Revascularization Of Islet Grafts Induced By Rat Vascular Endothelial Cell Transfected By VEGF₁₆₅

IntroductionIslet transplantation is considered a potentially curative treatment for diabetic mellitus. However, this protocol depends on the infusion of sufficiently large amount of islets requiring two or more cadaveric pancreas. Apart from immune rejection, another critical limitation to islet transplantation is the rate and extent of islet revascularization. Isolated islets are severd from their native vascular network such that after transplantation, the survival and function of islet grafts must depend on the reestablishment of new vessels within the grafts. Rapid and adequate islet revascularization may be critical for the survival and function of the transplanted islets. It is estimated that <30% of islet mass becomes stably engrafted. We used rat vascular endothelial cell transfected by VEGF₁₆₅ to elevated the VEGF production in the site of islet transplantation, observed that whether the elevated VEGF expression could stimulate the angiogenesis of the grafts. These results provided proof - of — principle that local expression of an-giogenic molecules can enhanced islet revascularization, thereby improving the outcome of marginal islet transplantation with better glycemic control in diabetic rats.Material and MethodsThe wistar rats (BW150 ±20g) were purchased from Animal Center of China Medical University. Diabetic rats were induced by streptozocin ( STZ, ip, 60mg/kg) , and diabetes was defined as the nonfasting blood glucose level wasgreater than 16. 8mmol/L on two consecutive measurement. The blood glucose level was measured by the meter( SURESTEP-TM PLUS, LIFESCAN).Islet Isolation: Islets were isolated and purified according to modified Minnesota program. After intraductal infusion of 10 - 12ml cold Hank' s balanced solution containing 1. 5mg/ml of type V collagenase ( C9263 , sigma) , pancreas were surgically procured and digested at 37℃ for 15 -20min. During the digestion, the pancreas should be observed closely, and stopped digestion by cold H ank's solution when the emulsion appeared. The islets were purified by discon-tinous Ficoll density gradient (25% , 23% , 20. 5% and 11% ) centrifugation (4℃ , 3000rpm × 10min). The distinct islets were collected and washed. The islets free of acinar cells, vessels, lymph nodes and duct were used for transplantation.Experiment one: The diabetic rats were divided into two groups, the control group and experimental group for transplant ( n = 10). The degree of purity was defined as dithizone ( DTZ, 140mmol/L) staining. Insulin secretion in response to glucose challenge in vitro defined the isolated islets function. 600 -800IE graft islets were handpicked and transplanted through spleen of the diabetic recipients. Blood glucose and insulin were evaluated after operation every other day. IVGTT was performed 10 days after transplant. HE and immunohisto-chemical stain of Insulin - 6 for liver and spleen of recipients were used to evaluate the histology and islet grafts.Experiment two: The rats were divided into three groups: the control group: normal vascular endothelial cell (VEC) were transplanted under the capsule of the kidney; the control vector group: the VEC transfected by the pIRES2 - EGFP plasmid were transplanted; experimental group: the VEC transfected by the pIRES2 - EGFP/VEGF₁₆₅ plasmid were transplanted ( n = 10). The EGFP expression and positive ration were observed by fluorescence microscope. Flurocytometery were used to calculate the transfection efficiency. The VEGF mRNA expression were detected by RT - PCR. Immunohistochemical stain of human VEGF₁₆₅ were used to determine the expression of the VEGF in the VEC. HE stain for kidney were used to evaluate the histology and angiogenesis.Experiment three: The diabetic recipients were divided into three groups.The control group: the islet were transplanted under the capsule of the kidney; the VEC group: the VEC were transplanted together with the islets; the experimental group: the VEC transfected by pIRES2 — EGFP/VEGF₁₆₅ plasmid were transplanted ( n = 10 ). 300IEQ islets which considered as marginal grafts were transplanted. Blood glucose and insulin were evaluated after operation every other day. IVGTT was performed 10 days after transplant. HE and immunohisto-chemical stain of Insulin - 6, VEGF and CD34 for kidney were used to evaluate the histology of islet grafts. The MVD were calculated.Data are expressed as mean and SD or SEM. The significant of difference was evaluated by t test. A p value <0.05 was considered statistical significant.ResultsExperiment one: 300 - 400IE islets were procured from one donor rat. The degree of purity was 85% - 90%. Insulin secretion response to glucose challenge in vitro showed the mean value of insulin in the low - glucose medium was 68. 4 ±9. 1μIU/ml, while that of high - glucose medium was 382. 9 ±8. 9μIU/ml, the SI was 5. 59 ±0. 62, that means the grafts functioned well. The blood glucose level and the insulin level of plasma of the diabetic recipient restored to normal 3 days after transplantation. The IVGTT showed no significant difference in both the amplitude of blood glucose induction and the kinetics of the blood glucose decline between the recipient and the normal non - diabetic rats. The K value of the IVGTT after transplant was similar to the normal ones. The recipient spleen were normal with some special cells of red plasma in HE stain in the sinus of the spleen. These special cells were positive ones in immu-nohistochemical stain of Insulin -6. The similar cells were also seen in the portal area of the liver.Experiment two; EGFP expression were detected in the control vector group and experimental group, with the positive ratio of 12% - 14%. There were no EGFP expression in the control ones. The transfected ratio was 13.06% determined by flurocytometery. The positive stained cells of immunohistochemi-cal stain of VEGF were observed in the experimental group, with the rate of12% - 14%. But none in the control and control vector groups. RT - PCR showed VEGF₁₆₅ mRNA expression in the experimental ones. 14 days after operation , the microvsacular network had formed under the capsule of the experimental rats'kindeys. Histology result showed obvious microvascular had formed , with plentiful blood sinus in the VEGF transfected VEC. But no vessels were seen in the two other groups, though the transplanted VEC were still alive.Experiment three: The blood glucose level and the insulin level of plasma of the diabetic recipient restored to normal 3 days after transplantation. In contrast, diabetic rats receiving the same islets without or with normal VEC displayed moderate hyperglycemia ( 13mmol/L — 14mmol/L ) , no significant difference between the two groups ( p >0. 05 ). The same with the level of plasma insulin. The IVGTT showed both the amplitude of blood glucose induction and the kinetics of the blood glucose of the experimental ones had restored to normal. The K value of the IVGTT after transplant was similar to the normal ones. In contrast, the level of blood glucose increased obviously after injected glucose, after 90min, hyperglycemia were still not corrected. HE and immuno-histochemical stain showed that large amount of islet grafts were seen under the capsule of the kidney, which were positive stained by insulin - 6Ab and VEGF Ab. In the mass of the cell, the VEC positive stained by CD34 were observed, the MVD value of the islet grafts was 74. 3 6. 74. The similar mass were also seen in the two other groups, but with few positive cells stained by insulin - 6 Ab and CD34 Ab . No positive cells stained by VEGF appeared in groups. The MVD of the two groups were 11.43 2.22 and 10.9 2.45 respectively.Conclusion1. In situ infusion, collagenase digestion, purify by Ficoll gradient centrifu-gation are the effective methods for islets isolation.2. The islets transplanted through spleen is a simple and feasible method, with the well functioning grafts which stay in the splenic sinus and hepatic portal area.3. The human VEGF₁₆₅ gene could be transferred to rat vascular endothelial...