Published papers

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.

Abstract Axial length is the primary determinant of eye size, and it is elongated in myopia. However, the underlying mechanism of the onset and progression of axial elongation remain unclear. Here, we show that endoplasmic reticulum (ER) stress in sclera is an essential regulator of axial elongation in myopia development through activation of both PERK and ATF6 axis followed by scleral collagen remodeling. Mice with lens-induced myopia (LIM) showed ER stress in sclera. Pharmacological interventions for ER stress could induce or inhibit myopia progression. LIM activated all IRE1, PERK and ATF6 axis, and pharmacological inhibition of both PERK and ATF6 suppressed myopia progression, which was confirmed by knocking down above two genes via CRISPR/Cas9 system. LIM dramatically changed the expression of scleral collagen genes responsible for ER stress. Furthermore, collagen fiber thinning and expression of dysregulated collagens in LIM were ameliorated by 4-PBA administration. We demonstrate that scleral ER stress and PERK/ATF6 pathway controls axial elongation during the myopia development in vivo model and 4-PBA eye drop is promising drug for myopia suppression/treatment.

Background: myopia prevalence is high among Japanese schoolchildren, but the underlying causes are unclear. Objective: To examine the distributions of ocular biometry and refraction and their associations with lifestyle variables among Japanese schoolchildren. Methods: This cross-sectional school-based study included 2140 children aged 3–14 years in Tokyo, Japan, and evaluated the distributions under non-cycloplegic conditions and the associated environmental factors. Results: The prevalence of spherical equivalent (SE) ≤−0.75 diopter among preschoolers (aged 3–6 years), elementary school students (aged 6–11 years), and junior high school students (aged 12–14 years) was 49.7%, 72.4%, and 87.7%, respectively. Multiple linear regression analyses showed that the time spent using digital devices was associated positively with lens thickness (β = 0.010; p < 0.050) but not SE, axial length, or vitreous chamber depth. The time spent reading was associated negatively with lens thickness (β = −0.012; p < 0.050), SE (β = −0.152; p < 0.010), axial length (β = 0.110; p < 0.001), and vitreous chamber depth (β = 0.110; p < 0.001). Conclusions: The data indicated that almost half of preschoolers may be myopic. The association with the lens thickness differed depending on the type of near work performed.

Retinal ischemia–reperfusion (I/R) injury is a common cause of visual impairment. To date, no effective treatment is available for retinal I/R injury. In addition, the precise pathological mechanisms still need to be established. Recently, pemafibrate, a peroxisome proliferator-activated receptor α (PPARα) modulator, was shown to be a promising drug for retinal ischemia. However, the role of pemafibrate in preventing retinal I/R injury has not been documented. Here, we investigated how retinal degeneration occurs in a mouse model of retinal I/R injury by elevation of intraocular pressure and examined whether pemafibrate could be beneficial against retinal degeneration. Adult mice were orally administered pemafibrate (0.5 mg/kg/day) for 4 days, followed by retinal I/R injury. The mice were continuously administered pemafibrate once every day until the end of the experiments. Retinal functional changes were measured using electroretinography. Retina, liver, and serum samples were used for western blotting, quantitative PCR, immunohistochemistry, or enzyme linked immunosorbent assay. Retinal degeneration induced by retinal inflammation was prevented by pemafibrate administration. Pemafibrate administration increased the hepatic PPARα target gene expression and serum levels of fibroblast growth factor 21, a neuroprotective molecule in the eye. The expression of hypoxia-response and pro-and anti-apoptotic/inflammatory genes increased in the retina following retinal I/R injury; however, these changes were modulated by pemafibrate administration. In conclusion, pemafibrate is a promising preventive drug for ischemic retinopathies.

