While an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-1 (VCAM-1) but also involves other molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 (IL-1) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-alpha-induced VCAM-1 and E-selectin are significantly reduced by physiological concentrations of HDLs. An an HDL concentration of 1 mg/mL apolipoprotein A-I, the protein expressions of VCAM-1, ICAM-1, and E-selectin were inhibited by 89.6 +/- 0.4% (mean +/-SD, n=4), 64.8 +/- 1.0%, and 79.2 +/- 0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-alpha receptor. HDLs were effective when added from 16 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-alpha-induced expression of ICAM-1 by human foreskin fibroblasts, suggesting that the effect is cell-type restricted.(ABSTRACT TRUNCATED AT 250 WORDS)
The signaling pathways that couple tumor necrosis factor-␣ (TNF␣) receptors to functional, especially inf lammatory, responses have remained elusive. We report here that TNF␣ induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNF␣ resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dosedependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNF␣ on endothelial cells leading to extracellular signal-regulated kinases and NF-B activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNF␣-induced extracellular signal-regulated kinases and NF-B activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNF␣-induced endothelial cell activation.Tumor necrosis factor-␣ (TNF␣) was originally described for its antitumor activity, but is now recognized to be one of the most pleiotropic cytokines in mediating systemic inflammatory and immune responses (1, 2). A major site for these TNF␣ actions is the vascular endothelium, where TNF␣ triggers endothelial cells to secrete various cytokines and induces or enhances the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 and E-selectin (3). The regulated expression of these adhesion molecules is essential for the recruitment of circulating blood cells to the endothelium during the inflammatory and immune responses (3-5).TNF␣ activity is exerted through binding two distinct membrane receptors, p55 (TNF␣-R1) and p75 (TNF␣-R2). Engagement of the TNF␣ receptors results in recruitment of two distinct classes of receptor-associated proteins, one the TRADD, FADD͞MORT1 and RIP family, and the other, the TRAF family (6-8). Both of these appear to couple TNF␣ receptors to downstream signaling cascades such as cysteine proteases and NF-B activation to regulate cell proliferation, differentiation, and programmed cell death (6). Recently, the lipid second messenger, ceramide, has also received attention in TNF␣ signaling (6, 9). TNF␣ stimulates the activation of sphingomyelinase, yielding ceramide that, in turn, can induce apoptosis and may play a role in apoptotic signaling in various cell types (6, 9). In addition, ceramide can be subsequently metabolized to sphingosine and sphingosine 1-phosphate (S1P), via ceramidase and sphingosine kinase (SKase) activation, respectively (10). These sphingomyelin metabolites were also proposed to play a variety of roles in regulation of cellular activities such as calcium mobilization, cell motility, and mitogenesis (9, 10). In this study, we demonstrate that TNF␣ promoted generation of ceramide that was...
Objective-To study the acceptor specificity for human ABCG1 (hABCG1)-mediated cholesterol efflux. Methods and Results-Cells overexpressing hABCG1 were created in Chinese Hamster Ovary (CHO-K1) cells and characterized in terms of lipid composition. hABCG1 expressed in these cells formed homodimers and was mostly present intracellularly. Cholesterol efflux from hABCG1 cells to HDL 2 and HDL 3 was increased but not to lipid-free apolipoproteins. A range of phospholipid containing acceptors apart from high-density lipoprotein (HDL) subclasses were also efficient in mediating ABCG1-dependent export of cholesterol. Importantly, a buoyant phospholipidcontaining fraction generated from incubation of lipid-free apoA-I with macrophages was nearly as efficient as HDL 2 . The capacity of acceptors to induce ABCG1-mediated efflux was strongly correlated with their total phospholipid content, suggesting that acceptor phospholipids drive ABCG1-mediated efflux. Most importantly, acceptors for ABCG1-mediated cholesterol export could be generated from incubation of cells with lipid-free apoA-I through the action of ABCA1 alone. Conclusions-These
Abstract-The preferred extracellular acceptor of cell phospholipids and unesterified cholesterol in the process mediated by the ATP-binding cassette A1 (ABCA1) transporter is a monomolecular, prebeta-migrating, lipid-poor or lipid-free form of apolipoprotein (apo) A-I. This monomolecular form of apoA-I is quite distinct from the prebeta-migrating, discoidal high-density lipoprotein (HDL) that contains two or three molecules of apoA-I per particle and which are present as minor components of the HDL fraction in human plasma. The mechanism of the ABCA1-mediated efflux of phospholipid and cholesterol from cells has been studied extensively. In contrast, much less attention has been given to the origin and subsequent metabolism of the acceptor lipid-free/lipid-poor apoA-I. There is a substantial body of evidence from studies conducted in vitro that a monomolecular, lipid-free/lipid-poor form of apoA-I dissociates from HDL during the remodeling of HDLs by plasma factors such as cholesteryl ester transfer protein, hepatic lipase, and phospholipid transfer protein. The rate at which apoA-I dissociates from HDL is influenced by the phospholipid composition of the particles and by the presence of apoA-II. This review describes current knowledge regarding the formation, metabolism, and regulation of monomolecular, lipid-free/lipid-poor apoA-I in plasma.
Spherical high density lipoproteins (HDL) † predominate in human plasma. However, little information exists on the structure of the most common HDL protein, apolipoprotein (apo) A-I, in spheres vs. better studied discoidal forms. We produced spherical HDL by incubating reconstituted discoidal HDL with physiological plasmaremodeling enzymes and compared apoA-I structure in discs and spheres of comparable diameter (79 -80 and 93-96 Å). Using cross-linking chemistry and mass spectrometry, we determined that the general structural organization of apoA-I was overall similar between discs and spheres, regardless of diameter. This was the case despite the fact that the 93 Å spheres contained three molecules of apoA-I per particle compared with only two in the discs. Thus, apoA-I adopts a consistent general structural framework in HDL particles-irrespective of shape, size and the number of apoA-Is present. Furthermore, a similar cross-linking pattern was demonstrated in HDL particles isolated from human serum. We propose the first experiment-based molecular model of apoA-I in spherical HDL particles. This model provides a new foundation for understanding how apoA-I structure modulates HDL function and metabolism.sphere ͉ disk G iven the inverse correlation between high density lipoprotein (HDL) levels and cardiovascular disease, a key question in vascular biology relates to how apolipoproteins modulate the metabolism and function of HDL. Significant evidence supports a role for HDL in the process of reverse cholesterol transport whereby lipids and cholesterol in the vessel wall are transported to the liver for catabolism. However, because of a lack of information on HDL structure and the molecular basis of its interactions with other proteins, our understanding of HDL metabolism and function is at a basic stage.The ''glue'' that holds most HDL particles together is apolipoprotein (apo)A-I, a highly ␣-helical, 28-kDa polypeptide. It comprises some 70% of HDL protein mass, making it the clear starting point for deriving a basic understanding of HDL structure. In humans, apoA-I is primarily present in two major spherical HDL species, HDL 2 (d ϭ 1.063-1.125 g/ml) and HDL 3 (d ϭ 1.125-1.210 g/ml) with diameters ranging from 70 to 120 Å. More minor, but clearly important, HDL subspecies include lipid-poor apoA-I and nascent discoidal particles (reviewed in ref. 1). Highly reactive but low abundance discoidal HDLs are critical intermediates between lipid-poor apoA-I and mature spherical HDL. Easily produced in vitro, they have been heavily used for structural studies (2). Despite some debates on details of certain regions of apoA-I in discs, the majority of recent theoretical and experimental data supports the so-called ''double belt'' model (3). In this scheme, each of two ring-shaped apoA-I molecules wrap around a leaflet of a disk-like patch of lipid bilayer in an anti-parallel orientation.Despite their abundance in plasma, much less is known about the structure of apoA-I in spherical particles. They contain a neutral lipid...
Objective-Type 2 diabetes is characterized by impaired -cell secretory function, insulin resistance, reduced high-density lipoprotein (HDL) levels, and increased cardiovascular risk. Given the current interest in therapeutic interventions that raise HDLs levels, this study investigates the effects of HDLs on insulin secretion from -cells. Methods and Results-Incubation of Min6 cells and primary islets under basal or high-glucose conditions with either apolipoprotein (apo) A-I or apoA-II in the lipid-free form, as a constituent of discoidal reconstituted HDLs (rHDLs), or with HDLs isolated from human plasma increased insulin secretion up to 5-fold in a calcium-dependent manner. The increase was time and concentration dependent. It was also K ATP channel and glucose metabolism dependent under high-glucose, but not low-glucose, conditions. The lipid-free apolipoprotein-mediated increase in insulin secretion was ATP binding cassette (ABC) transporter A1 and scavenger receptor-B1 dependent. The rHDL-mediated increase in insulin secretion was ABCG1 dependent. Exposure of -cells to lipid-free apolipoproteins also increased insulin mRNA expression and insulin secretion without significantly depleting intracellular insulin or cholesterol levels. Conclusion-These results establish that lipid-free and lipid-associated apoA-I and apoA-II increase -cell insulin secretion and indicate that interventions that raise HDLs levels may be beneficial in type 2 diabetes. -Cell dysfunction and insulin resistance are major pathophysiological characteristics of type 2 diabetes. Although insulin resistance occurs early in disease onset, it causes diabetes only when -cell dysfunction is also present. [1][2][3] The UK Prospective Diabetes Study found that at the time of disease diagnosis, -cell secretory function was about 50% of normal and predicted that this was the culmination of at least 10 years of prior functional deterioration. 4 This progressive decline in -cell function eventually leads to -cell loss by apoptosis. 1,5 See accompanying article on page 1497Normoglycemia is maintained in insulin-resistant individuals by -cell compensation, which enhances insulin secretion. Type 2 diabetes develops when -cells can no longer maintain this compensatory response. The reasons why -cells become dysfunctional in some individuals but not in others have not been elucidated. Recent evidence suggests that cholesterol accumulation compromises -cell function and reduces insulin secretion and that this can be alleviated by depleting the cells of cholesterol. 6 As high-density lipoproteins (HDLs) are the predominant acceptors of cell cholesterol, it follows that they may be important for maintaining normal -cell function and insulin secretion. Indeed, it has recently been reported that infusing supraphysiological (80 mg/kg) doses of discoidal reconstituted HDLs (rHDL) containing apolipoprotein (apo) A-I complexed with phosphatidylcholine, (A-I)rHDL, into humans with type 2 diabetes increases plasma insulin levels, reduces plasma...
However, not all studies show a clear association between HDL-cholesterol and cardiovascular disease. 4,5 The observation that the capacity of serum to promote macrophage Molecular Medicine© 2015 American Heart Association, Inc. Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. Methods and Results:We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. Conclusions:
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