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Progressive aniridia associated keratopathy is worsening visual acuity of congenital aniridia subjects lifelong. Restoration of PAX6 expression in PAX6 haploinsufficient limbal epithelial cells could be one therapeutic option. In a previous study using aniridia-like CRISPR/Cas9 genome-edited corneal epithelial cells, the antipsychotic drugs duloxetine and ritanserin increased PAX6 mRNA and protein expression. Our purpose was to investigate the effect of duloxetine and ritanserin on cultured primary limbal epithelial cells (pLECs) without and with PAX6 knockdown. pLECs were isolated from 11 aniridia patients and corneoscleral rims of 8 healthy human donors and were treated with 5 µM duloxetine or ritanserin for 24 hours. In addition, pLECs were transfected with small interfering RNA (siRNA) (PAX6 knockdown) in the siRNA-based aniridia cell model and were also treated by 5 µM duloxetine or ritanserin for 24 hours. Gene and protein expression were analyzed using qPCR and Western blot. In both primary aniridia limbal epithelial cells and the siRNA-based aniridia cell model, the expression of PAX6 at the transcriptional or translational level did not show significant changes through duloxetine or ritanserin treatment (p > 0.5). The target genes of PAX6 such as KRT3, KRT12, DSG1 , ALDH1A1, ADH7 , FABP5 , ABCG2 also did not change significantly (p ≥ 0.2). Our study shows that primary cultures of limbal epithelial cells from both aniridia patients and healthy donors were unresponsive to drug treatment. Therefore, our data suggest that different aniridia cell models or cell culture conditions exhibit varying responses to duloxetine and ritanserin. The use of in vivo models could further enhance our understanding of duloxetine and ritanserin treatment in aniridia-associated keratopathy.

Haploinsufficiency of the PAX6 gene is the primary pathogenic mechanism underlying classical congenital aniridia. Notably, at least 50% of patients with this condition develop glaucoma. Prostaglandin analogues, such as travoprost, are widely used to lower intraocular pressure in this patient population. At the same time, limbal epithelial cells (LECs) are believed to play a key role in the development and progression of aniridia-associated keratopathy (AAK). Therefore, the aim of this study was to investigate the effects of travoprost on cell viability, proliferation, migration, and the expression of key genes and proteins in both normal and PAX6-knockdown LECs. Primary human LECs were isolated from seven corneal donors. To simulate the haploinsufficient state characteristic of congenital aniridia, a PAX6-knockdown model, using siRNA was employed. LECs were treated with 0.039-40 μg/mL travoprost concentration for 20 minutes. Cell viability was assessed using the XTT assay, while cell proliferation was evaluated by the BrdU assay. Based on XTT results, 0.156 and 0.313 μg/mL travoprost were selected for further measurements in both LECs and PAX6-knockdown LECs. A scratch assay was conducted to measure cell migration. The expression levels of PAX6, FOSL2, MAPKs, inflammatory markers, caspase-3, and MMP9 were analyzed at both the gene and protein levels using qRT-PCR, Western blot, and ELISA. Travoprost significantly reduced LEC viability at 0.156 μg/mL (p = 0.028), while only higher concentrations (20 and 40 μg/mL) inhibited significantly LEC proliferation (p ≤ 0.004). PAX6-knockdown LECs exhibited reduced migration compared to control cells (p ≤ 0.046); however, treatment with 0.313 μg/mL travoprost significantly enhanced their migration (p = 0.047), accompanied by upregulation of JNK1/2 protein (p = 0.039) and MMP9 mRNA and protein levels (p = 0.021, p = 0.027). PAX6 knockdown led to suppression of inflammation-related genes (p ≤ 0.031) and travoprost did not exacerbate inflammatory responses (p ≥ 0.155). Additionally, 0.313 μg/mL travoprost significantly increased caspase-3 protein levels in PAX6-knockdown LECs (p = 0.044). Travoprost, at specific concentrations, can reduce the viability and proliferation of limbal epithelial cells. At 0.313 μg/mL, it significantly upregulates JNK1/2 and MMP9 expression, thereby enhancing the migratory capacity of PAX6-knockdown LECs. These findings may offer valuable insights for the selection of antiglaucomatous medications in patients with congenital aniridia.

Congenital aniridia is a rare eye disease characterized by loss of PAX6 protein leading to aniridia-associated keratopathy that significantly reduces vision. The miR-204-5p is a possible regulator of PAX6 function and here we evaluate its effect in multiple in vitro and in vivo models. In vitro, miR-204-5p overexpression suppressed vascular factor ANGPT1 in human limbal stem cells (T-LSC) and Pax6-knockdown LSC (mut-LSC), and in primary human limbal epithelial cells (LEC) at the gene and protein levels and following LPS stimulation. However, miR-204-5p inhibited VEGFA expression only in mut-LSCs and LPS-stimulated LEC. Also, miR-204-5p increased PAX6 expression in mut-LSC and differentiated corneal epithelial cells, but not in LEC. Topical miR-204-5p after LPS-induced keratitis in mice failed to suppress Vegfa , Angpt1 , Il-1β , and Tnf-α or rescue Pax6 levels in contrast to in vitro results, although it significantly reduced the inflammatory infiltrate in the cornea. In Pax6 Sey/+ aniridia mice, miR-204-5p did not rescue PAX6 levels or suppress Vegfa , Angpt1 , or inhibit the ERK1/2 pathway. While short-term miR-204-5p treatment effectively suppresses VEGFA and ANGPT1 and enhances PAX6 expression in multiple corneal epithelia, effects are variable across primary and immortalized cells. Effects of longer-term in vivo treatment, however, require further study.

Aniridia is a rare congenital disorder in which there is a variable degree of hypoplasia or the absence of iris tissue associated with multiple other ocular changes, some present from birth and some arising progressively over time. Most cases are associated with dominantly inherited mutations or deletions of the PAX6 gene. This article will review the clinical manifestations, the molecular basis including genotype-phenotype correlations, diagnostic approaches and management of aniridia.

In congenital aniridia, not only limbal epithelial cells but also limbal stromal cells may contribute to the development of aniridia associated keratopathy (AAK). Secondary glaucoma affects 50-75% of patients with congenital aniridia, and prostaglandin analogs are commonly used for conservative treatment. This study aimed to explore the effect of travoprost on corneal limbal stromal cells from healthy (LSCs) and congenital aniridia subjects (AN-LSCs), in vitro. Cells were extracted from aniridia (AN-LSCs) (n=7) and healthy donors (LSCs) (n=7). In culture, the cells were treated with travoprost at concentrations ranging from 0.039-40 μg/mL for 20 minutes. Cell viability, proliferation and migration were determined to assess the effect of travoprost on AN-LSCs and LSCs. Analysis of inflammation-, retinoic acid signaling-, and apoptosis-related genes and proteins was performed using qPCR, Western blot, and ELISA. One-way ANOVA was used to analyze cell viability and proliferation. The Mann-Whitney test was applied to compare between-group differences, while the Friedman test was used to assess within-group differences. Both in LSCs and AN-LSCs, travoprost treatment at 0.078 μg/mL and higher concentrations significantly reduced cell viability (p≤0.033; p<0.001) and proliferation decreased both in LSCs and AN-LSCs at 40 μg/mL travoprost concentration (p=0.006; p=0.002). At 6 and 12 hours, 0.313 μg/mL travoprost significantly increased the migration rate of AN-LSCs (p=0.021; p=0.021). AN-LSCs displayed lower PAX6 and JNK (MAPK8) mRNA (p<0.001) but higher MMP-3, MMP-9, ADH7, FABP5 and VEGFA mRNA levels (p≤0.037) than LSCs. PTGFR and JNK mRNA levels, MMP9 and ADH7 protein levels increased significantly in AN-LSCs after 0.313 μg/mL travoprost treatment (p≤0.039), while NF-κB and ADH7 protein levels decreased significantly in LSCs using 0.313 μg/mL travoprost (p=0.039; p<0.001). Travoprost may affect viability, proliferation, and migration of both LSCs and AN-LSCs, with AN-LSCs exhibiting greater sensitivity than LSCs. Additionally, travoprost may regulate MMP-9 expression in AN-LSCs via the JNK signaling pathway. Furthermore, in AN-LSCs, travoprost treatment does not lead to a decrease in NF-κB and ADH7 protein levels.

Aniridia-associated keratopathy (AAK) results from the paired box 6 (PAX6) gene haploinsufficiency, leading to depletion of limbal epithelial stem cells, persistent inflammation and a gradual loss of vision. Although fibroblasts are acknowledged as sentinel cells initiating corneal inflammation, their contribution in the chronic inflammatory state of AAK remains unexplored. Our study aims to compare PAX6 and inflammatory cytokine expression in limbal fibroblast cells (LFCs) of corneal donors and aniridia patients (AN-LFCs). Furthermore, we subjected LFCs and AN-LFCs to LPS and cobalt chloride (CoCl2), to investigate specific inflammatory reactions. Following isolation and culture, primary LFCs (n = 7) and AN-LFCs (n = 7) were subjected to a 24-hour treatment with E. coli LPS or a 48-hour exposure to CoCl2 as a chemical oxidative stress (OS) inducer. Subsequently, PAX6 as well as IL-1β, IL-6, TNF-α, and VEGF gene expression was examined by qPCR. Corresponding protein levels were assessed by ELISA from the cell culture supernatants. AN-LFCs exhibited similar PAX6 mRNA levels as normal LFCs (p ≥ 0.38). Notably, LPS treatment (p ≤ 0.02), but not CoCl2, significantly enhanced PAX6 expression. Untreated AN-LFCs showed higher IL-6 mRNA (p = 0.002) and protein levels (p = 0.009) but lower TNF-α protein expression (p = 0.016) than LFCs. When exposed to LPS, AN-LFCs exhibited higher IL-1β mRNA (p = 0.0008), as well as IL-6 protein expression (p = 0.029) than normal LFCs. Under OS, an elevated IL-6 protein (p = 0.010) was observed in AN-LFCs compared to LFCs. Our study revealed inflammatory gene and protein expression alterations in AN-LFCs, that could be involved in the stromal niche perturbations seen in AAK.

Background Aniridia is a rare panocular disease caused by gene mutation in the PAX6, which is essential for eye development. Aniridia is inherited in an autosomal dominant manner, but its phenotype can vary significantly among individuals with the same mutation. Animal models, such as drosophila, zebrafish, and rodents, have been used to study aniridia through Pax6 deletions. Recently, patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro, providing new insights into therapeutic strategies. Methods In this study, corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations, allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived samples. Results Untargeted metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts, with amino acid metabolism pathways being consistently enriched in AAK organoids. Conclusions This study emphasizes the profound impact of AAK mutations on metabolism, particularly in amino acid biosynthesis and energy metabolism pathways.

In the present study, we evaluate gene and protein expression levels in an in vitro siRNA-mediated PAX6 knockdown limbal epithelial cell (LEC) model after RA treatment. This study aims to investigate the direct effects of active RA products and their association with key regulators of the RA signaling pathway in siRNA PAX6 knockdown LECs, providing further insights into the potential role of RA signaling in AAK pathogenesis. Primary human limbal epithelial cells (LECs) were subjected to siRNA-mediated PAX6 knockdown to mimic PAX6 deletion in congenital aniridia (n = 8). Following knockdown, 0 µM, 1 µM, and 5 µM all-trans retinoic acid (RA) treatments were applied to both the siRNA PAX6 control and knockdown groups. After 48 hours of incubation, the mRNA expression levels of paired box 6 (PAX6), alcohol dehydrogenase 7 (ADH7), retinol dehydrogenase 10 (RDH10), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), cytochrome P450 family 26 subfamily A member 1 (CYP26A1), retinol-binding protein 1 (RBP1), cellular retinoic acid-binding protein 2 (CRABP2), fatty acid-binding protein 5 (FABP5), retinoid X receptor alpha (RXRA), retinoid X receptor beta (RXRB), retinoic acid receptor alpha (RARA), retinoic acid receptor beta (RARB), peroxisome proliferator-activated receptor gamma (PPARG), and vascular endothelial growth factor A (VEGFA) were analyzed using qPCR. Protein expression levels were assessed using ELISA or Western blot, while cell proliferation rates were measured using the BrdU assay. PAX6, ADH7, ALDH1A1, FABP5 mRNA levels and PAX6, ADH7, ALDH1A1, FABP5, PPARG2, RARB protein levels were significantly lower in the PAX6 knockdown group, than in controls (p ≤ 0.018). PPARG mRNA level was significantly higher in the PAX6 knockdown group than in controls (p = 0.012). ALDH1A1 mRNA expression was significantly downregulated using 5 µM RA treatment in the control group (p = 0.038). CYP26A1 mRNA expression was upregulated using 1 µM and 5 µM RA treatment in both the PAX6 control (p < 0.001; p < 0.001) and the PAX6 knockdown group (p = 0.001; p = 0.002). CRABP2 mRNA expression in the PAX6 knockdown group (p = 0.02) and CRABP2 protein expression in both groups were downregulated using 5 µM RA concentration (p = 0.003; p = 0.02). Protein expression of RXRA was downregulated to 5 µM RA treatment in the controls (p = 0.007). mRNA expression of RARA in the PAX6 knockdown groups (p = 0.023) and mRNA expression of RARB in both groups (p = 0.007, p < 0.001) were downregulated to 5 µM RA treatment. RARB protein expression was downregulated to 1 µM and 5 µM RA treatment (p = 0.02, p = 0.004) in the controls. VEGFA mRNA expression in PAX6 controls was upregulated using 5 µM RA (p = 0.041). Cell proliferation rate was downregulated in PAX6 knockdown groups compared to the controls and downregulated using 5 µM RA concentration only in the controls (p < 0.001, p = 0.025). Our results reveal a reduced proliferation rate in PAX6 knockdown LECs, along with a less pronounced downregulation of proliferation in response to increased RA concentration. Additionally, the study highlights altered expression of key regulators in the RA signaling pathway, influenced by both PAX6 activity and RA treatment. These findings suggest a potential disruption in RA-mediated cellular regulation in PAX6-deficient LECs.

Congenital aniridia is marked by substantial inflammatory changes to the ocular surface. However the exact mechanisms of epithelial-stromal interaction are not fully understood. The purpose of this study was to investigate inflammatory cytokine expression in limbal epithelial cells and fibroblasts following exposure to each other’s conditioned medium (CM). Healthy primary limbal epithelial cells (pLECs) and healthy (LFC) or aniridia primary limbal fibroblasts (AN-LFC) were isolated. A PAX6-deficient limbal epithelial cell line (mut-LSCs) modeled aniridia. pLECs underwent siRNA-mediated PAX6 knockdown (siPAX6 pLECs) with control cells transfected with non-specific siRNA (siCtrl pLECs). siCtrl and siPAX6 pLECs were treated with LFC-CM and AN-LFC-CM for 24 hours, while LFC and AN-LFC were treated with pLECs-CM and mut-LSCs-CM for 48 hours. Gene and protein expression of IL-1β, IL-6, IL-8, TNF-α and VEGF-A were measured using qPCR and ELISA. Except for an increased IL-8 protein expression in siPAX6 pLECs treated with LFC-CM, gene and protein levels of inflammatory biomarkers remained unchanged in siCtrl and siPAX6 pLECs, regardless of treatment with LFC-CM or AN-LFC-CM. In LFCs, pLECs-CM decreased TNF-α mRNA and IL-8 protein, while increasing IL-1β, IL-6, IL-8 mRNA and IL-6 protein. LFCs treated with mut-LSCs-CM showed decreased TNF-α mRNA and increased IL-6 protein. AN-LFCs treated with pLECs-CM showed increased IL-6, IL-8 and VEGF-A mRNA and IL-6 protein. mut-LSCs-CM did not alter AN-LFC expression. Limbal fibroblasts’ secretome minimally inflames limbal epithelial cells, suggesting a supportive niche role. In contrast, pLECs-CM induces a stronger fibroblast response, indicating abnormal interactions in congenital aniridia.

Aniridia is a rare congenital condition of abnormal eye development arising principally from heterozygous mutation of the PAX6 gene. Among the multiple complications arising in the eye, aniridia-associated keratopathy (AAK) is a severe vision-impairing condition of the cornea associated with a progressive limbal stem cell deficiency that lacks suitable treatment options. Current mouse models of aniridia do not accurately represent the onset and progression dynamics of human AAK, hindering therapy development. Here, we performed deep phenotyping of a haploinsufficient Pax6 +/– small-eye (Sey) mouse model on the 129S1/SvImJ background, which exhibits key features of mild presentation at birth and progressive AAK with aging, mimicking human disease. The model exhibits a slowly progressing AAK phenotype and provides insights into the disease, including disturbed basal epithelial cell organization, function, and marker expression; persistent postnatal lymphangiogenesis; disrupted corneal innervation patterns; and persisting yet altered limbal stem cell marker expression with age. The model recapitulates many of the known features of human disease, enabling investigation of underlying disease mechanisms and, importantly, access to a well-defined temporal window for evaluating future therapeutics.