bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of glutathione-dependent DNA double-strand breaks by the novel anticancer drug brostallicin
Authors: Guirouilh-Barbat Josée , Zhang Yong-Wei , Pommier Yves
Published: July 2009

Brostallicin is a DNA minor groove binder in phase II clinical trials. Here, we show that brostallicin induces γ-H2AX nuclear foci that colocalize with 53BP1 and are dependent on glutathione, as shown by inhibition of those γ-H2AX foci by l-buthionine sulfoximine. To differentiate brostallicin from the clinically approved minor groove binder trabectedin (ecteinascidin 743), we tested whether the brostallicin-induced γ-H2AX and antiproliferative responses were dependent on nucleotide excision repair and found that, unlike trabectedin, they are not. Additionally, brostallicin retained activity in the trabectedin-resistant HCT116-ER5 cell line. Induction of γ-H2AX foci by brostallicin was partially dependent on the repair nuclease Mre11. Pretreatment with aphidicolin partially reduced brostallicin-induced γ-H2AX foci, suggesting that brostallicin induces both replication-associated and replication-independent DNA damage. Replication-associated DNA damage was further shown by the colocalization of γ-H2AX foci with replication foci and by the rapid inhibition of DNA synthesis and accumulation of cells in S phase in response to brostallicin. In addition, brostallicin was able to induce lower intensity γ-H2AX foci in human circulating lymphocytes. Together, our results indicate that brostallicin induces DNA double-strand breaks and suggest γ-H2AX as a pharmacodynamic biomarker for brostallicin. [Mol Cancer Ther 2009;8(7):1985–94]
Determinants for the efficiency of anticancer drugs targeting either Aurora-A or Aurora-B kinases in human colon carcinoma cells
Authors: Kaestner Phillip , Stolz Ailine , Bastians Holger
Published: July 2009

The mitotic Aurora kinases, including Aurora-A and Aurora- B, are attractive novel targets for anticancer therapy, and inhibitory drugs have been developed that are currently undergoing clinical trials. However, the molecular mechanisms how these drugs induce tumor cell death are poorly understood. We have addressed this question by comparing the requirements for an efficient induction of apoptosis in response to MLN8054, a selective inhibitor of Aurora-A, and the selective Aurora-B inhibitor ZM447439 in human colon carcinoma cells. By using various isogenic knockout as well as inducible colon carcinoma cell lines, we found that treatment with MLN8054 induces defects in mitotic spindle assembly, which causes a transient spindle checkpoint–dependent mitotic arrest. This cell cycle arrest is not maintained due to the activity of MLN8054 to override the spindle checkpoint. Subsequently, MLN8054-treated cells exit from mitosis and activate a p53-dependent postmitotic G1 checkpoint, which subsequently induces p21 and Bax, leading to G1 arrest followed by the induction of apoptosis. In contrast, inhibition of Aurora-B by ZM447439 also interferes with normal chromosome alignment during mitosis and overrides the mitotic spindle checkpoint but allows a subsequent endoreduplication, although ZM447439 potently activates the p53-dependent postmitotic G1 checkpoint. Moreover, the ZM447439-induced endoreduplication is a prerequisite for the efficiency of the drug. Thus, our results obtained in human colon carcinoma cells indicate that although both Aurora kinase inhibitors are potent inducers of tumor cell death, the pathways leading to the induction of apoptosis in response to these drugs are distinct. [Mol Cancer Ther 2009;8(7):2046–56]
Zalypsis (PM00104) is a potent inducer of γ-H2AX foci and reveals the importance of the C ring of trabectedin for transcription-coupled repair inhibition
Authors: Guirouilh-Barbat Josée , Antony Smitha , Pommier Yves
Published: July 2009

Zalypsis (PM00104) is a novel tetrahydroisoquinoline alkaloid related to trabectedin [ecteinascidin 743 (Et743)]. Et743 and PM00104 have similar A and B rings but differ in their C rings. The present study shows that Et743 and PM00104 differ in at least two ways: in their DNA binding properties and nucleotide excision repair (NER) dependency for cellular targeting. DNase I footprinting shows that the two drugs bind DNA differentially. We also found that, in contrast to Et743, the antiproliferative activity of PM00104 does not depend on transcription-coupled NER. Accordingly, PM00104 induces γ-H2AX foci with the same efficiency in NER-deficient or NER-proficient cells. Moreover, the formation of γ-H2AX foci is replication dependent for PM00104, whereas it is both transcription and replication dependent in the case of Et743. These findings show the importance of the C ring structure of tetrahydroisoquinoline ecteinascidin derivatives for NER targeting. Finally, PM00104 exerts antiproliferative activity at nanomolar concentrations and induces γ-H2AX response in two Ewing's sarcoma cell lines, suggesting that γ-H2AX could serve as a pharmacodynamic biomarker for the clinical development of PM00104. [Mol Cancer Ther 2009;8(7):2007–14]
Nuclear Factor-κB Inhibition by Parthenolide Potentiates the Efficacy of Taxol in Non–Small Cell Lung Cancer In vitro and In vivo
Authors: Zhang Dianliang , Qiu Lin , Jin Xianqing , Guo Zhenhua , Guo Chunbao
Published: July 2009

