Identification of a novel agrin-dependent pathway in cell signaling and adhesion within the erythroid niche.

Establishment of cell-cell adhesion is crucial in embryonic development as well as within the stem cell niches of an adult. Adhesion between macrophages and erythroblasts is required for the formation of erythroblastic islands, specialized niches where erythroblasts proliferate and differentiate to produce red blood cells throughout life. The Eph family is the largest known family of receptor tyrosine kinases (RTKs) and controls cell adhesion, migration, invasion and morphology by modulating integrin and adhesion molecule activity and by modifying the actin cytoskeleton. Here, we identify the proteoglycan agrin as a novel regulator of Eph receptor signaling and characterize a novel mechanism controlling cell-cell adhesion and red cell development within the erythroid niche. We demonstrate that agrin induces clustering and activation of EphB1 receptors on developing erythroblasts, leading to the activation of α5ß1 integrins. In agreement, agrin knockout mice display severe anemia owing to defective adhesion to macrophages and impaired maturation of erythroid cells. These results position agrin-EphB1 as a novel key signaling couple regulating cell adhesion and erythropoiesis.

Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1.

Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases.

Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques.

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1-2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin.

Cell proliferation, apoptosis and mitochondrial damage in rat B50 neuronal cells after cisplatin treatment.

OBJECTIVES: Cisplatin (cisPt) is used as a chemotherapeutic agent for the treatment of a variety of human tumours; more recently, it has been demonstrated that tumour cell exposure to cisPt ultimately results in apoptosis, but the mechanism by which nuclear cisPt/DNA generates the cytoplasmic cascade of events involved has not been clarified. We have investigated the effects of cisPt on proliferation in the neuronal cell line B50, with particular attention being given to understand whether mitochondria are a target of cisPt and their involvement in the apoptotic process. MATERIALS AND METHODS: Rat neuronal B50 cells were used to investigate the mechanisms of cisPt-induced cytotoxicity; this line has been used as a model system for neurotoxicity in vivo. RESULTS: Changes in proliferation, induction of apoptosis, activation of caspase-3 and DNA fragmentation were observed in the cells, as well as morphological and biochemical alterations of mithocondria. Activation of caspase-9 confirmed that mitochondria are a target of cisPt. CONCLUSION: CisPt exerts cytotoxic effects in the neuronal B50 cell line via a caspase-dependent pathway with mitochondria being central relay stations.

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage.

Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group-i.e. acetate-to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells.

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10(-5) M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm2). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24-72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.

Nucleolus disassembly in mitosis and apoptosis: dynamic redistribution of phosphorylated-c-Myc, fibrillarin and Ki-67.

The nucleolus may undergo disassembly either reversibly during mitosis, or irreversibly in apoptosis, thus allowing the redistribution of the nucleolar proteins. We investigated here by immunocytochemistry the fate of three representative proteins, namely phosphorylated c-Myc, fibrillarin and Ki-67, and found that they behave independently in both processes: they relocate in distinct compartments during mitosis, whereas during apoptosis they may either be cleaved (Ki-67) or be extruded into the cytoplasm with a different kinetics and following an ordered, non chaotic program. The separation of these nucleolar proteins which occurs in early apoptotic nuclei continues also in the cytoplasm, and culminates in the final formation of apoptotic blebs containing different nucleolar proteins: this evidence confirms that the apoptotic bodies may be variable in size, content and surface reactivity, and include heterogeneous aggregates of nuclear proteins and/or nucleic acids.

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.

Administration of the antitumor drug mitoguazone protects normal thymocytes against spontaneous and etoposide-induced apoptosis.

The suggestion has been made that polyamines may be involved in the control of cell death, since exceedingly high or low levels induce apoptosis in different cell systems. For a deeper insight into the relationship between apoptosis and polyamine metabolism, we investigated in vitro the effect on rat thymocytes of mitoguazone (MGBG, which inhibits S-adenosylmethionine decarboxylase, i.e. a key enzyme in the polyamine biosynthetic pathway). Thymocytes were selected as an especially suitable model system, since they undergo spontaneous apoptosis in vivo and can be easily induced to apoptose in vitro by etoposide, used here as an apoptogenic agent. MGBG protected thymocytes from both spontaneous and drug-induced apoptosis, and this protective effect was associated with a decrease in polyamine oxidase activity and total polyamine levels.

MPTP-induced increase in c-Fos- and c-Jun-like immunoreactivity in the monkey cerebellum.

The transcription factors c-Fos and c-Jun have been described to be overexpressed following many pathological stimuli, but whether they are required for neurodegeneration or neuroprotection is still open. In the present report, we analyzed the role of c-Fos and c-Jun proteins in Purkinje cell degeneration caused by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the monkey cerebellum, and determined the neuroprotective effect of the antioxidant drug a-dihydroergocryptine (DHEC), whose prior and simultaneous administration reduced the MPTP-induced neuronal loss in the substantia nigra. Immunocytochemistry for c-Fos- and c-Jun-like proteins showed persistent increased staining in Purkinje cells of MPTP-treated monkeys. The staining was greatly reduced in animals receiving DHEC. Similar results were observed in white matter glial cells after immunoreaction for c-Fos. The results suggest that, at least as far as the cerebellum is concerned, the increase in c-Fos and c-Jun expression correlate with cell damage, rather than with preservation.

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate.

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.

Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism.

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.

Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update.

Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. The best known poly(ADP-ribosylating) enzyme, PARP-1, is a DNA nick sensor and uses betaNAD(+) to form polymers of ADP-ribose which are further bound to nuclear protein acceptors. To strictly regulate poly(ADP-ribose) turnover, its degradation is assured by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 plays two opposite roles: its stimulation leads to poly(ADP-ribose) synthesis, whereas caspases cause PARP-1 cleavage and inactivation. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. The fate and the possible role of these fragments during apoptosis will be discussed.

Morphological changes in the frog cerebellar cortex after unilateral section of the statoacustic nerve.

To investigate a possible role of the cerebellum in vestibular compensation that follows a lesion to the vestibular apparatus, the morphological changes of the cerebellar cortex of adult frogs following unilateral statoacustic nerve section was analyzed by means of electron microscopy starting from 3 days after the neurectomy for up to 6 months. On the ipsilateral side, massive abnormality was found in all layers at early postsurgical intervals. This involved both nerve fibers and cell bodies. Fibers often appeared condensed or vacuolated with poorly compacted myelin sheath. Cells had electronlucent and vacuolated cytoplasm to varying extent. Alterations became less conspicuous after 30 days and after 60 days altered nerve cells were no longer present. On the contralateral side, only a few Purkinje and granule cells were affected at early postsurgical stages. This may derive from the fact that, in the frog, some of the vestibular primary afferents reach contralateral cerebellar cortex. At 30 days, alterations had substantially progressed, and at 60 days they involved all the cortical layers. Fiber debris was present in the granular and molecular layers and numerous Purkinje cells were electrondense and shrunken. This lateness in alteration may be a consequence of the prolonged silence of the vestibular nucleus contralateral to the lesion. At 4 and 6 months the tissue architecture was normal.

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

Poly(ADP-ribose) polymerase-1 (PARP-1) plays the active role of "nick sensor" during DNA repair and apoptosis, when it synthesizes ADP-ribose from NAD(+) in the presence of DNA strand breaks. Moreover, PARP-1 becomes a target of apoptotic caspases, which originate two proteolytic fragments of 89 and 24 kDa. The precise relationship between PARP-1 activation and degradation during apoptosis is still a matter of debate. In human Hep-2 cells driven to apoptosis by actinomycin D, we have monitored PARP-1 activity by the mAb 10H, which is specific for the ADP-ribose polymers, and we have observed that poly(ADP-ribose) synthesis is a very early response to the apoptotic stimulus. The analysis of the presence and fate of the p89 proteolytic fragment revealed that PARP-1 proteolysis by caspases is concomitant with poly(ADP-ribose) synthesis and that p89 migrates from the nucleus into the cytoplasm in late apoptotic cells with advanced nuclear fragmentation.

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells.

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.

Development of the anatomical alteration of the cerebellar fissura prima.

The development of the naturally occurring malformation of the cerebellar fissura prima was monitored in rats starting from 4 days of life to the adulthood. The first sign of the malformation was evident at 10 days of life and consisted of an interruption of the pia mater and the fusion of the external granular layers on the two sides of the fissura. Later, nests of apparently mature granule cells could be seen to be encircled by cells of the external granular layer and to be connected to the granule cell layer by thin bridges of cells. Calretinin immunoreactive fibers followed the bridges of cells to reach the ectopic masses of cells. Towards the end of histogenesis and in adult animals, brush cells and Golgi cells were present in the ectopic masses of granule cells. The latter appeared to contribute to the formation of normal glomeruli, as in the orthotopic granule cell layer. In addition, bundles of parallel fibers crossed the boundary between the molecular layers on the two side of the fissure, thus suggesting that parallel fibers can contact Purkinje cells of the opposite folium.

Distribution of calretinin-like immunoreactivity in the brain of Rana esculenta.

The distribution of calretinin-like immunoreactivity has been analyzed in the brain of Rana esculenta. Several neurons of nuclei belonging to sensory pathways, subhabenular area and left habenula were immunopositive. Immunoreactivity was present in fibers of motor and sensory pathways, thalamus, tegmentum and isthmus. The immunolabeling pattern partially overlapped that previously described in the rat. However, in comparison with the rat, fewer cells and fibers were immunoreactive and there were less positive brain nuclei. especially in the pallium, septum and striatum, that were totally negative. Taking into consideration that these regions are rather simple in the frog, the presence of calretinin seems to be consistent with the degree of complexity of brain areas and segregation of different nuclei.