Published online before print February 1, 2007, doi: 10.1161/01.ATV.0000259358.31234.37
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© 2007 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Anti–LOX-1 Rescues Endothelial Function in Coronary Arterioles in Atherosclerotic ApoE Knockout Mice
From the Department of Veterinary Physiology & Pharmacology (X.X., X.G., C.Z.), Texas A&M University, College Station, Tex; the Department of Physiology (B.J.P.), LSU Health Sciences Center, New Orleans, La; and the Department of Physiology (J.-M.C.), Peking Union Medical College, Beijing, P.R. China.
Correspondence to Cuihua Zhang, MD, PhD, Department of Veterinary Physiology & Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466. E-mail czhang{at}cvm.tamu.edu
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Abstract |
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Background— We hypothesized that atherosclerosis inhibits NO-mediated endothelium-dependent dilation of coronary arterioles through interaction of ox-LDL with its receptor, LOX-1, through the production of O2ÿ– in endothelial cells.
Methods and Results— We assessed the role of ox-LDL in endothelial dysfunction in a murine model of atherosclerosis (ApoE KO mice). Coronary arterioles from WT control and ApoE KO mice were isolated and pressurized without flow. Although dilation of vessels to endothelium-independent vasodilator SNP was not altered between ApoE KO and WT mice, dilation to the endothelium-dependent agonist, ACh was reduced in ApoE KO versus WT mice. Impaired vasodilation to ACh in ApoE KO mice is partially restored by NAD(P)H oxidase inhibitor, apocynin or DPI. Messenger RNA expression for NAD(P)H oxidases was higher in ApoE KO mice than that in WT and anti–LOX-1 treated ApoE KO mice. Anti–LOX-1, given in vivo, restored NO-mediated coronary arteriolar dilation in ApoE KO mice, but did not affect the endothelium-dependent vasodilation in controls.
Conclusions— These results suggest that ox-LDL impairs endothelium-dependent NO-mediated dilation of coronary arterioles by activation of a signaling cascade involving LOX-1 and NAD(P)H oxidase expression.
We examined the role of LOX-1 in endothelial dysfunction in ApoE KO mice. Impaired vasodilation to ACh in ApoE KO mice is partially restored by NAD(P)H oxidase inhibitor. Anti–LOX-1 restored vasodilation to ACh; reduced mRNA expression of NAD(P)H oxidase and O2ÿ– production. LOX-1 is implicated in endothelial dysfunction.
Key Words: atherosclerosis • coronary artery disease • endothelial function • microcirculation • reactive oxygen species
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Introduction |
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Atherosclerosis accounts for over 500 000 deaths annually in the United States. Diseases related to atherosclerosis such as myocardial infarction and stroke account for the majority of deaths in industrialized countries.1 Atherosclerosis is a complex, multifactorial disease with both genetic and environmental determinants. In patients with cardiovascular risk factors such as hypercholesterolemia, hypertension, or aging, endothelial dysfunction precedes the development of atherosclerosis and predisposes to the development of structural vascular changes.1 Endothelial dysfunction is one of the earliest manifestations of atherosclerosis, and the assessment of endothelial function may be a useful prognostic tool for coronary artery disease.2 The oxidized low-density lipoprotein (ox-LDL) is recognized as a major cause of endothelial dysfunction in atherogenesis.2 Ox-LDL has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO) and mediates several of its biological effects via lectin-like ox-LDL receptor-1 (LOX-1).3 Recent studies show that LOX-1 expression is upregulated in atherosclerotic tissues from rabbits and humans.4
The induced expression of LOX-1 in endothelial cells may provide a molecular link for incorporation of ox-LDL into the cells, resultant cellular activation, dysfunction, and injury. In addition, ox-LDL bound to LOX-1 induces the generation of superoxide anions (O2ÿ–)5 that inactivate NO and activate NF-
B,6 CD40/CD40L,7 and subsequently induce upregulation in the expression of vasoconstrictive molecules (ET-1), adhesion molecules (P-selectin, vascular cell adhesion molecule [VCAM]-1, intercellular adhesion molecule-1 [ICAM-1]), and chemokines [MCP-1])8 via gene transcription in endothelial cells. Recently, apolipoprotein-E gene knockout (ApoE KO) mice, which lack the gene encoding apolipoprotein-E, have been shown to develop hyperlipidemia and atherosclerosis similar to humans. Moreover, these mice develop hypertension and show endothelial dysfunction. Accordingly, we hypothesized that inhibition of LOX-1 will remediate endothelial dysfunction in atherosclerosis. Therefore, the purpose of our study was to document the cellular and molecular mechanisms involved in ox-LDL/LOX-1 in coronary microcirculation during atherosclerosis by studying NO-mediated vasodilation to acetylcholine (ACh) in isolated and pressurized coronary arterioles, O2ÿ– production, and expression of NAD(P)H oxidase with and without neutralizing antibodies to LOX-1.
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Methods |
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Mouse Model
The procedures followed were in accordance with approved guidelines set by the Laboratory Animal Care Committee at Texas A&M University. To understand the pathophysiological alteration of atherosclerosis and to assess the potential role of ox-LDL/LOX-1 in coronary microcirculation during atherosclerosis, mice 8 to 10 weeks old of either sex, ApoE KO9 and wild-type (WT, C57BL/6) control mice (untreated with chemicals or antibodies) were obtained from the Jackson Laboratory (Bar Harbor, Me). To accelerate lesion formation in ApoE KO mice, all animals were treated with a Western type diet (adjusted calories diet; Harlan Teklad TD 88137; 42% from milk fat, 0.15% cholesterol) for 12 weeks administered with chow. Animals had free access to water, were maintained at 24°C, and kept at a 12 hour light/dark cycle. Food and drug intake were monitored during the entire study. Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The control (WT) mice match the same backgrounds of Ape KO mice.
LOX-1 Neutralization
The neutralizing antibody to LOX-15 used in these studies is specific rat anti-mouse antibody (R&D, Anti-mouse LOX-1/SR-E1 Antibody, Catalog Number: AF1564), which blocks the binding of ox-LDL to LOX-1. After 12 weeks high fat diet, both control (WT) and atherosclerotic (ApoE KO) mice received either normal mouse IgG (nonimmune IgG, control antibody; 16 µg protein/mL, 0.1 mL/mouse; i.p.), or the neutralizing antibodies to LOX-1 (3 doses of Anti-mouse LOX-1/SR-E1 Antibody: 1 µg/mL, 8 µg/mL or16 µg protein/mL, 0.1 mL/mouse; i.p.) for a week.
Protein Expression of eNOS by Western Blot Analyses
For Western blot analysis, coronary arterioles (4 to 6 vessels per sample) were separately homogenized and sonicated in lysis buffer (Cellytic MT Mammalian Tissue Lysis/Extraction Reagent, Sigma). Protein concentrations were assessed with use of BCA Protein Assay Kit (Pierce), and equal amounts of protein (40 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond, Amersham). eNOS protein expression was detected by Western blot analysis with use of eNOS primary antibody (Abcam) in 3 groups of WT, ApoE KO, ApoE KO mice treated with anti–LOX-1 (16 µg protein/mL, 0.1 mL/mouse; 7 days, i.p.). Horseradish peroxidase-conjugated goat anti-mouse was used as the secondary antibody (Abcam). Signals were visualized by enhanced chemiluminescence (ECL, Amersham), and quantified by Quantity One (BioRad Versadoc imaging system).
Functional Assessment of Isolated Coronary Arterioles
The techniques for identification and isolation of coronary microvessels were described in detail previously.10,11 The heart was excised and immediately placed in cold (4°C) saline solution. Each coronary arteriole (50 to 100 µm in internal diameter) was carefully isolated and then used in the functional and molecular studies described below. To determine the response of coronary arterioles to ACh, vessels were cannulated with glass micropipettes and pressurized to 60 cm H2O intraluminal pressure without flow. After developing a stable basal tone (ie, spontaneous constriction to 60% to 70% of maximal diameter), the experimental interventions were performed. The concentration–diameter relationships for an activator of endothelium-dependent NO-mediated vasodilation, ACh (0.1 nmol/L to 10 µmol/L) and NO donor, sodium nitroprusside (SNP, 0.1 nmol/L to 10 µmol/L) were then established. The contributions of the NO pathway in these vasodilations were examined by treating the vessels with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 µmol/L, 30-minute incubation).
To determine whether LOX-1 was playing a role in atherosclerosis, endothelial-dependent and -independent dilation was assessed in coronary arterioles from either control antibody for LOX-1 (nonimmune IgG), or anti–LOX-1 IgG-treated mice (details refer to method-LOX-1 neutralization above). We administered ox-LDL (0.5 mg/protein/mL, intraluminal incubation for 60 minutes)12 and assessed responses of microvessels to ACh to show the acute administration of ox-LDL could mimic the responses of atherosclerosis. The vasodilations in the presence of ox-LDL were further examined in control mice treated with anti–LOX-1.
To determine the role of LOX-1 and O2ÿ– anions in atherosclerosis, the above vasodilatory functions were examined in the presence of free radical scavenger, TEMPOL (3 mmol/L), NAD(P)H oxidase inhibitor, apocynin (100 µmol/L), or diphenyleneiodonium (DPI, 10 µmol/L). The contribution of xanthine oxidase and the mitochondrial respiratory chain in generating O2ÿ– in atherosclerosis were assessed by treating the vessels with a xanthine oxidase inhibitor oxypurinol (100 µmol/L), or the mitochondrial respiratory chain inhibitor rotenone (1 µmol/L), separately. All drugs were administered extraluminally or intraluminally for 60-minute incubation.
Expression of LOX-1 and NAD(P)H Oxidase Subunits by Real-Time Polymerase Chain Reaction
Total RNA was extracted from left ventricular coronary arteries using Trizol reagent (Life Technologies Inc), and was processed directly to cDNA synthesis using the SuperScript III Reverse Transcriptase (Life Technologies Inc). The primers of NAD(P)H oxidase subunits, p22-phox, p40-phox, p-47-phox, p67-phox, NOX-1, NOX-2 (gp91-phox), and NOX-4, were designed (primer 3 software) and synthesized (Qiagen). cDNA was amplified using qRT-PCR Kit with SYBR Green (Life Technologies Inc). Data are calculated by 2–
CT method13 and are presented as fold change of transcripts for LOX-1 gene and NAD(P)H oxidase gene in ApoE KO mice normalized to internal control (ß-actin), compared with control mice (defined as 1.0-fold).
Measurement of Superoxide (O2ÿ–) by Electron Paramagnetic Resonance Spectroscopy
A 10% vessel homogenate was prepared in a 50 mmol/L phosphate buffer containing 0.01 mmol/L EDTA. The supernatants containing 2 mmol/L CP-H (1-hydrox-3carboxypyrrolidine) were incubated for 30 minutes at 37° and frozen quickly in liquid nitrogen (LN2). EPR spectroscopy was performed at room temperature using a Bruker EMX spectrometer and 1-mm diameter capillaries. O2ÿ– quantitation from the EPR spectra was determined by double integration of the peaks, with reference to a standard curve generated from horse radish peroxidase generation of the anion from standard solutions of hydrogen peroxide, using p-acetamidophenol as the cosubstrate,10 then normalized by protein concentration.
Chemicals
All drugs were obtained from Sigma, except as specifically stated. ACh, SNP, L-NMMA, TEMPOL, and apocynin were dissolved in PSS for functional studies and in PBS for fluorescence detection. ox-LDL was prepared as reported previously,12 DPI and rotenone were dissolved in DMSO; oxypurinol was dissolved in water. These drugs were then diluted in PSS to obtain the desired final concentration. The final concentration of DMSO in the tissue bath was 0.03%. Vehicle control studies indicated that these final concentrations of solvent had no effect on the arteriolar function.
Data Analysis
At the end of each experiment, the vessel was relaxed with 100-µmol/L SNP to obtain its maximal diameter at 60 cm H2O intraluminal pressure.10,11 All diameter changes in response to agonists were normalized to the vasodilation in response to 100 µmol/L SNP and expressed as a percentage of maximal dilation. All data are presented as mean±SEM, except as specifically stated (eg, as mean±SD for molecular study). Statistical comparisons of vasomotor responses under various treatments were performed with one-way or two-way ANOVA and intergroup differences were tested with Bonferonni Inequality. Significance was accepted at P<0.05.
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Results |
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Expression of LOX-1
Figure 1A shows the elevations of LOX-1 mRNA expression—over 6-fold higher in ApoE KO mice compared with control (WT), but anti–LOX-1 markedly reduced the LOX-1 expression in ApoE KO mice.
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Protein Expression of eNOS
Western blotting (Figure 1B) shows decreased expression of eNOS in ApoE KO mice as a percentage of control, eg, 200% represents a doubling of expression. The protein expression of eNOS was lower in coronary arterioles of ApoE KO than in control mice, but eNOS expression in ApoE KO mice treated with anti–LOX-1 was similar compared with the protein expression of eNOS in control mice. The results in Figure 1 corroborate the functional results shown below.
Effects of Atherosclerosis on NO-Mediated Vasodilation to ACh
The basal tone was stable over time at 1 hour, 2 hours, and 3 hours during the experiments. No significant differences in the baseline characteristics of the isolated coronary arterioles from control and ApoE KO mice were detected (Table). All drugs did not affect the basal tone in these functional studies (data not shown). Isolated coronary arterioles from control mice dilated dose-dependently to the endothelium-dependent agonist, ACh (Figure 2A). Administration of NOS inhibitor L-NMMA (10 µmol/L) reduced the vasodilatory responses to ACh compared with controls. In a similar manner as L-NMMA, ACh-induced vasodilation was impaired in ApoE KO mice (Figure 2A). Nonimmune IgG did not affect the vasodilation in control or ApoE KO mice (Figure 2A). Function of smooth muscle was preserved, because SNP-induced vasodilation was equivalent in both WT control and ApoE KO mice (Figure 2B).
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Role of LOX-1 in Atherosclerosis-Induced Vascular Dysfunction
Neutralizing antibodies to LOX-1 (1, 8, or 16 µg/mL, 0.1 mL/mouse, i.p. for 7 days) dose-dependently restored NO-mediated coronary arteriolar dilation in ApoE KO mice without affecting the dilation in the WT control mice (Figure 3A). ACh-induced dilation was significantly blunted by ox-LDL (Figure 3B) or L-NMMA (Figure 2A), but combined treatment (ox-LDL+L-NMMA) did not induce further abrogation of endothelial dilation (Figure 3B). Furthermore, anti-LOX-1 prevented ox-LDL-induced impaired vasodilation in control mice (Figure 3B).
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Roles of Superoxide, NAD(P)H Oxidase, Xanthine Oxidase, and Mitochondrial Respiratory Chain in Atherosclerosis-Induced Vascular Dysfunction
In ApoE KO mice, administered free radical scavenger, TEMPOL (3 mmol/L), NAD(P)H oxidase inhibitor, apocynin (100 µmol/L), or DPI (10 µmol/L) maintained vasodilation to ACh (Figure 4), but xanthine oxidase inhibitor, oxypurinol (100 µmol/L, n=4), or mitochondrial respiratory chain inhibitor, rotenone (1 µmol/L, n=4) did not (data not shown). Furthermore, oxypurinol (n=4), rotenone (n=5), or apocynin (n=4) did not affect ACh-induced vasodilation in control mice (data not shown).
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Atherosclerosis-Induced Superoxide Production in Murine Coronary Arterioles
The production of O2ÿ–, as quantified by EPR spectroscopy, increased in ApoE KO mice compared with control mice, and administration of anti–LOX-1 (16 µg protein/mL, 0.1 mL/mouse) or apocynin (100 mg/kg/d) for 7 days (i.p.) reduced the expression of O2ÿ– to the level observed in control mice (Figure 5). Anti–LOX-1 or DPI (10 mg/kg/d, i.p. for 7 days) prevented ox-LDL–induced O2ÿ– production in control mice (Figure 5).
