Permanent Senescence Via p16 Leads to Guttae Formation in an In Vitro Human Corneal Endothelial Cell Model

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by corneal endothelial cell (CEnC) degeneration and excessive extracellular matrix (ECM) deposition. FECD is an age-related disorder manifested by upregulation of senescence markers. However, the association between senescence and pathological ECM accumulation remains unclear. This study investigated whether senescence mediated cell-cycle arrest drives aberrant ECM deposition and guttae formation in FECD. Methods: A chronic stress (CS) model was established by concurrently exposing immortalized human CEnCs to ultraviolet-A light (UVA; 25 J/cm2) and 4-hydroxyestradiol (4OHE2; 20 µM) on days 1, 6, and 21, followed by approximately a 60-day recovery period. In vitro guttae were characterized using brightfield microscopy, scanning electron microscopy (SEM), immunofluorescence, and compared with FECD specimens. Cell cycle dynamics, senescence markers, and ECM expression were assessed using flow-cytometry, cell sorting, immunostaining, ELISA, and RT-PCR. Results: In vitro guttae displayed characteristic morphology and ultrastructural similarity to FECD guttae. CS-exposed CEnCs showed significant CDKN2A upregulation (42-fold, P < 0.01) and increased p16 positivity (63 ± 12% vs. 7 ± 3% in controls, P < 0.0001), indicating senescence. Flow cytometry revealed accumulation of CS cells in G0/G1 (74 ± 6% vs. 57 ± 3%, P < 0.0001) phase. G0/G1 sorted p16+ cells exhibited upregulation of COL4A1 (188-fold, P < 0.0001) and LAMB1 (98-fold, P < 0.05). Immunostaining confirmed p16 colocalization with COL4A1 and LAMB1 in CS cells and FECD tissues. ELISA showed elevated levels of TGFBIp in the conditioned media obtained from CS-exposed cells. Conclusions: G0/G1-arrested p16+ve senescent CEnCs promote pathological ECM deposition and guttae formation. This model, integrating environmental, hormonal, and aging factors, provides a biologically relevant platform for studying FECD and testing therapeutics.