IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Cone cell dysfunction attenuates retinal neovascularization in oxygen-induced retinopathy mouse model.

Aberrant neovascularization is the most common feature in retinopathy of prematurity (ROP), which leads to the retinal detachment and visual defects in neonates with a low gestational age eventually. Understanding the regulation of inappropriate angiogenic signaling benefits individuals at-risk. Recently, neural activity originating from the specific neural activity has been considered to contribute to retinal angiogenesis. Here, we explored the impact of cone cell dysfunction on oxygen-induced retinopathy (OIR), a mouse model commonly employed to understand retinal diseases associated with abnormal blood vessel growth, using the Gnat2cpfl3 (cone photoreceptor function loss-3) strain of mice (regardless of the sex), which is known for its inherent cone cell dysfunction. We found that the retinal avascular area, hypoxic area, and neovascular area were significantly attenuated in Gnat2cpfl3 OIR mice compared to those in C57BL/6 OIR mice. Moreover, the HIF-1α/VEGF axis was also reduced in Gnat2cpfl3 OIR mice. Collectively, our results indicated that cone cell dysfunction, as observed in Gnat2cpfl3 OIR mice, leads to attenuated retinal neovascularization. This finding suggests that retinal neural activity may precede and potentially influence the onset of pathological neovascularization.

Blockade of mTORC1-NOX signaling pathway inhibits TGF-ß1-mediated senescence-like structural alterations of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) undergoes characteristic structural changes and epithelial-mesenchymal transition (EMT) during normal aging, which are exacerbated in age-related macular degeneration (AMD). Although the pathogenic mechanisms of aging and AMD remain unclear, transforming growth factor-ß1 (TGF-ß1) is known to induce oxidative stress, morphometric changes, and EMT as a senescence-promoting factor. In this study, we examined whether intravitreal injection of TGF-ß1 into the mouse eye elicits senescence-like morphological alterations in the RPE and if this can be prevented by suppressing mammalian target of rapamycin complex 1 (mTORC1) or NADPH oxidase (NOX) signaling. We verified that intravitreal TGF-ß1-induced stress fiber formation and EMT in RPE cells, along with age-associated morphometric changes, including increased variation in cell size and reduced cell density. In RPE cells, exogenous TGF-ß1 increased endogenous expression of TGF-ß1 and upregulated Smad3-ERK1/2-mTORC1 signaling, increasing reactive oxygen species (ROS) production and EMT. We demonstrated that inhibition of the mTORC1-NOX4 pathway by pretreatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-dependent protein kinase, or GKT137831, a NOX1/4 inhibitor, decreased ROS generation, prevented stress fiber formation, attenuated EMT, and improved the regularity of the RPE structure in vitro and in vivo. These results suggest that intravitreal TGF-ß1 injection could be used as a screening model to investigate the aging-related structural and functional changes to the RPE. Furthermore, the regulation of TGF-ß-mTORC1-NOX signaling could be a potential therapeutic target for reducing pathogenic alterations in aged RPE and AMD.

Antitumor Activity of Novel Signal Transducer and Activator of Transcription 3 Inhibitors on Retinoblastoma.

Signal transducer and activator of transcription 3 (STAT3) is a plausible therapeutic target in the treatment of retinoblastoma, the most common intraocular malignant tumor in children. STAT3, a transcription factor of several genes related to tumorigenesis, is activated in retinoblastoma tumors as well as other cancers. In this study, we investigated the structure-activity relationship of a library of STAT3 inhibitors, including a novel series of derivatives of the previously reported compound with a Michael acceptor (compound 1). We chose two novel STAT3 inhibitors, compounds 11 and 15, from the library based on their inhibitory effects on the phosphorylation and transcription activity of STAT3. These STAT3 inhibitors effectively suppressed the phosphorylation of STAT3 and inhibited the expression of STAT3-related genes CCND1, CDKN1A, BCL2, BCL2L1, BIRC5, MYC, MMP1, MMP9, and VEGFA Intraocularly administered STAT3 inhibitors decreased the degree of tumor formation in the vitreous cavity of BALB/c nude mice of an orthotopic transplantation model. It is noteworthy that compounds 11 and 15 did not induce in vitro and in vivo toxicity on retinal constituent cells and retinal tissues, respectively, despite their potent antitumor effects. We suggest that these novel STAT3 inhibitors be used in the treatment of retinoblastoma. SIGNIFICANCE STATEMENT: The current study suggests the novel STAT3 inhibitors with Michael acceptors possess antitumor activity on retinoblastoma, the most common intraocular cancer in children. Based on detailed structure-activity relationship studies, we found a 4-fluoro and 3-trifluoro analog (compound 11) and a monochloro analog (compound 15) of the parental compound (compound 1) inhibited STAT3 phosphorylation, leading to suppressed retinoblastoma in vitro and in vivo.

