Zinc regulates reactive oxygen species generation in platelets.

Vascular complications resulting from atherosclerosis development are a major cause of death. Reactive oxygen species (ROS) are produced by platelets during activation, and have been demonstrated to positively regulate platelet activatory responses. Zn2+ is also an important hemostatic cofactor in platelets, acting both as a platelet agonist and second messenger. Whilst the effect of Zn2+-dependent signaling mechanisms on ROS production in nucleated cells has been demonstrated, comparable roles in platelets have yet to be investigated. In this study we investigated the relationship between fluctuations in cytosolic Zn2 [Zn2+]i and platelet ROS production. Agonist-evoked ROS production, GSH levels and GPx activity are abrogated in platelets treated with the Zn2+-chelator, TPEN. Conversely, increasing platelet [Zn2+]i using Zn2+ ionophores potentiated ROS generation and decreased GSH levels and GPx activity. Zn2+-dependent ROS production was sensitive to pretreatment with DPI or mitoTEMPO, NADPH oxidase and mitochondria inhibitors respectively. Increasing [Zn2+]i resulted in increases of Erk1/2 and JNK phosphorylation. Our data are consistent with a functional association between [Zn2+]i and ROS production in platelets that could influence thrombus formation in a clinical context.

The cannabinoid WIN 55,212-2 prevents neuroendocrine differentiation of LNCaP prostate cancer cells.

BACKGROUND: Neuroendocrine (NE) differentiation represents a common feature of prostate cancer and is associated with accelerated disease progression and poor clinical outcome. Nowadays, there is no treatment for this aggressive form of prostate cancer. The aim of this study was to determine the influence of the cannabinoid WIN 55,212-2 (WIN, a non-selective cannabinoid CB1 and CB2 receptor agonist) on the NE differentiation of prostate cancer cells. METHODS: NE differentiation of prostate cancer LNCaP cells was induced by serum deprivation or by incubation with interleukin-6, for 6 days. Levels of NE markers and signaling proteins were determined by western blotting. Levels of cannabinoid receptors were determined by quantitative PCR. The involvement of signaling cascades was investigated by pharmacological inhibition and small interfering RNA. RESULTS: The differentiated LNCaP cells exhibited neurite outgrowth, and increased the expression of the typical NE markers neuron-specific enolase and ßIII tubulin (ßIII Tub). Treatment with 3 µM WIN inhibited NK differentiation of LNCaP cells. The cannabinoid WIN downregulated the PI3K/Akt/mTOR signaling pathway, resulting in NE differentiation inhibition. In addition, an activation of AMP-activated protein kinase (AMPK) was observed in WIN-treated cells, which correlated with a decrease in the NE markers expression. Our results also show that during NE differentiation the expression of cannabinoid receptors CB1 and CB2 dramatically decreases. CONCLUSIONS: Taken together, we demonstrate that PI3K/Akt/AMPK might be an important axis modulating NE differentiation of prostate cancer that is blocked by the cannabinoid WIN, pointing to a therapeutic potential of cannabinoids against NE prostate cancer.

Reactive oxygen species: physiological roles in the regulation of vascular cells.

Reactive oxygen species (ROS) are now appreciated to play several important roles in a number of biological processes and regulate cell physiology and function. ROS are a heterogeneous chemical class that includes radicals, such as superoxide ion (O2(•-)), hydroxyl radical (OH(•)) and nitric oxide (NO(•)), and non-radicals, such as hydrogen peroxide (H2O2), singlet oxygen ((1)O2), hypochlorous acid (HOCl), and peroxynitrite (NO3 (-)). In the cardiovascular system, besides playing a critical role in the development and progression of vasculopathies and other important pathologies such as congestive heart failure, atherosclerosis and thrombosis, ROS also regulate physiological processes. Evidence from a wealth of cardiovascular research studies suggests that ROS act as second messengers and play an essential role in vascular homeostasis by influencing discrete signal transduction pathways in various systems and cell types. They are produced throughout the vascular system, regulate differentiation and contractility of vascular smooth muscle cells, control vascular endothelial cell proliferation and migration, mediate platelet activation and haemostasis, and significantly contribute to the immune response. Our understanding of ROS chemistry and cell biology has evolved to the point of realizing that different ROS have distinct and important roles in cardiovascular physiology. This review will outline sources, functions and molecular mechanisms of action of different ROS in the cardiovascular system and will describe their emerging role in healthy cardiovascular physiology and homeostasis.

Involvement of PPARγ in the antitumoral action of cannabinoids on hepatocellular carcinoma.

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.

The novel NOX inhibitor 2-acetylphenothiazine impairs collagen-dependent thrombus formation in a GPVI-dependent manner.

BACKGROUND AND PURPOSE: NADPH oxidases (NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2-acetylphenothiazine (2-APT) on human platelet functional responses and intracellular signaling pathways. EXPERIMENTAL APPROACH: The generation of superoxide ions was assessed by single cell imaging on adhering platelets using dihydroethidium (DHE), while other reactive oxygen species (ROS) were detected with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)-DCFDA). Whole blood thrombus formation, washed platelet aggregation, integrin αIIbß3 inside-out signalling, Syk phosphorylation and PKC activation were analysed to understand the functional consequences of NOX inhibition by 2-APT in platelets. KEY RESULTS: Superoxide ion generation stimulated by platelet adhesion on collagen and fibrinogen was significantly inhibited by 2-APT in concentration-dependent manner (IC(50) = 306 nM and 227 nM, respectively), whereas cumulative ROS accumulation was not affected by this pharmacological agent. 2-APT also abolished collagen-dependent whole blood thrombus formation and washed platelet aggregation in response to collagen but not thrombin. The activation of integrin αIIbß3 and PKC in response to the GPVI-specific agonist collagen-related peptide (CRP) was significantly reduced, whereas the same responses to thrombin were not significantly affected by 2-APT. Finally, Syk activation in response to collagen but not thrombin was inhibited by 2-APT. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that 2-APT attenuates GPVI-specific signalling and is a novel inhibitor of collagen-induced platelet responses. Therefore, NOXs could represent a novel target for the discovery of anti-thrombotic drugs.

Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids--a novel family of potential anticancer agents--on the growth of HCC. We found that Δ(9)-tetrahydrocannabinol (Δ(9)-THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB(2)) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB(2) receptor. We also found that Δ(9)-THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine-threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase ß was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that Δ(9)-THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC.

Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: involvement of CB2.

BACKGROUND: We have previously shown that cannabinoids induce growth inhibition and apoptosis in prostate cancer PC-3 cells, which express high levels of cannabinoid receptor types 1 and 2 (CB(1) and CB(2)). In this study, we investigated the role of CB(2) receptor in the anti-proliferative action of cannabinoids and the signal transduction triggered by receptor ligation. METHODS: The human prostate cancer cell lines, namely PC-3, DU-145 and LNCaP, were used for this study. Cell proliferation was measured using MTT proliferation assay, [(3)H]-thymidine incorporation assay and cell-cycle study by flow cytometry. Ceramide quantification was performed using the DAG kinase method. The CB(2) receptor was silenced with specific small interfering RNA, and was blocked pharmacologically with SR 144528. In vivo studies were conducted by the induction of prostate xenograft tumours in nude mice. RESULTS: We found that the anandamide analogue, R(+)-Methanandamide (MET), as well as JWH-015, a synthetic CB(2) agonist, exerted anti-proliferative effects in PC-3 cells. R(+)-Methanandamide- and JWH-015-induced cell death was rescued by treatment with the CB(2) receptor antagonist, SR 144528. Downregulation of CB(2) expression reversed the effects of JWH-015, confirming the involvement of CB(2) in the pro-apoptotic effect of cannabinoids. Further analysing the mechanism of JWH-015-induced cell growth inhibition, we found that JWH-015 triggered a de novo synthesis of ceramide, which was involved in cannabinoid-induced cell death, insofar as blocking ceramide synthesis with Fumonisin B1 reduced cell death. Signalling pathways activated by JWH-015 included JNK (c-Jun N-terminal kinase) activation and Akt inhibition. In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice. CONCLUSIONS: This study defines the involvement of CB(2)-mediated signalling in the in vivo and in vitro growth inhibition of prostate cancer cells and suggests that CB(2) agonists have potential therapeutic interest and deserve to be explored in the management of prostate cancer.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola) / Desenvolvimento embrionário de Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola)

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

O desenvolvimento embrionário dos tricladidos é conhecido, fundamentalmente, por estudos realizados em espécies de Dendrocoelum, Planaria, Polycelis e, mais recentemente, Schmidtea. O presente estudo descreve o desenvolvimento embrionário de Girardia tigrina (Girard, 1850), a partir de análises realizadas em cortes histológicos seriados e semifinos de glicol-metacrilato, ao microscópio óptico. Noventa e quatro casulos foram coletados no período entre a postura e a eclosão, em intervalos de até vinte e quatro horas. A seqüência das modificações morfológicas no embrião permitiu a identificação de nove estágios embrionários. Na postura dos casulos, envoltos por uma cápsula rígida, observam-se numerosos embriões dispersos entre grande quantidade de células vitelinas. No estágio I (aproximadamente até 6 horas após a postura), as células vitelinas e as embrionárias mostram uma distribuição aleatória. O estágio II (entre 12 e 24 horas após a postura) caracteriza-se pela formação de agrupamentos de blastômeros, os quais posteriormente formam uma enteroblástula. Aproximadamente dois dias após a postura (estágio III), ocorre a formação da epiderme e do sistema digestivo embrionário, sendo que este último degenera no estágio seguinte. O estágio V (até o quarto dia após a postura) caracteriza-se pela formação da epiderme definitiva. Entre o quarto e o sexto dia posteriores à postura, começa a organogênese dos órgãos internos definitivos (estágio VI). Aproximadamente catorze dias após a postura (estágio IX), completa-se a formação do sistema nervoso. Neste estágio, o embrião já apresenta características similares aos espécimes juvenis. A eclosão de Girardia tigrina ocorre entre doze e vinte e dois dias após a postura dos casulos.

Assessment of the potential of progenitor stem cells extracted from human peripheral blood for seeding a novel vascular graft material.

OBJECTIVE: A novel nanocomposite has recently been developed based on polyhedral oligomeric silsesquioxane attached by direct reaction onto a urethane segment, as a potential vascular graft material; its trade name is UCL-Nano. The UCL-Nano has been demonstrated to have similar viscoelastic properties to the walls of a natural artery, to be resistant to degradation and to be able to sustain endothelial cell seeding. Human peripheral blood contains both circulating endothelial cells and endothelial progenitor cells, which may be suitable for conduit seeding. The aim of this study was to develop a system with the potential to deliver an endothelial cell-seeded bypass graft in a realistic time frame. MATERIALS AND METHODS: Endothelial progenitor cells and circulating endothelial cells were isolated from human peripheral blood and were characterized by fluorescent-activated cell sorting, reverse transcriptase-polymerase chain reaction and immunohistochemistry. Isolated cells were seeded on nanocomposite and were maintained in culture for 35 days. RESULTS: The UCL-Nano was successfully seeded with cells and a confluent cell layer was achieved after 14-day culture. Cells remained viable and confluent on the nanocomposite for 35 days. CONCLUSION: In conclusion, these results suggest that this process has potential both for a realistic and achievable two-stage seeding process for vascular bypass grafts and for the potential development of a device, with the aim of achieving in situ seeding once implanted.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola).

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

Improving the sampling technique of arterialized capillary samples to obtain more accurate PaO2 measurements.

Arterialized earlobe capillary blood samples (ELCS) have been used as a measurement of blood gas status for over 20 years. There is general acceptance that there is a strong correlation and limits of agreement between arterial and arterialized blood samples with respect to pH and PaCO2. Although the correlation between the arterial and arterialized PaO2 is good, the limits of agreement poor. Our aim was to improve the accuracy of this technique in the measurement of PaO2 by simultaneously monitoring the oxygen saturation by pulse oximetry whilst taking an ELCS. We hypothesize that significant discrepancies between the SaO2 and SpO2 highlight either a poorly arterialized sample or an over aerated sample from air bubbles. We compared the SpO2 with the SaO2 of an arterial sample from 27 inpatients. We used the limits of agreement between these samples to define the degree of discordance we would accept between SaO2 and SpO2 before repeat ELCS. Subsequently, 252 consecutive patients attending our respiratory physiology unit over a six-month period had an ELCS and simultaneous SpO2. If there was a discrepancy between SaO2 and SpO2 of > 2% the ELCS was repeated. There was a good correlation and limits of agreement between the SpO2 and arterial SaO2 (r = 0.97, mean difference +/- 95% limits of agreement: 0.34 +/- 2.68). A difference of more than 2% between arterialized SaO2 and SpO2 was identified in 21 patients out of 252 (8.3%) with SaO2 higher in two and lower in 19 (r = 0.96, mean difference +/- 95% limits of agreement: 0.66 +/- 3.1). Repeat ELCS of these 21 samples reduced this discrepancy improving the concordance of the measurements (r = 0.98, mean difference +/- 95% limits of agreement: 0.47 +/- 1.0). In one case a difference of 3% remained between the saturations. We conclude that the addition of simultaneous pulse oximetry with ELCS will identify rogue measurements in about 8% of cases highlighting the need for repeat samples and thus increasing the accuracy of the measurement of PaO2 by ELCS.

[Perforated gastric ulcer in the child: a rare complication, a case report]. / Perforation d'ulcère gastrique de l'enfant: une complication rare, à propos d'un cas.

UNLABELLED: Perforated gastric ulcer is unusual in children. We report a case in a girl with an unexpected evolution. CASE REPORT: A 13-year-old girl was admitted for abdominal pain. She had no particular personal history but her father had a perforated ulcer. On admission she was not painful, her abdomen was soft on palpation. The white blood cell count was 1.7 x 10(3)/mm3. A right pneumoperitoneum was seen on an abdominal X-ray film. Because of her good general status and the normalization of the abdominal X ray film six hours later, no surgical exploration was performed. On the fourth day, a gastrointestinal endoscopy showed an anterior gastric ulcer which was perforated. Biopsies did not isolate H. pylori. The patient was given a treatment with amoxicillin-metronidazole (7 d) and oméprazole (7 weeks). An endoscopic control, one month later, showed a total healing of the gastric ulcer. CONCLUSION: Peptic ulcerations and their complications are underdiagnosed in childhood. This could lead to delay in diagnosis or inappropriate treatment specially in case of perforation.

