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Background and Aim: Retinopathy of prematurity (ROP) is the major ocular problem of preterm infants that occurs with abnormal proliferation of immature retinal vessels. Although pentoxifylline (PTX) was reported to inhibit vasculogenesis and neovascularization in experimental studies, there is no clinical data about the effects of PTX treatment on the development and severity of ROP. This clinical study aimed to investigate the possible effects of PTX on the development of ROP., Materials and Methods: A single-centre retrospective study was conducted including preterm infants who were hospitalised in the neonatal intensive care unit between 2015-2017 years. Infants were divided into two groups in terms of PTX administration for adjuvant therapy, as PTX and non-PTX groups., Results: A total of 211 infants were included in the study [gestational age 29 (27-31) weeks, birth weight 1140 (960-1340) g]. From these, 97 infants (46%) were given PTX treatment. The two groups were similar in terms of demographic data and baseline clinical characteristics. Any stage of ROP was detected in 47.4% of infants in the PTX group, which was significantly higher than those in the non-PTX group (27.2%) ( p  = 0.002). The incidence of advanced-stage ROP in the PTX group (10.3%) was also higher than in the non-PTX group (2.6%) ( p  = 0.021). Repeated usage of PTX was not found to be related to the development of ROP ( p  = 0.059). The time of PTX administration was similar between the ROP and no-ROP groups (median; one vs one week, p  = 0.825). Surfactant therapy, duration of hospital stay, and PTX treatment were found as significant risk factors for ROP in the logistic regression analysis., Conclusions: In contrast to the experimental studies and also promising results of PTX treatment in some neonatal morbidities, it may be associated with increased incidence and stage of ROP.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4 EC-KO ) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

Purpose: Pathological blood vessel growth in the eye is implicated in several diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. The limits of current disease therapies have created the need to identify and characterize new antiangiogenic drugs. Here, we identify the secreted chemorepellent semaphorin-3fa (Sema3fa) as an endogenous anti-angiogenic in the eye., Methods: We generated a CRISPR/Cas9 sema3fa zebrafish mutant line, sema3faca304/304. We assessed the retinal and choroidal vasculature in both larval and adult wild-type and sema3fa mutant zebrafish., Results: We find sema3fa mRNA is expressed by the ciliary marginal zone, neural retina, and retinal pigment epithelium of zebrafish larvae as choroidal vascularization emerges and the hyaloid/retinal vasculature is remodeled. The hyaloid vessels of sema3fa mutants develop appropriately but fail to remodel during the larval period, with adult mutants exhibiting a denser network of capillaries in the retinal periphery than seen in wild-type. The choroid vasculature is also defective in that it develops precociously, and aberrant, leaky sprouts are present in the normally avascular outer retina of both sema3faca304/304 larvae and adult fish., Conclusions: Sema3fa is a key endogenous signal for maintaining an avascular retina and preventing pathologic vascularization. Furthermore, we provide a new experimentally accessible model for studying choroid neovascularization (CNV) resulting from primary changes in the retinal environment that lead to downstream vessel infiltration.

Background: To prospectively investigate retinal vascular changes in patients undergoing epiretinal membrane (ERM) and internal limiting membrane (ILM) peeling using swept source optical coherence tomography angiography (SSOCTA)., Methods: Consecutive patients were grouped based on ERM severity and followed using SSOCTA up to month 3 after surgical intervention. Superficial and deep foveal avascular zone (s/dFAZ) as well as foveal and parafoveal vessel density (VD) were correlated with ERM severity and visual acuity. Differences between groups were evaluated., Results: Significant correlations were found between ERM severity and baseline sFAZ, dFAZ and best corrected visual acuity (BCVA), central retinal subfield thickness (CST) and ΔCST (r = -0.52, r = -0.43, r = -0.42, r = 0.58, r = 0.39; all p<0.05). Vascular flow parameters did not correlate with age, peeling size, pseudophakia or CST, but correlated with intraretinal cysts presence. No associations of BCVA with any of the OCTA parameters across time were found. Significant differences between ERM severity groups 1 and 2 were found for sFAZ at baseline (p = 0.005) and at the 3-month follow-up (p = 0.014), and for dFAZ at baseline (p = 0.017). Superficial foveal and parafoveal VD were not significantly different between groups (all p>0.05)., Conclusions: This study clearly shows that ERM severity based on ERM staging has to be taken into account when undertaking studies in patients with idiopathic ERM using SSOCTA. Further, specific changes in the superficial and deep retinal vasculature in eyes undergoing ERM and ILM peeling were found. However, the clinical usefulness and prognostic value for post-surgical treatment BCVA of the SSOCTA-derived variables (sFAZ and dFAZ area, as well as foveal and parafoveal VD) used remains questionable., Competing Interests: The authors have declared that no competing interests exist.

The hormone prolactin (PRL) is emerging as an important regulator of ocular blood vessels. PRL is pro-angiogenic and acquires anti-angiogenic properties after undergoing proteolytic cleavage to the PRL fragment, vasoinhibin. The vascularisation of the rodent retina develops after birth when it rapidly expands until completion at the end of the first postnatal week. Exposure of newborn mice to high oxygen levels lowers the rate of blood vessel growth. In the present study, we investigated whether PRL treatment modifies the vascularisation of the retina in newborn mice exposed to high oxygen or to normoxia and whether the retinal conversion of PRL to vasoinhibin may be altered in the neonate. Newborn mice and their nursing mothers were subjected to 75% oxygen or to normoxia from postnatal day (P) 6 to P8 (group 1) or from P2 to P5 (group 2). PRL (2 µg g -1 , i.p., twice a day) or vehicle was injected from P5 to P8 in group 1 and from P1 to P5 in group 2. PRL treatment reduced the retinal inhibition of blood vessel growth and the increase in vascular regression induced by hyperoxia as revealed by immunofluorescence staining of blood vessels and the expression of angiogenesis and apoptosis markers. The pro-angiogenic effect may involve a reduced conversion of PRL to vasoinhibin. Incubation of PRL with retinal extracts showed reduced activity of the PRL-cleaving protease, cathepsin D, in the neonate vs the adult retina that was further reduced under hyperoxia. PRL and the PRL receptor mRNA were expressed at higher levels in the retina at P8 than in the adult, whereas endogenous PRL was undetectable in the circulation at P8. We conclude that PRL has a pro-angiogenic effect in the neonate retina as a result of its reduced conversion to vasoinhibin and that PRL produced by the retina may help promote physiological vascularisation after birth., (© 2020 British Society for Neuroendocrinology.)

