Table 1.
Detailed information on the included subjects with congenital aniridia is provided. The table includes the aniridia-associated keratopathy (AAK) grade according to Lagali et al. [11 25], as well as gender and age.
Table 2.
Detailed information on the healthy corneal donors used in this study.
Table 3.
Specifications of Real-Time PCR Primers.
Table 4.
Flow cytometry antibodies and detection reagents.
Fig 1.
Apoptotic rates of limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs), after treatment with 17.5 mM and 70 mM glucose for 48 hours.
LSCs and AN-LSCs were cultured in medium containing either 17.5 mM or 70 mM glucose for 48 hours, then stained with Annexin V-APC/PI and analyzed by flow cytometry. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; significant p values are indicated. Flow cytometry revealed that apoptosis was significantly reduced in both LSCs (p = 0.0170) and AN-LSCs (p = 0.0181) after 48 hours of treatment with 70 mM glucose compared to 17.5 mM.
Fig 2.
mRNA levels of CASP family markers (A-E), BCL2 family markers (F-H) and other apoptosis-related markers (I-M) in limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs), after treatment with 17.5 mM and 70 mM glucose for 48 hours.
CASP3, CASP7, CASP8, CASP9 and CASP10 mRNA levels (A-E), BCL2, BAX and BID mRNA levels (F-H) and CDKN1A, CDKN1B, TNFα, XIAP and BIRC5 mRNA levels (I-M) are shown. Values are expressed on a logarithmic (log₂) scale as geometric mean ± geometric standard deviation. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; significant p values are indicated. Following 70 mM glucose treatment, CASP8 and CASP10 mRNA levels were significantly reduced in both LSCs (p = 0.0448) and AN-LSCs (p = 0.0171), while XIAP and BIRC5 were significantly upregulated (p = 0.0375; p = 0.0442). In AN-LSCs, CASP3 and CDKN1A mRNA levels were also significantly decreased under high glucose compared to 17.5 mM (p = 0.0331). Additionally, under 70 mM glucose conditions, BAX mRNA levels were significantly lower in AN-LSCs than in LSCs (p = 0.0255).
Fig 3.
Protein levels of Caspase family markers in limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs), after treatment with 17.5 mM and 70 mM glucose for 48 hours, using flow cytometry (A-E).
Protein levels of Caspase-3, Caspase-7, Caspase-8, Caspase-9, and Caspase-10 are shown. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; significant p values are indicated. Representative histograms display primary antibody staining in green and staining with the corresponding secondary antibody alone (negative control) in red. MFI: mean fluorescence intensity, normalized to the secondary antibody control. In AN-LSCs, the protein levels of Caspase-3 and Caspase-8 were significantly reduced following 70 mM glucose treatment compared to 17.5 mM (p ≤ 0.0257).
Fig 4.
Protein levels of Bcl-2 family markers in limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs), after treatment with 17.5 mM and 70 mM glucose for 48 hours, using flow cytometry (A-C).
Protein levels of Bcl-2, Bax, and Bid are shown. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; significant p values are indicated. Representative histograms display primary antibody staining in green, while staining with the corresponding secondary antibody alone (negative control) is shown in red. MFI: mean fluorescence intensity, normalized to the secondary antibody control. In AN-LSCs, Bcl-2 protein levels were significantly upregulated under 70 mM glucose treatment compared to 17.5 mM (p = 0.0284).
Fig 5.
Protein levels of other apoptosis-related markers in limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs), after treatment with 17.5 mM and 70 mM glucose for 48 hours, using flow cytometry (A-E) and ELISA (F).
Protein levels of p21, p27, XIAP, TNF-α, and Survivin are shown. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; significant p values are indicated. Representative histograms display primary antibody staining in green, with the corresponding secondary antibody alone (negative control) shown in red. MFI: mean fluorescence intensity, normalized to the secondary antibody control. Following 70 mM glucose treatment, XIAP protein levels increased significantly in both LSCs (p = 0.0451) and AN-LSCs (p = 0.0134), while Survivin protein levels showed a significant rise in AN-LSCs (p = 0.0467). Nevertheless, none of the other analyzed protein levels, assessed by flow cytometry or ELISA, changed significantly (p ≥ 0.2239).
Fig 6.
Acute high-glucose exposure induces anti-apoptotic signaling in human limbal stromal cells of healthy subjects and patients with congenital aniridia.
Schematic representation of the effects of high glucose (70 mM, 48 hours) on apoptotic signaling in human limbal stromal cells (LSCs) and aniridia-LSCs (AN-LSCs). Elevated glucose suppresses the expression of pro-apoptotic factors, including CASP8, CASP10, BAX, and CASP3, while simultaneously enhancing the expression of anti-apoptotic molecules such as BCL-2, XIAP, and Survivin. In AN-LSCs, these effects are even more pronounced, with notable reductions in executioner Caspase-3 and the cell cycle inhibitor p21 (CDKN1A), indicating a more robust anti-apoptotic adaptation in these cells. This coordinated molecular shift contributes to the inhibition of apoptosis and promotes cell survival, particularly in congenital aniridia-derived limbal stromal cells. The figure illustrates the shift in balance from apoptotic signaling toward a survival-promoting profile under acute metabolic stress.