184 research outputs found
Wnt/beta-catenin signaling controls development of the blood–brain barrier
No full textThe blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown
Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature
Get PDFVascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans
Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes
No full textDevelopment of the retinal vasculature is controlled by a hierarchy of interactions among retinal neurons, astrocytes and blood vessels. Retinal neurons release platelet-derived growth factor (PDGFA) to stimulate proliferation of astrocytes, which in turn stimulate blood vessel growth by secreting vascular endothelial cell growth factor (VEGF). Presumably, there must be counteractive mechanisms for limiting astrocyte proliferation and VEGF production to prevent runaway angiogenesis. Here, we present evidence that the developing vessels provide feedback signals that trigger astrocyte differentiation - marked by cessation of cell division, upregulation of glial fibrillary acidic protein (GFAP) and downregulation of VEGF. We prevented retinal vessel development by raising newborn mice in a high-oxygen atmosphere, which leads, paradoxically, to retinal hypoxia (confirmed by using the oxygen-sensing reagent EF5). The forced absence of vessels caused prolonged astrocyte proliferation and inhibited astrocyte differentiation in vivo. We could reproduce these effects by culturing retinal astrocytes in a low oxygen atmosphere, raising the possibility that blood-borne oxygen itself might induce astrocyte differentiation and indirectly prevent further elaboration of the vascular network
NRP1 acts cell autonomously in endothelium to promote tip cell function during sprouting angiogenesis
No full textNeuropilin (NRP) 1 is a receptor for the vascular endothelial growth factor (VEGF)-A and is essential for normal angiogenesis. Previous in vitro experiments identified NRP1 interactions with VEGF-A's main signaling receptor VEGFR2 within endothelial cells, but also between nonendothelial NRP1 and endothelial VEGFR2. Consistent with an endothelial role for NRP1 in angiogenesis, we found that VEGFR2 and NRP1 were coexpressed in endothelial tip and stalk cells in the developing brain. In addition, NRP1 was expressed on two cell types that interact with growing brain vessels-the neural progenitors that secrete VEGF-A to stimulate tip cell activity and the pro-angiogenic macrophages that promote tip cell anastomosis. Selective targeting of Nrp1 in each of these cell types demonstrated that neural progenitor-and macrophage-derived NRP1 were dispensable, whereas endothelial NRP1 was essential for normal brain vessel growth. NRP1 therefore promotes brain angiogenesis cell autonomously in endothelium, independently of heterotypic interactions with nonendothelial cells. Genetic mosaic analyses demonstrated a key role for NRP1 in endothelial tip rather than stalk cells during vessel sprouting. Thus, NRP1-expressing endothelial cells attained the tip cell position when competing with NRP1-negative endothelial cells in chimeric vessel sprouts. Taken together, these findings demonstrate that NRP1 promotes endothelial tip cell function during angiogenesis
The role of inflammation in retinal ischaemia
Get PDFPurpose: The immune cascade is known to contribute to the pathology seen in oxygen induced retinopathy (OIR), however its exact role is poorly defined. We hypothesised that immune cell activation worsens hypoxia and exacerbates the neovascular sequelae seen in ischaemic retinopathy. Methods: We assess the effect of intraperitoneal lipopolysaccharide (IP LPS) injection in OIR mice. Neovascularisation was assessed by measuring the avascular area and neovascular tuft area at P17. Hypoxia was assessed using vessel tortuosity and EF5 hypoxia staining at P14. The activated retinal inflammatory cell population was characterised using immunohistochemistry and flow cytometry. Transgenic mice were bred to delete different subpopulations of inflammatory cells to define their role in hypoxia modulation. RNA sequencing performed on retinal tissue analysed the effect of systemic LPS on retinal cytokines and angiogenesis markers. Results: IP LPS injection at P12 in OIR mice significantly reduced neovascularisation at P17 and hypoxia at P14. Immunohistochemistry revealed an influx of round, CD11b+, lectin stained cells into the retina of LPS treated mice, which on flow cytometry were identified as myeloid cells, being predominantly Cd11b+Ly6Ghi neutrophils. Experimental depletion of the myeloid lineage was achieved using ROSA26eGFP-DTALysMcre, CCL2 and CCR2 knockout mouse lines and anti-CCR2 antibody MC21. However, when these mice were LPS treated, the effects on hypoxia readouts caused by LPS were still seen. Transcriptional profiling of the retina (using RNAseq) revealed a large upregulation in IL1β in the central, hypoxic retina of LPS treated mice. Injection of IL1β at P12 also mimicked the effects of LPS, suggesting that IL1β is a key mediator of the hypoxia reducing effect LPS has in the OIR model. Conclusions: These findings are counterintuitive to the current literature and provide new insight into the role the immune system has on regulating oxygen demand in the retina. This novel approach to reducing hypoxia has the potential to lead to novel therapies targeting hypoxia and preventing neovascularisation in ischaemic eye disease
VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia
Get PDFVascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. it controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells
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