PURPOSE: To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 27, in retinal pigment epithelium (RPE), since RPE injury may be an important feature of age-related macular degeneration (ARMD). METHODS: Retinal cross sections from eyes of Lewis rats were examined for Hsp27 in vivo by immunohistochemistry, and in vitro expression of Hsp27 in human ARPE-19 cells was determined by Northern and Western blot analysis. Oxidant-mediated injury was performed by exposing ARPE-19 cells to myeloperoxidase and hydrogen peroxide. Cell lines stably expressing green fluorescent protein (GFP) targeted to the cell membrane were used to study injury-induced membrane blebbing, and XTT conversion was used to detect cell viability. RESULTS: High level of Hsp27 expression was detected in vivo in ganglion cells, RPE, and photoreceptor outer segments of rat retina. ARPE-19 cells also expressed high levels of Hsp27 in vitro. Oxidative injury in ARPE-19 cells resulted in transcriptional and translational activation of Hsp27 and induced extensive membrane blebbing. A high level of Hsp 27 protein was detected within membrane blebs. Increased expression of Hsp27 was also observed in differentiated ARPE-19 cells when compared with dividing cells. Higher Hsp27 levels in differentiated RPE cells correlated with increased viability and phenotypically different blebbing after exposure to the injury stimulus. In addition, sublethal injury doses caused a moderate amount of membrane blebbing, which was well tolerated by differentiated ARPE-19 cells. CONCLUSIONS: These results indicate that Hsp27 may be an important component of the RPE injury response and may contribute to injury-induced membrane blebbing in differentiated RPE cells. It is hypothesized that Hsp27 levels may play a role in disease states in the retina, such as ARMD.
PURPOSE: To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 27, in retinal pigment epithelium (RPE), since RPE injury may be an important feature of age-related macular degeneration (ARMD). METHODS: Retinal cross sections from eyes of Lewis rats were examined for Hsp27 in vivo by immunohistochemistry, and in vitro expression of Hsp27 in human ARPE-19 cells was determined by Northern and Western blot analysis. Oxidant-mediated injury was performed by exposing ARPE-19 cells to myeloperoxidase and hydrogen peroxide. Cell lines stably expressing green fluorescent protein (GFP) targeted to the cell membrane were used to study injury-induced membrane blebbing, and XTT conversion was used to detect cell viability. RESULTS: High level of Hsp27 expression was detected in vivo in ganglion cells, RPE, and photoreceptor outer segments of rat retina. ARPE-19 cells also expressed high levels of Hsp27 in vitro. Oxidative injury in ARPE-19 cells resulted in transcriptional and translational activation of Hsp27 and induced extensive membrane blebbing. A high level of Hsp 27 protein was detected within membrane blebs. Increased expression of Hsp27 was also observed in differentiated ARPE-19 cells when compared with dividing cells. Higher Hsp27 levels in differentiated RPE cells correlated with increased viability and phenotypically different blebbing after exposure to the injury stimulus. In addition, sublethal injury doses caused a moderate amount of membrane blebbing, which was well tolerated by differentiated ARPE-19 cells. CONCLUSIONS: These results indicate that Hsp27 may be an important component of the RPE injury response and may contribute to injury-induced membrane blebbing in differentiated RPE cells. It is hypothesized that Hsp27 levels may play a role in disease states in the retina, such as ARMD.
Authors: Andrea Russo; Raffaele Turano; Francesco Morescalchi; Elena Gambicorti; Anna Cancarini; Sarah Duse; Ciro Costagliola; Francesco Semeraro Journal: Graefes Arch Clin Exp Ophthalmol Date: 2017-03-10 Impact factor: 3.117
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