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Keywords:

  • Apoptosis;
  • islets;
  • mitochondria-dependent;
  • PFC;
  • preservation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Islet transplantation is a treatment option for type I diabetic patients. Preservation of human pancreata prior to islet isolation using two-layer method with perfluorocarbon (PFC) and University of Wisconsin solution (UW) results in twofold increase in islet yields. The objective of this study was to determine the mechanism by which islets undergo apoptosis and determine PFC's effects on this process. Gene array analysis was used to analyze the expression of pro- and anti-apoptotic genes in islets isolated from pancreata preserved under varying conditions. A 12-fold increase in the expression of inhibitor of apoptosis (IAP) and survivin was observed in islets isolated from pancreata preserved in PFC. This was accompanied by decreased expression of BAD (3.7-fold), BAX (2.7-fold) and caspases (5.2-fold). Levels of activated caspase-9 (77.98%), caspase-2 (61.5%), caspase-3 (68.3%) and caspase-8 (37.2%) were also reduced. ‘Rescue’ of pancreata after storage (12 h) in UW by preservation using PFC also resulted in a down-regulation of pro-apoptotic genes and inhibition of caspase activation. Apoptosis observed in islets from all groups was mainly mitochondria-dependent, mediated by change in redox potential initiated by hypoxia. We demonstrate that reduction in hypoxia of pancreata preserved using PFC leads to significant up-regulation of anti-apoptotic and inhibition of pro-apoptotic genes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Islet transplantation is now considered an emerging treatment option for select patients with type I diabetes. Since the landmark report from Edmonton (1), many centers have initiated corticosteroid-free immunosuppressive protocols for successful clinical islet transplantation. The minimally invasive nature of the islet transplant procedure makes it a desirable alternative to whole-organ pancreas transplantation. While the initial results of islet transplantation using the Edmonton protocol are promising, the need for a large number of islets to achieve insulin independence necessitates the use of multiple pancreata per patient, since currently only a fraction of the islets in a whole pancreas are successfully isolated. Limited islet yields are due in part to the effects of cold preservation on the pancreata combined with the effects of the isolation process itself. In addition, recent results describing follow-up of long-term islet transplants indicate that the majority of recipients lose some islet graft function over time raising the possibility that additional islet transplants may become necessary (2). Thus, the need to initially isolate healthy islets remains paramount to the field.

Prolonged cold storage of the organs has been hypothesized to induce hypoxia and oxidative stress (3,4). This, in turn, results in death of the isolated islets by apoptosis (5,6). The use of perfluorocarbon (PFC), together with University of Wisconsin (UW) solution, has been previously described as a way of improving islet recovery from animal pancreata (7,8). Results obtained from our facility, as well as other centers, have clearly demonstrated that storage of the pancreata in PFC significantly increases the yield of islets obtained from these organs. However, the mechanism by which PFC improves islet yield is unknown. Understanding of this mechanism could aid in designing more effective strategies for donor management, organ preservation and islet isolation techniques.

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. Apoptosis was originally described by Kerr et al. as an event characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation and degradation of DNA (9). Two major pathways for the induction of apoptosis—extrinsic (TNF pathway) and intrinsic (mitochondrial pathway)—have been identified (10). Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in ischemic injury and apoptosis induction. Oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly (11). Mitochondria-dependent apoptosis has been shown to require release of cytochrome C from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as ‘caspases’ (12). Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome C release and protect against oxidative stress-induced apoptosis (13).

In this study, we analyzed the pathway by which cell death occurs in islets isolated from human deceased donors. We also identified the mechanism by which preservation of pancreata in PFC inhibits islet apoptosis, resulting in increased yields and improved function. Our results demonstrate that preservation of pancreata in PFC reduces hypoxia and results in up-regulation of anti-apoptotic gene expression with a concomitant down-regulation of pro-apoptotic gene expression.

