- Email: [email protected]
Enhanced Gene Transfer to Pancreatic Islets Using Glucagon-Like Peptide-1
Enhanced Gene Transfer to Pancreatic Islets Using Glucagon-Like Peptide-1 B.W. Lee,* M.-H. Kim,* H.Y. Chae, H.J. Hwang, D. Kang, and S.H. Ihm ABSTRACT Objective. The efficient transfer of genes into intact islets is difficult since islets exist as clusters of differentiated cells with little replication potential. Cell proliferation in response to growth factors is known to be accompanied by loosening of cell-to-cell contacts and increasing paracellular permeability. In this study, we investigated whether gene delivery into intact islet cells was facilitated by modulating -cell proliferation. Methods. Isolated rat islets were pretreated with glucagon-like peptide (GLP)-1 or human growth hormone for 24 hours, or with 300 mg/dL of glucose for 48 hours before transduction with a suboptimal dose of recombinant adenoviral vector expressing green fluorescent protein (GFP) and -galactosidase (multiplicity of infection of 25). Transduction efficiency was assessed by measuring -galactosidase activity and GFP expression using enzyme-linked immunosorbent assay, flow cytometry, and fluorescence microscopy. The numbers of 7-aminoactinomycin D-positive dead cells and 5-ethynyl-2-deoxyuridine (EdU)-positive proliferating cells were also monitored using flow cytometry and fluorescence microscopy. Results. The transduction efficiency of rat islet cells by a suboptimal dose of viral vector was significantly improved by GLP-1 pretreatment, accompanied by enhanced cell viability and cell proliferation. An increased GFP expression in islet cells after GLP-1 pretreatment was observed among the increased numbers of EdU-positive proliferating cells. Conclusion. Pretreatment of rat islets with GLP-1 enhanced the transduction efficiency of an adenoviral vector, reducing viral dose burden while improving islet cell viability. From a therapeutic standpoint, genetic modification of pancreatic islets combined with GLP-1 pretreatment may be a promising option for ex vivo gene therapy prior to islet transplantation. ANCREATIC ISLET TRANSPLANTATION is a promising therapeutic intervention for insulin-deficient diabetes. One significant obstacle to successful islet transplantation is an early and profound loss of transplanted islets from hypoxic and inflammatory insults during the first few days
P
after implantation.1– 4 To improve survival rates of transplanted islets, ex vivo cytoprotective gene transfer strategies have been investigated.5– 8 However, the efficient transfer of genes into intact islets is difficult since islet cells exist as clusters of differentiated cells with very little replication
From the Department of Internal Medicine (B.W.L., H.Y.C., H.J.H., S.-H.I.), Hallym University College of Medicine, Chuncheon, Korea; and Ilsong Institute of Life Science (M.-H.K., D.K.), Hallym University, Anyang, Korea. *B.W.L. and M.-H.K. contributed equally. Current address for B.W. Lee is Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea.
This work was supported by research grants from the Innovative Research Institute for Cell Therapy, Republic of Korea (A062260) and from Hallym University Medical Center Research Fund (01-2009-14) to S.H.I. Address reprint requests to Sung-Hee Ihm, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896 Pyungchon-dong, Dongan-gu, Anyang, Kyonggi-do 431-070, Korea. E-mail: [email protected]
© 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 591–596 (2013)
0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.10.040 591
592
potential.9 Even with adenoviral vectors, islet transfection efficiency is low, and only cells located in the peripheral region of an islet are targeted.7,10 The successful application of gene therapy to islet modification is currently limited by inefficient gene delivery. Several recent studies indicate that functional -cell mass is dynamic, maintaining euglycemia through a balance of apoptosis and proliferation. Islet cell numbers can increase in response to insulin demand, certain growth factors, and hormones.11–14 This property has been demonstrated in both physiological and pathophysiological conditions, such as pregnancy and obesity.15,16 Many exogenous and endogenous growth factors, including prolactin, placental lactogen, hepatocyte growth factor, parathyroid hormonerelated protein, glucagon-like peptide (GLP)-1, exendin-4, and insulin-like growth factor-I, are known to increase -cell proliferation in vivo and in vitro.17–26 Glucose can also play a key regulatory role in pancreatic -cell plasticity, as a potent stimulus of -cell growth both in vivo and in vitro.27–30 It is known that cell proliferation in response to growth factors is accompanied by loosening of cell-to-cell contact and increasing paracellular permeability.31,32 Thus, we hypothesized that gene delivery into intact islet cells could be facilitated by modulating -cell proliferation potential with these agents. In this study, we investigated the effect of growth hormone, GLP-1, and high glucose on cell proliferation and gene transduction efficiency in pancreatic -cells. MATERIALS AND METHODS Islet Isolation Pancreatic islets were isolated from male Sprague-Dawley rats (200 –250 g) by digestion using 1 mg/mL collagenase P (Roche, Indianapolis, Ind, USA), separated by discontinuous Ficoll gradient purification (Biochrom AG, Berlin, Germany), and cultured free-floating in Medium 199 (Gibco, Grand Island, NY, USA). Individual islets were collected using micropipettes.
