Inhibitory effect of methylmercury on migration and tube formation by cultured human vascular endothelial cells

 The effect of methylmercury chloride (MeHg) on migration and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was quantitatively analyzed. The distance of endothelial cell outgrowth from the scraped edge of a monolayer was measured. HUVEC outgrowth was inhibited by MeHg (1.0–5.0 μM) treatment in a dose-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 μM MeHg for 24 h inhibited tube formation dose-dependently. These results suggest that migration and tube formation by HUVECs are susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function, which may be involved in the pathogenesis of arteriosclerosis. Received: 29 July 1994 / Accepted: 22 November 1994     

Effect of lead on tube formation by cultured human vascular endothelial cells

 The effect of lead acetate (Pb) on the growth of and tube formation by cultured human umbilical vascular endothelial cells (HUVEC) was examined. HUVEC were collected by enzymatic digestion with collagenase. The number of viable cells of HUVEC was negligibly affected by cultivation with Pb at concentrations of 1 – 100 μM, but was slightly reduced by cultivation at 500 μM. Tube formation was studied by culturing the cells on a gelled basement membrane matrix (Matrigel). Treatment of HUVEC with 0.1 – 50.0 μM Pb for 24 h inhibited tube formation dose-dependently. The length of tube formation decreased time-dependently with 1.0 μM Pb. These findings suggest that Pb inhibits the formation of a capillary network by HUVEC, and that Pb could be injurious to endothelial cell function. Received: 23 January 1995 / Accepted: 20 March 1995     

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.     

Tryptophan 387 and 390 residues in ADAMTS13 are crucial to the ability of vascular tube formation and cell migration of endothelial cells

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) was mainly generated and secreted from endothelial cells (ECs). Our previous study showed that tryptophan (Trp) residues at 387 and 390 in ADAMTS13 are required for its secretion and enzymatic activity. However, the effects on its host cell as well as the potential mechanism have not been clear. The aim of the study was to examine the effects of Trp residues 387 and 390 of ADAMTS13 on the biological processes of ECs. Herein, Trp was substituted with alanine in ADAMTS13 to generate ADAMTS13 mutants at 387 (W387A), 390 (W390A), and double mutants at 387 and 390 (2WA), respectively. We found that substitution mutation impaired vascular endothelial growth factor (VEGF) secretion and the downstream JAK1/STAT3 activation, the binding ability to Von Willebrand factor, cell proliferation, migration, and vascular tube formation. Overall, our study concluded that Trp³⁸⁷ and Trp³⁹⁰ of ADAMTS13 play vital roles in the biological function of ECs.     

Inhibition of farnesyltransferase reduces angiogenesis by interrupting endothelial cell migration

Inhibitors of farnesyltransferase (FTI) have been developed for cancer treatment for more than a decade. Aside from being a therapeutic target in tumor cells, little is known about the role of farnesyltransferase (FTase) in other physiological processes. In this study, we revealed the involvement of FTase in angiogenesis and showed that FTI inhibited angiogenesis by directly acting on endothelial cells. Inhibition of FTase interrupted cell migration in vitro and in vivo. In addition, we found that FTase was important for cell polarization, cell spreading and pseudopodia formation. We also found that FTase interacted with microtubule end binding protein 1 (EB1) and that this interaction was critical for the localization of EB1 to microtubule tips. Our findings thus offer novel insight into the functions of FTase in endothelial cells and provide valuable information for the use of FTI in cancer therapy.     

Dykellic acid inhibits cell migration and tube formation by RhoA-GTP expression

Dykellic acid, a novel factor initially identified from the culture broth of Westerdykella multispora F50733, has been shown to inhibit matrix metalloprotease 9 activity, caspase-3 activity, B cell proliferation and LPS-induced IgM production, suggesting that this factor may have anti-cancer effects. In an effort to further address the possible anti-tumoral effects of dykellic acid, we used wound healing, invasion and RhoA-GTP assays to examine the effects of dykellic acid on cell migration, invasion and angiogenesis. Our results revealed that dykellic acid dose-dependently inhibits B16 cell migration and motility, and inhibits HUVEC tube formation. Western blot analysis of the active form of RhoA (RhoA-GTP) showed that dykellic acid treatment decreased the levels of RhoA-GTP. These findings collectively suggest that dykellic acid may have both anti-metastatic and anti-angiogenic acitivites, and provides the first evidence for the involvement of RhoA in dykellic acid-induced effects.     

Alkylphospholipids inhibit capillary-like endothelial tube formation in vitro: antiangiogenic properties of a new class of antitumor agents

Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.     

丹酚酸B对血管内皮细胞迁移及管腔形成的影响

目的探讨丹酚酸B(Salvianolic acid B,Sal B)对人脐静脉内皮细胞增殖、迁移和管腔形成的作用,了解其对血管新生的影响和作用机制。方法采用3H参入法检测Sal B对HUVECS增殖作用;Transwell观察Sal B对HUVECS迁移的影响;用体外毛细血管管腔结构形成试验检测Sal B对HUVECS管腔形成的影响;分别采用酶联免疫吸附测定法(ELISA)和Westen blot法检测Sal B处理后HUVECS分泌血管内皮细胞生长因子(VEGF)水平和VEGF表达的变化。结果3H参入法,结果显示Sal B对HUVECS增殖具有促进作用;Transwell试验显示,Sal B可以促进HUVECS迁移;管腔形成实验显示,与空白组比较,Sal B可以使HUVECS管腔形成数增多,管腔长度增长。经Sal B处理后,与空白组比较,HUVECS上清中VEGF含量增加,VEGF表达上调。结论Sal B对HUVECS增殖,迁移和管腔形成有促进作用,其作用机制可能是通过上调HUVECS中VEGF的表达,从而促进血管新生。     

Strategies for the synthesis of the novel antitumor agent peloruside A

The microtubule-stabilizing agent (+)-peloruside A has emerged as a potential therapeutic agent for the treatment of cancer. Two total syntheses have been published and these reports have stimulated additional studies to advance the methodology and strategies for accessing this molecular architecture. This review details the biological data, modeling and conformation analyses, and synthetic studies toward the synthesis of (+)-peloruside A, that were reported prior to December 2007.     

Phosphatidylinositol inhibits vascular endothelial growth factor-A--induced migration of human umbilical vein endothelial cells

Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 microg/ml) inhibited VEGF-A-induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 microg/ml inhibited the VEGF-A-induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.     

Dissociation between the Ca(2+) signal and tube formation induced by vascular endothelial growth factor in bovine aortic endothelial cells

Previous studies from our laboratory as well as from others have suggested that the thiazolidinediones have the capacity to act as insulinomimetic agents, especially in the liver. In order to further characterize this insulinomimetic action, we evaluated the effect of troglitazone, a representative thiazolidinedione, on lactate- and glucagon-stimulated gluconeogenesis, in the presence or absence of insulin (10 nM) in isolated rat hepatocytes. The antigluconeogenic effect of troglitazone under basal (lactate-stimulated) conditions was found to be due to an elevation in the fructose 2,6-bisphosphate content, which was, however, not mediated by an activation of 6-phosphofructo 2-kinase. Troglitazone (125 and 250 microM) in the absence of insulin, produced a dose-dependent reduction in glucagon-stimulated gluconeogenesis, thereby suggesting an insulinomimetic effect. In addition, troglitazone (125 and 250 microM), in combination with insulin, produced an additive inhibition of gluconeogenesis during glucagon-stimulated conditions. However, unlike insulin, the metabolic mechanism responsible for these effects (in the presence or absence of insulin) does not involve fructose 2,6-bisphosphate.     

Inhibition by nicotine of the formation of prostacyclin-like activity in rabbit and human vascular tissue.

1 Rings of vascular tissue (from rabbit aorta or human peripheral vein) were incubated at room temperature in Tyrode solution in the absence or presence of nicotine or indomethacin. 2 Addition of portions of the incubates to human platelet-rich plasma (HPRP) elicited a decrease in adenosine 5'-diphosphate (ADP)-induced platelet aggregation in this plasma. Authentic prostacyclin (PGI2) also induced such a decrease. The decreased aggregation amplitudes that followed the addition of the vascular tissue incubates and of PGI2 were equally potentiated by theophylline (10(-4) M). 3 Both nicotine and indomethacin counteracted the formation of platelet anti-aggregatory activity in the vascular tissue incubates. The IC50S of nicotine and of indomethacin on the formation of platelet antiaggregatory activity were 2 X 10(-5) M and 6 X 10(-6) M, respectively. 4 Nicotine failed to affect the platelet anti-aggregatory effect induced by authentic PGI2 in HPRP. 5 It is concluded that nicotine counteracts the formation of platelet anti-aggregatory activity in rabbit aorta and human peripheral vein by eliciting an inhibitory effect on the bioformation of prostacyclin in these types of vascular tissue.     

Tricyclic antidepressant amitriptyline inhibits autophagic flux and prevents tube formation in vascular endothelial cells

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The ex vivo aortic ring angiogenesis and in vitro‐cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM‐autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis.     

Inhibition of vascular endothelial growth factor expression and production by triptolide

Triptolide, the major component of the diterpenoids of the Chinese herb Tripterygium wilfordii Hook f. (Celastraceae), inhibited vascular endothelial growth factor expression and secretion in endothelial cells treated by 12-O-tetradecanoylphorbol 13-acetate dose-dependently. This effect may be one of the mechanisms underlying the therapeutic effects of triptolide on rheumatoid arthritis.     

