Integration of Primary Endocrine Cells and Supportive Cells Using Functionalized Silk Promotes the Formation of Prevascularized Islet-like Clusters.

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons.

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of trans­nosology pathways in diabetes and its co-morbidities.

Silk matrices promote formation of insulin-secreting islet-like clusters.

Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.

Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices.

Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.

A common polymorphism in the promoter region of the TNFSF4 gene is associated with lower allele-specific expression and risk of myocardial infarction.

BACKGROUND: The TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression. METHODS AND RESULTS: Based on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele. CONCLUSIONS: Our data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI.

Pathophysiological, genetic and gene expression features of a novel rodent model of the cardio-metabolic syndrome.

BACKGROUND: Complex etiology and pathogenesis of pathophysiological components of the cardio-metabolic syndrome have been demonstrated in humans and animal models. METHODOLOGY/PRINCIPAL FINDINGS: We have generated extensive physiological, genetic and genome-wide gene expression profiles in a congenic strain of the spontaneously diabetic Goto-Kakizaki (GK) rat containing a large region (110 cM, 170 Mb) of rat chromosome 1 (RNO1), which covers diabetes and obesity quantitative trait loci (QTL), introgressed onto the genetic background of the normoglycaemic Brown Norway (BN) strain. This novel disease model, which by the length of the congenic region closely mirrors the situation of a chromosome substitution strain, exhibits a wide range of abnormalities directly relevant to components of the cardio-metabolic syndrome and diabetes complications, including hyperglycaemia, hyperinsulinaemia, enhanced insulin secretion both in vivo and in vitro, insulin resistance, hypertriglyceridemia and altered pancreatic and renal histological structures. Gene transcription data in kidney, liver, skeletal muscle and white adipose tissue indicate that a disproportionately high number (43-83%) of genes differentially expressed between congenic and BN rats map to the GK genomic interval targeted in the congenic strain, which represents less than 5% of the total length of the rat genome. Genotype analysis of single nucleotide polymorphisms (SNPs) in strains genetically related to the GK highlights clusters of conserved and strain-specific variants in RNO1 that can assist the identification of naturally occurring variants isolated in diabetic and hypertensive strains when different phenotype selection procedures were applied. CONCLUSIONS: Our results emphasize the importance of rat congenic models for defining the impact of genetic variants in well-characterised QTL regions on in vivo pathophysiological features and cis-/trans- regulation of gene expression. The congenic strain reported here provides a novel and sustainable model for investigating the pathogenesis and genetic basis of risks factors for the cardio-metabolic syndrome.

Human genetic evidence that OX40 is implicated in myocardial infarction.

We recently showed that genetic variants in OX40L are associated with myocardial infarction (MI) and severity of coronary artery disease in human. A number of studies also suggest a possible role for OX40 (the OX40L receptor) as a factor contributing to atherosclerosis. In the present study, the OX40 gene was screened for variants associated with precocious MI, using individuals with MI before the age of 60 and controls. Despite the fact that the OX40 gene is highly conserved between species and that relatively few common genetic variants were encountered, an association with MI was seen for a polymorphism in intron 5 (rs2298212). In silico investigation suggested that genetic variation (rs2298211), linked to this intronic variant, is possibly affecting spliceosome function. Our results provide evidence that variants in human OX40 might influence susceptibility to MI. The relevance of these findings is supported by the vital functions fulfilled by OX40 in mammals as reflected by the high level of evolutionary conservation.

Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility.

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P