Intersegmental Vessels Research Articles

The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development

Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.

Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2.

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

In vivo Screening of Natural Products Against Angiogenesis and Mechanisms of Anti-Angiogenic Activity of Deoxysappanone B 7,4'-Dimethyl Ether.

IntroductionThe aim of this study was to screen the leading compounds of natural origin with anti-angiogenic potential and to investigate their anti-angiogenic mechanism preliminarily.Materials and MethodsAn initial screening of 240 compounds from the Natural Products Collection of MicroSource was performed using the transgenic zebrafish strain Tg [fli1a: enhanced green fluorescent protein (EGFP)]y1. The zebrafish embryos at 24 h post-fertilization were exposed to the natural compounds for an additional 24 h; then, morphological changes in the intersegmental vessels (ISVs) were observed and quantified under a fluorescence microscope. The expression profiles of angiogenesis-related genes in the zebrafish embryos were detected using quantitative real-time PCR.ResultsFive compounds were identified with potential anti-angiogenic activity on the zebrafish embryogenesis. Among them, deoxysappanone B 7.4ʹ-dimethyl ether (Deox B 7,4) showed anti-angiogenic activity on the formation of ISVs in a dose-dependent manner. The inhibition of ISV formation reached up to 99.64% at 5 μM Deox B 7,4. The expression of delta-like ligand 4 (dll4), hes-related family basic helix-loop-helix transcription factor with YRPW motif 2, ephrin B2, fibroblast growth factor receptor (fgfr) 3, cyclooxygenase-2, protein tyrosine phosphatase, receptor type B (ptp-rb), phosphoinositide-3-kinase regulatory subunit 2, slit guidance ligand (slit) 2, slit3, roundabout guidance receptor (robo) 1, robo2, and robo4 were down-regulated, while vascular endothelial growth factor receptor-2, fgfr 1, and matrix metallopeptidase 9 were up-regulated in the zebrafish embryos treated with Deox B 7,4.ConclusionDeox B 7,4 has a therapeutic potential for the treatment of angiogenesis-dependent diseases and may exert anti-angiogenic activities by suppressing the slit2/robo1/2, slit3/robo4, cox2/ptp-rb/pik3r2, and dll4/hey2/efnb2a signaling pathways as well as activation of vegfr-2/fgfr1/mmp9.

Cardenolide-rich fraction of Pergularia tomentosa as a novel Antiangiogenic agent mainly targeting endothelial cell migration.

Angiogenesis related abnormalities underlie several life-threatening disorders. Despite approved therapies, scientists have yet to develop highly efficient, low cost approaches with minimal side effects. We evaluated the antiangiogenic activity of 50% hydroalcoholic extracts of Pergularia tomentosa L. root and aerial parts along with their EtOAc and water fractions, in vivo and in vitro. Transgenic zebrafish line Tg(fli1:EGFP) was used for in vivo assay and human umbilical vein endothelial cell (HUVEC) migration test along with possibility of tube formation were performed as in vitro tests. Furthermore, microvasculature in chicken chorioallantoic membrane (CAM) was assessed under P. tomentosa treatment. The fractionation of the 50% hydroalcoholic extracts was led to the identification of the best active fraction in this study. The metabolite profiling of the active fraction was also carried out using LC-HRESIMS analysis. Pergularia tomentosa markedly inhibited intersegmental vessel (ISV) formation at 48h post-fertilization (hpf) embryos in zebrafish. The water fraction of root hydroalcoholic extract (PtR2), showed strong antiangiogenic effect with minimal adverse viability impacts. Over 80% of embryos showed more than 50% inhibition in their ISV development at 20 and 40μg/mL. PtR2 at 20μg/mL substantially reduced human umbilical vein endothelial cell (HUVEC) migration up to 40%, considerable destruction of the formed tubes in the tube formation and microvasculature in CAM assays. Immunocytochemistry showed a marked reduction in vascular endothelial cadherin (VE-cadherin) abundance at cell junctions concurrent with substantial reduction of phospho-Akt (p-Akt) and β-catenin protein expressions. Phytochemical profile of PtR2 showed a rich source of cardenolide structures, including ghalakinoside, calactin and calotropin derivatives. Thus, the P. tomentosa cardenolide-rich fraction (PtR2) may hold a considerable promise for an antiangiogenic impact by impairment of endothelial cell (EC) migration and viability. Graphical abstract.

Cabozantinib, a Multityrosine Kinase Inhibitor of MET and VEGF Receptors Which Suppresses Mouse Laser-Induced Choroidal Neovascularization.

Choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries and is particularly associated with age-related macular degeneration (AMD). Cabozantinib (CBZ) hinders the activation of multiple receptor tyrosine kinases involved in tumor angiogenesis, such as hepatocyte growth factor receptor (MET) and vascular endothelial growth factor receptor 2 (VEGFR2). We aimed to investigate the role and mechanism of CBZ in a mouse laser-induced CNV model. In zebrafish embryos, CBZ perturbed intersegmental vessel (ISV) formation without obvious neurodevelopment impairment. In the mouse laser-induced CNV model, phosphorylated hepatocyte growth factor receptor (p-MET) and phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2) were increased in the CNV region. CBZ intravitreal injection or oral gavage alleviated CNV leakage and the CNV lesion area without obvious intraocular toxicity, as well as disturbed the phosphorylation of MET and VEGFR2. Additionally, CBZ downregulated the expression of the hepatocyte growth factor (HGF) with no effect on the expression of the vascular endothelial growth factor (VEGF). CBZ downregulated HGF, p-MET, and p-VEGFR2 expressions in vitro, as well as inhibited the proliferation, migration, and tube formation of b-End3 cells. In summary, CBZ alleviates mouse CNV formation possibly via inhibiting the activation of MET and VEGFR2. The findings provide a novel potential therapy method for CNV patients.

Novel technique for identification of the pulmonary intersegmental plane using manual jet ventilation during pulmonary segmentectomy.

IntroductionFor successful pulmonary segmentectomy, the identification of boundaries between segments is important. Previous measures include tracing the intersegmental vessels by staining with a dye via the affected pulmonary artery or bronchus and inflating with oxygen via a high frequency ventilator. However, problems with these methods have been reported.AimWe developed a novel method using a manual jet ventilator (MJV) and investigated its efficacy in identification of the pulmonary intersegmental plane.Material and methodsPatients underwent MJV for pulmonary segmentectomy in the period from January 2013 to December 2017 at our institution. The patients’ characteristics, resected segments, availability of clear resection planes, and complications associated with MJV from medical records were investigated. A questionnaire survey was conducted with the surgeons on the effectiveness of lung segment identification using MJV.ResultsOf 199 cases of planned pulmonary segmentectomy, 171 cases with descriptions of identified intersegmental planes were analyzed. Of these, 152 (89%) cases showed a clear boundary. There were 19 cases where the exact boundaries were not clearly identified, but segmentectomy was still performed. Furthermore, we found that identification of the right upper lobes was difficult (p = 0.0028). A subjective questionnaire was answered by the 12 surgeons who performed the procedures. All 12 responded that MJV was very effective or effective regarding clarity, safety, shorter identification time, and shorter resection time.ConclusionsMJV enabled surgeons to more easily and safely identify the pulmonary intersegmental plane, thereby suggesting that MJV has clinical significance during pulmonary segmentectomy.

Glycine exerts dose-dependent biphasic effects on vascular development of zebrafish embryos

Glycine, a non-essential amino acid, is involved in both angiogenesis and anti-angiogenesis. We hypothesized that glycine would exert dose-dependent different effects on angiogenesis. In this study, we investigated the effects of a broad range of concentrations of glycine on vascular development using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos. Effects of glycine transporter (GlyT) inhibitors (sarcosine and bitopertin) and a glycine receptor (GlyR) inhibitor (strychnine) were also examined in embryos in the absence or presence of glycine. After exposure to glycine and inhibitors, intersegmental vessels (ISVs) were observed by fluorescent microscopy. Low concentrations of glycine promoted the development of ISVs, whereas high concentrations reduced it. These effects of glycine could generally be reversed by treatment with GlyT and GlyR inhibitors. Furthermore, expressions of vascular endothelial growth factor (VEGF) (an angiogenic factor) and nitric oxide synthase (NOS) (an enzyme for nitric oxide synthesis) were associated with the dose-dependent effects of glycine. Our results suggest that glycine exerts dose-dependent biphasic effects on vascular development, which rely on GlyTs and GlyRs, and correlate with the expression of VEGF and NOS genes. At low concentrations, glycine acted as an angiogenic factor. In contrast, at high concentrations, glycine induced anti-angiogenesis. This evidence provides a novel insight into glycine as a unique target in angiogenic and anti-angiogenic therapy.

