High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase (original) (raw)
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15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells
Lipids, 2014
Caveolae are cholesterol and glycosphingolipidsenriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenasemediated modification of HDL 3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL 3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL. Keywords High density lipoprotein Á Endothelial cells Á 15-Lipoxygenase Á Caveolae Á Nitric oxide Abbreviations 15LO 15-Lipoxygenase ABCG1 ATP binding cassette transporter G1 CAV-1 Caveolin-1 eNOS Endothelial nitric oxide synthase HDL High density lipoprotein HUVEC Human umbilical vein endothelial cell(s) LOX-1 Lectin-like oxidized low-density lipoprotein receptor-1 MAPK Mitogen-activated protein kinase NO Nitric oxide S1P3
Journal of Biological Chemistry, 2000
Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 g/ml high density lipoprotein (HDL) prevented oxLDLinduced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 g/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.
Journal of Biological Chemistry, 2002
Recently it has been demonstrated that high density lipoprotein (HDL) binding to scavenger receptors, class B, type I (SR-BI) stimulates endothelial nitric-oxide synthase (eNOS) activity. In the present studies we used a Chinese hamster ovary cell system and a human microvascular endothelial cell line to confirm that HDL stimulates eNOS activity in a SR-BI-dependent manner. Importantly, we have extended these studies to examine the mechanism whereby HDL binding to SR-BI stimulates eNOS. eNOS can be stimulated by an increase in intracellular calcium, by phosphorylation by Akt kinase, or by an increase in intracellular ceramide. Calcium imagining studies and experiments with the calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N, N,N-tetraacetic acid tetra(acetoxymethyl) ester demonstrated that HDL binding to SR-BI does not induce an increase in intracellular calcium. Antibodies specific for activated Akt kinase demonstrated that HDL binding to SR-BI does not induce Akt kinase activation. However, HDL binding to SR-BI caused a reversible increase in intracellular ceramide levels from 97 ؎ 14 pmol/mg of protein to 501 ؎ 21 pmol/mg of protein. In addition, C 2-ceramide stimulated eNOS to the same extent as HDL, whereas C 2-dihydroceramide did not stimulate eNOS. We conclude that HDL binding to SR-BI stimulates eNOS by increasing intracellular ceramide levels and is independent of an increase in intracellular calcium or Akt kinase phosphorylation.
Journal of Biological Chemistry, 2003
High density lipoprotein (HDL) activates endothelial nitric oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or MAP kinase. In the present study we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I (SR-BI). HDL (10-50 µg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. PI3 kinase inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the non-receptor tyrosine kinase, src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and MEK inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, src-mediated signaling which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme. by guest on June 4, 2016 http://www.jbc.org/ Downloaded from Recently Li and colleagues also reported that HDL binding to SR-BI activates eNOS (5). The HDL-induced increase in NO production may be critical to the atheroprotective features of HDL since diminished bioavailablity of endotheliumderived NO has a key role in the early pathogenesis of hypercholesterolemiainduced vascular disease and atherosclerosis (6-8). However, the mechanisms by which HDL activates eNOS are yet to be clarified. eNOS is one of three isoenzymes which convert L-arginine to L-citrulline plus NO. The activity of eNOS is regulated by complex signal transduction pathways that involve various phosphorylation events and protein-protein interactions. Many stimuli modulate eNOS activity by activating kinases which alter the phosphorylation of the enzyme (9-15). Akt kinase (also known as PKB) by guest on June 4, 2016 http://www.jbc.org/ Downloaded from activates eNOS by directly phosphorylating the enzyme at serine-1179 (Ser-1179) (19). Akt itself is phosphorylated and activated by phosphoinositide 3kinase (PI3 kinase), which in turn is activated by a tyrosine kinase (TK). Both receptor TK and non-receptor TK are involved in PI3 kinase-Akt mediated eNOS activation by various agonists . In contrast to Ser-1179, phosphorylation of threonine-497 (Thr-497) of eNOS attenuates enzyme activity . eNOS is also modulated by MAP kinases (23,24) and unlike Akt, the effect of MAP kinases on eNOS activity can be either positive or negative (9,25-27). The role of kinase cascades in signaling by HDL from SR-BI to eNOS is entirely unknown.
Atherosclerosis, 2005
The antiatherogenic role of high-density lipoprotein (HDL) has been related to its ability to increase the activity of endothelial nitric oxide synthase (eNOS) and to protect low-density lipoprotein (LDL) against oxidative modification. The present study was aimed to determine whether and how HDL antagonizes oxidized LDL (oxLDL) that has been formed and accumulated in circulation. Pre-infusion of rats with HDL effectively prevented oxLDL-induced renal vascular constriction. Consistently, pre-incubation of human saphenous vein endothelial cells with HDL (100 g/ml) reversed the oxLDL-induced suppression of endothelium-dependent cyclic-GMP production in co-cultured smooth muscle cells. However, the changes of Akt phosphorylation and eNOS activity in endothelial cells in response to lipoprotein treatments under our assay condition were not significant. Intriguingly, pretreatment of human umbilical vein endothelial cells with HDL (50 g/ml) for only 30 s effectively reduced the level of free radicals generated by oxLDL or H 2 O 2 . In kidneys of living rats, renal arterial infusion of oxLDL greatly enhanced ischemia/reperfusion-induced free radicals, which could be attenuated by HDL pretreatment. We conclude that HDL may antagonize oxLDL on endothelial function through an Akt-independent pathway in which HDL preserves nitric oxide bioactivity by attenuating oxLDL-triggered free radical generation.
