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Cytoprotection of pancreatic islets before and early after transplantation using gene therapy
Kidney International, Vol. 61, Symposium 1 (2002), pp. S79–S84
Cytoprotection of pancreatic islets before and early after transplantation using gene therapy JUAN L. CONTRERAS, GUADALUPE BILBAO, CHERYL A. SMYTH, DEVIN E. ECKHOFF, XIAO L. JIANG, STACIE JENKINS, FRANCIS T. THOMAS, DAVID T. CURIEL, and JUDITH M. THOMAS Transplant Center and Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA
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].
Insulin dependent diabetes mellitus (IDDM) is a disorder of insulin deficiency secondary to pancreatic -cell loss. Both the prevalence and incidence of diabetes in the U.S. have escalated in the last 25 years. IDDM is a chronic disease that may prompt a variety of complications with widespread morbidity and premature mortality. IDDM is the leading cause of blindness and kidney failure among adults in the U.S. IDDM is conventionally treated by exogenous insulin, however, the majority of patients develop one or more end-organ complications during their lives. Pancreas organ transplantation, on the other hand, can permanently reverse diabetes, but success is constrained by morbidity associated with major surgery Key words: islet transplantation, gene therapy, Bcl-2 gene.
2002 by the International Society of Nephrology
and by low availability of suitable donor organs. Pancreatic islet transplantation (PIT) by intraportal vein infusion can circumvent surgical disadvantages of organ transplantation but clinical application of PIT has been offset by low rates of success [3–5]. The discrepancy between duration of transplant function with whole pancreas organs and purified islets suggest a fundamental biologic difference in these grafts. Although isolated PIT is susceptible to complex biologic insults incurred during organ procurement and preservation, initial re-vascularization, proinflammatory cytokine release, immune rejection, and the diabetogenic effect of immunosuppressive drugs, the isolation procedure itself appears to induce profound changes in islet structure and function [3, 6–10]. Tissue-specific survival factors and/or the removal of the extracellular matrix, may be responsible for eliminating cells that end up in an ectopic location (i.e., anoikis) [10–12]. The final outcome of these processes is destruction of the transplanted islets, mainly by programmed cell death or apoptosis (Fig. 1). Experimental studies have demonstrated a significant selective decrease in the -cell mass early after transplantation in purified canine islet autografts and in human islets transplanted into nude mice [13, 14]. Apoptosis is a highly regulated process of cell death and is controlled through the expression of specific genes that are conserved in nematodes through mammals. Among these, the Bcl-2 gene family is one of the most important [15]. The protein encoded by the Bcl-2 gene has been implicated in the prolongation of cell survival by blocking apoptosis and necrosis. Gene transfer studies have shown that over-expression of Bcl-2 can protect cells from death induced by a wide variety of diverse insults and stimuli [10, 15–25]. Given the functional importance of Bcl-2 gene, it constitutes a prime target for therapeutic intervention in numerous disease states. Bcl-2 is expressed in pancreatic islets and its possible that contributes to the survival capacity of the islets. Over-
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Fig. 1. Vulnerability of pancreatic islets. Isolated pancreatic islets are susceptible to complex biological insults incurred during pancreas procurement and islet isolation, poor vascularization early after transplantation, exposure to proinflammatory cytokines and other immune mediated injuries, hyperglycemia, and the use of diabetogenic immunosuppressive drugs. The final outcome of these processes is destruction of the transplanted islets mainly by apoptosis.
