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【摘要】 Endothelial cell dysfunction (ECD) is emerging as a common denominator for diverse cardiovascular abnormalities associated with inhibition of endothelial nitric oxide (NO) synthase (eNOS). Elevated levels of asymmetric dimethylarginine (ADMA), a potent eNOS inhibitor, are common in renal failure and may contribute to ECD. Through DNA microarray screening of genes modulated in human umbilical vein endothelial cells (HUVEC) by N G -nitro- L -arginine methyl ester ( L -NAME), we found a 1.8-fold increase in low-density lipoprotein receptor-1 (LOX-1) expression. LOX-1 is a major endothelial receptor for oxidized low-density lipoproteins (OxLDL) and is assumed to play a role in the initiation and progression of atherosclerosis. Here, we confirmed the upregulation of LOX-1 mRNA and protein level by quantitative RT-PCR and Western blot analysis. Increased expression of LOX-1 was associated with the accumulation of DiI-labeled OxLDL (DiI-OxLDL) in ADMA- and L -NAME-pretreated HUVEC. To evaluate the contribution of LOX-1 in ADMA-induced accumulation of OxLDL by HUVEC, we used the competitive receptor inhibitor, soluble LOX-1. Treatment of HUVEC with soluble LOX-1 was associated with an approximately two- to threefold inhibition of DiI-OxLDL uptake in L -NAME- or ADMA-treated HUVEC. In conclusion, ADMA- or L -NAME-induced NO deficiency leads to the increased expression of LOX-1 mRNA and protein in HUVEC, which in turn results in the accumulation of OxLDL. Competition with LOX-1-soluble extracellular domain reduces OxLDL accumulation. In summary, elevated ADMA levels, i.e., in patients with renal failure, may be responsible for endothelial accumulation of OxLDL via upregulated LOX-1 receptor, thus contributing to endothelial lipidosis and dysfunction.
【关键词】 asymmetric dimethylarginine endothelial dysfunction chronic renal failure
ENDOTHELIAL DYSFUNCTION IS emerging as a common denominator for diverse cardiovascular abnormalities, such as atherosclerosis, diabetes, hypertension, and renal failure. Inhibition of endothelial nitric oxide (NO) synthase (eNOS) is one of the hallmarks of developing endothelial cell dysfunction ( 2, 9, 15, 37 ). Impaired endothelium-dependent vasorelaxation precedes the development of clinical manifestations of the disease and is detectable even before angiographic manifestations become apparent ( 2, 9, 14, 20, 36 ). One of the mechanisms leading to endothelial dysfunction is the accumulation of an endogenous inhibitor of eNOS, asymmetric dimethylarginine (ADMA) ( 1, 8, 13, 31, 43, 46 ). Plasma levels of ADMA are elevated in patients with chronic renal failure ( 11, 21, 22, 32, 34, 44, 49 ), hypercholesterolemia ( 8, 9, 29, 47 ), occlusive vascular disease, and hypertension ( 1, 5, 14, 16 - 18, 20, 42, 43, 48 ) and associated with reduced NO production and impaired endothelium-dependent vasodilation. Inhibition of NOS by ADMA increases endothelial oxidative stress and upregulates the expression of redox-sensitive genes that encode for endothelial adhesion molecules, similar to that observed in early atherogenesis ( 48 ).
In an attempt to screen for the consequences of NO inhibition, in preliminary studies we examined human umbilical vein endothelial cells (HUVEC) treated with the nonselective NOS inhibitor N G -nitro- L -arginine methyl ester ( L -NAME), with complementary DNA microarrays (Clontech) and, among other findings, detected an increased expression of mRNA encoding lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) compared with untreated control cells. It has previously been demonstrated that LOX-1 facilitates uptake and mediates several biological effects of oxidized low-density lipoproteins (OxLDL) in endothelial cells ( 38 ). Specifically, OxLDL binding to LOX-1 results in a significant increase in the generation of reactive oxygen species (ROS) that facilitate the oxidation of native LDL or partially oxidized LDL and can, in turn, upregulate LOX-1 expression and contribute to further ROS generation in endothelial cells ( 7 ). Furthermore, upregulation of LOX-1 by OxLDL is associated with decreased eNOS expression and MAPK activity in human coronary artery endothelial cells ( 36 ). However, it is not known whether changes in NO and/or ROS could feed back and modulate LOX-1 expression, particularly in the conditions of NO deficiency, as it develops in patients with chronic renal failure and elevated ADMA levels. Therefore, the purpose of this study was to validate the above cDNA microarray finding and analyze the potential role of NO deficiency, as induced by a more pathophysiologically relevant inhibitor, ADMA, on LOX-1 expression and LOX-1 receptor-mediated functions in endothelial cells. Our findings indicate that ADMA potently induces LOX-1 expression, resulting in the augmented uptake of modified LDL by endothelial cells, thus contributing to endothelial lipidosis and initiating early atherosclerosis.
MATERIALS AND METHODS
Cell culture. HUVEC were used between passages 3 and 8 and cultured in endothelial cell basal medium-2 (EBM-2 medium; Clonetics, San Diego, CA) containing 2% FBS. ADMA or L -NAME was added to the culture medium for 24 h before the experiments.
Cardiovascular microarray analysis. Microarray analysis of differential gene expression between two mRNA populations, control and L -NAME-treated HUVEC, was performed using an Atlas cardiovascular microarray (Clontech) composed of 588 human cardiovascular-related cDNAs on positively charged nylon membranes according to the manufacturer's instructions. cDNA levels from 0.5 mM L -NAME-treated HUVEC (24-h exposure) were compared with untreated cells at passage 3. The design of the cDNA array and the complete list of genes are available at www.bdbiosciences.com/clontech/atlas/genetics/7734_1_HuCardio.pdf.
Relative quantitative RT-PCR. Oligonucleotide primers were designed to amplify LOX-1 mRNA from HUVEC cells. The sequence of the sense primer was 5'-ACAGATCTCAGCCCGGCAACAAGCA-3', and the sequence of the antisense primer was 5'-GGGAGACAGCGCCTCGGACTCTAAAT-3', generating a product of 463 bp. Total RNA was isolated from HUVEC with TRIzol total RNA isolation reagent (Life Technologies BRL), and the mRNA was then reverse-transcribed to cDNA with AMV reverse transcriptase and amplified with expand high-fidelity enzyme mix, which were provided in the Titan One Tube RT-PCR System (Boehringer Mannheim, Indianapolis, IN). Equal amounts of mRNA (2 µg) from different samples were used in one 25-µl reaction containing 1 x RT-PCR reaction buffer, 0.2 mM dNTP, 5 mM DTT, and 0.5 µM each primer. The RT-PCR profile consisted of a 30-min incubation at 50°C and a 2-min denaturation at 94°C and was then followed by 30 s of denaturation at 94°C, 1 min of annealing at 60°C, and 2 min of elongation at 72°C. There were 30 amplification cycles for LOX-1, confirmed in pilot experiments as an exponential phase of amplification. An 18S mRNA internal control was also coamplified by incorporating 18S/18S competition mRNA primer (Ambison, Austin, TX) in another reaction tube. The appropriate 18S:18S competimer ratio and optimal number of PCR cycles were in accord with the manufacturer's recommendations. Internal standard primers for 18S generated a product of 489 bp. Products were analyzed by running 10% of the reaction mixture on a 2% agarose gel with ethidium bromide staining. The ratios of LOX-1 to 18 sRNA were obtained in at least four independent experiments. Optical densities of ethidium bromide-stained DNA bands were quantitated using National Institutes of Health IMAGE software.
Western blotting. Cells were lysed in the ice-cold lysis buffer of the following composition: 20 mM Tris, 140 mM NaCl, 1 mM EDTA, complete miniprotease inhibitor cocktail, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM NaF, and 1 mM orthovanadate, pH 7.8. The protein concentration of the lysates was determined with a Pierce bicinchonic protein assay against BSA standards. The samples were diluted with SDS sample buffer and stored at -20°C. Twenty micrograms of total cellular protein were electrophoretically separated in a 4-20% Tris-glycine gel (Invitrogen) and electroblotted to Immobilon-P membranes (Millipore). The membranes were blocked with 4% BSA in PBS for 1 h, incubated with the primary antibodies for 1 h [a mouse monoclonal anti-LOX-1 antibody, dilution 1:1,000 with 0.1% BSA or a mouse monoclonal anti- -tubulin antibody (Sigma), dilution 1:1,000], and incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Amersham) for 30 min.
