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There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets. Diabetes 54: [1755][1756][1757][1758][1759][1760][1761][1762] 2005

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Exocytosis of insulin is dependent on the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins in the B-cells. We assessed insulin release as well as gene and protein expression of SNARE complex protein in isolated pancreatic islets of type 2 diabetic patients (n ‫؍‬ 4) and nondiabetic control subjects (n ‫؍‬ 4). In islets from the diabetic patients, insulin responses to 8.3 and 16.7 mmol/l glucose were markedly reduced compared with control islets (4.7 ؎ 0.3 and 8.4 ؎ 1.8 vs. 17.5 ؎ 0.1 and 24.3 ؎ 1.2 U ⅐ islet ؊1 ⅐ h ؊1 , respectively; P < 0.001). Western blot analysis revealed decreased amounts of islet SNARE complex and SNARE-modulating proteins in diabetes: syntaxin-1A (21 ؎ 5% of control levels), SNAP-25 (12 ؎ 4%), VAMP-2 (7 ؎ 4%), nSec1 (Munc 18; 34 ؎ 13%), Munc 13-1 (27 ؎ 4%), and synaptophysin (64 ؎ 7%). Microarray gene chip analysis, confirmed by quantitative PCR, showed that gene expression was decreased in diabetes islets: syntaxin-1A (27 ؎ 2% of control levels), SNAP-25 (31 ؎ 7%), VAMP-2 (18 ؎ 3%), nSec1 (27 ؎ 5%), synaptotagmin V (24 ؎ 2%), and synaptophysin (12 ؎ 2%). In conclusion, these data support the view that decreased islet RNA and protein expression of SNARE and SNAREmodulating proteins plays a role in impaired insulin secretion in type 2 diabetic patients. It remains unclear, however, to which extent this defect is primary or secondary to, e.g., glucotoxicity. Diabetes 55: [435][436][437][438][439][440] 2006 I n addition to insulin resistance, impaired insulin response to glucose appears to be a prerequisite for type 2 diabetes to develop (1,2). For practical and ethical reasons, most studies of molecular mechanisms behind this functional B-cell defect have been performed in animal models of the disease. One such rodent is the Goto-Kakizaki (GK) rat, which is nonobese with moderate hyperglycemia on a background of greatly impaired insulin secretion and mildly to moderately decreased insulin sensitivity (3-5). Several metabolic abnormalities with potential impact on B-cell secretory function have been demonstrated in the pancreatic islets of GK rats (4,6 -9). Exocytosis of insulin is critically dependent on the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins in the B-cells (10 -12). We have shown that expression of several of the SNARE complex proteins, such as VAMP-2, syntaxin-1A, SNAP-25, and nSec1 (Munc18), were decreased by ϳ40% in GK versus control rat islets (13,14), and similar impairments were also found in islets of the fa/fa Zucker rat (15). Moreover, in GK rat islets, dysregulation of SNARE complex protein expression was evident, because their compensatory increase by high-glucose exposure was abrogated (13).Here, we have investigated the role of the exocytotic SNARE complex proteins and several SNARE-modulating proteins in pancreatic islets obtained from patients with type 2 diabetes compared with nondiabetic control subjects. For that purpose, we studied the gene expression by Affymetrix microarray chip...

OBJECTIVE—In clinical islet transplantation, the instant blood-mediated inflammatory reaction (IBMIR) is a major factor contributing to the poor initial engraftment of the islets. This reaction is triggered by tissue factor and monocyte chemoattractant protein (MCP)-1, expressed by the transplanted pancreatic islets when the islets come in contact with blood in the portal vein. All currently identified systemic inhibitors of the IBMIR are associated with a significantly increased risk of bleeding or other side effects. To avoid systemic treatment, the aim of the present study was to render the islet graft blood biocompatible by applying a continuous heparin coating to the islet surface. RESEARCH DESIGN AND METHODS—A biotin/avidin technique was used to conjugate preformed heparin complexes to the surface of pancreatic islets. This endothelial-like coating was achieved by conjugating barely 40 IU heparin per full-size clinical islet transplant. RESULTS—Both in an in vitro loop model and in an allogeneic porcine model of clinical islet transplantation, this heparin coating provided protection against the IBMIR. Culturing heparinized islets for 24 h did not affect insulin release after glucose challenge, and heparin-coated islets cured diabetic mice in a manner similar to untreated islets. CONCLUSIONS—This novel pretreatment procedure prevents intraportal thrombosis and efficiently inhibits the IBMIR without increasing the bleeding risk and, unlike other pretreatment procedures (e.g., gene therapy), without inducing acute or chronic toxicity in the islets.