Research Design and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Pancreata preservation

Pancreata from 24 deceased donors were procured using standard surgical techniques including in situ arterial and portal flush with UW solution. The criteria for donor pancreas acceptance were: multi-organ donor, adequate in situ hypothermic perfusion with UW solution for perfusion and cold storage, minimum 25 years to 70 years of age, hospitalization stay < 96 h, less than 10 min of warm ischemia time, no elevation of serum creatinine levels by more than 50% of the initial value or elevation of transaminases to levels greater than two times the normal values. Pancreata were stored in 1 L jars at 4°C for transportation to the islet processing facility at Washington University in St. Louis. Group A pancreata were preserved immediately in 1000 mL of UW solution. Group B pancreata were preserved using the two-layer method with 333 mL of pre-oxygenated PFC as the bottom layer and approximately 500–600 mL of UW solution as the top layer. The pancreata were placed at the interface between these two solutions. Group C pancreata were shipped to the islet processing facility in 1000 mL of UW solution but then switched to two-layer preservation with 333 mL of pre-oxygenated PFC as bottom layer and approximately 500–600 mL of fresh UW solution as the top layer for 8 h prior to processing. Pancreata were assigned to the individual groups at the discretion of the procuring agency depending on their standard procurement procedures. Pancreata were either preserved in UW solution or in UW solution/PFC depending on the availability of PFC at the specific organ procurement organization.

Islet isolation

Islets were isolated from the pancreata using the modified Ricordi method. Briefly, the pancreata were distended with 350 mL of liberase and pulmozyme mixture blend at 37°C. Then the pancreata were transferred to the Ricordi chamber and digestion was performed at 37°C. Cold Hank's balanced salt solution (HBSS) containing 2.5% human serum albumin was added to the preparation to inhibit enzymatic digestion. The islets were purified using a continuous ficoll gradient using COBE2991 cell processor. The viability of the isolated islets was assessed using a fluorescent dye inclusion/exclusion principle. The basal and glucose-stimulated insulin secretion was measured using a Modified Eizirik's method. Briefly, 10 islet equivalents (IEQs) were exposed to basal media, 1.7 and 16.7 mM glucose. The secreted insulin was quantitated using an Insulin Enzyme immunoassay (14). In vivo function of the isolated islets was assessed by transplanting 3000 IEQs under kidney capsule of a diabetic-SCID mouse to prove that the islets were functionally active and measuring their potency in restoring normoglycemia (15).

Gene array analysis

Expression profiles of the different pro- and anti-apoptotic genes in the isolated islets were analyzed using an apoptotic gene array as per manufacturers' recommendation. Briefly, total RNA was extracted from 5000 IEQs using Trizol reagent. The RNA was reverse-transcribed and radiolabeled using AmpoLabeling-LPR Kit (Super Array Biosciences, Frederick, MD, USA). The radiolabeled probes were hybridized with GEArray Q series Human Apoptosis Gene Array (Super Array Biosciences) overnight at 60°C. Membranes were washed twice with 2× SSC/1% SDS and twice with 0.2× SSC/0.5% SDS and then exposed to x-ray film. After overnight exposure at −70°C, the film was developed. The data obtained from the membranes were analyzed using the Gene Array Analyzer Software (Super Array Biosciences). The data were normalized using GAPDH. Genes not expressed in at least 6 of the 8 samples in each group were excluded from further analysis.

Western blot analysis

Expression levels of the pro- and anti-apoptotic genes were analyzed in the isolated islets by western blot analysis using specific antibodies. Protein extracts from the islets were extracted using protein extraction reagent, Mper reagent (Pierce Biochemicals, Rockford, IL, USA). The protein concentration was estimated using BCA protein assay (Pierce Biochemicals). The protein extracts (50 μg/lane) were electrophoresed on 4–12% NuPage Bis-Tris system (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk solution and then probed with anti-survivin, anti-cIAP2, anti-Bcl2, anti-phospo-Bcl2, anti-cytochrome C, anti-Apaf1 and anti-AIF1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 2 h at room temperature. Peroxidase-conjugated goat anti-mouse IgG (Organon Teknika Corp., West Chester, PA, USA) was used as the secondary antibody and the blots were developed by Supersignal West Pico Chemiluminescent Substrate kit (Pierce Biochemicals). The blots were then exposed to x-ray film for 30 min and processed. Densitometric analysis of the bands was performed using QuantityOne software (Biorad, Hercules, CA, USA). The expression levels of the genes were presented as density of the gene of interest/density of the β-actin.

Caspase profiling

The levels of activated caspases in the isolated islets were quantitated using BD ApoAlert™ Caspase Profiling Plate (BD Biosciences, San Jose, CA, USA). Briefly, 1000 IEQs were lysed with chilled lysis buffer and mixed with 2× reaction buffer and loaded onto the caspase profiling plates. The plates were incubated for 2 h at 37°C and fluorometric detection of caspase activity was read with a 380-nm excitation filter and 460-nm emission filter.

Statistical analysis

Data are reported as mean ± SD. Differences between multiple experimental groups were compared by analysis of variance (ANOVA) followed by Tukey's multiple comparison test. A value of p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Preservation in PFC increases islet yield and viability

The yield of isolated islets, donor characteristics and viability of the islet preparations is summarized in Table 1. No significant difference in the blood glucose levels, body mass index or age was observed between the three different groups. No significant correlation could be established between the amount or the duration of vasoreactive medicines administered in the donors to the yield of isolated islets. In addition, measurements of continued acceptable function of other intra-abdominal end organs like kidney and liver (measured by creatinine and transaminase stability) further indicated that the vasopressors administered in the donors exerted no significant effect on the pancreas.

Table 1.  Increased islet yields in pancreata preserved immediately in UW/PFC
 Group AGroup BGroup C
  1. *p < 0.05

No of Isolations888
PFC storage (h)08.8 ± 2.48.5 ± 2.1
Total cold ischemia time8.9 ± 0.711.0 ± 2.226.6 ± 4.9
Body mass index27.7 ± 6.527.8 ± 7.826.9 ± 6.1
Cause of death2 MVA, 3 CVA and 3 ICH4 CVA and 4 ICH1MVA, 4 CVA and 3 ICH
Pancreata weight92.2 ± 25.8100.1 ± 24.988.4+ 18.5
Total IE234583 ± 59812466195 ± 168866*228000 ± 77083
Pre-purification yield330762 ± 70485657334 ± 234569323760 ± 97867
Percentage recovery58.6 ± 17.359 ± 11.757.6 ± 18.5
IE/g2499 ± 12384987 ± 1538*2194 ± 1121
Purity79.6 ± 20.280.4 ± 8.569.4 ± 25.8
Islet index0.9 ± 0.30.7 ± 0.30.8 ± 0.3
Islet viability (%)89.5 ± 4.296.1 ± 2.487.8 ± 9.2
Stimulation index2.8 ± 2.22.7 ± 1.42.4 ± 1.0

Preservation of pancreata in UW/PFC resulted in a significant increase in the islet yield per gram of pancreata compared to the control group preserved in UW solution alone (4987 ± 1538 vs. 2499 ± 1238, p < 0.05). Moreover, the yield of islets preserved in PFC even after prolonged cold storage, resulted in yields equivalent to that obtained in pancreata with short cold ischemia (2194 ± 1121 vs. 2499 ± 1238, p ≥ 0.0.5). In comparison, the islet yield from pancreas preserved for >16 h in UW solution alone was 148 862 ± 46245 (data not shown). No significant difference in the immediate insulin secretory capacity was observed within the groups. Performance in the in vivo functional assay demonstrated no significant differences between the three groups.

Preservation in PFC increases anti-apoptotic and decreases pro-apoptotic gene expression

Apoptosis is a major pathway through which islets undergo death after isolation and has been considered as a major factor for primary graft nonfunction (16,17). The concerted action of pro- and anti-apoptotic genes determine the overall fate of the cell. Analysis of the islets isolated from pancreata in PFC showed consistent expression of the following pro-apoptotic genes: bad (0.68 ± 0.46, sample/β-actin), bax (0.58 ± 0.43), caspases (2.9 ± 1.4), TRAF5 (1.03 ± 0.48), TRAF6 (1.55 ± 0.82), TRIP (0.6 ± 0.19), TNF-β/LTB (0.9 ± 0.44) (Figure 1). These results indicate that apoptosis in the isolated islets is through both mitochondria-dependent and -independent pathways. As shown in Figure 1, islets isolated from Group B (p < 0.05) displayed a (12.7 ± 5.2)-fold increase in IAP2 expression compared to a (2.9 ± 2.5)-fold increase in islets isolated from Group C (p < 0.05). Similarly a (12.4 ± 5.8)-fold increase in expression of survivin was observed in islets isolated from Group B whereas no significant difference in expression of survivin was observed in islets from Group C. In contrast, the expression levels of the pro-apoptotic genes bad (3.7 ± 1.4 fold), bax (2.7 ± 0.9 fold), caspases (5.2 ± 3.8 fold) were significantly down-regulated in islets isolated from pancreata stored in PFC (Group B). Similarly, a reduction in the expression levels of pro-apoptotic genes bad (1.4 ± 0.8 fold), bax (1.2 ± 0.4 fold), caspases (1.5 ± 1.0 fold), TRAF5 (1.2 ± 0.5 fold) and TNF-β/LTB (1.7 ± 0.7 fold) was observed in islets isolated from pancreata preserved in PFC after prolonged cold storage (Figure 1). These data indicate that storage of the pancreata in PFC results in increased expression of the anti-apoptotic genes and decreased expression of pro-apoptotic genes resulting in less cell death by apoptosis.