Purpose: To quantify the macular microvasculature in healthy children of various ages by using optical coherence tomography angiography (OCTA)., Design: Prospective cross-sectional study., Methods: A total of 333 normal children from 4 to 16 years old were included. OCTA was performed on a 3- × 3-mm area centered on the macular region. Vascular density, perfusion density, fovea avascular zone (FAZ) area, FAZ perimeter, and FAZ acircularity index (AI) were measured and adjusted for axial length. Differences were compared among various ages., Results: Among the different age groups, both macular vascular density and perfusion density increased with age (P < .0001 and P = .0028, respectively). After adjustments were made for the spherical equivalent (SE) and axial length, macular vascular density was significantly associated with age (r = 0.183; P = .001) No factors were significantly correlated with the perfusion density after adjustment for the age, SE, or axial length. The FAZ area and FAZ perimeter did not change among groups of different ages. Nevertheless, the AI of FAZ in the 4.00-6.99-year-old group was smaller to that of the 13.00-15.99-year-old group (P = .03). Younger children had significantly higher rates of nonconsecutive vessels branched toward the macular center (P = .0002) and vascular loops contributing to irregular shapes of FAZ (P = .024)., Conclusions: Macular vascular density and perfusion density continuously increase with age in children. Despite the fact that FAZ area and perimeter did not change, the microstructure of FAZ pruned and tended to form a smooth and regular avascular area during development., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Humans carrying homozygous loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), develop osteoporosis and a defective retinal vasculature known as familial exudative vitreoretinopathy (FEVR) due to disruption of the Wnt signaling pathway. The purpose of this study was to use CRISPR-Cas9-mediated gene editing to create strains of Lrp5-deficient rats and to determine whether knockout of Lrp5 resulted in phenotypes that model the bone and retina pathology in LRP5-deficient humans. Knockout of Lrp 5 in rats produced low bone mass, decreased bone mineral density, and decreased bone size. The superficial retinal vasculature of Lrp5-deficient rats was sparse and disorganized, with extensive exudates and decreases in vascularized area, vessel length, and branch point density. This study showed that Lrp 5 could be predictably knocked out in rats using CRISPR-Cas9, causing the expression of bone and retinal phenotypes that will be useful for studying the role of Wnt signaling in bone and retina development and for research on the treatment of osteoporosis and FEVR.

This study investigates the hyaloid vascular regression and its relationship to the retinal and choroidal vascular developments using optical coherence tomography angiography (OCTA). Normal and oxygen-induced retinopathy (OIR) rat eyes at postnatal day 15, 18, 21, and 24 were longitudinally imaged using OCTA. At each day, two consecutive imaging for visualizing the hyaloid vasculature and the retinal and choroidal vasculatures were conducted. The hyaloid vessel volume and the retinal and choroidal vessel densities were measured. The hyaloid vessel volumes gradually decreased during the regression, although the OIR eyes exhibited large vessel volumes at all time points. A spatial relationship between persistent hyaloid vasculature and retardation of underlying retinal vascular development was observed in the OIR eyes. Furthermore, anti-vascular endothelial growth factor (VEGF) was administered intravitreally to additional OIR eyes to observe its effect on the vascular regression and development. The VEGF injection to OIR eyes showed reduced persistent hyaloid vessels in the injected eyes as well as in the non-injected fellow eyes. This study presents longitudinal imaging of intraocular vasculatures in the developing eye and shows the utility of OCTA that can be widely used in studies of vascular development and regression and preclinical evaluation of new anti-angiogenic drugs.

Ischemic proliferative retinopathy (IPR), such as proliferative diabetic retinopathy (PDR), retinal vein occlusion and retinopathy of prematurity is a major cause of vision loss. Our previous studies demonstrated that periostin (PN) and tenascin-C (TNC) are involved in the pathogenesis of IPR. However, the interactive role of PN and TNC in angiogenesis associated with IPR remain unknown. We found significant correlation between concentrations of PN and TNC in PDR vitreous humor. mRNA and protein expression of PN and TNC were found in pre-retinal fibrovascular membranes excised from PDR patients. Interleukin-13 (IL-13) promoted mRNA and protein expression of PN and TNC, and co-immunoprecipitation assay revealed binding between PN and TNC in human microvascular endothelial cells (HRECs). IL-13 promoted angiogenic functions of HRECs. Single inhibition of PN or TNC and their dual inhibition by siRNA suppressed the up-regulated angiogenic functions. Pathological pre-retinal neovessels of oxygen-induced retinopathy (OIR) mice were attenuated in PN knock-out, TNC knock-out and dual knock-out mice compared to wild-type mice. Both in vitro and in vivo, PN inhibition had a stronger inhibitory effect on angiogenesis compared to TNC inhibition, and had a similar effect to dual inhibition of PN and TNC. Furthermore, PN knock-out mice showed scant TNC expression in pre-retinal neovessels of OIR retinas. Our findings suggest that interaction of PN and TNC facilitates pre-retinal angiogenesis, and PN is an effective therapeutic target for IPR such as PDR.

Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFβ1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFβ1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFβ1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFβ1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFβ1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.

Crumbs proteins are transmembrane proteins that regulate cellular apico-basal polarity. Animals carrying mutated crb1 present retinal vascular abnormalities; this mutation is associated with progressive retinal degeneration with intraretinal cystoid fluid collection in humans. This study aimed to evaluate a potential role of crumbs proteins in retinal vascular development and maintenance. We demonstrated that crumbs homologues (CRBs) were differentially expressed and changed dramatically during mouse retinal vascular development. Intravitreal injection of CRB1 and CRB2 siRNA induced delayed development of the deep capillary plexus and premature development of the intermediate capillary plexus, resulting in disrupted vascular integrity. However, microfluidic chip assay using human retinal endothelial cells revealed that CRBs do not directly affect in vitro retinal angiogenesis. CRBs control retinal angiogenesis by regulating neuroglial vascular endothelial growth factor-A (VEGFA) and matrix metalloproteinase-3 expression. These findings demonstrate a pivotal role of CRBs in providing critical neurotrophic support through normal layered vascular network development and maintenance. This implies that preserving CRBs and restoring layered retinal vascular networks could be novel targets for preventing vision-threatening retinal diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

Aim: Evidence suggests microvascular dysfunction (wider retinal venules and narrower arterioles) in schizophrenia (SCZ) and bipolar disorder (BD). The vascular development is synchronous with neuronal development in the retina and brain. The retinal vessel trajectory is related to retinal nerve fiber layer thinning and cerebrovascular abnormalities in SCZ and BD and has not yet been examined. Hence, in this study we examined the retinal vascular trajectory in SCZ and BD in comparison with healthy volunteers (HV)., Methods: Retinal images were acquired from 100 HV, SCZ patients, and BD patients, respectively, with a non-mydriatic fundus camera. Images were quantified to obtain the retinal arterial and venous trajectories using a validated, semiautomated algorithm. Analysis of covariance and regression analyses were conducted to examine group differences. A supervised machine-learning ensemble of bagged-trees method was used for automated classification of trajectory values., Results: There was a significant difference among groups in both the retinal venous trajectory (HV: 0.17 ± 0.08; SCZ: 0.25 ± 0.17; BD: 0.27 ± 0.20; P < 0.001) and the arterial trajectory (HV: 0.34 ± 0.15; SCZ: 0.29 ± 0.10; BD: 0.29 ± 0.11; P = 0.003) even after adjusting for age and sex (P < 0.001). On post-hoc analysis, the SCZ and BD groups differed from the HV on retinal venous and arterial trajectories, but there was no difference between SCZ and BD patients. The machine learning showed an accuracy of 86% and 73% for classifying HV versus SCZ and BD, respectively., Conclusion: Smaller trajectories of retinal arteries indicate wider and flatter curves in SCZ and BD. Considering the relation between retinal/cerebral vasculatures and retinal nerve fiber layer thinness, the retinal vascular trajectory is a potential marker for SCZ and BD. As a relatively affordable investigation, retinal fundus photography should be further explored in SCZ and BD as a potential screening measure., (© 2019 The Authors. Psychiatry and Clinical Neurosciences © 2019 Japanese Society of Psychiatry and Neurology.)

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.

Purpose: To investigate the longitudinal findings of fundus features and spectral-domain optical coherence tomography (SD-OCT) to characterize the morphologic features in a mouse model of defective glutamate/aspartate transporter (GLAST -/- mice)., Materials and Methods: The fundus findings and SD-OCT images were longitudinally recorded at five time points from postnatal (P) 22 to P156 in GLAST -/- mice. As a control wild type, age-matched C57BL/6J mice were employed. The mouse retina was subdivided into five layers, and the thickness of each layer was longitudinally measured by InSight® using SD-OCT pictures. The SD-OCT findings were compared with the histologic appearances. The diameter of the retinal blood vessels was measured by the ImageJ® software program using SD-OCT images. The data were statistically compared between both age-matched mouse groups., Results: The retinal blood vessels appeared more dilated in GLAST -/- mice than in wild-type mice. This tendency was statistically significant at all time points after P44 by analyses using SD-OCT images. The ganglion cell complex (GCC) and outer nuclear layer (ONL) were significantly thinner in GLAST -/- mice at all time points after P80 than in the wild-type mice. This tendency was more clearly indicated by SD-OCT than histologic sections., Discussion: In the present study, we found for the first time the dilation of the retinal blood vessels and the thinning of the ONL in GLAST -/- mice, in addition to the thinning of the GCC., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Takayuki Gonome et al.)

Background: Aging of the human retina is accompanied by oxidative stress that exerts profound changes in the retinal neurons. It is unknown if oxidative stress influences the cellular components of the retinal vessels in some ways., Methods: We examined changes in retinal vessels in human donor eyes (age: 35-94 years; N=18) by light and transmission electron microscopy, TUNEL and immunohistochemistry for biomarkers of vascular smooth muscle cells (SMC; actin), oxidative stress (4-hydroxy 2-nonenal [HNE] and nitrotyrosine), microglia (Iba-1) and vessels (isolectin B 4 )., Results: The earliest changes in the endothelium and pericytes of capillaries are apparent from the seventh decade. With aging, there is clear loss of organelles and cytoplasmic filaments, and a progressive thickening of the endothelial and pericyte basal lamina. Loss of filaments, accumulation of lipofuscin and autophagic vacuoles are significant events in aging pericytes and SMC. Actin immunolabelling reveals discontinuity in arterial SMC layers during eighth decade, indicating partial degeneration of SMC. This is followed by hyalinization, with degeneration of the endothelium and SMC in arteries and arterioles of the nerve fibre layer (NFL) and ganglion cell layer in ninth decade. Iba-1 positive microglia were in close contact with the damaged vessels in inner retina, and their cytoplasm was rich in lysosomes. HNE immunoreactivity, but not of nitrotyrosine, was detected in aged vessels from seventh decade onwards, suggesting that lipid peroxidation is a major problem of aged vessels. However, TUNEL positivity seen during this period was limited to few arteries and venules of NFL., Conclusion: This study shows prominent age-related alterations of the pericytes and SMC of retinal vessels. These changes may limit the energy supply to the neurons and be responsible for age-related loss of neurons of the inner retina., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Neuron function relies on and instructs the development and precise organization of neurovascular units that in turn support circuit activity. However, our understanding of the molecular cues that regulate this relationship remains sparse. Using a high-throughput screening pipeline, we recently identified several new regulators of vascular patterning. Among these was the potassium channel tetramerization domain-containing protein 7 (KCTD7). Mutations in KCTD7 are associated with progressive myoclonic epilepsy, but how KCTD7 regulates neural development and function remains poorly understood. To begin to identify such mechanisms, we focus on mouse retina, a tractable part of the central nervous system that contains precisely ordered neuron subtypes supported by a trilaminar vascular network. We find that deletion of Kctd7 induces defective patterning of the adult retina vascular network, resulting in increased branching, vessel length, and lacunarity. These alterations reflect early and specific defects in vessel development, as emergence of the superficial and deep vascular layers were delayed. These defects are likely due to a role for Kctd7 in inner retina neurons. Kctd7 is absent from vessels but present in neurons in the inner retina, and its deletion resulted in a corresponding increase in the number of bipolar cells in development and increased vessel branching in adults. These alterations were accompanied by retinal function deficits. Together, these data suggest that neuronal Kctd7 drives growth and patterning of the vasculature and that neurovascular interactions may participate in the pathogenesis of KCTD7-related human diseases., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The link between in utero and early life insults and the development of chronic illness remains to be fully understood, but there is increasing data to indicate that microvasculature pathology plays an important mechanistic role. Currently available data indicate that retinal microvasculature changes are detectable in children as young as six years of age, however, there are no data for younger children. We present retinal microvasculature measurement from the first two years of life. Retinal images suitable for analysis were available from 18 infants in our proof-of-concept study. The mean and standard deviation (SD) for birth weight and gestation was 3410 (384) g and 39.1(1.4) weeks, respectively. Retinal vessel calibres were summarized as the mean(SD) central retinal arteriolar equivalent (CRAE) at six months of age was 156 (32) μm, increased to 175 (75) μm by 12 months and a slightly declined by 24 months of age to 168 (50) μm. In a similar pattern, mean(SD) central retinal venular equivalent (CRVE) at six months was 211 (19) μm, increased to 238 (25) μm by 12 months of age followed by a slight decline at 24 months of age to 222 (36) μm. The arterio-venous ratio and tortuosity index remained the same at 6, 12 and 24 months. Findings from this study could help future investigators better understand early microvasculature changes and adaptation that occur early in life., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7-P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.

The mouse retina provides an excellent model for studying angiogenesis. Recent advancements in high-throughput microscopy and image analysis provide great tools to visualize and describe the complexity of the retinal vascular architecture in a detailed and comprehensive way. Most developmental studies have focused on only a few parameters mostly in the inner-most layers that do not describe the entirety of the three-dimensional vascular network. Here, we analyzed the entire three-dimensional retinal vascular architecture and its growth and remodeling starting from the age of postnatal day 3 to 4 months in mice. We show plexus specific characteristics of the vasculature in terms of vascular tissue fraction, branching and length of the blood vessels, and distance and distribution between single capillaries. Such detailed knowledge is of particular interest, as it has become apparent that disease-specific mechanisms and treatments affect the retinal vasculature often in a plexus specific way.

Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreER TM -eGFP) is a suitable model to study the fate of retina-resident PCs., Material and Methods: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers., Results: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreER TM -eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4-P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10-P12). The number of reporter-positive PCs doubled using homozygous animals., Conclusion: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreER TM -eGFP mice and additionally by mating the NG2CreER TM with a different reporter mouse to increase the low recombination efficiency.

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Retinal microvessels can be visualized non-invasively and mirror the status of the cerebral microvasculature., Aims: To investigate whether in young children born prematurely or at term cognitive performance is related to retinal microvascular traits., Study Design, Subjects: In 93 prematurely born infants (birth weight < 1000 g) and 87 controls born at term, we measured head circumference (HC) and determined intelligence quotient (IQ) by combining matrix reasoning and spatial span (Wechsler Non-Verbal test, Dutch version) and post-processed retinal photographs using Singapore I Vessel Assessment software (version 3.6)., Outcome Measures, Results: Compared with controls, cases had smaller HC (51.7 vs 53.4 cm; p < 0.001), lower IQ (93.9 vs 109.2; p < 0.001), smaller retinal arteriolar (CRAE; 162.7 vs 174.0 μm; p < 0.001) and venular (CRVE; 234.9 vs 242.8 μm; p = 0.003) diameters and CRAE/CRVE ratio (0.69 vs 0.72; p = 0.001). A 1-SD decrease in CRAE was associated with smaller HC (-0.53 cm; p < 0.001) and lower total IQ (-3.74; p < 0.001), matrix reasoning (-1.77; p = 0.004) and spatial span (-2.03; p = 0.002). These associations persisted after adjustment for sex and age and risk factors for cognitive impairment, including blood pressure, body mass index and parental educational attainment., Conclusions: HC, total IQ, matrix reasoning and spatial span decrease with smaller retinal arteriolar diameter. Our findings suggest that maldevelopment of the cerebral microcirculation, as mirrored by the retinal microvasculature, has lasting effects on the growth of the brain and cognitive performance of prematurely born children., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis., (© 2017 Wiley Periodicals, Inc.)

Background: We investigated whether cardiometabolic risk factors measured in serum (total cholesterol [TC], high-density lipoprotein [HDL], triglyceride, leptin, insulin, glucose and insulin resistance) are associated with the retinal microvasculature, a marker of cardiovascular aging, in healthy children and adolescents. Moreover, we tested whether these associations are due to direct biological effects or more indirectly due to adiposity-related side effects., Methods: We recruited 168 healthy Flemish children (7-16 years old, 54.8% boys). Blood samples and retinal photographs were taken during clinical examinations. Arteriolar and venular vessel calibers were calculated using a semi-automated computer program. Multivariable regression models were used and adjusted for age, sex, mean arterial pressure (MAP) and alternate retinal caliber. In a second step, we adjusted for body mass index z-score (zBMI)., Results: Only continuous serum leptin was associated with retinal parameters, i.e. wider arterioles; however, this disappeared after adjustment for zBMI. Children with high cardiometabolic risk factors (>90th percentile for serum leptin, insulin and insulin resistance) had wider arterioles compared to children with lower concentrations, even after additional adjustment for zBMI. No significant associations were found with lipids., Conclusions: In this healthy population of children and adolescents, the hormones insulin and leptin and insulin resistance were associated with retinal microvasculature alterations, mainly in children with high cardiometabolic factors (>90th percentile), while lipids were not. These associations were independent of zBMI.

