Our aim was to examine the expression of PAX6 and keratocyte-specific markers in human limbal stromal cells (LSCs) in congenital aniridia (AN) and in healthy corneas, in vitro. Primary human LSCs were extracted from individuals with aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8). The cells were cultured in either normal-glucose serum-containing cell culture medium (NGSC-medium) or low-glucose serum-free cell culture medium (LGSF-medium). Analysis of PAX6 and keratocyte-specific markers was conducted using qPCR and Western blotting. The keratocyte-specific markers included Collagen I (COL1A1), Collagen III (COL3A1), Collagen V (COL5A1), α-smooth muscle actin (ACTA2), Aldehyde Dehydrogenase 3 Family, Member A1 (ALDH3A1), Keratocan (KER), Lumican (LUM), and CD34. PAX6 mRNA expression exhibited a significant decrease in AN-LSCs compared to LSCs in both NGSC- and LGSF-medium (p = 0.04; p = 0.014). There was a marked reduction in COL5A1 mRNA expression (p = 0.011), accompanied by notably higher ALDH3A1 and KER mRNA levels (p = 0.007; p = 0.013) in AN-LSCs compared to LSCs when using NGSC-medium. In LGSF-medium, AN-LSCs showed a significant increase in COL1A1 and COL5A1 mRNA expression compared to LSCs (p = 0.048; p = 0.002). Moreover, COL1A1 and α-SMA protein expression were significantly elevated in AN-LSCs compared to LSCs in LGSF-medium (p = 0.048, p = 0.008). Our investigation affirms the altered expression of PAX6 and keratocyte-specific markers in AN-LSCs relative to healthy controls. Both NGSC- and LGSF-medium exerted distinct effects on both LSCs and AN-LSCs. The observed variations in PAX6 and keratocyte-specific marker expression in AN-LSCs may play a pivotal role in the development and progression of aniridia-associated keratopathy.

Introduction In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. Methods Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. Results In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). Conclusions The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.

Purpose To investigate the effect of Rose Bengal photodynamic therapy (RB-PDT) on viability and proliferation of human limbal epithelial stem cells (T-LSCs), human corneal epithelial cells (HCE-T), human limbal fibroblasts (LFCs), and human normal and keratoconus fibroblasts (HCFs and KC-HCFs) in vitro. Methods T-LSCs and HCE-T cell lines were used in this research. LFCs were isolated from healthy donor corneal limbi (n = 5), HCFs from healthy human donor corneas (n = 5), and KC-HCFs from penetrating keratoplasties of keratoconus patients (n = 5). After cell culture, RB-PDT was performed using 0.001% RB concentration and 565 nm wavelength illumination with 0.14 to 0.7 J/cm2 fluence. The XTT and the BrdU assays were used to assess cell viability and proliferation 24 h after RB-PDT. Results RB or illumination alone did not change cell viability or proliferation in any of the cell types (p ≥ 0.1). However, following RB-PDT, viability decreased significantly from 0.17 J/cm2 fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm2 fluence in T-LSCs (p < 0.001), HCE-T (p < 0.05), and LFCs ((p < 0.0001). Cell proliferation decreased significantly from 0.14 J/cm2 fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm2 fluence in HCFs (p < 0.05). Regarding LFCs proliferation, no values could be determined by the BrdU assay. Conclusions Though RB-PDT seems to be a safe and effective treatment method in vivo, its dose-dependent phototoxicity on corneal epithelial and stromal cells has to be respected. The data and experimental parameters applied in this study may provide a reliable reference for future investigations.

Purpose: 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. Materials and methods: 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. Results: 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). Conclusions: 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.

MicroRNA-204-5p (miR-204-5p) is a critical regulator of differentiation, structural maintenance, and inflammation in limbal epithelial cells (LECs). This study examined the role of miR-204-5p in modulating the gene expression related to transcription factors, cell structure, extracellular matrix remodeling, and retinoic acid signaling under normal and lipopolysaccharide (LPS)-induced inflammatory conditions. Using qPCR, we analyzed the mRNA levels of FOSL2, FOXC1, Meis2, PPARγ, ABCG2, PTGES2, IL-1β, IL-6, KRT3, KRT12, MMP2, MMP9, RARA, RARB, RXRA, RXRB, CRABP2, RBP1, RDH10, ADH7, ADH1A1, FABP5, CYP1B1, and CYP26A1, while changes in protein levels were assessed via Western blot or ELISA. Our data revealed that the overexpression of miR-204-5p reduced the mRNA levels of FOXC1, KRT12, and RDH10 under normal and inflammatory conditions (p ≤ 0.039). Additionally, it decreased FOSL2 and RXRA mRNA under normal conditions (p = 0.006, p = 0.011) and KRT3 and FABP5 mRNA under inflammatory conditions (p = 0.010, p = 0.001). The IL-6 mRNA expression was significantly increased following the LPS treatment in cells overexpressing miR-204-5p (p = 0.029). A protein analysis revealed significant reductions in FOXC1 and KRT3 in the miR-204-5p-transfected cells during LPS-induced inflammation (p = 0.020, p = 0.030). These findings suggest that miR-204-5p modulates genes critical to the differentiation, migration, and inflammatory response of LECs. The modulation of FOXC1 and KRT3 by miR-204-5p highlights these proteins as novel targets under inflammatory conditions.

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.