Involvement of matrix metalloproteinase-9 in platelet-activating factor-induced angiogenesis

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-kappaB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-kappaB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-kappaB inhibitors. Transfected NF-kappaB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-kappaB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-kappaB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-kappaB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.     

Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibiting the matrix-degrading proteases

Flavonoids have been proposed to act as chemopreventive agents in numerous epidemiological studies and have been shown to inhibit angiogenesis and proliferation of tumor cells and endothelial cells in vitro. Angiogenesis requires tightly controlled extracellular matrix degradation mediated by extracellular proteolytic enzymes including matrix metalloproteinases (MMPs) and serine proteases, in particular, the urokinase-type plasminogen activator (uPA)-plasmin system. In this study, we have investigated the antiangiogenic mechanism of the flavonoids, genistein, apigenin, and 3-hydroxyflavone in a human umbilical vein endothelial cell (HUVEC) model. The stimulation of serum-starved HUVECs with vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF) caused marked increase in MMP-1 production and induced the pro-MMP-2 activation accompanied by the increase in MT1-MMP expression. However, pretreatment with flavonoids before VEGF/bFGF stimulation completely abolished the VEGF/bFGF-stimulated increase in MMP-1 and MT1-MMP expression and pro-MMP-2 activation. Genistein blocked VEGF/bFGF-stimulated increase in TIMP-1 expression and decrease in TIMP-2 expression. Apigenin and 3-hydroxyflavone further decreased TIMP-1 expression below basal level and completely abolished TIMP-2 expression. VEGF and bFGF stimulation also significantly induced uPA expression, most strikingly the level of 33 kDa uPA, and increased the expression of PA inhibitor (PAI)-1. Genistein, apigenin, and 3-hydroxyflavone effectively blocked the generation of 33 kDa uPA, and further decreased the activity of the 55 kDa uPA and the expression of PAI-1 below the basal level. In conclusion, these data suggest that genistein, apigenin, and 3-hydroxyflavone inhibit in vitro angiogenesis, in part via preventing VEGF/bFGF-induced MMP-1 and uPA expression and the activation of pro-MMP-2, and via modulating their inhibitors, TIMP-1 and -2, and PAI-1.     

膜型基质金属蛋白酶-1在不同内皮细胞株中的表达差异

为研究膜型基质金属蛋白酶-1（membrane-type matrix metalloproteinase-1, MT1-MMP）在血管生物学中的作用机制,比较了3株常用的内皮细胞株：人微血管内皮细胞株HMEC-1、人脐静脉内皮细胞株ECV304和EAhy926中MT1-MMP及与其功能相关的MMP-2,TIMP-2的表达差异.实时PCR 和流式细胞术检测HMEC-1、EAhy926和ECV304中MT1-MMP/MMP-2/TIMP-2的表达,明胶酶谱法分析各细胞株上清中MMP-2的酶活.实时PCR结果显示,3株细胞均表达MT1-MMP与TIMP-2,MT1-MMP在EAhy926中表达最高,TIMP-2在ECV304中表达最高,而仅在EAhy926中检测到MMP-2的表达.流式细胞术和酶谱的结果与PCR结果基本一致.MT1-MMP和MMP-2在典型的大血管内皮细胞株EAhy926中高表达可能与该细胞独特的来源、表型特点和功能有关.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Platelet-activating factor induces matrix metalloproteinase-9 expression through Ca(2+)- or PI3K-dependent signaling pathway in a human vascular endothelial cell line

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of a variety of angiogenic factors, via the nuclear factor (NF)-kappaB activation. Recently, we reported that PAF upregulates MMP-9 expression in a NF-kappaB-dependent manner. In this study, we investigated the signaling pathway involved in PAF-induced MMP-9 expression in ECV304 cells. Our current data indicate that the Ca(2+)- or phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is necessary for PAF-induced MMP-9 expression. Furthermore, PAF-induced NF-kappaB activation was blocked by selective inhibitors of Ca(2+), PI3K, or extracellular signal-regulated kinase (ERK). Our results suggest that PAF-induced MMP-9 expression, in a NF-kappaB-dependent manner, is regulated by Ca(2+), PI3K and ERK signaling pathways.     

Oxygen tension regulates the maturation of the blood-brain barrier.

