Oxygen-induced Retinopathy Retinas Research Articles

Aberrant Retinal Angiogenesis, Structure, and Synaptic Morphology in an in vivo Mouse Model of ROP

ObjectiveRetinopathy of prematurity (ROP) is a condition of aberrant retinal vascularization in premature infants. Vascular endothelial growth factor (VEGF) is a major angiogenic factor in both physiologic and pathologic retinal angiogenesis including ROP. However, cellular changes that take place in ROP are not fully understood. The aim of this study is to understand changes in retinal morphology, vasculature, and structure in mice with oxygen‐induced retinopathy (OIR), a model for ROP.MethodsTwenty‐two mice underwent hyperoxia exposure at 77% to induce OIR, while twenty‐three age‐matched control mice were raised in room air (RA). After anesthesia, pupils were dilated and fluorescein was injected intraperitoneally. After in vivo imaging at P19, P24, P32, and P47, eyes were isolated and fixed in Bouin's fixative for morphological analysis by H&E staining, and paraformaldehyde for immunohistochemistry. For immunohistochemistry, markers for photoreceptor presynapses (PSD95), bipolar cells (PKCalpha), microglia (iba1), and vascularization (VEGF) were used to investigate synaptic abnormalities, inflammation, and vascularization. To detect apoptosis, ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore) was used.ResultsOIR mice had a vascular‐avascular retinal phenotype, with more dilated veins and more tortuous arteries than RA mice in early ages. The OIR retinas showed disorganized synaptic interactions between photoreceptor cells and bipolar cells, with significant thinning in the ganglion cell, inner nuclear, inner plexiform, and outer plexiform layers. Retinal cross‐sections showed altered structure in OIR mice, with dilated blood vessels and neovascularization in the inner retina. There was significantly increased microglia activity in ganglion cell, inner nuclear, and inner plexiform layers. There was increased apoptosis in OIR mice, which decreased over time, but persisted until P47. There was increased VEGF expression in bipolar cells. TUNEL staining showed increased apoptosis in OIR mice than RA mice at P19 (P<0.001), P24 (P<0.001), P32 (P<0.001), and P47 (P<0.001). Apoptosis decreased over time with developmental maturity of the OIR mice, but persisted at P47.Conclusion: Our results demonstrate patchy vascular–avascular phenotype, defective synaptic interactions and persistent apoptosis in the OIR retina, which affected retinal vascularization, and structure in the long term. Our study aids the understanding of the cellular and molecular complexities of the retina following oxidative stress, and could lead to potential new therapeutic targets.Support or Funding InformationDepartment of Pediatrics, University of Wisconsin, Madison (OJM), National Institutes of Health (R01 EY022086) (AI), and Timothy William Trout Professorship in Eye Research (AI)

Melatonin attenuated retinal neovascularization and neuroglial dysfunction by inhibition of HIF-1α-VEGF pathway in oxygen-induced retinopathy mice.

Retinopathy of prematurity (ROP) is a retinopathy characterized by retinal neovascularization (RNV) occurring in preterm infants treated with high concentrations of oxygen and may lead to blindness in severe cases. Currently, anti-VEGF therapy is a major treatment for ROP, but it is costly and may cause serious complications. The previous study has demonstrated that melatonin exerted neuroprotective effect against retinal ganglion cell death induced by hypoxia in neonatal rats. However, whether melatonin is anti-angiogenic and neuroglial protective in the progression of ROP remains unknown. Thus, this study was to investigate the effect of melatonin on RNV and neuroglia in the retina of oxygen-induced retinopathy (OIR) mice. The results showed a reduction in retinal vascular leakage in OIR mice after melatonin treatment. Besides, the size of retinal neovascular and avascular areas, the number of preretinal neovascular cell nuclei, and the number of proliferative vascular endothelial cells within the neovascular area were significantly decreased in mice treated with melatonin. After oxygen-induced injury, the density of astrocytes was decreased, accompanied by morphologic and functional changes of astrocytes. Besides, retinal microglia were also activated. Meanwhile, the levels of inflammatory factors were elevated. However, these pathologic processes were all hindered by melatonin treatment. Furthermore, HIF-1α-VEGF pathway was activated in the retina of OIR mice, yet was suppressed in melatonin-treated OIR mice retinas. In conclusion, melatonin prevented pathologic neovascularization, protected neuroglial cells, and exerts anti-inflammation effect via inhibition of HIF-1α-VEGF pathway in OIR retinas, suggesting that melatonin could be a promising therapeutic agent for ROP.

