AICAR upregulates ABCA1/ABCG1 expression in the retinal pigment epithelium and reduces Bruch's membrane lipid deposit in ApoE deficient mice.

The etiology of age-related macular degeneration (AMD) is diverse; however, recent evidence suggests that the lipid metabolism-cholesterol pathway might be associated with the pathophysiology of AMD. The ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are essential for the formation of high-density lipoprotein (HDL) and the regulation of macrophage cholesterol efflux. The failure of retinal or retinal pigment epithelium (RPE) cholesterol efflux to remove excess intracellular lipids causes morphological and functional damage to the retina. In this study, we investigated whether treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMP-activated protein kinase (AMPK) activator, improves RPE cholesterol efflux and Bruch's membrane (BM) lipid deposits. The protein and mRNA levels of ABCA1 and ABCG1 in ARPE-19 cells and retinal and RPE/choroid tissue from apolipoprotein E-deficient (ApoE-/-) mice were evaluated after 24 weeks of AICAR treatment. The cholesterol efflux capacity of ARPE-19 cells and the cholesterol-accepting capacity of apoB-depleted serum from mice were measured. The thickness of the BM and the degree of lipid deposition were evaluated using electron microscopy. AICAR treatment increased the phosphorylation of AMPK and the protein and mRNA expression of ABCA1 and ABCG1 in vitro. It promoted cholesterol efflux from ARPE-19 cells and upregulated the protein and mRNA levels of ABCA1 and ABCG1 in the retina and RPE in vivo. ApoB-depleted serum from the AICAR-treated group showed enhanced cholesterol-accepting capacity. Long-term treatment with AICAR reduced BM thickening and lipid deposition in ApoE-/- mice. In conclusion, AICAR treatment increased the expression of lipid transporters in the retina and RPE in vivo, facilitated intracellular cholesterol efflux from the RPE in vitro, and improved the functionality of HDL to accept cholesterol effluxed from the cell, possibly via AMPK activation. Collectively, these effects might contribute to the improvement of early age-related pathologic changes in the BM. Pharmacological improvement of RPE cholesterol efflux via AMPK activation may be a potential treatment strategy for AMD.

A case of melanoma-associated retinopathy with autoantibodies against TRPM1.

PURPOSE: To report a case of melanoma-associated retinopathy (MAR) with autoantibodies against the transient receptor potential cation channel, subfamily M, member 1 (TRPM1) with asymmetric severe vision loss. METHODS: We evaluated a patient with heel skin melanoma showing progressive vision loss in both eyes confirmed with a baseline ophthalmic examination, fluorescein angiography, spectral domain optical coherence tomography (OCT), visual field test, and full-field electroretinogram (ERG). Immunofluorescence assays and western blot analysis revealed autoantibodies in the patient's serum. RESULTS: The patient's best-corrected visual acuities were 20/50 in the right eye and hand motion in the left eye. Visual field test showed severely depressed visual fields especially in the left eye. Fluorescein angiography and OCT revealed extrafoveal choroidal neovascularization in the left eye. The patient had an electronegative ERG, suggesting MAR, and autoantibodies against TRPM1 and aldolase C were detected in the patient's blood sample. CONCLUSIONS: The clinical features of MAR patients with positive anti-TRPM1 autoantibodies can be manifested as severe vision loss, and the identification of autoantibodies can be helpful for confirming the diagnosis.

Anti-VEGF PolysiRNA Polyplex for the Treatment of Choroidal Neovascularization.

