Development Of Retinal Vasculature Research Articles

Macrophages play a crucial role in vascular smooth muscle cell coverage.

The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFβ-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively active Foxo1 overexpression, substantial accumulation of TGFβ1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFβ-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.

Copper oxide nanoparticles suppress retinal angiogenesis via inducing endothelial cell cuproptosis.

Background: Copper oxide nanoparticles (CuO NPs) exhibit antitumor activity; however, their potential as anantiangiogenesis agent is unknown. Materials & methods: The antiangiogenesis properties of CuO NPs were evaluated in vitro and in vivo and the underlying mechanism was examined using RNAsequencing and metabolomic analyses. Results: CuO NPs inhibited endothelial cell function in vitro. They also mitigated retinal vasculature development and alleviated pathological retinal angiogenesis in vivo. RNAsequencing and metabolomic analyses revealed that CuO NPs disrupt the tricarboxylicacid cycle and induce cuproptosis, which was further supported by evaluating cuproptosis-related metabolites and proteins. Conclusion: CuO NPs may be aneffective antiangiogenic agent for the treatment of retinal angiogenesis.

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Andrographolide suppresses hypoxia-induced embryonic hyaloid vascular system development through HIF-1a/VEGFR2 signaling pathway.

Ocular abnormalities and the development of retinal vasculature may cause postnatal retinopathy. In the past decade, tremendous progress has been made in identifying the mechanisms that regulate retina vasculature. However, the means of regulating embryonic hyaloid vasculature development is largely unknown. This study aims to determine whether and how andrographolide regulates embryonic hyaloid vasculature development. Murine embryonic retinas were used in this study. Whole mount isolectin B4 (IB4) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence staining (IF) were performed to determine whether andrographolide is critical for embryonic hyaloid vasculature development. BrdU incorporation assay, Boyden chamber migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay were performed to evaluate whether andrographolide regulates the proliferation and migration of vascular endothelial cells. Molecular docking simulation and Co-immunoprecipitation assay were used to observe protein interaction. Hypoxia conditions exist in murine embryonic retinas. Hypoxia induces HIF-1a expression; high-expressed HIF-1a interacts with VEGFR2, resulting in the activation of the VEGF signaling pathway. Andrographolide suppresses hypoxia-induced HIF-1a expression and, at least in part, interrupts the interaction between HIF-1a and VEGFR2, causing inhibiting endothelial proliferation and migration, eventually inhibiting embryonic hyaloid vasculature development. Our data demonstrated that andrographolide plays a critical role in regulating embryonic hyaloid vasculature development.

Cystathionine-γ-lyase overexpression modulates oxidized nicotinamide adenine dinucleotide biosynthesis and enhances neovascularization

ObjectiveHydrogen sulfide is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-γ-lyase (CGL). CGL-dependent hydrogen sulfide production is required for neovascularization in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have not yet been elucidated. MethodsMale whole body CGL-overexpressing transgenic (CGLTg) mice and wild-type (WT) littermates (C57BL/6J) were subjected to the hindlimb ischemia model (age, 10-12 weeks). Functional recovery was assessed via the treadmill exercise endurance test. Leg perfusion was measured by laser Doppler imaging and vascular endothelial-cadherin immunostaining. To examine the angiogenic potential, aortic ring sprouting assay and postnatal mouse retinal vasculature development studies were performed. Finally, comparative metabolomics analysis, oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) analysis, and quantitative real-time polymerase chain reaction were performed on CGLWT and CGLTg gastrocnemius muscle. ResultsThe restoration of blood flow occurred more rapidly in CGLTg mice. Compared with the CGLWT mice, the median ± standard deviation running distance and time were increased for the CGLTg mice after femoral artery ligation (159 ± 53 m vs 291 ± 74 m [P < .005] and 17 ± 4 minutes vs 27 ± 5 minutes [P < .05], respectively). Consistently, in the CGLTg ischemic gastrocnemius muscle, the capillary density was increased fourfold (0.05 ± 0.02 vs 0.20 ± 0.12; P < .005). Ex vivo, the endothelial cell (EC) sprouting length was increased in aorta isolated from CGLTg mice, especially when cultured in VEGFA (vascular endothelial growth factor A)-only media (63 ± 2 pixels vs 146 ± 52 pixels; P < .05). Metabolomics analysis demonstrated a higher level of niacinamide, a precursor of NAD+/NADH in the muscle of CGLTg mice (61.4 × 106 ± 5.9 × 106 vs 72.4 ± 7.7 × 106 area under the curve; P < .05). Similarly, the NAD+ salvage pathway gene expression was increased in CGLTg gastrocnemius muscle. Finally, CGL overexpression or supplementation with the NAD+ precursor nicotinamide mononucleotide improved EC migration in vitro (wound closure: control, 35% ± 9%; CGL, 55% ± 11%; nicotinamide mononucleotide, 42% ± 13%; P < .05). ConclusionsOur results have demonstrated that CGL overexpression improves the neovascularization of skeletal muscle on hindlimb ischemia. These effects correlated with changes in the NAD pathway, which improved EC migration.

