Measuring potential effects of the developmental burden associated with the vertebrate notochord.

The notochord functions primarily as a supporting tissue to maintain the anteroposterior axis of primitive chordates, a function that is replaced entirely by the vertebral column in many vertebrates. The notochord still appears during vertebrate embryogenesis and plays a crucial role in the developmental pattern formation of surrounding structures, such as the somites and neural tube, providing the basis for the vertebrate body plan. The indispensable role of the notochord has often been referred to as the developmental burden and used to explain the evolutionary conservation of notochord; however, the existence of this burden has not been successfully exemplified so far. Since the adaptive value of target tissues appears to result in the evolutionary conservation of upstream structures through the developmental burden, we performed comparative gene expression profiling of the notochord, somites, and neural tube during the mid-embryonic stages in turtles and chicken to measure their evolutionary conservation. When compared with the somites and neural tube, overall gene expression profiles in the notochord showed significantly lower or merely comparable levels of conservation. However, genes involved in inductive signalings, such as the sonic hedgehog (Shh) cascade and the formation of functional primary cilia, showed relatively higher levels of conservation in all the three structures analyzed. Collectively, these results suggest that shh signals are critical as the inductive source and receiving structures, possibly constituting the inter-dependencies of developmental burden.

Craniofacial development of hagfishes and the evolution of vertebrates.

Cyclostomes, the living jawless vertebrates including hagfishes and lampreys, represent the most basal lineage of vertebrates. Although the monophyly of cyclostomes has been supported by recent molecular analyses, the phenotypic traits of hagfishes, especially the lack of some vertebrate-defining features and the reported endodermal origin of the adenohypophysis, have been interpreted as hagfishes exhibiting a more ancestral state than those of all other vertebrates. Furthermore, the adult anatomy of hagfishes cannot be compared easily with that of lampreys. Here we describe the craniofacial development of a series of staged hagfish embryos, which shows that their adenohypophysis arises ectodermally, consistent with the molecular phylogenetic data. This finding also allowed us to identify a pan-cyclostome pattern, one not shared by jawed vertebrates. Comparative analyses indicated that many of the hagfish-specific traits can be explained by changes secondarily introduced into the hagfish lineage. We also propose a possibility that the pan-cyclostome pattern may reflect the ancestral programme for the craniofacial development of all living vertebrates.

Expansion of the neck reconstituted the shoulder-diaphragm in amniote evolution.

The neck acquired flexibility through modifications of the head-trunk interface in vertebrate evolution. Although developmental programs for the neck musculoskeletal system have attracted the attention of evolutionary developmental biologists, how the heart, shoulder and surrounding tissues are modified during development has remained unclear. Here we show, through observation of the lateral plate mesoderm at cranial somite levels in chicken-quail chimeras, that the deep part of the lateral body wall is moved concomitant with the caudal transposition of the heart, resulting in the infolding of the expanded cervical lateral body wall into the thorax. Judging from the brachial plexus pattern, an equivalent infolding also appears to take place in mammalian and turtle embryos. In mammals, this infolding process is particularly important because it separates the diaphragm from the shoulder muscle mass. In turtles, the expansion of the cervical lateral body wall affects morphogenesis of the shoulder. Our findings highlight the cellular expansion in developing amniote necks that incidentally brought about the novel adaptive traits.

Effect of polyp regression and reduction on treatment efficacy in polypoidal choroidal vasculopathy treated with aflibercept.

Intravitreal injection of aflibercept (IVA) has successfully treated polypoidal choroidal vasculopathy (PCV), and polyp morphology is an important indicator of treatment efficacy. However, many studies have not reported the presence or absence of polyp regression and treatment outcomes, and few studies have reported polyp reduction and treatment outcomes in cases with residual polyps. We retrospectively measured the polyp area on indocyanine green angiography images before and after the IVA loading phase and investigated the regression and reduction of polyps and treatment outcomes of 81 eyes with PCV treated with IVA. We investigated the relationship between the presence or absence of complete regression of polyps and the percentage change in the polyp area and treatment outcomes. Eyes with complete polyp regression had significantly better visual acuity improvements compared with baseline at 12 months (P = 0.0108), fewer treatments (P = 0.0024), fewer recurrences during 12-months follow-up (P = 0.0010), and more "dry maculas" at 3 months (P = 0.0048) than eyes in which polyp regression did not occur. A significant correlation was seen only between the percentage of polyp regression and visual acuity at 3 months (P = 0.0395). Regarding IVA therapy for PCV, the presence or absence of complete polyp regression at the end of the loading phase affected the treatment outcome, whereas the degree of polyp reduction in cases of residual polyps had no effect.

