IL-23 Priming Enhances the Neuroprotective Effects of MSC-Derived Exosomes in Treating Retinal Degeneration.

Purpose: Neuroinflammation is a characteristic feature of neurodegenerative diseases. Mesenchymal stem cell-derived exosomes (MSC-exo) have shown neuroprotective effects through immunoregulation, but the therapeutic efficacy remains unsatisfactory. This study aims to enhance the neuroprotective capacity of MSC-exo through IL-23 priming for treating retinal degeneration in mice. Methods: MSC were primed with IL-23 stimulation in vitro, and subsequently, exosomes (MSC-exo and IL-23-MSC-exo) were isolated and characterized. Two retinal degenerative disease models (NaIO3-induced mice and rd10 mice) received intravitreal injections of these exosomes. The efficacy of exosomes was assessed by examining retinal structural and functional recovery. Furthermore, exosomal microRNA (miRNA) sequencing was conducted, and the effects of exosomes on the M1 and M2 microglial phenotype shift were evaluated. Results: IL-23-primed MSC-derived exosomes (IL-23-MSC-exo) exhibited enhanced capability in protecting photoreceptor cells and retinal pigment epithelium (RPE) cells against degenerative damage and fostering the restoration of retinal neural function in both NaIO3-induced retinal degeneration mice and rd10 mice when compared with MSC-exo. The exosomal miRNA suppression via Drosha knockdown in IL-23-primed MSC would abolish the neuroprotective role of IL-23-MSC-exo, highlighting the miRNA-dependent mechanism. Bioinformatic analysis, along with further in vivo biological studies, revealed that IL-23 priming induced a set of anti-inflammatory miRNAs in MSC-exo, prompting the transition of M1 to M2 microglial polarization. Conclusions: IL-23 priming presents as a potential avenue for amplifying the immunomodulatory and neuroprotective effects of MSC-exo in treating retinal degeneration.

Urolithin A promotes p62-dependent lysophagy to prevent acute retinal neurodegeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death. METHODS: A combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question. RESULTS: UA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy. CONCLUSIONS: Collectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.

Tamoxifen protects photoreceptors in the sodium iodate model.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice.

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Temporary Upregulation of Nrf2 by Naringenin Alleviates Oxidative Damage in the Retina and ARPE-19 Cells.

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO3-induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO3-treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway.

Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells.

Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of retinal pigment epithelium (RPE) cells. However, the mechanism of RPE cell death induced by SI remains unclear. In this study, we investigated the necrotic features of cultured human retinal pigment epithelium (ARPE-19) cells treated with SI and found that apoptosis or necroptosis was not the major death pathway. Instead, the death process was accompanied by significant elevation of intracellular labile iron level, ROS, and lipid peroxides which recapitulated the key features of ferroptosis. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) partially prevented SI-induced cell death. Further studies revealed that SI treatment did not alter GPX4 (glutathione peroxidase 4) expression, but led to the depletion of reduced thiol groups, mainly intracellular GSH (reduced glutathione) and cysteine. The study on iron trafficking demonstrated that iron influx was not altered by SI treatment but iron efflux increased, indicating that the increase in labile iron was likely due to the release of sequestered iron. This hypothesis was verified by showing that SI directly promoted the release of labile iron from a cell-free lysate. We propose that SI depletes GSH, increases ROS, releases labile iron, and boosts lipid damage, which in turn results in ferroptosis in ARPE-19 cells.

Protective Effects of Fermented Paprika (Capsicum annuum L.) on Sodium Iodate-Induced Retinal Damage.