Giant Y79 retinoblastoma cells contain functionally active T-type calcium channels.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (-54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

Bispecific anti-mPDGFRß x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRß expressing cells.

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRß) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRß expressing cells. Multiple anti-mPDGFRß antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.

Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.

Tumor Environment of Retinoblastoma, Intraocular Cancer.

Retinoblastoma, an intraocular cancer primarily affecting children, interacts with surrounding intraocular and extraocular structures in the development and progression. Subretinal and vitreous seeds are characteristic features of retinoblastoma, which result from the interaction between the tumor and its environment at the levels of tissue and microenvironment. The retina and vitreous affect the disease course and responses to treatment options. Also, neighboring cells in the retina and physicochemical properties of the tumor microenvironment are related to the biological activities of retinoblastoma tumors. Researches focusing on the tumor environment of retinoblastoma will lead to the development of more effective treatment options, which can revolutionize the prognosis of patients with retinoblastoma.

Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood-retinal barrier in diabetic retinopathy.

Inner and outer blood-retinal barriers (BRBs), mainly composed of retinal endothelial cells and retinal pigment epithelial (RPE) cells, respectively, maintain the integrity of the retinal tissues. In this study, we aimed to investigate the mechanisms of the outer BRB disruption regarding the interaction between RPE and microglia. In mice with high-fat diet-induced obesity and streptozotocin-induced hyperglycemia, microglia accumulated on the RPE layer, as in those after intravitreal injection of interleukin (IL)-6, which is elevated in ocular fluids of patients with diabetic retinopathy. Although IL-6 did not directly affect the levels of zonula occludens (ZO)-1 and occludin in RPE cells, IL-6 increased VEGFA mRNA in RPE cells to recruit microglial cells. In microglial cells, IL-6 upregulated the mRNA levels of MCP1, MIP1A, and MIP1B, to amplify the recruitment of microglial cells. In this manner, IL-6 modulated RPE and microglial cells to attract microglial cells on RPE cells. Furthermore, IL-6-treated microglial cells produced and secreted tumor necrosis factor (TNF)-α, which activated NF-κB and decreased the levels of ZO-1 in RPE cells. As STAT3 inhibition reversed the effects of IL-6-treated microglial cells on the RPE monolayer in vitro, it reduced the recruitment of microglial cells and the production of TNF-α in RPE tissues in streptozotocin-treated mice. Taken together, IL-6-treated RPE and microglial cells amplified the recruitment of microglial cells and IL-6-treated microglial cells produced TNF-α to disrupt the outer BRB in diabetic retinopathy.

Outcomes of Proton Beam Radiation Therapy for Retinoblastoma With Vitreous Seeds.

Vitreous seeds are the most challenging aspect in the management of retinoblastoma. We report the outcomes of treatment with proton beam radiation therapy (PBRT) for retinoblastoma with vitreous seeds in naive or previously treated eyes. In this retrospective case series, we analyzed data of 4 retinoblastoma patients with vitreous seeds who received PBRT at the Proton Therapy Center, National Cancer Center in Korea between June 2007 and August 2017. All 4 eyes treated by PBRT were classified as group D according to the International Classification of Retinoblastoma (ICRB) criteria, and the vitreous seeds, as class 3 (clouds). The tumor and vitreous seeds regressed in 2 eyes, and globe salvage was achieved in these 2 eyes (50%). The post-PBRT ophthalmologic follow-up time of these 2 preserved eyes was 12 and 50 months, respectively. Visual acuity measurements of the successfully treated patients were 20/40 and 20/600. No radiation-associated malignancies were noted. In conclusion, PBRT successfully treated vitreous seeds classified as clouds in half of the cases, and successfully treated patients who retained useful vision. Therefore, PBRT might be a viable treatment option for vitreous seeds in patients with retinoblastoma.

Quantitative Proteomics Reveals ß2 Integrin-mediated Cytoskeletal Rearrangement in Vascular Endothelial Growth Factor (VEGF)-induced Retinal Vascular Hyperpermeability.

Retinal vascular hyperpermeability causes macular edema, leading to visual deterioration in retinal diseases such as diabetic retinopathy and retinal vascular occlusion. Dysregulation of junction integrity between endothelial cells by vascular endothelial growth factor (VEGF) was shown to cause retinal vascular hyperpermeability. Accordingly, anti-VEGF agents have been used to treat retinal vascular hyperpermeability. However, they can confer potential toxicity through their deleterious effects on maintenance and survival of neuronal and endothelial cells in the retina. Thus, it is important to identify novel therapeutic targets for retinal vascular hyperpermeability other than VEGF. Here, we prepared murine retinas showing VEGF-induced vascular leakage from superficial retinal vascular plexus and prevention of VEGF-induced leakage by anti-VEGF antibody treatment. We then performed comprehensive proteome profiling of these samples and identified retinal proteins for which abundances were differentially expressed by VEGF, but such alterations were inhibited by anti-VEGF antibody. Functional enrichment and network analyses of these proteins revealed the ß2 integrin pathway, which can prevent dysregulation of junction integrity between endothelial cells through cytoskeletal rearrangement, as a potential therapeutic target for retinal vascular hyperpermeability. Finally, we experimentally demonstrated that inhibition of the ß2 integrin pathway salvaged VEGF-induced retinal vascular hyperpermeability, supporting its validity as an alternative therapeutic target to anti-VEGF agents.

Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity.

Bare gold nanospheres have been shown to have anti-angiogenic effects but are optically unfavorable because their resonant wavelength lies in the visible spectrum. Here, we design gold nanodisks with a higher scattering capability than gold nanorods and with a resonant wavelength at near-infrared region - the area where the source of light utilized by optical coherence tomography (OCT) lies. With a physical synthesis system, we then fabricate 160-nm-sized gold nanodisks exhibiting resonant wavelength at 830 nm. The synthesized nanoparticles were successfully visualized in in vivo OCT at concentrations as low as 1 pM. After demonstrating their binding ability to vascular endothelial growth factor (VEGF), we show that they suppress VEGF-induced migration of endothelial cells. Finally, we demonstrate that intravitreally injected gold nanodisks attenuate neovascularization of oxygen-induced retinopathy in mice, in a dose dependent manner, such that they are cleared from the vitreous within 2 weeks without histologic or electrophysiologic toxicity.

Size, surface charge, and shape determine therapeutic effects of nanoparticles on brain and retinal diseases.

Nanoparticles can be valuable therapeutic options to overcome physical barriers to reach central nervous system. Systemically administered nanoparticles can pass through blood-neural barriers; whereas, locally injected nanoparticles directly reach neuronal and perineuronal cells. In this review, we highlight the importance of size, surface charge, and shape of nanoparticles in determining therapeutic effects on brain and retinal diseases. These features affect overall processes of delivery of nanoparticles: in vivo stability in blood and other body fluids, clearance via mononuclear phagocyte system, attachment with target cells, and penetration into target cells. Furthermore, they are also determinants of nano-bio interfaces: they determine corona formation with proteins in body fluids. Taken together, we emphasize the importance of considerations on characteristics of nanoparticles more suitable for the treatment of brain and retinal diseases in the development of nanoparticle-based therapeutics. FROM THE CLINICAL EDITOR: The central nervous system (CNS) remains an area where drug access and delivery are difficult clinically due to the blood brain barrier. With advances in nanotechnology, many researchers have designed and produced nanoparticle-based systems in an attempt to solve this problem. In this concise review, the authors described the current status of drug delivery to the CNS, based on particle size and shape. This article should stimulate more research to be done on future drug design.

Anti-angiogenic effect of bare titanium dioxide nanoparticles on pathologic neovascularization without unbearable toxicity.

Local application requires fewer nanoparticles than systemic delivery to achieve effective concentration. In this study, we investigated the potential toxicity and efficacy of bare titanium dioxide (TiO2) nanoparticles by local administration into the eye. Mono-disperse, 20nm-size TiO2 nanoparticles did not affect the viability of retinal constituent cells within certain range of concentrations (~1.30µg/mL). Furthermore, local delivery of TiO2 nanoparticles did not induce any significant toxicity at the level of gene expression and histologic integrity in the retina of C57BL/6 mice. Interestingly, at the low concentration (130ng/mL) without definite toxicity, these nanoparticles suppressed in vitro angiogenesis processes and in vivo retinal neovascularization in oxygen-induced retinopathy mice when they are administered intravitreally. Taken together, our results demonstrate that even TiO2 nanoparticles can be safely utilized for the treatment of retinal diseases at the adequate concentration levels, especially through local administration. FROM THE CLINICAL EDITOR: In this paper the local application of titanium dioxide is described as a local treatment for retinal diseases associated with neovascularization. While these nanoparticles have known systemic toxicity, this work demonstrates that when applied locally in a mouse model, they can be used without observable toxicity even in their native forms.

Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium.

Transplantation of mitochondria derived from mesenchymal stem cells (MSCs) has emerged as a new treatment method to improve mitochondrial dysfunction and alleviate cell impairment. Interest in using extrinsic mitochondrial transplantation as a therapeutic approach has been increasing because it has been confirmed to be effective in treating various diseases related to mitochondrial dysfunction, including ischemia, cardiovascular disease, and toxic damage. To support this application, we conducted an experiment to deliver external mitochondria to retinal pigment epithelial cells treated with oligomeric amyloid-beta (oAß). Externally delivered amyloid-beta internalizes into cells and interacts with mitochondria, resulting in mitochondrial dysfunction and intracellular damage, including increased reactive oxygen species and destruction of tight junction proteins. Externally delivered mitochondria were confirmed to alleviate mitochondrial dysfunction and tight junction protein disruption as well as improve internalized oAß clearance. These results were also confirmed in a mouse model in vivo. Overall, these findings indicate that the transfer of external mitochondria isolated from MSCs has potential as a new treatment method for age-related macular degeneration, which involves oAß-induced changes to the retinal pigment epithelium.

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.

Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs.

BACKGROUND: With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. METHODS: We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer's solution containing carboplatin and melphalan after the injection of retinoblastoma cells. RESULTS: The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. CONCLUSIONS: This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.

Nerve growth factor-mediated vascular endothelial growth factor expression of astrocyte in retinal vascular development.

The angiogenic aspect of neurotrophins and their receptors rather than the neuroscientific aspect has been focused. However, their role in retinal vascular development is underdiscovered. The purpose of this study is to understand the role of neurotrophin receptors in retinal vascular development and the mechanisms of their action. To identify the expression of tropomyosin receptor kinase receptor (Trk) in developing retina, tissues of 4, 8, 12, 16 and 26 day-old mice were prepared for experiments. Immunohistochemistry and immunofluorescence double staining against glial fibrillary acidic protein and type IV collagen were performed. TrkA was expressed mainly along the vessel structure in inner part of retina, especially in retinal astrocyte. In cultured primary astrocyte, recombinant nerve growth factor (NGF) was used to activate TrkA. NGF induced the phosphorylation of TrkA, and it also enhanced the level of activated Akt and vascular endothelial growth factor (VEGF) mRNA. Inhibition of phosphoinositide 3-kinase (PI3K) reversed the NGF-induced activation of these two molecules. This study demonstrated that TrkA activation on NGF leads to VEGF elevation by PI3K-Akt pathway and therefore suggested that TrkA could be a stimulator of retinal vascular development.

NK cell-associated antigen expression in retinoblastoma animal model.

Natural killer (NK) cells are critical components of our immune system. Herein, we for the first time analyzed the expression and localization of the activating receptor NK cell lectin-like receptor gene 2D (NKG2D) ligands, HLA-G, MICA, MICA/B, and ULBP-2 in orthotopic transplantation models of retinoblastoma. Interestingly, HLA-G and MICA/B were expressed in retinoblastoma cell, whereas MICA and ULBP-2 were not detected. Moreover, HLA-G and MICA/B were primarily detected in proliferative area of the tumor periphery with high Ki-67 immunostaining. Our results suggest that NKG2D ligands are differentially expressed in retinoblastoma, which would play a crucial role in immunomodulation in retinoblastoma.