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Islet Graft Survival and Function: Concomitant Culture and Transplantation With Vascular Endothelial Cells in Diabetic Rats

Pan, Xiaoming1,2,3; Xue, Wujun1,2; Li, Yang1; Feng, Xinshun1; Tian, Xiaohui1,2; Ding, Chenguang1

doi: 10.1097/TP.0b013e3182356ca7
Basic and Experimental Research
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SDC

Background. Human islet transplantation is a great potential therapy for type I diabetes. To investigate islet graft survival and function, we recently showed the improved effects after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats.

Methods. ECs were isolated, and the viability of isolated islets was assessed in two groups (standard culture group and co-culture group with ECs). Then streptozotocin-induced diabetic rats were divided into four groups before islet transplantation as follows: group A with infusion of islet grafts; group B with combined vascular ECs and islet grafts; groups C and D as controls with single ECs infusion and phosphate-buffered saline injection, respectively. Blood glucose and insulin concentrations were measured daily. Expression of vascular endothelial growth factor was investigated by immunohistochemical staining. The mean microvascular density was also calculated.

Results. More than 90% of acridine orange-propidium iodide staining positive islets demonstrated normal morphology while co-cultured with ECs for 7 days. Compared with standard control, insulin release assays showed a significantly higher simulation index in co-culture group except for the first day (P<0.05). After transplantation, there was a significant difference in concentrations of blood glucose and insulin among these groups after 3 days (P<0.05). The mean microvascular density in co-culture group was significantly higher than that in single islet group (P=0.04).

Conclusion. Co-culture with ECs in vitro could improve the survival and function of isolated rat islet, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats.

SUPPLEMENTAL DIGITAL CONTENT IS AVAILABLE IN THE TEXT.

1 Center of Nephropathy, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an City, Shaanxi Province, People's Republic of China.

2 Center of Organ Transplantation, Institute of Organ Transplantation, Xi'an Jiaotong University, Xi'an City, Shaanxi Province, People's Republic of China.

This work was supported by the Natural Science Foundation of Shaanxi Province (grant no. SJ08C201), the National Natural Science Foundation of China (grant no. 30972947), Clinical Key Disciplines of National Public Health Department (2007 years), and Major Scientific and Technological Special Projects of Shaanxi Province (grant no. 2007ZDKG-67).

The authors declare no conflicts of interest.

3 Address correspondence to: Xiaoming Pan, M.D., Center of Nephropathy, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, West 277 Xiaozai Road, Xi'an City, Shaanxi Province 710061, People's Republic of China.

E-mail: panxiaoming@medmail.com.cn

X.P. and W.X. participated in the research design and writing of the manuscript; Y.L., X.F., X.T., and C.D. participated in performance of the research; and X.P. and C.D. participated in the data analysis.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal's Web site (www.transplantjournal.com). A combined file of all SDC is available as SDC 1 (http://links.lww.com/TP/A546).

Received 16 May 2011. Revision requested 31 May 2011.

Accepted 30 August 2011.

Human islet transplantation is a great potential therapy for type I diabetes (1, 2). However, this approach has limited clinical applications because of the lack of cadaver donors, host immune rejection, primary nonfunction, and cell death of islets after the transplantation (3–5). A major obstacle is that a great number of islets die in the first day posttransplantation (6). To achieve successful islet transplantation, primary nonfunction of islets has to be eliminated. It is one possible reason for this islet death that isolation procedures sever vascular connections of islets, and islets remain avascular during the culture and first few days posttransplantation (7, 8). Therefore, unlike the whole pancreas transplantation, the process of revascularization is required for islet grafts to establish adequate microvascular blood supply. Revascularization of the transplanted islets is of great importance for the survival and function of islet grafts. To solve these problems, it is necessary to elucidate mechanisms affecting islet graft function. We and others have shown that vascular endothelial growth factor (VEGF) expression promotes new blood vessel formation and improves the outcome of islet transplantation (9, 10). Effective prevention of islet destruction after transplantation requires not only revascularization of islets but also abrogation of cytokine-mediated islet cell death and dysfunction triggered by immune and inflammatory reactions. However, little is known about the interaction between vascular endothelial cells (ECs) and islet cells. Moreover, few investigators have noticed that ECs remained in the islets after islet isolation are lost after 7 days of culture (11). Therefore, we programmed co-culture and co-transplantation of islets with ECs to improve their function and survival in diabetic rats.

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RESULTS

Identification of ECs

Using inverted fluorescence microscope (Olympus IX70, Japan), cellular morphology revealed that the isolated cells were ECs. Except using the EC-specific cell surface marker CD-31, ECs were characterized by their expression of factor VIII antigen (von Willebrand, immunohistochemical S-P; Fig. 1A). The morphology showed a paved monolayer with a periplast, demonstrating a characteristic buffy positive reaction (12).