The neural cells and factors determining normal vascular growth are not well defined even though vision-threatening neovessel growth, a major cause of blindness in retinopathy of prematurity (ROP) (and diabetic retinopathy), is driven by delayed normal vascular growth. We here examined whether hyperglycemia and low adiponectin (APN) levels delayed normal retinal vascularization, driven primarily by dysregulated photoreceptor metabolism. In premature infants, low APN levels correlated with hyperglycemia and delayed retinal vascular formation. Experimentally in a neonatal mouse model of postnatal hyperglycemia modeling early ROP, hyperglycemia caused photoreceptor dysfunction and delayed neurovascular maturation associated with changes in the APN pathway; recombinant mouse APN or APN receptor agonist AdipoRon treatment normalized vascular growth. APN deficiency decreased retinal mitochondrial metabolic enzyme levels particularly in photoreceptors, suppressed retinal vascular development, and decreased photoreceptor platelet-derived growth factor ( Pdgfb ). APN pathway activation reversed these effects. Blockade of mitochondrial respiration abolished AdipoRon-induced Pdgfb increase in photoreceptors. Photoreceptor knockdown of Pdgfb delayed retinal vascular formation. Stimulation of the APN pathway might prevent hyperglycemia-associated retinal abnormalities and suppress phase I ROP in premature infants., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Vitamin D provides a significant benefit to human health, and its deficiency has been linked to a variety of diseases including cancer. Vitamin D exhibits anticancer effects perhaps through inhibition of angiogenesis. We previously showed that the active form of vitamin D (1, 25(OH)2D3; calcitriol) is a potent inhibitor of angiogenesis in mouse model of oxygen-induced ischemic retinopathy (OIR). Many of vitamin D's actions are mediated through vitamin D receptor (VDR). However, the role VDR expression plays in vascular development and inhibition of neovascularization by 1, 25(OH)2D3 remains unknown. Here using wild type (Vdr +/+) and Vdr-deficient (Vdr -/-) mice, we determined the impact of Vdr expression on postnatal development of retinal vasculature and retinal neovascularization during OIR. We observed no significant effect on postnatal retinal vascular development in Vdr -/- mice up to postnatal day 21 (P21) compared with Vdr +/+ mice. However, we observed an increase in density of pericytes (PC) and a decrease in density of endothelial cells (EC) in P42 Vdr -/- mice compared with Vdr +/+ mice, resulting in a significant decrease in the EC/PC ratio. Although we observed no significant impact on vessel obliteration and retinal neovascularization in Vdr -/- mice compared with Vdr +/+ mice during OIR, the VDR expression was essential for inhibition of retinal neovascularization by 1, 25(OH)2D3. In addition, the adverse impact of 1, 25(OH)2D3 treatment on the mouse bodyweight was also dependent on VDR expression. Thus, VDR expression plays a significant role during retinal vascular development, especially during maturation of retinal vasculature by promoting PC quiescence and EC survival, and inhibition of ischemia-mediated retinal neovascularization by 1, 25(OH)2D3.

Objective: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown., Approach and Results: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice., Conclusions: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis., (© 2017 American Heart Association, Inc.)

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

Mediating the expansion of vascular beds in many physiological and pathological settings, angiogenesis requires dynamic changes in endothelial cell behavior. However, the molecular mechanisms governing endothelial cell activity during different phases of vascular growth, remodeling, maturation, and quiescence remain elusive. Here, we characterize dynamic gene expression changes during postnatal development and identify critical angiogenic factors in mouse retinal endothelial cells. Using actively translating transcriptome analysis and in silico computational analyses, we determine candidate regulators controlling endothelial cell behavior at different developmental stages. We further show that one of the identified candidates, the transcription factor MafB, controls endothelial sprouting in vitro and in vivo, and perform an integrative analysis of RNA-Seq and ChIP-Seq data to define putative direct MafB targets, which are activated or repressed by the transcriptional regulator. Together, our results identify novel cell-autonomous regulatory mechanisms controlling sprouting angiogenesis.Angiogenesis is a complex process that requires coordinated changes in endothelial cell behavior. Here the authors use Ribo-tag and RNA-Seq to determine temporal profiles of transcriptional activity during postnatal retinal angiogenesis, identifying transcriptional regulators of the process.

Retinopathy of prematurity (ROP) is the leading cause of childhood blindness, but current anti-VEGF therapy is concerned with delayed retinal vasculature, eye, and brain development of preterm infants. The clinical observation of reduced ROP severity in premature infants after caffeine treatment for apnea suggests that caffeine may protect against ROP. Here, we demonstrate that caffeine did not interfere with normal retinal vascularization development but selectively protected against oxygen-induced retinopathy (OIR) in mice. Moreover, caffeine attenuated not only hypoxia-induced pathologic angiogenesis, but also hyperoxia-induced vaso-obliteration, which suggests a novel protection window by caffeine. At the hyperoxic phase, caffeine reduced oxygen-induced neural apoptosis by adenosine A 2A receptor (A 2A R)-dependent mechanism, as revealed by combined caffeine and A 2A R-knockout treatment. At the hypoxic phase, caffeine reduced microglial activation and enhanced tip cell formation by A 2A R-dependent and -independent mechanisms, as combined caffeine and A 2A R knockout produced additive and nearly full protection against OIR. Together with clinical use of caffeine in neonates, our demonstration of the selective protection against OIR, effective therapeutic window, adenosine receptor mechanisms, and neuroglial involvement provide the direct evidence of the novel effects of caffeine therapy in the prevention and treatment of ROP.-Zhang, S., Zhou, R., Li, B., Li, H., Wang, Y., Gu, X., Tang, L., Wang, C., Zhong, D., Ge, Y., Huo, Y., Lin, J., Liu, X.-L., Chen, J.-F. Caffeine preferentially protects against oxygen-induced retinopathy., (© FASEB.)

Angiogenesis and blood-brain barrier formation are required for normal central nervous system (CNS) function. Both processes are controlled by Wnt or Norrin (NDP) ligands, Frizzled (FZD) receptors, and β-catenin-dependent signalling in vascular endothelial cells. In the retina, FZD4 and the ligand NDP are critical mediators of signalling and are mutated in familial exudative vitreoretinopathy. Here, we report that NDP is a potent trigger of FZD4 ubiquitination and induces internalization of the NDP receptor complex into the endo-lysosomal compartment. Inhibition of ubiquitinated cargo transport through the multivesicular body (MVB) pathway using a dominant negative ESCRT (endosomal sorting complexes required for transport) component VPS4 EQ strongly impairs NDP/FZD4 signalling in vitro and recapitulates CNS angiogenesis and blood-CNS-barrier defects caused by impaired vascular β-catenin signalling in mice. These findings provide evidence for an important role of FZD4 endocytosis in NDP/FZD4 signalling and in CNS vascular biology and disease.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally. In recent years, studies have shown that the origins of CVD may be traced to vascular and metabolic processes in early life. Retinal vascular imaging is a new technology that allows detailed non-invasive in vivo assessment and monitoring of the microvasculature. In this systematic review, we described the application of retinal vascular imaging in children and adolescents, and we examined the use of retinal vascular imaging in understanding CVD risk in early life. We reviewed all publications with quantitative retinal vascular assessment in two databases: PubMed and Scopus. Early life CVD risk factors were classified into four groups: birth risk factors, environmental risk factors, systemic risk factors and conditions linked to future CVD development. Retinal vascular changes were associated with lower birth weight, shorter gestational age, low-fibre and high-sugar diet, lesser physical activity, parental hypertension history, childhood hypertension, childhood overweight/obesity, childhood depression/anxiety and childhood type 1 diabetes mellitus. In summary, there is increasing evidence supporting the view that structural changes in the retinal microvasculature are associated with CVD risk factors in early life. Thus, the retina is a useful site for pre-clinical assessment of microvascular processes that may underlie the future development of CVD in adulthood., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

G protein-coupled receptor (GPCR) signalling, including that involving apelin (APLN) and its receptor APLNR, is known to be important in vascular development. How this ligand-receptor pair regulates the downstream signalling cascades in this context remains poorly understood. Here, we show that mice with Apln, Aplnr or endothelial-specific Aplnr deletion develop profound retinal vascular defects, which are at least in part due to dysregulated increase in endothelial CXCR4 expression. Endothelial CXCR4 is negatively regulated by miR-139-5p, whose transcription is in turn induced by laminar flow and APLN/APLNR signalling. Inhibition of miR-139-5p in vivo partially phenocopies the retinal vascular defects of APLN/APLNR deficiency. Pharmacological inhibition of CXCR4 signalling or augmentation of the miR-139-5p-CXCR4 axis can ameliorate the vascular phenotype of APLN/APLNR deficient state. Overall, we identify an important microRNA-mediated GPCR crosstalk, which plays a key role in vascular development.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of VEGF., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy., (© 2015 AlphaMed Press.)

Vascular abnormalities in the eye are the leading cause of many forms of inherited and acquired human blindness. Loss-of-function mutations in the Wnt-binding co-receptor LRP5 leads to aberrant ocular vascularization and loss of vision in genetic disorders such as osteoporosis-pseudoglioma syndrome. The canonical Wnt-β-catenin pathway is known to regulate retinal vascular development. However, it is unclear what precise role LPR5 plays in this process. Here, we show that loss of LRP5 function in mice causes retinal hypovascularization during development as well as retinal neovascularization in adulthood with disorganized and leaky vessels. Using a highly specific Flk1-CreBreier line for vascular endothelial cells, together with several genetic models, we demonstrate that loss of endothelium-derived LRP5 recapitulates the retinal vascular defects in Lrp5-/- mice. In addition, restoring LRP5 function only in endothelial cells in Lrp5-/- mice rescues their retinal vascular abnormalities. Furthermore, we show that retinal vascularization is regulated by LRP5 in a dosage dependent manner and does not depend on LRP6. Our study provides the first direct evidence that endothelium-derived LRP5 is both necessary and sufficient to mediate its critical role in the development and maintenance of retinal vasculature.

Delayed revascularization of ischemic neural tissue is a major impediment to preservation of function in central nervous system (CNS) diseases including stroke and ischemic retinopathies. Therapeutic strategies allowing rapid revascularization are greatly needed to reduce ischemia-induced cellular damage and suppress harmful pathologic neovascularization. However, key mechanisms governing vascular recovery in ischemic CNS, including regulatory molecules governing the transition from tissue injury to tissue repair, are largely unknown. NF-E2-related factor 2 (Nrf2) is a major stress-response transcription factor well known for its cell-intrinsic cytoprotective function. However, its role in cell-cell crosstalk is less appreciated. Here we report that Nrf2 is highly activated in ischemic retina and promotes revascularization by modulating neurons in their paracrine regulation of endothelial cells. Global Nrf2 deficiency strongly suppresses retinal revascularization and increases pathologic neovascularization in a mouse model of ischemic retinopathy. Conditional knockout studies demonstrate a major role for neuronal Nrf2 in vascular regrowth into avascular retina. Deletion of neuronal Nrf2 results in semaphorin 6A (Sema6A) induction in hypoxic/ischemic retinal ganglion cells in a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent fashion. Sema6A expression increases in avascular inner retina and colocalizes with Nrf2 in human fetal eyes. Extracellular Sema6A leads to dose-dependent suppression of the migratory phenotype of endothelial cells through activation of Notch signaling. Lentiviral-mediated delivery of Sema6A small hairpin RNA (shRNA) abrogates the defective retinal revascularization in Nrf2-deficient mice. Importantly, pharmacologic Nrf2 activation promotes reparative angiogenesis and suppresses pathologic neovascularization. Our findings reveal a unique function of Nrf2 in reprogramming ischemic tissue toward neurovascular repair via Sema6A regulation, providing a potential therapeutic strategy for ischemic retinal and CNS diseases.

Vascular development and maintenance are controlled by a complex transcriptional program, which integrates both extracellular and intracellular signals in endothelial cells. Here we study the roles of three closely related SoxF family transcription factors-Sox7, Sox17, and Sox18 -in the developing and mature mouse vasculature using targeted gene deletion on a mixed C57/129/CD1 genetic background. In the retinal vasculature, each SoxF gene exhibits a distinctive pattern of expression in different classes of blood vessels. On a mixed genetic background, vascular endothelial-specific deletion of individual SoxF genes has little or no effect on vascular architecture or differentiation, a result that can be explained by overlapping function and by reciprocal regulation of gene expression between Sox7 and Sox17. By contrast, combined deletion of Sox7, Sox17, and Sox18 at the onset of retinal angiogenesis leads to a dense capillary plexus with a nearly complete loss of radial arteries and veins, whereas the presence of a single Sox17 allele largely restores arterial identity, as determined by vascular smooth muscle cell coverage. In the developing retina, expression of all three SoxF genes is reduced in the absence of Norrin/Frizzled4-mediated canonical Wnt signaling, but SoxF gene expression is unaffected by reduced VEGF signaling in response to deletion of Neuropilin1 (Npn1). In adulthood, Sox7, Sox17, and Sox18 act in a largely redundant manner to maintain blood vessel function, as adult onset vascular endothelial-specific deletion of all three SoxF genes leads to massive edema despite nearly normal vascular architecture. These data reveal critical and partially redundant roles for Sox7, Sox17 and Sox18 in vascular growth, differentiation, and maintenance.

Objective: The aim of the study was to examine the associations of birth outcomes and longitudinally measured fetal and infant growth patterns with retinal vessel calibers in childhood., Methods: In a population-based prospective cohort study among 4122 children, we measured growth characteristics in second and third trimester of pregnancy, at birth, and at 6, 12, 24, 36, 48, and 72 months. At the age of 6 years, we measured retinal arteriolar and venular calibers from digitized retinal photographs., Results: We observed that compared with term-born children, those born preterm had narrower retinal arteriolar caliber [differences -0.46 standard deviation score (95% confidence interval -0.77 to -0.15) and -0.24 standard deviation score (95% confidence interval -0.42 to -0.05) for children born <34 and 34-37 weeks of gestation, respectively]. Children born with a low birth weight (<2500  g) had narrower retinal arteriolar caliber than children with a normal birth weight, but this association was fully explained by gestational age at birth. Accelerated infant growth until 24 months was associated with narrow retinal arteriolar caliber, especially among preterm-born children (P < 0.05). Early growth measures were not associated with retinal venular caliber., Conclusion: Preterm birth and accelerated infant growth are associated with narrower retinal arteriolar caliber in childhood. Whether these microvascular adaptations explain the well known associations of fetal and infant characteristics with cardiovascular disease in later life should be further studied.

Objective: To investigate retinal maturation in premature infants (gestation age <37 weeks). using computer-assisted indirect ophthalmoscope imaging., Methods: Premature infants at postmenstrual age 33-46 weeks, who underwent fundus examinations using computer-aided indirect ophthalmoscopy, were stratified into seven postmenstrual-age groups. Images of macular morphology, peripheral retinal vascularization and fundus pigmentation were compared., Results: The study included 268 infants in the following postmenstrual-age groups: 33-34 weeks (n = 19), 35-36 weeks (n = 37), 37-38 weeks (n = 49), 39-40 weeks (n = 55), 41-42 weeks (n = 49), 43-44 weeks (n = 34), and 45-46 weeks (n = 25). The macula matured with increasing postmenstrual age. A mature macula was observed in 92% of infants at 45-46 weeks. Complete vascularization was achieved at 41-42 weeks in the nasal retina and at 43-44 weeks in the temporal retina. The number of retinas with normal pigmentation increased with postmenstrual age (rising to 84% of infants at postmenstrual age 45-46 weeks)., Conclusions: Following premature birth, macular morphology, retinal vascularization and retinal pigmentation continue to develop. This study provides reference images of normal retinal development in premature infants., (© The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Key Points: We provide evidence for two distinct functions of the NADPH oxidase Nox4 in angiogenesis using Nox4 knockout mice. First, Nox4 maintains vascular endothelial growth factor expression and prevents an increase in angiopoietin 1 expression, thereby contributing to angiogenesis in exercise. Second, deletion of Nox4, via an enhanced angiopoietin 1 expression, contributes to stabilization of new formed vessels and prevents an exacerbated neo-angiogenesis in oxygen-induced retinopathy. By contrast, Nox4 does not influence developmental angiogenesis., Abstract: By producing H2 O2 , the NADPH oxidase Nox4 is involved in hypoxia-induced angiogenesis, as present in vascular remodelling of the hypertrophic heart or blood flow recovery after hind limb ischaemia. In the present study, we hypothesized that Nox4 contributes to proper capillary growth in the retina and in exercised muscles and investigated this in wild-type and Nox4(-/-) mice. Exercise, as induced by voluntary running in a running wheel or forced running on a treadmill, stimulated capillary growth in wild-type but not Nox4(-/-) mice. As an underlying mechanism, we identified both vascular endothelial growth factor (VEGF) expression to be reduced and angiopoietin 1 (Ang1) expression to be increased in response to Nox4 knockout. To differentiate the two factors, oxygen-induced retinopathy was investigated. In this model, deletion of Nox4 protected from neo-angiogenesis and stabilized the network of regrown vessels, which is a typical feature of Ang1. However the angiogenesis in the developing retina was similar between Nox4(-/-) and wild-type mice. Thus, Nox4 contributes to exercise- and hypoxia-induced angiogenesis through a dual mechanism of maintaining VEGF and preventing Ang-1 expression, whereas the developmental angiogenesis is Nox4 independent., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Extracellular matrix (ECM) plays a central role in angiogenesis. ECM degrading enzymes breakdown the pre-existing vascular basement membrane at an early stage of angiogenesis and subsequently degrade stromal ECM as the new vessels invade into tissues. Conversely certain ECM components including collagen, fibronectin or fibrin are required for endothelial cell migration and tube morphogenesis. As the new vessels form they lay down a basement membrane that surrounds the endothelial tubes and is essential for their stability. In the rodent eye the transient expression of fibronectin and matricellular proteins plays a key role in retinal vascular development. In pathological retinal angiogenesis, such as in proliferative diabetic retinopathy, preretinal neovascularization occurs where new blood vessels invade the cortical vitreous gel and these blood vessels require vitreous collagen for their growth. The vitreous is normally anti-angiogenic and contains endogenous ECM inhibitors of angiogenesis including opticin and thombospondins, and ECM fragments such as endostatin. In preretinal neovascularization, the combined anti-angiogenic effects of these molecules are overcome by an excess of growth factors such as vascular endothelial growth factor-A, and new vessels grow into the vitreous with potentially blinding sequelae., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin β1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that β1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin β1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin β1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

Here we report that VEGF-A and IGF-1 differ in their ability to stabilize newly formed blood vessels and endothelial cell tubes. Although VEGF-A failed to support an enduring vascular response, IGF-1 stabilized neovessels generated from primary endothelial cells derived from various vascular beds and mouse retinal explants. In these experimental systems, destabilization/regression was driven by lysophosphatidic acid (LPA). Because previous studies have established that Erk antagonizes LPA-mediated regression, we considered whether Erk was an essential component of IGF-dependent stabilization. Indeed, IGF-1 lost its ability to stabilize neovessels when the Erk pathway was inhibited pharmacologically. Furthermore, stabilization was associated with prolonged Erk activity. In the presence of IGF-1, Erk activity persisted longer than in the presence of VEGF or LPA alone. These studies reveal that VEGF and IGF-1 can have distinct inputs in the angiogenic process. In contrast to VEGF, IGF-1 stabilizes neovessels, which is dependent on Erk activity and associated with prolonged activation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Angiogenesis, the formation of new blood vessels, is regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). VEGFR2 is abundant in the tip cells of angiogenic sprouts, where VEGF/VEGFR2 functions upstream of the delta-like ligand 4 (DLL4)/Notch signal transduction pathway. VEGFR3 is expressed in all endothelia and is indispensable for angiogenesis during early embryonic development. In adults, VEGFR3 is expressed in angiogenic blood vessels and some fenestrated endothelia. VEGFR3 is abundant in endothelial tip cells, where it activates Notch signaling, facilitating the conversion of tip cells to stalk cells during the stabilization of vascular branches. Subsequently, Notch activation suppresses VEGFR3 expression in a negative feedback loop. Here we used conditional deletions and a Notch pathway inhibitor to investigate the cross-talk between VEGFR2, VEGFR3, and Notch in vivo. We show that postnatal angiogenesis requires VEGFR2 signaling also in the absence of Notch or VEGFR3, and that even small amounts of VEGFR2 are able to sustain angiogenesis to some extent. We found that VEGFR2 is required independently of VEGFR3 for endothelial DLL4 up-regulation and angiogenic sprouting, and for VEGFR3 functions in angiogenesis. In contrast, VEGFR2 deletion had no effect, whereas VEGFR3 was essential for postnatal lymphangiogenesis, and even for lymphatic vessel maintenance in adult skin. Knowledge of these interactions and the signaling functions of VEGFRs in blood vessels and lymphatic vessels is essential for the therapeutic manipulation of the vascular system, especially when considering multitargeted antiangiogenic treatments.

We aimed to determine the role of age-related changes in the mammalian target of rapamycin (mTOR) activity in endothelial cell growth during retinal vascular development in mice. Mice were administered the mTOR inhibitor rapamycin as follows: (i) for 6 days from postnatal day 0 (P0) to P5, (ii) for 2 days on P6 and P7, and (iii) for 2 days on P12 and P13. For comparison, we examined the effects of KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on retinal vascular development. The retinal vasculature and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTOR activity, were evaluated using immunohistochemistry. Vascularization was delayed and capillary density was reduced in mice administered rapamycin from P0 to P5 compared to the vehicle-treated mice. Rapamycin administration on P6 and P7 decreased the vascular density but did not significantly delay the radial vascular growth. Rapamycin administration on P12 and P13 did not significantly affect the retinal superficial blood vessels. Immunoreactivity for pS6 was detected in both endothelial cells in the vascular front and non-vascular cells in the retinal parenchyma, and rapamycin markedly diminished the pS6 immunoreactivity. KRN633 administration on P0 and P1 completely inhibited retinal vascularization. The effects of KRN633 on retinal blood vessels decreased in magnitude in an age-dependent manner. These results suggest that the mTOR pathway in endothelial cells activated by VEGF contributes to physiologic vascular development, and that the mTOR pathway in endothelial cells is modulated in a postnatal age-dependent manner., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To define the role of hypoxia and vascular endothelial growth factor (VEGF) in modifying the pattern, density, and permeability of the retinal vasculature in mouse models in which Norrin/Frizzled4 signaling is impaired., Methods: Retinal vascular structure was analyzed in mice with mutation of Ndp (the gene coding for Norrin) or Frizzle4 (Fz4) with or without three additional perturbations: (1) retinal hyperoxia and reduction of VEGF, (2) reduced induction of VEGF in response to hypoxia, or (3) reduced responsiveness of vascular endothelial cells (ECs) to VEGF. These perturbations were produced, respectively, by (1) genetic ablation of rod photoreceptors in the retinal degeneration 1 (rd1) mutant background, (2) conditional deletion of the gene coding for hypoxia-inducible factor (HIF)-2alpha either in all neural retina cells or specifically in Müller glia, and (3) conditional deletion of the VEGF coreceptor neuropilin1 (NRP1) in ECs., Results: All three conditions reduced vascular proliferation. Eliminating HIF2-alpha in Müller glia blocked VEGF induction in the inner nuclear layer, identifying HIF2-alpha as the transcription factor responsible for the hypoxia response in these cells. When Norrin/Frizzled4 signaling was eliminated, a secondary elevation in VEGF levels was required to compromise the barrier to transendothelial movement of high molecular weight compounds., Conclusions: In the absence of Norrin or Frizzled4, the vascular phenotype is determined by the primary defect in Norrin/Frizzled4 signaling (i.e., canonical Wnt signaling) and compensatory responses resulting from hypoxia. This work may be useful in guiding therapeutic strategies for the treatment of familial exudative vitreoretinopathy (FEVR)., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)