image

Figure 1. Preservation in PFC increases expression of anti-apoptotic genes and decreases pro-apoptotic genes. Analysis of apoptotic gene expression profiles by gene array analysis demonstrated a significant increase in the expression of IAP and survivin (p < 0.05) in islets from pancreata preserved in PFC. There was also a significant decrease in the levels of anti-apoptotic genes. The mean fold expression of the individual genes are expressed as bars (sample/β-actin). Open bars represent the islets from pancreata preserved in UW (Group A), closed bars represent the islets from pancreata preserved immediately in PFC (Group B) and bars with stripes represent the islets from pancreata preserved in PFC after cold ischemia (Group C).

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Reduction in activated caspase levels in islets isolated from PFC stored pancreata

Activation of caspases is an important hallmark of apoptosis (18). To study the effect of PFC on islet apoptosis, the levels of the activated caspases in the isolated islets were determined. The levels of active caspase 9 in seven of the eight islets isolated from pancreata preserved in PFC (Group B) were reduced by 78.0 + 28.7% compared to controls (Figure 2). Similarly, the levels of caspase 2 (61.5%), caspase 3 (68.3%) and caspase 8 (37.2%) were significantly reduced in the islets isolated from pancreata preserved in PFC (Group B) compared to controls preserved in UW solution (Group A) (Figure 2). However, in islets isolated from pancreata preserved in PFC after cold storage in UW solution (Group C) the mean levels of activated caspase 2 (0.71 vs. 0.65), caspase 3 (1.05 vs. 1.04), caspase 8 (0.48 vs. 0.62) and caspase 9 (0.85 vs. 0.66) were similar to that observed in the islets isolated from control pancreata (Group A). The results indicated that preservation of pancreata in the PFC prior to islet isolation results in significant reduction of apoptosis.

image

Figure 2. Significant reduction in the levels of active initiator and effector caspases in islets from pancreata preserved in PFC. Caspase profiling demonstrated a significant reduction in the levels of active caspase 2, caspase 3, caspase 8 and caspase 9 levels in islets isolated from pancreata preserved in PFC. The bars represent the mean optical densities at 460 nm. Open bars represent the islets from pancreata preserved in UW (Group A), closed bars represent the islets from pancreata preserved immediately in PFC (Group B) and bars with stripes represent the islets from pancreata preserved in PFC after cold ischemia (Group C).*p < 0.05

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Increased levels of activated Bcl2 in islets from pancreata preserved in PFC

The Bcl2 family of intracellular proteins plays an important role in regulation of caspase activation. Activation and function of Bcl2 has been shown to be regulated by phosphorylation (19). Therefore, the levels of native and phosporylated Bcl2 in the samples were analyzed by Western blot. No significant difference in the levels of native Bcl2 was observed in the islets isolated from any of the three groups. However, as shown in Figure 3, (3.9 ± 0.9)-fold (p < 0.05) increase in the levels of phosporylated Bcl2 was observed in six of the eight islets from pancreata preserved in PFC compared to controls preserved in UW alone. Similarly, (2.4 ± 0.6)-fold (p < 0.05) increase in the levels of activated Bcl2 was observed in five of the eight islets isolated from pancreata stored in PFC after prolonged cold storage. The results indicate that preservation of pancreata in PFC increases the levels of active Bcl2 in the isolated islets leading to the inhibition of apoptosis.