Galectin-1 expression imprints a neurovascular phenotype in proliferative retinopathies and delineates responses to anti-VEGF.

Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1−/−) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.

Pathogenic Role of microRNA-21 in Diabetic Retinopathy Through Downregulation of PPARα.

Fenofibrate, a specific agonist of peroxisome proliferator–activated receptor-α (PPARα), displays robust therapeutic effects on diabetic retinopathy (DR) in patients with type 2 diabetes. Our recent studies have shown that PPARα is downregulated in the diabetic retina, which contributes to the pathogenesis of DR. However, the mechanism for diabetes-induced downregulation of PPARα remains unknown. We investigated the role of microRNA-21 (miR-21) in regulating PPARα in DR. miR-21 was overexpressed, while PPARα levels were decreased in the retina of db/db mice, a model of type 2 diabetes. Such alterations were also observed in palmitate-treated retinal endothelial cells. miR-21 targeted PPARα by inhibiting its mRNA translation. Knockout of miR-21 prevented the decrease of PPARα, alleviated microvascular damage, ameliorated inflammation, and reduced cell apoptosis in the retina of db/db mice. Intravitreal injection of miR-21 inhibitor attenuated PPARα downregulation and ameliorated retinal inflammation in db/db mice. Further, retinal miR-21 levels were increased, while PPARα levels were decreased in oxygen-induced retinopathy (OIR). Knockout of miR-21 prevented PPARα downregulation and ameliorated retinal neovascularization and inflammation in OIR retinas. In conclusion, diabetes-induced overexpression of miR-21 in the retina is at least partly responsible for PPARα downregulation in DR. Targeting miR-21 may represent a novel therapeutic strategy for DR.

Silencing of galectin-1 inhibits retinal neovascularization and ameliorates retinal hypoxia in a murine model of oxygen-induced ischemic retinopathy

Aberrant neovascularization is a consequence of inappropriate angiogenic signaling and contributes to several diseases. Although many regulators of pathogenic angiogenesis have been identified, the understanding of this process remains incomplete. Galectin-1 (Gal-1), as a homodimeric protein with a single carbohydrate-recognition domain, is implicated in several pathologic processes, including angiogenesis; however, its involvement in retinal neovascularization (RNV) remains unknown. Here, we investigated the anti-angiogenic effect of silencing Gal-1 through intravitreal injection in a mouse model of oxygen-induced retinopathy (OIR). Our results revealed that Gal-1 was overexpressed and closely related to retinal neo-vessels in OIR retinas. After silencing Gal-1 via intravitreal injection of adenoviral-Gal-1-RNA interference (Ad-Gal-1-RNAi), RNV and retinal hypoxia were significantly attenuated, indicating the anti-angiogenic effect of Gal-1 inhibition. Western blot analysis and real-time polymerase chain reaction indicated that the expression of both neuropilin-1 (Nrp-1) and B cell lymphoma-2 (Bcl-2) decreased after intravitreal injection of Ad-Gal-1-RNAi, implying the possible involvement of Nrp-1 and Bcl-2 in Gal-1-related angiogenic processes. Additionally, whole-mount fluorescence and hematoxylin and eosin staining showed that intravitreal injection of Ad-Gal-1-RNAi did not significantly disrupt the retinal vasculature and neuronal structure of room air mice. Moreover, Ad-Gal-1-RNAi transfer promoted retinal vascular sprouting and increased retinal vascular perfusion, likely through decreased phosphorylation of myosin phosphatase target protein-1. Collectively, our results demonstrated that Gal-1 functions as an important regulator in RNV and offers a promising strategy for the treatment of RNV diseases, such as proliferative diabetic retinopathy and retinopathy of prematurity.

Apolipoprotein E2 and E3, but Not E4, Promote Retinal Pathologic Neovascularization.

To determine the relationship between the different isoforms of apolipoprotein E (ApoE) and retinal neovascularization. The concentrations of ApoE and VEGF in vitreous humor samples with either a macular hole (MH), or diabetic macular edema (DME), or proliferative diabetic retinopathy (PDR) with or without intravitreal injection of bevacizumab (IVB) were measured by ELISA. The effects of each isoform of ApoE on human retinal microvascular endothelial cells (HRMECs) in culture or on the retina of oxygen-induced retinopathy (OIR) mice were investigated. The concentrations of ApoE and VEGF were significantly higher in the vitreous humor of patients with PDR and DME than in patients with an MH. There was a significant positive correlation between the concentrations of ApoE and VEGF in vitreous humor of patients. In vitro assays showed that ApoE2 and ApoE3, but not ApoE4, promoted the VEGF-induced cell proliferation and migration. In vivo assays showed that intravitreal injections of ApoE2 and ApoE3 increased the number and area of nodes in the retina of OIR mice. Moreover, ApoE was expressed in the vascular endothelial cell in both normal and OIR retinas, but their expression levels were different at postnatal day (P) 12 and P17. These results demonstrate that ApoE2 and ApoE3, but not ApoE4, have proangiogenic effects, and the increased expression of ApoE in the vitreous humor of patients with PDR and DME indicates that ApoE2 and ApoE3 are involved in the development of retinal neovascularization in eyes.

Retinal expression of small non-coding RNAs in a murine model of proliferative retinopathy.

Ocular neovascularization is a leading cause of blindness in proliferative retinopathy. Small non-coding RNAs (sncRNAs) play critical roles in both vascular and neuronal development of the retina through post-transcriptional regulation of target gene expression. To identify the function and therapeutic potential of sncRNAs in retinopathy, we assessed the expression profile of retinal sncRNAs in a mouse model of oxygen-induced retinopathy (OIR) with pathologic proliferation of neovessels. Approximately 2% of all analyzed sncRNAs were significantly altered in OIR retinas compared with normoxic controls. Twenty three microRNAs with substantial up- or down-regulation were identified, including miR-351, -762, -210, 145, -155, -129-5p, -150, -203, and -375, which were further analyzed for their potential target genes in angiogenic, hypoxic, and immune response-related pathways. In addition, nineteen small nucleolar RNAs also revealed differential expression in OIR retinas compared with control retinas. A decrease of overall microRNA expression in OIR retinas was consistent with reduced microRNA processing enzyme Dicer, and increased expression of Alu element in OIR. Together, our findings elucidated a group of differentially expressed sncRNAs in a murine model of proliferative retinopathy. These sncRNAs may exert critical post-transcriptional regulatory roles in regulating pathological neovascularization in eye diseases.

Antiangiogenic effect of betaine on pathologic retinal neovascularization via suppression of reactive oxygen species mediated vascular endothelial growth factor signaling

Reactive oxygen species (ROS) as well as vascular endothelial growth factor (VEGF) play important roles in pathologic retinal neovascularization. We investigated whether betaine inhibits pathologic retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR). Betaine was intravitreally injected in OIR mice at postnatal day (P) 14. At P17, the neovascular tufts area in OIR retina was analyzed. Intravitreal injection of betaine (200μM) effectively reduced the neovascular tufts area in OIR retina (68.0±6.7% of the control eyes, P<0.05). Even in a high concentration (2mM), betaine never induced any retinal toxicity or cytotoxicity. Betaine significantly inhibited VEGF-induced proliferation, migration, and tube formation in human retinal microvascular endothelial cells (HRMECs). Betaine suppressed VEGF-induced VEGFR-2, Akt and ERK phosphorylation in HRMECs. In human brain astrocytes, betaine reduced tBH-induced ROS production, and subsequently attenuated tBH-induced VEGFA mRNA transcription via suppression of ROS. Our data suggest that betaine has an anti-angiogenic effect on pathologic retinal neovascularization via suppression of ROS mediated VEGF signaling. Betaine could be a potent anti-angiogenic agent to treat pathologic retinal neovascularization.

Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy.

Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization.

Vulnerability of Dopaminergic Amacrine Cells to Chronic Ischemia in a Mouse Model of Oxygen-Induced Retinopathy.

PurposeRetinal dopamine deficiency is a potential cause of myopia and visual deficits in retinopathy of prematurity (ROP). We investigated the cellular mechanisms responsible for lowered levels of retinal dopamine in an oxygen-induced retinopathy (OIR) mouse model of ROP.MethodsRetinopathy was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12. Oxygen-induced retinopathy and age-matched control mice were euthanized at P12, P17, P25, or P42 to P50. Immunohistochemistry, electrophysiology, and biochemical approaches were used to determine the effect of OIR on the structure and function of dopaminergic amacrine cells (DACs).ResultsThe total number of DACs was unchanged in OIR retinas at P12 despite significant capillary dropout in the central retina. However, a significant loss of DACs was observed in P17 OIR retinas (in which neovascularization was maximal), with the cell loss being more profound in the central (avascular) than in the peripheral (neovascular) regions. Cell loss was persistent in both regions at P25, at which time retinal neovascularization had regressed. At P42, the percentage of DACs lost (54%) was comparable to the percent decrease in total dopamine content (53%). Additionally, it was found that DACs recorded in OIR retinas at P42 to P50 had a complete dendritic field and exhibited relatively normal spontaneous and light-induced electrical activity.ConclusionsThe results suggest that remaining DACs are structurally and functionally intact and that loss of DACs is primarily responsible for the decreased levels of retinal dopamine observed after OIR.

Hydrogen Sulfide Contributes to Retinal Neovascularization in Ischemia-Induced Retinopathy.

PurposeHydrogen sulfide (H2S) is an endogenous gaseous signaling molecule with significant pathophysiological importance, but its role in retinal neovascular diseases is unknown. Hydrogen sulfide is generated from L-cysteine by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and/or 3-mercaptopyruvate sulfurtransferase (3-MST). The aim of this study was to investigate the role of H2S in retinal neovascularization (NV) in ischemia-induced retinopathy.MethodsStudies were performed in a murine model of oxygen-induced retinopathy (OIR). Hydrogen sulfide was detected with a fluorescent assay. Western blots and immunohistochemistry were used to assess the changes of H2S-producing enzymes. Gene deletion and pharmacologic inhibition were used to investigate the role of H2S in retinal NV.ResultsHydrogen sulfide production was markedly increased in retinas from OIR mice compared with those from room air (RA) controls. Cystathionine-β-synthase and CSE were significantly increased in OIR retinas, whereas 3-MST was not changed. Cystathionine-β-synthase was expressed throughout the neuronal retina and upregulated in neurons and glia during OIR. Cystathionine-γ-lyase was also localized to multiple retinal layers. Its immunoreactivity was prominently increased in neovascular tufts in OIR. Pharmacologic inhibition of CBS/CSE or genetic deletion of CSE significantly reduced retinal NV in OIR.ConclusionsOur data indicate that the H2S-generating enzymes/H2S contributes to retinal NV in ischemia-induced retinopathy and suggest that blocking this pathway may provide novel therapeutic approaches for the treatment of proliferative retinopathy.