Choroidal neovascularization (CNV) is a major cause of severe vision loss in patients with age-related macular degeneration (AMD). Present ocular siRNA delivery technology is limited due to poor delivery through the retina to the choroid, where CNV originates. Our goal was to develop an optimized nanosized polyRNAi-based therapeutic delivery system to the subretinal space. We developed it by siRNA multimerization (polysiRNA) followed by coating with branched polyethylenimine and hyaluronic acid, and then evaluated its efficacy in vitro and in vivo. The polysiRNA polyplex showed a narrow size distribution (260.7 ± 43.27 nm) and negative charge (-4.98 ± 0.47 mV) owing to the hyaluronic acid outer layer. In vitro uptake of the polysiRNA polyplex by human ARPE cells was discovered, and the direct inhibition of VEGF mRNA translation was confirmed in B16F10 cells. The intravitreally administered polysiRNA polyplex overcame both the vitreous and retina barriers in vivo and reached the subretinal space efficiently. Intravitreal injection of the polysiRNA polyplex was not toxic to the retina in histopathology. Furthermore, intravitreal injections of the polysiRNA polyplex at both 1 and 7 days after laser photocoagulation inhibited laser-induced choroidal neovascularization, compared to that of the control (p < 0.05). These results suggest that anti-VEGF polysiRNA polyplexes show great potential in delivering multimeric RNAi-based therapeutics to treat retinal or choroidal disorders.

Neonatal systemic inflammation in rats alters retinal vessel development and simulates pathologic features of retinopathy of prematurity.

BACKGROUND: Alteration of retinal angiogenesis during development leads to retinopathy of prematurity (ROP) in preterm infants, which is a leading cause of visual impairment in children. A number of clinical studies have reported higher rates of ROP in infants who had perinatal infections or inflammation, suggesting that exposure of the developing retina to inflammation may disturb retinal vessel development. Thus, we investigated the effects of systemic inflammation on retinal vessel development and retinal inflammation in neonatal rats. METHODS: To induce systemic inflammation, we intraperitoneally injected 100 µl lipopolysaccharide (LPS, 0.25 mg/ml) or the same volume of normal saline in rat pups on postnatal days 1, 3, and 5. The retinas were extracted on postnatal days 7 and 14, and subjected to assays for retinal vessels, inflammatory cells and molecules, and apoptosis. RESULTS: We found that intraperitoneal injection of LPS impaired retinal vessel development by decreasing vessel extension, reducing capillary density, and inducing localized overgrowth of abnormal retinal vessels and dilated peripheral vascular ridge, all of which are characteristic findings of ROP. Also, a large number of CD11c+ inflammatory cells and astrocytes were localized in the lesion of abnormal vessels. Further analysis revealed that the number of major histocompatibility complex (MHC) class IIloCD68loCD11bloCD11chi cells in the retina was higher in LPS-treated rats compared to controls. Similarly, the levels of TNF-α, IL-1ß, and IL-12a were increased in LPS-treated retina. Also, apoptosis was increased in the inner retinal layer where retinal vessels are located. CONCLUSIONS: Our data demonstrate that systemic LPS-induced inflammation elicits retinal inflammation and impairs retinal angiogenesis in neonatal rats, implicating perinatal inflammation in the pathogenesis of ROP.

Vascularized tissue on mesh-assisted platform (VT-MAP): a novel approach for diverse organoid size culture and tailored cancer drug response analysis.

This study presents the vascularized tissue on mesh-assisted platform (VT-MAP), a novel microfluidic in vitro model that uses an open microfluidic principle for cultivating vascularized organoids. Addressing the gap in 3D high-throughput platforms for drug response analysis, the VT-MAP can host tumor clusters of various sizes, allowing for precise, size-dependent drug interaction assessments. Key features include capability for forming versatile co-culture conditions (EC, fibroblasts and colon cancer organoids) that enhance tumor organoid viability and a perfusable vessel network that ensures efficient drug delivery and maintenance of organoid health. The VT-MAP enables the culture and analysis of organoids across a diverse size spectrum, from tens of microns to several millimeters. The VT-MAP addresses the inconsistencies in traditional organoid testing related to organoid size, which significantly impacts drug response and viability. Its ability to handle various organoid sizes leads to results that more accurately reflect patient-derived xenograft (PDX) models and differ markedly from traditional in vitro well plate-based methods. We introduce a novel image analysis algorithm that allows for quantitative analysis of organoid size-dependent drug responses, marking a significant step forward in replicating PDX models. The PDX sample from a positive responder exhibited a significant reduction in cell viability across all organoid sizes when exposed to chemotherapeutic agents (5-FU, oxaliplatin, and irinotecan), as expected for cytotoxic drugs. In sharp contrast, PDX samples of a negative responder showed little to no change in viability in smaller clusters and only a slight reduction in larger clusters. This differential response, accurately replicated in the VT-MAP, underscores its ability to generate data that align with PDX models and in vivo findings. Its capacity to handle various organoid sizes leads to results that more accurately reflect PDX models and differ markedly from traditional in vitro methods. The platform's distinct advantage lies in demonstrating how organoid size can critically influence drug response, revealing insights into cancer biology previously unattainable with conventional techniques.

Magnetically Controlled Intraocular Delivery of Dexamethasone Using Silica-Coated Magnetic Nanoparticles.

Although the number of patients with eye diseases is increasing, efficient drug delivery to the posterior segment of the eyeball remains challenging. The reasons include the unique anatomy of the eyeball, the blood-aqueous barrier, the blood-retina barrier, and drug elimination via the anterior chamber and uveoscleral routes. Solutions to these obstacles for therapeutic delivery to the posterior segment will increase the efficacy, efficiency, and safety of ophthalmic treatment. Micro/nanorobots are promising tools to deliver therapeutics to the retina under the direction of an external magnetic field. Although many groups have evaluated potential uses of micro/nanorobots in retinal treatment, most experiments have been performed under idealized in vitro laboratory conditions and thus do not fully demonstrate the clinical feasibility of this approach. This study examined the use of magnetic nanoparticles (MNPs) to deliver dexamethasone, a drug widely used in retinal disease treatment. The MNPs allowed sustainable drug release and successful magnetic manipulation inside bovine vitreous humor and the vitreous humor of living rabbits. Therefore, controlled drug distribution via magnetic manipulation of MNPs is a promising strategy for targeted drug delivery to the retina.

FITC-Labeled RGD Peptides as Novel Contrast Agents for Functional Fluorescent Angiographic Detection of Retinal and Choroidal Neovascularization.

The development of choroidal neovascularization (CNV) is a crucial factor in the pathophysiology and prognosis of exudative age-related macular degeneration (AMD). Therefore, the detection of CNV is essential for establishing an appropriate diagnosis and treatment plan. Current ophthalmic imaging techniques, such as fundus fluorescent angiography and optical coherence tomography, have limitations in accurately visualizing CNV lesions and expressing CNV activity, owing to issues such as excessive dye leakage with pooling and the inability to provide functional information. Here, using the arginine-glycine-aspartic acid (RGD) peptide's affinity for integrin αvß3, which is expressed in the neovascular endothelial cells in ocular tissues, we propose the use of fluorescein isothiocyanate (FITC)-labeled RGD peptide as a novel dye for effective molecular imaging of CNV. FITC-labeled RGD peptides (FITC-RGD2), prepared by bioconjugation of one FITC molecule with two RGD peptides, demonstrated better visualization and precise localization of CNV lesions than conventional fluorescein dyes in laser-induced CNV rodent models, as assessed using various imaging techniques, including a commercially available clinical fundus camera (Optos). These results suggest that FITC-RGD2 can serve as an effective novel dye for the diagnosis of neovascular retinal diseases, including AMD, by enabling early detection and treatment of disease occurrence and recurrence after treatment.

Intravitreal injectable hydrogel rods with long-acting bevacizumab delivery to the retina.

