In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice

Angiogenesis is the main mechanism of vascular remodeling during late development and, after birth, in wound healing. Perturbations of angiogenesis occur in cancer, diabetes, ischemia, and inflammation. While much progress has been made in identifying factors that control angiogenesis, the understanding of the precise molecular mechanisms involved is incomplete. Here we identify a small GTPase, Rap1b, as a positive regulator of angiogenesis. Rap1b-deficient mice had a decreased level of Matrigel plug and neonatal retinal neovascularization, and aortas isolated from Rap1b-deficient animals had a reduced microvessel sprouting response to 2 major physiological regulators of angiogenesis: vascular endothelial growth factor (VEGF) and basic fibroblasts growth factor (bFGF), indicating an intrinsic defect in endothelial cells. Proliferation of retinal endothelial cells in situ and in vitro migration of lung endothelial cells isolated from Rap1b-deficient mice were inhibited. At the molecular level, activation of 2 MAP kinases, p38 MAPK and p42/44 ERK, important regulators of endothelial migration and proliferation, was decreased in Rap1b-deficient endothelial cells in response to VEGF stimulation. These studies provide evidence that Rap1b is required for normal angiogenesis and reveal a novel role of Rap1 in regulation of proangiogenic signaling in endothelial cells.     

血管内皮、碱性成纤维细胞生长因子表达与非小细胞肺癌患者生存期的关系

目的 探讨血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)在非小细胞肺癌(NSCLC)中的表达及其与患者生存期的关系。方法 应用免疫组织化学方法检测96例NSCLC组织中VEGF、bFGF蛋白水平的表达，并探讨二者表达与患者生存期的关系。结果 NSCLC中VEGF、bFGF表达阳性率分别为51．0％(49／96)和58．3％(56／96)，二者具有相关性(P=0．041)，与患者生存期之间无统计学意义。结论 VEGF、bFGF在NSCLC血管形成中起重要作用，但不是NSCLC独立的预后因素。     

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.     

Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and activation of platelets

Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, alpha(IIb)beta(3), inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the alpha(IIb)beta(3) integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases. (Blood. 2000;96:4216-4221)     

Role of growth factors in coronary morphogenesis

This communication briefly reviews the role of angiogenic growth factors in myocardial vessel formation during development. The earliest signs of vascularization are the migration and differentiation of angioblasts from the epicardium and subepicardium into the myocardium. A regulator of this process is vascular endothelial growth factor (VEGF), which is probably triggered by hypoxia. The subsequent formation of vascular tubes is regulated by multiple growth factors: VEGF family members, fibroblast growth factors (FGFs), and angiopoietins and their receptors. Our studies on explanted quail hearts reveal that these growth factors are interdependent. We also have shown that a harmonic interplay of growth factors characterizes early postnatal development in rats. Neutralizing antibodies to either basic FGF (bFGF) or VEGF inhibit capillary formation, whereas arteriolar growth is markedly inhibited by bFGF, but not VEGF, neutralizing antibodies. Arteriolar diameter is also increased when anti-bFGF and anti-VEGF are administered in combination. Thus, the hierarchical development of the arteriolar vasculature depends on both of these growth factors; however, the establishment of arterioles, as reflected by length density, is dependent on bFGF but not on VEG. Finally, stretch of cardiac myocytes and endothelial cells serves as a stimulus for increases in growth factor and receptor proteins. We have shown that cyclic stretch of either cell type increases VEGF, and that endothelial cells respond to stretch by up-regulation of VEGF receptor-2 (VEGFR-2), and Tie-2 receptor. These results indicate that both mechanical and metabolic factors are primary stimuli for coronary angiogenesis.     