The membranes were then washed three times with 0.1% Tween 20 in PBS, pH 7.4, for 5 min each, and protein-antibody conjugates were detected by chemiluminescence (Super Signal CL-HRP, Pierce Chemical).
Quantitative fluorescence microscopy and NO production measurement. NO production was measured using quantitative fluorescence microscopy of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate; NO-sensitive fluorescent dye)-loaded HUVEC stimulated with a calcium ionophore, A-23187. HUVEC were cultured on sterile fibronectin-coated glass-bottom 30-mm petri dishes ( 12, 23 ). Cells were examined using a Photon Technology International spectrofluorometer attached to a Nikon Diaphot fluorescence microscope at excitation/emission wavelengths of 485 and 530 nm, respectively. Fluorescence labeling of HUVEC was accomplished by incubation with 1 nM DAF-FM diacetate for 45 min ( 23, 26 ). HUVEC were subjected to different concentrations of L -NAME and ADMA for 5 min, baseline DAF fluorescence was recorded, and the cells were treated with 1 µM calcium ionophore A-23187 on the stage of the microscope to test for the completeness of inhibition of NO production. Data are expressed as fluorescence intensity (arbitrary units).
Lipoprotein modification and labeling. The LDL was oxidized by incubating 200 µg/ml of LDL in PBS containing 5 µmol/l CuSO 4 at 37°C for 27 h ( 33 ). This procedure resulted in an electrophoretic mobility for oxidized LDL of 1.0 relative to albumin and 4.0 relative to native LDL ( 31 ). Fluorescent labeling of LDL was accomplished by adding 75 µl of 3 mg/ml DiI in DMSO to 4 mg of oxidized LDL ( 33 ). The mixture was incubated under sterile conditions at 37°C for 8 h. Labeled lipoproteins were isolated by ultracentrifugation (100,000 g for 4 h). This procedure typically resulted in incorporation of 5-15 µg DiI/mg LDL protein.
Preparation of soluble LOX-1, LOX-Fc. The cDNA coding the extracellular domain of bovine LOX-1 (180-810) tagged with the Bam HI site was subcloned into the Bam HI site of pCd5lneg1 ( 41 ). A stable transformant of LOX-Fc (BLOX-Fc-CHO) was developed as described ( 41 ). LOX-Fc was prepared from the conditioned medium of BLOX-Fc-CHO with protein A-agarose (Bio-Rad) column chromatography.
DiI-OxLDL uptake in soluble LOX-treated cells. HUVEC were cultured on sterile fibronectin-coated glass coverslips. HUVEC were treated with indicated concentrations of ADMA or L -NAME in serum-containing EBM-2 for 24 h and incubated with 50 µg/ml of LOX-Fc in the culture medium at 37°C for 2 h. Cells were then washed, fluorescent lipoprotein (10 µg/ml DiI-OxLDL) was added, and incubations continued for 12 h in serum-containing medium. HUVEC were plated at a density permissive of 100% confluence on the day of treatment with DiI-OxLDL. HUVEC were washed with ice-cold PBS, pH 7.4, and immediately fixed with 4% paraformaldehyde. Cells were examined using fluorescence microscopy. Data are expressed as fluorescence intensity (arbitrary units) of cell-retained fluorophores and normalized to the background fluorescence of untreated HUVEC.
Statistical analyses. Data are expressed as means ± SE. Comparisons among the groups were performed using ANOVA, one-way ANOVA, followed by Tukey's posttest for multiple comparisons. The means of the two groups were compared by Student's t -test. A statistically significant difference was accepted at P < 0.05.
RESULTS
Induction of LOX-1 gene and protein expression by L -NAME and ADMA in HUVEC. In the preliminary screening of cardiovascular-relevant genes (588 genes) modulated in HUVEC by treatment for 24 h with a NOS inhibitor, 500 µM L -NAME, the LOX-1 message level (among several differentially expressed genes) was upregulated 1.8-fold, above the confidence level of the technique (1.5-fold change; Fig. 1 A ). To confirm these findings, we cultured HUVEC in the presence of 500, 300, and 150 µM L -NAME or 50, 30, and 15 µM ADMA for 24 h before RNA isolation. We found that 15 µM ADMA and 300 µM L -NAME maximally induced LOX-1 gene expression; therefore, in the following experiments, these concentrations of NOS inhibitors were used. LOX-1 mRNA level was increased 2.5-fold after 24-h treatment with 15 µM ADMA ( P < 0.001) and 1.9-fold after 24-h exposure to 300 µM L -NAME ( P < 0.01; Fig. 1 B ).
Fig. 1. Abundance of low-density lipoprotein receptor-1 (LOX-1) in human umbilical vein endothelial cells (HUVEC) treated with asymmetric dimethylarginine (ADMA) or N G -nitro- L -arginine methyl ester ( L -NAME). A : LOX-1 mRNA abundance in control and L -NAME-treated HUVEC. L -NAME was applied at a concentration of 500 µM for 24 h. Total RNA was extracted and subjected to hybridization on "cardiovascular" microarrays. L -NAME treatment resulted in a 1.8-fold increase in LOX-1 message. B : representative relative quantitative RT-PCR of LOX-1 mRNA levels in the control, 500, 300, and 150 µM L -NAME- or 50, 30, and 15 µM ADMA-treated HUVEC for 24 h ( lanes 1 - 7, respectively), which were normalized to 18S internal standard mRNA level (representative of 3 independent experiments). * P < 0.01 vs. control. ** P < 0.05 vs. control. *** P < 0.001 vs. control. C : representative Western blot analysis of LOX-1 expression in control, 24-h L -NAME- and ADMA-treated HUVEC. Cell proteins were separated by 4-20% Tris-glycine gel electrophoresis, and LOX-1 (or tubulin) was immunodetected with the monoclonal antibody (representative of 3 independent experiments). * P < 0.05 vs. control. ** P < 0.01 vs. control. *** P < 0.001 vs. control.
The abundance of LOX-1 protein upon induction with ADMA or L -NAME was examined by Western blot analysis at 24 h of continuous exposure to 15 µM ADMA or 300 µM L -NAME. LOX-1 protein expression increased 2.7-fold ( P < 0.001) after 24-h exposure to ADMA and 1.9-fold ( P < 0.05) after L -NAME ( Fig. 1 C ).
Inhibition of NO production and induction of LOX-1 protein expression by L -NAME and ADMA in HUVEC. To elucidate whether these particular concentrations of L -NAME and ADMA, which effectively upregulated LOX-1 expression in HUVEC, were able to suppress NO production by HUVEC, we used quantitative fluorescence microscopy of cells loaded with NO-sensitive fluorescent dye, DAF-FM diacetate. DAF-FM diacetate-loaded HUVEC were pretreated with 300, 50, and 15 µM L -NAME or 15, 10, and 3 µM ADMA and eNOS was stimulated with 1 µM calcium ionophore A-23187 ( Fig. 2, C and D ). Addition of this calcium ionophore resulted in robust NO production in control cells and cells pretreated with 15 µM L -NAME or 3 µM ADMA, suggesting that inhibition of eNOS by these concentrations of L -NAME/ADMA was marginal. In contrast, pretreatment with 300 µM L -NAME or 15 µM ADMA was associated with a profound suppression of NO production, as judged from the lack of an increase in DAF-FM diacetate fluorescence ( Fig. 2, C and D ). Using Western blot analysis, we found that the same concentrations, 15 µM ADMA and 300 µM L -NAME, maximally induced LOX-1 gene expression but had no effect on eNOS expression ( Fig. 2, A and B ). Thus, although the concentration of ADMA employed in our experiments is quite high, it is within the pathophysiologically relevant range. According to Sydow et al. ( 46 ), ADMA levels can be increased up to 10-fold in the serum of patients with chronic renal failure.
