Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34⁺ hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2–3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.     

Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve

Subcellular fractionation of bovine splenic nerves, which consist mainly of sympathetic nerve fibers, has been useful for characterizing cellular organelles en route to the terminal. In the present study we have characterized the subcellular distribution of both secretory and membrane proteins. A newly discovered chromogranin-like protein, NESP55, was found in large dense-core vesicles. The endogenous processing of NESP55 was comparable to that of chromogranins but more limited than that of secretogranin II and chromogranin B. For membrane proteins three major types of distribution were found. The amine carrier VMAT2 was confined to large dense-core vesicles. VAMP or synaptobrevin was present both in large dense-core vesicles and in lighter vesicles, whereas SNAP-25, syntaxin, and two types (N and L) of Ca2+ channels were found in a special population of lighter vesicles but were not present in large dense-core vesicles or at the most in very low concentrations. The plasma membrane norepinephrine transporter was apparently present in a separate type of vesicle, but this requires further study. These results further characterize vesicles en route to the terminal and establish for the first time that peptides involved in exocytosis (syntaxin, SNAP-25, and N- and L-type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense-core vesicles indicates that the formation of small dense-core vesicles in the terminals requires a reuse of membrane components of large dense-core vesicles.     

Characterization of surface sugars on algal cells with fluorescein isothiocyanate-conjugated lectins

Summary. We used qualitative and quantitative fluorescence microscopy of the fluorescein isothiocyanate-conjugated lectins Concanavalin A, phytohaemagglutinin-erythroagglutinin, pokeweed mitogen, and peanut agglutinin to examine sugar composition on the cell surface and cell-associated mucilage (where present) in a number of cultured and environmental algae. Lectin-binding activity was markedly different between laboratory-cultured and environmental samples of the same species. Sugar composition of the cyanobacterium Anabaena cylindrica varied with growth cycle, although no clear pattern of change was observed. Akinetes typically showed lectin-binding activity higher than that of the vegetative cells or heterocysts throughout the growth cycle. Algae with mucilage showed greater lectin binding, indicating that mucilage contained more surface sugars accessible to the lectin probe compared with the cell wall surface. A low level of galactose and N-acetyl galactosamine (detected by peanut agglutinin) was associated with the surface mucilage of most algal species. Relatively high amounts of mannose, glucose, and N-acetyl glucosamine (detected by Concanavalin A, phytohaemagglutinin, and pokeweed mitogen) were also present. Lectin binding was shown to be a highly specific and sensitive approach to the examination of cell surface chemistry of both cultured and environmental algae and to the study of biodiversity in phytoplankton. Correspondence: School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, U.K.     

The potential use of cultured cells to detect non-neuronal targets of neuropeptide action

Although many studies have described the localization and possible neuromodulatory role of neuropeptides little attempt has been made to determine whether glial cells are possible targets of neuropeptides' actions. The use of primary cell cultures derived from neonatal rat central and peripheral nervous system may provide a means of assaying for such effects and gaining a better understanding of glial cells' roles in nervous system function.     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.     

Selective labeling and monitoring pH changes of lysosomes in living cells with fluorogenic pH sensors

New BODIPY-based pH probes have been designed with excitation and emission wavelengths suitable for fluorescence microscopy and flow cytometry. These pH probes are cell-permeable, selectively label lysosomes, and can be used for noninvasive monitoring of lysosomal pH changes during physiological and pathological processes.     

Immunohistochemical localization of chromogranin A and B in endocrine cells of the alimentary tract of the adult lizard Podarcis sicula

The distribution, argyrophilia, and the possible amine/peptide co-localizations in endocrine cells immunoreactive (IR) to antisera against chromogranin A (CgA) and chromogranin B (CgB) in the alimentary tract of the lizard Podarcis sicula have been investigated using novel monoclonal antibodies. Many CgA-IR and CgB-IR cells were found in the tract, except in the distal small intestine. Almost all chromogranin-IR cells (Cgs-IR) were also argyrophilic with parallel intensity. Some CgA-IR and CgB-IR cells did not display co-localized amines or peptides. CgA or CgB or both were found co-localized, with some local differences, in almost all serotonin-IR, histamine-IR, substance P-IR and gastric peptide tyrosine tyrosine (PYY)-IR cells. Moreover, both Cgs were co-localized only in some somatostatin-IR cells, whereas neurotensin-IR, gastrin/cholecystokinin-IR, pancreatic polypeptide-IR and intestinal PYY-IR cells did not show any co-localization with Cgs. The presence of Cgs in the endocrine cells was heterogeneous with regard to the complex interrelationship with their amine/peptide content. Consequently, Cgs cannot be considered as universal markers of all endocrine cell types.     

Immunohistochemical localization of urotensin I and other neuropeptides in the caudal neurosecretory system of three species of teleosts and two species of elasmobranchs

Summary In three species of teleosts — carp Cyprinus carpio; grass carp Ctenopharyngodon idella; and crucian carp Carassius auratus — the caudal neurosecretory system displays small, medium-sized and large neurons. Urotensin I (UI)-immunoreactive and UI-nonreactive neurons were found in all three groups; in general, the number of the latter neurons exceeded that of the former. Noteworthy are: (i) UI-immunoreactive fibers in the caudal spinal cord and (ii) dense accumulations of UI-immunoreactive product around the capillaries of the urophysis. In two species of elasmobranchs — cat shark Heterodontus japonicas and swell shark Cephaloscyllium umbratile — neurosecretory neurons decreased in size in rostro-caudal direction. Most of the neurosecretory perikarya, their axons and the corresponding neurohemal areas were UI-immunoreactive, but a small number of secretory neurons was devoid of immunoreaction. Oxytocin, arginine vasopressin, substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide and gastrin-releasing peptide were not detected in the caudal neurosecretory system of the carp.     

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog,Rana esculenta

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were costored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method     

LysoTracker is a marker of differentiated alveolar type II cells

Background

LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.

Methods

Mouse lung cells were sorted on the basis of CD45^negCD31^negEpCAM^posLysoTracker^pos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.

Results

The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker^pos AT2 cells generated SP-C^pos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.

Conclusions

This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.     

Immunohistochemical Distribution of RGS7 Protein and Cellular Selectivity in Colocalizing with Gαq Proteins in the Adult Rat Brain

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein-coupled receptor signal transducer for phospholipase Cβ-mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Gαq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.     

Organelle-selective energy transfer: a fluorescent indicator of intracellular environment

A dye cassette fluoresces green (ca 520 nm) in the cytoplasm, endoplasmic reticulum (ER), and lysosomes, but red in mitochondria, that is, it illustrates ‘organelle specific energy transfer’. This phenomenon may open new horizons in intracellular imaging.     

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca^2 +-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca^2 + also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca^2 +.     

Scanning electron microscopy of bone cells in culture

Summary Embryonic and young rat bone cells have been grown in culture and examined in the scanning electron microscope (SEM). Compared with cells fixed in situ and taken directly from the animal, the cultured osteoblastic cells were smoother, flatter and more extensive and showed tighter intercellular contacts. Some matrix is formed in culture and undergoes at least partial mineralization as judged by the accumulation of Ca and P measured by energy dispersive x-ray analysis. Findings concerning the morphology of the collagen arrangement were indecisive. Some superficial cells, free of surrounding matrix, resembled osteocytes in normal in vivo bone. This may indicate that a proportion of the extracellular matrix produced by the cultured cells failed to polymerise into recognizable bone matrix, and that osteocytic morphology is not dependent upon the physical characteristics of the bone matrix.     

Existence of serotonin and neuropeptides-immunoreactive endocrine cells in the small and large intestines of the mole-rats (Spalax leucodon)

The present study was conducted to clarify the regional distribution and relative frequency of endocrine cells secreting serotonin, substance P (SP), cholecystokinin-8 (CCK-8), vasoactive intestinal polypeptide (VIP) and neurotensin in the small and large intestine of the mole-rats (Spalax leucodon), by specific immunohistochemical methods. In the small and large intestine of mole-rats (Spalax leucodon), serotonin, SP and VIP were identified with various frequencies, but CCK-8 and neurotensin were not observed. Most of the IR cells in the small and large intestine were located in the intestinal crypt and epithelium however, they were more frequency in the intestinal crypt. Serotonin-IR cells were detected throughout the whole intestinal tract, predominantly in the duodenum and colon. SP-IR cells were demonstrated throughout the whole intestinal tract except for the ileum and rectum with highest frequencies in the cecum. VIP-IR cells were found in all parts of the small intestine except for the large intestine.In conclusion, the general distribution patterns and relative frequency of intestinal endocrine cells of the mole-rats (Spalax leucodon) was similar to those of some rodent species. However, some species-dependent unique distributions and frequencies characteristics of endocrine cells were also observed in the present study.     

In vitro expression of natriuretic peptides in cardiomyocytes differentiated from monkey embryonic stem cells

Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis.     

Isolation of multicellular complexes of follicular dendritic cells and lymphocytes: Immunophenotypical characterization,electron microscopy and culture studies

Summary The morphological and phenotypical features of multicellular complexes formed by follicular dendritic cells and lymphocytes (FDC-LC) isolated from human hyperplastic tonsils and adenoids are described. FDC-LC obtained with this procedure were morphologically and immuno-phenotypically heterogeneous. In one type of FDCLC, probably obtained from germinal centers, the lymphocytes exhibited ultrastructural features of centroblasts and centrocytes. In a second type, likely derived from follicular mantles, the enclosed lymphocytes were small in size and characterized by a condensed chromatin pattern. Similar heterogeneity was observed by immuno-phenotypical analysis, which revealed a prevalence of IgD+, CD3-, MT2+ small lymphocytes in a high proportion of FDC-LC. Both types of FDC-LC contained desmoplakin immunoreactivity in a typical punctate pattern corresponding to intercellular junctions when tested with a specific antibody. These findings confirm the importance of FDC in maintaining the follicular structure and also suggest that the different zones forming lymphoid follicles (mantle zone and germinal center) are formed by lymphocytes gathered in single domains by cytoplasmic processes of FDC. These domains have strong resistance to mechanical stress, such as that used in isolation procedures. FDC-LC have also been maintained as organized multicellular clusters for short periods (more than 48 h) in agarose gel cultures.Supported in part by: Ministero Pubblica Istruzione, Italy, and A.I.R.C., Milano, Italy