Abstract
Purpose :
Cross-linked actin networks (CLAN) are web-like actin structures frequently observed in glaucomatous trabecular meshwork (TM) cells or TM cells treated with TGFβ2 or dexamethasone (DEX). We determined the effect of CLAN formation on cell proliferation and cell senescence in primary human TM (pHTM) cells.
Methods :
Several characterized pHTM cell strains were treated with or without 5ng/ml TGFβ2 for 7 days. At the end of treatment, the cells were fixed and immunostained for markers of cell proliferation (Ki67) and senescence (p21, H2AX, and β-gal). The cells were also co-stained with DAPI for nuclei detection and Phalloidin for F-actin/CLAN visualization. The data were analyzed using Student’s t-tests/ranked tests. P values <0.05 were considered significant.
Results :
The control pHTM cells had significantly less CLAN+ cells than those treated with 5ng/ml TGFβ2 (0.33 ± 0.43% vs 11.3 ± 6.42%, n=22, p < 0.001). Ki67 expression was not significantly different between control and TGFβ2 groups (5.9 ± 0.56% vs 11.1 ± 7.3%, n = 5, p = 0.189). Ki67 was only observed in CLAN- cells in both groups. p21 expression was significantly higher in the control group compared to the TGFβ2 group (87.5 ± 3.8% vs 70.3 ± 6.9%, n = 5, p = 0.001). In addition, all CLAN+ pHTM cells expressed p21 (100%). H2AX expression was significantly lower in the control group compared to the TGFβ2 group (13.3 ± 8.5% vs 27.3 ± 9.6%, n = 6, p = 0.023). Among CLAN+ cells, H2AX was expressed in 62.8 ± 16.3% of cells. β-gal was expressed significantly more in the control group than in TGFβ2 group (32.7 ± 5.4% vs 22.6 ± 5.9%, n = 6, p = 0.007). Among CLAN+ cells, β Gal was expressed in 33.1 ± 5.81% of cells.
Conclusions :
CLAN formation seems to be associated with decreased pHTM cell proliferation and increased cell senescence. In addition, TGFβ2 decreased cell senescence markers p21 and β-gal but increased β-gal in CLAN- pHTM cells. Further research is needed to understand whether a causation relation exists between CLAN and cell proliferation/senescence.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.