Fig. 2. Effect of L -NAME and ADMA on LOX-1, endothelial nitric oxide synthase (eNOS) protein expression, and NO production in HUVEC. A : representative Western blot of LOX-1 and eNOS expression in control, 24-h 300, 50, and 15 µM L -NAME-treated HUVEC. Cell proteins were separated by 4-20% Tris-glycine gel electrophoresis, and LOX-1 eNOS (or tubulin) was immunodetected with the monoclonal antibody. B : representative Western blot of LOX-1 and eNOS expression in control, 24-h 15, 10, and 3 µM ADMA-treated HUVEC. Cell proteins were separated by 4-20% Tris-glycine gel electrophoresis, and LOX-1 eNOS (or tubulin) was immunodetected with the monoclonal antibody. C : dose dependence of L -NAME-modulated NO production in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM)-loaded HUVEC cells stimulated by Ca ionofore A-23187 (arrow). D : dose dependence of ADMA-modulated NO production in DAF-FM-loaded HUVEC cells stimulated by A-23187 (arrow). a.u., Arbitrary units.
Soluble LOX-Fc inhibits the accumulation of OxLDL in L -NAME- or ADMA-treated HUVEC. Previously, it has been demonstrated that LOX-1 can be proteolytically cleaved and its extracellular lectin-like domain released as a soluble form (LOX-Fc) that is capable of interacting with LOX-1 natural ligands, thus inhibiting their binding to the cognate receptor. To determine whether LOX-1 function in HUVEC can be inhibited by LOX-Fc, DiI-labeled OxLDL accumulation by HUVEC was measured in LOX-Fc-pretreated cells. HUVEC were cultured in the presence of 15 µM ADMA or 300 µM L -NAME for 24 h, treated with LOX-Fc for 2 h, and further incubated with DiI-OxLDL for 12 h. Unbound ligand was removed by repeated washing, cells were fixed with 4% paraformaldehyde, and the amount of ligand associated with cells was determined using quantitative fluorescence microscopy. As shown in Fig. 3, treatment of HUVEC with ADMA or L -NAME substantially increased DiI-OxLDL uptake by endothelial cells (2.4-fold, P < 0.001, and 2-fold, P < 0.001, respectively). The DiI-OxLDL accumulation was decreased by LOX-Fc: 3.3-fold ( P < 0.001) in ADMA-treated HUVEC and 3.9-fold ( P < 0.001) in L -NAME-treated cells ( Fig. 4 ). These data further support the conclusion that LOX-1 plays a major role in the uptake of OxLDL.
Fig. 3. L -NAME- and ADMA-enhanced oxidized low-density lipoprotein (OxLDL) uptake in HUVEC is inhibited by soluble LOX-1. Accumulation of OxLDL in HUVEC treated with 300 µM L -NAME or 15 µM ADMA compared with control is shown. Fifty microliters of LOX-Fc were incubated with 10 µg/ml of DiI-labeled OxLDL for 12 h at 37°C. Cells were then washed, fixed, and examined microscopically (magnification x 600). Data analysis was performed using Universal Imaging software.
Fig. 4. Uptake of OxLDL in HUVEC modulated by LOX-Fc. Cells were treated with 300 µM L -NAME or 15 µM ADMA and compared with control. * P < 0.05 vs. control and LOX-Fc-exposed HUVEC. ** P < 0.001 vs. corresponding control and L -NAME-treated HUVEC, and control and ADMA-treated, unexposed HUVEC.
Inhibitory effects of NO donors and SOD mimetics on the upregulated LOX-1 protein expression in L -NAME- or ADMA-treated HUVEC. To dissect the role of NO deficiency and/or ROS excess, both associated with ADMA/ L -NAME inhibition of eNOS, in the induction of LOX-1 expression in HUVEC, cells were treated with 300 µM L -NAME and 15 µM ADMA and supplemented with NO donor N -hydroxy- L -arginine (NOHA) and SOD mimetic (MnTBAP) for 24 h. Cell lysates were examined by Western blot analysis using anti-LOX-1 antibody. As expected, treatment with either L -NAME or ADMA upregulated LOX-1 expression ( Fig. 5 ). Pretreatment of these cells with 2.5 µM MnTBAP significantly suppressed LOX-1 upregulation in L -NAME- or ADMA-exposed cells. NOHA alone was effective only in HUVEC treated with ADMA ( P < 0.001 vs. ADMA-treated cells). However, no additive effect of 100 µM NOHA pretreatment was observed ( Fig. 5 ), suggesting that it is the oxidative stress that is mainly responsible for LOX-1 upregulation in ADMA- or L -NAME-treated HUVEC.
Fig. 5. Effects of NO donors and SOD mimetics on upregulated LOX-1 protein expression in L -NAME- and ADMA-treated HUVEC. Cells were treated with 300 µM L -NAME and 15 µM ADMA and/or supplemented with NO donor N -hydroxy- L -arginine (NOHA) and SOD mimetic (MnTBAP) for 24 h, and lysates were blotted with LOX-1 antibody. The treatment with either L -NAME or ADMA resulted in the upregulation of LOX-1 expression. MnTBAP alone significantly suppressed the LOX-1 upregulation in L -NAME- or ADMA-treated HUVEC; NOHA alone was effective only in ADMA-treated HUVEC, with no additive effect afforded by NOHA. * P < 0.001 vs. corresponding control and L -NAME/ADMA-treated HUVEC, control, and ADMA-treated HUVEC exposed to MnTBAP, NOHA, or MnTBAP and NOHA. ** P < 0.01 vs. corresponding control and L -NAME-treated HUVEC exposed to MnTBAP or MnTBAP and NOHA.
DISCUSSION
Endothelial dysfunction, typically associated with reduced NO bioavailability in vivo, plays a critical role in atherogenesis. Screening HUVEC with complementary DNA microarrays for modulation of gene expression after application of a NOS inhibitor, L -NAME, revealed that LOX-1, a major receptor for OxLDL in endothelial cells, was upregulated.
LOX-1 belongs to the C-type lectin-like protein superfamily and is initially synthesized as a 40-kDa precursor protein with a N-linked high-mannose-type carbohydrate, which is further glycosylated and processed into a 50-kDa mature form. Like other scavenger receptors ( 40 ), LOX-1 has a wide spectrum of physiological ligands, including OxLDL, anionic phospholipids, aged/apoptotic cells, activated platelets, and bacteria ( 4, 6, 24, 25, 30, 35, 45 ). The LOX-1 gene is a so-called immediate early gene that is dynamically modulated by several factors in vitro and in vivo ( 25, 28 ). LOX-1 expression is induced by stimuli such as inflammatory cytokines, OxLDL per se, tumor necrosis factor-, transforming growth factor-, and ANG II in vitro, and some proatherogenic factors in vivo ( 6, 24, 25, 28 ).
We confirmed the cDNA microarray findings by the observation of upregulated LOX-1 mRNA and protein expression in L -NAME- and ADMA-exposed HUVEC. The significant increase in LOX-1 expression in the above in vitro studies suggests that elevated plasma levels of this natural eNOS inhibitor, ADMA, found in patients with chronic renal failure and in hypercholesterolemia, may play a pathophysiological role in the development of endothelial dysfunction and early atherosclerosis. Interestingly, it has recently been found that the phenylacetic acid (PAA), a degradation product of phenylalanine, another potent inhibitor of NOS, also accumulates in patients with end-stage renal disease ( 19 ). In healthy subjects, PAA was not detectable in plasma, whereas in patients with end-stage renal failure, plasma concentrations of PAA were 3.49 ± 0.33 mmol/l ( 19 ). According to Sydow et al. ( 46 ), ADMA inhibits the conversion of L -(guanidino- 15 N 2 )arginine to 15 N-nitrite (a specific index of NOS activity) at concentrations of 5 µM in human endothelial cells. ADMA levels at concentrations between 1 and 10 µM have been shown to reduce NOS activity in rat mesentery and cerebral blood vessels in vitro. In our study, we used pathophysiological concentrations of ADMA, usually encountered in patients with chronic renal diseases, when plasma levels of ADMA are increased up to 5- to 10-fold.
It has previously been demonstrated that in the presence of serum, LOX-1 exhibits two types of binding affinities for OxLDL ( 6, 24, 25 ). The high-affinity binding component has the lowest K d value among the known receptors for OxLDL. Some unknown factors in the serum appear to change the binding of OxLDL to LOX-1 from low affinity to high affinity ( 6, 7, 25, 34 ). LOX-1-mediated action of OxLDL induces a decrease in NO release, a significant increase in the generation of ROS in endothelial cells, and the increased expression of adhesion molecules, all hallmarks of endothelial dysfunction ( 7 ). Moreover, the OxLDL-mediated decrease in eNOS expression and increase in the expression of adhesion molecules were reduced by LOX-1 antisense oligonucleotides ( 7 ). ROS produced by the ligation of LOX-1 could facilitate the oxidation of LDL or partially oxidized LDL, which in turn could upregulate LOX-1 expression ( 7, 36 ) and contribute to further O 2 - generation and continued inactivation of NO. Our findings offer an additional aspect of LOX-1 regulation in pathological conditions when NO production is reduced by retained NOS inhibitors: both ADMA and L -NAME are important inducers of LOX-1 expression. Increased expression of LOX-1 by ADMA and L -NAME was associated with two- to threefold increased uptake of OxLDL. LOX-1 can be proteolytically cleaved and released from the plasma membrane in the soluble form ( 6, 27 ). Soluble LOX-1 has a molecular mass of 35 kDa. Purification of soluble LOX-1 and the NH 2 -terminal amino acid sequencing identified the two cleavage sites (Arg86-Ser87 and Lys89-Ser90), both of which are located in the membrane-proximal lectin-like extracellular domain of LOX-1, which is responsible for the recognition for OxLDL ( 6, 27 ). The conserved COOH-terminal residues of the lectin-like domain are involved in binding OxLDL, in particular, the large loop between the third and fourth cysteine, as well as the COOH-terminal end residues, which play critical roles in OxLDL binding. Using this competitive inhibitor of LOX-1, we further confirmed the primary role of this particular receptor for OxLDL in its accumulation by the endothelium. Accumulation of OxLDL was decreased by LOX-Fc: 3.3-fold in ADMA-treated HUVEC and 3.9-fold in L -NAME-treated HUVEC.
Furthermore, our findings offer the possible mechanisms that involved the upregulation LOX-1 under conditions of elevated ADMA levels. Using NO donors and SOD mimetics, we attempted to prevent the induction of LOX-1 expression by HUVEC. Although a SOD mimetic significantly suppressed the LOX-1 upregulation in L -NAME- or ADMA-exposed cells, NOHA was effective only in ADMA-exposed HUVEC and no additive effect by this NO donor was found, suggesting that ROS are critically responsible for LOX-1 upregulation in these conditions. Our hypothesis on the predominant ROS-induced upregulation of LOX-1 in endothelial cells exposed to eNOS inhibition is summarized in Fig. 6. There is ample evidence that NOS can function as a NO-generating or as an O 2 - -generating enzyme, the latter being a result of uncoupling of oxygen reduction and arginine oxidation by NOS ( 10 ). It was postulated that ADMA is an important endogenous regulator of the L -arginine/NO pathway ( 5, 11, 29 ). Elevated ADMA levels and subsequent inhibition of NO generation caused eNOS uncoupling with elevated superoxide production. LOX-1 promoter possesses binding sites for numerous transcription factors, including AP-1, AP-2, NF-IL6, STAT family, and NF- B ( 3, 39, 50 ). We speculate that increased ADMA/ L -NAME levels may cause eNOS uncoupling that, in turn, stimulated the NF- B-responsive element of OLR-1 gene, which led to upregulation of LOX-1 expression by endothelial cells ( Fig. 6 ).
Fig. 6. Hypothetical summary of L -NAME/ADMA-induced upregulation of LOX-1 through deficient NO production vis-a-vis an oxidative stress pathway.
In summary, ADMA-associated NO deficiency leads to the induction of LOX-1 in human endothelial cells. NO deficiency and the ensuing upregulation of LOX-1 trigger the accumulation of OxLDL by the endothelium. The elevated ADMA levels, as seen in patients with chronic renal failure and several cardiovasacular diseases, may therefore be responsible for endothelial accumulation of OxLDL via increased expression of LOX-1 receptor, thus further aggravating endothelial dysfunction. Inhibition of LOX-1 gene expression or OxLDL uptake with a competitive inhibitor, soluble LOX-1, respectively, reduces OxLDL accumulation by the endothelium and may provide future potential therapeutic strategies in protecting the endothelium against lipidosis, endothelial dysfunction, and progressive atherosclerotic lesions.
GRANTS
These studies were supported in part by National Institutes of Health Grants DK-54602 and DK-52783.
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作者单位：1 Department of Medicine, Renal Research Institute, and Division of Nephrology, New York Medical College, Valhalla, New York 10595; and 2 Department of Bioscience, National Cardiovascular Center Research Institute, Osaka 565-856 Japan