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1.
Exp Dermatol ; 33(1): e14955, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37897068

RESUMEN

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.


Asunto(s)
Cafeína , Pirazinas , Envejecimiento de la Piel , Humanos , Recién Nacido , Cafeína/farmacología , Senoterapéuticos , Técnicas de Cultivo de Órganos , Proyectos Piloto , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Piel/metabolismo , Envejecimiento , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Biomarcadores/metabolismo
3.
Methods Mol Biol ; 2154: 249-254, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32314223

RESUMEN

Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo models have been used in the past. Here we describe in detail the methodology of the human skin punch-in-a-punch ex vivo wound healing model.


Asunto(s)
Biomarcadores , Cicatrización de Heridas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Piel/metabolismo , Piel/patología , Cicatrización de Heridas/genética
4.
EMBO Rep ; 20(8): e47047, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31379129

RESUMEN

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.


Asunto(s)
Vasos Sanguíneos/citología , Encéfalo/citología , Células Endoteliales/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica/genética , Pez Cebra/embriología , Actinas/antagonistas & inhibidores , Actinas/genética , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestructura , Encéfalo/irrigación sanguínea , Encéfalo/embriología , Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Circulación Cerebrovascular/genética , Embrión no Mamífero , Células Endoteliales/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Miosina Tipo II/antagonistas & inhibidores , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Óxido Nítrico/metabolismo , Orgánulos/metabolismo , Orgánulos/ultraestructura , Polimerizacion/efectos de los fármacos , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal , Tiazolidinas/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
5.
Elife ; 62017 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-28498105

RESUMEN

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.


Asunto(s)
Encéfalo/embriología , Endocitosis , Células Endoteliales/metabolismo , Sustancias Macromoleculares/metabolismo , Pez Cebra/embriología , Animales
6.
Arterioscler Thromb Vasc Biol ; 35(8): 1815-25, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26069237

RESUMEN

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.


Asunto(s)
Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Neovascularización Retiniana/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Endocitosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas , Interferencia de ARN , Neovascularización Retiniana/genética , Neovascularización Retiniana/fisiopatología , Transducción de Señal , Transfección
7.
Nat Commun ; 6: 6429, 2015 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-25752958

RESUMEN

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.


Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Endotelio/crecimiento & desarrollo , Integrina beta1/metabolismo , Uniones Intercelulares/fisiología , Morfogénesis/fisiología , Neovascularización Fisiológica/fisiología , Vasos Retinianos/crecimiento & desarrollo , Animales , Western Blotting , Encéfalo/anatomía & histología , Proliferación Celular , Endotelio/metabolismo , Marcación de Gen , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Inmunoprecipitación , Uniones Intercelulares/metabolismo , Ratones , Microscopía Electrónica , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Vasos Retinianos/ultraestructura
8.
Nat Cell Biol ; 15(3): 249-60, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23354168

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Moléculas de Adhesión Celular/metabolismo , Endocitosis/fisiología , Endotelio Vascular/citología , Efrina-B2/metabolismo , Neovascularización Fisiológica , Proteína Quinasa C/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Endotelio Vascular/metabolismo , Efrina-B2/antagonistas & inhibidores , Efrina-B2/genética , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Transgénicos , Morfogénesis , Fosforilación , Proteína Quinasa C/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Retina/citología , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
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