Oral Bruton tyrosine kinase inhibitors selectively block atherosclerotic plaque-triggered thrombus formation in humans.

Interaction of von Willebrand factor (VWF) with platelet glycoprotein Ib (GPIb) and interaction of collagen with GPVI are essential for thrombus formation on ruptured or eroded atherosclerotic plaques (atherothrombosis). GPIb and GPVI signal through Bruton tyrosine kinase (Btk), which can be blocked irreversibly by oral application of ibrutinib, an established therapy for chronic lymphocytic leukemia (CLL) with long-term safety. We found that ibrutinib and the novel Btk inhibitors acalabrutinib and ONO/GS-4059 block GPVI-dependent static platelet aggregation in blood exposed to human plaque homogenate and collagen but not to ADP or arachidonic acid. Moreover, Btk inhibitors prevented platelet thrombus formation on human atherosclerotic plaque homogenate and plaque tissue sections from arterially flowing blood, whereas integrin α2ß1 and VWF-dependent platelet adhesion to collagen, which is important for physiologic hemostasis, was not affected. This plaque-selective platelet inhibition was also observed in CLL patients taking 450 mg of ibrutinib and in volunteers after much lower and intermittent dosing of the drug. We conclude that Btk inhibitors, by targeting GPIb and GPVI signal transduction, suppress platelet thrombus accretion from flowing blood on atherosclerotic plaque but spare hemostatic platelet function. Btk inhibitors hold promise as the first culprit lesion-focused oral antiplatelet drugs and are effective at low doses.

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry.

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ168 h 3.2 log10 cfu cm-2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.

Development of a New Monomer for the Synthesis of Intrinsic Antimicrobial Polymers with Enhanced Material Properties.

The use of biocidal compounds in polymers is steadily increasing because it is one solution to the need for safety and hygiene. It is possible to incorporate an antimicrobial moiety to a polymer. These polymers are referred to as intrinsic antimicrobial. The biocidal action results from contact of the polymer to the microorganisms, with no release of active molecules. This is particularly important in critical fields like food technology, medicine and ventilation technology, where migration or leaching is crucial and undesirable. The isomers N-(1,1-dimethylethyl)-4-ethenyl-benzenamine and N-(1,1-dimethyl-ethyl)-3-ethenyl-benzenamine (TBAMS) are novel (Co-)Monomers for intrinsic anti-microbial polymers. The secondary amines were prepared and polymerized to the corresponding water insoluble polymer. The antimicrobial activity was analyzed by the test method JIS Z 2801:2000. Investigations revealed a high antimicrobial activity against Staphylococcus aureus and Escherichia coli with a reduction level of >4.5 log10 units. Furthermore, scanning electron microscopy (SEM) of E. coli. in contact with the polymer indicates a bactericidal action which is caused by disruption of the bacteria cell membranes, leading to lysis of the cells.

Modeling and Phenotyping Acute and Chronic Type 2 Diabetes Mellitus In Vitro in Rodent Heart and Skeletal Muscle Cells.

Type 2 diabetes (T2D) has a complex pathophysiology which makes modeling the disease difficult. We aimed to develop a novel model for simulating T2D in vitro, including hyperglycemia, hyperlipidemia, and variably elevated insulin levels targeting muscle cells. We investigated insulin resistance (IR), cellular respiration, mitochondrial morphometry, and the associated function in different T2D-mimicking conditions in rodent skeletal (C2C12) and cardiac (H9C2) myotubes. The physiological controls included 5 mM of glucose with 20 mM of mannitol as osmotic controls. To mimic hyperglycemia, cells were exposed to 25 mM of glucose. Further treatments included insulin, palmitate, or both. After short-term (24 h) or long-term (96 h) exposure, we performed radioactive glucose uptake and mitochondrial function assays. The mitochondrial size and relative frequencies were assessed with morphometric analyses using electron micrographs. C2C12 and H9C2 cells that were treated short- or long-term with insulin and/or palmitate and HG showed IR. C2C12 myotubes exposed to T2D-mimicking conditions showed significantly decreased ATP-linked respiration and spare respiratory capacity and less cytoplasmic area occupied by mitochondria, implying mitochondrial dysfunction. In contrast, the H9C2 myotubes showed elevated ATP-linked and maximal respiration and increased cytoplasmic area occupied by mitochondria, indicating a better adaptation to stress and compensatory lipid oxidation in a T2D environment. Both cell lines displayed elevated fractions of swollen/vacuolated mitochondria after T2D-mimicking treatments. Our stable and reproducible in vitro model of T2D rapidly induced IR, changes in the ATP-linked respiration, shifts in energetic phenotypes, and mitochondrial morphology, which are comparable to the muscles of patients suffering from T2D. Thus, our model should allow for the study of disease mechanisms and potential new targets and allow for the screening of candidate therapeutic compounds.

Phytosterols reduce cholesterol absorption by inhibition of 27-hydroxycholesterol generation, liver X receptor α activation, and expression of the basolateral sterol exporter ATP-binding cassette A1 in Caco-2 enterocytes.

Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 µmol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar (3)H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXRα) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXRα-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXRα activation in human enterocytes. Exogenous LXRα agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXRα-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXRα-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXRα antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol.

Responsivity of the Striatal Dopamine System to Methylphenidate-A Within-Subject I-123-ß-CIT-SPECT Study in Male Children and Adolescents With Attention-Deficit/Hyperactivity Disorder.

Background: Methylphenidate (MPH) is the first-line pharmacological treatment of attention-deficit/hyperactivity disorder (ADHD). MPH binds to the dopamine (DA) transporter (DAT), which has high density in the striatum. Assessments of the striatal dopamine transporter by single positron emission computed tomography (SPECT) in childhood and adolescent patients are rare but can provide insight on how the effects of MPH affect DAT availability. The aim of our within-subject study was to investigate the effect of MPH on DAT availability and how responsivity to MPH in DAT availability is linked to clinical symptoms and cognitive functioning. Methods: Thirteen adolescent male patients (9-16 years) with a diagnosis of ADHD according to the DSM-IV and long-term stimulant medication (for at least 6 months) with MPH were assessed twice within 7 days using SPECT after application of I-123-ß-CIT to examine DAT binding potential (DAT BP). SPECT measures took place in an on- and off-MPH status balanced for order across participants. A virtual reality continuous performance test was performed at each time point. Further clinical symptoms were assessed for baseline off-MPH. Results: On-MPH status was associated with a highly significant change (-29.9%) of striatal DAT BP as compared to off-MPH (t = -4.12, p = 0.002). A more pronounced change in striatal DAT BP was associated with higher off-MPH attentional and externalizing symptom ratings (Pearson r = 0.68, p = 0.01). Striatal DAT BP off-MPH, but not on-MPH, was associated with higher symptom ratings (Pearson r = 0.56, p = 0.04). Conclusion: Our findings corroborate previous reports from mainly adult samples that MPH changes striatal DAT BP availability and suggest higher off-MPH DAT BP, likely reflecting low baseline DA levels, as a marker of symptom severity.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Positron emission tomography has a high negative predictive value for progression or early relapse for patients with residual disease after first-line chemotherapy in advanced-stage Hodgkin lymphoma.

In the HD15 trial of the German Hodgkin Study Group, the negative predictive value (NPV) of positron emission tomography (PET) using [(18)F]-fluorodeoxyglucose in advanced-stage Hodgkin lymphoma (HL) was evaluated. A total of 817 patients were enrolled and randomly assigned to receive BEACOPP-based chemotherapy. After completion of chemotherapy, residual disease measuring more than or equal to 2.5 cm in diameter was assessed by PET in 311 patients. The NPV of PET was defined as the proportion of PET(-) patients without progression, relapse, or irradiation within 12 months after PET review panel. The progression-free survival was 96% for PET(-) patients (95% confidence interval [CI], 94%-99%) and 86% for PET(+) patients (95% CI, 78%-95%, P = .011). The NPV for PET in this analysis was 94% (95% CI, 91%-97%). Thus, consolidation radiotherapy can be omitted in PET(-) patients with residual disease without increasing the risk for progression or early relapse compared with patients in complete remission. The impact of this finding on the overall survival at 5 years must be awaited. Until then, response adapted therapy guided by PET for HL patients seems to be a promising approach that should be further evaluated in clinical trials. This trial is registered at http://isrctn.org study as #ISRCTN32443041.

Selective and rapid monitoring of dual platelet inhibition by aspirin and P2Y12 antagonists by using multiple electrode aggregometry.

