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Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells
R Agarwal1, M Talati, W Lambert
1Department of Anatomy and Cell Biology and The North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA.
Abstract:
A gradual loss of cells occurs within the human trabecular meshwork during normal aging and appears to be increased in patients with primary open-angle glaucoma. The exact mechanism by which cells are lost in either condition is not known, however phagocytosis, cell migration and cell death have been suggested. Apoptosis is one method by which cell death can occur. We have examined the modulators for apoptosis within the human trabecular meshwork using both cell lines and ex-vivo dissected trabecular meshwork tissues obtained from normal donors. Using RT-PCR it was shown that mRNA for several modulators of apoptosis (Fas, Bcl-2, Bcl-xl, Bax, and ICE) are expressed by both cell lines and ex-vivo tissues. Apoptosis was stimulated to occur by treating cell lines with a monoclonal antibody (IgM) to Fas. Apoptosis was verified via morphological changes to the cells, transferase-mediated dUTP nick-end labeling TUNEL Immunofluorescence, and DNA laddering. Control cells exposed to IgM did not undergo apoptosis. These results represent the first report of apoptosis modulators within the human trabecular meshwork and demonstrate that human trabecular meshwork cells can be stimulated to undergo apoptosis via the Fas/FasL pathway.
Insights
Human trabecular meshwork cells undergo apoptosis via the Fas/FasL pathway. This study identifies apoptosis modulators in trabecular meshwork cells, offering insights into glaucoma pathogenesis.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Cell loss in the human trabecular meshwork (HTM) occurs during aging and is elevated in primary open-angle glaucoma.
- Mechanisms of HTM cell loss, including phagocytosis, migration, and cell death, are not fully understood.
- Apoptosis, a form of programmed cell death, is a potential contributor to HTM cell loss.
Purpose of the Study:
- To investigate the presence and role of apoptosis modulators in the human trabecular meshwork.
- To determine if HTM cells can be induced to undergo apoptosis.
- To explore the Fas/FasL pathway's involvement in HTM apoptosis.
Main Methods:
- Utilized reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA expression of apoptosis modulators (Fas, Bcl-2, Bcl-xl, Bax, ICE) in HTM cell lines and ex-vivo tissues.
- Stimulated apoptosis in HTM cell lines using a monoclonal antibody (IgM) targeting Fas.
- Verified apoptosis through morphological assessment, TUNEL immunofluorescence, and DNA laddering assays.
Main Results:
- mRNA for key apoptosis modulators, including Fas, Bcl-2, Bcl-xl, Bax, and ICE, were detected in both HTM cell lines and ex-vivo tissues.
- Treatment with anti-Fas IgM antibody successfully induced apoptosis in HTM cell lines, confirmed by multiple assays.
- Control cells treated with IgM did not exhibit signs of apoptosis, indicating Fas specificity.
Conclusions:
- This study provides the first evidence of apoptosis modulators within the human trabecular meshwork.
- Human trabecular meshwork cells possess the machinery to undergo apoptosis.
- The Fas/FasL pathway can be activated to induce apoptosis in human trabecular meshwork cells, suggesting its potential role in glaucoma.