Diabetic retinopathy (DR) is a complication of diabetes and one of the leading causes of vision loss worldwide. Despite extensive efforts to reduce visual impairment, the prevalence of DR is still increasing. The initial pathophysiology of DR includes damage to vascular endothelial cells and loss of pericytes. Ensuing hypoxic responses trigger the expression of vascular endothelial growth factor (VEGF) and other pro-angiogenic factors. At present, the most effective treatment for DR and diabetic macular edema (DME) is the control of blood glucose levels. More advanced cases require laser, anti-VEGF therapy, steroid, and vitrectomy. Pan-retinal photocoagulation for non-proliferative diabetic retinopathy (NPDR) is well established and has demonstrated promising outcomes for preventing the progressive stage of DR. Furthermore, the efficacy of laser therapies such as grid and subthreshold diode laser micropulse photocoagulation (SDM) for DME has been re-ported. Vitrectomy has been performed for vitreous hemorrhage and tractional retinal detachment for patients with PDR. In addition, anti-VEGF treatment has been widely used for DME, and re-cently its potential to prevent the progression of PDR has been remarked. Even with these treat-ments, many patients with DR lose their vision and suffer from potential side effects. Thus, we need alternative treatments to address these limitations. In recent years, the relationship between DR, lipid metabolism, and inflammation has been featured. Research in diabetic animal models points to peroxisome proliferator-activated receptor alpha (PPARα) activation in cellular metabolism and inflammation by oral fenofibrate and/or pemafibrate as a promising target for DR. In this paper, we review the status of existing therapies, summarize PPARα activation therapies for DR, and discuss their potentials as promising DR treatments.

PURPOSE: To describe the functional phenotypic features of East Asian patients with RP1L1-associated occult macular dystrophy (i.e., Miyake disease). DESIGN: An international multi-center retrospective cohort study. METHODS: Twenty-eight participants (53 eyes) with Miyake disease were enrolled at three centres: in Japan, China, and Korea. Ophthalmological examinations including spectral-domain optic coherence tomography (SD-OCT) and multifocal electroretinogram (mfERG) were performed. Patients were classified into three functional groups based on mfERG: Group 1, paracentral dysfunction with relatively preserved central/peripheral function; Group 2, homogeneous central dysfunction with preserved peripheral function; and Group 3, widespread dysfunction over the recorded area. Three functional phenotypes were compared in clinical parameters and SD-OCT morphological classification (severe phenotype, blurred/flat ellipsoid zone and absence of the interdigitation zone; mild phenotype, preserved ellipsoid zone). RESULTS: There were eight eyes in Group 1, 40 eyes in Group 2, and five eyes in Group 3. The patients in Group 1 showed significantly later onset (P=.005) and shorter disease duration (P=.002), compared with those in Group 2. All eight eyes in Group 1 showed the mild morphological phenotype, while 43/45 eyes in Groups 2 and 3 presented the severe phenotype, which identified a significant association between the functional grouping and the morphological classification (P<.001). CONCLUSIONS: A spectrum of functional phenotypes of Miyake disease was first documented with identifying three functional subtypes. Patients with paracentral dysfunction had the mildest phenotype, and those with homogeneous central or widespread dysfunction showed overlapping clinical findings with severe photoreceptor changes, suggesting various extents of visual impairment.

PURPOSE: To investigate the risk factors for the development of proliferative vitreoretinopathy grade C (PVR-C), independent of prior surgical invasion. METHODS: Patients who underwent surgery for rhegmatogenous retinal detachment were prospectively registered with the Japan-Retinal Detachment Registry, organized by the Japanese Retina and Vitreous Society, between February 2016 and March 2017. Data obtained from general ophthalmic examinations performed before and at 1, 3, and 6 months after surgery were analyzed. RESULTS: We included 2013 eyes of 2013 patients (men, 1326 (65.9%); mean age, 55.2 ± 15.2 years) from amongst 3446 registered patients. Preoperative PVR-C was observed in 3.6% of patients. Propensity score matching revealed that a shorter axial length (AL) was a risk factor for preoperative PVR-C (OR (Odds Ratio), 0.81; 95% CI (Confidence Interval), 0.69 to 0.96; p = 0.015), which was a risk factor for surgical failure (OR, 4.22; 95% CI, 1.12 to 15.93; p = 0.034); the association was particularly significant for eyes with an AL < 25.0 mm (p = 0.016), while it was insignificant for eyes with an AL ≥ 25.0 mm. CONCLUSIONS: A shorter AL was related to the development of PVR-C before surgical invasion. Our results will help elucidate the fundamental pathogenesis of PVR and caution clinicians to meticulously examine eyes with a shorter AL to detect retinal detachment before PVR development.

