VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular 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

Neuropilin 1 Involvement in Choroidal and Retinal Neovascularisation

Purpose Inhibiting VEGF is the gold standard treatment for neovascular age-related macular degeneration (AMD). It is also effective in preventing retinal oedema and neovascularisation (NV) in diabetic retinopathy (DR) and retinal vein occlusions (RVO). Neuropilin 1 (Nrp1) is a co-receptor for VEGF and many other growth factors, and therefore a possible alternative drug target in intra ocular neovascular disease. Here we assessed choroidal and retinal NV in an inducible, endothelial specific knock out model for Nrp1. Methods Crossing Nrp1 floxed mice with Pdgfb-CreERT2 mice produced tamoxifen-inducible, endothelial specific Nrp1 knock out mice (Nrp1ΔEC) and Cre-negative, control littermates. Cre-recombinase activity was confirmed in the Ai3(RCL-EYFP) reporter strain. Animals were subjected to laser-induced CNV (532 nm) and spectral domain-optical coherence tomography (SD-OCT) was performed immediately after laser and at day 7. Fluorescein angiography (FA) evaluated leakage and postmortem lectin staining in flat mounted RPE/choroid complexes was also used to measure CNV. Furthermore, retinal neovascularisation in the oxygen induced retinopathy (OIR) model was assessed by immunohistochemistry in retinal flatmounts. Results In vivo FA, OCT and post-mortem lectin staining showed a statistically significant reduction in leakage (p<0.05), CNV volume (p<0.05) and CNV area (p<0.05) in the Nrp1ΔEC mice compared to their Cre-negative littermates. Also the OIR model showed reduced retinal NV in the mutant animals compared to wild types (p<0.001). Conclusion We have demonstrated reduced choroidal and retinal NV in animals that lack endothelial Nrp1, confirming a role of Nrp1 in those processes. Therefore, Nrp1 may be a promising drug target for neovascular diseases in the eye

Loss of Müller's cells and photoreceptors in macular telangiectasia type 2

PURPOSE: To correlate postmortem histology from a patient with macular telangiectasia (MacTel) type 2 with previously recorded clinical imaging data. DESIGN: Observational clinicopathologic case report. METHODS: The distribution of retinal blood vessels was used to map the location of serial wax sections in color fundus and optical coherence tomography (OCT) images. Fluorescent immunohistochemistry was used to visualize markers for Müller's cells (vimentin and retinaldehyde-binding protein 1), photoreceptors (L-M opsin, rhodopsin, and cytochrome oxidase 2), and the outer limiting membrane (OLM) (zonula occludens 1 and occludin). MAIN OUTCOME MEASURES: Distribution of specific markers in immunohistochemistry on retinal sections through the fovea in relation to clinical data. RESULTS: The clinically recorded region of macular pigment loss in the macula correlated well with Müller's cell depletion. The OCT data showed a loss of the photoreceptor inner segment/outer segment (IS/OS) junction in the central retina, which correlated well with rod loss but not with cone loss. Markers for the OLM were lost where Müller's cells were lost. CONCLUSIONS: We have confirmed our previous finding of Müller's cell loss in MacTel type 2 and have shown that the area of Müller's cell loss matches the area of macular pigment depletion. In this patient, the IS/OS junction seen by OCT was absent in a region where rods were depleted but cones were still present

Expression of neonatal Fc receptor in the eye

PURPOSE: The neonatal Fc receptor (FcRn) plays a critical role in the homeostasis and degradation of immunoglobulin G (IgG). It mediates the transport of IgG across epithelial cell barriers and recycles IgG in endothelial cells back into the bloodstream. These functions critically depend on the binding of FcRn to the Fc domain of IgG. The half-life and distribution of intravitreally injected anti-VEGF molecules containing IgG-Fc domains might therefore be affected by FcRn expressed in the eye. In order to establish whether FcRn-Fc(IgG) interactions may occur in the eye, we studied the mRNA and protein distribution of FcRn in postmortem ocular tissue. METHODS: We used qPCR to study mRNA expression of the transmembrane chain of FcRn (FCGRT) in retina, optic nerve, RPE/choroid plexus, ciliary body/iris plexus, lens, cornea, and conjunctiva isolated from mouse, rat, pig, and human postmortem eyes and used immunohistochemistry to determine the pattern of FcRn expression in FCGRT-transgenic mouse and human eyes. RESULTS: In all four tested species, Fcgrt mRNA was expressed in the retina, RPE/choroid, and the ciliary body/iris, while immunohistochemistry documented FcRn protein expression in the ciliary body epithelium, macrophages, and endothelial cells in the retinal and choroidal vasculature. CONCLUSIONS: Our results demonstrate that FcRn has the potential to interact with IgG-Fc domains in the ciliary epithelium and retinal and choroidal vasculature, which might affect the half-life and distribution of intravitreally injected Fc-carrying molecules

Retinal vasculature development in health and disease

Development of the retinal vasculature is based on highly coordinated signalling between different cell types of the retina, integrating internal metabolic requirements with external influences such as the supply of oxygen and nutrients. The developing mouse retinal vasculature is a useful model system to study these interactions because it is experimentally accessible for intra ocular injections and genetic manipulations, can be easily imaged and develops in a similar fashion to that of humans. Research using this model has provided insights about general principles of angiogenesis as well as pathologies that affect the developing retinal vasculature. In this review, we discuss recent advances in our understanding of the molecular and cellular mechanisms that govern the interactions between neurons, glial and vascular cells in the developing retina. This includes a review of mechanisms that shape the retinal vasculature, such as sprouting angiogenesis, vascular network remodelling and vessel maturation. We also explore how the disruption of these processes in mice can lead to pathology - such as oxygen induced retinopathy - and how this translates to human retinopathy of prematurity

Synergistic effect of vascular endothelial growth factor gene inactivation in endothelial cells and skeletal myofibres on muscle enzyme activity, capillary supply and endurance exercise in mice

NEW FINDINGS: What is the central question of this study? Dose VEGF expressed by both endothelial cells and skeletal myofibers maintain the number of skeletal muscle capillaries and regulate endurance exercise. What is the main finding and its importance? VEGF expressed by both endothelial cells and skeletal myofibers is not essential for maintaining capillary number but does contribute to exercise performance. ABSTRACT: Many chronic diseases lead to exercise intolerance, with loss of skeletal muscle capillaries. While many muscle cell types (myofibers, satellite cells, endothelial cells, macrophages and fibroblasts) express VEGF, most muscle VEGF is stored in myofibers vesicles which can release VEGF to signal VEGF receptor-expressing cells. VEGF gene ablation in myofibers or endothelial cells alone does not cause capillary regression. We hypothesized that simultaneously deleting endothelial cell (EC) and skeletal myofiber (Skm) VEGF would cause capillary regression and impair exercise performance. This was tested in adult mice by simultaneous conditional deletion of the VEGF gene (Skm/EC-VEGF-/- mice) through the use of VEGFLoxP, HSA-Cre-ERT2 and PDGFb-iCre-ERT2 transgenes. These double-deletion mice were compared to three control groups - WT, EC VEGF deletion alone and myofiber VEGF deletion alone. Three weeks after initiating gene deletion, Skm/EC-VEGF-/- mice, but not SkmVEGF-/- or EC-VEGF-/- mice, reached exhaustion 40 minutes sooner than WT mice in treadmill tests (p = 0.002). WT, SkmVEGF-/-, and EC-VEGF-/-, but not Skm/EC-VEGF-/- mice, gained weight over the three weeks. Capillary density, fiber area and capillary:fiber ratio in soleus, plantaris, gastrocnemius and cardiac papillary muscle were similar across the groups. Phosphofructokinase and pyruvate dehydrogenase activities increased only in Skm/EC-VEGF-/- mice. These data suggest that deletion of VEGF signaling simultaneously in endothelial cells and myofibers, while reducing treadmill endurance and despite compensatory augmentation of glycolysis, is not required for muscle capillary maintenance. Reduced endurance remains unexplained, but may possibly be related to a role for VEGF in controlling perfusion of contracting muscle

Spatial distribution of metabolites in the retina and its relevance to studies of metabolic retinal disorders

Introduction: The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. Objectives: To better understand the properties of different retinal areas we studied the differential distribution of metabolites across the retina. Methods: We conducted an untargeted metabolomics analysis on full-thickness punches from three different regions (macula, temporal peri-macula and periphery) of healthy primate retina. Results: Nearly half of all metabolites identified showed differential abundance in at least one comparison between the three regions. Furthermore, mapping metabolomics results from macula-specific eye diseases onto our region-specific metabolite distributions revealed differential abundance defining systemic metabolic dysregulations that were region specific. Conclusions: The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Our results may help to better understand the pathobiology of retinal diseases with region specificity

Intraretinal pigmented cells in retinal degenerative disease

PURPOSE: Invasion of pigmented cells into the retina occurs in retinal degenerative diseases, such as macular telangiectasia type 2 (MacTel) and retinitis pigmentosa (RP). These intraretinal pigmented cells may be derived from the retinal pigment epithelium (RPE), but differences and similarities between intraretinal pigmented cells and RPE have so far not been well characterised.Clinicopathologic case report. METHOD: Here, we compared intraretinal pigment cells with RPE cells by immunohistochemistry. Immunohistological stains for classic RPE markers (RPE65, CRALBP and KRT18) and blood vessel markers (lectin and collagen 4) were done on sections from postmortem eye tissue from two MacTel donors, an RP donor and a control donor. MAIN OUTCOME MEASURES: Presence of specific immunohistochemistry markers on intraretinal pigmented and RPE cells. RESULTS: We found that intraretinal pigmented cells did not express RPE65 and CRALBP, with a small subset expressing them weakly. However, they all expressed KRT18, which was also present in normal RPE cells. Interestingly, we also found clusters of KRT18-positive cells in the retina that were not pigmented. CONCLUSIONS: Our findings suggest that RPE cells invading the retina dedifferentiate (losing classic RPE markers) and can be pigmented or unpigmented. Therefore, the number of RPE cells invading the retina in retinal degenerative disease may be underappreciated by funduscopy

Damage and fluctuations induce loops in optimal transport networks

Leaf venation is a pervasive example of a complex biological network, endowing leaves with a transport system and mechanical resilience. Transport networks optimized for efficiency have been shown to be trees, i.e. loopless. However, dicotyledon leaf venation has a large number of closed loops, which are functional and able to transport fluid in the event of damage to any vein, including the primary veins. Inspired by leaf venation, we study two possible reasons for the existence of a high density of loops in transport networks: resilience to damage and fluctuations in load. In the first case, we seek the optimal transport network in the presence of random damage by averaging over damage to each link. In the second case, we seek the network that optimizes transport when the load is sparsely distributed: at any given time most sinks are closed. We find that both criteria lead to the presence of loops in the optimum state

Macular telangiectasia type 2 - Visual acuity, disease endstage and the MacTel Area. MacTel Project Report No. 8

Purpose: To report the visual acuity measures from the MacTel registry study and to investigate and describe phenotypic findings in eyes with substantial vision loss due to MacTel type 2 Design: Cross-sectional multi-center study. Subjects: Participants of the Natural History Observation (and Registry) of MacTel Study. Methods: Best–corrected visual acuity (BCVA) data, retinal imaging data and clinical data were accessed from the MacTel study databases in May 2019. Main Outcome Measures: Frequency distribution of BCVA and its relation to age. Morphological changes in eyes with very late disease stages, defined by a BCVA ≤ 20/200. Average retinal thickness of ETDRS fields on OCT. Dimensions of the area affected by MacTel (MacTel area). Results: BCVA was ≤20/50 in 37.3% and ≤20/200 in 3.8% of 4449 eyes of 2248 patients. 18.4% and 0.7% of all patients had bilateral BCVA ≤20/50 and ≤20/200, respectively. There was an asymmetry between right and left eyes (median BCVA 71 versus 74 letters), a finding supported by more advanced morphological changes in right eyes. BCVA correlated with participant’s age, but the effect size was small. If a neovascularization or macular hole was present, bilateral occurrence was frequent (33% or 17%, respectively), and BCVA was >20/200 (79% or 78% respectively) or ≥20/50 (26% or 13%, respectively). Eyes with advanced disease (BCVA ≤20/200) showed the following characteristics: 1) Atrophy of the foveal photoreceptor layer with or without associated subretinal fibrosis; 2) an affected area, termed here the “MacTel area”, limited to a horizontal diameter not exceeding the distance between the temporal optic disc margin and foveal center, and the vertical diameter not exceeding approximately 0.85 times this distance. Exceptions were eyes with large active or inactive neovascular membranes; 3) reduced retinal thickness measures within the MacTel area; and 4) less frequent retinal greying and more frequent hyperpigmentations compared to eyes with better BCVA. Conclusions: Severe vision loss is rare in MacTel and is related to photoreceptor atrophy in most people. Results indicate disease asymmetry with slightly worse vision and more advanced disease manifestation in right eyes. MacTel-related neurodegeneration does not spread beyond the limits of the “MacTel area”