RESUMO
An 8-month-old girl referred from her pediatrician with a diagnosis of neurofibromatosis type 1 (NF1) presented with an enlarged cloudy cornea of the left eye and a swollen left side of the face. Her left eye had intraocular pressure (IOP) of 21 mm Hg, corneal diameter of 16 mm, ectropion uvea, cup:disk ratio of 0.9, axial length of 28.06 mm, and S-shaped upper lid deformity. Uneventful combined trabeculotomy-trabeculectomy with mitomycin C was performed. On postoperative day 1, there was a new total hyphema that persisted for 2 weeks. An anterior chamber washout was performed, revealing the source of bleeding to be a persistent tunica vasculosa lentis along the zonules of the lens. Viscotamponade was performed, and the corneal wounds were closed, with the ocular tension slightly elevated. Bleeding did not recur for the following 5 months, and IOP was controlled until final follow-up.
Assuntos
Hifema , Pressão Intraocular , Neurofibromatose 1 , Trabeculectomia , Humanos , Feminino , Hifema/etiologia , Hifema/diagnóstico , Pressão Intraocular/fisiologia , Neurofibromatose 1/complicações , Neurofibromatose 1/diagnóstico , Lactente , Glaucoma/etiologia , Glaucoma/cirurgia , Glaucoma/diagnóstico , Cristalino/cirurgia , Complicações Pós-Operatórias , Mitomicina/administração & dosagem , Mitomicina/uso terapêutico , Doenças do Cristalino/diagnóstico , Doenças do Cristalino/etiologia , Doenças do Cristalino/cirurgia , Estruturas Embrionárias , Vasos Retinianos/embriologiaRESUMO
Throughout the central nervous system, astrocytes adopt precisely ordered spatial arrangements of their somata and arbors, which facilitate their many important functions. Astrocyte pattern formation is particularly important in the retina, where astrocytes serve as a template that dictates the pattern of developing retinal vasculature. Thus, if astrocyte patterning is disturbed, there are severe consequences for retinal angiogenesis and ultimately for vision - as seen in diseases such as retinopathy of prematurity. Here we discuss key steps in development of the retinal astrocyte population. We describe how fundamental developmental forces - their birth, migration, proliferation, and death - sculpt astrocytes into a template that guides angiogenesis. We further address the radical changes in the cellular and molecular composition of the astrocyte network that occur upon completion of angiogenesis, paving the way for their adult functions in support of retinal ganglion cell axons. Understanding development of retinal astrocytes may elucidate pattern formation mechanisms that are deployed broadly by other axon-associated astrocyte populations.
Assuntos
Astrócitos/fisiologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Animais , Axônios/fisiologia , Morte Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Humanos , Neovascularização Fisiológica , Fibras Nervosas/fisiologia , Retina/citologia , Retina/embriologia , Células Ganglionares da Retina/fisiologia , Vasos Retinianos/embriologia , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/fisiologia , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/fisiopatologiaRESUMO
Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Mamíferos , Neovascularização Fisiológica , Retina/embriologia , Vasos Retinianos/embriologia , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/embriologia , Camundongos , Camundongos Transgênicos , Proteína Smad1/genética , Proteína Smad5/genéticaRESUMO
Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of ß-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.
Assuntos
Células Endoteliais/metabolismo , Neovascularização Fisiológica , Vasos Retinianos/metabolismo , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vasos Retinianos/citologia , Vasos Retinianos/embriologia , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.
Assuntos
Receptores Frizzled/fisiologia , Retina/patologia , Vasos Retinianos/patologia , Remodelação Vascular/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Modelos Animais de Doenças , Embrião não Mamífero , Vitreorretinopatias Exsudativas Familiares/diagnóstico , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/patologia , Estudos de Viabilidade , Receptores Frizzled/genética , Humanos , Neovascularização Patológica/embriologia , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Retina/diagnóstico por imagem , Retina/embriologia , Retina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/embriologia , Vasos Retinianos/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.
Assuntos
Endotélio Vascular/metabolismo , Neovascularização Fisiológica , Proteína rhoA de Ligação ao GTP/deficiência , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Embrião de Mamíferos , Desenvolvimento Embrionário , Endotélio Vascular/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Camundongos Transgênicos , Vasos Retinianos/embriologia , Vasos Retinianos/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaAssuntos
Angiografia , Olho/embriologia , Vasos Retinianos/embriologia , Tomografia de Coerência Óptica , Angiografia/métodos , Animais , Olho/irrigação sanguínea , Olho/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Vasos Retinianos/diagnóstico por imagem , Tomografia de Coerência Óptica/métodosRESUMO
During development, the fetal vasculature nourishes the developing lens and retina, and it subsequently regresses after the formation of the retinal vessels. Persistent fetal vasculature (PFV) occurs as a result of a failure of fetal ocular vasculature to undergo normal programmed involution, which leads to blindness or serious loss of vision. Persistent fetal vasculature is responsible for as much as 5% of childhood blindness in western countries. The regulatory mechanisms responsible for fetal vascular regress remain obscure, as do the underlying causes of the failure of regression. Because of recent advancements in microinvasive surgical techniques, the early treatment of PFV has become safer and more effective, thus paving the way for the development of a future new treatment strategy. In this review, clinical and imaging manifestations of PFV and the progress in the treatment of PFV are highlighted.
Assuntos
Cegueira/etiologia , Procedimentos Cirúrgicos Oftalmológicos/métodos , Vítreo Primário Hiperplásico Persistente , Catarata/etiologia , Extração de Catarata , Diagnóstico Diferencial , Olho/irrigação sanguínea , Humanos , Cristalino/embriologia , Vítreo Primário Hiperplásico Persistente/complicações , Vítreo Primário Hiperplásico Persistente/patologia , Vítreo Primário Hiperplásico Persistente/cirurgia , Retina/embriologia , Vasos Retinianos/embriologiaRESUMO
The retina is one of the most metabolically active tissues in the body, consuming high levels of oxygen and nutrients. A well-organized ocular vascular system adapts to meet the metabolic requirements of the retina to ensure visual function. Pathological conditions affect growth of the blood vessels in the eye. Understanding the neuronal biological processes that govern retinal vascular development is of interest for translational researchers and clinicians to develop preventive and interventional therapeutics for vascular eye diseases that address early drivers of abnormal vascular growth. This review summarizes the current knowledge of the cellular and molecular processes governing both physiological and pathological retinal vascular development, which is dependent on the interaction among retinal cell populations, including neurons, glia, immune cells, and vascular endothelial cells. We also review animal models currently used for studying retinal vascular development.
Assuntos
Retina/embriologia , Retina/fisiologia , Doenças Retinianas/fisiopatologia , Vasos Retinianos/embriologia , Vasos Retinianos/patologia , Animais , Modelos Animais de Doenças , Humanos , Neovascularização Fisiológica/fisiologiaRESUMO
The development of the ocular vasculatures is perfectly synchronized to provide the nutritional and oxygen requirements of the forming human eye. The fetal vasculature of vitreous, which includes the hyaloid vasculature, vasa hyaloidea propria, and tunica vasculosa lentis, initially develops around 4-6 weeks gestation (WG) by hemo-vasculogenesis (development of blood and blood vessels from a common progenitor, the hemangioblast). This transient fetal vasculature expands around 12 WG by angiogenesis (budding from primordial vessels) and remains until a retinal vasculature begins to form. The fetal vasculature then regresses by apoptosis with the assistance of macrophages/hyalocytes. The human choroidal vasculature also forms by a similar process and will supply nutrients and oxygen to outer retina. This lobular vasculature develops in a dense collagenous tissue juxtaposed with a cell constitutively producing vascular endothelial growth factor (VEGF), the retinal pigment epithelium. This epithelial/endothelial relationship is critical in maintaining the function of this vasculature throughout life and maintaining it's fenestrated state. The lobular capillary system (choriocapillaris) develops first by hemo-vasculogenesis and then the intermediate choroidal blood vessels form by angiogenesis, budding from the choriocapillaris. The human retinal vasculature is the last to develop. It develops by vasculogenesis, assembly of CXCR4+/CD39+ angioblasts or vascular progenitors perhaps using Muller cell Notch1 or axonal neuropilinin-1 for guidance of semaphorin 3A-expressing angioblasts. The fovea never develops a retinal vasculature, which is probably due to the foveal avascular zone area of retina expressing high levels of antiangiogenic factors. From these studies, it is apparent that development of the mouse ocular vasculatures is not representative of the development of the human fetal, choroidal and retinal vasculatures.
Assuntos
Corioide/irrigação sanguínea , Retina/embriologia , Vasos Retinianos/embriologia , Corpo Vítreo/irrigação sanguínea , Corioide/embriologia , Humanos , Neovascularização Patológica/embriologia , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/embriologiaRESUMO
The population of infants at risk for retinopathy of prematurity (ROP) varies by world region; in countries with well developed neonatal intensive care services, the highest risk infants are those born at less than 28 weeks gestational age (GA) and less than 1 kg at birth, while, in regions where many aspects of neonatal intensive and ophthalmological care are not routinely available, more mature infants up to 2000 g at birth and 37 weeks GA are also at risk for severe ROP. Treatment options for both groups of patients include standard retinal laser photocoagulation or, more recently, intravitreal anti-VEGF drugs. In addition to detection and treatment of ROP, this review highlights new opportunities created by telemedicine, where screening and diagnosis of ROP in remote locations can be undertaken by non-ophthalmologists using digital fundus cameras. The ophthalmological care of the ROP infant is undertaken in the wider context of neonatal care and general wellbeing of the infant. Because of this context, this review takes a multi-disciplinary perspective with contributions from retinal vascular biologists, pediatric ophthalmologists, an epidemiologist and a neonatologist. This review highlights the latest insights regarding cellular and molecular mechanisms in the formation of the retinal vasculature in the human infant, pathogenesis of ROP, detection and treatment of severe ROP, the risks and benefits of anti-VEGF therapy, the identification of new therapies over the horizon, and the optimal neonatal care regimen for best ROP outcomes, and the benefits and pitfalls of telemedicine in the remote screening and diagnosis of ROP, all of which have the potential to improve ROP outcomes.
Assuntos
Retinopatia da Prematuridade , Inibidores da Angiogênese/uso terapêutico , Humanos , Lactente , Recém-Nascido , Terapia a Laser/métodos , Programas de Rastreamento , Vasos Retinianos/embriologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/fisiopatologia , Retinopatia da Prematuridade/terapia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , VitrectomiaRESUMO
Retinal vasculature develops in a highly orchestrated three-dimensional (3-D) sequence. The stages of retinal vascularization are highly susceptible to oxygen perturbations. We demonstrate that optical tissue clearing of intact rat retinas and light-sheet microscopy provides rapid 3-D characterization of vascular complexity during retinal development. Compared with flat mount preparations that dissect the retina and primarily image the outermost vascular layers, intact cleared retinas imaged using light-sheet fluorescence microscopy display changes in the 3-D retinal vasculature rapidly without the need for point scanning techniques. Using a severe model of retinal vascular disruption, we demonstrate that a simple metric based on Sholl analysis captures the vascular changes observed during retinal development in 3-D. Taken together, these results provide a methodology for rapidly quantifying the 3-D development of the entire rodent retinal vasculature.
Assuntos
Microscopia de Fluorescência , Retina/embriologia , Vasos Retinianos/embriologia , Animais , RatosRESUMO
The superficial ocular vasculature of the embryonic zebrafish develops in a highly stereotypic manner and hence provides a convenient model for studying molecular mechanisms that regulate vascular patterning. We have used transgenic zebrafish embryos in which all endothelial cells express enhanced Green Fluorescent Protein and small molecule inhibitors to examine the contribution of two signaling pathways, vascular endothelial growth factor (VEGF) and Hedgehog (Hh) pathways, to the development of the superficial system. We find that most, but not all vessels of the superficial system depend on VEGF signaling for their growth. Hh signaling appears to limit superficial vessel growth over the dorsal eye and is required to promote superficial vessel growth over the ventral eye. These effects of Hh signaling are indirect. Our initial analyses of factors that regulate growth and patterning of superficial ocular vessels suggest that early patterning events in the embryo during organogenesis stages could influence vascular patterning later on. By studying development of specific vascular systems it should be possible to identify new roles for signaling pathways in regulating vascular development.
Assuntos
Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Cristalino/embriologia , Vasos Retinianos/embriologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal , Olho/irrigação sanguínea , Proteínas Hedgehog/genética , Cristalino/irrigação sanguínea , Ligantes , Neovascularização Fisiológica/fisiologia , Organogênese , Fenótipo , Transdução de Sinais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In lower vertebrates, retinal stem cells (RSCs) capable of producing all retinal cell types are a resource for retinal tissue growth throughout life. However, the embryonic origin of RSCs remains largely elusive. Using a Zebrabow-based clonal analysis, we characterized the RSC niche in the ciliary marginal zone of zebrafish retina and illustrate that blood vessels associated with RSCs are required for the maintenance of actively proliferating RSCs. Full lineage analysis of RSC progenitors reveals lineage patterns of RSC production. Moreover, in vivo lineage analysis demonstrates that these RSC progenitors are the direct descendants of a set of bipotent progenitors in the medial epithelial layer of developing optic vesicles, suggesting the involvement of the mixed-lineage states in the RSC lineage specification.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/fisiologia , Retina/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proliferação de Células , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo , Retina/embriologia , Retina/metabolismo , Vasos Retinianos/embriologia , Vasos Retinianos/fisiologia , Nicho de Células-Tronco , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: Angiogenesis is a hallmark of embryonic development and various ischemic and inflammatory diseases. Prostaglandin E2 receptor subtype 3 (EP3) plays an important role in pathophysiologic angiogenesis; however, the precise mechanisms remain unknown. Here, we investigated the role of EP3 in zebra fish embryo and mouse retina angiogenesis and evaluated the underlying mechanisms. APPROACH AND RESULTS: The EP3 receptor was highly expressed in the vasculature in both zebra fish embryos and murine fetal retinas. Pharmacological inhibition or genetic deletion of EP3 significantly reduced vasculature formation in zebra fish embryos and mouse retinas. Further characterization revealed reduced filopodia extension of tip cells in embryonic retinas in EP3-deficient mice. EP3 deletion activated Notch activity by upregulation of delta-like ligand 4 expression in endothelial cells (ECs). Inhibition of Notch signaling rescued the angiogenic defects in EP3-deficient mouse retinas. Moreover, EP3 deficiency led to a significant increase in ß-catenin phosphorylation at Ser675 and nuclear accumulation of ß-catenin in ECs. Knockdown or inhibition of ß-catenin restored the impaired sprouting angiogenesis resulting from EP3 deficiency in ECs. The EP3 receptor depressed protein kinase A activity in ECs by coupling to Gαi. Inhibition of protein kinase A activity significantly reduced Ser675 phosphorylation and nuclear translocation of ß-catenin, abolished the increased delta-like ligand 4 expression, and subsequently restored the impaired angiogenic capacity of EP3-deficient ECs both in vitro and in vivo. CONCLUSIONS: Activation of the EP3 receptor facilitates sprouting angiogenesis through protein kinase A-dependent Notch signaling, suggesting that EP3 and its downstream pathways maybe potential therapeutic targets in the treatment of ischemic diseases.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptores Notch/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Neovascularização Retiniana , Vasos Retinianos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dinoprostona/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Proteínas de Membrana/metabolismo , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea , Fosforilação , Interferência de RNA , Receptores de Prostaglandina E Subtipo EP3/deficiência , Receptores de Prostaglandina E Subtipo EP3/genética , Vasos Retinianos/embriologia , Transdução de Sinais , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , beta Catenina/genéticaRESUMO
Familial exudative vitreoretinopathy (FEVR) is characterized by delayed retinal vascular development, which promotes hypoxia-induced pathologic vessels. In severe cases FEVR may lead to retinal detachment and visual impairment. Genetic studies linked FEVR with mutations in Wnt signaling ligand or receptors, including low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we investigated ocular pathologies in a Lrp5 knockout (Lrp5(-/-)) mouse model of FEVR and explored whether treatment with a pharmacologic Wnt activator lithium could bypass the genetic defects, thereby protecting against eye pathologies. Lrp5(-/-) mice displayed significantly delayed retinal vascular development, absence of deep layer retinal vessels, leading to increased levels of vascular endothelial growth factor and subsequent pathologic glomeruloid vessels, as well as decreased inner retinal visual function. Lithium treatment in Lrp5(-/-) mice significantly restored the delayed development of retinal vasculature and the intralaminar capillary networks, suppressed formation of pathologic glomeruloid structures, and promoted hyaloid vessel regression. Moreover, lithium treatment partially rescued inner-retinal visual function and increased retinal thickness. These protective effects of lithium were largely mediated through restoration of canonical Wnt signaling in Lrp5(-/-) retina. Lithium treatment also substantially increased vascular tubular formation in LRP5-deficient endothelial cells. These findings suggest that pharmacologic activation of Wnt signaling may help treat ocular pathologies in FEVR and potentially other defective Wnt signaling-related diseases.
Assuntos
Lítio/farmacologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Doenças Retinianas/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Oftalmopatias Hereditárias , Vitreorretinopatias Exsudativas Familiares , Feminino , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Retina/efeitos dos fármacos , Retina/embriologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/embriologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis, the formation of new blood vessels by remodeling and growth of pre-existing vessels, is a highly orchestrated process that requires a tight balance between pro-angiogenic and anti-angiogenic factors and the integration of their corresponding signaling networks. The family of Rho GTPases, including RhoA, Rac1, and Cdc42, play a central role in many cell biological processes that involve cytoskeletal changes and cell movement. Specifically for Rac1, we have shown that excision of Rac1 using a Tie2-Cre animal line results in embryonic lethality in midgestation (embryonic day (E) 9.5), with multiple vascular defects. However, Tie2-Cre can be also expressed during vasculogenesis, prior to angiogenesis, and is active in some hematopoietic precursors that can affect vessel formation. To circumvent these limitations, we have now conditionally deleted Rac1 in a temporally controlled and endothelial-restricted fashion using Cdh5(PAC)-iCreERT2 transgenic mice. In this highly controlled experimental in vivo system, we now show that Rac1 is required for embryonic vascular integrity and angiogenesis, and for the formation of superficial and deep vascular networks in the post-natal developing retina, the latter involving a novel specific function for Rac1 in vertical blood vessel sprouting. Aligned with these findings, we show that RAC1 is spatially involved in endothelial cell migration, invasion, and radial sprouting activities in 3D collagen matrix in vitro models. Hence, Rac1 and its downstream molecules may represent potential anti-angiogeneic therapeutic targets for the treatment of many human diseases that involve aberrant neovascularization and blood vessel overgrowth.
Assuntos
Células Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica , Neuropeptídeos/fisiologia , Retina/embriologia , Vasos Retinianos/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Alelos , Animais , Movimento Celular , Endotélio Vascular/metabolismo , Feminino , Genes Reporter , Genótipo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/genética , RNA Interferente Pequeno/metabolismo , Vasos Retinianos/embriologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular 'tubulogenesis' are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1(-/-) ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC-EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.
Assuntos
Proteínas de Transporte/metabolismo , Neovascularização Fisiológica , Vasos Retinianos/embriologia , Vasos Retinianos/metabolismo , Envelhecimento/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Feminino , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , GravidezRESUMO
The Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain unclear. Here, we assess the cellular mechanisms by which Cdc42 regulates mammalian vascular morphogenesis and maintenance. In vivo deletion of Cdc42 in embryonic ECs (Cdc42(Tie2KO)) results in blocked lumen formation and endothelial tearing, leading to lethality of mutant embryos by E9-10 due to failed blood circulation. Similarly, inducible deletion of Cdc42 (Cdc42(Cad5KO)) at mid-gestation blocks angiogenic tubulogenesis. By contrast, deletion of Cdc42 in postnatal retinal vessels leads to aberrant vascular remodeling and sprouting, as well as markedly reduced filopodia formation. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. We also identify Cdc42 effectors Pak2/4 and N-WASP, as well as the actomyosin machinery, to be crucial for EC actin organization. This work supports the notion of Cdc42 as a central regulator of the cellular machinery in ECs that drives blood vessel formation.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Aorta/metabolismo , Apoptose , Vasos Sanguíneos/citologia , Adesão Celular , Polaridade Celular , Proliferação de Células , Sobrevivência Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Feminino , Deleção de Genes , Integrases/metabolismo , Camundongos Knockout , Modelos Biológicos , Gravidez , Pseudópodes/metabolismo , Receptor TIE-2/metabolismo , Vasos Retinianos/embriologia , Vasos Retinianos/metabolismo , Saco Vitelino/irrigação sanguínea , Saco Vitelino/metabolismoRESUMO
BACKGROUND: Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. METHODS AND RESULTS: Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a US Food and Drug Administration-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction in ICH severity. CONCLUSIONS: Our data are the first to show therapeutic prevention in vivo of ICH resulting from Col4a1 mutation and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations.