Elisa Maillard

Prevention of adhesion and promotion of pseudoislets formation from a β-cell line by fluorocarbon emulsions

Transplantation of pancreatic islets from cadaveric human donors is a promising therapy for type 1 diabetes. By reducing hyper/hypoglycaemic events, this therapy can strongly improve the quality of life of patients. There have been substantial advances in the islet-transplantation protocol (the Edmonton Protocol) through an improved islet-isolation method and the use of steroid-free immunosuppressants. Nevertheless, several difficulties continue to limit this approach. These include the gradual loss of islet graft functionality, side effects related to the use of immunosuppressants and, last but not least, the small number of potential donors. An alternate approach to solving the problem of the shortage of human islets consists of the transplantation of b-cell lines or b-cell surrogates. For this purpose, sources of b cells, such as pig and mouse pancreas and human and mouse embryonic stem cells are being investigated. Established bcell lines, such as the mouse insulinoma-6 (MIN6) cells offer several advantages over isolated islet material. The native pancreatic islet tissue is composed of various cell types (a, b and d cells). Furthermore, the sensitivity to glucose of b cells from freshly isolated islets exhibits considerable variability. In contrast, the MIN6 cell line provides a homogenous b-cell population whose insulin secretion responds physiologically to glucose stimulation. Under classical culture conditions, MIN6 cells adhere to uncoated, negatively charged tissue culture plastic to form mono ACHTUNGTRENNUNGlayers, but when grown on collagen-derived substrates, they form islet-like structures (pseudoislets) after 6–8 days of culture. Due to a lack of cell-to-cell contact, b cells from mono ACHTUNGTRENNUNGlayers are considerably less responsive to glucose and other nutrients than b cells arranged as pseudoislets. Hence, pseudoislets offer a better experimental model for studies of b-cell functionality than monolayer cells. In this study, we report that a perfluorocarbon (PFC) emulsion is able to prevent the adhesion of MIN6 cells onto negatively charged tissue culture plastic. Furthermore, the cells were seen to rapidly aggregate into well-formed pseudoislets. Owing to the specific properties of PFCs mentioned below, this approach to pseudoislet formation has the advantage of being nonimmunogenic, in contrast to the collagen-derived substrate approach. Owing to exceptional biological inertness, excellent spreading properties, high oxygen solubility and extremely low solubility in water, PFCs are being investigated for various biomedical applications. These include, in particular, intravascular oxygen transport, diagnosis, lung-surfactant replacement and drug delivery. Within the framework of the present investigation, the oxygen-carrying capacity of PFCs is also expected to prevent the hypoxia that contributes to the failure of post-transplant survival. This study represents an initial step whose aim is to establish the effects of PFCs on the viability and morphology of the MIN6 cell line. A PFC emulsion stabilized with Pluronic F-68 has been shown to reduce the endothelial adherence of sickled red cells. In that study, however, the reduction of adherence was also obtained with a PFC-free aqueous solution of Pluronic F68. The result was, therefore, attributed to a lubricating effect of the surfactant. In the present study, two hydrocarbon (HC) oil emulsions (Intralipid@ and a soybean oil (SBO) emulsion) that use the same surfactant as the PFC emulsion, provided the appropriate controls for assessing the effect of the PFC. Other studies have shown a modification of cell adhesion by using either pure liquid PFCs (cell culture at a liquid/liquid interface) or PFC-functionalized solid substrates. In both studies, a tendency to reduction of cell adhesion and promotion of spherical cell shape (reduced cell spreading) was observed. In the liquid/liquid system, cell growth was in the form of island monolayers, rather than in a continuous sheet monolayer of cells. In the present study, a 70% w/v-concentrated perfluorooctyl bromide (C8F17Br) emulsion, emulsified with egg yolk phospholipids (EYP, 4.67% w/v) and stabilized by a semifluorinated alkane, C6F13C10H21 (F6H10, 3.27% w/v), was used. [27] This PFC emulsion has a small average droplet size (~80 nm average diameter after terminal heat sterilization) and a narrow particlesize distribution. The mean diameter of the PFC droplets was only 100 nm after five months of storage at 4 8C. This PFC emulsion as well as the HC emulsions (Intralipid and a 45% w/v SBO emulsion) used as controls were diluted with an equal volume of twice-concentrated culture medium (DMEM) and added to the culture wells (see Experimental Section). The MIN6 cells were then incubated in DMEM containing various percentages (v/v) of these 1:1 diluted emulsions. For clarity, the results are expressed in terms of % v/v of the 1:1 diluted emulsions in the culture medium as well as, in the case of the PFC emulsion, % w/v of PFC in the total DMEM+emulsion volume. The effect of the PFC and HC emulsions on MIN6 cell cultures was investigated by counting the number of cells that remained adhered to the substrate. This number was compared with that obtained for control experiments in which the cells [a] Dr. M. Sanchez Dominguez, Dr. M. P. Krafft Syst mes Organis s Fluor s Finalit s Th rapeutiques (SOFFT). Institut Charles Sadron (CNRS) 6 rue Boussingault. 67083 Strasbourg, Cedex (France) Fax: (+33)388-414-099 E-mail : krafft@ics.u-strasbg.fr [b] E. Maillard, Dr. S. Sigrist, Dr. A. Belcourt Centre Europ en d’Etude du Diab te (CeeD) 1 boulevard Ren Leriche 67200 Strasbourg (France) Fax: (+33)390-201-219 E-mail : severine.sigrist@wanadoo.fr

Pro-Inflammatory and Pro-Oxidant Status of Pancreatic Islet In Vitro Is Controlled by TLR-4 and HO-1 Pathways

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.

Extracellular matrix proteins involved in pseudoislets formation

Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

Improvement of rat islet viability during transplantation: validation of pharmacological approach to induce VEGF overexpression.

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.108 pfu/pancreas) or were incubated in the presence of DFO (10 μmol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific.

Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in β-cell lines

Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in β-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). β-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 μmol/L of DFO (103.95 ± 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected β-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation.

Portal or subcutaneous insulin infusion: efficacy and impact on liver inflammation

Intraperitoneal insulin allows physiological portal insulin administration and first‐pass hepatic insulin extraction, but the impact on liver metabolism and inflammation is unknown. Our objective was to compare the impact, on metabolic control and liver function, of the same dose of insulin administered either intraperitoneally or subcutaneously during continuous infusion in diabetic rats. Wistar rats were randomly divided into 4 groups: control (C), untreated diabetic (streptozotocin, 100 mg/kg) and diabetic rats treated by continual subcutaneous Insuplant® infusion (CSII) and continual intraperitoneal Insuplant® infusion (CPII) of 2 UI/200 g/day (via an osmotic mini‐pump for 1–4 weeks). Insulin signalling pathways were analysed through hepatic expression of growth hormone receptor and phosphorylated insulin receptor substrate 1. Metabolic control was determined by measurement of body weight, blood glucose and fructosamine. Liver function was assessed by measuring insulin‐like growth factor‐1 (IGF‐1), with global inflammation assessed by levels of alpha‐2‐macroglobulin (α2M) and lipid peroxidation in plasma. Liver inflammation was evaluated by quantification of hepatic macrophage infiltration and reactive oxygen species production. CPII induced a better improvement in metabolic control and liver function than CSII, producing a significant decrease in blood glucose and fructosamine, coupled with increased IGF‐1 and hepatic glycogen storage. Moreover, liver oxidative stress and liver inflammation were reduced. Such observations indicate that the same insulin level in CPII improves glucose control and hepatic glucose metabolism and function, attenuating the hepatic inflammatory response to diabetes. These data demonstrate the importance of focusing on therapeutics to allow first‐pass hepatic insulin extraction or prevent diabetic complications.

Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells.

The in vitro methods currently used to screen bioactive compounds focus on the use of a single model of oxidative stress. However, this simplistic view may lead to conflicting results. The aim of this study was to evaluate the antioxidant properties of two natural extracts (a mix of red wine polyphenols (RWPs) and epigallocatechin gallate (EGCG)) with three models of oxidative stress induced with hydrogen peroxide (H2O2), a mixture of hypoxanthine and xanthine oxidase (HX/XO), or streptozotocin (STZ) in RINm5F beta cells. We employed multiple approaches to validate their potential as therapeutic treatment options, including cell viability, reactive oxygen species production, and antioxidant enzymes expression. All three oxidative stresses induced a decrease in cell viability and an increase in apoptosis, whereas the level of ROS production was variable depending on the type of stress. The highest level of ROS was found for the HX/XO-induced stress, an increase that was reflected by higher expression antioxidant enzymes. Further, both antioxidant compounds presented beneficial effects during oxidative stress, but EGCG appeared to be a more efficient antioxidant. These data indicate that the efficiency of natural antioxidants is dependent on both the nature of the compound and the type of oxidative stress generated.

In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation

Introduction This study investigated the angiogenic properties of liraglutide in vitro and in vivo and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR). Materials and Methods Rat pancreatic islets were incubated in vitro with 10 μmol/L of liraglutide (Lira) for 12, 24 and 48 h. Islet viability was studied by fluorescein diacetate/propidium iodide staining and their function was assessed by glucose stimulation. The angiogenic effect of liraglutide was determined in vitro by the measure of vascular endothelial growth factor (VEGF) secretion using enzyme-linked immunosorbent assay and by the evaluation of VEGF and platelet-derived growth factor-α (PDGFα) expression with quantitative polymerase chain reaction technic. Then, in vitro and in vivo, angiogenic property of Lira was evaluated using immunofluorescence staining targeting the cluster of differentiation 31 (CD31). To understand angiogenic mechanisms involved by Lira, HIF-1α and mTOR activation were studied using western blotting. In vivo, islets (1000/kg body-weight) were transplanted into diabetic (streptozotocin) Lewis rats. Metabolic control was assessed for 1 month by measuring body-weight gain and fasting blood glucose. Results Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, versus control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion versus control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. In vivo, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001). Conclusion The beneficial effects of liraglutide on islets appeared to be linked to its angiogenic properties. These findings indicated that glucagon-like peptide-1 analogues could be used to improve transplanted islet revascularisation.

Featured Article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy

Long-term insulin delivery can reduce blood glucose variability in diabetic patients. In this study, its impact on oxidative stress status, inflammation, and liver injury was investigated. Diabetes was induced in Wistar rats with a single dose of streptozotocin (100 mg/kg). Untreated rats and rats administered Insuplant® (2 UI/200 g/day) through a subcutaneous osmotic pump for one or four weeks were compared with non-diabetic controls. Body weight, fructosamine level, total cholesterol, Insulin Growth Factor-1 (IGF-1) level, lipid peroxidation, and total antioxidant capacity were measured. Hepatic injury was determined through the measurement of glycogen content, reactive oxygen species (ROS) production, and macrophage infiltration. Liver oxidative stress status was evaluated through the measurement of superoxide dismutase (SOD), catalase (CAT), and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) expression, and p38 mitogen-activated protein kinase (p38MAPK) activation. Induction of diabetes led to increased plasma oxidative stress and inflammation. Moreover, ROS production and macrophage infiltration increased in addition to SOD, CAT, and NADPH oxidase expression. Intensive insulin therapy improved metabolic control in diabetic animals as seen by a restoration of hepatic glycogen, plasma IGF-1 levels, and a decrease in plasma oxidative stress. However, insulin treatment did not result in a decrease in acute inflammation in diabetic rats as seen by continued ROS production and macrophage infiltration in the liver, and a decrease of p38MAPK activation. These results suggest that the onset of diabetes induces liver oxidative stress and inflammation, and that subcutaneous insulin administration cannot completely reverse these changes. Targeting oxidative stress and/or inflammation in diabetic patients could be an interesting strategy to improve therapeutic options.

Comparison of Perfluorodecalin and HEMOXCell as Oxygen Carriers for Islet Oxygenation in an In Vitro Model of Encapsulation.

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)/cm2 and under 2% pO2 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.