Our study indicated that the cultured islet cells showed good dithizone-stained morphology within 1 week. The co-cultured islets still demonstrated morphological integrity until 2 weeks, whereas standard cultured islets displayed irregularity and structure ablation. Moreover, compared with standard cultured islet cells, we observed more particles, zymogen granules, relatively integrated nuclear structure and unchanged mitochondrial cristae in co-cultured islet β-cells at the end of the first week. However, the standard cultured islets displayed early apoptotic signs and reduced zymogen granules, even though the positive AO/PI staining was more than 90%. The possible reason for these morphological changes was that cytokine might be tightly related to islet graft function and structure in addition to extracellular matrix contributions. ECs secrete cytokines, such as VEGF. These cytokines promote proliferation, inhibit differentiation, and finally improve islet graft function and structure.
In recent years, isolated islets without prior culture show characteristic beneficial for transplantation. With short period of culture before transplantation in various media, islet transplantation has achieved acceptable results (14), especially with the techniques to create islet-EC grafts (15, 16). Co-cultured islets and attached ECs become a composite layer. Subsequently, ECs and islets grow until they reach confluence.
In addition to the morphological changes, we observed a significantly higher simulation index in the ECs-islets co-culture group except for the first day compared with the standard culture group. For this possible reason, we presumed that islet secretory function was declined in the standard culture group because of the lack of EC cytokine stimulation. Another explanation was likely related with collagen IV, which is a ubiquitous component of basement membranes, the sheet-like matrix that underlies epithelial and ECs. It is secreted by ECs and interacts with integrin a1β1 in fetal β-cells to potentiate insulin secretion. This functional interaction between ECs and β-cells is important for the postnatal maturation of β-cells (17). These data suggest that co-culture with ECs can improve survival and function of islets.
By either morphology observation or insulin release assay, we showed that ECs, co-cultured in vitro with islets, played a positive role and improved the survival and function of isolated rat islets. In recent years, more and more evidence revealed this direct or indirect interaction between ECs and islet cells. By comparing human islets with islets isolated from normal and hDAF transgenic pigs, Bennet et al. (18) reported that ECs express high levels of complement regulatory proteins on islets of Langerhans, including decay-accelerating factor, MCP and CD59. Langlois et al. (19) recently reported that desferrioxamine, binding free iron in the bloodstream and enhancing its elimination in the urine, stimulates VEGF overexpression and increases islet vascularization, and it is used as a new drug to improve islet viability during transplantation. Being a biologically active surface for isolated islets, ECs express numerous varieties of counteracting proteins, such as heparan sulfate. ECs inhibit platelet aggregation by releasing various acting factor, such as prostacyclin, thrombomodulin, platelet-derived ADP, and nitric oxide. As mentioned earlier, the interaction between ECs and islet cells is a complex process. Our data showed consistent results with release of various factors, especially VEGF.
In our experiment, we selected Sprague-Dawley rats as both donors and recipients. This type of islet transplantation belongs to the isotransplant model, and it has nothing to do with immune rejection. It just took 2 days for fasting blood glucose level to return to the normal level in rats receiving concomitant administration of islets and ECs. Moreover, it seemed that it was a better glucose control therapy compared with the group receiving only islets. Meanwhile, the insulin concentration in islets-ECs co-transplantation group peaked at the fifth day posttransplantation, and then it was maintained at a higher level between 12.36±0.57 and 11.22±0.79 μIU/mL. In contrast, the insulin concentration was increased only 2 days in islet single transplantation group, and then it was fixed at a relatively lower level during the rest of time, ranging from 6.92±1.03 to 4.02±0.68 μIU/mL. In addition, the diabetic rats in this group exhibited moderate hyperglycemia. The microvascular density in co-transplantation group was markedly higher than that in islet single transplantation group.
