Elevated anti-apoptotic shift in primary human aniridia limbal stromal cells following 48 hours supraphysiological
Shanhe Liu1,2, Shuailin Li1,2, Shao-Lun Hsu1,2
1Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Saarland, Germany.
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.
