A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 µl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 µl/cm(2) in 24-well (10 µl) or 96-well (2 µl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

Transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis C virus infection in vitro.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. CONCLUSION: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo.

Uptake and trafficking of liposomes to the endoplasmic reticulum.

Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low-density lipoprotein receptors for endocytosis and enter cells through a caveolin- and microtubule-dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22-25 min, which was confirmed by fluorescence-dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER-assembling entities, such as the ER-budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV-1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.

High-fat meals rich in EPA plus DHA compared with DHA only have differential effects on postprandial lipemia and plasma 8-isoprostane F2α concentrations relative to a control high-oleic acid meal: a randomized controlled trial.

BACKGROUND: Eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) supplementation has beneficial cardiovascular effects, but postprandial influences of these individual fatty acids are unclear. OBJECTIVES: The primary objective was to determine the vascular effects of EPA + DHA compared with DHA only during postprandial lipemia relative to control high-oleic acid meals; the secondary objective was to characterize the effects of linoleic acid-enriched high-fat meals relative to the control meal. DESIGN: We conducted a randomized, controlled, double-blind crossover trial of 4 high-fat (75-g) meals containing 1) high-oleic acid sunflower oil (HOS; control), 2) HOS + fish oil (FO; 5 g EPA and DHA), 3) HOS + algal oil (AO; 5 g DHA), and 4) high-linoleic acid sunflower oil (HLS) in 16 healthy men (aged 35-70 y) with higher than optimal fasting triacylglycerol concentrations (mean ± SD triacylglycerol, 1.9 ± 0.5 mmol/L). RESULTS: Elevations in triacylglycerol concentration relative to baseline were slightly reduced after FO and HLS compared with the HOS control (P < 0.05). The characteristic decrease from baseline in plasma nonesterified fatty acids after a mixed meal was inhibited after AO (Δ 0-3 h, P < 0.05). HLS increased the augmentation index compared with the other test meals (P < 0.05), although the digital volume pulse-reflection index was not significantly different. Plasma 8-isoprostane F2α analysis revealed opposing effects of FO (increased) and AO (reduced) compared with the control (P < 0.05). No differences in nitric oxide metabolites were observed. CONCLUSIONS: These data show differential postprandial 8-isoprostane F2α responses to high-fat meals containing EPA + DHA-rich fish oil compared with DHA-rich AO, but these differences were not associated with consistent effects on postprandial vascular function or lipemia. More detailed analyses of polyunsaturated fatty acid-derived lipid mediators are required to determine possible divergent functional effects of single meals rich in either DHA or EPA. This trial was registered at clinicaltrials.gov as NCT01618071.