Retinal Neovascularization In Mice Research Articles

Postnatal hyperglycemia alters amino acid profile in retinas (model of Phase I ROP)

Nutritional deprivation occurring in most preterm infants postnatally can induce hyperglycemia, a significant and independent risk factor for suppressing physiological retinal vascularization (Phase I retinopathy of prematurity (ROP)), leading to compensatory but pathological neovascularization. Amino acid supplementation reduces retinal neovascularization in mice. Little is known about amino acid contribution to Phase I ROP. In mice modeling hyperglycemia-associated Phase I ROP, we found significant changes in retinal amino acids (including most decreased L-leucine, L-isoleucine, and L-valine). Parenteral L-isoleucine suppressed physiological retinal vascularization. In premature infants, severe ROP was associated with a higher mean intake of parenteral versus enteral amino acids in the first two weeks of life after adjustment for treatment group, gestational age at birth, birth weight, and sex. The number of days with parenteral amino acids support independently predicted severe ROP. Further understanding and modulating amino acids may help improve nutritional intervention and prevent Phase I ROP.

Comparative study of Curcumin and Lucentis on retinal neovascularization

Introduction To investigate the effect of Curcumin on retinal neovascularization in mice with oxygen-induced lesions. Methods 7-day-old (P7) C57/BL6J mice were randomly divided into control group, OIR group, DMSO group, 100 mg/kg, 50 mg/kg and 25 mg/kg curcumin group and Lucentis group (15 mice per group). Mice in the experimental group were fed for 5 days in high oxygen partial pressure environment, and then in normal oxygen air environment for another 5 days. Corresponding interventions were given at 12-16 days of age (P12-16). At 17 days of age (P17), the eyeball was removed and the retina was paved with Isolectin GS-IB4 fluorescence staining. Real-time PCR was used to detect VEGF mRNA levels in tissues and cells. The protein expression level of VEGF was detected by Western blot. Results Immunofluorescence showed that curcumin injection could significantly reduce the formation of retinal neovascularization and astrocyte injury in OIR, and 100 mg/kg curcumin group had the best effect. Compared with the control group, mRNA and protein expression of VEGF in retina of mice in OIR and DMSO groups were significantly up-regulated (P<0.05); Compared with OIR group, curcumin group and Lucentis group were down-regulated (P<0.05). The protein expression and mRNA level of VEGF in HRCECs of curcumin group decreased with the increase of curcumin concentration, and the effect of curcumin group at 80μmol/L was similar to that of Lucentis group. In the HRCECs cultured with the same concentration of curcumin, the protein expression and mRNA level of VEGF decreased with the prolongation of drug intervention time. Conclusion Curcumin can down-regulate the expression of VEGF in retinal tissues and cells, thereby inhibiting retinal neovascularization and HRCECs cell proliferation.

MicroRNA-124-3p Attenuated Retinal Neovascularization in Oxygen-Induced Retinopathy Mice by Inhibiting the Dysfunction of Retinal Neuroglial Cells through STAT3 Pathway.

MicroRNA (miRNA) is a non-coding RNA that can regulate the expression of many target genes, and it is widely involved in various important physiological activities. MiR-124-3p was found to associate with the normal development of retinal vessels in our previous study, but the mechanism of its anti-angiogenic effect on pathological retinal neovascularization still needed to be explored. Therefore, this study aimed to investigate the effect and mechanism of miR-124-3p on retinal neovascularization in mice with oxygen-induced retinopathy (OIR). Here, we found that intravitreal injection of miR-124-3p agomir attenuated pathological retinal neovascularization in OIR mice. Moreover, miR-124-3p preserved the astrocytic template, inhibited reactive gliosis, and reduced the inflammatory response as well as necroptosis. Furthermore, miR-124-3p inhibited the signal transducer and activator of transcription 3 (STAT3) pathway and decreased the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor. Taken together, our results revealed that miR-124-3p inhibited retinal neovascularization and neuroglial dysfunction by targeting STAT3 in OIR mice.

P07-010-23 Docosahexaenoic Acid in Combination With Arachidonic Acid Alleviates Retinal Neovascularization in Mice Modeling Retinopathy of Prematurity

P07-010-23 Docosahexaenoic Acid in Combination With Arachidonic Acid Alleviates Retinal Neovascularization in Mice Modeling Retinopathy of Prematurity

Mechanism of piR-1245/PIWI-like protein-2 regulating Janus kinase-2/signal transducer and activator of transcription-3/vascular endothelial growth factor signaling pathway in retinal neovascularization.

Inhibiting retinal neovascularization is the optimal strategy for the treatment of retina-related diseases, but there is currently no effective treatment for retinal neovascularization. P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) is a type of small non-coding RNA implicated in a variety of diseases. In this study, we found that the expression of piR-1245 and the interacting protein PIWIL2 were remarkably increased in human retinal endothelial cells cultured in a hypoxic environment, and cell apoptosis, migration, tube formation and proliferation were remarkably enhanced in these cells. Knocking down piR-1245 inhibited the above phenomena. After intervention by a p-JAK2 activator, piR-1245 decreased the expression of hypoxia inducible factor-1α and vascular endothelial growth factor through the JAK2/STAT3 pathway. For in vivo analysis, 7-day-old newborn mice were raised in 75 ± 2% hyperoxia for 5 days and then piR-1245 in the retina was knocked down. In these mice, the number of newly formed vessels in the retina was decreased, the expressions of inflammation-related proteins were reduced, the number of apoptotic cells in the retina was decreased, the JAK2/STAT3 pathway was inhibited, and the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor were decreased. Injection of the JAK2 inhibitor JAK2/TYK2-IN-1 into the vitreous cavity inhibited retinal neovascularization in mice and reduced expression of hypoxia inducible factor-1α and vascular endothelial growth factor. These findings suggest that piR-1245 activates the JAK2/STAT3 pathway, regulates the expression of hypoxia inducible factor-1α and vascular endothelial growth factor, and promotes retinal neovascularization. Therefore, piR-1245 may be a new therapeutic target for retinal neovascularization.

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization.

To investigate whether ultrasound-targeted cationic microbubbles (CMBs) destruction could deliver endostatin-green fluorescent protein (GFP) plasmids efficiently to the human retinal endothelial cells (HRECs) and inhibit retinal neovascularization in mice. CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy. Experiments assessing HRECs migration and vascular formation were performed to evaluate gene therapy's efficiency in vitro. A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl, Bcl-2, vascular endothelial growth factor (VEGF) and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction (qPCR). The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5, 14, and 28d after treatment. The gene expression of endostatin was the highest in the group of the CMBs. Besides, the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro. In vivo, retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups. The research illustrates ultrasound-targeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

PAI-1 is a vascular cell-specific HIF-2-dependent angiogenic factor that promotes retinal neovascularization in diabetic patients.

For patients with proliferative diabetic retinopathy (PDR) who do not respond adequately to pan-retinal laser photocoagulation (PRP) or anti–vascular endothelial growth factor (VEGF) therapies, we hypothesized that vascular cells within neovascular tissue secrete autocrine/paracrine angiogenic factors that promote disease progression. To identify these factors, we performed multiplex ELISA angiogenesis arrays on aqueous fluid from PDR patients who responded inadequately to anti-VEGF therapy and/or PRP and identified plasminogen activator inhibitor-1 (PAI-1). PAI-1 expression was increased in vitreous biopsies and neovascular tissue from PDR eyes, limited to retinal vascular cells, regulated by the transcription factor hypoxia-inducible factor (HIF)-2α, and necessary and sufficient to stimulate angiogenesis. Using a pharmacologic inhibitor of HIF-2α (PT-2385) or nanoparticle-mediated RNA interference targeting Pai1, we demonstrate that the HIF-2α/PAI-1 axis is necessary for the development of retinal neovascularization in mice. These results suggest that targeting HIF-2α/PAI-1 will be an effective adjunct therapy for the treatment of PDR patients.

The Long-Noncoding RNA TUG1 Regulates Oxygen-Induced Retinal Neovascularization in Mice via MiR-299.

PurposeThe oxygen-induced retinal neovascularization mouse model closely approximates pathological changes associated with human retinal neovascularization-associated diseases, including retinopathies. We used this model and human retinal endothelial cells (HRECs) under hypoxia to explore the relationship between taurine upregulated gene-1 (TUG1), vascular endothelial growth factor (VEGF), and miR-299-3p on retinopathy of prematurity (ROP).MethodsAn oxygen-induced retinopathy (OIR) mouse model was established; the mice were divided into a normal control group, OIR group, TUG1 control group (lentivirus control), and TUG1-knockdown group. The apoptosis of retinal cells was evaluated using a TUNEL assay. Angiogenic, apoptotic, and inflammatory factors were detected by Western blot, immunohistochemistry, and immunofluorescence analyses. HRECs were cultured under hypoxia and assessed for VEGF expression, apoptosis, tubule formation, and migration ability. The relationship between TUG1, VEGF, and miR-299-3p was detected via a dual luciferase reporter gene assay.ResultsIntravitreal injection of TUG1 lentivirus reduced the inflammatory response in the mouse retinal tissue and markedly reduced pathological changes in the retina. Overexpression of miR-299 in HRECs reduced the apoptosis rate, tube formation, and migration ability of hypoxia-treated cells, thereby inhibiting the formation of new blood vessels. The dual luciferase reporter gene assay suggested that miR-299 has binding sites for TUG1 and VEGF.ConclusionsTUG1 reduces the expression of VEGFA by competitively adsorbing miR-299-3p and facilitates the regulation of retinal neovascularization, suggesting that it may serve as a new therapeutic target for retinal neovascular diseases.

HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease.

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.

Evogliptin, a dipeptidyl peptidase-4 inhibitor, attenuates pathological retinal angiogenesis by suppressing vascular endothelial growth factor-induced Arf6 activation

Dipeptidyl peptidase-4 (DPP-4) inhibitors are used for the treatment of type 2 diabetes mellitus (DM). Recent studies have shown that beyond their effect in lowing glucose, DPP-4 inhibitors mitigate DM-related microvascular complications, such as diabetic retinopathy. However, the mechanism by which pathological retinal neovascularization, a major clinical manifestation of diabetic retinopathy, is inhibited is unclear. This study sought to examine the effects of evogliptin, a potent DPP-4 inhibitor, on pathological retinal neovascularization in mice and elucidate the mechanism by which evogliptin inhibits angiogenesis mediated by vascular endothelial growth factor (VEGF), a key factor in the vascular pathogenesis of proliferative diabetic retinopathy (PDR). In a murine model of PDR, an intravitreal injection of evogliptin significantly suppressed aberrant retinal neovascularization. In human endothelial cells, evogliptin reduced VEGF-induced angiogenesis. Western blot analysis showed that evogliptin inhibited the phosphorylation of signaling molecules associated with VEGF-induced cell adhesion and migration. Moreover, evogliptin substantially inhibited the VEGF-induced activation of adenosine 5′-diphosphate ribosylation factor 6 (Arf6), a small guanosine 5′-triphosphatase (GTPase) that regulates VEGF receptor 2 signal transduction. Direct activation of Arf6 using a chemical inhibitor of Arf-directed GTPase-activating protein completely abrogated the inhibitory effect of evogliptin on VEGF-induced activation of the angiogenic signaling pathway, which suggests that evogliptin suppresses VEGF-induced angiogenesis by blocking Arf6 activation. Our results provide insights into the molecular mechanism of the direct inhibitory effect of the DPP-4 inhibitor evogliptin on pathological retinal neovascularization. In addition to its glucose-lowering effect, the antiangiogenic effect of evogliptin could also render it beneficial for individuals with PDR.

Hepatocyte growth factor is upregulated in ischemic retina and contributes to retinal vascular leakage and neovascularization.

In patients with macular edema due to ischemic retinopathy, aqueous levels of hepatocyte growth factor (HGF) correlate with edema severity. We tested whether HGF expression and activity in mice with oxygen‐induced ischemic retinopathy supports a role in macular edema. In ischemic retina, HGF was increased in endogenous cells and macrophages associated with retinal neovascularization (NV). HGF activator was increased in and around retinal vessels potentially providing vascular targeting. One day after intravitreous injection of HGF, VE‐cadherin was reduced and albumin levels in retina and vitreous were significantly increased indicating vascular leakage. Injection of VEGF caused higher levels of vitreous albumin than HGF, and co‐injection of both growth factors caused significantly higher levels than either alone. HGF increased the number of macrophages on the retinal surface, which was blocked by anti‐c‐Met and abrogated in chemokine (C‐C motif) ligand 2 (CCL2)−/− mice. Injection of anti‐c‐Met significantly decreased leakage within 24 hours and after 5 days it reduced retinal NV in mice with ischemic retinopathy, but had no effect on choroidal NV. These data indicate that HGF is a pro‐permeability, pro‐inflammatory, and pro‐angiogenic factor and along with its activator is increased in ischemic retina providing support for a potential role of HGF in macular edema in ischemic retinopathies.

Lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor inhibits retinal neovascularization in mice of oxygen-induced retinopathy

Objective To investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR). Methods One hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P<0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P<0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P<0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P<0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P<0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P<0.05). model group and Vec group (P<0.05). Conclusion Intravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1. Key words: Retinal neovascularization/preventionc Polypyrimidine traet-binding protein/drug effects; Lentivirus infections; NF-E2-related factor 2; Heme oxygenase (decyclizing); Animal experimentation

MiR-191 inhibits oxygen-induced retinal neovascularization in mice

Objective To observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR). Methods Eighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue. Results In the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20, P<0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71, P<0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60; P<0.05). Conclusion Intravitreal injection of LV- 191 inhibits RNV in mice model of OIR possibly through up-regulating p21. Key words: Retinal neovascularization/prevention c Lentivirus infections; Animal experimentation

Anti-secretogranin III therapy of oxygen-induced retinopathy with optimal safety.

Retinopathy of prematurity (ROP) with pathological retinal neovascularization is the most common cause of blindness in children. ROP is currently treated with laser therapy or cryotherapy, both of which may adversely affect the peripheral vision with limited efficacy. Owing to the susceptibility of the developing retina and vasculatures to pharmacological intervention, there is currently no approved drug therapy for ROP in preterm infants. Secretogranin III (Scg3) was recently discovered as a highly disease-restricted angiogenic factor, and a Scg3-neutralizing monoclonal antibody (mAb) was reported with high efficacy to alleviate oxygen-induced retinopathy (OIR) in mice, a surrogate model of ROP. Herein we independently investigated the efficacy of anti-Scg3 mAb in OIR mice and characterized its safety in neonatal mice. We developed a new Scg3-neutralizing mAb recognizing a distinct epitope and independently established the therapeutic activity of anti-Scg3 therapy to alleviate OIR-induced pathological retinal neovascularization in mice. Importantly, anti-Scg3 mAb showed no detectable adverse effects on electroretinography and developing retinal vasculature. Furthermore, systemic anti-Scg3 mAb induced no renal tubular injury or abnormality in kidney vessel development and body weight gain of neonatal mice. In contrast, anti-vascular endothelial growth factor drug aflibercept showed significant side effects in neonatal mice. These results suggest that anti-Scg3 mAb may have the safety and efficacy profiles required for ROP therapy.

Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice.

Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.

Expression and significance of the Hedgehog signal transduction pathway in oxygen-induced retinal neovascularization in mice.

AimThe aim of the study was to investigate the signal transduction mechanism of Hedgehog–vascular endothelial growth factor in oxygen-induced retinopathy (OIR) and the effects of cyclopamine on OIR.MethodsAn OIR model was established in C57BL/6J mice exposed to hyperoxia. Two hundred mice were randomly divided into a control group, an OIR group, an OIR-control group (treated with isometric phosphate-buffered saline by intravitreal injection), and a cyclopamine group (treated with cyclopamine by intravitreal injection), with 50 mice in each group. The retinal vascular morphology was observed using adenosine diphosphatase and number counting using hematoxylin and eosin-stained image. Quantitative real-time quantitative polymerase chain reaction was used to detect mRNA expression. Protein location and expression were evaluated using immunohistochemistry and Western blot.ResultsThe OIR group and OIR-control group demonstrated large-area pathological neovascularization and nonperfused area when compared with the control group (both P<0.05). The area of nonperfusion and neovascularization in the cyclopamine group was significantly reduced compared with the OIR and OIR-control groups (both P<0.05). Compared with the control group, the OIR and OIR-control groups had more vascular endothelial cells breaking through the inner limiting membrane. The number of new blood vessel endothelial cell nuclei in the cyclopamine group was significantly reduced (both P<0.05) when compared with the OIR and OIR-control groups. The mRNA and protein expressions of Smoothened, Gli1, and vascular endothelial growth factor in the signal pathway of the OIR and OIR-control groups were significantly higher than those of the control group; however, in the cyclopamine group, these factors were reduced when compared with the OIR and OIR-control groups (all P<0.05).ConclusionOur data suggest that abnormal expression of the Hedgehog signaling pathway may be closely associated with the formation of OIR. Inhibiting the Smoothened receptor using cyclopamine could control retinal neovascularization, providing new ideas and measures for the prevention of oxygen-induced retinal neovascularization.

Inhibitory effects of Genistein on hyperoxia-induced retinal neovascularization

Objective To investigate the effect of Genistein, a tyrosinase inhibitor, on retinal neovascularization in mice. Methods Thirty-six 7-day-old C57BL/6J mice were randomly assigned into hyperoxia-induced group, Genistein group, DMSO group and normal control group.The mice in the hyperoxia-induced group, Genistein group and DMSO group were fed in a static chamber with the oxygen volume fraction (75±2)% for 5 days and then sent back to natural environment for 5 days to establish retinal neovascular models, and 1 μl Genistein diluted by 5% dimethyl sulfoxide (DMSO) (400 mg/L) and 1 μl DMSO were intravitreally injected in the 12-day-old mice of Genistein group and DMSO group, respectively.The mice in the normal control group were bred in natural environment.The fluorescence angiography was carried out in 17-day-old mice (2 mice in each group) to prepare the whole retinal flatmounts, and the morphology of retinal vessels was observed under the fluorescence microscope.The other mice in various groups were sacrificed and the retinas were collected.The expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in mRNA and protein levels were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The use and care of the mice complied with regulations for the management of laboratory animals. Results Retinal vessels were normal in the mice of normal control group.In the mice of the hyperoxia-induced group and DMSO group, retinal vessels were tortuous, and neovacularization and non-perfusion areas were visible.In the Genistein group, retinal vessels were clearly visible, but non-perfusion areas were exhibited.The relative expression levels of VEGF mRNA in retinas were 0.64±0.25, 0.37±0.23, 0.03±0.02 and 0.04±0.02, and the relative expression levels of bFGF mRNA in retinas were 21.40±3.07, 17.22±2.63, 0.52±0.25 and 0.67±0.23, in the hyperoxia-induced group, DMSO group, Genistein group and normal control group, and the expressions of VEGF and bFGF in mRNA level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P<0.05). The protein expression levels of VEGF and bFGF were 1.01±0.05 and 0.97±0.06 in the hyperoxia-induced group, 1.06±0.07 and 1.03±0.08 in the DMSO group, 0.73±0.05 and 0.76±0.07 in the Genistein group, 0.52±0.05 and 0.56±0.05 in the normal control group.The expressions of VEGF and bFGF in protein level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P<0.05). Conclusions Genistein can inhibit hyperoxia-induced retinal neovascularization may be by down-regulating the expressions of VEGF and bFGF in retinas. Key words: Angiogenesis inhibitors/pharmacology; Neovascularization, retinal; hyperoxia; Vascular endothelial growth factor; Fibroblast growth factor

The Nicotinic Cholinergic Pathway Contributes to Retinal Neovascularization in a Mouse Model of Retinopathy of Prematurity.

PurposeTo investigate the role of nicotinic acetylcholine receptors (nAChRs) in retinal vascular development and ischemia-induced retinal neovascularization (NV).MethodsThe expression of nAChR subtypes and VEGF signaling pathway components was assessed in mice with and without oxygen-induced ischemic retinopathy by comparing expression levels at postnatal day (P) 14 and P17 in mice exposed to 75% oxygen from P7 to P12 and returned to room air versus mice pups that were exposed to ambient oxygen levels during the same period. The effect of topical or intraocular injection of mecamylamine, a nonspecific nAChR antagonist, or targeted deletion of α7- or α9-nAChRs on ischemia-induced retinal NV was determined by comparing the amount of retinal NV at P17 in these mice versus appropriate controls.ResultsThe expression of nAChR subunits and components of the VEGF signaling pathways was increased in ischemic retina. Topical application or intraocular injection of mecamylamine decreased retinal NV in this model. Mecamylamine had no effect on normal retinal vascular development or on revascularization of the central retinal area of nonperfusion in mice with ischemic retinopathy. Targeted deletion of α9, but not α7, nAChR receptor subunits reduced retinal NV in mice with ischemic retinopathy.ConclusionThese data suggest that nAChR signaling, primarily through the α9 nAChR subunit, contributes to ischemia-induced retinal NV, but not retinal vascular development. Mecamylamine or a specific α9 nAChR antagonist could be considered for treatment of retinopathy of prematurity and other ischemic retinopathies.

Anti–Vascular Endothelial Growth Factor Agents in the Treatment of Retinal Disease: From Bench to Bedside

The association of retinal hypoxia with retinal neovascularization has been recognized for decades, causing Michaelson to postulate in 1948 that a factor secreted by hypoxic retina was involved. The isolation of vascular endothelial growth factor (VEGF), characterization of its angiogenic activity, and demonstration that its expression was increased in hypoxic tissue made it a prime candidate. Intraocular levels of VEGF are elevated in patients with retinal or iris neovascularization, and VEGF-specific antagonists markedly suppress retinal neovascularization in mice and primates with ischemic retinopathy. Vascular endothelial growth factor antagonists also suppress choroidal neovascularization, and transgenic expression of VEGF in the retina of mice causes subretinal neovascularization. Clinical trials using a VEGF antagonist that blocks all isoforms of VEGF-A in patients with neovascular age-related macular degeneration (nAMD) demonstrated dramatic benefit. Similar results have been obtained with 2 other VEGF antagonists. Retinal hypoxia also contributes to diabetic macular edema (DME), and because of the absence of good animal models, small clinical trials were used to test the role of VEGF. The results clearly implicated VEGF as a major contributor to DME and have been confirmed by several large multicenter trials. A similar strategy demonstrated that VEGF is a major contributor to macular edema resulting from retinal vein occlusion, also confirmed in multicenter trials. Secondary outcomes in these large clinical trials have shown that VEGF inhibition improves retinal hemorrhages, retinal vessel closure, and progression of nonproliferative diabetic retinopathy. Anti-VEGF agents also provide therapeutic benefits in proliferative diabetic retinopathy. Thus, the development of VEGF antagonists has revolutionized the treatment of nAMD, diabetic retinopathy, and other ischemic retinopathies, but in many patients, the upregulation of VEGF is prolonged. Although the molecular signaling by which hypoxia and some other insults lead to upregulation of VEGF has been elucidated, it has not yet led to a treatment that reliably reduces the production of VEGF, necessitating continued neutralization by repeated intraocular injections of VEGF antagonists in many patients. The next horizon in the evolution of anti-VEGF therapy is the development of longer-acting agents or delivery platforms that provide sustained neutralization with fewer injections.

Promoting effect of Toll like receptor 4 on oxygen-induced retinal neovascularization and its mechanism

Background Studies showed that Toll like receptors (TLRs) play an important role in the development of retinal angiogenesis.However, the effect and mechanism of TLR4 affecting angiogenesis in oxygen induced retinopathy (OIR) had not been clarified yet. Objective This study aimed to investigate the mechanism of TLR4 in oxygen induced retinal neovascularization in mice. Methods Eighteen 7-day-old (P7) C57BL/6J mice were randomly divided into normoxic group, OIR model group and OIR+ lipopolysaccharide (LPS)-EB group.OIR models were established by exposing the mice with maternal mouse to 75% oxygen environment for 5 days, then 1 mg/kg LPS-EBO and PBS were intraperitoneally injected in P12 mice in the OIR+ LPS-EB group and OIR model group, respectively.Whole retinal flatmounts of P17 mice were prepared and Lectin staining was performed to calculate the ratio of avascular and neovascular area to retina area.The frozen sections of the posterior ocular segment were prepared, and the number of activated microglial cell was detected by immunofluorescence technique, and the expressions of CD11b and TLR4 in the microglial cells as well as the levels of vascular endothelial growth factor (VEGF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) were assayed by fluorescence double labeling method.The use and care of the experimental animals complied with ARVO Statement. Results The distribution and morphology of retinal vessels were normal in P17 mice of the normoxic group, and avascular and new blood vessels cluster were found in the mice of the OIR group and OIR+ LPS-EB group.The ratio of avascular area was (18.47±1.32)% and that of the new blood vessel area was (3.29±0.85)% in the OIR+ LPS-EB group, which were increased in comparison with (15.78±1.44)% and (1.77±0.19)% of the OIR group (t=3.36, P=0.01; t=4.22, P=0.00). Immunofluorescence results displayed a few Iba1+ microglia cells in the normoxic group, the cells number were significantly higher in the OIR+ LPS-EB group than that in the OIR group ([95.50±4.77]/5 mm versus [74.83±4.17]/5 mm; t=8.00, P<0.01). TLR4+ microglia cells were few in the normoxic group, and the cells number were evidently higher in the OIR+ LPS-EB group than that in the OIR group ([49.50±6.38]/5 mm versus [28.17±6.24]/5 mm, t=5.86, P<0.01). The number of microglia cells coexpressing CD11b with VEGF/IL-1β/TNF-α were (1.17±0.75) /5 mm, 0 and 0 in P17 mice of the normoxic group, respectively, and the cells number in the OIR+ LPS-EB group was more than that in the OIR group ([44.50±8.78]/5 mm versus [28.50±5.61]/5 mm, F=44.01, P<0.01; [24.10±6.49]/5 mm versus [16.00±3.46]/5 mm, F=11.31, P<0.01; [33.83±14.82]/5 mm versus [23.00±2.83]/5 mm, t=19.92, P<0.01). Conclusions TLR4 promotes retinal neovascularization probably by activating specific signaling pathways downstream of microglia cells and accelerating the release of vascular growth factors and inflammatory factors. Key words: Toll like receptor 4; Retinal neovascularization; angiogenesis; Ischemia/pathology; Neuroglia/metabolism; Disease models, animal; Mice, inbred C57BL; Inflammation mediators/metabolism; Signal transduction