Investigative Ophthalmology & Visual Science

PurposeMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in many inflammatory and fibrotic diseases; however, its role in primary open-angle glaucoma (POAG) and trabecular meshwork (TM) dysfunction remains unknown. In this study, we investigated whether MIF-CD74 signaling regulates TM pathobiology through modulation of the transcription factor, Blimp-1, and downstream cytoskeletal reorganization and extracellular matrix (ECM) remodeling. MethodPrimary human TM cells (HTMC) were exposed to glaucomatous stressors, including TGF-{beta}2, rMIF, or a pro-inflammatory milieu. Expression of MIF, its receptor CD74, and Blimp-1 was measured by qPCR and immunoblotting. ECM proteins and phosphorylated myosin-light chain (pMLC) were evaluated by immunofluorescence staining. In vivo, MIF-CD74 and Blimp-1 expression were examined in the TM/anterior segment (AS) tissue of Tg.CreMYOCY437H and lentiviral (LV)-TGF-{beta}2-induced ocular hypertension (OHT) mouse models. Functional involvement of MIF signaling in TM pathobiology was examined using the irreversible MIF inhibitor 4-IPP and the immunomodulatory metabolites agmatine and thiamine. ResultsGlaucomatous stressors significantly upregulated MIF and CD74 expression with concomitant suppression of Blimp-1 in HTMC. Similarly, TM/AS tissue from both OHT models (Tg.CreMYOCY437H and LV-TGF-{beta}2) demonstrated increased MIF-CD74 expression accompanied by reduced Blimp-1 levels. Activation of MIF-CD74 signaling triggered pro-inflammatory and cell death pathways and promoted ECM remodeling, characterized by increased fibrotic protein expression and enhanced RhoA/ROCK-mediated MLC phosphorylation, indicating modulation of TM contractility. Pharmacological inhibition of MIF attenuated inflammatory signaling, reduced ECM deposition and cytoskeletal remodeling, and suppressed RhoA/ROCK/MLC activation, restoring a protective TM phenotype. ConclusionOur findings identify MIF-CD74 signaling as a previously unrecognized regulator of TM dysfunction in POAG. MIF-mediated suppression of Blimp-1 mechanistically links inflammatory signaling to cytoskeletal contractility and fibrotic ECM remodeling, key determinants of aqueous humor outflow resistance. Targeting the MIF-CD74/Blimp-1 axis may represent a novel therapeutic strategy to restore TM homeostasis and reduce intraocular pressure in glaucoma.

Structured AbstractO_ST_ABSPurposeC_ST_ABSRPE oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. MethodsWe altered the availability of O2 to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production, 13C6-glucose flux, hypoxia inducible factor (HIF-1) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation. ResultsMedium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. ConclusionsDespite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood". As RPE-centric diseases like age-related macular degeneration (AMD) involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.

Conjunctival in vitro models present a valuable system to investigate conjunctival tissue homeostasis and pathologies. Combinations of collagen and fibroblasts as a stroma equivalent and the supplementation with serum have been reported to promote the differentiation of epithelial cells. However, how the individual factors affect differentiation of ocular surface cells is insufficiently understood. In this study, we analyzed the effect of serum concentration, a collagen matrix, and fibroblasts on conjunctival differentiation in a 3D in vitro model. For this purpose, we developed a computational analysis pipeline for the quantification of optical coherence tomography (OCT) data sets, allowing a time resolved, non-invasive assessment of conjunctiva epithelium differentiation, including goblet cell density. High-resolution dynamic full-field OCT (D-FFOCT) was employed to verify the identity of goblet cells. Conjunctival markers were further analyzed via histology, real-time quantitative PCR, and ELISA to confirm the data of the OCT analysis pipeline. We found that serum is required to induce epithelial differentiation while higher concentrations of 5 - 10% impaired epithelial development. The culture on a collagen matrix increased conjunctival markers upon stimulation with serum, while the co-culture with fibroblasts increased epithelial stratification. Increased serum concentration resulted in the increased occurrence of goblet cells of up to 20 cells/mm{superscript 2}. Altogether, the complementary analyses confirmed the quantified OCT data. Summarized, we identified the combination of serum (3%), collagen, and fibroblasts as a condition resulting in the highest physiological resemblance. Altogether, our study emphasizes the need for fine-tuning of culture conditions for 3D in vitro models. SIGNIFICANCE STATEMENTPhysiologically relevant in vitro conjunctiva models are essential for studying ocular surface homeostasis and disease. Our study refines 3D conjunctival culture conditions to more closely resemble native tissue by systematically identifying serum concentration, collagen scaffolds and fibroblasts as key drivers of epithelial differentiation. By applying non-invasive optical coherence tomography analysis, we enable longitudinal assessment of tissue maturation, addressing the need for non-destructive, repeatable tissue analysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/698626v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@13efafaorg.highwire.dtl.DTLVardef@1cf4529org.highwire.dtl.DTLVardef@c5c63forg.highwire.dtl.DTLVardef@5a85c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

In response to central nervous system (CNS) injury, tissue resident immune cells such as microglia and circulating systemic neutrophils are often first responders. The degree to which these cells interact in response to CNS damage is poorly understood, and even less so, in the neural retina which poses a challenge for high resolution imaging in vivo. In this study, we deploy fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) to study microglia and neutrophils in mice. We simultaneously track immune cell dynamics using label-free phase-contrast AOSLO at micron-level resolution. Retinal lesions were induced with 488 nm light focused onto photoreceptor (PR) outer segments. These lesions focally ablated PRs, with minimal collateral damage to cells above and below the plane of focus. We used in vivo (AOSLO, SLO and OCT) imaging to reveal the natural history of the microglial and neutrophil response from minutes-to-months after injury. While microglia showed dynamic and progressive immune response with cells migrating into the injury locus within 1-day after injury, neutrophils were not recruited despite close proximity to vessels carrying neutrophils only microns away. Post-mortem confocal microscopy confirmed in vivo findings. This work illustrates that microglial activation does not recruit neutrophils in response to acute, focal loss of PRs, a condition encountered in many retinal diseases.

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEM, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

PurposeImpairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically-used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction. MethodsPrimary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. ResultsSimvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and extracellular fibronectin deposition. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. ConclusionsYAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

PurposeCross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM), yet their role in ocular hypertension remains unclear. We used a human TM cell line that spontaneously forms fluorescently-labeled CLANs (GTM3L) to explore the origin of CLANs, developed techniques to increase CLAN incidence in GMT3L cells, and computationally studied the biomechanical properties of CLAN-containing cells. MethodsGTM3L cells were fluorescently sorted for viral copy number analysis. CLAN incidence was increased by (i) differential sorting of cells by adhesion, (ii) cell deswelling, and (iii) cell selection based on cell stiffness. GTM3L cells were also cultured on glass or soft hydrogel to determine substrate stiffness effects on CLAN incidence. Computational models were constructed to mimic and study the biomechanical properties of CLANs. ResultsAll GTM3L cells had an average of 1 viral copy per cell. LifeAct-GFP expression level did not affect CLAN incidence rate, but CLAN rate was increased from [~]0.28% to [~]50% by a combination of adhesion selection, cell deswelling, and cell stiffness-based sorting. Further, GTM3L cells formed more CLANs on a stiff vs. a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. ConclusionsIt is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the effect of CLANs on TM biomechanics and mechanobiology as well as the etiology of CLAN formation in the TM.

C-C chemokine receptor type 3 (CCR3) has been linked with age-related macular degeneration (AMD) pathologies. Specifically, its function as an immune modulator in AMD remains unclear. To address this question, we investigated the impact of CCR3 inhibition on inflammation, a key driver of AMD pathologies, by assessing inflammatory cytokines and infiltrating immune cells in two models of ocular inflammation. Mice were orally dosed twice a day with AKST4290, a CCR3 small molecular inhibitor, in the sodium iodate (NaIO3) and myelin oligodendrocyte glycoprotein (MOG) models. A combination of autoradiography and analytical chemistry techniques were used to assess drug concentration and distribution. Bead-based multiplexing technology was used to determine cytokine concentrations, and flow cytometry and immunohistochemistry was used to ascertain ocular immune-cell composition. CCR3 expression was detected in the retinal pigment epithelium (RPE)/choroid complex where AKST4290 was found to preferentially accumulate at sustained levels. In the NaIO3 model, inhibition of CCR3 with AKST4290 significantly decreased both the concentration of specific chemokines and the number of multiple populations of infiltrating peripheral immune cell. Furthermore, effects of CCR3 inhibition on immune cell infiltration were confirmed in the MOG model. These data demonstrate that CCR3 inhibition strongly modulates local inflammation by impacting both cytokine concentrations and immune cell composition in ocular diseases. Moreover, these findings together with the known role of CCR3 in promoting pathologic angiogenesis implicate a pleiotropic role for CCR3 in AMD.

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

The lacrimal gland produces the tear films aqueous component, which moistens and nourishes the ocular surface to maintain eye health. Reduced production of this component leads to dry eye disease, which affects over 250 million people worldwide. Despite the impact on patients, the availability of primary human material to study underlying disease mechanisms is severely limited and there is no cell model available for human lacrimal gland epithelial cells. After insertion of an SV40 antigen into primary human lacrimal gland epithelial cells, we selected, expanded, and characterized three epithelial cell clones from a female lacrimal gland donor. We show their epithelial character at genomic (PCR and RNAseq) and protein (immunofluorescence) levels and grow these cells in a 3D cell spheroid model. Here, we report the development of an immortalized human lacrimal gland epithelial cell line that improves accessibility to study the molecular pathogenesis mechanisms of dry eye disease and link them to causal treatments. We show the expression of typical lacrimal gland epithelial cell marker genes (e.g. PAX6, FOXC1, AQP5, CSTB, and CST6) and describe the feasibility of the cells to form 2D cell sheets and 3D cell spheroids. We successfully established immortalized human lacrimal gland cells with epithelial character. In the future, the integration of these cells into larger studies holds great potential for advancing our understanding of dry eye disease and its underlying cellular mechanisms.

PurposeInflammation and oxidative stress are contributing factors to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell penetrating peptide from the kinase inhibitory region of intracellular checkpoint inhibitor Suppressor of Cytokine Signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD. MethodsWe used Anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS) and TNF to stimulate inflammation, and paraquat to induce mitochondrial oxidative stress. We used a human RPE cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation by microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring nuclear translocation of NF-{kappa}B p65 and MAP kinase p38, and we used qRT-PCR and Western blots to evaluate induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins by microscopy and measurement of transepithelial electrical resistance. We used intraperitoneal injection of sodium iodate to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury as assessed by fundoscopy, Optical Coherence Tomography (OCT) and histology. ResultsR9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE and the loss of transepithelial electrical resistance were prevented in the presence of R9-SOCS3-KIR. R9-SOCS3-KIR prevented the increased expression of genes related to inflammation in response to C5a treatment. R9-SOCS3-KIR also blocked lipopolysaccharide (LPS) induction of cyclooxygenase and inflammatory markers including IL-6, MCP1, COX-1 and IL-1{beta}. R9-SOCS3-KIR prevented paraquat mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop instillation of R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate. ConclusionR9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used model of RPE/retina oxidative injury. Since this peptide can be administered by corneal instillation, this treatment may offer a convenient way to slow the progression of ocular diseases arising from inflammation and chronic oxidative stress.

PURPOSEARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODSARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTSARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONSARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. TRANSLATIONAL RELEVANCEThis study contributes to mounting evidence for changes in ARPE19 cell physiology with increasing passage number. This information is of value for the continued use of ARPE19 cells as a model system for RPE biology and the development of therapeutics.

Current retinal pigment epithelium (RPE) cell replacement strategies in trials for age-related macular degeneration (AMD) are based on either pluripotent stem cell (PSC) or adult RPE stem cell (RPESC) sources. We used Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) to simultaneously assess single-cell transcriptomic and surface protein information, comparing these two RPE sources. Both RPESC-RPE and PSC-RPE expressed key RPE markers and exhibited cellular heterogeneity. However, RPESC-RPE had higher expression of genes related to mature retinal functions, whereas PSC-RPE had greater expression of genes involved in stem cell development and differentiation. We identified two surface proteins that distinguished the cell types. The "dont eat me" signal, CD24, was detected robustly on adult RPESC-RPE cells, while CD57 was detected on most PSC-RPE cells. The differences in gene and surface protein expression suggest that the two RPE sources differ in functional, adhesion, and immunomodulatory properties, which may impact transplantation outcomes.

Induced pluripotent stem cell-derived retinal organoids (ROs) have become promising personalized models to study inherited retinal diseases (IRDs) and develop new innovative therapies. Although ROs mimic key retinal features and show some light responsiveness, their differentiation and maturation remain limited and lengthy. We hypothesize that standard dark culture conditions limit the expression of genes and proteins related to retinal function, delaying differentiation and complicating disease modeling. Therefore, we investigated whether daily light exposure could promote photoreceptor maturation. ROs were exposed to six hours of daily light starting from day 70 in vitro. Light conditioning led to enhanced photoreceptor maturation, specifically an increase in rod photoreceptors, a late-born cell type, without signs of increased stress or cell death. Our findings suggest that daily light exposure enhances RO differentiation, opening new avenues to investigate molecular and cellular phenotypes in IRDs and accelerate therapy development in more relevant functional models.

PurposeGlaucoma, a multifactorial ocular neuropathy, can lead to irreversible vision loss. Diagnosis involves assessing optic cupping (increased cup-to-disc ratios) and structural changes (like retinal nerve fiber layer thinning) through clinical imaging. Elevated intraocular pressure (IOP) is commonly associated with glaucoma, but not always. However, understanding disease progression is hindered by limited access to donor ocular tissue and consistent clinical data. We hypothesize that the proteome of aqueous humor and plasma may be altered in disease and correlates with clinical parameters such as IOP and cup-to-disc ratios. MethodsAqueous humor (AH) and plasma samples were collected from 36 glaucoma patients (17 male, 19 female), and 35 non-glaucomatous control patients (16 male, 19 female) undergoing cataract surgery. The protein profile was compared using the SOMAscan(R) assay system for proteome profiling. From glaucomatous donors, correlations between IOP and cup- to-disc ratios to proteome differences were determined. ResultsOverall proteomics profiles between both AH and plasma were compared by combining all samples (glaucoma and non-glaucoma) and then performing correlation analyses. This study revealed similar protein abundance in the two biological fluids. Additionally, it identified different abundance of proteins in plasma and AH between glaucoma and non- glaucoma samples. The differential proteins identified were involved in pathways related to vascular integrity, inflammation, immune response, cell adhesion, and complement activation. Generally, glaucomatous AH showed higher protein levels. Neurofilament light chain (NEFL) protein correlated with elevated IOP and inflammatory markers, but not with cup-to-disc ratios. ConclusionsTogether, our data demonstrate that the proteins identified in this study from glaucomatous donors correspond to markers of neurodegeneration and those that may inhibit cell proliferation or disrupt vascular integrity.

PurposeMyopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRAs weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOxTM14, CAS:198696-03-6, referred as floRA), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues. MethodsO_ST_ABSBindingC_ST_ABSWe assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I) and retinol binding protein 4 (RBP4). Direct visualizationWild-type C57BL/6J mice were euthanized, eyes enucleated, and wedges containing sclera and choroid incubated for specific durations in 50 M floRA+BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ. ResultsAssociation constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2-13 x 105 M-1, indicating non-specific binding. floRA could be visualized in sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei). ConclusionsfloRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I and RBP4. Considering these proteins relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement.

BACKGROUND/OBJECTIVESThe cornea, the transparent outer layer of the eye, is avascular and composed of three layers: a self-renewing epithelium, a transparent stroma rich in extracellular matrix (ECM) and an endothelium. This study aimed to profile ECM proteins including proteoglycans, from human decellularized extracellular matrix (hdECM) proteins using proteomics analysis. SUBJECTS/METHODSThree independent batches of lyophilised hdECM samples were subjected to proteomics analysis. Samples were processed by in-gel trypsin digestion and analysed on a Q-Exactive mass spectrometer. Protein identification and label free quantification were performed using Proteome Discoverer 2.2.0.388 RESULTSProteomic profiling identified an average of 18 proteins per batch, with 13 consistently present across all samples. Key proteins, TGF-{beta}, keratocan, fibrillin and collagen XII, were abundant, highlighting their role in inflammation regulation, collagen organisation, and matrix remodelling. Proteoglycans such as docorins and lumican were also detected indicating their contribution to ECM structure and maintenance of corneal transparency. Network and Pathway analysis revealed involvement in ECM organization, integrin interactions, glycosaminoglycan metabolism, and collagen fibril assembly. These proteins are critical for preserving corneal architecture, modulating cell behaviour, and supporting processed such as signalling, migration, angiogenesis, and tissue repair particularly through proper collagen spacing essential for corneal transparency. This study, to the best of our knowledge, represents the first comprehensive profiling of extracellular matrix proteins from decellularised human corneas.

Induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells, closely resembling healthy RPE, offer valuable insights for retinal disease modelling. This study evaluates immature and mature phenotypes of iPSC-RPE and ARPE-19 cells, comparing RPE characteristics, cell-associated complement profiles, and TGF-{beta}1-mediated stress responses across transcript expression, secretion, and protein levels. Statistical analyses were performed using independent means t-test and Wilcoxon rank-sum test. Mature iPSC-RPE cells exhibited characteristic RPE-morphology, with elevated secretion of complement components (C3, FH/FHL-1, FI), unique FB secretion, and apical complement localisation. Intracellular C3 processing revealed cleavage products different from known blood-derived C3 fragments and showed maturation-dependent differences; only mature iPSC-RPE cells secreted active C3 forms (C3(H2O), C3a) and exhibited the intact C3 {beta}-chain. Mature ARPE-19 and iPSC-RPE cells demonstrated resistance to TGF-{beta}1 treatment, which reduced complement secretion without affecting C3a release. In conclusion, ARPE-19 and iPSC-RPE cells demonstrated local production of complement components and maturation-dependent intracellular processing of C3 into active forms. These findings highlight the impact of RPE maturation on local C3 activation, providing a basis for future studies on C3 functionality in the RPE and its potential pathological effects. Impact statementCell state-dependent local processing of complement C3 in retinal pigment epithelium cells generates active C3 forms that may evade anti-C3 therapies, influencing age-related macular degeneration and treatment outcomes. Funding statementThis project has received funding from the PRO RETINA, Amberg, Germany, under grant agreement number "Pro-Re/Projekt/Schikora - Pauly.05-2022", and Deutsche Forschungsgemeinschaft (DFG), under grant agreement number "498244102 (MA 6139/5-1)". Open access funding provided by the Open Access Publishing Fund of University Marburg. Funding sources were not involved in study design, data collection and interpretation, or the decision to submit the work for publication. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/636432v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@6c63b3org.highwire.dtl.DTLVardef@58010org.highwire.dtl.DTLVardef@11ab625org.highwire.dtl.DTLVardef@ac4924_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeIn age-related macular degeneration (AMD) and Sorsbys fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating {beta}-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate {beta}-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. MethodsWe probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13C6-glucose and 13C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). ResultsACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsbys fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. ConclusionsFirsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.