Adenoviral Vectors and Transduction The recombinant adenoviral vector Ad-EGFP-LacZ expresses green fluorescent protein (GFP) and -galactosidase (LacZ) under the control of the cytomegalovirus promoter. To prepare adenoviruses, Ad-EGFP-LacZ was propagated in HEK293 cells and purified by the AdEasy system (Stratagene, La Jolla, Calif, USA). Multiplicity of infection (MOI) was determined by optical density at 260 nm and by plaque assay. MOI calculations assumed 1000 cells per islet equivalent. Transduction was accomplished by incubating aliquots of isolated islets in serum-free medium for 4 hours in the presence of virus at different MOIs ranging from 0 to 200 with agitation every 1 hour. To examine the effect of hormones and high glucose on cell proliferation and transduction efficiency, islets were pretreated with 10 nmol/L of GLP-1 or 1 g/mL of human growth hormone (hGH; (Sigma-Aldrich, St Louis, Mo, USA) for 24 hr, or with 300 mg/dL of glucose for 48 hours, before transduction at an MOI of 25.
Assessment of Transduction Efficiency -galactosidase activity assay. After 24 hours of transduction with Ad-EGFP-LacZ, -galactosidase activity was measured in islet cell
LEE, KIM, CHAE ET AL lysates with 2-N- phenyl--D-galactopyranoside as a colorimetric substrate, according to the method previously described.33 Briefly, after transduction, islets were washed twice with phosphate-buffered saline and lysed in buffer containing protease inhibitors. The lysate was reacted with substrate buffer (60 mmol/L Na2HPO4, 40 mmol/L NaH2PO4, 1 mg/mL 2-N-phenyl--D-galactoside, 2.7 L/mL -mercaptoethanol) in a 96-well plate for 6 hours at 37°C. The color development was stopped by the addition of 1 mmol/L Na2CO3, and the absorbance of the samples was read at 420 nm. Protein determination was performed with a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, Ill, USA). B-galactosidase activity of the samples was expressed relative to that of mocktransduced islet cell lysate. GFP fluorescence. Islets were examined for GFP expression 24 hours after transduction using fluorescence microscopy. In addition, a quantitative analysis of transduction was performed on disassociated islet cells (dissociation buffer; Gibco, Grand Island, NY, USA) by acquiring 50,000 cells per treatment on a flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA) to determine the percentage of cells expressing GFP. All experiments were performed in triplicate.
Quantification of Islet Cell Death and Proliferation Transduced islets were harvested and dissociated into a single-cell suspension. Quantitative analysis of cell death was performed on dissociated islet cells by flow cytometry using 7-aminoactinomycin D (7-AAD, Sigma) and at least 50,000 cells per sample. To measure the percentage of proliferating cells in islet cell suspensions, 5-ethynyl-2-deoxyuridine (Edu; Click-iT EdU, Invitrogen, Eugene, Ore, USA) was used to stain cells for flow cytometry. Intact transduced islets were also examined for EdU incorporation using fluorescence microscopy.
Statistical Analysis Statistical analysis was performed using PRISM (GraphPad Software Inc, San Diego, Calif, USA). Results are expressed as means ⫾ standard deviations. A one-way analysis of variance, Bonferroni post hoc multiple comparison test, and Student t test were used where appropriate to compare variables. Statistical significance was defined P values below .05.
RESULTS Effect of Adenoviral Dose on Transduction Efficiency into Islets
To identify appropriate vector doses for the cell proliferation experiment, aliquots of freshly isolated rat islets were transduced with the Ad-EGFP-LacZ vector at MOIs of 12.5, 25, 50, 100, or 200 or were mock transduced with buffer. After 4 hours of transduction, islets were harvested and -galactosidase activity was measured in islet cell lysates to determine the efficiency of islet cell transduction. Under these experimental conditions, Ad-EGFP-LacZ transduction of islets was dependent upon viral dose up to 100 MOI (Fig 1). Using a higher viral dose (200 MOI) was somewhat detrimental to gene transfer into rat islets. The vector showed minimal transduction efficiency at MOIs of 12.5 and 25, and significantly higher efficiency at MOIs of 50 and 100, as compared to the mock-transduced control (both P ⬍ .001). In subsequent experiments, we used an MOI of
ENHANCED GENE TRANSFER
Fig 1. Transduction efficiency of the Ad-EGFP-LacZ vector at various multiplicity of infection (MOI) in rat islets, as assessed by -galactosidase (LacZ) activity. The transduction efficiency was minimal at MOIs of 12.5 and 25 and significantly higher than the mock-transduced control at MOIs of 50 and 100. *P ⬍ .001.
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dure, islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at an MOI of 25. They were dispersed to single islet cells, and stained with 7-AAD or EdU to determine viability and proliferation status, respectively. In the flow cytometry analysis, the transduction efficiency of rat islets pretreated with 10 nmol/L of GLP-1 was significantly higher than that of control islets (P ⬍ .01, Fig 4A), confirming our results obtained by -galactosidase assay and fluorescence microscopy. The number of 7-AAD-positive dead islet cells was significantly decreased with GLP-1 pretreatment (P ⬍ 0.05, Fig 4B), and the number of EdU-positive proliferating cells was significantly increased (P ⬍ .05, Fig 4C). Thus, the transduction efficiency into rat islet cells of a suboptimal dose of viral vector was significantly improved by GLP-1 pretreatment and was accompanied by enhanced cell viability and cell proliferation. Interestingly, the increased GFP expression in GLP-1 pretreated islet cells was observed among the increased EdU-positive proliferating cells (Fig 5). DISCUSSION
25 as a suboptimal viral dose in order to examine the effect of hGH, GLP-1, and high glucose on islet transduction efficiency. Effect of hGH, GLP-1, and High Glucose on the Transduction Efficiency Into Islets
To examine effects on transduction efficiency, islets were pretreated with 1 g/mL of hGH or 10 nmol/L of GLP-1 for 24 hours, or with 300 mg/dL of glucose for 48 hours, alone or in combination, before transduction with the Ad-EGFPLacZ vector. At an MOI of 25, pretreatment with GLP-1, high glucose, or GLP-1 and high glucose led to significantly higher gene transduction efficiency in -galactosidase activity assay, as compared with untreated control islets (Fig 2). Transduction efficiency after pretreatment with the combination of GLP-1 and high glucose was higher than with glucose alone (P ⬍ .01), but not significantly different from that seen with GLP-1 pretreatment alone. Ad-EGFP-LacZ transduction at an MOI of 25 in GLP-1 or GLP-1/high glucose-pretreated islets was comparable to that seen at an MOI of 100 without pretreatment (Fig 1). These results demonstrate strong GLP-1-mediated enhancement of gene transfer into rat islets. The effect of hGH, GLP-1, and high glucose on transduction efficiency in islets was also assessed by GFP expression (Fig 3). In concordance with the -galactosidase activity, rat islets without pretreatment showed very low GFP expression at an MOI of 25. Pretreatment with GLP-1 or GLP-1/high glucose resulted in noticeably higher GFP expression. Effect of GLP-1 on Islet Cell Viability and Proliferation
To examine the effects of GLP-1 pretreatment on islet cell viability and proliferation during the transduction proce-
Ex vivo genetic modification of pancreatic islets to enhance survival during early posttransplant period is a plausible strategy in islet transplantation. At present, the lack of an efficient and safe vector system is a bottleneck in the
Fig 2. Transduction efficiency of the Ad-EGFP-LacZ vector at an MOI of 25 in rat islets, as assessed by -galactosidase activity. Islets were pretreated with 1 g/mL of human growth hormne (hGH) or 10 nmol/L of glucagon-like peptide (GLP)-1 for 24 hours, or 300 mg/dL of glucose for 48 hours, alone or in combination, prior to transduction. Pretreatment with GLP-1 or high glucose led to significantly higher gene transduction efficiency as compared with the controls. Transduction efficiency after pretreatment with the combination of GLP-1 and high glucose was higher than with glucose alone, but not significantly different from that seen with GLP-1 pretreatment alone. *P ⬍ .001; **P ⬍ .01.
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LEE, KIM, CHAE ET AL
Fig 3. Transduction efficiency of the Ad-EGFP-LacZ vector at a multiplicity of infection of 25 in rat islets, as assessed by green fluorescent protein (GFP) expression. Under fluorescence microscopy, rat islets without pretreatment showed very low GFP expression. Pretreatment with glucagon-like peptide (GLP)-1 or GLP-1/high glucose resulted in noticeably higher GFP expression. hGH, human growth hormone.
development of gene transfer to pancreatic islets. Thus, the successful application of gene therapy in clinical islet transplantation depends mainly on the development of gene delivery vectors. Adenoviral vectors, which have been commonly used for gene transfer, show higher transfection efficiency in islets than do nonviral carriers, although the latter raise fewer safety issues.7,10,34 Previous studies have reported that in vitro transduction of intact rat and human islets using adenoviral vectors promoted GFP expression in 30% of cells, at most.35 Moreover, confocal sectioning of intact islets demonstrated that only cells located in the outermost layer were successfully targeted. Islets are compact clusters of about 1000 rarely dividing cells. In normal islet morphology, the cells of the inner core are much less exposed to transfection reagents than are cells in the peripheral region, due to diffusion barriers. To overcome
this physical constraint of accessibility, islets have often been dissociated to single cells before transduction, resulting in improved transduction efficiency.34,36 However, islet dispersion prior to genetic modification to enhance islet survival does not seem to be a viable option in clinical islet transplantation. It is known that cell proliferation in response to growth factors is accompanied by loosening of cell-to-cell contact and increasing paracellular permeability.31,32 In this study, we tested the hypothesis that gene delivery into intact islet cells would be facilitated by modulating -cell proliferation with hormones or high glucose, using an adenoviral-mediated gene delivery system to transfer EGFP and LacZ genes to rodent islets. Ad-EGFP-LacZ transduction of islets showed a viral dose-dependent increase up to an MOI of 100 MOI; an MOI of 25 was a suboptimal viral dose for islet trans-
Fig 4. Effect of glucagon-like peptide (GLP)-1 on Ad-EGFP-LacZ transduction, islet cell viability and proliferation. Islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at a multiplicity of infection of 25, and were dispersed to single islet cells, followed by 7-aminoactinomycin D (7-AAD) or 5-ethynyl-2-deoxyuridine (EdU) staining. (A) In a flow cytometry analysis, the transduction efficiency of rat islets pretreated with 10 nmol/L of GLP-1 was higher than that of untreated control islets. (B) 7-AAD-positive dead islet cells were decreased by GLP-1 pretreatment. (C) EdU-positive proliferating islet cells were increased by GLP-1 pretreatment. *P ⬍ .01; **P ⬍ .05.
ENHANCED GENE TRANSFER
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Fig 5. Effect of glucagon-like peptide (GLP)-1 on Ad-EGFPLacZ transduction and cell proliferation in rat islets. Islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at a multiplicity of infection of 25, and stained with 5-ethynyl-2deoxyuridine (EdU). (A) Under fluorescence microscopy, rat islets without pretreatment showed very low GFP expression and scanty EdU-positive cells. (B) GLP-1 pretreatment increased both GFP expression of islet cells and numbers of EdU-positive proliferating cells.
duction. At the MOI, pretreatment of islets with GLP-1, high glucose, or the combination of GLP-1 and high glucose led to significantly higher gene transduction efficiencies, when assessed by either -galactosidase activity or GFP expression. While combined pretreatment with GLP-1/high glucose led to higher efficiency than did glucose alone (P ⬍ .01), it was not significantly different from GLP-1 pretreatment, indicating the GLP-1 is more effective in enhancing transduction efficiency than is glucose. Interestingly, transduction at an MOI of 25 in GLP-1 or GLP-1/high glucose-pretreated islets was comparable to that of an MOI of 100 without pretreatment (Fig 1). Thus, GLP-1 treatment of islets before transduction may reduce the optimal adenoviral dose for efficient target gene delivery. Considering that the immunogenicity and possible toxicity of high adenoviral vector titers are the main limiting factors in their application to clinical transplantation, GLP-1 pretreatment of islets to reduce viral load for transduction may make adenoviral vectors competitive with nonviral vectors in terms of safety. In our results, GLP-1 treatment of rat islets at 10 nmol/L for 24 hours increased the number of EdU-positive proliferating islet cells, while enhancing the transduction efficiency of rat islet cells with a suboptimal dose of viral vector. Because adenoviral vectors transfect both dividing and nondividing cells, we assume that increased cell proliferation leads to enhanced adenoviral vector accessibility to islet cells through cell-proliferation-associated loosening of cell-to-cell contacts and increasing paracellular permeability. As GLP-1 has been shown not only to stimulate -cell proliferation, but also to have antiapoptotic effects on these cells,37,38 we examined the effect of GLP-1 pretreatment on islet cell viability during the transduction procedure. In a FACS analysis, 7-AAD-positive dead islet cells were signif-
icantly decreased by GLP-1 pretreatment, showing an additional beneficial effect of GLP-1 in islet transduction. Under our experimental conditions, although GLP-1 pretreatment significantly improved the transduction efficiency of rat islet cells with a suboptimal dose of adenoviral vector, transduction efficiency was still low, as shown by GFP FACS analysis. However, depending on the target gene chosen and the therapeutic intervention intended, this degree of transduction efficiency with improved islet viability and reduced safety issues could suffice. For example, the modification of pancreatic islets with this protocol may be more feasible with genes for secretory proteins than with those for cell-resident proteins. Further studies to determine if GLP-1 pretreatment continues to enhance transduction efficiency above a 25 MOI dose of adenoviral vector is needed. In conclusion, the pretreatment of rat islets with GLP-1 enhanced the transduction efficiency of an adenoviral vector, reducing adenoviral dose burden while improving islet viability. From a therapeutic standpoint, genetic modification of pancreatic islets combined with GLP-1 pretreatment may be a promising option for ex vivo gene therapy in islet transplantation.
REFERENCES 1. Korsgren O, Lundgren T, Felldin M, et al. Optimising islet engraftment is critical for successful clinical islet transplantation. Diabetologia. 2008;51:227. 2. Davalli AM, Scaglia L, Zangen DH, et al. Vulnerability of islets in the immediate posttransplantation period. Dynamic changes in structure and function. Diabetes. 1996;45:1161. 3. Biarnes M, Montolio M, Nacher V, et al. Beta-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes. 2002;51:66.
596 ´ , Stroka DM, et al. Apoptosis in hypoxic 4. Moritz W, Meier F human pancreatic islets correlates with HIF-1 alpha expression. FASEB J. 2002;16:745. 5. Levine F. Gene therapy for diabetes: strategies for beta-cell modification and replacement. Diabetes Metab Rev. 1997;13:209. 6. Narang AS, Mahato RI. Biological and biomaterial approaches for improved islet transplantation. Pharmacol Rev. 2006; 58:194. 7. Lai Y, Drobinskaya I, Kolossov E, et al. Genetic modification of cells for transplantation. Adv Drug Deliv Rev. 2008;60:146. 8. Lee BW, Lee M, Chae HY, et al. Effect of hypoxia-inducible VEGF gene expression on revascularization and graft 1 function in mouse islet transplantation. Transpl Int. 2011;24:307. 9. Teta M, Long SY, Wartschow LM, et al. Very slow turnover of -cells in aged adult mice. Diabetes. 2005;54:2557. 10. Sigalla J, David A, Anegon I, et al. Adenovirus-mediated gene transfer into isolated mouse adult pancreatic islets: normal beta-cell function despite induction of an anti-adenovirus immune response. Hum Gene Ther. 1997;8:1625. 11. Bonner-Weir S. Life and death of the pancreatic beta cells. Trends Endocrinol Metab. 2000;11:375. 12. Teta M, Rankin MM, Long SY, et al. Growth and regeneration of adult  cells does not involve specialized progenitors. Dev Cell. 2007;12:817. 13. Dor Y, Brown J, Martinez OI, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature. 2004;429:41. 14. Scaglia L, Cahill CJ, Finegood DT, et al. Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat. Endocrinology. 1997;138:1736. 15. Parsons JA, Brelje TC, Sorenson RL. Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology. 1992;130:1459. 16. Montanya E, Nacher V, Biarnes M, et al. Linear correlation between -cell mass and body weight throughout the lifespan in Lewis rats: role of -cell hyperplasia and hypertrophy. Diabetes. 2000;49:1341. 17. Nielsen JH, Galsgaard ED, Møldrup A, et al. Regulation of beta-cell mass by hormones and growth factors. Diabetes. 2001; 50(Suppl 1):S25. 18. Freemark M, Avril I, Fleenor D, et al. Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology. 2002;143:1378. 19. Vasavada RC, Garcia-Ocaña A, Zawalich WS, et al. Targeted expression of placental lactogen in the  cells of transgenic mice results in  cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem. 2000;275:15399. 20. Fujinaka Y, Sipula D, Garcia-Ocana A, et al. Characterization of mice doubly transgenic for parathyroid hormone-related protein and murine placental lactogen: a novel role for placental lactogen in pancreatic -cell survival. Diabetes. 2004;53:3120. 21. García-Ocaña A, Vasavada RC, Cebrian A, et al. Transgenic overexpression of hepatocyte growth factor in the - cell markedly improves islet function and islet transplant outcomes in mice. Diabetes. 2001;50:2752.
LEE, KIM, CHAE ET AL 22. Villanueva-Peñacarrillo ML, Cancelas J, de Miguel F, et al. Parathyroid hormone-related peptide stimulates DNA synthesis and insulin secretion in pancreatic islets. J Endocrinol. 1999;163: 403. 23. Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes. 2000;49:741. 24. Friedrichsen BN, Neubauer N, Lee YC, et al. Stimulation of pancreatic beta-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways. J Endocrinol. 2006;188:481. 25. Buteau J, Foisy S, Rhodes CJ, et al. Protein kinase Czeta activation mediates glucagon-like peptide-1-induced pancreatic -cell proliferation. Diabetes. 2001;50:2237. 26. Beith JL, Alejandro EU, Johnson JD. Insulin stimulates primary beta-cell proliferation via Raf-1 kinase. Endocrinology. 2008;149:2251. 27. Bernard C, Thibault C, Berthault MF, et al. Pancreatic -cell regeneration after 48-h glucose infusion in mildly diabetic rats is not correlated with functional improvement. Diabetes. 1998;47: 1058. 28. Paris M, Bernard-Kargar C, Berthault MF, et al. Specific and combined effects of insulin and glucose on functional pancreatic beta-cell mass in vivo in adult rats. Endocrinology. 2003;144:2717. 29. Bonner-Weir S, Deery D, Leahy JL, et al. Compensatory growth of pancreatic beta-cells in adult rats after short-term glucose infusion. Diabetes. 1989;38:49. 30. Alonso LC, Yokoe T, Zhang P, et al. Glucose infusion in mice. A new model to induce beta-cell replication. Diabetes. 2007;56:1792. 31. Gumbiner BM, Yamada KM. Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol. 1995;7:615. 32. González-Mariscal L, Tapia R, Chamorro D. Crosstalk of tight junction components with signaling pathways. Biochim Biophys Acta. 2008;1778:729. 33. McKay L, Miller A 3rd, Sandine WE, et al. Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses. J Bacteriol. 1970;102:804. 34. Leibowitz G, Beattie GM, Kafri T, et al. Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes. 1999;48:745. 35. Barbu AR, Bodin B, Welsh M, et al. A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans. Diabetologia. 2006;49:2388. 36. Barbu AR, Akusjärvi G, Welsh N. Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response. Endocrinology. 2005;146:2406. 37. Li Y, Hansotia T, Yusta B, et al. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem. 2003;278:471. 38. Brubaker PL, Drucker DJ. Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology. 2004;145:2653.
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after implantation.1– 4 To improve survival rates of transplanted islets, ex vivo cytoprotective gene transfer strategies have been investigated.5– 8 However, the efficient transfer of genes into intact islets is difficult since islet cells exist as clusters of differentiated cells with very little replication
From the Department of Internal Medicine (B.W.L., H.Y.C., H.J.H., S.-H.I.), Hallym University College of Medicine, Chuncheon, Korea; and Ilsong Institute of Life Science (M.-H.K., D.K.), Hallym University, Anyang, Korea. *B.W.L. and M.-H.K. contributed equally. Current address for B.W. Lee is Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea.
This work was supported by research grants from the Innovative Research Institute for Cell Therapy, Republic of Korea (A062260) and from Hallym University Medical Center Research Fund (01-2009-14) to S.H.I. Address reprint requests to Sung-Hee Ihm, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896 Pyungchon-dong, Dongan-gu, Anyang, Kyonggi-do 431-070, Korea. E-mail: [email protected]
© 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 591–596 (2013)
0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.10.040 591
592
potential.9 Even with adenoviral vectors, islet transfection efficiency is low, and only cells located in the peripheral region of an islet are targeted.7,10 The successful application of gene therapy to islet modification is currently limited by inefficient gene delivery. Several recent studies indicate that functional -cell mass is dynamic, maintaining euglycemia through a balance of apoptosis and proliferation. Islet cell numbers can increase in response to insulin demand, certain growth factors, and hormones.11–14 This property has been demonstrated in both physiological and pathophysiological conditions, such as pregnancy and obesity.15,16 Many exogenous and endogenous growth factors, including prolactin, placental lactogen, hepatocyte growth factor, parathyroid hormonerelated protein, glucagon-like peptide (GLP)-1, exendin-4, and insulin-like growth factor-I, are known to increase -cell proliferation in vivo and in vitro.17–26 Glucose can also play a key regulatory role in pancreatic -cell plasticity, as a potent stimulus of -cell growth both in vivo and in vitro.27–30 It is known that cell proliferation in response to growth factors is accompanied by loosening of cell-to-cell contact and increasing paracellular permeability.31,32 Thus, we hypothesized that gene delivery into intact islet cells could be facilitated by modulating -cell proliferation potential with these agents. In this study, we investigated the effect of growth hormone, GLP-1, and high glucose on cell proliferation and gene transduction efficiency in pancreatic -cells. MATERIALS AND METHODS Islet Isolation Pancreatic islets were isolated from male Sprague-Dawley rats (200 –250 g) by digestion using 1 mg/mL collagenase P (Roche, Indianapolis, Ind, USA), separated by discontinuous Ficoll gradient purification (Biochrom AG, Berlin, Germany), and cultured free-floating in Medium 199 (Gibco, Grand Island, NY, USA). Individual islets were collected using micropipettes.
Adenoviral Vectors and Transduction The recombinant adenoviral vector Ad-EGFP-LacZ expresses green fluorescent protein (GFP) and -galactosidase (LacZ) under the control of the cytomegalovirus promoter. To prepare adenoviruses, Ad-EGFP-LacZ was propagated in HEK293 cells and purified by the AdEasy system (Stratagene, La Jolla, Calif, USA). Multiplicity of infection (MOI) was determined by optical density at 260 nm and by plaque assay. MOI calculations assumed 1000 cells per islet equivalent. Transduction was accomplished by incubating aliquots of isolated islets in serum-free medium for 4 hours in the presence of virus at different MOIs ranging from 0 to 200 with agitation every 1 hour. To examine the effect of hormones and high glucose on cell proliferation and transduction efficiency, islets were pretreated with 10 nmol/L of GLP-1 or 1 g/mL of human growth hormone (hGH; (Sigma-Aldrich, St Louis, Mo, USA) for 24 hr, or with 300 mg/dL of glucose for 48 hours, before transduction at an MOI of 25.
Assessment of Transduction Efficiency -galactosidase activity assay. After 24 hours of transduction with Ad-EGFP-LacZ, -galactosidase activity was measured in islet cell
LEE, KIM, CHAE ET AL lysates with 2-N- phenyl--D-galactopyranoside as a colorimetric substrate, according to the method previously described.33 Briefly, after transduction, islets were washed twice with phosphate-buffered saline and lysed in buffer containing protease inhibitors. The lysate was reacted with substrate buffer (60 mmol/L Na2HPO4, 40 mmol/L NaH2PO4, 1 mg/mL 2-N-phenyl--D-galactoside, 2.7 L/mL -mercaptoethanol) in a 96-well plate for 6 hours at 37°C. The color development was stopped by the addition of 1 mmol/L Na2CO3, and the absorbance of the samples was read at 420 nm. Protein determination was performed with a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, Ill, USA). B-galactosidase activity of the samples was expressed relative to that of mocktransduced islet cell lysate. GFP fluorescence. Islets were examined for GFP expression 24 hours after transduction using fluorescence microscopy. In addition, a quantitative analysis of transduction was performed on disassociated islet cells (dissociation buffer; Gibco, Grand Island, NY, USA) by acquiring 50,000 cells per treatment on a flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA) to determine the percentage of cells expressing GFP. All experiments were performed in triplicate.
Quantification of Islet Cell Death and Proliferation Transduced islets were harvested and dissociated into a single-cell suspension. Quantitative analysis of cell death was performed on dissociated islet cells by flow cytometry using 7-aminoactinomycin D (7-AAD, Sigma) and at least 50,000 cells per sample. To measure the percentage of proliferating cells in islet cell suspensions, 5-ethynyl-2-deoxyuridine (Edu; Click-iT EdU, Invitrogen, Eugene, Ore, USA) was used to stain cells for flow cytometry. Intact transduced islets were also examined for EdU incorporation using fluorescence microscopy.
Statistical Analysis Statistical analysis was performed using PRISM (GraphPad Software Inc, San Diego, Calif, USA). Results are expressed as means ⫾ standard deviations. A one-way analysis of variance, Bonferroni post hoc multiple comparison test, and Student t test were used where appropriate to compare variables. Statistical significance was defined P values below .05.