FIGURE 1.

FIGURE 1.

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Observation of Islets

Dithizone-stained islet cells appeared reddish-orange, and the morphology of islets was better, as manifested in both the standard culture group and co-culture group with ECs. Various sized and round, elliptical, or irregular cells were observed under the microscope. Cell clusters were easily identifiable. Cells showed good morphology within the first week in both two groups. However, islets in standard culture group displayed irregularity and loose resolution at 2 weeks, whereas islets in co-culture group still maintained integrity (Fig. 1B and C). Figure 1(D) shows that both live cells (with green fluorescence) and dead cells (with red fluorescence) could be observed under an inverted fluorescence microscope after acridine orange-propidium iodide (AO/PI) staining. Green fluorescence was observed in most cells, suggesting the high survival rate in islet cells. Figure 1(E) shows that green fluorescent could be observed from about 90% cells in the co-culture group on the seventh day using AO/PI staining. By transmission electron microscopy analysis, β-cells we detected in co-culture group were with more discrete particles and zymogen granules than that of single culture group, besides nuclear structures were relatively compressed at 7 days in co-culture group. However, islet cells displayed early apoptosis and reduced zymogen granules in single culture group (Fig. 1F–H). In the mass, there were some increased mitochondria, but no obvious difference in mitochondrial cristae was observed in both groups.

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Insulin Release Assay

After cultured islets (150±50 mm in diameter) were preincubated for 12 hr in 1 mL Roswell Park Memorial Institute (RPMI) 1640 medium containing 2.7 mmol/L glucose, islets were incubated with RPMI 1640 medium containing 3.3 mmol/L glucose or 16.7 mmol/L glucose for serial 60 min. Data showed that insulin secretion was increased (13). We simultaneously detected 20 cultured islet samples for insulin release assay in both culture groups. When the incubation process was finished, the supernatants were subjected to insulin radioimmunoassay. The islets stimulation index was determined by the ratio of the insulin secretion stimulated by the high versus low glucose solutions. Compared with stimulated insulin secretion of the two culture groups in vitro, there was a significant difference in periods of 5 to 14 days (P<0.05), whereas no obvious difference was observed within the first 5 days (P>0.05; Fig. 2; see Table 1, SDC 2, http://links.lww.com/TP/A547). In addition, the simulation index of the co-culture group was significantly higher than that of the standard group within the first 3 days except for the first day (P<0.05) and the periods of 5 to 14 days (P<0.01; Fig. 3; see Table 2, SDC 3, http://links.lww.com/TP/A548).

FIGURE 2.

FIGURE 2.

FIGURE 3.

FIGURE 3.

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Posttransplantation Effects of Islet Grafts in Diabetic Rats

The nuclei morphology of islet cell is different from renocortical cell. We used hematoxylin-eosin staining to detect the viable islet cell 7 days after transplantation. More islet cells in co-culture group were found under renal capsule. On the contrary, few islet cells were detected in standard culture group. In co-culture group, the expression of insulin, CD31, and von Willebrand factor were detected as brown-yellow lumpy masses of cells on the seventh day posttransplantation, while they were significantly lower in standard culture group (Fig. 1I–P). After grouped islets were transplanted into streptozotocin (STZ)-induced diabetic rats, we monitored the blood glucose level and insulin concentration daily. We observed a statistically significant difference between groups A and B (P<0.05). The fasting blood glucose level in group A was not back to the normal level until the third day posttransplantation, and from then they fluctuated between 10.15±0.74 and 14.934±0.94 mmol/L. However, blood glucose level returned to the normal level at 2 days posttransplantation in group B. In addition, it was described as a good result of blood glucose control because the fasting blood glucose level fluctuated between 5.38±0.66 and 6.36±1.01 mmol/L (Fig. 4; see Table 3, SDC 4, http://links.lww.com/TP/A549). Meanwhile, the insulin concentration in group B peaked at the fifth day posttransplantation, then it was maintained at a high level between 12.36±0.57 and 11.22±0.79 μIU/mL. Moreover, in group A, the insulin concentration was increased only for 2 days and then it was fixed at a relatively lower level between 6.92±1.03 and 4.02±0.68 μIU/mL till the 14th day (Fig. 5; see Table 4, SDC 5, http://links.lww.com/TP/A550). Furthermore, mean microvascular density in group B was higher than that in group A (12.58 [1.81] vs. 10.38 [0.97]; P=0.04).

FIGURE 4.

FIGURE 4.

FIGURE 5.

FIGURE 5.

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DISCUSSION

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.

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MATERIALS AND METHODS

Animal Models

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.

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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).

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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.

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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.

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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).

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Histology Studies

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).

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Statistical Analysis

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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Keywords:

Islet transplantation; Cell culture; Endothelial cells; Revascularization; Rat

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