Intersegmental Vessel Formation Research Articles

Exploring the anti-angiogenic properties of diosgenin saponin from Trigonella graecum (fenugreek): A study on Zebrafish embryos

Background: Angiogenesis plays a role in both physiological and pathological processes such as wound healing, embryonic development, and cancer metastasis, and it is the process by which new blood vessels are formed from existing ones. Diosgenin is a naturally occurring steroidal sapogenin found in plants such as yam and fenugreek, which is a compound of interest because of its pharmacological properties, including anti-carcinogenic effects. Zebrafish embryos are used due to their translucent nature, which facilitates the direct observation of vascular development. Aims and Objectives: The purpose of this study is to utilize zebrafish embryos to examine the impact of diosgenin on angiogenesis. It specifically aims to determine whether diosgenin could inhibit the development of inter-segmental vessels (ISV) in zebrafish embryos and also to investigate its effect on vascular endothelial growth factor (VEGF-A), a key regulator of angiogenesis. Materials and Methods: To explore the anti-angiogenic properties of diosgenin, we performed in vivo experiments using the zebrafish angiogenesis model. This involved staining red blood cells with O-dianisidine and conducting semiquantitative real-time polymerase chain reaction analysis to assess VEGF-A mRNA expression levels. Results: Diosgenin was shown to hinder the growth and formation of ISV in dosage-dependent development using a red blood cell staining assay when compared to the positive control, SU5416. In addition, there was a significant decrease in VEGF-A expression. Conclusion: In the presence of diosgenin, there is an evidence of inhibition of ISV development and suppression of VEGF mRNA expression. These findings underscore the therapeutic potential of diosgenin for targeting angiogenesis in cancer. However, further research is needed to understand the mechanism underlying diosgenin’s effect and its clinical relevance.

Investigation of Kelussia Odoratissima and Angelica Sinensis Similarities in Zebrafish-based In-vivo Bioactivity Assays and Their Chemical Composition.

Kelussia odoratissima and Angelica sinensis are two medicinal plants commonly used in Iran and China, respectively. They have been used in their indigenous traditional medicine, for various diseases including, blood refining, inflammation, cold, flu, stress, cardiovascular diseases, and nervous disorders. This study was conducted to evaluate the volatile oil composition of K. odoratissima leaves (KVL) and A. sinensis root (AVR); we also examined the biological activity of essential oils (EOs) and hydroalcoholic extracts of both plants using two different transgenic zebrafish (Danio rerio) models: angiogenesis and pancreatic beta cell (pBC) regeneration models. Both EOs were isolated by hydrodistillation and analysed by GC and GC/MS. For viability tests, larvae were treated with different concentrations of extracts to determine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a dose-dependent manner for their biological activity using tissue-specific transgenic zebrafish Tg(fli-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups. Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7% of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that both EOs blocked ISVs formation in the Tg (fli-1: EGFP) embryos increased to 10% of the control value, while both hydroalcoholic extracts did not show any anti-angiogenesis effects in these embryos. In addition, AVR has been shown to significantly induce PBC regeneration following ablation in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5'-N-ethylcarboxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity of both EOs could be attributed to the phthalide structures while for the PBC regenerative activity, other compounds including β-Thujaplicinol, exclusively existing in AVR, might be effective. Although the genera, organs, and origin of these plants are different, their similar chemical composition and biological activities make them valuable resources for further investigation in basic medical and pharmaceutical science.

Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Sinularin Induces Oxidative Stress-Mediated Apoptosis and Mitochondrial Dysfunction, and Inhibits Angiogenesis in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a cancer of largely unknown cause that leads to a 5-year survival rate of approximately 7% in the United States. Current treatment strategies are not effective, indicating a strong need for the development of novel therapies. In this study, the outcomes of sinularin, a marine-derived product, were evaluated against GBM. Our cellular studies using GBM cells revealed that sinularin induces cell death. The measured half maximal inhibitory concentrations (IC50) values ranged from 30 to 6 μM at 24–72 h. Cell death was induced via the generation of ROS leading to mitochondria-mediated apoptosis. This was evidenced by annexin V/propidium iodine staining and an upregulation of cleaved forms of the pro-apoptotic proteins caspase 9, 3, and PARP, and supported by CellROXTM Green, MitoSOXTM Red, and CM-H2DCFDA staining methods. In addition, we observed a downregulation of the antioxidant enzymes SOD1/2 and thioredoxin. Upon treatment with sinularin at the ~IC50 concentration, mitochondrial respiration capacities were significantly reduced, as shown by measuring the oxygen consumption rates and enzymatic complexes of oxidative phosphorylation. Intriguingly, sinularin significantly inhibited indicators of angiogenesis such as vessel tube formation, cell migration, and cell mobility in human umbilical vein endothelial cells or the fusion cell line EA.Hy926. Lastly, in a transgenic zebrafish model, intersegmental vessel formation was also significantly inhibited by sinularin treatment. These findings indicate that sinularin exerts anti-brain cancer properties that include apoptosis induction but also antiangiogenesis.

Temporal-spatial low shear stress induces heterogenous distribution of hematopoietic stem cell budding in zebrafish.

Hematopoietic stem/progenitor cells (HSPCs) originate from endothelial cells (ECs) localized on the ventral side of the dorsal aorta (DA), and hemodynamic parameters may suffer sharp changes in DA at HSPCs development stage for intersegmental vessel formation. However, the temporal-spatial shear stress parameters and biomechanics mechanisms of HSPC budding remain unknown. Here, we found that the hematopoietic endothelium (HE) in the aorta-gonad-mesonephros was heterogeneous; that is, HEs were mainly distributed at the ventral side of the vascular bifurcation in zebrafish embryos, which was found to show low shear stress (LSS) through numerical simulation analysis. Furthermore, HSPCs localized in the posterior somite of aorta-gonad-mesonephros with slow velocity. On the temporal scale, there was a slow velocity and LSS during HE budding from 36h post-fertilization and decreased shear stress with drug expanded HSPC numbers. Mechanistically, matrix metalloproteinase (MMP) expression and macrophage chemotaxis were significantly increased in HEs by RNA-seq. After treatment with an MMP13 inhibitor, HSPCs were significantly reduced in both the aorta-gonad-mesonephros and caudal hematopoietic tissue in embryos. Our results show that HSPC budding is heterogeneous, and the mechanism is that physiological LSS controls the emergence of HSPCs by promoting the accumulation of macrophages and subsequent MMP expression.

Marine Bromophenol Bis(2,3,6-Tribromo-4,5-Dihydroxybenzyl)ether Inhibits Angiogenesis in Human Umbilical Vein Endothelial Cells and Reduces Vasculogenic Mimicry in Human Lung Cancer A549 Cells.

Angiogenesis, including the growth of new capillary blood vessels from existing ones and the malignant tumors cells formed vasculogenic mimicry, is quite important for the tumor metastasis. Anti-angiogenesis is one of the significant therapies in tumor treatment, while the clinical angiogenesis inhibitors usually exhibit endothelial cells dysfunction and drug resistance. Bis(2,3,6-tribromo-4,5-dihydroxybenzyl)ether (BTDE), a marine algae-derived bromophenol compound, has shown various biological activities, however, its anti-angiogenesis function remains unknown. The present study illustrated that BTDE had anti-angiogenesis effect in vitro through inhibiting human umbilical vein endothelial cells migration, invasion, tube formation, and the activity of matrix metalloproteinases 9 (MMP9), and in vivo BTDE also blocked intersegmental vessel formation in zebrafish embryos. Moreover, BTDE inhibited the migration, invasion, and vasculogenic mimicry formation of lung cancer cell A549. All these results indicated that BTDE could be used as a potential candidate in anti-angiogenesis for the treatment of cancer.

