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      Cells Derived from Regenerated Endothelium of the Porcine Coronary Artery Contain More Oxidized Forms of Apolipoprotein-B-100 without a Modification in the Uptake of Oxidized LDL

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          Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1’-dioctadecyl-3,3,3’,3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC<sub>50</sub> and E<sub>max</sub>, were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation.

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          Most cited references 6

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          The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis.

          RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.
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            LOX-1, the receptor for oxidized low-density lipoprotein identified from endothelial cells: implications in endothelial dysfunction and atherosclerosis.

            Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) was initially identified as the major receptor for oxidized LDL (OxLDL) in endothelial cells. Its inducible expression in macrophages and smooth muscle cell was also observed. LOX-1 is a Type II membrane protein with a typical C-type lectin structure at the extracellular C-terminus. It can be cleaved by an unknown protease at the extracellular juxtamembrane region to release the soluble form of LOX-1. The extracellular domains of LOX-1 are post-translationally modified by N-linked glycosylation. Mutagenesis studies revealed that the lectin domain of LOX-1 is the functional domain that recognizes the LOX-1 ligand. The C-terminal end residues and several conserved positively charged residues spanning the lectin domain are essential for OxLDL binding. LOX-1 activation by OxLDL causes endothelial changes that are characterized by activation of nuclear factor-kappaB through an increased reactive oxygen species, subsequent induction of adhesion molecules, and endothelial apoptosis. In vitro, expression of LOX-1 is induced by many inflammatory cytokines, oxidative stress, hemodynamic stimuli, and OxLDL. In vivo, the expression is enhanced in pro-atherogenic settings including, hypertension, hyperlipidemia, and diabetes, and, indeed, is accumulated in the atherosclerotic and glomerulosclerotic lesions. LOX-1 binds multiple classes of ligands that are implicated in the pathogenesis of atherosclerosis. Besides OxLDL, LOX-1 can recognize apoptotic/aged cells, activated platelets, and bacteria, implying versatile physiological functions. Taken together, all these findings support the possible contribution of LOX-1 to the pathogenesis of vascular disorders, particularly atherosclerosis. Development of antagonists for LOX-1 might be a good therapeutic approach to vascular diseases.
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              A colorimetric microtiter assay for the quantitation of cytokine activity on adherent cells in tissue culture.

               Yona Keisari (1992)
              A colorimetric microtiter assay was developed for the quantitation of adherent cells in culture, which is based on the staining of cells with a commercially available staining kit. Adherent L929, A375 cell lines and human monocytes were stained with Hemacolor reagents and the color eluted with SDS 0.5% was determined spectrophotometrically with an ELISA plate reader at 630 nm. The method enabled the detection of cells and was linear up to 3 x 10(4) L929 cells/well. The Hemacolor staining assay was compared to the crystal violet staining assay and the MTT reduction assay, and was found to be sensitive, accurate and reproducible, and has the advantage of enabling microscopic inspection of the stained cells prior to color elution. The assay was found to be suitable for the determination of cytotoxic cytokines, and the enumeration of adherent monocytes. This method might be also used for the quantitation of cytotoxic drugs, and the cytophatic activity of viruses.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                August 2003
                26 September 2003
                : 40
                : 4
                : 389-398
                aInstitut de Recherches Servier, Suresnes, et bInstitut de Recherches International Servier, Courbevoie, France
                72817 J Vasc Res 2003;40:389–398
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 2, References: 43, Pages: 10
                Research Paper


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