image

Figure 3. Preservation in PFC increases the active phospoBcl2, cIAP2 and survivin levels in islets. The levels of functional bcl2, cIAP2 and survivin in the isolated islets were analyzed by Western blot using a phospo-bcl2 specific monoclonal antibody, anti-cIAP2 antibody or anti-survivin antibody. Open bars represent the islets from pancreata preserved in UW (Group A), closed bars represent the islets from pancreata preserved immediately in PFC (Group B) and bars with stripes represent the islets from pancreata preserved in PFC after cold ischemia (Group C). The bars represent the mean relative levels of protein in the isolated islets (protein of interest/β-actin). A significant increase in p-bcl2 was observed in islets isolated from Group B (3.9 fold) and Group C (2.4 fold) compared to islets from Group A (p < 0.05). The levels of cIAP2 were significantly elevated in islets from Group B compared to islets from Group A or Group C. Whereas the levels of survivin were 3–5-fold lower in the islets from Group A or Group C compared to the levels in islets from Group C (*p < 0.05).

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Preservation in PFC upregulates the expression of cIAP2

The inhibitor of apoptosis (IAP) gene family encodes a group of proteins, many of which have been shown to directly inhibit the activity of specific subsets of caspases. IAP proteins (IAPs) are currently considered as the only known direct physiologic caspase inhibitors (20,21). Analysis of cIAP expression demonstrated a 2 ± 0.67 fold (p < 0.05) increase in the expression of cIAP2 in seven of the eight islet preparations from pancreata preserved in PFC compared to controls (Figure 3). Preservation in PFC after prolonged cold storage resulted in a 1.2 ± 0.4 fold increased expression of cIPA2 in three of the eight islet preparations.

Cytosolic survivin levels are higher in islets isolated from pancreata preserved in PFC

Survivin, a member of the IAP family plays a crucial role in the regulation of apoptosis and cell mitosis. Survivin released from the mitochondria into the cytosol has been shown to inhibit caspase activation and regulate apoptosis induction (22). Analysis of survivin expression demonstrated that the levels of survivin were increased (2.7 ± 0.9 fold, p < 0.05) in the cytosol of islets isolated from seven of the eight pancreata stored in PFC compared to islets isolated from pancreata not preserved in PFC (Figure 3). However, the levels of survivin were lower (2 ± 0.4 folds) in islets isolated from pancreata preserved in PFC after prolonged cold storage.

Preservation in PFC reduces the levels of cytochrome C released from the mitochondria

Hypoxia and changes in the redox potential result in loss of mitochondrial membrane integrity and release of cytochrome C into the cytosol (23). Analysis of the levels of cytosolic cytochrome C in the isolated islets demonstrated that there was a 40 ± 10% (p < 0.05) reduction in the levels of cytochrome C in islets isolated from pancreata preserved in PFC compared to islets isolated from pancreata preserved in UW solution alone (Figure 4). In islets isolated from pancreata preserved in PFC after cold storage the levels of cytosolic cytochrome C (seven of eight islets) were reduced by 13 ± 9.8% compared to the controls (Figure 4). These results demonstrate that preservation of pancreata in PFC results in reduction of hypoxia and release of cytochrome C from the mitochondria, a central event in apoptosis induction.

image

Figure 4. Significant reduction in levels of cytosolic cytochrome C, and Apaf1 in islets isolated from pancreata preserved in PFC. The levels of cytochrome C and Apaf1 in the cytosolic extracts of the isolated islets were analyzed by Western blot. The bars represent the mean levels of cytochrome C and Apaf1 in the cytosol of isolated islets compared to β-actin levels. Open bars represent the islets from pancreata preserved in UW (Group A), closed bars represent the islets from pancreata preserved immediately in PFC (Group B) and bars with stripes represent the islets from pancreata preserved in PFC after cold ischemia (Group C). A significant decrease in the levels of cytochrome C released from the mitochondria into the cytosol of isolated islets was observed in Group B compared to Groups A and C (*p < 0.05). Whereas a significant decrease was observed in the levels of Apaf1 released in the cytosol of isolated islets from Groups B and C in comparison to Group A (p < 0.05).

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Preservation of pancreata in PFC reduces the levels of cytosolic ApaF1

ApaF1 is a crucial component of the apoptasome which links the cytochrome C released from the mitochondria to caspase activation (24). Analysis of the cytosolic extracts of islets showed that there was a 61.5 ± 21.8% (p < 0.05) reduction in levels of cytosolic Apaf1 (Figure 4) in islets from pancreata preserved in PFC compared to preservation in UW solution alone. Preservation of pancreata in PFC even after prolonged cold storage also resulted in a 42.7 ± 14.4% (p < 0.05) reduction in the levels of cytosolic Apaf1 compared to the control islets (Figure 4) demonstrating that preservation of PFC reduces hypoxia, preserving mitochondrial membrane integrity.

Cytosolic AIF1 levels are lower in islets preserved in PFC

When cells are induced to undergo apoptosis, AIF is released from mitochondria to the cytosol and to the nucleus. The release of AIF occurs concomitantly with the dissipation of the mitochondrial inner transmembrane potential, and shortly before the release of cytochrome C (25,26). AIF is a caspase-independent effector of apoptosis that acts beyond the Bcl2-controlled checkpoint of cell death (27). AIF has been shown to cause the dissipation of the mitochondrial transmembrane potential and the release of cytochrome C, followed by the condensation of nuclear chromatin and the exposure of phosphatidylserine on the plasma membrane surface. Analysis of AIF1 levels demonstrated that preservation of pancreata in PFC resulted in a 57.3 ± 13% (p < 0.05) reduction in the levels of cytosolic AIF1 compared to the controls (Figure 5). Preservation in PFC after prolonged cold storage also resulted in the reduction of cytosolic AIF1 (15.5 ± 12.4%, Figure 5). These results indicate that preservation of pancreata in PFC leads to stabilization of the mitochondrial inner transmembrane potential and inhibition of apoptosis in the isolated islets.

image

Figure 5. Levels of AIF1 are significantly lower in islets from pancreata preserved in PFC. The levels of AIF1 in the isolated islets were analyzed by Western blot. The bars represent the mean fold expression levels of AIF1 in the isolated islets (AIF1/β actin). The levels of AIF1 were significantly lower in the islets isolated from Group A compared to those from Group B or Group C (*p < 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Islet cell transplantation is an attractive treatment option for select individuals with type I diabetes. Islets isolated from more than one donor organ are often essential to restore normoglycemia in these patients and this need for islets from multiple donors is one barrier to wider application of this therapy. Better donor management, improved organ preservation, optimal islet processing, pre-transplant islet modifications, alternative transplant techniques and novel immunosuppressive agents or immune modulation have all been proposed as methods to enhance the success of islet transplantation (28–33). Islet cell death during pancreata procurement, storage and isolation is an impediment to achieving success with a single islet donor. Apoptosis, initiated by hypoxic metabolic stress and various cytokines, has shown to be the major pathways through which islets undergo cell death (16,33). This cell loss has been shown to be initiated shortly after islet isolation with detectable apoptosis within 48 h of isolation (16).

The cascade of ischemic injury during the process of pancreas procurement and storage is hypothesized to be due to hypoxia. Hypoxia in the pancreas may lead to decrease in the mitochondrial potential leading to the release of cytochrome C and Apaf1 into the cytosol. This, in turn, may activate the initiator caspase 9, leading to caspase 3 activation and apoptosis in the isolated islets. Prevention of hypoxia by preservation of the pancreata in PFC can lead to improved oxygenation resulting in an increased expression of anti-apoptotic genes and cell survival. Preliminary studies on measurements of dissolved oxygen concentration in the UW solution during pancreata preservation demonstrated 80% decline in the oxygen concentration compared to the original dissolved oxygen concentration of the UW solution. Significantly, when pancreata were preserved in PFC/UW solutions, there was only a 23% drop in the dissolved oxygen concentration. This suggests that during the process of pancreata preservation, the oxygen released from the PFC may replenish the oxygen consumed by the pancreata.

Results from this study demonstrated that there was a significant increase in the cytosolic levels of Apaf1, AIF1 and cytochrome C in the isolated islets indicating apoptotic cell death was mainly mediated by the change in mitochondrial potential. These observations are similar to those observed in a rat islet model where it was demonstrated that apoptosis in islets is mainly mediated by the mitochondrial pathway (34). In this study a significant increase in the levels of activated caspases 2, 3 and 9 was observed in the islets isolated from pancreas preserved in UW solution. Preservation of the pancreas using the two layer (UW/PFC) was found to decrease the levels of these activated caspases (34).