expression of Bcl-2 in human islets and in insulinoma cells prevents apoptosis induced by IL-1, IFN-␥, and TNF-␣ and also induces cytoprotection from hypoxia [20, 22]. Replication-deficient adenoviral vectors are the most efficient vectors currently available for transducing nondividing cells and can be produced in high titers; their epichromosomal location diminishes the risk of insertional mutagenesis. E-1 deleted adenovirus vectors have been tested in PI [26, 27]. Recently, we developed a replication-deficient adenoviral vector encoding the human anti-apoptotic Bcl-2 gene [16]. Gene transfer of Bcl-2 induced cytoprotection of endothelial cells and liver grafts during routine preservation in University of Wisconsin solution for organ transplantation and after reperfusion [17–19]. Over-expression of Bcl-2 decreases the injury associated with an immune response generated by adenoviral vectors after in vivo gene transfer to the liver [16, 28]. In our laboratory, we evaluated the potential of genetic modification of nonhuman primate (NHP) pancreatic islets by gene transfer of Bcl-2 as an approach to prevent loss of pancreatic -cells before and early after transplantation. Numerous publications have demonstrated that adenoviruses are very efficient at infecting human and rodent islets ex vivo [26, 27, 29]. However, no experience in gene transfer in nonhuman primate islets has been reported. Initial experiments in our laboratory demonstrated that ⬎95% of the cells in non-human primate islets expressed the transgene after genetic modification with adenoviral vectors at 500 pfu/cell (Fig. 2A, B, C, and D). Bcl-2 expression after gene transfer with an adenoviral vector (AdBcl-2) was confirmed by Western blot (Fig. 2E). An immunohistochemical analysis after genetic modification
of PI with AdBcl-2 showed that ⬎95% of the insulin and non-insulin producing cells over-express Bcl-2 compared with only 10% of the cells without genetic modification (data not shown). After demonstration of high efficient gene transfer in NHP islets, we sought the potential cytoprotective properties of the Bcl-2 gene. DNA fragmentation is the biochemical marker of apoptosis [15]. As previously reported in human islets, apoptosis is a frequent event during standard culture before transplantation [7, 9, 10, 30]. Islet cell DNA fragmentation assessed by ELISA assay during culture time is shown in Figure 3. A significant reduction in DNA fragmentation was observed at 7 days in culture in PI genetically modified to overexpress Bcl-2 compared with AdCMVLacZ (irrelevant gene) or untransfected islets (P ⬍ 0.05). These results demonstrate the cytoprotective properties of the antiapoptotic Bcl-2 gene in NHP islets. Initial experiments in our laboratory demonstrated that 2000 Islet Equivalents (IEQ) NHP islets per mouse transplanted into the portal vein is the “optimal” dose for restoration of normoglycemia one day after the transplant in streptozotocin (STZ)-induced SCID mice. Reducing the number of unmodified islets per mouse to 1000 IEQ, 58% of the recipients were found euglycemic on day 1 postransplant, and only 8% on day 100 (Fig. 4). Similar results were observed in recipients of islets that over-express an irrelevant gene (euglycemia in 50% on day 1, 8% on day 100). A significant difference in the percentage of euglycemic animals early and late post-transplant was observed in recipients of islets that over-expressed Bcl-2 (Fig. 4, P ⬍ 0.005 compared with AdCMVLacZ or unmodified islets). All recipients of a “marginal dose” of Bcl-2 islets presented euglycemia the day after the transplant and 83% on day 100. These results demonstrate the ability of Bcl-2 transfected islets to reverse diabetes in STZ-induced SCID mice even after the transplant of a marginal number of cells. A more sophisticated form of metabolic evaluation of the transplanted islets is demonstrated in Figure 5. The glucose disposal rate (Kg), a measure of functional islet mass, became progressively reduced over time, indicating loss of islet mass [31, 32]. By 30 days after the transplant, glucose disposal rate was reduced to pre-transplant diabetic values in the two control groups (unmodified islets or islets transfected with LacZ). In contrast, no significant change in the capacity to dispose glucose was observed in animals that received islets transduced with Bcl-2. One of the current limitations in clinical islet transplantation is the availability of cadaver organs [2, 3, 5]. After demonstration of the cytoprotective effects of Bcl-2, next we sought the islet mass required to establish stable euglycemia after transplantation. Most of the diabetic recipients that received 2000 IEQ were found euglycemic 3 and 15 days post-transplantation. Reducing
Contreras et al: Cytoprotection of pancreatic islets
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Fig. 2. Transfection efficiency of adenoviral vectors in non-human primate pancreatic islets. NHP islets were infected ex vivo with different concentrations of an E1-deleted adenoviral vector encoding -galactosidase (A ⫽ none, B ⫽ 10 pfu/cell, C ⫽ 100 pfu/cell, and D 500 ⫽ pfu/cell). An immunohistochemical analysis demonstrated that more than 95% of the cells expressed the reporter gene (LacZ). Bcl-2 expression after gene transfer with similar E1-deleted adenoviral vector encoding the human Bcl-2 gene was confirmed by Western blot (E). Immunoblot analysis was performed as described in Methods. Lane 1: positive control; Lane 2: NHP islets transfected with an adenovirus vector encoding an irrelevant gene (LacZ); Lane 3: NHP islets transfected with AdCMVhBcl-2.
Fig. 3. Bcl-2 transfected islets analysis for spontaneous apoptosis during culture by ELISA. NHP islets were incubated without virus (mock), AdCMVLacZ, or AdCMVhBcl-2 and cultured as described in Methods. DNA fragmentation was assessed at 7 days. Data are mean ⫾ SE for islets from 3 different isolations and are expressed as enrichment factors, which represent the ratio of optical densities for control and experimental conditions. *P ⬍ 0.05 vs. AdCMVLacZ and vs. Mock.
the number of islets to 1000 IEQ, 55–58% of the animals that received unmodified islets or islets transfected with LacZ were euglycemic compared with 100% of the animals that received islets transfected with Bcl-2. With 500 IEQ, none of the animals that received unmodified islets or islets treated with AdCMVLacZ were euglycemic compared with 100% of the animals that received islets treated
with AdCMVBcl-2. Similar results were observed at 15 days post-transplant These results have a significant clinical implication because gene transfer of Bcl-2 potentially will reduce the number of cells per recipient required to achieve euglycemia without exogenous insulin administration. In summary, targeting the apoptosis pathway with gene transfer of Bcl-2 is an attractive cytoprotective alternative in pancreatic islets during culture time and early after transplantation. Gene transfer of anti-apoptotic genes is a potential new therapeutic approach for successful islet transplantation. METHODS Animals Normal, 4–6 kg male rhesus monkeys (Maccaca mulatta) were obtained from breeding colonies at LABS (Yamassee, SC), University of Wisconsin (Madison, WI), and Hazelton (Alice, TX). Male SCID mice (Jackson Laboratory, Bar Harbor, ME), 9 to 12 weeks old (25–30 g), were fed on a laboratory diet with water and food ad libitum. Surgical and nonsurgical procedures were carried out in accordance the National Institutes of Health Guide for the Care and Use of Primates under supervision of the Institutional Animal Care and Use Committee. SCID mice were made diabetic by a single intraperi-
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Fig. 4. Stable reversal of blood glucose in streptozotocin induced diabetic SCID mice after transplantation of Bcl-2 transfected NHP islets. Animals received a suboptimal dose of 1000 IEQ/mouse into the portal vein (N ⫽ 12 per group). NHP islets were not exposed to virus (Mock) or transfected with AdCMVLacZ or AdCMVhBcl-2. Tail vein blood glucose was determined as described in Methods.
Fig. 5. Recipients of Bcl-2 transfected NHP islets presented stable islet mass after transplantation. Glucose disposal rate after an intraperitoneal glucose tolerance test was performed as described in methods on day –2 (before the transplant), 3, 10, 15, and 30 after intraportal transplantation of 1000 IEQ/mouse. NHP islets were not transfected (mock or transfected with AdCMVLacZ or AdCMVhBcl-2). Tail vein blood glucose was determined after a glucose challenge as described in Methods. Data are means ⫾ SE for 6 animals from 3 different islet preparations.
toneal injection of freshly reconstituted 200 mg/kg of streptozotocin (Sigma). Recipient blood glucose levels were monitored by glucose oxidase strips using a Boehringer-Mannheim Accu Check III (blood obtained from the tail vein). Diabetes was defined as blood glucose levels ⬎300 mg/dL for 3 consecutive days. Normoglycemia was defined as non-fasting blood glucose ⬍200 mg/dL. Islet isolation Pancreata were procured from normal monkeys as described [33]. The pancreatic duct was cannulated to facili-
tate Liberase-HI (Boehringer-Mannheim, Indianapolis, IN) digestion. The islets were separated by the semiautomated technique and purified using a COBE-2991 Cell processor (COBE, Lakewood, CO). The time between pancreas procurement and islet isolation was routinely ⬍ 2 hours. The isolated islets were incubated in supplemented CMRL 1066 (Mediatech) and 10% heat-inactivated fetal calf serum (FCS) at 26⬚C with 5% CO2 for 12 hours before the gene transfer. Islet Equivalent number was assessed after dithizone staining and counting under the inverted microscope as described. The viability was determined by staining with ethidium bromide and acridine orange, then determined by two-color fluorescence microscopy. The routine number of IEQ/pancreas was 112,066 ⫾ 46,149 IEQ/donor. Only preparations with viability and purity ⬎90% were used. All the experiments were performed on the islets derived from a single donor in triplicate and repeated on at least four occasions from subsequent donors. Generation of a recombinant adenovirus vector and gene transfer A recombinant, E1 deleted adenovirus carrying the human Bcl-2 gene under the control of the cytomegalovirus promoter (AdCMVhBcl-2) was constructed as previously described [16]. A similar recombinant adenovirus encoding the reporter gene E coli ⫺galactosidase (AdCMVLacZ) was used as a control. A plaque assay on HeLa cells confirmed the absence of spontaneously generated replication competent adenovirus. Pancreatic islets were washed twice in CMRL 1066, supplemented as described previously with ⬍2% of FCS. 1000 or 2000 IEQ were infected in 24-well tissue plates at 500 particle forming units/cell in a minimum volume of 0.5 mL at 37⬚C. After 2 hours, CMRL 1066 supplemented as previously with 10% FCS was added and cultured overnight at 26⬚C and 5% CO2. After the infection, the islets
Contreras et al: Cytoprotection of pancreatic islets
were washed (3⫻) with CMRL 1066 solution and cultured for an additional 12 hours before being tested for further experiments. Untransfected islets were processed in parallel. For the long-term culture experiments, the medium and well plates were changed every second day. Islet viability after gene transfer was assessed as previously described. Transfection efficiency and transgene expression
8. 9. 10. 11.
Transfection rate was determined by the -galactosidase (Lac-Z) histochemical staining assay according to the instruction of the manufacturer. Immunoblot analysis of human Bcl-2 protein expression was performed as described [16, 19].
13.
Quantification of islet apoptosis by detection of DNA fragmentation by ELISA assay
15.
DNA fragmentation was measured using a sandwich enzyme-linked immunosorbent assay (ELISA, BoehringerMannheim) which detects cytosolic histone-associated DNA fragments (mono or oligonucleosomes) following the instruction of the manufacturer. Results were expressed as enrichment factor (EF), which represents the ratio of optical densities for control and experimental conditions. Islet transplantation and functional testing
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After genetic modification, 1000 or 2000 IEQ/mouse were transplanted into the portal vein as previously described [34]. For the Intraperitoneal Glucose Tolerance Test, after an overnight fast, mice were injected intraperitoneally with glucose (1.0 g/kg body weight). Blood samples were taken at various time points (0–60 min). Blood glucose concentrations were determined as previously described.
22.
Reprint requests to Judith M. Thomas, 1808 7th Avenue South, 563 Boshell Diabetes Building, Birmingham, AL 35294. E-mail: jthomas@ms_surgery.uab.edu
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REFERENCES 1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoidfree immunosuppressive regimen. N Engl J Med 343:230–238, 2000 2. Zwillich T: Diabetes research: Islet transplants not yet ready for prime time. Science 289:531–533, 2000 3. Hering B, Ricordi C: Results, research priorities, and reason for optimism: Islet transplantation for patients with type 1 diabetes. Graft 2:12–27, 2000 4. Kenyon N S., Ranuncoli A., Masetti M., Chatzipetrou M., Ricordi C: Islet transplantation: Present and future perspectives. Diabetes Metab Rev 14:303–313, 1998 5. Weir GC, Bonner-Weir S: Scientific and political impediments to successful islet transplantation. Diabetes 46:1247–1256, 1997 6. Carlsson PO, Liss P, Andersson A, Jansson L: Measurements of oxygen tension in native and transplanted rat pancreatic islets. Diabetes 47:1027–1032, 1998 7. Paraskevas S, Aikin R, Duguid WP, et al: Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of
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Langerhans: Implications for cultured islet survival. FEBS Lett 455:203–208, 1999 Rabinovitch A, Suarez-Pinzon WL, Shi Y, Morgan AR, Bleackley RC: DNA fragmentation is an early event in cytokineinduced islet beta-cell destruction. Diabetologia 37:733–738, 1994 Rosenberg L, Wang R, Paraskevas S, Maysinger D: Structural and functional changes resulting from islet isolation lead to islet cell death. Surgery 126:393–398, 1999 Thomas FT, Contreras JL, Bilbao G, et al: Anoikis, extracellular matrix, and apoptosis factors in isolated cell transplantation. Surgery 126:299–304, 1999 Frisch SM, Francis H: Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619–626, 1994 Frisch SM, Ruoslahti E: Integrins and anoikis. Curr Opin Cell Biol 9:701–706, 1997 Alejandro R, Cutfield RG, Shienvold FL, et al: Natural history of intrahepatic canine islet cell autografts. J Clin Invest 78:1339– 1348, 1986 Davalli AM, Ogawa Y, Ricordi C, et al: A selective decrease in the beta cell mass of human islets transplanted into diabetic nude mice. Transplantation 59:817–820, 1995 Kroemer G: The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3:614–620, 1997 Bilbao G, Contreras JL, Zhang HG, et al: Adenovirus-mediated gene expression in vivo is enhanced by the antiapoptotic bcl-2 gene. J Virol 73:6992–7000, 1999 Bilbao G, Contreras JL, Gomez-Navarro J, et al: Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation. Transplantation 67:775– 783, 1999 Bilbao G, Contreras JL, Eckhoff DE, et al: Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 230:185– 193, 1999 Bilbao G, Contreras JL, Mikheeva G, et al: Genetic cytoprotection of human endothelial cells during preservation time with an adenoviral vector encoding the anti-apoptotic human Bcl-2 gene. Transplant Proc 31:1012–1015, 1999 Dupraz P, Rinsch C, Pralong WF, et al: Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion. Gene Ther 6:1160–1169, 1999 Lacronique V, Mignon A, Fabre M, et al: Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat Med 2:80–86, 1996 Rabinovitch A, Suarez-Pinson W, Strynadka K, et al: Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes 48:1223–1229, 1999 Shimazaki K, Urabe M, Monahan J, et al: Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: Prospects for gene therapy of ischemia-induced neuronal death. Gene Ther 7:1244–1249, 2000 Shimizu S, Eguchi Y, Kosaka H, et al: Prevention of hypoxiainduced cell death by Bcl-2 and Bcl-xL. Nature 374:811–813, 1995 Yamabe K, Shimizu S, Kamiike W, Matsuda H: Prevention of hypoxic liver cell necrosis by in vivo human bcl-2 gene transfection. Biochem Biophys Res Commun 243:217–223, 1998 Leibowitz G, Beattie GM, Kafri T, et al: Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48:745– 753, 1999 Muruve DA, Manfro RC, Strom TB, Libermann TA: Ex vivo adenovirus-mediated gene delivery leads to long-term expression in pancreatic islet transplants. Transplantation 64:542–546, 1997 Lieber A, He CY, Meuse L, et al: Inhibition of NF-kappaB activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver. J Virol 72:9267–9277, 1998 Levine, F. Gene therapy for diabetes: Strategies for beta-cell modification and replacement. Diabetes Metab Rev 13:209–246, 1997 Paraskevas S, Duguid WP, Maysinger D, et al: Apoptosis occurs in freshly isolated human islets under standard culture conditions. Transplant Proc 29:750–752, 1997
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31. Elmer DS, Hataway DK, Bashar AA, et al: Use of glucose disappearance rates (kG) to monitor endocrine function of pancreas allografts. Clin Transplant 12:56–64, 1998 32. Teuscher AU, Kendall DM, Smets YF, et al: Successful islet autotransplantation in humans: Functional insulin secretory reserve as an estimate of surviving islet cell mass. Diabetes 47:324– 330, 1998
33. Thomas, FT, Ricordi C, Contreras JL, et al: Reversal of naturally occuring diabetes in primates by unmodified islet xenografts without chronic immunosuppression. Transplantation 67:846–854, 1999 34. Xenos ES, Stevens RB, Sutherland DE, et al: The role of nitric oxide in IL-1 beta-mediated dysfunction of rodent islets of Langerhans: Implications for the function of intrahepatic islet grafts. Transplantation 57:1208–1212, 1994
Cytoprotection of pancreatic islets before and early after transplantation using gene therapy JUAN L. CONTRERAS, GUADALUPE BILBAO, CHERYL A. SMYTH, DEVIN E. ECKHOFF, XIAO L. JIANG, STACIE JENKINS, FRANCIS T. THOMAS, DAVID T. CURIEL, and JUDITH M. THOMAS Transplant Center and Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA
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].
Insulin dependent diabetes mellitus (IDDM) is a disorder of insulin deficiency secondary to pancreatic -cell loss. Both the prevalence and incidence of diabetes in the U.S. have escalated in the last 25 years. IDDM is a chronic disease that may prompt a variety of complications with widespread morbidity and premature mortality. IDDM is the leading cause of blindness and kidney failure among adults in the U.S. IDDM is conventionally treated by exogenous insulin, however, the majority of patients develop one or more end-organ complications during their lives. Pancreas organ transplantation, on the other hand, can permanently reverse diabetes, but success is constrained by morbidity associated with major surgery Key words: islet transplantation, gene therapy, Bcl-2 gene.
2002 by the International Society of Nephrology
and by low availability of suitable donor organs. Pancreatic islet transplantation (PIT) by intraportal vein infusion can circumvent surgical disadvantages of organ transplantation but clinical application of PIT has been offset by low rates of success [3–5]. The discrepancy between duration of transplant function with whole pancreas organs and purified islets suggest a fundamental biologic difference in these grafts. Although isolated PIT is susceptible to complex biologic insults incurred during organ procurement and preservation, initial re-vascularization, proinflammatory cytokine release, immune rejection, and the diabetogenic effect of immunosuppressive drugs, the isolation procedure itself appears to induce profound changes in islet structure and function [3, 6–10]. Tissue-specific survival factors and/or the removal of the extracellular matrix, may be responsible for eliminating cells that end up in an ectopic location (i.e., anoikis) [10–12]. The final outcome of these processes is destruction of the transplanted islets, mainly by programmed cell death or apoptosis (Fig. 1). Experimental studies have demonstrated a significant selective decrease in the -cell mass early after transplantation in purified canine islet autografts and in human islets transplanted into nude mice [13, 14]. Apoptosis is a highly regulated process of cell death and is controlled through the expression of specific genes that are conserved in nematodes through mammals. Among these, the Bcl-2 gene family is one of the most important [15]. The protein encoded by the Bcl-2 gene has been implicated in the prolongation of cell survival by blocking apoptosis and necrosis. Gene transfer studies have shown that over-expression of Bcl-2 can protect cells from death induced by a wide variety of diverse insults and stimuli [10, 15–25]. Given the functional importance of Bcl-2 gene, it constitutes a prime target for therapeutic intervention in numerous disease states. Bcl-2 is expressed in pancreatic islets and its possible that contributes to the survival capacity of the islets. Over-
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Contreras et al: Cytoprotection of pancreatic islets
Fig. 1. Vulnerability of pancreatic islets. Isolated pancreatic islets are susceptible to complex biological insults incurred during pancreas procurement and islet isolation, poor vascularization early after transplantation, exposure to proinflammatory cytokines and other immune mediated injuries, hyperglycemia, and the use of diabetogenic immunosuppressive drugs. The final outcome of these processes is destruction of the transplanted islets mainly by apoptosis.
expression of Bcl-2 in human islets and in insulinoma cells prevents apoptosis induced by IL-1, IFN-␥, and TNF-␣ and also induces cytoprotection from hypoxia [20, 22]. Replication-deficient adenoviral vectors are the most efficient vectors currently available for transducing nondividing cells and can be produced in high titers; their epichromosomal location diminishes the risk of insertional mutagenesis. E-1 deleted adenovirus vectors have been tested in PI [26, 27]. Recently, we developed a replication-deficient adenoviral vector encoding the human anti-apoptotic Bcl-2 gene [16]. Gene transfer of Bcl-2 induced cytoprotection of endothelial cells and liver grafts during routine preservation in University of Wisconsin solution for organ transplantation and after reperfusion [17–19]. Over-expression of Bcl-2 decreases the injury associated with an immune response generated by adenoviral vectors after in vivo gene transfer to the liver [16, 28]. In our laboratory, we evaluated the potential of genetic modification of nonhuman primate (NHP) pancreatic islets by gene transfer of Bcl-2 as an approach to prevent loss of pancreatic -cells before and early after transplantation. Numerous publications have demonstrated that adenoviruses are very efficient at infecting human and rodent islets ex vivo [26, 27, 29]. However, no experience in gene transfer in nonhuman primate islets has been reported. Initial experiments in our laboratory demonstrated that ⬎95% of the cells in non-human primate islets expressed the transgene after genetic modification with adenoviral vectors at 500 pfu/cell (Fig. 2A, B, C, and D). Bcl-2 expression after gene transfer with an adenoviral vector (AdBcl-2) was confirmed by Western blot (Fig. 2E). An immunohistochemical analysis after genetic modification
of PI with AdBcl-2 showed that ⬎95% of the insulin and non-insulin producing cells over-express Bcl-2 compared with only 10% of the cells without genetic modification (data not shown). After demonstration of high efficient gene transfer in NHP islets, we sought the potential cytoprotective properties of the Bcl-2 gene. DNA fragmentation is the biochemical marker of apoptosis [15]. As previously reported in human islets, apoptosis is a frequent event during standard culture before transplantation [7, 9, 10, 30]. Islet cell DNA fragmentation assessed by ELISA assay during culture time is shown in Figure 3. A significant reduction in DNA fragmentation was observed at 7 days in culture in PI genetically modified to overexpress Bcl-2 compared with AdCMVLacZ (irrelevant gene) or untransfected islets (P ⬍ 0.05). These results demonstrate the cytoprotective properties of the antiapoptotic Bcl-2 gene in NHP