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1.
Circ Res ; 133(4): 333-349, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37462027

RESUMO

BACKGROUND: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. METHODS: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. RESULTS: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. CONCLUSIONS: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.


Assuntos
Vasos Linfáticos , Proteínas Monoméricas de Ligação ao GTP , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quilomícrons/metabolismo , Vasos Linfáticos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Permeabilidade Capilar
2.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34504019

RESUMO

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.


Assuntos
Artérias Carótidas/fisiologia , Doenças das Artérias Carótidas/prevenção & controle , Células Endoteliais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Estresse Mecânico , Remodelação Vascular , Animais , Artérias Carótidas/citologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Células Endoteliais/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
3.
J Am Soc Nephrol ; 32(9): 2255-2272, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34341180

RESUMO

BACKGROUND: Kidney function requires continuous blood filtration by glomerular capillaries. Disruption of glomerular vascular development or maintenance contributes to the pathogenesis of kidney diseases, but the signaling events regulating renal endothelium development remain incompletely understood. Here, we discovered a novel role of Slit2-Robo signaling in glomerular vascularization. Slit2 is a secreted polypeptide that binds to transmembrane Robo receptors and regulates axon guidance as well as ureteric bud branching and angiogenesis. METHODS: We performed Slit2-alkaline phosphatase binding to kidney cryosections from mice with or without tamoxifen-inducible Slit2 or Robo1 and -2 deletions, and we characterized the phenotypes using immunohistochemistry, electron microscopy, and functional intravenous dye perfusion analysis. RESULTS: Only the glomerular endothelium, but no other renal endothelial compartment, responded to Slit2 in the developing kidney vasculature. Induced Slit2 gene deletion or Slit2 ligand trap at birth affected nephrogenesis and inhibited vascularization of developing glomeruli by reducing endothelial proliferation and migration, leading to defective cortical glomerular perfusion and abnormal podocyte differentiation. Global and endothelial-specific Robo deletion showed that both endothelial and epithelial Robo receptors contributed to glomerular vascularization. CONCLUSIONS: Our study provides new insights into the signaling pathways involved in glomerular vascular development and identifies Slit2 as a potential tool to enhance glomerular angiogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Glomérulos Renais/irrigação sanguínea , Néfrons/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Animais , Animais Recém-Nascidos , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/patologia , Camundongos , Néfrons/patologia , Transdução de Sinais , Proteínas Roundabout
4.
Circ Res ; 120(9): 1414-1425, 2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28298294

RESUMO

RATIONALE: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.


Assuntos
Permeabilidade Capilar , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Células Endoteliais/metabolismo , Pulmão/irrigação sanguínea , Vasos Retinianos/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Junções Aderentes/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Carcinoma Pulmonar de Lewis/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Feminino , Genótipo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Neovascularização Fisiológica , Fenótipo , Vasos Retinianos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Proc Natl Acad Sci U S A ; 112(3): 761-6, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25561555

RESUMO

Angiogenesis, the formation of new blood vessels, is regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). VEGFR2 is abundant in the tip cells of angiogenic sprouts, where VEGF/VEGFR2 functions upstream of the delta-like ligand 4 (DLL4)/Notch signal transduction pathway. VEGFR3 is expressed in all endothelia and is indispensable for angiogenesis during early embryonic development. In adults, VEGFR3 is expressed in angiogenic blood vessels and some fenestrated endothelia. VEGFR3 is abundant in endothelial tip cells, where it activates Notch signaling, facilitating the conversion of tip cells to stalk cells during the stabilization of vascular branches. Subsequently, Notch activation suppresses VEGFR3 expression in a negative feedback loop. Here we used conditional deletions and a Notch pathway inhibitor to investigate the cross-talk between VEGFR2, VEGFR3, and Notch in vivo. We show that postnatal angiogenesis requires VEGFR2 signaling also in the absence of Notch or VEGFR3, and that even small amounts of VEGFR2 are able to sustain angiogenesis to some extent. We found that VEGFR2 is required independently of VEGFR3 for endothelial DLL4 up-regulation and angiogenic sprouting, and for VEGFR3 functions in angiogenesis. In contrast, VEGFR2 deletion had no effect, whereas VEGFR3 was essential for postnatal lymphangiogenesis, and even for lymphatic vessel maintenance in adult skin. Knowledge of these interactions and the signaling functions of VEGFRs in blood vessels and lymphatic vessels is essential for the therapeutic manipulation of the vascular system, especially when considering multitargeted antiangiogenic treatments.


Assuntos
Neovascularização Fisiológica , Vasos Retinianos/crescimento & desenvolvimento , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Camundongos
6.
Development ; 140(7): 1497-506, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23462469

RESUMO

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.


Assuntos
Vasos Sanguíneos/embriologia , Sistema Linfático/embriologia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Alelos , Animais , Animais Geneticamente Modificados , Comunicação Autócrina/genética , Comunicação Autócrina/fisiologia , Vasos Sanguíneos/crescimento & desenvolvimento , Movimento Celular/genética , Movimento Celular/fisiologia , Códon sem Sentido/fisiologia , Embrião não Mamífero , Feminino , Sistema Linfático/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Comunicação Parácrina/genética , Comunicação Parácrina/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
7.
Blood ; 122(5): 658-65, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23803710

RESUMO

Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissue-specific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranially to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PlGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PlGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization.


Assuntos
Sistema Nervoso Central/irrigação sanguínea , Revascularização Cerebral , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas da Gravidez/fisiologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Neoplasias do Sistema Nervoso Central/etiologia , Neoplasias do Sistema Nervoso Central/genética , Encefalite/etiologia , Encefalite/genética , Feminino , Terapia Genética/métodos , Hemangioma/etiologia , Hemangioma/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/genética , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas da Gravidez/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/efeitos adversos , Fator C de Crescimento do Endotélio Vascular/genética
8.
Nature ; 454(7204): 656-60, 2008 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-18594512

RESUMO

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.


Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Regulação para Baixo , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/genética , Receptores Notch/metabolismo , Transdução de Sinais
9.
Pulm Circ ; 12(4): e12167, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36532314

RESUMO

Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how ß-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to ß-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of ß-arrestin 1 and ß-arrestin 2. We found that ß-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of ß-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. ß-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, ß-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. ß-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of ß-arrestin 1 and ß-arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial ß-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development.

10.
Dev Cell ; 57(19): 2321-2333.e9, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36220082

RESUMO

Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.


Assuntos
Polaridade Celular , Células Endoteliais , Junções Aderentes/metabolismo , Animais , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Endoteliais/metabolismo , Camundongos , Morfogênese , Retina/metabolismo
11.
Dev Cell ; 56(15): 2237-2251.e6, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34273276

RESUMO

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here, we show that diving tip cells invading the mouse neuroretina (D-tip cells) are distinct from tip cells guiding the superficial retinal vascular plexus (S-tip cells). D-tip cells have a unique transcriptional signature, including high TGF-ß signaling, and they begin to acquire blood-retina barrier properties. Endothelial deletion of TGF-ß receptor I (Alk5) inhibits D-tip cell identity acquisition and deep vascular plexus formation. Loss of endothelial ALK5, but not of the canonical SMAD effectors, leads to aberrant contractile pericyte differentiation and hemorrhagic vascular malformations. Oxygen-induced retinopathy vasculature exhibits S-like tip cells, and Alk5 deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that non-canonical-TGF-ß signaling could improve retinal revascularization and neural function in ischemic retinopathy.


Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Retina/fisiologia , Neovascularização Retiniana/metabolismo , Animais , Células Endoteliais/metabolismo , Endotélio Vascular , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Retina/citologia , Retina/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
12.
Front Physiol ; 11: 509, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547411

RESUMO

Lymphatic endothelial cells (LECs) lining lymphatic vessels develop specialized cell-cell junctions that are crucial for the maintenance of vessel integrity and proper lymphatic vascular functions. Successful lymphatic drainage requires a division of labor between lymphatic capillaries that take up lymph via open "button-like" junctions, and collectors that transport lymph to veins, which have tight "zipper-like" junctions that prevent lymph leakage. In recent years, progress has been made in the understanding of these specialized junctions, as a result of the application of state-of-the-art imaging tools and novel transgenic animal models. In this review, we discuss lymphatic development and mechanisms governing junction remodeling between button and zipper-like states in LECs. Understanding lymphatic junction remodeling is important in order to unravel lymphatic drainage regulation in obesity and inflammatory diseases and may pave the way towards future novel therapeutic interventions.

