Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas

Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells

BACKGROUND: In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential. METHODOLOGY/PRINCIPAL FINDINGS: Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types. CONCLUSIONS/SIGNIFICANCE: These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells

In Vitro Proliferation of Adult Human Beta-Cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide

Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts

<p>Abstract</p> <p>Background</p> <p>Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples.</p> <p>Results</p> <p>The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay.</p> <p>Conclusions</p> <p>This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.</p

The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Endothelium-Derived Netrin-4 Supports Pancreatic Epithelial Cell Adhesion and Differentiation through Integrins α2β1 and α3β1

BACKGROUND: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1 and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. CONCLUSIONS/SIGNIFICANCE: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins

Preoperative Red Cell Distribution Width and 30-day mortality in older patients undergoing non-cardiac surgery: a retrospective cohort observational study

Increased red cell distribution width (RDW) is associated with poorer outcomes in various patient populations. We investigated the association between preoperative RDW and anaemia on 30-day postoperative mortality among elderly patients undergoing non-cardiac surgery. Medical records of 24,579 patients aged 65 and older who underwent surgery under anaesthesia between 1 January 2012 and 31 October 2016 were retrospectively analysed. Patients who died within 30 days had higher median RDW (15.0%) than those who were alive (13.4%). Based on multivariate logistic regression, in our cohort of elderly patients undergoing non-cardiac surgery, moderate/severe preoperative anaemia (aOR 1.61, p = 0.04) and high preoperative RDW levels in the 3rd quartile (>13.4% and ≤14.3%) and 4th quartile (>14.3%) were significantly associated with increased odds of 30-day mortality - (aOR 2.12, p = 0.02) and (aOR 2.85, p = 0.001) respectively, after adjusting for the effects of transfusion, surgical severity, priority of surgery, and comorbidities. Patients with high RDW, defined as >15.7% (90th centile), and preoperative anaemia have higher odds of 30-day mortality compared to patients with anaemia and normal RDW. Thus, preoperative RDW independently increases risk of 30-day postoperative mortality, and future risk stratification strategies should include RDW as a factor

Renal function at the time of a myocardial infarction maintains prognostic value for more than 10 years

<p>Abstract</p> <p>Background</p> <p>Renal function is an important predictor of mortality in patients with myocardial infarction (MI), but changes in the impact over time have not been well described.</p> <p>We examined the importance of renal function by estimated GFR (eGFR) and se-creatinine as an independent long-term prognostic factor.</p> <p>Methods</p> <p>Prospective follow-up of 6653 consecutive MI patients screened for entry in the Trandolapril Cardiac Evaluation (TRACE) study. The patients were analysed by Kaplan-Meier survival analysis, landmark analysis and Cox proportional hazard models. Outcome measure was all-cause mortality.</p> <p>Results</p> <p>An eGFR below 60 ml per minute per 1.73 m², consistent with chronic renal disease, was present in 42% of the patients. We divided the patients into 4 groups according to eGFR. Overall, Cox proportional-hazards models showed that eGFR was a significant prognostic factor in the two groups with the lowest eGFR, hazard ratio 1,72 (confidence interval (CI) 1,56-1,91) in the group with the lowest eGFR. Using the eGFR group with normal renal function as reference, we observed an incremental rise in hazard ratio. We divided the follow-up period in 2-year intervals. Landmark analysis showed that eGFR at the time of screening continued to show prognostic effect until 16 years of follow-up. By multivariable Cox regression analysis, the prognostic effect of eGFR persisted for 12 years and of se-creatinine for 10 years. When comparing the lowest group of eGFR with the group with normal eGFR, prognostic significance was present in the entire period of follow-up with a hazard ratio between 1,97 (CI 1,65-2,35) and 1,35 (CI 0,99-1,84) in the 2-year periods.</p> <p>Conclusions</p> <p>One estimate of renal function is a strong and independent long-term prognostic factor for 10-12 years following a MI.</p