Previous studies suggest that during the development of pancreas, ECs produce an instructive signal to participate in the process of pancreatic endocrine differentiation and morphogenesis (20, 21). Furthermore, not only the ECs– endocrine cells signaling system but also the functional blood vessels are required in pancreatic development (22). Because pancreatic islets capillary network is disrupted during the islet isolation process, revascularization is one of the critical steps. It is possible for the transplanted pancreatic islets to function well and deliver insulin to peripheral tissues and to supply oxygen and other nutrients for the islets. Both our early studies (10) and this study also revealed that the development of vascularized islet grafts was accompanied with islet morphogenesis. These data suggest that coordinated interactions of islet grafts and ECs were required by vascularized islet, and ECs-islet cells communication was a crucial factor. Therefore, our present results should be correlated with various cell factors, especially VEGF, which is one of the well-established angiogenesis-stimulating cytokines both in vitro and in vivo.
In current practice of islet transplantation, 60% of recipients remained insulin independent for 1 year (23–25). However, a lot of limitations existed in this procedure. For a successful treatment, it required a significant amount of acinar tissue with the isolated islets (isolated from two or more pancreases). It has been reported that more than 60% of the transplanted islets fail to engraft (23, 26, 27) because of a number of factors, including the insufficient nutrient/oxygen supply to engrafted islets and incompatibility of blood with the islet surface (28). The improvement of islet vascularization is one way to improve islet engraftment and lower the number of required acinar tissue. Some studies have confirmed that islet function and survival can be improved by increasing vessel density around transplanted islets by interaction with angiogeneic growth factors and co-transplantation with mesenchymal stem cells (29–32). Another way to achieve angiogenesis is through transplantation of ECs. ECs drive new blood vessel formation that can support functional cells. It has been reported that human umbilical vein ECs co-transplanted with supporting fibroblasts or mesenchymal precursor cells integrate with the host vasculature and promote the survival of co-transplanted skeletal, skin, and human embryonic stem cell-derived myocardial tissue (33–37). Consistent with this result, we also obtained the same data in our research. Therefore, it should continue to be evaluated as a complementary alternative for isolated islet transplantation.
In conclusion, co-culture with ECs in vitro could improve the survival and function of isolated rat islets, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats. However, it was necessary to further investigate the outcomes of co-transplantation of vascular ECs and islets.
MATERIALS AND METHODS
All animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. To render diabetic phenotype, rats were intraperitoneally injected with 1% STZ (Sigma, w/v) at a dosage of 65 mg/kg body weight, and then they were starved for 4 hr in the next 3 consecutive days (38). Glucose level of more than 16.8 mmol/L was defined as hyperglycemia using tail vein blood after STZ injection, and the average of two consecutive tests was recorded. In addition, body weight was regularly monitored.
Isolation of Islets of Langerhans and Culture
Pancreatic islets were isolated from Sprague-Dawley rats (250–300 g body weight; Animal Lab of Xi'an Jiaotong University, China). Rats were starved overnight before the abdomen was opened. Then pancreas were digested with 10 mL of 1 mg/mL collagenase P (pH 7.4, Roche Diagnostics, Germany) at 38°C for 20 min, and a discontinuous Ficoll (Type 400DL; Sigma, St. Louis, MO) gradient purification was followed (39). The suspended islets were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 2 mmol/L l-glutamine (Sigma), 100 IU/mL penicillin (Sigma), and 100 μg/mL streptomycin (Sigma). Culture medium was changed every 2 days. To simultaneously visualize the live and dead islet cells, islet viability was assessed using a double AO/PI fluorochrome (Sigma) staining (26).
Isolation of Vascular ECs and Culture
Hearts and lungs were aseptically dissected from the chest cavity of exsanguinated rats and placed in a petri dish containing phosphate-buffered saline (PBS) solution. The lung was dissected away by cutting the pulmonary vasculature, and the heart was perfused with PBS. The aorta was removed from the heart. Connective tissue surrounding the vessel exterior was teased away, and the vessel was briefly washed in PBS. The aorta was then rinsed again with PBS and carefully dissected to expose the interluminal surface. Subsequently, the aorta was cut into several small segments. These pieces were incubated with collagenase solution (1 mg/mL of collagenase II [Sigma]) at 37°C for 1 hr, and a centrifugation at 1000 rpm for 10 min was followed. After rinsing with PBS, the ECs of aorta were resuspended in RPMI 1640 culture medium containing 10% FBS, 1% glucose (w/v), and 0.5% streptomycin-penicillin solution (10,000 U/mL, Invitrogen Corp, Carlsbad, CA). ECs were incubated at 37°C in a humidified circumstance with 5% CO2. The culture medium was regularly changed with an average of 2 days.