Comparison of Loading Dose between Aflibercept and Faricimab for Neovascular Age-Related Macular Degeneration.

BACKGROUND: Recently, faricimab was approved as the new drug for neovascular age-related macular degeneration (nAMD). We lack the knowledge to choose between the existing drug and this new drug to use for treatment-naïve nAMD cases. In this study, we compared the functional and morphologic effects in loading dose between patients with treatment-naïve nAMD treated with either intravitreal aflibercept (IVA) or intravitreal faricimab (IVF) injection in a clinical setting. METHOD: This retrospective study included 30 eyes of 28 patients who started treatment with IVA between June and September 2022 and 30 eyes of 29 patients who were administered IVF between October 2022 and March 2023. All patients received three monthly IVA or IVF. The best corrected visual acuity (BCVA), central retinal thickness (CRT), and the proportion of eyes with residual exudative change at baseline and 1,2, and 3 months after initial treatment were compared between the groups. RESULTS: The mean BCVA significantly improved from pre-treatment after the loading dose in the IVA group (0.46 ± 0.46-0.36 ± 0.37, p = 0.0047) but not in the IVF group (0.46 ± 0.41-0.44 ± 0.45, p = 0.60). The mean CRT significantly improved in both groups. The proportion of eyes with residual exudative change was greater in the IVF group than in the IVA group 2 months after the first treatment (p = 0.026). The analysis of cases that achieved complete resolution of exudative changes after the loading dose showed that the IVA group had a significant improvement in the BCVA, whereas the IVF group did not (p = 0.0047 and 0.20, respectively). CONCLUSIONS: Although both IVA and IVF significantly improved CRT, the BCVA improved significantly in the IVA group but not in the IVF group.

Prevalence, Associated Factors, and Inter-Eye Differences of Refractive Errors in a Population-Based Japanese Cohort: The Tohoku Medical Megabank Eye Study.

PURPOSE: To investigate the prevalence, associated factors, and inter-eye differences of myopia and astigmatism in an adult Japanese population-based cohort. METHODS: A total of 4282 participants from the Tohoku Medical Megabank Organization Eye Study (ToMMo Eye Study) underwent comprehensive ocular examinations as well as extensive physiological tests and a lifestyle questionnaire. The spherical equivalent (SE) and cylinder power were obtained as refractive parameters. The age- and gender-stratified prevalences of high myopia (SE < -5D), myopia (SE < -0.5D), hyperopia (SE > 0.5D), astigmatism (cylinder power < -0.5D), and anisometropia (SE difference >1D) were calculated. Multivariable analyses were performed to identify associated factors for refractive error (RE). Distribution and associated factors of the inter-eye difference in RE were also investigated. RESULTS: The age-adjusted prevalence of high myopia, myopia, hyperopia, astigmatism, and anisometropia was 15.9%, 63.5%, 14.7%, 51.1%, and 14.7%, respectively. Both myopia and high myopia were more prevalent in the younger age group, while astigmatism was more prevalent in the older age group. Age, education, blood pressure, intraocular pressure, and corneal thickness are significantly associated with myopic refraction. Age, gender, intraocular pressure, and corneal thickness are correlated with astigmatism. Older age was associated with against-the-rule astigmatism. Older age, myopia, and longer education showed a significant correlation with large inter-eye differences in SERE. CONCLUSIONS: This study demonstrated the high prevalence of myopia in young Japanese, which may be caused by a generational shift. This study also confirmed the influence of age and education on both the prevalence and inter-eye differences of RE.

Non-parsimonious evolution of hagfish Dlx genes.

