Vascular endothelial growth factor (VEGF) is a key mediator of exudative age-related macular degeneration (eAMD), yet non-invasive biomarkers for disease monitoring remain limited. This study evaluates VEGF levels in human tear fluid as a potential biomarker for eAMD and investigates the molecular dynamics of VEGF in a laser-induced choroidal neovascularization (lCNV) mouse model. Tear VEGF levels were quantified using proximity qPCR immunoassays in eAMD patients (n = 29) and healthy controls (n = 21) and correlated with optical coherence tomography (OCT) findings. Molecular analyses, including immunohistochemistry, gene expression profiling, and phosphorylation assays, were conducted on choroid–retinal pigment epithelium (RPE) and lacrimal gland (LG) tissues from lCNV mice (n = 25). Tear VEGF levels were significantly elevated in eAMD patients, correlating with disease severity. Females exhibited higher VEGF levels, a pattern not replicated in the mouse model. In lCNV mice, VEGF overexpression originated from the choroid–RPE, driven by hypoxic and inflammatory signaling, with no significant LG contribution. Increased VEGF, IL-6, and vimentin expression, along with NF-κB and STAT3 activation, were observed. These findings suggest that tear VEGF is a promising non-invasive biomarker for eAMD, warranting further validation for clinical application in disease monitoring and treatment optimization.

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia‐induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel ex vivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co‐immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co‐immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low‐density lipoprotein receptor‐related protein‐1 (LRP‐1), resulting in inhibition of β‐catenin–mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells ex vivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial‐specific mechanism of β‐catenin–mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non‐canonical β‐catenin downstream effects mediated by LRP‐1/uPAR interaction.

The human retinal pigment epithelium (RPE) cell line ARPE‐19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE‐specific laminin (LN) and nicotinamide (NAM) could improve ARPE‐19 redifferentiation to resemble mature RPE and improve the assessment of RPE‐specific gene therapy strategies. ARPE‐19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521‐coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno‐associated virus (AAV)1 or AAV2, carrying a VMD2‐driven GFP, were assessed at 2‐ and 4‐weeks post‐plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin‐1 and RPE 65 was promoted concomitantly with a reduction of several epithelial‐mesenchymal transition markers, such as TNF‐α, TGF‐β, CDH2, and vimentin. Redifferentiated ARPE‐19 transduced at low multiplicity of infection of both AAV1‐ and AAV2‐VMD2‐GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post‐plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE‐19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE‐19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral‐mediated RPE‐specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE‐centered diseases.

The Microphthalmia-associated transcription factor (MITF) plays a critical role in retinal pigment epithelium (RPE) development and function. Dysfunctional autophagy and lysosomal degradation in the RPE have been implicated in age-related retinal degeneration, yet the contribution of MITF to these pathways remains incompletely understood. Here, we show that reduced Mitf expression impairs autophagy in mouse and human RPE cells. Primary RPE cells from Mitf ^mi-vga9/+ heterozygotes mice displayed altered autophagic flux characterized by accumulation of LC3B-II and p62, while MITF knockdown in human ARPE-19 cells promoted autophagosome accumulation. Ultrastructural analysis further revealed age-dependent accumulation of autolysosomes and lipofuscin-like granules in mutant RPE cells. In addition, expression of autophagy-related genes was altered in mutant RPE tissue, supporting disrupted lysosomal-autophagic homeostasis. Together, our findings identify MITF as an important regulator of autophagy in the RPE and suggest that impaired MITF-dependent homeostasis may contribute to retinal degeneration.

Glaucoma is a leading cause of irreversible blindness worldwide, yet available treatments fail to prevent disease progression for all patients. It is characterized by a progressive dysfunction and loss of retinal ganglion cells. Neuroinflammation has been recognized as an underlying neurodegenerative mechanism of glaucoma in animal models and human post-mortem samples, and targeting neuroinflammation may provide additional means to neuroprotection. Galectin-3, a pro-inflammatory mediator encoded by the LGALS3 gene in humans, holds promise as a treatable target as its pharmacological and genetic inhibition is neuroprotective in multiple models of experimental glaucoma. However, the role of Galectin-3 in glaucoma remains unclear, particularly whether its emergence is a consequence of degeneration, or occurs at earlier time points. To address these knowledge gaps, we labeled IBA1, GFAP, and Galectin-3 in retina sections at early glaucoma stages in the rat bead glaucoma model, and in human retina from glaucoma donors. In the rat, IBA1 volume, but not GFAP, increased at an early, pre-degenerative timepoint. Accompanying this, we identified a significant increase of Galectin-3/IBA1 colocalization compared to control at the same timepoint, supporting the upregulation of Galectin-3 in early inflammation, preceding retinal ganglion cell degeneration in experimental glaucoma. However, a significant increase in Galectin-3/GFAP colocalization compared to control at the same timepoint in the rat additionally associates Galectin-3 production with astrocytes and Müller glia. This Galectin-3 to Müller glia relationship was significantly pronounced in human glaucomatous retina, predominating over microglia co-labelling. We further demonstarted that human MIO-M1 Müller glia in vitro express Galectin-3, but this is not altered in response to glaucoma relevant stimuli (TNF-α or mild-metabolic stress from rotenone). Instead, Galectin-3 expression was altered in phagocytosing states from exposure to E. coli particles, brain synaptosomes, or apoptotic neuronal debris. These findings provide further insight into Galectin-3 and gross inflammatory responses in glaucoma pathology.