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Am J Physiol Heart Circ Physiol 287: H782-H790, 2004. First published March 11, 2004; doi:10.1152/ajpheart.00822.2003
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Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation

I. V. Smirnova,1 M. Kajstura,2 T. Sawamura,3 and M. S. Goligorsky1

Departments of 1Medicine and 2Pathology, Renal Research Institute and Division of Nephrology, New York Medical College, Valhalla, New York 10595; and 3Department of Bioscience, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan

Submitted 26 August 2003 ; accepted in final form 8 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intimal infiltration by monocytes and accumulation of lipids represent a critical step in the formation of fatty streaks during atherogenesis. Because elevated plasma levels of asymmetric dimethylarginine (ADMA), a potent nitric oxide (NO) synthase (NOS) inhibitor, are prevalent in diverse cardiovascular diseases, the goal of this study was to examine the contribution of NO deficiency to macrophage lipid accumulation. Inhibition of NO synthesis in PMA-primed human monocytic leukemia HL-60 cells resulted in a twofold increase in expression of the receptor for oxidized LDL (OxLDL), termed the lectin-like OxLDL receptor (LOX-1). Blockade of inducible NOS in activated macrophages resulted in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-OxLDL accumulation and imparted macrophages with a foamy appearance as detected with oil-red O lipid staining. ADMA (15 µM) or NG-nitro-L-arginine methyl ester (L-NAME, 300 µM), both of which suppress inducible NOS activity, increased oil-red staining 1.9- and 2.8-fold, respectively. Macrophages treated with ADMA or L-NAME showed a 2.4-fold increase in accumulation of DiI-OxLDL. To examine the role of LOX-1 in this process, we used small interfering RNA (siRNA) duplex-mediated LOX-1 gene silencing. LOX-1 expression was suppressed twofold by siRNA as shown by Western blot analysis. This suppression was associated with a two- to fourfold decrease in DiI-OxLDL uptake as identified by fluorescence microscopy and decreased oil-red O staining by activated macrophages. In conclusion, accumulation of ADMA (a competitive inhibitor of NOS) in patients with chronic renal failure may be responsible for upregulation of LOX-1 receptor and increased OxLDL uptake, thus contributing to lipidosis and foam cell formation. The data illustrate an additional nonendothelial mode of antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation by macrophages.

lectin-like oxidized low-density lipoprotein receptor-1; nitric oxide; renal failure; atherogenesis

THERE IS GROWING AWARENESS that a potent inhibitor of nitric oxide (NO) synthases (NOS), namely, asymmetric dimethylarginine (ADMA), is accumulated in diverse diseases associated with accelerated atherosclerosis (1, 3, 6, 7, 13, 21, 45, 56). The plasma levels of ADMA are ~1 µmol/l in healthy humans and are elevated more than twofold in hypercholesterolemic individuals (3, 5, 56). In elderly patients with peripheral arterial disease and generalized atherosclerosis, plasma ADMA levels range from 2.5 to 3.5 µmol/l and correlate with the severity of vascular disease (2, 3, 5, 27). In patients with chronic renal failure, which is a condition associated with an ~20-fold increase in risk for fatal cardiovascular complications, plasma ADMA levels may be elevated ninefold (5, 16, 28, 29, 43, 46, 53, 57). Interestingly, it was recently demonstrated that yet another inhibitor of NOS, phenylacetic acid (PAA), which is a degradation product of phenylalanine, accumulates in plasma of patients with end-stage renal failure (22). Collectively, these data indicate that the activity of NOS in these patients may be significantly suppressed. Although there is a certain amount of direct evidence to support the presence of stimulated expression of inducible NOS (iNOS) in atherosclerosis, which is associated with foam cells (20, 23, 33, 34), no study has confirmed the roles of elevated ADMA levels or NO deficiency in foam cell formation and modified LDL uptake by macrophages during chronic renal failure.

Foam cell formation occurs as a result of unregulated uptake of modified lipoproteins by scavenger receptors with deposition of cholesterol esters in the cytoplasm (38, 42, 55). The emerging concept that the lectin-like, oxidized, low-density lipoprotein receptor (LOX-1), which is the major scavenger receptor for oxidized LDL (OxLDL) in endothelial cells (54), plays an important role in the development of endothelial dysfunction and early atherogenesis is the subject of considerable interest (11). This hypothesis is based on several key findings as follows: 1) upregulation of LOX-1 and uptake of OxLDL suppresses NO production by endothelial cells (12), 2) upregulation of LOX-1 increases adhesion of monocytes to the endothelium under static and flow conditions (18), and 3) LOX-1 is upregulated in activated macrophages and is abundant in atherosclerotic plaques (41). These findings suggest the potential involvement of LOX-1 in foam cell formation (41). The possibility of an existing synergism between upregulated LOX-1 and defective NO synthesis in activated macrophages, although highly plausible, has not been explored. Our findings demonstrate that inhibition of NO synthesis by elevated ADMA levels in activated macrophages leads to upregulation of LOX-1, which is associated with increased uptake of oxidized LDL and rapid formation of lipid-laden cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Suspensions of human monocytic leukemia HL-60 cells were maintained in RPMI 1640 medium that contained 20% fetal bovine serum. Differentiation of HL-60 cells into monocytes/macrophages was stimulated with 10 nM PMA for 4–5 days. ADMA or NG-nitro-L-arginine methyl ester (L-NAME) was added to the cell culture medium for 24–48 h before experiments were performed.

Western blotting. Cells were lysed in ice-cold lysis buffer that contained the following: 20 mM Tris·HCl, 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. Protein concentration of the lysates was determined with Pierce bicinchoninic protein assay against BSA standards. The samples were diluted with SDS sample buffer and stored at –20°C. Total cellular protein (20 µg) was 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 primary antibodies for 1 h [1:1,000 dilutions of mouse monoclonal LOX-1 antibody and mouse monoclonal {beta}-tubulin antibody (Sigma)], 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).

Fluorescence-activated cell sorting analysis and NO production measurement. NO production was measured using fluorescence-activated cell sorting (FACS) analysis in HL-60 cells loaded with the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate). Suspensions of HL-60 cells were maintained in RPMI 1640 medium that contained 20% fetal bovine serum. Differentiation of HL-60 cells into monocytes/macrophages was stimulated with 10 nM PMA for 4 days. ADMA or L-NAME was added to the cell culture medium for 24 h before experiments were performed. Fluorescence labeling of PMA-differentiated HL-60 cells was accomplished by incubation with 1 nM DAF-FM diacetate for 45 min (30, 36). Cells were washed three times by centrifugation at 1,500 rpm. L-NAME (300 µM) or ADMA (15 µM) was added in the washing buffer. We analyzed 10,000 cells/sample using a Becton-Dickinson FACSCalibur cytometer equipped with a 488-nm laser. Emitted light was measured in control and treated cells. Signal overlap between the fluorescence 1 (DAF-FM) and fluorescence 2 (phycoerythrin) channels was compensated for appropriately. Data were expressed as arbitrary fluorescence units, and histograms were plotted using CELLQuest software (Becton-Dickinson).

RNA interference and in vitro small interfering RNA transfection. Small interfering RNA (siRNA) sequences targeting the OLR1 gene (National Center for Biotechnology Information accession number NM_002543) were synthesized by researchers at Dharmacon Research (Lafayette, CO) using SMART selection. HL-60 cells were transfected with siRNAs using the GeneSilencer siRNA transfection protocol (Gene Therapy Systems). Four days before transfection, HL-60 cells were treated with 10 nM PMA and then plated on polylysine-coated coverslips 1 day before transfection; this insured that the cells were 50–70% confluent on the day of transfection. The GeneSilencer reagent was diluted in serum-free RPMI medium according to the manufacturer's (Gene Therapy Systems) protocol. HL-60 cells were transfected with siRNAs at a concentration of 0.25 µM. Control cells were incubated with RPMI alone without siRNA or with and without GeneSilencer reagent. The siRNA-GeneSilencer mixture was added to cells growing in serum-free RPMI, and cells were incubated at 37°C for 72 h. L-NAME and ADMA were added to the culture medium of growing cells after 48 h of incubation with siRNA. Cells were harvested 72 h after transfection for protein expression or were studied using fluorescence microscopy.

Lipoprotein modification and labeling. LDL was oxidized by incubating 200 µg/ml LDL in PBS that contained 5 µmol/l CuSO4 at 37°C for 27 h (44). This procedure resulted in an electrophoretic mobility for oxidized LDL of 1.0 relative to albumin and of 4.0 relative to native LDL (44). Fluorescent labeling of LDL was performed by adding 75 µl of 3 mg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in dimethyl sulfoxide to 4 mg (protein) of oxidized LDL. 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 of DiI per milligram of LDL protein.

DiI-OxLDL uptake assays. Uptake of DiI-OxLDL was used to assess the functional consequences of upregulated LOX-1 expression. HL-60 cells were cultured on polylysine-coated sterile coverslips. The siRNA-transfected and -nontransfected, primed HL-60 cells were treated with the indicated concentrations of ADMA or L-NAME in serum that contained RPMI for 24 h. Cells were then washed, fluorescent lipoprotein (DiI-OxLDL, 10 µg/ml) was added, and incubation continued for 24 h in the serum-containing medium. HL-60 cells were washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) and immediately fixed with 4% paraformaldehyde. Cells were examined using fluorescence microscopy (Nikon Diaphot) and image analysis (Universal Imaging). Data are expressed as fluorescence intensity (in arbitrary units) of DiI-OxLDL.

