G M Tezel1, G M Seigel, M B Wax. 1. Washington University School of Medicine Department of Ophthalmology & Visual Sciences Missouri, St. Louis 63110, USA.
Abstract
PURPOSE: To study effects of cell density on retinal cell survival. METHODS: Apoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantitative analysis of apoptotic cells was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique and flow cytometry. Cytoskeleton was examined using immunocytochemistry and specific staining of actin by phalloidin and DNase I. In addition, alterations in the cytoskeletal proteins, bcl-2 family of proteins and hsp27 were studied using western blotting. RESULTS: Incubation of the cells under apoptotic stimuli caused higher rates of apoptosis in lower density cultures as determined by TUNEL technique and flow cytometric analysis. Both morphologic examination of cytoskeleton and western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultures compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bax was lower in lower density cultures compared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bax expression increased in both lower and higher density cultures. However, we observed that the expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli. CONCLUSIONS: These findings demonstrate that retinal cells are more resistant to apoptosis in higher density cultures, independent of the inducer. This might be partly due to protective activity of endogenous hsp27 in the cells at higher density, which contributes to cytoskeletal integrity in response to apoptotic stimuli.
PURPOSE: To study effects of cell density on retinal cell survival. METHODS: Apoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantitative analysis of apoptotic cells was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique and flow cytometry. Cytoskeleton was examined using immunocytochemistry and specific staining of actin by phalloidin and DNase I. In addition, alterations in the cytoskeletal proteins, bcl-2 family of proteins and hsp27 were studied using western blotting. RESULTS: Incubation of the cells under apoptotic stimuli caused higher rates of apoptosis in lower density cultures as determined by TUNEL technique and flow cytometric analysis. Both morphologic examination of cytoskeleton and western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultures compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bax was lower in lower density cultures compared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bax expression increased in both lower and higher density cultures. However, we observed that the expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli. CONCLUSIONS: These findings demonstrate that retinal cells are more resistant to apoptosis in higher density cultures, independent of the inducer. This might be partly due to protective activity of endogenous hsp27 in the cells at higher density, which contributes to cytoskeletal integrity in response to apoptotic stimuli.
Authors: Miryam A Fragoso; Amit K Patel; Rei E I Nakamura; Hyun Yi; Krishna Surapaneni; Abigail S Hackam Journal: PLoS One Date: 2012-10-04 Impact factor: 3.240