Somite morphogenesis is required for axial blood vessel formation during zebrafish embryogenesis.

Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate toward the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid-induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.

Zebrafish vascular quantification: a tool for quantification of three-dimensional zebrafish cerebrovascular architecture by automated image analysis.

Zebrafish transgenic lines and light sheet fluorescence microscopy allow in-depth insights into three-dimensional vascular development in vivo. However, quantification of the zebrafish cerebral vasculature in 3D remains highly challenging. Here, we describe and test an image analysis workflow for 3D quantification of the total or regional zebrafish brain vasculature, called zebrafish vasculature quantification (ZVQ). It provides the first landmark- or object-based vascular inter-sample registration of the zebrafish cerebral vasculature, producing population average maps allowing rapid assessment of intra- and inter-group vascular anatomy. ZVQ also extracts a range of quantitative vascular parameters from a user-specified region of interest, including volume, surface area, density, branching points, length, radius and complexity. Application of ZVQ to 13 experimental conditions, including embryonic development, pharmacological manipulations and morpholino-induced gene knockdown, shows that ZVQ is robust, allows extraction of biologically relevant information and quantification of vascular alteration, and can provide novel insights into vascular biology. To allow dissemination, the code for quantification, a graphical user interface and workflow documentation are provided. Together, ZVQ provides the first open-source quantitative approach to assess the 3D cerebrovascular architecture in zebrafish.

Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells.

Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

In-vivo 3D imaging of Zebrafish's intersegmental vessel development by a bi-directional light-sheet illumination microscope.

Precise quantification of vascular developments in Zebrafish requires continuous in-vivo 3D imaging. Here we employed a bi-directional light-sheet illumination microscope to characterize the development process of Zebrafish's intersegmental vessels. A Virtual Reality-based method was used to measure the lengths of intersegmental vessels (ISVs). The quantified growth rates of typical ISVs can be plotted, and unusual growth of some specific vessels was also observed.

Vandetanib versus Cabozantinib in Medullary Thyroid Carcinoma: A Focus on Anti-Angiogenic Effects in Zebrafish Model.

Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REarranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti-tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ-CRC-1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. In vivo, the anti-angiogenic potential of VAN and CAB is evaluated in Tg(fli1a:EGFP)y1 transgenic fluorescent zebrafish embryos by analyzing the effects on the physiological development of the sub-intestinal vein plexus and the tumor-induced angiogenesis after TT and MZ-CRC-1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ-CRC-1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ-CRC-1 and CAB in TT cells. Regarding zebrafish, both drugs inhibit angiogenesis in a dose-dependent manner, in particular CAB shows a more potent anti-angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti-angiogenic activity of CAB appears to be more relevant.

Zebrafish modeling mimics developmental phenotype of patients with RAPGEF1 mutation.

RAPGEF1 is a guanine nucleotide exchange factor responsible for transmitting extracellular signals to the Ras family of GTPase located at the inside of membrane. Here, we report for the first time a homozygous mutation of RAPGEF1 in a consanguineous family with two siblings affected by neuropsychiatric disorder. To confirm the correlation of the mutation and the phenotype, we utilized in silico analysis and established a zebrafish model. Survival rate was reduced in the rapgef1a-knockdown model, and the zebrafish showed global morphological abnormalities, particularly of brain and blood vessels. Co-application of human RAPGEF1 wildtype mRNA effectively rescued the abnormal phenotype, while that of RAPGEF1 mRNA carrying the human mutation did not. This work is the first report of a human Mendelian disease associated with RAPGEF1 and the first report of a zebrafish model built for this gene. The phenotype of zebrafish model provides further evidence that defective RAPGEF1 may lead to global developmental delay in human patients.

Biomass-derived cellulose nanoparticles display considerable neurotoxicity in zebrafish.

The widespread use of nanomaterials poses a great threat to human living environments. Among them, biomass-derived cellulose nanoparticle (CN) is one of the widely used nanomaterials. To date, the toxicity of CNs during embryonic development remains undetermined. In this study, we exposed zebrafish embryos to cellulose nanofibrils (CNFs) and cellulose nanocrystals (CNCs) to evaluate the toxicity of these CNs. Exposure to CNFs or CNCs below 30 mg/ml exhibited no dose-dependent increases in malformation and mortality in zebrafish embryos. Then we demonstrated that CNs were highly enriched in zebrafish embryo via imaging analyses of embryos treated with FITC-coupled CNCs. In addition, we found that CNF or CNC exposure resulted in compromised motor ability of zebrafish larva. Furthermore, it was revealed that the differentiation and the morphogenesis of motor neurons were significantly interrupted. While, blood vessels were normally patterned, suggesting the specific neurotoxicity of these nanomaterials. Transcriptome sequencing assay showed that the neurotoxicity of CNs in the motor neurons might be attributed to the expression alteration of neural genes. In summary, we discovered the neurotoxicity of CNs for the first time.

Morphometric evaluation of Japanese quail embryos and their extraembryonic vascular networks exposed to low-frequency magnetic field with two different intensities.

