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Progress Report 2013

KFO252_long

Progress Report 2013: Project 9 - Engraftment, function and beta cell regulation in the adrenal transplant model

Background

Engraftment and re-vascularization are key factors in human pancreatic islet transplantation. The microenvironment of the adrenal gland features potentially ideal characteristics as a novel transplant site which will be analyzed here using pre-clinical models of islet transplantation. A key focus is directed at the unique propensities of the adrenal microenvironment as a niche for islet engraftment such as high oxygen tension and local anti-inflammatory and immunosuppressive factors.  Our aim is a significant reduction of the transplanted islet mass necessary for achieving normoglycemia by improving survival and function in a newly established adrenal cell transplant model and a large animal diabetes model. The project focuses on in vitro cell-cell interaction of adrenal and islet cells and in vivo analysis of functional integration and augmentation of β-cell mass within the adrenal gland. 

Achievements

For in vitro analysis of islet-adrenal cell interaction, a novel co-culture system of isolated islets and adrenal cells was developed to specifically analyze islet viability and functional potency as well as antioxidative environment mediated by adrenal cells. We could demonstrate the beneficial alliance of different endocrine cell types to promote islet viability and function as a basis for in vivo studies. Therefore, isolated rat islets were transplanted to the intraadrenal transplant site of diabetic nude mice and compared to the classic model of kidney capsule transplantation. Graft function and integration were tested by metabolic follow-up and immunohistochemical staining of intraadrenal grafts. A rapid decrease in blood glucose levels occurred in both transplantation models and all animals reached normoglycemia within the first days after transplantation. Transplanted islet mass was then titrated down within a minimal mass model and shows first evidence, that the intra-adrenal transplant site allows for significant reduction in islet mass to achieve stable normoglycemia. Analysis of explanted islet grafts show a remarkably close integration of islet grafts in the adrenal cortex-medulla region with time-dependent formation of a dense vascular network. This beneficial effect of adrenal-islet interaction could be increased by pre-conditional treatment with a potent growth-hormone-releasing hormone (GHRH) agonist MR403. This agent has been tested on insulinoma cells, isolated rat islets, and adrenal β-cell co-cultures in vitro. MR403 caused a significant increase in cell viability and proliferation and revealed an antiapoptotic effect on insulinoma cells. Viability of rat islets was increased after treatment with the agonist and in co-culture with adrenal cells. Furthermore, we could show changes in vascular gene expression in response to lipoprotein apheresis and in the context of arteriovenous differentiation of endothelial cells. There might be also additional links between growth hormones and oxidative stress in human diseases states. Currently, we are analyzing the oxidative stress in adrenal and endothelial cells. In summary, in these studies we could demonstrate that the adrenal may indeed be a highly attractive site for islet transplantation and that GHRH analogs might allow further reduction of the islet mass needed to reverse diabetes. 

For translation of the intra-adrenal transplantation model to large animals, our mini-pig model for diabetes has been technically improved and evaluated for further transplant studies.  

Completion of objectives

1. The functional characterization of islet-adrenal cell interaction has been completed in vitro and localization of intra-adrenal grafts studied in vivo satisfying objective 1.

2. The immunohistochemical studies on islet integration and vascularization in the mouse model are on-going, partial results were presented and published. Also metabolic studies on long-term survival and augmentation of beta cell mass are completed in the small animal model. 

3. The pre-clinical model of diabetic minipigs has been established, first therapeutic proof of principle trials have been successfully performed.