Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin

BACKGROUND: The role of dietary components in cancer progression and metastasis is an emerging field of clinical importance. Many stages of cancer progression involve carbohydrate-mediated recognition processes. We therefore studied the effects of high pH- and temperature-modified citrus pectin (MCP), a nondigestible, water-soluble polysaccharide fiber derived from citrus fruit that specifically inhibits the carbohydrate-binding protein galectin-3, on tumor growth and metastasis in vivo and on galectin-3-mediated functions in vitro. METHODS: In vivo tumor growth, angiogenesis, and metastasis were studied in athymic mice that had been fed with MCP in their drinking water and then injected orthotopically with human breast carcinoma cells (MDA-MB-435) into the mammary fat pad region or with human colon carcinoma cells (LSLiM6) into the cecum. Galectin-3-mediated functions during tumor angiogenesis in vitro were studied by assessing the effect of MCP on capillary tube formation by human umbilical vein endothelial cells (HUVECs) in Matrigel. The effects of MCP on galectin-3-induced HUVEC chemotaxis and on HUVEC binding to MDA-MB-435 cells in vitro were studied using Boyden chamber and labeling assays, respectively. The data were analyzed by two-sided Student's t test or Fisher's protected least-significant-difference test. RESULTS: Tumor growth, angiogenesis, and spontaneous metastasis in vivo were statistically significantly reduced in mice fed MCP. In vitro, MCP inhibited HUVEC morphogenesis (capillary tube formation) in a dose-dependent manner. In vitro, MCP inhibited the binding of galectin-3 to HUVECs: At concentrations of 0.1% and 0.25%, MCP inhibited the binding of galectin-3 (10 micro g/mL) to HUVECs by 72.1% (P =.038) and 95.8% (P =.025), respectively, and at a concentration of 0.25% it inhibited the binding of galectin-3 (1 micro g/mL) to HUVECs by 100% (P =.032). MCP blocked chemotaxis of HUVECs toward galectin-3 in a dose-dependent manner, reducing it by 68% at 0.005% (P<.001) and inhibiting it completely at 0.1% (P<.001). Finally, MCP also inhibited adhesion of MDA-MB-435 cells, which express galectin-3, to HUVECs in a dose-dependent manner. CONCLUSIONS: MCP, given orally, inhibits carbohydrate-mediated tumor growth, angiogenesis, and metastasis in vivo, presumably via its effects on galectin-3 function. These data stress the importance of dietary carbohydrate compounds as agents for the prevention and/or treatment of cancer.     

In vivo inhibition of angiogenesis by a soluble form of melanotransferrin

Melanotransferrin (MTf), the membrane-bound human melanoma antigen p97, binds to plasminogen and stimulates its activation, thus regulating a crucial step involved in angiogenesis. In our study, a truncated soluble form of MTf (sMTf) inhibits, in a dose-dependent manner, the in vitro tubulogenesis of human umbilical vessel endothelial cells (HUVEC) induced by media conditioned with MTf-expressing cells. Following these results, we used the in vivo Matrigel plug angiogenesis assay to investigate whether truncated sMTf could inhibit neovascularization in mice. We found that basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and MTf-expressing cells strongly stimulate in vivo Matrigel neovascularization. However, subcutaneous (s.c.) administration of truncated sMTf inhibits both bFGF- and VEGF- as well as human melanoma SK-Mel-28-induced angiogenesis. This inhibition was dependent on the truncated sMTf concentration and reached maximal inhibition at 40 mg/kg/week. Furthermore, we found that a single s.c. injection of truncated sMTf into nude mice at 5 mg/kg produced a maximal blood concentration of approximately 40 nM, which is comparable with the level required to inhibit in vitro HUVEC tubulogenesis. Overall, our results strongly suggest that s.c. administration of truncated sMTf may provide a novel therapeutic strategy for the treatment of angiogenesis-related disorders.     

Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts

Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis.     

