Intracellular mechanisms in the activation of human platelets by low-density lipoproteins.
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ABSTRACT:
SUBMITTER: Andrews HE
PROVIDER: S-EPMC1147741 | biostudies-other | 1987 Mar
REPOSITORIES: biostudies-other
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Project description:Platelets and lipoproteins play a crucial role in atherogenesis, in part by their ability to modulate inflammation and oxidative stress. While oxidized low density lipoproteins (OxLDL) play a central role in the development of this disease, high density lipoproteins (HDL) represent an atheroprotective factor of utmost importance. As platelet function is remarkably sensitive to the influence of plasma lipoproteins, it was the aim of this study to clarify if HDL are able to counteract the stimulating effects of OxLDL with special emphasis on aspects of platelet function that are relevant to inflammation. Therefore, HDL were tested for their ability to interfere with pro-thrombotic and pro-inflammatory aspects of platelet function. We are able to show that HDL significantly impaired OxLDL-induced platelet aggregation and adhesion. In gel-filtered platelets, HDL decreased both the formation of reactive oxygen species and CD40L expression. Furthermore, HDL strongly interfered with OxLDL-induced formation of platelet-neutrophil aggregates in whole blood, suggesting that platelets represent a relevant and sensitive target for HDL. The finding that HDL effectively competed with the binding of OxLDL to the platelet surface might contribute to their atheroprotective and antithrombotic properties.
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Project description:BACKGROUND:Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages. RESULTS:Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-?1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-?B) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment. CONCLUSIONS:The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-?1- and NF-?B-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.
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Project description:This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.
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Project description:BackgroundTraditionally, the impact of lipoproteins on vascular disease has been evaluated in light of their quantity, that is, cholesterol content, in plasma. However, recent studies of high-density lipoproteins (HDLs) have focused on functionality with regard to atheroprotection. For example, bioassays have emerged to assess the ability of HDL, in its near native plasma environment, to promote cholesterol removal (efflux) from cells. As a result, attention has focused on developing plasma-based assays for other putative HDL protective functions including protecting low-density lipoproteins (LDLs) from oxidative damage.ObjectiveTo determine the feasibility of such an assay in a complex sample such as plasma, we evaluated the contribution of HDL vs other plasma factors in preventing LDL oxidation.MethodsWe separated normolipidemic human plasma by gel filtration chromatography and assessed each fraction for its ability to prevent LDL modification by water soluble radical and copper-initiated oxidation mechanisms.ResultsUsing proteomics and selective precipitation methods, we identified major antioxidative contributions for fibrinogen, immunoglobulin G, albumin, and small soluble molecules like uric acid and ascorbate, with albumin being especially dominant in copper-initiated mechanisms. HDL particles were minor contributors (∼1%-2%) to the antioxidant capacity of plasma, irrespective of oxidation mechanism.ConclusionsGiven the overwhelming background of antioxidant capacity inherent to highly abundant plasma proteins, specific bioassays of HDL antioxidative function will likely require its complete separation from plasma.
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Project description:Very low-density lipoprotein (VLDL) is the main plasma carrier of triacylglycerol that is elevated in pathological conditions such as diabetes, metabolic syndrome, obesity and dyslipidemia. How variations in triacylglycerol levels influence structural stability and remodeling of VLDL and its metabolic product, low-density lipoproteins (LDL), is unknown. We applied a biochemical and biophysical approach using lipoprotein remodeling by lipoprotein lipase and cholesterol ester transfer protein, along with thermal denaturation that mimics key aspects of lipoprotein remodeling in vivo. The results revealed that increasing the triacylglycerol content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased triacylglycerol content in LDL promotes lipoprotein remodeling and fusion. These effects were observed in single-donor lipoproteins from healthy subjects enriched in exogenous triolein, in single-donor lipoproteins from healthy subjects with naturally occurring differences in endogenous triacylglycerol, and in LDL and VLDL from pooled plasma of diabetic and normolipidemic patients. Consequently, triacylglycerol-induced destabilization is a general property of plasma lipoproteins. This destabilization reflects a direct effect of triacylglycerol on lipoproteins. Moreover, we show that TG can act indirectly by increasing lipoprotein susceptibility to oxidation and lipolysis and thereby promoting the generation of free fatty acids that augment fusion. These in vitro findings are relevant to lipoprotein remodeling and fusion in vivo. In fact, fusion of LDL and VLDL enhances their retention in the arterial wall and, according to the response-to-retention hypothesis, triggers atherosclerosis. Therefore, enhanced fusion of triacylglycerol-rich lipoproteins suggests a new causative link between elevated plasma triacylglycerol and atherosclerosis.
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