The burden of neurodegenerative diseases in the central nervous system (CNS) is increasing globally. There are various risk factors for the development and progression of CNS diseases, such as inflammatory responses and metabolic derangements. Thus, curing CNS diseases requires the modulation of damaging signaling pathways through a multitude of mechanisms. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors (PPARα, PPARβ/δ, and PPARγ), and they work as master sensors and modulators of cellular metabolism. In this regard, PPARs have recently been suggested as promising therapeutic targets for suppressing the development of CNS diseases and their progressions. While the therapeutic role of PPARγ modulation in CNS diseases has been well reviewed, the role of PPARα modulation in these diseases has not been comprehensively summarized. The current review focuses on the therapeutic roles of PPARα modulation in CNS diseases, including those affecting the brain, spinal cord, and eye, with recent advances. Our review will enable more comprehensive therapeutic approaches to modulate PPARα for the prevention of and protection from various CNS diseases.

Retinal degeneration is a progressive retinal damage in ocular vascular diseases. There are several reasons for this, such as occlusion of arteries or veins, diabetic retinopathy, or hereditary retinal diseases. To study pathological mechanisms of retinal degeneration, it is required to develop experimentally reproducible and clinically relevant models. In our previous studies, we developed a murine model of retinal hypoperfusion by unilateral common carotid artery occlusion (UCCAO) which mimics the pathophysiology of ocular ischemic syndrome (OIS) in humans, and described broad pathological mechanisms in the retina after UCCAO. However, there still remain missing pieces of the ocular pathologic process by UCCAO. In this study, we examined those unfound mechanisms. UCCAO was performed on adult mice. Ocular dysfunctions, histological deficits, and inflammation were examined after UCCAO, compared with sham-operated mice. Evaluation values were analyzed by electrophysiological, histological, and molecular biological methods. Eyelid drooping was permanently seen after UCCAO. Induction time point of acute reversible cataract under anesthesia was shortened. Retinal/visual dysfunctions were detected 2-4 weeks after UCCAO. Specifically, scotopic b-wave was more affected than a-wave, with the dysfunction of photopic b-wave. Impaired oscillatory potentials and visual evoked potential were constantly observed. Pathological Müller gliosis/inflammation was featured with NeuN-positive cell loss in the ganglion cell layer. Axial length, intraocular pressure, pupillary light reflex, and retinal pigment epithelium/choroidal thickness were not changed by UCCAO. A murine model of retinal ischemia by UCCAO can be useful for studying a series of degenerative process in the ischemic retina.

In recent years, the number of patients with age-related macular degeneration (AMD) is increasing worldwide along with increased life expectancy. Currently, the standard treatment for wet-AMD is intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs. The upstream of VEGF is hypoxia-inducible factor (HIF), a master regulator of hypoxia-responsive genes responsive to acute and chronic hypoxia. HIF activation induces various pathological pro-angiogenic gene expressions including VEGF under retinal hypoxia, ultimately leading to the development of ocular ischemic neovascular diseases. In this regard, HIF is considered as a promising therapeutic target in ocular ischemic diseases. In clinical ophthalmology, abnormal hypofluorescent areas have been detected in the late-phase of indocyanine green angiography, which are thought to be lipid deposits at the level of Bruch’s membrane to choriocapillaris in vitreoretinal diseases. These deposits may interfere with the oxygen and nutrients that should be supplied to the retinal pigment epithelium, and that HIF/VEGF is highly suspected to be expressed in the hypoxic retinal pigment epithelium, leading to neovascularization. In this review, we comprehensively summarize pathophysiology of AMD-related ocular diseases with the HIF/VEGF pathway from basic and clinic researches with recent findings.

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.

The purpose of this study was to investigate the association between dry eye disease (DED) and myopia by evaluating higher order aberrations (HOAs) and choroidal thickness (CT). We recruited 72 myopic children with DED symptoms (mean age 12.8 years), measured the tear film breakup time (TBUT), corneal/intraocular/total ocular HOAs, CT, and axial length (AL), administered lifestyle questionnaires, and evaluated the relationships among TBUT, HOAs, CT, and AL. The TBUT was correlated significantly with the corneal HOAs and intraocular HOAs but not with the total ocular HOAs. Multiple regression analyses showed that the AL was associated significantly with the TBUT (β = − 0.067, P = 0.004), the intraocular HOAs, and total ocular HOAs but not with the corneal HOAs. The CT was associated significantly with the TBUT and AL (β = 9.15 and − 7.85, respectively; P < 0.001 and = 0.01, respectively). Our data suggested the association between DED and myopia might be independent of the HOAs. We showed that the TBUT was associated with the CT, which is related to the AL. Because the parasympathetic nervous system affects the lacrimal glands and CT, the parasympathetic nervous system might be a common upstream factor in the association between DED and myopia.

Fundus fluorescent angiography is a standard examination in Japan that can directly visualize the circulatory failure in diabetic retinopathy but is not used in Western countries. In this study, we examine the relationship between the non-perfusion area in fundus fluorescent angiography and the progression of diabetic retinopathy. We evaluated 22 eyes between 22 patients who had their first fundus fluorescent angiography during a clinical episode at Keio University Hospital from January 2012 to May 2015, were diagnosed as having preproliferative diabetic retinopathy, and could be followed for at least three years. The non-perfusion area index (%) in nine segmented fundi in the initial fundus fluorescent angiography was calculated, and the progression to proliferative diabetic retinopathy over three years was evaluated. Three out of the 22 eyes (13.6%) developed proliferative diabetic retinopathy over three years. The non-perfusion area index for the initial fundus fluorescent angiography was significantly associated with progression to proliferative diabetic retinopathy. The non-perfusion area index in the posterior pole was most strongly correlated with the progression to proliferative diabetic retinopathy. Thus, the non-perfusion area index in the posterior pole among those with preproliferative diabetic retinopathy may predict the progression to proliferative diabetic retinopathy in the subsequent three years.

Ocular ischemic syndrome (OIS) is one of the severe ocular disorders occurring from stenosis or occlusion of the carotid arteries. As the ophthalmic artery is derived from the branch of the carotid artery, stenosis or occlusion of the carotid arteries could induce chronic ocular hypoperfusion, finally leading to the development of OIS. To date, the pathophysiology of OIS is still not clearly unraveled. To better explore the pathophysiology of OIS, several experimental models have been developed in rats and mice. Surgical occlusion or stenosis of common carotid arteries or internal carotid arteries was conducted bilaterally or unilaterally for model development. In this regard, final ischemic outcomes in the eye varied depending on the surgical procedure, even though similar findings on ocular hypoperfusion could be observed. In the current review, we provide an overview of the pathophysiology of OIS from various experimental models, as well as several clinical cases. Moreover, we cover the status of current therapies for OIS along with promising preclinical treatments with recent advances. Our review will enable more comprehensive therapeutic approaches to prevent the development and/or progression of OIS.

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.

Purpose: To characterize and monitor the clinical and electrophysiological features of a Chinese patient with KCNV2 retinopathy. Methods: A 17-year-old Chinese male with the diagnosis of cone dystrophy with supernormal rod response (CDSRR) was followed-up for 5 years, with full ophthalmological examinations, including decimal best corrected visual acuity (BCVA), fundus photography, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinogram (ERG). Genetic screening was performed to detect the sequence variations in the retinal dystrophy associated genes in the patient and his parents. Results: The patient demonstrated the characteristic full-field electroretinography (ERG) features of CDSRR, namely a profound enlargement of the dark-adapted ERG b-wave amplitude with increasing flash strength and a broadened a-wave trough; this case also had undetectable light-adapted ERGs. A BCVA of 0.15 was maintained over 5 years in both eyes; while progressive macular atrophy was identified. Molecular genetic analyses revealed two novel disease-causing KCNV2 variants in compound heterozygous state: c.1408 G > C (p.Gly470Arg) and c.1500 C > G (p.Tyr500Ter). Conclusions: This is the first long-term case study of an East Asian patient with molecularly confirmed CDSRR. The progressive atrophy with maintained VA demonstrated in this case will be valuable for increasing the understanding of the natural course of KCNV2 retinopathy and it will help in counselling patients with this disease.

Retinal ischemia is a leading cause of irreversible blindness worldwide. Inner retinal dysfunction including loss of retinal ganglion cells is encountered in a number of retinal ischemic disorders. We previously reported administration of two different hypoxia-inducible factor (HIF) inhibitors exerted neuroprotective effects in a murine model of retinal ischemia/reperfusion (I/R) which mimics these disorders, as inner retinal degeneration could be involved in pathological HIF induction. However, this notion needs further investigation. Therefore, in this study, we attempted to use retina-specific Hif-1α conditional knockout (cKO) mice to uncover this notion more clearly under the same condition. Hif-1α cKO mice showed inner retinal neurodegeneration to a lesser extent than control mice. Hif-1α depletion in a murine 661W retinal cell line reduced cell death under pseudohypoxic and hypoxic conditions. Among hypoxia-related genes, the expression of BCL2 19 kDa protein-interacting protein 3 (Bnip3) was substantially upregulated in the inner retinal layer after retinal I/R. In this regard, we further examined Bnip3 depletion in retinal neurons in vitro and in vivo and found the similar neuroprotective effects. Our results support the notion that the HIF-1α/BNIP3 pathway may have a critical role in inner retinal neurodegeneration, which can be linked with the development of new promising therapeutics for inner retinal ischemic disorders.

Myopia is increasing worldwide and its preventable measure should urgently be pursued. N-3 polyunsaturated fatty acids (PUFAs) have been reported to have various effects such as vasodilative and anti-inflammatory, which myopia may be involved in. This study is to investigate the inhibitory effect of PUFAs on myopia progression. A lens-induced myopia (LIM) model was prepared using C57B L6/J 3-week-old mice, which were equipped with a -30 diopter lens to the right eye. Chows containing two different ratios of n-3/n-6 PUFA were administered to the mice, and myopic shifts were confirmed in choroidal thickness, refraction, and axial length in the n-3 PUFA-enriched chow group after 5 weeks. To exclude the possibility that the other ingredients in the chow may have taken the suppressive effect, fat-1 transgenic mice, which can produce n-3 PUFAs endogenously, demonstrated significant suppression of myopia. To identify what elements in n-3 PUFAs took effects on myopia suppression, enucleated eyes were used for targeted lipidomic analysis, and eicosapentaenoic acid (EPA) were characteristically distributed. Administration of EPA to the LIM model confirmed the inhibitory effect on choroidal thinning and myopia progression. Subsequently, to identify the elements and the metabolites of fatty acids effective on myopia suppression, targeted lipidomic analysis was performed and it demonstrated that metabolites of EPA were involved in myopia suppression, whereas prostaglandin E2 and 14,15-dihydrotestosterone were associated with progression of myopia. In conclusion, EPA and its metabolites are related to myopia suppression and inhibition of choroidal thinning.

The myopia epidemic has become a global public health problem. Although myopia is progressing worldwide, the recent coronavirus infections 2019 (COVID-19) outbreak has spurred myopia progression. The current evidence-based treatments for humans are atropine eye drops, optical treatment with defocus, use of orthokeratology, extending proximity working distance, pausing from near work every half hour and increased time outside the home. Studies on myopia using animal models have been conducted for more than 40 years. In recent years, new mechanisms of myopia suppression have been revealed from animal experiments such as inflammation control, intraocular pressure control, light control, and the activity of early growth response protein 1 control. This mini-review provides a summary of the scientific evidence currently available on the control of myopia, and the possible treatments mitigating myopia.