Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY Diabetes is a major co-morbidity of cystic fibrosis (CF), affecting 20% of adolescents and 40-50% of adults with CF. CF-related diabetes (CFRD) is associated with worse disease outcomes and a 4-fold increase in mortality relative to CF patients without diabetes. Impaired insulin release is the key defect underlying CFRD, but its etiology is poorly understood. Pancreatic ductal epithelial cells (PDECs) are the major source of pancreatic CFTR and are the initiating site of CF pancreas pathology, which leads to destruction of pancreatic acini (in 85% of CF cases). Islets are relatively preserved in the CF pancreas. However, islet morphology is profoundly altered, including increased α cells (although glucagon release is impaired in CF), and loss of islet capillaries and macrophages. These cell types support normal β cell function and so their disruption in CF likely contributes to insulin deficiency. Recent studies, including our preliminary data, show that knockdown/inhibition of CFTR in PDECs can induce islet dysfunction (impaired insulin release). However, the relative contribution of direct PDEC-islet effects vs. those occurring indirectly via pancreatic acinar cells or other islet cells are unknown. Moreover, the underlying mechanisms are entirely unexplored. We hypothesize that CFTR-defective human PDECs exert detrimental effects on β cells, both directly and indirectly, to impair insulin release and β cell health. We will perform studies in primary human cells (PDECs, islet β-cells, α- cells, acinar cells, islet endothelial cells and macrophages). We will also use CF donor pluripotent stem cell (PSC)-derived PDECs. Specific Aim 1: To determine whether CFTR-defective PDECs impair insulin release via direct effects on islet β-cells. Transcriptomic and proteomic analysis will be done on PDECs and PDEC-derived extracellular vesicles using primary or hPSC-derived PDECs ± CFTR mutations. From these data, we will identify key pathways that mediate cross talk between PDECs and β cells. The impact of those pathways will then be tested in intervention studies using islets and primary human β cells. Specific Aim 2: To determine whether CFTR-defective PDECs impair insulin release indirectly. We will recapitulate the known toxic effects of CFTR-defective PDECs on acinar cells using an in vitro system and determine whether acinar elicited factors can themselves impair β cell function. Next, we will systematically interrogate the effect of CFTR-defective PDECs and/or acinar cells previously exposed to those PDECS on the function, identity and viability of islet α cells, endothelial cells and macrophages. Specific Aim 3: Effect of PDEC-CFTR knockdown on insulin release in human pancreas slices. Pancreas slices, which preserve the 3D arrangement of pancreatic cell types, will be used to determine the effect of PDEC-CFTR loss on insulin release over time, along with concurrent measurement of the viability/function of acinar cells and α-cells along with morphological changes in islet endothelial cells and macrophages.
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