Knowledge and practice regarding retinopathy of prematurity among paediatricians

Background: Retinopathy of prematurity, a vision threatening disease of the preterm occurs due to abnormal retinal vasculature development. Early detection and timely management, prevents blindness. Paediatricians, the primary care providers, require good knowledge of risk factors, time for screening, and referral to an ophthalmologist for their management. Objectives: To study knowledge and referral practices for retinopathy of prematurity followed by registered paediatricians in Nepal. Methods: This study was a questionnaire-based descriptive cross-sectional study among 81 paediatricians of Nepal from October 2021 to December 2021. A self-administered questionnaire was sent to all registered paediatricians in Nepal via Google Forms. The paediatricians who did not respond were excluded from the study. Descriptive statistics, frequency tables, and percentages were calculated using SPSS. Ethical clearance was obtained from Institutional Review Committee of Kathmandu Medical College. Results: Eighty-one paediatricians participated in the study. All of them were aware of retinopathy of prematurity. Eighty (98.8%) were aware that low gestational age was an important risk factor. Only 23 (28.4%) of the paediatricians stated that the first screening should be done within 30 days of life. Eighty (98.8%) paediatricians responded that retinopathy of prematurity is treatable. Lack of well-equipped transportation system for sick babies to eye centres was the major barrier reported. Conclusion: Awareness regarding risk factors, screening, and referral protocols for retinopathy of prematurity among paediatricians is needed. Multidisciplinary team, formulation and implementation of guidelines to prevent blindness secondary to retinopathy of prematurity is important.

Disruption of CD73-Derived and Equilibrative Nucleoside Transporter 1–Mediated Adenosine Signaling Exacerbates Oxygen-Induced Retinopathy

Retinopathy of prematurity (ROP) is characterized by pathologic angiogenesis in retina, and it remains a leading cause of blindness in children. Although enhanced extracellular adenosine is markedly increased in response to retinal hypoxia, adenosine acting at the A1 and A2A receptors oppositely affects pathologic angiogenesis. In the oxygen-induced retinopathy (OIR) model of ROP, we demonstrated herein that pharmacologic and genetic inactivation of CD73 (the key 5'-ectonucleotidase for extracellular generation of adenosine) did not affect normal retinal vasculature development but exacerbated intravitreal neovascularization at postnatal day (P) 17 and delayed revascularization at P21 of OIR. This exacerbated damage to retinal vessels by CD73 inactivation was associated with increased cellular apoptosis and microglial activation but decreased astrocyte function at P17 of OIR. Furthermore, pharmacologic blockade of equilibrative nucleoside transporter 1/2 (ENT1/2; bidirectional transport for controlling the balance of intracellular and extracellular adenosine) by 6-nitrobenzylthioinosine aggravated pathologic angiogenesis at P17 of OIR. Last, pharmacologic blockade of ENT1/2 and genetic inactivation of CD73 also aggravated avascular areas at the hyperoxia phase (P12) of OIR. Thus, disruption of CD73-derived extracellular adenosine or ENT1/2-mediated transport of adenosine flux across membrane aggravated the damage to retinal vessels. These findings support that adenosine is an endogenous protective regulator that limits oxygen-induced retinopathy, and enhancing extracellular adenosine signaling represents a novel neuroprotection strategy for ROP by targeting CD73 and ENT1/2 activities.

