Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans.

ABSTRACT

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.