Hypoxia has a preeminent deleterious effect on organ preservation and islet yield. Indeed, islets as well as other ells/tissues with high oxygen consumption rates such as, retina and neurons, are extremely sensitive to hypoxia. Hypoxic conditions are introduced as early as organ preservation and are 1 of the causes of islet loss during and after islet isolation and purification. The objective of this application is to increase the quality and viability of transplantable islets by improving 2 critical phases of islet transplantation;islet isolation and islet culture conditions. There is substantial evidence indicating that hypoxia induces activation of signaling pathways of p38 and JNK stress mitogen activated protein kinases (MAPK) and pro-inflammatory transcription factor nuclear factor kappa B (NFKB). The activation of these cell-death signaling pathways triggers apoptosis and subsequent islet destruction. It is our working hypothesis that an integrated strategy consisting of antagonizing hypoxia, and simultaneous inhibition of downstream signaling pathways during islet isolation will result in a significant improvement in islet yield and potency. A promising approach of cytoprotection against hypoxia is over expression of oxygen binding proteins such as, neuroglobin (Ngb). Ngb is a recently discovered member of the globin vertebrate superfamily, intracellular heme-Fe-proteins that bind reversibly to oxygen. Ngb is upregulated under hypoxia conditions and is selectively expressed in retina, neurons and pancreatic beta-cells. It is currently believed that the biological effects of Ngb are mediated in part by its powerful scavenging of reactive oxygen species (ROS). We are proposing to execute the integrated strategy with protein transduction, a technology that we previously demonstrated to be very efficient for transient expression of full-length proteins and peptides in islets. Our strategy is based on the combination of oxygen delivery and ROS scavenging provided by Ngb TAT-fusion protein and the inhibition of p38, JNK and NFicB signaling pathways. To validate this hypothesis and achieve the objectives of this application we are proposing 3 specific aims. The first 2 aims will provide information of cytoprotection of islets in culture, a step currently adopted in islet transplantation protocols.
The third aim will render information regarding cytoprotection of islets during islet isolation.
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| Pileggi, Antonello; Ricordi, Camillo (2013) A new home for pancreatic islet transplants: the bone marrow. Diabetes 62:3333-5 |
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| Fornoni, A; Pileggi, A; Molano, R D et al. (2008) Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation. Diabetologia 51:298-308 |
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| Klein, Dagmar; Mendoza, Valeska; Pileggi, Antonello et al. (2005) Delivery of TAT/PTD-fused proteins/peptides to islets via pancreatic duct. Cell Transplant 14:241-8 |
| Klein, Dagmar; Ribeiro, Melina M; Mendoza, Valeska et al. (2004) Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets. Biochem Biophys Res Commun 323:473-8 |
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