In this study, we have examined the molecular events induced by parthenolide, a sesquiterpene lactone, and explored possible mechanisms of resistance and sensitization of tumor cells to Taxol. We showed that parthenolide could antagonize Taxol-mediated nuclear factor-κB (NF-κB) nuclear translocation and activation and Bcl-xl up-regulation by selectively targeting I-κB kinase activity. In A549 cells, inhibition of nuclear factor-κB by parthenolide resulted in activation of the mitochondrial death pathway to promote cytochrome c release and caspase 3 and 9 activation. In contrast, Taxol alone induced apoptosis via a pathway independent of mitochondria cytochrome c cascade. In addition, depletion of Bcl-xl rescued the apoptotic response to Taxol. Moreover, treatment with parthenolide increased the efficacy of the Taxol-induced inhibition of A549 tumor xenografts in mice. This study elucidated the cellular responses induced by parthenolide that decrease the threshold of mitochodria-dependent apoptosis in the treatment of non–small cell lung cancer cells. (Mol Cancer Res 2009;7(7):1139–49)
Therapeutic Inhibition of the Epidermal Growth Factor Receptor in High-Grade Gliomas: Where Do We Stand?
Authors: Karpel-Massler Georg , Schmidt Ursula , Unterberg Andreas , Halatsch Marc-Eric
Published: July 2009

High-grade gliomas account for the majority of intra-axial brain tumors. Despite abundant therapeutic efforts, clinical outcome is still poor. Thus, new therapeutic approaches are intensely being investigated. Overexpression of the epidermal growth factor receptor (HER1/EGFR) is found in various epithelial tumors and represents one of the most common molecular abnormalities seen in high-grade gliomas. Dysregulated HER1/EGFR is found in 40% to 50% of glioblastoma, the most malignant subtype of glioma. Several agents such as tyrosine kinase (TK) inhibitors, antibodies, radio-immuno conjugates, ligand-toxin conjugates, or RNA-based agents have been developed to target HER1/EGFR or its mutant form, EGFRvIII. To date, most agents are in various stages of clinical development. Clinical data are sparse but most advanced for TK inhibitors. Although data from experimental studies seem promising, proof of a significant clinical benefit is still missing. Among the problems that have to be further addressed is the prediction of the individual patient's response to HER1/EGFR-targeted therapeutics based on molecular determinants. It is quite possible that blocking HER1/EGFR alone will not sufficiently translate into a clinical benefit. Therefore, a multiple target approach concomitantly aimed at different molecular sites might be a favorable concept. This review focuses on current HER1/EGFR-targeted therapeutics and their development for high-grade gliomas. (Mol Cancer Res 2009;7(7):1000–12)
Cyclooxygenase-2 Up-Regulates Ataxia Telangiectasia and Rad3 Related through Extracellular Signal-Regulated Kinase Activation
Authors: Kim Young Mee , Lee Eun Jung , Park Soo-Yeon , Cho Kwan Ho , Kim Joo Young , Pyo Hongryull
Published: July 2009

Cyclooxygenase-2 (COX-2) overexpression caused prolonged G2 arrest after exposure to ionizing radiation (IR) in our previous study. We were therefore interested in investigating the function of COX-2 in the G2 checkpoint pathway. Interestingly, we found that cells in which COX-2 is overexpressed showed up-regulated ataxia telangiectasia and Rad3 related (ATR) expression compared with control cells. In this study, we investigated the mechanism of ATR up-regulation by COX-2 and tested our hypothesis that COX-2–induced extracellular signal-regulated kinase (ERK) activation mediates up-regulation of ATR by COX-2. To investigate the relationship between COX-2 and ATR, we used two stable COX-2–overexpressing cancer cell lines (HCT116–COX-2 and H460–COX-2), a COX-2 knockdown A549 lung cancer cell line (AS), and an ATR knockdown HCT116 cell line. Cells were treated with various drugs [celecoxib, prostaglandin E2 (PGE2), PD98059, U0126, and hydroxyurea] and were then analyzed using reverse transcription-PCR, confocal microscopy, Western blotting, and clonogenic assay. COX-2–overexpressing cells were shown to have increased ERK phosphorylation and ATR expression compared with control cells, whereas AS cells were shown to have decreased levels of phospho-ERK and ATR. In addition, exogenously administered PGE2 increased ERK phosphorylation. Inhibition of ERK phosphorylation decreased ATR expression in both HCT116–COX-2 and A549 cells. HCT116–COX-2 cells were resistant to IR or hydroxyurea compared with HCT116-Mock cells, whereas administration of ATR shRNA showed the opposite effect. COX-2 stimulates ERK phosphorylation via PGE2. This COX-2–induced ERK activation seems to increase ATR expression and activity in endogenous COX-2–overexpressing cancer cells as well as in COX-2–overexpressing stable cell lines. (Mol Cancer Res 2009;7(7):1158–68)
Targeting of Tumor Blood Vessels: A Phage-Displayed Tumor-Homing Peptide Specifically Binds to Matrix Metalloproteinase-2-Processed Collagen IV and Blocks Angiogenesis In vivo
Authors: Mueller Jan , Gaertner Florian C. , Blechert Birgit , Janssen Klaus-Peter , Essler Markus
Published: July 2009

Proteolytic degradation of the basement membrane by the matrix metalloproteinase-2 and -9 is an essential step in tumor angiogenesis. On proteolytic degradation, cryptic sites in collagen IV are formed, which serve as a migration signal for endothelial cells and are specific for angiogenic blood vessels. The aim of this study was to generate peptides that bind specifically to proteolytically processed collagen IV and to test whether these peptides accumulate in tumor vasculature and are able to block angiogenesis. To obtain such peptides, we performed a combined in vivo and in vitro phage display screen using a recombinant phage-displayed peptide library. We found a phage displaying the peptide sequence TLTYTWS that specifically binds to collagen IV modified by matrix metalloproteinase-2. We then tested the ability of the phage to bind to the vasculature in xenograft tumors and found indeed a significant accumulation of the phage in tumors but not in control organs. The tumor homing of the TLTYTWS phage is specific, as it can be blocked by coinjection chemically synthesized cognate peptide. Moreover, TLTYTWS peptide inhibits angiogenesis in an in vivo assay in a concentration-dependent manner and significantly reduces endothelial differentiation in vitro. In conclusion, we report about a novel tumor-homing peptide that specifically binds to proteolytically processed collagen IV, accumulates in tumors, and blocks angiogenesis. This peptide may be a new useful tool for diagnostic and therapeutic procedures in oncology.(Mol Cancer Res 2009;7(7):1078–85)
CXCR4 and Matrix Metalloproteinase-1 Are Elevated in Breast Carcinoma–Associated Fibroblasts and in Normal Mammary Fibroblasts Exposed to Factors Secreted by Breast Cancer Cells
Authors: Eck Sarah M. , Côté Anik L. , Winkelman William D. , Brinckerhoff Constance E.
Published: July 2009

The complex molecular communications that occur between neoplastic and stromal cells within the tumor microenvironment play an integral role in breast cancer pathogenesis. Carcinoma-associated fibroblasts (CAF) produce tumor-enhancing factors and have been strongly implicated in breast cancer development. Similar to the way in which tumors have been compared with “wounds that never heal,” CAFs have been equated to activated fibroblasts, which are present in inflammatory environments, in which they aid in wound healing through tissue remodeling and repair. Matrix metalloproteinase-1 (MMP-1) and G protein–coupled receptor, CXCR4, are elevated in these activated fibroblasts, in which they facilitate angiogenesis and matrix degradation, processes that are also vital to breast cancer metastasis. In this study, we investigated MMP-1 and CXCR4 expression in normal human mammary fibroblasts (HMF) exposed to soluble breast cancer factors. Historically, elevated CXCR4 expression is associated with breast cancer cells. However, we show that soluble factors secreted by SUM102 breast cancer cells stimulated the expression of MMP-1 and CXCR4 in HMFs. As a result, these stromal cells acquired an invasive and migratory phenotype. To confirm the clinical relevancy of our findings, we analyzed CAFs obtained from primary breast cancers. These cells also displayed elevated MMP-1 and CXCR4 levels compared with counterpart fibroblasts, and were more invasive and migratory. Together, our data suggest that soluble breast cancer factors initiate the transdifferentiation of normal HMFs to tumor-promoting CAFs, and that through the induction of MMP-1 and CXCR4 levels, these cells exhibit an invasive and migratory phenotype.(Mol Cancer Res 2009;7(7):1033–44)
Matrix Protein CCN1 Is Critical for Prostate Carcinoma Cell Proliferation and TRAIL-Induced Apoptosis
Authors: Franzen Carrie A. , Chen Chih-Chiun , Todorović Viktor , Juric Vladislava , Monzon Ricardo I. , Lau Lester F.
Published: July 2009

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins αvβ3 and α6β4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Cα-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy. (Mol Cancer Res 2009;7(7):1045–55)
Requirement of Phosphatidylinositol(3,4,5)Trisphosphate in Phosphatidylinositol 3-Kinase-Induced Oncogenic Transformation
Authors: Denley Adam , Gymnopoulos Marco , Kang Sohye , Mitchell Christina , Vogt Peter K.
Published: July 2009

Phosphatidylinositol 3-kinases (PI3K) are divided into three classes, which differ in their substrates and products. Class I generates the inositol phospholipids PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 referred as PIP, PIP2, and PIP3, respectively. Class II produces PIP and PIP2, and class III generates only PIP. Substrate and product differences of the three classes are determined by the activation loops of their catalytic domains. Substitution of the class I activation loop with either class II or III activation loop results in a corresponding change of substrate preference and product restriction. We have evaluated such activation loop substitutions to show that oncogenic activity of class I PI3K is linked to the ability to produce PIP3. We further show that reduction of cellular PIP3 levels by the 5′-phosphatase PIPP interferes with PI3K-induced oncogenic transformation. PIPP also attenuates signaling through Akt and target of rapamycin. Class III PI3K fails to induce oncogenic transformation. Likewise, a constitutively membrane-bound class I PI3K mutant retaining only the protein kinase is unable to induce transformation. We conclude that PIP3 is an essential component of PI3K-mediated oncogenesis and that inability to generate PIP3 abolishes oncogenic potential. (Mol Cancer Res 2009;7(7):1132–8)
Genetic contribution to variation in atherothrombotic phenotypes in the Asian Indian population
Authors: Khadrinarasimhaih Natesha , Kanjilal Saikat , Mukerjee Manjari , Kakkar Vijay , Rao Veena
Published: July 2009

