| BackgroundAngiogenesis,the formation of new blood vessels from existing host vessels,plays a key role in tumorigenesis by providing oxygen and nutrients to rapidly growing tumor tissues.Angiogenesis,therefore,can be divided into two main stages:the first stage is the induction of the "angiogenic Switch" by inducing the new tumor blood vessel from the host vessels;the second stage is the tumor vessel network formation by inducing vessel network from the existing new tumor blood vessel.Thus,the angiogenic switch is the first step of the angiogenesis.The angiogenic switch is a key step of the transition of tumor tissues from an avascular one to a vascular one,and the occurrence can be understood as whether the first new blood vessel has grown in the tumor.However,due to the limitation of the research system,angiogenic switch cannot be accuratly differentiated from the angiogenesis to conduct a detailed study.In other words,the process of tumor inducing the first vessel sprouting has not been studied in vivo and the exact mechanism of the angiogenic switch is still unclear.Now,the pathways and molecules involved in the angiogenic switch are mainly derived from theoretical speculation.It is generally believed that when the tumor grows to 1-2mm3,hypoxia activates the HIF pathway,upregulates the expression of vascular growth factors,such as vascular endothelial growth factor(VEGF)and induces the angiogenesis to promote tumor growth.VEGF is a important pro-angiogenic factor.VEGF family proteins include VEGFA,VEGFB,VEGFC,VEGFD and placental growth factor(PlGF).VEGFA is one of the best studied members of the family and also the target of numerous anti-tumor therapies.VEGFA activates different downstream signaling pathways and mediates different cell fates by binding to vascular endothelial growth factor receptor 2(VEGFR2),including promoting cell proliferation by phospholipase C γ(PLCγ)and extracellular signal regulated kinase 1/2(ERK1/2),mediating cell migration by focal plaque kinase(FAK)and promoting cell survival by the phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT).A large number of studies have shown that VEGF expression is mainly regulated by hypoxia and hypoxia-inducible factors(HIFs),but an increasing number of studies have shown that it can also be regulated by pathways that do not depend on hypoxia or HIFs.Restriction of amino acid intake promotes VEGF expression,while the deletion of amylon-tRNA synthase up-regulates VEGF through the unfolded protein response(UPR)pathway.In addition,the general control nonderepressible 2(GCN2)/activating transcription factor 4(ATF4)pathway and the oestrogen-related receptor alpha(ERRα)/peroxisome activated receptor coactivator 1 alpha(PGC-1α)pathway can also promote the expression of VEGF.Thus,the angiogenic switch may also be regulated by other pathways independent of hypoxia or HIFs.Zebrafish is a promising model for angiogenesis research.Zebrafish embryos are small in size and can be observed directly with high resolution microscope within 30 days after fertilization.The transparency of zebrafish embryos and the availability of vascular-specific transgenic reporter lines with an enhanced green fluorescent protein(EGFP)in all blood vessels throughout embryogenesis allow easy intravital imaging of vessels.We have established a zebrafish angiogenic switch model with the vascularspecific transgenic reporter lines.This model can be used to dynamically visualize the angiogenic switch(the process of first tumor vessel sprouting)and study the mechanism of the angiogenic switch in vivo with the high resolution microscope.MethodsThe zebrafish angiogenic switch model was established by inoculation of red fluorescent labeled B16 mammalian melanoma cells(B 16-Red)into perivitelline space of Tg(flk1:EGFP)zebrafish embryos,which’s blood vessel labeled with green fluorescence.The process of tumor tissues inducing vessel sprouting from host blood vessels to promote the angiogenic switch can be observed.Hypoxia probe was used to detect hypoxia during the angiogenic switch in zebrafish xenograft.Small interfering RNAs(siRNA)were used to silence the expression of Hif1α.The successfully silenced tumor cells have been implanted in the perivitelline space of transgenic zebrafish embryo to establish an angiogenic switch model,and the effect of Hifla on the angiogenic switch can be observed and studied.Plasmid vectors were constructed for the knockout or overexpression of Vegfa,and tumor cells successfully knocked out or overexpressed Vegfa were inoculated into the perivitelline space of transgenic zebrafish embryos to establish an angiogenic switch model to observe