Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization.

In response to many apoptotic stimuli, oligomerization of Bax is essential for mitochondrial outer membrane permeabilization and the ensuing release of cytochrome c. These events are accompanied by mitochondrial fission that appears to require Drp1, a large GTPase of the dynamin superfamily. Loss of Drp1 leads to decreased cytochrome c release by a mechanism that is poorly understood. Here we show that Drp1 stimulates tBid-induced Bax oligomerization and cytochrome c release by promoting tethering and hemifusion of membranes in vitro. This function of Drp1 is independent of its GTPase activity and relies on arginine 247 and the presence of cardiolipin in membranes. In cells, overexpression of Drp1 R247A/E delays Bax oligomerization and cell death. Our findings uncover a function of Drp1 and provide insight into the mechanism of Bax oligomerization.

Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis.

Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Targeting Endothelial Connexin37 Reduces Angiogenesis and Decreases Tumor Growth.

Connexin37 (Cx37) and Cx40 form intercellular channels between endothelial cells (EC), which contribute to the regulation of the functions of vessels. We previously documented the participation of both Cx in developmental angiogenesis and have further shown that loss of Cx40 decreases the growth of different tumors. Here, we report that loss of Cx37 reduces (1) the in vitro proliferation of primary human EC; (2) the vascularization of subcutaneously implanted matrigel plugs in Cx37-/- mice or in WT using matrigel plugs supplemented with a peptide targeting Cx37 channels; (3) tumor angiogenesis; and (4) the growth of TC-1 and B16 tumors, resulting in a longer mice survival. We further document that Cx37 and Cx40 function in a collaborative manner to promote tumor growth, inasmuch as the injection of a peptide targeting Cx40 into Cx37-/- mice decreased the growth of TC-1 tumors to a larger extent than after loss of Cx37. This loss did not alter vessel perfusion, mural cells coverage and tumor hypoxia compared to tumors grown in WT mice. The data show that Cx37 is relevant for the control of EC proliferation and growth in different tumor models, suggesting that it may be a target, alone or in combination with Cx40, in the development of anti-tumoral treatments.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Connexins: key mediators of endocrine function.

The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane "hemi-channels," for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing ß-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

Gap junction proteins are key drivers of endocrine function.

It has long been known that the main secretory cells of exocrine and endocrine glands are connected by gap junctions, made by a variety of connexin species that ensure their electrical and metabolic coupling. Experiments in culture systems and animal models have since provided increasing evidence that connexin signaling contributes to control the biosynthesis and release of secretory products, as well as to the life and death of secretory cells. More recently, genetic studies have further provided the first lines of evidence that connexins also control the function of human glands, which are central to the pathogenesis of major endocrine diseases. Here, we summarize the recent information gathered on connexin signaling in these systems, since the last reviews on the topic, with particular regard to the pancreatic beta cells which produce insulin, and the renal cells which produce renin. These cells are keys to the development of various forms of diabetes and hypertension, respectively, and combine to account for the exploding, worldwide prevalence of the metabolic syndrome. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Targeting Cx40 (Connexin40) Expression or Function Reduces Angiogenesis in the Developing Mouse Retina.

OBJECTIVE: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown. APPROACH AND RESULTS: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice. CONCLUSIONS: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress.

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.

Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

Orthodontically induced cervical root resorption in humans is associated with the amount of tooth movement.

INTRODUCTION: The aim of this study was to investigate the variation in the amount of the orthodontically induced cervical root resorption and the association with several factors, such as the amount of tooth displacement, location of tooth in the maxilla or mandible, and presence of an interference that may influence the amount of root resorption. SUBJECTS AND METHODS: This study included 30 subjects (20 females, 10 males) with an age range of 11.3 to 43.0 years. Using a standardized experimental orthodontic tooth movement, 59 premolars were moved buccaly during 8 weeks with application of 1 N force. Fifty-eight contralateral premolars not subjected to orthodontic tooth movement served as controls. At the end of the experimental period the teeth were carefully extracted, scanned in a micro-computed tomography scanner with a resolution of 9 µm, and the reconstructed images were processed for volumetric evaluation of resorption craters at the cervical part of the root surface. Data were analyzed using unpaired t-test and the Pearson's correlation. RESULTS: Higher amount of cervical root resorption was detected in the orthodontically moved teeth (0.00055 mm3) compared to controls (0.00003 mm3; P < 0.001). Moderate correlation was found between root resorption in the two experimental teeth within the same individual (R = 0.421, P = 0.023). Teeth located in the mandible presented more resorption than those in the maxilla (P = 0.046). The amount of root resorption was correlated to the amount of tooth movement (R = 0.318, P = 0.016). CONCLUSION: Application of a 1 N force over a 2-month period provokes severe root resorption at the compression cervical sites. Resorption is correlated with the amount of tooth movement and the location of the teeth.

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.

AIMS/HYPOTHESIS: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. METHODS: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. RESULTS: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. CONCLUSIONS/INTERPRETATION: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.

Involvement of long non-coding RNAs in beta cell failure at the onset of type 1 diabetes in NOD mice.

AIMS/HYPOTHESIS: Exposure of pancreatic beta cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. As little is known about their role in insulin-secreting cells, this study aimed to evaluate their contribution to beta cell dysfunction. METHODS: The expression of lncRNAs was determined by microarray in the MIN6 beta cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells. RESULTS: MIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase in these lncRNAs was also seen in prediabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favoured beta cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB). CONCLUSIONS/INTERPRETATION: Our study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitises beta cells to apoptosis, probably contributing to their failure during the initial phases of the disease.

Targeting GLP-1 receptors for repeated magnetic resonance imaging differentiates graded losses of pancreatic beta cells in mice.

AIMS/HYPOTHESIS: Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model. METHODS: We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5 T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively. RESULTS: We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells. CONCLUSIONS/INTERPRETATION: The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.

Slow potentials encode intercellular coupling and insulin demand in pancreatic beta cells.

AIMS/HYPOTHESIS: Ion fluxes constitute a major integrative signal in beta cells that leads to insulin secretion and regulation of gene expression. Understanding these electrical signals is important for deciphering the endogenous algorithms used by islets to attain homeostasis and for the design of new sensors for monitoring beta cell function. METHODS: Mouse and human islets were cultured on multielectrode arrays (MEAs) for 3-13 days. Extracellular electrical activities received on each electrode were continuously amplified and recorded for offline characterisation. RESULTS: Differential band-pass filtering of MEA recordings of mouse islets showed two extracellular voltage waveforms: action potentials (lasting 40-60 ms) and very robust slow potentials (SPs, lasting 800-1,500 ms), the latter of which have not been described previously. The frequency of SPs directly correlated with glucose concentration, peaked at 10 mmol/l glucose and was further augmented by picomolar concentrations of glucagon-like peptide-1. SPs required the closure of ATP-dependent potassium channels as they were induced by glucose or glibenclamide but were not elicited by KCl-induced depolarisation. Pharmacological tools and the use of beta cell specific knockout mice showed that SPs reflected cell coupling via connexin 36. Moreover, increasing and decreasing glucose ramps showed hysteresis with reduced glucose sensitivity during the decreasing phase. SPs were also observed in human islets and could be continuously recorded over 24 h. CONCLUSIONS/INTERPRETATION: This novel electrical signature reflects the syncytial function of the islets and is specific to beta cells. Moreover, the observed hysteresis provides evidence for an endogenous algorithm naturally present in islets to protect against hypoglycaemia.

Multimodal image coregistration and inducible selective cell ablation to evaluate imaging ligands.

We combined multimodal imaging (bioluminescence, X-ray computed tomography, and PET), tomographic reconstruction of bioluminescent sources, and two unique, complementary models to evaluate three previously synthesized PET radiotracers thought to target pancreatic beta cells. The three radiotracers {[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]FP-DTBZ), [(18)F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline ((18)F-AV-266), and (2S,3R,11bR)-9-(3-fluoropropoxy)-2-(hydroxymethyl)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol ((18)F-AV-300)} bind vesicular monoamine transporter 2. Tomographic reconstruction of the bioluminescent signal in mice expressing luciferase only in pancreatic beta cells was used to delineate the pancreas and was coregistered with PET and X-ray computed tomography images. This strategy enabled unambiguous identification of the pancreas on PET images, permitting accurate quantification of the pancreatic PET signal. We show here that, after conditional, specific, and rapid mouse beta-cell ablation, beta-cell loss was detected by bioluminescence imaging but not by PET imaging, given that the pancreatic signal provided by three PET radiotracers was not altered. To determine whether these ligands bound human beta cells in vivo, we imaged mice transplanted with luciferase-expressing human islets. The human islets were imaged by bioluminescence but not with the PET ligands, indicating that these vesicular monoamine transporter 2-directed ligands did not specifically bind beta cells. These data demonstrate the utility of coregistered multimodal imaging as a platform for evaluation and validation of candidate ligands for imaging islets.

Glucose-stimulated insulin secretion: the hierarchy of its multiple cellular and subcellular mechanisms.

Glucose-stimulated insulin secretion is ensured by multiple molecular, cellular and tissue events. In this issue of Diabetologia, Low et al (DOI: 10.1007/s00125-013-3019-5 ) have taken an important new step towards understanding the hierarchical organisation of these events, by monitoring in vitro the individual exocytosis of multiple beta cells within intact mouse islets. The authors show that glucose stimulation markedly increases the number of exocytotic events per cell and, to a lesser extent, the number of beta cells contributing to this event. In this commentary we discuss these novel observations and propose that metabolic and electrical coupling of islet beta cells is responsible for a more homogeneous glucose-induced secretory response of cells in an intact islet as compared with isolated beta cells.

Connexin-dependent signaling in neuro-hormonal systems.

The advent of multicellular organisms was accompanied by the development of short- and long-range chemical signalling systems, including those provided by the nervous and endocrine systems. In turn, the cells of these two systems have developed mechanisms for interacting with both adjacent and distant cells. With evolution, such mechanisms have diversified to become integrated in a complex regulatory network, whereby individual endocrine and neuro-endocrine cells sense the state of activity of their neighbors and, accordingly, regulate their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the (neuro)hormone-producing cells of all endocrine glands and secretory regions of the central nervous system so far investigated in vertebrates. This review summarizes the distribution of connexins in the mammalian (neuro)endocrine systems, and what we know about the participation of these proteins on hormone secretion, the life of the producing cells, and the action of (neuro)hormones on specific targets. The data gathered since the last reviews on the topic are summarized, with particular emphasis on the roles of Cx36 in the function of the insulin-producing beta cells of the endocrine pancreas, and of Cx40 in that of the renin-producing juxta-glomerular epithelioid cells of the kidney cortex. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.