Identification of de novo EP300 and PLAU variants in a patient with Rubinstein\u2013Taybi syndrome-related arterial vasculopathy and skeletal anomaly

Rubinstein–Taybi syndrome (RSTS) is a human genetic disorder characterized by distinctive craniofacial features, broad thumbs and halluces, and intellectual disability. Mutations in the CREB binding protein (CREBBP) and E1A binding protein p300 (EP300) are the known causes of RSTS disease. EP300 regulates transcription via chromatin remodeling and plays an important role in cell proliferation and differentiation. Plasminogen activator, urokinase (PLAU) encodes a serine protease that converts plasminogen to plasmin and is involved in several biological processes such as the proteolysis of extracellular matrix-remodeling proteins and the promotion of vascular permeability and angiogenesis. Recently, we discovered a patient who presented with RSTS-related skeletal anomaly and peripheral arterial vasculopathy. To investigate the genetic cause of the disease, we performed trio whole genome sequencing of the genomic DNA from the proband and the proband’s parents. We identified two de novo variants coined c.1760T>G (p.Leu587Arg) and c.664G>A (p.Ala222Thr) in EP300 and PLAU, respectively. Furthermore, functional loss of EP300a and PLAUb in zebrafish synergistically affected the intersegmental vessel formation and resulted in the vascular occlusion phenotype. Therefore, we hypothesize that the de novo EP300 variant may have caused RSTS, while both the identified EP300 and PLAU variants may have contributed to the patient’s vascular phenotype.

Evaluation of Toxicity and Antiangiogenic Activity of Murraya koenigii Leaf Extracts in Zebrafish

Abstract Introduction This study aimed to evaluate the toxicity and the antiangiogenic activity of Murraya koenigii leaf extracts in zebrafish. Materials and Methods Crude aqueous and ethanol extracts of M. koenigii leaves were obtained following a standard solvent-based extraction method. The toxicity of these extracts on zebrafish embryonic development was determined and the LC50 values were calculated. Finally, the antiangiogenic activity of the M. koenigii leaf extracts was evaluated in a transgenic zebrafish (Tg [fli1: GFP]) that express GFP under the control of fli1, a transcription factor involved in vasculogenesis. Results The LC50 value of the aqueous extract of M. koenigii leaves was much larger (2,450 ppm) compared with that of ethanol extract (9.85 ppm), suggesting a lower toxicity of the aqueous extract. The antiangiogenic activity was evident only in aqueous extract, with significant effects in formation of intersegmental vessels and subintestinal veins in treated Tg (fli1: GFP) zebrafish embryos. Conclusion This study demonstrated the biological activity of M. koenigii leaf extracts as angiogenesis suppressor, suggesting its therapeutic potential as an angiogenesis inhibitor. Identification of active components from natural sources with potent antiangiogenic activity could lead to the development of anticancer agents.

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.

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.

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.

Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Higher Anti-angiogenesis Activity, Better Cellular Uptake and Longer Half-life of a Novel Glyco-modified Endostatin by Polysulfated Heparin.

Endostatin (ES) is a promising anti-angiogenesis protein and has been approved for the treatment of non-small cell lung cancer, but short half-life, poor stability and nonspecific delivery caused great pain to patients and produced unsatisfactory treatment effectiveness. In this work, in order to overcome these disadvantages, ES was covalently modified by polysulfated heparin (PSH) with the expectancy of longer half-life, higher anti-angiogenesis activity and better cellular uptake. To characterize the cellular uptake, flow cytometry and confocal laser scanning microscopy were used to study the intracellular localization of fluorescein isothiocyanate-labeled ES and PSH-ES in EAhy926 endothelial cells. Zebrafish model was used to study the anti-angiogenesis activities of ES and its derivatives in vivo. The 125I-radiolabeled ES and PSH-ES were administered to healthy BALC/c mice for the pharmacokinetics study. Compared with ES, better cellular uptake effects were detected in PSH-ES group. Both ES and PSH-ES showed inhibition on the intersegmental vessels formation, while PSH-ES displayed a higher one. The half-life of PSH-ES was lengthened and area under the curve (AUC) was increased. At the same time, ES and PSH-ES were both widely and rapidly distributed in the lungs, livers, kidneys and hearts with little difference. The results indicated that PSH displayed good properties as a novel glyco-modifier for protein and peptide. The results also showed that PSH-ES displayed better cellular uptake, higher antiangiogenesis activity and prolonged half-life, which would lead to better anti-tumour effects.

Identification of mundoserone by zebrafish in vivo screening as a natural product with anti-angiogenic activity.

The present study aimed to screen natural products with anti-angiogenic potential from the Natural Products Collection of MicroSource. The anti-angiogenic activity of 240 natural products was assessed using the zebrafish line Tg(fli1a: EGFP)y1. At 24 h post-fertilization, the embryos were treated with the library compounds for 24 h and, the morphology of the intersegmental vessels (ISVs) was then assessed using a fluorescence microscope, followed by counting of ISVs and calculation of the inhibition ratio. The expression of angiogenesis-associated genes was determined by quantitative polymerase chain reaction. The results indicated that mundoserone inhibited ISV formation in zebrafish embryos in a dose-dependent manner, with a significant anti-angiogenic activity observed at a concentration of 10 µM, leading to an ISV inhibition ratio of 73.6±1.3%. Mundoserone significantly reduced the expression of slit guidance ligand 3 (SLIT3), roundabout guidance receptor 1 (ROBO1) and −2, fibroblast growth factor receptor (FGFR)2 and −3, as well as protein tyrosine phosphatase, receptor type B (PTP-RB), but increased the expression of NOTCH1A. Accordingly, mundoserone may be an effective angiogenic inhibitor, which acts via downregulation of SLIT/ROBO1 and FGFR/PTP-RB, and upregulation of NOTCH1A signaling.

Abstract 15: PA28γ contributes to tumor angiogenesis through regulation of IL-6 and CCL2 in oral squamous cell carcinoma

Abstract Background: PA28γ has been shown to promote tumor development and progression. Although PA28γ has been suggested to play a role in tumor angiogenesis, direct evidence for the molecular mechanisms underlying this phenomenon have not been investigated. Methods: Oral squamous cell carcinoma (OSCC) stable cell lines with varying levels of PA28γ expression were constructed. EA.hy926 human endothelial cells were co-cultured with these lines to evaluate the influence of PA28γ on EA.hy926 cell proliferation, migration, invasion, and tube formation. Using a subcutaneous xenotransplantation tumor model of under-expressing PA28γ HSC-3 cells, we explored effects on tumor angiogenesis in nude mice. We performed screens of the different angiogenesis-related genes and cytokines being expressed using PCR and cytokine chips to identify candidates with the potential to affect tumor angiogenesis. Then, candidates were verified using Western blot, and neutralization tests immunohistochemistry. Finally human OSCC clinical cohort was used to validate. Results: PA28γ could enhance the ability of OSCC cells to interact with endothelial cells, thereby recruit endothelial cells and induce angiogenesis. Ablation of PA28γ inhibits tumor-induced angiogenesis in xenografts mice model in vivo. In vivo zebrafish model experiments showed that depleted PA28γ remarkably blocked the formation of intersegmental vessels in zebrafish embryos. Furthermore, Angiogenesis-related PCR, cytokine chips and Western blot analysis indicated that PA28γ through regulate IL-6 and CCL2 expression, thereby subsequent activation of the Stat3/VEGF and PI3K/MMP-1 axis to promote angiogenesis. Neutralization or block results IL-6 and CCL2 has an essential role in PA28γ-controlled promotes of angiogenesis. In addition, clinical OSCC cohort validate that PA28γ positively correlates with IL-6 or CCL2 expression. Conclusion: Collectively, our data show that PA28γ contributes to the tumor angiogenesis, by regulating IL-6 and CCL2. PA28γ thus represents a novel therapeutic target for treating PA28γ-positive OSCC cancers. Citation Format: Xin Zeng, Jing Li, Jiajia Liu, Ning Ji, Hui Feng, Qianming Chen. PA28γ contributes to tumor angiogenesis through regulation of IL-6 and CCL2 in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 15.

Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress.

Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Moderate activation of UPR is required for maintaining endoplasmic reticulum (ER) homeostasis and profoundly contributes to tumorigenesis. Defects in UPR signaling are implicated in the attenuation of various malignant phenotypes including cell proliferation, migration, and invasion, as well as angiogenesis. This suggests UPR as a promising target in cancer therapy. The pharmacological effects of the plant Scindapsus cf. hederaceus on human cancer cell lines is not understood. In this study, we identified an ethyl acetate extract from Scindapsus cf. hederaceus (SH-EAE), which markedly altered the protein expression of UPR-related genes in human non-small cell lung cancer (NSCLC) cells. Treatment with the SH-EAE led to the dose-dependent suppression of colony forming ability of both H1299 and H460 cells, but not markedly in normal bronchial epithelial BEAS-2B cells. SH-EAE treatment also attenuated the migration and invasion ability of H1299 and H460 cells. Moreover, SH-EAE strikingly suppressed the protein expression of two ER stress sensors, including inositol requiring enzyme-1α (IRE-1α) and protein kinase R-like ER kinase (PERK), and antagonized the induction of C/EBP homologous protein (CHOP) expression by thapsigargin, an ER stress inducer. SH-EAE induced the formation of massive vacuoles which are probably derived from ER. Importantly, SH-EAE impaired the formation of intersegmental vessels (ISV) in zebrafish larvae, an index of angiogenesis, but had no apparent effect on the rate of larval development. Together, our findings demonstrate, for the first time, that the ability of SH-EAE specifically targets the two sensors of UPR, with significant anti-proliferation and anti-migration activities as a crude extract in human NSCLC cells. Our finding also indicates potential applications of SH-EAE in preventing UPR activation in response to Tg-induced ER stress. We suggest that SH-EAE attenuates UPR adaptive pathways for rendering the NSCLC cells intolerant to ER stress.

Anti-angiogenic activity of para-coumaric acid methyl ester on HUVECs in vitro and zebrafish in vivo

BackgroundPara-coumaric acid methyl ester (pCAME) is one of the bioactive components of Costus speciosus (Koen) Sm. (Zingiberaceae). This plant is traditionally used in Asia to treat catarrhal fevers, worms, dyspepsia, and skin diseases. PurposeTo investigate the anti-angiogenic activity of pCAME and its molecular mechanism of action. Study designWe investigated the anti-angiogenic activity of pCAME on human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish (Danio rerio) in vivo. MethodsIn vitro cell proliferation, would healing, migration and tube formation assays were used, along with in vivo physiological angiogenic vessel formation, tumor-induced angiogenic vessel formation assays on zebrafish model. qRT-PCR and RNA-seq were also used for the target investigation. ResultspCAME could inhibit the proliferation, would healing, migration and tube formation of HUVECs, disrupt the physiological formation of intersegmental vessels (ISVs) and the subintestinal vessels (SIVs) of zebrafish embryos, and inhibit tumor angiogenesis in the zebrafish cell-line derived xenograft (zCDX) model of SGC-7901 in a dose-dependent manner. Mechanistic studies revealed that pCAME inhibited vegf/vegfr2 and ang/tie signaling pathways in zebrafish by quantitative RT-PCR analysis, and regulated multi-signaling pathways involving immune, inflammation and angiogenesis in SGC-7901 zCDX model by RNA-seq analysis. ConclusionpCAME may be a multi-target anti-angiogenic drug candidate and hold great potential for developing novel therapeutic strategy for cancer treatment.

Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo.

Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angio-genesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear.MethodsIn this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using Tg(fli:EGFP) transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined.ResultsWe found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling.ConclusionFor the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research.

Ponatinib exerts anti-angiogenic effects in the zebrafish and human umbilical vein endothelial cells via blocking VEGFR signaling pathway.

Angiogenesis is a hallmark for cancer development because it is essential for cancer growth and provides the route for cancer cell migration (metastasis). Understanding the mechanism of angiogenesis and developing drugs that target the process has therefore been a major focus for research on cancer therapy. In this study, we screened 114 FDA-approved anti-cancer drugs for their effects on angiogenesis in the zebrafish. Among those with positive effects, we chose to focus on Ponatinib (AP24534; Iclusig®) for further investigation. Ponatinib is an inhibitor of the tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), and its clinical trial has been approved by FDA for the treatment of the disease. In recent clinical trials, however, some side effects have been reported for Ponatinib, mostly on blood vessel disorders, raising the possibility that this drug may influence angiogenesis. In this study, we demonstrated that Ponatinib was able to suppress the formation of intersegmental vessels (ISV) and subintestinal vessels (SIV) in the zebrafish larvae. The anti-angiogenic effect of Ponatinib was further validated by other bioassays in human umbilical vein endothelial cells (HUVECs), including cell proliferation and migration, tube formation, and wound healing. Further experiments showed that Ponatinib inhibited VEGF-induced VEGFR2 phosphorylation and its downstream signaling pathways including Akt/eNOS/NO pathway and MAPK pathways (ERK and p38MAPK). Taken together, these results suggest that inhibition of VEGF signaling at its receptor level and downstream pathways may likely be responsible for the antiangiogenic activity of Ponatinib.

Lmo2 (LIM-Domain-Only 2) Modulates Sphk1 (Sphingosine Kinase) and Promotes Endothelial Cell Migration

Lmo (LIM-domain-only)2 transcription factor is involved in hematopoiesis and vascular remodeling. Sphk (sphingosine kinase)1 phosphorylates sphingosine to S1P (sphingosine-1-phosphate). We hypothesized that Lmo2 regulates Sphk1 to promote endothelial cell (EC) migration and vascular development. APPROACH AND RESULTS: Lmo2 and Sphk1 knockdown (KD) were performed in Tg(fli1:EGFP) y1 zebrafish and in human umbilical vein EC. Rescue of phenotypes or overexpression of these factors were achieved using mRNA encoding Lmo2 or Sphk1. EC proliferation in vivo was assessed by BrdU (bromodeoxyuridine) immunostaining and fluorescence-activated cell sorter analysis of dissociated Tg(fli1:EGFP) y1 embryos. Cell migration was assessed by scratch assay in human umbilical vein EC and mouse aortic rings. Lmo2 interactions with Sphk1 promoter were assessed by ChIP-PCR (chromatin immunoprecipitation-polymerase chain reaction). Lmo2 or Sphk1 KD reduced number and length of intersegmental vessels. There was no reduction in the numbers of GFP+ (green fluorescent protein) ECs after Lmo2 KD. However, reduced numbers of BrdU+GFP+ nuclei were observed along the dysmorphic intersegmental vessels, accumulating instead at the sprouting origin of the intersegmental vessels. This anomaly was likely because of impaired EC migration, which was confirmed in migration assays using Lmo2 KD human umbilical vein ECs and mouse aortic rings. Both in vivo and in vitro, Lmo2 KD reduced Sphk1 gene expression, associated with less Lmo2 binding to the Sphk1 promoter as assessed by ChIP-PCR. Sphk1 mRNA rescued the Lmo2 KD phenotype. Our data showed that Lmo2 is necessary for Sphk1 gene expression in ECs. Lmo2 KD reduced Lmo2-Sphk1 gene interaction, impaired intersegmental vessels formation, and reduced cell migration. We identified for the first time Sphk1 as downstream effector of Lmo2.