Inhibition of polyamine oxidase reduces hyperoxia‐mediated neurovascular injury in retina

Retinopathy of prematurity (ROP) is a potentially blinding eye disease that affects premature infants and often leads to lifelong vision impairment. Hyperoxia treatment is shown to induce neurovascular damage in the developing retina. Using an oxygen‐induced retinopathy (OIR) mouse model for ROP, we have demonstrated that deletion of the arginase 2 reduced hyperoxia–induced retinal neurodegeneration through the regulation of polyamine metabolism. The present study was undertaken to study the impact of polyamine oxidase (PAO) inhibitor, MDL 72527, on hyperoxia‐induced retinal neurovascular injury in the OIR retina. Newborn wild‐type (WT) mice pups were exposed to 70% oxygen from postnatal day (P) 7 to P12. Pups were treated daily starting at P6 with either vehicle or MDL 72527 (39 mg/kg of body weight, in saline, i.p.) and sacrificed at different stages of hyperoxia treatment. Retinal cryostat sections were used for neurodegeneration studies using immunolabeling of retinal neurons. Analysis of vaso‐obliteration (N=10–12), vessel sprouting (N=7–9) and microglial activation (N=6) were performed on retinal flat mounts. Mixed rat retinal neuronal cultures (7 DIV) were subjected to normoxia (21%) or hyperoxia (50%) with or without MDL 72527 at different time points (N=6–8). These were processed for cellular signaling analysis using Western blotting. Immunolabeling studies using synaptophysin antibody showed the presence of improved synaptic contacts in MDL 72527‐treated OIR retina (P12) compared to vehicle treated groups. Analysis of retinal flat mounts showed significantly reduced a vascular area at P9 and P12( p&lt;0.05) and increased vascular sprouting at P9 (p&lt;0.01) in MDL 72527‐treated compared to vehicle‐treated OIR retinas after 2 or 5 days of hyperoxia. These results demonstrate reduced vascular injury in OIR retina in response to PAO inhibition. Immunolabeling studies using Iba‐1 antibody showed that activation of microglia was significantly reduced and number of resting microglia were increased in the OIR retina with MDL treatment (p&lt;0.05). Analysis of signaling pathways in retinal neurons in response to hyperoxia showed activation of neuro‐inflammatory signaling studied by increased p‐NFkB, p‐ERK1/2 and cytochrome‐C levels, which were significantly reduced by MDL treatment. Our data suggest that PAO inhibition offers a new therapeutic target towards hyperoxia‐induced retinal neurovascular injury.Support or Funding InformationThese studies are supported by AHA 11SDG7440088, Culver VDI Pilot Grant Award and NEI‐R01‐EY011766.

Expression changes of brain-derived neurotrophic factor in retina of oxygen-induced retinopathy

Background Neovascular diseases such as retinopathy of prematurity often leads to irreversible vision loss.The study on oxidative damage mechanism is becoming more and more important.Whether brain derived neurotrophic factor (BNDF) has protection to retinal ganglion cells(RGCs) has few research reports. Objective The study was to investigate the expression changes of BDNFin mouse retinas of oxygen-induced retinopathy (OIR). Methods Thirty SPF C57BL/6J immature rats were divided into OIR group and normal control group and to fifteen rats for each group.OIR models were established by raising 7-day-old (P7) mice together with maternal mouse in (75±3)% oxygen environment for 5 days and then returned to the normal air environment, and the mice of the normal control group were raised in the normal air environment.The P17 mice were sacrificed for retinal histopathological examination by hematoxylin and eosin staining, and the number of vascular endothelial cell nucleus extending the inner limiting membrane was counted.Whole retinal mounts were prepared after fluorescein isothiacyanate-dextran (FITC-dextran) (9 ml/kg) was retrosorbitally injected, and the distribution of retinal vessels was observed in P17 mice.The relative expression levels of BNDF in retinas were detected in P13, P15, P17 mice, and the results were compared between the normal control group and the OIR group. Results Histopathological examination showed that retinal inner limiting membrane was smooth in the normal control group, but a lots of vascular endothelial cell nucleus extending the inner limiting membrane were seen under the optical microscope in the OIR group.The number of the vascular endothelial cell nucleus extending the inner limiting membrane was 1.70±0.68 in the normal group and 45.3±3.13 in the OIR group, showing significant difference between them (t=86.5, P=0.00). Whole retinal mount revealed that normal retinal vessels and network-like capillaries were exhibited in the mice in the normal group, while tortuous vessels, capillary loss and non-perfusion areas were revealed in the OIR group on the whole retina mounts.The relative expressing levels of BDNF in retinas were 263.992±9.451 and 218.432±9.710 in P15 and P17 mice in the OIR group, which were significantly higher than 230.324±7.779 and 115.846±7.305 in the normal control group (t=14.2, 42.3, P<0.05). Conclusions OIR can be inhibited by increasing the expression of BNDF. Key words: Brain-derived neurotrophic factor; Hyperoxia/complications; Retinal neovascularization/pathology; Disease models, animal; Mice, inbred C57BL

M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR). Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow-derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation. The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow-derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs. These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

MicroRNA-184 modulates canonical Wnt signaling through the regulation of frizzled-7 expression in the retina with ischemia-induced neovascularization

Aberrant activation of Wnt signaling contributes to ischemia-induced retinal neovascularization in oxygen-induced retinopathy (OIR), although the underlying mechanism is so far unclear. Here, we show that microRNA-184 (miR-184) is significantly down-regulated in the retina of OIR mice, and miR-184 negatively modulates Wnt signaling both in vivo and in vitro. Furthermore, we show that the Wnt receptor, frizzled-7, is a downstream target of miR-184, and delivery of miR-184 mimic inhibits Wnt signaling in the OIR retina. These results suggest that decreased levels of miR-184 are responsible, at least in part, for the aberrant activation of Wnt signaling in ischemia-induced retinal neovascularization.

Activated complement classical pathway in a murine model of oxygen-induced retinopathy.

To investigate whether the complement system is involved in a murine model of oxygen-induced retinopathy (OIR). Forty C57BL/6J newborn mice were divided randomly into OIR group and control group. OIR was induced by exposing mice to 75%±2% oxygen from postnatal 7d (P7) to P12 and then recovered in room air. For the control group, the litters were raised in room air. At the postnatal 17d (P17), gene expressions of the complement components of the classical pathway (CP), the mannose-binding lectin (MBL) pathway and the alternative pathway (AP) in the retina were determined by quantitative real-time polymerase chain reaction (RT-PCR). Retinal protein expressions of the key components in the CP were examined by Western blotting. Whole mounted retina in the OIR mice showed area of central hypoperfusion in both superficial and deep layers and neovascular tufts in the periphery. The expressions of C1qb and C4b genes in the OIR retina were significantly higher than those of the controls. The expression of retinal complement factor B (CFB) gene in OIR mice was significantly lower than those of the controls. However, the expressions of C3 and complement factor H (CFH) genes were higher. The protein synthesis of the key components involved in the CP (C1q, C4 and C3) were also significantly higher in OIR mouse retina. Although MBL-associated serine protease 1 (MASP1) and MASP2 were detected in both the OIR and the control groups, the expressions were weak and the difference between the two groups was not significant. Our data suggest that the complement system CP is activated during the pathogenesis of murine model of OIR.

Endothelial TWIST1 promotes pathological ocular angiogenesis.

