In vitro head-to-head comparison of anticoagulation properties of two heparin brands in a human blood miniature mock loop.

OBJECTIVES: The first aim was the development of a human blood miniature mock-loop system consisting of 2 identical extracorporeal circuits, which enable systematic head-to-head comparisons of test substances. In a second step, we evaluated the suitability of the mock-loop system, by comparing 2 different brands of heparin (ROTEXMEDICA vs B.BRAUN), which have showed different anticoagulation capacities in the clinic. METHODS: For 1 experiment (18 in total), blood of the same healthy human donor was divided into 2 portions (2 × 50 ml), heparinized with 37.5 IU⋅ml-1 of the competing test substances and diluted to a haematocrit value of 20-25%. Each mock loop was filled with 70 ml, and in vivo heparin degradation was simulated in 3 different groups by protamine application, representing 0%, 50% and 100% heparin antagonization. At baseline, 5, 60, 120, 240 and 360 min, blood samples were taken to perform thromboelastometry, flow cytometry, haemolysis and general haemostasis analysis. RESULTS: Blood pressure, blood flow and blood temperature within the loops remained stable for 6 h in all groups. After 6 h, in the 100% antagonized ROTEXMEDICA heparin group, significantly increased haemolysis (148.7 ± 80 mg⋅dl-1 vs 57.5 ± 15.8 mg⋅dl-1), activated platelets (8 ± 3.8% vs 3.3 ± 0.7%), D-dimers (7376 ± 7144 ng ml-1 vs 576.2 ± 190 ng ml-1) and fulminant blood clots were detected. CONCLUSIONS: Our in vitro system is suitable for the detection of reduced anticoagulation capacity of a test drug, which was reported in vivo previously.

Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

Characterization of extracellular vesicles derived from cardiac cells in an in vitro model of preconditioning.

Preconditioning is a promising technique to protect the heart from ischaemia-reperfusion injury. In this context, the crosstalk between different cardiac cell types and especially the exchange of cardioprotective mediators has come into the focus of current research. Recently, extracellular vesicles (EVs), nano-sized structures, emerged as possible communication mediators. They are taken up by recipient cells and can alter gene expression or activate intracellular signal cascades. It has been shown that all cardiac cell types are able to secrete EVs, but so far the influence of an in vitro preconditioning stimulus on EV concentration and composition has not been investigated. Therefore, we stimulated primary cardiac myocytes and fibroblasts from neonatal rats, as well as H9c2 cells, with two known in vitro preconditioning stimuli: hypoxia or isoflurane. EVs were isolated from cell culture supernatants 48 h after stimulation by differential centrifugation and size exclusion chromatography. They were characterized by transmission electron microscopy, tunable resistive pulse sensing, miRNA array and Western blot analysis. The detected EVs had the typical cup-shaped morphology and a size of about 150 nm. No significant differences in EV concentration were observed between the different groups. The protein and miRNA load was affected by in vitro preconditioning with isoflurane or hypoxia. EV markers like Alix, CD63, flotillin-1 and especially heat shock protein 70 were significantly up-regulated by the treatments. Several miRNAs like miR-92b-3p, miR-761 and miR-101a-5p were also significantly affected. A migration assay confirmed the physiological benefit of these EVs. Taken together, our findings show that a model of in vitro preconditioning of cardiac cells does not influence EV concentration but strongly regulates the EV cargo and affects migration. This might indicate a role for EV-mediated communication in isoflurane- and hypoxia-induced in vitro preconditioning.

Selenium and its supplementation in cardiovascular disease--what do we know?

The trace element selenium is of high importance for many of the body's regulatory and metabolic functions. Balanced selenium levels are essential, whereas dysregulation can cause harm. A rapidly increasing number of studies characterizes the wide range of selenium dependent functions in the human body and elucidates the complex and multiple physiological and pathophysiological interactions of selenium and selenoproteins. For the majority of selenium dependent enzymes, several biological functions have already been identified, like regulation of the inflammatory response, antioxidant properties and the proliferation/differentiation of immune cells. Although the potential role of selenium in the development and progression of cardiovascular disease has been investigated for decades, both observational and interventional studies of selenium supplementation remain inconclusive and are considered in this review. This review covers current knowledge of the role of selenium and selenoproteins in the human body and its functional role in the cardiovascular system. The relationships between selenium intake/status and various health outcomes, in particular cardiomyopathy, myocardial ischemia/infarction and reperfusion injury are reviewed. We describe, in depth, selenium as a biomarker in coronary heart disease and highlight the significance of selenium supplementation for patients undergoing cardiac surgery.