173 research outputs found
Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature
No full textVascular 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
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
Mapping the Molecular Landscape of Early Diabetic Retinopathy: A Multi-layered Analysis of the Human Retina
No full textBackground: The early stages of diabetic retinopathy are not well understood, and studies in humans are limited by the scarcity of post-mortem retinal tissue, variable post-mortem delays, and differences among donors. Under these real-world conditions, many spatial and sequencing methods that work well in model systems either fail or behave unpredictably. This thesis defines the technical limits of multilayer molecular profiling in the human diabetic retina and develops a decision framework to guide future studies. /
Methods: Three approaches were tested on FFPE and OCT-embedded human retina: high-plex immunohistochemistry (IBEX), RNA-focused spatial methods (Light-Seq and HCR-RNA-FISH), and genome-wide DNA methylation profiling using the Infinium MethylationEPIC v2.0 array. I judged feasibility by whether signals were reproducible across donors, whether there were clear quality-control readouts (RIN/DV200 for RNA, DIN for DNA), and whether the differential methylation patterns were biologically interpretable. /
Results: In archival retina, IBEX and spatial RNA methods were usable but not reliable as first-line discovery tools. IBEX provided informative maps of blood vessels and glial cells, but it was limited by antibody specificity, antigen retrieval conditions, autofluorescence, and donor-to-donor variability. Several spatial transcriptomics methods were tested and found to be unsuitable due to poor mRNA quality impacting on reverse transcription and library preparation; only short-amplicon imaging on promptly fixed tissue was consistently robust. In contrast, genome-wide DNA methylation profiling with the MethylationEPIC v2 array was technically robust and reproduced validated signals, including changes in the ten-eleven translocation (TET) pathway, which plays a crucial role in DNA demethylation and epigenetic regulation.
epigenome‑wide significance. /
Conclusion: IBEX, spatial RNA, and DNA methylation provide complementary but not equivalent information in the human post-mortem retina. Under typical tissue constraints, DNA methylation emerged as the most robust discovery readout. In a small pilot study this approach detected differences between diabetic and control samples in stress response and demethylation pathways and indicated heterogeneous epigenetic age acceleration in diabetic retina, but did not reach epigenome wide significance. On the other hand, IBEX and spatial RNA were better suited to localise and testing selected pathways in high-quality samples. Overall, this thesis established a framework that links tissue quality to method choice and defines realistic performance limits for each assay type in human post-mortem retina. This is already influencing local practice. RNA/DNA quality control, in situ screening, and the MethylationEPIC v2 analysis pipeline are now used in our institute to triage donor tissue and design new studies. More broadly, this thesis offers a platform for planning studies using scarce and variable human retinal tissue
VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia
No full textVascular 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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