Paolo Meda

Production of Chemokines by Perivascular Adipose Tissue: A Role in the Pathogenesis of Atherosclerosis?

Objective—Obesity is associated with an increased risk for cardiovascular disease. Although it is known that white adipose tissue (WAT) produces numerous proinflammatory and proatherogenic cytokines and chemokines, it is unclear whether adipose-derived chemotactic signals affect the chronic inflammation in atherosclerosis. Methods and Results—Histological examination showed that perivascular WAT (pWAT) is in close proximity to vascular walls, particularly at sites that have a tendency to develop atherosclerosis. In rodents, the amount of pWAT is markedly increased by a high-fat diet. At a functional level, supernatant from subcutaneous and pWAT strongly induced the chemotaxis of peripheral blood leukocytes. The migration of granulocytes and monocytes was mostly mediated by interleukin-8 and monocyte chemoattractant protein-1, respectively, whereas both chemokines contributed to the migration of activated T cells. Moreover, pWAT produces these chemokines, as shown by immunohistochemistry and by explant culture. The accumulation of macrophages and T cells at the interface between pWAT and the adventitia of human atherosclerotic aortas may reflect this prochemotactic activity of pWAT. Conclusions—Human pWAT has chemotactic properties through the secretion of different chemokines, and we propose that pWAT might contribute to the progression of obesity-associated atherosclerosis.

Expression of connexin36 in the adult and developing rat brain.

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

EphA-Ephrin-A-Mediated β Cell Communication Regulates Insulin Secretion from Pancreatic Islets

In vertebrates, beta cells are aggregated in the form of pancreatic islets. Within these islets, communication between beta cells inhibits basal insulin secretion and enhances glucose-stimulated insulin secretion, thus contributing to glucose homeostasis during fasting and feeding. In the search for the underlying molecular mechanism, we have discovered that beta cells communicate via ephrin-As and EphAs. We provide evidence that ephrin-A5 is required for glucose-stimulated insulin secretion. We further show that EphA-ephrin-A-mediated beta cell communication is bidirectional: EphA forward signaling inhibits insulin secretion, whereas ephrin-A reverse signaling stimulates insulin secretion. EphA forward signaling is downregulated in response to glucose, which indicates that, under basal conditions, beta cells use EphA forward signaling to suppress insulin secretion and that, under stimulatory conditions, they shift to ephrin-A reverse signaling to enhance insulin secretion. Thus, we explain how beta cell communication in pancreatic islets conversely affects basal and glucose-stimulated insulin secretion to improve glucose homeostasis.

Involvement of MicroRNAs in the Cytotoxic Effects Exerted by Proinflammatory Cytokines on Pancreatic β-Cells

OBJECTIVE Pancreatic β-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated β-cell cytotoxicity. RESEARCH DESIGN AND METHODS We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in β-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated β-cell failure by modifying their expression in insulin-secreting MIN6 cells. RESULTS We found that IL-1β and TNF-α induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1β exposure. Moreover, anti–miR-34a and anti–miR-146a treatment protected MIN6 cells from cytokine-triggered cell death. CONCLUSIONS Our data identify miR-21, miR-34a, and miR-146a as novel players in β-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice.

Heterogeneity and contact-dependent regulation of hormone secretion by individual B cells☆

A reverse hemolytic plaque assay was developed to visualize insulin release from individual adult pancreatic B cells. Cells obtained by mechanical dispersion of isolated rat islets of Langerhans were mixed with protein A-coated sheep red blood cells and incubated in the presence of an anti-insulin serum, under conditions known to affect insulin release. The cell mixture was further incubated with complement and finally fixed. Insulin release was revealed by the presence of hemolytic plaques which resulted from the complement-mediated lysis of red blood cells bearing insulin-anti-insulin complexes bound to protein A. Quantitation of hemolytic plaques around trypan blue-unstained and immunohistochemically identified B cells showed that stimulation of insulin release results in the recruitment of increasing numbers of secreting B cells as well as in the enhanced response of individual B cells. Reverse changes occur upon inhibition of insulin release. Comparison of freshly dispersed and one-day-cultured preparations did not reveal significant differences in the secretory response of undamaged B cells. In both preparations, single B cells responded to secretagogues in smaller proportions and to a lesser extent than clusters in which B cells had either maintained or restored contacts and junctional communication with their neighbours. However, the overall preponderant response of clusters was less than expected from the number of individually secreting B cells they contained. The data show that B cells are heterogeneous in terms of their ability to release insulin and provide evidence that cell-to-cell adhesion and/or junctional communication regulate hormone secretion from individual B cells.

Regulation of PKD by the MAPK p38δ in Insulin Secretion and Glucose Homeostasis

Summary Dysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Connexins, pannexins, innexins: novel roles of "hemi-channels"

The advent of multicellular organisms, some 800 million years ago, necessitated the development of mechanisms for cell-to-cell synchronization and for the spread of signals across increasingly large cell populations [168, 185]. Many structures and mechanisms have evolved to achieve such functions [4, 15]. Among these mechanisms, one which is prominent in both the invertebrate and the vertebrate world, across the entire phylogenetic scale, involves the transmembrane flux of large cytosolic and extracellular molecules [4, 15, 65, 66, 69–71, 121, 128, 129, 147, 154, 163]. These fluxes, in turn, are dependent on the formation of specific channels that in all animal classes are made by tetra-span integral membrane proteins [65, 66, 69–71, 121, 128, 129, 147, 154, 163] (Fig. 1). Fig. 1 Innexins, pannexins, and connexins form different types of gap junctional and “hemi-channels.” Invertebrates express many different innexins (purple; 25 isoforms in C. elegans) that may form either gap junction channels for cell-to-cell ... Keywords: Gap junctions, Connexons, Pannexons, Innexons, Membrane channels, Ca2+, ATP, Glutamate Three junctional protein families form membrane channels permeable to large molecules Early electrophysiological and electron microscopy studies converged in the realization that gap junctions, the membrane domains that concentrate intramembrane particles at sites of close membrane apposition, were the physical substrate of cell-to-cell communication in both invertebrate [59] and vertebrate tissues [139] (Fig. 1). The finding that similar drugs (the long-chain alcohols heptanol and octanol) and conditions (intracellular acidification) inhibited intercellular communication in both invertebrate and vertebrate systems [58, 85] was taken as further support that all gap junctions had a similar structure and function. Still, the different size of intramembrane particles, their different partition into the P- and the E-fracture faces of the cell membrane, and the different width of the gap space delineated by the two interacting membranes, suggested that the proteins making vertebrate gap junctions were different from those making the invertebrate structures [96]. These differences have functional consequences, as most elegantly demonstrated by co-culturing cell lines from different animal species. In these experiments, heterotypic coupling was shown between insect cells, as well as between different types of vertebrate cells, whereas virtually no coupling was observed between cells of phylogenetically distant species [48]. There is no longer any question that vertebrate gap junction channels are made by various combinations of different connexin proteins [66, 69–71]. The 20–21 isoforms of this family in rodents and man differ in size, but share a similar membrane topography. Thus, all connexins feature four transmembrane domains connected by two extracellular loops, each comprising three highly conserved Cys residues, a cytoplasmic loop, and both N and C termini in the cytosol (Fig. 2). The difference in size of the different connexin isoforms is essentially due to a different length of the cytoplasmic loop and/or the C terminus [39, 64, 65, 69–71, 109]. Current nomenclature (Table 1) designates connexin proteins as nCxZ, where n is the species (e.g., h, m, r for human, mouse, rat, respectively), and Z is the predicted molecular weight, in kDa. The genes encoding connexin proteins are named according to subgroups, in the order of discovery (e.g., Gjb1 encodes Cx32, Gjb2 encodes Cx26, Gja1 encodes Cx43, etc). Fig. 2 Membrane topography of the proteins forming gap junctions and “hemi-channels.” All invertebrate innexins, vertebrate ortholog pannexins, and nonhomologous vertebrate connexins feature four transmembrane domains connected by two extracellular ... Table 1 The family of human connexins Attempts to identify the proteins making invertebrate gap junctions initially revealed the proteins Ogre, Passover, Uncoordinated, and Shaking B in Drosophila and Caenorhabditis (hence the original OPUS acronym to name these proteins) with no primary sequence homology to connexins [128, 129, 195]. Since that time, more than 25 other junctional proteins revealing significant similarities have been identified in C. elegans, and it is clear that many other forms are expressed in other invertebrate species [75, 128, 129, 195] (Table 2). Collectively, these proteins have been named innexins to stress their invertebrate distribution and their role, analogous to that of connexins, in the formation of gap junctions (Fig. 1). Strikingly, innexins also share with connexins a similar structure and membrane topography (Fig. 2), even though the two sets of proteins have no homology in their primary amino acid sequence [75, 128, 129, 195]. Notably, connexins display three conserved Cys within each of the two extracellular loops, whereas innexins only carry two such residues. Thus, it is curious in retrospect that hydra development was reported to be blocked by an antibody prepared against Cx32 [53], that other antibodies have detected connexin-like proteins in anemone [112] and other marine invertebrates [5], and that junctional proteins isolated from hepatopancreas of crayfish and lobster were reported to have a sequence similar to that of rat liver connexins [51]. Table 2 A present view of the growing family of invertebrate innexins Sequencing of mammalian genomes has revealed a third family comprising only three genes that code for proteins with a primary sequence showing about 20% similarity to that of innexins [121, 154, 195]. On this basis, these proteins were thought to represent vertebrate homologs of the innexins, and were termed pannexins (Table 3) to encompass both invertebrate and vertebrate members [121, 154]. Like connexins and innexins, all three pannexins display N- and C-terminal domains within the cytoplasm, large extracellular and cytoplasmic loop domains, and four membrane spanning segments (Fig. 2). Like innexins but in contrast to connexins, pannexins contain two Cys residues in each extracellular loop [121, 154]. Furthermore, and in marked contrast with both innexins and connexins, pannexins display consensus sequences for glycosylation [13, 14, 125] (Fig. 2). The distribution of Pnx1 (Pnx1), the most studied form, is widespread and, in most types of cells and tissues, largely overlaps with that of connexins [17, 8]. While at least some phenotypes resulting from loss of specific connexin species are not compensated by pannexin changes [136, 146], suggesting a different function of the two protein families, in other cases, the effects of Pnx1 transfection mimicked that of Cx43, implicating a comparable/overlapping role of these two proteins [93]. Table 3 The family of human pannexins Connexins and innexins form cell-to-cell channels at gap junctional regions of the cell membrane Expression and deletion studies in a variety of systems have established that connexin hexamers, termed connexons, concentrate at gap junction domains of the cell membrane, where the intercellular space is reduced to a gap 2–3 nm wide. At these sites, the connexons of one cell align with, and strongly bind to the connexons of an adjacent cell, establishing a continuous intercellular hydrophilic pathway (Fig. 1) for the cell-to-cell exchange of multiple types of cytosolic molecules [64, 65, 69–71, 155]. The functional importance of this electrical and metabolic coupling is shown by a variety of striking and tissue-specific phenotypes that can be experimentally induced after overexpression or knock-out of individual connexin isoforms, as well as after the knock-in replacement of one isoform by another [92, 192]. It is further stressed by the identification of a number of diseases that are undoubtedly linked to connexin mutations [43, 57, 90, 94, 127, 143]. A variety of other diseases are thought to be due to altered amounts and/or function of these gap junction proteins [21, 108, 153]. Similarly, innexins oligomerize to form innexons that cluster at gap junctions of invertebrate cells (Fig. 1). Functional expression studies in paired Xenopus oocytes demonstrated that several, even though not all innexins also formed intercellular channels [8, 95, 166], and that at least some innexin mutations give rise to phenotypes expected for lack of gap junction-mediated intercellular communication [10, 25, 27, 37, 179]. In fact, it was on the basis of such dysfunctional phenotypes that the OPUS gene family was first identified. In contrast, and in spite of an initial report [18], pannexons appear unable to form sizable amounts of cell-to-cell channels under most conditions [76, 146, 147, 163] (Fig. 1). This lack of formation of functional gap junctions is likely due to the glycosylation of the extracellular loops of pannexins [13, 14, 125], which, as mentioned above, is not observed for either connexins or innexins. Still, forced expression of pannexins in paired Xenopus oocytes increased the conductance of the junctional cell membrane to current carrying ions in a way that can only be accounted for by the formation of pannexin cell-to-cell channels [13].

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia by Pseudomonas aeruginosa

ABSTRACT The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.

Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells

To assess whether and how specifically contact influences the functioning of differentiated cells, we have studied the secretion of adult pancreatic B-cells as a function of aggregation to either homologous B-cells or other heterologous endocrine islet cell types, all present in a mixed cell suspension. Using an immunological plaque assay for insulin, we have quantitated the proportion of single and aggregated B-cells inducing the formation of a hemolytic plaque (a reflection of the size of the secreting cell population) and the area of these plaques (a reflection of the hormonal output of individual cells or aggregates) after a 30-min stimulation by 16.7 mM glucose. By taking into account the number of B-cells within the aggregates, we have calculated from these data the insulin output on a per B-cell basis. We show here that the homologous contact between companion B-cells promotes the recruitment of secreting B-cells and increases their individual secretion of insulin twofold over that of single B-cells. By contrast, heterologous B- to non-B-cell contact was not effective in enhancing the recruitment of secreting B-cells and in promoting their individual secretion. These findings show that a highly differentiated cell function, such as insulin secretion, is controlled specifically by homologous cell to cell contacts.