| The success of islet transplantation is hampered by variability of islet isolations and the considerable loss of islets early after transplantation. Causes of these are the lot-to-lot variability of isolation enzymes and the inadequate revascularisation of islets. It has been shown that VEGF plays a crucial role in this process. The aim of my thesis is to study a new two-component enzyme blend for human islet isolations and to assess the effects of VEGF-A upregulation in beta-cells on islet cell revascularisation and function after transplantation. We compared the results of human islet isolations performed either with Collagenase NB1/Neutral Protease NB1 enzyme combination (group I, n=9) or with traditional Liberase HI (group II, n=9). Islet yield, morphology, apoptosis, in vitro and in vivo functions were analysed. Islets from RIP-VEGF transgenic mice were isolated, studied in vitro, then transplanted into chemically diabetic mice. Moreover, we modified the CDM3D beta cells using a lentiviral vector to promote secretion of VEGF-A in a Tetracycline (TC)-controlled manner (CDM3D-TET-VEGF cells). In vitro VEGF secretion, angiogenesis and stimulated insulin secretion were assessed. The cells were transplanted into syngeneic STZ-diabetic mice to assess the effects of this controlled VEGF expression in vivo. Time to normoglycaemia, IPGTT, graft vascular density were evaluated. Total IEQ/gr of pancreas was higher, islet morphology was improved and there was a higher proportion of free and intact islets with less apoptosis when the new NB1 enzyme was used (232). The time for diabetic mice to return to normoglycemia and the stimulated plasma glucose clearence were significantly accelerated in mice grafted with RIP-VEGF islets or CDM3D-TET-VEGF cells (233). VEGF delivery resulted in well defined TC controlled angiogenesis in vitro. There was a significant increase in vascular density of grafted CDM3D-TET-VEGF cells versus control cells. VEGF was only needed during the first 2-3 weeks after transplantation, when removed, normoglycemia and graft vascularisation were maintained (234). The most important benefit of the studied new enzyme blend is that it makes the human islet isolations adjusted to the characteristics of a given pancreas possible. On the other hand, TC-regulated temporal expression of VEGF using a gene therapy could present a novel way to improve early revascularisation and engraftment after islet cell transplantation. |