Coronary artery disease (CAD) is a multifactorial disease, and family history is the best available tool to assess gene-environment interaction. This study addressed the heritability of quantitative traits, namely lipid, coagulation/fibrinolysis and pro-inflammatory markers in the ongoing family-based Indian Atherosclerosis Research Study and assessed the effect of the type/lineage of CAD family history on inheritance patterns in the highrisk Indian population. A total of 518 families comprising 2,305 individuals were recruited in phase I of the IARS; of these, 1,195 individuals from 220 families were included in the heritability analysis. With the exception of leptin, all phenotypes exhibited significant age- and sex-adjusted heritability (p<0.0001). Amongst all the phenotypes analysed after adjustment for confounding factors, the significantly higher heritability estimates of triglycerides (0.53, p<0.0001), lipoprotein (a) (0.83, p<0.0001) and interleukin-6 (0.46, p<0.0001) with low spouse pair correlations identifies them as possible CAD risk factors. Families with parental history of CAD had onset of CAD symptoms at much younger ages with significantly higher heritability of proinflammatory markers, whereas in families with sibling history of CAD, more risk factors were present at significantly higher levels. Triglycerides, lipoprotein (a) and interleukin-6 appear to be promising atherothrombotic candidate phenotypes in this population. Genes controlling these phenotypes are possible candidate genes linked with CAD. An informed understanding and incorporation of ‘family history’ as a screening tool may help in the prevention and pre-emption of CAD.
Cross validation of the Multiple Electrode Aggregometry
Authors: Siller-Matula Jolanta , Gouya Ghazaleh , Wolzt Michael , Jilma Bernd
Published: July 2009

Several test systems exist for assessment of platelet function in patients under clopidogrel or aspirin therapy. The objective was to cross-validate the Multiple Electrode Aggregometry (MEA) with three other methods used for determining platelet reactivity under treatment with clopidogrel and aspirin. Platelet function was assessed by the MEA, Vasodilator Stimulated Phosphoprotein (VASP) phosphorylation assay, Platelet Function Analyzer-100 (PFA-100) and the Cone and Platelet Analyzer. Measurements were performed in blood from nine healthy volunteers at baseline, 2, 4, 6 and 72 hours after clopidogrel and aspirin loading. The apparent effect size for clopidogrel and aspirin was greatest for the MEA: treatment induced a 19-fold difference in the arachidonic acid-induced platelet aggregation and an 11-fold difference in the adenosine diphosphate-induced platelet aggregation before/after treatment. For comparison, aspirin and clopidogrel induced only 2.0– to 2.6 -fold changes in other tests (VASP assay, Cone and Platelet Analyzer and PFA-100). Maximal effects were seen 2 hours after aspirin loading and shorter than 72 hours after clopidogrel loading. In conclusion, aspirin and clopidogrel produce stronger signals in the MEA compared to several other methods.
Prognostic impact of haemostatic derangements in chronic heart failure
Authors: Vene Nina , Salobir Barbara , Šebeštjen Miran , Šabovic Mišo , Keber Irena , Jug Borut
Published: July 2009

Heart failure is characterised by activation of haemostasis. We sought to explore the prognostic impact of deranged haemostasis in chronic heart failure. In stable, optimally managed outpatients with chronic heart failure, baseline levels of prothrombin fragment F1+2, D-dimer, and tPA and PAI-1 antigens were determined. Clinical follow-up was obtained and the rate of events (heart failure related deaths or hospitalisations) was recorded. We included 195 patients [32.3% female, NYHA class II (66.2%) or III (33.8%), mean age 71 years]. During a median follow up of 693 (interquartile range [IQR] 574–788) days, 63 (30.9%) patients experienced an event; those with an event had higher levels of tPA antigen (median 11.8 [IQR 8.7–14.0] vs. 9.4 [7.9–12.1] µg/l; p=0.033) and D-dimer (938 [485–1269] vs. 620 [37–1076] µg/l; p=0.018). However, on Cox multivariate analysis, only tPA levels above optimal cut-off value of 10.2 µg/l (but not D-dimer) emerged as an independent predictor of prognosis (HRadjusted 2.695, 95% confidence interval 1.233–5.363; p=0.017). Our findings suggest that elevated tPA antigen levels are an independent prognostic predictor in patients with chronic stable heart failure.
Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3β in platelets
Authors: Hayashi Hideki , Sudo Toshiki
Published: July 2009

Elevating intracellular cAMP has been shown to inhibit platelet function. cAMP interferes with platelet-activating signals which lead to aggregation inhibition, but the precise mechanism is unclear.The present study examined if cAMP-elevating agents inhibited phosphatidylinositol 3-kinase (PI3-kinase) signaling in rat platelets by immunoblotting. Akt is one of the key molecules downstream of PI3K, and is phosphorylated by collagen stimulation. The phosphodiesterase-3 (PDE3) inhibitors cilostamide and cilostazol, and the adenylate cyclase activator forskolin, inhibited collagen-induced Akt phosphorylation at Ser473.The inhibitory effects of these cAMP-elevating agents on Akt phosphorylation were unchanged in the presence of the PKA (cyclic AMP-dependent protein kinase) inhibitor H-89. These effects were consistent with inhibition of platelet aggregation. It is known that inhibition of Akt phosphorylation leads to inhibition of phosphorylation of glycogen synthase kinase 3-beta (GSK-3β), which is an effector of Akt, but cAMP-elevating agents stimulated GSK-3βphosphorylation at Ser9.The PKA inhibitor H-89 attenuated GSK-3βphosphorylation.The cAMP-elevating agents cilostamide, cilostazol and forskolin did not directly affect the enzyme activity of PI3-kinase.These results suggested that cAMP-elevating agents have two effects on PI3K signalling: inhibition of Akt phosphorylation independent of PKA; and stimulation of GSK-3β phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMPelevating agents on platelet function.
Effect of natural killer T cell activation on initiation of atherosclerosis
Authors: van Puijvelde Gijs , van Wanrooij Eva , Hauer Arnaud , de Vos Paula , van Berkel Theo , Kuiper Johan
Published: July 2009

It has been shown that natural killer T (NKT) cell activation accelerates atherosclerosis in apoE−/− mice. ApoE is,however,an important mediator in the presentation of lipids which may complicate conclusions on the role of NKT cells in atherosclerosis. Treatment of LDLr−/− mice with α-GalCer during Western-type diet feeding is therefore of interest. Atherosclerosis was induced by Western-type diet feeding and collar placement around the carotid arteries in both LDLr−/− and apoE−/− mice. Subsequently, the mice were treated twice a week with α-GalCer. This resulted in an 84% reduction in plaque size in LDLr−/− mice (P<0.05), while no effect was observed in apoE−/− mice. In-vitro incubation of splenocytes with α-GalCer showed that LDLr−/− splenocytes proliferated stronger than apoE−/− splenocytes. This is reflected in a larger increase in production of cytokines and especially IL-10 after in-vitro stimulation with α-GalCer in LDLr−/− mice compared with apoE−/− splenocytes. Additionally, feeding a Western-type diet for 1.5 weeks induced a strong increase in the number of NKT cells in LDLr−/− mice and this increase was slower and less prominent in apoE−/− mice. Administration of α-GalCer to LDLr−/− mice in combination with Western-type diet feeding reduced plaque formation, but this effect was not seen in apoE−/− mice. This may be explained by the decreased presentation of lipids on CD1d molecules due to the lack of apoE. In this study we proved for the first time that NKT cells may also act in an atheroprotective manner.
The vulnerable patient: Refocusing on the plaque?
Authors: Heeneman Sylvia , Daemen Mat , Eijgelaar Wouter
Published: July 2009

The term ‘vulnerable plaque’ is used to refer to the lesions that are prone to rupture and may cause life-threatening events like acute coronary syndrome or stroke.The study of the vulnerable plaque phenotype and its detection has attracted increasing interest over the past decades. During this time, there have been some remarkable transitions in the paradigm on methods to identify patients at risk or patients to treat.Whereas formerly, the key factors used to determine an individual’s risk were primarily population-based traditional risk factors such as age, sex, body mass index, hypertension etc., new approaches are based on conditional risk factors that represent an individual’s current risk of suffering a cardiovascular event.These population based risk factors fall short in predicting near-future events in a high-risk individual. In the early 2000s, the focus of research into surrogate markers for cardiovascular event prediction shifted from the vulnerable plaque to the identification of the vulnerable patient.This new paradigm stimulated a number of new initiatives that aimed to identify vulnerable patients by testing systemic biomarkers that could identify patients at high risk for cardiovascular events. A second research paradigm is refocusing on the plaque by searching for plaque-derived biomarkers and non-invasive imaging modalities to assess characteristics of a plaque that determine its vulnerability.Although both concepts are attractive, they still need proper validation in large multicenter cohorts, while cost-effectiveness arguments also need to be assessed.
Structural analysis of eight novel and 112 previously reported missense mutations in the interactive FXI mutation database reveals new insight on FXI deficiency
Authors: Saunders Rebecca , Shiltagh Nuha , Gomez Keith , Mellars Gillian , Cooper Carolyn , Perry David , Tuddenham Edward , Perkins Stephen
Published: July 2009

Factor XI (FXI) functions in blood coagulation. FXI is composed of four apple (Ap) domains and a serine protease (SP) domain. Deficiency of FXI leads to an injury-related bleeding disorder, which is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C).The number of mutations previously reported in our interactive web database (http://www.FactorXI.org) is now significantly increased to 183 through our new patient studies and from literature surveys. Eight novel missense mutations give a total of 120 throughout the FXI gene (F11).The most abundant defects in FXI are revealed to be those from low-protein plasma levels (Type I: CRM-) that originate from protein misfolding, rather than from functional defects (Type II: CRM+). A total of 70 Ap missense mutations were analysed using a consensus Ap domain structure generated from the FXI dimer crystal structure. This showed that all parts of the Ap domain were affected.The 47 SP missense mutations were also distributed throughout the SP domain structure.The periphery of the Ap β-sheet structure is sensitive to structural perturbation caused by residue changes throughout the Ap domain, yet this β-sheet is crucial for FXI dimer formation. Residues located at the Ap4:Ap4 interface in the dimer are much less directly involved. We conclude that the abundance of Type I defects in FXI results from the sensitivity of the Ap domain folding to residue changes within this, and discuss how structural knowledge of the mutations improves our understanding of FXI deficiencies.
Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level Functional and modelling studies
Authors: Crescente Marilena , Jessen Gisela , Momi Stefania , Höltje Hans-Dieter , Gresele Paolo , de Gaetano Giovanni , Cerletti Chiara
Published: July 2009

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets.We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB2. Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex,and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of lowdose aspirin.
Comparison of recombinant and plasma-derived antithrombin biodistribution in a rabbit model
Authors: Berry Leslie , Thong Bruce , Chan Anthony
Published: July 2009

Antithrombin (AT) is a native plasma protein that acts as the main inhibitor of enzymes generated by the coagulation cascade. In extreme thrombotic conditions, consumption of plasma AT can make treatment with AT-associated heparin therapies less effective. Supplementation with recombinant human AT (rhAT) has shown promise but altered pharmacokinetics were observed when comparing stable heparin complexes of the plasma-derived AT (pAT) and rhAT proteins. To understand the differential clearance mechanisms,biodistribution of rhAT and pAT was determined. 125I-labelled ATryn (rhAT) or Kybernin P (pAT) was intravenously injected into rabbits. At various time points, animals were sacrificed and organs analysed for bound radioactivity. 131I-albumin, injected shortly before termination, was used as a marker for trapped blood. Levels of circulating protein + metabolites were significantly less for rhAT than pAT (p < 0.001) and removal of acid soluble fragments confirmed that differences were due to more rapid rhAT clearance. More rhAT (28% dose) than pAT (3% dose) was liver-associated by the earliest time points, corresponding to elevated rhAT degradation products in urine/feces. However, at intermediate times (4 hours), rhAT showed significantly increased arterial and venous uptake over pAT (p ≤0.001).These vessel wall interactions accounted for the primary differences between clearance of rhAT and pAT during these time periods. Overall, circulating rhAT is more rapidly lost to the liver and vessels than pAT. Increased vessel wall binding may facilitate rhAT treatment of vascular thrombosis.
Internal quality control of prothrombin time in primary care: comparing the use of patient split samples with lyophilised control materials
Authors: Petersen Per , Sølvik Una , Sandberg Sverre , Stavelin Anne
Published: July 2009

Many primary care laboratories use point-of-care (POC) instruments to monitor patients on anticoagulant treatment. The internal analytical quality control of these instruments is often assessed by analysing lyophilised control materials and/or by sending patient samples to a local hospital laboratory for comparison (split sample).The aim of this study was to evaluate the utility of these two models of prothrombin time quality control. The models were evaluated by power functions created by computer simulations based on empirical data from 18 primary care laboratories using the POC instruments Thrombotrack, CoaguChek S, or Hemochron Jr. Signature. The control rules 12S, 13S, exponential weighted moving average, and the deviation limits of ± 10% and ± 20% were evaluated by their probability of error detection and false rejections. The total within-lab coefficient of variation was 3.8% and 6.9% for Thrombotrack, 8.9% and 10.5% for CoaguChek S, and 9.4% and 14.8% for Hemochron Jr. Signature for the control sample measurements and the split sample measurements, respectively. The probability of error detection was higher using a lyophilised control material than a patient split sample for all three instruments, whereas the probability of false rejection was similar. A higher probability of error detection occurred when lyophilised control material was used compared with the patient split samples; therefore, lyophilised control material should be used for internal analytical quality control of prothrombin time in primary health care.
Variability of INR in patients on stable long-term treatment with phenprocoumon and acenocoumarol and implications for analytical quality requirements
Authors: van Geest-Daalderop Johanna H. H. , Péquériaux Nathalie C. V. , van den Besselaar Anton M. H. P.
Published: July 2009

