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Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Division of Endocrinology, BST E-1140, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}msx.dept-med.pitt.edu.
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Abstract |
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Introduction |
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Hayek, Otonkoski, and colleagues (4, 5) have demonstrated that the addition of hepatocyte growth factor (HGF) to rodent or human islets in culture induces islet cell proliferation. We have shown that, when delivered to the pancreatic ß-cell of transgenic mice, HGF increases ß-cell proliferation, ß-cell number, islet number, islet size, and overall islet mass (6, 7). Moreover, HGF gene transfer increased the expression of three key ß-cell genes, glucokinase, Glut-2 (the ß-cell glucose transporter), and insulin (6, 7). These coordinated events led to enhanced glucose-stimulated insulin secretion in vitro, mild fasting hypoglycemia, and superior glucose tolerance in HGF transgenic mice compared with their normal littermates (6). More recently, we have demonstrated that delivery of HGF using adenovirus to otherwise normal murine islets markedly improves the outcomes and function of islets transplanted into diabetic, immunodeficient, severe combined immunodeficiency (SCID) mice (8). In that study, we also demonstrated that HGF has potent prosurvival effects on transplanted murine islets (8). This prosurvival effect of HGF is mediated by phosphatidylinositol 3-kinase (8). Thus, HGF appears to be an ideal growth factor for enhancing islet transplant outcomes: it increases ß-cell proliferation (4, 5, 6, 7); enhances ß-cell survival in a transplant setting (8); increases the expression of glucokinase, Glut-2, and insulin, and thereby enhances glucose-stimulated insulin secretion (7); and has demonstrated efficacy in two transplant models in mice (7, 8).
Our prior studies were performed in immunodeficient mice, in which allograft, immunosuppressant, and adenoviral immunological problems with islet allograft gene therapy were attenuated or not operative. The main question addressed by the current study was the following: What is the efficacy of adenoviral delivery of HGF in a setting that more closely resembles that which occurs in humans? We therefore developed a rat model that mimics several key features of the currently employed Edmonton protocol. First, we employed an allogeneic system, using Lewis rat islets as donors and Sprague Dawley rats as recipients. Second, we employed the portal delivery of islets, as is employed in human islet transplants (1, 2), instead of the renal subcapsular graft we have employed in prior studies (7, 8). Third, we used the same glucocorticoid-free combination of immunosuppressive agents employed in the Edmonton studies: low-dose tacrolimus (FK-506), sirolimus (rapamycin), daclizumab (IL-2 receptor monoclonal antibody) (1, 2).
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Materials and Methods |
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Islet graft donors
Male Lewis rats (Charles River Laboratories) (6–8 wk old; body weight, 250–300 g) were used as islet donors. Islets were isolated as previously described by Ricordi and Rastellini (9), with some modifications. Briefly, after general anesthesia, a midline abdominal incision was performed, and the pancreas was exposed and injected through the pancreatic duct with 15 ml of 1.7 mg/ml collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) in Hank’s buffered saline solution (HBSS) (Life Technologies, Inc., Long Island, NY). The pancreas was surgically removed and incubated at 37 C for 17 min, and then passed through a 500-µm wire mesh. The digested pancreas was rinsed with HBSS, and islets were separated by density gradient in Histopaque (Sigma-Aldrich). After several washes with HBSS, islets were hand picked under a microscope. To standardize the islet mass to be transplanted, one islet equivalent (IE) was defined as one 125-µm-diameter islet. Before transplantation, islets were placed in culture overnight in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 2 mmol/liter L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and 25 mmol/liter HEPES buffer (Mediatech, Herndon, VA) at 37 C and 5% CO2.
Portal vein islet transplantation
A midline laparotomy was performed in the diabetic rats under isofluorane general anesthesia. Islets were then slowly injected into the portal vein via the superior mesenteric vein in a volume of 400 µl PBS solution. After infusion, the syringe was rinsed several times by repeated aspiration and reinfusion of portal vein blood. Manual compression of the injection site followed removal of the needle to minimize bleeding. Tail blood glucose levels were measured under nonfasting conditions at d 1, 2, 3, 5, and 7, and then weekly or biweekly, as shown in the figures. Blood samples were weekly obtained from a tail vein for plasma insulin determination and biochemistry. Plasma insulin determinations were performed by RIA per the manufacturer’s specifications (Linco Research, St. Louis, MO).
Immunosuppression regimen
Immunosuppression consisted of low-dose tacrolimus (FK-506) (0.5 mg/kg·d im; Fujisawa, Deerfield, IL), sirolimus (rapamycin) (2 mg/kg sc, every 48 h; Wyeth-Ayerst, Princeton, NJ) and daclizumab (2 mg/kg iv at d 0; 1 mg/kg at d 15; Roche, Nutley, NJ), as described in the figures. These doses were selected to mimic the doses used in the human Edmonton protocol. Doses were calculated based on previous reports (10, 11, 12, 13, 14). Whole-blood tacrolimus concentrations were measured by microparticle enzyme immunoassay technology using the IMX tacrolimus assay (Abbott Laboratories, Abbott Park, IL), and whole-blood rapamycin levels were measured in the Clinical Chemistry Laboratory at the University of Pittsburgh Medical Center using HPLC mass spectrometry.
Adenovirus construction and islet transduction
Ad5 adenoviral vectors encoding murine HGF and ß-galactosidase (LacZ) were prepared as we have described in detail previously (8), using the methods of Becker et al. (15). Isolated islets were infected with purified adenovirus in RPMI 1640 for 1 h at 37 C (at a multiplicity of infection of 250, assuming 
1000 cells/islet), rinsed with RPMI 1640 containing 1% penicillin/streptomycin and 10% fetal bovine serum, and incubated in 0.5 ml of the same medium for 18–24 h at 37 C on the day before transplant.
Relative semiquantitative PCR
Total islet RNA was isolated, and PCR was performed as described in detail previously (6, 7, 8).
Western blot for HGF
Western blotting of isolated islets for murine HGF was performed as described previously (8).
Histology and insulin staining and insulin determinations
Livers were removed on d 25, fixed overnight in Bouin’s solution, and embedded in paraffin. Five-micrometer sections were obtained and stained with hematoxylin and eosin using standard techniques. Immunostaining for insulin and visualization of the staining was achieved using the species-appropriate avidin-biotin complex system as previously described (6, 7, 8).
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Results |
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, institution of the Edmonton protocol in normal Sprague Dawley rats (solid line) resulted in the development of frank diabetes by d 10. This contrasted with the findings in normal control rats that remained euglycemic under the same conditions (dotted line). It also contrasted with findings in rats treated with sirolimus plus daclizumab (dashed line), which also remained euglycemic. These findings indicate that the Edmonton protocol, in the doses employed herein, causes diabetes in normal rats. Importantly, the trough tacrolimus concentrations were 6 ± 1.5 ng/ml (mean ± SE), in the low therapeutic range for humans, and trough rapamycin concentrations were 8.7 ± 2.5 ng/ml (mean ± SE), also in the range reported by the Edmonton group in humans (1, 2).
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, insulin concentrations remained normal in the control, euglycemic control rats. In contrast, insulin levels rose in the sirolimus- plus daclizumab-treated rats. These rats also remained euglycemic (Fig. 1
), indicating that the combination of sirolimus and daclizumab in the doses used, causes insulin resistance, but, as a result of compensatory ß-cell insulin secretion, did not result in diabetes. The rats that received the full triple-drug regimen displayed higher insulin concentrations than their normal controls, indicating that the triple-drug regimen, too, causes insulin resistance in normal rats. Importantly, these animals displayed lower insulin concentrations than the sirolimus-plus-daclizumab animals, and also displayed frank diabetes (Fig. 2). These findings indicate that the addition of tacrolimus to the sirolimus-plus-daclizumab combination results in a failure of ß-cells to compensate for insulin resistance induced by the Edmonton regimen.
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, Lewis rat islets at a dose of 15 IE/g were portally transplanted into STZ-diabetic Sprague Dawley rats randomly assigned to receive either the Edmonton agents or no immunosuppressive drugs. As can be seen in Fig. 3A, rats not receiving immunosuppression progressed transiently toward normal blood glucose levels for approximately 1 wk, and then became diabetic, consistent with rejection by the Sprague Dawley rats of the allogeneic Lewis islets. In contrast, islet allografts in the setting of the three-drug immunosuppression regimen functioned until the end of the experiment on d 25. These results indicate that the Edmonton immunosuppression regimen was indeed effectively immunosuppressive.
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, the blood glucose concentrations were so close to normal that an improvement would be difficult to clearly discern. To develop a platform from which a clear improvement could be determined, we further reduced the dose of transplanted islets to 10 IE/g. As can be seen in Fig. 3B, in rats receiving no immunosuppression, as in the prior experiment in A, transplanted islets ceased functioning by d 10, presumably reflecting rejection. In contrast, in rats receiving 10 IE/g, glycemic control was actually worsened by immunosuppression. The results in Fig. 3 extend the observations in Figs. 1 and 2 demonstrating that the three-drug regimen is detrimental not only to endogenous islet function but also to transplanted islets.
Ad.HGF effectively transduces rat islets
To examine the ability of Ad.HGF and Ad.lacZ to transduce rat islets, we examined HGF expression by RT-PCR in Lewis rat islets exposed to no virus, to Ad.lacZ, or to Ad.HGF for 24 h. Ad.lacZ transduction resulted in strong ß-galactosidase staining not present in normal islets (data not shown). As can be seen in Fig. 4A, RT-PCR products representing HGF are present in far greater quantities in Ad.HGF-transduced islets than in normal or Ad.lacZ-transduced islets. Similarly, at the protein level, Ad.HGF effectively induced HGF expression, as shown in Fig. 4B. By 48 h, HGF protein is easily visible in Ad.HGF-transduced islets, and by 72 h, HGF protein expression is increased further. In contrast, as shown, neither islets infected with Ad.lacZ nor uninfected islets displayed visible HGF expression under these conditions. Collectively, these results indicate that Ad.HGF transduction at the multiplicity of infection and under the conditions employed can lead to expression of HGF in rat islets.
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, and the ability to introduce HGF into islets using Ad.HGF, we next combined these approaches. As shown in Fig. 5, normal uninfected and Ad.lacZ-transduced Lewis rat allograft transplants at 10 IE/g had only a transitory effect on blood glucose. These results are almost identical with those in Fig. 3B. In striking contrast, Ad.HGF introduction into this otherwise-inadequate islet mass markedly enhanced graft function. As can be seen in Fig. 3B, random, nonfasting glucose concentrations in the rats receiving the 10 IE/g portal Ad.HGF-transduced allografts rapidly approached normal and remained in the 200–250 mg/dl range for the duration of the experiment. Importantly, this degree of glucose control was comparable with that observed in normal rats receiving the Edmonton agents (Fig. 1), and was superior to the 250–300 mg/dl range achieved using 50% larger (15 IE/g) doses of normal islets in Fig. 3A.
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are shown in Fig. 6. As anticipated from the glucose results, plasma insulin concentrations were markedly higher in the rats receiving the Ad.HGF than in rats receiving equivalent numbers of either Ad.lacZ or normal islets. Together with the glucose results in Fig. 5, these results indicate that the introduction of HGF using an adenoviral vector markedly improves portal allograft function, not only compared with Ad.lacZ-transduced islets, but also compared with normal islets.
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. This figure illustrates several points. First, abundant insulin-staining ß-cell clusters can be observed in the livers of the 10 IE/g Ad.HGF animals. Second, in the presence of immunosuppression, there is no evidence of an inflammatory response to either the islet allograft or the adenovirus used to transduce the islets. Third, livers from animals transplanted with Ad.HGF-transduced islets, compared with Ad.lacZ islets, contained larger numbers of islets. This is shown quantitatively in Fig. 8, where the number of islets per liver section is seen to be more than 2-fold higher in those livers that received Ad.HGF-transduced islets than in controls. This estimate is of course an underestimate if one considers that the average size of the residual transplanted islets in the Ad.HGF livers is significantly larger than that in the Ad.lacZ livers (Fig. 7).
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and 5) were repeated and combined using the following three groups of rats, each of which received the Edmonton agents as in Figs. 1 and 5: 8 normal, nondiabetic rats treated with the Edmonton immunosuppressive regimen, and 5 Ad.lacZ and 10 Ad.HGF Lewis marginal (10 IE/g)-islet allografts into STZ-diabetic Sprague Dawley recipients. However, instead of sacrificing the animals on d 25, they were observed indefinitely. As can be seen in Fig. 9, the new control group of STZ-diabetic animals receiving 10 IE/g, the marginal islet mass, and Ad.lacZ (open squares) remained diabetic, confirming the results of briefer studies shown in Figs. 3B and 5. All five of these animals had died by wk 17 of the experiment. In marked contrast, pretreatment of this same marginal islet mass with Ad.HGF (Fig. 9, filled squares) again dramatically and rapidly improved blood random blood glucose values, as had been observed in earlier experiments (Fig. 5) over a 25-d time period. However, it can now be appreciated that the duration of this improvement compared with the controls is long-lasting: the Ad.HGF animals displayed blood glucose concentrations some 200 mg/dl lower than the Ad.lacZ animals until wk 16–18, by which time all of the Ad.lacZ animals had died. Thus, the improvement in control lasted at least five months, approximately one fifth of the 2-yr life span of a rat.
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, normal rats exposed to the Edmonton reagents (open circles) again promptly developed diabetes as had occurred in Fig. 1 during the 25-d experiment. However, as seen in Fig. 9, with more prolonged exposure to the Edmonton regimen, the diabetes became even more severe, such that by 16 wk, these animals had nonfasting blood glucose concentrations of approximately 400 mg/dl. These studies confirm and underscore the observations in Fig. 1 indicating that these agents, even when used in doses that are associated with normal therapeutic blood levels, are diabetogenic in rats. Importantly, the juxtaposition of the Ad.HGF rats and the normal Sprague Dawley rats in Fig. 9, together with earlier data in the SCID mouse HGF transplant model (7, 8), which does not receive immunosuppressive drugs and that does not develop diabetes, makes it clear that the diabetes in the Ad.HGF and the normal Sprague Dawley rats in Figs. 5 and 9 is a result of the immunosuppressive drugs. |
Discussion |
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HGF is an ideal candidate for enhancing islet function for the reasons summarized in the introductory section. These prior findings in immunodeficient mice beg three important additional questions: 1) Could this adenoviral HGF enhancement be applied to a model employing the portal delivery site employed in human islet transplantation? 2) Would the improvement in function also be apparent under the more adverse conditions of allograft immunity, adenovirus-directed immunity, and the potential diabetogenic toxicities of immunosuppressive agents? and 3) Will the improvement be maintained over the long term?
The first concern was to select a vector for use in islet gene delivery. Adenovirus has been associated with a number of adverse features, and is viewed by many as a vector that can be used to provide proof of principle regarding the efficacy of gene therapy, but is unlikely to be useful in ultimate human gene therapy applications (16). Negative features are that it evokes a host immune response that can cause anaphylaxis, as well as host immune system-driven destruction of the target cells that express adenoviral proteins. Moreover, adenovirus is a nonintegrating vector, and expression of the gene product in question would therefore have a limited duration of expression. Although these may be negative considerations in some settings, in the relatively unique setting of islet transplantation, they may be positive attributes. Because islet transplantation requires immunosuppression, immune attack on adenovirus-transduced islet grafts will also likely be attenuated. Moreover, because the vast majority of transplanted islet cells die within the first day after transplantation (17), transient expression of an agent that promotes islet engraftment during the first week may be all that is necessary. The lack of integration may be an advantage as well, because random integration into tumor suppressor or other key cellular genes will not occur. Importantly, because the virus is administered ex vivo, the viral load delivered to the patient would be small, reducing concerns regarding anaphylaxis. Finally, immunosuppression would also presumably attenuate host allergic reactions. Thus, we believe that adenovirus is worthy of consideration in human islet transplant settings.
One could reasonably ask why one must deliver adenovirus using gene therapy approaches to islets, rather than by systemic injection in the setting of islet transplant. HGF has been administered using such an approach (18), but systemic administration and generalized overexpression using the metallothionein promoter in mice is limited by host toxicity, and local doses to the islet obtained by viral delivery would be higher than those achievable using systemic administration routes. Thus, viral delivery to islets before transplant would appear to be more attractive than systemic administration of HGF to recipients.
One reason for moving from standard immunosuppressive regimens to the three agents that comprise the Edmonton protocol was the need to avoid ß-cell toxicity and insulin resistance induced by the high-dose tacrolimus and glucocorticoids (1, 2, 19, 20, 21). Our studies clearly demonstrate, to our surprise, that low-dose tacrolimus and sirolimus, with plasma levels comparable with those desired in humans, is indeed associated with ß-cell toxicity and marked insulin resistance in rats. We cannot be certain from this study in rats whether induction of diabetes might occur in normal humans treated with the Edmonton cocktail, or whether this may be a rat-specific effect. This question merits consideration in humans. We presume, but have not proven, that the diabetogenic and immunosuppressive effects observed in the current study resulted from sirolimus and tacrolimus, because daclizumab is an antihuman CD25 antibody, and was presumably inert in the current study. These observations suggest that there is room for further improvement in designing and implementing immunosuppressive strategies. However, from the perspective of developing model systems with which to rigorously test islet transplant approaches, these results are ideal, because they allow one to test the efficacy of genetically enhanced islets in an adverse, potentially real-world situation.
