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Reperfusion-Preconditioned Hepatocytes Improves Islet Function and Survival
Coculture With Ischemia/Reperfusion-Preconditioned Hepatocytes Improves Islet Function and Survival J.G. Leea,b, J.I. Leec, J.Y. Kimb, Y. Chob, K.H. Huha,b, B.S. Kimb,d, M.S. Kima,b, Y.S. Kima,b, and D.J. Jooa,b,* a Department of Surgery, Yonsei University College of Medicine, Seoul, Korea; bResearch Institute for Transplantation, Yonsei University College of Medicine, Seoul, Korea; cDepartment of Veterinary Obstetrics and Theriogenology, College of Veterinary Medicine and Regenerative Medicine Laboratory, Center for Stem Cell Research, Institute of Biomedical Science and Technology (IBST), Konkuk University, Seoul, Korea; and dDivision of Nephrology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea
ABSTRACT In clinical islet transplantation, hepatic ischemia and insufficient neovascularization of transplanted islets are barriers to islet survival and function. However, hepatocytes have a potency to protect themselves against ischemia. We hypothesized that ischemia/reperfusion preconditioning (IRP) of hepatocytes might beneficially affect islet cells in a coculture system. Primary islets were cocultured with primary hepatocytes, and hepatocyte IRP was conducted by subjecting cells to hypoxic conditions for single 15-minute/30-minute hypoxia, or 2 tandem 15-minute/30-minute hypoxic treatments (hypoxic-normoxic-hypoxic). We show that gene expression levels of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), transforming growth factor-a (TGF-a), and TGF-b1 in hepatocytes were increased by IRP. IRP hepatocytes secreted hepatocyte growth factor and insulin-like growth factor-1. Coculture of islets with IRP hepatocytes enhanced islet insulin secretion in glucose challenge test and expression of the survival-related gene Bcl-2 and the regenerating gene-1a (Reg-1a). Islets cocultured with the 30-minute double-IRP hepatocytes displayed significantly higher viability in the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase dUTP nick end labeling stain compared with that of islets subjected to 30 minutes of hypoxia. These results suggest that islet coculture with IRP hepatocytes can improve islet survival and insulin secretion.
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CLINICAL research group in Edmonton developed a successful human islet transplantation protocol in 2000, in which islets are transplanted into the liver through the portal vein [1]. However, early graft failure is a major hurdle for successful intraportal islet transplantation, although the mechanisms remain unclear. These mechanisms may be mediated by immunological, mechanical, or structural factors, or ischemia-reperfusion injury. In cell transplantation, individual cells are exposed to an environment with insufficient oxygenation and vascularization. Thus, hepatic ischemia and insufficient neovascularization of transplanted islets are major barriers to early graft survival and function [2]. Yin et al [2] revealed that the islet emboli injected through portal vein block blood flow to capillary beds, which leads to local hypoxia, expression of ª 2018 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 50, 3887e3894 (2018)
proinflammatory cytokines, and apoptosis and necrosis of the surrounding liver tissue. They showed 10 minutes of ischemic-preconditioning of mice liver prior to islet transplantation prevents early graft failure from ischemia injury.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-R1A1A1012734). *Address correspondence to Dong Jin Joo, MD, PhD, Department of Surgery, Yonsei University College of Medicine 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea. Tel: þ82-2-2228-2131, Fax: þ82-2-313-8289 E-mail: djjoo@ yuhs.ac 0041-1345/18 https://doi.org/10.1016/j.transproceed.2018.07.007
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According to this report, escaping ischemic liver injury can create optimal environment for transplanted islets. Hepatocytes are well known for their regenerative capacities [3e5]; they have protective mechanisms against ischemic- and T-cell-mediated injury, which involves transforming growth factor-b1 (TGF-b1), interleukin (IL)-6-gp, and GP130-signal transducer and activator or transcription 3 (STAT3)-mediated signaling pathway [6,7]. Transplanted islet cells encounter an environment that is surrounded with hepatocytes during intraportal engraftment of clinically transplanted islets. No studies have addressed whether the protective or regenerative effects of hepatocytes also affect transplanted islets. Coculture methods of islet cells with different cell types, such as Sertoli and stem cells, have been investigated as a way to improve islet cell function [8,9]. These cells do not reflect the clinical environment of intraportal islet transplantation. Hepatic ischemia and insufficient neovascularization of transplanted islets are obstacles to the long-term survival and insulin-secreting function of islets [2]. Thus, coculture with hepatocytes under ischemic conditions may mimic the environment encountered during intraportal islet transplantation. The protective effects of IL-6 and STAT3 on ischemia and reperfusion injuries in affected livers and cardiac muscles during ischemic preconditioning are well known [10,11]. However, it is unclear whether these hepatocyte properties affect cells from other organs. We hypothesized that ischemia/reperfusion-preconditioned (IRP) hepatocytes could enhance islet cell survival and function in coculture conditions. Cocultured pancreatic islets have been reported to affect hepatocyte growth and differentiation [12,13]. Thus, cocultured islets and hepatocytes should symbiotically support each other. To evaluate the potential beneficial effects of hepatocytes on islet cells, we cocultured primary islet cells with IRP hepatocytes. We observed that cocultured IRP hepatocytes improved the function and survival-related markers of islet cells.
MATERIALS AND METHODS Primary Cell Isolation Male Sprague Dawley (SD) rats were used for hepatocyte and islet isolation. The rats were maintained according to the ethical guidelines of the institution, and the Animal Investigation Committee of Yonsei University approved the experimental protocol. For hepatocyte isolation, the SD rats were anesthetized using a combination of 50 mg/kg of ketamine (Yuhan Co, Seoul, South Korea) and 6.7 mg/kg of xylazine (Bayer Korea Ltd, Seoul, South Korea) or 15 mg/kg of zoletil (Virbac Lab, Carros, France) and 9.3 mg/kg of xylazine, which were injected intraperitoneally. The hepatic portal vein was cannulated with a 20-gauge catheter and perfused with 250 mL Ca2þ-free liver perfusion medium at a rate of 25 mL/min for 10 minutes. The suprahepatic vena cava was immediately cut to flush the perfusion fluid. We then perfused liver digestion medium at the same rate and volume as specified above. The digested liver was transferred to a culture dish filled with cold hepatocyte wash medium, and cells were dispersed by manually
LEE, LEE, KIM ET AL shaking. Subsequently, the suspended hepatocyte cells were filtered through a 100-mm mesh filter and centrifuged at 50 g for 5 minutes. Viable cells were identified by trypan blue staining. Islets were isolated as described previously [14]. Pancreata were injected and expanded with 10 mL of 1 mg/mL collagenase P (Roche, Indianapolis, Ind, United States) in Hank’s balanced salt solution (Invitrogen, Carlsbad, Calif, United States) and then digested for 16 minutes. The digestion was stopped with cold Hank’s balanced salt solution containing 5% fetal bovine serum. The islets were passed through a 500-mm mesh filter and purified on a Histopaque density gradient (Sigma-Aldrich, St Louis, Mo, United States). The collected islets were purified twice by manual selection. Cells were maintained in RPMI1640 (Invitrogen Co, Carlsbad, Calif, Untied States), supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100mg/mL streptomycin at 37 C in a 5% CO2 humidified atmosphere. D-glucose concentration of the media was 2 g/L.
Hepatocyte Ischemia/Reperfusion Preconditioning and Coculture With Islets Hepatocyte IRP was performed as follows: isolated hepatocytes were incubated under hypoxic conditions in a modular incubator chamber (Billups-Rothenverg, Del Mar, Calif, United States) with 5% CO2 in a humidified atmosphere. Five different conditions were implemented: 1. normoxia (control), 2. hypoxia for 15 minutes (15-minute ischemia injury), 3. hypoxia for 15 minutes, normoxia for 15 minutes, and hypoxia for another 15 minutes (double 15-minute IRP), 4. hypoxia for 30 minutes (30-minute ischemia injury), and 5. hypoxia for 30 minutes, normoxia for 30 minutes, and hypoxia for another 30 minutes (double 30-minute IRP). For treatment with conditioned media, the collected media were centrifuged at 8000 rpm for 3 minutes, and the pelleted debris was discarded. Next, the supernatants were immediately transferred into the new sample wells containing the experimentally isolated islets. For Transwell (Costar, Corning, NY, United States) cultures, experimental islets were transferred into wells containing hepatocytes at the endpoints of the ischemic treatments.
Western Blot Analysis The expression levels of p-STAT3 and STAT3 were measured in IRP hepatocytes by western blotting. Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif, United States) and lysed on ice for 10 minutes in buffer (BioSource, Camarillo, Calif, United States) supplemented with a protease inhibitor cocktail (Sigma). The lysates were centrifuged at 12,000 g for 15 minutes at 4 C after trituration on ice through a syringe fitted with a 26-gauge needle. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, Calif, United States). Aliquots of 20 mg per sample were mixed with loading buffer (60 mM Tris-HCl, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue), separated on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour with 5% nonfat dry milk before incubating with an antibody against either p-STAT3 or STAT3 (Cell Signaling, Beverly, Mass, United States). The blots were washed and then incubated for 1 hour at room temperature with horseradish peroxidaseeconjugated anti-rabbit or anti-goat antibody. Antibody binding was visualized by enhanced chemiluminescence (Amersham ECL, GE Healthcare, United Kingdom), and the images were detected and quantified by densitometry on a Bio-Rad densitometer. Representative blots were selected from a set of 3 independent experiments.
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Glucose Challenge Test of Cocultured Islets Function of islets cocultured with IRP hepatocytes was tested by 1-hour exposure to different concentrations of glucose media (100mg/dL and 400mg/dL). Insulin-secreting ability of cocultured islets was measured by glucose challenge test expressed as the stimulation index (SI). This static short-term insulin secretion test allowed the calculation of an SI (SI ¼ [insulin secretion at 400 mg glucose/dL]/[insulin secretion at 100 mg glucose/dL]).
3889 Real-Time PCR System (Applied Biosystems, Foster City, Calif, United States) according to the manufacturer’s protocol. Gene expression levels are reported as fold-change in the level of transcript relative to the level of GAPDH.
Immunofluorescence Staining
Islet survival, after coculture with IRP hepatocytes or treatment with exogenous recombinant HGF and IGF-1 treatments, was determined by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays as follows: 1 mg/mL of MTT was added to each well, and samples were incubated at 37 C in an incubator with humidified 5% CO2 atmosphere for 2 hours. Then, extraction buffer (20% SDS, 50% N, N-dimethylformamide, pH 4.7) was added, and samples were incubated overnight at 37 C. The optical density of samples was measured with a microplate reader (SpectraMax 340, Molecular Devices Co, Calif, United States) at a wavelength of 562 nm.
BCL-2 and REG-1a proteins were examined by immunofluorescent staining. Islets cocultured with IRP hepatocytes were fixed in 4% paraformaldehyde for 2 days. Next, the islets were dehydrated overnight in 10%, 20%, and 30% sucrose. Islet slices were snapfrozen in an optimal cutting-temperature solution, 4 mm sections were cut, and sections were mounted on slides. Subsequently, the sections were permeabilized with 0.1% Triton X-100 (SigmaAldrich, St Louis, Mo, United States) and blocked with 0.5% bovine serum albumin (Invitrogen, Carlsbad, Calif, United States) in PBS. The slides were incubated overnight at 4 C with a primary polyclonal antibody against BCL-2 (Santa Cruz Biotechnology) or REG-1a (USCN Life Science, Wuhan, China) at 1:100 dilution in blocking solution. The slides were washed and incubated with an Alexa Fluor 488-conjugated antimouse antibody (BCL-2) or Alexa Fluor 568-conjugated anti-mouse antibody (REG-1a) (Invitrogen) for 1 hour at room temperature.
RNA Isolation and Real-Time Polymerase Chain Reaction
Statistical Analysis
IRP hepatocytes and cocultured islets cells were rinsed twice with cold PBS and treated with TRIzol reagent (Invitrogen) to extract mRNA. Chloroform was added to the lysed cells, and the cells were incubated for 15 minutes. The samples were then centrifuged at 13,000 g for 15 minutes at 4 C. The aqueous phase of each sample was transferred to a new tube, mixed with isopropyl alcohol to precipitate RNA, and the precipitated RNA was pelleted by centrifugation at 13,000 g for 10 minutes at 4 C. The extracted RNA was washed twice with 75% ethanol in RNase-free water. Finally, each isolated RNA sample was dissolved in 20 mL of RNase-free water and stored at 70 C before use. Quantities of total RNA were measured with a NanoDrop spectrophotometer (NanoDrop Technologies, Inc, Wilmington, Del, United States). Each reverse transcriptase reaction (AccuPower RT Premix Kit, Invitrogen, Co, Carlsbad, Calif, United States) included 1 mg of total RNA for cDNA synthesis. Total RNA was reverse-transcribed with Oligo-dT according to an M-MLV Reverse Transcriptase (Invitrogen) protocol to synthesize cDNA. The cDNA samples were analyzed by real-time polymerase chain reaction (PCR) with the following primers: GAPDH, Forward 5’TCCCTCAAGATTGTCAGCAA-3’, Reverse 5’-AGATCCACAAACGGATACATT-3’; Insulin-like growth factor 1 (Igf-1), Forward 5’-CAGTTCGTGTGTGGACCAAG-3’, Reverse 5’TCAGCGGAGCACAGTACATC-3’; Hepatocyte growth factor (Hgf), Forward 5’-CGAGCTATCGCGGTAAAGAC-3’, Reverse 5’-TGTAGCTTTCACCGTTGCAG-3’; Transforming growth factor-alpha (Tgf-a), Forward 5’-AGCATGTGTCTGCCACTCTG3’, Reverse 5’-TGGATCAGCACACAGGTGAT-3’; Transforming growth factor-beta 1 (Tgf-b1), Forward 5’-ATACGCCTGAGTGGCTGTCT-3’, Reverse 5’-TGGGACTGATCCCATTGATT-3’; Regenerating islet-derived 1 alpha (Reg-1a), Forward 5’CCTGATGGTCCTTTCTCCAA-3’, Reverse 5’-CAGAGAGGCCAGAAAGTTGC-3’; B-cell lymphoma 2 (Bcl-2), Forward 5’CGACTTTGCAGAGATGTCCA-3’, Reverse 5’-ATGCCGGTTCAGGTACTCAG-3’; and Insulin 1 (Ins-1), Forward 5’CACCTTTGTGGTCCTCAC-3’, Reverse 5’-AGTTGGTAGAGGGAGCAG-3’. Real-time PCR was performed with SYBR Premix Ex Taq (Takara, Tokyo, Japan) on an ABI StepOnePlus
The results are presented as means (SDs). Mean group values were compared using t tests and SigmaPlot ver. 2.0 (SYSTAT, Chicago, Ill, United States). P values less than .05 were considered statistically significant.
Islet Survival-Related Marker Measurements
RESULTS Regeneration-Related Protein and Gene Expression of IRP Hepatocytes
In the hepatocyte analysis, phosphorylated STAT3 (p-STAT3) levels were significantly higher in the 15- and 30minute double-IRP groups than in the control groups immediately after IRP exposure and after 1 day in culture. The p-STAT3 level remained elevated until day 1, but the difference between p-STAT3 levels in the IRP and control groups declined at day 1 (Fig 1A). The Igf-1, Hgf, Tgf-a, and Tgf-b1 gene expression levels were evaluated by conducting real-time PCR analyses on day 0 and day 1 after IRP. No significant differences in Igf-1 expression levels were detected immediately after ischemic treatment for 15 and 30 minutes or after double-IRP treatments (Fig 1B). However, Igf-1, Tgf-a, and Tgf-b1 expression was relatively induced in both IRP groups. After 24 hours, Igf-1 expression was significantly increased in the 30-minute double-IRP group, and the gene expression levels of Hgf, Tgf-a, and Tgf-b1 were significantly higher in both IRP groups compared with those in the other groups. Secreted Factors From IRP Hepatocytes
To evaluate the secretory function of IRP hepatocytes, we measured HGF, IGF-1, TGF-a, and TGF-b1 protein levels in culture supernatants by performing enzyme-linked immunosorbent assay (ELISA). Although there were no significant increases in TGF-a or TGF-b1 in the treated groups compared with the control group, HGF and IGF-1
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Fig 1. Regeneration-related protein and gene expression. (A) p-STAT3 protein expression in ischemia/reperfusion preconditioning (IRP) hepatocytes. p-STAT3 protein known as a cell regeneration significantly increased by IRP. Both conditions of 15 m 2 and 30 m 2 IRP increased p-STAT3 protein expression more than control and single ischemia preconditioned group in day 0 and 1. (B) Real-time polymerase chain reaction (PCR) analysis of gene expression in IRP hepatocytes on day 0 and 1. Gene expressions of Hgf, Tgf-a, and Tgf-b1 were significantly increased by each of 15 m 2 and 30 m 2 IRP. The Igf-1 gene expression showed delayed increase at day 1 in 30 m 2 IRP. n ¼ 5, *P < .05 compared with the control group.
increased with longer preconditioning times (Fig 2A). This delayed IGF-1 secretion was correlated with delayed gene expression of Igf-1 (Fig 1B). Effects of Secreted Factors From IRP Hepatocytes on Islet Insulin Secretion
ELISA analyses were performed to assess insulin secretion from islets cocultured with hepatocytes. Figure 2B shows the levels of insulin secretion from islets that were cocultured with preconditioned hepatocytes in transwells and the percentages of conditioned medium. All islets were evaluated at day 1 or 2 after IRP. On both days 1 and 2, cells grown under transwell conditions with preconditioned hepatocytes showed significantly higher levels of insulin secretion in the 15- and 30-minute double-IRP conditions than the control group. To check effects of secreted elements from IRP hepatocytes, islets were put on a conditioned media originating from the same culture media
of IRP hepatocytes, but hepatocytes were removed after culture. Islets under the conditioned media showed significantly higher insulin secretion than control group except islets under the medium from day 2 for the 15-minute double-IRP condition. This result revealed that the secretary elements from the IRP hepatocytes affected insulin secretion of islets. Evaluation of the Beneficial Effects on Insulin Secretion Function and Islet Survival
Glucose challenge tests of the islets were expressed by SI. SI was measured from the islets cocultured with hepatocytes under different time condition of IRP. Islets cocultured with 15 m and 15 m 2 IRP hepatocytes showed higher SI than the control group, but there was no significantly increased SI in 30 m and 30 m 2 IRP groups (Fig 3A). Real-time PCR analysis was used to evaluate the survivalrelated gene expression levels of Bcl-2, Tgf- b1, Reg-1a, and
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Fig 2. Secreted factors from ischemia/reperfusion preconditioning (IRP) hepatocytes. (A) Hepatocyte growth factor (HGF) and insulinlike growth factor (IGF)-1 secretion, as determined by supernatant protein levels, tended to increase depending on the duration of preconditioning treatment. HGF secretion increased by 30 m 2 IRP at day 1 and 2. IGF-1 increased by each of 15 m 2 and 30 m 2 IRP at only day 2. (B) Islets cultured in the conditioned media that had same condition but removing hepatocytes after culture showed similar insulin secretion as islets cocultured with hepatocytes in the transwell condition. This revealed that the secretary elements from the IRP hepatocytes affected insulin secretion function of islets. *P < .05 compared with the control group.
Ins-1 in islets cocultured with preconditioned hepatocytes. Bcl-2 and Reg-1a gene expression levels significantly increased after both 15- and 30-minute double-IRP treatments, and Tgf-b1 expression was significantly higher in cells treated with 15-minute double-IRP than under other conditions. Ins-1 gene tended to express more in IRP treatment but there were no significant differences among the treatment groups (Fig 3B). Immunofluorescence labeling was performed to evaluate the expression of the survival-related proteins BCL-2 and REG-1a in cocultured islets with IRP hepatocytes. The 15-minute double-IRP group was strongly positive for BCL2 compared with the control group. The 15- and 30-minute double-IRP groups expressed significantly higher REG-1a than the control group (Fig 3C). We conducted MTT assays to confirm the islets’ cell viability under each condition. The 15- and 30-minute hypoxia-injured groups displayed more apoptosis in MTT assay on days 1 and 2. By contrast, the 15- and 30minute double-IRP groups showed more cell viability on day 1 and 2. Besides, the 15- and 30-minute double-IRP
groups showed significantly greater cell viability, according to the MTT assay compared with the 15- and 30-minute hypoxia-only treatment groups (P < .05, Fig 4A). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay performed in 15- and 30-minute double-IRP groups showed less apoptotic islets than control group (Fig 4B). DISCUSSION
In this study, we investigated the interactions between cocultured hepatocytes and islets. A previous study reported that IRP was beneficial for hepatocyte regeneration and STAT-3 activation and prevented ischemic reperfusion injury [2,15]. Our previous work used the Hep-G2 and RIN5F cell lines, showed that ischemia increased IL-6/STAT-3 pathway activation in Hep-G2 cells, and showed that coculture with hypoxic-preconditioned Hep-G2 cells increased insulin secretion in cocultured RIN-5F cell lines [16]. We subjected primary hepatocytes to longer IRP treatments, but were unable to increase the IRP time in the
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Fig 3. Insulin secretion function and survivalrelated gene/protein expression in cocultured islets. (A) Glucose challenge test. Islets cocultured with 15 m and 15 m 2 ischemia/reperfusion preconditioning (IRP) hepatocytes showed higher Stimulation Index than the control group. (B) Bcl2 and Reg-1a gene expression of islets cocultured with hepatocytes in transwell were significantly increased by each of 15 m 2 and 30 m 2 IRP. Tgf-b1 genes were significantly increased in 15 m 2 IRP. (C) Cells were immunolabeled for B-cell lymphoma (BCL)-2 and regenerating gene (REG)-1a. The 15 m 2 IRP group labeled strongly for BCL-2 (red) compared with the control group. The 15 m 2 and 30 m 2 IRP groups labeled strongly for REG-1a (green) compared with the control group. Hoechst staining is shown in blue ( 200). *P < .05 compared with the control group. The length of white bar is 100 mm.