The oxygen tension during the development of vascular systems influences vascular vessel formation through regulating angiogenesis. We studied the effect of hypoxia/reoxygenation (H/R) to explain its role in concert with astrocytes involvement in the development of the blood-brain barrier (BBB). On the basis of the fact that the disappearance of hypoxic regions and the decreased expression of vascular endothelial growth factor (VEGF) were observed by immunohistochemistry in a development-dependent manner in rat cerebral cortex, we examined the effects of astrocytes on the BBB-like properties of ECV304 cells by exposing astrocytes to H/R. Conditioned medium of reoxygenated astrocytes inhibited [(3)H]thymidine incorporation and tube formation of ECV 304 cells. When astrocytes were exposed to reoxygenation, the expression of VEGF was reduced, whereas the expression of angiopoietin-1 and thrombospondin-1 was enhanced. Moreover, [(3)H]sucrose permeability assay revealed that astrocytes enhance the barrier function of ECV 304 cells in coculture model within 5 h of reoxygenation. Correspondingly, the occludin expression of ECV 304 cells was slightly increased by the conditioned medium of reoxygenated astrocytes. In conclusion, our study suggests that reoxygenation of astrocytes may act as a significant driving force for the maturation of the BBB during brain development through oxygen-regulated gene(s).     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling

Isoliquiritigenin (ISL, 4,2′,4′-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0–20 μmol/L ISL with or without 10 μg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-α2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-α2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Anti-angiogenic effects of trabectedin (Yondelis; ET-743) on human breast cancer cells

Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata, has been shown to have antitumor effects. In this study, we assessed the possible anti-angiogenic effects of trabectedin on human umbilical vein endothelial cells (HUVECs) and breast cancer cell lines. An XTT cell viability assay was used to determine cytotoxicity. A scratch assay was used to detect the migration of cells after trabectedin treatment. Angiogenic cytokine profiles of breast cancer cell lines, before and after treatment with trabectedin, were investigated using an angiogenesis antibody array. Changes in mRNA expression levels of VEGF were evaluated using qRT-PCR. Trabectedin inhibited the viability of HUVECs and breast cancer cells in a concentration- and time-dependent manner. The migration of both HUVECs and breast cancer cells was suppressed by trabectedin treatment. Angiogenic cytokines which are known to regulate tumorigenicity and angiogenesis, such as GM-CSF, IGFBP-2, VEGF, and uPA, were inhibited, while several anti-angiogenic cytokines such as TIMP-1 and Serpin E1were induced in breast cancer cells. Furthermore, expression levels of VEGF mRNA were inhibited in all breast cancer cells tested. Although additional studies are needed to elucidate the molecular mechanisms underlying the anti-angiogenic activity of trabectedin, our results suggest that trabectedin may act as a potential anti-angiogenic agent in breast cancer cells.     

丹参单体IH764-3通过下调H2O2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达

目的：研究丹参单体IH764—3对H2O2刺激的肝星状细胞（HSC）基质金属蛋白酶-13（MMP-13）、基质金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响以及此过程中粘着斑激酶（FAK）的变化。方法：应用RT-PCR方法检测MMP-13及FAKmRNA表达,原位杂交方法检测TIMP-1mRNA水平,Western blotting技术检测FAK及TIMP-1蛋白表达。结果：IH764—3干预组的MMP-13mRNA在2h的表达强度明显上调,而TIMP-1mRNA表达明显受抑,FAKmRNA表达强度明显下调;IH764—3干预24h组FAK及TIMP-1蛋白表达受抑制。结论：丹参单体IH764—3可以诱导MMP-13表达,抑制TIMP-1表达,下调FAK表达是其中的机制之一。     

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2 h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.

Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.     

裂解型腺病毒Ad-E1A对人脐静脉内皮细胞的影响

以QBI-293A细胞基因组DNA为模板,PCR扩增E1A基因,酶切连接到pAdTrack-CMV转移质粒上,pAdTrack-CMV-E1A经PmeI线性化后,与pAdEasy-1共转化大肠杆菌BJ5183,筛选重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-E1A,经PacI线性化,脂质体转染QBI-293A细胞,获得裂解型腺病毒Ad-E1A。裂解型腺病毒Ad-E1A在ECV304细胞内复制裂解,抑制细胞的生长,并可以降低VEGF的表达,探讨了Ad-E1A可能通过抑制ECV304细胞NF-κB的激活而引起细胞生长抑制的机制,说明Ad-E1A具有抑制肿瘤转移的功能。     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.