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EMBO Rep ; 20(8): e47047, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31379129


We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Elife ; 62017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28498105


The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Encéfalo/embriologia , Endocitose , Células Endoteliais/metabolismo , Substâncias Macromoleculares/metabolismo , Peixe-Zebra/embriologia , Animais
Arterioscler Thromb Vasc Biol ; 35(8): 1815-25, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26069237


OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling is a major regulator of physiological and pathological angiogenesis. VEGF receptor activity is strongly controlled by endocytosis, which can terminate or enhance signal transduction in the angiogenic endothelium, but the exact molecular regulation of these processes remains incompletely understood. We have therefore examined the function of Numb family clathrin-associated sorting proteins in angiogenesis. APPROACH AND RESULTS: We show that Numb proteins are expressed by endothelial cells during retinal angiogenesis in mice. Inducible inactivation of the Numb/Numbl genes in the postnatal endothelium led to impaired vessel growth, reduced endothelial proliferation and sprouting, and decreased VEGF receptor activation. Biochemistry and cell biology experiments established that Numb can interact with VEGFR2 and VEGFR3 and controls VEGF receptor activation in response to ligand stimulation. Experiments in cultured endothelial cells showed that Numb proteins counteract VEGF receptor degradation and promote VEGFR2 recycling back to the plasma membrane. CONCLUSIONS: Numb proteins control VEGF receptor endocytosis, signaling, and recycling in endothelial cells, which promotes the angiogenic growth of blood vessels.

Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endocitose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Transporte Proteico , Interferência de RNA , Neovascularização Retiniana/genética , Neovascularização Retiniana/fisiopatologia , Transdução de Sinais , Transfecção
Nat Commun ; 6: 6429, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25752958


Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin ß1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that ß1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin ß1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin ß1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

Antígenos CD/metabolismo , Caderinas/metabolismo , Endotélio/crescimento & desenvolvimento , Integrina beta1/metabolismo , Junções Intercelulares/fisiologia , Morfogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Vasos Retinianos/crescimento & desenvolvimento , Animais , Western Blotting , Encéfalo/anatomia & histologia , Proliferação de Células , Endotélio/metabolismo , Marcação de Genes , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Imunoprecipitação , Junções Intercelulares/metabolismo , Camundongos , Microscopia Eletrônica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/ultraestrutura
Nat Cell Biol ; 15(3): 249-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23354168


Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Moléculas de Adesão Celular/metabolismo , Endocitose/fisiologia , Endotélio Vascular/citologia , Efrina-B2/metabolismo , Neovascularização Fisiológica , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Western Blotting , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Movimento Celular , Proliferação de Células , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/metabolismo , Efrina-B2/antagonistas & inibidores , Efrina-B2/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Transgênicos , Morfogênese , Fosforilação , Proteína Quinase C/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Retina/citologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz