Roles of metformin-mediated girdin expression in metastasis of epithelial ovarian cancer.

Antimetastatic effect of Metformin has been documented in epithelial ovarian cancer (EOC). Presently, we investigated the regulatory mechanism of Metformin in EOC metastasis. First, Girdin was significantly enhanced in EOC tumorous tissues and cell lines. Seconded, knockdown of Girdin significantly suppressed EOC cell viability, migration, and invasion, while upregulation of Girdin produced the opposite effects in vitro and facilitated lung metastasis in EOC cell xenograft in vivo. In addition, we confirmed that the inhibitory effect of Metformin on Girdin expression. Mechanistically, the oncogenic effects of Girdin could be reversed by LY294002 (an AKT pathway inhibitor) and Metformin. These results suggested that Metformin attenuated EOC metastasis through Girdin and targeting Girdin may be a promising therapeutic strategy for EOC in the future.

An Auxin Transport Inhibitor Targets Villin-Mediated Actin Dynamics to Regulate Polar Auxin Transport.

Auxin transport inhibitors are essential tools for understanding auxin-dependent plant development. One mode of inhibition affects actin dynamics; however, the underlying mechanisms remain unclear. In this study, we characterized the action of 2,3,5-triiodobenzoic acid (TIBA) on actin dynamics in greater mechanistic detail. By surveying mutants for candidate actin-binding proteins with reduced TIBA sensitivity, we determined that Arabidopsis (Arabidopsis thaliana) villins contribute to TIBA action. By directly interacting with the C-terminal headpiece domain of villins, TIBA causes villin to oligomerize, driving excessive bundling of actin filaments. The resulting changes in actin dynamics impair auxin transport by disrupting the trafficking of PIN-FORMED auxin efflux carriers and reducing their levels at the plasma membrane. Collectively, our study provides mechanistic insight into the link between the actin cytoskeleton, vesicle trafficking, and auxin transport.

The inhibitory effect of curcumin via fascin suppression through JAK/STAT3 pathway on metastasis and recurrence of ovary cancer cells.

BACKGROUND: Fascin is an actin-binding protein and highly expressed in ovarian cancer cells. It is associated with metastasis of cancer and may be a useful prognostic factor. Anticancer activity of curcumin is related to its effect on several signaling mechanisms. Although there have been many reports regarding the anticancer properties of curcumin, its inhibitory effects on migration and invasion of ovarian cancer cells, particularly in the context of fascin expression, have not been reported. The purpose of this study was to investigate the effect of curcumin on fascin expression in ovarian cancer cells and to propose a possible mechanism for the anticancer activity of curcumin through reduced fascin expression. METHODS: SKOV3, human epithelial ovary cancer cell line, was cultured with curcumin at various dose and duration. The fascin was quantified using cell viability test and Western blot. To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. Attachment assay, migration assay and invasion assay were analyzed to approve the change of cellular invasiveness of ovary cancer after curcumin. To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed. RESULTS: MTS assays showed that cell viability was different at various concentration of curcumin, and as concentration increased, cell viability tended to decrease. Curcumin appears to suppress fascin expression, even with a minimal concentration and short exposure time. Also, curcumin may suppress fascin expression in ovarian cancer cells through STAT3 downregulation. The attachment assay, migration assay and invasion assay of the ovarian cancer cells exhibited a statistically significant decrease. Immunofluorescence revealed a change of cell shape from a typical form of uninfluenced cells to a more polygonal appearance, with a significant reduction in filopodia formation. CONCLUSIONS: Curcumin reduces fascin expression through JAK/STAT3 pathway inhibition, which interferes with the cellular interactions essential for the metastasis and recurrence of ovarian cancer cells. Higher curcumin concentrations and longer exposure times concomitantly decreased fascin expression.

Protective effects of DPP-4 inhibitor on podocyte injury in glomerular diseases.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) is a serine protease that inhibits the degradation of glucagon-like peptide 1. DPP-4 inhibitors are used worldwide to treat type 2 diabetes mellitus and were recently shown to have pleiotropic effects such as anti-oxidant, anti-inflammatory, and anti-fibrotic actions. DPP-4 inhibitors improve albuminuria and renal injury including glomerular damage independent of its hypoglycemic effect. Although DPP-4 is mainly expressed in the kidney, the physiological function of DPP-4 remains unclear. METHODS: The localization of renal DPP-4 activity was determined in human renal biopsy specimens with glycyl-1-prolyl-4-methoxy-2-naphthylamide and the effects of a DPP-4 inhibitor were examined in human cultured podocyte. RESULTS: DPP-4 activity under normal conditions was observed in some Bowman's capsular epithelial cells and proximal tubules, but not in the glomerulus. DPP-4 activity was observed in crescent formation in anti-neutrophil myeloperoxidase cytoplasmic antigen antibody nephritis, nodular lesions in diabetic nephropathy, and some podocytes in focal segmental glomerulosclerosis. Notably, the DPP-4 inhibitor saxagliptin suppressed DPP-4 activity in podocytes and the proximal tubules. To assess the effect of DPP-4 inhibitor on podocytes, human cultured podocytes were injured by Adriamycin, which increased DPP-4 activity; this activity was dose-dependently suppressed by saxagliptin. Treatment with saxagliptin maintained the structure of synaptopodin and RhoA. Saxagliptin also improved the detachment of podocytes. CONCLUSIONS: DPP-4 activity induces degradation of synaptopodin and reduction of RhoA, resulting in destruction of the podocyte cytoskeleton. Saxagliptin may have pleiotropic effects to prevent podocyte injury.

Flightless-I mediates the repression of estrogen receptor α target gene expression by the glucocorticoid receptor in MCF-7 cells.

The human homologue of flightless-I (FLII) belong to the gelsolin protein family and contain a gelsolin-like domain at the C-terminus and a leucine-rich repeat (LRR) domain at the N-terminus. FLII regulates estrogen receptor alpha (ERα) and glucocorticoid receptor (GR)-mediated transcription by direct interaction through different domains, suggestive of its potential role in the crosstalk between the ERα and GR signaling pathway. Here, we demonstrate that FLII plays a critical role in GR-mediated repression of ERα target gene expression. In FLII-depleted cells, the reduction in 17-ß-estradiol (E2)-induced ERα occupancy following treatment with dexamethasone (Dex) at the estrogen responsive element (ERE) site of the ERα target gene was significantly inhibited. The ERE binding of GR by the cotreatment with E2 and Dex was significantly inhibited by FLII depletion, indicating that FLII is required for the recruitment of GR at the ERE sites of ERα target genes. In addition, the recruitment of ERα-induced FLII to ERE sites was significantly reduced by Dex treatment. In protein binding assays, GR inhibited the E2-induced interaction between ERα and FLII, suggesting that GR interferes with the binding of ERα and FLII at the ERα target genes, resulting in the release of ERα and FLII from EREs. Taken together, our data reveal an unknown mechanism by which the transcription coactivator FLII regulates the GR-mediated repression of ERα target gene expression in MCF-7 cells.

Neuroprotective effects of SMTP-44D in mice stroke model in relation to neurovascular unit and trophic coupling.

Stachybotrys microspora triprenyl phenol (SMTP)-44D has both anti-oxidative and anti-inflammatory activities, but its efficacy has not been proved in relation to the pathological changes of neurovascular unit (NVU) and neurovascular trophic coupling (NVTC) in ischemic stroke. Here, the present study was designed to assess the efficacies of SMTP-44D, moreover, compared with the standard neuroprotective reagent edaravone in ischemic brains. ICR mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min, SMTP-44D (10 mg/kg) or edaravone (3 mg/kg) was intravenously administrated through subclavian vein just after the reperfusion, and these mice were examined at 1, 3, and 7 d after reperfusion. Compared with the vehicle group, SMTP-44D treatment revealed obvious ameliorations in clinical scores and infarct volume, meanwhile, markedly suppressed the accumulations of 4-HNE, 8-OHdG, nitrotyrosine, RAGE, TNF-α, Iba-1, and cleaved caspase-3 after tMCAO. In addition, SMTP-44D significantly prevented the dissociation of NVU and improved the intensity of NAGO/BDNF and the number of BDNF/TrkB and BDNF/NeuN double positive cells. These effects of SMTP-44D in reducing oxidative and inflammatory stresses were similar to or stronger than those of edaravone. The present study demonstrated that SMTP-44D showed strong anti-oxidative, anti-inflammatory, and anti-apoptotic effects, moreover, the drug also significantly improved the NVU damage and NVTC in the ischemic brain.

Effects of Post-Treatment Hydrogen Gas Inhalation on Uveitis Induced by Endotoxin in Rats.

BACKGROUND Molecular hydrogen (H2) has been widely reported to have benefiicial effects in diverse animal models and human disease through reduction of oxidative stress and inflammation. The aim of this study was to investigate whether hydrogen gas could ameliorate endotoxin-induced uveitis (EIU) in rats. MATERIAL AND METHODS Male Sprague-Dawley rats were divided into a normal group, a model group, a nitrogen-oxygen (N-O) group, and a hydrogen-oxygen (H-O) group. EIU was induced in rats of the latter 3 groups by injection of lipopolysaccharide (LPS). After that, rats in the N-O group inhaled a gas mixture of 67% N2 and 33% O2, while those in the H-O group inhaled a gas mixture of 67% H2 and 33% O2. All rats were graded according to the signs of uveitis after electroretinography (ERG) examination. Protein concentration in the aqueous humor (AqH) was measured. Furthermore, hematoxylin-eosin staining and immunostaining of anti-ionized calcium-binding adapter molecule 1 (Iba1) in the iris and ciliary body (ICB) were carried out. RESULTS No statistically significant differences existed in the graded score of uveitis and the b-wave peak time in the Dark-adapted 3.0 ERG among the model, N-O, and H-O groups (P>0.05), while rats of the H-O group showed a lower concentration of AqH protein than that of the model or N-O group (P<0.05). The number of the infiltrating cells in the ICB of rats from the H-O group was not significantly different from that of the model or N-O group (P>0.05), while the activation of microglia cells in the H-O group was somewhat reduced (P<0.05). CONCLUSIONS Post-treatment hydrogen gas inhalation did not ameliorate the clinical signs, or reduce the infiltrating cells of EIU. However, it inhibited the elevation of protein in the AqH and reduced the microglia activation.

Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease.

OBJECTIVE: Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing. METHODS: Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, ß-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients. RESULTS: In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis. CONCLUSIONS: These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

Does Pantoprazole Affect the On-Treatment Platelet Reactivity in Patients With Acute STEMI Treated With ADP Receptor Blockers?-A Pilot Prospective Study.

BACKGROUND: Proton pump inhibition (PPI) administrated together with adenosine diphosphate (ADP) receptor blockers (ADPRB) significantly reduces the risk of gastrointestinal bleeding. Nevertheless, there is a heated discussion about an interaction between PPI and ADPRB that leads to high on-treatment platelet reactivity (HTPR). STUDY QUESTION: Is there a relationship between pantoprazole PPI and HTPR on ADPRB therapy in patients with acute ST-elevation myocardial infarction (STEMI). METHODS: Single center pilot study in patients with acute STEMI was performed. This study enrolled totally 87 patients (34 clopidogrel-treated and 53 new ADPRB-treated patients). Pantoprazole was administrated in 33 patients. HTPR was detected with ADP-induced light transmission aggregometry and vasodilator-stimulated phosphoprotein phosphorylation analysis. Samples were taken before coronary angiography (sample 1) and on the next day after the procedure (sample 2). RESULTS: No significant differences were found in pantoprazole-treated patients and patients without PPI neither in sample 1 (59.2 ± 29.5% vs. 54.9 ± 22.7%, P = 0.49) nor in sample 2 (43.8 ± 27.2% vs. 37.0 ± 22.9%, P = 0.30). Similarly, there were no significant differences in the platelet reactivity index of vasodilator-stimulated phosphoprotein phosphorylation in both samples (sample 1: 53.3 ± 29.8% vs. 65.0 ± 20.5%, P = 0.11; sample 2: 30.8 ± 27.1% vs. 40.6 ± 27.5%, P = 0.19). A comparison of clopidogrel and new ADP receptor blockers in patients on pantoprazole PPI did not reveal significant differences in on-treatment platelet reactivity. CONCLUSIONS: This study did not reveal interaction between pantoprazole and ADPRB in patients with acute STEMI.

Morphine decreases ticagrelor concentrations but not its antiplatelet effects: a randomized trial in healthy volunteers.

BACKGROUND: Our recent drug interaction trial with clopidogrel shows that morphine decreases the concentrations and pharmacodynamic effects of clopidogrel, which could lead to treatment failure in susceptible individuals. We hypothesized that the pharmacodynamic consequences of drug-drug interactions would be less between morphine and ticagrelor. MATERIALS AND METHODS: Twenty-four healthy subjects received a loading dose of 180 mg ticagrelor together with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, crossover trial. Pharmacokinetics were determined by liquid chromatography tandem mass spectrometry, and ticagrelor pharmacodynamic effects were measured by platelet function tests (whole blood platelet aggregation: multiplate, platelet plug formation: PFA-100, vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay). RESULTS: Concomitant i.v. injection of morphine slows drug resorption of ticagrelor and its active metabolite (P < 0·05) by 1 h and decreases plasma levels of ticagrelor and its active metabolite by 25-31% (P ≤ 0·03) and the drug exposure (area under the curve) by 22-23% (P ≤ 0·01). Importantly, however, the pharmacodynamic effects of ticagrelor on platelet aggregation in whole blood, platelet plug formation and VASP phosphorylation are not affected by morphine. CONCLUSIONS: Morphine co-administration moderately decreases ticagrelor plasma concentrations but does not inhibit its pharmacodynamic effects in healthy volunteers within 6 h after drug administration. Limitations of our trial include the investigation in healthy volunteers under standardized conditions, which does not necessarily reflect a realistic emergency scenario.

Kelch-Like Protein 2 Mediates Angiotensin II-With No Lysine 3 Signaling in the Regulation of Vascular Tonus.

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target.

Effect of BMP7 on podocyte transdifferentiation and Smad7 expression induced by hyperglycemia.

OBJECTIVE: To investigate the effect of BMP7 on the transdifferentiation and Smad7 expression of podocytes induced by high glucose in vitro and to explore its possible protective mechanisms. METHODS: Mouse podocytes were cultured and divided into normal glucose group (NG), high glucose group (HG), mannitol group, NG+BMP7 group, and HG+BMP7 group. Real-time PCR and Western blot were applied respectively to detect the mRNA and protein expression levels of synaptopodin, desmin, and Smad7. RESULTS: The cells significantly up-regulated the mRNA and protein expression of desmin and reduced the expression of both synaptopodin and Smad7 after 48 hours (vs. NG, p < 0.01). BMP7 dramatically suppressed the mRNA and protein expression of desmin and protected the expression of synaptopodin and Smad7 after incubation with high glucose for 48 hours (vs. HG, p < 0.01). CONCLUSIONS: BMP7 can inhibit the epithelial-to-mesenchymal cell transformation (EMT) of podocytes induced by high glucose; Smad7 may mediate the blunting effects of BMP7 on high glucose in podocytes.

Cyclic AMP prevents decrease of phosphorylated ezrin/radixin/moesin and chloride intracellular channel 5 expressions in injured podocytes.

BACKGROUND: Our previous in vitro studies suggested that cyclin AMP (cAMP) signaling protects against podocyte injury. However, the molecular mechanisms remain unknown. The aim of the present study was to explore the role of forskolin, an agonist for adenylate cyclase, on ezrin/radixin/moesin (ERM) phosphorylation and chloride intracellular channel 5 (CLIC5) expressions in injured podocytes. METHODS: ADR nephrosis model were induced by adriamycin (ADR) injection in BalB/C mice. Parts of ADR nephrosis mice were pretreated with forskolin. Albuminuria was estimated by urine Coomassie blue stain. Nephrin, synaptopodin, CLIC5, phosphorylated ERM and podocalyxin were measured by confocal microscopy. CLIC5 and phosphorylated ERM also were studied using western blotting. RhoA and Rac1 were estimated by G-Lisa kit. RESULTS: We found that forskolin partially alleviated albuminuria and width of foot processes. Nephrin, synaptopodin, phosphorylated-ERM (p-ERM) and CLIC5 expression were decreased in ADR mice, which were improved by forskolin pretreatment. In vitro studies, pretreatment of podocytes with pCPT-cAMP(PKA-selective cAMP analogue)prevented puromycin aminonucleoside (PAN)-induced CLIC5 downregulation. 8-pCPT-2'-O-Me-cAMP (2Me-cAMP, an Epac-selective cAMP analogue) blocked PAN-induced p-ERM downregulation. PAN inhibited RhoA activation in podocytes, which could be prevented by pCPT-cAMP pretreatment. Y-27632, a Rho inhibitor, decreased CLIC5 expression in podocytes. CONCLUSION: Activation cAMP signaling might attenuate albuminuria in ADR-induced nephrosis mice. Different downstream signaling pathway might mediate cAMP protection on CLIC5 and p-ERM expression, respectively.

Suppression of actopaxin impairs hepatocellular carcinoma metastasis through modulation of cell migration and invasion.

UNLABELLED: Early reports suggested that actopaxin, a member of the focal adhesion proteins, regulates cell migration. Here we investigated whether actopaxin is involved in hepatocellular carcinoma (HCC) progression and metastasis. We examined actopaxin expression in human HCC samples using immunohistochemistry and western blotting. The functional and molecular effect of actopaxin was studied in vitro by overexpression in a nonmetastatic HCC cell line, as well as repression in a metastatic cell line. The in vivo effect of actopaxin repression was studied in nonobese diabetic and severe combined immunodeficient mice. We found that actopaxin was frequently overexpressed in human HCC patients and its overexpression positively correlated with tumor size, stage, and metastasis. Actopaxin expression also correlated with the metastatic potential of HCC cell lines. Actopaxin overexpression induced the invasion and migration ability of nonmetastatic HCC cells, whereas down-regulation of actopaxin reverted the invasive phenotypes and metastatic potential of metastatic HCC cells through regulating the protein expression of certain focal adhesion proteins including ILK, PINCH, paxillin, and cdc42, as well as regulating the epithelial-mesenchymal transition pathway. Furthermore, there was a close association between actopaxin and CD29. HCC cells with stronger CD29 expression showed a higher actopaxin level, whereas actopaxin repression attenuated CD29 activity. Finally, actopaxin down-regulation enhanced the chemosensitivity of HCC cells towards oxaliplatin treatment by way of a collective result of suppression of survivin protein, ß-catenin, and mammalian target of rapamycin pathways and up-regulation of p53. CONCLUSION: This study provides concrete evidence of a significant role of actopaxin in HCC progression and metastasis, by way of regulation of cell invasiveness and motility, an epithelial-mesenchymal transition process, and chemosensitivity to cytotoxic drugs.

Synthetic chondramide A analogues stabilize filamentous actin and block invasion by Toxoplasma gondii.

Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the ß-tyrosine moiety blocked parasite growth on host cell monolayers with EC50 values that ranged from 0.3 to 1.3 µM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.

Comparison between initial and chronic response to clopidogrel therapy after coronary stenting for acute coronary syndrome and influence on clinical outcomes.

BACKGROUND: Studies have addressed the benefit of tailored therapy based on initial response to clopidogrel loading dose. However, the appropriate timing for platelet testing remains uncertain. METHODS: The present study was performed to compare initial clopidogrel response after 600 mg loading dose and 1-month platelet response and their relationship with ischemic and bleedings events. A total of 475 patients with non-ST-segment elevation acute coronary syndrome undergoing percutaneous coronary intervention have been included in the present study. All patients were treated with 600 mg clopidogrel followed by 150 mg daily. Clopidogrel low response was defined by high on-treatment platelet reactivity (HPR) with vasoactive stimulated phosphoprotein >50%, and "hyperresponse," as platelet reactivity index vasoactive stimulated phosphoprotein (PRI VASP) <95th percentile after 600 mg. RESULTS: After 600 mg, 210 patients were identified with HPR (44%), and 23 patients (5%), with hyperresponse (PRI VASP <8%). At 1 month on 150 mg clopidogrel daily, 184 patients (39%) had HPR (39%), 14 patients (3 %) had hyperresponse, and mean PRI VASP was significantly lower (43% ± 19% vs 46% ± 21%, P = .04). At 1 month, among the 210 patients with HPR after 600 mg, 127 (60%) remained, whereas among the 265 patients responders after 600 mg, only 57 (22%) remained with HPR (60% vs 22%, P < .0001). Initial response was significantly associated with risk of stent thrombosis and bleeding complications, whereas 1-month assessment was only linked with bleeding events. CONCLUSION: In conclusion, the present study showed that initial clopidogrel response in patients with acute coronary syndrome is not a reliable predictor of response to maintenance therapy and their values for prediction of clinical outcome are likely to be different.

The distribution of fibrillin-2 and LTBP-2, and their co-localisation with fibrillin-1 in adult bovine tail disc.

We investigated the distribution of fibrillin-2 and LTBP-2 (latent TGF-ß binding protein-2) in the intervertebral disc of the adult bovine tail. The association of fibrillin-2 and of LTBP-2 with fibrillin-1 was examined by dual immunofluorescence staining. Both fibrillin-2 and LTBP-2 were found extensively distributed in all regions of the disc with the organisation of the network varying significantly region to region. In the outer annulus fibrosus (OAF) both fibrillin-2 and LTBP-2 co-localised with fibrillin-1 forming fibres running parallel to the collagen fibres of the lamellae with the microfibrillar network staining densely in between the adjacent lamellae and also at the boundaries of the collagen bundle compartments. In the inner annulus fibrosus (IAF) and nucleus pulposus (NP), co-localised fibrillin-1,2 and LTBP-2 formed a chondron-like structure around the cell. By contrast, the inter-territorial matrix of the IAF and NP contained a dense network of fibrillin-2 but only sparse/filamentous fibres of fibrillin-1 and LTBP-2. Dual immunostaining revealed that in this region, fibrillin-2 was highly colocalised with elastin. The LTBP-2 network co-localised well with that of fibrillin-1 in all regions and indeed is reported to bind strongly to fibrillin-1. However, interestingly LTBP-2 but not fibrillin-1 or fibrillin-2 was removed by hyaluronidase but not collagenase pre-digestion. Our results suggest that fibrillin-2 and LTBP-2 could play an important role in disc function.

Justification of 150 mg clopidogrel in patients with high on-clopidogrel platelet reactivity.

BACKGROUND: The GRAVITAS trial showed that 150 mg clopidogrel did not improve outcome in patients with high on-clopidogrel platelet reactivity (HPR) screened by the VerifyNow assay. We aimed to determine the impact of 150 mg clopidogrel in stable angina patients with HPR identified with conventional aggregometry (LTA). MATERIALS AND METHODS: Clopidogrel-naive stable angina patients before ad hoc percutaneous coronary intervention were recruited into a randomized, double-blind, placebo-controlled trial (NCT00638326). Twelve to 24 h after the 600-mg loading dose of clopidogrel, ADP(5µM)-stimulated maximal (AGGmax), late platelet aggregation (AGGlate) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-PRI) were evaluated. Patients with HPR (AGGmax ≥ 34%) were randomly allocated to 75 or 150 mg clopidogrel after 4 weeks. After control platelet function measurements at day 28, 75 mg clopidogrel was administered to all patients until 1 year. RESULTS: The study was prematurely terminated at the stage of 200 enroled patients. Administration of 150 mg clopidogrel significantly reduced platelet aggregation (AGGmax: 45·0 ± 6·8 vs. 33·8 ± 15·1, P < 0·01; AGGlate: 27·1 ± 14·7 vs. 13·8 ± 18·0, P < 0·01) and VASP-PRI (57·5 ± 15·2 vs. 37·2 ± 17·1; P < 0·01), while platelet reactivity remained unchanged in patients with HPR receiving 75 mg clopidogrel. The higher maintenance dose of clopidogrel was associated with a significant reduction in cardiovascular (CV) death and myocardial infarction (MI) (0% vs. 11·4%, P = 0·04) and in CV death, MI or target vessel revascularization (24·6% vs. 3·1%; P = 0·01) during 1 year. CONCLUSIONS: One-month administration of 150 mg maintenance dose of clopidogrel reduces platelet reactivity and might decrease the risk of thrombo-ischaemic complications in stable angina patients with HPR identified by LTA.

Slingshot isoform-specific regulation of cofilin-mediated vascular smooth muscle cell migration and neointima formation.

OBJECTIVE: We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. METHODS AND RESULTS: Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the "wound," and platelet-derived growth factor (PDGF)-induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. CONCLUSION: These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

1,8 Cineole and Ellagic acid inhibit hepatocarcinogenesis via upregulation of MiR-122 and suppression of TGF-ß1, FSCN1, Vimentin, VEGF, and MMP-9.

Hepatocellular carcinoma (HCC) is one of the most burdened tumors worldwide, with a complex and multifactorial pathogenesis. Current treatment approaches involve different molecular targets. Phytochemicals have shown considerable promise in the prevention and treatment of HCC. We investigated the efficacy of two natural components, 1,8 cineole (Cin) and ellagic acid (EA), against diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) induced HCC in rats. DEN/2-AAF showed deterioration of hepatic cells with an impaired functional capacity of the liver. In addition, the levels of tumor markers including alpha-fetoprotein, arginase-1, alpha-L-fucosidase, and ferritin were significantly increased, whereas the hepatic miR-122 level was significantly decreased in induced-HCC rats. Interestingly, treatment with Cin (100mg/kg) and EA (60mg/kg) powerfully restored these biochemical alterations. Moreover, Cin and EA treatment exhibited significant downregulation in transforming growth factor beta-1 (TGF-ß1), Fascin-1 (FSCN1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and epithelial-mesenchymal transition (EMT) key marker, vimentin, along with a restoration of histopathological findings compared to HCC group. Such effects were comparable to Doxorubicin (DOX) (2mg/kg); however, a little additive effect was evident through combining these phytochemicals with DOX. Altogether, this study highlighted 1,8 cineole and ellagic acid for the first time as promising phytochemicals for the treatment of hepatocarcinogenesis via regulating multiple targets.