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The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Human second trimester Amniotic Fluid Stem Cells (hAFSCs) harbour the potential to differentiate into cells of each of the three germ layers and to form Embryoid Body (EB)-like aggregates, without inducing teratoma formation and with no ethical concerns. However, in spite of the number of reports on hAFSCs-EBs and their characterization, a thorough evaluation in light and electron microscopy of morphological and morphometric features of hAFSCs-EBs development in vitro has not been reported yet. Apart from a superficial layer of epithelial-like flat cells, displaying rare microvilli on the free surface, hAFSCs-EBs enclose inner material, abundant in vesicles and secretory granules, showing early characteristics of connective extracellular matrix dispersed among different types of inner cells. The observation of a number of microvesicles mainly represented by microparticles and, to a lower extent, by exosomes indicates the presence of a complex cellular communication system within this structure. According to morphological analysis, after 7 days of in vitro culture hAFSCs-EB appears as a well-organized corpuscle, sufficiently young to be a carrier of stemness and at the same time, when appropriately stimulated, able to differentiate. In fact, 7-day hAFSCs-EB represents itself an initial cellular transformation towards a specialized structure both in recording and in providing different stimuli from the surrounding environment, organizing structures and cells towards a differentiation fate.

The present study was aimed at identifying a new scaffold/stem cell combination useful to treat large bone defects. Human amniotic fluid stem cells (AFSCs) were expanded in vitro, labeled with a fluorescent cell-permeable dye (PKH26) and transplanted in vivo in a femoral injured rat model. The femoral defect was left untreated (control rats) or filled with hydroxyapatite (HA; natural nanocrystalline carbonated hydroxyapatite-Orthoss®) scaffold alone or loaded with PKH26-labeled AFSCs. All animals were killed 3 weeks after implantation. Both gross anatomy and histological observations revealed a major bone regenerative response in rat specimens treated with HA scaffold, alone or supplemented with AFSCs. Samples injected with HA plus AFSCs displayed the presence of abundant fibrotic tissue, the formation of periosteal woven bone, and an increased presence of blood vessels in the bone marrow, with still fluorescent AFSCs in close proximity. These observations provide evidence that natural HA plus AFSCs represents a promising alternative therapeutic strategy to autologous bone grafting procedures.

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1‐R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC‐derived hepatocyte‐like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.

Multidrug resistance (MDR) protein 1, which is also known as permeability glycoprotein (Pgp), and tissue factor (TF) are recurrently overexpressed on the surface of cancer cells, likely in response to stimuli such as chemotherapy. Microparticles (MPs) released from cancer cells into the bloodstream express tumour markers on their surface that may be useful as predictive biomarkers for evaluating disease progression. The present study measured the level of TF/factor VII (FVII)-dependent coagulation of MPs isolated from the plasma of cancer patients with various tumours, who were undergoing chemotherapy. Furthermore, Pgp expression on the surface of MPs was evaluated by immunohistochemistry. A total of 50 cancer patients, as well as 10 healthy volunteers, were enrolled in the present study. MP-associated TF/FVII-dependent coagulation pathways were evaluated as the effect of an anti-FVII antibody on the time to thrombin generation, as compared with controls treated with saline. The significantly lengthened times of coagulation [obtained in 20/50 samples (36.5 ± 16%) after treatment with anti-FVIIa when compared with controls] suggest the presence of TF activity is associated with circulating MPs. Furthermore, the 20 MP/TF-positive samples were associated with Pgp overexpression on their surface. Conversely, in the remaining samples (n=30), treatment with the anti-FVIIa antibody did not significantly lengthen the time to clotting (

IgA nephropathy (IgAN) exhibits a variable course ranging from a benign condition to progressive renal failure with a high proportion of patients undergoing end-stage renal disease in the long term. It is unclear how to predict the risk of this progression. Mesangial C4d deposition has been found to be associated with a poor prognosis. Our aim was to search histological lesions with possible prognostic value in IgAN biopsies performed at the time of the diagnosis. Clinical and laboratory records of 44 patients undergoing renal biopsy were reviewed. IgAN was diagnosed in 32 patients and minimal change disease (MCD) in 12. C4d deposition, glomerular endothelial cell area, glomerular and interstitial macrophages were evaluated in all biopsies. Clinical and laboratory data were available in all IgAN patients at the time of diagnosis and in 21 patients at the end of follow-up (mean follow-up 48.09 ± 19.69 months). All IgAN and MCD biopsies were C4d negative. The number of glomerular macrophages and the area of glomerular endothelial cells in IgAN were greater than in MCD (P

Morphological features of granulosa cells can reflect their functional status. The present study was aimed at comparing possible differences in the fine structure of human granulosa cells exposed to gonadotrophin-releasing hormone (GnRH) agonist or antagonist treatment during ovarian stimulation. Cells were obtained from follicular aspirates of 21 women treated with recombinant follicle-stimulating hormone (rFSH) plus either a GnRH agonist or a GnRH antagonist. Conventional light microscopy procedures and computerized image analysis systems were used to identify different cell type morphological patterns and to quantify different cells distribution. Two morphologically distinct granulosa cell populations, defined as large/pale and small/dark cells, were identified and a different distribution in the two groups of women under investigation was found: a significantly higher percentage in large/pale cells was detected in the agonist-treated women (P0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P0.05). Ultrastructural observations showed the presence in both cell populations of typical hallmarks of steroidogenic cells, highlighting signs of functional activity in the large/pale cell population. Further investigations are needed to define the possible clinical significance of these morphological findings.

Background: Hypoxia and aging determine on mammalian cells a stress response which implies modified production of oxidants, reactive oxygen species or reactive nitrogen species at the mitochondrial level, interfering with cell-signaling proteins and inducing mitochondrial damage, apoptosis occurrence and functional consequences. Objective: Here we report the effects of hypoxia on the in vivo morphological and biochemical response of young and aged Wistar rat hearts. Methods: Left ventricles were excised from each experimental point and processed. Investigations of vascular endothelial growth factor (VEGF) expression and apoptotic events, mitochondrial damage, were performed by light and electron microscopy, respectively; endothelial, inducible and neuronal NOS, PKCα, pPKCα, caspase-3 expression and Apaf-1/cytochrome c complex formation were assessed by Western blotting and co-immunoprecipitation analyses, respectively. Results: Besides morphological modifications, which confirm mitochondrial suffering upon hypoxia exposure in both young and aged hearts, the role played by PKCα in controlling nitric oxide synthase (NOS) protein level was investigated. Downstream PKCα activation, a dramatic iNOS expression increase, concomitant to enhanced apoptotic cell percentage and Apaf-1/cytochrome c co-immunoprecipitation, is evident in the hypoxic young, suggesting iNOS-mediated activation of the mitochondrial apoptotic pathway. Conclusions: Moreover, overexpression of iNOS and VEGF in the hypoxic young rat hearts suggests that an increased VEGF level may allow coordinated development of the lymphatic and blood vasculature, necessary for fluid homeostasis and to counteract oxidative stress. Thus the inhibition of such growth factor proposes new therapeutic possibilities for diseases associated to vascular function and for solid tumors which show pathological angiogenesis and lymphoangiogenesis.

Shear stress is determined by three physical components described in a famous triad: blood flow, blood viscosity and vessel geometry. Through the direct action on endothelium, shear stress is able to radically interfere with endothelial properties and the physiology of the vascular wall. Endothelial cells (ECs) have also to sustain biochemical stresses represented by chemokines, growth factors, cytokines, complement, hormones, nitric oxide (NO), oxygen and reactive oxygen species (ROS). Many growth factors, cytokines, chemokines, hormones, and chemical substances, like NO, act and regulate endothelium functions and homeostasis. Among these cytokines Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) has been assigned a regulatory role in ECs physiology and physiopathology. Thus, the aim of this review is to provide a general overview of the endothelial response pathways after different types of biomechanical and biochemical stress in in vitro models and to analyze the crucial role of TRAIL under pathological conditions of the cardiocirculatory system like atherosclerosis, coronary artery disease, and diabetes.

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age-related diseases. The rise in ROS levels might constitute a stress-stimulus activating specific redox-sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1alpha (HIF-1alpha)--the master regulator of oxygen homeostasis--concomitant with p66(Shc) activation and reduced IkBalpha phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF-1alpha levels, combined with a low level of IkBalpha phosphorylation, a decrease in IAP-1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF-1alpha activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia.

Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1lowmutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize granules because von Willebrand factor is barely detectable in mutant MKs, and P-selectin, although normally expressed, is found frequently associated with the demarcation membrane system (DMS) instead of within granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in GATA-1low platelets. Mutant MKs are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process to establish contact with MKs by fusing their membrane with those of the DMS. As a result, 16% (in spleen) to 34% (in marrow) of GATA-1low MKs contain 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1low MKs have morphologic features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathologic neutrophil emperipolesis may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenvironment. (Blood. 2004;104:3573-3580)

Ionizing radiation induces a series of multiple intracellular events which can lead to activation of caspases, cytoplasmic proteases involved in the occurrence of apoptosis. The response of leukemic cells to ionizing radiation is amplified when they have been pre-treated with the anticancer drug etoposide, therefore the aim of this work has been to establish the lowest etoposide concentration combined with the lowest ionizing radiation dose to obtain the best antineoplastic response. Two leukemic cell lines, HL-60 and Jurkat, employed in this study, demonstrated different sensitivities to ionizing radiation and to etoposide treatment, with Jurkat T cells requiring a higher dose (1 μM) to display cell cycle perturbation and apoptotic DNA damage similar to those seen in HL-60. We hypothesize that this kind of response could be mediated by mitochondrial release of apoptogenic factors and by SAPK/JNK metabolic pathway activation, both leading to caspase-3 cleavage. All in all these results provide insight into the sensitivity or resistance of leukemic cells to antineoplastic agents and identify molecular targets for rational therapeutic intervention strategies.

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neoΔHS or GATA-1low mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue+ mast cells and apoptotic metachromatic− mast cell precursors in connective tissues and peritoneal lavage and numerous (60–70% of all the progenitors) “unique” trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

During ageing, the occurrence of apoptosis is due to a progressive impairment of normal functions, leading to eliminate redundant, damaged or infected cells. Here we report that also in myocardial tissue, ageing, besides reduction of the number of myocytes and of specialized conduction tissue cells, reduction in Ca(++) transport across the membrane, includes the establishment of apoptosis. In particular, the occurrence of this process, which is less represented than we would have expected, is mediated by the balance between the well known Bcl-2 protein family members, Bad, Bax and Bcl-2, related to the pathway PI-3-kinase/AKT-1, which is known to deliver a survival signal. In fact, aged myocardial cells disclose a suboptimal response, which underlines the possibility that they can become more sensitive to damaging factors or diseases, more frequently occurring during ageing, probably due to an exploited molecular control of apoptosis.

Endometriosis and urinary calculosis can co-occur. Clinical studies have shown that both painful and non-painful endometriosis in women are associated with enhanced pain and referred muscle hyperalgesia from urinary calculosis, but the mechanisms underlying this phenomenon are still poorly understood. The aim of this study was to develop an animal model adequate to explore this viscero-visceral interaction in standardized conditions. Using a model of endometriosis previously developed to study reduced fertility and vaginal hyperalgesia, endometriosis (endo) or shamendometriosis (sham-endo) was induced in rats by autotransplantation of small pieces of uterus (or, for sham-endo, fat) on cascade mesenteric arteries, ovary, and abdominal wall. After the endometrial, but not the fat autografts had produced fluid-filled cysts (3 weeks), urinary calculosis was induced by implanting an artificial stone into one ureter. Pain behaviors were monitored by continuous 24-h videotape recordings before and after stone implantation. Referred muscle hyperalgesia was assessed by measuring vocalization thresholds to electrical stimulation of the oblique musculature (L1 dermatome). The data were compared with previously reported data from rats that had received only the stone. Neither endo nor sham-endo alone induced pain behaviors. Following stone implantation, in endo rats compared to sham-endo and stone-only rats, pain behaviors specifically associated with urinary calculosis were significantly increased and new pain behaviors specifically associated with uterine pathology became evident. Muscle hyperalgesia was also significantly increased. To explore the relationship between the amount of endometriosis and that of ureteral pain behavior, two separate groups of endo rats were treated with either a standard non-steroidal anti-inflammatory drugs (ketoprofen) or placebo from the 12th to the 18th day after endometriosis induction. The stone was implanted on the 21st day. Ketoprofen treatment compared to placebo significantly reduced the size of the cysts and both ureteral and uterine pain behaviors post-stone implantation. The size of the cysts showed a significant linear correlation with the poststone ureteral pain behaviors. In conclusion, endo increased pain crises and muscle hyperalgesia typically induced by a ureteral calculosis, and the ureteral calculosis revealed additional pain behaviors typically induced by uterine pathophysiology; and this enhancement was a function of the degree of endometriosis. This result closely reproduces the condition observed in humans and could be due to a phenomenon of ‘viscero-visceral’ hyperalgesia, in which increased input from the cyst implantation sites to common spinal cord segments (T10-L1) facilitates the central effect of input from the urinary tract. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

The present investigation was aimed at studying the effects of dimethylsulfoxide (DMSO) in combination with high dose (15 and 60 Gy) ionising radiation on the growth and differentiation of murine erythroleukemia cells (MEL). The incubation with DMSO was performed for 96 h starting immediately after exposure to radiation and resulted only in a slight inhibition of cell growth and in a high increase in cell death with the induction of both necrosis and apoptosis. The enhancement of radiation cytotoxicity was directly related to dose, time in culture and degree of differentiation as demonstrated by the severe and multiple aberrations observed in light and electron microscopy. Of interest was the observation in induced cells of a marked rearrangement of the plasma membrane architecture as well as that of the nuclear envelope, with a massive translocation and/or decrease in the nuclear pore complexes.

Amniotic fluid stem cells (AFSCs) harbour the potential to differentiate into cells of any of the three germ layers and to form embryoid bodies (EBs) without inducing teratoma formation (De Coppi et al., 2007; Antonucci et al., 2012). However, no previous findings have been reported regarding embryoid body in vitro development and ultrastructural organization. Thus, this was the aim of our study. Amniotic fluid samples were obtained from women undergoing amniocentesis for prenatal diagnosis at 16-19 weeks of pregnancy after written informed consent and the local ethical committee approval. Human AFSCs were cultured up to the 8th passage and analysed with RT-PCR for the expression of pluripotency markers. Some cellular pools were cultured in suspension in uncoated Petri dishes (hanging drop method) to obtain EB formation. After 5 days of culture, the appearance of EBs of different size was observed with phase contrast microscopy and monitored up to 10-15 days of culture. In parallel, EB standard embedding in paraffin for light microscopy or in epoxy resin for transmission electron microscopy was performed. RT-PCR analysis revealed the presence of classical markers of pluripotency (OCT4, NANOG, SOX2) in AFSCs at the 2th-8th passage, whereas specific markers of the three embryonic germ layers were detected in EB specimens. Morphological assays of three-dimensional aggregates demonstrated the presence of solid structures only at the beginning of the culture whereas signs of apoptotic cell death accompanied by the secretion of an amorphous substance were soon detected. These features were preliminary to the development, at later culture time intervals, of an inner hollow cavity surrounded by a crown of flat cells displaying a number of electron dense granules and highly resembling trophoblastic cells., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.

During heart development, cell hyperplasia and hypertrophy are the main mechanisms by which cardiac mass grows. Both these processes along with programmed cell death lead to complete growth and function. In addition, since the establishment of cardiac function depends on the relationship between oxygen supply and demand, we investigated some of the molecular mechanisms at the basis of rat myocardial cell response to hypoxic stress at different times of neonatal life. In particular, the role played by hypertrophic and survival factors like NF-kB and IAP-1 (Inhibiting Apoptosis Protein) and by death factors ASK-1 (Apoptosis Signal Regulating Kinase), JNK/SAPK (Jun-N-Terminal-Kinase/Stress-Activated Protein Kinase) pathways in regulating caspase-3 expression and activity has been evaluated by immunohistochemical and Western blotting analyses, respectively. Level of phosphorylation of IkBalpha and IAP-1 expression were substantial in 8-day-old hypoxic hearts, suggesting the persistence of NF-kB driven hypertrophic signal along with a rescue attempt against hypoxic stress. In contrast, ASK-1 mediated JNK/SAPK activation, regulating Bcl(2) levels, allows Bax homodimerization and caspase-3 activation in the same experimental conditions. Thus, a regulation carried out by NF-kB and JNK/SAPK pathways on caspase-3 activation at day 8 of neonatal life can be suggested as the main factor for the heart 'adaptive' response to hypoxia.

The development and growth of the rat heart implies hyperplasia, which stops at birth, and hypertrophy, allowing cardiac mass to grow in response to programmed genetic events along with to haemodynamic overload. Moreover, hypertrophy is accomplished to apoptosis which controls the final number of myocardial cells, deletes vestigial structures, and takes part in remodelling the organ. Since at the basis of all these processes, which lead to the complete development of the heart, the activation of specific signalling pathways underlies, attention has been addressed to the role played in vivo by Protein Kinase C zeta (PKC zeta) in regulating NF-kB signalling system and intrinsic mitochondrial apoptotic route at days 1, 4, 10 and 22 of rat life. In fact, a role has been assigned to PKC zeta in indirectly phosphorylating IKBa, which peaks between 10 and 22 days, through a IKK determining, in turn, NF-kB activation, concomitantly to cytochrome c/Apaf 1 co-localization in the cytoplasm and caspase-9/caspase-3 activation, which leads to the occurrence of apoptosis. Thus a key role for PKC zeta in regulating the hypertrophic and apoptotic events leading to establishment of complete function in rat neonatal heart is here suggested.

Exposure of rats to intermittent hypoxia determines different responses at tissue and cell level. Heart mainly undergoes the effects of hypoxic injury and its response is determined both by the relationship between oxygen supply and demand and by its functional state. Since molecular mechanisms mediate cells sensing and response to low O(2) concentration, here we explore the role played by Protein Kinase C alpha (PKC alpha) in the signal transduction mechanisms leading to the occurrence of morphological responses in rat neonatal, young and old heart subjected to intermittent hypoxia. Along with a key role for hypoxia inducible factor and vascular endothelial growth factor in the occurrence of continuous state of dynamic adaptation of vasculature, PKC alpha presumably phosphorylates IkBalpha in rat normoxic and hypoxic neonatal hearts, supporting the hypothesis of a rescue strategy carried out against hypoxia, together with an hypertrophic response. In hypoxic young heart PKC alpha activation, paralleled by sustained Bax homodimerization and caspase-3 activation, along with reduced p-IKBalpha and Inhibiting Apoptosis Protein (IAP) expression, suggests that the young early and deeply undergoes the effects of lowered oxygen tension. In addition, since no modifications concerning PKC alpha driven signalling system are evidenced in both the experimental conditions, we suggest an oxygen impaired sensing during ageing.

From birth to aging the heart undergoes functional changes reflecting biochemical and ultrastructural modifications which imply apoptosis. This is a physiological process resulting from genetic programs closely associated with development and aging. During development apoptosis eliminates redundant cells leading to heart remodeling, while during aging it eliminates damaged or exhausted cells. In the present paper we analyze some molecular mechanisms involved with heart morphological modifications, especially in the neonatal heart which displays different features in the subendocardial and myocardial area. The high number of subendocardial apoptotic cells and the inverted ratio of Bcl-2/Bax molecule expression in the two heart compartments led us to hypothesize a different metabolism in the myocardium as compared with subendocardium. Moreover, we propose that PKC zeta may mediate this different response by activating Nf-kB pathway and by maintaining the balance between hypertrophic growth and apoptosis involved with remodeling of neonatal heart. Further, we underline that in the aged heart, where this pathway is not activated, such balance is not maintained.

The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in Inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (INOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of INOS. Expression of INOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). INOS expression was evaluated through immunohistochemical techniques (Ab anti-INOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of INOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for INOS in the same experimental conditions. In addition, the INOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 ± 0.01 vs 0.02 ± 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating INOS.

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase-3 activation, we paid attention to the influence exerted by Protein kinase C δ on transcription factor NF-κB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC δ/NF-κB pathway activation, probably mediated by novel IKKs and a role for PI-3-kinase in activating PKC δ at Thr 505 by PDK-1 phosphorylation is suggested. In addition, since caspase-3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF-κB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF-κB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings. J. Cell. Biochem. 89: 956–963, 2003. © 2003 Wiley-Liss, Inc.

Murine erythroleukemia cells (Friend) respond to ionizing radiation with the activation and nuclear translocation of p85alpha subunit of phosphatidylinositol-3-kinase (PI-3-kinase) which mediates the downstream activation and nuclear translocation of atypical Protein kinase C zeta (PKC zeta). This event occurs mainly upon high dose of ionizing radiation (15 Gy) and is concomitant to an increase in BrdU incorporation, which probably accounts for a predominant repair DNA synthesis. Following treatment with wortmannin, a relatively specific inhibitor of PI-3-kinase, both an increased number of apoptotic cells and the inhibition of protein kinase C zeta translocation were detected. Altogether the evidence suggests a potential role of the PI-3-kinase/PKC zeta pathway in protecting Friend cells from ionizing radiation-induced apoptosis offering PKC zeta for consideration as possible target of pharmacological treatments.

Friend erythroleukemia cells require high doses (15 Gy) of ionizing radiation to display a reduced rate of proliferation and an increased number of dead cells. Since ionizing radiation can activate several signaling pathways at the plasma membrane which can lead to the nuclear translocation of a number of proteins, we looked at the intranuclear signaling system activated by Protein Kinases C, being this family of enzymes involved in the regulation of cell growth and death. Our results show an early and dose-dependent increased activity of ζ and e isoforms, although PKC ζ is the only isoform significantly active and translocated into the nuclear compartment upon low (1.5 Gy) and high (15 Gy) radiation doses. These observations are concomitant and consistent with an increase in the anti-apoptotic protein Bcl-2 level upon both radiation doses. Our results point at the involvement of the PKC pathway in the survival response to ionizing radiation of this peculiar cell line, offering PKC ζ for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent. © 2002 Wiley-Liss, Inc.

The response of Jurkat T cells to ionizing radiation (IR) includes cell cycle arrest and DNA damage, which lead to the occurrence of apoptosis. Here, we try to elucidate some of the early intracellular signals which control the induction of such a process upon IR exposure, addressing to examine the specific role of several PKC isoforms (delta, epsilon, zeta) and their subcellular distribution. Attention has been focused on the connections between nuclear PKC delta activation and the expression of cell death regulators (Bcl-2 family proteins Bad, Bax and Bcl-2) and cell death effector caspase-3 (CPP32) which lead to the cleavage of cytoskeletal and nuclear proteins and induction of apoptosis. Altogether these results let us to conclude that PKC delta, potentiating the pro-apoptotic effect of caspase 3, plays a key role in the cellular response to IR and thus can be considered a molecular target for therapy.

The biological actions of tumor necrosis factor alpha (TNF-alpha) are mediated by two cell surface receptors, TNFR-1 and TNFR-2. These receptors do not display protein tyrosine kinase activity. Nevertheless, an early TNF-induced activation of specific tyrosine kinases has been reported as an important cue to the cellular response to this cytokine. Here we present evidence that TNF-alpha induces the activation of the cytoplasmic Janus tyrosine kinases Jak1 and Tyk2 in both human healthy peripheral and lymphoma B cells. This event was accompanied by the recruitment of a specific set of latent cytosolic transcription factors, Stat3 and Stat5b. Furthermore, Jak1 coprecipitated with TNFR-1 after TNF-alpha treatment. These data suggest that at least in human B cells this cytokine can exert its biological effects through the Jak-Stat signaling pathway and that such signals are initiated through an interaction between TNFR-1 and Jak 1.

