Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background

Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.

Methodology

Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.

Conclusion

Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.     

Hypoxia-induced metastasis model in embryonic zebrafish

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.     

Context-dependent proangiogenic function of bone morphogenetic protein signaling is mediated by disabled homolog 2

Bone morphogenetic proteins (BMPs) have diverse functions during development in vertebrates. We have recently shown that BMP2 signaling promotes venous-specific angiogenesis in zebrafish embryos. However, factors that confer a context-dependent proangiogenic function of BMP2 signaling within endothelial cells need to be identified. Here, we report that Disabled homolog 2 (Dab2), a cargo-specific adaptor protein for Clathrin, is essential to mediate the proangiogenic function of BMP2 signaling. We find that inhibition of Dab2 attenuates internalization of BMP receptors and abrogates the proangiogenic effects of BMP signaling in endothelial cells. Moreover, inhibition of Dab2 decreases phosphorylation of SMAD-1, 5, and 8, indicating that Dab2 plays an essential role in determining the outcome of BMP signaling within endothelial cells and may provide a molecular basis for a context-dependent proangiogenic function of BMP2 signaling.     

Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.     

A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2⁺ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals.     

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.     

Dual blockade of VEGFR1 and VEGFR2 by a novel peptide abrogates VEGF-driven angiogenesis,tumor growth,and metastasis through PI3K/AKT and MAPK/ERK1/2 pathway

Background

Neutralization of vascular endothelial growth factor receptor 1 (VEGFR1) and/or VEGFR2 is a widely used means of inhibiting tumor angiogenesis.

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma.     

Zebrafish embryo, a tool to study tumor angiogenesis

Zebrafish (Danio rerio) represents a powerful model system in cancer research. Recent observations have shown the possibility to exploit zebrafish to investigate tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Experimental models have been established in zebrafish adults, juveniles, and embryos, each one with its own advantages and disadvantages. Novel genetic tools and high resolution in vivo imaging techniques are also becoming available in zebrafish. It is anticipated that zebrafish will represent an important tool for chemical discovery and gene targeting in tumor angiogenesis. This review focuses on the recently developed tumor angiogenesis models in zebrafish, with particular emphasis to tumor engrafting in zebrafish embryos.     

GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.     

Nicotine induces cyclooxygenase-2 and vascular endothelial growth factor receptor-2 in association with tumor-associated invasion and angiogenesis in gastric cancer

Blockade of angiogenesis is a promising strategy to suppress tumor growth, invasion, and metastasis. Vascular endothelial growth factor (VEGF), which binds to tyrosine kinase receptors [VEGF receptors (VEGFR) 1 and 2], is the mediator of angiogenesis and mitogen for endothelial cells. Cyclooxygenase-2 (COX-2) plays an important role in the promoting action of nicotine on gastric cancer growth. However, the action of nicotine and the relationship between COX-2 and VEGF/VEGFR system in tumorigenesis remain undefined. In this study, the effects of nicotine in tumor angiogenesis, invasiveness, and metastasis were studied with sponge implantation and Matrigel membrane models. Nicotine (200 microg/mL) stimulated gastric cancer cell proliferation, which was blocked by SC-236 (a highly selective COX-2 inhibitor) and CBO-P11 (a VEGFR inhibitor). This was associated with decreased VEGF levels as well as VEGFR-2 but not VEGFR-1 expression. Topical injection of nicotine enhanced tumor-associated vascularization, with a concomitant increase in VEGF levels in sponge implants. Again, application of SC-236 (2 mg/kg) and CBO-P11 (0.4 mg/kg) partially attenuated vascularization by approximately 30%. Furthermore, nicotine enhanced tumor cell invasion through the Matrigel membrane by 4-fold and promoted migration of human umbilical vein endothelial cells in a cocultured system with gastric cancer cells. The activity of matrix metalloproteinases 2 and 9 and protein expressions of plasminogen activators (urokinase-type plasminogen activator and its receptor), which are the indicators of invasion and migration processes, were increased by nicotine but blocked by COX-2 and VEGFR inhibitors. Taken together, our results reveal that the promoting action of nicotine on angiogenesis, tumor invasion, and metastasis is COX-2/VEGF/VEGFR dependent.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce.PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway.MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis.ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1.ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.     

Proangiogenic function of CD40 ligand-CD40 interactions

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.     

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9735-y) contains supplementary material, which is available to authorized users.     

FLT4 基因在斑马鱼早期发育过程中的表达

目的:研究FLT4(VEGFR3)基因在斑马鱼早期发育过程中的表达。方法:提取斑马鱼胚胎的总RNA,制备地高辛标记的FLT4RNA反义探针,WISH(整胚胎原位杂交)研究FLT4在斑马鱼早期发育过程中的表达。结果:成功合成FLT4基因探针,获得FLT4基因在斑马鱼早期发育过程中的表达情况:FlT4在2-cell、32-cell、oblong、shield期前普遍性表达(0.75h、1.7h、3.7h、6h);24h在头部表达较多,特别是在ICM(intermediate cell mass,中间细胞群)区处有特异性表达;48h、72h在头部表达均较高表达。结论:FLT4在早期参与了血管的形成和造血的发生,在脑部和肾小管的发育中可能起到了重要作用。