Within each patient treated with vitamin K antagonist (VKA), variation of the international normalised ratio (INR) occurs over the treatment period. The purpose of the present study was to assess INR variation in selected patients on long-term treatment in whom the dose of VKA was not changed.This type of variation is considered as “biological variation” which is caused by many factors but not VKA dose changes or other medication. Four groups of long-term patients were examined: each group with a different VKA (acenocoumarol or phenprocoumon) or a different target intensity (INR 2.0–3.5 or 2.5–4.0). All patients were monitored with the same PT system (Hepato Quick, STA-R Evolution coagulation instrument) by one laboratory.The variation of the INR within each patient was expressed as coefficient of variation (CV, in %).The CV was corrected for the average imprecision of the INR measurement (CV, 2.4%). The mean corrected CV values for the four groups were: 10.9% (acenocoumarol, target INR 2.0–3.5); 10.5% (acenocoumarol, target INR 2.5–4.0); 10.4% (phenprocoumon, target INR 2.0–3.5); 9.1% (phenprocoumon, target INR 2.5–4.0). The analytical performance goal for the INR measurement (imprecision) can be derived from the within-subject biological variation. Desirable INR imprecision goals are <4.9% and <5.3% CV for monitoring of phenprocoumon and acenocoumarol, respectively. These goals were achieved using the aforesaid PT system.
Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies
Authors: Livnat Tami , Tamarin Ilia , Mor Yoram , Winckler Harry , Horowitz Zeev , Korianski Yoseph , Bar-Zakay Barak , Salomon Ophira , Seligsohn Uri
Published: July 2009

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 µg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 µg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 µg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.
New ECG Criteria in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy
Authors: Cox Moniek G.P.J. , van der Smagt Jasper J. , Wilde Arthur A.M. , Wiesfeld Ans C.P. , Atsma Douwe E. , Nelen Marcel R. , Rodriguez Luz-Maria , Loh Peter , Cramer Maarten J. , Doevendans Pieter A. , van Tintelen J. Peter , de Bakker Jacques M.T. , Hauer Richard N.W.
Published: July 2009

Background—

Desmosomal changes, electric uncoupling, and surviving myocardial bundles in fibrofatty tissue characterize arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Resultant activation delay is pivotal for reentry and thereby ventricular tachycardia (VT). Current task force criteria (TFC) for diagnosis have limited sensitivity. The aim of this study was to assess the diagnostic value of additional criteria on activation delay and VT to improve identification of affected individuals.

Methods and Results—

ECG criteria were studied, while off drugs, in 50 index patients with proven ARVD/C according to TFC (TFC ≥4 points) and 33 patients with probable ARVD/C (TFC 3 points, or TFC3), being 21 index patients and 12 family members of proven ARVD/C patients. Newly proposed additional criteria are (1) prolonged terminal activation duration in V
1
-V
3
, an indicator of activation delay, (2) VT with left bundle-branch block morphology and superior axis, and (3) multiple VT morphologies. All index patients were screened for mutations in ARVD/C-related genes encoding desmosomal proteins. Altogether, 23 of 33 (70%) TFC3 patients fulfilled ARVD/C diagnosis when newly proposed criteria were applied additionally to current TFC. VT with left bundle-branch block morphology and superior axis or multiple VT morphologies were recorded in 12 and 9 of 33 TFC3 patients, respectively, all being index patients. When applying prolonged terminal activation duration additionally to TFC on depolarization/conduction abnormalities, 14 (42%) TFC3 patients fulfilled ARVD/C diagnosis. Results were not significantly different between mutation carriers and noncarriers.

Conclusions—

Adding the newly proposed criteria to current TFC for ARVD/C will improve identification of affected individuals importantly, independent of outcome of DNA analyses.
IL-1β Inhibits TGFβ in the Temporomandibular Joint
Authors: Lim W.H. , Toothman J. , Miller J.H. , Tallents R.H. , Brouxhon S.M. , Olschowka M.E. , Kyrkanides S.
Published: July 2009

Similarly to humans, healthy, wild-type mice develop osteoarthritis, including of the temporomandibular joint (TMJ), as a result of aging. Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα, are known to contribute to the development of osteoarthritis, whereas TGFβ has been associated with articular regeneration. We hypothesized that a balance between IL-1β and TGFβ underlies the development of TMJ osteoarthritis, whereby IL-1β signaling down-regulates TGFβ expression as part of disease pathology. Our studies in wild-type mice, as well as the Col1-IL1βXAT mouse model of osteoarthritis, demonstrated an inverse correlation between IL-1β and TGFβ expression in the TMJ. IL-1β etiologically correlated with joint pathology, whereas TGFβ expression associated with IL-1β down-regulation and improvement of articular pathology. Better understanding of the underlying inflammatory processes during disease will potentially enable us to harness inflammation for orofacial tissue regeneration.
Structural Integrity of Enamel: Experimental and Modeling
Authors: Xie Z. , Swain M.V , Hoffman M.J.
Published: July 2009

Tooth enamel is the hardest tissue in the human body and is directly responsible for dental function. Due to its non-regenerative nature, enamel is unable to heal and repair itself biologically after damage. We hypothesized that with its unique microstructure, enamel possesses excellent resistance to contact-induced damage, regardless of loading direction. By combining instrumented indentation tests with microstructural analysis, we report that enamel can absorb indentation energy through shear deformation within its protein layers between apatite crystallites. Moreover, a near-isotropic inelastic response was observed when we analyzed indentation data in directions either perpendicular or parallel to the path of enamel prisms. An “effective” crystal orientation angle, 33°–34°, was derived for enamel microstructure, independent of the loading direction. These findings will help guide the design of the nanostructural architecture of dental restorative materials.
Dietary Carbohydrates and Dental-Systemic Diseases
Authors: Hujoel P.
Published: July 2009

