Konstantin Bloch

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Enhanced Oxygen Supply Improves Islet Viability in a New Bioartificial Pancreas

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)—the “β-Air”—that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immunoprotective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.

Vascularization of wide pore agarose–gelatin cryogel scaffolds implanted subcutaneously in diabetic and non-diabetic mice

Polymeric scaffolds have been reported to promote angiogenesis, facilitating oxygen delivery; however, little is known about the effect of diabetes on the neo-vascularization of implanted polymeric scaffolds at subcutaneous (SC) sites. In this study we compare the effect of diabetes on scaffold vascularization following SC implantation into diabetic and non-diabetic mice. Wide pore agarose cryogel scaffolds with grafted gelatin were prepared by a two-step freezing procedure and subsequent thawing. The scaffolds were implanted subcutaneously into streptozoticin-induced diabetic mice and control, non-diabetic mice. The vascularization process was estimated using histological sections, in which endothelial cells were identified by Von Willebrand factor (vWF) and CD31 antigen staining and the pericyte layer was confirmed by alpha-smooth muscle actin (alpha-SMA) visualization. Comparative analysis showed a similar thickness of fibrous capsules around the vascularized scaffolds in both diabetic and non-diabetic animals. Intensive staining for alpha-SMA indicated the formation of mature blood vessels in the surrounding fibrous capsule and tissue invading the scaffold area. No statistically significant differences in capillary density and area occupied by blood vessels were found between diabetic and non-diabetic mice. In conclusion, the present study shows no adverse effects of diabetes on new blood vessel formation in SC implanted agarose cryogel scaffolds with grafted gelatin.

The Novel Multifunctional, Iron-Chelating Drugs M30 and HLA20 Protect Pancreatic β-Cell Lines from Oxidative Stress Damage

Increasing evidence suggests that oxidative stress (OS)-induced pancreatic β-cell impairments is involved in diabetes and diabetic complications. Our group has recently synthesized two multifunctional nontoxic, lipophilic, iron-chelating drugs, 5-{N-methyl-N-propargylaminomethyl}-8-hydroxyquinoline (M30) and 5-{4-propargylpiperazin-1-ylmethyl}-8-hydroxyquinoline (HLA20), for the treatment of various OS-mediated pathogeneses. These compounds contain the N-propargylamine cytoprotective moiety of the antiparkinsonian drug rasagiline (Azilect) and the iron-complexing component 8-hydroxyquinoline. The aim of this research was to evaluate the protective effect of the multifunctional iron-chelating drugs on rat insulin-producing pancreatic β-cells (INS-1E and RINm) against OS-induced cytotoxicity. We found that M30 and HLA20 markedly and dose-dependently inhibited H2O2-induced cytotoxicity, associated with decreased intracellular reactive oxygen species formation and increased catalase activity. In accordance, the catalase inhibitor 3-amino-1,2,4-triazol blocked the protective action of M30 against H2O2-induced damage. Both compounds significantly increased the levels of the iron-responsive protein transferrin receptor indicating their iron-chelating effect. Further mechanistic studies showed that M30 and HLA20 attenuated H2O2-induced mitochondrial membrane potential loss, decreased the release of cytochrome c into the cytoplasm, and inhibited the activation of caspase-3, suggesting that these drugs may produce cytoprotective effects via the preservation of mitochondrial function. These results indicate that the novel drugs, M30 and HLA20 display significant cytoprotective activity against OS-induced cytotoxicity in insulin producing β-cells, which might be of therapeutic use in the treatment of diabetes mellitus.

Neonatal pig islets induce a lower T-cell response than adult pig islets in IDDM patients.

BACKGROUND Pancreatic pig islets may provide a substitute in the future for difficult to obtain human islets for transplantation in insulin-dependent diabetes millitus (IDDM) patients. However, the immune response to xenografts may significantly hamper this approach. Because neonatal tissue is believed to be less immunogenic, we examined whether the T-cell response to neonatal pig islets differs from the response to adult islets. METHODS The T-cell proliferative response to different concentrations of sonicated neonatal and adult pig islets, as well as to insulin and mitogens, was tested in 21 recent onset IDDM patients and 21 healthy controls. We determined the presence of various circulating islet autoantibodies and their association with the T-cell response in IDDM patients. RESULTS In the IDDM patients, sonicated adult pig islets (at 1 microg protein/ml) induced a significantly higher frequency (12 of 21 vs. 1 of 21, p<0.001) and magnitude (2.58+/-0.44 vs. 1.38+/-0.13, p<0.02) of positive T-cell responses than neonatal islets at the same concentration. Similar results were obtained with a 10-fold higher concentration of islet sonicate. There was no significant association between the individual T-cell responses and the presence of circulating autoantibodies in IDDM patients. CONCLUSION These results indicate that neonatal pig islets induce a lower T-cell reactivity than adult islets, suggesting that the neonatal tissue may be immunologically more suitable for future islet xenotransplantation.

Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-producing RINm Cells

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and [3H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Induction of beta-cell resistance to hypoxia and technologies for oxygen delivery to transplanted pancreatic islets.

Hypoxia is believed to be a crucial factor involved in cell adaptation to environmental stress. Islet transplantation, especially with immunoisolated islets, interrupts vascular connections, resulting in the substantially decreased delivery of oxygen and nutrients to islet cells. Insulin‐producing pancreatic beta cells are known to be highly susceptible to oxygen deficiency. Such susceptibility to hypoxia is believed to be one of the main causes of beta‐cell death in the post‐transplantation period. Different strategies have been developed for the protection of beta cells against hypoxic injury and for oxygen delivery to transplanted islets. The enhancement of beta‐cell defense properties against hypoxia has been achieved using various techniques such as gene transfection, drug supplementation, co‐culturing with stem cells and cell selection. Technologies for oxygen delivery to transplanted islets include local neovascularization of subcutaneous sites, electrochemical and photosynthetic oxygen generation, oxygen refuelling of bio‐artificial pancreas and whole body oxygenation by using hyperbaric therapy. Progress in the field of oxygen technologies for islet transplantation requires a multidisciplinary approach to explore and optimize the interaction between components of the biological system and different technological processes. This review article focuses mainly on the recently developed strategies for oxygenation and protection from hypoxic injury – to achieve stable and long‐term normoglycaemia in diabetic patients with transplanted pancreatic islets. Copyright

Selection of insulin-producing rat insulinoma (RINm) cells with improved resistance to oxidative stress

The defense system against reactive oxygen species is believed to be crucial for the survival of insulin-producing cells after various injuries. The aim of our study was to select a subpopulation of insulin-producing RINm cells with higher resistance to oxidative stress. The cells resistant to hydrogen peroxide (RINmHP) were obtained by repeated exposure of parental RINm cells to 100 and 200 microM hydrogen peroxide (HP). The increased resistance of RINmHP cells to HP was confirmed by three different cytotoxicity assays. In addition, the selected cells also were resistant to the cytotoxic effect of activated rat splenocytes compared to parental cells. The half-life of HP in the RINmHP cell culture medium was about 2.5 times lower than that of the parental cells, corresponding to the increased level of catalase expression and activity in selected cells. The increased defense property of the selected cells was not associated with any significant changes in insulin content and insulin response to a mixture of glucose with isobutyl methyl xanthine or potassium chloride. In conclusion, repeated exposure to HP induces selection of RINm cells with improved resistance to oxidative stress. This improved defense characteristic probably is due to an increased level of catalase expression and activity in the selected cells.

Islet transplantation in a subarachnoid cavity surrounding olfactory bulb of diabetic rats.

Olfactory Bulb of Diabetic Rats C linical trials indicate that islet transplantation is a potential successful treatment for patients with type 1 and a subgroup of patients with type 2 diabetes. Usually, the liver is used as a site for islet transplantation. However, islet transplantations in this site are hampered by insufficient oxygenation, cell rejection, and procedure-related complications. The subarachnoid cavity, filled with cerebrospinal fluids, was considered as a potential site for cell transplantation because of immunoprivileged properties, excellent oxygen and nutrition supply, efficient metabolic waste removal, relatively large volume, and easy access. However, little has been published on the islet transplantation in this site. In the past, normoglycemia has been achieved in diabetic rats after syngeneic and allogeneic islet transplantation in the cerebrospinal fluids of cerebral ventricles (1, 2) or spinal cord (3). In this study, we investigated glucose homeostasis in diabetic rats after islet transplantation in a subarachnoid cavity surrounding the olfactory bulb. The olfactory bulb is known to be enriched with insulin receptor, displayed the highest transport rate for insulin in the brain (4), and plays an important role in the development of different neurodegenerative diseases, particularly in Alzheimer and Parkinson diseases (5). It has been shown that decreased brain insulin levels and/or signaling are associated with impaired learning, memory, and various neurodegenerative diseases (6). A recent clinical trial has indicated that intranasal delivery of insulin through the olfactory pathway may constitute a promising therapy for diabetesand age-related neurodegenerative disorders (7). With this rationale, we studied the different parameters of glucose homeostasis and islet morphology after islet transplantation in a subarachnoid cavity of diabetic rats (see Materials and Methods). This transplantation procedure enabled the assembly of grafted islets directly onto the glomeruli of the olfactory bulb. As shown in Figure 1A, all severely diabetic rats transplanted with 3000 islet equivalents achieved normoglycemia within the first 2 days, which was maintained for 2 months after transplantation. One month and two months after transplantation, the rats demonstrated normal glucose tolerance typical for healthy animals (Fig. 1B). These data are well correlated with the normalization of blood C-peptide level found in transplanted animals (mean [SD], 526 [90], 56 [47], and 372 [129] pmol/L in intact, diabetic, and transplanted rats, respectively). In contrast, the nontransplanted diabetic animals were permanently hyperglycemic, showing impaired glucose tolerance, decreased level of blood C-peptide, and body weight loss. They died within the first month after diabetes induction.

Sensitivity of HaCat keratinocytes to diabetogenic toxins.

Metabolic, genetic and environmental factors very likely play an important role in the development of skin lesions in diabetes mellitus. While these lesions are involved in secondary diabetes complications, various diabetogenic genotoxic agents may induce direct skin damage. In the present study we examined the potential of known diabetogenic agents (streptozotocin (STZ) and alloxan (AL)), with different mechanisms of action, for induction of direct injury in an immortal human keratinocyte HaCat cell line. In contrast to STZ, which induces alkylation of DNA, a genotoxic effect of AL is achieved through reactive oxygen species. We found that HaCat cells are highly sensitive to STZ, but not to AL. At a concentration of 10mM STZ, cell viability decreased to 32 +/-13% of control (P<0.05), as compared to 82 +/-14% with 10mM of AL. Cells treated with 10 and 20mM STZ showed a significant increase in apoptosis (3.9- and 6.7-fold), but not in necrosis, compared to naive cells (P<0.05). In contrast to STZ, no increase in apoptotic and necrotic cell death was observed after AL treatment. Pretreatment with non-metabolizable 3-O-methyl glucose (3-OMG), which can blockade glucose transporter, or with poly(ADP-ribose) polymerase inhibitors (nicotinamide or 3-aminobenzamide), did not protect keratinocytes from STZ injury. Our results show that STZ, but not AL, is highly toxic to the HaCat cell line. Unlike insulin-producing cells, STZ-induced injury of immortal human keratinocyte HaCat cells is independent of the glucose transporters as well as of the activation of poly(ADP-ribose) polymerase.