Oxygen-induced Retinopathy Research Articles

Overexpression of miR-181a-5p inhibits retinal neovascularization through endocan and the ERK1/2 signaling pathway.

Retinal neovascularization (RNV) is a common pathological feature of angiogenesis‐related retinopathy. Endocan inhibition has previously been reported to suppress RNV in oxygen‐induced retinopathy (OIR); however, its molecular mechanisms remain to be elucidated. Here, we investigated the role and mechanism of endocan in OIR. We established an OIR mouse model and detected aberrant endocan overexpression in OIR mouse retinas. Endocan inhibition through small interfering RNA or a neutralizing antibody inhibited vascular endothelial growth factor‐induced cell survival, cell proliferation, and tube formation in human retinal endothelial cells in vitro and reduced the RNV area in vivo. Using RNA sequencing, a luciferase reporter assay, and bioinformatics analyses, we identified endocan as a microRNA‐181a‐5p target gene. The antiangiogenic effect of miR‐181a‐5p on RNV was verified by intravitreal injection, and we showed that this involved the extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) signaling pathway. Collectively, our data demonstrate that miR‐181a‐5p/endocan regulates retinal angiogenesis through the ERK1/2 signaling pathway and might represent an attractive therapeutic strategy for RNV.

Issue Cover (May 2020)

Journal of NeurochemistryVolume 153, Issue 3 Issue CoverFree Access Issue Cover (May 2020) First published: 27 April 2020 https://doi.org/10.1111/jnc.14743 Read the full article: ‘VEGF-Trap is a potent modulator of vasoregenerative responses and protects dopaminergic amacrine network integrity in degenerative ischemic neovascular retinopathy’ by J. E. Rojo Arias, M. Economopoulou, D. A. Juárez López, A, Kurzbach, K. H. Au Yeung, V. Englmaier, M. Merdausl, M. Schaarschmidt, M. Ader, H. Morawietz, R. H. W. Funk, J. Jászai, (J. Neurochem. 2020, vol. 153 (3), pp. 390–412) on doi: 10.1111/jnc.14875 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Front cover: Ocular microvasculopathies compromise vision in diseases as retinopathy of prematurity and proliferative diabetic retinopathy. Using the oxygen induced retinopathy mouse model, we investigated the effects of aflibercept in modulating the aberrant retinal microvascular response to ischemia. Image content: In this image, a magnified view of a flat-mounted retina from a 17 day old mouse subjected to the oxygen-induced retinopathy protocol and subsequently treated with aflibercept is shown. FITC-labeled dextran (150 kDa) was injected intra-peritoneally to animals 2 hours prior to sacrifice and was detected in the retinal microvasculature residing primarily within the vascular lumen (green). By contrast, immunolabeling of the tissue with an anti-human IgG antibody (magenta) and visualization of cell nuclei with DAPI (white) revealed that Aflibercept is also detected in blood vessel walls, suggesting it can be transported across retinal endothelial cells under ischemic conditions. Albeit slow in nature, this controlled transport might be central to achieving the functional benefits in vision we report in our study. Read the full article ‘VEGF-Trap is a potent modulator of vasoregenerative responses and protects dopaminergic amacrine network integrity in degenerative ischemic neovascular retinopathy’ by J. E. Rojo Arias, M. Economopoulou, D. A. Juárez López, A, Kurzbach, K. H. Au Yeung, V. Englmaier, M. Merdausl, M. Schaarschmidt, M. Ader, H. Morawietz, R. H. W. Funk, J. Jászai, (J. Neurochem. 2020, vol. 153 (3), pp. 390–412) on doi: 10.1111/jnc.14875 Volume153, Issue3May 2020 RelatedInformation

Vascular permeability in retinopathy is regulated by VEGFR2 Y949 signaling to VE-cadherin

Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact.

PPARα-Dependent Effects of Palmitoylethanolamide Against Retinal Neovascularization and Fibrosis

PurposePathological neovascularization and fibrosis are common pathological changes of many retinal diseases, such as proliferative retinopathy (PR) and age-related macular degeneration (AMD). Treatment modalities for these pathological changes are limited. The purpose of the present study was to test the effects of palmitoylethanolamide (PEA), an endocannabinoid mimetic amide, on retinal neovascularization and fibrosis and to determine its molecular mechanism of action.MethodsA rat Müller cell line (rMC-1), a mouse model of oxygen-induced retinopathy (OIR), and the very-low-density lipoprotein receptor (VLDLR) knockout mouse model were used. PEA was intraperitoneally injected or orally administrated in animal models. Inflammation and profibrotic changes were evaluated by western blot analysis. Glial fibrillary acidic protein (GFAP) and peroxisome proliferator-activated receptor alpha (PPARα) were measured by RT-PCR and western blot analysis.ResultsProfibrotic changes were present in OIR and Vldlr−/− retinas. PEA significantly alleviated inflammation and inhibited neovascularization in OIR and Vldlr−/− retinas and suppressed profibrotic changes in OIR and Vldlr−/− retinas. Moreover, PEA potently suppressed Müller gliosis in these retinas. In rMC-1 cells, PEA suppressed Müller gliosis, reduced inflammatory cytokines, and attenuated profibrotic changes. Further, both mRNA and protein levels of PPARα were elevated in the retina under PEA treatment, and the effects of PEA were abolished in Pparα−/− OIR mice.ConclusionsPEA reduced retinal neovascularization and fibrotic changes and suppressed Müller gliosis in experimental PR and neovascular AMD by activating PPARα. PEA may be a potential treatment for retinopathies with pathological neovascularization and fibrosis.

Hypoxia-Inducible Factor Inhibitors Derived from Marine Products Suppress a Murine Model of Neovascular Retinopathy.

Neovascular retinal degenerative diseases are the leading causes of blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat these diseases currently. However, recent reports indicate that long term suppression of VEGF in the eye is associated with chorioretinal atrophy. Therefore, a physiological amount of VEGF is required for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcriptional factor upstream of VEGF. We previously reported that HIF regulated pathological angiogenesis in the retina of murine models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Most of the known HIF inhibitors are anti-cancer agents which may have systemic adverse effects in for clinical use; thus, there is a need for safer and less invasive HIF inhibitors. In this study, we screened marine products, especially fish ingredients, and found that six species of fish had HIF inhibitory effects. Among them, administration of Decapterus tabl ingredients significantly suppressed retinal neovascular tufts by inhibiting HIF expression in a murine oxygen-induced retinopathy model. These results indicate that particular fish ingredients can act as anti-angiogenic agents in retinal neovascularization diseases.

Endothelial Yes-Associated Protein 1 Promotes Astrocyte Proliferation and Maturation via Cytoplasmic Leukemia Inhibitory Factor Secretion in Oxygen-Induced Retinopathy.

PurposePurpose The role of endothelial Yes-associated protein 1 (YAP) in the pathogenesis of retinal angiogenesis and the astrocyte network in the mouse oxygen-induced retinopathy (OIR) model is unknown.MethodsFor in vivo studies, OIR was induced in conditional endothelial YAP knockout mice and their wild-type littermates. Retinal vascularization and the astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro experiments were performed in astrocytes cultured with human microvascular endothelial cell-1–conditioned medium to analyze the mechanisms underlying the effect of endothelial YAP on astrocytes.ResultsEndothelial YAP deletion not only impaired retinal blood vessels, but also caused a sparse and disrupted astrocyte network in response to OIR. Levels of the immature astrocyte marker (platelet-derived growth factor A) in the retina were substantially increased owing to YAP deficiency, suggesting a possible failure in astrocyte maturation, whereas retinal expression of leukemia inhibitory factor (LIF) was decreased. In vitro studies suggested that loss or overexpression of YAP resulted in elevated or decreased LIF secretion by human microvascular endothelial cell-1, respectively. Increased LIF levels in the culture medium promoted astrocyte maturation and proliferation and rescued YAP inhibition-induced astrocyte loss. Finally, activating YAP could protect against the pathology of the astrocyte network and even suppress pathologic retinal vascularization in control OIR mice, but not in endothelial YAP-deficient OIR mice.ConclusionsEndothelial YAP regulation of LIF secretion is required for normalized astrocyte network formation in OIR, thereby providing a novel target for protecting the astrocyte network and thus benefiting retinal blood vessels.

ASK1-Mediated Phosphorylation Blocks HDAC6Ubiquitination and Degradation to Drive the Disassembly of Photoreceptor Connecting Cilia.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. However, the pathogenesis and molecular mechanisms underlying ROP remain elusive. Herein, using the oxygen-induced retinopathy (OIR) mouse model of ROP, we demonstrate that disassembly of photoreceptor connecting cilia is an early event in response to oxygen changes. Histone deacetylase 6 (HDAC6) is upregulated in the retina of OIR mice and accumulates in the transition zone of connecting cilia. We also show that in response to oxygen changes, apoptosis signal-regulating kinase 1 (ASK1) is activated and phosphorylates HDAC6, blocking its ubiquitination by von Hippel-Lindau and subsequent degradation by the proteasome. Moreover, depletion of HDAC6 or inhibition of the ASK1/HDAC6 axis protects mice from oxygen-change-induced pathological changes of photoreceptors. These findings reveal a critical role for ASK1/HDAC6-mediated connecting cilium disassembly in the OIR mouse model of ROP and suggest a potential value of ASK1/HDAC6-targeted agents for prevention of this disease.

Inhibition of retinal neovascularization by a PEDF-derived nonapeptide in newborn mice subjected to oxygen-induced ischemic retinopathy

Retinopathy of prematurity (ROP) is a growing cause of lifelong blindness and visual defects as improved neonatal care worldwide increases survival in very-low-birthweight preterm newborns. Advancing ROP is managed by laser surgery or a single intravitreal injection of anti-VEGF, typically at 33–36 weeks gestational age. While newer methods of scanning and telemedicine improve monitoring ROP, the above interventions are more difficult to deliver in developing countries. There is also concern as to laser-induced detachment and adverse developmental effects in newborns of anti-VEGF treatment, spurring a search for alternative means of mitigating ROP. Pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor appears late in gestation, is undetected in 25–28 week vitreous, but present at full term. Its absence may contribute to ROP upon transition from high-to-ambient oxygen environment or with intermittent hypoxia. We recently described antiangiogenic PEDF-derived small peptides which inhibit choroidal neovascularization, and suggested that their target may be laminin receptor, 67LR. The latter has been implicated in oxygen-induced ischemic retinopathy (OIR). Here we examined the effect of a nonapeptide, PEDF 336, in a newborn mouse OIR model. Neovascularization was significantly decreased in a dose-responsive manner by single intravitreal (IVT) injections of 1.25–7.5 μg/eye (1.0–6.0 nmol/eye). By contrast, anti-mouse VEGFA164 was only effective at 25 ng/eye, with limited dose-response. Combination of anti-VEGFA164 with PEDF 336 gave only the poorer anti-VEGF response while abrogating the robust inhibition seen with peptide-alone, suggesting a need for VEGF in sensitizing the endothelium to the peptide. VEGF stimulated 67LR presentation on endothelial cells, which was decreased in the presence of PEDF 336. Mouse and rabbit eyes showed no histopathology or inflammation after IVT peptide injection. Thus, PEDF 336 is a potential ROP therapeutic, but is not expected to be beneficial in combination with anti-VEGF.

PKCθ‐JunB axis via upregulation of VEGFR3 expression mediates VEGFR2‐induced pathological retinal neovascularization

Pathological retinal neovascularization is the most common cause of vision loss. PKCθ has been shown to play a role in type 2 diabetes, that is linked to retinal neovascularization. Based on these findings, in this study, we have determined the role of PKCθ in retinal neovascularization using human retinal microvascular endothelial cells (HRMVECs) and oxygen‐induced retinopathy (OIR) as models. VEGFA induced PKCθ phosphorylation in a time dependent manner in HRMVECs and down regulation of its levels attenuated VEGFA‐induced HRMVECs migration, sprouting and tube formation but not DNA synthesis. Furthermore, genetic deletion of PKCθ in C57BL/6 mice inhibited hypoxia‐induced retinal endothelial cell proliferation, tip cell formation, sprouting and neovascularization. We also observed that VEGFA induces VEGFR3 expression via PKCθ‐dependent JunB induction in the regulation of HRMVEC migration, sprouting, and tube formation in vitro and OIR‐induced retinal endothelial cell proliferation, tip cell formation, sprouting and neovascularization in vivo. In addition, PKCθ‐JunB axis modulates VEGFA‐induced VEGFR3 promoter activity and its expression downstream to VEGFR2 activation both in vitro and in vivo. Depletion of VEGFR2 or 3 levels attenuated VEGFA‐induced HRMVEC migration, sprouting and tube formation in vitro and retinal angiogenesis in vivo and it appears that these events were dependent on STAT3 activation. In addition, VEGFR3 appears to be mediating retinal neovascularization in a ligand dependent and independent manner downstream to VEGFR2 activation. Together, these observations infer that PKCθ‐dependent JunB‐mediated VEGFR3 expression targeting STAT3 activation is required for VEGFA‐VEGFR2 induced pathological retinal neovascularization.Support or Funding InformationThis work was supported by a grant EY04856 from NIH to GNR.

IL‐33/ST2L mediated ADAMTS10 activation regulate retinal endothelial cell sprouting and neovascularization in a murine model of hypoxia‐induced retinopathy

Pathological retinal neovascularization is a clinical manifestation of various proliferative retinopathies, including retinopathy of prematurity (ROP), diabetic retinopathy, and the wet form of macular degeneration. A characteristic feature of these conditions is the presence of hypoxic areas and over‐expression of the proangiogenic vascular endothelial growth factor (VEGF). Treatments of these neovascular diseases using laser or anti‐VEGF therapies not only induce various local and systemic side effects but also are only partially effective. Here, using a murine model of oxygen‐induced retinopathy, we found that either intravitreal injections of IL‐33 siRNA or genetic deletion of IL‐33 resulted in reduced neovascularization with no significant changes in the avascular area. These findings suggest a role for IL‐33 in pathological neovascularization with little or no effect on the normal retinal repair. Interestingly, we observed that along with IL‐33 activation, hypoxia also induces expression of ST2 transmembrane receptor (ST2L), both at mRNA and protein levels, with little or no effect on secretory ST2 (sST2) levels. Our in vitro experiments using human retinal microvascular endothelial cells as a model suggest the importance of IL‐33/ST2L‐signaling on endothelial cell migration, tube formation, and sprouting. Besides it, our in vitro and in vivo findings provide evidence that IL‐33/ST2L‐signaling via ADAMTS10 activation facilitates sprout formation and invasion of connective tissue through controlled degradation of extracellular matrix proteins (ECMs). These results strongly suggest that IL‐33/ST2L‐signaling plays a key role in retinal neovascularization and thus represents a new pharmacological target for the treatment of diseases where excessive neovascularization is the underlying pathology.Support or Funding InformationThe research is supported by NIH EY029709 grant..

IPSC-derived endothelial cell response to hypoxia via SDF1a/CXCR4 axis facilitates incorporation to revascularize ischemic retina.

Ischemic retinopathies are major causes of blindness worldwide. Local hypoxia created by loss of vascular supply leads to tissue injury and aberrant neovascularization in the retina. There is a great need for therapies that enhance revascularization of hypoxic neuroretinal tissue. To test the therapeutic feasibility of human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) for the treatment of ischemic retinopathies, we compared the angiogenic potential of hiPSC-ECs with mature human retinal endothelial cells (HRECs) in response to hypoxia. hiPSC-ECs formed more robust and complex vascular networks in collagen gels, whereas HRECs displayed minimal sprouting. The cells were further tested in the mouse oxygen-induced retinopathy (OIR) model. Retinas with hiPSC-EC injection showed colocalization with host vessels, whereas HRECs lacked such responses. hiPSC-ECs markedly reduced vaso-obliteration and pathological neovascularization. This beneficial effect of hiPSC-ECs was explained by the stromal cell-derived factor-1a (SDF1a)/CXCR4 axis; hiPSC-ECs exhibited much higher cell-surface expression of CXCR4 than HRECs and greater chemotaxis toward SDF1a-embedded 3D collagen hydrogel. Furthermore, treatment with neutralizing antibody to CXCR4 abolished recruitment of hiPSCs in the OIR model. These findings suggest superior angiogenic potential of hiPSC-ECs under hypoxia and underscore the importance of SDF1a/CXCR4 in the reparative function of hiPSC-ECs in ischemic diseases.

Novel Anti-Interleukin-1β Therapy Preserves Retinal Integrity: A Longitudinal Investigation Using OCT Imaging and Automated Retinal Segmentation in Small Rodents.

Retinopathy of prematurity (ROP) is the leading cause of blindness in neonates. Inflammation, in particular interleukin-1β (IL-1β), is increased in early stages of the disorder, and contributes to inner and outer retinal vasoobliteration in the oxygen-induced retinopathy (OIR) model of ROP. A small peptide antagonist of IL-1 receptor, composed of the amino acid sequence, rytvela, has been shown to exert beneficial anti-inflammatory effects without compromising immunovigilance-related NF-κB in reproductive tissues. We conducted a longitudinal study to determine the efficacy of “rytvela” in preserving the integrity of the retina in OIR model, using optical coherence tomography (OCT) which provides high-resolution cross-sectional imaging of ocular structures in vivo. Sprague–Dawley rats subjected to OIR and treated or not with “rytvela” were compared to IL-1 receptor antagonist (Kineret). OCT imaging and custom automated segmentation algorithm used to measure retinal thickness (RT) were obtained at P14 and P30; gold-standard immunohistochemistry (IHC) was used to confirm retinal anatomical changes. OCT revealed significant retinal thinning in untreated animals by P30, confirmed by IHC; these changes were coherently associated with increased apoptosis. Both rytvela and Kineret subsided apoptosis and preserved RT. As anticipated, Kineret diminished both SAPK/JNK and NF-κB axes, whereas rytvela selectively abated the former which resulted in preserved monocyte phagocytic function. Altogether, OCT imaging with automated segmentation is a reliable non-invasive approach to study longitudinally retinal pathology in small animal models of retinopathy.

MicroRNA-18a-5p Administration Suppresses Retinal Neovascularization by Targeting FGF1 and HIF1A

Pathologic ocular neovascularization commonly results in visual impairment or even blindness in numerous fundus diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD). MicroRNAs regulate angiogenesis through modulating target genes and disease progression, making them a new class of targets for drug discovery. In this study, we investigated the potential role of miR-18a-5p in retinal neovascularization using a mouse model of oxygen-induced proliferative retinopathy (OIR). We found that miR-18a-5p was highly expressed in the retina of pups as well as retinal endothelial cells, and was consistently down-regulated during retinal development. On the other hand, miR-18a-5p was increased significantly during pathologic neovascularization in the retinas of OIR mice. Moreover, intravitreal administration of miRNA mimic, agomiR-18a-5p, significantly suppressed retinal neovascularization in OIR models. Accordingly, agomir-18a-5p markedly suppressed human retinal microvascular endothelial cell (HRMEC) function including proliferation, migration, and tube formation ability. Additionally, we demonstrated that miR-18a-5p directly down-regulated known vascular growth factors, fibroblast growth factor 1 (FGF1) and hypoxia-inducible factor 1-alpha (HIF1A), as the target genes. In conclusion, miR-18a-5p may be a useful drug target for pathologic ocular neovascularization.

CCAAT/Enhancer-Binding Protein β Mediates Oxygen-Induced Retinal Neovascularization via Retinal Vascular Damage and Vascular Endothelial Growth Factor.

Objective To evaluate the role of CCAAT/enhancer-binding protein β (C/EBP β (C/EBP Methods Rats with OIR were exposed to alternating hypoxic and hyperopic conditions for 14 days. Then, the rats with OIR were assigned randomly to groups that received intravitreal injections of either shRNA lentiviral particles targeting C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP Results In OIR rats, the expression levels of C/EBP β (C/EBP P < 0.01). The p-C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP P < 0.01). The p-C/EBP β (C/EBP β (C/EBP β (C/EBP P < 0.01). The p-C/EBP Conclusions C/EBP β shRNA inhibits RNV in OIR. A potential mechanism may be that the activity of C/EBP β increases with its overexpression, which in turn aggravates the amount of the retinal vascular damage and promotes transcription of VEGF. C/EBP β might be a new therapeutic target for preventing RNV.β (C/EBP β (C/EBP β (C/EBP

Temporospatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model.

Myeloid cells such as resident retinal microglia (MG) or infiltrating blood-derived macrophages (Mϕ) accumulate in areas of retinal ischemia and neovascularization (RNV) and modulate neovascular eye disease. Their temporospatial distribution and biological function in this process, however, remain unclarified. Using state-of-the-art methods, including cell-specific reporter mice and high-throughput RNA sequencing (RNA Seq), this study determined the extent of MG proliferation and Mϕ infiltration in areas with retinal ischemia and RNV in Cx3cr1CreERT2 :Rosa26-tdTomato mice and examined the transcriptional profile of MG in the mouse model of oxygen-induced retinopathy (OIR). For RNA Seq, tdTomato-positive retinal MG were sorted by flow cytometry followed by Gene ontology (GO) cluster analysis. Furthermore, intraperitoneal injections of the cell proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) were performed from postnatal day (p) 12 to p16. We found that MG is the predominant myeloid cell population while Mϕ rarely appears in areas of RNV. Thirty percent of retinal MG in areas of RNV were EdU-positive indicating a considerable local MG cell expansion. GO cluster analysis revealed an enrichment of clusters related to cell division, tubulin binding, ATPase activity, protein kinase regulatory activity, and chemokine receptor binding in MG in the OIR model compared to untreated controls. In conclusion, activated retinal MG alter their transcriptional profile, exhibit considerable proliferative ability and are by far the most frequent myeloid cell population in areas of ischemia and RNV in the OIR model thus presenting a potential target for future therapeutic approaches.

Neutralization of Bombina variegata peptide 8 suppresses retinal neovascularization in two different murine models: The oxygen-induced retinopathy model and the rhodopsin promoter/VEGF transgenic mouse model

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Antagonist of nucleolin, N6L, inhibits neovascularization in mouse models of retinopathies.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Nox (NADPH Oxidase) 1, Nox4, and Nox5 Promote Vascular Permeability and Neovascularization in Retinopathy.

Hypertension is a risk factor for the vascular permeability and neovascularization that threatens vision in diabetic retinopathy. Excess reactive oxygen species derived from the Nox (NADPH oxidase) isoforms, Nox1 and Nox4, contributes to vasculopathy in diabetic retinopathy; however, if Nox1/4 inhibition is beneficial in hypertensive diabetic retinopathy is unknown. Here, we determined that diabetic spontaneously hypertensive rats had exacerbated retinal vascular permeability and expression of angiogenic and inflammatory factors, compared with normotensive diabetic Wistar Kyoto rats. GKT136901, a specific dual inhibitor of Nox1 and Nox4, prevented these events in diabetic Wistar Kyoto rats and spontaneously hypertensive rats. Retinal neovascularization does not develop in diabetic rodents, and therefore, the oxygen-induced retinopathy model is used to evaluate this pathology. We previously demonstrated that Nox1/4 inhibition reduced retinal neovascularization in oxygen-induced retinopathy. However, although Nox5 is expressed in human retina, its contribution to retinopathy has not been studied in vivo, largely due to its absence from the rodent genome. We generated transgenic mice with inducible human Nox5 expressed in endothelial cells (vascular endothelial-cadherin+Nox5+ mice). In vascular endothelial-cadherin+Nox5+ mice with oxygen-induced retinopathy, retinal vascular permeability and neovascularization, as well as the expression of angiogenic and inflammatory factors, were increased compared with wild-type littermates. In bovine retinal endothelial cells, which express Nox1, Nox4, and Nox5, Nox1/4 inhibition, as well as Nox5 silencing RNA, reduced the high glucose-induced upregulation of oxidative stress, angiogenic, and inflammatory factors. Collectively, these data indicate the potential of Nox1, Nox4, and Nox5 inhibition to reduce vision-threatening damage to the retinal vasculature.

Inhibition of mTOR via an AAV-Delivered shRNA Tested in a Rat OIR Model as a Potential Antiangiogenic Gene Therapy.

PurposeRecent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR).MethodsSprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition.ResultsCompared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders.ConclusionsTaken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.

The effect of desflurane on retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

Retinopathy of prematurity (ROP) is an ocular disorder that primarily occurs in premature infants and is the most common cause of vision impairment. This study examined the effect of desflurane on angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Mice were randomly allocated to the control (C), ROP control (Rc), or ROP with desflurane exposure (Rd) group. To induce ROP, 7-day-old mice were exposed to 75% oxygen in a chamber for 5days [postnatal days (P) 7-12], and thereafter returned to room air. Age-matched mice exposed to room air formed the C group. The Rd group was exposed to 8% desflurane for 2h on P12, P13, and P14 with 40% oxygen. To observe changes in angiogenesis of the retina, mice were sacrificed at P16. The ratio of avascular area/total retinal area was not changed significantly in the Rd group, compared to the Rc group. The expression of endothelial growth factor A (VEGF-A) and hypoxia inducible factor-1α (HIF-1α) in the Rd group and Rc group was not significantly different. Desflurane does not have a significant influence on retinal angiogenesis via HIF-1α and VEGF-A expression in the OIR mouse model. However, these findings are not directly applicable to premature infants, and it is thus necessary to perform further studies to determine the effect of desflurane on angiogenesis.