Animals developed or in an embryonic stage, are constantly subjected to magnetic pollution generated by electrical and electronic devices. Several researches have used the bird embryo as an experimental model to evaluate the action of magnetic field (MF) and electromagnetic field (EMF). This study proposed to perform a morphometric evaluation in the embryos and in the blood vascular network of the yolk sac membranes (YSM) of Japanese quail (Coturnix japonica) exposed to the 60 Hz MF with two different intensities (0.16 and 0.65 mT). A total of 30 eggs were used, 10 eggs were used for each assay. Each assay formed a group (control group, group submitted to the MF of 0.16 mT and 0.65 mT). The images of the skeletonized vascular network of YSM were evaluated by two methods of fractal dimension: box-counting dimension (Dbc) and information dimension (Dinf). The embryos were evaluated by body mass, percentage cephalic length and body area. The fractal dimensions revealed no difference among groups. There were no significant differences in relation to embryonic body mass among groups. However, the embryos exposed to 0.65 mT MF presented a smaller embryonic body development (body area and percentage cephalic length). In conclusion, 0.16 mT and 0.65 mT magnetic fields were not able to generate significant effects on vasculogenesis and angiogenesis. However, the embryos exposed to 6 h of magnetic field with 0.65 mT intensity and 60 Hz frequency showed a decrease in embryonic body development.

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.

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

PURPOSE: 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. METHODS: 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. RESULTS: Pergularia tomentosa markedly inhibited intersegmental vessel (ISV) formation at 48 h 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 40 µg/mL. PtR2 at 20 µg/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 ß-catenin protein expressions. Phytochemical profile of PtR2 showed a rich source of cardenolide structures, including ghalakinoside, calactin and calotropin derivatives. CONCLUSION: 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.

Attenuation of cadmium-induced vascular toxicity by pro-angiogenic nanorods.

Cadmium (Cd) is a common heavy metal that causes major environmental pollution with adverse effects on human health and well-being. Exposure to Cd is known to exhibit detrimental consequences on all the vital organ systems of the body, especially the vascular system. Certain approaches using anti-oxidants and chelating agents have been demonstrated previously to mitigate Cd-induced toxicity. However, these approaches are associated with their own limitations. In this context, there is a critical need for the development of alternative treatment strategies to address the conditions associated with Cd-poisoning. One such novel approach is the application of nanomedicine which is well-known to resolve several health complications by improving disease therapy. Recently, our group demonstrated the role of europium hydroxide nanorods (EHN) in promoting vascular growth using in vitro and in vivo assay systems. Therefore, in the present study, we have evaluated the effect of EHN on health of endothelial cells (EA.hy926) and fibroblasts (NIH 3T3) intoxicated by Cd. The results revealed that EHN significantly improved the viability of EA.hy926 and NIH 3T3 cells, reduced apoptotic cell population, increased nitric oxide (NO) production and promoted blood vasculature development in the chick embryo model, which were hampered due to Cd insult. Molecular studies demonstrated the reduced expression of tumor suppressor (p53) and elevated anti-apoptotic protein (Bcl-xL) levels along with enhanced NO production through endothelial nitric oxide synthase (eNOS) activation as the plausible mechanisms underlying protective role of EHN against Cd-induced vascular toxicity. Considering the above observations, we strongly believe that EHN could be a potential nanomedicine approach for overcoming Cd-induced toxicity by improving vascular health and functioning.

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.

Influence of a 1000 Times Weakened Magnetic Field on Embryogenesis and Ontogenesis of the Japanese Quail in Several Generations.

The paper presents experimental data on the influence of a 1000-fold weakening of the Earth's magnetic field on the embryonic and postembryonic development of the Japanese quail in three generations. It has been shown that the weakening of the earth's magnetic field by a factor of 1000 affects the formation of blood vessels in Japanese quail embryos, in particular, causing a decrease in angiogenesis in seven-day-old embryos of both the first generation (F1) and the next two ones (F2 and F3). Pathological and anatomical studies of embryos of different ages in three generations have revealed various pathologies associated with vascular system disorders, as well as disorders in the development of the beak and eyes. In the ontogenesis of F3 quails, there is a decrease in the hatchability of chicks.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Tongnao 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 STUDY: Recently, 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 METHODS: We 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. RESULTS: TND 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. CONCLUSION: Our 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.

AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo.