Oil-red O staining. The siRNA-transfected and -nontransfected HL-60 cells were cultured on polylysine-coated glass coverslips, and cells were treated with 300 µM L-NAME and 15 µM ADMA for 24 h. The cells were washed three times with PBS, fixed with 4% paraformaldehyde, and stained with oil-red O and hematoxylin as previously described (25, 37).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of LOX-1 protein expression by ADMA and L-NAME in HL-60 cells. We modeled the specific effects of NO deficiency on LOX-1 expression in HL-60 cells by exposing the cells to NOS inhibitors. HL-60 cells were cultured for 4 days in the presence of 10 nM PMA and were then treated with different concentrations of L-NAME or ADMA for an additional 24 h before protein isolation. Using Western blot analysis, we found that 15 µM ADMA and 300 µM L-NAME maximally induced LOX-1 gene expression in PMA-differentiated HL-60 cells (Fig. 1). LOX-1 protein expression was increased by 25% in control PMA-differentiated HL-60 cells compared with undifferentiated cells and showed robust increases of ~217% after exposure to 15 µM ADMA and ~138% after exposure to 300 µM L-NAME (Fig. 1). Immunodetectable iNOS was faintly detectable in nonprimed HL-60 cells but was readily detectable in PMA-differentiated HL-60 cells. Treatment with L-NAME or ADMA did not modify iNOS expression in PMA-differentiated macrophages within the time frame of these experiments (Fig. 1).



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  		Fig. 1. Western blot analysis of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in human monocytic leukemia cell line HL-60. Data presented show nondifferentiated HL-60 cells, control PMA-differentiated HL-60 cells, NG-nitro-L-arginine methyl ester (L-NAME)-treated differentiated cells, and asymmetric dimethylarginine (ADMA)-treated differentiated HL-60 cells. Cell proteins were separated by 4–20% Tris-glycine gel electrophoresis, and LOX-1 was immunodetected with the monoclonal antibody (representative of 3 independent experiments). iNOS, inducible nitric oxide synthase. *P < 0.01 vs. PMA-differentiated HL-60 cells; **P < 0.001 vs. PMA-differentiated HL-60 cells.

 
Inhibition of NO production by L-NAME and ADMA in HL-60 cells. To elucidate whether the above maximally effective (in LOX-1 expression) concentrations of L-NAME and ADMA were indeed associated with suppression of NO production, we used FACS analysis of cells loaded with the NO-sensitive fluorescent indicator DAF-FM diacetate. DAF-FM diacetate-loaded HL-60 cells were pretreated with PMA to induce iNOS and were either treated or were not treated with 300 µM L-NAME or 15 µM ADMA for 24 h before the experiments were performed (Fig. 2, C and D). We found a robust increase in NO production in PMA-differentiated HL-60 cells compared with nondifferentiated HL-60 cells (Fig. 2, A and B). Pretreatment with 300 µM L-NAME or 15 µM ADMA was associated with a profound suppression of NO production in a subpopulation of macrophages that expressed the highest iNOS activity (Fig. 2, C and D). These differences in NO generation between primed HL-60 cells exposed and not exposed to NOS inhibitors were compared using Kolmogorov-Smirnov analysis and were found to be statistically significant (P < 0.001). On the other hand, despite the 20-fold difference in the concentrations of L-NAME and ADMA, the extent of iNOS suppression appeared to be comparable. This is surprising, because L-NAME has a much more potent inhibitory effect on the isolated enzyme in vitro. However, in in vivo systems, EC50 results might be quite different. For instance, Boger et al. (4) demonstrated that in cholesterol-fed rabbits, administration of L-NAME "did not induce a further significant increase in intima/media ratio as compared to the cholesterol group. Intima/media ratios were significantly correlated to the ADMA levels in individual rabbits." These data clearly indicate that ADMA (even at concentration of 2.2 uM) exerted a pro-atherogenic effect over and above that of cholesterol feeding, whereas administration of L-NAME did not. One of the peculiarities of in vivo action of these two inhibitors is related to their discrepant effects on the y+ carrier and intracellular L-arginine import. Closs et al. (9) demonstrated that ADMA potently suppresses the y+ transporter, whereas L-NAME does not (9). This implies that in activated macrophages with already-enhanced L-arginine utilization, inhibition of its import by ADMA could have an additive effect. Indeed, Chan et al. (10) showed that the end effect of ADMA is enhanced twofold in the presence of reduced L-arginine concentration (10). The above scenario may provide a plausible explanation of our findings. Although the concentration of ADMA employed in our experiments was quite high, it was within the pathophysiologically relevant range. According to Boger et al. (4–6), ADMA levels can be increased up to 10-fold in the serum of patients with chronic renal failure.



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  		Fig. 2. Effects of L-NAME and ADMA on nitric oxide (NO) production in differentiated HL-60 cells. NO production in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate)-loaded HL-60 cells was measured using flow cytometry analysis in suspensions of HL-60 cells stimulated with 10 nM PMA for 4 days. ADMA or L-NAME was added to the cell culture medium for 24 h before experiments. With the use of a Becton-Dickinson FACSCalibur cytometer equipped with a 488-nm laser, 10,000 cells/sample were analyzed. A: representative fluorescence-activated cell sorting (FACS) data of PMA-differentiated control HL-60 cells not labeled with DAF-FM diacetate. FL1, fluorescence 1. B: relative NO production in 1 nM DAF-FM diacetate-loaded PMA-differentiated HL-60 cells. Note the subpopulation of robust NO-synthesizing cells (M2), which is predominantly suppressed in the subsequent panels. C: relative NO production in 1 nM DAF-FM-loaded PMA-differentiated HL-60 cells treated for 24 h with 300 µM L-NAME (solid tracing on gray background of B). D: relative NO production in 1 nM DAF-FM-loaded PMA-differentiated HL-60 cells treated for 24 h with 15 µM ADMA (solid tracing on gray background of B). Data are presented as arbitrary fluorescence units and histograms. P < 0.001 vs. corresponding control and L-NAME-exposed HL-60 cells or control and ADMA-exposed HL-60 cells by Kolmogorov-Smirnov test.

 
Specific inhibition of LOX-1 protein expression by siRNAs. To determine the effects of siRNA on LOX-1 expression levels, ADMA- or L-NAME-treated and -untreated primed HL-60 cells were examined by Western blot analysis. Treatment of HL-60 cells with siRNA produced a marginal decrease in LOX-1 expression in nondifferentiated and differentiated cells but resulted in a threefold decrease in LOX-1 expression in L-NAME- and ADMA-treated differentiated HL-60 cells as judged from the results of Western blot analysis (Fig. 3).



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  		Fig. 3. Knockdown of LOX-1 by directed small interfering RNA (siRNA). Effects of siRNA transfection on LOX-1 protein levels in L-NAME (300 µM)-treated, ADMA (15 µM)-treated, and untreated HL-60 cells. Western blot analysis of LOX-1 protein was carried out 48 h after the beginning of transfection of HL-60 cells with 0.25 µM siRNA. {beta}-Tubulin served as a loading control. Control cells were incubated with RPMI 1640 alone (no siRNA or GeneSilencer reagent). Data are representative of two replicate experiments.

 
Uptake of OxLDL by ADMA- or L-NAME-treated HL-60 cells. To determine whether upregulated expression of LOX-1 by ADMA and L-NAME is correlated with enhanced uptake of OxLDL, DiI-labeled OxLDL internalization into HL-60 cells was measured in siRNA-transfected and -nontransfected cells. HL-60 cells were cultured in the presence of L-NAME or ADMA for 24 h before incubation with the fluorescent LOX-1 ligand DiI-OxLDL for 24 h in serum-containing medium. Unbound ligand was removed by repeated washings, cells were fixed with 4% paraformaldehyde, and the amount of ligand associated with cells was determined. As shown in Fig. 4, treatment of HL-60 cells with L-NAME or ADMA substantially induced DiI-OxLDL uptake with HL-60 cells. This effect of ADMA on OxLDL was maximal at a concentration of 15 µM (2.45-fold increase compared with control; P < 0.001). L-NAME (300 µM) resulted in a 2.36-fold (P < 0.001) increase in fluorescence intensity compared with control (Figs. 4 and 5). The DiI-OxLDL accumulation was decreased by LOX-1-directed siRNA by 2.6-fold (P < 0.01) in L-NAME-treated and 3.8-fold (P < 0.001) in ADMA-treated HL-60 cells (Fig. 5). Furthermore, DiI-OxLDL uptake was decreased by 2.4-fold in nonprimed compared with PMA-differentiated HL-60 cells and was downregulated by LOX-1-directed siRNA (Fig. 5), which suggests that HL-60 cells exhibit a constitutive expression of LOX-1 that may be involved in the accumulation of modified LDL even under basal conditions (see Figs. 4 and 5).



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  		Fig. 4. L-NAME and ADMA enhance specific oxidized LDL (OxLDL) uptake in HL-60 cells mediated by LOX-1. Representative images of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled OxLDL accumulation in undifferentiated HL-60 cells (A and B), control differentiated untreated cells (C and D), differentiated cells treated with 300 µM L-NAME (E and F), and differentiated cells treated with 15 µM ADMA (G and H). LOX-1-directed siRNA transfected (B, D, F, and H) and nontransfected (A, C, E, and G) HL-60 cells were incubated for 24 h at 37°C with 10 µg/ml OxLDL. Cells were then washed, fixed, and examined with a x60 objective using phase-contrast or a fluorescein filter set.

 


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  		Fig. 5. Uptake of DiI-OxLDL by HL-60 cells was measured in nondifferentiated untreated cells, control differentiated untreated cells, differentiated cells treated with 300 µM L-NAME, and differentiated cells treated with 15 µM ADMA. LOX-1-directed siRNA-transfected and nontransfected HL-60 cells were incubated for 24 h at 37°C with 10 µg/ml OxLDL. ^P < 0.01 vs. corresponding nondifferentiated nontransfected and transfected HL-60 cells; *P < 0.001 vs. corresponding nondifferentiated untreated nontransfected HL-60 cells and PMA-differentiated nontransfected control HL-60 cells; **P < 0.001 vs. corresponding control PMA-differentiated nontransfected cells and L-NAME- or ADMA-treated differentiated nontransfected cells; ***P < 0.001 vs. corresponding nontransfected differentiated and transfected differentiated HL-60 cells.

 
Effects of L-NAME and ADMA on LOX-1-receptor expression and foam cell formation in HL-60 macrophages. To examine the effects of L-NAME and ADMA on the transformation of PMA-treated HL-60 macrophages into foam cells, staining with oil-red O was performed. Oil-red O staining showed that the stimulatory effects of L-NAME and ADMA on LOX-1 expression were accompanied by the appearance of accumulation of intracellular lipid droplets typical of foam cell formation (Figs. 6 and 7). Oil-red O staining was decreased 1.6-fold by LOX-1-directed siRNA in unactivated HL-60 cells (Fig. 7), which suggests that HL-60 cells express LOX-1 constitutively. After L-NAME treatment, the intensity of oil-red O staining was increased 2.8-fold (P < 0.001) and was suppressed 2.2-fold (P < 0.001) by LOX-1-directed siRNA (see Fig. 6). ADMA resulted in a 1.9-fold (P < 0.001) increase in oil-red O staining, and this effect was suppressed by LOX-1-directed siRNA (see Figs. 6 and 7).



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  		Fig. 6. Gallery of images of HL-60 cells treated with 300 µM L-NAME and 15 µM ADMA and stained with oil-red O. Accumulation of oil-red O in nondifferentiated untreated cells (A and B), control differentiated untreated cells (C and D), differentiated treated cells with 300 µM L-NAME (E and F), and differentiated treated cells with 15 µM ADMA (G and H) is shown. LOX-1-directed siRNA transfected (B, D, F, and H) and nontransfected (A, C, E, and G) HL-60 cells were stained with oil-red O and examined with a x60 objective.

 


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  		Fig. 7. Semiquantitative image analysis of oil-red O (OR-O) uptake by HL-60 cells subjected to the indicated treatments. *P < 0.001 vs. corresponding nondifferentiated untreated nontransfected HL-60 cells and PMA-differentiated nontransfected control HL-60 cells; ^P < 0.01 vs. corresponding nondifferentiated nontransfected and transfected HL-60 cells; **P < 0.001 vs. corresponding control PMA-differentiated nontransfected cells and L-NAME- or ADMA-treated differentiated nontransfected cells; ***P < 0.001 vs. corresponding nontransfected differentiated and transfected differentiated HL-60 cells.

 
Inhibitory effects of NO donors and SOD mimetics on upregulated LOX-1 protein expression in L-NAME- or ADMA-treated human umbilical vein endothelial cells. To dissect the roles of NO deficiency and/or excessive generation of reactive oxygen species (ROS), both of which are associated with inhibition of iNOS by ADMA or L-NAME, in the induction of LOX-1 expression in PMA-differentiated HL-60 cells, cells were supplemented with 100 µM of the NO donor N-hydroxy-L-arginine (NOHA) and/or 2.5 µM of the SOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) for 24 h during the standard incubation with 300 µM L-NAME or 15 µM ADMA. In addition, differentiated HL-60 cells were also subjected to 100 µM L-arginine. Cell lysates were examined by Western blot analysis using anti-LOX-1 antibody. As expected, treatment with either L-NAME or ADMA increased LOX-1 expression (Fig. 8). Pretreatment of these cells with 2.5 µM MnTBAP or 100 µM NOHA significantly suppressed LOX-1 upregulation in L-NAME- and ADMA-exposed cells (Fig. 8). No additive effects of pretreatment by NOHA and MnTBAP were observed in L-NAME-exposed HL-60 cells, whereas an additive effect was found in ADMA-treated cells, which suggests that both nitrosative and oxidative stresses are responsible for LOX-1 expression in ADMA-treated PMA-differentiated HL-60 cells (Fig. 9). Intriguingly, treatment with L-arginine also inhibited LOX-1, which suggests that the substrate deficiency may be in part responsible for the sustained decrease in NO synthesis in activated macrophages (see Fig. 8).



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  		Fig. 8. Effects of NO donor and SOD mimetic on LOX-1 expression in L-NAME- or ADMA-treated HL-60 cells. Cells were treated with 300 µM L-NAME or 15 µM ADMA and/or were supplemented with NO donor N-hydroxy-L-arginine (NOHA) and/or SOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) for 24 h, and lysates were blotted with LOX-1 antibody. Treatment with either L-NAME or ADMA resulted in the upregulation of LOX-1 expression. MnTBAP alone significantly suppressed the LOX-1 upregulation in ADMA-treated HL-60 cells, whereas NOHA downregulated the LOX-1 upregulation in L-NAME-exposed cells. No additive effect was afforded by NOHA in L-NAME-exposed HL-60 cells, but an additive effect was found in ADMA-exposed HL-60 cells.

 


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  		Fig. 9. Hypothetical summary of L-NAME- and ADMA-induced upregulation of LOX-1 in macrophages through deficient NO production vis a vis oxidative and nitrosative stress pathways.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This work shows for the first time that the NOS inhibitors ADMA and L-NAME increase the abundance of LOX-1, the dominant endothelial scavenger receptor for OxLDL, and lead to accumulation of OxLDL and foam cell formation in PMA-primed HL-60 cells. Previously it was demonstrated that circulating human blood monocytes or undifferentiated THP-1 cells express only trace levels of LOX-1 transcripts and protein, but LOX-1 was upregulated together with other scavenger receptors in both cell types after differentiation (26, 41, 50). In THP-1 cells, LOX-1 mRNA expression was time- and dose-dependently induced as early as 12 h after treatment with 10 nM PMA and remained elevated for at least 7 days (41). Interestingly, these results appear to correlate with previous reports that scavenger receptor A (SR-A) displays time- and concentration-dependent induction in PMA-treated THP-1 cells (15). In addition, expression of LOX-1 mRNA was significantly upregulated in a time- and concentration-dependent manner by the inflammatory cytokine TNF-{alpha} in murine peritoneal macrophages (50). Our results strongly indicate that the nonprimed human monocytic leukemia cell line HL-60 is characterized by the constitutive expression of LOX-1 that can mediate basal scavenging of modified LDL by these cells. The LOX-1 protein expression increased by 25% in PMA-differentiated HL-60 cells compared with undifferentiated monocytic cells. After 24 h of exposure to ADMA, LOX-1 showed a robust increase of ~217% in PMA-differentiated HL-60 cells and ~138% after 24 h of exposure of L-NAME of differentiated cells compared with control cells. In addition, upregulation of LOX-1 was associated with a greater than twofold increased accumulation of DiI-OxLDL in macrophages subjected to ADMA or L-NAME compared with untreated differentiated HL-60 cells. LOX-1-directed siRNA decreased by two- to threefold the uptake of DiI-OxLDL under these conditions. Thus the finding of ADMA-induced upregulation of LOX-1 in macrophages implicates this circulating NOS inhibitor, which is consistently elevated in diverse cardiovascular diseases and chronic renal failure, in the progression of atherosclerosis.

The appearance of lipid-laden foam cells is one of the hallmarks of fatty streaks and atherosclerotic plaques. The transformation of macrophages into lipid-laden foam cells is most likely the result of receptor-mediated uptake of cholesterol-rich particles. Several mechanisms are known to contribute to the monocyte scavenging of OxLDL (60). Monocytes and macrophages possess receptors for OxLDL including SR-A (the first OxLDL receptor to be characterized and cloned; Ref. 35), scavenger receptors BI, CD36, CD68, and LOX-1, and the scavenger receptor for phosphatidylserine and oxidized lipoprotein (3, 14, 24, 40, 52). Therefore, macrophage transformation into the foam cell can potentially be governed by several lipoprotein receptors that mediate the uptake of cholesterol-rich particles (14, 41). The contribution of LOX-1 to this process remains uncertain. Our results indicate that blockade of the iNOS synthesis of NO by activated macrophages rendered macrophages with a foamy appearance as detected with oil-red O lipid staining: ADMA or L-NAME increased oil-red O staining 1.9- and 2.8-fold, respectively. The siRNA duplex-mediated LOX-1 gene silencing decreased approximately twofold the oil-red O staining of activated macrophages, which suggests that LOX-1 is predominantly involved in the regulation of the foamy appearance by macrophages. These findings provide direct evidence that LOX-1 is a specific receptor for OxLDL, which plays a critical role in foam cell formation especially when iNOS is inhibited.

LOX-1 was originally purified as a receptor for OxLDL (11, 44) and is highly expressed by vascular endothelial cells, macrophages, and vascular smooth muscle cells in atherosclerotic lesions at various stages in humans and rabbits (31, 32, 40, 47, 50, 51). LOX-1 is a type II membrane glycoprotein with an extracellular COOH-terminal lectin domain that is initially synthesized as a 40-kDa precursor protein with an N-linked high-mannose-type carbohydrate and is further glycosylated and processed into a 50-kDa mature form (11, 51). Importantly, the LOX-1 gene is a so-called immediate early gene that possess NF-{kappa}B response element (54) and is dynamically modulated by TNF-{alpha} (50), transforming growth factor-{beta} (15), angiotensin II (49), endothelin-1 (49), peroxisome proliferator-activated receptor-{alpha} (8), oxidized LDL and phorbol esters in vitro (11, 41), and proinflammatory conditions, oxidative stress, and hemodynamic abnormalities in vivo (11). The precise mechanism(s) by which increased ADMA level and reduced NO production induce the foamy appearance of macrophages remains unknown. It is well established, however, that NO may act as an antiatherogenic molecule (12, 19, 22, 39). Reduced NO availability was shown to be associated with increased oxidative stress in endothelial cells (12) and to in turn activate the transcription factor NF-{kappa}B (12, 48) and lead to enhanced endothelial adhesiveness associated with expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 (12, 48). Present data extend this view of an antiatherogenic effect of NO on macrophages: LOX-1 is upregulated under conditions when NO production is reduced. It was previously demonstrated that binding of OxLDL to LOX-1 results in a significant increase in the generation of ROS by endothelial cells (12). The increased binding of OxLDL to LOX-1 and ROS production could facilitate the oxidation of native LDL or partially oxidized LDL accumulated via other scavenger receptors and could in turn upregulate LOX-1 expression (12, 48) and contribute to further accumulation of Ox-LDL, O2–· generation, and peroxynitrite formation.

Using an NO donor and a SOD mimetic, we attempted to prevent the induction of LOX-1 expression by differentiated HL-60. The SOD mimetic and NO donor significantly suppressed LOX-1 upregulation in L-NAME- and ADMA-treated macrophages (see Fig. 8). No additive effect of pretreatment by the SOD mimetic was observed in L-NAME-exposed differentiated HL-60 cells, whereas an additive effect was found in ADMA-treated cells; this suggests that both nitrosative and oxidative stresses are responsible for LOX-1 expression in ADMA-treated differentiated HL-60 cells. The possibility for the development of substrate deficiency in activated macrophages is supported by the finding that L-arginine partially prevented LOX-1 overexpression in activated HL-60 cells. Our hypothesis on the mechanisms of LOX-1 induction in macrophages exposed to iNOS inhibition is summarized in Fig. 9.

NO has been shown to have both preventive and promotional effects on cell demise by either turning on apoptotic pathways or inhibiting several components of the proapoptotic cascade (17, 19, 58, 59). NO causes a G1 cell cycle arrest in tight correlation with p53 expression, transactivation, and subsequent upregulation of p21Waf1/Cip1 (58). Interestingly, expression of p21Waf1/Cip1 may prevent initiation of apoptosis at relatively low concentrations of NO. At high concentrations of NO, the activation of proapoptotic signaling systems such as p53 accumulation, opening of permeability transition pores, and cytochrome c leakage into the cytoplasm activate caspases and set in motion the NO-mediated apoptotic cell death (58). In macrophages, the consensus is that iNOS induction promotes cell apoptosis (17, 58). It is tempting to speculate that our findings of accumulated foam cell formation in ADMA- and L-NAME-exposed macrophages is consistent with the scenario that iNOS inhibition could prevent the apoptotic clearance of lipid-laden macrophages. In summary, our data offer an additional putative mechanism for ADMA-induced accelerated atherosclerosis in patients with chronic renal failure, namely, via induction of LOX-1 expression and increased OxLDL uptake by macrophages. Together with previous demonstrations that OxLDL uptake leads to increased synthesis of ADMA that affects endothelial cell-monocyte interactions, the data suggest the existence of a vicious circle from inhibition of NO production and subsequent increase in OxLDL accumulation and further increases in the synthesis of the NOS inhibitor ADMA, which then accelerates foam cell formation. The data illustrate an additional nonendothelial mode of the antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation in macrophages.


