thrombospondin-1 on fatty acid uptake, nitric oxide signaling, and caspase activation in vascular cells

ABT-510 is a potent mimetic of an anti-angiogenic sequence from the second type 1 repeat of thrombospondin-1. ABT-510 and the original d-Ile mimetic from which it was derived, GDGV(dI)TRIR, are similarly active for inhibiting vascular outgrowth in a B16 melanoma explant assay. Because GDGV(dI)TRIR and thrombospondin-1 modulate nitric oxide signaling by inhibiting the fatty translocase activity of CD36, we examined the ability ABT-510 to modulate fatty acid uptake into vascular cells and downstream nitric oxide/cGMP signaling. Remarkably, ABT-510 is less active than GDGV(dI)TRIR for inhibiting myristic acid uptake into both endothelial and vascular smooth muscle cells. Correspondingly, ABT-510 is less potent than GDGV(dI)TRIR for blocking a myristate-stimulated increase in cell adhesion to collagen and nitric oxide-driven accumulation of cGMP. ABT-510 at concentrations sufficient to inhibit CD36 fatty acid translocase activity synergizes with thrombin in aggregating platelets and blunts the activity of NO to delay aggregation, but again less than GDGV(dI)TRIR. In contrast, ABT-510 is more potent than GDGV(dI)TRIR for inducing caspase activation in vascular cells. Thus, we propose that ABT-510 is a drug with at least two mechanisms of action, and its potent anti-tumor activity may be in part independent of CD36 fatty acid translocase inhibition.     

Recent developments in the inhibition of angiogenesis: examples from studies on platelet factor-4 and the VEGF/VEGFR system

Inhibition of angiogenesis is an important strategy to block tumor growth and invasion. We discuss herein results from our ongoing investigations on platelet factor-4 (PF-4) and the VEGF/VEGFR system. Platelet factor-4 (PF-4) is an anti-angiogenic ELR-negative chemokine. PF-4 inhibits endothelial cell proliferation and migration, and angiogenesis in vitro and in vivo. We have studied the structure and anti-angiogenic activities of a C-terminal fragment of PF-4 named PF-4 CTF. This molecule retains anti-angiogenic activity, blocks the interaction of angiogenesis factors with their receptors and may also be improved by mutation or domain-swapping. It seems, therefore, to be a good candidate for further development. Furthermore, we have developed a cyclic vascular endothelial growth inhibitor (Cyclo VEGI) from the structure of VEGF-A. In aqueous solution, cyclo-VEGI adopts an alpha helix conformation. Cyclo-VEGI inhibits binding of iodinated VEGF(165) to endothelial cells and angiogenesis. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival.