M. Pinget

Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice.

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 ± 6.9 μm in diabetic mice and 34.4 ± 3.9 μm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 ± 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.

In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles.

PLGA nanoparticles (NPs) are largely developed for biological applications but little is known about their uptake. Therefore, we focused our study on the modalities of insulin-loaded PLGA NPs transport across Caco-2 monolayers, and their hypoglycaemic effect on diabetic rats. Insulin-loaded PLGA NPs were formulated by a double emulsion solvent evaporation process. NPs mean diameter was between 130 and 180 nm. NPs were smooth and spherical with an entrapment efficiency above 80%. Fluorescently labeled NPs were incubated with Caco-2 cells to study the process of uptake and the intracellular fate by flow cytometry and confocal laser scanning microscopy. The kinetic of absorption was time-dependent and occurred by clathrin-mediated endocytosis. The intracellular traffic led to a basolateral exocytosis of NPs. In vitro studies and in vivo intraduodenal administration to diabetic rats showed that NPs were resistant in intestinal conditions long enough to allow both the intestinal absorption of NPs and the delivery of functional insulin in bloodstream. The resulting in vivo hypoglycaemic effect was similar to a long-acting insulin one. As no effect on glycaemia occurred after oral administration, further studies need to be conducted to protect NPs from the degradation occurring at the enteric level.

Diffusion properties of an artificial membrane used for Langerhans islets encapsulation: an in vitro test

Glucose and insulin permeability of an artificial membrane (AN69, HOSPAL, Sweden) used for Langerhans islets encapsulation were investigated. In vivo, a 1 and 7 d intraperitoneal implantation of the AN69 membrane in rats induced a loss of permeability towards glucose and insulin probably due to a protein-coating performed after implantation. In vitro, a protein-coating of the AN69 membrane with fetal calf serum solution reproduced similar results. Thus this in vitro test which mimicks in vivo conditions should be proposed to evaluate rapidly the physicochemical properties of a membrane suitable for pancreatic islets encapsulation.

Influence of corona surface treatment on the properties of an artificial membrane used for Langerhans islets encapsulation: permeability and biocompatibility studies

An artificial membrane (AN69 Hospal) suitable for pancreatic islets encapsulation was submitted to a physicochemical treatment (corona discharge) to improve its insulin permeability. This effect depends on the duration of the electrical discharge (expressed as the speed of a conveyor belt) and the distance between the electrodes and the membrane. Among the various treatments tested, the most efficient (distance of 5 cm and a speed of 2 cm s-1) produced a three-fold increase in insulin diffusion. This improvement persisted after a protein-coating test which mimics in vivo conditions. At 1 y after the peritoneal implantation, the corona-treated membrane remained biocompatible. Thus, corona discharge treatment may serve to optimize the properties of artificial membranes used for pancreatic islets encapsulation.

Perfluorocarbon emulsions prevent hypoxia of pancreatic β-cells.

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.

Overexpression of Vascular Endothelial Growth Factor In Vitro Using Deferoxamine: A New Drug to Increase Islet Vascularization During Transplantation

During pancreatic islet transplantation, delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in cell death and early graft failure. Deferoxamine (DFO), an iron chelator, increases vascular endothelial growth factor (VEGF) expression in cells. The aim of this work was to study the effect of DFO on beta-cell and pancreatic islet viability as well as VEGF expression. beta-cell lines from rat insulinoma (Rin m5f) and primary cultures of pancreatic islets from Wistar rats were incubated with DFO (10, 100, and 1000 micromol/L). The viability was evaluated using fluorescein diacetate/propidium iodide for dying pancreatic islets and using cell titers for Rin m5f. Expression of VEGF messenger RNA (mRNA) was quantified using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, VEGF secretion was determined using enzyme-linked immunosorbent assays at 1 to 3 days after treatment. The addition of 10 micromol/L of DFO preserved Rin m5F viability at 24 hours after treatment (10 micromol/L; 101.33% +/- 5.66%; n = 7). However, 100 and 1000 micromol/L of DFO induced cell death (68.92% +/- 5.83% and 65.89% +/- 5.83%, respectively; n = 4). In the same way, viability of pancreatic islets in the presence of DFO was preserved. RT-PCR analysis showed stimulation of VEGF mRNA in the presence of 10 micromol/L of DFO in islets at 3 days after culture. Finally, 10 micromol/L of DFO stimulated secretion of VEGF 7.95 +/- 0.84 versus 1.80 +/- 1.10 pg/microg total protein with 10 micromol/L of DFO in rat islets at 3 days after culture, n = 3; P < .001). The use of DFO to stimulate VEGF expression and increase islet vascularization may be a realistic approach to improve islet viability during transplantation.

Influence of acinar tissue contamination on encapsulated pancreatic islets: morphological and functional studies.

BACKGROUND The present study concerns the influence of acinar contamination on pancreatic islets encapsulated in an artificial membrane (AN 69). METHODS Pure, handpicked pancreatic islets were contaminated by the addition of acinar tissue (ratio, 1:1). The morphological aspect and insulin release of both pure (n=12) and contaminated (n=12) encapsulated islets were assessed after 10 days of culture or implantation in the peritoneal cavity of rats. RESULTS After implantation, the encapsulated islets, irrespective of their purity, were totally altered, whereas the morphological aspect of the cultivated islets remained intact only in the absence of acinar tissue contamination. This contamination induced a significant decrease in the stimulation index of insulin release. The stimulation index decreased by 42% for fresh islets and by 52% and 34% for cultivated and implanted islets, respectively, without modification in their basal release of insulin. CONCLUSIONS The acinar tissue proved detrimental to the encapsulated implanted and cultivated islets.

Design, characterisation, and bioefficiency of insulin-chitosan nanoparticles after stabilisation by freeze-drying or cross-linking.

Insulin delivery by oral route would be ideal, but has no effect, due to the harsh conditions of the gastrointestinal tract. Protection of insulin using encapsulation in self-assembled particles is a promising approach. However, the lack of stability of this kind of particles in biological environments induces a low bioavailability of encapsulated insulin after oral administration. The objective of this work was to evaluate the effect of two stabilisation strategies alone or combined, freeze-drying and cross-linking, on insulin-loaded chitosan NPs, and to determine their bioefficiency in vitro and in vivo. NPs were prepared by complex coacervation between insulin and chitosan, stabilised either by cross linking with sodium tripolyphosphate solution (TPP), by freeze-drying or both treatments. In vitro bioefficiency NP uptake was evaluated by flow cytometry on epithelial models (Caco-2/RevHT29MTX (mucus secreting cells)). In vivo, NPs were injected via catheter in the peritoneum or duodenum on insulinopenic rats. Freeze-drying increased in size and charge (+15% vs control 412 ± 7 nm; + 36 ± 0.3 mV) in comparison with cross linking which decreased NP size (-25%) without impacting the NP charge. When combined the consecutive treatments reduced NPs size and increased charges as compared to standard level. Freeze drying is necessary to prevent the destruction of NP in intestinal environment in comparison with no freeze dryed one where 60% of NP were destroyed after 2h. Additionally freeze drying combined with cross linking treatments improved bioefficiency of NP with uptake in cell increased when mucus is present. Combination of both treatment showed a protection of insulin in vivo, with a reduction of glycemia when NPs were administrated. This work showed that the combination of freeze drying and cross linking treatment is necessary to stabilize (freeze-drying) and increase bioefficiency (cross-linking) of self assembled NP in the delivery of insulin in vitro and in vivo.

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.