islets. Initial experiments in our laboratory demonstrated that 2000 Islet Equivalents (IEQ) NHP islets per mouse transplanted into the portal vein is the “optimal” dose for restoration of normoglycemia one day after the transplant in streptozotocin (STZ)-induced SCID mice. Reducing the number of unmodified islets per mouse to 1000 IEQ, 58% of the recipients were found euglycemic on day 1 postransplant, and only 8% on day 100 (Fig. 4). Similar results were observed in recipients of islets that over-express an irrelevant gene (euglycemia in 50% on day 1, 8% on day 100). A significant difference in the percentage of euglycemic animals early and late post-transplant was observed in recipients of islets that over-expressed Bcl-2 (Fig. 4, P ⬍ 0.005 compared with AdCMVLacZ or unmodified islets). All recipients of a “marginal dose” of Bcl-2 islets presented euglycemia the day after the transplant and 83% on day 100. These results demonstrate the ability of Bcl-2 transfected islets to reverse diabetes in STZ-induced SCID mice even after the transplant of a marginal number of cells. A more sophisticated form of metabolic evaluation of the transplanted islets is demonstrated in Figure 5. The glucose disposal rate (Kg), a measure of functional islet mass, became progressively reduced over time, indicating loss of islet mass [31, 32]. By 30 days after the transplant, glucose disposal rate was reduced to pre-transplant diabetic values in the two control groups (unmodified islets or islets transfected with LacZ). In contrast, no significant change in the capacity to dispose glucose was observed in animals that received islets transduced with Bcl-2. One of the current limitations in clinical islet transplantation is the availability of cadaver organs [2, 3, 5]. After demonstration of the cytoprotective effects of Bcl-2, next we sought the islet mass required to establish stable euglycemia after transplantation. Most of the diabetic recipients that received 2000 IEQ were found euglycemic 3 and 15 days post-transplantation. Reducing
Contreras et al: Cytoprotection of pancreatic islets
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Fig. 2. Transfection efficiency of adenoviral vectors in non-human primate pancreatic islets. NHP islets were infected ex vivo with different concentrations of an E1-deleted adenoviral vector encoding -galactosidase (A ⫽ none, B ⫽ 10 pfu/cell, C ⫽ 100 pfu/cell, and D 500 ⫽ pfu/cell). An immunohistochemical analysis demonstrated that more than 95% of the cells expressed the reporter gene (LacZ). Bcl-2 expression after gene transfer with similar E1-deleted adenoviral vector encoding the human Bcl-2 gene was confirmed by Western blot (E). Immunoblot analysis was performed as described in Methods. Lane 1: positive control; Lane 2: NHP islets transfected with an adenovirus vector encoding an irrelevant gene (LacZ); Lane 3: NHP islets transfected with AdCMVhBcl-2.
Fig. 3. Bcl-2 transfected islets analysis for spontaneous apoptosis during culture by ELISA. NHP islets were incubated without virus (mock), AdCMVLacZ, or AdCMVhBcl-2 and cultured as described in Methods. DNA fragmentation was assessed at 7 days. Data are mean ⫾ SE for islets from 3 different isolations and are expressed as enrichment factors, which represent the ratio of optical densities for control and experimental conditions. *P ⬍ 0.05 vs. AdCMVLacZ and vs. Mock.
the number of islets to 1000 IEQ, 55–58% of the animals that received unmodified islets or islets transfected with LacZ were euglycemic compared with 100% of the animals that received islets transfected with Bcl-2. With 500 IEQ, none of the animals that received unmodified islets or islets treated with AdCMVLacZ were euglycemic compared with 100% of the animals that received islets treated
with AdCMVBcl-2. Similar results were observed at 15 days post-transplant These results have a significant clinical implication because gene transfer of Bcl-2 potentially will reduce the number of cells per recipient required to achieve euglycemia without exogenous insulin administration. In summary, targeting the apoptosis pathway with gene transfer of Bcl-2 is an attractive cytoprotective alternative in pancreatic islets during culture time and early after transplantation. Gene transfer of anti-apoptotic genes is a potential new therapeutic approach for successful islet transplantation. METHODS Animals Normal, 4–6 kg male rhesus monkeys (Maccaca mulatta) were obtained from breeding colonies at LABS (Yamassee, SC), University of Wisconsin (Madison, WI), and Hazelton (Alice, TX). Male SCID mice (Jackson Laboratory, Bar Harbor, ME), 9 to 12 weeks old (25–30 g), were fed on a laboratory diet with water and food ad libitum. Surgical and nonsurgical procedures were carried out in accordance the National Institutes of Health Guide for the Care and Use of Primates under supervision of the Institutional Animal Care and Use Committee. SCID mice were made diabetic by a single intraperi-
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Fig. 4. Stable reversal of blood glucose in streptozotocin induced diabetic SCID mice after transplantation of Bcl-2 transfected NHP islets. Animals received a suboptimal dose of 1000 IEQ/mouse into the portal vein (N ⫽ 12 per group). NHP islets were not exposed to virus (Mock) or transfected with AdCMVLacZ or AdCMVhBcl-2. Tail vein blood glucose was determined as described in Methods.
Fig. 5. Recipients of Bcl-2 transfected NHP islets presented stable islet mass after transplantation. Glucose disposal rate after an intraperitoneal glucose tolerance test was performed as described in methods on day –2 (before the transplant), 3, 10, 15, and 30 after intraportal transplantation of 1000 IEQ/mouse. NHP islets were not transfected (mock or transfected with AdCMVLacZ or AdCMVhBcl-2). Tail vein blood glucose was determined after a glucose challenge as described in Methods. Data are means ⫾ SE for 6 animals from 3 different islet preparations.
toneal injection of freshly reconstituted 200 mg/kg of streptozotocin (Sigma). Recipient blood glucose levels were monitored by glucose oxidase strips using a Boehringer-Mannheim Accu Check III (blood obtained from the tail vein). Diabetes was defined as blood glucose levels ⬎300 mg/dL for 3 consecutive days. Normoglycemia was defined as non-fasting blood glucose ⬍200 mg/dL. Islet isolation Pancreata were procured from normal monkeys as described [33]. The pancreatic duct was cannulated to facili-
tate Liberase-HI (Boehringer-Mannheim, Indianapolis, IN) digestion. The islets were separated by the semiautomated technique and purified using a COBE-2991 Cell processor (COBE, Lakewood, CO). The time between pancreas procurement and islet isolation was routinely ⬍ 2 hours. The isolated islets were incubated in supplemented CMRL 1066 (Mediatech) and 10% heat-inactivated fetal calf serum (FCS) at 26⬚C with 5% CO2 for 12 hours before the gene transfer. Islet Equivalent number was assessed after dithizone staining and counting under the inverted microscope as described. The viability was determined by staining with ethidium bromide and acridine orange, then determined by two-color fluorescence microscopy. The routine number of IEQ/pancreas was 112,066 ⫾ 46,149 IEQ/donor. Only preparations with viability and purity ⬎90% were used. All the experiments were performed on the islets derived from a single donor in triplicate and repeated on at least four occasions from subsequent donors. Generation of a recombinant adenovirus vector and gene transfer A recombinant, E1 deleted adenovirus carrying the human Bcl-2 gene under the control of the cytomegalovirus promoter (AdCMVhBcl-2) was constructed as previously described [16]. A similar recombinant adenovirus encoding the reporter gene E coli ⫺galactosidase (AdCMVLacZ) was used as a control. A plaque assay on HeLa cells confirmed the absence of spontaneously generated replication competent adenovirus. Pancreatic islets were washed twice in CMRL 1066, supplemented as described previously with ⬍2% of FCS. 1000 or 2000 IEQ were infected in 24-well tissue plates at 500 particle forming units/cell in a minimum volume of 0.5 mL at 37⬚C. After 2 hours, CMRL 1066 supplemented as previously with 10% FCS was added and cultured overnight at 26⬚C and 5% CO2. After the infection, the islets
Contreras et al: Cytoprotection of pancreatic islets
were washed (3⫻) with CMRL 1066 solution and cultured for an additional 12 hours before being tested for further experiments. Untransfected islets were processed in parallel. For the long-term culture experiments, the medium and well plates were changed every second day. Islet viability after gene transfer was assessed as previously described. Transfection efficiency and transgene expression
8. 9. 10. 11.
Transfection rate was determined by the -galactosidase (Lac-Z) histochemical staining assay according to the instruction of the manufacturer. Immunoblot analysis of human Bcl-2 protein expression was performed as described [16, 19].
13.
Quantification of islet apoptosis by detection of DNA fragmentation by ELISA assay
15.
DNA fragmentation was measured using a sandwich enzyme-linked immunosorbent assay (ELISA, BoehringerMannheim) which detects cytosolic histone-associated DNA fragments (mono or oligonucleosomes) following the instruction of the manufacturer. Results were expressed as enrichment factor (EF), which represents the ratio of optical densities for control and experimental conditions. Islet transplantation and functional testing
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16. 17.
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19.
After genetic modification, 1000 or 2000 IEQ/mouse were transplanted into the portal vein as previously described [34]. For the Intraperitoneal Glucose Tolerance Test, after an overnight fast, mice were injected intraperitoneally with glucose (1.0 g/kg body weight). Blood samples were taken at various time points (0–60 min). Blood glucose concentrations were determined as previously described.
22.
Reprint requests to Judith M. Thomas, 1808 7th Avenue South, 563 Boshell Diabetes Building, Birmingham, AL 35294. E-mail: jthomas@ms_surgery.uab.edu
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