Bioengineering with Endothelial Progenitor Cells Improves the Vascular Engraftment of Transplanted Human Islets.

Pancreatic islets isolated for transplantation are disconnected from their vascular supply and need to establish a new functional network posttransplantation. Due to poor revascularization, prevailing hypoxia with correlating increased apoptosis rates in experimental studies can be observed for months posttransplantation. Endothelial progenitor cells (EPCs) are bone marrow-derived cells that promote neovascularization. The present study tested the hypothesis that EPCs, isolated from human umbilical cord blood, could be coated to human islet surfaces and be used to promote islet vascular engraftment. Control or EPC bioengineered human islets were transplanted into the renal subcapsular space of nonobese diabetic/severe combined immunodeficiency mice. Four weeks posttransplantation, graft blood perfusion and oxygen tension were measured using laser Doppler flowmetry and Clark microelectrodes, respectively. Vessel functionality was also assessed by in vivo confocal imaging. The vascular density and the respective contribution of human and recipient endothelium were assessed immunohistochemically by staining for human and mouse CD31. Islet grafts with EPCs had substantially higher blood perfusion and oxygen tension than control transplants. Furthermore, analysis of the vascular network of the grafts revealed that grafts containing EPC bioengineered islets had a superior vascular density compared with control grafts, with functional chimeric blood vessels. We conclude that a simple procedure of surface coating with EPCs provides a possibility to improve the vascular engraftment of transplanted human islets. Established protocols are also easily applicable for intraportal islet transplantation in order to obtain a novel directed cellular therapy at the site of implantation in the liver.

Pancreatic islet blood flow and its measurement.

Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting ß-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future.

Extensive Loss of Islet Mass Beyond the First Day After Intraportal Human Islet Transplantation in a Mouse Model.

Clinical islet transplantation is characterized by a progressive deterioration of islet graft function, which renders many patients once again dependent on exogenous insulin administration within a couple of years. In this study, we aimed to investigate possible engraftment factors limiting the survival and viability of experimentally transplanted human islets beyond the first day after their transplantation to the liver. Human islets were transplanted into the liver of nude mice and characterized 1 or 30 days after transplantation by immunohistochemistry. The factors assessed were endocrine mass, cellular death, hypoxia, vascular density and amyloid formation in the transplanted islets. One day posttransplantation, necrotic cells, as well as apoptotic cells, were commonly observed. In contrast to necrotic death, apoptosis rates remained high 1 month posttransplantation, and the total islet mass was reduced by more than 50% between 1 and 30 days posttransplantation. Islet mass at 30 days posttransplantation correlated negatively to apoptotic death. Vascular density within the transplanted islets remained less than 30% of that in native human islets up to 30 days posttransplantation and was associated with prevailing hypoxia. Amyloid formation was rarely observed in the 1-day-old transplants, but was commonly observed in the 30-day-old islet transplants. We conclude that substantial islet cell death occurs beyond the immediate posttransplantation phase, particularly through apoptotic events. Concomitant low vascularization with prevailing hypoxia and progressive amyloid development was observed in the human islet grafts. Strategies to improve engraftment at the intraportal site or change of implantation site in the clinical setting are needed.

Co-transplantation of human pancreatic islets with post-migratory neural crest stem cells increases ß-cell proliferation and vascular and neural regrowth.

CONTEXT: Neural crest stem cells (NCSCs) are capable of substantially improving murine islet function by promoting ß-cell proliferation. OBJECTIVE: The present study aimed to investigate the potential of NCSCs to stimulate human ß-cell proliferation, and improve neural and vascular engraftment of human islets. DESIGN, SETTING, AND SUBJECTS: Human pancreatic islets from 18 brain-dead cadaveric donors (age range, 19-78 y) were obtained through the Nordic Network for Clinical Islet Transplantation. ß-cell proliferation and graft function was investigated at our experimental laboratory. INTERVENTION AND MAIN OUTCOME MEASURES: Human islets were transplanted, either alone or together with spheres of NCSCs. ß-cell proliferation, as well as islet neural and vascular densities, were assessed by immunohistochemistry. Graft blood perfusion and oxygen tension were measured using laser-Doppler flowmetry and Clark microelectrodes, respectively. RESULTS: Two days posttransplantation, the number of Ki67-positive ß-cells was doubled in human islets that had been exposed to NCSCs. Similar findings were obtained in vitro, as well as with EdU as proliferation marker. Four weeks posttransplantation, NCSC-exposed human islet grafts had much higher neural and vascular densities. The newly formed blood vessels were also functional, given that these human islets had a substantially higher blood perfusion and oxygen tension when compared with control transplants. CONCLUSION: We conclude that exposure to NCSCs stimulates human ß-cell proliferation, and that these cells improve both the neural and vascular engraftment of transplanted human islets. NCSCs are a promising cellular therapy for translation into clinical use.

Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury.

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.

The therapeutic role of endothelial progenitor cells in Type 1 diabetes mellitus.

Pancreatic ß-cells sense and adjust the blood glucose level by secretion of insulin. In Type 1 diabetes mellitus, these insulin-producing cells are destroyed, leaving the patients incapable of regulating blood glucose homeostasis. At the time of diagnosis, most patients still have 20-30% of their original ß-cell mass remaining. These residual ß-cells are targets for intervention therapies aimed at preventing further autoimmune destruction, in addition to increasing the number of existing ß-cells. Such a therapeutic option is highly desirable since it may lead to a full recovery of newly diagnosed patients, with no need for further treatment with immunosuppressant drugs or exogenous insulin administration. In this article, we propose that endothelial progenitor cells, a cell type known to promote and support neovascularization following endothelial injury, may be used as part of a combinational stem cell therapy aimed to improve the vascularization, survival and proliferation of ß-cells.