and study the influence of Vegfa on the angiogenic switch.The microtumor tissues before and after the angiogenic switch were obtained by microsurgery,and the transcriptome of the microtumor tumor tissues was sequenced by single cell sequencing technology.The transcriptome changes were analyzed by bioinformatics method to obtain the changed genes and pathways during the angiogenic switch.3D tumor cell sphere model was established to simulate the growth of tumor tissues in vitro and verify the changed genes and pathways during the angiogenic switch.Protein translation in tumor cells was inhibited by puromycin or cycloheximide,and thus the effect of protein translation inhibition on Vegfa expression could be observed and studied under non-hypoxic conditions in vitro.Puromycin inhibited protein translation,and siRNA interfered the expression of Vegfa in tumor cells.Then the tumor cells were transplanted in the perivitelline space of transgenic zebrafish embryo to establish an angiogenic switch model.Thus we can observe and study the influence of protein translation inhibition and interference of the expression of Vegfa on the angiogenic switch,and their relationship during the angiogenic switch.A mouse xenograft model was established to observe and study the effect of protein translation inhibition on angiogenesis.Inhibition of the transcription of 3D tumor spheres and tumor cells with αAmanitin to determine whether the inhibition of protein translation increases Vegfa transcription,or stabilizes Vegfa mRNA.Zebrafish xenograft models were exposured to the single-target inhibitor Nintedanib(VEGFR2)and Tivantinib(c-Met)or dual-target inhibitor Cabozantinib(VEGFR2/c-Met)to observe and study the influence of VEGFR2 and c-Met on the angiogenic switch.Results1.The establishment of the zebrafish angiogenic switch model.The zebrafish angiogenic switch model was established by inoculation of red fluorescent labeled B16 mammalian melanoma cells into perivitelline space of Tg(flk1:EGFP)zebrafish embryo,which’s blood vessel labeled with green fluorescence,to observe the tumor tissues inducing the sprouting of the first blood vessel from the host blood vessels,and then gradually forming vessel network.The process of tumor tissues inducing the first blood vessl from the host vessels is called angiogenic switch.2.Hypoxia was not the major stimulator of the angiogenic switch.The tumor cells incubated with hypoxia reagent cultured under hypoxic(1%)showed hypoxic fluorescent signals,but no fluorescence was detected in the hypoxia reagent-incubated transplant microtumors.It indicated that no significant hypoxia had occurred during the angiogenic switch.We knocked down Hifla by siRNA,transplanted Hifla knockdown tumor cells in zebrafish and found no significant effect on the angiogenic switch.The tumor vessel branches and length showed no significant changes in zebrafish with Hifla knockdown tumors compared with controls.It indicated that Hif1α had no significant influence on the angiogenic switch.In addition,the expression of Car9/CA9 and Slc2a1/GLUT-1(HIF target genes)also showed no significant changes during the angiogenic switch in transplant microtumors.The gene set enrichment analysis(GSEA)of the transcriptome of the transplant microtumors showed no significant change in the HIF-1 pathway,and in the expression of HIF target genes during the angiogenic switch,including Car,Slc2a1,Lgsf8,Kcnj12,Macrodl,Eno3,Tmem38a,Relt,Hn1,Kansl1,Gch1 and Endog.It indicated that hypoxia was not the major stimulator of the angiogenic switch.3.Angiogenic switch depends on Vegfa.The expression of Vegfa was significantly increased in the transcriptome and in the dissected transplant microtumors during the angiogenic switch.The zebrafish angiogenic switch models with tumor cells transfected with plasmid to knock out or overexpress Vegfa were established.Vegfa knockout in transplant microtumors prevented even the first tumor vessel sprouting,while overexpression of Vegfa promoted an earlier onset of the angiogenic switch.Compared with control tumors,tumors in which Vegfa was knocked out had fewer vessel branches and shorter vessel lengths,while the tumors in which the Vegfa was overexpressed had more vessel branches and longer vessel lengths.It suggested that angiogenic switch depends on Vegfa.4.Protein translation decreases during the angiogenic switch.GSEA analysis revealed that 5 signaling pathways showed significant downregulation during the angiogenic switch,including aminoacyl-trnas biosynthesis,galactose metabolism,amino sugar and nucleotide sugar metabolism,proteasome,and fatty acid elongation.Aminoacyl-trnas biosynthesis pathway is a key signal pathway for protein translation.The genes expression of aminoacyl-trnas synthetases of the aminoacyl-trnas biosynthesis pathway were significantly decreased,such as Cars,Mars and Kars.Aminoacyl-trnas biosynthesis pathway genes combine amino acids with tRNA to form aminoacyl-trnas,which transports amino acids for protein synthesis.The metabolites of the galactose,amino sugar and nucleotide sugar provide precursors of protein glycosylation and the proteasome is responsible for protein degradation.The downregulated pathway activity indicated that protein translation may decrease during the angiogenic switch.Gene ontology analysis(GO)found that the changes of biological process were mainly concentrated in the protein translation,the changes of molecular function mainly included ribosome binding,and the changes of cell components mainly reflected in the ribosome subunits during the angiogenic switch.The results showed that the transcriptome changes during the angiogenic switch were mainly related to protein translation.Western blot analysis of the protein translation marker eIF2α(Eukaryotic Initiation Factor 2)revealed an increase of it’s phosphorylation,suggesting that the protein translation of the xenografts decreased during the angiogenic switch.5.Inhibition of protein translation of tumor cells promotes Vegfa transcription independent of Hif1αThe inhibition of protein translation by puromycin or cycloheximide in tumor cells or 3D tumor spheres significantly increased Vegfa expression,which were reversed after the addition of α-Amanitin(inhibite the transcription).The results showed that the inhibition of protein translation mainly increased Vegfa transcription rather than Vegfa mRNA stability.Puromycin-mediated inhibition of protein translation significantly increased the expression Vegfa and knockdown of Hif1α significantly decreased the expression Vegfa in tumor cells.However,knockdown of Hif1α While inhibiting protein translation by puromycin in tumor cells also significantly increased the expression of Vegfa as the same as puromycin treatment.It suggested that knockdown of Hifla did not affect the increase of Vegfa transcription induced by puromycin-mediated inhibition of protein translation.6.Inhibition of protein translation promotes angiogenic switch depending on Vegfa.zebrafish angiogenic switch models were established with puromycin treated tumor cells to inhibit protein translation.The ability of zebrafish xenograft inducing the angiogenic switch was significantly enhanced.Both the length of tumor vessel and the ratio of vessel area versus tumor area were increased significantly.However,knockdown of Vegfa while treating tumor cells with puromycin eliminated the effect of promoting the angiogenic switch.The length of tumor vessel and the ratio of vessel area versus tumor area were similar to those of the control group.These results indicated that the inhibition of protein translation promotes angiogenic switch depending on Vegfa.7.Vegfa may promote the angiogenic switch by VEGFR2 and c-Met.Nintedanib(VEGFR2 inhibitor)inhibited the formation of tumor vessel network,but did not affect the angiogenic switch.The vessel branches and length of nintedanib treated tumors did not change significantly during the angiogenic switch,but decreased significantly in the vessel network stage.Tivantinib,a c-Met inhibitor,has a similar effect.Nintedanib combined with tivantinib or cabozantinib(inhibite VEGFR2 and cMet)significantly inhibited the angiogenic switch and vessel network formation.The vessel branches and length significantly decreased during both the angiogenic switch and the vessel network formation.These results indicated that Vegfa may promotes the angiogenic switch by VEGFR2 and c-Met.ConclusionIn this study,we observe and study the angiogenic switch directly,real-time,and dynamically in vivo with the zebrafish angiogenic switch model.It was found that there was no significant hypoxia signal during the angiogenic switch,and knockdown of Hifla also did not affect angiogenic switch.But Vegfa played a key role in the angiogenic switch.Futhermore,the protein translation of tumor cells promoted Vegfa transcription and Vegfa promoted the angiogenic switch by VEGFR2 and c-Met.In this study,an in vivo zebrafish angiogenic switch model was established to explore the role and relationship of hypoxia,Hif1α,Vegfa,protein translation,VEGFR2 and c-Met during the angiogenic switch,and to provide dynamic and visual in vivo evidence about the angiogenic switch.Thus,it provids theoretical basis for the treatment targeting tumor vessels. |
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