Sodium 4-phenylbutyrate protects against liver ischemia reperfusion injury by inhibition of endoplasmic reticulum-stress mediated apoptosis.

BACKGROUND: Evidence is emerging that the endoplasmic reticulum (ER) participates in initiation of apoptosis induced by the unfolded protein response and by aberrant Ca(++) signaling during cellular stress such as ischemia/reperfusion injury (I/R injury). ER-induced apoptosis involves the activation of caspase-12 and C/EBP homologous protein (CHOP), and the shutdown of translation initiated by phosphorylation of eIF2alpha. Sodium 4-phenylbutyrate (PBA) is a low molecular weight fatty acid that acts as a chemical chaperone reducing the load of mutant or unfolded proteins retained in the ER during cellular stress and also exerting anti-inflammatory activity. It has been used successfully for treatment of urea cycle disorders and sickle cell disease. Thus, we hypothesized that PBA may reduce ER-induced apoptosis triggered by I/R injury to the liver. METHODS: Groups of male C57BL/6 mice were subjected to warm ischemia (70% of the liver mass, 45 minutes). Serum aspartate aminotransferase was assessed 6 hours after reperfusion; apoptosis was evaluated by enzyme-linked immunosorbent assays of caspase-12 and plasma tumor necrosis factor alpha, Western blot analyses of eIF2alpha, and reverse transcriptase-polymerase chain reaction of CHOP expression. RESULTS: A dose-dependent decrease in aspartate aminotransferase was demonstrated in mice given intraperitoneal PBA (1 hour before and 12 hours after reperfusion), compared with vehicle-treated controls; this effect was associated with reduced pyknosis, parenchymal hemorrhages, and neutrophil infiltrates in PBA-treated mice, compared with controls. In a lethal model of total liver I/R injury, all vehicle-treated controls died within 3 days after reperfusion. In contrast, 50% survival (>30 days) was observed in animals given PBA. The beneficial effects of PBA were associated with a greater than 45% reduction in apoptosis, decreased ER-mediated apoptosis characterized by significant reduction in caspase-12 activation, and reduced levels of both phosphorylated eIF2alpha and CHOP. Significant reductions in plasma levels of tumor necrosis factor alpha and liver myeloperoxidase content were demonstrated after PBA treatment. CONCLUSIONS: Reduction in ER stress-induced hepatocellular injury was achieved by the administration of PBA. Targeting the ER-associated cell death pathway might offer a novel approach to reduce I/R injury to the liver.

Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation.

Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2 kDa) as well as 5 kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6 kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.

Activated protein C preserves functional islet mass after intraportal transplantation: a novel link between endothelial cell activation, thrombosis, inflammation, and islet cell death.

Clinical studies indicate that significant loss of functional islet mass occurs in the peritransplant period. Islets are injured as a result of detrimental effects of brain death, pancreas preservation, islet isolation, hypoxia, hyperglycemia, and immune-mediated events. In addition, recent studies demonstrated that islets are injured as a result of their exposure to blood and of activation of intrahepatic endothelial and Kupffer cells, resulting in inflammation and thrombosis. Activated protein C (APC) is an anticoagulant enzyme that also exerts anti-inflammatory and antiapoptotic activities by acting directly on cells. Here, we report that exogenous administration of recombinant murine APC (mAPC) significantly reduced loss of functional islet mass after intraportal transplantation in diabetic mice. Animals given mAPC exhibited better glucose control, higher glucose disposal rates, and higher arginine-stimulated acute insulin release. These effects were associated with reduced plasma proinsulin, intrahepatic fibrin deposition, and islet apoptosis early after the transplant. In vitro and in vivo data demonstrated that mAPC treatment was associated with a significant reduction of proinflammatory cytokine release after exposure of hepatic endothelial cells to islets. mAPC treatment also prevented endothelial cell activation and dysfunction elicited by intrahepatic embolization of isolated islets inherent to pancreatic islet transplantation (PIT). This study demonstrates multiple remarkable beneficial effects of mAPC for PIT and suggests that APC therapy may enhance the therapeutic efficacy of PIT in diabetic patients.

A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets.

BACKGROUND: Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2. METHODS: Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT). RESULTS: No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time. CONCLUSIONS: Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.

Enhanced isolated pancreatic islet recovery and functionality in rats by 17beta-estradiol treatment of brain death donors.

BACKGROUND: Current isolation techniques recover only 20% to 50% of the pancreatic islets. Brain death (BD) is characterized by activation of proinflammatory cytokines (PICs) with reduced islet yields and functionality. We previously reported that 17beta-estradiol (E2) induces cytoprotection to human islets exposed to PICs. Furthermore, inhibition of PIC release has been demonstrated after E2 treatment. In the present study, we evaluated if E2 treatment to BD donors would improve pancreatic islet recovery and functionality. METHODS: BD was induced in male, 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. Rats were mechanically ventilated for 6 hours. Only rats with mean arterial blood pressure > 75 mm Hg were used. Animals (n = 6) received E2 (1 mg/kg/iv immediately after BD induction), vehicle (V), or the combination of 17beta-estradiol and a selective estrogen receptor antagonist ICI 182,780 (ICI, 3 mg/kg/ip/1 hour before BD induction). Islet viability was determined by ethidium bromide-acridine orange. PICs were assessed by ELISA. Islet functionality was determined by static incubation and glucose disposal rate (Kg) after intraportal transplantation (3000 islet equivalent[IEQ]/syngeneic streptozotocin-induced diabetic rat). RESULTS: A 2- to 3-fold reduction in TNF-alpha, IL-1beta, and IL-6 was demonstrated in BD donors given E2; this effect reversed by ICI 182,780. Pancreatic sections from control BD donors presented 26.5% +/- 4% TUNEL-positive beta-cells compared with 15.1% +/- 3% in 17beta-estradio-treated animals. Islet recovery was enhanced in E2-treated donors (1233.4 +/- 123 IEQ/pancreas) compared with controls (725 +/- 224 IEQ, P < .05). Islet viability was significantly enhanced by E2. Higher islet functionality was demonstrated in vitro and in vivo after transplantation in islets recovered from E2-treated BD donors. CONCLUSIONS: Islet recovery and functionality in vitro and in vivo were significantly improved by 17beta-estradiol treatment to BD donors. These observations may lead to strategies to reduce the effects of BD on isolated islets and improve the results in clinical islet transplantation.

Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice.

BACKGROUND: Ischemia/reperfusion injury (I/R injury) of the liver remains a significant problem during liver surgery and transplantation. I/R injury is associated with liver apoptosis, which is mediated by death receptors such as Fas and tumor necrosis factor alpha (TNF-alpha), and/or mitochondrial dysfunction induced by cellular stress. Caspase-8 is presumed to be the apex of the death-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" proteases in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8 and caspase-3 siRNA in a murine model of liver I/R injury. METHODS: In C57BL/6 mice, 45% or 70% of the liver mass was clamped for 90 minutes. For survival analysis, total hepatic ischemia was induced for 45 minutes. In vivo delivery of siRNA was performed via the portal vein by high-volume injection (0.5 nmol of siRNA in 1 mL containing 10% lipiodol) 60 minutes before ischemia. As a control, animals received either vehicle or non-sense siRNA (siRNA-scrambled). RESULTS: Liver uptake of siRNA was analyzed in transgenic mice who express beta-galactosidase (beta-gal) (C57BL/6J-TgN(MTn-LacZ)204Bri) after administration of siRNA-LacZ. A 3- to 4-fold decrease in beta-gal activity was accomplished at 0.5 nmol. No significant change in beta-gal activity was demonstrated in mice receiving non-sense siRNA. Immunohistochemical studies found that 60% of the liver cells efficiently took up siRNA. Significant reduction in serum aspartate transaminase was found in animals treated with siRNA caspase-8 or caspase-3 compared with siRNA-scrambed or vehicle-treated controls. More than a 60% reduction in caspase-8 and caspase-3 gene expression and activities was accomplished after siRNA administration. Animals treated with siRNA presented lower infiltration of polymorphonuclear leukocytes and better preservation of the liver architecture compared with controls. All of the control mice subjected to total liver ischemia died within 5 days. In contrast, 30% of the animals given siRNA caspase-8 and 50% of those treated with siRNA caspase-3 survived indefinitely (>30 days). CONCLUSIONS: Small interfering RNA targeted to caspase-8 and caspase-3 provided significant protection against I/R injury to the liver. This approach could be therapeutic in liver transplantation and other conditions associated with I/R injury to the liver.

Brain death significantly reduces isolated pancreatic islet yields and functionality in vitro and in vivo after transplantation in rats.

Although approximately 1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n = 6). In controls, the catheter was not inflated (n = 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-alpha, IL-1beta, and IL-6 mRNA was noted in the pancreas. Brain death donors presented lower insulin release after glucose stimulation assessed by in situ perfusion of the pancreas. Islet recovery was reduced in brain death donors compared with controls (at 6 h 602.3 +/- 233.4 vs. 1,792.5 +/- 325.4 islet equivalents, respectively; P < 0.05). Islet viability assessed in dissociated islet cells and in intact cultured islets was reduced in islets recovered from brain death donors, an effect associated with higher nuclear activities of NF-kappaB p50, c-Jun, and ATF-2. Islet functionality evaluated in vitro by static incubation and in vivo after intraportal transplantation in syngeneic streptozotocin-induced diabetic rats was significantly reduced in preparations obtained from brain death donors. In conclusion, brain death significantly reduced islet yields and functionality. These observations may lead to strategies to reduce the effects of brain death on pancreatic islets and improve the results in clinical transplantation.

Suppression of the c-Jun N-terminal kinase pathway by 17beta-estradiol can preserve human islet functional mass from proinflammatory cytokine-induced destruction.

BACKGROUND: The c-Jun N-terminal kinase (JNK) activation occurs after islet isolation, oxidative stress, and proinflammatory cytokine (PIC) exposure to beta-cells. Previous studies demonstrated that 17beta-estradiol modulates the activity of JNK; therefore we assessed the effects of 17beta-estradiol on JNK activation on islet survival and function after transplantation. METHODS: Isolated human pancreatic islets were incubated with PIC and 17beta-estradiol. Viability was analyzed by a colorimetric assay, islet mass by DNA content, JNK activity by Western blots, AP-1 nuclear activity with a promoter-Luciferase AP-1 responsive construct, and c-Fos, Jun-D, and ATF-2 nuclear activities by an enzyme-linked immunosorbent assay. Islet functionality was evaluated after transplantation in streptozotocin-induced diabetic NOD-SCID mice. RESULTS: The 17beta-estradiol enhanced islet viability and islet mass after exposure to PIC. A significant reduction in JNK activation occurred in islets treated with 17beta-estradiol, compared with controls, an effect partially dependent on estrogen receptors. The 17beta-estradiol induced a significant reduction in nuclear AP-1, c-fos, Jun-D, and ATF-2 activities. Animals that received 17beta-estradiol-treated islets had better islet functionality compared with saline solution-treated controls. CONCLUSIONS: The 17beta-estradiol improved isolated human pancreatic islets survival after PIC exposure by inhibition of JNK. These effects were associated with reduction in JNK targets, including the nuclear activities of transcription factors AP-1, c-Jun, c-Fos, Jun-D and ATF-2, involved in apoptosis in beta-cells. The 17beta-estradiol therapy may improve the results in clinical transplantation.

Peripheral mobilization of recipient bone marrow-derived endothelial progenitor cells enhances pancreatic islet revascularization and engraftment after intraportal transplantation.

