Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function
Autoři:
Krishana S. Sankar aff001; Svetlana M. Altamentova aff002; Jonathan V. Rocheleau aff001
Působiště autorů:
Department of Physiology, University of Toronto, Toronto, Ontario, Canada
aff001; Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
aff002; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
aff003; Department of Medicine, University of Toronto, Toronto, Ontario, Canada
aff004
Vyšlo v časopise:
PLoS ONE 14(10)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0222424
Souhrn
Background
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.
Methods
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.
Results
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.
Conclusions
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.
Klíčová slova:
Enzyme metabolism – Glucose metabolism – Hypoxia – Insulin – Insulin secretion – Microfluidics – Mitochondria – Medical hypoxia
Zdroje
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