Retinal vascular diseases such as neovascular age-related macular degeneration (nAMD) are the leading cause of blindness worldwide. They can be treated with intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents by inhibiting VEGF which is a major agent of abnormal blood vessel growth. However, because of drug's short half-life, clinical treatment often requires monthly repeated intravitreal injections, causing treatment burden and undertreatment. Among various kinds of drug carriers, in situ forming hydrogels have been studied as potential intravitreal drug carriers for the high drug loading, easy injection, controlled drug release, and protection of encapsulated drugs from the environment. However, gelation time, crosslinking degree, and drug release patterns following injection of a liquid that will be subsequently gelled in situ are susceptible to be hindered by dilution of the hydrogel precursor solution with body fluids (e.g., blood or vitreous). Here, we report an injectable pre-crosslinked hydrogel rod to overcome the limitations of in situ forming hydrogels and to extend intravitreal half-life of anti-VEGF for reducing intraocular injection frequency. Hydrogel rods can be simply prepared using in situ forming hydrogels, and injectable using a designed rod injector. The adjustable crosslinking degree of hydrogel rods easily controlled bevacizumab release profiles in a sustained manner. Compared with in situ forming hydrogels, hydrogel rods effectively reduced initial burst release, and showed sustained release with long-term drug efficacy in vitro. From the 4-month in vivo pharmacokinetic analysis, following the intravitreal injection of hydrogel rods, the half-life of bevacizumab in the vitreous and retina was significantly extended, and drug elimination to aqueous humor was effectively reduced. Finally, intraocular stability, degradation, and inflammatory response of hydrogel rods were evaluated. We expect that the hydrogel rod can be a potential drug delivery system for the treatment of nAMD and other conditions that need long-term and local sustained drug administration. STATEMENT OF SIGNIFICANCE: Herein, we report an injectable pre-crosslinked hydrogel rod based on an in situ forming hydrogel to achieve intravitreal long-acting anti-VEGF delivery to reduce injection frequency and improve the long-term visual outcomes of patients with retinal vascular diseases. Hydrogel rods were readily prepared using removable molds and injected using customized injectors. Compared to the in situ forming hydrogel, hydrogel rods showed significantly reduced initial burst release, controllable release profiles for several months, physical stability, and a long-acting anti-angiogenic effect. Animal studies demonstrated that the hydrogel rods dramatically prolonged the intraocular drug half-life while significantly reducing drug elimination for up to four months. Moreover, the biodegradability and safety of the hydrogel rods suggest their suitability as an advanced intravitreal DDS for treating retinal vascular diseases.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Plasma Antiretinal Autoantibody Profiling and Diagnostic Efficacy in Patients With Autoimmune Retinopathy.

PURPOSE: To evaluate plasma antiretinal autoantibody (ARA) profiling and diagnostic efficacy for autoimmune retinopathy (AIR). DESIGN: A multicenter, diagnostic evaluation study. METHODS: Forty-nine patients with a clinical diagnosis of AIR, disease controls including 20 patients with retinitis pigmentosa (RP), and 30 normal controls were included. Plasma samples from patients were analyzed for the presence of 6 ARAs, including recoverin, α-enolase, carbonic anhydrase II, heat shock protein 60, aldolase C, and cone-rod homeobox/cone-rod retinal dystrophy 2 using western blotting. RESULTS: Autoantibody detection rates against cone-rod homeobox/cone-rod retinal dystrophy 2, heat shock protein 60, and aldolase C in AIR were 67.3%, 40.8%, and 42.9%, respectively, which were higher than those in RP and normal controls (P < .001, P < .001, and P = .007, respectively), but recoverin, α-enolase, and carbonic anhydrase II were not different from other control groups (P = .117, P = .774, and P = .467, respectively). Among ARAs, antirecoverin antibody was the most specific, as it was found in 3 (6.1%) patients with AIR and none of the control groups. As the number of detected ARAs increased, the probability of AIR increased (odds ratio: 1.913; P < .001; 95% confidence interval: 1.456-2.785). The positive number of ARAs was significantly higher when photoreceptor disruption was observed on optical coherence tomography, or severe dysfunction was observed in electroretinography (P = .022 and P = .029, respectively). CONCLUSIONS: The profiles of ARAs in the AIR group were different from those in the RP and normal controls. The higher number of positive ARAs suggests a higher possibility of AIR diagnosis. ARAs should be used as adjunct tools for the clinical diagnosis of AIR.

Permeability of the Retina and RPE-Choroid-Sclera to Three Ophthalmic Drugs and the Associated Factors.

In this study, Retina-RPE-Choroid-Sclera (RCS) and RPE-Choroid-Sclera (CS) were prepared by scraping them off neural retina, and using the Ussing chamber we measured the average time-concentration values in the acceptor chamber across five isolated rabbit tissues for each drug molecule. We determined the outward direction permeability of the RCS and CS and calculated the neural retina permeability. The permeability coefficients of RCS and CS were as follows: ganciclovir, 13.78 ± 5.82 and 23.22 ± 9.74; brimonidine, 15.34 ± 7.64 and 31.56 ± 12.46; bevacizumab, 0.0136 ± 0.0059 and 0.0612 ± 0.0264 (×10-6 cm/s). The calculated permeability coefficients of the neural retina were as follows: ganciclovir, 33.89 ± 12.64; brimonidine, 29.83 ± 11.58; bevacizumab, 0.0205 ± 0.0074 (×10-6 cm/s). Between brimonidine and ganciclovir, lipophilic brimonidine presented better RCS and CS permeability, whereas ganciclovir showed better calculated neural retinal permeability. The large molecular weight drug bevacizumab demonstrated a much lower permeability than brimonidine and ganciclovir. In conclusion, the ophthalmic drug permeability of RCS and CS is affected by the molecular weight and lipophilicity, and influences the intravitreal half-life.

A Fully Human Monoclonal Antibody Targeting cKIT Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Intraocular Distribution and Kinetics of Intravitreally Injected Antibodies and Nanoparticles in Rabbit Eyes.

Purpose: To investigate the intraocular distribution and kinetics of antibodies and nanoparticles in the experimental model. Methods: Antibodies (whole IgG 149kDa, antigen-binding fragments 48.39 kDa) and four kinds of nondegradable nanoparticles (25, 50, 200, and 250 nm) were intravitreally injected in the right eye of New Zealand white rabbits. The average optical density and concentration were used to measure intraocular distribution and kinetics. Results: After intravitreal injection, antibodies were distributed throughout the vitreous humor and eliminated gradually into anterior and posterior routes. Fluorescence intensity decreased 1 day after injection and was not detected 25 days after injection. The nondegradable nanoparticles migrated posteriorly to the retina 7 days after injection onward and anteriorly to the aqueous humor from 1 hour to 1 day after injection. The fluorescence intensity of the nanoparticles was relatively stable in the vitreous humor, compared to antibodies. Nanoparticles accumulated on the internal limiting membrane of the retina with no penetration into deeper retinal tissue, whereas the smaller size 25 nm nanoparticles passed across the ciliary body and moved into choroid, retina, and suprachoroidal space. A gradual decrease of nanoparticles by their sizes in the vitreous after 30 days after injection was described as the percentage ratio: 61.1% (25 nm), 69.1% (50 nm), 78.6% (200nm), and 85.3% (250 nm). Conclusions: Our study revealed the in vivo intraocular distribution and kinetics of antibodies and nanoparticles with diverse sizes and the result might help to develop newer intraocular drugs and drug delivery systems to treat retinal diseases. Translational Relevance: These experimental results can be valuable data for human research.

Intraocular Pharmacokinetics of 10-fold Intravitreal Ranibizumab Injection Dose in Rabbits.