Angiogenesis in acute promyelocytic leukemia: induction by vascular endothelial growth factor and inhibition by all-trans retinoic acid

Recent studies indicate that angiogenesis is important in the pathogenesis of leukemias, apart from its well-established role in solid tumors. In this study, the possible role of angiogenesis in acute promyelocytic leukemia (APL) was explored. Bone marrow trephine biopsies from patients with APL showed significantly increased microvessel density and hot spot density compared with normal control bone marrow biopsies. To identify the mediators of angiogenesis in APL, quantitative and functional assays were performed using the NB4 APL cell line as a model system. Conditioned media (CM) from the NB4 cells strongly stimulated endothelial cell migration. CM from the NB4 cells contained high levels of vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor (bFGF). Most important, the addition of neutralizing VEGF antibodies completely inhibited the ability of NB4 CM to stimulate endothelial cell migration, suggesting that APL angiogenesis is mediated by VEGF. The effect of all-trans retinoic acid (ATRA) on APL angiogenesis was then studied. ATRA therapy resulted in a decrease in bone marrow microvessel density and hot spot density. CM from ATRA-treated APL cells did not stimulate endothelial cell migration. Finally, quantitative assays showed that ATRA treatment resulted in the abrogation of VEGF production by the NB4 cells. These results show that there is increased angiogenesis and VEGF production in APL and that ATRA therapy inhibits VEGF production and suppresses angiogenesis. The addition of specific antiangiogenic agents to differentiation therapy or chemotherapy should be explored. (Blood. 2001;97:3919-3924)     

胰腺星状细胞对血管内皮细胞增殖的影响

目的研究人胰腺星状细胞(PSC)在体内、外对血管内皮细胞增殖的影响。方法使用含10%小牛血清的M199培养基培养人脐静脉内皮细胞(HUVEC);含10%小牛血清的DMEM/F12细胞培养基培养PSC,检测前24 h换无血清培养基。逆转录-聚合酶链反应(RT-PCR)和Western印迹法检测血管内皮生长因子(VEGF)、白细胞介素(IL)-8和碱性纤维母细胞生长因子(bFGF)mRNA及蛋白在PSC中的表达,酶联免疫吸附试验检测PSC上清液中上述因子的含量;应用流式细胞技术和四甲基偶氮唑盐(MTT)比色法检测PSC上清液对HUVEC的增殖作用。同时,应用小管形成实验和鸡胚尿囊膜(CAM)模型研究PSC上清液对血管内皮细胞体内外血管生成的影响。结果PSC能独立合成并分泌VEGF、IL- 8及bFGF,流式细胞及MTT结果提示其上清液能使HUVEC的S期比例增加,有丝分裂增多。在体外血管形成研究中,PSC上清液能明显地促血管内皮细胞增殖并形成中空的管状结构;在体内CAM模型中,PSC上清液能使血管数目增加,管径增粗。结论PSC在体内外具有促血管生成作用。这一过程可能与其合成的VEGF、IL-8及bFGF等血管活性因子相关。PSC可能参与胰腺肿瘤的早期血行转移过程。     

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.     

Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2

Neuropilin-1 (NRP-1) is a type 1 membrane protein that binds the axon guidance factors belonging to the class-3 semaforin family. In endothelial cells, NRP-1 serves as a co-receptor for vascular endothelial growth factor (VEGF) and regulates VEGF receptor 2 (VEGFR-2)-dependent angiogenesis. Although gene-targeting studies documenting embryonic lethality in NRP-1 null mice have demonstrated a critical role for NRP-1 in vascular development, the activities of NRP-1 in mature endothelial cells have been incompletely defined. Using RNA interference-mediated silencing of NRP-1 or VEGFR-2 in primary human endothelial cells, we confirm that NRP-1 modulates VEGFR-2 signaling-dependent mitogenic functions of VEGF. Importantly, we now show that NRP-1 regulates endothelial cell adhesion to extracellular matrix proteins independently of VEGFR-2. Based on its dual role as an enhancer of VEGF activity and a mediator of endothelial cell adhesiveness described here, NRP-1 emerges as a promising molecular target for the development of antiangiogenic drugs.     

沙利度胺对人脐静脉内皮细胞增殖和血管生成的影响

目的 探讨沙利度胺治疗血管发育不良所致消化道出血的机制.方法 体外培养人脐静脉内皮细胞至对数生长期,分为空白对照组、溶剂对照组(二甲基亚砜)和不同浓度(10、20、40、60、80、100μg/ml)沙利度胺组,根据加或不加成纤维细胞生长因子(bFGF,10 ng/ml),共分为16组.刺激72 h后,MTT法检测细胞增殖情况,酶联免疫吸附法和实时定量PCR法测定血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)表达.结果 加或不加bFGF刺激,中、高浓度(≥40/μg/ml)沙利度胺均能抑制人脐静脉内皮细胞增殖.未加bFGF刺激时.20μg/ml沙利度胺能明显抑制VEGF表达.加bFGF刺激时,10 μg/ml沙利度胺即能明显抑制VEGF表达.未检出TNF-α表达.结论 体外实验中,沙利度胺能抑制人脐静脉内皮细胞增殖和VEGF表达,从而抑制血管生成,达到治疗血管发育不良所致消化道出血的目的 .     

Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.     

Angiogenesis in the thyroid gland

Angiogenesis is the mechanism of blood vessel formation after the first few days of embryogenesis, and is essential for all tissue growth. In adults, angiogenesis occurs in the thyroid during disease processes including goitre, Graves' disease, thyroiditis and cancer. The molecular mechanisms controlling angiogenesis are becoming clearer, and therapy targeting these processes is coming closer to clinical fruition. Both promoters and inhibitors of angiogenesis have been identified in the thyroid, including vascular endothelial growth factor (VEGF), fibroblast growth factor, and thrombospondin. This commentary will review the understanding of the control of angiogenesis within the context of the thyroid gland, and review the pre-eminent role of VEGF as the angiogenic signal from the follicular cells to the endothelial cells.     

Modeling and simulation of microfluid effects on deformation behavior of a red blood cell in a capillary

BackgroundAngiogenesis plays an essential role in the growth and metastatic development of tumours. Recent in vitro studies have reported bisphosphonates as having anti-angiogenic properties. They have been shown to inhibit proliferation, induce apoptosis and decrease capillary-like tube formation, but often the in vitro concentrations and dosing schedules used do not reflect drug pharmacokinetics or clinical dosing regimens.Materials and methodsHuman umbilical vein endothelial cells were exposed to physiologically relevant doses of the bisphosphonate ibandronate, mimicking the clinical administration of oral ibandronate (1 h daily dosing over 8 days at concentrations ranging from 1–10 μM). Cellular growth characteristics were then assessed.ResultsLow-dose ibandronate (1.25–2 μM) significantly reduced endothelial cell growth, while 2 μM ibandronate also significantly reduced capillary-like tube formation and increased apoptosis of endothelial cells compared to untreated cells. There was no significant difference in activity with doses above 2 μM. However, inhibiting bFGF stimulated cell growth increased VEGF expression.ConclusionThis work has demonstrated that repeated low-dose drug administration (metronomic therapy) of ibandronate has certain anti-angiogenic properties by inhibiting the stimulatory effects of bFGF. However targeting the inhibition of bFGF alone is unlikely to be a successful approach for completely inhibiting angiogenesis due to the interplay between bFGF and VEGF.     

Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma.

Angiogenesis is in part related to mast cells. However, the biological significance of mast cells within lung carcinoma remains unclear. Immunohistochemistry was used to stain for tryptase, CD34 and vascular endothelial growth factor (VEGF) in 85 cases of stage I nonsmall cell lung carcinoma. VEGF was found in 33 of 53 adenocarcinomas and 14 of 32 squamous cell carcinomas. Cases of adenocarcinoma had significantly higher mast cell counts than those of squamous cell carcinoma. In adenocarcinoma, mast cell counts in VEGF-positive tumours were significantly higher than in VEGF-negative tumours, whereas in squamous cell carcinoma they were not. Good correlation was observed between intratumoural mast cell counts and microvessel counts. Double staining showed most intratumoural mast cells expressed VEGF. Importantly, only in lung adenocarcinoma, members in the high mast cell count group had significantly worse prognosis than those in the low mast cell count group. It is concluded that tumour-released vascular endothelial growth factors may be related to mast cell accumulation, intratumoural mast cells may produce vascular endothelial growth factor, and stromal mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma.