Abstract
Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin.
Highlights
Pancreatic islet beta-cells sense blood glucose and secrete insulin directly into the blood stream [1]
endothelial cell (EC) morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static and microfluidic flow culture
The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin
Summary
Pancreatic islet beta-cells sense blood glucose and secrete insulin directly into the blood stream [1]. Pancreatic islets maintain their vasculature immediately after being harvested, it is well established that during ex vivo culture EC density decreases to ~50% in the first day and is almost completely gone by the fourth day [2] The loss of these cells in culture severely limits studying the interaction between beta-cells and ECs. Islet ECs are involved in the revascularization of transplanted tissue for the treatment for type 1 diabetes yet the EC of donor islets are likely lost due to the common practice of culturing donor islets for 48h to diminish tissue inflammation prior to transplantation [2,3,4]. We aim to determine the impact of hypoxia on islet vascularization
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