Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

A phenotypic switch in the dispersal strategy of breast cancer cells selected for metastatic colonization.

An important question in cancer evolution concerns which traits make a cell likely to successfully metastasize. Cell motility phenotypes, mediated by cell shape change, are strong candidates. We experimentally evolved breast cancer cells in vitro for metastatic capability, using selective regimes designed to simulate stages of metastasis, then quantified their motility behaviours using computer vision. All evolved lines showed changes to motility phenotypes, and we have identified a previously unknown density-dependent motility phenotype only seen in cells selected for colonization of decellularized lung tissue. These cells increase their rate of morphological change with an increase in migration speed when local cell density is high. However, when the local cell density is low, we find the opposite relationship: the rate of morphological change decreases with an increase in migration speed. Neither the ancestral population, nor cells selected for their ability to escape or invade extracellular matrix-like environments, displays this dynamic behavioural switch. Our results suggest that cells capable of distant-site colonization may be characterized by dynamic morphological phenotypes and the capacity to respond to the local social environment.

Talin is a substrate for SUMOylation in migrating cancer cells.

Focal adhesions (FAs) play an important role in cancer cell migration and metastasis by linking the actin cytoskeleton to the extracellular matrix, allowing the cell to generate traction. SUMOylation is a post-translational modification of proteins on lysine residues that can affect protein localisation, turnover and protein-protein interactions. In this study, we demonstrate that talin, a key component of FAs, can be post-translationally modified by SUMOylation in MDA-MB-231 breast cancer cells and U2OS osteosarcoma cells. Furthermore we demonstrate that SUMOylation regulates the dynamic activities of FAs including their number, size and turnover rate. Inhibiting SUMOylation significantly reduced the speed of cell migration. The identification of talin as a SUMO target provides insight into the mechanisms regulating focal adhesion formation and turnover and potentially identifies a novel mechanism underlying cell migration.

Compressed collagen and decellularized tissue - novel components in a pipeline approach for the study of cancer metastasis.

BACKGROUND: Metastasis is a complex process which is difficult to study and model. Experimental ingenuity is therefore essential when seeking to elucidate the biological mechanisms involved. Typically, in vitro models of metastasis have been overly simplistic, lacking the characteristic elements of the tumour microenvironment, whereas in vivo models are expensive, requiring specialist resources. Here we propose a pipeline approach for the study of cell migration and colonization, two critical steps in the metastatic cascade. METHODS: We used a range of extracellular matrix derived contexts to facilitate a progressive approach to the observation and quantification of cell behaviour in 2D, 3D and at border zones between dimensions. At the simplest level, cells were set onto collagen-coated plastic or encapsulated within a collagen matrix. To enhance this, a collagen compression technique provided a stiffened, denser substrate which could be used as a 2D surface or to encapsulate cells. Decellularized tissue from the chorioallantoic membrane of the developing chicken embryo was used to provide a more structured, biologically relevant extracellular matrix-based context in which cell behaviour could then be compared with its in vivo counterpart. RESULTS: Cell behaviour could be observed and quantified within each context using standard laboratory techniques of microscopy and immunostaining, affording the opportunity for comparison and contrast of behaviour across the whole range of contexts. In particular, the temporal constraints of the in vivo CAM were removed when cells were cultured on the decellularized CAM, allowing for much longer-term cell colonization and cell-cell interaction. CONCLUSIONS: Together the assays within this pipeline provide the opportunity for the study of cell behaviour in a replicable way across multiple environments. The assays can be set up and analysed using easily available resources and standard laboratory equipment. We believe this offers the potential for the detailed study of cell migration and colonization of tissue, essential steps in the metastatic cascade. Also, we propose that the pipeline could be used in the wider arena of cell culture in general with the increasingly more complex contexts allowing cell behaviours and interactions to be explored in a stepwise fashion in an integrated way.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro.

Alkyl esters of p-hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20 ± 2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10(-8) M 17ß-oestradiol, 1-5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben. Long-term exposure (20 ± 2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and ß-catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis.

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

Age-related changes in speed and mechanism of adult skeletal muscle stem cell migration.

Skeletal muscle undergoes a progressive age-related loss in mass and function. Preservation of muscle mass depends in part on satellite cells, the resident stem cells of skeletal muscle. Reduced satellite cell function may contribute to the age-associated decrease in muscle mass. Here, we focused on characterizing the effect of age on satellite cell migration. We report that aged satellite cells migrate at less than half the speed of young cells. In addition, aged cells show abnormal membrane extension and retraction characteristics required for amoeboid-based cell migration. Aged satellite cells displayed low levels of integrin expression. By deploying a mathematical model approach to investigate mechanism of migration, we have found that young satellite cells move in a random "memoryless" manner, whereas old cells demonstrate superdiffusive tendencies. Most importantly, we show that nitric oxide, a key regulator of cell migration, reversed the loss in migration speed and reinstated the unbiased mechanism of movement in aged satellite cells. Finally, we found that although hepatocyte growth factor increased the rate of aged satellite cell movement, it did not restore the memoryless migration characteristics displayed in young cells. Our study shows that satellite cell migration, a key component of skeletal muscle regeneration, is compromised during aging. However, we propose clinically approved drugs could be used to overcome these detrimental changes.

Nitric Oxide Synthase Potentiates the Resistance of Cancer Cell Lines to Anticancer Chemotherapeutics.

BACKGROUND: Despite the advancement in the fields of medical science and molecular biology, cancer is still the leading cause of death worldwide. Chemotherapy is a choice for treatment; however, the acquisition of chemoresistance is a major impediment for cancer management. Many mechanisms have been postulated regarding the acquisition of chemo-resistance in breast cancer and the impact on cellular signalling and the induction of apoptosis in tumour cells. The mechanism of the apoptotic mutation ofp53 and bcl-2 proteins is commonly associated with increased resistance to apoptosis and, therein, to chemotherapy. OBJECTIVES: The current study was aimed to investigate A172 and MDA-MB-231 cancer cells'sensitivity against chemotherapeutic drugs, including cisplatin, doxorubicin, and paclitaxel with different doses. Moreover, it estimates resistance of cancer cells by evaluating Nitric Oxide Synthase (NOS) expression and evaluate its correlation with the expression profile proteins of the apoptosis regulating Bcl-2 family. METHODS: Dose-dependent sensitivity to cisplatin, doxorubicin or paclitaxel was evaluated on spheroid cultured A172 and MDA-MB-231 cells lines, was measured by time-lapse microscopy over a 72h period. Expressions of two Nitric Oxide (NO) synthases isoforms (iNOS, eNOS), anti-apoptotic (Bcl-2, phospho-Bcl-2, Mcl-1, and Bcl-xL) and proapoptotic (BID, Bim, Bok, Bad, Puma, and Bax) were evaluated by Western blot. The effect of NO modulation on antiand pro-apoptotic molecule expression was also studied using Western blot. RESULTS: A172 cells show more resistance to chemotherapy drugs than MDA-MB-231 cancer cells, therefore, they need higher doses for apoptosis. Resistance of gliomas might be returned to higher significant expression of endothelial eNOS expression. It was clear that there is not a significant effect of NO modulation on the expression of pro- andantiapoptotic proteins on both cell lines. CONCLUSION: The present work provides a putative mechanism for the acquisition of drug resistance in breast cancer and glioma, which might be significant for clinical outcomes.

mTOR modulates resistance to gemcitabine in lung cancer in an MTORC2 dependent mechanism.

BACKGROUND: Lung cancer has a poor prognosis partly due to a lack of response to treatments such as the chemotherapy drug gemcitabine. Combinations of chemotherapy drugs with signal transduction inhibitors may be more effective treatments. In this study we have investigated the impact of targeting the mTOR signalling pathway on the efficacy of gemcitabine in different cancer cell lines. METHODS: Time-lapse microscopy, immuno-staining, and western blot techniques were used to evaluate the efficacy of applied treatments either in measuring phosphorylation levels of mTOR down-stream targets or in tracking down the fate of targeted cells. Reactive oxygen species and relative levels of protein phosphorylation were also quantified. For comparison between treated groups t-test and analysis of variance test were applied. RESULTS: Our data showed that mTORC1 has no role in sensitising A549 lung cancer cells to gemcitabine. However, targeting mTORC1/2 with the pharmacological inhibitor torin1 or by over-expressing Deptor, the negative regulator of mTOR signalling, sensitised these cells to gemcitabine. Silencing mTORC2, but not mTORC1, induced apoptosis and significantly improved the apoptosis-inducing effects of gemcitabine. Results also suggest that Rictor is required to maintain cell survival through modulating p38α, ERK1/2, RSK1/2/3 and the transcription factor STAT3. Multiple cell line comparisons revealed that PANC-1 pancreatic cancer cells were also sensitive to mTOR inhibition, but MCF7 breast cancer, MCF10A breast epithelial and H727 lung cancer cell lines were more resistant to the treatment. CONCLUSIONS: Inhibition of mTORC2 may have benefits in the treatment of gemcitabine resistant cancers, and the genetic background of the cell line may determine its response to mTOR inhibition.

Roles of Endocytic Processes and Early Endosomes on Focal Adhesion Dynamics in MDA-MB-231 Cells.

BACKGROUND: Focal adhesion (FA) play a critical role in many biological processes which include cell survival and cell migration. They serve as cellular anchor, allowing cells to stay attached to the extracellular matrix (ECM), and can also regulate cellular transduction. Previously, it has been suggested that vesicles such as endosomes could interact directly with FA or be implicated in their turnover. In this study, we investigated whether there is a relationship between FA and the early endocytic machinery in MDA-MB-231 cells. METHODS: In this study, cell culture, transfection, time laps confocal microscopies, immunocytochemistry, western blotting, Cell fractionation and immunoprecipitation techniques were performed. RESULTS: Cells acutely treated with Dynasore, an inhibitor of dynamin, or with Pitstop 2, an inhibitor of clathryn-dependent endocytosis showed a reduction in the expression of early endosome biomarkers such as Rab5 and EEA1. Additionally, cells treated with these endocytic inhibitors exhibited an increase number and size of FA, as well as an increase FA turnover duration. This data was consistent with the reduction of the speed of cell migration. We demonstrated that Rab5- and EEA1-positive early endosomes were found to be colocalized with internalized FA. CONCLUSION: The present study suggests that there is a link between FA and early endosome markers, which indicates that the early endosomes may be involved in FA dynamics.

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line.

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.

Cancer cell lines show high heritability for motility but not generation time.

Tumour evolution depends on heritable differences between cells in traits affecting cell survival or replication. It is well established that cancer cells are genetically and phenotypically heterogeneous; however, the extent to which this phenotypic variation is heritable is far less well explored. Here, we estimate the broad-sense heritability (H 2) of two cell traits related to cancer hallmarks--cell motility and generation time--within populations of four cancer cell lines in vitro and find that motility is strongly heritable. This heritability is stable across multiple cell generations, with heritability values at the high end of those measured for a range of traits in natural populations of animals or plants. These findings confirm a central assumption of cancer evolution, provide a first quantification of the evolvability of key traits in cancer cells and indicate that there is ample raw material for experimental evolution in cancer cell lines. Generation time, a trait directly affecting cell fitness, shows substantially lower values of heritability than cell speed, consistent with its having been under directional selection removing heritable variation.

Mathematical modelling of cell migration.

The complexity of biological systems creates challenges for fully understanding their behaviour. This is particularly true for cell migration which requires the co-ordinated activity of hundreds of individual components within cells. Mathematical modelling can help understand these complex systems by breaking the system into discrete steps which can then be interrogated in silico. In this review, we highlight scenarios in cell migration where mathematical modelling can be applied and discuss what types of modelling are most suited. Almost any aspect of cell migration is amenable to mathematical modelling from the modelling of intracellular processes such as chemokine receptor signalling and actin filament branching to larger scale processes such as the movement of individual cells or populations of cells through their environment. Two common ways of approaching this modelling are the use of models based on differential equations or agent-based modelling. The application of both these approaches to cell migration are discussed with specific examples along with common software tools to facilitate the process for non-mathematicians. We also highlight the challenges of modelling cell migration and the need for rigorous experimental work to effectively parameterise a model.

Isorhapontigenin, a resveratrol analogue selectively inhibits ADP-stimulated platelet activation.

Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85 µM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100 µM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50 > 100 µM) but inhibited collagen induced platelet aggregation at 50 µM and 100 µM. Isorhapontigenin also inhibited integrin αIIbß3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100 µM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85 µM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor.

Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation.

Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbß3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases.

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver.

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Trophoblast apoptosis is inhibited by hepatocyte growth factor through the Akt and beta-catenin mediated up-regulation of inducible nitric oxide synthase.

Excessive apoptosis of trophoblast cells is thought to be a contributing factor in complications of pregnancy such as pre-eclampsia. Hepatocyte growth factor (HGF) inhibits apoptosis in trophoblasts and we have investigated the signalling pathways through which this anti-apoptotic effect is mediated. Treatment of cells with HGF led to rapid phosphorylation of Akt while an Akt inhibitor blocked the protective effect of HGF. Glycogen synthase kinase-3beta (GSK-3beta) was found to be one of the downstream targets of Akt. HGF treatment inactivated GSK-3beta which in turn led to the activation of the transcription factor beta-catenin. Pharmacological inhibition of GSK-3beta, independently of HGF treatment, strongly increased both beta-catenin activity and cell survival, suggesting that beta-catenin alone has a pronounced anti-apoptotic effect. We also found that both HGF treatment and pharmacological activation of beta-catenin leads to increased expression of inducible nitric oxide synthase (iNOS). We suggest that the Akt mediated activation of beta-catenin leads to inhibition of trophoblast apoptosis following increased expression of iNOS.

Uterine spiral artery remodeling involves endothelial apoptosis induced by extravillous trophoblasts through Fas/FasL interactions.

OBJECTIVE: Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. METHODS AND RESULTS: Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. CONCLUSIONS: Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.

Propeptide-mediated inhibition of myostatin increases muscle mass through inhibiting proteolytic pathways in aged mice.

Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-ß family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss.