BMX‐001, a novel redox‐active metalloporphyrin,improves islet function and engraftment in a murine transplant model

Islet transplantation has become a well‐established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX‐001 (MnTnBuOE‐2‐PyP⁵⁺ [Mn(III) meso‐tetrakis‐(N‐ b ‐butoxyethylpyridinium‐2‐yl)porphyrin]) and its earlier derivative, BMX‐010 (MnTE‐2‐PyP [Mn(III) meso‐tetrakis‐(N‐methylpyridinium‐2‐yl)porphyrin]) could improve islet function and engraftment outcomes. Long‐term culture of human islets with BMX‐001, but not BMX‐010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24 hours with 34 μmol/L BMX‐001 exhibited improved insulin secretion (n = 3 isolations, P < .05) in response to glucose relative to control islets. In addition, 34 μmol/L BMX‐001–supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P < .05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34 μmol/L BMX‐001–treated islet recipients became euglycemic (n = 11 of 19) compared with 19% of nontreated control islet recipients (n = 3 of 19, P < .05). Of murine recipients receiving a marginal dose of human islets cultured with 34 μmol/L BMX‐001, 92% (n = 12 of 13) achieved euglycemia compared with 57% of control recipients (n = 8 of 14, P = .11). These results demonstrate that the administration of BMX‐001 enhances in vitro viability and augments murine marginal islet mass engraftment.     

Novel culture technique involving an histone deacetylase inhibitor reduces the marginal islet mass to correct streptozotocin-induced diabetes

Islet transplantation is limited by the difficulties in isolating the pancreatic islets from the cadaveric donor and maintaining them in culture. To increase islet viability and function after isolation, here we present a novel culture technique involving an histone deacetylase inhibitor (HDACi) to rejuvenate the isolated islets. Pancreatic islets were isolated from Sprague-Dawley (SD) rats and one group (FIs; freshly isolated islets) was used after overnight culture and the other group (RIs; rejuvenated islet) was subjected to rejuvenation culture procedure, which is composed of three discrete steps including degranulation, chromatin remodeling, and regranulation. FIs and RIs were compared with regard to intracellular insulin content, glucose-stimulated insulin secretion (GSIS) capacity, gene expression profile, viability and apoptosis rate under oxidative stresses, and the engraftment efficacy in the xenogeneic islet transplantation models. RIs have been shown to have 1.9 ± 0.28- and 1.7 ± 0.31-fold greater intracellular insulin content and GSIS capacity, respectively, than FIs. HDACi increased overall histone acetylation levels, with inducing increased expression of many genes including insulin 1, insulin 2, GLUT2, and Ogg1. This enhanced islet capacity resulted in more resistance against oxidative stresses and increase of the engraftment efficacy shown by reduction of twofold marginal mass of islets in xenogeneic transplantation model. In conclusion, a novel rejuvenating culture technique using HDACi as chromatin remodeling agents improved the function and viability of the freshly isolated islets, contributing to the reduction of islet mass for the control of hyperglycemia in islet transplantation.     

Immune checkpoint CD47 molecule engineered islets mitigate instant blood‐mediated inflammatory reaction and show improved engraftment following intraportal transplantation

Instant blood‐mediated inflammatory reaction (IBMIR) causes significant destruction of islets transplanted intraportally. Myeloid cells are a major culprit of IBMIR. Given the critical role of CD47 as a negative checkpoint for myeloid cells, we hypothesized that the presence of CD47 on islets will minimize graft loss by mitigating IBMIR. We herein report the generation of a chimeric construct, SA‐CD47, encompassing the extracellular domain of CD47 modified to include core streptavidin (SA). SA‐CD47 protein was expressed in insect cells and efficiently displayed on biotin‐modified mouse islet surface without a negative impact on their viability and function. Rat cells engineered with SA‐CD47 were refractory to phagocytosis by mouse macrophages. SA‐CD47‐engineered islets showed intact structure and minimal infiltration by CD11b⁺ granulocytes/macrophages as compared with SA‐engineered controls in an in vitro loop assay mitigating IBMIR. In a syngeneic marginal mass model of intraportal transplantation, SA‐CD47‐engineered islets showed better engraftment and function as compared with the SA‐control group (87.5% vs 14.3%). Engraftment was associated with low levels of intrahepatic inflammatory cells and mediators of islet destruction, including high‐mobility group box‐1, tissue factor, and IL‐1β. These findings support the use of CD47 as an innate immune checkpoint to mitigate IBMIR for enhanced islet engraftment with translational potential.     

Rapid Restoration of Vascularity and Oxygenation in Mouse and Human Islets Transplanted to Omentum May Contribute to Their Superior Function Compared to Intraportally Transplanted Islets

Transplantation of islets into the liver confers several site‐specific challenges, including a delayed vascularization and prevailing hypoxia. The greater omentum has in several experimental studies been suggested as an alternative implantation site for clinical use, but there has been no direct functional comparison to the liver. In this experimental study in mice, we characterized the engraftment of mouse and human islets in the omentum and compared engraftment and functional outcome with those in the intraportal site. The vascularization and innervation of the islets transplanted into the omentum were restored within the first month by paralleled ingrowth of capillaries and nerves. The hypoxic conditions in the islets early posttransplantation were transient and restricted to the first days. Newly formed blood vessels were fully functional, and the blood perfusion and oxygenation of the islets became similar to that of endogenous islets. Furthermore, islet grafts in the omentum showed at 1 month posttransplantation functional superiority to intraportally transplanted grafts. We conclude that in contrast to the liver the omentum provides excellent engraftment conditions for transplanted islets. Future studies in humans will be of great interest to investigate the capability of this site to also harbor larger grafts without interfering with islet functionality.     

The Introduction of Human Heme Oxygenase‐1 and Soluble Tumor Necrosis Factor‐α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase‐1 (HO‐1) or soluble tumor necrosis factor‐α receptor type I with human IgG₁ Fc (sTNF‐αR‐Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO‐1, sTNF‐αR‐Fc, sTNF‐αR‐Fc/HO‐1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood–derived CD34⁺ stem cells into NOD‐scid‐IL‐2Rγ^null mice. Both HO‐1 and sTNF‐αR‐Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig‐to‐humanized mice transplantation. The sTNF‐αR‐Fc/HO‐1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor‐α and IL‐6 in treatment groups; however, frequency of pig‐specific interferon‐γ–producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO‐1 or sTNF‐αR‐Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO‐1 or sTNF‐αR‐Fc transgenic pigs have potential for islet xenotransplantation.     

Early barriers to neonatal porcine islet engraftment in a dual transplant model

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for β cell replacement. Proof‐of‐concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood‐mediated inflammatory reaction and a thorough understanding of early xeno‐specific immune responses. A paucity of data exist comparing xeno‐specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])‐positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild‐type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3‐galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.     

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti‐inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long‐chain poly(ethylene glycol) (PEG) to mitigate detrimental host‐implant interactions. The effect of PEGylation on islet engraftment and long‐term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti‐thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC‐matched recipients and received MHC‐disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c‐peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.     

Improved outcomes of islet autotransplant after total pancreatectomy by combined blockade of IL‐1β and TNFα

The efficacy of islet transplant is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. We report the use of a combination of etanercept and anakinra (ANA+ETA) to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplant. The patients were divided into 3 groups: no treatment (control [CTL]), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C‐peptide, secretory unit of islet transplant objects (SUITO) index, and hemoglobin A_1c. Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin‐6, interleukin‐8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C‐peptide, glucose, hemoglobin A_1c, and SUITO index but without differences in insulin dose. These results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes.     

Improved transplantation outcome through delivery of DNA encoding secretion signal peptide‐linked glucagon‐like peptide‐1 into mouse islets

Glucagon‐like peptide‐1 (GLP‐1) stimulates cell proliferation and has anti‐apoptotic effects on pancreatic islet β cells. In our previous study, the transduction of mouse islets with a recombinant adenovirus containing GLP‐1 cDNA enhanced islet graft survival. In this study, we sought to deliver the GLP‐1 gene using a nonviral vector, which raises fewer safety issues in clinical application. We constructed a plasmid, pβ‐SP‐GLP‐1, in which a secretion signal peptide (SP) was inserted to increase GLP‐1 secretion, and transfected mouse islets using the nonviral carrier Effectene. Transfection of pβ‐SP‐GLP‐1 induced a significant increase in bioactive GLP‐1 levels in islet cultures. Islets transfected with pβ‐SP‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in pβ‐SP‐GLP‐1‐transfected islets. Diabetic syngeneic mice transplanted under the kidney capsule with a marginal mass of pβ‐SP‐GLP‐1‐transfected islets rapidly became normoglycemic, with 88% of recipients being normoglycemic at 30 days post‐transplantation compared with 52% of mice that received pβ‐transfected islet grafts (P < 0.05). Islet grafts retrieved 7 days after transplantation revealed that the pβ‐SP‐GLP‐1‐transfected group had significantly more Ki67‐positive cells as compared with the pβ‐transfected group. In conclusion, delivery of a plasmid containing a secretion SP and GLP‐1 cDNA using a nonviral carrier leads to efficient secretion of GLP‐1 in mouse islet cells, enhances islet cell survival during the early post‐transplant period, and improves islet transplantation outcome.     

Effect of hypoxia‐inducible VEGF gene expression on revascularization and graft function in mouse islet transplantation

For gene transfer strategies to improve islet engraftment, vascular endothelial growth factor (VEGF) expression should be regulated in a way that matches the transient nature of revascularization with simultaneously avoiding undesirable effects of overexpression. The aim of this study was to investigate the effects of hypoxia‐inducible VEGF gene transfer using the RTP801 promoter on islet grafts. We implanted pSV‐hVEGF transfected, pRTP801‐hVEGF transfected or nontransfected mouse islets under the kidney capsule of streptozotocin‐induced diabetic syngeneic mice. Human VEGF immunostaining of day 3 grafts revealed that the pRTP801‐hVEGF transfected group had higher hVEGF expression compared with the pSV‐hVEGF transfected group. BS‐1 staining of day 3 grafts from the pRTP801‐hVEGF transfected group showed the highest vascular density, which was comparable with day 6 grafts from the nontransfected group. In 360 islet equivalent (IEQ)‐transplantation which reverted hyperglycemia in all mice, the area under the curve of glucose levels during intraperitoneal glucose tolerance test 7 weeks post‐transplant was lower in mice transplanted with pRTP801‐hVEGF transfected grafts compared with mice transplanted with nontransfected grafts. In 220 IEQ‐transplantations, diabetic mice transplanted with pRTP801‐hVEGF islets became normoglycemic more rapidly compared with mice transplanted with pSV‐hVEGF or nontransfected islets, and diabetes reversal rate after 50 days was 90%, 68%, and 50%, respectively. In conclusion, our results indicate that regulated overexpression of hVEGF in a hypoxia‐inducible manner enhances islet vascular engraftment and preserves islet function overtime in transplants.     

Loss of end‐differentiated β‐cell phenotype following pancreatic islet transplantation

Replacement of pancreatic β‐cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life‐threatening hypoglycemia in type 1 diabetes. Although near‐normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β‐cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress‐induced β‐cell dysfunction in type 2 diabetes. Here we report loss of end‐differentiated β‐cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin‐positive cells had lost expression of the end‐differentiation marker, urocortin‐3, or appeared to co‐express the α‐cell marker, glucagon. In contrast, no insulin⁺/urocortin‐3^? cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end‐differentiated phenotype may facilitate β‐cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β‐cell sources, graft sites, and ultimately fully vascularized bioengineered insulin‐secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β‐cell phenotype has been maintained.     

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose–basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.     

缺氧致早期胰岛移植失败的原因及对策

胰腺移植和胰岛移植是目前惟一能治愈Ⅰ型糖尿病的方法.相对前者,胰岛移植是一种微创的技术,在手术操作及免疫抑制等方面更具有针对性,这使其成为近年Ⅰ型糖尿病治疗中令人瞩目的 焦点.然而现今的胰岛分离和提纯技术会导致低氧应激,这会严重损害胰岛β细胞的功能.同时,只有在重建血供的条件下,才能保证移植的胰岛细胞得到充足的氧供和营养底物以成功地定植于受体组织.这个过程需要7～14 d.在这期间,由于对缺氧有较强敏感性,β细胞会大量死亡(＞50%),特别是位于胰岛中心的细胞.文章综合文献及作者的实验经验,回顾了影响胰岛细胞定植的缺氧相关的因素以及减少缺氧诱导的细胞凋亡的可行对策.

Abstract：

Pancreas and islet transplantation is the only treatment that can cure type 1 diabetes mellitus. Less invasive and more targeted surgical and immunosuppressive regimens make islet transplantation a more attractive treatment for type 1 diabetes. Current methods of islet isolation and purification cause hypoxic stress to which β cells are extremely vulnerable. Transplanted islets need to re-establish their vascular system in order to obtain sufficient oxygen and nutrient supply for stable engraftment. However, this process takes at least 7- 14 days to complete. Massive (＞50%) β cells are dead before revascularization due to hypoxia, especially the core of the islets. Therefore, the obvious critical problem is the circulatory deficit to which the islets are susceptible in the immediate post-transplant period.In the current study, we reviewed various hypoxic-related insults to islets before complete engraftment, and feasible strategies to reduce hypoxic-induced apoptosis based on our experimental experiences together with that of others and investigated the possibility of revascularization in islet transplantation.     

Early Metabolic Markers That Anticipate Loss of Insulin Independence in Type 1 Diabetic Islet Allograft Recipients

The objective of this study was to identify predictors of insulin independence and to establish the best clinical tools to follow patients after pancreatic islet transplantation (PIT). Sequential metabolic responses to intravenous (I.V.) glucose (I.V. glucose tolerance test [IVGTT]), arginine and glucose‐potentiated arginine (glucose‐potentiated arginine‐induced insulin secretion [GPAIS]) were obtained from 30 patients. We determined the correlation between transplanted islet mass and islet engraftment and tested the ability of each assay to predict return to exogenous insulin therapy. We found transplanted islet mass within an average of 16 709 islet equivalents per kg body weight (IEQ/kg BW; range between 6602 and 29 614 IEQ/kg BW) to be a poor predictor of insulin independence at 1 year, having a poor correlation between transplanted islet mass and islet engraftment. Acute insulin response to IVGTT (AIR_GLU) and GPAIS (AIR_max) were the most accurate methods to determine suboptimal islet mass engraftment. AIR_GLU performed 3 months after transplant also proved to be a robust early metabolic marker to predict return to insulin therapy and its value was positively correlated with duration of insulin independence. In conclusion, AIR_GLU is an early metabolic assay capable of anticipating loss of insulin independence at 1 year in T1D patients undergoing PIT and constitutes a valuable, simple and reliable method to follow patients after transplant.     

Effect of glucagon-like peptide-1 gene expression on graft function in mouse islet transplantation

This study investigated the effect of local glucagon‐like peptide‐1 (GLP‐1) production within mouse islets on cytoprotection in vitro and in vivo by gene transfer of GLP‐1. Transduction of recombinant adenovirus vector expressing GLP‐1 (rAd‐GLP‐1) induced a significant increase in bioactive GLP‐1 in the mouse islet culture, whereas transduction with adenovirus vector expressing β‐galactosidase (rAd‐LacZ), as a control, had no effect on GLP‐1 secretion. Islets transduced with rAd‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in rAd‐GLP‐1‐transduced islets. When transplanted under the kidney capsule of diabetic syngeneic mice, islet grafts retrieved 4 or 7 days after transplantation revealed that the rAd‐GLP‐1‐transduced group had significantly more Ki67‐positive cells as compared with the rAd‐LacZ‐transduced group. Regarding blood glucose control, diabetic mice transplanted with a marginal mass of rAd‐GLP‐1‐transduced islets became normoglycemic more rapidly and 78% of the recipients were normoglycemic at 35 days post‐transplant, whereas only 48% of the mice transplanted with rAd‐LacZ‐transduced islets were normoglycemic (P < 0.05). In conclusion, delivery of the GLP‐1 gene to islets enhanced islet cell survival during the early post‐transplant period, and preserved islet mass and functions over time in the transplants.     

Metabolic aspects of neonatal rat islet hypoxia tolerance

Sensitivity of pancreatic islets to hypoxia is one of the most important of the obstacles responsible for their failure to survive within the recipients. The aim of this study was to compare the in vitro hypoxia tolerance of neonatal and adult rat islet cells and to study the glucose metabolism in these cells after exposure to hypoxia. Islet cells from both age categories were cultured in different hypoxic levels for 24 h and insulin secretion and some metabolites of glucose metabolism were analysed. Glucose‐stimulated insulin secretion decreased dramatically in both cell preparations in response to the decrease in oxygen level. The reduction of insulin secretion was more detectable in adult cells and started at 5% O₂, while a significant reduction was obtained at 1% O₂ in neonatal cells. Moreover, basal insulin release of neonatal cells showed an adaptation to hypoxia after a 4‐day culture in hypoxia. Intracellular pyruvate was higher in neonatal cells than in adult ones, while no difference in lactate level was observed between them. Similar results to that of pyruvate were observed for adenosine triphosphate (ATP) and the second messenger cyclic adenosine monophosphate (cAMP). The study reveals that neonatal rat islet cells are more hypoxia‐tolerant than the adult ones. The most obvious metabolic observation was that both pyruvate and lactate were actively produced in neonatal cells, while adult cells depended mainly on lactate production as an end‐product of glycolysis, indicating a more enhanced metabolic flexibility of neonatal cells to utilize the available oxygen and, at the same time, maintain metabolism anaerobically.     

