Tumor Necrosis Factor Related Apoptosis Inducing Ligand (Trail) in endothelial response to biomechanical and biochemical stresses in arteries.

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.

Features of endothelial dysfunction in umbilical cord vessels of women with gestational diabetes.

BACKGROUND AND AIMS: Gestational diabetes (GDM) is associated with increased oxidative stress and overexpression of inflammatory cytokines, both of which might lead to endothelial dysfunction and vascular disease. As such, GDM could be viewed as a sort of 'short lived' metabolic syndrome. As umbilical cord vessels represent a suitable model for the study of vascular alterations brought about by GDM, the aim of the present work was to characterize the phenotype of human umbilical vein endothelial cells (HUVECs) chronically exposed to hyperglycaemia and to a pro-inflammatory environment during pregnancy so as to identify molecular modifications of cellular homoeostasis eventually impacting on endothelial dysfunction. METHODS AND RESULT: Tissue specimens and HUVECs were obtained from umbilical cords of GDMand control women. As compared to controls, GD-HUVEC exhibited enhanced monocyte adhesion and vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1(ICAM-1) expression and exposure on plasma membrane after tumour necrosis factor-alpha(TNF-α) stimulation (Western blot, flow cytometer). As compared to control cells, GD-HUVEC in basal conditions exhibited enhanced monocyte adhesion, nitric oxide synthase (NOS) expression and activity (eNOS Real-Time polymerase chain reaction, Western Blot for eNOS total protein and monomers/dimers ratio, conversion of [3H]-L-arginine in [3H]-L-citrulline), increased O(-)(2)egeneration together with increased NT levels (immunofluorescence) and reduced NO bioavailability(guanosine 3',5'-monophosphate (cGMP) production, EIA). Furthermore, immunohistochemistry revealed increased eNOS and NT immunoreactivity in GD umbilical cords. CONCLUSION: Endothelial cells exposed in vivo even transiently to hyperglycaemia, oxidative stress and inflammation exhibit durable pro-atherogenic modifications.

Dual role of the caspase enzymes in satellite cells from aged and young subjects.

Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage.

Inhibition of P-glycoprotein-mediated transport by S-adenosylmethionine and cynarin in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells.

Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to protect normal cells from chemotherapy-induced cytotoxixity.

TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway.

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.

The effect of the plasticizer diethylhexyl phthalate on transport activity and expression of P-glycoprotein in parental and doxo-resistant human sarcoma cell lines.

Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 µM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 µM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 µM doxo in the presence of the lowest concentration of DEHP (3 µM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 µM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 µM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.

Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist.

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 (P<0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P<0.05). 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.

Viagra, surgery and anesthesia: a dangerous cocktail with a risk of blindness.

Since the launch in 1998 of the anti-impotence drug sildenafil (viagra), the American food and drug administration has identified 50 cases of drug-related blindness, the so-called nonarteritic anterior ischemic optic neuropathy. This, very serious, side effect frequently leads to sudden, mostly irreversible loss of vision, and there is no proven effective treatment to cure patients or to prevent recurrence. The mechanism of ischemic optic neuropathy is not clear, but it could be related to the fact that the ophthalmic and central retinal arteries have an autoregulation of their own blood flow without any autonomic nerve supply; vasoreactivity could be lower albeit efficient, and therefore more vulnerable to systemic modifications of the circulation. But decreased visual acuity and loss of visual ability also are, although uncommon, anesthesiological and surgical complications. These data are consistent with the hypothesis that sildenafil, surgery and anesthesia, taken together, could be a potentially dangerous cocktail of risk factors for sudden irreversible loss of vision. To reduce the risk, sildenafil use should be avoided at least one week before surgical operations, since the reported cases of blindness developed 36h after drug ingestion.

Peroxiredoxin genes are not induced in myeloid leukemia cells exposed to ionizing radiation.

Peroxiredoxins (Prx) comprise an extended family of small antioxidant proteins which conserve a thioredoxin-dependent catalytic function that can contribute to cell protection from reactive oxygen species (ROS). ROS generation is one of the deleterious intracellular effects of ionizing radiation, but the role of Prx during radiation treatment has not been extensively explored. Present experiments measure effects of ionizing radiation on expression of human Prx types I (PAGA), II (NKEF-B) and IV (AOE372) in human myeloid leukemia cells (K562). Prx gene transcription was analyzed by amplifying with RT-PCR cDNAs complementary to each Prx-specific coding sequence and by identifying the derived products with Southern blotting procedure. Transcripts of GAPDH were used as the endogenous standard for semi-quantitative comparisons. No consistent increase in Prx gene expression was detected at time intervals up to 72 h after gamma radiation doses that caused cell cycle arrest and nuclear damage (maximum 20 Gy). Immunoblots also were consistent with a prolonged expression or stability of the Prx I/II proteins. Similarly, a cytotoxic concentration of the oxidant hemin, which stimulates rapid hemoglobinization of K562 cells, caused no induction of Prx gene expression. Our results indicate a high Prx stability in human radio-resistant leukemia cells.

The effect of peribulbar block with ropivacaine on bi-hemispheric cerebral oxygen saturation in aged patients.

We evaluated the effects of peribulbar block for eye surgery on bi-hemispheric regional cerebral oxygenation (rSO2)) of aged patients. In 66 adult patients, peribulbar block was performed using ropivacaine 10 mg.ml(-1) with hyaluronidase 100 IU.ml(-1). Cerebral oxygenation was monitored using continuous non-invasive, near-infrared spectroscopy. The rSO2 data on the side where the eye block was performed were evaluated as eye block side values, whereas the data recorded on the other side were taken as control values. Mean rSO2 values on the side where regional block was performed were not significantly different from control values (p > 0.05). Nevertheless, in several patients, a slight desaturation in the cerebral hemisphere on the block side was detected. Therefore, in aged patients, peribulbar block with ropivacaine does not significantly modify bi-hemispheric rSO2, but rSO2 monitoring during peribulbar block should be a field of future research in aged patients with brain injury or disease.

PI-3-kinase/NF-kappaB mediated response of Jurkat T leukemic cells to two different chemotherapeutic drugs, etoposide and TRAIL.

Jurkat T leukemic cells respond to Etoposide, antineoplastic agent which targets the DNA unwinding enzyme, Topoisomerase II, and TNF-Related-Apoptosis-Inducing-Ligand (TRAIL), 34 kDa transmembrane protein, which displays minimal or no toxicity on normal cells and tissues, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an higher number of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1 and, in parallel, IkappaB alpha on both serine and tyrosine residues. On the other hand, in Etoposide Jurkat exposed cells Ser 32-36 phosphorylation of IkappaB alpha is not sufficient to overbalance the apoptotic fate of the cells, since Bax increase, IAP decrease, and caspase-3 activation determine the persistence of the apoptotic state along with the occurrence of cell death by necrosis. Thus, the existence of a balance between apoptotic and rescue response in 20% of cells surviving to TRAIL suggests the possibility of pushing it in favor of cell death in order to improve the yield of pharmacological strategies.

Caspase-3 is dually regulated by apoptogenic factors mitochondrial release and by SAPK/JNK metabolic pathway in leukemic cells exposed to etoposide-ionizing radiation combined treatment.

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 microM) 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.

Induction of oxidative stress in murine cell lines by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent bacterial mutagen produced during chlorination of drinking water, was tested for the induction of oxidative stress in two murine cell lines: NIH 3T3 (fibroblasts) and L929 (fibrosarcoma cells). Following 1 h MX treatment at concentrations between 100 and 1000 microM, cellular stress conditions were monitored by measuring reactive oxygen species formation (ROS) and reduced glutathione levels (GSH). The kinetics of ROS formation and GSH depletion was investigated from 10 min to 1 h. MX caused detachment of cells at 1000 microM in L929 cells and at 300 microM in NIH 3T3 cells but the viability of the cells, measured by the trypan blue assay, decreased only by 20 and 7%, respectively, in 1h. MX increased ROS production in L929 cells in a dose-dependent manner, by 120% at 500 microM of MX in 1 h. The maximum ROS production was attained already in 10min. In NIH 3T3 cells, the ROS production was slightly, but not statistically significantly stimulated at 200 microM between 20 and 60 min. Concomitantly, MX decreased the intracellular content of GSH dose-dependently in both cell lines, by 48% in L929 cells at 500 microM of MX and 32% in NIH 3T3 cells at 200 microM of MX in one hour. The majority of this GSH depletion had occurred in 10 min. These findings indicate that MX induces oxidative stress in mammalian cells in vitro though the sensitivity of cells may differ for this effect.

Protein kinase C zeta nuclear translocation mediates the occurrence of radioresistance in friend erythroleukemia cells.

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 zeta and epsilon isoforms, although PKC zeta 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 zeta for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent.

[Anastomotic dehiscence in colorectal surgery]. / La deiscenza delle anastomosi in chirurgia colo-rettale.

Anastomotic leakage represents the most dangerous complication in digestive surgery and, particularly, in colorectal one. Indeed a careful evaluation and modification of all possible risk factors and a correct procedure aren't often enough to overcome a potential postoperative leakage or at least material filtration. The Authors review their series to analyse the role of all possible general and local risk factors and to verify what conditions can suggest an two or three stages operation.

[Antibiotic and immunostimulating prophylaxis in colorectal surgery]. / Profilassi antibiotica e immunostimolante in chirurgia colo-rettale.

The Authors, after considering post-operatory sepsis as cause of failures and as an important economic damage, make among them a distinction according to the arising area and to the seriousness of their manifestations. They, later, describe corrective and prophylactic measures which in colo-rectal surgery ase campulsory assumptions for sepsis prevention. After relating their experience, Authors reaffirm short-term protocol validity and point out validity of alternation of large spectrum molecules in order to avoid possible arising of bacterial resistances.

Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation.

Ionizing radiation is one of the agents inducing activation of DNA repair, cell cycle arrest, apoptosis and cell death. Here we report evidence for an enhanced activity of DNA polymerase beta, one of the repair enzymes, concomitant to the activation of the pathway phosphatidylinositol-3-kinase/AKT-1 (PI-3-kinase/AKT-1), which delivers a survival signal in Friend erythroleukemia cells exposed to 15 Gy. Significantly, the preincubation of the cellls with PI-3-kinase inhibitors wortmannin and LY 294002, disactivating this pathway, sensitizes the cells to ionizing radiation by further reducing the rate of proliferation without substantial variations of the number of dead cells. Thus, we suggest a role for these enzymes in maintaining survival programs upon exposure to ionizing radiation and in giving to these cells a chance to recover from this stress.

Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)--mediated cytotoxicity by selective up-regulation of TRAIL-R1.

Cytotoxic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in different combinations with either a low (1.5 Gy) or a high (15 Gy) single dose of ionizing radiation (IR), was investigated on erythroleukemic cells (K562, HEL, Friend, primary leukemic erythroblasts) and on primary CD34(+)-derived normal erythroblasts. Human recombinant TRAIL alone variably affected the survival/growth of erythroleukemic cells; K562 cells were the most sensitive. Moreover, all erythroleukemic cells were radio-resistant, as demonstrated by the fact that cytotoxicity was evident only after treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were used in combination, an additive effect was noticed in all erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR was observed with both low and high IR doses and required the sequential treatment of IR 3 to 6 hours before the addition of TRAIL. Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary CD34(+)-derived normal erythroblasts when used alone, but their combination did not show any additive effect. Moreover, the cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was accompanied by the selective up-regulation of the surface expression of TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp (OMe)-CH(2) (z-VAD-fmk) caspase inhibitor. On the other hand, the surface expression of TRAIL-R1 in CD34(+)-derived normal erythroblasts was unaffected by IR, which induced the up-regulation of the decoy TRAIL-R3. These data demonstrate that treatment with IR provides an approach to selectively sensitize erythroleukemic cells, but not normal erythroblasts, to TRAIL-induced apoptosis through the functional up-regulation of TRAIL-R1.

Colonic metaplasia in the long-term follow-up of the ileal neobladder.

OBJECTIVE: To look for the presence of colonic metaplasia (CM), the shifting from sialomucins (SIs) to sulfomucins (SUs), considered as a probably premalignant lesion in the stomach in the mucosa of the ileal neobladder (IN). METHODS: 19 patients with IN were subjected to endoscopic biopsy; the samples were analyzed by means of histochemistry with high iron diamine, a test indicated to identify SIs and SUs. RESULTS: CM was never observed earlier than 1 year after the operation, was absent in 9/19 patients (mean follow-up 14 months) and present in 10/19 (mean follow-up 59 months). CONCLUSION: Time-dependent phenotypic changes, already described in the stomach as being premalignant, take place after constant contact with urine in the mucosa of the IN. It is at present unclear whether they may be defíned as only metaplastic or frankly preneoplastic; anyway, a careful follow-up remains indicated in all patients with íntestinal urinary diversions.