Johanna Henriksnäs

Clinical and Experimental Pancreatic Islet Transplantation to Striated Muscle: Establishment of a Vascular System Similar to that in Native Islets

OBJECTIVE Curing type 1 diabetes by transplanting pancreatic islets into the liver is associated with poor long-term outcome and graft failure at least partly due to inadequate graft revascularization. The aim of the current study was to evaluate striated muscle as a potential angiogenic site for islet transplantation. RESEARCH DESIGN AND METHODS The current study presents a new experimental model that is found to be applicable to clinical islet transplantation. Islets were implanted into striated muscle and intraislet vascular density and blood flow were visualized with intravital and confocal microscopy in mice and by magnetic resonance imaging in three autotransplanted pancreatectomized patients. Mice were rendered neutropenic by repeated injections of Gr-1 antibody, and diabetes was induced by alloxan treatment. RESULTS Contrary to liver-engrafted islets, islets transplanted to mouse muscle were revascularized with vessel densities and blood flow entirely comparable with those of islets within intact pancreas. Initiation of islet revascularization at the muscular site was dependent on neutrophils, and the function of islets transplanted to muscle was proven by curing diabetic mice. The experimental data were confirmed in autotransplanted patients where higher plasma volumes were measured in islets engrafted in forearm muscle compared with adjacent muscle tissue through high-resolution magnetic resonance imaging. CONCLUSIONS This study presents a novel paradigm in islet transplantation whereby recruited neutrophils are crucial for the functionally restored intraislet blood perfusion following transplantation to striated muscle under experimental and clinical situations.

The gastric mucus layers: constituents and regulation of accumulation

The mucus layer continuously covering the gastric mucosa consists of a loosely adherent layer that can be easily removed by suction, leaving a firmly adherent mucus layer attached to the epithelium. These two layers exhibit different gastroprotective roles; therefore, individual regulation of thickness and mucin composition were studied. Mucus thickness was measured in vivo with micropipettes in anesthetized mice [isoflurane; C57BL/6, Muc1-/-, inducible nitric oxide synthase (iNOS)-/-, and neuronal NOS (nNOS)-/-] and rats (inactin) after surgical exposure of the gastric mucosa. The two mucus layers covering the gastric mucosa were differently regulated. Luminal administration of PGE(2) increased the thickness of both layers, whereas luminal NO stimulated only firmly adherent mucus accumulation. A new gastroprotective role for iNOS was indicated since iNOS-deficient mice had thinner firmly adherent mucus layers and a lower mucus accumulation rate, whereas nNOS did not appear to be involved in mucus secretion. Downregulation of gastric mucus accumulation was observed in Muc1-/- mice. Both the firmly and loosely adherent mucus layers consisted of Muc5ac mucins. In conclusion, this study showed that, even though both the two mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated by luminal PGE(2) and NO. A new gastroprotective role for iNOS was indicated since iNOS-/- mice had a thinner firmly adherent mucus layer. In addition, a regulatory role of Muc1 was demonstrated since downregulation of gastric mucus accumulation was observed in Muc1-/- mice.

Markedly Decreased Blood Perfusion of Pancreatic Islets Transplanted Intraportally Into the Liver: Disruption of Islet Integrity Necessary for Islet Revascularization

Experimental studies indicate low revascularization of intraportally transplanted islets. This study aimed to quantify, for the first time, the blood perfusion of intrahepatically transplanted islets and elucidate necessary factors for proper islet graft revascularization at this site. Yellow chameleon protein 3.0 islets expressing fluorescent protein in all cells were transplanted. Graft blood perfusion was determined by microspheres. The vascular density and relative contribution of donor blood vessels in revascularization was evaluated using islets expressing green fluorescent protein under the Tie-2 promoter. Blood perfusion of intrahepatic islets was as a mean only 5% of that of native islets at 1-month posttransplantation. However, there was a marked heterogeneity where blood perfusion was less decreased in islets transplanted without prior culture and in many cases restored in islets with disrupted integrity. Analysis of vascular density showed that distorted islets were well revascularized, whereas islets still intact at 1-month posttransplantation were almost avascular. Few donor endothelial cells were observed in the new islet vasculature. The very low blood perfusion of intraportally transplanted islets is likely to predispose for ischemia and hamper islet function. Since donor endothelial cells do not expand posttransplantation, disruption of islet integrity is necessary for revascularization to occur by recipient blood vessels.

Oxygenation of islets and its role in transplantation.

Purpose of reviewTo summarize recent studies on the oxygenation of pancreatic islets and its role in islet transplantation. Recent findingsPancreatic islet cells are highly sensitive to hypoxic conditions. Hypoxia contributes to poor islet yield at isolation, as well as inflammatory events and cellular death during culture and early posttransplantation. Use of oxygen carriers, such as semifluorinated alkanes, during pancreas preservation and gas-permeable devices for islet culture and transport has in recent studies proven beneficial. Beta-cell death can be limited posttransplantation by targeting hypoxia-induced cellular pathways that cause apoptotic death. Owing to low revascularization, impaired oxygenation seems to prevail in intraportally transplanted islets. Means to improve revascularization, oxygenation and function of transplanted islets can be achieved not only by stimulating angiogenic factors, but also by decrease of angiostatic factors such as thrombospondin-1 in islets for transplantation. Moreover, bone-marrow-derived cells, such as mesenchymal stem cells and hematopoietic stem cells, can induce or contribute to increased revascularization. SummaryLow oxygenation of islets contributes to cellular death and dysfunction during preparation of islets for transplantation, as well as posttransplantation. Interventions at these different steps to ensure adequate oxygenation have the potential to improve the results of clinical islet transplantation.

Increased Recruitment but Impaired Function of Leukocytes during Inflammation in Mouse Models of Type 1 and Type 2 Diabetes

Background Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. Methodology/Principal Findings Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62±18% and 85±21%, respectively) and high fat diet-induced (77±25% and 86±17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3±1.0) compared to control (8.0±1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. Conclusions/Significance These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.

Perfluorohexyloctane improves long-term storage of rat pancreata for subsequent islet isolation.

Pancreas oxygenation by means of the hyperoxygen carrier perfluorodecalin (PFD) has been established to prevent ischemically induced damage from cold‐stored pancreata. However, large‐scale studies did not confirm the promising results that had been observed in smaller donor populations. This study assessed whether islet isolation from pancreata stored for prolonged periods can be improved by utilizing the new oxygen carrier perfluorohexyloctane (F6H8) characterized by lower gravity and higher lipophilicity than PFD. Subsequent to 24 h of storage in either oxygenated PFD or F6H8, the rat pancreata were assessed for the intrapancreatic partial oxygen pressure (pO2) and subsequently processed with current standard procedures. The intrapancreatic pO2 was nearly identical in rat pancreata stored either in PFD or F6H8. Nevertheless, rat islet isolation outcome was significantly increased in terms of yield, integrity, in vitro function and post‐transplant outcome after transplantation in diabetic nude mice when F6H8 was used as oxygen carrier. This proof‐of‐concept study demonstrated in rats that islet isolation performed after long‐term storage of oxygenated pancreatic tissue can be significantly improved if PFD was replaced by F6H8.

X-ray Microanalysis of Apical Fluid in Cystic Fibrosis Airway Epithelial Cell Lines

The ionic composition of the fluid lining the airways (airway surface liquid, ASL) in healthy subjects and patients with cystic fibrosis (CF) has been a matter of controversy. It has been attempted to resolve conflicting theories by using cell cultures, but published results show a wide variety of values for the ionic concentrations in the apical fluid in these cultures. To investigate CFTR-mediated HCO 3 -conductance and the role of HCO 3 - in regulating ASL pH we determined the pH of the fluid covering the apical surface of airway epithelial cells. A normal (16HBE14o - ) and a CF (CFBE41o - ) bronchial epithelial cell line were grown on membrane inserts in both a liquid-liquid interface culture system for 7 days, and in an air-liquid interface culture system for one month. The elemental composition of the fluid covering the apical surface was determined by X-ray microanalysis of frozen-hydrated specimens, or by X-ray microanalysis of Sephadex beads that had been equilibrated with the apical fluid. Analysis showed that the apical fluid had a Na + and Cl -concentration of about 80-100 mM and thus was slightly hypotonic. The ionic concentrations were somewhat higher in air-liquid interface than in liquid-liquid interface cultures. The apical fluid in CF cells had significantly higher concentrations of Na and Cl than that in control cultures. In control cultures, the concentrations of Na and Cl in the apical fluid increased if glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) was added to the apical medium. Exposing the cells to the metabolic inhibitor NaCN also resulted in a significant increase of the Na and Cl concentrations in the apical fluid. The results agree with the notion that these cell cultures are mainly absorptive cells, and that ion absorption by the CF cells is reduced compared to that in normal cells. The pH measurements of the fluid covering the apical part of cell cultures support the notion that bicarbonate ions may be transported by CFTR, and that this can be inhibited by specific CFTR inhibitors.

The α2-adrenoceptor antagonist yohimbine normalizes increased islet blood flow in GK rats: a model of type 2 diabetes.

Overexpression of α2A-adrenoceptors contributes to type 2 diabetes in GK rats. We aimed to investigate if α2-adrenoceptor inhibition affected islet blood flow in these rats. Anesthetized GK and Wistar-F rats were given the α2-adrenoceptor inhibitor yohimbine (2.5 mg/kg BW) intravenously. The GK rats had higher blood glucose and serum insulin concentrations than WF rats. Yohimbine affected neither of these values in WF rats, but decreased blood glucose and increased serum insulin concentrations in GK rats. Total pancreatic and islet blood flows, measured with microspheres, were increased in GK when compared to WF rats. Yohimbine affected none of the blood flow values in WF rats, whereas islet blood flow in GK rats was reduced to values similar to those seen in WF rats. Overexpression of α2-adrenoceptors may contribute to the increased islet blood flow seen in GK rats, and may be eligible for pharmacologic intervention.

Resistin increases islet blood flow and decreases subcutaneous adipose tissue blood flow in anaesthetized rats

Aim:  Resistin is an adipokine which has been suggested to participate in the induction of insulin resistance associated with type 2 diabetes. The aim of the present study was to investigate whether acute administration of resistin influences tissue blood perfusion in rats.

Endothelin-1 markedly decreases the blood perfusion of transplanted pancreatic islets in rats.

BACKGROUND Transplantation of insulin-producing β-cells is the only available curative treatment for type 1 diabetes. However, graft function declines within the first years after transplantation, which may reflect inadequate vascular engraftment. Endothelin-1 (ET-1) is a potent vasoconstrictor whose production is regulated by both hypoxia and inflammation. Moreover, the plasma concentration of ET-1 is elevated in patients with type 1 diabetes. The aim of this study was to investigate the gene expression and effects of ET-1 and its 2 receptor antagonists, BQ123 and BQ788, on blood flow in syngeneic rat islet transplants. METHODS Pancreatic islets from Wistar Furth rats were isolated and transplanted syngeneically under the kidney capsule. Transplant and kidney cortex blood flow was measured using laser Doppler flowmetry after administration of ET-1 via topical application, or after administration of BQ123 and BQ788 intravenously. The grafts and isolated islets were analyzed for mRNA expression of ET-1, ET(A) receptor, ET(B) receptor, and endothelin-converting enzyme 1 using by reverse-transcription polymerase chain reaction. RESULTS ET-1 markedly decreased transplant blood flow (77.5 ± 4.4% 1 minute after administration; n = 6), whereas neither BQ123 nor BQ788 had vascular effects. No differences in relative gene expression between the grafts and freshly isolated control islets were seen for ET-1 (0.65 ± 0.14 [n = 8] vs 0.79 ± 0.24 [n = 5]), ET(A) receptor (0.37 ± 0.14 [n = 8] vs 0.25 ± 0.04 [n =5]), ET(B) receptor (4.78 ± 1.43 [n = 8] vs 1.94 ± 0.32 [n = 5]), or endothelin converting enzyme 1 (7.25 ± 1.88 [n = 8] vs 11.83 ± 0.95 [n = 5]) when expressed as 2(-ΔCt). CONCLUSION Exogenous ET-1 strongly affects the blood perfusion of transplanted islets, and endogenous levels can, if up-regulated, contribute to graft failure.