Two contradictory hypotheses on the role of dietary carbohydrates in health and disease shape how dental-systemic associations are regarded. On one side, Cleave and Yudkin postulated that excessive dietary fermentable carbohydrate intake led—in the absence of dental interventions such as fluorides— first to dental diseases and then to systemic diseases. Under this hypothesis, dental and systemic diseases shared—as a common cause—a diet of excess fermentable carbohydrates. Dental diseases were regarded as an alarm bell for future systemic diseases, and restricting carbohydrate intake prevented both dental and systemic diseases. On the opposite side, Keys postulated the lipid hypothesis: that excessive dietary lipid intake caused systemic diseases. Keys advocated a diet high in fermentable carbohydrate for the benefit of general health, and dental diseases became regarded as local dietary side effects. Because general health takes precedence over dental health when it comes to dietary recommendations, dental diseases became viewed as local infections; interventions such as fluorides, sealants, oral hygiene, antimicrobials, and dental fillings became synonymous with maintaining dental health, and carbohydrates were no longer considered as a common cause for dental-systemic diseases. These opposing dietary hypotheses have increasingly been put to the test in clinical trials. The emerging trial results favor Cleave-Yudkin’s hypothesis and may affect preventive approaches for dental and systemic diseases.
Roles of the Primary Cilium Component Polaris in Synchondrosis Development
Authors: Ochiai T. , Nagayama M. , Nakamura T. , Morrison T. , Pilchak D. , Kondo N. , Hasegawa H. , Song B. , Serra R. , Pacifici M. , Koyama E.
Published: July 2009

Primary cilia regulate several developmental processes and mediate hedgehog signaling. To study their roles in cranial base development, we created conditional mouse mutants deficient in Polaris, a critical primary cilium component, in cartilage. Mutant post-natal cranial bases were deformed, and their synchondrosis growth plates were disorganized. Expression of Indian hedgehog, Patched-1, collagen X, and MMP-13 was reduced and accompanied by decreases in endochondral bone. Interestingly, there was excessive intramembranous ossification along the perichondrium, accompanied by excessive Patched-1 expression, suggesting that Ihh distribution was wider and responsible for such excessive response. Indeed, expression of heparan sulfate proteoglycans (HS-PGs), normally involved in restricting hedgehog distribution, was barely detectable in mutant synchondroses. Analyses of the data provides further evidence for the essential roles of primary cilia and hedgehog signaling in cranial base development and chondrocyte maturation, and point to a close interdependence between cilia and HS-PGs to delimit targets of hedgehog action in synchondroses.
PTHrP Induces Notch Signaling in Periodontal Ligament Cells
Authors: Nakao A. , Kajiya H. , Fukushima H. , Fukushima A. , Anan H. , Ozeki S. , Okabe K.
Published: July 2009

Periodontal ligament (PDL) cells are known to play important roles in tooth eruption and alveolar bone metabolism. We previously reported that PTHrP increases RANKL expression in human PDL cells, suggesting that it promotes odontoclastic root resorption during tooth eruption. While it is known that Notch-related genes play a key role during bone development, the role of the Notch signaling pathway in PDL cells during tooth and bone resorption is less clear. We hypothesized that PTHrP induces a Notch ligand in PDL cells and thereby regulates osteo- and odontoclastogenesis. We found that PTHrP increased Notch1 ligand Jagged1 expression in human PDL cells in a dose- and time-dependent manner. PTHrP-induced Jagged1 up-regulation was mediated by PKA activation, but not by PKC. Jagged1 also promoted RANKL-induced osteoclastogenesis. These results demonstrate that PTHrP induces Jagged1 expression in PDL cells, leading to osteo- and odontoclastogenesis, and thus likely promoting tooth and alveolar bone resorption.
Amphiregulin Induces Proliferative Activities in Osseous Dysplasia
Authors: Shigeishi H. , Yamaguchi S. , Mizuta K. , Nakakuki K. , Fujimoto S. , Amagasa T. , Kamata N.
Published: July 2009

Human osseous dysplasia (OD) is a benign fibro-osseous neoplasm of periodontal ligament origin in which normal bone is replaced with fibrous connective tissue containing abnormal bone or cementum. However, cellular differentiation and proliferation in OD have not been fully elucidated. In vitro culture systems have distinct advantages for analytical studies. Therefore, we established immortalized cell lines (OD-1) from OD lesions of the jaw from an individual with gnathodiaphyseal dysplasia (GDD). We hypothesized that OD-1 had a characteristic growth mechanism different from that of mineralized-associated cells such as osteoblasts. To clarify the difference of gene expression patterns between OD-1 and osteoblasts, we compared the profiles of genes expressed in the 2 cell types by microarray analysis. We identified amphiregulin to be highly expressed in OD-1 compared with osteoblasts and gingival fibroblasts. OD-1 showed proliferative activities regulated in an autocrine manner by amphiregulin, and amphiregulin may play a significant role in the proliferation of OD.