Inhibition of cell proliferation, invasion and migration by ursolic acid in human lung cancer cell lines

Ursolic acid (UA) is a pentacyclic triterpene naturally occurring in many plant foods. Apoptotic, anti-invasive and anti-migratory effects of UA at 2, 4, 8, or 16 μmol/L in human non-small cell lung cancer A549, H3255 and Calu-6 cell lines were examined. The impact of this compound upon associated biomarkers such as vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinase (MMP) was also evaluated. UA treatments concentration-dependently decreased cell viability, and lowered Na⁺-K⁺-ATPase activity (P < 0.05). This compound at 4–16 μmol/L concentration-dependently increased DNA fragmentation, and reduced VEGF and transforming growth factor beta1 levels in test cancer cells (P < 0.05). UA concentration-dependently suppressed ICAM-1 expression (P < 0.05). This compound significantly declined fibronectin expression (P < 0.05), but concentration-dependent effect was shown in H3255 cells only (P < 0.05). UA treatments significantly suppressed the expression of MMP-9 and MMP-2 (P < 0.05), and inhibited protein kinase C activity in test cell lines (P < 0.05). UA treatments also concentration-dependently reduced cell invasion (P < 0.05); however, this compound at 4–16 μmol/L significantly decreased cell migration (P < 0.05), and concentration-dependent effect was shown in A549 and Calu-6 cells (P < 0.05). These findings suggested that this triterpene was a potent anti-lung cancer agent, and it might be able to retard invasion and metastasis of lung cancer cells.     

Effects of the microtubule stabilizing agent peloruside A on the proteome of HL-60 cells

Peloruside A, isolated from the marine sponge Mycale hentscheli, has a similar mechanism of action to paclitaxel (Taxol?), a drug used clinically to treat tumors of the breast, ovary and lung. Paclitaxel and peloruside stabilize the polymerized form of tubulin and arrest cells in G₂/M of the cell cycle. We have therefore used two-dimensional electrophoresis of proteins to examine the effect of peloruside A on the human HL-60 promyeloid leukemic cell line. Our goals included investigation whether affected proteins could be mapped onto pathways that predicted the cellular effects of this compound. In response to 100 nM peloruside A treatment for 24 h, seventeen identified proteins showed significant change in abundance with fourteen increases and three decreases. Use of Ingenuity Pathways Analysis confirmed that peloruside A affected pathways consistent with the known effects on microtubules and apoptosis. Our results also indicate a potential role of c-Myc in the cellular actions of peloruside consistent with an induction of aneuploidy seen at low concentrations of peloruside.     

Inhibition of vascular endothelial growth factor-induced angiogenesis by resveratrol through interruption of Src-dependent vascular endothelial cadherin tyrosine phosphorylation

Resveratrol, a polyphenolic compound found in grapes and other fruits, has been reported to inhibit angiogenesis with an as yet elusive mechanism. Here, we investigate the detailed mechanism by which resveratrol inhibits vascular endothelial growth factor (VEGF)-induced angiogenic effects in human umbilical endothelial cells (HUVECs). Exposure of HUVECs to 1 to 2.5 muM resveratrol significantly blocked VEGF-mediated migration and tube formation but not cell proliferation. Under the same concentrations, resveratrol failed to affect VEGF-stimulated activation of VEGF receptor, extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase, and Akt. Of interest, resveratrol, at the dose of 1 or 2.5 muM, effectively abrogated VEGF-mediated tyrosine phosphorylation of vascular endothelial (VE)-cadherin and its complex partner, beta-catenin. This inhibitory effect of resveratrol reflected on the retention of VE-cadherin at cell-cell contacts as demonstrated by immunofluorescence. Src kinase assay showed that VEGF-induced endogenous Src kinase activation was strongly inhibited by 1 and 2.5 muM resveratrol. Supportively, inhibition of Src activity by overexpression of Csk resulted in attenuation of the tyrosine phosphorylation of VE-cadherin and endothelial cell (EC) tube formation. Again, transfection with v-Src, an active form of Src, could reverse resveratrol inhibition of VE-cadherin tyrosine phosphorylation and EC tube formation. Reactive oxygen species (ROS) has been shown to be involved in VE-cadherin phosphorylation and its related functions. Flow cytometric analysis showed that VEGF stimulated an evident increase of peroxide, which was strongly attenuated by resveratrol. In addition, antioxidant N-acetyl-cysteine was demonstrated to strongly inhibit VEGF-mediated Src activation, VE-cadherin tyrosine phosphorylation, and HUVEC tube formation. Together, our data suggest that resveratrol inhibition of VEGF-induced angiogenesis was mediated by disruption of ROS-dependent Src kinase activation and the subsequent VE-cadherin tyrosine phosphorylation.