13.
Science ; 361(6402): 599-603, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-30093598

RESUMO

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream through intestinal lymphatic vessels called lacteals. Here, we show that preventing lacteal chylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 (Nrp1) and Vascular endothelial growth factor receptor 1 (Vegfr1; also known as Flt1) renders mice resistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-A bioavailability and signaling through VEGFR2, inducing lacteal junction zippering and chylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 and vascular endothelial (VE)-cadherin signaling inhibition rescued chylomicron transport in the mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherin anchors prevented chylomicron uptake in wild-type mice. These data suggest that lacteal junctions may be targets for preventing dietary fat uptake.


Assuntos
Quilomícrons/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/metabolismo , Neuropilina-1/genética , Obesidade/etiologia , Obesidade/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Antígenos CD/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/metabolismo , Quilomícrons/efeitos adversos , Gorduras na Dieta/efeitos adversos , Enterócitos/metabolismo , Deleção de Genes , Absorção Intestinal/genética , Absorção Intestinal/fisiologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
EMBO Mol Med ; 7(11): 1418-25, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26459520

RESUMO

Vascular endothelial growth factor C (VEGF-C) binding to its tyrosine kinase receptor VEGFR-3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF-C/VEGFR-3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF-C in adult vasculature are unknown. We show here that VEGF-C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF-C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The Vegfc-deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high-fat diet, the Vegfc-deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism.


Assuntos
Intestinos/fisiologia , Metabolismo dos Lipídeos , Vasos Linfáticos/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Deleção de Genes , Camundongos , Camundongos Knockout , Fator C de Crescimento do Endotélio Vascular/genética
15.
J Clin Invest ; 124(9): 3975-86, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25061878

RESUMO

In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.


Assuntos
Humor Aquoso/fisiologia , Córnea/irrigação sanguínea , Vasos Linfáticos/fisiologia , Fator C de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Movimento Celular , Proliferação de Células , Células Endoteliais/fisiologia , Humanos , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL
16.
J Clin Invest ; 124(2): 824-34, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24430181

RESUMO

The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.


Assuntos
Angiopoietina-1/antagonistas & inibidores , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Deleção de Genes , Receptor de TIE-1/genética , Vasos Retinianos/patologia , Inibidores da Angiogênese/química , Angiopoietina-1/metabolismo , Animais , Apoptose , Sobrevivência Celular , Homozigoto , Ligantes , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neovascularização Patológica , Fenótipo , Receptor de TIE-1/fisiologia , Receptor TIE-2/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
17.
Sci Signal ; 6(282): ra52, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23821770

RESUMO

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1P) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1B) did not. Unlike P-L1B and VEGF-B, B-L1P and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1P also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.


Assuntos
Proteínas da Gravidez/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Western Blotting , Linhagem Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Fator de Crescimento Placentário , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator B de Crescimento do Endotélio Vascular/química , Fator B de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
18.
J Natl Cancer Inst ; 104(6): 461-75, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22343031

RESUMO

BACKGROUND: Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized. METHODS: We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided. RESULTS: Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm(2), difference = 32.67 mm(2), 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell-cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell-cell junctions in endothelial-tumor cell cocultures. CONCLUSION: Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell-cell junctions.


Assuntos
Inibidores da Angiogênese/farmacologia , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/metabolismo , Endotélio Vascular/efeitos dos fármacos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/farmacologia , Hipóxia Celular , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Junções Intercelulares/patologia , Neoplasias Pulmonares/secundário , Linfangiogênese , Metástase Linfática , Melanoma/irrigação sanguínea , Melanoma/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Neovascularização Patológica/tratamento farmacológico , Receptor TIE-2/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transplante Heterólogo , Regulação para Cima
19.
Nat Cell Biol ; 13(10): 1202-13, 2011 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-21909098

RESUMO

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.


Assuntos
Células Endoteliais/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica , Receptores Notch/metabolismo , Vasos Retinianos/metabolismo , Rombencéfalo/irrigação sanguínea , Transdução de Sinais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos/farmacologia , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transdução Genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/genética , Fator B de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
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