BACKGROUND: Pancreatic islet transplantation has been validated as a treatment for type 1 diabetes. However, a high number of islets is required to establish euglycemia. Transplantation of islets leads to loss of islet vasculature, which requires revascularization to ensure adequate survival. Islet vascular density in transplanted islets is markedly decreased compared with endogenous islets. The feasibility of revascularization of ischemic tissues by mobilizing endothelial progenitor cells or angioblasts has been demonstrated. Therefore, we investigated the therapeutic potential of angioblast mobilization for stimulation of islet revascularization and therefore engraftment after transplantation. METHODS: FVB/NJ mice underwent bone marrow transplantation from transgenic mice constitutively expressing beta-galactosidase encoded by LacZ under regulation of the endothelial cell-specific promoter TIE-2 (FEV/NJ-TIE-2-LacZ). Three weeks after reconstitution, animals received an intrahepatic islet syngeneic infusion (FVB/NJ donors). The contribution of angioblasts into sites of islet revascularization was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), beta-galactosidase (beta-gal) activity, and immunohistochemistry. Islet vascular density was assessed morphometrically followed by in situ BS-1 lectin staining and functional islet mass after transplantation by metabolic studies. Angioblasts were mobilized with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.5 microg/day/7 days). RESULTS: An islet dose-dependent increase in beta-gal was demonstrated after transplantation. These results were confirmed by RT-PCR and immunohistochemistry. GM-CSF increased the number of peripheral angioblasts and their localization into sites of islet revascularization. A significant increase in islet vascular density was observed in animals treated with GM-CSF versus controls. Higher functional islet mass was demonstrated in animals treated with GM-CSF. CONCLUSIONS: Augmentation of angioblasts in the peripheral circulation resulted in higher islet vascular density and engraftment. This novel strategy may improve the results in clinical islet transplantation.

Double genetic modification of adenovirus fiber with RGD polylysine motifs significantly enhances gene transfer to isolated human pancreatic islets.

BACKGROUND: New strategies for improving durable functional islet mass will be instrumental in facilitating islet transplantation as a cure for type 1 diabetes mellitus. The ability to transfer immunoregulatory or cytoprotective genes into pancreatic islets may enhance survival. Adenoviral vectors (Ad5) have been used widely to deliver therapeutic genes to different tissues. Limitations associated with the use of Ad5 for gene therapy are related to the reliance of the virus on the presence of its primary receptor, the transient nature of the transgene expression, and the immediate inflammatory and immune response elicited by the infection. Because the arginine-glycine-aspartame (RGD) and polylysine (pK7) motifs have been shown to enhance Ad5 infection through an Ad5 receptor-independent pathway, we hypothesized that they could act additively to improve infectivity and reduce toxicity to isolated human pancreatic islets (IHPI). METHODS: Hand-picked IHPI were infected with nonmodified Ad5, single-modified Ad5 with RGD (Ad5RGD) or pK7 (ad5pK7), and Ad5RGDpK7. Transfection efficiency was evaluated by green fluorescent protein and luciferase expression. Apoptosis was assessed using a quantitative assay, activation of caspase 3 by a colorimetric assay, nuclear factor (NF)-kappaB nuclear translocation using a promoter-luciferase NF-kappaB responsive construct, regulated on activation normal T-cell expressed and secreted (RANTES) by enzyme-linked immunosorbent assay. In vivo functionality was evaluated after transplantation into diabetic nonobese diabetic severe combined immunodeficiency mice. RESULTS: Compared with unmodified and singly-modified Ad5 vectors, Ad5RGDpK7 demonstrated the highest infectivity. After the infection of IHPI with adenoviral vectors using the minimal dose required to infect greater than 80% of the islet cells (Ad5, 500 viral particles [VP]/cell; Ad5RGD and Ad5pK7, 10 VP/cell; Ad5RGDpK7, 0.1 VP/cell), islets infected with Ad5RGDpK7 presented a significant reduction in apoptosis, NF-kappaB nuclear translocation, RANTES expression, and higher glucose disposal rate; reduced Ad5-driven specific Th1 and antibody response were also observed. CONCLUSIONS: Ad5RGDpK7 exhibited higher transfection efficiency, allowing a significant reduction in the viral dose required to infect greater than 80% of the islet cells. The reduction in the viral dose was associated with reduced toxicity, inflammation, and immune responses related to Ad5 infection. This strategy may thus be used to successfully modify isolated pancreatic islets.

Coupling endoplasmic reticulum stress to cell death program in isolated human pancreatic islets: effects of gene transfer of Bcl-2.