primary cell experiments because of the subsequent reduction of primary hepatocyte viability. Thus, double-IRP conditions for 15 and 30 minutes were defined as the IRP model with respect to primary hepatocyte survival. As a result, primary hepatocyte viability was comparable with that of the control group as demonstrated by the MTT assay and TUNEL stain. First, to confirm our IRP model properly works to stimulate regenerative factors from the hepatocytes against ischemia-reperfusion injury, we checked p-STAT-3 protein
and tissue regenerationerelated gene expressions. Hepatocytes are well characterized in the literature for their regenerative capacities [3e5]; they have protective mechanisms against ischemia-reperfusion injuries that involve transforming growth factor-b1 (TGF-b1), interleukin (IL)-6, and the GP130 signal transducer and STAT3-mediated signaling pathway [6,7]. Our results confirmed that the levels of p-STAT3 rapidly increased immediately after IRP treatment and remained elevated until day 1, but the difference between IRP and control groups decreased
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Fig 4. Islet survival analysis. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays indicated that islets had greater viability in the 15 m 2 and 30 m 2 ischemia/reperfusion preconditioning (IRP) groups than in the hypoxia-alone groups. (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain of control, 15 m 2, and 30 m 2 IRP groups. *P < .05 compared with the control group at day 2. yP < .05 compared with each ischemia-alone group.
thereafter. This result is reasonable because the JAK-STAT pathway is triggered by ischemia but later stabilizes [17]. We confirmed that primary hepatocytes displayed the same condition of p-STAT3 by conducting an in vitro study under IRP. The STAT3 pathway and IGF-1, HGF, TGF-a, and TGFb1 gene expression are important for tissue regeneration and organ protection after ischemia-reperfusion injury [18]. In the current study, the gene expression levels of Igf-1 on day 1 and the expression levels of Hgf and Tgf-b1 on days 0 and 1 were upregulated in the IRP hepatocytes. The expression levels of these genes differed from those in groups treated with ischemia alone, which had expression levels similar to those in the control group. The expression levels of Hgf, Tgf-a, and Tgf-b1, after 15 and 30 minutes of double-IRP treatment on day 1 were significantly higher than those in cells treated for 15 and 30 minutes with ischemia injury alone. This suggests that IRP induced the synthesis of protective factors in hepatocytes that were not induced by ischemia injury alone. We measured the survival-related gene expression in islets cocultured with IRP-hepatocytes in transwells. The effects of TGF-b1 on liver regeneration or protection against ischemic injury remain controversial [19,20]. However, TGF-b1 expression is thought be associated with survival from IRP-mediated injury. In the current study, Tgfb1 gene expression increased in cocultured islets, and Reg-1a expression was significantly upregulated in cocultured islets, which are involved in beta cell growth. Reg-1a was originally isolated from a cDNA library derived from regenerated rat pancreatic islets, which represents islet cell regeneration [21]. Our results suggest that a secretory factor from the cocultured IRP hepatocytes affects islet gene expression associated with regeneration. However, no significant differences in the Ins-1 gene expression were
observed in islets after coculture with primary IRP hepatocytes. This is a limitation of the current study. Ins-1 gene expression was higher after prolonged IRP duration, but this correlation was not statistically significant. Although there were no significant differences in the Ins-1 gene expression, the levels of secreted insulin in the transwell cocultures, measured by ELISA, significantly increased in response to double-IRP treatment for 15 and 30 minutes. Thus, we conclude that IRP hepatocytes improve insulin secretion from cocultured islets. Bcl-2 is an antiapoptotic marker for pancreatic beta cells [22]. In the current study, Bcl-2 gene expression was significantly higher in islets cocultured with IRP hepatocytes compared with that in the control group. We also showed that the levels of BCL-2 and REG-1a proteins increased in the cocultured islets. The MTT assays revealed that islets cocultured for 15 and 30 minutes with double-IRP hepatocytes had significantly higher viability than the hypoxiaalone group. In addition, TUNEL stain in the IRP group showed less apoptosis than the control group. This result supports our hypothesis that IRP hepatocytes provide survival benefits to cocultured islets. However, a limitation of this study is that we could not determine in vivo survival. Therefore, further in vivo investigations will be necessary to prove this result. Ischemia and ischemia-reperfusion are inevitable in clinical islet transplantation. Ischemia-injured islets and islets surrounded by ischemia-injured hepatocytes cannot survive in the long term, but our data suggest that regeneration power of IRP hepatocytes beneficially affects cocultured islet survival. We are currently developing further in vivo studies and potential clinical applications. In conclusion, coculture of islets with IRP hepatocytes can prevent ischemia or ischemia/reperfusion-related
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injuries and improve insulin-secreting capacities and survival in cultured islets. ACKNOWLEDGMENT This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012-R1A1A1012734).
REFERENCES [1] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230e8. [2] Yin D, Ding JW, Shen J, Ma L, Hara M, Chong A. Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning. Am J Transplant 2006;6:60e8. [3] Kavtiashvili KG, Gabuniia UA, Khundadze TO, Dugladze DI, Chumburidze RA, Khananashvili ShD. The stimulation of regenerative processes in the liver in acute liver failure. Arkh Patol 1990;52:47e52. [4] Matsumoto K, Nakamura T. Hepatocyte growth factor: molecular structure and implications for a central role in liver regeneration. J Gastroenterol Hepatol 1991;6:509e19. [5] Kim RD, Kim JS, Watanabe G, Mohuczy D, Behrns KE. Liver regeneration and the atrophy-hypertrophy complex. Semin Intervent Radiol 2008;25:92e103. [6] Teoh N, Leclercq I, Pena AD, Farrell G. Low-dose TNFalpha protects against hepatic ischemia-reperfusion injury in mice: implications for preconditioning. Hepatology 2003;37:118e28. [7] Klein C, Wüstefeld T, Assmus U, et al. The IL-6-gp130STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury. J Clin Invest 2005;115:860e9. [8] Teng Y, Xue WJ, Ding XM, et al. Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro. Chin Med J (Engl) 2005;118:1857e62. [9] Banerjee I, Sharma N, Yarmush M. Impact of co-culture on pancreatic differentiation of embryonic stem cells. J Tissue Eng Regen Med 2011;5:313e23.
LEE, LEE, KIM ET AL [10] Duval H, Mbatchi SF, Grandadam S, et al. Reperfusion stress induced during intermittent selective clamping accelerates rat liver regeneration through JNK pathway. J Hepatol 2010;52:560e9. [11] Matsumoto T, O’Malley K, Efron PA, et al. Interleukin-6 and STAT3 protect the liver from hepatic ischemia and reperfusion injury during ischemic preconditioning. Surgery 2006;140:793e802. [12] Lee KW, Lee SK, Joh JW, et al. Influence of pancreatic islets on spheroid formation and functions of hepatocytes in hepatocyte-pancreatic islet spheroid culture. Tissue Eng 2004;10: 965e77. [13] Kaufmann PM, Fiegel HC, Kneser U, Pollok JM, Kluth D, Rogiers X. Influence of pancreatic islets on growth and differentiation of hepatocytes in co-culture. Tissue Eng 1999;5:583e96. [14] Kim JY, Lee JI, Jeong JH, et al. Improved yield and functional parameters of rat pancreas islets isolated under intramuscular anesthesia. Cell Transplant 2010;19:743e50. [15] Butler KL, Huffman LC, Koch SE, Hahn HS, Gwathmey JK. STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium. Am J Physiol Heart Circ Physiol 2006;291:H797e803. [16] Joo DJ, Kim JY, Lee KI, et al. Impact of coculture with ischemic preconditioned hepatocellular carcinoma cell line (HepG2) cells on insulin secreting function of rat insulin-secreting cell line (RIN-5F) cells. Transplant Proc 2012;44:1099e103. [17] Soond SM, Latchman DS, Stephanou A. STAT signalling in the heart and cardioprotection. Expert Rev Mol Med 2006;8:1e16. [18] Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res 2004;61:448e60. [19] Kim M, Park SW, Kim M, D’Agati VD, Lee HT. Isoflurane post-conditioning protects against intestinal ischemia-reperfusion injury and multiorgan dysfunction via transforming growth factorbeta1 generation. Ann Surg 2012;255:492e503. [20] Feng M, Wang Q, Zhang F, Lu L. Ex vivo induced regulatory T cells regulate inflammatory response of Kupffer cells by TGF-beta and attenuate liver ischemia reperfusion injury. Int Immunopharmacol 2012;12:189e96. [21] Terazono K, Yamamoto H, Takasawa S, et al. A novel gene activated in regenerating islets. J Biol Chem 1988;263:2111e4. [22] Soria B, Gauthier BR. Dual trade of Bcl-2 and Bcl-xL in islet physiology: balancing life and death with metabolism secretion coupling. Diabetes 2013;62:18e21.
ABSTRACT In clinical islet transplantation, hepatic ischemia and insufficient neovascularization of transplanted islets are barriers to islet survival and function. However, hepatocytes have a potency to protect themselves against ischemia. We hypothesized that ischemia/reperfusion preconditioning (IRP) of hepatocytes might beneficially affect islet cells in a coculture system. Primary islets were cocultured with primary hepatocytes, and hepatocyte IRP was conducted by subjecting cells to hypoxic conditions for single 15-minute/30-minute hypoxia, or 2 tandem 15-minute/30-minute hypoxic treatments (hypoxic-normoxic-hypoxic). We show that gene expression levels of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), transforming growth factor-a (TGF-a), and TGF-b1 in hepatocytes were increased by IRP. IRP hepatocytes secreted hepatocyte growth factor and insulin-like growth factor-1. Coculture of islets with IRP hepatocytes enhanced islet insulin secretion in glucose challenge test and expression of the survival-related gene Bcl-2 and the regenerating gene-1a (Reg-1a). Islets cocultured with the 30-minute double-IRP hepatocytes displayed significantly higher viability in the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase dUTP nick end labeling stain compared with that of islets subjected to 30 minutes of hypoxia. These results suggest that islet coculture with IRP hepatocytes can improve islet survival and insulin secretion.
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CLINICAL research group in Edmonton developed a successful human islet transplantation protocol in 2000, in which islets are transplanted into the liver through the portal vein [1]. However, early graft failure is a major hurdle for successful intraportal islet transplantation, although the mechanisms remain unclear. These mechanisms may be mediated by immunological, mechanical, or structural factors, or ischemia-reperfusion injury. In cell transplantation, individual cells are exposed to an environment with insufficient oxygenation and vascularization. Thus, hepatic ischemia and insufficient neovascularization of transplanted islets are major barriers to early graft survival and function [2]. Yin et al [2] revealed that the islet emboli injected through portal vein block blood flow to capillary beds, which leads to local hypoxia, expression of ª 2018 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
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proinflammatory cytokines, and apoptosis and necrosis of the surrounding liver tissue. They showed 10 minutes of ischemic-preconditioning of mice liver prior to islet transplantation prevents early graft failure from ischemia injury.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-R1A1A1012734). *Address correspondence to Dong Jin Joo, MD, PhD, Department of Surgery, Yonsei University College of Medicine 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea. Tel: þ82-2-2228-2131, Fax: þ82-2-313-8289 E-mail: djjoo@ yuhs.ac 0041-1345/18 https://doi.org/10.1016/j.transproceed.2018.07.007
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According to this report, escaping ischemic liver injury can create optimal environment for transplanted islets. Hepatocytes are well known for their regenerative capacities [3e5]; they have protective mechanisms against ischemic- and T-cell-mediated injury, which involves transforming growth factor-b1 (TGF-b1), interleukin (IL)-6-gp, and GP130-signal transducer and activator or transcription 3 (STAT3)-mediated signaling pathway [6,7]. Transplanted islet cells encounter an environment that is surrounded with hepatocytes during intraportal engraftment of clinically transplanted islets. No studies have addressed whether the protective or regenerative effects of hepatocytes also affect transplanted islets. Coculture methods of islet cells with different cell types, such as Sertoli and stem cells, have been investigated as a way to improve islet cell function [8,9]. These cells do not reflect the clinical environment of intraportal islet transplantation. Hepatic ischemia and insufficient neovascularization of transplanted islets are obstacles to the long-term survival and insulin-secreting function of islets [2]. Thus, coculture with hepatocytes under ischemic conditions may mimic the environment encountered during intraportal islet transplantation. The protective effects of IL-6 and STAT3 on ischemia and reperfusion injuries in affected livers and cardiac muscles during ischemic preconditioning are well known [10,11]. However, it is unclear whether these hepatocyte properties affect cells from other organs. We hypothesized that ischemia/reperfusion-preconditioned (IRP) hepatocytes could enhance islet cell survival and function in coculture conditions. Cocultured pancreatic islets have been reported to affect hepatocyte growth and differentiation [12,13]. Thus, cocultured islets and hepatocytes should symbiotically support each other. To evaluate the potential beneficial effects of hepatocytes on islet cells, we cocultured primary islet cells with IRP hepatocytes. We observed that cocultured IRP hepatocytes improved the function and survival-related markers of islet cells.
MATERIALS AND METHODS Primary Cell Isolation Male Sprague Dawley (SD) rats were used for hepatocyte and islet isolation. The rats were maintained according to the ethical guidelines of the institution, and the Animal Investigation Committee of Yonsei University approved the experimental protocol. For hepatocyte isolation, the SD rats were anesthetized using a combination of 50 mg/kg of ketamine (Yuhan Co, Seoul, South Korea) and 6.7 mg/kg of xylazine (Bayer Korea Ltd, Seoul, South Korea) or 15 mg/kg of zoletil (Virbac Lab, Carros, France) and 9.3 mg/kg of xylazine, which were injected intraperitoneally. The hepatic portal vein was cannulated with a 20-gauge catheter and perfused with 250 mL Ca2þ-free liver perfusion medium at a rate of 25 mL/min for 10 minutes. The suprahepatic vena cava was immediately cut to flush the perfusion fluid. We then perfused liver digestion medium at the same rate and volume as specified above. The digested liver was transferred to a culture dish filled with cold hepatocyte wash medium, and cells were dispersed by manually
LEE, LEE, KIM ET AL shaking. Subsequently, the suspended hepatocyte cells were filtered through a 100-mm mesh filter and centrifuged at 50 g for 5 minutes. Viable cells were identified by trypan blue staining. Islets were isolated as described previously [14]. Pancreata were injected and expanded with 10 mL of 1 mg/mL collagenase P (Roche, Indianapolis, Ind, United States) in Hank’s balanced salt solution (Invitrogen, Carlsbad, Calif, United States) and then digested for 16 minutes. The digestion was stopped with cold Hank’s balanced salt solution containing 5% fetal bovine serum. The islets were passed through a 500-mm mesh filter and purified on a Histopaque density gradient (Sigma-Aldrich, St Louis, Mo, United States). The collected islets were purified twice by manual selection. Cells were maintained in RPMI1640 (Invitrogen Co, Carlsbad, Calif, Untied States), supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100mg/mL streptomycin at 37 C in a 5% CO2 humidified atmosphere. D-glucose concentration of the media was 2 g/L.
Hepatocyte Ischemia/Reperfusion Preconditioning and Coculture With Islets Hepatocyte IRP was performed as follows: isolated hepatocytes were incubated under hypoxic conditions in a modular incubator chamber (Billups-Rothenverg, Del Mar, Calif, United States) with 5% CO2 in a humidified atmosphere. Five different conditions were implemented: 1. normoxia (control), 2. hypoxia for 15 minutes (15-minute ischemia injury), 3. hypoxia for 15 minutes, normoxia for 15 minutes, and hypoxia for another 15 minutes (double 15-minute IRP), 4. hypoxia for 30 minutes (30-minute ischemia injury), and 5. hypoxia for 30 minutes, normoxia for 30 minutes, and hypoxia for another 30 minutes (double 30-minute IRP). For treatment with conditioned media, the collected media were centrifuged at 8000 rpm for 3 minutes, and the pelleted debris was discarded. Next, the supernatants were immediately transferred into the new sample wells containing the experimentally isolated islets. For Transwell (Costar, Corning, NY, United States) cultures, experimental islets were transferred into wells containing hepatocytes at the endpoints of the ischemic treatments.
Western Blot Analysis The expression levels of p-STAT3 and STAT3 were measured in IRP hepatocytes by western blotting. Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif, United States) and lysed on ice for 10 minutes in buffer (BioSource, Camarillo, Calif, United States) supplemented with a protease inhibitor cocktail (Sigma). The lysates were centrifuged at 12,000 g for 15 minutes at 4 C after trituration on ice through a syringe fitted with a 26-gauge needle. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, Calif, United States). Aliquots of 20 mg per sample were mixed with loading buffer (60 mM Tris-HCl, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue), separated on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour with 5% nonfat dry milk before incubating with an antibody against either p-STAT3 or STAT3 (Cell Signaling, Beverly, Mass, United States). The blots were washed and then incubated for 1 hour at room temperature with horseradish peroxidaseeconjugated anti-rabbit or anti-goat antibody. Antibody binding was visualized by enhanced chemiluminescence (Amersham ECL, GE Healthcare, United Kingdom), and the images were detected and quantified by densitometry on a Bio-Rad densitometer. Representative blots were selected from a set of 3 independent experiments.
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Glucose Challenge Test of Cocultured Islets Function of islets cocultured with IRP hepatocytes was tested by 1-hour exposure to different concentrations of glucose media (100mg/dL and 400mg/dL). Insulin-secreting ability of cocultured islets was measured by glucose challenge test expressed as the stimulation index (SI). This static short-term insulin secretion test allowed the calculation of an SI (SI ¼ [insulin secretion at 400 mg glucose/dL]/[insulin secretion at 100 mg glucose/dL]).
3889 Real-Time PCR System (Applied Biosystems, Foster City, Calif, United States) according to the manufacturer’s protocol. Gene expression levels are reported as fold-change in the level of transcript relative to the level of GAPDH.
Immunofluorescence Staining
Islet survival, after coculture with IRP hepatocytes or treatment with exogenous recombinant HGF and IGF-1 treatments, was determined by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays as follows: 1 mg/mL of MTT was added to each well, and samples were incubated at 37 C in an incubator with humidified 5% CO2 atmosphere for 2 hours. Then, extraction buffer (20% SDS, 50% N, N-dimethylformamide, pH 4.7) was added, and samples were incubated overnight at 37 C. The optical density of samples was measured with a microplate reader (SpectraMax 340, Molecular Devices Co, Calif, United States) at a wavelength of 562 nm.
BCL-2 and REG-1a proteins were examined by immunofluorescent staining. Islets cocultured with IRP hepatocytes were fixed in 4% paraformaldehyde for 2 days. Next, the islets were dehydrated overnight in 10%, 20%, and 30% sucrose. Islet slices were snapfrozen in an optimal cutting-temperature solution, 4 mm sections were cut, and sections were mounted on slides. Subsequently, the sections were permeabilized with 0.1% Triton X-100 (SigmaAldrich, St Louis, Mo, United States) and blocked with 0.5% bovine serum albumin (Invitrogen, Carlsbad, Calif, United States) in PBS. The slides were incubated overnight at 4 C with a primary polyclonal antibody against BCL-2 (Santa Cruz Biotechnology) or REG-1a (USCN Life Science, Wuhan, China) at 1:100 dilution in blocking solution. The slides were washed and incubated with an Alexa Fluor 488-conjugated antimouse antibody (BCL-2) or Alexa Fluor 568-conjugated anti-mouse antibody (REG-1a) (Invitrogen) for 1 hour at room temperature.
