The LCHADD Mouse Model Recapitulates Early-Stage Chorioretinopathy in LCHADD Patients.

Purpose: Recent studies have shown that the retinal pigment epithelium (RPE) relies on fatty acid oxidation (FAO) for energy, however, its role in overall retinal health is unknown. The only FAO disorder that presents with chorioretinopathy is long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). Studying the molecular mechanisms can lead to new treatments for patients and elucidate the role of FAO in the RPE. This paper characterizes the chorioretinopathy progression in a recently reported LCHADD mouse model. Methods: Visual assessments, such as optokinetic tracking and fundus imaging, were performed in wildtype (WT) and LCHADD mice at 3, 6, 10, and 12 months of age. Retinal morphology was analyzed in 12-month retinal cross-sections using hematoxylin and eosin (H&E), RPE65, CD68, and TUNEL staining, whereas RPE structure was assessed using transmission electron microscopy (TEM). Acylcarnitine profiles were measured in isolated RPE/sclera samples to determine if FAO was blocked. Bulk RNA-sequencing of 12 month old male WT mice and LCHADD RPE/sclera samples assessed gene expression changes. Results: LCHADD RPE/sclera samples had a 5- to 7-fold increase in long-chain hydroxyacylcarnitines compared to WT, suggesting an impaired LCHAD step in long-chain FAO. LCHADD mice have progressively decreased visual performance and increased RPE degeneration starting at 6 months. LCHADD RPE have an altered structure and a two-fold increase in macrophages in the subretinal space. Finally, LCHADD RPE/sclera have differentially expressed genes compared to WT, including downregulation of genes important for RPE function and angiogenesis. Conclusions: Overall, this LCHADD mouse model recapitulates early-stage chorioretinopathy seen in patients with LCHADD and is a useful model for studying LCHADD chorioretinopathy.

Exploring the choroidal vascular labyrinth and its molecular and structural roles in health and disease.

The choroid is a key player in maintaining ocular homeostasis and plays a role in a variety of chorioretinal diseases, many of which are poorly understood. Recent advances in the field of single-cell RNA sequencing have yielded valuable insights into the properties of choroidal endothelial cells (CECs). Here, we review the role of the choroid in various physiological and pathophysiological mechanisms, focusing on the role of CECs. We also discuss new insights regarding the phenotypic properties of CECs, CEC subpopulations, and the value of measuring transcriptomics in primary CEC cultures derived from post-mortem eyes. In addition, we discuss key phenotypic, structural, and functional differences that distinguish CECs from other endothelial cells such as retinal vascular endothelial cells. Understanding the specific clinical and molecular properties of the choroid will shed new light on the pathogenesis of the broad clinical range of chorioretinal diseases such as age-related macular degeneration, central serous chorioretinopathy and other diseases within the pachychoroid spectrum, uveitis, and diabetic choroidopathy. Although our knowledge is still relatively limited with respect to the clinical features and molecular pathways that underlie these chorioretinal diseases, we summarise new approaches and discuss future directions for gaining new insights into these sight-threatening diseases and highlight new therapeutic strategies such as pluripotent stem cell‒based technologies and gene therapy.

Heparanase Deficiency Is Associated with Disruption, Detachment, and Folding of the Retinal Pigment Epithelium.

PURPOSE: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. MATERIAL AND METHODS: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. RESULTS: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. CONCLUSIONS: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

3-Methyladenine Alleviates Experimental Subretinal Fibrosis by Inhibiting Macrophages and M2 Polarization Through the PI3K/Akt Pathway.

