Growth Of Intersegmental Vessels Research Articles

The angiogenesis-modulating effects of coumarin-derivatives

Coumarin is a natural compound that is rich in plants. Coumarin and its derivates were reported to have many biological activities, such as anti-bacterial, anti-tumor, and anti-coagulation. In this study, we examined the angiogenic modulating activities of six previously synthesized coumarin derivatives (Compound #1-#6) in zebrafish embryos and further confirmed them in a chick model. According to the survival rate in a zebrafish model, Compound #1 (100 %), #2 (82.5–100 %), and #4 (100 %) showed much less toxicity than Compound #3 (19.2–100 %), #5 (0–100 %), and #6 (0–100 %). Using a green blood vessel fluorescent transgenic fish Tg(fli1:egfp) to record the angiogenesis-modulating effects of Compound #1, #2, and #4, we found that Compound #2 had the highest effects in interfering intersegmental vessel growth, subintestinal vein growth, and caudal vein plexus remodeling. Chick chorioallantoic membrane (CAM) assay also showed that Compound #2 exposure led to a reduction of blood vessel growth. Real-time PCR experiments revealed that Compound #2 significantly changed the expression of vascular growth-related genes flt1, cdh5, and nrp1a in zebrafish. Based on our data from zebrafish and chick models, a new coumarin-derivative (Compound #2) possesses anti-angiogenic activity with low toxicity, but further investigation in mammal models is asked to confirm our findings.

Functional characterization of stap2b in zebrafish vascular development.

The genetic control and signaling pathways of vascular development are not comprehensively understood. Transcription factors Islet2 (Isl2) and nr2f1b are critical for vascular growth in zebrafish, and further transcriptome analysis has revealed potential targets regulated by isl2/nr2f1b. In this study, we focused on the potential activation gene signal-transducing adaptor protein 2b (stap2b) and revealed a novel role of stap2b in vascular development. stap2b mRNA was expressed in developing vessels, suggesting stap2b plays a role in vascularization. Knocking down stap2b expression by morpholino injection or Crispr-Cas9-generated stap2b mutants caused vascular defects, suggesting a role played by stap2b in controlling the patterning of intersegmental vessels (ISVs) and the caudal vein plexus (CVP). The vessel abnormalities associated with stap2b deficiency were found to be due to dysregulated cell migration and proliferation. The decreased expression of vascular-specific markers in stap2b morphants was consistent with the vascular defects observed. In contrast, overexpression of stap2b enhanced the growth of ISVs and reversed the vessel defects in stap2b morphants. These data suggest that stap2b is necessary and sufficient to promote vascular development. Finally, we examined the interaction between stap2b and multiple signaling. We showed that stap2b regulated ISV growth through the JAK-STAT pathway. Moreover, we found that stap2b was regulated by Notch signaling to control ISV growth, and stap2b interacted with bone morphogenetic protein signaling to contribute to CVP formation. Altogether, we demonstrated that stap2b acts downstream of the isl2/nr2f1b pathway to play a pivotal role in vascular development via interaction with multiple signaling pathways.

LC-MS Analysis of Ginsenosides in Different Parts of Panax quinquefolius and Their Potential for Coronary Disease Improvement.

Seven main ginsenosides, including ginsenoside Re, ginsenoside Rb1, pseudoginsenoside F11, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, and ginsenoside F2, were identified by LC-QTOF MS/MS from root, leaf and flower extracts of Panax quinquefolius. These extracts promoted intersegmental vessel growth in a zebrafish model, indicating their potential cardiovascular health benefits. Network pharmacology analysis was then conducted to reveal the potential mechanisms of ginsenoside activity in the treatment of coronary artery disease. GO and KEGG enrichment analyses elucidated that G protein-coupled receptors played a critical role in VEGF-mediated signal transduction and that the molecular pathways associated with ginsenoside activity are involved in neuroactive ligand-receptor interaction, cholesterol metabolism, the cGMP-PKG signaling pathway, etc. Moreover, VEGF, FGF2, and STAT3 were confirmed as the major targets inducing proliferation of endothelial cells and driving the pro-angiogenic process. Overall, ginsenosides could be potent nutraceutical agents that act to reduce the risks of cardiovascular disease. Our findings will provide a basis to utilize the whole P. quinquefolius plant in drugs and functional foods.

