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



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.

Klíčová slova:

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


1. Olsson R, Carlsson P-O. The pancreatic islet endothelial cell: Emerging roles in islet function and disease. Int J Biochem Cell Biol. 2006.

2. Nyqvist D, Köhler M, Wahlstedt H, Berggren PO. Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts. Diabetes. 2005.

3. Bretzel RG, Jahr H, Eckhard M, Martin I, Winter D, Brendel MD. Islet cell transplantation today. Langenbecks Arch Surg. 2007.

4. Brissova M, Powers AC. Revascularization of transplanted islets: Can it be Improved? Diabetes. 2008.

5. Sankar KS, Green BJ, Crocker AR, Verity JE, Altamentova SM, Rocheleau JV. Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells. PLoS One. 2011.

6. Peiris H, Bonder CS, Coates PTH, Keating DJ, Jessup CF. The β-cell/EC axis: How do islet cells talk to each other? Diabetes. 2014.

7. Zhang N, Richter A, Suriawinata J, Harbaran S, Altomonte J, Cong L, et al. Elevated Vascular Endothelial Growth Factor Production in Islets Improves Islet Graft Vascularization. Diabetes. 2004.

8. Narang AS, Cheng K, Henry J, Zhang C, Sabek O, Fraga D, et al. Vascular endothelial growth factor gene delivery for revascularization in transplanted human islets. Pharm Res. 2004.

9. Su D, Zhang N, He J, Qu S, Slusher S, Bottino R, et al. Angiopoietin-1 production in islets improves islet engraftment and protects islets from cytokine-induced apoptosis. Diabetes. 2007.

10. Olerud J, Johansson M, Lawler J, Welsh N, Carlsson PO. Improved vascular engraftment and graft function after inhibition of the angiostatic factor thrombospondin-1 in mouse pancreatic islets. Diabetes. 2008.

11. Olsson R, Maxhuni A, Carlsson PO. Revascularization of transplanted pancreatic islets following culture with stimulators of angiogenesis. Transplantation. 2006.

12. Richards OC, Raines SM, Attie AD. The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action. Endocrine Reviews. 2010.

13. Spilsbury K, Garrett KL, Shen WY, Constable IJ, Rakoczy PE. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol. 2000.

14. Crawford SE, Stellmach V, Murphy-Ullrich JE, Ribeiro SMF, Lawler J, Hynes RO, et al. Thrombospondin-1 is a major activator of TGF-β1 in vivo. Cell. 1998.

15. Brissova M, Fowler M, Wiebe P, Shostak A, Shiota M, Radhika A, et al. Intraislet Endothelial Cells Contribute to Revascularization of Transplanted Pancreatic Islets. Diabetes. 2004.

16. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006.

17. Stefan Y, Meda P, Neufeld M, Orci L. Stimulation of insulin secretion reveals heterogeneity of pancreatic B cells in vivo. J Clin Invest. 1987.

18. Rocheleau JV, Head WS, Nicholson WE, Powers AC, Piston DW. Pancreatic islet beta-cells transiently metabolize pyruvate. J Biol Chem. 2002.

19. Rocheleau JV., Head WS, Piston DW. Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J Biol Chem. 2004.

20. Kelly BD, Hackett SF, Hirota K, Oshima Y, Cai Z, Berg-Dixon S, et al. Cell Type-Specific Regulation of Angiogenic Growth Factor Gene Expression and Induction of Angiogenesis in Nonischemic Tissue by a Constitutively Active Form of Hypoxia-Inducible Factor 1. Circ Res. 2003.

21. Duffy DC, McDonald JC, Schueller OJA, Whitesides GM. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal Chem. 1998.

22. Hiriart M, Aguilar-Bryan L. Channel regulation of glucose sensing in the pancreatic beta-cell. Am J Physiol Endocrinol Metab. 2008.

23. Ardyanto TD, Osaki M, Tokuyasu N, Nagahama Y, Ito H. CoCl2-induced HIF-1alpha expression correlates with proliferation and apoptosis in MKN-1 cells: a possible role for the PI3K/Akt pathway. Int J Oncol. 2006.

24. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha J Biol Chem. 2003.

25. Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 2006.

26. RyFukuda O, Zhang H, Kim J whan, Shimoda L, Dang CV., Semenza GL. HIF-1 Regulates Cytochrome Oxidase Subunits to Optimize Efficiency of Respiration in Hypoxic Cells. Cell. 2007.

27. Sato Y. et al. Cellular Hypoxia of Pancreatic β-cell Due to High Levels of Oxygen Consumption for Insulin Secretion in vitro. The Journal of Biological Chemistry. 2011.

28. Kerendi F, Kirshbom PM, Halkos ME, Wang NP, Kin H, Jiang R, et al. Cobalt Chloride Pretreatment Attenuates Myocardial Apoptosis After Hypothermic Circulatory Arrest. Ann Thorac Surg. 2006.

29. Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell. 2006.

30. Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell. 1999.

31. Woolf AS, Yuan HT. Angiopoietin growth factors and Tie receptor tyrosine kinases in renal vascular development. Pediatr Nephrol. 2001.

32. Li J, Shworak NW, Simons M. Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1alpha-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites. J Cell Sci. 2002.

33. Hatanaka K, Lanahan AA, Murakami M, Simons M. Fibroblast growth factor signaling potentiates VE-cadherin stability at adherens junctions by regulating SHP2. PLoS One. 2012.

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2019 Číslo 10
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