Diseases of the outer retina, including age-related macular degeneration (AMD), are major cause of permanent visual damage. The pathogenesis of AMD involves oxidative stress and damage of the retinal pigment epithelium. Capsicum annuum L. (paprika) fruits have been known as a source of vitamins, carotenoids, phenolic compounds, and metabolites with a well-known antioxidant activity, which have positive effects on human health and protection against AMD and cataracts. In this study, we investigated whether paprika (fermented (FP), yellow, and orange colored) fermented with Lactobacillus (L.) plantarum could increase the protective effect of retinal degeneration using in vitro and in vivo models. FP significantly increased cell survival and reduced levels of lactate dehydrogenase as well as intracellular reactive oxygen species (ROS) increase in SI (sodium iodate, NaIO3)-treated human retinal pigment epithelial (ARPE-19) cells. We developed a model of retinal damage in C57BL/6 mice using SI (30 mg/kg) via intraperitoneal injection. Seven days after SI administration, deformation and a decrease in thickness were observed in the outer nuclear layer, but improved by FP treatment. FP administration protected the SI-mediated reduction of superoxide dismutase and glutathione levels in the serum and ocular tissues of mice. The overproduction of cleaved poly(ADP-Ribose) Polymerase (PARP)1, caspase-3 and -8 proteins were significantly protected by FP in SI-treated cells and ocular tissues. In addition, we evaluated the potentiating effects of FP on antioxidants and their underlying mechanisms in RAW 264.7 cells. Lipopolysaccharide (LPS)-induced nitrite increase was markedly blocked by FP treatment in RAW 264.7 cells. Furthermore, FP reduced LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 activation. The FP also enhanced the inhibitory effects on mitogen activated kinase signaling protein activation in ARPE-19 and RAW 264.7 cells and ocular tissues. There was no significant difference in total phenol and flavonoid content in paprika by fermentation, but the vitamin C content was increased in orange colored paprika, and protective effect against oxidative stress-mediated retinal damage was enhanced after fermentation. These results suggest that FP may be a potential candidate to protect against retinal degenerative diseases through the regulation of oxidative stress.

Combined Transplantation With Human Mesenchymal Stem Cells Improves Retinal Rescue Effect of Human Fetal RPE Cells in Retinal Degeneration Mouse Model.

Purpose: We verified whether fetal RPE (fRPE) cells and mesenchymal stem cells (MSCs) cotransplantation can combine the features of these two cell types and alleviate retinal degeneration in a retinal degenerative disease mouse model. Methods: Tail vein injection of sodium iodate (NaIO3) was conducted to establish the retinal degenerative disease mouse model. MSCs and fRPE cells were transplanted either separately or combined in the subretinal space of retinal degenerative disease animals. ERG, optical coherence tomography, histologic, and immunofluorescence analyses were performed. Furthermore, the expression level of Crx, rhodopsin, Iba1, F4/80, Caspase 3, nerve growth factor, and brain-derived neurotrophic factor were assessed to investigate the mechanisms involved in cell transplantation effects. Results: Cotransplantation of fRPE and MSC cells promoted significant improvements in ERG results and in the survival rate of transplanted cells. In addition, MSC and fRPE cell cotransplantation resulted in an increase in the thickness of the total retina, as well as in the outer and inner nuclear layers. Combined transplantation also upregulated the expression level of Crx and rhodopsin and downregulated caspase 3 expression, highlighting its better photoreceptor rescue effect in relation to the single cell type transplantation. Finally, combined transplantation suppressed the expression of Iba1 and F4/80 factors while increasing the endogenous expression of nerve growth factor and brain-derived nerve growth factor neurotrophic factors. These data suggest that MSC and fRPE cell cotransplantation is able to suppress immunoreactions and promote neurotrophic factor excretion. Conclusions: Combined transplantation of MSCs and fRPE cells results in a better retinal rescue effect than single cell type transplantation in NaIO3-induced retinopathy.

Oral administration of ferulic acid or ethyl ferulate attenuates retinal damage in sodium iodate-induced retinal degeneration mice.

Epidemiological studies indicate that the daily intake of antioxidants from a traditional Asian diet reduces the risk of developing age-related macular degeneration. Many of the phytochemicals that are abundant in whole grains exhibit a wide variety of biological activity such as antioxidant, anti-inflammatory, and neuroprotective effects. Ferulic acid (FA) is a phenolic acid found in vegetables and grains that has therapeutic potential for diabetes mellitus, Alzheimer's disease, and other diseases. We investigated the retinal protective effect of FA in a sodium iodate (NaIO3)-induced model of retinal degeneration. In a human retinal pigment epithelial cell line, FA attenuated H2O2-induced injury and lipopolysaccharide- or 7-ketocholesterol-induced inflammation. In mice, the oral administration of FA or its analog, ethyl ferulate, attenuated the morphological and functional features of NaIO3-induced retinal degeneration according to optical coherence tomography and electroretinography. Our results demonstrate that the oral administration of FA provides protective effects to the retina, suggesting that the intake of FA as a daily supplement or daily healthy diet containing rich vegetables and whole grains may prevent age-related macular degeneration.

Dental pulp stem cells therapy overcome photoreceptor cell death and protects the retina in a rat model of sodium iodate-induced retinal degeneration.

Blindness and vision loss contribute to irreversible retinal degeneration, and cellular therapy for retinal cell replacement has the potential to treat individuals who have lost light sensitive photoreceptors in the retina. Retinal cells are well characterized in function, and are a subject of interest in cellular replacement therapy of photoreceptors and the retinal pigment epithelium. However, retinal cell transplantation is limited by various factors, including the choice of potential stem cell source that can show variability in plasticity as well as host tissue integration. Dental pulp is one such source that contains an abundance of stem cells. In this study we used dental pulp-derived mesenchymal stem cells (DPSCs) to mitigate sodium iodate (NaIO3) insult in a rat model of retinal degeneration. Sprague-Dawley rats were first given an intravitreal injection of 3 × 105 DPSCs as well as a single systemic administration of NaIO3 (40 mg/kg). Electroretinography (ERG) was performed for the next two months and was followed-up by histological analysis. The ERG recordings showed protection of DPSC-treated retinas within 4 weeks, which was statistically significant (* P ≤ .05) compared to the control. Retinal thickness of the control was also found to be thinner (*** P ≤ .001). The DPSCs were found integrated in the photoreceptor layer through immunohistochemical staining. Our findings showed that DPSCs have the potential to moderate retinal degeneration. In conclusion, DPSCs are a potential source of stem cells in the field of eye stem cell therapy due to its protective effects against retinal degeneration.

Glycyrrhizin protects against sodium iodate-induced RPE and retinal injury though activation of AKT and Nrf2/HO-1 pathway.

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age-related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI-induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2-related factor2 (Nrf-2) and HO-1, thereby protecting RPE from SI-induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.

Sirt3 mediates the protective effect of hydrogen in inhibiting ROS-induced retinal senescence.

Hydrogen possesses antioxidative effects and cures numerous types of ophthalmopathy, but the mechanism of hydrogen on ROS-induced retinal senescence remains elusive. In this study, retinal morphology revealed that hydrogen reduced the number and size of vitreous black deposits in Bruch's membrane in NaIO3 mice. Hydrogen also reduced ROS levels in the retina as assessed by DHE staining. Moreover, this result was consistent with the downregulation of expression of the oxidative stress hallmark OGG1. These findings suggested that hydrogen can reduce retinal oxidative stress induced by NaIO3, and this result was further verified using the antioxidant ALCAR. Mechanistic analysis revealed that hydrogen significantly inhibited the downregulation of Sirt3 expression, and this notion was confirmed using AICAR, which restores Sirt3 expression and activity. Moreover, hydrogen reduced the expression of p53, p21 and p16 and the number of blue-green precipitations in the retinas of NaIO3 mice as assessed by SA-ß-gal staining. We also found that hydrogen decreased the expression of the DNA damage-related protein ATM, cyclinD1 and NF-κB but increased the expression of the DNA repair-related protein HMGB1, suggesting that hydrogen inhibits senescence in retinas of NaIO3 mice. Additionally, OCT examination revealed that hydrogen suppressed retinal high reflex formation significantly and prevented the retina from thinning. This result was supported by ERG assays that demonstrated that hydrogen prevented the reduction in a- and b-wave amplitude induced by NaIO3 in mice. Thus, our data suggest that hydrogen may inhibit retinal senescence by suppressing the downregulation of Sirt3 expression through reduced oxidative stress reactions.

Irreversible Photoreceptors and RPE Cells Damage by Intravenous Sodium Iodate in Mice Is Related to Macrophage Accumulation.

Purpose: To determine the mechanism causing degeneration of the retinal pigment epithelium (RPE) and photoreceptors in mice after an intravenous injection of sodium iodate (NaIO3). Methods: The time-dependent changes in NaIO3-induced retinal degeneration were determined by analyzing the retinal morphology by optical coherence tomographic (OCT) images, histological sections of the retina, physiology of the retina by electroretinography (ERG), and retinal blood flow by laser speckle flowgraphy. In addition, the expression of the genes associated with age-related macular degeneration in humans was assessed in the NaIO3-treated mice by RT-PCR. We also investigated whether macrophages were involved in the NaIO3-induced retinal degeneration. Results: The intravenous injection of 20 mg/kg NaIO3 altered the morphology of the RPE cells and the ERGs transiently. With 40 mg/kg of NaIO3, the degeneration of the RPE cells was still present at 28 days. Aggregated melanin granules were surrounded by zonula occludens protein 1 (ZO-1)-positive cells. In addition, 40 mg/kg of NaIO3 led to a reduction in the amplitudes of the a- and b-waves of the dark-adapted ERGs. Histological studies showed that macrophages had infiltrated the retina and were present around the altered RPE cells. Depletion of the macrophages by a prior injection of clodronate liposomes prevented the damage of the outer retina after the NaIO3 injection but not the RPE. Conclusions: The NaIO3-induced retinal damage was reversible at low concentrations but permanent at high concentrations of NaIO3. The accumulation of macrophages around the RPE cells caused the photoreceptor cell death.

Pigment Epithelium-derived Factor Protects Retinal Pigment Epithelial Cells Against Cytotoxicity "In Vitro".

Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.

The rescue effect of mesenchymal stem cell on sodium iodate-induced retinal pigment epithelial cell death through deactivation of NF-κB-mediated NLRP3 inflammasome.

Age-related macular degeneration (AMD) is a multifactorial disease resulting in the gradual loss of retinal pigment epithelium (RPE) and the permanent visual damage. Various risk factors, including oxidative stress, form a complex network at the confluence of inflammation. Mesenchymal stem cell (MSC) is a well-studied population of adult stem cell with strong neuroprotective and immunoregulatory properties. Here, we reported the protective effect of MSC on sodium iodate (NaIO3)-triggered RPE degeneration. Sodium iodate (NaIO3)-induced RPE cell death was remarkably reduced when cocultured with MSC. Inhibition of several cell death pathways mediated by mitochondrial instability and its subsequent caspase-1/3/8 activation was implicated in this process. In addition, NLRP3 inflammasome, the upstream of caspase-1 activation, was also found downregulated via suppressing its priming signal NF-κB pathway. Taken together, MSC protected against NaIO3-triggered RPE death via deactivating NF-κB-mediated NLRP3 inflammasome and maintaining mitochondrial integrity. This study highlights the significant role of MSC in modulating the proinflammatory environment of AMD, and suggests the clinical value of MSC in treating AMD as well as RPE replacement therapy.

Sodium Iodate-Induced Mouse Model of Age-Related Macular Degeneration Displayed Altered Expression Patterns of Sumoylation Enzymes E1, E2 and E3.

PURPOSE: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model. METHODS: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes. RESULTS: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas. CONCLUSION: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.

Monocyte infiltration and proliferation reestablish myeloid cell homeostasis in the mouse retina following retinal pigment epithelial cell injury.

Age-related macular degeneration (AMD), a leading contributor of vision loss, currently lacks comprehensive treatment. While AMD histopathology involves retinal pigment epithelium (RPE) injury associated with immune cell infiltration, the nature of immune cell responses to RPE injury remains undefined. We induced RPE injury pharmacologically and genetically in transgenic mouse models in which microglia and systemic monocytes were separately tagged, enabling a spatial and temporal dissection of the relative contributions of microglia vs. monocytes to post-injury changes. We found that myeloid cell responses to RPE injury occur in stages: (1) an early mobilization of endogenous microglia from the inner retina to the RPE layer, followed by (2) subsequent monocyte infiltration from the retinal vasculature into the inner retina that replenishes the local myeloid cell population in a CCR2-regulated manner. These altered distributions of myeloid cells post-injury were long-lived, with recruited monocytes acquiring the distribution, markers, and morphologies of neighboring endogenous microglia in a durable manner. These findings indicate the role played by infiltrating monocytes in maintaining myeloid cell homeostasis in the retina following AMD-relevant RPE injury and provide a foundation for understanding and therapeutically modulating immune aspects in retinal disease.

Protective Effect of Met12, a Small Peptide Inhibitor of Fas, on the Retinal Pigment Epithelium and Photoreceptor After Sodium Iodate Injury.

Purpose: A major problem in macular degeneration is the inability to reduce RPE and photoreceptor death. These cells die by necroptosis and apoptosis, respectively, but the upstream activator(s) of these death pathways is unknown. In this study, we use the sodium iodate (NaIO3) model of oxidative stress to test the hypothesis that activation of the Fas receptor contributes to the death of the RPE and photoreceptors. Methods: Sodium iodate was injected in Brown-Norway rats via femoral vein injection. Both in vivo (fundus photography, optical coherence tomography, and fluorescein angiography) and ex vivo (histology, immunohistochemistry, Western blot, and RT-PCR) analyses of the RPE and retina were conducted at baseline, as well as at various times post NaIO3 injection. The ability of intravitreal injection of Met12, a small peptide inhibitor of the Fas receptor, to prevent RPE and photoreceptor cell death was assessed. Results: Injection of NaIO3 led to Fas-mediated activation of both necroptosis and apoptosis in the RPE and photoreceptors, respectively. This was accompanied by a significant increase in the number of microglia/macrophages in the outer retina. Met12 significantly reduced the activation of the Fas-mediated death pathways, resulting in reduced RPE and photoreceptor death and a decreased immune response. Conclusions: Our results demonstrate that NaIO3 activates Fas-mediated cell death, both in the RPE and photoreceptor, and that a small peptide antagonist of the Fas receptor, Met12, significantly reduces the extent of this cell death. These findings suggest a role for Fas inhibition to protect the RPE and photoreceptors from death due to oxidative stress.

Resveratrol protects RPE cells from sodium iodate by modulating PPARα and PPARδ.

Selective killing of RPE cells in vivo by sodium iodate develops cardinal phenotypes of atrophic age-related macular degeneration. However, the molecular mechanisms are elusive. We tried to search for small cyto-protective molecules against sodium iodate and explore their mechanisms of action. Sodium iodate-mediated RPE cell death was associated with increased levels of reactive oxygen species (ROS) and IL-8. Resveratrol, a natural occurring polyphenol compound, was found to strongly protect RPE cells from sodium iodate with inhibition of production of ROS and IL-8. Resveratrol activated all isoforms of PPARs. Treatment with PPARα and PPARδ agonists inhibited sodium iodate-induced ROS production and protected RPE cells from sodium iodate. A PPARα antagonist significantly reduced resveratrol's protection of RPE cells from sodium iodate. Paradoxically, knocking down PPARδ also rendered RPE cells resistant to sodium iodate. Moreover, PPAR agonists reversed sodium iodate-induced production of IL-8. However, neutralizing extracellular IL-8 failed to protect RPE cells from sodium iodate. Taken together, these observations show that resveratrol protects RPE cells from sodium iodate injury through the activation of PPARα and alteration of PPARδ conformation. PPARα and δ modulators might ameliorate stress-induced RPE degeneration in vivo.

Long-term neuroprotective effects of NT-4-engineered mesenchymal stem cells injected intravitreally in a mouse model of acute retinal injury.

PURPOSE: Retinal degenerative diseases targeting the RPE and adjacent photoreceptors affect millions of people worldwide. The field of stem cell- and gene-based therapy holds great potential for the treatment of such diseases. The present study sought to graft genetically engineered mesenchymal stem cells (MSCs) that continuously produce neurotrophin-4 (NT-4) into the murine eye after the onset of acute retinal injury. METHODS: C57BL/6 mice were subjected to acute retinal damage using a low dose of sodium iodate (20 mg/kg of body weight), followed by intravitreal injection of lentivirally modified MSC-NT-4 into the right eye. At 3 months after the MSC transplantation grafted cell survival, retinal function and gene expression were analyzed. RESULTS: Immunofluorescence analysis confirmed that transplanted MSCs survived for at least 3 months after intravitreal injection and preferentially migrated toward sites of injury within the retina. MSC-NT-4 actively produced NT-4 in the injured retina and significantly protected damaged retinal cells, as evaluated by ERG and optical coherence tomography (OCT). Of importance, the long-term therapy with MSC-NT-4 was also associated with induction of prosurvival signaling, considerable overexpression of some subsets of transcripts, including several members of the crystallin ß-γ superfamily (Cryba4, Crybb3, Cryba2, Crybb1, Crybb2, Cryba1, and Crygc) and significant upregulation of biological processes associated with visual perception, sensory perception of light stimulus, eye development, sensory organ development, and system development. CONCLUSIONS: Transplantation of genetically modified MSCs that produce neurotrophic growth factors may represent a useful strategy for treatment of different forms of retinopathies in the future.