Pathological neovessel formation impacts many blinding vascular eye diseases. Identification of molecular signatures distinguishing pathological neovascularization from normal quiescent vessels is critical for developing new interventions. Twist-related protein 1 (TWIST1) is a transcription factor important in tumor and pulmonary angiogenesis. This study investigated the potential role of TWIST1 in modulating pathological ocular angiogenesis in mice. Twist1 expression and localization were analyzed in a mouse model of oxygen-induced retinopathy (OIR). Pathological ocular angiogenesis in Tie2-driven conditional Twist1 knockout mice were evaluated in both OIR and laser-induced choroidal neovascularization models. In addition, the effects of TWIST1 on angiogenesis and endothelial cell function were analyzed in sprouting assays of aortic rings and choroidal explants isolated from Twist1 knockout mice, and in human retinal microvascular endothelial cells treated with TWIST1 small interfering RNA (siRNA). TWIST1 is highly enriched in pathological neovessels in OIR retinas. Conditional Tie2-driven depletion of Twist1 significantly suppressed pathological neovessels in OIR without impacting developmental retinal angiogenesis. In a laser-induced choroidal neovascularization model, Twist1 deficiency also resulted in significantly smaller lesions with decreased vascular leakage. In addition, loss of Twist1 significantly decreased vascular sprouting in both aortic ring and choroid explants. Knockdown of TWIST1 in endothelial cells led to dampened expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased endothelial cell proliferation. Our study suggests that TWIST1 is a novel regulator of pathologic ocular angiogenesis and may represent a new molecular target for developing potential therapeutic treatments to suppress pathological neovascularization in vascular eye diseases.

Activation of the Endothelin System Mediates Pathological Angiogenesis during Ischemic Retinopathy

Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulation of angiogenic mediators, including vascular endothelial growth factor A (VEGFA) in OIR. Mice were exposed to 75% oxygen from post-natal day P7 to P12, treated with either vehicle or EDNRA antagonist BQ-123 or EDNRB antagonist BQ-788 on P12, and kept at room air from P12 to P17 (ischemic phase). RT-PCR analysis revealed increased levels of EDN2 and EDNRA mRNA, and Western blot analysis revealed increased EDN2 expression during the ischemic phase. EDNRA inhibition significantly increased vessel sprouting, resulting in enhanced physiological angiogenesis and decreased pathological NV, whereas EDNRB inhibition modestly improved vascular repair. OIR triggered significant increases in VEGFA protein and mRNA for delta-like ligand 4, apelin, angiopoietin-2, and monocyte chemoattractant protein-1. BQ-123 treatment significantly reduced these alterations. EDN2 expression was localized to retinal glia and pathological NV tufts of the OIR retinas. EDN2 also induced VEGFA protein expression in cultured astrocytes. In conclusion, inhibition of the EDNRA during OIR suppresses pathological NV and promotes physiological angiogenesis.

Expression of netrin-1 receptors in retina of oxygen-induced retinopathy in mice.

BackgroundNetrin-1 has been reported to promote retinal neovascularization in oxygen-induced retinopathy (OIR). However, netrin-1 receptors, which may mediate netrin-1 action during retinal neovascularization, have not been characterized. In this study, we investigated netrin-1 receptor subtype expression and associated changes in the retinas of mice with OIR.MethodsC57BL/6J mice were exposed to 75±2% oxygen for 5 days and then returned to normal air to induce retinal neovascularization. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to examine the expression of netrin-1 receptor subtypes in the mouse retinas. Double staining of netrin-1 receptor subtypes and isolectin B4 was used to determine the location of the netrin-1 receptor subtypes in the retinas. Inhibition of retinal neovascularization was achieved by UNC5B shRNA plasmid intravitreal injection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections.ResultsRT-PCR results showed that, except for UNC5A, netrin-1 receptor subtypes UNC5B, UNC5C, UNC5D, DCC, neogenin, and A2b were all expressed in the retinas of OIR mice 17 days after birth. Western blots showed that only UNC5B expression was significantly increased on that day, and immunofluorescence results showed that only UNC5B and neogenin were expressed in retinal vessels. Treatment of OIR mice with the UNC5B shRNA plasmid dramatically reduced neovascular tufts and neovascular outgrowth into the inner limiting membrane.ConclusionsUNC5B may promote retinal neovascularization in OIR mice.