Nitrobenzoate-Derived Compound X8 Impairs Vascular Development in Zebrafish.

Proper growth and patterning of blood vessels are critical for embryogenesis. Chemicals or environmental hormones may interfere with vascular growth and cause developmental defects. Nitrobenzoate-based compounds have been demonstrated to have a wide range of biological and pharmacological functions, leading to the development of numerous 4-nitrobenzoate derivatives for clinical application. In this study, we tested a novel nitrobenzoate-derived compound, X8, and investigated its effects on vascular development using zebrafish as a model organism. We first determined the survival rate of embryos after the addition of exogenous X8 (0.5, 1, 3, 5, and 10 μM) to the fish medium and determined a sublethal dose of 3 μM for use in further assays. We used transgenic fish to examine the effects of X8 treatment on vascular development. At 25–32 h postfertilization (hpf), X8 treatment impaired the growth of intersegmental vessels (ISVs) and caudal vein plexuses (CVPs). Moreover, X8-treated embryos exhibited pericardial edema and circulatory defects at 60–72 hpf, suggesting the effects of X8 in vasculature. Apoptosis tests showed that the vascular defects were likely caused by the inhibition of proliferation and migration. To investigate the molecular impacts underlying the defects in the vasculature of X8-treated fish, the expression levels of vascular markers, including ephrinb2, mrc1, and stabilin, were assessed, and the decreased expression of those genes was detected, indicating that X8 inhibited the expression of vascular genes. Finally, we showed that X8 treatment disrupted exogenous GS4012-induced angiogenesis in Tg(flk:egfp) zebrafish embryos. In addition, vascular defects were enhanced during cotreatment with X8 and the VEGFR2 inhibitor SU5416, suggesting that X8 treatment causes vascular defects mediated by disruption of VEGF/VEGFR2 signaling. Collectively, our findings indicate that X8 could be developed as a novel antiangiogenic agent.

Active perception during angiogenesis: filopodia speed up Notch selection of tip cells in silico and in vivo.

How do cells make efficient collective decisions during tissue morphogenesis? Humans and other organisms use feedback between movement and sensing known as ‘sensorimotor coordination’ or ‘active perception’ to inform behaviour, but active perception has not before been investigated at a cellular level within organs. Here we provide the first proof of concept in silico/in vivo study demonstrating that filopodia (actin-rich, dynamic, finger-like cell membrane protrusions) play an unexpected role in speeding up collective endothelial decisions during the time-constrained process of ‘tip cell’ selection during blood vessel formation (angiogenesis). We first validate simulation predictions in vivo with live imaging of zebrafish intersegmental vessel growth. Further simulation studies then indicate the effect is due to the coupled positive feedback between movement and sensing on filopodia conferring a bistable switch-like property to Notch lateral inhibition, ensuring tip selection is a rapid and robust process. We then employ measures from computational neuroscience to assess whether filopodia function as a primitive (basal) form of active perception and find evidence in support. By viewing cell behaviour through the ‘basal cognitive lens' we acquire a fresh perspective on the tip cell selection process, revealing a hidden, yet vital time-keeping role for filopodia. Finally, we discuss a myriad of new and exciting research directions stemming from our conceptual approach to interpreting cell behaviour.This article is part of the theme issue ‘Basal cognition: multicellularity, neurons and the cognitive lens’.

KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence.

Shear stress, a blood flow-induced frictional force, is essential in the control of endothelial cell (EC) homeostasis. High laminar shear stress (HLSS), as observed in straight parts of arteries, assures a quiescent non-activated endothelium through the induction of Krüppel-like transcription factors (KLFs). Connexin40 (Cx40)-mediated gap junctional communication is known to contribute to a healthy endothelium by propagating anti-inflammatory signals between ECs, however, the molecular basis of the transcriptional regulation of Cx40 as well as its downstream effectors remain poorly understood. Here, we show that flow-induced KLF4 regulated Cx40 expression in a mouse EC line. Chromatin immunoprecipitation in ECs revealed that KLF4 bound to three predicted KLF consensus binding sites in the Cx40 promoter. HLSS-dependent induction of Cx40 expression was confirmed in primary human ECs. The downstream effects of Cx40 modulation in ECs exposed to HLSS were elucidated by an unbiased transcriptomics approach. Cell cycle progression was identified as an important downstream target of Cx40 under HLSS. In agreement, an increase in the proportion of proliferating cell nuclear antigen (PCNA)-positive ECs and a decrease in the proportion of ECs in the G0/G1 phase were observed under HLSS after Cx40 silencing. Transfection of communication-incompetent HeLa cells with Cx40 demonstrated that the regulation of proliferation by Cx40 was not limited to ECs. Using a zebrafish model, we finally showed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessel in embryos knock-out for the Cx40 orthologs Cx41.8 and Cx45.6. Most significant effects were observed in embryos with a mutant Cx41.8 encoding for a channel with reduced gap junctional function. Faster intersegmental vessel growth in Cx41.8 mutant embryos was associated with increased EC proliferation as assessed by PH3 immunostaining. Our data shows a novel evolutionary-conserved role of flow-driven KLF4-dependent Cx40 expression in endothelial quiescence that may be relevant for the control of atherosclerosis and diseases involving sprouting angiogenesis.

Exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP) induces vascular toxicity through Nrf2-VEGF pathway in zebrafish and human umbilical vein endothelial cells

The growing production and extensive use of organophosphate flame retardants (OPFRs) have led to an increase in their environmental distribution and human exposure. Developmental toxicity is a major concern of OPFRs' adverse health effects. However, the impact of OPFRs exposure on vascular development and the toxicity pathway for developmental defects are poorly understood. In this study, we investigated the effects of exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a frequently detected OPFR, on early vascular development, and the possible role of nuclear factor erythroid 2-related factor (Nrf2)-dependent angiogenic pathway in TDCPP's vascular toxicity. TDCPP exposure at 300 and 500 μg/L impeded the growth of intersegmental vessels (ISV), a type of microvessels, as early as 30 hpf. Consistently, a similar pattern of decreased extension and remodeling of common cardinal vein (CCV), a typical macrovessel, was observed in zebrafish at 48 hpf and 72 hpf. Developing vasculature in zebrafish was more sensitive than general developmental parameters to TDCPP exposure. The expression of genes related to VEGF signaling pathway dose-dependently decreased in TDCPP-treated larvae. In in vitro experiments using human umbilical vein endothelial cells (HUVECs), the increased cell proliferation induced by VEGF was suppressed by TDCPP exposure in a dose-dependent fashion. In addition, we found a repression of Nrf2 expression and activity in TDCPP-treated larvae and HUVECs. Strikingly, the application of CDDO-Im, a potent Nrf2 activator, enhanced VEGF and protected against defective vascular development in zebrafish. Our results reveal that vascular impairment is a sensitive index for early exposure to TDCPP, which could be considered in the environmental risk assessment of OPFRs. The identification of Nrf2-mediating VEGF pathway provides new insight into the adverse outcome pathway (AOP) of OPFRs.

The function of Nsdhl during vascular development in zebrafish

Genetic conrol of vascular development and patterning in vertebrates is not fully understood. We used zebrafish as a model organism to identify transcription factors Islet2 (Isl2) and Nr2f1b required for the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP). We further identify NAD(P)H steroid dehydrogenase‐like (Nsdhl) gene is positively regulated by Ist2 /Nr2f1b. Nsdhl is a dehydrogenase that participates in sterol production in cholesterol biosynthesis. Excess cellular cholesterol often leads to abnormal proliferation and migration. In addition, recent study showed cholesterol efflux related to angiogenesis control.Here, we show Nsdhl is highly conserved in all species. Nsdhl mRNA is expressed in vessels, suggesting its roles in vascular development. Knockdown of Nsdhl expression by Morpholino (MO) disrupts the growth of ISV and CVP, suggesting the role of Nsdhl in controlling ISV and CVP growth. Consist with the defects in vascular development, we examined the expression of vascular markers flk1, mrc1, stabilin, flt4 and ephrinb2 and we found the remodeling the expression of vascular markers in nsdhlMO. To address whether the cell death contributes to the ISV and CVP defects in nsdhlMO, we performed AO staining and TUNEL assay. The data showed that vascular defects do not caused by cell death, but likely due to the impairment of proliferation and migration. We also revealed the relationship between Nsdhl and VEGF/BMP signals. Together, we showed that Nsdhl plays important role for vascular development in zebrafish.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the roles of connexin 32 in zebrafish vascular development

The process of vascular development in vertebrates are mainly divided into vasculogenesis and angiogenesis. We previously identified the transcription factors Islet2 (isl2) and Nr2f1b required for the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP) in zebrafish. Microarray analysis showed Connexin 32 is regulated by Isl2/Nr2f1b. Gap junction protein Connexins32 (Cx32) has been shown functions on the blood vessels in injury mice and enhances angiogenesis in cell‐based study, however, there is no reference on the vascular development in animal study. In addition, signal pathways related to cx32 and angiogenesis have not yet been clarified. In this study, we first showed cx32 mRNA is expressed is expressed in developing vessels from 18S to 30 hpf stages, suggesting its roles in vasculature. Knockdown of cx32 by morpholino injection causes vascular defects in ISV and CVP, suggesting the role of cx32 in vascular growth. TUNEL assay showed that vascular defects do not caused by cell death, but likely due to the impairment of endothelial cell proliferation and migration. Consistent with vascular growth defects, loss of cx32 affects the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, the overexpression of cx32 does not impair the vascular growth, but rescue the cx32 morphant, suggesting the specificity of morpholino knockdown. Finally, we examine the interaction between cx32 and multiple signals and observed that cx32 is regulated by VEGF signals for ISV growth and BMP signals for CVP patterning.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fine-tune regulation of carboxypeptidase N1 controls vascular patterning during zebrafish development

Vascular development is regulated by complicated signals and molecules in vertebrates. In this study, we characterized a novel function of carboxypeptidase N1 (Cpn1) in the vasculature. We show that cpn1 mRNA is expressed in developing vessels. The knockdown of cpn1 by morpholino injection impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting the role of cpn1 in vascular development. We showed that vascular defects are not caused by cell death but are due to the impairment of migration and proliferation. Consistent with vascular growth defects, loss of cpn1 affects the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, the overexpression of cpn1 impaired the growth of ISV and CVP, but the remodeling expression of vascular markers was different from the knockdown of cpn1, indicating the differential regulation mechanisms in cpn1-overexpressing embryos. We examine the interaction between cpn1 and multiple signals and observed that cpn1 is regulated by Notch/VEGF signals for ISV growth and likely regulates BMP signals for CVP patterning. In conclusion, we demonstrate that cpn1 has a critical role in the vascular development of zebrafish. We also reveal a fine-tune regulation of cpn1 that controls vascular patterning mediated by multiple signals.

Ftr82 Is Critical for Vascular Patterning during Zebrafish Development.

Cellular components and signaling pathways are required for the proper growth of blood vessels. Here, we report for the first time that a teleost-specific gene ftr82 (finTRIM family, member 82) plays a critical role in vasculature during zebrafish development. To date, there has been no description of tripartite motif proteins (TRIM) in vascular development, and the role of ftr82 is unknown. In this study, we found that ftr82 mRNA is expressed during the development of vessels, and loss of ftr82 by morpholino (MO) knockdown impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting that ftr82 plays a critical role in promoting ISV and CVP growth. We showed the specificity of ftr82 MO by analyzing ftr82 expression products and expressing ftr82 mRNA to rescue ftr82 morphants. We further showed that the knockdown of ftr82 reduced ISV cell numbers, suggesting that the growth impairment of vessels is likely due to a decrease of cell proliferation and migration, but not cell death. In addition, loss of ftr82 affects the expression of vascular markers, which is consistent with the defect of vascular growth. Finally, we showed that ftr82 likely interacts with vascular endothelial growth factor (VEGF) and Notch signaling. Together, we identify teleost-specific ftr82 as a vascular gene that plays an important role for vascular development in zebrafish.

PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish.

Micro/nanoparticles could cause adverse effects on cardiovascular system and increase the risk for cardiovascular disease-related events. Nanoparticles prepared from poly(ethylene glycol) (PEG)-b-poly(ε-caprolactone) (PCL), namely PEG-b-PCL, a widely studied biodegradable copolymer, are promising carriers for the drug delivery systems. However, it is unknown whether polymeric PEG-b-PCL nano-micelles give rise to potential complications of the cardiovascular system. Zebrafish were used as an in vivo model to evaluate the effects of PEG-b-PCL nano-micelle on cardiovascular development. The results showed that PEG-b-PCL nano-micelle caused embryo mortality as well as embryonic and larval malformations in a dose-dependent manner. To determine PEG-b-PCL nano-micelle effects on embryonic angiogenesis, a critical process in zebrafish cardiovascular development, growth of intersegmental vessels (ISVs) and caudal vessels (CVs) in flk1-GFP transgenic zebrafish embryos using fluorescent stereomicroscopy were examined. The expression of fetal liver kinase 1 (flk1), an angiogenic factor, by real-time quantitative polymerase chain reaction (qPCR) and in situ whole-mount hybridization were also analyzed. PEG-b-PCL nano-micelle decreased growth of ISVs and CVs, as well as reduced flk1 expression in a concentration-dependent manner. Parallel to the inhibitory effects on angiogenesis, PEG-b-PCL nano-micelle exposure upregulated p53 pro-apoptotic pathway and induced cellular apoptosis in angiogenic regions by qPCR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay. This study further showed that inhibiting p53 activity, either by pharmacological inhibitor or RNA interference, could abrogate the apoptosis and angiogenic defects caused by PEG-b-PCL nano-micelles, indicating that PEG-b-PCL nano-micelle inhibits angiogenesis by activating p53-mediated apoptosis. This study indicates that polymeric PEG-b-PCL nano-micelle could pose potential hazards to cardiovascular development.

Development and characterization of functionalized self-assembling nanopeptide on the effect of angiogenesis for injured brain tissue regeneration

Event Abstract Back to Event Development and characterization of functionalized self-assembling nanopeptide on the effect of angiogenesis for injured brain tissue regeneration Kai-Chiech Chang1, Yung-Jen Chuang2, Wen-Han Chang3, Ming-Yuan Huang3 and Tzu-Wei Wang1 1 National Tsing Hua University, Department of Materials Science and Engineering, Taiwan 2 National Tsing Hua University, Department of Medical Science & Institute of Bioinformatics and Structural Biology, Taiwan 3 Mackay Memorial Hospital, Department of Emergency, Taiwan Introduction: Tissue and organ regeneration has been regarded as an optimal therapeutic strategy for traumatic brain injury. In this study, we proposed a three-dimensional (3D) hydrogel matrix to facilitate neoangiogenesis around damaged brain wound region[1]-[3]. A functionalized peptide, RADA16-SVVYGLR (AcN-RADARADARADARADASVVYGLR-CONH2), is designed, synthesized, and self-assembles into nanofibers network to form hydrogel with fiber entanglements[4]. The hydrogel with mechanical stiffness provides a suitable substrate for encapsulated neural stem cells adhesion and differentiation. The functional peptide, SVVYGLR, had been shown to regulate growth of hemopoietic stem cells and to induce angiogenesis[5],[6]. From previous in vitro and in vivo study, the hydrogel made of RADA16-SVVYGLR was compatible for cells proliferation in zebrafish embryos development. We expect that functionalized peptide hydrogel would create a favorable microenvironment for neural tissue repair and serve as a potential therapeutic application in traumatic brain damages. Method: Peptide solution: All aqueous peptide solutions were prepared by dissolving in Milli-Q water, stored at 4°C. Physiochemical properties analyses: Peptide secondary structure, nanofibers morphology and hydrogel rheology were analyzed by circular dichroism (CD), transmission electron microscopy (TEM), and rheometer, respectively. In vivo study: Zebrafish line (Tg(kdr:EGFP)) were used to perform zebrafish embryo toxicity test (zFET) including analysis of the survival rate and the development of intersegmental vessels (ISVs). Results and Discussion: The CD spectrum showed RADA16 and RADA16-SVVYGLR were in the form of beta sheet with minimum at 216 nm and maximum at 196 nm (Fig. 1). Morphology of both peptides were nanofibers network, developing scaffolds favorable to cellular processes and molecules transportation (Fig. 2). As the CD and TEM results, the self-assembling mechanism was not changed dramatically after linking the motif on the terminal of RADA16. The rheology of hydrogels showed that G’ was higher than G’’, indicating the gel-like property. (Fig. 3) The survival rate of fish embryos treated with peptide hydrogel was similar to that in the control group (Fig. 4). After 72 hours post-fertilization, the phenotypic traits of ISVs growth were identical in fish treated with hydrogel and that in control group, showing the normal physiological growth of ISVs to the DLAVs and PCVs. (Fig. 5) The zFET showed toxicity was not observed. Conclusion: The self-assembling peptides in our study, characterized as beta sheet secondary structure with angiogenic sequence, formed into a gel-like 3D scaffold with nanofibers network which was a suitable matrix for NSCs proliferation and a biocompatible biomaterial by zebrafish animal model. The hydrogel scaffold by functionalized self-assembling peptide has a potential role in clinical application to improve tissue regeneration. Dr. Wen-Han Chang; Dr. Ming-Yuan Huang; Prof. Yung-Jen Chuang; NTHU-Mackay Memorial Hospital Joint Research Program (103N2770E1); Research funding support of Ministry of Science and Technology, R.O.C. (104-2628-E-007-002-MY3)

Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development.

BackgroundThe specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein identity in mice. In this study, we show that nr2f1b is important for vein and tip cell identity during zebrafish development.ResultsNr2f1b mRNA is expressed in ventral lateral mesoderm at 15S stage and in vessels at 24 hpf consistent with a role in early vascular specification. Morpholino knockdown of nr2f1b results in a decrease in both vein cell number and expression of the vein specific marker flt4 and mrc1, suggested its role in venous specification. We also show loss of nr2f1b reduced ISV cell number and impairs ISV growth, which is likely due to the impairment of angiogenic cells migration and/or proliferation by time-lapse imaging. Consequently, nr2f1b morphants showed pericardial edema and circulation defects. Overexpression of nr2f1b under the fli promoter increases the number of venous cells and ISV endothelial cells indicated the function of nr2f1b is required and necessary for vascular development. We further showed that nr2f1b likely interact with Notch signalling. nr2f1b expression is increased in rbpsuh morphants and DAPT-treatment embryos suggested nr2f1b is negatively regulated by Notch activity.ConclusionsWe show nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development, which is mediated by Notch signalings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0209-0) contains supplementary material, which is available to authorized users.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.

Follistatin‐BMP Signaling Regulated by Isl2/CoupTFIb is Critical for Vascular Development

While many regulators and signaling molecules controlling the process of vascular development are in zebrfish, genetic regulating the vein identity, intersegmental vessels (ISV) and cardinal vein plexus (CVP) patterning is still not fully understood. We previously identified the transcription factors isl2/coupTFIb required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. Microarray analysis showed highly overlapping downstream targets controlled by isl2/coupTFIb, suggesting that cooperatively regulate vascular development. In this study, we analyze the functional relevance of one potential target from array hits, follistatin 1a (fst1a). Fst1a is a bone morphogenetic protein (BMP) antagonist related to axis formation during the development and shown involved in venous angiogenesis. However, no description of fst1a functioning in vascular so far. The interaction of notch and BMP signalings to regulate vasculization is unknown.We first examine amino acid sequence comparison and show fst1a is highly conserved among the vertebrates. Our in‐situ hybridization results showed fst1a is expressed in the lateral plate mesoderm and vessels. Overexpressing fst1a driven by fli1 promoter in zebrafish embryos, we observe the vascular defects in the growth of ISV and CVP. We further address the genetic interaction between fst1a and isl2, we found fst1 expression is increased in isl2 morphants by in‐situ hybridization. In addition, knockdown of fst1a can partial rescue the vascular defects in isl2 morphants. We suggest fst1a likely regulated by isl2. Since fst1 involved in BMP and isl2 functions downstream of notch, we conclude fst1a‐BMP signaling play an important role to regulate vascular patterning and interacts with isl2/coupTFIb‐Notch signaling.

Nuclear receptor subfamily 2 group F member 1a (nr2f1a) is required for vascular development in zebrafish.

Genetic regulators and signaling pathways are important for the formation of blood vessels. Transcription factors controlling vein identity, intersegmental vessels (ISV) growth and caudal vein plexus (CVP) formation in zebrafish are little understood as yet. Here, we show the importance of the nuclear receptor subfamily member 1A (nr2f1a) in zebrafish vascular development. Amino acid sequence alignment and phylogenetic analysis of nr2f1a is highly conserved among the vertebrates. Our in situ hybridization results showed nr2f1a mRNA is expressed in the lateral plate mesoderm at 18 somite stage and in vessels at 24–30 hpf, suggesting its roles in vasculization. Consistent with this morpholino-based knockdown of nr2fla impaired ISV growth and failed to develop fenestrated vascular structure in CVP, suggesting that nr2f1a has important roles in controlling ISV and CVP growth. Consequently, nr2f1a morphants showed pericardial edema and circulation defects. We further demonstrated reduced ISV cells and decreased CVP endothelial cells sprouting in nr2f1a morphants, indicating the growth impairment of ISV and CVP is due to a decrease of cell proliferation and migration, but not results from cell death in endothelial cells after morpholino knockdown. To test molecular mechanisms and signals that are associated with nr2f1a, we examined the expression of vascular markers. We found that a loss of nr2f1a results in a decreased expression of vein/ISV specific markers, flt4, mrc1, vascular markers stabilin and ephrinb2. This indicates the regulatory role of nr2f1a in controlling vascular development. We further showed that nr2f1a likely interact with Notch signaling by examining nr2f1a expression in rbpsuh morphants and DAPT-treatment embryos. Together, we show nr2f1a plays a critical role for vascular development in zebrafish.

The zebrafish Tie2 signaling controls tip cell behaviors and acts synergistically with Vegf pathway in developmental angiogenesis.

Angiogenesis process in development is temporally accurate, and involves sprouting, subsequent endothelial cell proliferation, and migration. Tip cells, sensing the extracellular cues, play an important role in this process. Although it is known that several pathways including vascular endothelial growth factor (VEGF) and Notch control tip cell behaviors, the signaling process is largely unknown. Here we showed that an endothelial tyrosine kinase receptor-Tie2 was required for intersegmental vessel (ISV) growth and essential for the sprouting, migration, and proliferation of tip cells with morpholino knockdown strategy in zebrafish. Knockdown of vegf effectively reduced tie2 mRNA level, and tie2 knockdown efficiently blocked the vegf over-expression induced tyrosine kinase receptor-VEGFR1 (flk1) expression, which suggested that the function of Tie2 may be linked to the downstream of VEGF signaling pathway. Furthermore, we found that the embryos displayed normal ISV growth when injected with tie2 or vegf morpholino alone at a low dose, while co-knockdown of them resulted in a severe ISV defect, indicating a synergistic role in ISV formation. These observations demonstrate that Tie2 is an important regulator of tip cell behaviors. Moreover, these findings provide in vivo evidence that Tie2 acts coordinately with Vegf signaling to control angiogenesis.

3D quantitative analyses of angiogenic sprout growth dynamics

Zebrafish intersegmental vessel (ISV) growth is widely used to study angiogenesis and to screen drugs and toxins that perturb angiogenesis. Most current ISV growth assays observe the presence or absence of ISVs or perturbation of ISV morphology but do not measure growth dynamics. We have developed a four-dimensional (4D, space plus time) quantitative analysis of angiogenic sprout growth dynamics for characterization of both normal and perturbed growth. We tracked the positions of the ISV base and tip for each ISV sprout in 4D. Despite immobilization, zebrafish embryos translocated globally and non-uniformly during development. We used displacement of the ISV base and the angle between the ISV and the dorsal aorta to correct for displacement and rotation during development. From corrected tip cell coordinates, we computed average ISV trajectories. We fitted a quadratic curve to the average ISV trajectories to produce a canonical ISV trajectory for each experimental group, arsenic treated and untreated. From the canonical ISV trajectories, we computed curvature, average directed migration speed and directionality. Canonical trajectories from treated (arsenic exposed) and untreated groups differed in curvature, average directed migration speed and angle between the ISV and dorsal aorta. 4D analysis of angiogenic sprout growth dynamics: (1) Allows quantitative assessment of ISV growth dynamics and perturbation, and (2) provides critical inputs for computational models of angiogenesis.

Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry for Identification of In Vitro and In Vivo Metabolites of Bornyl Gallate in Rats

Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution using methanol and water containing 0.2% (V : V) formic acid as the mobile phase. Metabolites identification involved analyzing the retention behaviors, changes of molecular weights and MS/MS fragment patterns of BG and the metabolites. Five compounds were identified as isomers of hydroxylated BG metabolites in vitro. The major metabolites of BG in rat urine and plasma proved to be BG-O-glucuronide and O-methyl BG-O-glucuronide. The proposed method confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of BG.