Decline of CD34+ endothelial cells in both cultured and transplanted Islets of Langerhans

Chloe Rackham, Sinead Barry, Peter Jones, Aileen King. King’s College, London, United Kingdom.

Transplantation of islets of Langerhans has emerged as a promising therapy for the treatment of type 1 diabetes mellitus. The adaptation of the transplanted islets to the implantation organ with regard to revascularization, reinnervation, and reorganization of other stromal components (engraftment) is essential for the long term survival and function of the transplanted islets. During the isolation process, the blood supply to the islets is severed and thus after implantation islets must revascularize. The revascularization process is currently suboptimal, leading to an insufficient blood flow and consequently impaired oxygenation of the transplanted islets. This leads to hypoxia induced cell death and a decline in vital aspects of β cell function, including both insulin synthesis and secretion. Donor Islet Endothelial Cells (IECs) are thought to be important for the revascularization of transplanted islets. It has been reported that donor IECs die or de-differentiate in culture. Therefore, we have investigated the effect of a 3 day culture period on the expression of CD34 (a marker for capillary endothelial cells). Syngenic islet transplantations were carried out in which 200 islets were isolated from donor male C57BL/6 mice and transplanted underneath the kidney capsule of normoglycaemic recipient male C57BL/6 mice on the day of isolation. The islet grafts were removed at 2 weeks post transplantation; a time point at which the revascularization process is thought to be complete and at 3 days post transplantation in order to assess whether the in vivo environment is better for donor IEC survival than that in vitro. Immunohistochemistry, utilizing CD34 antisera, was used to quantify IECs. In vitro, the vascular density (number of IECs/mm2) of freshly isolated islets declined from 639 ± 28 to 327 ± 29, 150 ± 22 and 34 ± 8 in 1, 2 and 3 day cultured islets respectively; (mean ± SEM, n = 4. p<0.01 for fresh islets vs 1, 2 and 3 day cultured islets, One Way ANOVA). In the in vivo experiments, the vascular density of transplanted islets increased between 3 and 14 days (3 days: 29 ± 5, n=3 and 14 days: 449 ± 17, n=4, p<0.001 ANOVA). However, the vascular density at 3 and 14 days post transplantation was significantly reduced compared to islets within the native pancreas (864 ± 38, n = 4. p<0.001 vs 3 and 14 day transplanted islets, ANOVA). These studies indicate that the in vitro environment, as well as that in vivo, does not provide conditions that ensure the survival of donor IECs in the first few days after islet isolation. However, despite the numbers of IECs declining during the initial post transplantation period, the revascularization process increases the number of endothelial cells by 14 days. Unfortunately, this does not restore the microvasculature to the extent that is seen within the native pancreas.