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 (4.10(8) pfu/pancreas) or were incubated in the presence of DFO (10 mumol/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

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

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

International audienc

<i>In Vitro</i> and <i>In Vivo</i> Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation

<div><p>Introduction</p><p>This study investigated the angiogenic properties of liraglutide <i>in vitro</i> and <i>in vivo</i> and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR).</p><p>Materials and Methods</p><p>Rat pancreatic islets were incubated <i>in vitro</i> 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 <i>in vitro</i> 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, <i>in vitro</i> and <i>in vivo</i>, 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. <i>In vivo</i>, 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.</p><p>Results</p><p>Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, <i>versus</i> control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion <i>versus</i> control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. <i>In vivo</i>, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001).</p><p>Conclusion</p><p>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.</p></div

The angiogenic effects of liraglutide <i>in vivo</i>.

<p>(A) CD31 intensity toward islet surface of rats transplanted with control islets (CTL; black bar) as compared to animals grafted with islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity surrounding transplanted islets toward analysed surface in CTL (black bars) <i>versus</i> Lira (grey bars) groups (C) Insulin intensity toward islet surface in CTL islets (black bars) as compared to Lira (grey bar) (C) Immunostaining of insulin and endothelial cells 30 days post implantation for control islets (CTL; a, b, c, d) <i>versus</i> Lira (e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.</p

Anti-inflammatory and pro-oxidant status of pancreatic islet after isolation.

<p>Islets were incubated in M199 culture medium for 0, 12, 24 and 48 h. (A) Gene expression of Ho-1 was evaluated by qRT-PCR and protein expression of HO-1 was analyzed by Western blotting. (B) IL-10 release in supernatant was evaluated by ELISA. (C, D) ROS production was evaluated with DHE. Data shown are mean ± SEM and are representative of at least three independent experiments. *p<0,05; compared to T0 and N/A means that the values were not available.</p

Oral insulin delivery, the challenge to increase insulin bioavailability: Influence of surface charge in nanoparticle system

International audienceOral administration of insulin increases patient comfort and could improve glycemic control thanks to the hepatic first passage. However, challenges remain. The current approach uses poly (d, lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), an effective drug carrier system with a long acting profile. However, this system presents a bioavailability of less than 20% for insulin encapsulation. In this context, physico-chemical parameters like surface charge could play a critical role in NP uptake by the intestinal barrier. Therefore, we developed a simple method to modulate NP surface charge to test its impact on uptake in vitro and finally on NP efficiency in vivo. Various NPs were prepared in the presence (+) or absence (−) of polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), and/or coated with chitosan chloride. In vitro internalization was tested using epithelial culture of Caco-2 or using a co-culture (Caco-2/RevHT29MTX) by flow cytometry. NPs were then administered by oral route using a pharmaceutical complex vector (100 or 250 UI/kg) in a diabetic rat model.SDS-NPs (−42 ± 2 mV) were more negatively charged than −PVA-NPs (–22 ± 1 mV) and chitosan-coated NPs were highly positively charged (56 ± 2 mV) compared to +PVA particles (−2 ± 1 mV), which were uncharged. In the Caco-2 model, NP internalization was significantly improved by using negatively charged NPs (SDS NPs) compared to using classical NPs (+PVA NPs) and chitosan-coated NPs. Finally, the efficacy of insulin SDS-NPs was demonstrated in vivo (100 or 250 UI insulin/kg) with a reduction of blood glucose levels in diabetic rats. Formulation of negatively charged NPs represents a promising approach to improve NP uptake and insulin bioavailability for oral delivery

Metabolic control in diabetic rats after islet transplantation.

<p>(A) Body-weight gain <i>versus</i> t = 0 day in no transplanted diabetic rats called SHAM (filled diamond), in transplanted diabetic animals with untreated islets called control group (filled square) and in transplanted diabetic rats using treated islets with Lira called Lira group (filled triangle). (B) The mean body-weight gains in SHAM (white bar), control (CTL; black bar) and 10μmol/L of liraglutide (Lira; grey bar) groups over the entire experiment. (C) Fasting glycaemia in the SHAM (filled diamond), control (filled square) and Lira (filled triangle) groups at the indicated time-points. (D) Mean fasting glycaemia in SHAM (white bar), control (black bar) and Lira (grey bar) during the experiment. Results were expressed as means ± SEM. ***p<0.001; **p<0.01; *p<0.05.</p

The angiogenic effects of liraglutide <i>in vitro</i>.

<p>(A) Insulin intensity toward islet surface in control islets (CTL; black bar) as compared to islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity toward islet surface in CTL (black bar) <i>versus</i> Lira (grey bar) group (C) Immunostaining of insulin and endothelial cells after 24 h for control islets (CTL; a, b, c, d) <i>versus</i> islets cultured with Lira10μM (Lira; e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.</p