Ophthalmology
Abstract
Multiple members of the caveolae-associated protein (Cavin) family are implicated in angiogenesis. However, the specific role of CAVIN3 in pathological angiogenesis within the eye remains unclear. The present study demonstrated that CAVIN3 knockdown in endothelial cells (ECs) promoted vascular normalization in ocular pathological neovascularization. Elevated CAVIN3 expression was observed in the ECs of retinal pigment epithelium/choroid complexes from patients with neovascular age-related macular degeneration and fibrovascular membranes from patients with proliferative diabetic retinopathy. Additionally, upregulated Cavin3 expression was detected in laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) mouse models. In both OIR and CNV mice, Cavin3 knockdown inhibited pathological neovascularization. Cavin3 deficiency further disrupted EC proliferation and vascular sprouting, thereby promoting vascular normalization by partially restoring microenvironmental hypoxia and reestablishing pericyte-EC interactions. Mechanistically, we demonstrated that zinc finger E-box–binding homeobox 1 (ZEB1) regulated CAVIN3 transcription in ECs under hypoxic conditions. CAVIN3 deficiency modulated pathological vascularization by inhibiting ERK phosphorylation, which downregulated jagged 1 (JAG1) expression. Conclusively, this study elucidated the protective role of endothelial CAVIN3 deficiency in pathological neovascularization models, addressing a gap in understanding the regulatory role of Cavins in angiogenesis. These findings suggested a therapeutic direction for ocular neovascular diseases.
Authors
Weiqi Li, Yeran Zhang, Hongjing Zhu, Na Su, Ruxu Sun, Xiying Mao, Qin Yang, Songtao Yuan
Abstract
Inherited retinal degenerations (IRDs) are important causes of progressive, irreversible blindness. Hereditary macular diseases in particular are significant in their effect on the specialized, central cone photoreceptor-rich macula responsible for high resolution vision. Autosomal dominant Best vitelliform macular dystrophy (BVMD), caused by variants in the BEST1 gene, is one of the most common inherited macular dystrophies. Gene therapies have emerged as promising treatments for IRDs, but a lack of suitable animal models has hindered progress both in treatments and in understanding the mechanisms underlying macular diseases. Here, we report a Macaca fascicularis carrying a heterozygous potential pathogenic BEST1p.Q327E variant that disrupts the BEST1 ion channel by destabilizing the A195 helix, mirroring the structural perturbations seen in certain human pathological mutants. Longitudinal imaging over two years revealed progressive macular changes, including subfoveal cleft enlargement, lipid-rich deposit accumulation, retinal pigment epithelium (RPE) disruption, and central-to-peripheral photoreceptor degeneration, recapitulating early human BVMD pathology. Histopathology demonstrated diminished BEST1 expression, attenuation of the RPE-photoreceptor interface, and two distinct types of lipid deposits, including heretofore unappreciated cone mitochondrial-enriched lesions, highlighting selective cone mitochondria vulnerability. This first non-human primate model of inherited macular dystrophy links BEST1 mutations, mitochondrial dysfunction, and progressive macular degeneration, offering new insights into BVMD pathophysiology and highlighting its utility for studying disease progression and potential therapeutic interventions.
Authors
Wei Yi, Mingming Xu, Ying Xue, Yingxue Cao, Ziqi Yang, Lingli Zhou, Yang Zhou, Le Shi, Xiaomei Mai, Zehui Sun, Wenjie Qing, Yuying Li, Aolun Qing, Kaiwen Zhang, Lechun Ou, Shoudeng Chen, Elia J. Duh, Xialin Liu
Abstract
Degenerative retinal disorders leading to irreversible photoreceptor death are a common cause of blindness. Optogenetic gene therapy aims to restore vision in affected individuals by introducing light sensitive opsins into the surviving neurons of the inner retina. While up until now the main focus of optogenetic therapy has been on terminally blind individuals, treating at stages where residual native vision is present could have several advantages. Yet, it is still unknown how residual native and optogenetic vision would interact if present at the same time. Using transgenic mice expressing the optogenetic tool ReaChR in ON-bipolar cells, we herein examine this interaction through electroretinography (ERG) and visually evoked potentials (VEP). We find that optogenetic responses show a peculiar ERG signature and are enhanced in retinas without photoreceptor loss. Conversely, native responses are dampened in the presence of ReaChR. Moreover, in VEP recordings we find that optogenetic responses reach the cortex asynchronous to the native response. These findings should be taken into consideration when planning future clinical trials and may direct future preclinical research to optimize optogenetic approaches for visual restoration. The identified ERG signatures moreover may serve to track treatment efficiency in clinical trials.
Authors
Eleonora Carpentiero, Steven Hughes, Jessica Rodgers, Nermina Xhaferri, Sumit Biswas, Michael J. Gilhooley, Mark W. Hankins, Moritz Lindner
Abstract
Lipid metabolism is closely linked with antitumor immunity and autoimmune disorders. However, the precise role of lipid metabolism in uveitis pathogenesis is not clear. In our study, we analyzed the single-cell RNA-Seq (scRNA-Seq) data from cervical draining lymph nodes (CDLNs) of mice with experimental autoimmune uveitis (EAU), revealing an increased abundance of fatty acids in Th17 cells. Subsequent scRNA-Seq analysis identified the upregulation of DGAT1 expression in EAU and its marked reduction under various immunosuppressive agents. Suppression of DGAT1 prevented the conversion of fatty acids into neutral lipid droplets, resulting in the accumulation of lipid peroxidation and subsequent reduction in the proportion of Th17 cells. Inhibiting lipid peroxidation by Ferrostatin-1 effectively restored Th17 cell numbers that were decreased by DGAT1 inhibitor. Moreover, we validated the upregulation of DGAT1 in CD4+ T cells from patients with Vogt-Koyanagi-Harada (VKH) disease, a human uveitis. Inhibiting DGAT1 induced lipid peroxidation in human CD4+ T cells and reduced the proportion of Th17 cells. Collectively, our study focused on elucidating the regulatory mechanisms underlying Th17 cell survival and proposed that targeting DGAT1 may hold promise as a therapeutic approach for uveitis.
Authors
Tianfu Wang, Runping Duan, Zhaohuai Li, Bowen Zhang, Qi Jiang, Loujing Jiang, Jianjie Lv, Wenru Su, Lei Feng
Abstract
Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell-specific effects of LHON-linked mtDNA mutations remains poorly understood and there has been no effective treatment or cure for this disorder. Using a mice model bearing a LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell-specific deficiencies, especially in retinal ganglion cells (RGC), rods and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation-induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness, and increasing numbers of RGCs, photoreceptors and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide new insight into retina-specific pathophysiology of mitochondrial retinopathy arising from vitamin A deficiency and mitochondrial dysfunction induced by mtDNA mutation and step toward for therapeutic intervention for LHON and other mitochondrial retinopathy.
Authors
Cheng Ai, Huiying Li, Chunyan Wang, Yanchun Ji, Douglas C. Wallace, Junbin Qian, Yimin Zhu, Min-Xin Guan
Abstract
The high-temperature requirement A1 (HTRA1), a serine protease, has been demonstrated to play a pivotal role in the extracellular matrix (ECM) and has been reported to be associated with the pathogenesis of age-related macular degeneration (AMD). To delineate its role in the retina, the phenotype of homozygous Htra1-KO (Htra1–/–) mice was characterized to examine the effect of Htra1 loss on the retina and retinal pigment epithelium (RPE) with age. The ablation of Htra1 led to a significant reduction in rod and cone photoreceptor function, primary cone abnormalities followed by rods, and atrophy in the RPE compared with WT mice. Ultrastructural analysis of Htra1–/– mice revealed RPE and Bruch’s membrane (BM) abnormalities, including the presence of sub-RPE deposits at 5 months (m) that progressed with age accompanied by increased severity of pathology. Htra1–/– mice also displayed alterations in key markers for inflammation, autophagy, and lipid metabolism in the retina. These results highlight the crucial role of HTRA1 in the retina and RPE. Furthermore, this study allows for the Htra1–/– mouse model to be utilized for deciphering mechanisms that lead to sub-RPE deposit phenotypes including AMD.
Authors
Pooja Biswas, DaNae R. Woodard, T.J. Hollingsworth, Naheed W. Khan, Danielle R. Lazaro, Anne Marie Berry, Manisha Dagar, Yang Pan, Donita Garland, Peter X. Shaw, Chio Oka, Takeshi Iwata, Monica M. Jablonski, Radha Ayyagari
Abstract
Autoimmune uveitis (AU) is a sight-threatening ocular autoimmune disorder that often manifests as retinal vasculitis. Increased neutrophil infiltration around retinal vessels has been reported during the progression of AU, while how they function is not fully recognized. Neutrophil extracellular traps (NETs), produced by activated neutrophils, have been suggested to be detrimental in autoimmune diseases. Here, we found that NETs were elevated in patients with active AU, and this was verified in an experimental AU (EAU) mouse model. Depletion of neutrophils or degradation of NETs with deoxyribonuclease-I (DNase I) could decrease CD4+ effector T cell (Teff) infiltration in retina and spleen to alleviate EAU. Moreover, we found that the expression of adhesion molecules, selectin, and antigen-presenting molecules was elevated in EAU retina and in retinal microvascular endothelial cells (RMECs) cocultured with NETs. The stimulated RMECs further facilitated CD4+ T cell adhesion, activation, and differentiation into Teffs. Mechanistically, NETs trigger RMEC activation by hastening cell senescence through the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Slowing down senescence or inhibiting the cGAS/STING pathway in RMECs reduces the activation and differentiation of CD4+ T cells. These results suggest a deleterious role of NETs in AU. Targeting NETs would offer an effective therapeutic method.
Authors
Zuoyi Li, Zhuang Li, Yunwei Hu, Yanyan Xie, Yuxun Shi, Guanyu Chen, Jun Huang, Zhiqiang Xiao, Wenjie Zhu, Haixiang Huang, Minzhen Wang, Jianping Chen, Xiaoqing Chen, Dan Liang
Abstract
Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that accurately replicates these human POAG features has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing the outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant loss of retinal ganglion cells (RGCs) and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.
Authors
Balasankara Reddy Kaipa, Ramesh Kasetti, Yogapriya Sundaresan, Linya Li, Sam Yacoub, J. Cameron Millar, William Cho, Dorota Skowronska-Krawczyk, Prabhavathi Maddineni, Krzysztof Palczewski, Gulab S. Zode
Abstract
BACKGROUND. Current clinical sequencing methods cannot effectively detect DNA methylation and allele-specific variation to provide parent-of-origin information from the proband alone. Parent-of-origin effects can lead to differential disease and the inability to assign this in de novo cases limits prognostication in the majority of affected individuals with retinoblastoma, a hereditary cancer with suspected parent-of-origin effects. METHODS. To directly assign parent-of-origin in retinoblastoma patients, genomic DNA was extracted from blood samples for sequencing using a programmable, targeted single-molecule long-read DNA genomic and epigenomic approach. This allowed germline variant calling and simultaneous haplotype-resolved CpG methylation in subjects with familial (n=7) and de novo (n=9) retinoblastoma. RESULTS. Targeted long-read sequencing allowed phasing genomic variation with a differentially methylated region in intron 2 of the RB1 gene to confirm parent-of-origin in known familial samples. Leveraging this approach allowed us to directly assign parent-of-origin rapidly in simple and complex de novo cases from the proband alone. The ability to assign parent-of-origin in all cases of retinoblastoma showed that harboring disease-causing variants on the paternally inherited allele, whether arising familial or de novo, is associated with more advanced cancer staging at presentation and significantly greater risk of chemotherapy failure (P=0.002). CONCLUSION. This study demonstrates the diagnostic potential of multi-omic long-read profiling to unveil the parent-of-origin effect in hereditary cancer. The approach in this work will be instrumental in assigning parent-of-origin to other genetic diseases using local and distant imprinting signals in the genome. FUNDING. National Eye Institute, NIH (K08EY033789); Gerber Foundation; Research to Prevent Blindness
Authors
Andrew W. Stacey, Kenji Nakamichi, Jennifer Huey, Jeffrey Stevens, Natalie Waligorski, Erin E. Crotty, Russell N. Van Gelder, Debarshi Mustafi
Abstract
Despite recent advances in the treatment of thyroid eye disease thyroid-related eye disease (TED), marked gaps remain in our understanding of the underlying molecular mechanisms, particularly concerning the insulin-like growth factor-1 receptor (IGF-1R) pathway. To dissect the pathophysiology of TED, we used single-nucleus RNA-Seq to analyze orbital fat specimens from both patients with TED and matched individuals acting as controls. The analysis demonstrated a marked increase in the proportion of fibroblasts transitioning to adipogenesis in the orbital fat of patients with TED compared with that in control patients. This was associated with diverse alterations in immune cell composition. Significant alterations in the IGF-1R signaling pathway were noted between TED specimens and those from control patients, indicating a potential pathological mechanism driven by IGF-1R signaling abnormalities. Additionally, our data showed that linsitinib, a small-molecule inhibitor of IGF-1R, effectively reduced adipogenesis in TED orbital fibroblasts in vitro, suggesting its potential utility as a therapeutic agent. Our findings reveal that, beyond immune dysfunction, abnormal IGF-1R signaling leading to enhanced adipogenesis is a crucial pathogenic mechanism in TED.
Authors
Dong Won Kim, Soohyun Kim, Jeong Han, Karan Belday, Emily Li, Nicholas Mahoney, Seth Blackshaw, Fatemeh Rajaii
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