No abstract

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on β-cell lines and rat pancreatic islets. RINm5F β-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). β-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 μg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 μg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 μg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 μg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation.

In bioartificial pancreases (BP), the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2) in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ)/cm2) and cultured in normal atmospheric pressure (160 mmHg) as well as hypoxic conditions (15 mmHg) for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance.

Transplantation of encapsulated islets in a bioartificial pancreas is a promising alternative to free islet cell therapy to avoid immunosuppressive regimens. However, hypoxia, which can induce a rapid loss of islets, is a major limiting factor. The efficiency of oxygen delivery in an in vitro model of bioartificial pancreas involving hypoxia and confined conditions has never been investigated. Oxygen carriers such as perfluorocarbons and hemoglobin might improve oxygenation. To verify this hypothesis, this study aimed to identify the best candidate of perfluorodecalin (PFD) or HEMOXCell to reduce cellular hypoxia in a bioartificial pancreas in an in vitro model of encapsulation ex vivo. The survival, hypoxia, and inflammation markers and function of rat islets seeded at 600 islet equivalents (IEQ)/cm and under 2% pO were assessed in the presence of 50 μg/mL of HEMOXCell or 10% PFD with or without adenosine. Both PFD and HEMOXCell increased the cell viability and decreased markers of hypoxia (hypoxia-inducible factor mRNA and protein). In these culture conditions, adenosine had deleterious effects, including an increase in cyclooxygenase-2 and interleukin-6, in correlation with unregulated proinsulin release. Despite the effectiveness of PFD in decreasing hypoxia, no restoration of function was observed and only HEMOXCell had the capacity to restore insulin secretion to a normal level. Thus, it appeared that the decrease in cell hypoxia as well as the intrinsic superoxide dismutase activity of HEMOXCell were both mandatory to maintain islet function under hypoxia and confinement. In the context of islet encapsulation in a bioartificial pancreas, HEMOXCell is the candidate of choice for application in vivo.