IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Yuan H ，Chen S ，Duncan MR ，de Rivero Vaccari JP ，Keane RW ，Dalton Dietrich W ，Chou TH ，Benny M ，Schmidt AF ，Young K ，Park KK ，Porciatti V ，Elizabeth Hartnett M ，Wu S ... -  《-》

Up-regulation of VEGF by retinoic acid during hyperoxia prevents retinal neovascularization and retinopathy.

Retinopathy of prematurity (ROP) is directly associated with abnormal expression of retinal vascular endothelial growth factor (VEGF) in premature neonates. This study was to investigate whether the systemic administration of retinoic acid (RA) regulates retinal VEGF expression and prevents retinal neovascularization and retinopathy in the oxygen-induced retinopathy (OIR) mouse model. C57BL/6 mice were subjected to OIR by exposure to 75% oxygen from postnatal day (P) 7 to 12 of age. RA was intraperitoneally injected daily to pups from P6 to P9. Retinal whole mount staining and image analysis, immunostaining, Western blotting, quantitative RT-PCR, TUNEL assay, and electroretinography were performed to evaluate the effects of RA on VEGF expression, retinal neovascularization, and retinal neuron functions. Systemic administration of RA in OIR mice promoted retinal VEGF mRNA and protein expression in phase I; the stabilized level of VEGF in phase I supported retinal vascular development and counteracted vaso-obliteration in OIR mice. Subsequently, the excessive generation of VEGF in phase II was attenuated; the retinal vascular leakage and apoptotic cells were significantly ameliorated. As a result, RA significantly prevented the development of hypoxia-induced retinal neovascularization and retinopathy in OIR mice and improved the functional recovery of retinal neurons downstream of photoreceptor cells as measured by focal electroretinography. Systemic administration of RA regulates retinal VEGF expression and supports retinal vascular development in OIR mouse model. We propose that systemic administration of RA to extremely low birth weight, preterm infants during oxygen therapy could potentially be an effective therapeutic approach for the prevention of ROP.

Wang L ，Shi P ，Xu Z ，Li J ，Xie Y ，Mitton K ，Drenser K ，Yan Q ... -  《-》

Long-term evaluation of retinal morphology and function in a mouse model of oxygen-induced retinopathy.

Retinopathy of prematurity (ROP) is a condition of aberrant retinal vascularization in premature infants in response to high levels of oxygen used for critical care that can potentially cause blindness. Although therapies to mitigate vascular abnormalities are being evaluated, functional deficits often remain in patients with treated or regressed ROP. This study investigated long-term outcomes of hyperoxia on retinal morphology and function using a mouse model of oxygen-induced ischemic retinopathy (OIR). Twenty-two mice were exposed to 77% oxygen to induce OIR, while 23 age-matched control mice were raised in room air (RA). In vivo fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), and focal electroretinography (fERG) were performed at P19, P24, P32, and P47, followed by histological assessments of retinal morphology, gliosis, microglia activation, and apoptosis. FA in OIR mice showed capillary attrition despite peripheral revascularization. Inner retina thinning was detected with SD-OCT; outer and inner retinal dysfunction were demonstrated with fERG. Histology of the OIR mice exhibited a thin, disorganized structure. Immunohistochemistry showed increased gliosis, microglial activation, and apoptosis with increasing age from P19 to P47. The synapses between rod photoreceptor cells and rod bipolar cells were ectopically localized in the OIR mice. We demonstrated histological evidence of persistent ectopic synapses, prolonged cellular apoptosis, and gliosis in the OIR retina that corresponded with long-term in vivo evidence of capillary attrition, inner retinal thinning, and dysfunction despite full peripheral revascularization. Further studies on the mechanisms underlying these persistent phenotypes could enhance our understanding of ROP pathogenesis and lead to new therapeutic targets to preserve visual function in premature infants.

Mezu-Ndubuisi OJ ，Macke EL ，Kalavacherla R ，Nwaba AA ，Suscha A ，Zaitoun IS ，Ikeda A ，Sheibani N ... -  《MOLECULAR VISION》

Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.

Saito Y ，Geisen P ，Uppal A ，Hartnett ME ... -  《MOLECULAR VISION》

Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice.

Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.

Guo Y ，Qin J ，Sun R ，Hao P ，Jiang Z ，Wang Y ，Gao Z ，Zhang H ，Xie K ，Zhang W ... -  《-》