Pro-apoptotic shift in human aniridia-derived limbal stromal cells under prolonged high-glucose stress, In vitro

Abstract

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.