Sphingosine-1-phosphate receptor 1/5 selective agonist alleviates ocular vascular pathologies.

Ocular abnormal angiogenesis and edema are featured in several ocular diseases. S1P signaling via S1P1 likely is part of the negative feedback mechanism necessary to maintain vascular health. In this study, we conducted pharmacological experiments to determine whether ASP4058, a sphingosine 1-phosphate receptor 1/5 (S1P1/5) agonist, is useful in abnormal vascular pathology in the eye. First, human retinal microvascular endothelial cells (HRMECs) were examined using vascular endothelial growth factor (VEGF)-induced cell proliferation and hyperpermeability. ASP4058 showed high affinity and inhibited VEGF-induced proliferation and hyperpermeability of HRMECs. Furthermore, S1P1 expression and localization changes were examined in the murine laser-induced choroidal neovascularization (CNV) model, a mouse model of exudative age-related macular degeneration, and the efficacy of ASP4058 was verified. In the CNV model mice, S1P1 tended to decrease in expression immediately after laser irradiation and colocalized with endothelial cells and Müller glial cells. Oral administration of ASP4058 also suppressed vascular hyperpermeability and CNV, and the effect was comparable to that of the intravitreal administration of aflibercept, an anti-VEGF drug. Next, efficacy was also examined in a retinal vein occlusion (RVO) model in which retinal vascular permeability was increased. ASP4058 dose-dependently suppressed the intraretinal edema. In addition, it suppressed the expansion of the perfusion area observed in the RVO model. ASP4058 also suppressed the production of VEGF in the eye. Collectively, ASP4058 can be a potential therapeutic agent that normalizes abnormal vascular pathology, such as age-related macular degeneration and RVO, through its direct action on endothelial cells.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing.

BACKGROUND: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. METHODS: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. RESULTS: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. CONCLUSIONS: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Arctigenin Prevents Retinal Edema in a Murine Retinal Vein Occlusion Model.

Macular edema causes vision loss in patients with retinal vein occlusion (RVO) and diabetic macular edema (DME). The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is used for treatment; however, this therapy is invasive, and recurrence occurs in some cases. The establishment of a non-invasive treatment would help to solve these problems. Here, we focused on arctigenin, a lignan polyphenol found in burdock sprout, and has effects on inflammatory and microcirculatory when taken orally. We hypothesized that oral intake of arctigenin could be effective against retinal edema in RVO and DME. In this study, the degree of retinal edema by measuring the total retinal thickness using optical coherence tomography (OCT) and the thickness of the inner nuclear layer (INL) by hematoxylin-eosin (H&E) staining were investigated. Oral administration of arctigenin ameliorated retinal edema in an RVO murine model by inhibiting the decrease in occludin and vascular endothelial (VE)-cadherin. Moreover, in retinas with edema, arctigenin suppressed the induction of VEGF, tumor necrosis factor α (TNFα), and matrix metallopeptidase 9 (MMP9). Next, the effects of arctigenin on barrier function were assessed in human retinal microvascular endothelial cells (HRMECs) by measuring the trans-endothelial electrical resistance (TEER) and conducting fluorescein isothiocyanate (FITC)-dextran permeability assays. Arctigenin showed a protective effect against VEGF-induced barrier dysfunction. In addition, arctigenin inhibited the TNFα-mediated activation of the nuclear factor-kappaB (NF-κB)/p38 mitogen-activated protein kinase (MAPK) pathway. These results suggested that oral administration of arctigenin has beneficial effects on retinal edema by inhibiting vascular hyperpermeability in endothelial cells.

TRPV4 channels promote vascular permeability in retinal vascular disease.

This study aimed to determine the role of transient receptor potential vanilloid 4 (TRPV4), a calcium (Ca2+)-permeable cation channel, in the pathophysiology of retinal vascular disease. The retinal vein occlusion (RVO) murine model was created by irradiating retinal veins using lasers. TRPV4 expression and localization were evaluated in RVO mice retinas. In addition, we examined the effects of TRPV4 antagonists (RQ-00317310, HC-067047, GSK2193874, and GSK2798745) on retinal edema, blood flow, and ischemic areas in RVO mice. Furthermore, changes in the retinal expression of tumor necrosis factor (TNF)-α and aquaporin4 (AQP4) by RQ-00317310 were analyzed using Western blot. We also assessed the barrier integrity of epithelial cell monolayers using trans-endothelial electrical resistance (TEER) in Human Retinal Microvascular Endothelial Cells (HRMECs). The expression of TRPV4 was significantly increased and co-localized with glutamine synthetase (GS), a Müller glial marker, in the ganglion cell layer (GCL) of the RVO mice. Moreover, RQ-00317310 administration ameliorated the development of retinal edema and ischemia in RVO mice. In addition, the up regulation of TNF-α and down-regulation of AQP4 were lessened by the treatment with RQ-00317310. Treatment with GSK1016790A, a TRPV4 agonist, increased vascular permeability, while RQ-00317310 treatment decreased vascular endothelial growth factor (VEGF)- or TRPV4-induced retinal vascular hyperpermeability in HRMECs. These findings suggest that TRPV4 plays a role in the development of retinal edema and ischemia. Thus, TRPV4 could be a new therapeutic target against the pathological symptoms of retinal vascular diseases.

Timolol maleate, a ß blocker eye drop, improved edema in a retinal vein occlusion model.

Purpose: To investigate the therapeutic effects of eye drops, namely, timolol maleate, a ß-adrenergic receptor antagonist, and latanoprost, a prostaglandin F2α analog, on retinal edema in a murine retinal vein occlusion (RVO) model. Methods: An RVO model was established using laser-induced RVO in mice, which were administered timolol maleate and latanoprost eye drops several times after venous occlusion. Subsequently, the thickness of the inner nuclear layer (INL) and the expression levels of such genes as Vegf and Atf4, which are stress markers of the endoplasmic reticulum, were examined. Primary human cultured retinal microvascular endothelial cells (HRMECs) were treated with timolol under hypoxic conditions, after which the gene expression pattern was investigated. Importantly, an integrated stress response inhibitor (ISRIB) was used in the RVO model, he known ISRIB, which suppresses the expression of ATF4 in retinal edema. Results: Increased INL thickness was suppressed by timolol eye drops, as were the expressions of Vegf and Atf4, in the RVO model. However, latanoprost eye drops did not induce any change in INL thickness. In HRMECs, hypoxic stress and serum deprivation increased the Vegf and Atf4 expressions; in response, treatment with timolol suppressed the Vegf expression. Furthermore, the ISRIB decreased the Vegf expression pattern and edema formation, which are associated with RVO. Conclusions: These results indicate that timolol eye drops may be a potential option for RVO treatment.

Efficacy of an Anti-Semaphorin 3A Neutralizing Antibody in a Male Experimental Retinal Vein Occlusion Mouse Model.

Purpose: Semaphorin 3A (Sema3A) is a promising therapeutic target for macular edema in age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion (RVO). Anti-vascular endothelial growth factors (anti-VEGFs) are the current standard of care for many retinal diseases. This study investigated the Sema3A neutralizing antibody BI-X and/or anti-VEGF therapy (aflibercept) in an RVO mouse model. Treatment efficacy was examined and grouped by timing subsequent to the RVO mouse model induction: efficacy against the onset of intraretinal edema 1 day postinduction and protective effects at 7 days postinduction. Methods: We examined the changes in expression of Sema3A in the retina of an RVO mouse model. In addition, changes in expression of tumor necrosis factor (TNF)-α and semaphorin-related proteins (neuropilin-1 and plexin A1) in the retina upon treatment were analyzed by Western blotting. The effects of BI-X and/or aflibercept were evaluated using measures of retinal edema, blood flow, and thinning of the inner nuclear layer. Results: Induction of vein occlusion in the RVO mouse model significantly increased Sema3A expression in the retina, particularly in the inner nuclear layer. BI-X was effective as a monotherapy and in combination with anti-VEGF therapy, demonstrating a beneficial effect on intraretinal edema and retinal blood flow. Moreover, in the RVO mouse model, BI-X monotherapy normalized the changes in expression of TNF-α and semaphorin-related proteins. Conclusions: These findings support targeting Sema3A to treat intraretinal edema and retinal ischemia.

The pathological association between the anterior eye segment and the retina in a murine model of neovascular glaucoma.

Neovascular glaucoma (NVG) is caused by the formation of new blood vessels in the angle, iris, and cornea in retinal ischemic disease, such as proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), which can reduce the visual acuity. However, the pathophysiological symptoms of NVG are still not well understood because there is no model for the formation of NVG in the angle, iris, and cornea. The aim of this study was to investigate the involvement of NVG during ischemic disease, in a murine model of retinal ischemia. We evaluated the changes of the intraocular pressure (IOP) and pathological symptoms in the anterior eye segment and retina in this model, and the changes in the RNA or protein expression of vascular endothelial growth factor (VEGF) and fibrosis-related factors were analyzed in the retina and cornea by quantitative real-time polymerase chain reaction or western blot, respectively. Furthermore, we examined the changes in IOP after intravitreal injection of an anti-VEGF antibody. First, NVG formed in the retinal ischemic murine model, and the IOP was elevated in mice with NVG formation. Interestingly, VEGF expression was decreased in the retina but increased in the cornea in the murine model of NVG. On the other hand, fibrosis-related factors were increased in the retina and also significantly increased in the cornea in NVG. Moreover, the administration of anti-VEGF antibody immediately after vessel occlusion suppressed the increase in IOP, but administration at 7 days after vessel occlusion accelerated the increase in IOP. These findings suggest that the formation of NVG may be correlated with the pathological symptoms of retinal ischemic disease, via changes in VEGF and fibrosis-related factor expression.

Oral administration of NSP-116, a free radical scavenger, suppresses the symptoms of retinal vein occlusion in the murine model.

Retinal vein occlusion (RVO) is an intractable eye disease that results in reduced visual acuity, associated with retinal ischemia, hemorrhage, and edema. RVO results in excessive ROS production in the retina, causing inflammation and retinal edema. A free radical scavenger, 4-(4-acetylpiperazin-1-yl)-2-(1H-imidazole-1-yl) aniline (NSP-116), has been reported to demonstrate antioxidative effects and prevent ROS production in the retina. Therefore, NSP-116 may represent a useful drug for treating the pathological symptoms of RVO, such as retinal edema and ischemic symptoms. This study aimed to investigate the effects of NSP-116 in a murine model of RVO. We evaluated the thickness of the retinal layer and the size of the non-perfused area following the oral administration of NSP-116. Moreover, we used western blot analysis to examine the expression levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α, after NSP-116 administration, and examined the localization of 8-hydroxy-2'-deoxyguanosine (8-OHdG), by immunostaining. The findings indicate that NSP-116 suppressed retinal edema and expansion the non-perfused area by suppressing the increased expression of VEGF, TNF-α, and 8-OHdG in the murine RVO model. In conclusion, the oral administration of NSP-116 may serve as an effective pharmacological treatment for the pathological symptoms of RVO.

Establishment of a pigmented murine model abundant with characteristics of retinal vein occlusion.

Retinal vein occlusion (RVO) is a vascular disease that represents characteristic retinal hemorrhage and dilated retinal veins. Despite its clinical importance, its pathogenesis remains largely unknown because of limited opportunities to acquire human retinal samples. Therefore, an animal model that reproduces the clinical features of RVO patients is required for further investigation. In this study, we established a pigmented murine RVO model that reproduced characteristic fundus appearances similar to human RVO findings. Retinal edema in this model was observed in both optical coherence tomography and histological analysis, which is a clinically important outcome. With quantitative real-time PCR analysis on retinal samples, we revealed that the mRNA level of vascular endothelial growth factor (VEGF) increased in the retina induced RVO. Moreover, this retinal edema was reduced by intravitreal injection of anti-VEGF antibody. These results were consistent with human clinical knowledge and suggested that this model could be a useful tool for research into new therapeutic approaches.

Nrf2 Activator RS9 Suppresses Pathological Ocular Angiogenesis and Hyperpermeability.

Purpose: Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods: The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results: Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-ß, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions: These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.

Oral and ocular administration of crocetin prevents retinal edema in a murine retinal vein occlusion model.

Purpose: We investigated the effects of oral and ocular administration of crocetin in a murine retinal vein occlusion (RVO) model. Crocetin is a type of carotenoid contained in the fruit of gardenia (Gardenia jasminoides Ellis) and the stigma of saffron (Crocus staruts L). Methods: This study was performed on a murine RVO model, which was created by laser irradiation of retinal veins. We evaluated the retinal thickness after the oral administration of crocetin (100 mg/kg) 1 and 6 h before laser irradiation, and immediately, 6 h, 12 h, and 18 h after laser irradiation in the murine RVO model. In addition, we measured the retinal layer thickness after administration of crocetin eye drops (0.03% or 0.10%) immediately, 6 h, and 12 h after laser irradiation. Western blotting of retinal tissue was used to determine the expression levels of matrix metalloproteinase (MMP-9), tumor nuclear factor (TNF-α), and occludin after oral administration of crocetin. Results: Oral and ocular administration of crocetin improved retinal edema in the murine RVO model. Crocetin administration statistically significantly suppressed overexpression of MMP-9 and TNF-α, and reversed the reduction of occludin. Conclusions: These findings indicate that crocetin can protect retinal tight junctions by suppressing retinal edema through an anti-inflammatory effect, which suggests that crocetin may be useful for RVO disease.

Pathophysiological Role of VEGF on Retinal Edema and Nonperfused Areas in Mouse Eyes With Retinal Vein Occlusion.

Purpose: To determine the relationship between retinal morphologic changes and molecules involved in the changes after anti-VEGF treatment in the retina of retinal vein occlusion (RVO) murine model. Methods: The studies were performed on murine RVO model created by laser irradiation of retinal veins. The site of VEGF expression was determined by immunostaining, and the retinal thickness was measured in the images obtained by optical coherence tomography. The levels of VEGF-related and inflammatory factors after an intravitreal injection of anti-VEGF antibody immediately or 7 days after laser irradiation were determined by Western blotting. Results: The level of VEGF increased in all retinal layers 1 day after laser irradiation, and expression was higher in the partially perfused areas than in the completely nonperfused areas. In eyes with high expression level of VEGF, early administration of anti-VEGF antibody reduced the retinal thickness, and expressions of VEGF and inflammatory factors returned to normal levels. However, the levels of phosphorylated protein kinase B (AKT), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and endothelial nitric oxide synthase (eNOS) were increased by early administration of anti-VEGF antibody. On the other hand, in eyes with low concentration of VEGF, late injection of anti-VEGF antibody induced retinal thinning and the concentrations of phosphorylated AKT, ERK1/2, and eNOS were lower than that in normal group. Furthermore, anti-VEGF antibody lessened the reduction of aquaporin-4. Conclusions: These findings indicate that the effect of anti-VEGF antibody is most likely dependent on its effect on the intraocular VEGF levels.

Irreversible Photoreceptors and RPE Cells Damage by Intravenous Sodium Iodate in Mice Is Related to Macrophage Accumulation.

Purpose: To determine the mechanism causing degeneration of the retinal pigment epithelium (RPE) and photoreceptors in mice after an intravenous injection of sodium iodate (NaIO3). Methods: The time-dependent changes in NaIO3-induced retinal degeneration were determined by analyzing the retinal morphology by optical coherence tomographic (OCT) images, histological sections of the retina, physiology of the retina by electroretinography (ERG), and retinal blood flow by laser speckle flowgraphy. In addition, the expression of the genes associated with age-related macular degeneration in humans was assessed in the NaIO3-treated mice by RT-PCR. We also investigated whether macrophages were involved in the NaIO3-induced retinal degeneration. Results: The intravenous injection of 20 mg/kg NaIO3 altered the morphology of the RPE cells and the ERGs transiently. With 40 mg/kg of NaIO3, the degeneration of the RPE cells was still present at 28 days. Aggregated melanin granules were surrounded by zonula occludens protein 1 (ZO-1)-positive cells. In addition, 40 mg/kg of NaIO3 led to a reduction in the amplitudes of the a- and b-waves of the dark-adapted ERGs. Histological studies showed that macrophages had infiltrated the retina and were present around the altered RPE cells. Depletion of the macrophages by a prior injection of clodronate liposomes prevented the damage of the outer retina after the NaIO3 injection but not the RPE. Conclusions: The NaIO3-induced retinal damage was reversible at low concentrations but permanent at high concentrations of NaIO3. The accumulation of macrophages around the RPE cells caused the photoreceptor cell death.

Exposure to excessive blue LED light damages retinal pigment epithelium and photoreceptors of pigmented mice.

To determine the characteristics of the damages of the retinal pigment epithelium (RPE) and photoreceptors of pigmented mice induced by exposure to blue light emitting diode (LED) light, and to determine the mechanisms causing the damages. Exposure to blue LED light for 3 days induced retinal damage, and the characteristics of the damage differed from that induced by white fluorescent light exposure. Ophthalmoscopy showed that blue LED exposure for 3 days induced white spots on the retina, and histological examinations showed materials accumulated at the IS/OS junction of the photoreceptors. The accumulated materials were stained by ionized calcium binding adapter molecule-1 (Iba-1), a marker for macrophages. The debris was also positive for periodic acid-Schiff (PAS). An enlarging the area of RPE was detected just after the blue LED exposure especially around the optic nerve, and this led to a secondary degeneration of the photoreceptors. Exposure of pigmented mice to 3 consecutive days of blue LED light will cause RPE and photoreceptor damage. The damage led to an accumulation of macrophages and drusen-like materials around the outer segments of the photoreceptors. This blue light exposed model may be useful for investigating the pathogenesis of non-exudative age-related macular degeneration.

Effects of kallidinogenase on retinal edema and size of non-perfused areas in mice with retinal vein occlusion.

Kallidinogenase has been used to treat retinal vein occlusion (RVO) in patients, although there are no evidences on the effects of kallidinogenase on the retinal edema and the non-perfused areas in eyes with a RVO. We have established a murine RVO model with retinal edema and non-perfused areas. The purpose of this study was to evaluate the effects of kallidinogenase on the retinal edema and size of the non-perfused areas in the mouse RVO model. We evaluated the thickness of the retinal layers and size of the non-perfused areas, and the blood flow by laser speckle flowgraphy in RVO model. The effects of an intravenous injection of kallidinogenase on the retinal edema and size of the non-perfused areas were determined. In addition, the expressions of phosphorylated protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) were measured by Western blotting. Our results showed that kallidinogenase reduced the degree of retinal edema and size of the non-perfused areas by an increase in the blood flow in RVO model. Kallidinogenase also increased the levels of phosphorylated Akt and eNOS. These findings indicate that kallidinogenase acted through Akt/eNOS-dependent phosphorylation. Thus, kallidinogenase should be considered as a possible therapeutic agent for RVO patients.

A pharmacological approach in newly established retinal vein occlusion model.

The mechanism underlying the effects of anti-vascular endothelial growth factor (VEGF) antibody in retinal vein occlusion (RVO) treatment is poorly understood, partly due to the lack of RVO animal models that mimic clinical pathology. The aims of this study were to establish a suitable RVO model, clarify the pathogenic mechanisms, and evaluate the effects of anti-VEGF antibody in the model. Mouse retinal veins were occluded by laser photocoagulation after rose bengal injection. Reduction of the b/a wave amplitude ratio, retinal nonperfusion, cystoid edema, and hard exudates were observed after occlusion, and expression of RVO-related genes was altered. Administration of anti-VEGF antibody immediately, or 7 days, after occlusion resulted in reduction and increase of the nonperfused area, respectively. We conclude that the present model will be useful for clarification of the pathogenic mechanisms, and that the timing of anti-VEGF antibody administration is important for the successful amelioration of retinal nonperfusion.