A variety of toxic insults can result in endoplasmic reticulum (ER)-stress that ultimately leads to apoptosis. beta-cells have a highly developed ER due to a great commitment to insulin production. The present study was carried out to determine the role of ER-stress in isolated human pancreatic islet apoptosis, and the potential protective effects of Bcl-2. Isolated human islets were infected with an adenoviral vector encoding Bcl-2 and then exposed to brefeldin-A, tunicamycin, A23187 and pro-inflammatory cytokines. Activation of caspase-12 was analyzed by means of Western blots. Apoptosis was evaluated using a commercial quantitative assay. ER-stress-inducers promoted caspase-12 activation and apoptosis, effect reversed by overexpression of Bcl-2. Co-localization of caspase-12 and Bcl-2 in the microsomal islet fractions were demonstrated by means of Western blots. We can conclude that the current studies highlight the importance of Bcl-2 as an anti-apoptotic protein, and shed new light on the mechanisms underlying its cytoprotective effects on pancreatic islets.

Stable alpha- and beta-islet cell function after tolerance induction to pancreatic islet allografts in diabetic primates.

Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable alpha- and beta-cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long-term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional alpha- and beta-cell mass and metabolic function was assessed in streptozotocin (STZ)-induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14-day tolerance induction protocol based on F(Ab)2-IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2-IT or DSG alone] showed early 10-day function but all lost islet function by days 15-70. One recipient [F(Ab)2-IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ-induced diabetic primates resulted in restoration of normal alpha- and beta-cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2-IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.

17beta-Estradiol protects isolated human pancreatic islets against proinflammatory cytokine-induced cell death: molecular mechanisms and islet functionality.

INTRODUCTION: Proinflammatory cytokines (PIC) (interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha) are released after intraportal islet transplantation lead to functional suppression and islet apoptosis. Estradiol has been shown to promote survival of cells undergoing PIC-induced apoptosis. In this study, we evaluated the effects of estradiol on isolated human pancreatic islet (IHPI) survival after exposure to PIC and analyzed potential mechanisms of action. METHODS: Hand-picked, freshly isolated IHPI were incubated with PIC and estradiol. Viability was analyzed from single islet cells stained with ethidium bromide and acridine orange, apoptosis using a quantitative kit, NF-kappaB nuclear translocation using a promoter-Luciferase NF-kappaB responsive construct, mitochondrial permeability transition using the ApoAlert Mitochondrial kit, and caspase 9 by a fluorometric assay. In vitro functionality was examined by static incubation, and a limited number of islets were transplanted in nonobese diabetic, severe combined immunodeficient mice. RESULTS: 17beta-Estradiol induced a dose-dependent increase in islet viability, an effect partially reversed by the estrogen receptor antagonist ICI 182,780. In vitro, islets treated with estradiol presented higher stimulation index. Euglycemia was achieved in 6 of 12 animals that received estradiol-treated islets compared with 1 of 12 control animals. Lower NF-kappaB nuclear translocation, cytochrome release, and caspase 9 activation occurred in islets treated with estradiol. CONCLUSIONS: Estradiol promoted IHPI survival and improved functionality after PIC exposure in vitro and in vivo after transplantation. The molecular mechanisms involved included a decrease in NF-kappaB nuclear translocation, decrease in mitochondrial cytochrome release, and caspase 9 activation. The use of estradiol might be beneficial in clinical islet transplantation.

Simvastatin induces activation of the serine-threonine protein kinase AKT and increases survival of isolated human pancreatic islets.

BACKGROUND: Pancreatic islets are susceptible to myriad insults that occur during islet isolation and transplantation. Studies demonstrated the role of Akt in regulating pancreatic beta-cell growth and survival. Activation of Akt maintains Bad phosphorylation and prevents its binding to mitochondrial targets, decreases caspase-9 activity, and prevents the translocation of forkhead transcription factors (FKHR). Simvastatin activates Akt in mammalian cells; therefore, we investigated the role of simvastatin on human pancreatic islets (HPI) survival. METHODS: HPI were treated with simvastatin, with and without LY294002, an inhibitor of phosphoinositide 3-kinase. PI viability was examined with ethidium bromide-acridine orange, and apoptosis was examined using a quantitative assay. Akt, Bad, FKHR phosphorylation, and mitochondrial cytochrome release were analyzed by Western blots. Caspase-9 activity was assessed by a fluorometric assay. A limited number of HPI were transplanted after simvastatin treatment in diabetic NOD-SCID mice. RESULTS: Low levels of Akt phosphorylation (activation) were demonstrated early after islet isolation. Akt activation; increase in islet viability; and decrease in Bad phosphorylation, cytochrome release, caspase-9 activation, and translocation of FKHR were observed after simvastatin treatment, effects reversed by LY294002. Among recipients of islets without simvastatin, none demonstrated reversal of diabetes after the transplant. In contrast, 58% of the recipients given islets treated with simvastatin remained euglycemic 30 days after the transplant. CONCLUSIONS: Targeting the survival pathway with simvastatin exerts a cytoprotective effect on isolated PI. Activation of the Akt pathway is a potential new therapeutic approach to reduce loss of functional islet mass to bolster success in clinical islet transplantation.

Genetically engineered intracellular single-chain antibodies in gene therapy.

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.

17-Beta-estradiol protects the liver against warm ischemia/reperfusion injury and is associated with increased serum nitric oxide and decreased tumor necrosis factor-alpha.

BACKGROUND: Ischemia/reperfusion injury (I/R injury) to the liver can occur in low-flow states associated with trauma and shock and surgical procedures such as liver transplantation. Recent studies have shown that the administration of the female sex hormone 17-beta-estradiol after trauma-hemorrhage in animals restores depressed cardiac, hepatocellular, and immune functions. In this study we evaluated the effects of 17-beta-estradiol on I/R injury to the liver. METHODS: The medial lobe of the liver in normal male C57BL/6 mice was clamped at its base for 90 minutes. 17-Beta-estradiol was given 1 hour before I/R injury at 40 and 4000 microg/kg intraperitoneally. Biochemical analysis was performed, and liver biopsy specimens were obtained at 24 hours. RESULTS: A dose-dependent reduction in aspartate aminotransferase level was observed in animals (n = 8) given estradiol (243 +/- 23 IU/L) compared with saline-treated animals (902 +/- 42 IU/L, P <.001). The majority (90%) of the cytoprotective effect of estradiol was reverted by ICI 182,780 (a potent estrogen receptor antagonist). A significant increase in serum nitric oxide (NO) level was observed in animals given estradiol compared with controls; the effect was reversed by ICI 182,780 and N-nitro-L-arginine-methyl ester (an inhibitor of NO synthesis). A reduction in serum tumor necrosis factor-alpha was observed after injury in animals given estradiol compared with controls (30.2 +/- 11.1 vs 75.8 +/- 17.2 pg/mL, P <.001). Estradiol treatment significantly reduced liver necrosis, disintegration of hepatic cords, and neutrophil infiltration in an estrogen receptor-dependent manner. CONCLUSIONS: Estradiol administration significantly reduced injury after I/R to the liver, an effect that is mainly receptor-mediated and is associated with increased serum NO, decreased TNF-alpha, and decreased number of neutrophils in liver biopsy specimens. Estrogen therapy may be important in clinical conditions associated with I/R injury to the liver.

Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.

Cytoprotection of pancreatic islets before and early after transplantation using gene therapy.

Pancreatic islet transplantation (PIT) is an attractive alternative to insulin-dependent diabetes treatment but is not yet a clinical reality. The first few days after PIT are characterized by substantial pancreatic islet dysfunction and death. Apoptosis has been documented in PI after extracellular matrix removal, during culture time, after exposure to proinflammatory cytokines, hypoxic conditions before islet revascularization, and rejection. Targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, over-expression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation into diabetic SCID mice. Genetic modification of PI also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass post-transplant and reduce the high islet requirement currently needed for successful stable reversal of insulin-dependent diabetes [1, 2].