Inhibition of hypoxia-inducible factors suppresses subretinal fibrosis.

Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.

Therapeutic Effects of Anti-Inflammatory and Anti-Oxidant Nutritional Supplementation in Retinal Ischemic Diseases.

Appropriate nutrients are essential for cellular function. Dietary components can alter the risk of systemic metabolic diseases, including cardiovascular diseases, cancer, diabetes, and obesity, and can also affect retinal diseases, including age-related macular degeneration, diabetic retinopathy, and glaucoma. Dietary nutrients have been assessed for the prevention or treatment of retinal ischemic diseases and the diseases of aging. In this article, we review clinical and experimental evidence concerning the potential of some nutritional supplements to prevent or treat retinal ischemic diseases and provide further insights into the therapeutic effects of nutritional supplementation on retinopathies. We will review the roles of nutrients in preventing or protecting against retinal ischemic diseases.

Highly sensitive visual restoration and protection via ectopic expression of chimeric rhodopsin in mice.

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into Gloeobacter rhodopsin, thereby generating Gloeobacter and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

Suppressive effects of violet light transmission on myopia progression in a mouse model of lens-induced myopia.

The prevalence of myopia has been steadily increasing for several decades, and this condition can cause extensive medical and economic issues in society. Exposure to violet light (VL), a short wavelength (360-400 nm) of visible light from sunlight, has been suggested as an effective preventive and suppressive treatments for the development and progression of myopia. However, the clinical application of VL remains unclear. In this study, we aimed to investigate the preventive and suppressive effects of VL on myopia progression. Various transmittances of VL (40%, 70%, and 100%) were tested in C57BL/6J mice with lens-induced myopia (LIM). Changes in the refractive error, axial length, and choroid thickness during the 3-week LIM were measured. The myopic shift in refractive error and difference in axial length between the 0 and -30 diopter lens was lessened in a transmission-dependent manner. Choroidal thinning, which was observed in myopic conditions, was suppressed by VL exposure and affected by its transmission. The results suggest that myopia progression can be managed using VL transmittance. Therefore, these factors should be considered for the prevention and treatment of myopia.

Pemafibrate prevents choroidal neovascularization in a mouse model of neovascular age-related macular degeneration.

Background: Pathological choroidal neovascularization (CNV) is one of the major causes of visual impairment in neovascular age-related macular degeneration (AMD). CNV has been suppressed by using anti-vascular endothelial growth factor (VEGF) antibodies. However, some clinical cases have demonstrated the failure of anti-VEGF therapies. Furthermore, anti-VEGF agents might induce the development of ocular atrophy. Recently, peroxisome proliferator-activated receptor alpha (PPARα) activation using pemafibrate treatment was suggested as one of the promising therapeutic targets in the prevention of ocular ischemia. However, the preventive role of pemafibrate remains unclear in CNV. We aimed to examine the preventive role of pemafibrate on laser-induced pathological CNV. Methods: Adult male C57BL/6 mice were orally supplied pemafibrate (0.5 mg/kg) for four days, followed by laser irradiation. Then, pemafibrate was consecutively given to mice with the same condition. CNV was visualized with isolectin-IB4. The eye (retina and/or retinal pigment epithelium [RPE]-choroid), liver, and serum were used for biomolecular analyses. Results: We found that pemafibrate administration suppressed CNV volumes. Pemafibrate administration activated PPARα downstream genes in the liver and eye (especially, RPE-choroid). Furthermore, pemafibrate administration elevated serum fibroblast growth factor 21 levels and reduced serum levels of triglycerides. Conclusions: Our data suggest a promising pemafibrate therapy for suppressing CNV in AMD.

Axl, Immune Checkpoint Molecules and HIF Inhibitors from the Culture Broth of Lepista luscina.

Two compounds 1 and 2 were isolated from the culture broth of Lepista luscina. This is the first time that compound 1 was isolated from a natural source. The structure of compound 1 was identified via 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 along with 8-nitrotryptanthrin (4) were evaluated for their biological activities using the A549 lung cancer cell line. As a result, 1 and 2 inhibited the expression of Axl and immune checkpoint molecules. In addition, compounds 1, 2 and 4 were tested for HIF inhibitory activity. Compound 2 demonstrated statistically significant HIF inhibitory effects on NIH3T3 cells and 1 and 2 against ARPE19 cells.

HIF Inhibition Therapy in Ocular Diseases.

The uncontrolled growth of blood vessels is a major pathological factor in human eye diseases that can result in blindness. This effect is termed ocular neovascularization and is seen in diabetic retinopathy, age-related macular degeneration, glaucoma and retinopathy of prematurity. Current treatments for these diseases include laser photocoagulation, topical injection of corticosteroids, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents and vitreoretinal surgery. Although strategies to inhibit VEGF have proved to be dramatically successful in some clinical studies, there remains the possibility of significant adverse effects regarding the blockade of crucial physiological roles of VEGF and the invasive nature of the treatments. Moreover, it is evident that other pro-angiogenic factors also play important roles in the development of these diseases, as seen in cases in which anti-VEGF therapies have failed. Therefore, new types of effective treatments are required. In this review, we discuss a promising strategy for the treatment of ocular neovascular diseases, i.e., the inhibition of hypoxia-inducible factor (HIF), a master regulator of angiogenesis. We also summarize promising recently investigated HIF inhibitors as treatments for ocular diseases. This review will facilitate more comprehensive approaches to understanding the protective aspects of HIF inhibition in the prevention of ocular diseases.

Glucose levels between the anterior chamber of the eye and blood are correlated based on blood glucose dynamics.

Glycemic control is essential to manage metabolic diseases such as diabetes. Frequent measurements of systemic glucose levels with prompt managements can prevent organ damages. The eye is a glucose highly demanding organ in our body, and the anterior chamber (AC) in the eye has been suggested for a noninvasive blood glucose monitoring site. However, calculating blood glucose levels from measuring glucose levels in AC has been difficult and unclear. In this study, we aimed to examine glucose levels from AC and find a correlation with blood glucose levels. A total of 30 patients with cataracts (men and women, study 1; 7 and 3, study 2; 9 and 11) who visited Keio University Hospital from 2015 to 2018 and agreed to participate in this study were recruited. Glucose levels from AC and the blood were examined by a UV-hexokinase or H2O2-electrode method before/during the cataract surgery. These values were analyzed with regression analyses depending on the groups (blood glucose-ascending and descending groups). In the blood glucose-descending group, glucose levels from AC were strongly correlated with blood glucose levels (a high R2 value, 0.8636). However, the relatively moderate correlation was seen in the blood glucose-ascending group (a low R2 value, 0.5228). Taken together, we showed different correlation ratios on glucose levels between AC and the blood, based on blood glucose dynamics. Stacking data regarding this issue would enable establishing noninvasive blood glucose monitoring from measuring glucose levels in AC more correctly, which will be helpful for proper and prompt managements for glucose-mediated complications.

Retinal dysfunction induced in a mouse model of unilateral common carotid artery occlusion.

BACKGROUND: Retinal ischemic stresses are associated with the pathogenesis of various retinal vascular diseases. To investigate pathological mechanisms of retinal ischemia, reproducible, robust and clinically significant experimental rodent models are highly needed. Previously, we established a stable murine model of chronic hypoperfusion retinal injuries by permanent unilateral common carotid artery occlusion (UCCAO) and demonstrated chronic pathological processes in the ischemic retina after the occlusion; however, retinal functional deficits and other acute retinal ischemic injuries by UCCAO still remain obscure. In this study, we attempted to examine retinal functional changes as well as acute retinal ischemic alterations such as retinal thinning, gliosis and cell death after UCCAO. METHODS: Adult mice (male C57BL/6, 6-8 weeks old) were subjected to UCCAO in the right side, and retinal function was primarily measured using electroretinography for 14 days after the surgery. Furthermore, retinal thinning, gliosis and cell death were investigated using optical coherence tomography, immunohistochemistry and TUNEL assay, respectively. RESULTS: Functional deficits in the unilateral right retina started to be seen 7 days after the occlusion. Specifically, the amplitude of b-wave dramatically decreased while that of a-wave was slightly affected. 14 days after the occlusion, the amplitudes of both waves and oscillatory potentials were significantly detected decreased in the unilateral right retina. Even though a change in retinal thickness was not dramatically observed among all the eyes, retinal gliosis and cell death in the unilateral right retina were substantially observed after UCCAO. CONCLUSIONS: Along with previous retinal ischemic results in this model, UCCAO can stimulate retinal ischemia leading to functional, morphological and molecular changes in the retina. This model can be useful for the investigation of pathological mechanisms for human ischemic retinopathies and furthermore can be utilized to test new drugs for various ischemic ocular diseases.

Rice Bran and Vitamin B6 Suppress Pathological Neovascularization in a Murine Model of Age-Related Macular Degeneration as Novel HIF Inhibitors.

Pathological neovascularization in the eye is a leading cause of blindness in all age groups from retinopathy of prematurity (ROP) in children to age-related macular degeneration (AMD) in the elderly. Inhibiting neovascularization via antivascular endothelial growth factor (VEGF) drugs has been used for the effective treatment. However, anti-VEGF therapies may cause development of chorioretinal atrophy as they affect a physiological amount of VEGF essential for retinal homeostasis. Furthermore, anti-VEGF therapies are still ineffective in some cases, especially in patients with AMD. Hypoxia-inducible factor (HIF) is a strong regulator of VEGF induction under hypoxic and other stress conditions. Our previous reports have indicated that HIF is associated with pathological retinal neovascularization in murine models of ROP and AMD, and HIF inhibition suppresses neovascularization by reducing an abnormal increase in VEGF expression. Along with this, we attempted to find novel effective HIF inhibitors from natural foods of our daily lives. Food ingredients were screened for prospective HIF inhibitors in ocular cell lines of 661W and ARPE-19, and a murine AMD model was utilized for examining suppressive effects of the ingredients on retinal neovascularization. As a result, rice bran and its component, vitamin B6 showed inhibitory effects on HIF activation and suppressed VEGF mRNA induction under a CoCl2-induced pseudo-hypoxic condition. Dietary supplement of these significantly suppressed retinal neovascularization in the AMD model. These data suggest that rice bran could have promising therapeutic values in the management of pathological ocular neovascularization.

PPARα Agonist Oral Therapy in Diabetic Retinopathy.

Diabetic retinopathy (DR) is an eye condition that develops after chronically poorly-managed diabetes, and is presently the main cause for blindness on a global scale. Current treatments for DR such as laser photocoagulation, topical injection of corticosteroids, intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents and vitreoretinal surgery are only applicable at the late stages of DR and there are possibilities of significant adverse effects. Moreover, the forms of treatment available for DR are highly invasive to the eyes. Safer and more effective pharmacological treatments are required for DR treatment, in particular at an early stage. In this review, we cover recently investigated promising oral pharmacotherapies, the methods of which are safer, easier to use, patient-friendly and pain-free, in clinical studies. We especially focus on peroxisome proliferator-activator receptor alpha (PPARα) agonists in which experimental evidence suggests PPARα activation may be closely related to the attenuation of vascular damages, including lipid-induced toxicity, inflammation, an excess of free radical generation, endothelial dysfunction and angiogenesis. Furthermore, oral administration of selective peroxisome proliferator-activated receptor alpha modulator (SPPARMα) agonists may induce hepatic fibroblast growth factor 21 expression, indirectly resulting in retinal protection in animal studies. Our review will enable more comprehensive approaches for understanding protective roles of PPARα for the prevention of DR development.

A Fairy Chemical Suppresses Retinal Angiogenesis as a HIF Inhibitor.

Neovascular retinal degeneration is a leading cause of blindness in advanced countries. Anti-vascular endothelial growth factor (VEGF) drugs have been used for neovascular retinal diseases; however, anti-VEGF drugs may cause the development of chorioretinal atrophy in chronic therapy as they affect the physiological amount of VEGF needed for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcription factor inducing VEGF expression under hypoxic and other stress conditions. Previously, we demonstrated that HIF was involved with pathological retinal angiogenesis in murine models of oxygen-induced retinopathy (OIR), and pharmacological HIF inhibition prevented retinal neovascularization by reducing an ectopic amount of VEGF. Along with this, we attempted to find novel effective HIF inhibitors. Compounds originally isolated from mushroom-forming fungi were screened for prospective HIF inhibitors utilizing cell lines of 3T3, ARPE-19 and 661W. A murine OIR model was used to examine the anti-angiogenic effects of the compounds. As a result, 2-azahypoxanthine (AHX) showed an inhibitory effect on HIF activation and suppressed Vegf mRNA upregulation under CoCl2-induced pseudo-hypoxic conditions. Oral administration of AHX significantly suppressed retinal neovascular tufts in the OIR model. These data suggest that AHX could be a promising anti-angiogenic agent in retinal neovascularization by inhibiting HIF activation.

The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage.

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain-blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have similar protective effects against excitotoxicity.