BACKGROUND: The number of members of the Dlx gene family increased during the two rounds of whole-genome duplication that occurred in the common ancestor of the vertebrates. Because the Dlx genes are involved in the development of the cranial skeleton, brain, and sensory organs, their expression patterns have been analysed in various organisms in the context of evolutionary developmental biology. Six Dlx genes have been isolated in the lampreys, a group of living jawless vertebrates (cyclostomes), and their expression patterns analysed. However, little is known about the Dlx genes in the hagfish, the other cyclostome group, mainly because the embryological analysis of this animal is difficult. RESULTS: To identify the hagfish Dlx genes and describe their expression patterns, we cloned the cDNA from embryos of the Japanese inshore hagfish Eptatretus burgeri. Our results show that the hagfish has at least six Dlx genes and one pseudogene. In a phylogenetic analysis, the hagfish Dlx genes and those of the lampreys tended to be excluded from the clade of the gnathostome Dlx genes. In several cases, the lamprey Dlx genes clustered with the clade consisting of two hagfish genes, suggesting that independent gene duplications have occurred in the hagfish lineage. Analysis of the expression of these genes showed distinctive overlapping expression patterns in the cranial mesenchymal cells and the inner ear. CONCLUSIONS: Independent duplication, pseudogenization, and loss of the Dlx genes probably occurred in the hagfish lineage after its split from the other vertebrate lineages. This pattern is reminiscent of the non-parsimonious evolution of its morphological traits, including its inner ear and vertebrae, which indicate that this group is an early-branching lineage that diverged before those characters evolved.

Late development of hagfish vertebral elements.

It has been demonstrated recently that hagfishes, one of two groups of extant jawless vertebrates, have cartilaginous vertebral elements. Embryological and gene expression analyses have also shown that this group of animals develops a sclerotome, the potential primordium of the axial skeleton. However, it has not been shown unequivocally that the hagfish sclerotome truly differentiates into cartilage, because access to late-stage embryos and information about the cartilaginous extracellular matrix (ECM) are lacking for these animals. Here we investigated the expression patterns of the biglycan/decorin (BGN/DCN) gene in the inshore hagfish, Eptatretus burgeri. The homologue of this gene encodes the major noncollagenous component of the cartilaginous ECM among gnathostomes. We clearly identified the expression of this gene in adult vertebral tissues and in embryonic mesenchymal cells on the ventral aspect of the notochord. Taking into account that the sclerotome in the gnathostomes expresses BGN/DCN gene during the chondrogenesis, it is highly expected the hagfish BGN/DCN-positive mesenchymal cells are derived from the sclerotomes. We propose that hagfishes and gnathostomes share conserved developmental mechanisms not only in their somite differentiation, but also in chondrogenesis of their vertebral elements.

Development of the chondrocranium in hagfishes, with special reference to the early evolution of vertebrates.

Recent molecular phylogenetic analyses have shown that the modern jawless vertebrates, hagfishes and lampreys, are more closely related to each other than to the other vertebrates, constituting a monophyletic group, the cyclostomes. In terms of their developmental morphology as well, it is possible to identify an embryonic pattern in hagfish embryos that is common to cyclostomes but not shared by jawed vertebrate embryos. On the basis of this pan-cyclostome embryonic pattern, we describe the developmental sequence of the chondrocranium and associated structures in the hagfish species Eptatretus burgeri and E. atami. Our aim was to establish homologies of the skeletal elements among cyclostomes by comparison of the developmental patterns with a lamprey, Lethenteron reissneri, to characterize further the cyclostome morphotype and its diversification in early vertebrate evolution. We show that the hagfish and lamprey chondrocrania can be compared perfectly at the level of modules corresponding to the craniofacial primordia constituting the cyclostome morphotype. In the adult anatomy, however, there are many instances in which homology cannot be established at the level of single skeletal elements, mainly because of the apparently highly apomorphic nature of the hagfish cranium. Even at the craniofacial modular level, the chondrocrania of cyclostomes and those of jawed vertebrates display very few primary homologies and are therefore very difficult to compare. We also discuss the problem of the homology of a neurocranial element, the trabecula.

Macular Retinoschisis from Optic Disc without a Visible Optic Pit or Advanced Glaucomatous Cupping (No Optic Pit Retinoschisis [NOPIR]).

PURPOSE: To review eyes with peripapillary and macular retinoschisis without a visible optic pit or advanced glaucomatous optic atrophy, or No Optic Pit Retinoschisis (NOPIR). DESIGN: Retrospective multicenter case series. SUBJECTS: The study included 11 eyes of 11 patients. METHODS: Retrospective study of eyes with macular retinoschisis without a visible optic pit, advanced optic nerve head cupping, or macular leakage on fluorescein angiography. MAIN OUTCOME MEASURES: Visual acuity (VA), retinoschisis resolution, months to resolution, and recurrence of retinoschisis RESULTS: The mean age was 68.1 ± 17.6 years, mean intraocular pressure was 17.4 ± 3.8 mmHg, and the mean spherical equivalent refractive error was -3.1 ± 2.9 diopters. No subject had pathologic myopia. Seven subjects were treated for glaucoma, and 9 subjects had nerve fiber layer defects on OCT. All eyes had retinoschisis in the outer nuclear layer (ONL) in the nasal macula and extending to the edge of the optic disc, and 8 subjects had fovea-involving retinoschisis. Three nonfoveal and 4 fovea-involved eyes were observed, and 4 fovea-involved eyes with vision loss underwent surgery. Surgery involved preoperative juxtapapillary laser followed by vitrectomy and membrane and internal limiting membrane peeling with intraocular gas and face-down position. The mean baseline VA was significantly worse in the surgery group than that in the observation group (P = 0.020). Retinoschisis resolved and vision improved in all surgical cases. The mean resolution time for the surgery group was 2.75 ± 0.96 months, which was shorter than that for the observation group (28.0 ± 21.2 months; P = 0.014). No eye developed recurrence of the retinoschisis after surgery. CONCLUSIONS: Peripapillary and macular retinoschisis can develop in eyes without a visible optic pit or advanced glaucomatous cupping. Eyes without foveal involvement and those with foveal involvement but only mild decrease in vision can be observed for spontaneous resolution. If there is persistent foveal involvement with vision loss, surgery can improve vision by resolving the macular retinoschisis. Surgery for fovea-involved macular retinoschisis without a visible optic pit resulted in faster anatomic resolution and better vision recovery. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Geographic Atrophy Phenotypes in Subjects of Different Ethnicity: Asia-Pacific Ocular Imaging Society Work Group Report 3.

OBJECTIVE: To characterize geographic atrophy (GA) and evaluate differences between Asians and non-Asians. DESIGN: Multicenter, retrospective case series. PARTICIPANTS: Subjects aged ≥ 50 years with GA secondary to age-related macular degeneration in the absence of neovascularization in the study eye and follow-up of ≥ 2 years. METHODS: The GA lesion characterized at baseline and last follow-up based on multimodal imaging (fundus autofluorescence [FAF], near infrared [NIR], and spectral domain-OCT). Patients were grouped as either Asian or non-Asian. MAIN OUTCOME MEASURES: Comparison of (1) phenotypes of GA lesions (size, foveal involvement, number of foci, drusen background, and choroid background) and (2) growth rates of GA. RESULTS: A total of 144 patients (169 eyes) with distribution of 50.9% Asians and 49.1% non-Asians. The age and sex were similar between Asians and non-Asians (Asians: mean age, 77.2 ± 10.1 years, 47.9% female; non-Asians: mean age, 79.7 ± 8.4 years, 58.7% female). Asians exhibited thicker choroids (167 ± 74 versus [vs.] 134 ± 56 µm; P < 0.01) and lower prevalence of drusen (40.7% vs. 66.3%; P < 0.01). At baseline, the GA area was smaller in Asians vs. non-Asians (NIR, 3.7 ± 4.6 vs. 6.3 ± 6.8 mm2; P = 0.01: FAF, 2.4 ± 3.4 vs. 8.4 ± 9.6 mm2; P < 0.01). Asians had fewer GA foci (1.7 ± 1.3 vs. 2.7 ± 2.2; P < 0.01) compared to non-Asians. The proportion with diffused or banded FAF junctional zone pattern was similar between Asians and non-Asians (44.2% vs. 60.2%; P = 0.20). Asians had a slower GA lesion growth rate than non-Asians (NIR, 0.7 vs. 1.9 mm2/year; P < 0.01: FAF, 0.3 vs. 2.0 mm2/year; P < 0.01: NIR, 0.2 vs. 0.4 mm/year; P < 0.01 square root transformed: FAF, 0.1 vs. 0.3 mm/year; P < 0.01 square root transformed). The factors associated with GA lesion growth rate are (from the highest effect size) ethnicity, junctional zone FAF pattern, baseline GA area, and number of GA foci. Higher GA lesion growth rate was observed in both Asian and non-Asian subgroups, with drusen or lesion size and FAF patterns meeting inclusion criteria of recent therapeutic trials, but growth rate remained significantly slower in Asians. Eyes with baseline lesion ≥ 5 mm2 showed the highest growth rate, and the difference between ethnicities was no longer significant (2.6 vs. 3.3 mm2/year; P = 0.14). CONCLUSIONS: There are differences in GA lesion phenotype, associated features, and growth rate between Asians and non-Asian subjects. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Three-Dimensional Volume Calculation of Intrachoroidal Cavitation Using Deep-Learning-Based Noise Reduction of Optical Coherence Tomography.

Purpose: Intrachoroidal cavitations (ICCs) are peripapillary pathological lesions generally associated with high myopia that can cause visual field (VF) defects. The current study aimed to evaluate a three-dimensional (3D) volume parameter of ICCs segmented from volumetric swept-source optical coherence tomography (SS-OCT) images processed using deep learning (DL)-based noise reduction and to investigate its correlation with VF sensitivity. Methods: Thirteen eyes of 12 consecutive patients with peripapillary ICCs were enrolled. DL-based denoising and further analyses were applied to parapapillary 6 × 6-mm volumetric SS-OCT scans. Then, 3D ICC volume and two-dimensional depth and length measurements of the ICCs were calculated. The correlations between ICC parameters and VF sensitivity were investigated. Results: The ICCs were located in the inferior hemiretina in all eyes. ICC volume (P = 0.02; regression coefficient [RC], -0.007) and ICC length (P = 0.04; RC, -4.51) were negatively correlated with the VF mean deviation, whereas ICC depth (P = 0.15) was not. All of the parameters, including ICC volume (P = 0.01; RC, -0.004), ICC depth (P = 0.02; RC, -0.008), and ICC length (P = 0.045; RC, -2.11), were negatively correlated with the superior mean total deviation. Conclusions: We established the volume of ICCs as a new 3D parameter, and it reflected their influence on visual function. The automatic delineation and 3D rendering may lead to improved detection and pathological understanding of ICCs. Translational Relevance: This study demonstrated the correlation between the 3D volume of ICCs and VF sensitivity.

Characteristics of choroidal neovascularization in elderly eyes with high myopia not meeting the pathologic myopia definition.

The META-Analysis of Pathologic Myopia Study group proposed a new classification system for myopic maculopathy (MM) with pathologic myopia (PM) defined as MM equal to/more serious than diffuse atrophy or the presence of plus lesions and myopic choroidal neovascularization (mCNV) defined as CNV in the eye with PM. However, CNV in elderly eyes with high myopia (HM) not meeting the PM definition (high-myopia CNV) are not classified as age-related macular degeneration (nAMD) or mCNV. This retrospective study included 39 eyes with high-myopia CNV, 20 eyes with mCNV, and 20 eyes with AMD. All patients were at least 40 years old. We compared the clinical characteristics and treatment outcomes among three groups. The high-myopia CNV group had significantly more CNV types, shorter axial length and fewer lacquer cracks (P < 0.0001, respectively); larger baseline greatest linear dimension (P = 0.0002), more fellow-eye drusen (P = 0.0106), more men (P = 0.0029), and more treatments (24 months, P = 0.0098) compared to the mCNV group. Compared with the nAMD group, the high-myopia CNV group was significantly younger (P = 0.0041), and had fewer CNV types (P = 0.0316), more lacquer cracks (P = 0.0079) and fewer drusen (affected-eye, P = 0.0006 and fellow-eye, P = 0.0222), and fewer treatments (24 months, P = 0.0030). Because the CNV in elderly eyes with HM not meeting the PM definition is classified as combined mCNV and nAMD, the clinical and angiographic findings are critical to determine the treatment strategy.

