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UnlabelledAs neural structures grow in size and increase metabolic demand, the CNS vasculature undergoes extensive growth, remodeling, and maturation. Signals from neural tissue act on endothelial cells to stimulate blood vessel ingression, vessel patterning, and acquisition of mature brain vascular traits, most notably the blood-brain barrier. Using mouse genetic and in vitro approaches, we identified retinoic acid (RA) as an important regulator of brain vascular development via non-cell-autonomous and cell-autonomous regulation of endothelial WNT signaling. Our analysis of globally RA-deficient embryos (Rdh10 mutants) points to an important, non-cell-autonomous function for RA in the development of the vasculature in the neocortex. We demonstrate that Rdh10 mutants have severe defects in cerebrovascular development and that this phenotype correlates with near absence of endothelial WNT signaling, specifically in the cerebrovasculature, and substantially elevated expression of WNT inhibitors in the neocortex. We show that RA can suppress the expression of WNT inhibitors in neocortical progenitors. Analysis of vasculature in non-neocortical brain regions suggested that RA may have a separate, cell-autonomous function in brain endothelial cells to inhibit WNT signaling. Using both gain and loss of RA signaling approaches, we show that RA signaling in brain endothelial cells can inhibit WNT-β-catenin transcriptional activity and that this is required to moderate the expression of WNT target Sox17. From this, a model emerges in which RA acts upstream of the WNT pathway via non-cell-autonomous and cell-autonomous mechanisms to ensure the formation of an adequate and stable brain vascular plexus.Significance statementWork presented here provides novel insight into important yet little understood aspects of brain vascular development, implicating for the first time a factor upstream of endothelial WNT signaling. We show that RA is permissive for cerebrovascular growth via suppression of WNT inhibitor expression in the neocortex. RA also functions cell-autonomously in brain endothelial cells to modulate WNT signaling and its downstream target, Sox17. The significance of this is although endothelial WNT signaling is required for neurovascular development, too much endothelial WNT signaling, as well as overexpression of its target Sox17, are detrimental. Therefore, RA may act as a "brake" on endothelial WNT signaling and Sox17 to ensure normal brain vascular development.

In diabetic retinopathy (DR), pericyte dropout from capillary walls is believed to cause the breakdown of the blood-retina barrier (BRB), which subsequently leads to vision-threatening retinal edema. While various proinflammatory cytokines and chemokines are upregulated in eyes with DR, their distinct contributions to disease progression remain elusive. Here, we evaluated roles of stromal cell–derived factor-1α (SDF-1α) and its receptor CXCR4 in the BRB breakdown initiated by pericyte deficiency. After inhibition of pericyte recruitment to developing retinal vessels in neonatal mice, endothelial cells (ECs) upregulated the expression of SDF-1α. Administration of CXCR4 antagonists, or EC-specific disruption of the CXCR4 gene, similarly restored the BRB integrity, even in the absence of pericyte coverage. Furthermore, CXCR4 inhibition significantly decreased both the expression levels of proinflammatory genes (P < 0.05) and the infiltration of macrophages (P < 0.05) into pericyte-deficient retinas. Taken together, EC-derived SDF-1α induced by pericyte deficiency exacerbated inflammation through CXCR4 in an autocrine or paracrine manner and thereby induced macrophage infiltration and BRB breakdown. These findings suggest that the SDF-1α/CXCR4 signaling pathway may be a potential therapeutic target in DR.

PURPOSE: Macular telangiectasia Type 2 (MacTel) is a bilateral, progressive, potentially blinding retinal disease characterized by vascular and neurodegenerative signs, including an increased parafoveal reflectivity to blue light. Our aim was to investigate the relationship of this sign with other signs of macular telangiectasia Type 2 in multiple imaging modalities. METHODS: Participants were selected from the MacTel Type 2 study, based on a confirmed diagnosis and the availability of images. The extent of signs in blue-light reflectance, fluorescein angiographic, optical coherence tomographic, and single- and dual-wavelength autofluorescence images were analyzed. RESULTS: A well-defined abnormality of the perifovea is demonstrated by dual-wavelength autofluorescence and blue-light reflectance in early disease. The agreement in area size of the abnormalities in dual-wavelength autofluorescence and in blue-light reflectance images was excellent: for right eyes: ρ = 0.917 (P < 0.0001, 95% confidence interval 0.855-0.954, n = 46) and for left eyes: ρ = 0.952 (P < 0.0001, 95% confidence interval 0.916-0.973, n = 49). Other changes are less extensive initially and expand later to occupy that area and do not extend beyond it. CONCLUSION: Our findings indicate that abnormal metabolic handling of luteal pigment and physical changes giving rise to increased reflectance are widespread in the macula throughout the natural history of the disease, precede other changes, and are relevant to early diagnosis.

Background/AimMacular pigment optical density (MPOD) is centrally depleted early on in macular telangiectasia type 2 (MacTel). Contrast sensitivity (CS) might be related to MPOD, and thus impaired in early MacTel. The effect of low luminance was assessed on both CS and best corrected visual acuity (BCVA).MethodsThis is a cross-sectional study. Pelli-Robson charts were used for CS testing at 1 m in photopic (110 lux) and mesopic (1 lux) conditions. BCVA was tested with ETDRS charts and low luminance visual acuity (LLVA) with a 2.0 log unit neutral density filter. MPOD was obtained with dual-wavelength autofluorescence.ResultsOne hundred and three eyes of 52 patients with MacTel (mean±SD age 62.9±10.2, range 35–77) were compared with 34 healthy eyes of 17 controls (mean±SD age 65.2±7.4, range 53–78). CS was significantly lower in the eyes with MacTel. This impairment was higher in low light conditions (low light contrast sensitivity (LL-CS)). Eyes at the early stages of MacTel had significantly lower LL-CS than controls, but normal (photopic) CS. The results were similar but less pronounced for BCVA/LLVA. Decrease in CS was correlated with loss of MPOD.ConclusionsLow light conditions have a detrimental effect on visual performance in MacTel. Impaired CS might correlate with MPOD depletion as a pathognomonic finding in MacTel. Functional impairment might precede structural disintegration, indicating dysfunction at the cellular level. The applied tests might be useful as additional functional assessments in clinical routine and as outcome measures in future interventional clinical trials.