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1low mice) to acute (phenylhydrazine [PHZ]–induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1low mice are profoundly thrombocytopenic (platelet counts [× 109/L] 82.0 ± 28.0 vs 840 ± 170.0 of their control littermates, P

Here we provide evidence for a role of phosphatidylinositol-3-kinase (PI-3-kinase) and for its product phosphatidylinositol-3,4, 5-triphosphate (PI3,4,5P3) in the occurrence of the metabolic differentiation state induced by DMSO in murine Friend erythroleukemia cells. Of note, the activation of PI-3-kinase correlated with the modulation of the activation of another enzyme, the atypical protein kinase C zeta (aPKC zeta). In particular, the expression of PI-3-kinase was substantially unaffected by DMSO treatment while its phosphorylation and the production of PI3,4,5P3 was strongly increased within 24 h of DMSO. Such a result was paralleled by an evident phosphorylation of a PKC zeta. Treatment of the cells with the two unrelated PI-3-kinase inhibitors wortmannin and LY 294002 impaired the recovery of the number of differentiated cells, therefore indicating that PI-3-kinase might be involved in the induction of erythroid differentiation, possibly engaging a protein kinase C zeta as downstream effector.

The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.

The incorporation of 5-bromo-2′-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process.

Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.

GATA-1 is a transcription factor involved in the intrinsic control of erythroid, megakaryocytic and mast cell differentiation. Experimentally-induced deletion of the megakaryocytic (Mk)-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. The relationship, if any, between defective megakaryocytopoiesis and development of the disease is still poorly understood. Electron microscopy studies, coupled with immuno-electron microscopy, have allowed us to determine that the GATA-1low mutation blocks Mk maturation between stage I and II, resulting in accumulation of defective Mk in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize α-granules, since von Willebrand factor is barely detectable in mutant Mk while P-selectin, although expressed at normal level, is found mainly associated with the Demarcation Membrane System (DMS) rather than with granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in the megathrombocytes in the blood from GATA-1low mice. Both in marrow and spleen, mutant Mk are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process of establishing contact with the Mk by fusing their membrane with those of the DMS. As a result, 16 (in spleen) - 34 (in marrow) percent of GATA-1low Mk contain 1-3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1low Mk have morphological features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathological neutrophil emperipolesis represents one of the mechanisms leading to myelofibrosis by releasing Mk cytokines (TGF-β) and neutrophil proteases (myeloperoxidase) in the microenvironment which result, respectively, in marrow fibrosis and abnormal stem/progenitor cell trafficking.

Background The nasal mucosa plays a key role in conditioning the inhaled air and in regulating the immune response. These functions led many authors to recommend mucosal sparing techniques for the surgical management of inferior turbinate hypertrophy. However, the histological modifications of chronic diseases retain the inflammatory activity and prevent the nasal physiology restoration. It has been proved that the basal cells of the nasal mucosa are able to proliferate and to repair after cold-knife incision. The aim of this study was to demonstrate that the healing process after removal of the inferior turbinate mucosa with cold techniques results in a complete structural restoration. Methods A prospective study was performed in 18 patients who underwent Microdebrider inferior turbinoplasty (cold technique). Subjective and objective improvement of nasal patency was evaluated with visual analogue scale, rhinomanometry, videoendoscopy and mucociliary transport test. Pre- and post-operative biopsy specimens were taken from 7 patients to evaluate the healing process. Two samples were taken from two healthy patients as control. The specimens were processed for transmission electron microscopy analysis. Results Videoendoscopy showed reduction of lower turbinate after surgery. Nasal patency augmented and no adverse consequences were observed. After 4 months the nasal mucosa showed normal appearance, with restoration of the pseudostratified ciliated pattern, intercellular connections and normal cellular morphology. Fibrosis and submucosal edema disappeared. At longer time after operation (4 years) clinical improvement was confirmed. Conclusions The total removal of the nasal mucosa with cold techniques results in a complete restoration of the normal structure and permanent resolution of the chronic inflammation typical of hypertrophic rhinopathy.