RESULTS Effect of Adenoviral Dose on Transduction Efficiency into Islets
To identify appropriate vector doses for the cell proliferation experiment, aliquots of freshly isolated rat islets were transduced with the Ad-EGFP-LacZ vector at MOIs of 12.5, 25, 50, 100, or 200 or were mock transduced with buffer. After 4 hours of transduction, islets were harvested and -galactosidase activity was measured in islet cell lysates to determine the efficiency of islet cell transduction. Under these experimental conditions, Ad-EGFP-LacZ transduction of islets was dependent upon viral dose up to 100 MOI (Fig 1). Using a higher viral dose (200 MOI) was somewhat detrimental to gene transfer into rat islets. The vector showed minimal transduction efficiency at MOIs of 12.5 and 25, and significantly higher efficiency at MOIs of 50 and 100, as compared to the mock-transduced control (both P ⬍ .001). In subsequent experiments, we used an MOI of
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Fig 1. Transduction efficiency of the Ad-EGFP-LacZ vector at various multiplicity of infection (MOI) in rat islets, as assessed by -galactosidase (LacZ) activity. The transduction efficiency was minimal at MOIs of 12.5 and 25 and significantly higher than the mock-transduced control at MOIs of 50 and 100. *P ⬍ .001.
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dure, islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at an MOI of 25. They were dispersed to single islet cells, and stained with 7-AAD or EdU to determine viability and proliferation status, respectively. In the flow cytometry analysis, the transduction efficiency of rat islets pretreated with 10 nmol/L of GLP-1 was significantly higher than that of control islets (P ⬍ .01, Fig 4A), confirming our results obtained by -galactosidase assay and fluorescence microscopy. The number of 7-AAD-positive dead islet cells was significantly decreased with GLP-1 pretreatment (P ⬍ 0.05, Fig 4B), and the number of EdU-positive proliferating cells was significantly increased (P ⬍ .05, Fig 4C). Thus, the transduction efficiency into rat islet cells of a suboptimal dose of viral vector was significantly improved by GLP-1 pretreatment and was accompanied by enhanced cell viability and cell proliferation. Interestingly, the increased GFP expression in GLP-1 pretreated islet cells was observed among the increased EdU-positive proliferating cells (Fig 5). DISCUSSION
25 as a suboptimal viral dose in order to examine the effect of hGH, GLP-1, and high glucose on islet transduction efficiency. Effect of hGH, GLP-1, and High Glucose on the Transduction Efficiency Into Islets
To examine effects on transduction efficiency, islets were pretreated with 1 g/mL of hGH or 10 nmol/L of GLP-1 for 24 hours, or with 300 mg/dL of glucose for 48 hours, alone or in combination, before transduction with the Ad-EGFPLacZ vector. At an MOI of 25, pretreatment with GLP-1, high glucose, or GLP-1 and high glucose led to significantly higher gene transduction efficiency in -galactosidase activity assay, as compared with untreated control islets (Fig 2). Transduction efficiency after pretreatment with the combination of GLP-1 and high glucose was higher than with glucose alone (P ⬍ .01), but not significantly different from that seen with GLP-1 pretreatment alone. Ad-EGFP-LacZ transduction at an MOI of 25 in GLP-1 or GLP-1/high glucose-pretreated islets was comparable to that seen at an MOI of 100 without pretreatment (Fig 1). These results demonstrate strong GLP-1-mediated enhancement of gene transfer into rat islets. The effect of hGH, GLP-1, and high glucose on transduction efficiency in islets was also assessed by GFP expression (Fig 3). In concordance with the -galactosidase activity, rat islets without pretreatment showed very low GFP expression at an MOI of 25. Pretreatment with GLP-1 or GLP-1/high glucose resulted in noticeably higher GFP expression. Effect of GLP-1 on Islet Cell Viability and Proliferation
To examine the effects of GLP-1 pretreatment on islet cell viability and proliferation during the transduction proce-
Ex vivo genetic modification of pancreatic islets to enhance survival during early posttransplant period is a plausible strategy in islet transplantation. At present, the lack of an efficient and safe vector system is a bottleneck in the
Fig 2. Transduction efficiency of the Ad-EGFP-LacZ vector at an MOI of 25 in rat islets, as assessed by -galactosidase activity. Islets were pretreated with 1 g/mL of human growth hormne (hGH) or 10 nmol/L of glucagon-like peptide (GLP)-1 for 24 hours, or 300 mg/dL of glucose for 48 hours, alone or in combination, prior to transduction. Pretreatment with GLP-1 or high glucose led to significantly higher gene transduction efficiency as compared with the controls. Transduction efficiency after pretreatment with the combination of GLP-1 and high glucose was higher than with glucose alone, but not significantly different from that seen with GLP-1 pretreatment alone. *P ⬍ .001; **P ⬍ .01.
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Fig 3. Transduction efficiency of the Ad-EGFP-LacZ vector at a multiplicity of infection of 25 in rat islets, as assessed by green fluorescent protein (GFP) expression. Under fluorescence microscopy, rat islets without pretreatment showed very low GFP expression. Pretreatment with glucagon-like peptide (GLP)-1 or GLP-1/high glucose resulted in noticeably higher GFP expression. hGH, human growth hormone.
development of gene transfer to pancreatic islets. Thus, the successful application of gene therapy in clinical islet transplantation depends mainly on the development of gene delivery vectors. Adenoviral vectors, which have been commonly used for gene transfer, show higher transfection efficiency in islets than do nonviral carriers, although the latter raise fewer safety issues.7,10,34 Previous studies have reported that in vitro transduction of intact rat and human islets using adenoviral vectors promoted GFP expression in 30% of cells, at most.35 Moreover, confocal sectioning of intact islets demonstrated that only cells located in the outermost layer were successfully targeted. Islets are compact clusters of about 1000 rarely dividing cells. In normal islet morphology, the cells of the inner core are much less exposed to transfection reagents than are cells in the peripheral region, due to diffusion barriers. To overcome
this physical constraint of accessibility, islets have often been dissociated to single cells before transduction, resulting in improved transduction efficiency.34,36 However, islet dispersion prior to genetic modification to enhance islet survival does not seem to be a viable option in clinical islet transplantation. It is known that cell proliferation in response to growth factors is accompanied by loosening of cell-to-cell contact and increasing paracellular permeability.31,32 In this study, we tested the hypothesis that gene delivery into intact islet cells would be facilitated by modulating -cell proliferation with hormones or high glucose, using an adenoviral-mediated gene delivery system to transfer EGFP and LacZ genes to rodent islets. Ad-EGFP-LacZ transduction of islets showed a viral dose-dependent increase up to an MOI of 100 MOI; an MOI of 25 was a suboptimal viral dose for islet trans-
Fig 4. Effect of glucagon-like peptide (GLP)-1 on Ad-EGFP-LacZ transduction, islet cell viability and proliferation. Islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at a multiplicity of infection of 25, and were dispersed to single islet cells, followed by 7-aminoactinomycin D (7-AAD) or 5-ethynyl-2-deoxyuridine (EdU) staining. (A) In a flow cytometry analysis, the transduction efficiency of rat islets pretreated with 10 nmol/L of GLP-1 was higher than that of untreated control islets. (B) 7-AAD-positive dead islet cells were decreased by GLP-1 pretreatment. (C) EdU-positive proliferating islet cells were increased by GLP-1 pretreatment. *P ⬍ .01; **P ⬍ .05.
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Fig 5. Effect of glucagon-like peptide (GLP)-1 on Ad-EGFPLacZ transduction and cell proliferation in rat islets. Islets pretreated with 10 nmol/L of GLP-1 for 24 hours were transduced with Ad-EGFP-LacZ vector at a multiplicity of infection of 25, and stained with 5-ethynyl-2deoxyuridine (EdU). (A) Under fluorescence microscopy, rat islets without pretreatment showed very low GFP expression and scanty EdU-positive cells. (B) GLP-1 pretreatment increased both GFP expression of islet cells and numbers of EdU-positive proliferating cells.
duction. At the MOI, pretreatment of islets with GLP-1, high glucose, or the combination of GLP-1 and high glucose led to significantly higher gene transduction efficiencies, when assessed by either -galactosidase activity or GFP expression. While combined pretreatment with GLP-1/high glucose led to higher efficiency than did glucose alone (P ⬍ .01), it was not significantly different from GLP-1 pretreatment, indicating the GLP-1 is more effective in enhancing transduction efficiency than is glucose. Interestingly, transduction at an MOI of 25 in GLP-1 or GLP-1/high glucose-pretreated islets was comparable to that of an MOI of 100 without pretreatment (Fig 1). Thus, GLP-1 treatment of islets before transduction may reduce the optimal adenoviral dose for efficient target gene delivery. Considering that the immunogenicity and possible toxicity of high adenoviral vector titers are the main limiting factors in their application to clinical transplantation, GLP-1 pretreatment of islets to reduce viral load for transduction may make adenoviral vectors competitive with nonviral vectors in terms of safety. In our results, GLP-1 treatment of rat islets at 10 nmol/L for 24 hours increased the number of EdU-positive proliferating islet cells, while enhancing the transduction efficiency of rat islet cells with a suboptimal dose of viral vector. Because adenoviral vectors transfect both dividing and nondividing cells, we assume that increased cell proliferation leads to enhanced adenoviral vector accessibility to islet cells through cell-proliferation-associated loosening of cell-to-cell contacts and increasing paracellular permeability. As GLP-1 has been shown not only to stimulate -cell proliferation, but also to have antiapoptotic effects on these cells,37,38 we examined the effect of GLP-1 pretreatment on islet cell viability during the transduction procedure. In a FACS analysis, 7-AAD-positive dead islet cells were signif-
icantly decreased by GLP-1 pretreatment, showing an additional beneficial effect of GLP-1 in islet transduction. Under our experimental conditions, although GLP-1 pretreatment significantly improved the transduction efficiency of rat islet cells with a suboptimal dose of adenoviral vector, transduction efficiency was still low, as shown by GFP FACS analysis. However, depending on the target gene chosen and the therapeutic intervention intended, this degree of transduction efficiency with improved islet viability and reduced safety issues could suffice. For example, the modification of pancreatic islets with this protocol may be more feasible with genes for secretory proteins than with those for cell-resident proteins. Further studies to determine if GLP-1 pretreatment continues to enhance transduction efficiency above a 25 MOI dose of adenoviral vector is needed. In conclusion, the pretreatment of rat islets with GLP-1 enhanced the transduction efficiency of an adenoviral vector, reducing adenoviral dose burden while improving islet viability. From a therapeutic standpoint, genetic modification of pancreatic islets combined with GLP-1 pretreatment may be a promising option for ex vivo gene therapy in islet transplantation.
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