BACKGROUND: Poor platelet inhibition by aspirin or clopidogrel has been associated with adverse outcomes in patients with cardiovascular diseases. A reliable and facile assay to measure platelet inhibition after treatment with aspirin and a P2Y12 antagonist is lacking. Multiple electrode aggregometry (MEA), which is being increasingly used in clinical studies, is sensitive to platelet inhibition by aspirin and clopidogrel, but a critical evaluation of MEA monitoring of dual anti-platelet therapy with aspirin and P2Y12 antagonists is missing. DESIGN AND METHODS: By performing in vitro and ex vivo experiments, we evaluated in healthy subjects the feasibility of using MEA to monitor platelet inhibition of P2Y12 antagonists (clopidogrel in vivo, cangrelor in vitro) and aspirin (100 mg per day in vivo, and 1 mM or 5.4 mM in vitro) alone, and in combination. Statistical analyses were performed by the Mann-Whitney rank sum test, student' t-test, analysis of variance followed by the Holm-Sidak test, where appropriate. RESULTS: ADP-induced platelet aggregation in hirudin-anticoagulated blood was inhibited by 99.3 +/- 1.4% by in vitro addition of cangrelor (100 nM; p < 0.001) and by 64 +/- 35% by oral clopidogrel (600 mg) intake (p < 0.05; values are means +/- SD). Pre-incubation of blood with aspirin (1 mM) or oral aspirin intake (100 mg/day for 1 week) inhibited arachidonic acid (AA)-stimulated aggregation >95% and 100 +/- 3.2%, respectively (p < 0.01). Aspirin did not influence ADP-induced platelet aggregation, either in vitro or ex vivo. Oral intake of clopidogrel did not significantly reduce AA-induced aggregation, but P2Y12 blockade by cangrelor (100 nM) in vitro diminished AA-stimulated aggregation by 53 +/- 26% (p < 0.01). A feasibility study in healthy volunteers showed that dual anti-platelet drug intake (aspirin and clopidogrel) could be selectively monitored by MEA. CONCLUSIONS: Selective platelet inhibition by aspirin and P2Y12 antagonists alone and in combination can be rapidly measured by MEA. We suggest that dual anti-platelet therapy with these two types of anti-platelet drugs can be optimized individually by measuring platelet responsiveness to ADP and AA with MEA before and after drug intake.

Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood.

Glycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen ("pure fibrin"), recombinant fibrinogen ("recombinant fibrin"), or generated more physiologically from endogenous fibrinogen in plasma ("plasma fibrin") or by exposing TF-coated surfaces to flowing blood ("blood fibrin"). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.

Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) and artherosclerosis share common characteristics in their pathogenesis. In this study, we investigated the effects of lipoproteins like native (n)-LDL, oxidized (ox)-LDL and high-density lipoprotein (HDL) on advanced senescence, extracellular matrix accumulation, cell loss, and transforming growth factor-beta2 (TGF-beta2) expression in cultured human retinal pigment epithelial (RPE) cells. Primary human RPE cells were incubated with 10-100 microg/ml n-LDL, ox-LDL, and HDL for 24h. For determination of advanced senescence, beta-galactosidase staining was used. The induction of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) mRNA was quantified by real-time PCR. Cell loss was investigated by live dead assay. Expression of TGF-beta2 was analyzed by real-time PCR and ELISA assays. Ox-LDL accelerated dose-dependently the onset of RPE senescence, whereas LDL and HDL had no effect. LDL and ox-LDL led to induced expression of Fn, Laa1 and Col4a2, whereas HDL had no influence. Incubation of RPE cells with 100 microg/ml ox-LDL induced marked cell death compared to untreated control cells. Expression of TGF-beta2 was dose-dependently increased by LDL and ox-LDL. LDL and ox-LDL induced cellular changes in RPE cells in vitro, which may resemble pathogenic events of AMD. These results may provide further information about the effects of LDL and ox-LDL in the human RPE and their potential role in the pathogenesis of AMD.

Substantia nigra echogenicity: A structural correlate of functional impairment of the dopaminergic striatal projection in Parkinson's disease.