2001
Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O 2 .) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O 2. . ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O 2. generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O 2. formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolize L-arginine to L-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O 2. . Endothelium-dependent relaxation is impaired in animals with atherosclerosis (1-3), which has been linked to a decreased production and/or biological activity of endotheliumderived nitric oxide (NO) 1 (4, 5). Oxidative inactivation of NO is
International Journal of Molecular Sciences, 2019
The maintenance of physiological levels of nitric oxide (NO) produced by eNOS represents a key element for vascular endothelial homeostasis. On the other hand, NO overproduction, due to the activation of iNOS under different stress conditions, leads to endothelial dysfunction and, in the late stages, to the development of atherosclerosis. Oxidized LDLs (oxLDLs) represent the major candidates to trigger biomolecular processes accompanying endothelial dysfunction and vascular inflammation leading to atherosclerosis, though the pathophysiological mechanism still remains to be elucidated. Here, we summarize recent evidence suggesting that oxLDLs produce significant impairment in the modulation of the eNOS/iNOS machinery, downregulating eNOS via the HMGB1-TLR4-Caveolin-1 pathway. On the other hand, increased oxLDLs lead to sustained activation of the scavenger receptor LOX-1 and, subsequently, to NFkB activation, which, in turn, increases iNOS, leading to EC oxidative stress. Finally, th...
American Heart Journal, 2002
Background: Low levels of high-density lipoprotein (HDL) cholesterol increase the risk of coronary artery disease (CAD), and recent clinical studies suggest that interventions in low-HDL patients are beneficial. The purpose of this study was to examine the effect of increased HDL levels on endothelium-dependent vasodilation. Methods: We studied patients with CAD with a low-density lipoprotein (LDL) level of <100 mg/dL. Patients with an HDL level of <36 mg/dL were treated with niacin (n = 11), and patients with an HDL level of >36 mg/dL were followed as controls (n = 10). Baseline and 3-month follow-up studies of flow-mediated dilation (FMD) and blood lipid levels were obtained. Results: HDL levels increased from 30.1 ± 1.2 to 40.5 ± 1.2 mg/dL in the niacin-treated patients (P < .001) but remained unchanged in the control patients. At baseline, FMD was impaired in both the treated (6.5% ± 1%) and the control (7.3% ± 1%) patients compared with 10 healthy subjects (16% ± 2%, P < .01). After 3 months, FMD improved in the niacin-treated patients (11.8% ± 1%, P = .001) but remained unchanged in the control patients (6.2% ± 1%). Exposure of cultured human vascular endothelial cells to HDL in vitro enhanced expression of endothelial nitric oxide synthase (eNOS), as shown by immunoblotting. Conclusions: In patients with CAD and well-controlled LDL levels, elevation of HDL with niacin improves endothelial function. HDL increases eNOS protein expression in cultured vascular endothelial cells. Taken together, these observations suggest that HDL-mediated increases in eNOS expression may contribute to the observed enhancement in vasorelaxation and thus support a previously unrecognized mechanism for the beneficial cardiovascular effects of HDL.
Vascular Health and Risk Management, 2005
In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDL) exert several beneficial effects, including the prevention and correction of endothelial dysfunction. HDL promote endothelium proliferation and diminish endothelial apoptosis; they play a key role in vasorelaxation by increasing the release of nitric oxide and prostacyclin through the induction of the expression and the activity of endothelial nitric oxide synthase and the coupling of cyclooxygenase 2 and prostacyclin synthase. In addition, HDL affect coagulation, fibrynolisis, platelet adhesion, adhesion molecules, and protease expression, and they exert antioxidant activity. These effects are achieved at the gene expression level and are dependent on the activation of several intracellular signaling pathways, including PI3K/Akt, ERK1/2, PKC, and p38MAPK. The complexity of the signaling pathways modulated by HDL reflects the different effects of the components of this class of lipoproteins such as apolipoproteins or lipids on endothelial cell gene expression and the subsequent modulation of endothelial function observed. The in vivo relevance of these findings to endothelial recovery during physiological or pathological conditions remains to be addressed; nevertheless, the results of clinical studies with synthetic HDL, ApoA-I mimetics, and drugs that are becoming available that selectively affect HDL plasma levels and biological functions support the importance of the correction of endothelial function by HDL.
PloS one, 2016
Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggest...