Co-transplantation of endothelial progenitor cells and pancreatic islets to induce long-lasting normoglycemia in streptozotocin-treated diabetic rats

Abstract

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3-5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.

Figure 1. Characterization of EPCs.

Flow cytometric analysis of EPCs. (A) Percentage of expression of CD45, CD11b, CD90, CD44, CD31 and Endothelium markers were shown. Each column represents mean ± SD of six different clones. (B) mRNA levels of VEGF-A, ANG-1, PECAM-1 and HGF in cultured EPCs (***p<0.0001 VEGF-A versus ANG-1; ***p<0.0001 VEGF-A versus HGF; ***p<0.0001 ANG-1 versus PECAM-1; *p<0.05 ANG-1 versus HGF; ***p<0.0001 PECAM-1 versus HGF). Error bars are ± SEM. (C-F) In vitro functionality of EPCs was identified by positive staining for endothelial antibodies (Endothelium (C, green), Dil-Ac-LDL (D, red), nuclei (E, blue), merge (F)).(G, H) EPCs cobblestone-like morphology.

Figure 2. Effect on the glycemic levels of cell-based treatments.

Glycemic levels of 700 syn-IE + 500,000 EPCs (n = 11, red line) compared with 700 syn-IE (n = 5, black line) and with 500,000 EPCs alone (n = 4, green line). (A) Co-transplantation of 700 syn-IE+500,000 EPCs induced a faster decrease of blood glucose levels than the other treatments (***p<0.0001, **p<0.001; *p<0.05). One way ANOVA was carried out to compare different treatments (***p<0.001 700 syn-IE+500,000 EPCs versus 700 syn-IE; ***p<0.01 700 syn-IE+500,000 EPCs versus 500,000 EPCs alone). On day 15^th, the difference between 700 syn-IE+500,000 EPCs and 700 syn-IE was extremely significant (*** p<0.001). (B) Monitoring of blood glucose levels in the long-term showed that animals receiving 700 syn-IE+500,000 EPCs maintained normoglycemic values for the overall observation time, (6 months) compared to animals treated with 700 syn-IE alone (***p<0.001). (C) IPGTT in transplanted animals. Glucose clearance in the transplanted groups on the 15^th post transplantation day. The comparison of glucose clearance for animals receiving 700 syn-IE (black line), 500,000 EPCs alone (green line) and 700 syn-IE+500,000 EPCs (red line) with healthy (blue line) and diabetic animals (yellow line) showed a return to normoglycemia within 120 min since the glucose injection only for the co-transplanted and healthy groups. One way ANOVA to compare different treatments, showed that at 60, 90, 120 and 150 min. healthy and co-transplanted groups showed no statistically significant differences, while the two groups showed statistically significant differences compared to the diabetic group (***p<0.001). (D) Glucose clearance in the co-transplanted group at different time points. The glucose clearance was measured at 15, 30, 90 days after transplantation for animals receiving 700 IE+500,000 EPCs. One way ANOVA showed that the differences among the curves were not statistically significant. Error bars are +/− SEM. One way ANOVA was performed within each curve by comparing different time points versus day 0.

Figure 3. Histological assessment.

Islet stained by anti-insulin antibody (magenta) respectively one month (A) and six months (B) after transplantation. (C–D) Islet, in the centre of a liver lobule, in contact with a network of new capillaries, as suggested by a strong presence of GFP+ EPCs (yellow) and PECAM-1 (red), from a perilobular vein (*) for animals at 30 days from transplant. The 3D reconstruction (D), better explain the previous picture (C). The islet was shown in green, while the perilobular vein was red. Orange represented the capillary network, the magenta an arteriole and the EPCs were yellow. New formed vessel in the islets one month (E) and six months (F) after transplantation, white arrows indicated endothelial (red) GFP+ EPCs. The endothelial cells in E exhibit a higher shape of those in F.

Figure 4. Evaluation of pancreas and liver tissues.

(A) Viable islets in the pancreas of wild type animals as indicated by black arrows; (B) exocrine tissue without residual islets in a pancreas section of animals co-transplanted with GFP+ EPCs and wild type islets at 30 days after transplant. (C) Intact hepatic parenchyma of wild type animals; (D) liver tissue section of animals receiving GFP+ EPCs and wild type islets: image of a pancreatic islet at 30 days after transplant. No lymphocitic infiltration is evident.

Figure 5. Vessel morphologic parameters assessment.

(A) Endothelial thickness: average height(µm ± SEM) of the endothelial cells within the islets of wild type animals (black column), animals receiving islets alone (700 IE) (white column) and animals receiving 700 syn-IE plus 500,000 EPCs (IE/EPC) sacrificed at different time points (gray column)(***p<0.0001 at days 15, 30 and 120 versus control for IE/EPC group; ***p<0.0001 for 700 IE versus control). (B) Vessel density: average number (± SEM) of endothelial cells detected along a line in histological sections for wild-type animals, animals receiving islets alone (700 IE) (white column) and animals receiving 700 syn-IE plus 500,000 EPCs (IE/EPC) sacrificed at different time points (gray column) (***p<0.0001 at days 15, 30 and 120 versus control for IE/EPC group; ***p<0.0001 for 700 IE versus control).

Figure 6. Modulation of angiogenic genes.

mRNA levels of angiogenic factors in the livers of healthy animals (black bars), animals transplanted with 700 syn-IE (white bars) or 700 syn-IE+500,000 EPCs (grey bars) at different time points. (A) VEGF-A, ***p<0.0001 versus healthy. Compared to the control group the 700 syn-IE alone showed an higher increase (***p<0.0001) of VEGF-A expression than 700 syn-IE plus 500,000 EPCs(**p<0.001). Moreover statistical significant differences were found at different time point after transplantation inside the groups (700 syn-IE alone ***p<0.0001 day 7 versus day 15 and day 30 and ***p<0.0001 day 15 versus day 30; 700 syn-IE plus 500,000 EPCs **p<0.001 day 15 versus day 7 and day 30 and ***p<0.0001 day 15 versus day 180. (B) In ANG-1 expression no significant differences were found between the 700 syn-IE plus 500,000 EPCs group and healthy while at day 30 from transplant a significant increase was found in the 700 syn-IE alone group versus healthy (*p<0.01). Furthermore at day 30 a significant difference was found between 700 syn-IE alone and 700 syn-IE plus 500,000 EPCs one (**p = 0.0014). (C) PECAM-1, **p = 0.003 versus healthy. The column of gene expression relative to the animals treated with 700 syn-IE alone was not reported at 180 days, because the animals died within 45 days after transplantation due to lack of glycemic control. Error bars are +/− SEM.