Pro-atherogenic actions of signal transducer and activator of transcription 1 serine 727 phosphorylation in LDL receptor deficient mice via modulation of plaque inflammation.

Atherosclerosis is a chronic inflammatory disorder of the vasculature regulated by cytokines. We have previously shown that extracellular signal-regulated kinase-1/2 (ERK1/2) plays an important role in serine 727 phosphorylation of signal transducer and activator of transcription-1 (STAT1) transactivation domain, which is required for maximal interferon-γ signaling, and the regulation of modified LDL uptake by macrophages in vitro. Unfortunately, the roles of ERK1/2 and STAT1 serine 727 phosphorylation in atherosclerosis are poorly understood and were investigated using ERK1 deficient mice (ERK2 knockout mice die in utero) and STAT1 knock-in mice (serine 727 replaced by alanine; STAT1 S727A). Mouse Atherosclerosis RT² Profiler PCR Array analysis showed that ERK1 deficiency and STAT1 S727A modification produced significant changes in the expression of 18 and 49 genes, respectively, in bone marrow-derived macrophages, with 17 common regulated genes that included those that play key roles in inflammation and cell migration. Indeed, ERK1 deficiency and STAT1 S727A modification attenuated chemokine-driven migration of macrophages with the former also impacting proliferation and the latter phagocytosis. In LDL receptor deficient mice fed a high fat diet, both ERK1 deficiency and STAT1 S727A modification produced significant reduction in plaque lipid content, albeit at different time points. The STAT1 S727A modification additionally caused a significant reduction in plaque content of macrophages and CD3 T cells and diet-induced cardiac hypertrophy index. In addition, there was a significant increase in plasma IL-2 levels and a trend toward increase in plasma IL-5 levels. These studies demonstrate important roles of STAT1 S727 phosphorylation in particular in the regulation of atherosclerosis-associated macrophage processes in vitro together with plaque lipid content and inflammation in vivo, and support further assessment of its therapeutical potential.

Non-random distribution of vacuoles in Schizosaccharomyces pombe.

A central question in eukaryotic cell biology asks, during cell division, how is the growth and distribution of organelles regulated to ensure each daughter cell receives an appropriate amount. For vacuoles in budding yeast, there are well described organelle-to-cell size scaling trends as well as inheritance mechanisms involving highly coordinated movements. It is unclear whether such mechanisms are necessary in the symmetrically dividing fission yeast, Schizosaccharomyces pombe, in which random partitioning may be utilized to distribute vacuoles to daughter cells. To address the increasing need for high-throughput analysis, we are augmenting existing semi-automated image processing by developing fully automated machine learning methods for locating vacuoles and segmenting fission yeast cells from brightfield and fluorescence micrographs. All strains studied show qualitative correlations in vacuole-to-cell size scaling trends, i.e. vacuole volume, surface area, and number all increase with cell size. Furthermore, increasing vacuole number was found to be a consistent mechanism for the increase in total vacuole size in the cell. Vacuoles are not distributed evenly throughout the cell with respect to available cytoplasm. Rather, vacuoles show distinct peaks in distribution close to the nucleus, and this preferential localization was confirmed in mutants in which nucleus position is perturbed. Disruption of microtubules leads to quantitative changes in both vacuole size scaling trends and distribution patterns, indicating the microtubule cytoskeleton is a key mechanism for maintaining vacuole structure.

Towards computer-aided design of cellular structure.

Cells are complex machines with tremendous potential for applications in medicine and biotechnology. Although much effort has been devoted to engineering the metabolic, genetic, and signaling pathways of cells, methods for systematically engineering the physical structure of cells are less developed. Here we consider how coarse-grained models for cellular geometry at the organelle level can be used to build computer-aided design (CAD) tools for cellular structure.

Classroom sound can be used to classify teaching practices in college science courses.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

High-resolution restoration of 3D structures from widefield images with extreme low signal-to-noise-ratio.

Four-dimensional fluorescence microscopy--which records 3D image information as a function of time--provides an unbiased way of tracking dynamic behavior of subcellular components in living samples and capturing key events in complex macromolecular processes. Unfortunately, the combination of phototoxicity and photobleaching can severely limit the density or duration of sampling, thereby limiting the biological information that can be obtained. Although widefield microscopy provides a very light-efficient way of imaging, obtaining high-quality reconstructions requires deconvolution to remove optical aberrations. Unfortunately, most deconvolution methods perform very poorly at low signal-to-noise ratios, thereby requiring moderate photon doses to obtain acceptable resolution. We present a unique deconvolution method that combines an entropy-based regularization function with kernels that can exploit general spatial characteristics of the fluorescence image to push the required dose to extreme low levels, resulting in an enabling technology for high-resolution in vivo biological imaging.

Organelle size scaling of the budding yeast vacuole is tuned by membrane trafficking rates.

Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates.

(+)-Catechin Attenuates Multiple Atherosclerosis-Associated Processes In Vitro, Modulates Disease-Associated Risk Factors in C57BL/6J Mice and Reduces Atherogenesis in LDL Receptor Deficient Mice by Inhibiting Inflammation and Increasing Markers of Plaque Stability.

SCOPE: A prospective study of 34492 participants shows an inverse association between (+)-catechin intake and coronary heart disease. The effects of (+)-catechin on atherosclerosis and associated risk factors are poorly understood and are investigated. METHODS AND RESULTS: (+)-Catechin attenuates reactive oxygen species production in human macrophages, endothelial cells and vascular smooth muscle cells, chemokine-driven monocytic migration, and proliferation of human macrophages and their expression of several pro-atherogenic genes. (+)-Catechin also improves oxidized LDL-mediated mitochondrial membrane depolarization in endothelial cells and attenuates growth factor-induced smooth muscle cell migration. In C57BL/6J mice fed high fat diet (HFD) for 3 weeks, (+)-catechin attenuates plasma levels of triacylglycerol and interleukin (IL)-1ß and IL-2, produces anti-atherogenic changes in liver gene expression, and reduces levels of white blood cells, myeloid-derived suppressor cells, Lin- Sca+ c-Kit+ cells, and common lymphoid progenitor cells within the bone marrow. In LDL receptor deficient mice fed HFD for 12 weeks, (+)-catechin attenuates atherosclerotic plaque burden and inflammation with reduced macrophage content and increased markers of plaque stability; smooth muscle cell and collagen content. CONCLUSION: This study provides novel, detailed insights into the cardio-protective actions of (+)-catechin together with underlying molecular mechanisms and supports further assessments of its beneficial effects in human trials.

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides.

Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5'-coupled conjugate and the other the 3'-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.

Key Roles of Inflammation in Atherosclerosis: Mediators Involved in Orchestrating the Inflammatory Response and Its Resolution in the Disease Along with Therapeutic Avenues Targeting Inflammation.

Inflammation is a critical driver of all stages of atherosclerosis, from lesion development to plaque rupture. Cytokines are mediators of the immune response and in atherosclerosis, the balance of anti- and pro-inflammatory cytokines is tipped in favor of the latter, resulting in persistent and unresolved inflammation. Although reducing plasma cholesterol levels mainly via the use of statins has positively impacted patient outcomes and reduced mortality rates, the presence of significant residual inflammation and cardiovascular risk posttherapy emphasizes the prevailing risk of primary and secondary events driven by inflammation independently of hyperlipidemia. Given the dominant role of inflammation in driving pathogenesis, alternative therapeutic avenues beyond targeting lowering of plasma lipids are required. This chapter will discuss the role of inflammation and pro-inflammatory cytokines in driving atherogenesis and disease progression, the therapeutic potential of targeting cytokines for atherosclerosis and promising avenues in this area.

Atherosclerosis: Pathogenesis and Key Cellular Processes, Current and Emerging Therapies, Key Challenges, and Future Research Directions.

Atherosclerosis is the principal cause of cardiovascular disease that continues to be a substantial drain on healthcare systems, being responsible for about 31% of all global deaths. Atherogenesis is influenced by a range of factors, including oxidative stress, inflammation, hypertension, and hyperlipidemia, and is ultimately driven by the accumulation of low-density lipoprotein cholesterol within the arterial wall of medium and large arteries. Lipoprotein accumulation stimulates the infiltration of immune cells (such as monocytes/macrophages and T-lymphocytes), some of which take up the lipoprotein, leading to the formation of lipid-laden foam cells. Foam cell death results in increased accumulation of dead cells, cellular debris and extracellular cholesterol, forming a lipid-rich necrotic core. Vascular smooth muscle cells from the arterial media also migrate into the intima layer and proliferate, taking up the available lipids to become foam cells and producing extracellular matrix proteins such as collagen and elastin. Plaque progression is characterized by the formation of a fibrous cap composed of extracellular matrix proteins and smooth muscle cells, which acts to stabilize the atherosclerotic plaque. Degradation, thinning, and subsequent rupture of the fibrous cap leads to lumen-occlusive atherothrombosis, most commonly resulting in heart attack or stroke. This chapter describes the pathogenesis of atherosclerosis, current and emerging therapies, key challenges, and future directions of research.

Investigation of Mitochondrial Bioenergetic Profile and Dysfunction in Atherosclerosis.

Mitochondrial function and activity are key indicators of overall cell health and mitochondrial dysfunction is closely associated with disruptions in normal cellular function. Altered mitochondrial function and cellular metabolism has been implicated in processes involved in ageing and associated pathologies. In atherosclerosis, compromised mitochondrial respiration can promote plaque instability and other processes that encourage pathogenesis and dysfunction. For example, increasing respiration promotes vascular smooth muscle cell (VSMC) proliferation and attenuates macrophage and VSMC apoptosis. Use of Agilent Seahorse technology to study mitochondrial bioenergetics has largely replaced previous outdated methods which provided limited insight into mitochondrial function and were associated with various issues. This chapter describes the use of Seahorse Agilent technology (Mito Stress Test) to study key parameters of mitochondrial respiration on cultured cells relevant to atherosclerosis.

Probing Inflammasome Activation in Atherosclerosis.

Atherosclerosis is driven by chronic inflammation in all stages of the disease. Inflammation is fueled by elevated levels of pro-inflammatory cytokines. Interleukins (IL) are cytokines of particular importance in atherosclerosis, due to their key involvement in various pro-atherogenic processes, including infiltration of immune cells to the lesion, stimulation of the production of other pro-inflammatory mediators by other sources, and generation of lipid laden foam cells, all of which contribute to plaque development and progression. Various stimuli that are abundant in atherosclerotic plaques, including oxidized low-density lipoprotein, cholesterol crystals and reactive oxygen species can trigger inflammasome activation. Importantly, activation of the nucleotide oligomerization domain leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activates the caspase-1 protease and results in the generation and release of potent pro-inflammatory cytokines, IL-1ß and IL-18. Both cytokines are influential in driving chronic inflammation and atherogenesis. This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) and Western blot to quantify these cytokines in cell supernatant and lysate respectively, after stimulating inflammasome activation in cultured cells.

Evaluation of Plaque Burden and Lipid Content in Atherosclerotic Plaques.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, fibrous plaques within the arterial wall of medium and large arteries. Plaques prone to rupture are typically rich in lipids and pro-inflammatory markers. Cells within the plaque can take up lipids via different mechanisms leading to the formation and accumulation of lipid-rich foam cells, a key hallmark of the disease. Evaluation of plaque burden and lipid content is hence important to determine disease progression and severity. This chapter describes the most commonly used staining methods that enable visualization and analysis of mouse atherosclerotic plaques. These methods include en face preparation of mouse aorta, and staining sections of arteries using hematoxylin and eosin, Oil Red O, and Masson's Trichrome.

Monitoring Cellularity and Expression of Key Markers in Atherosclerotic Plaques.

Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same individual, and can vary depending on its anatomical location within the vascular bed. Early in the disease and throughout its progression, immune cells infiltrate the lesion, contributing to the plaque phenotype via different mechanisms. Detailed characterization of constituent cell populations within plaques is hence required for more accurate assessment of disease severity and inflammatory burden. A wide range of fluorophore-conjugated antibodies targeted to key cell types implicated in all stages of the disease are commercially available, enabling visualization of the dynamic cellular landscape present within lesions. This chapter describes the use of immunofluorescence staining of atherosclerotic plaque sections to study plaque cellularity and expression of key markers.

Survey of Approaches for Investigation of Atherosclerosis In Vivo.

Although in vitro model systems are useful for investigation of atherosclerosis-associated processes, they represent simplification of complex events that occur in vivo, which involve interactions between many different cell types together with their environment. The use of animal model systems is important for more in-depth insights of the molecular mechanisms underlying atherosclerosis and for identifying potential targets for agents that can prevent plaque formation and even reverse existing disease. This chapter will provide a survey of such animal models and associated techniques that are routinely used for research of atherosclerosis in vivo.

The Lab4P Consortium of Probiotics Attenuates Atherosclerosis in LDL Receptor Deficient Mice Fed a High Fat Diet and Causes Plaque Stabilization by Inhibiting Inflammation and Several Pro-Atherogenic Processes.

SCOPE: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduces diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and is therefore investigated. METHODS AND RESULTS: Atherosclerosis-associated parameters are analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increases plasma HDL and triglyceride levels and decreases LDL/VLDL levels. Lab4P also reduces plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increases smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays show that Lab4P alters the liver expression of 19 key disease-associated genes. Lab4P also decreases the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrates attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. CONCLUSION: This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials.

Protective effects of a unique combination of nutritionally active ingredients on risk factors and gene expression associated with atherosclerosis in C57BL/6J mice fed a high fat diet.

Atherosclerosis, an inflammatory disorder of the vasculature and the underlying cause of cardiovascular disease, is responsible for one in three global deaths. Consumption of active food ingredients such as omega-3 polyunsaturated fatty acids, flavanols and phytosterols has many beneficial effects on cardiovascular disease. However, their combined actions on the risk factors for atherosclerosis remains poorly understood. We have previously shown that a formulation containing each of these active components at physiologically relevant doses modulated several monocyte/macrophage processes associated with atherosclerosis in vitro, including inhibition of cytokine-induced pro-inflammatory gene expression, chemokine-driven monocyte migration, expression of M1 phenotype markers, and promotion of cholesterol efflux. The objectives of the present study were to investigate whether the protective actions of the formulation extended in vivo and to delineate the potential underlying mechanisms. The formulation produced several favourable changes, including higher plasma levels of HDL and reduced levels of macrophages and myeloid-derived suppressor cells in the bone marrow. The mRNA expression of liver-X-receptor-α, peroxisome proliferator-activated receptor-γ and superoxide dismutase-1 was induced in the liver and that of interferon-γ and the chemokine (C-X-C motif) ligand 1 decreased, thereby suggesting the potential mechanisms for many beneficial effects. Other changes were also observed such as increased plasma levels of triglycerides and lipid peroxidation that may reflect potential activation of brown fat. This study provides new insights into the protective actions and the potential underlying mechanisms of the formulation in vivo, particularly in relation to risk factors together with changes in systemic inflammation and hepatic lipid alterations associated with atherosclerosis and metabolic syndrome, and supports further assessments in human trials.

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers.

Membrane-membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, P(dock), that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, P(dock) shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion.

A perspective on targeting inflammation and cytokine actions in atherosclerosis.

Atherosclerosis, a chronic inflammatory disorder of the vasculature that results in cardiovascular disease, continues to pose a significant health and economic burden on modern society. Whilst inflammation has generally been accepted as the key driver of all stages of the disease, it was not until recently that inhibition of a specific proinflammatory cytokine (IL-1ß) yielded successful results in the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study trial. This article offers a perspective on targeting inflammation for atherosclerosis, focusing on results of recent Phase III clinical trials, and discusses other potential candidates together with future challenges and prospects.

DNA-tethered membranes formed by giant vesicle rupture.

We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17-FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.