Histological processing techniques for the study of Dugesiidae development (Platyhelminthes, Tricladida, Paludicola).

The objective of the present study was to adapt techniques for the histological processing of Dugesiidae cocoons for the study of embryo development. The cocoons were fixed with formalin, SUSA, Bouin or paraformaldehyde/glutaraldehyde and subsequently embedded in Paraplast or glycol methacrylate (Historesin). Paraplast embedding yielded reasonable results only after the cocoon was perforated or fixed for a prolonged period of time using softening techniques with acid solutions. When the SUSA or Bouin fixative and Historesin embedding techniques were used the results were good for light microscopical analysis. Fixation with paraformaldehyde/glutaraldehyde and glycol methacrylate embedding resulted in better tissue preservation, and did not require prolonged fixation or softening techniques. Thus, we suggest this technique for light microscopical analysis of embryo development in Dugesiidae.

Histological processing techniques for the study of Dugesiidae development (Platyhelminthes, Tricladida, Paludicola)

The objective of the present study was to adapt techniques for the histological processing of Dugesiidae cocoons for the study of embryo development. The cocoons were fixed with formalin, SUSA, Bouin or paraformaldehyde/glutaraldehyde and subsequently embedded in Paraplast or glycol methacrylate (Historesin). Paraplast embedding yielded reasonable results only after the cocoon was perforated or fixed for a prolonged period of time using softening techniques with acid solutions. When the SUSA or Bouin fixative and Historesin embedding techniques were used the results were good for light microscopical analysis. Fixation with paraformaldehyde/glutaraldehyde and glycol methacrylate embedding resulted in better tissue preservation, and did not require prolonged fixation or softening techniques. Thus, we suggest this technique for light microscopical analysis of embryo development in Dugesiidae

The effect of a time delay on the measurement of capillary blood gases.

Patients using domiciliary nasal ventilation, or long-term oxygen, require regular assessment which could be carried out in the home. Blood gas analysis may be regarded as an essential part of the assessment. The present study investigated the effect of a 1-h time delay on the measurement of capillary blood gases. Four samples of arterialized earlobe blood were collected from 15 outpatients. One sample was analysed immediately, the other three were stored on crushed ice and analysed at intervals of 30, 45, and 60 min post-collection. In order to examine any range effect, a wide range of PaO2 values were examined. The delay, at all levels, resulted in minor changes in the measurement of PaO2, which would be unlikely to alter clinical management. The technique might be used for the reliable assessment of patients in the home.

Comparison of peak oxygen consumption during cycle and treadmill exercise in severe chronic obstructive pulmonary disease.

BACKGROUND: In normal subjects treadmill exercise usually produces the greatest maximal oxygen consumption (VO2max). This may not be true for patients with severe chronic obstructive pulmonary disease (COPD) in whom bicycle exercise, which offers support for the shoulder girdle, may produce a higher oxygen consumption than treadmill exercise. The aim of this study was to determine which mode of exercise produced the greatest oxygen consumption in patients with severe COPD. METHODS: Eight patients with severe COPD (forced expiratory volume in one second (FEV1) more than three standardised residuals below predicted) exercised to a symptom limited maximum on a bicycle and on a treadmill on separate days. The workload on the bicycle wa increased by 10 watts each minute, and the treadmill gradient was increased by 2.5% alternate minutes whilst the speed remained constant. Measurements of oxygen consumption (VO2), ventilation (VE), heart rate, and oxygen saturation were made, and capillary blood gases were measured before and immediately after exercise. Lactate concentration was measured before and four minutes after exercise. RESULTS: There were no differences at peak exercise between the two forms of exercise for VO2 (median 11.7 and 12.2 ml/min/kg for bicycle and treadmill, respectively), for VE (median 26.6 and 25.0 l/min, respectively), and for heart rate (median 119 and 115 beats/min, respectively). The median lactate levels after bicycle exercise were higher than those after the treadmill (2.42 v 0.94 mmol/l). CONCLUSIONS: Although only a small number of patients was studied and individual variability was large, there was no clear difference between the two forms of exercise. Regular bicycle exercise was unfamiliar to this group of patients and generated the greatest lactate response. The results do not support the hypothesis that bicycle exercise will produce a better performance in patients with severe COPD, but the two modes of exercise cannot be used interchangeably.