PURPOSE To investigate the electrophysiological features of macular telangiectasia Type 2 and their relationship to structure as determined by optical coherence tomography imaging. METHODS Forty-two eyes from 21 patients enrolled in the Macular Telangiectasia Natural History Observation Study were reviewed. All patients had full-field and pattern electroretinography (ERG; PERG) with some patients additionally having multifocal electroretinography (mfERG; N = 15) or electrooculography (N = 12). Multiple linear regression modeling assessed the relationship between the ellipsoid zone break size on optical coherence tomography and the central mfERG response. RESULTS Full-field ERG and electrooculography were normal in all eyes. Six eyes (14%) from five patients had subnormal PERG P50 amplitudes. Twenty-two of 30 eyes (73%) had reduced central or paracentral stimulus on mfERG. There was a significant correlation between ellipsoid zone break size and both the P1 amplitude (R = 0.37, P = 0.002) and P1:N1 ratio (R = 0.32, P = 0.002) of the central response on mfERG. CONCLUSION The electrophysiological findings in macular telangiectasia Type 2 are those of localized central dysfunction and are consistent with the structural data available from imaging and histologic studies. The ellipsoid zone break size correlates with mfERG reduction. The reduced mfERG P1:N1 ratio is consistent with inner retinal dysfunction.

Purpose We provide a proof of concept for the detailed characterization of retinal capillary features and surrounding photoreceptor mosaic using a customized nonadaptive optics angiography imaging system. Methods High-resolution fluorescein angiography (FFA) and/or indocyanine green angiography (ICGA) images were obtained using a modified Heidelberg retina angiograph (HRA2) device with a reduced scan angle enabling 3° field of view. Colocalized images of the photoreceptor mosaic also were captured in vivo using the same instrument. Visibility of vascular subbranches were compared between high-resolution images and conventional fundus angiography (FA) with a 30° field of view. Results High-resolution angiographic and infrared images (3° × 3° field of view, a 10-fold magnification) were obtained in 10 participants. These included seven patients with various retinal diseases, including myopic degeneration, diabetic retinopathy, macular telangiectasia, and central serous chorioretinopathy, as well as three healthy controls. Images of the retinal vasculature down to the capillary level were obtained on angiography with the ability to visualize a mean 1.2 levels more subbranches compared to conventional FA. In addition, imaging of the photoreceptor cone mosaic, to a sufficient resolution to calculate cone density, was possible. Movement of blood cells within the vasculature also was discernible on infrared videography. Conclusions This exploratory study demonstrates that fast high-resolution angiography and cone visualization is feasible using a commercially available imaging system. Translational relevance This offers potential to better understand the relationship between the retinal neurovascular system in health and disease and the timing of therapeutic interventions in disease states.

Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.

Rationale: The dramatic upregulation of αvβ3-integrin that occurs in the vasculature during tumor growth has long suggested that the endothelial expression of this molecule is an ideal target for antiangiogenic therapy to treat cancer. This discovery led to the development of small-molecule inhibitors directed against αvβ3-integrin that are currently in clinical trials. In 2002, we reported that β3-integrin−knockout mice exhibit enhanced tumor growth and angiogenesis. However, as β3-integrin is expressed by a wide variety of cells, endothelial cell–specific contributions to tumor angiogenesis are muddied by the use of a global knockout of β3-integrin function. Objective: Our aim was to examine the endothelial-specific contribution β3-integrin makes to tumor growth and angiogenesis. Methods and Results: We have crossed β3-integrin–floxed (β3-floxed) mice to 2 endothelial-specific Cre models and examined angiogenic responses in vivo, ex vivo, and in vitro. We show that acute depletion of endothelial β3-integrin inhibits tumor growth and angiogenesis preventatively, but not in already established tumors. However, the effects are transient, and long-term depletion of the molecule is ineffective. Furthermore, long-term depletion of the molecule correlates with many molecular changes, such as reduced levels of focal adhesion kinase expression and a misbalance in focal adhesion kinase phosphorylation, which may lead to a release from the inhibitory effects of decreased endothelial β3-integrin expression. Conclusions: Our findings imply that timing and length of inhibition are critical factors that need to be considered when targeting the endothelial expression of β3-integrin to inhibit tumor growth and angiogenesis.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies.

Purpose: Retinal ischemia has been traditionally assessed by fluorescein angiography, visualizing perfused vessels. However, this method does not provide any information about nonperfused vessels, and although it is often assumed that vessels in ischemic areas regress, we know little about how nonperfused retinal vessels change over time. Here, we aim to learn more about the long-term fate of nonperfused vessels in the retinal vasculature.\ud \ud Methods: Optical coherence tomography (OCT) was used to visualize perfusion as well as structural properties of the retinal vasculature in patients suffering from retinal vascular occlusions. In addition, postmortem tissue from a patient with long standing (6 years) central retinal vein occlusion (CRVO) was investigated, using immunohistochemistry on whole-mount retina and paraffin sections to visualize blood vessel components.\ud \ud Results: Comparing OCT angiography with enface OCT images revealed that in ischemic areas of the retina, nonperfused, larger vessels could be detected as hyperreflective structures in enface OCT images. Furthermore, analysis of a postmortem tissue sample from a CRVO patient with a large nonperfused region in the macula, revealed preservation of the basement membrane from all retinal vessels, including nonperfused, acellular vessels of all calibers.\ud \ud Conclusions: Our data suggests long-term preservation of vascular basement membrane in ischemic retina. This has implications for therapeutic approaches aiming to alleviate retinal ischemia via the regeneration of damaged vessels.

There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease.

Tissues with high metabolic rates often use lipid as well as glucose for energy, conferring a survival advantage during feast and famine.1 Current dogma suggests that high-energy consuming photoreceptors depend on glucose.2,3 Here we show that retina also uses fatty acids (FA) β-oxidation for energy. Moreover, we identify a lipid sensor Ffar1 that curbs glucose uptake when FA are available. Very low-density lipoprotein receptor (VLDLR), expressed in tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived FA.4,5 Vldlr is present in photoreceptors.6 In Vldlr−/− retinas, Ffar1, sensing high circulating lipid levels despite decreased FA uptake5, suppresses glucose transporter Glut1. This impaired glucose entry into photoreceptors results in a dual lipid/glucose fuel shortage and reduction in the Krebs cycle intermediate α-ketoglutarate (KG). Low α-KG levels promote hypoxia-induced factor-1α (Hif1a) stabilization and vascular endothelial growth factor (Vegfa) secretion by starved Vldlr−/− photoreceptors, attracting neovessels to supply fuel. These aberrant vessels invading normally avascular photoreceptors in Vldlr−/− retinas are reminiscent of retinal angiomatous proliferation (RAP), a subset of neovascular age-related macular degeneration (AMD)7, associated with high vitreous VEGF levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in neovascular AMD and other retinal diseases.

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller cell ablation and the consequences of primary Müller cell dysfunction have been studied in adult mice. We found that selective ablation of Müller cells led to photoreceptor apoptosis, vascular telangiectasis, blood-retinal barrier breakdown and, later, intraretinal neovascularization. These changes were accompanied by impaired retinal function and an imbalance between vascular endothelial growth factor-A (VEGF-A) and pigment epithelium derived factor. Intravitreal injection of cilliary neurotrophic factor inhibited photoreceptor injury but had no effect on the vasculopathy. Conversely, inhibition of VEGF-A activity attenuated vascular leak but did not protect photoreceptors. Our findings show that Müller glial deficiency may be an important upstream cause of retinal neuronal and vascular pathologies in retinal diseases. Combined neuroprotective and anti-angiogenic therapies may be required to treat Müller cell deficiency in retinal diseases and in other parts of the central nervous system associated with glial dysfunction.

Retinal pathologies are frequently accompanied by retinal vascular responses, including the formation of new vessels by angiogenesis (neovascularization). Pathological vascular changes may also include less well characterized traits of vascular remodeling that are non-neovascular, such as vessel pruning and the emergence of dilated and tortuous vessel phenotypes (telangiectasis). The molecular mechanisms underlying neovascular growth versus non-neovascular remodeling are poorly understood. We therefore undertook to identify novel regulators of non-neovascular remodeling in the retina by using the dystrophic Royal College of Surgeons (RCS) rat and the retinal dystrophy 1 (RD1) mouse, both of which display pronounced non-neovascular remodeling. Gene expression profiling of isolated retinal vessels from these mutant rodent models and wild-type controls revealed 60 differentially expressed genes. These included the genes for apelin (Apln) and for its receptor (Aplnr), both of which were strongly up-regulated in the mutants. Crossing RD1 mice into an Apln-null background substantially reduced vascular telangiectasia. In contrast, Apln gene deletion had no effect in two models of neovascular pathology [laser-induced choroidal neovascularization and the very low density lipoprotein receptor (Vldlr)-knockout mouse]. These findings suggest that in these models apelin has minimal effect on sprouting retinal angiogenesis, but contributes significantly to pathogenic non-neovascular remodeling.

We used the comparative proteomic technique iTRAQ coupled with offline 2DLC-MS/MS to analyze a rare specimen of the poorly understood, potentially blinding ophthalmic condition Macular Telangiectasia type 2 (MacTel type 2). We refined the technique using an internal standard consisting of pooled samples for each iTRAQ experiment to allow for multiple comparisons between different regions of the retina and different tissue donors. A total of 594 nonredundant proteins were identified in the retina and 168 in the vitreous, of which approximately half were found in significantly different abundance in the various comparisons made. The most prominent differences were found within the glycolytic pathway, where 8 proteins were reduced in the diseased macula compared with peripheral retina of the same eye, and 10 were also reduced in comparison with the macula of a control eye. Furthermore, Müller cell-associated proteins, including GFAP, VIME, and GLNA, were also reduced in the diseased macula, consistent with a link between the glycolytic pathway and Müller cells. These changes were validated by Western blotting and immunohistochemical studies. Proteomic analysis of the vitreous revealed an increase of proteins that were reduced in the retina. This supports proteomic analysis of the more easily available vitreous, which may reveal retina-specific protein changes associated with disease. Furthermore, our study has highlighted changes in the glycolytic pathway as a possible component of MacTel type 2 pathobiology.

Objectives The ultrastructural appearance of retinal capillaries can yield important information about disease mechanisms, but is not well characterised in human post mortem samples. We therefore aimed to create a baseline for the appearance of capillaries and establish how this is influenced by post mortem fixation delays and donor age. Methods Electron microscopy was used to characterise retinal capillaries in 20 anonymous donors (with no known eye diseases) of various ages and with various post mortem fixation delays. In addition, samples from six patients with conditions that are known to affect the retinal vasculature (four cases of type 2 diabetes without diabetic retinopathy, one case of diabetic retinopathy and one case of macular telangiectasia type 2) were analysed. Results Vacuoles were found in capillary basement membranes at the vessel–glia interface in all samples, from both the normal and disease cases. Vacuole frequency increased with donor age but was not influenced by post mortem fixation delays. Conclusion Vacuoles in the basement membrane are a normal feature of adult human retinal capillaries and do not indicate disease. Their incidence increases with age and might be a contributing factor to late-onset pathologies of the retinal vasculature.

Ischaemic vascular disease in the retina may either leave retina permanently ischaemic with slow degradation of vision, or alternatively lead to proliferative vascular disease, which can also destroy vision. To investigate the molecular and cellular mechanisms that contribute to this pathology a mouse model has been studied extensively. The model is based on the exposure of mouse pups to hyperoxia during a phase when their retinal vasculature is still developing. This leads to capillary depletion, and upon return to room air, results in retinal ischaemia and proliferative vascular disease in the retinal vasculature (oxygen-induced retinopathy (OIR)). Numerous studies using this OIR model have revealed that the regulation of angiogenic factors and the influence of inflammatory cells play a pivotal role in the vascular pathogenesis. It has also been demonstrated in the OIR model that proliferative vascular disease is not the only possible outcome of ischaemia-induced angiogenesis in the retina, but that ischaemic areas in the retina can be revascularised with healthy blood vessels. Therefore, understanding the factors that control the balance between pathological and healthy angiogenesis in the OIR model may have important implications for human retinal ischaemic disease.

SummaryThe Notch pathway is a highly conserved signaling system that controls a diversity of growth, differentiation, and patterning processes. In growing blood vessels, sprouting of endothelial tip cells is inhibited by Notch signaling, which is activated by binding of the Notch receptor to its ligand Delta-like 4 (Dll4). Here, we show that the Notch ligand Jagged1 is a potent proangiogenic regulator in mice that antagonizes Dll4-Notch signaling in cells expressing Fringe family glycosyltransferases. Upon glycosylation of Notch, Dll4-Notch signaling is enhanced, whereas Jagged1 has weak signaling capacity and competes with Dll4. Our findings establish that the equilibrium between two Notch ligands with distinct spatial expression patterns and opposing functional roles regulates angiogenesis, a mechanism that might also apply to other Notch-controlled biological processes.

The 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/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-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 β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-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.

© 2016 Macmillan Publishers Limited. All rights reserve, Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation., The research of M.P. is supported by the Max Planck Society, the European Research Council (ERC) Starting Grant ANGIOMET (311546), the Deutsche Forschungsgemeinschaft (SFB 834), the Excellence Cluster Cardiopulmonary System (EXC 147/1), the Cluster of Excellence Macromolecular Complexes (EXC115), the Foundation Leducq Transatlantic Network (ARTEMIS), the LOEWE grant Ub-Net, and the European Molecular Biology Organization Young Investigator Programme. S.S. was supported by a PhD fellowship of the Belgian Science Policy (IWT) and I.M.A. is a recipient of a DOC-fFORTE fellowship of the Austrian Academy of Sciences. C.A.F was supported by a European Union FP7 Marie Curie Post-doctoral Fellowship and a FCT grant (IF/00412/2012). The research of K.R. is supported by an ERC Advanced Grant (268921) and C.K. received support from the National Institutes of Health K08CA090438. The research of H.G. is supported by Cancer Research UK, an ERC consolidator grant (REshape), the Foundation Leducq Transatlantic Network (ARTEMIS), the Lister Institute of Preventive Medicine and the British Council under the BIRAX Initiative. The work of P.C. is funded by long-term structural funding: Methusalem Funding by the Flemish Government, ERC Advanced Research Grant (269073), and FWO G.0834.13N from the Flanders Science Fund.

Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3)/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma. We thank Bart Vanhaesebroeck (UCL Cancer Institute, London) for p110 alphaflox and p110 alphaD933A mice, Dimitris Kioussis (National Institute for Medical Research, London) for Vav-Cre mice, Erwin Wagner for Vegfr2flox mice, Ian Roswell and the transgenic services at the LRI for help with establishing mouse lines, Henrik Ortsater for assistance with experiments, and staff at the LRI and Uppsala University animal units for animal husbandry. We also thank the light microscopy unit at the LRI and BioVis at Uppsala University for advice and help with experiments. This study was supported by Cancer Research UK (L.S., I.M.-C., and T.M.); EMBO Young Investigator Programme, the Kjell and Marta Beijer Foundation, and the Swedish Research Council (T.M.); Fundacion Alfonso Martin Escudero (I.M.-C.); Spanish Ministry of Education (FECYT grant, postdoctoral mobility contract EDU/2934/2009; B.L.); MRC G0501711 (M.F.); Deutsche Forschunsgemeinschaft (DFG SA 137/1-3; D.S.); Ministry of Science and Innovation of Spain (grants BIO2009-09488 and SAF2010-18765 to S.O. and grant SAF2010-15661 to M.G.); European Research Council (ERC-2010-AdG-268804 to K.A. and ERC-2011-AdG-294556 to C.B.); Knut and Alice Wallenberg Foundation and the Swedish Cancer Foundation (C.B.); the Leducq Foundation (11CVD03) (K.A.); and the Marie Curie ITN (Vessel, FP7-PEOPLE-2012-ITN; K.A., R.H.A., C.B., and M.G.). Sí

The mechanisms that control differentiation of immature blood vessels into either arteries or veins are not well understood. Because oxygen tension in arteries is higher than in veins, oxygen has the potential to be an instructive signal for artery/vein (AV) differentiation. We test this hypothesis by exposing newborn mice to moderate hypoxia (10% atmospheric oxygen) and studying AV differentiation in the developing retinal vasculature. Forming retinal arteries fail to express the artery-specific markers Delta-like 4 (Dll4) and EphrinB2 during hypoxia. However, other aspects of AV differentiation are retained such as high levels of alpha smooth muscle actin in arterial mural cells and vein-specific expression of the msr/apj gene. The capillary network between arteries and veins is denser, and capillaries expressing the venous marker msr/apj are found in territories normally occupied by arterial capillaries. Thus, it appears that high oxygen in arterial blood is required for arterial expression of Dll4 and EphrinB2, which could be involved in cell-cell repulsion pathways that dictate the normal segregation of arteries and veins.

During vascular development, Notch signalling plays important roles in cell-cell communication and cell fate decisions. We studied expression of Notch 1-4 and its ligand Delta-like 4 (Dll4) in the developing retinal vasculature. Dll4 mRNA is strongly expressed in endothelial cells at the very tips of growing vessels ('tip cells') and also in arteries, where it is expressed in a segmented 'tiger's tail' pattern. This implies that developing retinal arteries contain different types of endothelial cells, Dll4-positive and Dll4-negative. The Dll4-positive stripes do not correspond to any obvious morphological property of the vascular network but correlate to some extent with the distribution of platelet derived growth factor B (PDGF-B) mRNA. However, PDGF-B expression is neither as artery-specific nor as clearly segmented as Dll4. Possible target cells for Dll4 signalling are retinal astrocytes (Notch1 positive), arterial pericytes (Notch3 positive) or arterial endothelial cells themselves (Notch4 positive). However, there is no clear reciprocity of Notch and Dll4 expression that allows identification of the interacting cells. Nevertheless, Dll4 stripes are a novel property of immature arteries, the origin and function of which remain to be explained.

To design therapies for demyelinating diseases such as multiple sclerosis, it will be important to understand the mechanisms that control oligodendrocyte progenitor cell (OPC) numbers in the adult central nervous system (CNS). During development, OPC numbers are limited by the supply of platelet-derived growth factor-A (PDGF-A). Here, we examine the role of PDGF-A in regulating OPC numbers in normal and demyelinated adult CNS using transgenic mice that overexpress PDGF-A in astrocytes under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-PDGF-A mice). In adult GFAP-PDGF-A mice, there was a marked increase in OPC density, particularly in white matter tracts, indicating that the PDGF-A supply controls OPC numbers in the adult CNS as well as during development. To discover whether increasing PDGF expression increases the number of OPCs following demyelination and whether this enhances the efficiency of remyelination, we induced demyelination in GFAP-PDGF-A transgenic mice by intraspinal injection of lysolecithin or dietary administration of cuprizone. In both demyelinating models, OPC density within lesions was significantly increased compared to wild-type mice. However, morphological analysis of lysolecithin lesions did not reveal any difference in the time course or extent of remyelination between GFAP-PDGF-A and wild-type mice. We conclude that the availability of OPCs is not rate limiting for remyelination of focal demyelinated lesions in the mouse. Nevertheless, our experiments show that it is possible to increase OPC population density in demyelinated areas by artificially increasing the supply of PDGF.

Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondriarelated genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants. Copyright

The murine VEGF gene is alternatively transcribed to yield the VEGF(120), VEGF(164), and VEGF(188) isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF(164/164) mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF(120/120) mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF(188/188) mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF(164), was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

PURPOSE: Retinal iron accumulation is observed in a wide range of retinal degenerative diseases, including AMD. Previous work suggests that Muller glial cells may be important mediators of retinal iron transport, distribution, and regulation. A transgenic model of Muller cell loss recently demonstrated that primary Muller cell ablation leads to blood–retinal barrier leakage and photoreceptor degeneration, and it recapitulates clinical features observed in macular telangiectasia type 2 (MacTel2), a rare human disease that features Muller cell loss. We used this mouse model to determine the effect of Muller cell loss on retinal iron homeostasis. METHODS: Changes in total retinal iron levels after Muller cell ablation were measured using inductively coupled plasma mass spectrometry. Corresponding changes in the expression of iron flux and iron storage proteins were determined using quantitative PCR, Western analysis, and immunohistochemistry. RESULTS: Muller cell loss led to blood–retinal barrier breakdown and increased iron levels throughout the neurosensory retina. There were corresponding changes in mRNA and/or protein levels of ferritin, transferrin receptor, ferroportin, Zip8, and Zip14. There were also increased iron levels within the RPE of retinal sections from a patient with MacTel2 and both RPE and neurosensory retina of a patient with diabetic retinopathy, which, like MacTel2, causes retinal vascular leakage. CONCLUSION: This study shows that Muller cells and the blood–retinal barrier play pivotal roles in the regulation of retinal iron homeostasis. The retinal iron accumulation resulting from blood–retinal barrier dysfunction may contribute to retinal degeneration in this model and in diseases such as MacTel2 and diabetic retinopathy.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and thus contributes to many vasoproliferative retinopathies including retinopathy of prematurity. Based on the importance of canonical transient receptor potential (TRPC) channels in VEGF signaling, we firstly evaluated the expression of TRPC channels in mouse retina by reverse transcriptase-polymerase chain reaction. All seven TRPC channels were expressed in mouse retina. TRPC4 channels were chosen for further analysis based on their upregulation on hypoxic retina according to the GEO database under the identifier GSE19886. Interestingly, TRPC4 suppression by intravitreal injection of siRNA against mTRPC4 significantly inhibited retinal neovascularization. To further investigate the effect of TRPC4 suppression on neovascularization, human retina microvascular endothelial cells (HRMECs) that are responsible for initiating neovascularization in response to increased VEGF in OIR retina were transfected with siRNA against TRPC4. As we have expected, suppression of TRPC4 effectively inhibited VEGF-induced migration and tube formation as well. Further evaluation on VEGF signaling pathway by western blot analysis of signaling molecules discovered that VEGF-induced activation of ERK, p38 MAPK and AKT signaling pathways were inhibited by suppression of TRPC4. These findings suggest that suppression of TRPC4 could be an alternative therapeutic option for VEGF-induced retinal neovascularization.

Development and stabilization of a vascular plexus requires the coordination of multiple signalling processes. Wnt planar cell polarity (PCP) signalling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue-specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodelling and postnatal retinal vascular patterning, with a loss of two-dimensional polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signalling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

Neurodegenerative changes in the retina and retinal vasculature abnormalities are two closely linked processes. In addition, both of these processes have been linked to inflammation numerous times in humans and in model systems. We have studied Macular Telangiectasia type 2 (MacTel), which is an uncommon eye disease that is characterised by both neurodegenerative and vascular changes. However, histological analysis on post-mortem tissue provided no evidence for inflammation in the retina of MacTel patients. This demonstrates that inflammation is not necessarily linked to neurodegeneration and vascular changes. Commercial interest

During development retinal astrocytes emerge from the optic nerve as a proliferating and migrating cell population that spreads across the inner surface of the retina. This process is driven by retinal ganglion cells secreting platelet derived growth factor (PDGFA). The resulting retinal astrocyte network is essential for the developing retinal vasculature. The retinal astrocytes provide a critical substrate and trophic support via the secretion of vascular endothelial growth factor (VEGF) for the growing blood vessels. Commercial interest

Purpose To evaluate the agreement between clinical examination, spectral-domain ocular coherence tomography (SD OCT), and fluorescein angiography (FA) in diagnosing intraretinal microvascular abnormality (IRMA) and neovascularization elsewhere (NVE) and define the SD OCT features that differentiate NVEs from IRMAs. Design Retrospective study. Methods Data were collected from 23 lesions from 8 diabetic patients, seen from July 2012 through October 2013 at Moorfields Eye Hospital, United Kingdom. Main outcomes were SD OCT features and FA leakage of IRMA and neovascular complex. The agreement between 3 evaluations was analyzed by Fleiss' kappa. Results The following 5 SD OCT features significantly differentiated IRMAs from NVEs: (1) hyperreflective dots in superficial inner retina ( P = .002); (2) the outpouching of internal limiting membrane (ILM) ( P = .004); (3) the breach of ILM ( P = .004); (4) the breach of posterior hyaloid ( P = .0005); (5) hyperreflective dots in vitreous ( P = .008). The agreement was moderate between 3 evaluations (κ = 0.48, P = 7.11 × 10 −5 ) but substantial between clinical and SD OCT evaluation (κ = 0.72, P = .00055). There was no significant agreement between OCT evaluation and FA leakage (κ = 0.249, P = .232). Conclusions SD OCT will be a valuable adjunct in evaluating IRMA and NVE, since it can verify the histopathologic correlate. SD OCT provides subtle anatomic insights and may be more accurate than clinical examination or leakage on FA, our current method of diagnosing this important endpoint, which has implications in future trial design for proliferative diabetic retinopathy prevention.

Purpose Diabetic macular ischaemia (DMI) is an important cause of visual loss in patients with diabetes, but its relationship to the larger retinal vessels is unknown. We examined whether retinal vessel calibre is related to DMI. Methods Clinic-based case–control study of patients with type 2 diabetes. The presence and severity of DMI was assessed using Early Treatment Diabetic Retinopathy Study (ETDRS) protocols from fundus fluorescein angiographic (FFA) images. Custom software was used to quantify the greatest linear dimension and area of the foveal avascular zone (FAZ). Retinal vessel calibre was measured using a semi-automated software on fundus fluorescein images. Results Of 53 patients examined, 18 (34%), 18 (34%) and 17 (32%) had no/mild, moderate and severe DMI, respectively. Persons with moderate or severe DMI had narrower mean retinal arteriolar calibre than persons with no/mild DMI (140.6 μm 95% confidence interval (CI) 134.7, 146.4 versus 150.7 μm, 95% CI 142.5, 158, p = 0.04). The association remained after multivariate adjustment for age, gender, previous panretinal photocoagulation, neovascularization at the disc and elsewhere and diabetic retinopathy severity. Increased FAZ size was also associated with narrower arteriolar calibre. Retinal venular calibre and arteriole to venule ratio (AVR) were not associated with DMI. Conclusions Retinal arteriolar narrowing was associated with moderate-to-severe macular ischaemia in eyes with diabetic retinopathy. This suggests that larger vessels other than capillaries may also be associated with DMI.

Neurons synthesise and secrete many growth and survival factors but it is not usually clear whether they are released locally at the cell body or further afield from axons or axon terminals. Without this information, we cannot predict the site(s) of action or the biological functions of many neuron-derived factors. For example, can neuronal platelet-derived growth factor (PDGF) be secreted from axons and reach glial cells in nerve-fibre (white-matter) tracts? To address this question, we expressed PDGF-A in retinal ganglion neurons in transgenic mice and tested for release of PDGF from cell bodies in the retina and from axons in the optic nerve. In both the retina and optic nerve, there are glial cells that express PDGF receptor alpha (PDGFR alpha) [1] and divide in response to PDGF [2-5], so we could detect functional PDGF indirectly through the mitogenic response of glia at both locations. Expressing PDGF-A in neurons under the control of the neuron-specific enolase promoter (NSE-PDGF-A) resulted in a striking hyperplasia of retinal astrocytes, demonstrating that PDGF is secreted from the cell bodies of neurons in the retina [4]. In contrast, glial proliferation in the optic nerve was unaffected, indicating that PDGF is not released from axons. When PDGF was expressed directly in the optic nerve under the control of an astrocyte-specific promoter (GFAP-PDGF-A), oligodendrocyte progenitors hyperproliferated, resulting in a hypertrophic optic nerve. We conclude that PDGF is constitutively secreted from neuronal cell bodies in vivo, but not from axons in white-matter tracts.

Purpose To investigate the association between peripheral and central ischemia in diabetic retinopathy. Design Retrospective, cross-sectional. Methods Consecutive ultra-widefield fluorescein angiography images were collected from patients with diabetes over a 12-month period. Parameters quantified include the foveal avascular zone (FAZ) area, peripheral ischemic index, peripheral leakage index, and central retinal thickness measurements, as well as visual acuity. The peripheral ischemia or leakage index was calculated as the area of capillary nonperfusion or leakage, expressed as a percentage of the total retinal area. Results Forty-seven eyes of 47 patients were included. A moderate correlation was observed between the peripheral ischemia index and FAZ area (r = 0.49, P = .0001). A moderate correlation was also observed between the peripheral leakage index and FAZ area, but only in eyes that were laser naive (r = 0.44, P = .02). A thinner retina was observed in eyes with macular ischemia (217 ± 81.8 μm vs 272 ± 36.0 μm) ( P = .02), but not peripheral ischemia (258 ± 76.3 μm vs 276 ± 68.0 μm) ( P = .24). The relationships between different patterns of peripheral and central macular pathology and visual acuity were evaluated in a step-wise multivariable regression model, and the variables that remained independently associated were age (r = 0.33, P = .03), FAZ area (r = 0.45, P = .02), and central retinal thickness (r = 0.38, P = .01), (R 2 -adjusted = 0.36). Conclusions Ultra-widefield fluorescein angiography provides an insight into the relationships between diabetic vascular complications in the retinal periphery and central macula. Although we observed relationships between ischemia and vascular leakage in the macula and periphery, it was only macular ischemia and retinal thinning that was independently associated with a reduced visual function.

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.

Type XVIII collagen is important in the early phase of retinal vascular development and for the regression of the primary vasculature in the vitreous body after birth. We show here that the retina in Col18a1-/- mice becomes densely vascularized by anomalous anastomoses from the persistent hyaloid vasculature by day 10 after birth. In situ hybridizations revealed normal VEGF mRNA expression, but the phenotype of collagen XVIII deficient mice closely resembled that of mice expressing VEGF120 and VEGF188 isoforms only, suggesting that type XVIII collagen may be involved in VEGF function. Type XVIII collagen was found to be indispensable for angiogenesis in the eye, as also oxygen-induced neovascularization was less intense than normal in the Col18a1-/- mice. We observed a marked increase in the amount of retinal astrocytes in the Col18a1-/- mice. Whereas the retinal vessels of wild-type mice are covered by astrocytes and the regressing, thin hyaloid vessels are devoid of astrocytes, the retinal vessels in the Col18a1-/- mice were similarly covered by astrocytes but not the persistent hyaloid vessels in the vitreous body. Interestingly, double null mice lacking type XVIII collagen and its homologue type XV collagen had the persistent hyaloid vessels covered by astrocytes, including the parts located in the vitreous body. We thus hypothesize that type XV collagen is a regulator of glial cell recruitment around vessels and that type XVIII collagen regulates their proliferation.

Background— Cystic kidneys and vascular aneurysms are clinical manifestations seen in patients with polycystic kidney disease, a cilia-associated pathology (ciliopathy). Survivin overexpression is associated with cancer, but the clinical pathology associated with survivin downregulation or knockout has never been studied before. The present studies aim to examine whether and how cilia function ( Pkd1 or Pkd2 ) and structure ( Tg737 ) play a role in cystic kidney and aneurysm through survivin downregulation. Methods and Results— Cysts and aneurysms from polycystic kidney disease patients, Pkd mouse, and zebrafish models are characterized by chromosome instability and low survivin expression. This triggers cytokinesis defects and formation of nuclear polyploidy or aneuploidy. In vivo conditional mouse and zebrafish models confirm that survivin gene deletion in the kidneys results in a cystic phenotype. As in hypertensive Pkd1, Pkd2 , and Tg737 models, aneurysm formation can also be induced in vascular-specific normotensive survivin mice. Survivin knockout also contributes to abnormal oriented cell division in both kidney and vasculature. Furthermore, survivin expression and ciliary localization are regulated by flow-induced cilia activation through protein kinase C, Akt and nuclear factor-κB. Circumventing ciliary function by re-expressing survivin can rescue polycystic kidney disease phenotypes. Conclusions— For the first time, our studies offer a unifying mechanism that explains both renal and vascular phenotypes in polycystic kidney disease. Although primary cilia dysfunction accounts for aneurysm formation and hypertension, hypertension itself does not cause aneurysm. Furthermore, aneurysm formation and cyst formation share a common cellular and molecular pathway involving cilia function or structure, survivin expression, cytokinesis, cell ploidy, symmetrical cell division, and tissue architecture orientation.

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

The role for the endothelial sphingosine-1-phosphate 1 receptor (S1P 1 R) in acute kidney injury (AKI) remains unclear as germline endothelial S1P1R deletion is embryonically lethal. Here, we generated conditional endothelial S1P1R deficiency by crossing mice with floxed S1P1R with mice expressing a tamoxifen-inducible form of Cre recombinase under the transcriptional control of the platelet-derived growth factor-β ( Pdgfβ ) gene. Mice with tamoxifen-induced deletion of endothelial S1P1R had increased renal tubular necrosis, inflammation, and impaired vascular permeability, as well as exacerbated renal tubular apoptosis after ischemic AKI compared with tamoxifen-treated wild-type mice. Moreover, endothelial S1P1R deletion resulted in increased hepatic injury after ischemic AKI. As a potential mechanism for exacerbated renal injury, conditional endothelial S1P1R–null mice had markedly reduced endothelial HSP27 expression compared with wild-type mice. Cultured glomerular endothelial cells treated with a specific S1P1R antagonist (W146) for 3 days also showed reduced HSP27 expression compared with vehicle-treated cells. Finally, mice treated with W146 for 3 days also showed reduced endothelial HSP27 expression as well as exacerbated renal and hepatic injury after ischemic AKI. Thus, our studies demonstrate a protective role for endothelial S1P1R against ischemic AKI most likely by regulating endothelial barrier integrity and endothelial HSP27 expression.

Neuropilin (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.

Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.

Angiogenesis underlies the majority of eye diseases that result in catastrophic loss of vision. Recent evidence has implicated the integrins alpha v beta 3 and alpha v beta 5 in the angiogenic process. We examined the expression of alpha v beta 3 and alpha v beta 5 in neovascular ocular tissue from patients with subretinal neovascularization from age-related macular degeneration or the presumed ocular histoplasmosis syndrome or retinal neovascularization from proliferative diabetic retinopathy (PDR). Only alpha v beta 3 was observed on blood vessels in ocular tissues with active neovascularization from patients with age-related macular degeneration or presumed ocular histoplasmosis, whereas both alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR. Since we observed both integrins on vascular cells from tissues of patients with retinal neovascularization from PDR, we examined the effects of a systemically administered cyclic peptide antagonist of alpha v beta 3 and alpha v beta 5 on retinal angiogenesis in a murine model. This antagonist specifically blocked new blood vessel formation with no effect on established vessels. These results not only reinforce the concept that retinal and subretinal neovascular diseases are distinct pathological processes, but that antagonists of alpha v beta 3 and/or alpha v beta 5 may be effective in treating individuals with blinding eye disease associated with angiogenesis.

The myelin-associated glycoprotein (MAG) has been shown to be inhibitory for certain neurons in vitro (Mukhopadhyay et al. 1994; McKerracher et al. 1994). To investigate whether MAG is an inhibitory component in peripheral myelin in vivo, MAG-deficient mutant mice were cross-bred with C57BL/Wlds mice that have delayed lesion-induced myelin degeneration and axon regrowth. While in crushed nerves of C57BL/Wlds mice expressing MAG, only 16% of myelin sheaths were associated with regrowing axons, this number was doubled in MAG-deficient C57BL/Wlds mice. These observations suggest that the absence of MAG may contribute to the improved axonal regrowth in the double mutants. Therefore, degeneration of MAG-containing myelin might be an important prerequisite to optimize axonal regrowth after peripheral nerve injury.

It has recently been shown that mice deficient in the gene for myelin-associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon-myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination-induced Schwann cell proliferation and remyelination lead to the formation of so-called onion bulbs. Expression of tenascin-C, a molecule indicative of peripheral nerve degeneration, was up-regulated by axon-deprived Schwann cells and regenerating axons were occasionally seen. Myelin-associated glycoprotein thus appears to play a crucial role in the long-term maintenance of the integrity of both myelin and axons.

Schwann cells in the distal stumps of lesioned peripheral nerves strongly express the extracellular matrix glycoprotein tenascin-C. To gain insights into the relationship between Wallerian degeneration, lesion induced tenascin-C upregulation and regrowth of axons we have investigated C57BL/Wlds (C57BL/Ola) mice, a mutant in which Wallerian degeneration is considerably delayed. Since we found a distinct difference in the speed of Wallerian degeneration between muscle nerves and cutaneous nerves in 16-week-old C57BL/Wlds mice, as opposed to 6-week-old animals in which Wallerian degeneration is delayed in both, we chose the older animals for closer investigation. Five days post lesion tenascin-C was upregulated in the muscle branch (quadriceps) but not in the cutaneous branch (saphenous) of the femoral nerve in 16-week-old animals. In addition myelomonocytic cells displaying endogenous peroxidase activity invaded the muscle branch readily whereas they were absent from the cutaneous branch at this time. We could further show that it is only a subpopulation of axon-Schwann cell units (mainly muscle efferents) in the muscle branch which undergo Wallerian degeneration and upregulate tenascin-C at normal speed and that the remaining axon-Schwann cell units (mainly afferents) displayed delayed Wallerian degeneration and no tenascin-C expression. Regrowing axons could only be found in the tenascin-C-positive muscle branch where they always grew in association with axon-Schwann cell units undergoing Wallerian degeneration. These observations indicate a tight relationship between Wallerian degeneration, upregulation of tenascin-C expression and regrowth of axons, suggesting an involvement of tenascin-C in peripheral nerve regeneration.

Wnt modulates glioma vascularization by regulating PDGF-B expression., Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.