Purpose: To explore the effects of 3-methyladenine (3-MA), a selective inhibitor of phosphatidylinositol-3-kinase (PI3K), on experimental subretinal fibrosis (SRF) in mice. Methods: The SRF mouse model was established by 532 nm laser photocoagulation at each fundus of mice on day 0. 3-MA was administered every 2 days from day 0 to 35. Immunofluorescence of choroidal flat mounts was performed to evaluate the size of SRF area, local macrophages, and polarization, respectively. Besides, Western blot analysis was carried out to assess the expression levels of macrophage polarization-related genes, Arg-1, Ym-1, and transforming growth factor-ß2 (TGF-ß2). Co-culture and migration experiments were used to demonstrate the inhibitory effect of 3-MA on fibroblasts. The gene knockout and Western blot analysis were used to explore the signal pathways related to macrophage polarization. Results: Compared with the control group, the 3-MA-treated group showed significantly less size of SRF area. 3-MA treatment reduced both circulating and local macrophages, and counteracted M2 polarization. Moreover, 3-MA inhibited fibroblast recruitment. Mechanistically, we proved that 3-MA inhibits macrophage M2 polarization by suppressing PI3K/Akt signal pathway rather than the PI3K-autophagy-related signal pathway. Conclusions: 3-MA exerts antifibrotic effects on experimental SRF by targeting circulating and local macrophages and M2 polarization, through PI3K/Akt signal pathway. These results support the potential use of 3-MA as a new therapeutic modality for SRF associated with neovascular age-related macular degeneration.

Mutation-Dependent Pathomechanisms Determine the Phenotype in the Bestrophinopathies.

Best vitelliform macular dystrophy (BD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and the autosomal recessive bestrophinopathy (ARB), together known as the bestrophinopathies, are caused by mutations in the bestrophin-1 (BEST1) gene affecting anion transport through the plasma membrane of the retinal pigment epithelium (RPE). To date, while no treatment exists a better understanding of BEST1-related pathogenesis may help to define therapeutic targets. Here, we systematically characterize functional consequences of mutant BEST1 in thirteen RPE patient cell lines differentiated from human induced pluripotent stem cells (hiPSCs). Both BD and ARB hiPSC-RPEs display a strong reduction of BEST1-mediated anion transport function compared to control, while ADVIRC mutations trigger an increased anion permeability suggesting a stabilized open state condition of channel gating. Furthermore, BD and ARB hiPSC-RPEs differ by the degree of mutant protein turnover and by the site of subcellular protein quality control with adverse effects on lysosomal pH only in the BD-related cell lines. The latter finding is consistent with an altered processing of catalytic enzymes in the lysosomes. The present study provides a deeper insight into distinct molecular mechanisms of the three bestrophinopathies facilitating functional categorization of the more than 300 known BEST1 mutations that result into the distinct retinal phenotypes.

Aldosterone as a mediator of severity in retinal vascular disease: Evidence and potential mechanisms.

Diabetic retinopathy (DR) and retinal vein occlusion (RVO) are the two most common retinal vascular diseases and are major causes of vision loss and blindness worldwide. Recent and ongoing development of medical therapies including anti-vascular endothelial growth factor and corticosteroid drugs for treatment of these diseases have greatly improved the care of afflicted patients. However, severe manifestations of retinal vascular disease result in persistent macular edema, progressive retinal ischemia and incomplete visual recovery. Additionally, choroidal vascular diseases including neovascular age-related macular degeneration (NVAMD) and central serous chorioretinopathy (CSCR) cause vision loss for which current treatments are incompletely effective in some cases and highly burdensome in others. In recent years, aldosterone has gained attention as a contributor to the various deleterious effects of retinal and choroidal vascular diseases via a variety of mechanisms in several retinal cell types. The following is a review of the role of aldosterone in retinal and choroidal vascular diseases as well as our current understanding of the mechanisms by which aldosterone mediates these effects.

Association of CD11b+ Monocytes and Anti-Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.

Importance: CD11b+ immune cells have been implicated in the formation of choroidal neovascularization in experimental studies on animals and disease-association studies on humans. However, the clinical importance of such observations remains unknown. Objective: To investigate whether the proportion of CD11b+ circulating monocytes is associated with the number of anti-vascular endothelial growth factor (anti-VEGF) injections in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). Design, Setting, and Participants: These observational cohort studies collected data from January 1, 2010, through December 31, 2013, and from January 1, 2015, through December 31, 2018. Fresh venous blood samples were acquired for flow cytometric immune studies in patients with neovascular AMD or PCV receiving treatment with aflibercept or ranibizumab as needed for 36 months. Patients (n = 81) without immune diseases were consecutively recruited from a single center in Denmark. Exposures: Proportion of CD11b+ circulating monocytes. Main Outcomes and Measures: The estimation of the number of intravitreal anti-VEGF injections given at 12, 24, and 36 months by the proportion of CD11b+ circulating monocytes and the correlation between these values. The angiogenic role of CD11b+ circulating monocytes was further evaluated by investigating the expression of the known proangiogenic receptor CCR2. Results: Eighty-one patients were included in the analysis (54% women; mean [SD] age, 76 [7] years). The proportion of CD11b+ monocytes at baseline positively estimated the future number of anti-VEGF injections at 12 (ρ = 0.77; 95% CI, 0.35-0.93; P = .004), 24 (ρ = 0.82; 95% CI, 0.44-0.95; P = .002), and 36 (ρ = 0.78; 95% CI, 0.34-0.94; P = .005) months. This association was also found retrospectively in a larger sample of patients with neovascular AMD at 12 (ρ = 0.46; 95% CI, 0.16-0.68; P = .004), 24 (ρ = 0.49; 95% CI, 0.20-0.70; P = .002), and 36 (ρ = 0.65; 95% CI, 0.41-0.80; P < .001) months and patients with PCV at 12 (ρ = 0.27; 95% CI, -0.28 to 0.68; P = .30), 24 (ρ = 0.60; 95% CI, 0.12-0.85; P = .02), and 36 (ρ = 0.70; 95% CI, 0.27-0.90; P = .005) months, suggesting that this association is not specific to AMD but rather reflects VEGF activity in neovascularization. CD11b+ monocytes highly coexpressed CCR2, an important monocytic marker of proangiogenic activity. Conclusions and Relevance: Results of this study demonstrated that the proportion of circulating CD11b+ monocytes estimated and correlated with the number of anti-VEGF injections in patients with neovascular AMD and PCV. Additional longitudinal studies are needed to determine whether these findings have clinical relevance to influence treatment algorithms or provide novel targets for medical therapy.

AUTOSOMAL DOMINANT VITREORETINOCHOROIDOPATHY: When Molecular Genetic Testing Helps Clinical Diagnosis.

PURPOSE: Autosomal dominant vitreoretinochoroidopathy is an extremely rare disease, which belongs to the BEST1-related disease spectrum. METHODS: Report of five patients with an initial diagnosis of atypical rod-cone dystrophy, for whom autosomal dominant vitreoretinochoroidopathy was retrospectively diagnosed on genetic results using targeted next-generation sequencing. Each patient had a comprehensive ophthalmic examination including multimodal retinal imaging and functional evaluation. RESULTS: Visual acuity ranged from <20/800 to 20/25. Two patients had narrowed angle with history of acute angle-closure glaucoma for one patient. Full-field electroretinogram showed severe reduction of both scotopic and photopic responses for 3/5 patients. Electrooculogram could be performed for one of the two patients with moderate alterations of full-field electroretinogram. It revealed severe light rise abnormalities with decreased Arden ratio (125% right eye, 145% left eye) in keeping with generalized severe dysfunction of the retinal pigment epithelium. On fundoscopy, the pathognomonic circumferential hyperpigmented band of the peripheral retina was totally absent in two patients. CONCLUSION: This report highlights the high phenotypic variability of autosomal dominant vitreoretinochoroidopathy, which may be misdiagnosed, especially in advanced forms with severe generalized photoreceptor dysfunction mimicking retinitis pigmentosa. Targeted next-generation sequencing can contribute to the proper clinical diagnosis, especially in case of atypical phenotypic features of autosomal dominant vitreoretinochoroidopathy.

Repeatability and reproducibility of retinal and choroidal thickness measurements in Diabetic Macular Edema using Swept-source Optical Coherence Tomography.

PURPOSE: To evaluate the repeatability and reproducibility of retinal and choroidal thickness measured with Swept source Optical Coherence Tomography (SS-OCT) in eyes with Diabetic Macular Edema (DME). METHODS: 42 DME eyes were imaged using SS-OCT standard Macular scanning protocols. Retinal and choroidal thickness were measured in the Total macular circle (TMC) and foveal central subfield (FCS) using device-integrated specific software. The coefficient of repeatability (CR) and intraclass correlation coefficient (ICC) were determined as a measure of repeatability and relative reliability within graders. Reproducibility was assessed using Bland-Altman plots and 95% limits of agreement (LoA) were determined as a measure of interobserver variability. RESULTS: Intragrader CR of retinal and choroidal thickness were 8.37 and 12.20 microns for TMC and 22.24 and 32.40 microns for FCS, and intergrader 95% LoA were 7.37-8.69 and -27.2-27.71 microns for TMC and -34.21-41.93 and -30.46-24.84 for FCS, respectively. Retinal and choroidal thickness showed very good intraobserver reliability for both TMC and FCS (ICC 0.99, LoA 0.98-0.99 in all cases). Intraobserver and interobserver variability for retinal and choroidal thickness was not significantly different for TMC (p = 0.98 and p = 0.90, p = 0.98 and p = 0.91) or FCS (p = 0.97 and p = 0.85, p = 0.78 and p = 0.73), respectively. CONCLUSIONS: Retinal and choroidal thickness in DME eyes can be quantified with good reliability, repeatability and reproducibility using new OCT devices that incorporate swept source technology. The technical advantages of this technology may provide new insights in the understanding of the choroidal changes related with DME.

Ocular Adnexal Amyloidosis: A Mass Spectrometric Analysis.

PURPOSE: Ocular adnexal amyloidosis (OAA) may represent localized manifestation of an underlying systemic process. Accurate identification of the amyloid fibrils can guide the systemic evaluation and treatment. The aim of this study was to characterize subtypes of OAA using immunohistochemistry and mass spectrometric analysis and to correlate with ocular involvement and systemic association. DESIGN: Retrospective case series. METHODS: Review of patients with OAA subtyped by immunohistochemistry and mass spectrometric analysis at the Cleveland Clinic from June 1995 to June 2017. RESULTS: While immunohistochemistry identified AL amyloid protein in 67% (4/6) of specimens tested, mass spectrometry identified AL amyloid protein in all specimens (10/10). AL lambda was identified in 5 (50%) samples, kappa in 3 (30%), and both kappa and lambda light chains in 2 (20%). The 5 cases of conjunctival amyloidosis were either AL lambda only (3 cases) or both lambda and kappa (2 cases). There were 3 cases that had associated systemic involvement. Two of these had eyelid skin involvement and AL kappa amyloidosis and the other patient had uveal involvement and AL lambda amyloidosis. CONCLUSIONS: Primary amyloidosis-AL is the most common form diagnosed by mass spectrometric analysis in patients with OAA. Immunohistochemistry is ineffective in the characterization of the amyloid deposits in a significant number of cases. Evaluation to exclude systemic involvement or associated underlying lymphoproliferative disorder is warranted.

Vitreous levels of apolipoprotein A1 and retinol binding protein 4 in human rhegmatogenous retinal detachment associated with choroidal detachment.

Purpose: This study aims to quantify the concentration of apolipoprotein A1 (APOA1) and retinol binding protein (RBP4) expressed in the vitreous humors of patients with rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD), rhegmatogenous retinal detachment (RRD), and idiopathic epimacular membrane (IEM). This study also aims to investigate the potential role of APOA1 and RBP4 as biomarkers of RRDCD. Methods: Enzyme-linked immunosorbent assay (ELISA) kits were used to obtain levels of APOA1 and RBP4 from the vitreous humor samples of 76 primary patients. These patients included 23 patients with RRDCD, 28 patients with RRD, and 24 patients with IEM. All patients were undergoing planned pars plana vitrectomy. The differences between the concentrations of the molecular biomarkers among different patient groups were analyzed using the Mann-Whitney U-test for nonparametric values and independent samples t-test or one-way ANOVA analysis for parametric data. The relationship between the molecular biomarkers, grades of proliferative vitreoretinopathy (PVR), and quadrants of retinal detachment were analyzed using nonparametric Spearman's rank correlation analysis. Results: The vitreous concentrations of APOA1 and RBP4 were statistically significantly higher in the RRDCD group compared to the RRD and IEM groups. Patients with severe PVR demonstrated a higher concentration of APOA1 and RBP4 compared to those with mild PVR, but this finding was not statistically significant. There was a statistically significant positive correlation between APOA1 and RBP4 in the RRDCD and RRD groups. Nonparametric Spearman's rank correlation analysis revealed that levels of APOA1 and RBP4 increased statistically significantly with an increasing number of detached retinal quadrants in the RRDCD and RRD groups. Conclusions: The findings of this study allude to the potential of APOA1 and RBP4 as specific biomarkers of RRDCD. The findings of this study may contribute to increased understanding regarding the role of APOA1 and RBP4 in RRDCD.

Polarization-Sensitive Optical Coherence Tomographic Documentation of Choroidal Melanin Loss in Chronic Vogt-Koyanagi-Harada Disease.

Purpose: Vogt-Koyanagi-Harada (VKH) disease is a systemic autoimmune disorder that affects organs with melanocytes. The sunset glow fundus (SGF) in VKH disease was evaluated with polarization-sensitive optical coherence tomography (PS-OCT). Methods: The study involved 28 eyes from 14 patients with chronic VKH disease, 21 eyes from 21 age-matched controls, and 22 eyes from 22 high-myopic patients with a tessellated fundus. VKH eyes were grouped into sunset or non-sunset groups on the basis of color fundus images. The presence of melanin in the choroid was determined by using the degree of polarization uniformity (DOPU) obtained by PS-OCT. The sunset glow index (SGI) was calculated by using color fundus images. Presence of an SGF was evaluated by using DOPU, SGI, subfoveal choroidal thicknesses, near-infrared images, and autofluorescence images at 488 nm (SW-AF) and 785 nm (NIR-AF). Results: There were 16 eyes in the sunset group and 12 eyes in the non-sunset group. For all eyes in the sunset group, the disappearance of choroidal melanin was clearly detected with PS-OCT. Percentage areas of low DOPU in the choroidal interstitial stroma of the sunset group were significantly lower than those of other groups and showed no overlap with other groups. The distribution of choroidal thicknesses and SGI in the sunset group substantially overlapped with other groups. The subjective analyses of the sunset and non-sunset groups, using near infrared, SW-AF, or NIR-AF, showed substantial inconsistencies with the PS-OCT results. Conclusions: PS-OCT provides an in vivo objective evaluation of choroidal melanin loss of the SGF in chronic VKH disease.

Retinopathy of prematurity: inflammation, choroidal degeneration, and novel promising therapeutic strategies.

Retinopathy of prematurity (ROP) is an important cause of childhood blindness globally, and the incidence is rising. The disease is characterized by initial arrested retinal vascularization followed by neovascularization and ensuing retinal detachment causing permanent visual loss. Although neovascularization can be effectively treated via retinal laser ablation, it is unknown which children are at risk of entering this vision-threatening phase of the disease. Laser ablation may itself induce visual field deficits, and there is therefore a need to identify targets for novel and less destructive treatments of ROP. Inflammation is considered a key contributor to the pathogenesis of ROP. A large proportion of preterm infants with ROP will have residual visual loss linked to loss of photoreceptor (PR) and the integrity of the retinal pigment epithelium (RPE) in the macular region. Recent studies using animal models of ROP suggest that choroidal degeneration may be associated with a loss of integrity of the outer retina, a phenomenon so far largely undescribed in ROP pathogenesis. In this review, we highlight inflammatory and neuron-derived factors related to ROP progression, as well, potential targets for new treatment strategies. We also introduce choroidal degeneration as a significant cause of residual visual loss following ROP. We propose that ROP should no longer be considered an inner retinal vasculopathy only, but also a disease of choroidal degeneration affecting both retinal pigment epithelium and photoreceptor integrity.

High-Resolution Adaptive Optics Retinal Image Analysis at Early Stage Central Areolar Choroidal Dystrophy With PRPH2 Mutation.

BACKGROUND AND OBJECTIVE: To report the clinical features of Japanese patients at Stage 1 and 2 of central areolar choroidal dystrophy (CACD). PATIENTS AND METHODS: Five family members had comprehensive ophthalmic examinations including adaptive optics (AO) retinal imaging. Mutation analysis of the PRPH2 gene was performed by Sanger sequencing. The protocol conformed to the tenets of the Declaration of Helsinki and was approved by the institutional review board of The Jikei University School of Medicine. RESULTS: Four family members had a heterozygous PRPH2 mutation, p.R172Q; however, one member with a mutation did not show any ophthalmological abnormalities. Two patients had mild parafoveal retinal dystrophy and a reduction of cone density determined by AO analysis. CONCLUSION: The results indicate that the parafoveal cone photoreceptors can be affected even at the early stage of CACD. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:1115-1126.].

Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1ß.

Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1ß receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1ß-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1ß as a potential therapeutic target in ROP.

Novel compound heterozygous variants in PLK4 identified in a patient with autosomal recessive microcephaly and chorioretinopathy.

It has been well documented that variants in genes encoding centrosomal proteins cause primary autosomal recessive microcephaly, although the association between centrosomal defects and the etiology of microcephaly syndromes is not fully understood. Polo-like kinase 4 (PLK4) is one of the centrosomal proteins required for centriole duplication. We here describe a patient with microcephaly and chorioretinopathy that harbors compound heterozygous missense variants, c.[442A>G]; [2336G>A], in the PLK4 gene. One of these variants, c.442A>G (p.(M148V)), resides in the kinase domain, and the other, c.2336G>A (p.(C779Y)), in the polo-box domain. Aberrant spindle formation was observed in a LCL derived from this patient. Overexpression experiments of the variant PLK4 proteins demonstrated that the p.(C779Y) but not the p.(M148V) had lost centriole overduplication ability. The altered mobility pattern of both variant proteins on a western blot further suggested alterations in post-translation modification. Our data lend support to the hypothesis that impaired centriole duplication caused by PLK4 variants may be involved in the etiology of microcephaly disorder.

Epithelial-mesenchymal transition of the retinal pigment epithelium causes choriocapillaris atrophy.

Epithelial-to-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is commonly observed at sites of choroidal neovascularization in patients suffering from age-related macular degeneration. To learn in an experimental model how RPE EMT affects the biology of the choroidal vasculature, we studied transgenic mice (ßB1-TGF-ß1) with ocular overexpression of transforming growth factor-ß1 (TGF-ß1). RPE EMT was detectable at postnatal day (P)1 and included marked structural and functional alterations such as loss of the outer blood-retina barrier and reduced mRNA expression of the RPE-characteristic molecules Rlbp1, Rpe65, Rbp1 and Vegfa. Moreover, vascular endothelial growth factor (VEGF) was not detectable by immunohistochemistry at the RPE/choroid interface, while RPE cells stained intensely for α-smooth muscle actin. The choriocapillaris, the characteristic choroidal capillary network adjacent to the RPE, developed normally and was not obviously changed in embryonic transgenic eyes but was absent at P1 indicating its atrophy. At around the same time, photoreceptors stopped to differentiate and photoreceptor apoptosis was abundant in the second week of life. Structural changes were also seen in the retinal vasculature of transgenic animals, which did not form intraretinal vessels, and the hyaloid vasculature, which did not regress. In addition, the amounts of retinal HIF-1α and its mRNA were markedly reduced. We conclude that high amounts of active TGF-ß1 in the mouse eye cause transdifferentiation of the RPE to a mesenchymal phenotype. The loss of epithelial differentiation leads to the diminished synthesis of RPE-characteristic molecules including that of VEGF. Lack of RPE-derived VEGF causes atrophy of the choriocapillaris, a scenario that disrupts photoreceptor differentiation and finally results in photoreceptor apoptosis. Lack of retinal vessel formation and of hyaloid vessel regression might be caused by the decrease in the metabolic requirements of the neuroretina leading to low amounts of retinal HIF-1α. In summary, our data indicate that failure of RPE differentiation may well precede and cause atrophy of the choriocapillaris. In contrast, RPE EMT is not sufficient to cause choroidal neovascularization.

Increased choroidal mast cells and their degranulation in age-related macular degeneration.

BACKGROUND/AIMS: Inflammation has been implicated in age-related macular degeneration (AMD). This study investigates the association of mast cells (MCs), a resident choroidal inflammatory cell, with pathological changes in AMD. METHODS: Human donor eyes included aged controls (n=10), clinically diagnosed with early AMD (n=8), geographic atrophy (GA, n=4) and exudative AMD (n=11). The choroids were excised and incubated for alkaline phosphatase (APase; blood vessels) and non-specific esterase activities (MCs). Degranulated (DG) and non-degranulated MCs in four areas of posterior choroid (nasal, non-macular, paramacular and submacular) were counted in flat mounts (4-6 fields/area). Choroids were subsequently embedded in JB-4 and sectioned for histological analyses. RESULTS: The number of MCs was significantly increased in all choroidal areas in early AMD (p=0.0006) and in paramacular area in exudative AMD (139.44±55.3 cells/mm(2); p=0.0091) and GA (199.08±82.0 cells/mm(2); p=0.0019) compared with the aged controls. DG MCs were also increased in paramacular (p=0.001) and submacular choroid (p=0.02) in all forms of AMD. Areas with the greatest numbers of DG MCs had loss of choriocapillaris (CC). Sections revealed that the MCs were widely distributed in Sattler's and Haller's layer in the choroidal stroma in aged controls, whereas MCs were frequently found in close proximity with CC in GA and exudative AMD and in choroidal neovascularisation (CNV). CONCLUSION: Increased MC numbers and degranulation were observed in all AMD choroids. These results suggest that MC degranulation may contribute to the pathogenesis of AMD: death of CC and retinal pigment epithelial and CNV formation. The proteolytic enzymes released from MC granules may result in thinning of AMD choroid.

Adrenomedullin: A potential therapeutic target for retinochoroidal disease.

Adrenomedullin (AM) is a 52-amino acid peptide with anti-inflammatory, anti-apoptotic, and anti-oxidative properties discovered in a human pheochromocytoma. It is a member of the calcitonin peptide superfamily, and its signal is mediated by calcitonin receptor-like receptor (CLR). CLR interacts with receptor activity-modifying proteins (RAMPs), among which RAMP-2 and RAMP-3 carry CLR from the endoplasmic reticulum to the cellular membrane to confer high affinity for AM. In addition to being implicated in a variety of systemic diseases, AM is a critical contributor to the pathogenesis of retinochoroidal disease. It is robustly upregulated in retinochoroidal disease models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularisation (CNV) as well as in human patients with retinochoroidal diseases. In this review, we discuss the most salient recent findings that strongly illustrate the role of AM in retinochoroidal disease. In the OIR model, AM was identified as a key angiogenic mediator of retinal vascularisation, and AM inhibition suppressed only pathological angiogenesis, not physiological angiogenesis. On the contrary, lesion size was larger in AM(+/-) CNV model mice, presumably due to the anti-inflammatory function of AM. Despite the success of anti-vascular endothelial growth factor agents for the treatment of retinochoroidal disease, therapeutic shortcomings remain. Finding ways to modulate AM activity will provide new treatment avenues. Potential treatment strategies modulating the action of AM and its signaling pathway have been studied extensively. AM and its signaling molecules are intriguing future treatment targets for retinochoroidal disease.

Molecular pathogenesis of retinal and choroidal vascular diseases.

There are two major types of ocular neovascularization that affect the retina, retinal neovascularization (NV) and subretinal or choroidal NV. Retinal NV occurs in a group of diseases referred to as ischemic retinopathies in which damage to retinal vessels results in retinal ischemia. Most prevalent of these are diabetic retinopathy and retinal vein occlusions. Subretinal and choroidal NV occur in diseases of the outer retina and Bruch's membrane, the most prevalent of which is age-related macular degeneration. Numerous studies in mouse models have helped to elucidate the molecular pathogenesis underlying retinal, subretinal, and choroidal NV. There is considerable overlap because the precipitating event in each is stabilization of hypoxia inducible factor-1 (HIF-1) which leads to upregulation of several hypoxia-regulated gene products, including vascular endothelial growth factor (VEGF), angiopoietin 2, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and several others. Stimulation of VEGF signaling and suppression of Tie2 by angiopoietin 2 and VE-PTP are critical for sprouting of retinal, subretinal, and choroidal NV, with perturbation of Bruch's membrane also needed for the latter. Additional HIF-1-regulated gene products cause further stimulation of the NV. It is difficult to model macular edema in animals and therefore proof-of-concept clinical trials were done and demonstrated that VEGF plays a central role and that suppression of Tie2 is also important. Neutralization of VEGF is currently the first line therapy for all of the above disease processes, but new treatments directed at some of the other molecular targets, particularly stabilization of Tie2, are likely to provide additional benefit for subretinal/choroidal NV and macular edema. In addition, the chronicity of these diseases as well as the implication of VEGF as a cause of retinal nonperfusion and progression of background diabetic retinopathy make sustained delivery approaches for VEGF antagonists a priority.