Purpose: To investigate intraocular pharmacokinetics of 10-fold dose of intravitreally injected ranibizumab compared with the conventional dose in an experimental model. Methods: Ranibizumab 30 µL at 10 mg/mL (conventional) and 100 mg/mL (10-fold) doses was injected separately into each eye of 28 rabbits. Ranibizumab concentrations in the aqueous humor, vitreous, and retina were estimated at each time period after injection, using enzyme-linked immunosorbent assay. The pharmacokinetic properties of ranibizumab were determined using a one-compartment model in all three ocular tissues. The time-concentration profile and predictive trends were plotted to determine drug efficacy in the retina. Results: Maximum concentrations after conventional and 10-fold dosing were observed in the retina at 1 and 4 days after injection, respectively. The half-life of ranibizumab after conventional and 10-fold dosing did not differ in the anterior chamber and vitreous, whereas the half-life was prolonged approximately twice with the 10-fold dose in the retina (36.74 h vs. 76.85 h) and serum (91.93 h vs. 179.01 h). Similarly, the estimated time for ranibizumab concentration in the retina over 27 ng/mL (minimum effective concentration of ranibizumab) was prolonged approximately twice with the 10-fold dose (1315 h [55 days] vs. 2393 h [100 days]). No adverse effects were observed in either group. Conclusions: The retinal half-life and concentration of ranibizumab in rabbit eyes were increased approximately twice after a 10-fold dose compared with the conventional dose. This finding indicates a possibility to lengthen the injection interval to improve the efficacy of ranibizumab in human eyes. Translational Relevance: Our results highlight the potential for clinical application of a high-dose (10-fold) of anti-VEGF agents to prolong the intravitreal injection intervals, simultaneously improving the drug efficacy.

In vivo imaging of the hyaloid vascular regression and retinal and choroidal vascular development in rat eyes using optical coherence tomography angiography.

This study investigates the hyaloid vascular regression and its relationship to the retinal and choroidal vascular developments using optical coherence tomography angiography (OCTA). Normal and oxygen-induced retinopathy (OIR) rat eyes at postnatal day 15, 18, 21, and 24 were longitudinally imaged using OCTA. At each day, two consecutive imaging for visualizing the hyaloid vasculature and the retinal and choroidal vasculatures were conducted. The hyaloid vessel volume and the retinal and choroidal vessel densities were measured. The hyaloid vessel volumes gradually decreased during the regression, although the OIR eyes exhibited large vessel volumes at all time points. A spatial relationship between persistent hyaloid vasculature and retardation of underlying retinal vascular development was observed in the OIR eyes. Furthermore, anti-vascular endothelial growth factor (VEGF) was administered intravitreally to additional OIR eyes to observe its effect on the vascular regression and development. The VEGF injection to OIR eyes showed reduced persistent hyaloid vessels in the injected eyes as well as in the non-injected fellow eyes. This study presents longitudinal imaging of intraocular vasculatures in the developing eye and shows the utility of OCTA that can be widely used in studies of vascular development and regression and preclinical evaluation of new anti-angiogenic drugs.

Preclinical Efficacy and Safety of VEGF-Grab, a Novel Anti-VEGF Drug, and Its Comparison to Aflibercept.

Purpose: VEGF-Grab is a novel anti-vascular endothelial growth factor (VEGF) candidate drug with higher affinity to both VEGF and placental growth factor (PlGF) compared to aflibercept. We investigated the preclinical efficacy of VEGF-Grab for ophthalmic therapy and compared it to that of aflibercept. Methods: The in vitro anti-VEGF efficacy of VEGF-Grab was determined using VEGF-induced cell proliferation/migration and tube formation assays. The in vivo antiangiogenic efficacy of intravitreal injection of either VEGF-Grab or aflibercept was evaluated using murine models of ocular angiogenesis: mouse oxygen-induced retinopathy (OIR) and rat laser-induced choroidal neovascularization (CNV). The in vivo retinal toxicity in the mouse eye resulting from the injection of either drug was evaluated with light and electron microscopy. Results: VEGF-Grab showed greater inhibition of VEGF-induced cell proliferation/migration than aflibercept, but it showed comparable inhibition of tube formation in vitro. In the in vivo OIR model, VEGF-Grab showed a comparable suppression of retinal neovascularization compared to aflibercept. Additionally, VEGF-Grab showed an efficacy similar to that of aflibercept in terms of CNV inhibition in the laser-induced CNV model. Histology and transmission electron microscopy showed no significant signs of toxicity in the mouse retina at 7 and 30 days following the intravitreal injection of VEGF-Grab or aflibercept. Conclusions: Compared to aflibercept, VEGF-Grab presented comparable in vivo antiangiogenic efficacy and superior in vitro anti-VEGF activity. The retinal safety profiles were comparable for the two drugs. Considering its known higher binding affinity to VEGF and PlGF compared to aflibercept, VEGF-Grab could be a potential candidate drug for neovascular retinal diseases and an alternative to aflibercept.

Preclinical SPECT Imaging of Choroidal Neovascularization in Mice Using Integrin-Binding [99mTc]IDA-D-[c(RGDfK)]2.

PURPOSE: Integrin ɑvß3, an adhesion molecule overexpressed in neovascular endothelial cells, is involved in ocular angiogenesis. Integrin ɑvß3-binding arginine-glycine-aspartic acid (RGD) peptide has been used to target and visualize new vessels. We explored the use of integrin ɑvß3-targeted RGD peptide ([99mTc]IDA-D-[c(RGDfK)]2) for in vivo molecular imaging of choroidal neovascularization (CNV). PROCEDURES: To induce CNV in animals, the right eyes of C57BL/6 mice were treated with retinal argon laser photocoagulation. CNV formation was confirmed on immunohistopathological examination of retinal and choroidal tissues. To explore the association of integrin with angiogenesis, integrin mRNA expression in the retinal and choroidal tissues was measured using real-time reverse transcriptase-polymerase chain reaction. For in vivo imaging, mice were intravenously injected with [99mTc]IDA-D-[c(RGDfK)]2 and single-photon emission computed tomography (SPECT) images of [99mTc]IDA-D-[c(RGDfK)]2 were obtained before laser induction (baseline) and at 1, 3, 7, and 14 days post-induction. CNV-induced regional alterations were measured using radiotracer uptake count. RESULTS: Immunohistopathological examination revealed that CNV lesions showed intense fluorescein isothiocyanate (FITC)-D-[c(RGDfK)]2 immunofluorescence, in contrast to the normal retina and choroid. Retinal integrin mRNA expression peaked at day 1 following CNV induction. On SPECT images using [99mTc]IDA-D-[c(RGDfK)]2, the radio-uptake count in eyes with CNV was significantly higher than in normal controls on days 1-7 (all p < 0.05), with a peak at day 3 representing the highest angiogenic activity. Our preclinical data demonstrated that [99mTc]IDA-D-[c(RGDfK)]2 can detect CNV and its associated angiogenesis in an animal model of CNV. CONCLUSIONS: SPECT imaging using an integrin ɑvß3-targeted RGD peptide radiotracer may be a useful tool for in vivo functional molecular imaging of CNV.

Oxygen-Induced Retinopathy and Choroidopathy: In Vivo Longitudinal Observation of Vascular Changes Using OCTA.

Purpose: The purpose of this study was to assess the retinal and choroidal vasculatures of an oxygen-induced retinopathy (OIR) rat model using optical coherence tomography angiography (OCTA) as well as to verify the performance of OCTA for visualizing in vivo vascular alterations, longitudinally and quantitatively. Methods: To induce OIR, Sprague Dawley rat pups were incubated in an 80% oxygen chamber from postnatal day 1 (P1) to P11 and returned to room air. OCTA imaging was performed in six eyes at P15, P18, P21, and P24. All eyes were imaged with ex vivo retinal flat mount immunofluorescence microscopy for comparison with OCTA. The areas of the neovascular tufts, retinal vessel tortuosities and diameters, and vessel densities of different retinal and choroidal layers were quantified. Results: The neovascular tufts were observed in two OIR eyes. The tuft areas decreased spontaneously from P18 to P24. The increase in arterial tortuosity and venous dilation were observed in the OIR eyes at P15 and P18. The retardation of vascular developments was observed in the deep vascular plexus and the choroidal layer in the OIR group while the superficial vascular plexus did not show developmental delay. Conclusions: This study demonstrates an application of OCTA for quantitative and longitudinal studies on in vivo vascular alterations, including neovascular tufts, increase in arterial tortuosity, venous dilation, and developmental delay in the OIR rat model.

Role of the Fc Region in the Vitreous Half-Life of Anti-VEGF Drugs.

Purpose: To identify the role of the fragment crystallizable (Fc) region in determining intraocular protein drug pharmacokinetics. Methods: We generated a new VEGF-Trap lacking the Fc region (FcfVEGF-Trap, MWt = 100 kDa) by replacing the Fc region of native VEGF-Trap (MWt = 145 kDa) with a dimerized coiled-coil domain. Forty-two rabbits were injected intravitreally with VEGF-Trap or FcfVEGF-Trap (n = 21 each) in one of the eyes, harvested at six time points (1 hour and 1, 2, 4, 14, and 30 days after injections). VEGF-Trap and FcfVEGF-Trap concentrations in the vitreous, aqueous humor, and retina/choroid were measured, and drug pharmacokinetic properties were analyzed. Results: In all three ocular compartments, the maximal concentrations for both FcfVEGF-Trap and VEGF-Trap were observed at 1 hour after injection. Half-lives of FcfVEGF-Trap in the vitreous and retina/choroid (145.02 and 102.12 hours, respectively) were 1.39 and 2.30 times longer than those of VEGF-Trap (103.99 and 44.42 hours, respectively). Total exposure of the aqueous humor and retina/choroid to FcfVEGF-Trap was 13.2% and 39% of the vitreous exposure, respectively, whereas VEGF-Trap concentrations were 25.2% and 26.2%, indicating that FcfVEGF-Trap shows a preference for posterior distribution and elimination. Conclusions: FcfVEGF-Trap, despite its lower molecular weight, showed longer half-lives in vitreous and retina/choroid than VEGF-Trap did, suggesting that Fc receptors in ocular tissues contribute to anti-VEGF drug elimination. Truncation or mutation of the Fc region can prolong the intraocular residence time of VEGF-Trap and possibly reduce the number of VEGF-Trap injections required in clinical practice.

Therapeutic effects of a novel siRNA-based anti-VEGF (siVEGF) nanoball for the treatment of choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries and is characterized by the development of choroidal neovascularization (CNV). Therapies for AMD have focused on suppressing angiogenic factors, such as vascular endothelial growth factor (VEGF), mainly via conventional anti-VEGF antibody agents. However, additional efforts have been made to develop effective small-interfering RNA (siRNA)-based intracellular therapeutic agents. In this study, we have manufactured a novel siRNA-based anti-VEGF nanoball (siVEGF NB). The siVEGF NB was composed of a siRNA hydrogel with a core of anti-VEGF sequence siRNA coated with branched PEI (bPEI) and hyaluronic acid (HA) in order by applying an electrical force. The novel siVEGF NBs, which were employed in a laser-induced CNV mouse model, were optimized as a retinal and choroidal delivery system through the vitreous humor to the sub-retinal space via CD44 receptor endocytosis on the inner limiting membrane, and showed therapeutic effects via pathways bypassing the TLR3-induced siRNA-class effect. The therapeutic effects of siVEGF NBs lasted for 2 weeks after intravitreal injection showing high targeting efficiency to the sub-retinal space. Thus, the newly developed siVEGF NB may have great potential for the delivery of RNAi-based therapeutics for ocular diseases, including AMD.