Anterior chamber flare and ciliochoroidal detachment using flare photometry and anterior segment optical coherence tomography in acute lupus choroidopathy: A case report.

PURPOSE: To report anterior chamber flare using laser flare photometry and ciliochoroidal detachment using anterior segment optical coherence tomography (AS-OCT) in a new onset acute lupus choroidopathy case. OBSERVATIONS: A 57-year-old woman with severe nephritis, pleural effusion, and ascites was referred to our ophthalmology clinic for rapid onset of bilateral blurred vision and eyelid swelling. She had a bilateral high-flared, shallow anterior chamber, and bilateral ciliochoroidal detachment, which was revealed using laser flare photometry and AS-OCT. She also had a serous retinal detachment and disc-macular retinoschisis with a thicker choroid and waved Bruch's membrane. Indocyanine green angiography (ICGA) demonstrated partial hypocyanescence in the early phase and multiple hypercyanescent spots at the intermediate to late phase, which are typical of lupus choroidopathy. Systemic lupus erythematosus was diagnosed, and after the administration of pulse methylprednisolone and pulse cyclophosphamide therapies, all eye findings completely resolved in a month, and all other signs and symptoms improved. CONCLUSIONS AND IMPORTANCE: Lupus choroidopathy, which is less common than retinopathy, might be under-diagnosed because of its difficult evaluation. Although ICGA is the gold standard for diagnosing lupus choroidopathy, a high flare of the anterior chamber and ciliochoroidal detachment might be different from lupus retinopathy. Laser flare photometry and AS-OCT can be non-invasive, helpful tools for the longitudinal evaluation of the patient's response to therapy.

Overview of the transcriptome profiles identified in hagfish, shark, and bichir: current issues arising from some nonmodel vertebrate taxa.

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the "Vertebrate TimeCapsule." The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database.

KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-ß2 suppression.

INTRODUCTION: Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-ß signalling pathway, which is involved in corneal epithelial wound healing. METHODS: HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-ß secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-ß2 treatment. RESULTS: KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-ß canonical signalling pathway, TGF-ß2 secretion by HCECs increased up to 5 folds, and several TGF-ß-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-ß signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-ß2 treatment. CONCLUSIONS: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-ß2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-ß signalling.

The Effect of Dietary Supplementation of Crocetin for Myopia Control in Children: A Randomized Clinical Trial.

The prevalence of myopia has been increasing in recent years. The natural carotenoid crocetin has been reported to suppress experimental myopia in mice. We evaluated the effects of crocetin on myopia suppression in children. A multicenter randomized double-blind placebo-controlled clinical trial was performed with 69 participants aged 6 to 12 years, whose cycloplegic spherical equivalent refractions (SER) were between -1.5 and -4.5 diopter (D). The participants were randomized to receive either a placebo or crocetin and followed up for 24 weeks. Axial length (AL) elongation and changes in SER were evaluated for 24 weeks. Both written informed assent from the participants and written informed consent from legal guardians were obtained in this study because the selection criteria of this trial included children aged between 6 and 12 years old. This trial was approved by the institutional review boards. A mixed-effects model was used for analysis, using both eyes. Two participants dropped out and 67 children completed this trial. The change in SER in the placebo group, -0.41 ± 0.05 D (mean ± standard deviation), was significantly more myopic compared to that in the crocetin group, -0.33 ± 0.05 D (p = 0.049). The AL elongation in the placebo group, 0.21 ± 0.02 mm, was significantly bigger than that in the crocetin group, 0.18 ± 0.02 mm (p = 0.046). In conclusion, dietary crocetin may have a suppressive effect on myopia progression in children, but large-scale studies are required in order to confirm this effect.