Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function

English version

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
prolekare.web.journal.doi_sk: 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:

Insulin – Glucose metabolism – Mitochondria – Insulin secretion – Hypoxia – Enzyme metabolism – Microfluidics – Medical hypoxia

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