Co-Culture Islets of Langerhans With Vascular ECs
Co-culture of islets and ECs was essentially established as described earlier. A total of 1000 to 3000 islet equivalent islets of langerhans were mixed with 0.3 to 0.5 million aortic ECs in 500 μL RPMI 1640 culture medium. Then the islets and ECs were incubated in 24-well plates at 37°C for 1 to 2 hr, and the culture was gently mixed once during the incubation. Subsequently, the islets and vascular ECs were transferred to 15-cm2 petri dishes, and they were cultured for 2 to 7 days (15). Co-cultured cells were then implanted under the capsule of left kidney in rats with STZ-induced diabetes.
Islet Co-Transplantation and Experimental Design
A total of approximately 500 islet equivalent islets were aspirated into polyethylene tubing (PE-50), pelleted by centrifugation for 2 min, and implanted under the left renal capsule on lateral aspect of the kidney. Twenty diabetic rats were divided into four groups based on the treatment as follows: group A: infusion of islet grafts; group B: concomitant transplantation of vascular ECs and islet grafts; groups C and D as controls: single ECs infusion and PBS injection. Blood glucose concentration was measured at 24 hr posttransplantation in fasted diabetic rats. Blood glucose level less than 7.8 mmol/L on 2 consecutive days was defined as the successful islet function. Serum insulin concentration was measured using chemiluminescence (AxSym assay; Abbott Laboratories, Abbott Park, IL) in fasted diabetic rats (40). The mean survival time of islet graft in each group was recorded. Within 14 days, renal tissue was sectioned and stained with VEGF antibodies using a hematoxylin or immunohistochemical method. Mean microvascular density was calculated (41).
Islet and ECs were collected from co-culture or graft tissues removed from the renal subcapsular spaces after animals were killed. Grafts were separately processed from each rat. Blood sample was obtained before the collection of islet grafts. Some graft tissues were fixed in 10% phosphate-buffered formalin overnight, and then they were embedded in paraffin and sectioned at 4.5 μm. Some tissues were fixed in freshly prepared 2% paraformaldehyde for electron microscopy preparation. The others were placed in RPMI 1640 medium supplemented with 10% FBS, kept on ice and then transferred into Eppendorf tubes containing 50 μL PBS with 0.2 mg/mL EDTA (Sigma). While on ice, the grafts were cut into small pieces with fine scissors, mechanically disrupted by syringe injection through progressively narrower gauge needles, and dissociated into single cells by incubation in the cell dissociation buffer at 37°C for 20 min. Islet grafts were stained with rabbit anti-rat insulin antibody (1:200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA) using an immunoperoxidase technique and then counterstained with hematoxylin. Isolated and cultured ECs were stained with EC-specific cell surface marker CD-31 and von Willebrand factor (Sigma). A two-color immunofluorescent histochemical method was used to stain the co-cultured or co-transplanted islets and ECs. The coded slides were visualized under light microscopy (IX71, Olympus Corporation, Tokyo, Japan). For electron microscopy analysis, islet graft tissues removed from the renal subcapsular spaces were fixed with 2.5% glutaraldehyde. After islets were located on a semithin section, 70-nm sections were placed on slot grids and analyzed by transmission electron microscopy (Hitachi H800 electron microscope, Japan).
Data were expressed as means±standard error. Two-tailed Student's t-tests were used to compare results between groups. Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL). P value less than 0.05 was considered as statistically significant.
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Islet transplantation; Cell culture; Endothelial cells; Revascularization; Rat
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