Characterization of Atherosclerotic Plaque Coating for Thrombosis Microfluidics Assays.

INTRODUCTION: Studying arterial thrombus formation by in vitro flow assays is a widely used approach. Incorporating human atherosclerotic plaque material as a thrombogenic surface in these assays represents a method to model the pathophysiological environment of thrombus formation upon plaque disruption. Up until now, achieving a homogeneous coating of plaque material and subsequent reproducible platelet adhesion has been challenging. Here, we characterized a novel method for coating of plaque material on glass coverslips for use in thrombosis microfluidic assays. METHODS: A homogenate of human atherosclerotic plaques was coated on glass coverslips by conventional manual droplet coating or by spin coating. Prior to coating, a subset of coverslips was plasma treated. Water contact angle measurements were performed as an indicator for the hydrophilicity of the coverslips. Homogeneity of plaque coatings was determined using profilometric analysis and scanning electron microscopy. Thrombogenicity of the plaque material was assessed in real time by microscopic imaging while perfusing whole blood at a shear rate of 1500 s-1 over the plaque material. RESULTS: Plasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. The most homogeneous plaque coating and highest platelet adhesion was obtained upon plasma treatment followed by spin coating of the plaque material. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation. CONCLUSION: Spin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when compared to conventional droplet coating on non-plasma treated coverslips. These properties are beneficial in ensuring the quality and reproducibility of flow experiments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00713-9.

Targeting of mitochondrial reactive oxygen species production does not avert lipid-induced insulin resistance in muscle tissue from mice.

AIMS/HYPOTHESIS: High-fat, high-sucrose diet (HF)-induced reactive oxygen species (ROS) levels are implicated in skeletal muscle insulin resistance and mitochondrial dysfunction. Here we investigated whether mitochondrial ROS sequestering can circumvent HF-induced oxidative stress; we also determined the impact of any reduced oxidative stress on muscle insulin sensitivity and mitochondrial function. METHODS: The Skulachev ion (plastoquinonyl decyltriphenylphosphonium) (SkQ), a mitochondria-specific antioxidant, was used to target ROS production in C2C12 muscle cells as well as in HF-fed (16 weeks old) male C57Bl/6 mice, compared with mice on low-fat chow diet (LF) or HF alone. Oxidative stress was measured as protein carbonylation levels. Glucose tolerance tests, glucose uptake assays and insulin-stimulated signalling were determined to assess muscle insulin sensitivity. Mitochondrial function was determined by high-resolution respirometry. RESULTS: SkQ treatment reduced oxidative stress in muscle cells (-23% p < 0.05), but did not improve insulin sensitivity and glucose uptake under insulin-resistant conditions. In HF mice, oxidative stress was elevated (56% vs LF p < 0.05), an effect completely blunted by SkQ. However, HF and HF+SkQ mice displayed impaired glucose tolerance (AUC HF up 33%, p < 0.001; HF+SkQ up 22%; p < 0.01 vs LF) and disrupted skeletal muscle insulin signalling. ROS sequestering did not improve mitochondrial function. CONCLUSIONS/INTERPRETATION: SkQ treatment reduced muscle mitochondrial ROS production and prevented HF-induced oxidative stress. Nonetheless, whole-body glucose tolerance, insulin-stimulated glucose uptake, muscle insulin signalling and mitochondrial function were not improved. These results suggest that HF-induced oxidative stress is not a prerequisite for the development of muscle insulin resistance.

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice.

OBJECTIVE: In conditions of continuous high-fat (HF) intake, the degree of saturation of the fatty acids (FAs) in the diet might have a crucial role in the onset of obesity and its metabolic complications. In particular, the FA composition of the diet might influence the storage form of lipids inside skeletal muscle. The aim of the present study was to examine whether the FA composition of HF diets differentially affects weight gain and accumulation of myocellular triacylglycerol (TAG) and diacylglycerol (DAG). Furthermore, we examined whether the FA composition of the diet was reflected in the composition of the myocellular lipid intermediates. DESIGN: C57Bl6 mice were fed HF diets (45% energy) mainly containing palm oil (PO), cocoa butter (CB), olive oil (OO) or safflower oil (SO; n=6 per group) for 8 weeks. A low-fat diet (10% energy, PO) was used as control. Body weight was monitored weekly. At the end of the dietary intervention, myocellular TAG and DAG content and profiles were measured. RESULTS: We here show that HF_CB prevented weight gain after 8 weeks of HF feeding. Furthermore, the HF diet rich in SO prevented the accumulation of both myocellular TAG and DAG. Interestingly, the FA composition of DAG and TAG in skeletal muscle was a reflection of the dietary FA composition. CONCLUSION: Already after a relatively short period, the dietary FA intake relates to the FA composition of the lipid metabolites in the muscle. A diet rich in polyunsaturated FAs seems to prevent myocellular lipid accumulation.

Predicting preparatory behaviours for condom use in female undergraduate students: a one-year follow-up study.

The objective of this study is to investigate whether the Theory of Planned Behaviour (i.e. attitude, subjective norm, perceived behavioural control and intention), fluctuations in motivation over time, and variables from the Prototype-Willingness Model (i.e. behavioural expectation and behavioural willingness to have unprotected sex) predict preparatory behaviours for condom use. Sixty-two female undergraduates completed baseline and one-year follow-up questionnaires. Having condoms at home and carrying condoms were predicted by behavioural willingness to have unsafe sex at baseline. Having bought condoms was predicted by the behavioural expectation to use condoms with new partners at baseline. Intention and fluctuations in motivation did not emerge as significant predictors of preparatory actions. Female undergraduates, who are more willing to have unprotected sex under risk-conducive circumstances, are also less likely to prepare adequately for condom use, and thereby increase their chances of encountering such situations. Overall, the findings are in support of the Prototype-Willingness Model.