Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination.

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age-related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser-induced model of choroidal neovascularization that models wet-AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co-expression of the citrullination enzyme peptidyl arginine deiminase-4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial-specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4-driven hypercitrullination may be a target for retinal fibrosis.

Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization.

Pathologic ocular neovascularization commonly causes blindness. It is critical to identify the factors altered in pathologically proliferating versus normally quiescent vessels to develop effective targeted therapeutics. MicroRNAs regulate both physiological and pathological angiogenesis through modulating expression of gene targets at the posttranscriptional level. However, it is not completely understood if specific microRNAs are altered in pathologic ocular blood vessels, influencing vascular eye diseases. Here we investigated the potential role of a specific microRNA, miR-150, in regulating ocular neovascularization. We found that miR-150 was highly expressed in normal quiescent retinal blood vessels and significantly suppressed in pathologic neovessels in a mouse model of oxygen-induced proliferative retinopathy. MiR-150 substantially decreased endothelial cell function including cell proliferation, migration, and tubular formation and specifically suppressed the expression of multiple angiogenic regulators, CXCR4, DLL4, and FZD4, in endothelial cells. Intravitreal injection of miR-150 mimic significantly decreased pathologic retinal neovascularization in vivo in both wild-type and miR-150 knockout mice. Loss of miR-150 significantly promoted angiogenesis in aortic rings and choroidal explants ex vivo and laser-induced choroidal neovascularization in vivo. In conclusion, miR-150 is specifically enriched in quiescent normal vessels and functions as an endothelium-specific endogenous inhibitor of pathologic ocular neovascularization.

Pharmacologic Activation of Wnt Signaling by Lithium Normalizes Retinal Vasculature in a Murine Model of Familial Exudative Vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is characterized by delayed retinal vascular development, which promotes hypoxia-induced pathologic vessels. In severe cases FEVR may lead to retinal detachment and visual impairment. Genetic studies linked FEVR with mutations in Wnt signaling ligand or receptors, including low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we investigated ocular pathologies in a Lrp5 knockout (Lrp5(-/-)) mouse model of FEVR and explored whether treatment with a pharmacologic Wnt activator lithium could bypass the genetic defects, thereby protecting against eye pathologies. Lrp5(-/-) mice displayed significantly delayed retinal vascular development, absence of deep layer retinal vessels, leading to increased levels of vascular endothelial growth factor and subsequent pathologic glomeruloid vessels, as well as decreased inner retinal visual function. Lithium treatment in Lrp5(-/-) mice significantly restored the delayed development of retinal vasculature and the intralaminar capillary networks, suppressed formation of pathologic glomeruloid structures, and promoted hyaloid vessel regression. Moreover, lithium treatment partially rescued inner-retinal visual function and increased retinal thickness. These protective effects of lithium were largely mediated through restoration of canonical Wnt signaling in Lrp5(-/-) retina. Lithium treatment also substantially increased vascular tubular formation in LRP5-deficient endothelial cells. These findings suggest that pharmacologic activation of Wnt signaling may help treat ocular pathologies in FEVR and potentially other defective Wnt signaling-related diseases.

Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization.

OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway.

Ocular and orbital tumors in childhood.

Pediatric tumors of the eye and orbit can be benign or malignant as well as congenital or acquired and are usually distinctively different than those seen in adults. Although most of these neoplasms are benign (eg, dermoid cyst, chalazion, molluscum), their location near and within a vital organ can result in serious dermatologic and ophthalmologic sequelae. Lesions discussed include vascular lesions, retinoblastomas (the most common primary pediatric intraocular malignancy), rhabdomyosarcoma (the most common primary pediatric orbital malignancy), Langerhans cell histiocytosis, and metastatic lesions to the orbit (neuroblastoma, Ewing sarcoma). Although cysts and ocular melanoma can occur within the pediatric population, these conditions are covered in other contributions in this issue of Clinics in Dermatology.

Efficacy, Safety, and Durability of Brolucizumab: An 8-Month Post-Marketing Surveillance Analysis.

Importance: Brolucizumab (Beovu®) is an anti-vascular endothelial growth factor (anti-VEGF) agent approved for the treatment of neovascular age-related macular degeneration (nvAMD). Brolucizumab was marketed for its noninferiority to aflibercept and its potential for greater durability. However, post-marketing utilization has been tempered by safety concerns. Objective: We evaluate the visual and anatomic efficacy of brolucizumab, examine changes in treatment intervals after switching to brolucizumab, and estimate the incidence of drug-related adverse events in the real world. Design Setting and Participants: This was a retrospective consecutive case series of 626 eyes (543 patients) with nvAMD treated with 1438 brolucizumab injections at a single retina practice between 10/1/2019 and 5/15/2020. Main Outcomes and Measures: Changes in visual acuity (VA); anatomic outcomes assessed by optical coherence tomography (OCT) including central subfield thickness (CST), macular volume (MV), presence of intraretinal fluid (IRF), subretinal fluid (SRF), and serous pigment epithelial detachment (sPED) on foveal line scans; treatment intervals before and after receiving brolucizumab; and the incidence of brolucizumab-related adverse events. Results: The majority of eyes (N = 531, 89.7%) had received prior anti-VEGF therapy with aflibercept, ranibizumab, and/or bevacizumab. VA improved in treatment-naïve eyes (+3.7 letters, p = 0.04), and was maintained in previously treated eyes. There were significant improvements in all anatomic outcomes in both groups (p < 0.001). We observed a 4.8% incidence of intraocular inflammation (IOI) and a 0.6% incidence of retinal vasculitis. The average treatment interval increased from 6.3 to 6.8 weeks (p = 0.001). Conclusions and Relevance: Brolucizumab treatment was associated with VA improvement in naïve eyes and maintenance of VA in previously treated eyes. Switching to brolucizumab was associated with improved anatomic outcomes and extended treatment intervals in most eyes. We observed a similar incidence of IOI and a lower incidence of retinal vasculitis compared to the Safety Review Committee's analysis of HAWK and HARRIER.

Clinical Features Associated with Acute Elevated Intraocular Pressure After Intravitreal Anti-VEGF Injections.

Purpose: To study the effects of intravitreal injection (IVI) of anti-VEGF (vascular endothelial growth factor) agents on intraocular pressure (IOP) and find associations with acute pressure spikes. Methods: This was a three-month, prospective study of patients receiving outpatient IVI of anti-VEGF agents for diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO) at the Acuity Eye Group Medical Centers. IOP was measured pre- and post-injection at 10-minute intervals up to 50 minutes after injection with a handheld tonometer. Patients with an IOP greater than 35 mmHg at 30 minutes received an anterior chamber paracentesis (ACP), while patients below 35 mmHg were monitored without intervention. Results: A total of 617 patients (51% female, 49% male) received IVI for DR (n = 199), AMD (n = 355), and RVO (n = 63). ACP was performed in 17 patients. Average pre-injection IOP was 16 ± 4 compared to 24 ± 7 mmHg for the non-ACP vs ACP group, respectively (mean ± standard deviation), p < 0.0001. IOP returned to baseline in 98% of patients at 50 minutes. A diagnosis of glaucoma and glaucoma suspect was more prevalent in the ACP group compared to the non-ACP group, 82.3% vs 14.2% and 17.6% vs 9.0%, respectively, p < 0.0001 and p > 0.05. Patients with a pre-injection IOP >25 mmHg and a history of glaucoma had a 58.3% rate of ACP. A 31-gauge needle had a higher mean increase in IOP from baseline compared to 30-gauge needle, p < 0.0001. Conclusion: IOP spikes are most significant in the first 10 minutes after IVI but typically resolve within the first hour. However, utilizing a smaller 31-gauge IVI in patients with a glaucoma history and pre-injection IOP >25 mmHg may be associated with significant IOP spikes lasting longer than 30 minutes.

FGF21 Administration Suppresses Retinal and Choroidal Neovascularization in Mice.

Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration: very-low-density lipoprotein-receptor-deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited tumor necrosis α (TNF-α) expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration.

SOCS3 in retinal neurons and glial cells suppresses VEGF signaling to prevent pathological neovascular growth.

Neurons and glial cells in the retina contribute to neovascularization, or the formation of abnormal new blood vessels, in proliferative retinopathy, a condition that can lead to vision loss or blindness. We identified a mechanism by which suppressor of cytokine signaling 3 (SOCS3) in neurons and glial cells prevents neovascularization. We found that Socs3 expression was increased in the retinal ganglion cell and inner nuclear layers after oxygen-induced retinopathy. Mice with Socs3 deficiency in neuronal and glial cells had substantially reduced vaso-obliterated retinal areas and increased pathological retinal neovascularization in response to oxygen-induced retinopathy, suggesting that loss of neuronal/glial SOCS3 increased both retinal vascular regrowth and pathological neovascularization. Furthermore, retinal expression of Vegfa (which encodes vascular endothelial growth factor A) was higher in these mice than in Socs3 flox/flox controls, indicating that neuronal and glial SOCS3 suppressed Vegfa expression during pathological conditions. Lack of neuronal and glial SOCS3 resulted in greater phosphorylation and activation of STAT3, which led to increased expression of its gene target Vegfa, and increased endothelial cell proliferation. In summary, SOCS3 in neurons and glial cells inhibited the STAT3-mediated secretion of VEGF from these cells, which suppresses endothelial cell activation, resulting in decreased endothelial cell proliferation and angiogenesis. These results suggest that neuronal and glial cell SOCS3 limits pathological retinal angiogenesis by suppressing VEGF signaling.