重组人血管内皮抑素对血管新生的影响研究

目的 探讨重组人血管内皮抑素（Endostar,恩度）对血管内皮细胞趋化、迁移、粘附、增殖及小管形成等与血管新生相关生物学行为的影响。方法 以原代培养的人脐静脉内皮细胞（HUVEC）为细胞模型,通过Boyden小室荧光定量分析、划痕试验、HUVEC荧光定量粘附分析、CFSE染色流式细胞术、CCK-8定量检测、小管形成试验和Matrigel栓试验研究恩度对HUVEC与血管新生相关的生物学行为的影响。结果 恩度在5～50 000ng／ml范围内可抑制血管内皮生长因子诱导HUVEC的迁移运动,且浓度为50ng／ml和500ng／ml时效果最明显;恩度在5～50 000ng／ml间可呈剂量依赖的方式抑制HUVEC向损伤部位的迁移。与0ng／ml相比,50、500和5000ng／ml 恩度处理的HUVEC的粘附率、增殖率及HUVEC形成网状小管结构的数量、面积和长度均降低,差异均有统计学意义（P＜0.05）; Matrigel栓实验结果显示,恩度在50～5000ng／ml间对SCID小鼠体内血管新生有明显的抑制作用。结论 恩度在细胞水平能抑制HUVEC与血管新生相关的生物学行为,包括HUVEC的趋化、迁移、粘附、增殖和小管形成;在动物水平能抑制SCID小鼠体内的血管新生,据此推断恩度能抑制血管新生。     

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

背景与目的：二氢杨梅素（dihydromyricetin，DHM）通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用，并研究其可能作用机制。方法：不同剂量DHM作用体外人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和胃癌细胞系MKN28后，采用细胞计数试剂盒（cell counting kit-8，CCK-8）法筛选无毒剂量的DHM，Transwell小室实验测定细胞侵袭；小管形成实验测定HUVEC体外血管生成能力；采用蛋白质印迹法（Western blot）检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、phospho-血管内皮生长因子受体（vascular endothelial growth factor receptor, VEGFR）2、细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）、phospho-c-Jun氨基端激酶（c-Jun NH2-terminal kinase，JNK）和phospho-p38表达水平；基质胶塞实验测定DHM对体内血管形成的影响；通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型，观察DHM给药对体内胃癌生长的影响，Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果：（0.5~2.5 μmol/L）的DHM对HUVEC和MKN28细胞生长无明显抑制作用，该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成，VEGFA（20 μg/L）可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果；DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降，而对p-p38和p-JNK的表达无明显影响；DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降；低剂量（30 mg/kg）DHM对裸鼠无明显的不良反应，但可抑制体内血管形成，同时有效延缓体内胃癌生长，体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用，但低剂量DHM对Ki-67无明显作用。结论：低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长，该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway by inositol pentakisphosphate results in antiangiogenic and antitumor effects

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.     

Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component in the root and rhizome of Rheum palmatum, is a tyrosine kinase inhibitor with a number of biological activities, including antitumor effects. Here, we examine the effects of emodin on vascular endothelial growth factor (VEGF)-A-induced angiogenesis, both in vitro and in vivo. In vitro, emodin dose-dependently inhibits proliferation, migration into the denuded area, invasion through a layer of Matrigel and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated with VEGF-A. Emodin also inhibits basic fibroblast growth factor-induced proliferation and migration of HUVECs and VEGF-A-induced tube formation of human dermal microvascular endothelial cells. Specifically, emodin induces the cell cycle arrest of HUVECs in the G0/G1 phase by suppressing cyclin D1 and E expression and retinoblastoma protein phosphorylation, and suppresses Matrigel invasion by inhibiting the basal secretion of matrix metalloproteinase-2 and VEGF-A-stimulated urokinase plasminogen activator receptor expression. Additionally, emodin effectively inhibits phosphorylation of VEGF-A receptor-2 (KDR/Flk-1) and downstream effector molecules, including focal adhesion kinase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, Akt and endothelial nitric oxide synthase. In vivo, emodin strongly suppresses neovessel formation in the chorioallantoic membrane of chick and VEGF-A-induced angiogenesis of the Matrigel plug in mice. Our data collectively demonstrate that emodin effectively inhibits VEGF-A-induced angiogenesis in vitro and in vivo. Moreover, inhibition of phosphorylation of KDR/Flk-1 and downstream effector molecules is a possible underlying mechanism of the anti-angiogenic activity of emodin. Based on these data, we propose that an interaction of emodin with KDR/Flk-1 may be involved in the inhibitory function of emodin toward VEGF-A-induced angiogenesis in vitro and responsible for its potent anti-angiogenic in vivo.     