Transcranial sonography (TCS) reveals abnormal spatial extension of substantia nigra (SN) echogenicity in a high proportion of patients with Parkinson's disease (PD). It has been proposed that this abnormality represents a structural trait that is mechanistically distinct from degeneration of dopaminergic nigrostriatal projection neurons. We sought to clarify the relationship between sonographic abnormalities of SN and dysfunction of striatal dopaminergic neurotransmission. We studied 50 patients with PD. The spatial extension of the echogenic SN area was compared with the activity of presynaptic striatal dopamine reuptake transporters, assessed in the same patients by I-123-2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane (beta-CIT) single-photon emission computed tomography (SPECT). Extension of echogenic SN area correlated (inversely) with striatal activity of presynaptic dopamine reuptake transporter in PD patients (R = -0.417; P = 0.003) and with the equivalent levodopa dose (R = 0.380; P = 0.006; linear regression analysis). Findings support the hypothesis that in PD abnormal extension of echogenic SN area provides a direct structural marker of degeneration of SN neurons. Therefore, in PD, TCS and beta-CIT assess pathophysiologically related phenomena.

Btk Inhibitors as First Oral Atherothrombosis-Selective Antiplatelet Drugs?

Bruton's tyrosine kinase (Btk) is essential for B cell differentiation and proliferation, but also platelets express Btk. Patients with X-linked agammaglobulinemia due to hereditary Btk deficiency do not show bleeding, but a mild bleeding tendency is observed in high dose therapy of B-cell malignancies with ibrutinib and novel second-generation irreversible Btk inhibitors (acalabrutinib and ONO/GS-4059). This review discusses recent studies that may explain this apparent paradox and gives mechanistic insights that suggest a unique potential of low dose irreversible Btk inhibitors as atherothrombosis-focused antiplatelet drugs.

Oral Bruton tyrosine kinase inhibitors block activation of the platelet Fc receptor CD32a (FcγRIIA): a new option in HIT?

Activation of the platelet Fc-receptor CD32a (FcγRIIA) is an early and crucial step in the pathogenesis of heparin-induced thrombocytopenia type II (HIT) that has not been therapeutically targeted. Downstream FcγRIIA Bruton tyrosine kinase (BTK) is activated; however, its role in Fc receptor-induced platelet activation is unknown. We explored the potential to prevent FcγRIIA-induced platelet activation by BTK inhibitors (BTKi's) approved (ibrutinib, acalabrutinib) or in clinical trials (zanubrutinib [BGB-3111] and tirabrutinib [ONO/GS-4059]) for B-cell malignancies, or in trials for autoimmune diseases (evobrutinib, fenebrutinib [GDC-0853]). We found that all BTKi's blocked platelet activation in blood after FcγRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcγRIIA cross-linking-induced platelet aggregation were for the irreversible BTKi's ibrutinib 0.08 µM, zanubrutinib 0.11 µM, acalabrutinib 0.38 µM, tirabrutinib 0.42 µM, evobrutinib 1.13 µM, and for the reversible BTKi fenebrutinib 0.011 µM. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKi's also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcγRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKi's. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcγRIIA activation. Platelet aggregation by adenosine 5'-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible BTKi's potently inhibit platelet activation by FcγRIIA in blood. This new rationale deserves testing in patients with HIT.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.

alpha-Tocopherol disturbs macrophage LXRalpha regulation of ABCA1/G1 and cholesterol handling.

Based on the oxidation hypothesis high doses of alpha-tocopherol have been advocated to prevent atherosclerosis, but clinical trials failed to demonstrate a benefit. As specific oxylipids activate PPARgamma and LXRalpha, master regulators of lipid metabolism and cholesterol exporters, we hypothesized, that high dose alpha-tocopherol might interfere with reverse cholesterol transport out of the vessel wall. Human THP-1 cells, a foam cell model, were preincubated with alpha-tocopherol or carrier before exposure to oxidized LDL, delipidated HDL or control buffer. Specific mRNAs were quantified by real-time RT-PCR, LXRalpha activation by a reporter gene assay and cellular cholesterol homeostasis by oxLDL and dHDL facilitated uptake and efflux assays. alpha-Tocopherol significantly reduced baseline expression and stimulation by oxLDL of LXRalpha activity, CD36, ABCA1, and ABCG1. alpha-Tocopherol also reversed the suppression of CD36 and ABCA1 by dHDL. Thus alpha-Tocopherol compromises cellular lipid scavenging and channelling of cholesterol into reverse transport out of the vessel wall.

Effect of chronic treatment with acetylsalicylic acid and clopidogrel on atheroprogression and atherothrombosis in ApoE-deficient mice in vivo.

Acetylsalicylic acid (ASA) and the thienopyridine clopidogrel are established anti-platelet drugs that significantly reduce secondary cardiovascular events in patients with manifest atherosclerosis. However, their impact on atherosclerotic lesion development remains controversial. Four-week-old ApoE-deficient mice were randomly assigned to four groups receiving a cholesterol diet together with either ASA (5 mg/kg), or clopidogrel (25 mg/kg), or a combination of both ASA and clopidogrel, or vehicle for 8-12 weeks. Using intravital microscopy we found that daily administration of ASA in combination with clopidogrel reduces platelet thrombus formation following rupture of atherosclerotic plaque in vivo by approximately 50%. However, therapy with ASA or clopidogrel alone, or in combination for a period of 8-12 weeks had no significant effect on adhesion of platelets to dysfunctional endothelial cells or on atherosclerotic lesion formation in the aortic root or the carotid artery. In conclusion, anti-platelet therapy is effective in reducing platelet adhesion and subsequent thrombus formation following rupture of atherosclerotic plaque in vivo. However, our data do not support a role of either drug in the primary prevention of atherosclerosis in ApoE-deficient mice.

Dimeric Glycoprotein VI Binds to Collagen but Not to Fibrin.

Platelet glycoprotein VI (GPVI) acts as a decisive collagen receptor in atherothrombosis. Besides collagen, injured atherosclerotic plaques expose tissue factor (TF) that triggers fibrin formation. Two recent studies reported that platelet GPVI also functions as fibrin receptor, which would importantly widen the mode of action of GPVI-targeted antithrombotic drugs. We studied the binding of two GPVI fusion proteins to fibrin under static and arterial flow conditions. Fibrin was prepared from purified fibrinogen or generated more physiologically from endogenous fibrinogen by coagulating plasma with thrombin. Fibrin formation was also triggered by exposing TF-coated surfaces or human atherosclerotic plaque slices to arterially flowing blood. By binding studies and advanced optical imaging, we found that recombinant dimeric GPVI-Fc fusion proteins with Fc from either IgG1 (GPVI-Fc1) or IgG2 (GPVI-Fc2) bound to collagen fibres, but neither to fibrin prepared from purified fibrinogen obtained from three suppliers, nor to physiological fibrin formed by thrombin in plasma or triggered by exposing TF or atherosclerotic plaque slices to arterially flowing blood. Our findings do not support a role of dimeric platelet GPVI as receptor for fibrin. This is important for the understanding of plaque-triggered platelet thrombus formation and is clinically relevant for future GPVI-targeting therapies with recombinant GPVI-Fc and anti-GPVI antibodies.

Oxidation products of stigmasterol interfere with the action of the female sex hormone 17beta-estradiol in cultured human breast and endometrium cell lines.

Phytosterols are constituents of plant membranes and are thus contained in low concentrations in vegetable products as well as at high concentrations in functional food designed to reduce serum cholesterol levels. Similar to ChOL, phytosterols are oxidized chemically in food and by biotransformation in vivo. Although oxyphytosterols have been detected in the serum of healthy human subjects, little is known of their biological activity. Therefore, the estrogenic and antiestrogenic activities of a mixture of six oxidation products of stigmasterol (oxy-StOL) were determined at the following endpoints: (i) the affinity to isolated human estrogen receptors (ER), (ii) the basal and 17beta-estradiol (E2)-induced expression of the alkaline phosphatase (AlP) in human endometrial adenocarcinoma (Ishikawa) cells, and (iii) the basal and E2-induced proliferation of human breast adenocarcinoma (MCF-7) cells. Oxy-StOL was able to replace E2 from human ERalpha and ERbeta and induced a weak estrogenic response in MCF-7 cells. Moreover, the E2-induced activity of the AlP in Ishikawa cells as well as the E2-induced proliferation of MCF-7 cells were decreased at noncytotoxic concentrations (up to 10 microM), indicating that at least one component of oxy-StOL represents an estrogen-active compound which might interfere with endogenous estrogens.