Pro-angiogenic activity of Tongnao decoction on HUVECs in vitro and zebrafish in vivo

Ethnopharmacological relevanceTongnao Decoction (TND) is a Chinese decoction approved and used in Jiangsu Province Hospital for the treatment of ischemic stroke. It shows conclusive efficiency in the improvement of neurologic impairment and activities of daily living of the patients. Aim of the studyRecently, angiogenesis has been recognized as a potential therapeutic strategy for treating cerebral ischemia. This study was aimed to provide comprehensive evidence for the pro-angiogenic effect of TND and characterize the underlying mechanism. Materials and methodsWe firstly established the chemical fingerprinting of TND. Then, the in vitro pro-angiogenic activities of TND were tested on human umbilical vein endothelial cells (HUVECs) through cell viability, wound healing and tube formation assays. The in vivo pro-angiogenic effects were evaluated on transgenic zebrafish embryos [Tg (fli-1: EGFP)] through the formation of intersegmental vessels (ISVs), subintestinal vessels (SIVs) and central arteries (CtAs). Lastly, the potential mechanisms of TND were analyzed by a blocking assay with eight pathways-specific kinase inhibitors. ResultsTND promoted the proliferation, migration and tube formation of HUVECs. TND also rescued the impairment of ISVs, SIVs and CtAs caused by VRI in a dose-dependent manner in zebrafish embryos. TND could activate vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K) – protein kinase B (Akt) and Raf – mitogen-activated protein kinase1/2 (MEK1/2) – extracellular regulated kinase 1/2 (ERK1/2) signaling pathways. ConclusionOur study firstly demonstrated the pro-angiogenic activities of TND. Our work provided evidences for the clinical usage of TND in restoring neurovascular function through promoting angiogenesis in the ischemic cerebral microvascular.

A systematical comparison of anti-angiogenesis and anti-cancer efficacy of ramucirumab, apatinib, regorafenib and cabozantinib in zebrafish model

AimsGastric cancer (GC) is one of the most common malignant tumors in the world. Anti-angiogenic therapy is a useful strategy for the treatment of advanced GC. This study was aimed to systemically compare the anti-angiogenesis, anti-cancer efficacy, as well as the safety of four known anti-angiogenic drugs, namely ramucirumab, apatinib, regorafenib and cabozantinib. Main methodsAnti-angiogenic effect was evaluated for the intersegmental vessels (ISVs) and subintestinal veins (SIVs) formation in the Tg (fli-1: EGFP) zebrafish embryos. Anti-cancer efficacy was tested for the in vivo cell proliferation in cell line derived tumor xenograft (CDX) model based on Tg (fli-1: EGFP) zebrafish embryos. Key findingsAll four drugs exhibited anti-angiogenic abilities and tumor inhibition effects in fli-1: EGFP transgenic zebrafish. Using zebrafish xenografted model, we found that effectiveness of ramucirumab in anti-GC-proliferation is better than apatinib, regorafenib and cabozantinib. The combination of anti-angiogenic drugs and cisplatin showed no significant benefit in tumors. Meanwhile, toxicity assay showed that all tested anti-angiogenic drugs could cause cardiovascular-related side effects. The therapeutic index (LD50/ED50) of cabozantinib is higher than apatinib and regorafenib, suggesting a potential as an anti-GC drug. SignificanceThe comparison of GC-related anti-angiogenic drugs was first reported. It was found that cabozantinib had a potential as an anti-GC drug. Zebrafish model was an ideal animal model for the research of anti-angiogenic behaviors.

Isosorbide mononitrate promotes angiogenesis in embryonic development of zebrafish

Coronary heart disease (CHD) is a leading cause of death worldwide, and angiogenesis plays important roles in CHD. Thus, in the present study, the angiogenic efficacy of four common cardiovascular medicines (aspirin, pravastatin, metoprolol and isosorbide mononitrate (ISMN)) was determined by the number and length of zebrafish intersegmental vessels (ISVs) after immersing zebrafish embryos in different medicines. Results showed that ISMN significantly increased the length and number of ISVs. ISMN is a long-acting nitrate ester drug. It has been used as a vasodilator to dilate arteries and veins to reduce the cardiac preload and postload. However, the effect of ISMN on angiogenesis remains unclear. Thus, by in vitro experiments, the angiogenic mechanism of ISMN was evaluated through detecting the viability and proliferation of human umbilical vein endothelial cells (HUVECs) and the expression of angiogenesis-related genes and miRNAs. Results indicated that ISMN could increase the viability and proliferation of HUVECs by decreasing apoptosis, and elevated the expressions of vedf, kdrl, pdgfr in zebrafish embryos. Furthermore, the expressions of miR-126, miR-130a and miR-210 were also regulated in ISMN-treated HUVECs. In conclusion, ISMN could promote angiogenesis in zebrafish embryos and HUVECs, implying ISMN may be a potential therapeutic in treating angiogenesis-related diseases.

