Insight into the cellular effects of nitrated phospholipids: Evidence for pleiotropic mechanisms of action.

Nitrated phospholipids have been recently identified in biological systems and showed to display anti-oxidant and anti-inflammatory potential in models of inflammation in vitro. Here, we have explored the effects of nitrated 1-palmitoyl-2-oleyl-phosphatidyl choline (NO2-POPC) in cellular models. We have observed that NO2-POPC, but not POPC, induces cellular changes consisting in cytoskeletal rearrangement and cell shrinking, and ultimately, loss of cell adhesion or impaired cell attachment. NO2-POPC releases NO in vitro and induces accumulation of NO in cells. Nevertheless, the effects of NO2-POPC are not superimposable with those of NO donors, which points to distinctive mechanisms of action. Notably, they show a stronger parallelism, although not complete overlap, with the effects of nitrated fatty acids. Interestingly, redistribution of vimentin by NO2-POPC is attenuated in a C328S mutant, thus indicating that this residue may be a target for direct or indirect modification in NO2-POPC-treated cells. Additionally, NO2-POPC interacts with several typical lipoxidation targets in vitro, including vimentin and PPARγ constructs, likely through cysteine residues. Therefore, nitrated phospholipids emerge as potential novel electrophilic lipid mediators with selective actions.

Afadin and RhoA control pancreatic endocrine mass via lumen morphogenesis.

Proper lumen morphogenesis during pancreas development is critical to endocrine and exocrine cell fate. Recent studies showed that a central network of lumens (termed core), but not the surrounding terminal branches (termed periphery), produces most islet endocrine cells. To date, it remains unclear how pancreatic lumens form and remodel and which aspects of lumen morphogenesis influence cell fate. Importantly, models testing the function of the central lumen network as an endocrine niche are lacking. Here, we identify mechanisms underlying lumen formation and remodeling and show that central lumen network morphogenesis impacts pancreatic endocrine mass. We show that loss of the scaffolding protein Afadin disrupts de novo lumenogenesis and lumen continuity in the tip epithelium. Codepletion of the actomyosin regulator RhoA and Afadin results in defects in the central lumens and arrests lumen remodeling. This arrest leads to prolonged perdurance of the central lumen network over developmental time and expansion of the endocrine progenitor population and, eventually, endocrine mass. Our study uncovers essential roles of Afadin and RhoA in pancreatic central lumen morphogenesis, which subsequently determines endocrine cell mass.

Topographic distribution of serotonin-immunoreactive urethral endocrine cells and their relationship with calcitonin gene-related peptide-immunoreactive nerves in male rats.

We investigated the topographic distribution and morphology of serotonin (5-HT)-immunoreactive endocrine cells in the urethra of male rats, and focused on their relationship with peptidergic nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP). Urethral endocrine cells immunoreactive for 5-HT were densely distributed in the epithelial layers of the prostatic part, but were sparsely distributed in the membranous and spongy parts of urethra. Distribution of urethral endocrine cells with 5-HT immunoreactivity in the prostatic part was restricted from the internal urethral orifice to the region of seminal colliculus. 5-HT-immunoreactive endocrine cells were also observed in the ductal epithelial layers of coagulating glands, prostatic glands, and seminal vesicles. 5-HT-immunoreactive endocrine cells were triangular or flask in shape and possessed an apical projection extending toward the urethral lumen, and basal or lateral protrusions intruding between other epithelial cells were also detected in some cells. Double immunolabeling for 5-HT and CGRP revealed that CGRP-immunoreactive nerve fibers attached to urethral endocrine cells with 5-HT immunoreactivity in the prostatic part. These results suggest that urethral endocrine cells may release 5-HT in response to luminal stimuli, and that these cells and CGRP-immunoreactive nerves may regulate each other by an axon reflex mechanism.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Immunohistochemical identification of the endocrine cells in the pancreatic islets of the camel, horse and cattle

This study considers the distribution of various endocrine cells in islets of Langerhans in the pancreas of several species of domestic animal, including the dromedary camel (Camelus dromedarius) using immunohistochemistry, and relates our observations with reference to the well-documented general histology of the mammalian pancreas. The pancreatic islets were observed as compact areas of pale cells surrounded by darker presumably exocrine tissue. The most distinct delineation of the islets from the surrounding acini was in the horse and the least was in cattle. Insulin-immunoreactive cells (β-cells) were most abundant followed by glucagon- (α-), somatostatin- (∆-), and pancreatic polypeptide-immunoreactive (F- or PP-) cells in decreasing order, in all species except cattle where PP-cells were second to β-cells in their distribution. The most prominent special pattern was observed in the distribution of α- and β- cells in the pancreatic islet of the horse where α-cells were located in the center of the islet surrounded by β-cells. In the camel, β-cells were distributed throughout the islet in the center and the periphery. Alpha cells were mostly observed as clumps in the periphery area. Clumps of small number of ∆-cells and a few PP cells were found throughout the islet. In cattle, β-cells were distributed throughout the islets. Other cells occupied a more peripheral location. The physical differences in distribution of endocrine cells might result in differences in the need and interaction of hormones to each other in different species

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Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas.

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8-20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8-15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14-20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/exocrine granules.

Immunoelectron microscopic observation of chicken glucagon-like peptide (GLP)-1-containing cells in tissues derived from thin section, paraffin block and conventional method.

The purpose of the present study was to investigate the possibility of immunoelectron microscopic observation of endocrine cells in paraffin-embedded tissues. The procedure, which involves reprocessing from sliced tissues and immunohistochemical staining by colloidal-gold immunolabeling of paraffin sections from paraffin blocks, was able to reveal the fine characteristics of secretory granules containing glucagon-like peptide-1. Morphometric analyses of the secretory granules showed no significant differences between the reprocessing procedure and a conventional post-embedding procedure, which was performed as a control. The reprocessing procedure has some advantages besides providing information on secretory granules containing the amino acid peptide. For example, the same cell can be observed under both a light microscope and the electron microscope. In addition, the high-electron densities of silver-enhanced gold particles are easily recognized, and the boundary between the profile of the granules and the immunogold labeling is clearly shown at the electron microscopic level. Furthermore, the procedure, which is inexpensive and does not require special devices, can effectively use precious samples that are already paraffin-embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues. To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method for sliced paraffin sections of gut endocrine cells.

Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826).

This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.

Destruction of tissue, cells and organelles in type 1 diabetic rats presented at macromolecular resolution.

Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution. The resource includes six datasets, totalling 25.000 micrographs, annotated for cellular and ultrastructural changes during autoimmune diabetes. Granulocytes are attracted to the endocrine tissue, followed by extravasation of a pleiotrophy of leukocytes. Subcellullar changes in beta cells include endoplasmic reticulum stress, insulin degranulation and glycogen accumulation. Rare findings include erythrocyte extravasation and nuclear actin-like fibers. While we focus on a rat model of autoimmune diabetes, our approach is general applicable.

[Morphogenesis and reaction to hypoxia of atrial myoendocrine cells in chick embryos (Gallus gallus)].

The ultrastructural and stereomorphometrical study of the right atrium of chick embryos at the 14th day of incubation has shown the cardiomyocytes to divide by mitosis and to be at different stages of differentiation. In the cytoplasm of some muscle cells we detected secretory granules that by their sizes and morphology can be classified into the forming, mature, and dissolved forms. By the 18th day of incubation most cardiomyocytes are already differentiated, and the number of secretory granules in them rises. Under conditions of hypoxia, after 3 days, in myoendocrine cells there are noted signs of accelerated release of the peptides synthesized earlier and accumulated in granules, while after one week - of enhancement of their synthesis. It can be concluded that in chick embryos, beginning from at least the 14th incubation day, the system of natriuretic heart peptides takes part in regulation of hemodynamics and of water-salt balance and responds to hypoxia.

Analysis of the morphology and distribution of argentaffin, argyrophil and insulin-immunoreactive endocrine cells in the small intestine of the adult opossum Didelphis aurita (Wied-Neuwied, 1826).

The aim of this study was to identify and quantify the argyrophil, argentaffin and insulin-immunoreactive cells (IIC) in the small intestine of the opossum Didelphis aurita. Seven adult male specimens of opossums were investigated. The animals were captured, and their blood insulin levels were determined. After euthanasia, fragments of the small intestine were processed for light microscopy and transmission electron microscopy, and submitted to histochemistry and immunohistochemistry for identification of argyrophil and argentaffin endocrine cells, and IIC. Argyrophil and argentaffin cells were identified in the intestinal villi and Liberkühn crypts, whereas IIC were present exclusively in the crypts. Ultrastructure of the IIC revealed cytoplasmic granules of different sizes and electron densities. The numbers of IIC per mm(2) in the duodenum and jejunum were higher than in the ileum (p<0.05). The animals had low levels of blood insulin (2.8 ± 0.78 µIU/ml). There was no correlation between insulin levels and the number of IIC in the small intestine. The IIC presented secretory granules, elongated and variable morphology. It is believed that insulin secretion by the IIC may influence the proliferation of cells in the Liberkühn crypts, and local glucose homeostasis, primarily in animals with low serum insulin levels, such as the opossum.

[Ultrastructural changes and regeneration of the endocrine apparatus of the human gastric mucosal glandular epithelium in patients with chronic erosive gastritis].

The aim of this investigation was to study the structure and regeneration of the endocrine apparatus of the human gastric mucosal glandular epithelium. Using electron microscopy, the mucosal biopsy specimens obtained from 14 patients with chronic erosive Helicobacter pylori-associated gastritis, were studied. The most pronounced changes were seen both in the numbers and ultrastructure of G- and P-endocrinocytes. The changes were detected in the nucleus structure, endocrine granule and polysome content, and he mitochondrial structure. The regeneration of the endocrine cells took place through the differentiation of the committed precursors via the "agranular" cell stage, transformation of the exocrine cells into the endocrine ones, and as a result of the formation of the epithelial cords on the erosion surfaces that consisted of the cells in diverse differentiation stages (from the undifferentiated to specialized cells of all the endocrine and exocrine types).

Cell organization of the rat pars tuberalis. Evidence for open communication between pars tuberalis cells, cerebrospinal fluid and tanycytes.

The pars tuberalis (PT) is the only pituitary region in close contact with the medial-basal hypothalamus and bathed by cerebrospinal fluid (CSF). Although PT has long been recognized as an endocrine gland, certain aspects of its structure remain obscure. The present investigation has been designed to gain information concerning (1) the cellular organization of PT, (2) the PT/median eminence spatial relationship and (3) the exposure of various cell compartments of PT to CSF. Non-endocrine cells (S100-reactive) appear as the organizer of the PT architecture. The apical poles of these cells line large cistern-like cavities and the processes of these cells establish a close spatial relationship with PT-specific secretory cells, portal capillaries and tanycytes. The cisterns are also endowed with clusters of ciliated cells and with a highly electron-dense and PAS-reactive content. The unique spatial organization of endocrine and non-endocrine cells of the PT supports a functional relationship between both cell populations. PT endocrine cells display a hallmark of PT-specific cells, namely, the paranuclear spot, which is a complex structure involving the Golgi apparatus, a large pool of immature secretory granules and a centriole from which originates a single 9+0 cilium projecting to the intercellular channels. Horseradish peroxidase (HRP) injected into the CSF readily reaches the intercellular channels of PT and the inner channel of the single cilium and is incorporated by the endocytic machinery of the secretory cells. The PT endocrine cells, through their single 9+0 cilium, may act as sensors of the CSF. HRP also reaches the lumen of the cisterns, indicating that this PT compartment is also exposed to CSF. PT endocrine cells establish direct cell-to-cell contacts with hypothalamic beta(1) tanycytes, suggesting a second means of brain-PT communication.

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells.

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

In vivo characterization of transplanted human embryonic stem cell-derived pancreatic endocrine islet cells.

OBJECTIVES: Islet-like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin-induced diabetic immuno-incompetent mice. MATERIALS AND METHODS: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). RESULTS: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C-peptide and glucagon, and for the ductal epithelial marker cytokeratin-19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas-specific cell markers was maintained for 70 days post-transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C-peptide in response to an oral bolus of glucose. CONCLUSIONS: hESC-derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C-peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin-producing cells for transplantation into patients with type 1 diabetes.

Chromogranin A-, serotonin-, synaptophysin- and vascular endothelial growth factor-positive endocrine cells and the prognosis of colorectal cancer: an immunohistochemical and ultrastructural study.

BACKGROUND AND AIM: Endocrine differentiation in colorectal adenocarcinoma has been reported but its significance as a prognostic marker remains uncertain. The aim of the present study was to analyze the prognostic significance of endocrine differentiation in colorectal cancer. METHODS: The presence of endocrine cells (EC) was determined in 137 colorectal cancers using light and electron immunohistochemistry and the immunogold method with chromogranin A, serotonin and synaptophysin. Vascular endothelial growth factor (VEGF) expression in tumor biopsies was also analyzed applying anti-VEGF antibodies. RESULTS: EC labeled with at least one of the studied markers were detected in 47 (34.3%) primary colorectal cancers (30% chromogranin A-positive, 33% synaptophysin-positive and 18% serotonin-positive). In 23% of tumor biopsies, VEGF-positive EC were also detected. The immunostaining on serial sections showed that some chromogranin A-, synaptophysin- or serotonin-positive EC also contained VEGF immune deposits. By the immunogold method, the presence of VEGF was localized to the granules of EC. Tumors with VEGF-positive EC appeared to have significantly higher vascularization, detected as systematic microvessel density (28.89 vs 15.22 vessels/mm(2), P = 0.044, Mann-Whitney U-test) compared to those without VEGF-positive EC. Ultrastructurally, EC in the tumor tissue displayed some features different from those in the normal colon. The survival analyses revealed that patients with EC in primary tumor tissues had a worse prognosis after surgical therapy than those without endocrine cell differentiation (P < 0.05, log-rank test). CONCLUSIONS: Endocrine differentiation is not an uncommon event in primary colorectal cancer and it could be a useful marker for a worse prognosis after the surgical therapy. Tumors positive for VEGF and containing VEGF-positive EC have higher vascularization, which probably also contributes to the unfavorable prognosis of patients.