Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.     

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.     

Zebrafish models to study hypoxia-induced pathological angiogenesis in malignant and nonmalignant diseases

Most in vivo preclinical disease models are based on mouse and other mammalian systems. However, these rodent-based model systems have considerable limitations to recapitulate clinical situations in human patients. Zebrafish have been widely used to study embryonic development, behavior, tissue regeneration, and genetic defects. Additionally, zebrafish also provides an opportunity to screen chemical compounds that target a specific cell population for drug development. Owing to the availability of various genetically manipulated strains of zebrafish, immune privilege during early embryonic development, transparency of the embryos, and easy and precise setup of hypoxia equipment, we have developed several disease models in both embryonic and adult zebrafish, focusing on studying the role of angiogenesis in pathological settings. These zebrafish disease models are complementary to the existing mouse models, allowing us to study clinically relevant processes in cancer and nonmalignant diseases, which otherwise would be difficult to study in mice. For example, dissemination and invasion of single human or mouse tumor cells from the primary site in association with tumor angiogenesis can be studied under normoxia or hypoxia in zebrafish embryos. Hypoxia-induced retinopathy in the adult zebrafish recapitulates the clinical situation of retinopathy development in diabetic patients or age-related macular degeneration. These zebrafish disease models offer exciting opportunities to understand the mechanisms of disease development, progression, and development of more effective drugs for therapeutic intervention.     

Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.     

Characterization of vascular mural cells during zebrafish development

Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origin and development of vascular MCs has been hampered by difficulties in observing these cells in vivo and performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA) and anterior mesenteric arteries (AMA) of developing animals, and that they also contribute to the outflow tract of the developing heart and ventral aorta (VA). Genetic data indicate that the vascular MCs of the LDA and AMA do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. We further show that zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of vascular smooth muscle cells and pericytes found in higher vertebrates. These data establish the zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MC-associated diseases.     

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2^E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.     

Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.     

Alteration of developmental and pathological retinal angiogenesis in angptl4-deficient mice

Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.     

慢性低氧所致肺动脉高压对大鼠肺血管平滑肌细胞及成纤维细胞中蛋白激酶CβI的膜转位和蛋白表达量的影响

目的通过观察慢性低氧所致肺动脉高压对大鼠肺血管平滑肌细胞及成纤维细胞中蛋白激酶CBI（PKCβI）的膜转位和蛋白表达量的影响,初步探讨PKCpI在慢性低氧诱导大鼠肺动脉高压的发生、发展过程中所起的作用。方法建立慢性常压低氧肺动脉高压大鼠模型,将雄性SD大鼠随机分为正常对照组、低氧1d、3d、7d、14d和21d组,应用蛋白免疫印迹和免疫组化技术检测肺动脉高压形成过程中大鼠肺血管平滑肌细胞及成纤维细胞中PKCβI的膜转位和蛋白表达水平。结果（1）RVSP和RV／（LV＋S）比值较正常对照组明显增加（P〈0．05）,低氧后3d、7d、14d和21d后大鼠肺血管明显增厚;（2）大鼠肺血管平滑肌细胞和成纤维细胞均有PKCβI的表达,且低氧14d后PKCβI的蛋白表达量较正常对照组相比降低（P〈0．05）。结论PKCβI蛋白表达量的下调可能参与了慢性低氧诱导的大鼠肺动脉高压肺血管重塑的发生、发展过程。     

Hypoxia-induced metastasis model in embryonic zebrafish

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.     

In vivo monitoring the dynamic process of acute retinal hemorrhage and repair in zebrafish with spectral‐domain optical coherence tomography

Retina, the only light sensor in the human eye, is hidden and extremely fragile. Optimized animal models and efficient imaging techniques are very important for the study of retinopathy. In this work, the rapid retinal injury process and the long‐term retinal repair process were in vivo continuously evaluated with a novel imaging technology spectral‐domain optical coherence tomography (SD‐OCT) in a unique animal model zebrafish. Acute retinal injury was constructed on adult zebrafish by needle injection surgery. SD‐OCT imaging was carried out immediately after the mechanical injury. The retinal hemorrhage, which lasted only 5 seconds, could be visualized dynamically by SD‐OCT. The process of blood clearance and retinal repair was also evaluated because SD‐OCT imaging is nondestructive. Both SD‐OCT imaging results and behavioral analyzing results demonstrated that zebrafish retina could be repaired by itself within 15 days, which was confirmed by the results of pathological experiment.     

p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stroke.     

A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio)

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.     

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.     

Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis

Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of omega-3- and omega-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy. We show that increasing omega-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive omega-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of omega-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-alpha. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of omega-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in omega-3-PUFA, and premature infants lack the important transfer from the mother to the infant of omega-3-PUFA that normally occurs in the third trimester of pregnancy. Supplementing omega-3-PUFA intake may be of benefit in preventing retinopathy.     

NO as a mediator during the early development of the cardiovascular system in the zebrafish

As a general pattern innervation of the cardiovascular system appears late during development in vertebrate embryos, and cardiovascular control may be achieved by hormonal activity in early stages. However, very little is known about the onset of NO-responsiveness during development, which in adult vertebrates is known to play a key function in many physiological processes such as control of vascular tone, neurotransmission, macrophage activity, and angiogenesis. Analysis of the effect of NO on the cardiovascular system in zebrafish (Danio rerio) embryos and larvae revealed almost no effect on cardiac activity during chronic exposure to NO-producing chemicals, whereas vascular reactivity was observed in veins and arteries of the zebrafish in early developmental stages (5-6 days post fertilization). Chronic exposure also modified the development of the vascular system. The presence of an NO donor (sodium nitroprusside) did not change the patterning of the vascular bed, but it induced an earlier appearance of some blood vessels in the trunk region of the zebrafish larvae. The data reveal that NO plays an important role in the development of the cardiovascular system and in the ontogeny of the cardiovascular control system in fish.     

成年斑马鱼脊髓损伤修复中脑gdnf和nos基因的表达

成年斑马鱼(Danio rerio)具有很强的脊髓损伤后自主修复的能力, 但目前其机制不明。为了研究斑马鱼中脑组织对脊髓再生的影响, 文章应用成年斑马鱼脊髓损伤模型, 采用实时定量PCR方法和原位杂交技术, 检测了斑马鱼脑中胶质细胞源性神经营养因子(gdnf)和一氧化氮合酶(nos)基因在脊髓损伤后4 h、12 h、6 d、11 d的表达情况, 展示了这两种基因在斑马鱼脑内不同核团的动态表达变化。结果显示, 成年斑马鱼脊髓损伤后, 神经营养因子gdnf基因在损伤急性期(4 h、12 h)和神经修复期(6 d、11 d)于斑马鱼脑内呈现显著性升高(P<0.05),而一氧化氮合酶基因nos的表达于损伤急性期显著性升高 (P<0.05), 随后下降, 并在修复期 (11 d)显著降低(P<0.05)。这表明, 脊髓损伤后, 高表达gdnf基因同时低表达nos基因的脑环境给脊髓损伤提供了良好的神经再生微环境, 从而可能促进轴突的再生长及运动能力的恢复。