RNA Isolation and Real-Time Polymerase Chain Reaction
Statistical Analysis
IRP hepatocytes and cocultured islets cells were rinsed twice with cold PBS and treated with TRIzol reagent (Invitrogen) to extract mRNA. Chloroform was added to the lysed cells, and the cells were incubated for 15 minutes. The samples were then centrifuged at 13,000 g for 15 minutes at 4 C. The aqueous phase of each sample was transferred to a new tube, mixed with isopropyl alcohol to precipitate RNA, and the precipitated RNA was pelleted by centrifugation at 13,000 g for 10 minutes at 4 C. The extracted RNA was washed twice with 75% ethanol in RNase-free water. Finally, each isolated RNA sample was dissolved in 20 mL of RNase-free water and stored at 70 C before use. Quantities of total RNA were measured with a NanoDrop spectrophotometer (NanoDrop Technologies, Inc, Wilmington, Del, United States). Each reverse transcriptase reaction (AccuPower RT Premix Kit, Invitrogen, Co, Carlsbad, Calif, United States) included 1 mg of total RNA for cDNA synthesis. Total RNA was reverse-transcribed with Oligo-dT according to an M-MLV Reverse Transcriptase (Invitrogen) protocol to synthesize cDNA. The cDNA samples were analyzed by real-time polymerase chain reaction (PCR) with the following primers: GAPDH, Forward 5’TCCCTCAAGATTGTCAGCAA-3’, Reverse 5’-AGATCCACAAACGGATACATT-3’; Insulin-like growth factor 1 (Igf-1), Forward 5’-CAGTTCGTGTGTGGACCAAG-3’, Reverse 5’TCAGCGGAGCACAGTACATC-3’; Hepatocyte growth factor (Hgf), Forward 5’-CGAGCTATCGCGGTAAAGAC-3’, Reverse 5’-TGTAGCTTTCACCGTTGCAG-3’; Transforming growth factor-alpha (Tgf-a), Forward 5’-AGCATGTGTCTGCCACTCTG3’, Reverse 5’-TGGATCAGCACACAGGTGAT-3’; Transforming growth factor-beta 1 (Tgf-b1), Forward 5’-ATACGCCTGAGTGGCTGTCT-3’, Reverse 5’-TGGGACTGATCCCATTGATT-3’; Regenerating islet-derived 1 alpha (Reg-1a), Forward 5’CCTGATGGTCCTTTCTCCAA-3’, Reverse 5’-CAGAGAGGCCAGAAAGTTGC-3’; B-cell lymphoma 2 (Bcl-2), Forward 5’CGACTTTGCAGAGATGTCCA-3’, Reverse 5’-ATGCCGGTTCAGGTACTCAG-3’; and Insulin 1 (Ins-1), Forward 5’CACCTTTGTGGTCCTCAC-3’, Reverse 5’-AGTTGGTAGAGGGAGCAG-3’. Real-time PCR was performed with SYBR Premix Ex Taq (Takara, Tokyo, Japan) on an ABI StepOnePlus
The results are presented as means (SDs). Mean group values were compared using t tests and SigmaPlot ver. 2.0 (SYSTAT, Chicago, Ill, United States). P values less than .05 were considered statistically significant.
Islet Survival-Related Marker Measurements
RESULTS Regeneration-Related Protein and Gene Expression of IRP Hepatocytes
In the hepatocyte analysis, phosphorylated STAT3 (p-STAT3) levels were significantly higher in the 15- and 30minute double-IRP groups than in the control groups immediately after IRP exposure and after 1 day in culture. The p-STAT3 level remained elevated until day 1, but the difference between p-STAT3 levels in the IRP and control groups declined at day 1 (Fig 1A). The Igf-1, Hgf, Tgf-a, and Tgf-b1 gene expression levels were evaluated by conducting real-time PCR analyses on day 0 and day 1 after IRP. No significant differences in Igf-1 expression levels were detected immediately after ischemic treatment for 15 and 30 minutes or after double-IRP treatments (Fig 1B). However, Igf-1, Tgf-a, and Tgf-b1 expression was relatively induced in both IRP groups. After 24 hours, Igf-1 expression was significantly increased in the 30-minute double-IRP group, and the gene expression levels of Hgf, Tgf-a, and Tgf-b1 were significantly higher in both IRP groups compared with those in the other groups. Secreted Factors From IRP Hepatocytes
To evaluate the secretory function of IRP hepatocytes, we measured HGF, IGF-1, TGF-a, and TGF-b1 protein levels in culture supernatants by performing enzyme-linked immunosorbent assay (ELISA). Although there were no significant increases in TGF-a or TGF-b1 in the treated groups compared with the control group, HGF and IGF-1
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Fig 1. Regeneration-related protein and gene expression. (A) p-STAT3 protein expression in ischemia/reperfusion preconditioning (IRP) hepatocytes. p-STAT3 protein known as a cell regeneration significantly increased by IRP. Both conditions of 15 m 2 and 30 m 2 IRP increased p-STAT3 protein expression more than control and single ischemia preconditioned group in day 0 and 1. (B) Real-time polymerase chain reaction (PCR) analysis of gene expression in IRP hepatocytes on day 0 and 1. Gene expressions of Hgf, Tgf-a, and Tgf-b1 were significantly increased by each of 15 m 2 and 30 m 2 IRP. The Igf-1 gene expression showed delayed increase at day 1 in 30 m 2 IRP. n ¼ 5, *P < .05 compared with the control group.
increased with longer preconditioning times (Fig 2A). This delayed IGF-1 secretion was correlated with delayed gene expression of Igf-1 (Fig 1B). Effects of Secreted Factors From IRP Hepatocytes on Islet Insulin Secretion
ELISA analyses were performed to assess insulin secretion from islets cocultured with hepatocytes. Figure 2B shows the levels of insulin secretion from islets that were cocultured with preconditioned hepatocytes in transwells and the percentages of conditioned medium. All islets were evaluated at day 1 or 2 after IRP. On both days 1 and 2, cells grown under transwell conditions with preconditioned hepatocytes showed significantly higher levels of insulin secretion in the 15- and 30-minute double-IRP conditions than the control group. To check effects of secreted elements from IRP hepatocytes, islets were put on a conditioned media originating from the same culture media
of IRP hepatocytes, but hepatocytes were removed after culture. Islets under the conditioned media showed significantly higher insulin secretion than control group except islets under the medium from day 2 for the 15-minute double-IRP condition. This result revealed that the secretary elements from the IRP hepatocytes affected insulin secretion of islets. Evaluation of the Beneficial Effects on Insulin Secretion Function and Islet Survival
Glucose challenge tests of the islets were expressed by SI. SI was measured from the islets cocultured with hepatocytes under different time condition of IRP. Islets cocultured with 15 m and 15 m 2 IRP hepatocytes showed higher SI than the control group, but there was no significantly increased SI in 30 m and 30 m 2 IRP groups (Fig 3A). Real-time PCR analysis was used to evaluate the survivalrelated gene expression levels of Bcl-2, Tgf- b1, Reg-1a, and
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Fig 2. Secreted factors from ischemia/reperfusion preconditioning (IRP) hepatocytes. (A) Hepatocyte growth factor (HGF) and insulinlike growth factor (IGF)-1 secretion, as determined by supernatant protein levels, tended to increase depending on the duration of preconditioning treatment. HGF secretion increased by 30 m 2 IRP at day 1 and 2. IGF-1 increased by each of 15 m 2 and 30 m 2 IRP at only day 2. (B) Islets cultured in the conditioned media that had same condition but removing hepatocytes after culture showed similar insulin secretion as islets cocultured with hepatocytes in the transwell condition. This revealed that the secretary elements from the IRP hepatocytes affected insulin secretion function of islets. *P < .05 compared with the control group.
Ins-1 in islets cocultured with preconditioned hepatocytes. Bcl-2 and Reg-1a gene expression levels significantly increased after both 15- and 30-minute double-IRP treatments, and Tgf-b1 expression was significantly higher in cells treated with 15-minute double-IRP than under other conditions. Ins-1 gene tended to express more in IRP treatment but there were no significant differences among the treatment groups (Fig 3B). Immunofluorescence labeling was performed to evaluate the expression of the survival-related proteins BCL-2 and REG-1a in cocultured islets with IRP hepatocytes. The 15-minute double-IRP group was strongly positive for BCL2 compared with the control group. The 15- and 30-minute double-IRP groups expressed significantly higher REG-1a than the control group (Fig 3C). We conducted MTT assays to confirm the islets’ cell viability under each condition. The 15- and 30-minute hypoxia-injured groups displayed more apoptosis in MTT assay on days 1 and 2. By contrast, the 15- and 30minute double-IRP groups showed more cell viability on day 1 and 2. Besides, the 15- and 30-minute double-IRP
groups showed significantly greater cell viability, according to the MTT assay compared with the 15- and 30-minute hypoxia-only treatment groups (P < .05, Fig 4A). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay performed in 15- and 30-minute double-IRP groups showed less apoptotic islets than control group (Fig 4B). DISCUSSION
In this study, we investigated the interactions between cocultured hepatocytes and islets. A previous study reported that IRP was beneficial for hepatocyte regeneration and STAT-3 activation and prevented ischemic reperfusion injury [2,15]. Our previous work used the Hep-G2 and RIN5F cell lines, showed that ischemia increased IL-6/STAT-3 pathway activation in Hep-G2 cells, and showed that coculture with hypoxic-preconditioned Hep-G2 cells increased insulin secretion in cocultured RIN-5F cell lines [16]. We subjected primary hepatocytes to longer IRP treatments, but were unable to increase the IRP time in the
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Fig 3. Insulin secretion function and survivalrelated gene/protein expression in cocultured islets. (A) Glucose challenge test. Islets cocultured with 15 m and 15 m 2 ischemia/reperfusion preconditioning (IRP) hepatocytes showed higher Stimulation Index than the control group. (B) Bcl2 and Reg-1a gene expression of islets cocultured with hepatocytes in transwell were significantly increased by each of 15 m 2 and 30 m 2 IRP. Tgf-b1 genes were significantly increased in 15 m 2 IRP. (C) Cells were immunolabeled for B-cell lymphoma (BCL)-2 and regenerating gene (REG)-1a. The 15 m 2 IRP group labeled strongly for BCL-2 (red) compared with the control group. The 15 m 2 and 30 m 2 IRP groups labeled strongly for REG-1a (green) compared with the control group. Hoechst staining is shown in blue ( 200). *P < .05 compared with the control group. The length of white bar is 100 mm.
primary cell experiments because of the subsequent reduction of primary hepatocyte viability. Thus, double-IRP conditions for 15 and 30 minutes were defined as the IRP model with respect to primary hepatocyte survival. As a result, primary hepatocyte viability was comparable with that of the control group as demonstrated by the MTT assay and TUNEL stain. First, to confirm our IRP model properly works to stimulate regenerative factors from the hepatocytes against ischemia-reperfusion injury, we checked p-STAT-3 protein
and tissue regenerationerelated gene expressions. Hepatocytes are well characterized in the literature for their regenerative capacities [3e5]; they have protective mechanisms against ischemia-reperfusion injuries that involve transforming growth factor-b1 (TGF-b1), interleukin (IL)-6, and the GP130 signal transducer and STAT3-mediated signaling pathway [6,7]. Our results confirmed that the levels of p-STAT3 rapidly increased immediately after IRP treatment and remained elevated until day 1, but the difference between IRP and control groups decreased
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Fig 4. Islet survival analysis. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays indicated that islets had greater viability in the 15 m 2 and 30 m 2 ischemia/reperfusion preconditioning (IRP) groups than in the hypoxia-alone groups. (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain of control, 15 m 2, and 30 m 2 IRP groups. *P < .05 compared with the control group at day 2. yP < .05 compared with each ischemia-alone group.
thereafter. This result is reasonable because the JAK-STAT pathway is triggered by ischemia but later stabilizes [17]. We confirmed that primary hepatocytes displayed the same condition of p-STAT3 by conducting an in vitro study under IRP. The STAT3 pathway and IGF-1, HGF, TGF-a, and TGFb1 gene expression are important for tissue regeneration and organ protection after ischemia-reperfusion injury [18]. In the current study, the gene expression levels of Igf-1 on day 1 and the expression levels of Hgf and Tgf-b1 on days 0 and 1 were upregulated in the IRP hepatocytes. The expression levels of these genes differed from those in groups treated with ischemia alone, which had expression levels similar to those in the control group. The expression levels of Hgf, Tgf-a, and Tgf-b1, after 15 and 30 minutes of double-IRP treatment on day 1 were significantly higher than those in cells treated for 15 and 30 minutes with ischemia injury alone. This suggests that IRP induced the synthesis of protective factors in hepatocytes that were not induced by ischemia injury alone. We measured the survival-related gene expression in islets cocultured with IRP-hepatocytes in transwells. The effects of TGF-b1 on liver regeneration or protection against ischemic injury remain controversial [19,20]. However, TGF-b1 expression is thought be associated with survival from IRP-mediated injury. In the current study, Tgfb1 gene expression increased in cocultured islets, and Reg-1a expression was significantly upregulated in cocultured islets, which are involved in beta cell growth. Reg-1a was originally isolated from a cDNA library derived from regenerated rat pancreatic islets, which represents islet cell regeneration [21]. Our results suggest that a secretory factor from the cocultured IRP hepatocytes affects islet gene expression associated with regeneration. However, no significant differences in the Ins-1 gene expression were
observed in islets after coculture with primary IRP hepatocytes. This is a limitation of the current study. Ins-1 gene expression was higher after prolonged IRP duration, but this correlation was not statistically significant. Although there were no significant differences in the Ins-1 gene expression, the levels of secreted insulin in the transwell cocultures, measured by ELISA, significantly increased in response to double-IRP treatment for 15 and 30 minutes. Thus, we conclude that IRP hepatocytes improve insulin secretion from cocultured islets. Bcl-2 is an antiapoptotic marker for pancreatic beta cells [22]. In the current study, Bcl-2 gene expression was significantly higher in islets cocultured with IRP hepatocytes compared with that in the control group. We also showed that the levels of BCL-2 and REG-1a proteins increased in the cocultured islets. The MTT assays revealed that islets cocultured for 15 and 30 minutes with double-IRP hepatocytes had significantly higher viability than the hypoxiaalone group. In addition, TUNEL stain in the IRP group showed less apoptosis than the control group. This result supports our hypothesis that IRP hepatocytes provide survival benefits to cocultured islets. However, a limitation of this study is that we could not determine in vivo survival. Therefore, further in vivo investigations will be necessary to prove this result. Ischemia and ischemia-reperfusion are inevitable in clinical islet transplantation. Ischemia-injured islets and islets surrounded by ischemia-injured hepatocytes cannot survive in the long term, but our data suggest that regeneration power of IRP hepatocytes beneficially affects cocultured islet survival. We are currently developing further in vivo studies and potential clinical applications. In conclusion, coculture of islets with IRP hepatocytes can prevent ischemia or ischemia/reperfusion-related
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injuries and improve insulin-secreting capacities and survival in cultured islets. ACKNOWLEDGMENT This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012-R1A1A1012734).
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