Emergent role of SARAF and store-operated Ca2+ entry in angiogenesis

Angiogenesis is a multistep process that controls endothelial cell (EC) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promotes signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated Ca2+ entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor in this process. The aim of this study is to examine the role of SARAF in angiogenesis. In vitro angiogenesis was studied using human umbilical endothelial cells (HUVECs) for tube formation assay and vessel sprouting using rat aortic ring by Matrigel assay supplemented with endothelial cell basal medium enriched with different growth factors (VEGF, FGF, b-EGF, and IGF). HUVECs migration was evaluated by wound healing assay, and HUVECs proliferation using Ki67+ marker. Ex vivo angiogenesis was examined by whole mount mice retina on P6 in neonatal mice injected with increasing concentrations of a SOCE inhibitor, GSK-7975A, on P3, P4, and P5. We observed that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as HUVEC tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice since it reduces vessel length and the number of junctions while it increases lacunarity. Moreover, we found that knockdown of SARAF using siRNA impairs VEGF-mediated [Ca2+]i increase and HUVEC tube formation, proliferation, and migration. Our data show for the first that SOCE inhibition prevents angiogenesis using different approaches and we provide evidence indicating that SARAF plays a critical role in angiogenesis.

The effect of AAV-mediated downregulation of Claudin-3 on the development of mouse retinal vasculature

Retinal vascular development is a very tightly regulated and organized process of vessel formation and regression to generate the mature vasculature system. Claudin-3 has been found to be required for the normal development of the neural retina and its vessels in zebrafish in our recent study. In this study, we investigated whether Claudin-3 played a role in the development of mouse retinal vasculature. Immunofluorescent staining was performed to detect the expression and localization of Claudin-3 in the mouse retina. Intravitreal injection of a recombinant adeno-associated virus (AAV) expressing a short hairpin RNA targeting Claudin-3 mRNA was performed to down-regulate Claudin-3 expression in retina in neonatal (Postnatal Day 3, P3) C57BL/6J mice. Retinal vessels were examined by isolectin B4 immunofluorescent staining on the whole-mount retinas and frozen retinal sections at P10. The apoptotic retinal ganglion cells (RGCs) were measured by TdT-mediated dUTP nick-end labelling (TUNEL) staining. Vascular endothelial growth factor A (VEGF-A) expression was detected by immunofluorescent staining. The protein levels of Claudin-3, VEGF-A and B cell lymphoma 2 (Bcl-2) were evaluated by Western blot at P7, P10 and P14. We found that Claudin-3 mainly expressed in the RGCs and progressively increased during the retinal development. The AAV-mediated downregulation of Claudin-3 at P3 impeded the development of retinal deep vascularization of P10 mouse, but without effect on the development of the retinal superficial plexus. Claudin-3 knockdown increased RGC apoptosis and reduced the expression of VEGF-A and Bcl-2 in the retinas. These results suggested that the downregulation of Claudin-3 induced RGC apoptosis and impeded the mouse retinal vascular development by downregulating the levels of VEGF-A and Bcl-2.

OCT-Angiography Findings in Patients with Amblyopia: Comparison between Healthy Controls, Treatment-Responsive, and Treatment-Unresponsive Amblyopic Patients.

There is no consensus on whether amblyopia affects the retinal vascular plexus and morphology. Previous studies focused on the differences between amblyopic patients and normal controls without evaluating amblyopic eyes after patching. To evaluate differences in the superficial vascular density of amblyopic eyes, normal eyes, and amblyopic eyes reaching normal BCVA after patch therapy, OCTA was used. All patients underwent a comprehensive ophthalmological examination, including visual acuity, refraction, ocular motility tests, and anterior and posterior segment examination. OCTA was performed by an expert physician using the Zeiss Cirrus 5000-HD-OCT Angioplex (Carl Zeiss, Meditec, Inc., Dublin, OH, USA). OCTA scans were performed using a 3 × 3 mm2 and 6 × 6 mm2 fovea-centered image setting. The mean outer macular vessel density in the previously amblyopic group was 19.15 ± 0.51%. This was statistically significantly higher than in both the amblyopic group (18.70 ± 1.14%) and the normal controls (18.18 ± 1.40%) (p = 0.014). The previously amblyopic group also significantly differed from both normal controls and amblyopic eyes with regards to the inner (p = 0.011), outer (p = 0.006), and full (p = 0.003) macular perfusion. Finally, linear regression analysis revealed that BCVA was linearly correlated to outer perfusion in amblyopic (p = 0.003) and ex amblyopic eyes (p < 0.001). Considering the cross-sectional nature of our study, from our results, we can only hypothesize a possible correlation between light stimulation and retinal vasculature development. However, further longitudinal studies are needed to support this hypothesis.

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

[Figure: see text].

SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.

Angiogenesis is a multistep process that controls endothelial cells (ECs) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promote signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated calcium entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor, and Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), in angiogenesis. Here, we show that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as human umbilical vein endothelial cell (HUVEC) tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice, since it reduces vessel length and the number of junctions, while it increases lacunarity. Moreover, we find that SARAF and Orai1 are involved in VEGF-mediated [Ca2+]i increase, and their knockdown using siRNA impairs HUVEC tube formation, proliferation, and migration. Finally, immunostaining and in situ proximity ligation assays indicate that SARAF likely interacts with Orai1 in HUVECs. Therefore, these findings show for the first time a functional interaction between SARAF and Orai1 in ECs and highlight their essential role in different steps of the angiogenesis process.

Endothelium-specific deletion of Nox4 delays retinal vascular development and mitigates pathological angiogenesis.

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 (Nox4EC-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.

Novel variants in familial exudative vitreoretinopathy patients with KIF11 mutations and the Genotype–Phenotype correlation

Familial exudative vitreoretinopathy (FEVR) is an inherited disease characterized by abnormal development of retinal vasculature. KIF11 mutations were identified to be associated with FEVR in recent years. The purpose of this study was to investigate novel variants and describe associated ocular and extraocular phenotypes in FEVR patients with KIF11 mutations. Herein, 417 probands with clinical diagnosis of FEVR were enrolled. Genetic testing and ophthalmic examinations were performed in all subjects, and the genotype–phenotype correlation was analyzed. Overall, KIF11 mutation was identified in nine probands (9/417, 2.2%) among the patients with FEVR phenotype. There were six males and three females whose median age was six months (range: four months to six years old) at first visit. Among the detected mutations, five (55.6%) were frameshift, two (22.2%) were missense, one (11.1%) nonsense, and one (11.1%) splicing. Seven of these KIF11 mutations were detected as novel. Four (4/9, 44.4%) of the mutations were de novo. Clinical examinations showed that: four probands presented with bilateral falciform retinal fold; two with bilateral tractional retinal detachment; one was observed tractional retinal detachment in one eye and retinal fold in the other eye; one had falciform retinal fold in one eye and chorioretinal atrophy in the other eye; one exhibited rhegmatogenous retinal detachment in the left eye. Six of the probands were detected to have microcephaly. In conclusion: Most (5/9,55.6%) of the causative mutations were frameshift, and nearly half (4/9, 44.4%) of the mutations were de novo. Most (8/9, 88.9%) patients with KIF11 mutations showed typical ocular manifestations of severe FEVR. Majority (6/9, 66.7%) of the probands had a KIF11 mutation and were detected to have microcephaly. Seven of these harbored KIF11 mutations detected to be novel.

Role of Smad4 from ocular surface ectoderm in retinal vasculature development.

To investigate how signals from lens regulate retinal vascular development and neovascularization. Le-Cre transgenic mouse line was employed to inactivate Smad4 in the surface ectoderm selectively. Standard histological and whole-mount retina staining were employed to reveal morphological changes of retinal vasculature in Smad4 defective eye. cDNA microarray and subsequent analyses were conducted to investigate the molecular mechanism underlying the vascular phenotype. Quantitative polymerase chain reaction (qPCR) was carried out to verify the microarrays results. We found that inactivation of Smad4 specifically on surface ectoderm leads to a variety of retinal vasculature anomalies. Microarray analyses and qPCR revealed that Sema3c, Sema3e, Nrp1, Tie1, Sox7, Sox17, and Sox18 are significantly affected in the knockout retinas at different developmental stages, suggesting that ocular surface ectoderm-derived Smad4 can signal to the retina and regulates various angiogenic signaling in the retina. Our data suggest that the cross-talk between ocular surface ectoderm and retina is important for retinal vasculature development, and Smad4 regulates various signaling associated with sprouting angiogenesis, vascular remodeling and maturation in the retina of mice.

Calcitriol and non-calcemic vitamin D analogue, 22-oxacalcitriol, attenuate developmental and pathological choroidal vasculature angiogenesis ex vivo and in vivo.

Aberrant ocular angiogenesis can underpin vision loss in leading causes of blindness, including neovascular age-related macular degeneration and proliferative diabetic retinopathy. Current pharmacological interventions require repeated invasive administrations, may lack efficacy and are associated with poor patient compliance and tachyphylaxis. Vitamin D has de novo anti-angiogenic properties. Here, our aim was to validate the ocular anti-angiogenic activity of biologically active vitamin D, calcitriol, and selected vitamin D analogue, 22-oxacalcitriol. Calcitriol induced a significant reduction in ex vivo mouse choroidal fragment sprouting. Viability studies in a human RPE cell line suggested non-calcemic vitamin D analogues including 22-oxacalcitriol have less off-target anti-proliferative activity compared to calcitriol and other analogues. Thereafter, the anti-angiogenic activity of 22-oxacalcitriol was demonstrated in an ex vivo mouse choroidal fragment sprouting assay. In zebrafish larvae, 22-oxacalcitriol was found to be anti-angiogenic, inducing a dose-dependent reduction in choriocapillaris development. Subcutaneously administered calcitriol failed to attenuate mouse retinal vasculature development. However, calcitriol and 22-oxacalcitriol administered intraperitoneally, significantly attenuated lesion volume in the laser-induced choroidal neovascularisation mouse model. In summary, calcitriol and 22-oxacalcitriol attenuate ex vivo and in vivo choroidal vasculature angiogenesis. Therefore, vitamin D may have potential as an interventional treatment for ophthalmic neovascular indications.

Transmembrane protein 215 promotes angiogenesis by maintaining endothelial cell survival

Sprouting angiogenesis is a major form of neovascularization of tissues suffering from hypoxia and other related stress. Endothelial cells (ECs) undergo proliferation, differentiation, programmed death, and migration during angiogenic sprouting, but the underlying molecular mechanisms regulating ECs in angiogenesis have been incompletely elucidated. Here we report that the transmembrane protein 215 (TMEM215) is involved in angiogenesis by regulating EC survival. The murine TMEM215 gene, which possesses two transcriptional starting sites as determined by 5′‐rapid amplification of complementary DNA (cDNA) ends (RACE), encodes a two‐pass TMEM. The TMEM215 transcripts were detected in ECs in addition to other tissues by quantitative reverse transcription‐polymerase chain reaction. Immunofluorescence showed that TMEM215 was expressed in the vasculature in retina, liver, and tumor, and colocalized with EC markers. We show that knockdown of TMEM215 in ECs induced strong cell death of ECs in vitro without affecting cell proliferation and migration, suggesting that TMEM215 was required for EC survival. Downregulation of TMEM215 expression compromised lumen formation and sprouting capacities of ECs in vitro. Moreover, intravitreous injection of TMEM215 small interfering RNA resulted in delayed and abnormal development of retinal vasculature with poor perfusion. These results identified TMEM215 as a novel molecule involved in angiogenesis by regulating the survival of ECs.

An FEVR-associated mutation in ZNF408 alters the expression of genes involved in the development of vasculature.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder hallmarked by an abnormal development of retinal vasculature. A missense mutation in ZNF408 (p.H455Y) was reported to underlie autosomal dominant FEVR in a large Dutch family, and ZNF408 was shown to play a role in the development of vasculature. Nonetheless, little is known about the molecular mechanism of ZNF408-associated FEVR. To investigate this, an in vitro model of ZNF408-associated FEVR was generated by overexpressing wild-type and p.H455Y ZNF408 in human umbilical vein endothelial cells. Cells overexpressing mutant ZNF408 were unable to form a capillary-like network in an in vitro tube formation assay, thereby mimicking the clinical feature observed in patients with FEVR. Intriguingly, transcriptome analysis revealed that genes involved in the development of vasculature were deregulated by the p.H455Y mutation. Chromatin immunoprecipitation showed that p.H455Y ZNF408 has reduced DNA-binding ability, as compared to the wild-type protein. The fact that the p.H455Y mutation disrupts the expression of genes important for the development of vasculature sheds further light on the molecular mechanisms underlying ZNF408-associated FEVR.

Adenosine A2A receptor antagonists act at the hyperoxic phase to confer protection against retinopathy

BackgroundRetinopathy of prematurity (ROP) remains a major cause of childhood blindness and current laser photocoagulation and anti-VEGF antibody treatments are associated with reduced peripheral vision and possible delayed development of retinal vasculatures and neurons. In this study, we advanced the translational potential of adenosine A2A receptor (A2AR) antagonists as a novel therapeutic strategy for selectively controlling pathological retinal neovascularization in oxygen-induced retinopathy (OIR) model of ROP.MethodsDeveloping C57BL/6 mice were exposed to 75% oxygen from postnatal (P) day 7 to P12 and to room air from P12 to P17 and treated with KW6002 or vehicle at different postnatal developmental stages. Retinal vascularization was examined by whole-mount fluorescence and cross-sectional hematoxylin-eosin staining. Cellular proliferation, astrocyte and microglial activation, and tip cell function were investigated by isolectin staining and immunohistochemistry. Apoptosis was analyzed by TUNEL assay. The effects of oxygen exposure and KW6002 treatment were analyzed by two-way ANOVA or Kruskal-Wallis test or independent Student’s t-test or Mann-Whitney U test.ResultsThe A2AR antagonist KW6002 (P7-P17) did not affect normal postnatal development of retinal vasculature, but selectively reduced avascular areas and neovascularization, with the reduced cellular apoptosis and proliferation, and enhanced astrocyte and tip cell functions in OIR. Importantly, contrary to our prediction that A2AR antagonists were most effective at the hypoxic phase with aberrantly increased adenosine-A2AR signaling, we discovered that the A2AR antagonist KW6002 mainly acted at the hyperoxic phase to confer protection against OIR as KW6002 treatment at P7-P12 (but not P12-P17) conferred protection against OIR; this protection was observed as early as P9 with reduced avascular areas and reduced cellular apoptosis and reversal of eNOS mRNA down-regulation in retina of OIR.ConclusionsAs ROP being a biphasic disease, our identification of the hyperoxic phase as the effective window, together with selective and robust protection against pathological (but not physiological) angiogenesis, elevates A2AR antagonists as a novel therapeutic strategy for ROP treatment.

Cross-Inhibition of Norrin and TGF-β Signaling Modulates Development of Retinal and Choroidal Vasculature.

Norrin is essential for the formation of the retinal vasculature during development and promotes its repair after damage via activation of Wnt/β-catenin signaling. Since retinal TGF-β signaling has essentially opposite effects on the retinal vasculature we investigated if and how Norrin inhibits TGF-β signaling, and vice versa. Eyes from transgenic mice with an overexpression of Norrin (βB1-Norrin) and/or active TGF-β (βB1-TGF-β1) in the lens were generated and analyzed by light microscopy, immunohistochemistry, and TUNEL. Further on, protein as well as mRNA levels were investigated by Western blot analyses and real-time RT-PCR, respectively. In βB1-TGF-β1 mice, the lack of retinal vascular development and choriocapillaris maintenance was rescued when transgenic Norrin was additionally overexpressed in the eye. In addition, retinal Wnt/β-catenin signaling and the levels of SMAD7, an inhibitor of the canonical TGF-β pathway, were substantially suppressed in retinae of βB1-TGF-β1 mice. In contrast, Norrin normalized Wnt/β-catenin signaling and SMAD7 levels in double transgenic mice. Moreover, in retinae of βB1-TGF-β1 mice, the amounts of phosphorylated SMAD3, a downstream mediator of TGF-β signaling, were increased compared to those of βB1-Norrin/βB1-TGF-β1 mice. In vitro, Norrin substantially reduced the TGF-β-mediated induction of target genes, an effect that was blocked by Dickkopf-1, a specific inhibitor of Wnt/β-catenin signaling. High amounts of TGF-β in the eye cause a substantial reduction in the activity of Wnt/β-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-β signaling.