Poor bioavailability and limited efficacy are challenges associated with using andrographolide as a therapeutic agent. We recently synthesized AGS-30, a new andrographolide derivative, in our laboratory. In this study we investigated the potential anti-tumor effect of AGS-30 and the underlying mechanisms, particularly those related to angiogenesis. Results from our in vitro experiments showed that AGS-30 exerted anti-angiogenic effects by inhibiting endothelial cell proliferation, migration, invasion, and tube formation. Phosphorylation and activation of angiogenesis-related signaling molecules (e.g., vascular endothelial growth factor [VEGF] receptor 2, mitogen-activated protein kinase kinase 1/2, extracellular signal-regulated kinase 1/2, mechanistic target of rapamycin [mTOR], protein kinase B [Akt], and p38) were markedly reduced by AGS-30. Meanwhile, AGS-30 potently inhibited cell proliferation and phosphorylation of cell survival-related proteins (e.g., Akt, mTOR, and ERK1/2) and decreased the expression of VEGF in HT-29 colon cancer cells. AGS-30 blocked microvessel sprouting in a rat aortic ring model and blood vessel formation in zebrafish embryos and a mouse Matrigel plug model. Additionally, AGS-30 suppressed tumor growth and angiogenesis in HT-29 colon cancer cell xenografts in nude mice. These effects were not observed when same concentration of andrographolide, the parent compound of AGS-30, was used. Thus, AGS-30 exerted a strong antitumor effect by inhibiting tumor cell growth and angiogenesis and is a candidate compound for the treatment of cancer.

Toxic effects of flufenoxuron on development and vascular formation during zebrafish embryogenesis.

Flufenoxuron, a chitin synthesis inhibitor that is widely used in developed countries as an insecticide, is rarely degraded in the environment. In addition to that in insects, flufenoxuron-mediated non-targeted death in organisms such as lizards and bees has been reported. However, the toxic effects of this compound on vascular development during embryogenesis, as well as the underlying mechanism, have not yet been elucidated. In the present study, we assessed abnormal development and cardiovascular damage induced by flufenoxuron in zebrafish embryos. Exposed zebrafish had malformed eyes and pathological characteristics such as heart and yolk sac edema. In accordance with developmental inhibition, cell cycle regulatory genes were dysregulated in zebrafish embryos upon exposure to flufenoxuron. We also discovered that this agent can disrupt vascular formation by interfering with angiogenesis-associated genes including the genes encoding vascular endothelial growth factor Aa (vegfaa), vegfc, fms-related tyrosine kinase 1 (flt1), and flt4 in zebrafish embryos. These anti-angiogenic effects of flufenoxuron were further verified using a well-known angiogenesis model, namely human umbilical vein endothelial cells. In conclusion, our results suggest that flufenoxuron inhibits overall development and angiogenesis during embryogenesis.

Crinamine Induces Apoptosis and Inhibits Proliferation, Migration, and Angiogenesis in Cervical Cancer SiHa Cells.

Crinumasiaticum is a perennial herb widely distributed in many warmer regions, including Thailand, and is well-known for its medicinal and ornamental values. Crinum alkaloids contain numerous compounds, such as crinamine. Even though its mechanism of action is still unknown, crinamine was previously shown to possess anticancer activity. In this study, we demonstrate that crinamine was more cytotoxic to cervical cancer cells than normal cells. It also inhibited anchorage-independent tumor spheroid growth more effectively than existing chemotherapeutic drugs carboplatin and 5-fluorouracil or the CDK9 inhibitor FIT-039. Additionally, unlike cisplatin, crinamine induced apoptosis without promoting DNA double-strand breaks. It suppressed cervical cancer cell migration by inhibiting the expression of positive regulators of epithelial-mesenchymal transition SNAI1 and VIM. Importantly, crinamine also exerted anti-angiogenic activities by inhibiting secretion of VEGF-A protein in cervical cancer cells and blood vessel development in zebrafish embryos. Gene expression analysis revealed that its mechanism of action might be attributed, in part, to downregulation of cancer-related genes, such as AKT1, BCL2L1, CCND1, CDK4, PLK1, and RHOA. Our findings provide a first insight into crinamine's anticancer activity, highlighting its potential use as an alternative bioactive compound for cervical cancer chemoprevention and therapy.

Two Compounds Isolated From Ganglioside GM1 Promote Angiogenesis in Zebrafish.

Ganglioside has been implicated to play important roles in modulating various cell signaling and biological functions. However, the functional analysis of a single ganglioside in a zebrafish model is so far lacking. In this study, we investigated the angiogenic effects of 2 monosialoganglioside compounds isolated from GM1 in zebrafish embryos. First, we showed the tested compounds are adequate safe. Then, we found that these compounds exhibited significant proangiogenic effect through enhancement of endothelial cell proliferation, migration, and differentiation. Furthermore, the 2 compounds were proved to promote angiogenesis through, at least partially, modulating the level of Notch signaling. This study provides the novel insights into the clinical application of the 2 ganglioside compounds and GM1.

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.

A Reliable Preclinical Model to Study the Impact of Cigarette Smoke in Development and Disease.

The World Health Organization has estimated that, worldwide, cigarette smoking has caused more than 100 million deaths in the last century, a number that is expected to increase in the future. Understanding cigarette smoke toxicity is key for research and development of proper public health policies. The current challenge is to establish a reliable preclinical model to evaluate the effects of cigarette smoke. In this work, we describe a simple method that allows for quantifying the toxic effects of cigarette smoke using zebrafish. Here, viability of larvae and adult fish, as well as the effects of cigarette smoke extracts on vascular development and tissue regeneration, can be easily assayed. © 2019 by John Wiley & Sons, Inc.