Retinal Cells Research Articles

Posterior eye delivery of angiogenesis-inhibiting RNA nanoparticles via subconjunctival injection

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.

Tricetin suppresses the cell migration and BMP-6 expression through p38 signaling pathways in human retinal pigment epithelium cells.

Proliferative vitreoretinopathy (PVR) is a visual-threatening disease, which cause from the migration of retinal pigment epithelium (RPE). Tricetin, a family of flavonoids, can inhibit the metastasis of several cancers. Herein, we aim to evaluate the possible effect of tricetin on inhibiting ARPE-19 cells migration. The Boyden chamber assay, wound healing assay, RNA sequencing, and Western blot analysis were applied in our experiment. The results revealed that tricetin inhibited the cell migration abilities of ARPE-19 cells. Moreover, using RNA sequencing technology, we revealed that tricetin repressed bone morphogenetic protein-6 (BMP-6) gene expressions in ARPE-19 cells. Overexpression of BMP-6 resulted in significant restoration of cell migration capabilities of tricetin-treated ARPE-19 cells. Furthermore, tricetin suppressed the phosphorylation of the p38 signaling pathway. Moreover, blocking the p38 pathway also inhibits BMP-6 expression and migration in the ARPE-19 cells. In conclusion, this study revealed that tricetin inhibits the ARPE-19 cell migration mainly via the suppression of BMP-6 expression and p38 signaling pathway.

Investigating the impact of empagliflozin on the retina of diabetic mice.

Diabetic retinopathy (DR) frequently results in compromised visual function, with hyperglycemia-induced disruption of the blood-retinal barrier (BRB) through various pathways as a critical mechanism. Existing DR treatments fail to address early and potentially reversible microvascular alterations. This study examined the effects of empagliflozin (EMPA), a selective Sodium-glucose transporter 2 (SGLT2) inhibitor, on the retina of db/db mice. The objective of this study is to investigate the potential role of EMPA in the prevention and delay of DR. db/db mice were randomly assigned to either the EMPA treatment group (db/db + Emp) or the model group (db/db), while C57 mice served as the normal control group (C57). Mice in the db/db + Emp group received EMPA for eight weeks. Body weight, fasting blood glucose (FBG), and blood VEGF were subsequently measured in all mice, along with the detection of specific inflammatory factors and BRB proteins in the retina. Retinal SGLT2 protein expression was compared using immunohistochemical analysis, and BRB structural changes were observed via electron microscopy. EMPA reduced FBG, blood VEGF, and retinal inflammatory factors TNF-α, IL-6, and VEGF levels in the eye tissues of db/db mice. EMPA also increased Claudin-1, Occludin-1, and ZO-1 levels while decreasing ICAM-1 and Fibronectin, thereby preserving BRB function in db/db mice. Immunohistochemistry revealed that EMPA reduced SGLT2 expression in the retina of diabetic mice, and electron microscopy demonstrated that EMPA diminished tight junction damage between retinal vascular endothelial cells and prevented retinal vascular basement membrane thickening in diabetic mice. EMPA mitigates inflammation and preserves BRB structure and function, suggesting that it may prevent DR or serve as an effective early treatment for DR.

Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1β (IL-1β) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

Reduced Thickness of the Retina in de novo Parkinson's Disease Shows A Distinct Pattern, Different from Glaucoma.

Parkinson's disease (PD) patients experience visual symptoms and retinal degeneration. Studies using optical coherence tomography (OCT) have shown reduced thickness of the retina in PD, also a key characteristic of glaucoma. To identify the presence and pattern of retinal changes in de novo, treatment-naive PD patients compared to healthy controls (HC) and early primary open angle glaucoma (POAG) patients. Macular OCT data (10×10 mm) were collected from HC, PD, and early POAG patients, at the University Medical Center Groningen. Bayesian informative hypotheses statistical analyses were carried out comparing HC, PD-, and POAG patients, within each retinal cell layer. In total 100 HC, 121 PD, and 78 POAG patients were included. We showed significant reduced thickness of the inner plexiform layer and retinal pigment epithelium in PD compared to HC. POAG patients presented with a significantly thinner retinal nerve fiber layer, ganglion cell layer, inner plexiform layer, outer plexiform layer, and outer photoreceptor and subretinal virtual space compared to PD. Only the outer segment layer and retinal pigment epithelium were significantly thinner in PD compared to POAG. De novo PD patients show reduced thickness of the retina compared to HC, especially of the inner plexiform layer, which differs significantly from POAG, showing a more extensive and widespread pattern of reduced thickness across layers. OCT is a useful tool to detect retinal changes in de novo PD, but its specificity versus other neurodegenerative disorders has to be established.

A novel GCaMP6f-RCS rat model for studying electrical stimulation in the degenerated retina.

Background: Retinal prostheses aim to restore vision by electrically stimulating the remaining viable retinal cells in Retinal Degeneration (RD) cases. Research in this field necessitates a comprehensive analysis of retinal ganglion cells' (RGCs) responses to assess the obtained visual acuity and quality. Here we present a novel animal model which facilitates the optical recording of RGCs activity in an RD rat. This model can significantly enhance the functional evaluation of vision restoration treatments. Methods: The development of the novel rat model is based on crossbreeding a retinal degenerated Royal College of Surgeons (RCS) rat with a transgenic line expressing the genetic calcium indicator GCaMP6f in the RGCs. Characterization of the model was achieved using Optical Coherence Tomography (OCT) imaging, histology, and electroretinography (ERG) at the ages of 4, 8, and 12weeks. Additionally, optical recordings of RGCs function in response to ex-vivo subretinal electrical stimulations were performed. Results: Histological investigations confirmed the high expression of GCaMP6f in the RGCs and minimal expression in the inner nuclear layer (INL). OCT imaging and histological studies revealed the expected gradual retinal degeneration, as evident by the decrease in retinal thickness with age and the formation of subretinal debris. This degeneration was further confirmed by ERG recordings, which demonstrated a significant decrease in the b-wave amplitude throughout the degeneration process, culminating in its absence at 12weeks in the GCaMP6f-RCS rat. Importantly, the feasibility of investigating subretinal stimulation was demonstrated, revealing a consistent increase in activation threshold throughout degeneration. Furthermore, an increase in the diameter of the activated area with increasing currents was observed. The spatial spread of the activation area in the GCaMP6f-RCS rat was found to be smaller and exhibited faster activation dynamics compared with the GCaMP6f-LE strain. Conclusion: This novel animal model offers an opportunity to deepen our understanding of prosthetically induced retinal responses, potentially leading to significant advancements in prosthetic interventions in visual impairments.

SUMOylation of GMFB regulates its stability and function in retinal pigment epithelial cells under hyperglycemia

BackgroundGlia maturation factor beta (GMFB) is a growth and differentiation factor that acts as an intracellular regulator of signal transduction pathways. The small ubiquitin-related modifier (SUMO) modification, SUMOylation, is a posttranslational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. However, the relationship between GMFB and SUMOylation is unclear. ResultsHere, we report for the first time that GMFB and SUMO1 are markedly increased in retinal pigment epithelial (RPE) cells at the early stage of diabetes mellitus (DM) under hyperglycemia. The GMFΒ protein could be mono-SUMOylated by SUMO1 at the K20, K35, K58 or K97 sites. SUMOylation of GMFB led to its increased protein stability and subcellular translocation. Furthermore, deSUMOylation of GMFΒ downregulates multiple signaling pathways, including the Jak-STAT signaling pathway, p38 pathway and NF-kappa B signaling pathway. ConclusionsThis work provides novel insight into the role of SUMOylated GMFB in RPE cells and provides a novel therapeutic target for diabetic retinopathy (DR).

Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycaemia.

In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive. Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant down-regulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycaemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycaemic zebrafish model. The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs. EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycaemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.

TIN2-mediated reduction of mitophagy induces RPE senescence under high glucose

The telomere-associated protein TIN2 localizes to both telomeres and mitochondria. Nevertheless, the impact of TIN2 on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. This research aims to examine the role of TIN2 in the senescence of RPE and its potential as a therapeutic target. Western blotting and immunofluorescence staining were utilized to identify TIN2 expression and mitophagy. RT-qPCR was employed to identify senescent associated secretory phenotype (SASP) in ARPE-19 cells infected with TIN2 overexpression. To examine mitochondria and the cellular senescence of RPE, TEM, SA-β-gal staining, and cell cycle analysis were used. The impact of TIN2 was examined using OCT and immunohistochemistry in mice. DHE staining and ZO-1 immunofluorescence were applied to detect RPE oxidative stress and tight junctions. Our research revealed that increased mitochondria-localized TIN2 aggravated the cellular senescence of RPE cells both in vivo and in vitro under hyperglycemia. TIN2 overexpression stimulated the mTOR signaling pathway in ARPE-19 cells and exacerbated the inhibition of mitophagy levels under high glucose, which can be remedied through the mTOR inhibitor, rapamycin. Knockdown of TIN2 significantly reduced senescence and mitochondrial oxidative stress in ARPE-19 cells under high glucose and restored retinal thickness and RPE cell tight junctions in DR mice. Our study indicates that increased mitochondria-localized TIN2 induced cellular senescence in RPE via compromised mitophagy and activated mTOR signaling. These results propose that targeting TIN2 could potentially serve as a therapeutic strategy in the treatment of DR.

Neuroprotective and anti-inflammatory effects of eicosane on glutamate and NMDA-induced retinal ganglion cell injury.

To investigate the protective effects, antioxidant potential, and anti-inflammatory mechanisms of eicosane on glutamate-induced cell damage and on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) injury in a mouse model of glaucoma. The protective effects of eicosane on the rat R28 retinal precursor cell line were assessed using cell counting kit-8 assays and Hoechst-propidium iodide staining. Intracellular reactive oxygen species (ROS) production was measured using the fluorescent probe 2'-7'-dichlorofluorescin diacetate and flow cytometry. The protective role of eicosane on NMDA-induced RGC injury in a mouse glaucoma model was determined by immunostaining of frozen sections of retina. The effects of eicosane on the metabolome of the retina in mice with NMDA-induced RGC damage were evaluated by liquid chromatography-mass spectroscopy (LC-MS) and untargeted metabolomics analyses. Eicosane treatment significantly attenuated glutamate-induced damage to R28 cells in vitro. Eicosane also protected RGCs against NMDA-induced injury in a mouse glaucoma model. Untargeted metabolomics analyses showed that eicosane increased multiple metabolites, including L-arginine and L-carnitine, in the retina. Eicosane has protective effects, antioxidant potential, and anti-inflammatory properties in an in vitro model of glutamate-induced cell damage and in an in vivo model of NMDA-induced RGC injury in mouse glaucoma through modulation of L-arginine and/or L-carnitine metabolism.

Neuron image segmentation based on convolution and BN fusion and multi-input feature fusion

The segmentation of neuronal morphology in electron microscopy images is crucial for the analysis and understanding of neuronal function. However, most of the existing segmentation methods are not suitable for challenging datasets where the neuronal structure is contaminated by noise or has interrupted parts. In this paper, we propose a segmentation method based on deep learning to determine the location information of neurons and reduce the influence of image noise in the data. Specifically, we adapt our neuron dataset based on UNet by using convolution with BN fusion and multi-input feature fusion. The method is named REDAFNet. The model simplifies the model structure and enhances the generalization ability by fusing the convolution layer and BN layer. The noise interference in the data was reduced by multi-input feature fusion, and the ability to understand and express the data was enhanced. The method takes a neuron image as input and its pixel segmentation map as output. Experimental results show that the segmentation accuracy of the proposed method is 91.96%, 93.86% and 80.25% on the ISBI2012 dataset, U-RISC retinal neuron dataset and N2DH-GOWT1 stem cell dataset, respectively. Compared with the existing segmentation methods, the proposed method can extract more complete feature information and achieve more accurate segmentation.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

CD200R promotes high glucose-induced oxidative stress and damage in human retinal pigment epithelial cells by activating the mTOR signaling pathway

Diabetic retinopathy (DR) is established as the primary cause of visual impairment and preventable blindness, posing significant social and economic burdens on healthcare systems worldwide. Oxidative stress has been identified as a major contributor to DR, yet the precise role of the transmembrane glycoprotein CD200R in this context remains elusive. We studied human retinal pigment epithelia ARPE-19 cells to investigate the role of CD200R in high-glucose (HG) induced oxidative stress. Under HG conditions, we found a significant increase in CD200R expression in a time-dependent pattern. Conversely, knockdown of CD200R effectively alleviated oxidative stress and restored cell viability in HG-treated ARPE-19 cells, a phenomenon corroborated by the addition of a reactive oxygen species (ROS) scavenger. Exploration of the AKT/mTOR signaling pathway confirmed its mediating role regarding CD200R knockdown suppression of the expression of key proteins induced by HG conditions. Additionally, we found that the inhibition of mTOR signaling with Rapamycin effectively countered HG-induced oxidative stress in ARPE-19 cells, suggesting a promising therapeutic target against oxidative stress in the context of DR. This study establishes the crucial role of CD200R in HG-induced oxidative stress and identifies potential therapeutic avenues for the treatment of DR.

Intraocular mRNA delivery with endogenous MmPEG10-based virus-like particles

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy.

AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Abstract Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death. However, there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation. Adeno-associated virus (AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa. The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function. To do this, we injected retinal degeneration 10 (rd10) mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark- and light-adapted electroretinogram, optical coherence tomography, and immunofluorescence. Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment, and the results from this analysis were verified by real-time polymerase chain reaction and western blotting. AAV2-PDE6B injection significantly upregulated PDE6β expression, preserved electroretinogram responses, and preserved outer nuclear layer thickness in rd10 mice. Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception, and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice. Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways. Furthermore, the phototransduction-related proteins Pde6α, Rom1, Rho, Aldh1a1, and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment. Finally, Bax/Bcl-2, p-ERK/ERK, and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment. Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

A potent bioreducible ionizable lipid nanoparticle enables siRNA delivery for retinal neovascularization inhibition

Small interfering RNA (siRNA) is emerging as a promising treatment for retinal neovascularization due to its specific inhibition of the expression of target genes. However, the clinical translation of siRNA drugs is hindered by the efficiency and safety of delivery vectors. Here, we describe the properties of a new bioreducible ionizable lipid nanoparticle (LNP) 2N12H, which is based on a rationally designed novel ionizable lipid called 2N12B. 2N12H exhibited degradation in response to the mimic cytoplasmic glutathione condition and ionization with a pKa value of 6.5, which remaining neutral at pH 7.4. At a nitrogen to phosphorus ratio of 5, 2N12H efficiently encapsulated and protected siRNA from degradation. Compared to the commercial vehicle Lipofectamine 2000, 2N12H demonstrated similar silencing efficiency and improved safety in the in vitro cell experiments. 2N12H/siVEGFA reduced the expression of VEGFA in retinal pigment epithelium cells and mouse retina, consequently suppressing cell migration and retinal neovascularization. In the mouse model, the therapeutic effect of 2N12H/siVEGFA was comparable to that of the clinical drug ranibizumab. Together, these results suggest the potential of this novel ionizable LNP to facilitate the development of nonviral ocular gene delivery systems.

Sustained Release Formulation of Hydroxypropyl-β-cyclodextrin Eye Drops Using Xanthan Gum.

Bietti's crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-β-cyclodextrin (HP-β-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-β-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-β-CyD. Our results suggest that HP-β-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-β-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-β-CyD alone. Furthermore, the slow release of HP-β-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-β-CyD, and that HP-β-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 μM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.