CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.     

Ghrelin protects human umbilical vein endothelial cells against high glucose-induced apoptosis via mTOR/P70S6K signaling pathway

Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin has an anti-apoptotic effect in several cell types. However, the molecule mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly understood. In this study, we investigated the intracellular mechanisms responsible for anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In addition, ghrelin protected HUVECs against high glucose induced apoptosis by increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin produces a protective effect on HUVECs through activating GHS-R1a and mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis caused by high glucose.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

Notch1信号通路激活在低氧诱导人脐静脉内皮细胞血管形成中的作用

目的观察低氧条件下HIF-1α/VEGF/Notch信号通路在人脐静脉内皮细胞（HUVEC）血管生成中的作用。 方法将HUVEC进行常氧和低氧[二氯化钴（CoCl2）,200 μmol/L]诱导,再将常氧和低氧处理的HUVEC应用Notch1信号通路的抑制剂DAPT （30 μmol/L,24 h）和激活剂JAG-1 （30 μmol/L,24 h）干预。通过体外小管形成实验观察低氧对HUVEC血管生成能力的影响。应用RT-PCR和Western blot检测HUVEC中低氧诱导因子-1α （HIF-1α）、血管内皮生长因子（VEGF）、基质金属蛋白酶-9 （MMP-9）和Notch1信号分子（Notch1、Dell4和JAG-1）的mRNA和蛋白表达。通过Transwell迁移实验和伤口愈合实验观察低氧、DAPT、JAG-1对HUVEC迁移能力的影响。应用MTT法检测低氧及Notch1对HUVEC增殖的影响。两组间比较采用t检验,采用析因设计方差分析低氧和DAPT以及低氧和JAG-1对HUVEC迁移能力、距离、小管形成能力和细胞增殖的交互作用。 结果与常氧组比较,低氧组小管总长[（8.18±0.62）mm比（15.43±1.32）mm]增高,差异具有统计学意义（P < 0.05）。与常氧组比较,低氧组的HIF-1α、VEGF、MMP-9、Notch1、Dell4和JAG-1的mRNA相对表达量和蛋白相对表达量（1.01±0.03比4.43±0.35,1.02±0.03比3.55±0.28,0.98±0.04比3.24±0.25,1.01±0.03比3.22±0.25,0.99±0.02比2.89±0.22,1.02±0.04比2.43±0.19,0.98±0.01比3.13±0.24,0.98±0.02比2.67±0.21,0.97±0.03比2.45±0.19,1.01±0.03比2.44±0.19,1.00±0.04比2.30±0.18,1.03±0.05比2.27±0.18）均升高,差异有统计学意义（P均< 0.05）。Transwell迁移实验和伤口愈合实验显示,低氧条件下,DAPT干预使HUVEC的迁移能力降低,JAG-1干预使HUVEC的迁移能力升高（P均< 0.05）。小管形成和MTT法测定显示,低氧条件下,DAPT干预使HUVEC的小管形成能力和细胞增殖能力降低,JAG-1干预使HUVEC的小管形成能力和细胞增殖能力升高（P均< 0.05）。析因设计的方差分析结果显示,低氧和JAG-1对迁移细胞数、小管形成和细胞增殖能力交互作用具有协同作用（P < 0.05）。 结论低氧可通过激活HIF-1α/VEGF/Notch1信号通路提高HUVEC的血管生成能力、迁移能力和细胞增殖能力。     

Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by diallyl disulfide (DADS)

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behaviour are very critical during angiogenesis. We analyzed the effect of diallyl disulfide (DADS) on angiogenesis in in vitro models using human umbilical vein endothelial cells (HUVECs). DADS significantly inhibited endothelial cell migration, invasion and tube formation. (3)H-thymidine proliferation assay clearly showed the inhibitory effect of DADS on the proliferation of HUVECs in vitro. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, zymography was performed to determine whether DADS affected protease activity. Gelatin zymographic analysis showed the inhibitory effect of DADS on the activation of matrix metalloproteinases-MMP-2 and MMP-9. These findings suggest that DADS acts as an angiogenesis inhibitor by inhibiting the activation of matrix metalloproteinases during endothelial morphogenesis.     

Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.     

睾酮对人血管内皮细胞产生NO、tPA和PAI-1的影响

目的:观察不同浓度睾酮对人血管内皮细胞生长、产生舒张因子及纤溶活性的影响.方法:体外培养人脐静脉内皮细胞(HUVEC),分为五个浓度睾酮组及单纯培养基对照组.做MTT实验观察睾酮对HUVEC生长的影响;还原酶法测定各组HUVEC释放NO量;ELISA法测定各组培养基中纤溶酶原激活物(tPA)及其抑制物(PAI-1)含量.结果:3×10-10mol/L-3×10-8mol/L睾酮组与对照组相比细胞生长良好,无明显差别;而大于生理剂量的两组(3×10-6～3×10-1mol/L)3 d后细胞生长明显受到抑制(P＜0.05).各浓度睾酮组产生NO量与对照组无明显区别.而3×10-10 mol/L～3×10-8 mol/L睾酮组tPA含量明显高于对照组(P＜0.01);大剂量组tPA产生明显减少(P＜0.01).所有实验组的PAI-1含量均明显降低.结论:生理及略低于生理剂量的睾酮对HUVEC生长及释放NO无不利影响,且增加纤溶活性.说明生理剂量睾酮对血管内皮功能、心血管系统有一定的保护作用,有利于防止动脉粥样硬化的发生、发展.     

Antiapoptotic effect of ouabain on human umbilical vein endothelial cells

The present study investigates the effect of ouabain on caspase-3 activation in human umbilical vein endothelial cells (HUVEC). Ouabain (EC(50) 20 nM) reduced caspase-3 activity in HUVEC treated for 24h in a medium deprived of fibroblast growth factor (FGF). Incubation for 5h in the absence of both FGF and serum produced an increase in caspase-3 activity that was completely abolished by 100 nM ouabain. Pretreatment with the phosphatidylinositol 3 kinase (PI-3K) inhibitor, wortmannin, prevented the protective effect of ouabain against serum deprivation. Furthermore, Western blotting analysis revealed an increase in phosphorylation of extracellular signal-regulated kinases (ERK-1 and ERK-2) induced by 100nM ouabain in serum-deprived cells. In accord, pretreatment of HUVEC with PD98059, inhibitor of the ERK pathway, abrogated the effect of ouabain. Our results show that ouabain has an antiapoptotic effect on HUVEC through the activation of PI-3K and ERK dependent pathways.     

Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite abundantly stored in platelets and released upon platelet activation. Recently, S1P has been postulated for its potential roles in angiogenesis. In this study, we provided several lines of evidence showing that S1P has angiogenic activity. In vitro, S1P stimulated DNA synthesis and chemotactic motility of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, reaching a near maximum at 1 microM. S1P also significantly induced tube formation of HUVECs on Matrigel. Matrigel plug assay in mice revealed that S1P promotes angiogenesis in vivo. In addition, exposure of HUVECs to S1P led to rapid activation of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in a pertussis toxin (PTX)-sensitive manner. Notably, HUVEC migration and tube formation in response to S1P were completely blocked by pretreatment with PTX. Further, the MEK inhibitor U0126 markedly inhibited S1P-induced tube formation but S1P-induced migration was not affected by inhibition of ERK and p38 MAPK. Taken together, these results indicate that S1P induces angiogenesis predominantly via G(i) protein-coupled receptors in endothelial cells and suggest that S1P may act as an important modulator of platelet-induced angiogenesis.     

Green tea constituent epigallocatechin-3-gallate inhibits angiogenic differentiation of human endothelial cells

Several independent research studies have shown that consumption of green tea reduces the development of cancer in many animal models. Epidemiological observations, though inconclusive, are suggesting that green tea consumption may also reduce the risk of some cancers in humans. These anti-carcinogenic effects of green tea have been attributed to its constituent polyphenols. Angiogenesis is a crucial step in the growth and metastasis of cancers. We have investigated the effect of the major polyphenolic constituent of green tea, epigallocatechin-3-gallate (EGCG), on the tube formation of human umbilical vein endothelial cells (HUVEC) on matrigel. Tube formation was inhibited by treatment both prior to plating and after plating endothelial cells on matrigel. EGCG treatment also was found to reduce the migration of endothelial cells in matrigel plug model. The role of matrix metalloproteinases (MMP) has been shown to play an important role during angiogenesis. Zymography was performed to determine if EGCG had any effect on MMPs. Zymographs of EGCG-treated culture supernatants modulated the gelatinolytic activities of secreted proteinases indicating that EGCG may be exerting its inhibitory effect by regulating proteinases. These findings suggest that EGCG acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.     

Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells

Lipopolysaccharide (LPS), as an important proinflammatory agent, targets the endothelium. However, almost all in vitro experiments of the effect of LPS on vascular endothelial cells (VECs) were performed under an artificially decreased concentration of serum that was not enough to maintain the cell growth for a long time. The mechanism underlying LPS action on VECs cultured in a nutrient‐rich condition is not clear. To address this question and mimic the in vivo condition, we investigated the effect of LPS on VEC autophagy, which is involved in numerous physiological processes. The effect of LPS on microtubule‐associated protein 1 light chain 3 (LC3) distribution, LC3‐II accumulation and p62 degradation showed that LPS effectively induced autophagy in VECs cultured in the presence of 20% serum. To understand the mechanism by which LPS triggers the cell autophagy, we first investigated the effects of LPS on the expression of BIRC2 (cIAP1), a well‐known apoptosis inhibitor, and on the kinase activity of mammalian target of rapamycin (mTOR) and nuclear translocation of p53. LPS increased BIRC2 expression in a dose‐ and time‐dependent manner and elevated the intranuclear level of p53 but had no effect on the mTOR pathway when it triggered VEC autophagy. Furthermore, knockdown of BIRC2 by RNA interference inhibited the autophagy and the translocation of p53 to nuclei induced by LPS. These data suggest a novel role for BIRC2 in LPS‐induced autophagy in VECs. J. Cell. Physiol. 225: 174–179, 2010. © 2010 Wiley‐Liss, Inc.     

Recruitment of human umbilical vein endothelial cells and human primary fibroblasts into experimental tumors growing in SCID mice

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.