Anti-angiogenic effects of a nutrient mixture on human umbilical vein endothelial cells

Matrix metalloproteinases (MMPs) have been recognized as key players in the degradation of the extracellular matrix (ECM) by migration and proliferation of endothelial cells and their subsequent invasion of the underlying stroma. The prevention of ECM degradation through the inhibition of MMP activity has been shown to be a promising therapeutic approach to block the invasion that occurs during angiogenesis. In previous studies, we demonstrated the anti-tumor effect of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, arginine, N-acetyl cysteine, selenium, copper and manganese on various tumor cell lines in vivo and in vitro. The aim of the present study was to determine whether this mixture has anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs). At near confluence, the HUVEC cell cultures were tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose for proliferation, migration, MMP expression, and invasion. Cell proliferation was evaluated by MTT assay, invasion potential by Matrigel invasion, MMP expression by gelatinase zymography, and cell migration by a 2 mm-wide scratch in plates. For tube formation, HUVECs were cultured in previously polymerized Matrigel. NM inhibited HUVEC migration, MMP expression and invasion through Matrigel in a dose-dependent manner. Zymography showed a dose-dependent inhibition of MMP-2 expression with virtual total inhibition at a 500 microg/ml concentration. Invasion through Matrigel was totally inhibited at 500 microg/ml NM. NM reduced cell migration by scratch test in a dose-dependent fashion with total inhibition at a 500 microg/ml concentration. NM also inhibited the tube formation of HUVECs, but did not significantly inhibit cell proliferation. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation with anti-angiogenic effects, such as inhibiting vascular tube formation and endothelial cell invasion and migration.     

Induction of spindle cell morphology in human vascular endothelial cells by human herpesvirus 8-encoded viral FLICE inhibitory protein K13

Human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus, is linked to the development of Kaposi's sarcoma, a disease characterized by the presence of distinctive proliferating spindle-like cells. Although HHV8 can induce spindle cell transformation of vascular endothelial cells in vitro, the viral gene(s) responsible for this phenotype remain to be identified. We demonstrate that expression of HHV8-encoded viral Fas-associated death domain protein-like IL-1beta-converting enzyme inhibitory protein K13 is sufficient to induce spindle cell phenotype in human umbilical vein endothelial cells (HUVEC), which is associated with the activation of the nuclear factor-kappaB (NF-kappaB) pathway and can be blocked by Bay-11-7082, a specific inhibitor of this pathway. K13 induces the expression of several genes known to be upregulated in HHV8-transformed vascular endothelial cells, such as interleukin (IL)-6, IL-8, CXC ligand 3 (CXCL3), orphan G protein coupled receptor (RDC1), cyclooxygenase-2 (COX-2) and dual-specificity phosphatase 5 (DUSP5). Furthermore, similar to K13, HHV8-induced spindle cell transformation of HUVEC is associated with NF-kappaB activation and can be blocked by Bay-11-7082. Thus, ectopic expression of a single latent gene of HHV8 is sufficient for the acquisition of spindle cell phenotype by vascular endothelial cells and NF-kappaB activation plays an essential role in this process.     

Antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin: an orally bioavailable heat shock protein 90 modulator.

PURPOSE: The purpose of this study was to investigate the antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; NSC707545), a water-soluble benzoquinone ansamycin. EXPERIMENTAL DESIGN: The activity of 17-DMAG, in vivo, was evaluated for inhibition of fibroblast growth factor (FGF)-2-induced angiogenesis in s.c. implanted Matrigel in mice. In vitro, the activity of 17-DMAG on endothelial cells (human umbilical vein endothelial cells; HUVEC) was tested in FGF-2; and vascular endothelial growth factor (VEGF)-induced proliferation and apoptosis, motility, and extracellular matrix invasion; and on the alignment of capillary like structures in Matrigel. The protein level of heat shock protein (Hsp)90 and client proteins was examined by Western blot in FGF-2 and VEGF-stimulated HUVEC. RESULTS: Daily oral administration of 17-DMAG affected the angiogenic response in Matrigel in a dose-dependent manner. The hemoglobin content in the Matrigel implants was significantly inhibited, and the histological analysis confirmed a decrease of CD31(+) endothelial cells and of structures organized in cord and erythrocyte-containing vessels. In vitro, the compound inhibited dose-dependently the migration and the extracellular matrix-invasiveness of HUVEC and their capacity to form capillary like structures in Matrigel. 17-DMAG treatment also inhibited FGF-2 and VEGF-induced HUVEC proliferation and resulted in apoptosis. Accordingly, the expression of Hsp90 direct client proteins (pAkt and c-Raf-1) or their downstream substrates including pERK was also affected. 17-DMAG consistently increased the expression of Hsp70. Throughout the study similar results were obtained with 17-allylamino-17-demethoxygeldanamycin (17-AAG; NSC330507), the analog compound currently undergoing clinical trials. CONCLUSIONS: We show that the Hsp90 targeting agents 17-DMAG and 17-AAG inhibit angiogenesis. The strong effects on endothelial cell functions, in vitro, indicate that the antiangiogenic activity of 17-DMAG/17-AAG could also be due to a direct effect on endothelial cells. The oral bioavailability of 17-DMAG might be of advantage in investigating the potential of this compound in clinical trials with antiangiogenic as well as antiproliferative endpoints.     

Influence of proinflammatory cytokines on the adhesion of human colon carcinoma cells to lung microvascular endothelium

In this experimental study, the influence of surgery-induced proinflammatory cytokines on tumor recurrence in the lung was investigated. A reproducible human in vitro assay was developed to study the adhesion of HT29 colon carcinoma cells to monolayers of microvascular endothelial cells of the lung (HMVECs-L) or human umbilical venous endothelial cells (HUVECs). Preincubation of HMVECs-L with maximally active concentrations of IL-1beta and TNF-alpha, but not with IL-6, resulted in at least 250% adhesion compared to control adhesion (p 相似文献   

PRL-3 initiates tumor angiogenesis by recruiting endothelial cells in vitro and in vivo

We show here that PRL-3 protein is expressed in fetal heart, developing blood vessels, and pre-erythrocytes but not in their mature counterparts. These observations imply that PRL-3 may be involved in the early development of the circulatory system. Because PRL-3 mRNA had been reported to be consistently elevated in metastatic samples derived from colorectal cancers, we attempted to investigate if PRL-3 might be involved in tumor angiogenesis and if PRL-3-expressing cells could cross-talk to human umbilical vascular endothelial cells (HUVEC) by using an in vitro coculture system. HUVECs were grown with fibroblasts, which were later overlaid with PRL-3-expressing cells. We observed that both PRL-3-expressing Chinese hamster ovary (CHO) cells and PRL-3-expressing DLD-1 human colon cancer cells could redirect the migration of HUVECs toward them; in addition, PRL-3-expressing DLD-1 cells could enhance HUVEC vascular formation. In vivo injection of PRL-3-expressing CHO cells into nude mice to form local tumors resulted in the recruitment of host endothelial cells into the tumors and initiation of angiogenesis. We further showed that PRL-3-expressing cells reduced interleukin-4 (IL-4) expression levels and thus attenuated IL-4 inhibitory effects on the HUVEC vasculature. Our findings provide direct evidence that PRL-3 may be involved in triggering angiogenesis and establishing microvasculature and it may serve as an attractive therapeutic target with respect to both angiogenesis and cancer metastasis.     

Lysophosphatidic acid receptor 2/3-mediated IL-8-dependent angiogenesis in cervical cancer cells

The expression of lysophosphatidic acid (LPA)-specific receptors in cervical cancer has not been clearly defined. In this study, we identified LPA1, LPA2 and LPA3 receptors' mRNA in SiHa, HeLa and CaSki cell lines by RT-PCR. These receptors were not associated with tumor cell proliferation in vitro. We then used a xenograph animal model to evaluate the effects of these receptors on in vivo cervical cancer tumorigenicity. When SiHa cells with different receptor expression patterns were seeded on the backs of SCID mice, the resulting knockout of both LPA2 and LPA3 significantly attenuated tumor growth; this decrease in tumor growth was found to be linked with decreased angiogenesis (microvessel density), suggesting that LPA2 and LPA3 are crucial for in vivo tumor growth through an angiogenic mechanism. We further investigated this mechanism of LPA receptor 2/3-mediated angiogenic capability by analyzing angiogenic factors in protein lysates from receptor knockout tumors, by detecting interleukin (IL-8) mRNA expression after treating with siRNA, by evaluating the biological role of LPA-enhanced IL-8 via endothelial cell tube formation, monolayer permeability, migration and cell growth assays, and by IL-8 knockout xenograft mice modeling. We found that the angiogenesis is mediated through IL-8. Finally, we evaluated the regulation pathways involved in LPA-induced IL-8 expression. We found that LPA receptor 2/3-mediated IL-8 expression occurs through Gi/PI3K/AKT, Gi/PKC and IκB/NF-κB signaling. In conclusion, we propose that LPA2 and LPA3 might play an important role in cervical cancer tumor growth through IL-8-dependent angiogenesis.     

(-)-Epigallocatechin gallate inhibits membrane-type 1 matrix metalloproteinase, MT1-MMP, and tumor angiogenesis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), which hydrolyzes type I collagen and activates MMP-2, are deeply involved in angiogenesis as well as in tumor cell invasion and metastasis. We previously screened a number of natural and synthetic compounds to obtain a specific inhibitor of MT1-MMP and observed that (-)-epigallocatechin gallate (EGCG) has a potent and distinct inhibitory activity against MT1-MMP. In the present study, we investigated the effect of EGCG on tumor angiogenesis. EGCG significantly inhibited the invasion of human umbilical vein endothelial cells (HUVECs) at the concentration of 10 microM. This effect was not due to the toxicity of EGCG since this concentration of EGCG did not affect the HUVEC growth. Furthermore, morphological change of HUVEC at this concentration of EGCG was not observed under confocal laser scanning microscopy. EGCG suppressed tube formation by HUVECs in vitro and angiogenesis in vivo by using dorsal air sac model. Finally, we observed that both colon 26 NL17 carcinoma and Meth A sarcoma growth was suppressed in these tumor-bearing mice by EGCG administration, at least partly though the inhibition of angiogenesis.     

Hispidulin, a small flavonoid molecule, suppresses the angiogenesis and growth of human pancreatic cancer by targeting vascular endothelial growth factor receptor 2-mediated PI3K/Akt/mTOR signaling pathway

Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti-inflammatory and anti-oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs in a dose-dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF-induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice. To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF-triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal-regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis.     

白头翁醇提物抑制血管生成的体外实验研究

目的 探讨白头翁醇提物对体外血管生成的抑制作用及其可能机制。方法 采用MTT法观察白头翁醇提物对人脐静脉血管内皮细胞（HUVEC）和人肠癌LoVo细胞增殖的影响;通过Transwell小室趋化实验、体外小管形成实验观察白头翁醇提物对HUVEC迁移、形成血管能力的影响;采用流式细胞仪检测白头翁醇提物对HUVEC的凋亡率及细胞周期的影响。结果 2～8μg／ml的白头翁醇提物作用48h对HUVEC的增殖抑制率为21.6％～72.0％,对LoVo细胞的增殖抑制率为13.2％～22.9％;体外小管形成实验发现,2～8μg／ml白头翁醇提物作用24h,HUVEC小管形成数目减少,且管腔不完整,与对照组比较差异有统计学意义（P＜0001）。经2～8μg／ml白头翁醇提物处理12h,HUVEC迁移数明显少于对照组（P＜0001）。2、4、8μg／ml白头翁醇提物作用于HUVEC细胞48h后,其凋亡率分别为952％、1864％和2007％,对照组为6.25％。8μg／ml白头翁醇提物作用于HUVEC细胞24h后,细胞周期停滞在G2／M期。结论 白头翁醇提物在体外能有效抑制血管生成,其机制可能与抑制HUVEC增殖、迁移和小管形成,诱导HUVEC凋亡,抑制HUVEC有丝分裂有关。     

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia.

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40 degrees C for 6-12 h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1beta, MIG). This is in contrast to the stimulatory effects of TNF-alpha or 43 degrees C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the alpha4beta7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.     

Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models

Insulin-like growth factor II (IGF-II), highly expressed in a number of human tumours, has been recently known to promote neovascularization in vivo. Yet, the detailed mechanism by which IGF-II induces angiogenesis has not been well defined. In the present study, we explored an angiogenic activity of IGF-II in in vitro angiogenesis model. Human umbilical vein endothelial cells (HUVECs) treated with IGF-II rapidly aligned and formed a capillary-like network on Matrigel. In chemotaxis assay, IGF-II remarkably increased migration of HUVECs. A rapid and transient activation of p38 mitogen-activated protein kinase (p38 MAPK) and p125 focal adhesion kinase (p125FAK) phosphorylation was detected in HUVECs exposed to IGF-II. IGF-II also stimulated invasion of HUVECs through a polycarbonate filter coated with Matrigel. Quantitative gelatin-based zymography identified that matrix metalloproteinase-2 (MMP-2) activity generated from HUVECs was increased by IGF-II. This induction of MMP-2 activity was correlated with Northern blot analysis, showing in HUVECs that IGF-II increased the expression of MMP-2 mRNA, while it did not affect that of TIMP-2, a tissue inhibitor of MMP-2. These results provide the evidence that IGF-II directly induces angiogenesis by stimulating migration and morphological differentiation of endothelial cells, and suggest that IGF-II may play a crucial role in the progression of tumorigenesis by promoting the deleterious neovascularization.