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Expression of NAD(P)H Oxidase
Real-time PCR shows mRNA expression of NAD(P)H oxidase from left ventricular coronary arterioles in control and ApoE KO mice (Figure 6). The mRNA expression (expressed as percent of control) for NAD(P)H oxidase (p47-phox, p67-phox, NOX-2, and NOX-4) were higher in ApoE KO mice than those in control mice, but anti–LOX-1 significantly attenuated the expression of NAD(P)H oxidase in ApoE KO mice. But mRNA expression of NOX-1 (Figure 6A), p22-phox, and p40-phox (data not shown) were similar in ApoE KO and control mice.
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Discussion |
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Our results suggest that increased expression of LOX-1, along with enhanced amounts of ox-LDL, induces endothelial dysfunction via activation of NAD(P)H oxidase and production of O2ÿ– in atherosclerosis. Importantly, our findings are based on the following observations: antibody neutralization of LOX-1 prevented coronary endothelial dysfunction, reduced O2ÿ– generation and expression of LOX-1, but increased protein expression of eNOS in ApoE KO mice. Blockade of NAD(P)H oxidase mimicked the actions of anti–LOX-1 on O2ÿ– production and endothelial function in ApoE KO mice. Molecular evidence indicated that the expression of LOX-1 and NAD(P)H oxidase subunits p47-phox, p67-phox, NOX-2, and NOX-4, were significantly increased, but anti-LOX-1 attenuated the mRNA expression of LOX-1 and NAD(P)H oxidase subunits in ApoE KO mice. Our findings show LOX-1 has a primary role in mechanism(s) underlying endothelial dysfunction in atherosclerosis.
The Role of LOX-1 in Endothelial Dysfunction in Atherosclerosis
LOX-1 was first identified and cloned by Sawamura et al3 as a mammalian endothelial receptor for ox-LDL. The induced expression of LOX-1 in endothelial cells may provide a molecular link for incorporation of ox-LDL into the cells, resultant cellular activation, dysfunction, and injury. Early human carotid atherosclerotic plaques display increased levels of LOX-1 mRNA with greater endothelial LOX-1 expression when compared with advanced lesions.14 Ox-LDL is recognized as a major cause of endothelial dysfunction in atherogenesis.2 Ox-LDL elicits endothelial dysfunction, favoring generation of reactive oxygen species (ROS), inhibition of NO synthesis, and enhancement of monocyte adhesion to activated endothelial cells. In addition, ox-LDL is involved in inducing smooth muscle cell migration proliferation, and is avidly ingested by macrophages, resulting in foam cell formation. The proapoptotic nature of ox-LDL has also been suggested to contribute to plaque instability, as it is cytotoxic to vascular endothelial cells, smooth muscle cells, and macrophages. Our results showed that ox-LDL via LOX interactions impaired ACh-induced vasodilation in ApoE KO mice. Importantly, anti–LOX-1 prevented ox-LDL–induced impaired vasodilation in WT control mice, which further establishes that anti–LOX-1 blocks the effects of ox-LDL. Our results also indicated that protein expression of eNOS was diminished in ApoE KO mice; however, eNOS expression in anti–LOX-1 treated ApoE KO mice is similar compared with the protein expression of eNOS in WT control mice, which further clarified the mechanisms underlying the endothelial protective actions of the anti–LOX-1.
Ox-LDL has been observed to induce abnormalities in endothelial function, which may be relevant for the progression of atherosclerotic lesions. Functional alterations of the endothelial cells may be involved in the reduction of vasodilation, in response to stimuli that induce NO release, in isolated arteries exposed to ox-LDL.5 Neutralizing LOX-1 monoclonal antibody can block LOX-1–mediated cellular uptake of ox-LDL. Endothelial cells mediate the uptake of ox-LDL by the recently identified lectin-like ox-LDL receptor-1, which accumulates in atherosclerotic lesions. LOX-1 expression is increased in atherosclerotic tissues from rabbits and humans.4,14 Our findings showed that mRNA expression of LOX-1 in left ventricular coronary arterioles was elevated in ApoE KO mice. Consistently, our results show a connection between the impaired dilation and LOX-1 in atherosclerosis because anti–LOX-1 prevented this impairment. The responses of arterioles to endothelium-dependent and endothelium-independent dilators showed that ox-LDL/LOX-1 was playing a pivotal role in endothelial dysfunction in atherosclerotic mice. Importantly, neutralizing antibodies to LOX-1 influenced/restored vascular function under the experimental conditions in ApoE KO mice. Our results indicate that ACh-induced NO-mediated vasodilation is impaired in atherosclerotic mouse model and inhibition of LOX-1 rescued endothelial function in atherosclerosis. This is the first demonstration that the antibody to neutralize LOX-1 rescues endothelial dysfunction in atherosclerosis and this may prove useful in clinical therapy of atherosclerosis.
The Role of Superoxide in LOX-Induced Endothelial Dysfunction
LOX-1 has been postulated to be an ox-LDL endothelial sensor that regulates cellular and morphological vascular changes in the vessel wall. Stimulation of the endothelial monolayer by binding of ox-LDL to LOX-1 produces additional ROS,15 generating a positive feedback loop for further LDL oxidization. ROS induce activation of key transcription factors including NF-B,15 which regulates gene expression for proinflammatory and adhesion molecules. Once oxidant stress is invoked, characteristic pathophysiologic features ensue, namely adverse vessel reactivity, vascular smooth muscle cell (VSMC) proliferation, macrophage adhesion, platelet activation, and lipid peroxidation.16 Increased inactivation of NO by O2ÿ– is thought to contribute to impaired endothelium-dependent vasodilation in patients with coronary artery disease.17
Endothelium-dependent vasodilation is profoundly impaired in animal models because of increased vascular oxidative stress.9 These notions were confirmed by the findings in our present study. We found atherosclerosis increased O2ÿ– production, and anti–LOX-1 or apocynin reduced the production of O2ÿ– to the level observed in control mice; and anti–LOX-1 or DPI prevented ox-LDL induced O2ÿ– production in control mice. Moreover, the NAD(P)H oxidase inhibitors, apocynin and DPI, restored atherosclerosis-impaired vasodilation, but xanthine oxidase inhibitor, allopurinol, or mitochondrial respiratory chain inhibitor rotenone, did not. Scavenging O2ÿ– with TEMPOL also restored dilation. These results indicate LOX-1 is a key molecule in atherosclerosis, evoking O2ÿ– production, and implicating NAD(P)H oxidase as the major source of O2ÿ–.
The mRNA expression for NAD(P)H oxidase subunits were higher in ApoE KO mice than those in control mice. Furthermore, anti–LOX-1 significantly reduced the expression of NAD(P)H oxidase in ApoE KO mice. This observation suggests that part of the vicious cycle of atherosclerosis is that oxidative stress begets further oxidative stress; anti–LOX-1 breaks this vicious cycle by preventing the contribution of ox-LDL to endothelial oxidative stress. Thus, the oxidative stress induced by ox-LDL seems to beget further oxidative stress, which may be why the pathology of endothelial dysfunction, once initiated, seems to evolve into vascular disease.
In conclusion, endothelial dysfunction in atherosclerosis is mediated, at least in part, via the interaction of ox-LDL with its receptor, LOX-1, which in turn stimulates endothelial generation of O2ÿ– radicals by activation of NAD(P)H oxidase. This knowledge improves our understanding of the mechanism(s) underlying endothelial dysfunction, defined as blunted endothelial dilation of coronary arterioles, during atherosclerosis. The results of this study may contribute to the development of novel adjunctive therapies using anti–ox-LDL and/or anti–LOX-1 antibodies or soluble receptors to prevent endothelial dysfunction following onset of atherosclerosis.
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Acknowledgments |
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Sources of Funding
This study was supported by grants from Pfizer Atorvastatin Research Award (2004-37), American Heart Association Scientist Development Grant (110350047A), NIH grants (RO1-HL077566 and RO1-HL085119) to Dr Zhang.
Disclosures
None.
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Footnotes |
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X.X. and X.G. contributed equally to this study.
Original received September 9, 2006; final version accepted January 19, 2007.
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