Results from our studies presented in Table 1 demonstrate that preservation of pancreata immediately in PFC/UW solution resulted in a twofold increase in the number of islets isolated per gram of pancreata compared to pancreata preserved in UW solution alone (4987 vs. 2499). Moreover when pancreas was preserved for >16 h in UW solution alone the number of islets per gram of pancreas processed decreased by 48.6% to 1285. These observations are similar to those reported by others (5,35,36), where it was demonstrated that preservation of pancreata in PFC results in increase in the islet yield. Although both human and animal studies have shown that preservation of pancreata with the two-layer method (PFC and UW) results in increased islet yield and function, the mechanism of action remains undetermined.

Results presented in this study (Figure 4) demonstrate that islets from pancreata preserved in PFC have significantly decreased cytosolic levels of cytochrome C and Apaf1 when compared to control islets, indicating that apoptosis in isolated islets is mainly mediated through the mitochondrial pathway. Apoptosis has been shown to be the major pathway through which cell death occurs after ischemic injury (37–39). Cold ischemia during traditional organ preservation has been shown to result in hypoxia (40) and induction of apoptosis is mitochondria dependent (41,42). Hypoxia has been shown to decrease the mitochondrial potential leading to the release of cytochrome C. The released cytochrome C binds with the Apaf1/caspase 9 complex to initiate apoptosis (24). Our observations indicate that preservation of pancreata in PFC reduces organ hypoxia during cold preservation, resulting in preservation of the mitochondrial redox potential and reduction in apoptosis mediated through the mitochondrial pathway.

Analysis of phosphorylated Bcl2 protein expression (Figure 3) demonstrated that in islets isolated from PFC-preserved pancreata, there was a three-fold increase in the levels of active Bcl2 compared to controls. The Blc2 family of proteins, comprised of the anti-apoptotic genes Bcl2, Bcl-xL, Bcl-w, A1 and Mcl1 as well as the pro-apoptotic members Bax and the BH3-only families, has been shown to play a central role in the regulation of apoptosis (43). Analysis of these proteins in isolated islets demonstrated that preservation in PFC resulted in a decrease in the expression of bad and bax, with no difference in the expression of Bcl2 when compared to control islets. Translational control and post-translational modifications of the Bcl2 family of proteins have been shown to play a crucial role in regulating apoptosis. Phosporylation of the Bcl2 at serine 80 is an important event required for the anti-apoptotic function of Bcl2 (44). Intermittent hypoxia and reoxygenation has been shown to induce protection through protein kinase C activation (45), through phosporylation of Bcl2 (46). Over-expression of Bcl2 both in human and in rodent islets has been shown to inhibit apoptosis (47,48). Similar to these studies, our results indicate that preservation of pancreata in PFC lead to activation of protein kinase C and other stress-related kinases resulting in phosporylation of Bcl2 and inhibition of apoptosis. This, in turn, could account for the increased islet yields from pancreata preserved in PFC.

A significant increase in the expression of cIAP2 and survivin was observed in islets isolated from pancreata preserved in PFC compared to control islets (Figure 3). Increased expression of IAP2 following hypoxia as well as overexpression of xIAP in β-cells (49) act by both HIF-1-dependent and independent mechanisms to inhibit apoptosis and promote cell survival (50,51). Survivin expression has also been shown to indicate a favorable remodeling pattern after acute ischemic injury (52,53). Our results demonstrate that reduction of hypoxia by preservation of pancreata in PFC leads to inhibition of apoptosis through preservation of mitochondrial transmembrane potential and increased expression of anti-apoptotics genes Bcl2, survivin and cIAP2.

In conclusion, we have shown that preservation of the pancreas in PFC results in the suppression of hypoxia-induced apoptosis mediated through the mitochondrial pathway. Inhibition of apoptosis by preservation in PFC is mediated by increased levels of the phosporylated bcl-2, cIAP2 and survivin, which might aid in the preservation of the mitochondrial potential and prevent the release of cytochrome C and Apaf1 into the cytosol. Moreover, the observation that preservation of pancreata in PFC even after prolonged cold storage in UW alone indicates that reoxygenation by PFC may rescue islets from undergoing apoptotic cell death. The mechanisms of islet apoptosis induction presented in this study provide additional avenues to increase the likelihood of restoring normoglycemia in recipients with islets isolated from a single donor.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by NIH RR16597, NIH 2P60DK20579, JDRF 1–2005–333 (TM). SR is recipient of JDRF Fellowship 3–2005–231. WC is supported by NIH HL66196.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Design and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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