Abstract
Purpose :
Fuchs endothelial corneal dystrophy(FECD) is a female predominant disorder caused by genetic and environmental factors. We have shown that Ultraviolet-A(UV) light causes DNA damage and senescence leading to a greater FECD phenotype in mice and that FECD cells re-enter the cell cycle(CC) and become arrested in G2/M. As the role of CC arrest and SASP in the development of FECD remains elusive, we investigated the development of FECD characteristics in each phase of the CC and the effect of SASP in response to DNA damage induced by UV and reactive estrogen metabolite(4OHE2).
Methods :
Telomerase-immortalized CEnCs, were treated with UV(25J/cm2)+4OHE2(20uM) for 24h and the SASP was collected at the end of the treatment. DNA damage(yH2AX and p53) and senescence(p21) was analyzed and compared with untreated controls by immunofluorescence and western blot. Flow cytometry was used for CC analysis. CEnCs from G0/G1 and G2/M were sorted and analyzed for senescence(CDKN1A), EMT(SNAI1/2,TGFBI), fibrosis(ACTA2,COL1A1) and guttae components(CLU,COL4A1) by RT-PCR. CEnCs were cultured for 24 hours with SASP and checked for senescent, EMT and fibrosis by RT-PCR. Mann-Whitney test was used for statistics.
Results :
Greater DNA damage(yH2AX+) was detected in the treated(14±3%) CEnCs compared to the controls(0.6±0.9%)(p<0.05); whereas p53 was expressed only in the treated cells. UV+4OHE2 caused a shift from G0/G1 to G2/M arrest(53±14%) compared to untreated controls(19±2%)(p<0.001) and led to increased p21 nuclear staining(9±2%) compared to the controls(0.3±0.5%)(p<0.05). Cell sorting detected >2 fold upregulation of CDKN1A,SNAI1,TGFBI,ACTA2,CLU,COL4A1 genes in G2/M vs G0/G1. SASP treated cells showed significant upregulation of SNAI1/2,TGFBI,COL1A1,ACTA2 but not CDKN1A, indicating that acute SASP is insufficient to induce senescence.
Conclusions :
UV+4OHE2 aggravates DNA damage and activates the p53-p21 axis initiating G2/M arrest. Although senescence was activated with acute stress, the generated SASP led mostly to EMT phenotype and early fibrosis, indicating that chronic stress is likely necessary to induce secretory mediators for senescence and profibrotic response seen in guttae formation. Selectively targeting G2/M arrested cells may provide a novel therapeutic strategy to preclude senescence and possibly fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.