Ophthalmology
Abstract
Macular edema (ME) can cause profound vision impairment and occurs in several prevalent retinal diseases, including diabetic retinopathy (DR), choroidal neovascularization (CNV), retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterized Tspan12 KODBM (Disrupted Barrier Maintenance) mice, a mouse model of cystoid edema generated by genetically and pharmacologically manipulating beta-catenin-dependent norrin/frizzled4 (FZD4) signaling. We assessed BRB function, cystoid edema, ERG, and microglia activation outcomes in an aging study with WT, C1qa KO, Tspan12 KODBM, and Tspan12 KODBM;C1qa KO compound mutant mice. Phenotypic analyses and cell-based experiments indicated that C1QA contributes to maintaining basal β-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12 KODBM;C1qa compound mutant mice. Activation of β-catenin-dependent signaling by a FZD4/LRP5 agonist antibody modality achieved complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/frizzled4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides novel insights into the contribution of C1QA to BRB maintenance.
Authors
Lingling Zhang, Jacklyn Levey, Md. Abedin, Ha-Neul Jo, Emmanuel Odame, Miranda Howe, Kaia L. Douglas, Elise Thoreen, Scott W. McPherson, Heidi Roehrich, Somasekar Seshagiri, Stephane Angers, Zhe Chen, Harald J. Junge
Abstract
Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.
Authors
Valentin J. Weber, Alice Reschigna, Maximilian J. Gerhardt, Thomas Heigl, Klara S. Hinrichsmeyer, Sander van den Engel, Dina Y. Otify, Zoran Gavrilov, Frank Blaser, Isabelle Meneau, Christian Betz, Hanno J. Bolz, Martin Biel, Stylianos Michalakis, Elvir Becirovic
Abstract
Recurrent acute anterior uveitis is a frequent extra-articular manifestation of the axial spondyloarthropathies (AxSpA); chronic inflammatory diseases affecting the spine, enthesis, peripheral joints, skin, and gastrointestinal tract. Pathology in AxSpA has been associated with local tissue-resident populations of interleukin (IL)-23 responsive lymphoid cells. Here we characterize a population of ocular T cell defined by CD3+CD4-CD8-CD69+gdTCR+IL-23R+ that reside within the anterior uvea as an ocular entheseal analogue of the mouse eye. Localised cytokine expression demonstrates that uveal IL-23R+ IL-17A-producing cells are both necessary and sufficient to drive uveitis in response to IL-23. This T cell population is also present in humans, occupying extravascular tissues of the anterior uveal compartment. Consistent with the concept of IL-23 as a unifying mediator in AxSpA, we present evidence that IL-23 can also act locally on tissue resident T cells in the anterior compartment of the eye at sites analogous to the enthesis to drive ocular inflammation.
Authors
Robert Hedley, Amy Ward, Colin J. Chu, Sarah E. Coupland, Serafim Kiriakidis, Peter C. Taylor, Stephanie G. Dakin, ORBIT Research Consortium, Christopher D. Buckley, Jonathan Sherlock, Andrew D. Dick, David A. Copland
Abstract
The loss of integrity of the blood retina barrier (BRB) is a key pathological hallmark of vision-threatening complications in diabetic retinopathy (DR). Although DR is considered a microvascular disease, mounting evidence from mouse models and patients show that inflammation is closely connected with microvasculopathy. Inflammatory responses during retinal pathophysiology are often orchestrated by microglia, resident innate immune cells of the retina. However, the precise role of microglia activity during DR pathogenesis remains elusive. Here, we used an anti PDGFRβ antibody and inducible endothelial cell-specific PDGFB-KO during postnatal development of retinal vasculature to reproduce key features of DR pathology in mice. In addition, we applied a minocycline therapy to modulate retinal inflammation. Postnatal depletion of pericytes or loss of PDGFB in retinal vessels altered BRB integrity, triggered secretion of angiogenic and inflammatory factors with concomitant microglia reactivity, which was sustained in mature retinas. Microglia reactivity was accompanied by upregulation of disease-associated genes. Notably, minocycline attenuated the cycle of inflammatory responses in young and mature retinas, thereby preserving retinal vascular and structural integrity in mice. Together, our findings suggest that immunomodulation of microglia-driven inflammatory responses preserves retinal vasculature and maintains BRB integrity in two different mouse models of human DR.
Authors
Urbanus Muthai Kinuthia, Christoph Möhle, Ralf H. Adams, Thomas Langmann
Abstract
High myopia (HM) and posterior staphyloma (PS) are major causes of vision loss worldwide. Genetic and environmental factors, especially light exposure, influence myopia. This study shows that LRP2 (Low-density lipoprotein-related receptor type 2) levels are decreased in the vitreous of patients with HM and PS, and that in human donor eyes affected by PS, LRP2 expression was reduced in the neural retina and retinal pigment epithelium (RPE), with morphologic changes similar to those observed in the Foxg1-Cre-Lrp2lox/lox mouse that also develops PS. In human iPSc-derived RPE cells (iRPE), LRP2 silencing regulated genes involved in eye and neuronal development, visual perception, tissue remodeling, hormone metabolism and RPE structure. Its expression increased under light exposure, particularly red light, but was downregulated by cortisol. These findings establish a link between LRP2, myopization, and environmental factors, highlighting its crucial role in nonsyndromic HM and PS. LRP2 appears to be a promising therapeutic target for high myopia treatment.
Authors
Kimberley Delaunay, Emilie Picard, Patricia Lassiaz, Laurent Jonet, Vidjea Cannaya, José Maria Ruiz-Moreno, Kentaro Kojima, Henrik Vorum, Bent Honoré, Jorge R. Medrano, Lasse Jørgensen Cehofski, Eric Pussard, Renata Kozyraki, Alicia Torriglia, Olivier Cases, Francine Behar-Cohen
Abstract
Background: Traumatic optic neuropathy (TON) is a leading cause of blindness following closed traumatic brain injury, with no effective treatments available. Previous interventional clinical trials were complicated by its low prevalence, variability in neurodegenerative severity, and unavailability of reliable biomarkers. Methods: We analyzed data from 1226 patients enrolled in the prospective National Multi-Center Collaborative Clinical Research Program of China (2017-2024) to establish a clinical profile and identify non-invasive biomarkers for neurodegenerative severity. Subgroup analysis of monocular TON patients revealed potential biomarkers including visual functional parameters, inner retinal thickness, and time post-injury. Results: The ganglion cell complex (GCC) thickness showed a strong correlation with retinal ganglion cell somata (R² = 0.87, p < .0001) and axon density (R² = 0.89, p < .0001) in a clinically relevant large animal model. Computational analysis demonstrated that using GCC thickness as a biomarker could substantially enhance the statistical power of clinical trials (by up to 4.5-fold), as confirmed by real-world data. Conclusion: This study presented the largest epidemiological analysis of TON to date and established GCC thickness as a crucial biomarker for stratifying disease severity and improving the efficiency of clinical trials. Trial registration: Chinese Clinical Trial Registry (ChiCTR-OOC-17013437). Funding: National Key R&D Program of China (Grant No.2022YFA1105500); Key Science and Technology Program of Wenzhou (Grant No.ZY2022021); National Natural Science Foundation of China (Grant No.82471080).
Authors
YiKui Zhang, BoYue Xu, ShiWei Huang, ZhaoHui Shi, Wei Xiong, Ruijun Wang, GuiQin Liu, Linlin Chen, ZhenHua Ge, YongJie Zhang, HongLei Liu, BaoYun Jia, Chunxia Wang, HaiHong Shi, Jun Kang, NingYu An, Shuirui Huang, De-Fu Chen, Shenghai Huang, YuTing Luo, MingYue Liu, ZhuoWei Wang, Zhonghao Yu, Jingwei Zheng, Wentao Yan, Gen Li, Hao Chen, XingGuang Deng, Shihui Wei, YunHai Tu, EnDe Wu, Kang Zhang, Wencan Wu
Abstract
Autologous photoreceptor cell replacement is one of the most promising strategies currently being developed for the treatment of patients with inherited retinal degenerative blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of transplantable photoreceptors required for these therapies. However, retinal organoids contain other retinal cell types, including bipolar, horizontal and amacrine cells, which are unneeded and may reduce the potency of the final therapeutic product. Therefore, approaches for isolating fate committed photoreceptor cells from dissociated retinal organoids are desirable. In this work, we present partial dissociation, a technique which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. We demonstrate up to 90% photoreceptor cell purity by simply selecting cell fractions liberated from retinal organoids during enzymatic digestion in the absence of mechanical dissociation. As the presented approach relies on the use of standard plasticware and commercially available cGMP compliant reagents, we believe that it is ideal for use in the preparation of clinical photoreceptor cell replacement therapies.
Authors
Nicholas E. Stone, Laura R. Bohrer, Nathaniel K. Mullin, Alexander Berthold, Allison T. Wright, Ian C. Han, Edwin M. Stone, Robert F. Mullins, Budd A. Tucker
Abstract
Dry age-related macular degeneration (AMD) is a leading cause of untreatable vision loss. In advanced cases, retinal pigment epithelium (RPE) cell loss occurs alongside photoreceptor and choriocapillaris degeneration. We hypothesized that an RPE-patch would mitigate photoreceptor and choriocapillaris degeneration to restore vision. An induced pluripotent stem cell–derived RPE (iRPE) patch was developed using a clinically compatible manufacturing process by maturing iRPE cells on a biodegradable poly(lactic-co-glycolic acid) (PLGA) scaffold. To compare outcomes, we developed a surgical procedure for immediate sequential delivery of PLGA-iRPE and/or PLGA-only patches in the subretinal space of a pig model of laser-induced outer retinal degeneration. Deep learning algorithm-based optical coherence tomography (OCT) image segmentation verified preservation of the photoreceptors over the areas of PLGA-iRPE–transplanted retina and not in laser-injured or PLGA-only–transplanted retina. Adaptive optics imaging of individual cone photoreceptors further supported this finding. OCT-angiography revealed choriocapillaris regeneration in PLGA-iRPE– and not in PLGA-only–transplanted retinas. Our data, obtained using clinically relevant techniques, verified that PLGA-iRPE supports photoreceptor survival and regenerates choriocapillaris in a laser-injured pig retina. Sequential delivery of two 8 mm2 transplants allows for testing of surgical feasibility and safety of the double dose. This work allows one surgery to treat larger and noncontiguous retinal degeneration areas.
Authors
Rohan Gupta, Irina Bunea, Bruno Alvisio, Francesca Barone, Rishabh Gupta, Dara Baker, Haohua Qian, Elena Daniele, Casey G. Contreary, Jair Montford, Ruchi Sharma, Arvydas Maminishkis, Mandeep S. Singh, Maria Teresa Magone De Quadros Costa, Amir H. Kashani, Juan Amaral, Kapil Bharti
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
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