AR-42: A Pan-HDAC Inhibitor with Antitumor and Antiangiogenic Activities in Esophageal Squamous Cell Carcinoma.

PurposeEsophageal squamous cell carcinoma (ESCC) is a refractory malignancy with high morbidity and mortality. Thus, there is an urgent need to find effective targets and agents for ESCC treatment. The purpose of this study was to assess the anti-ESCC effects of a pan-histone deacetylase (HDAC) inhibitor AR-42 and its mechanisms of action.MethodsImmunohistochemical staining was performed to detect HDAC1 expression in ESCC and adjacent tissue samples. MTT assay, Edu cell proliferation test, flow cytometry, and subcutaneous xenograft were used to assess the anti-ESCC effects of AR-42; furthermore, the antiangiogenic activity of AR-42 was evaluated using endothelial cell migration, invasion, and tube formation assays as well as zebrafish angiogenesis assay. Western blot analysis was performed to explore the underlying mechanism of the anti-ESCC activity of AR-42.ResultsHDAC1-positive expression was much higher in ESCC cells than in paracancerous tissues, and the elevated HDAC1 expression was a strong indicator of lymph node metastasis and a more advanced TNM stage of ESCC. Moreover, AR-42 potently suppressed ESCC cell growth through cellular proliferation inhibition and apoptosis induction. Moreover, AR-42 displayed a moderate antiangiogenic activity, and it could significantly inhibit the migration, invasion and tubulogenesis of human umbilical vein endothelial cells as well as intersegmental vessel formation in zebrafish at micromolar concentrations. More importantly, the inhibitory activity of AR-42 on ESCC cells and angiogenesis could also be observed in the TE-1 xenograft model. Further studies showed that AR-42 exerts its anti-ESCC effects mainly by upregulating the expression of p21 and blocking the transduction of multiple signaling cascades related to tumor growth, especially Stat3-mediated signaling.ConclusionOverall, AR-42 has significant potency for inhibiting ESCC cell growth and shows moderate effect in suppressing angiogenesis, displaying strong anti-ESCC effects in vitro and in vivo. Thus, AR-42 deserves further evaluation as a potential candidate for ESCC therapy.

Ibuprofen and diclofenac impair the cardiovascular development of zebrafish (Danio rerio) at low concentrations

The non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac are highly prescribed worldwide and their presence in aquatic system may pose a potential risk to aquatic organisms. Here, we systematically assessed their cardiovascular disruptive effects in zebrafish (Danio rerio) at environmentally relevant concentrations between 0.04 and 25.0 μg/L. Ibuprofen significantly increased the cardiac outputs of zebrafish embryos at actual concentrations of 0.91, 4.3 and 21.9 μg/L. It up-regulated the blood cell velocity, total blood flow and down-regulated the blood cell density at concentrations of 4.3 μg/L and higher. In comparison, diclofenac led to inhibition of spontaneous muscle contractions and decreased hatching rate of zebrafish embryos at the highest concentration (24.1 μg/L), while it had negligible effects on the cardiac physiology and hemodynamics. Transcriptional analysis of biomarker genes involved in cardiovascular physiology, such as the significantly up-regulated nppa and nkx2.5 expressions response to ibuprofen but not to diclofenac, is consistent with these observations. In addition, both ibuprofen and diclofenac altered the morphology of intersegmental vessels at high concentrations. Our results revealed unexpected cardiovascular functional alterations of NSAIDs to fish at environmental or slightly higher than surface water concentrations and thus provided novel insights into the understanding of their potential environmental risks.

Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo.

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5–2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5–4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Pro-angiogenesis effect and transcriptome profile of Shuxinyin formula in zebrafish

BackgroundAngiogenesis plays a critical role in ischemia disease like coronary heart disease. Shunxinyin formula has been developed for treating coronary heart disease according to the principle of traditional Chinese medicine while its underlying mechanism is not fully elucidated. PurposeHere, we hypothesize Shuxinyin formula could promote angiogenesis and microcirculation, and the underlying mechanism is also investigated. MethodsWe established the chemical profile of Shuxinyin (SXY) extract utilizing a UHPLC-Q/Exactive analysis system and evaluated its pro-angiogenesis effect in zebrafish model. The underlying mechanisms were investigated by combination of pharmacological experiments with transcriptome analysis in zebrafish. Zebrafish treated with VEGF was served as the positive control in present study. ResultsWe found SXY significantly enhanced the sub-intestinal vessel plexus (SIVs) growth in zebrafish. Co-treatment and post-treatment SXY attenuated VEGF receptor tyrosine kinase inhibitor II (VRI)-induced deficiency of intersegmental vessels (ISVs) in a concentration dependent manner. Post-treatment VEGF, which is a well-known angiogenesis driver, also partially ameliorated VRI-induced ISVs deficiency. In addition, SXY inhibited the down-regulation of VEGF receptors, including kdr, flt1 and kdrl, induced by VRI in zebrafish. The pro-angiogenesis effect of SXY on VRI-induced ISVs deficiency was suppressed by PI3K and JNK inhibitors, and Akt inhibitor abolished the pro-angiogenesis effect of SXY. The transcriptome profile of SXY preventing from VRI-induced vascular growth deficiency revealed that the underlying mechanisms were also co-related to cell junction, apoptosis and autophagy. ConclusionWe could conclude that SXY presented pro-angiogenesis effect and the action mechanisms were involved in VEGF/PI3K/Akt/MAPK signaling pathways, cell junction, apoptosis and autophagy.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

Developmental exposure to BDE-99 hinders cerebrovascular growth and disturbs vascular barrier formation in zebrafish larvae

Polybrominated diphenyl ethers (PBDEs) are distributed throughout the environment. Despite a moratorium on their use, concentrations of PBDEs in the atmosphere and in residential environments remain high due to their persistence. The environmental health risks remain concerning and one of the major adverse effects is neurodevelopmental toxicity. However, the early response and effects of PBDEs exposure on the developing brain remain unknown. In the present study, we investigated the impacts of 2,2′,4,4′,5-pentabrominated diphenyl ether (BDE-99) on vascular growth and vascular barrier function with an emphasis on cerebral blood vessels, in the early life stages, using a zebrafish model. No general toxicity was observed in exposing zebrafish larvae to 0-0.5 μM BDE-99 at 72 hpf. BDE-99 exposure resulted in neither general toxicity nor pronounced developmental impairment in somatic blood vessels, including intersegmental vessels (ISV) and common cardinal veins (CCV). Meanwhile, both 0.05 μM and 0.5 μM of BDE-99 reduced cerebrovascular density as well as down-regulation of VEGFA and VEGFR2 in the head. In addition, BDE-99 exposure increased vascular leakage, both in cerebral and truncal vasculature at 72 hpf. The accentuated vascular permeability was observed in the head. The mRNA levels of genes encoding tight junction molecules decreased in the BDE-99-exposed larvae, and more robust reductions in Cldn5, Zo1 and Jam were detected in the head than in the trunk. Moreover, proinflammatory factors including TNF-α, IL-1β and ICAM-1 were induced, and the expression of neurodevelopment-related genes was suppressed in the head following BDE-99 exposure. Taken together, these results reveal that developmental exposure to BDE-99 impedes cerebrovascular growth and disturbs vascular barrier formation. The cerebral vasculature in developing zebrafish, a more sensitive target for BDE-99, may be a promising tool for the assessment of the early neurodevelopmental effects due to PBDEs exposure.

Endoglin is a conserved regulator of vasculogenesis in zebrafish - implications for hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is a progressive vascular disease with high mortality and prevalence. There is no effective treatment of HHT due to the lack of comprehensive knowledge of its underlying pathological mechanisms. The majority of HHT1 patients carry endoglin (ENG) mutations. Here, we used Danio rerio (zebrafish) as an in vivo model to investigate the effects of endoglin knockdown on vascular development. According to phylogenetic analyses and amino acid sequence similarity analyses, we confirmed that endoglin is conserved in vertebrates and descended from a single common ancestor. Endoglin is highly expressed in the vasculature beginning at the segmentation period in zebrafish. Upon endoglin knockdown by morpholinos, we observed disruption in the intersegmental vessels (ISVs) and decreased expression of several vascular markers. RNA sequencing (RNA-Seq) results implied that the BMP-binding endothelial regulator (bmper) is a gene affected by endoglin knockdown. Rescue experiments demonstrated that overexpression of bmper significantly increased the number of endothelial cells (ECs) and reduced the defects at ISVs in zebrafish. Moreover, there was enhanced tube formation in ENG mutant ECs derived from a HHT patient after human recombinant BMPER (hrBMPER) stimulation. Taken together, our results suggest that bmper, a potential downstream gene of ENG, could be targeted to improve vascular integrity in HHT.

Clec14a genetically interacts with Etv2 and Vegf signaling during vasculogenesis and angiogenesis in zebrafish

BackgroundC-lectin family 14 Member A (Clec14a) is a transmembrane protein specifically expressed in vascular endothelial cells during embryogenesis. Previous in vitro and in vivo studies have provided conflicting data regarding Clec14a role in promoting or inhibiting angiogenesis, therefore its functional role in vascular development remains poorly understood.ResultsHere we have generated a novel clec14a mutant allele in zebrafish embryos using TALEN genome editing. clec14a mutant embryos exhibit partial defects and delay in the sprouting of intersegmental vessels. These defects in angiogenesis are greatly increased upon the knockdown of a structurally related C1qr protein. Furthermore, a partial knockdown of an ETS transcription factor Etv2 results in a synergistic effect with the clec14a mutation and inhibits expression of early vascular markers in endothelial progenitor cells, arguing that clec14a is involved in promoting vasculogenesis. In addition, Clec14a genetically interacts with Vegfa signaling. A partial knockdown of Vegfaa function in the clec14a mutant background resulted in a synergistic inhibition of intersegmental vessel sprouting.ConclusionsThese results argue that clec14a is involved in both vasculogenesis and angiogenesis, and suggest that Clec14a genetically interacts with Etv2 and Vegf signaling during vascular development in zebrafish embryos.

Study the function of GTPase related genes wasf1 and ect2 in zebrafish vascular development

Vascular development is important in vertebrates and regulated by complex signaling pathways. In the process of forming vessels are mainly divided into vasculogenesis and angiogenesis. Zebrafish is a powerful vertebrate model organism to study vascular development. Our previous study in zebrafish identified the transcription factors nr2f1b/islet2 required for blood vessels, mainly focus on the patterning of intersegmental vessels (ISV) and caudal vein plexus (CVP). The further microarray results showed that the expression of several GTPase related genes was regulated by nr2f1b/islet2, and we are interested in how those GTPase related genes contribute to vascular patterning in zebrafish.In this study, we reported two novel GTPase related genes wasf1 and ect2 that investigated their significance for the vascular development. In‐situ hybridization show that ect2 and wasf1 mRNA is expressed in developing vessels. Knockdown of ect2 and wasf1 by morpholino microinjection cause the defect in blood vessel formation. TUNEL assay and AO staining data show that vascular defects do not caused by cell death, but likely due to the impairment of proliferation and/or migration. Consistent with the defect in vascularture, loss of ect2 or wasf1 reduced the expression of the vascular markers. We further perform Western blotting to examine the relations between wasf1/ ect2 and major angiogentic VEGF/ BMP signals. We showed that GTPase related proteins wasf1 and ect2 play an important roles of vascular development mediated by VEGF/ BMP signalings in zebrafish.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pro-angiogenic activity of isoliquiritin on HUVECs in vitro and zebrafish in vivo through Raf/MEK signaling pathway

AimsLiquorice is a widely used herbal medicine for treating various diseases native to southern Europe and parts of Asia. Isoliquiritin (ISL), a licorice root-derived flavonoid, has been reported to exhibit antioxidant, anti-inflammatory, anti-genotoxic activity and anti-depression activities. This study was aimed to explore the pro-angiogenic activity of ISL and explicate the underlying mechanism. Main methodsIn vitro, ISL-treated human umbilical vein endothelial cells (HUVECs) were analyzed for cell viability, cell migration and tube formation. In vivo, pro-angiogenic effects were evaluated for the intersegmental vessels (ISVs) formation in transgenic zebrafish embryos [Tg(fli-1: EGFP)]. Furthermore, a blocking assay with eight pathways-specific kinase inhibitors were also used to determine the potential pro-angiogenic mechanism of ISL. Key findingsISL counteracted tyrosine kinase inhibitor II (VRI)-induced endothelial cell apoptosis and promoted cell migration and tube formation in HUVECs. ISL markedly rescued ISVs loss induced by VRI in zebrafish embryos, probably by activating vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K), Raf and mitogen-activated protein kinase (MEK)-dependent signaling pathways. SignificanceOur study first discovered and confirmed the pro-angiogenic activity of ISL both in HUVECs and zebrafish. Thus, ISL could be developed as a potential therapeutic agent by the role of pro-angiogenic activity for the treatment of cardiovascular diseases, cerebrovascular diseases and other vascular diseases.