Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular ...integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.
Retinitis pigmentosa occurs due to mutations that cause rod photoreceptor degeneration. Once most rods are lost, gradual degeneration of cone photoreceptors occurs. Oxidative damage and abnormal ...glucose metabolism have been implicated as contributors to cone photoreceptor death. Herein, we show increased phosphorylation of key enzymes of glucose metabolism in the retinas of rd10 mice, a model of RP, and retinas of wild type mice with paraquat-induced oxidative stress, thereby inhibiting these key enzymes. Dietary supplementation with glucose and pyruvate failed to overcome the inhibition, but increased reducing equivalents in the retina and improved cone function and survival. Dichloroacetate reversed the increased phosphorylation of pyruvate dehydrogenase in rd10 retina and increased histone acetylation and levels of TP53-induced glycolysis and apoptosis regulator (TIGAR), which redirected glucose metabolism toward the pentose phosphate pathway. These data indicate that oxidative stress induced damage can be reversed by shifting glycolytic intermediates toward the pentose phosphate pathway which increases reducing equivalents and provides photoreceptor protection.
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•Oxidative stress inhibits key enzymes in glucose metabolism pathway in the retina.•Oxidative stress inhibits TIGAR production in the retina and is reversed by NAC.•Supplementation with glycolytic substrates and DCA upregulate TIGAR production.•TIGAR directs substrates into the PPP pathway•PPP pathway produces reducing equivalents, protecting retina from oxidative stress.
There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route ...of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth ...factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch’s membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In
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mice which spontaneously develop subretinal NV, intraocular injection of 1.85 μg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in
Tet/opsin/VEGF
double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 μg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.
Hypoxia-inducible factor-1 (HIF-1) has been implicated in the pathogenesis of choroidal neovascularization (NV) and is an appealing target because it increases multiple pro-angiogenic proteins and ...their receptors. Acriflavine (ACF) binds HIF-1α and HIF-2α preventing binding to HIF-1β and inhibiting transcriptional activity of HIF-1 and HIF-2. Delivery of ACF to the eye by multiple routes strongly, but transiently, suppresses choroidal NV. We overcame design challenges and loaded highly water soluble ACF into poly(lactic-co-glycolic acid) (PLGA) microparticles (PLGA-ACF MPs) that release ACF in vitro for up to 60 days. Intravitreous injection of PLGA-ACF MPs in mice suppressed choroidal NV for at least 9 weeks and suprachoroidal injection of PLGA-ACF in rats suppressed choroidal NV for at least 18 weeks. Intravitreous, but not suprachoroidal injection, of PLGA-ACF MPs containing 38 μg of ACF in rabbits resulted in modest reduction of full-field electroretinogram (ERG) function. Over the span of 28 days after suprachoroidal injection of PLGA-ACF MP, rabbits had normal appearing retinas on fundus photographs, normal electroretinogram scotopic a- and b-wave amplitudes, no increase in intraocular pressure, and normal retinal histology. The active component of ACF, trypaflavine, had steady-state levels in the low nM range in RPE/choroid > retina for at least 16 weeks with a gradient from the side of the eye where the injection was done to the opposite side. These data suggest that suprachoroidal injection of PLGA-ACF MPs has the potential to provide a durable new treatment for retinal and choroidal vascular diseases.
Suprachoriodal delivery of PLGA-ACF microparticles delivers pharmacologically relevant levels of ACF throughout the eye that significantly reduces development of laser-induced choroidal neovascularization in rats. The site of suprachoroidal injections is the same as that for intravitreous injections but the depth of penetration is different. Display omitted
Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire
−/− mice fail to express self-antigens in the thymus, ...exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire
−/− retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire
−/− retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire
−/− retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.
ABSTRACT—Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor ...(VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1α–null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1α is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.
Retinitis pigmentosa (RP) is caused by many different mutations that promote the degeneration of rod photoreceptors and have no direct effect on cones. After the majority of rods have died cone ...photoreceptors begin to slowly degenerate. Oxidative damage contributes to cone cell death and it has been hypothesized that tissue hyperoxia due to reduced oxygen consumption from the loss of rods is what initiates oxidative stress. Herein, we demonstrate in animal models of RP that reduction of retinal hyperoxia by reducing inspired oxygen to continuous breathing of 11% O2 reduced the generation of superoxide radicals in the retina and preserved cone structure and function. These data indicate that retinal hyperoxia is the initiating event that promotes oxidative damage, loss of cone function, and cone degeneration in the RP retina.
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•In RP, after rods die from a mutation, cones are exposed to hyperoxia.•Hyperoxia causes activation of NADPH oxidase, promoting oxidative damage in cones.•Oxidative damage causes loss of function and eventual death of cones.•Reduced inspired oxygen in mice with RP reduces tissue hyperoxia.•Reduced Hyperoxia reduces oxidative damage and promotes cone survival and function.
Transcriptome is the entire repertoire of transcripts present in a cell at any particular time. We undertook a next-generation whole transcriptome sequencing approach to gain insight into the ...transcriptional landscape of the developing mouse lens.
We ascertained mouse lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools.
Mapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens at the above-mentioned six developmental stages. Of these, 46 genes exhibited a 40-fold differential (higher or lower) expression at one the five developmental stages (E18, P0, P3, P6, and P9) compared with their expression level at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens at six developmental time points. Of these, 49 miRNAs manifested an 8-fold differential (higher or lower) expression at one the five developmental stages, as mentioned above compared with their expression level at E15.
We report a comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for the development of the ocular lens and maintenance of its transparency.
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