One key feature of the current rat model is that it employs or defines a minimal mass or marginal mass of islets. This is critically important, because, as described by Inverardi, Ricordi, and others (22, 23, 24, 25), one needs to define the limits of adequacy to improve upon them. Thus, in the current model, the 15 IE/g model (Fig. 3A) allows one to clearly define the efficacy of the immunosuppression in protecting islet allografts, and therefore the relevance of the immunosuppressive regimen employed, but the dose of islets is too high to clearly examine enhanced efficacy. Therefore, the 10 IE/g dose, which in the setting of immunosuppression (Fig. 3B) leads to almost no improvement in glycemic control, serves as an ideal platform or minimal model from which to assess improved efficacy.
With this background, Ad.HGF gene delivery was strikingly effective, as shown in Figs. 5–9. Through the addition of Ad.HGF to the 10 IE/g model, glycemic control was far superior to that observed in diabetic control rats receiving normal or Ad.lacZ islets at a dose of 10 IE/g, and was comparable with otherwise completely normal rats treated with the Edmonton regimen (Figs. 1 and 9). Indeed, the outcome in these rats was far superior even to rats receiving 15 IE/g. Although it is difficult to quantitate the improvement in efficacy from these observations, one could reasonably argue that they are at least twice as effective as normal islets. These results are similar to those observed in the rat insulin promoter-HGF islet transplant model, where 250 rat insulin promoter-HGF islets functioned as well as 500 IE from normal islets (7). In addition, the improvement compared with control Ad.lacZ islets was long-lasting, up to 5 months. Given that the life span of a rat is 2 yr, this is approximately one fifth of the life span of a rat, and conceivably could correspond to 10–20 yr in a human. The long-term findings may also indicate that a self-renewing population of ß-cells or their precursors has been established. Importantly, it seems likely that the duration of efficacy of the Ad.HGF therapy was limited by the diabetogenic effect of the immunosuppressive agents, and not by intrinsic failure of the Ad.HGF strategy, because SCID mice receiving HGF-expressing islet grafts, once normalized, do not appear to develop diabetes (7, 8). Thus, moving from calcineurin inhibitors to other immunosuppressive regimens in the future may permit even longer efficacy of the HGF effects.
We do not know from these studies which cells within the islet are transduced by the Ad.HGF virus, or which cells secrete HGF. We have previously reported that, in mouse islets infected with Ad.lacZ, approximately 30% of the islets cells are transduced (8), and we assume that this applies to the Ad.HGF-transduced islets as well. Importantly, because HGF is a secreted factor, and because the promoter used is the cytomegalovirus promoter, it is not necessary that the Ad.HGF transduce ß-cells specifically. Any cell within the islet environment—endothelial cells, 
-cells, fibroblasts, ß-cells, ductal cells, or others—could in theory serve as a local producer of HGF.
Because the islets employed in the current study were delivered to the liver, one might ask whether the delivery of Ad.HGF to the liver might account for some of the improvement in glucose control observed. We believe that this is not a liver-specific effect, for the same degree of improvement in glycemia was observed when the Ad.HGF-transduced islets were delivered to the renal capsule in the SCID mouse model described previously (8). Moreover, the efficacy of the Ad.HGF appears to result from direct effects on the islet graft and not on the residual ß-cells in the host pancreas, because we have previously demonstrated in the mouse renal capsule graft model that unilateral nephrectomy completely reverses the effect (7, 8).
As described in the introductory section, HGF stimulates ß-cell proliferation, enhances ß-cell survival, and improves ß-cell function (glucose-stimulated insulin secretion). We cannot determine from the studies described herein which of these potential mechanisms, or what combination, is responsible for the enhanced engraftment and function observed.
In summary, adenoviral delivery of HGF enhances the function of transplanted islets in vivo in an Edmonton rat model that faithfully captures the key elements of human islet transplantation: portal delivery of islets, allograft immunity, immunosuppression, ß-cell toxicity, and insulin resistance. Further studies are necessary to determine whether this approach can be applied to human islets, to accurately quantitate the extent of improvement, to optimize the conditions of exposure to Ad.HGF, to identify more effective and less toxic immunosuppressive strategies, and to identify additional factors such as epidermal growth factor, betacellulin, glucagon-like peptide-1, PTHrP, or others that may display similar effects in this system, or that may act synergistically with HGF to further enhance islet graft performance.
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Acknowledgments |
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Footnotes |
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Abbreviations: HBSS, Hanks’ buffered saline solution; HGF, hepatocyte growth factor; IE, islet equivalent; SCID, severe combined immunodeficiency; STZ, streptozotocin.
Received August 18, 2003.
Accepted for publication September 29, 2003.
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