Self-assembling peptide nanofiber as potential substrates in islet transplantation

Hypoxia and reoxygenation (H/R)-induced damage often happens soon after islets are transplantation. The process of islet isolation and purification causes the rapid onset of hypoxia. We sought to develop a functional scaffold to sustain the structure and function of islets as well as to recover some of the surface molecules damaged during isolation, seeking to improve islet transplantation outcomes. Self-assembling peptide nanofiber (SAPNF), a new type of substrate has been shown to be an excellent biological material for neuronal cell culture and tissue engineering in animals. In this study, we investigated the protective effect of SAPNF on damage to rat islets. Freshly prepared rat islets from male Sprague-Dawley rats were seeded in plates coated with (SAPNF-treated group) or without (control group) SAPNF. The islets were then divided into two groups culture under normoxia for 7 days versus exposure to hypoxia (<1% O₂) for 6 hours followed by reoxygenation for 24 hours. The results showed that SAPNF exhibited improving effects on viability and function of cultured islets, protecting the one from H/R-induced damage. In both groups, the stimulation index of SAPNF-treated groups were about two times the controls. SAPNF treatment decreased apoptotic rates of islet cells. These results suggested the usefulness of SAPNF to maintain the viability and function of rat pancreatic islets. SAPNF may be a potential scaffold for clinical islet transplantation.     

Heat shock preconditioning impairs revascularization of freely transplanted pancreatic islets

BACKGROUND: Revascularization of freely transplanted pancreatic islets is essential for appropriate graft function and survival. During the first days after transplantation, however, islet transplants are avascular, and successful engraftment is believed to be markedly hampered by hypoxia-induced tissue injury. Because heat shock has been shown to induce cell resistance against hypoxia, it seems reasonable to stress pancreatic islets by heat before transplantation. In contrast, hypoxia is a major stimulus for angiogenesis, and thus heat shock preconditioning-induced resistance against hypoxia may decrease stimulation of angiogenesis. The authors therefore studied in vivo whether heat shock preconditioning of isolated islets affects angiogenesis and revascularization after free transplantation. METHODS: After collagenase isolation, heat shock-preconditioned islets (42 degrees C for 30 min) were transplanted syngeneically into nontreated skinfold chambers of Syrian hamsters. In a second group of animals, nontreated islets were transplanted into heat shock-preconditioned chambers. Nontreated islets transplanted into nontreated chambers served as controls. Islet angiogenesis and revascularization were quantitatively analyzed during 14 days after transplantation using intravital fluorescence microscopy. Expression of heat shock proteins (HSP) was confirmed by immunohistochemistry and Western blotting. RESULTS: Immunohistochemistry revealed expression of HSP32 (heme oxygenase [HO]-1), HSP72, and also intracellular insulin in isolated and transplanted pancreatic islets. Western blot analysis showed enhanced HSP32 but slightly decreased HSP72 expression in heat shock-preconditioned islets when compared with controls. Intravital microscopy revealed appropriate vascularization of control islets within 14 days after transplantation. Heat shock preconditioning of the host tissue (i.e., the skinfold chambers) did not affect islet vascularization when compared with controls. In contrast, heat shock preconditioning of the isolated islets resulted in a significantly (P < 0.05) impaired take rate, a reduced (P < 0.05) size of the newly formed microvascular network, and thus a smaller area (P < 0.05) of microvascularly perfused endocrine tissue. CONCLUSION: These data suggest that heat shock preconditioning of isolated pancreatic islets before transplantation impairs the process of graft angiogenesis and revascularization. Therefore, transient exposure of isolated islets to heat may not be considered a promising tool to improve the outcome of islet transplantation.