Purpose: The aim of this study was to investigate apoptosis in primary aniridia limbal stromal cells (LSCs) and to assess changes in the expression of genes and proteins associated with the apoptotic pathway in response to cobalt chloride (CoCl₂)-induced hypoxic stress, in vitro. Methods: Primary human limbal stromal cells were isolated from the limbal region of both aniridia (AN-LSCs; n = 8) and healthy (LSCs; n = 8) donors. The cells were treated with 0 µM, 50 µM, and 75 µM CoCl₂ for 48 hours. Apoptosis in each group was assessed by Flow cytometry (FC). The expression levels of apoptosis-related genes, including CASP 3/7/8/9/10, BCL2, BID, BAX, CDKN1A (p21), CDKN1B (p27), TNFα, XIAP, and BIRC5 (Survivin), were measured by qPCR. Protein level of these markers was analyzed by FC. TNFα protein expression in the supernatant was quantified using ELISA. Results: Flow cytometry analysis revealed a significantly higher apoptosis rate in AN-LSCs compared to LSCs (p < 0.0001). In AN-LSCs, treatment with 75 µM CoCl₂ further increased the apoptosis rate compared to untreated AN-LSCs (p = 0.047). BCL2 mRNA levels (p = 0.0291), Caspase-8 (p = 0.0341), Caspase-10 (p = 0.0085), Bcl-2 (p = 0.0014), XIAP (p = 0.0003) and Survivin (p = 0.0074) protein levels were significantly higher in LSCs than in AN-LSCs. Conversely, Caspase-3 (p = 0.0366), Caspase-9 (p = 0.0354), p21 (p = 0.0003), and p27 (p = 0.0164) protein levels were significantly higher in AN-LSCs than in LSCs. In LSCs, exposure to 75 µM CoCl₂ led to a reduction in BCL2 mRNA (p = 0.0102) and protein levels (p = 0.0484), accompanied by an increase in CDKN1B mRNA level (p = 0.0265). In AN-LSCs, 75 µM CoCl₂ treatment resulted in a decrease in CASP3 (p = 0.049), CASP7 (p = 0.041) and BCL2 (p = 0.0218) mRNA and Bcl-2 protein levels (p = 0.0405) and an increase of TNF-α protein levels in the cell culture supernatant (p = 0.0251). Conclusions: The apoptosis rate of LSCs from patients with congenital aniridia is higher than that of the control group, accompanied by alterations in multiple apoptosis-related markers. Additionally, CoCl₂-induced hypoxic stress further increases apoptosis in AN-LSCs and leads to changes in the expression of Caspase 3, Caspase 7, Bcl-2, and CDKN1B (p27). Further research is needed to elucidate the potential therapeutic targets in AAK, with the aim of preventing or slowing the progression of aniridia-associated keratopathy.

This study aimed to evaluate FOXC1-mediated regulatory mechanisms on gene and protein expression profiles in primary human limbal epithelial cells (pLECs) using siRNA-mediated FOXC1 knockdown under basal conditions and following lipopolysaccharide (LPS) and interleukin-1β (IL-1β)-induced inflammatory conditions. The gene expression related to inflammation, epithelial differentiation, cell proliferation and remodeling, and retinoic acid metabolism was analyzed using qPCR. Corresponding protein levels were assessed through Western blotting and ELISA. FOXC1 silencing significantly downregulated epithelial differentiation markers KRT12 and KRT13 at the mRNA and protein levels (p ≤ 0.045), whereas KRT3 and KRT19 were unaffected. Inflammatory signaling was markedly altered, with a reduced IL-6 and IL-8 mRNA expression (p ≤ 0.029), increased IL-1α expression (p ≤ 0.015), and condition-dependent changes in IL-6 and IL-8 protein secretion. CCL2 was increased at the mRNA level only (p = 0.007). VEGFA mRNA was consistently reduced (p ≤ 0.022) without corresponding protein changes, while TGF-β protein was increased under non-inflammatory and LPS conditions (p ≤ 0.011). Genes involved in retinoid metabolism, including CYP1B1, FABP5, CRABP2, RDH10, STRA6, and ALDH3A1, were significantly downregulated (p ≤ 0.037), with reduced CRABP2 and RDH10 protein levels (p ≤ 0.017) and a decreased FABP5/CRABP2 ratio under IL-1β stimulation (p = 0.006). FOXC1 knockdown affected proliferation-related genes, with decreased FOSL2 (p = 0.048) and increased MKi67 (p = 0.006). FOXC1 silencing disrupts epithelial differentiation, inflammatory signaling, retinoid metabolism, and selected proliferation-related pathways at the transcriptional level, with more selective effects on protein levels. Such changes may potentially predispose the ocular surface to lineage instability, fibrosis, and impaired regenerative capacity.

Metabolic stress can profoundly influence cell survival pathways in limbal stromal cells (LSCs). This study investigated the effects of prolonged supraphysiological glucose exposure on apoptotic responses in both healthy LSCs and LSCs derived from patients with congenital aniridia (AN-LSCs). Primary human LSCs (n = 12) and AN-LSCs (n = 8) were cultured under high-glucose conditions (70 mM) for either 48 or 72 h. Apoptotic cell populations were quantified using Annexin V/PI flow cytometry. Gene expression of key apoptosis-related markers-CASP3/7/8/9/10, BCL2, BAX, BID, CDKN1A, CDKN1B, XIAP, BIRC5, and TNFα-was assessed by qPCR, and corresponding protein levels were determined via flow cytometry and ELISA. Both cell types showed a significant increase in apoptosis after 72 h of high-glucose exposure compared to 48 h (LSCs: p = 0.0021; AN-LSCs: p = 0.0017). At the mRNA level, CASP3 was upregulated in both cell types (p = 0.0210; p = 0.0396), while XIAP was downregulated (p = 0.0312; p = 0.0141). In LSCs, CASP10 and CDKN1A (p21) mRNA levels increased (p = 0.0369; p = 0.0495), whereas BAX expression decreased (p = 0.0097). In AN-LSCs, CASP7 level increased (p = 0.0032), BIRC5 (Survivin) expression decreased (p = 0.0113). Protein analysis confirmed increased levels of Caspase-3, Caspase-7, and p21 in both groups after 72 h (p ≤ 0.0305), accompanied by a significant decrease in XIAP (p = 0.0078; p = 0.0348). Additionally, Survivin protein was markedly reduced in AN-LSCs after prolonged treatment (p = 0.0182). Prolonged high-glucose exposure induces a shift in LSCs from an initial protective state to enhanced apoptotic signaling. This transition, marked by increased pro-apoptotic and reduced anti-apoptotic markers, suggests that sustained metabolic stress can override early survival mechanisms in both LSCs and AN-LSCs. These insights may help guide the development of targeted therapies for aniridia-associated keratopathy.

Purpose: To establish and comprehensively characterize immortalized limbal epithelial cell lines derived from patients with PAX6 haploinsufficiency-associated congenital aniridia, as well as from a healthy donor. These well-defined cell models provide a reliable and reproducible platform for long-term experimental studies, facilitating mechanistic investigations and the development and evaluation of novel therapeutic approaches. Methods: Primary limbal epithelial cells were isolated from biopsies of two patients with distinct PAX6 variants and from a healthy donor. Immortalization was performed by InSCREENex GmbH. The resulting cell lines were characterized using microscopy, BrdU assay, qPCR, flow cytometry, immunocytochemistry, and mRNA sequencing. Results: Immortalized aniridia and control cell lines displayed typical polygonal epithelial morphology and comparable proliferation rates. Total PAX6 mRNA and protein levels were similar among groups; however, nuclear PAX6 immunosignals were significantly reduced in aniridia-derived lines. Expression of ABCG2, TP63, FOSL2, ALDH1A1, and FABP5 showed no significant differences, except for reduced ΔNp63α protein levels in one aniridia line. mRNA sequencing detected more than 14,000 transcripts, including subsets uniquely expressed in control and aniridia-derived lines. Conclusions: Immortalized aniridia limbal epithelial cell lines preserve key epithelial characteristics and overall transcriptomic similarity to controls while exhibiting disease-relevant molecular alterations. These cell lines represent models of PAX6-associated ocular surface disease.

Purpose: Previous work demonstrated that supraphysiological glucose remodels TGF-β1 and NF-κB signaling in human limbal stromal cells (LSCs) and congenital aniridia-derived LSCs (AN-LSCs). The present study investigated whether the same metabolic stress also alters apoptotic pathways in these cells. Methods: Primary LSCs (n = 12) and AN-LSCs (n = 8) were cultured for 48 hours in DMEM containing either normal (17.5 mM) or high (70 mM) glucose. Apoptosis was quantified by Annexin V/propidium iodide (PI) flow cytometry (FC). Expression levels of apoptosis-related genes—including CASP3/7/8/9/10, BCL2, BID, BAX, CDKN1A (p21), CDKN1B (p27), TNFα, XIAP, and BIRC5 (Survivin)—were assessed by qPCR. Protein levels of these markers were analyzed by FC, and TNFα protein concentrations in culture supernatants were measured by ELISA. Results: High glucose significantly reduced the proportion of apoptotic cells in both LSCs (p = 0.0170) and AN-LSCs (p = 0.0181). In both cell types, CASP8 (p = 0.0448; p = 0.0171) and CASP10 (p = 0.0001; p = 0.0007) mRNA levels decreased, while XIAP (p = 0.0375; p = 0.0442) and BIRC5 (p = 0.0196; p = 0.0003) were upregulated. AN-LSCs additionally showed reductions in CASP3 (p = 0.0138) and CDKN1A (p = 0.0331), and exhibited lower BAX levels than LSCs under high glucose (p = 0.0255). Protein analysis corroborated these findings in AN-LSCs: Caspase-3 (p = 0.0154) and Caspase-8 (p = 0.0257) decreased, while Bcl-2 (p = 0.0284) and Survivin (p = 0.0467) levels increased. XIAP protein levels rose in both LSCs (p = 0.0451) and AN-LSCs (p = 0.0134). Conclusions: A 48-hour exposure to 70 mM glucose induces a marked anti-apoptotic shift in human limbal stromal cells, more pronounced in cells derived from congenital aniridia patients. Together with previous evidence on TGF-β1 and NF-κB regulation, these findings suggest that limbal cells may mount an early protective response to metabolic stress, which could be harnessed therapeutically to manage aniridia-associated keratopathy through coordinated survival and stress pathways.