Hyperglycemia Leads to Müller Cell Death through Induction of Pyroptosis-Like Mechanisms

Abstract

Purpose: : Müller cell dysfunction in diabetes has clearly been identified. Whether these dysfunctions lead to Müller cell death in vivo is unclear to date. We have previously demonstrated that high glucose leads to caspase-1 activation and interleukin-1β secretion in Müller cells suggesting the activation of a pyroptosis-like type of cell death characterized by elevated caspase-1 activity and lack of common downstream pro-apoptotic markers. Therefore, this study was focused on identifying Müller cell loss in the diabetic retina and determining a potential high glucose-induced pyroptotic cell death mechanism in Müller cells.

Methods: : Mice (C57Bl6) carrying a Müller cell specific GFAP-GFP tag were made diabetic using streptozotocin (STZ) or left normal. At 7 months of diabetes, retinas of diabetic and control animals were isolated and number of Müller cells was determined. For in vitro studies, Müller cells were treated with normal (5mM) glucose, high (25mM) glucose, or high glucose plus the caspase-1 inhibitor YVAD-fmk (100µM) medium for up to 96 hours. To examine caspase-1 dependent caspase-6 and-3 activation patterns, caspase activity assays were performed. Western Blots analysis was used to determine activation of pro-apoptotic proteins like p53 and ICAD. TUNEL and trypan blue assays were performed to determine cell viability.

Results: : Diabetes led to a significant 14.6 ± 3.7% loss of Müller cells at 7 months of diabetes. High glucose-induced caspase-6 and caspase-3-like activities in Müller cells were dependent on caspase-1 activation. Inhibition of caspase-1 activity prevented high glucose-induced p53 upregulation by 98 ± 1.5%. Despite a small but significant high glucose-induced caspase-3-like activity, ICAD was not cleaved and no TUNEL positive cells were detectable. In contrast, high glucose increased the number of trypan blue positive cells by 2.3 ± 0.7 fold which was prevented by caspase-1 inhibition.

Conclusions: : These results indicate hyperglycemia induces Müller cell death by a pyroptosis-like cell death mechanism.

Keywords: diabetic retinopathy • Muller cells • apoptosis/cell death