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This study was supported in part by National Institutes of Health Grants DK-54602 and DK-52783.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Goligorsky, Renal Research Institute, Basic Sciences Bldg., Rm. C23, New York Medical College, Valhalla, NY 10595 (E-mail: Michael_Goligorsky{at}nymc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Achan V, Broadhead M, Malaki M, Whitley GS, Leiper J, MacAllister R, and Vallance P. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 23: 1455–1459, 2003.[Abstract/Free Full Text]
  2. Anderson TJ. Nitric oxide, atherosclerosis and the clinical relevance of endothelial dysfunction. Heart Fail Rev 8: 71–86, 2003.[CrossRef][ISI][Medline]
  3. Boger RH and Bode-Boger SM. Asymmetric dimethylarginine, derangements of the endothelial nitric oxide synthase pathway, and cardiovascular diseases. Semin Thromb Hemost 26: 539–545, 2000.[CrossRef][ISI][Medline]
  4. Boger RH, Bode-Boger SM, Kienke S, Stan AC, Nafe R, and Frolich JC. Dietary L-arginine decreases myointimal cell proliferation and vascular monocyte accumulation in cholesterol-fed rabbits. Atherosclerosis 136: 67–77, 1998.[CrossRef][ISI][Medline]
  5. Boger RH, Bode-Boger SM, Tsao PS, Lin PS, Chan JR, and Cooke JP. An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes. J Am Coll Cardiol 36: 2287–2295, 2000.[Abstract/Free Full Text]
  6. Boger RH, Vallance, P, and Cooke JP. Asymmetric dimethylarginine (ADMA): a key regulator of nitric oxide synthase. Atheroscler Suppl 4: 1–3, 2003.[Medline]
  7. Boger RH, Sydow K, Borlak J, Thum T, Lenzen H, Schubert B, Tsikas D, and Bode-Boger SM. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases. Circ Res 87: 99–105, 2000.[Abstract/Free Full Text]
  8. Cabrero A, Cubero M, Llaverias G, Jove M, Planavila A, Alegret M, Sanchez R, Laguna JC, and Carrera MV. Differential effects of peroxisome proliferator-activated receptor activators on the mRNA levels of genes involved in lipid metabolism in primary human monocyte-derived macrophages. Metabolism 52: 652–657, 2003.[CrossRef][ISI][Medline]
  9. Closs EI, Basha FZ, Habermeier A, and orstermann U. Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B. Nitric Oxide 1: 65–73, 1997.[CrossRef][ISI][Medline]
  10. Chan JR, Boger RH, Bode-Boger SM, Tangphao O, Tsao PS, Blaschke TF, and Cooke JP. Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans. Arterioscler Thromb Vasc Biol 20: 1040–1046, 2000.[Abstract/Free Full Text]
  11. Chen M, Masaki T, and Sawamura T. LOX-1, the receptor for oxidized low-density lipoprotein identified from endothelial cells: implications in endothelial dysfunction and atherosclerosis. Pharmacol Ther 95: 89–100, 2002.[CrossRef][ISI][Medline]
  12. Cominacini L, Rigoni A, Pasini AF, Garbin U, Davoli A, Campagnola M, Pastorino AM, Lo Cascio V, and Sawamura T. The binding of oxidized low-density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells through an increased production of superoxide. J Biol Chem 276: 13750–13755, 2001.[Abstract/Free Full Text]
  13. Cooke JP. Does ADMA cause endothelial dysfunction? Arterioscler Thromb Vasc Biol 20: 2032–2037, 2000.[Abstract/Free Full Text]
  14. De Villiers WJ and Smart EJ. Macrophage scavenger receptors and foam cell formation. J Leukoc Biol 66: 740–746, 1999.[Abstract]
  15. Draude G and Lorenz RL. TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages. Am J Physiol Heart Circ Physiol 278: H1042–H1048, 2000.[Abstract/Free Full Text]
  16. Fliser D, Kielstein JT, Haller H, and Bode-Boger SM. Asymmetric dimethylarginine: a cardiovascular risk factor in renal disease? Kidney Int Suppl 37–40, 2003.
  17. Gotoh T, Oyadomari S, Mori K, and Mori M. Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J Biol Chem 277: 12343–12350, 2002.[Abstract/Free Full Text]
  18. Hayashida K, Kume N, Minami M, and Kita T. Lectin-like oxidized LDL receptor-1 (LOX-1) supports adhesion of mononuclear leukocytes and a monocyte-like cell line THP-1 cells under static and flow conditions. FEBS Lett 511: 133–138, 2002.[CrossRef][ISI][Medline]
  19. Heinloth A, Brune B, Fischer B, and Galle J. Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation. Atherosclerosis 162: 93–101, 2002.[CrossRef][ISI][Medline]
  20. Hunter GC, Henderson AM, Westerband A, Kobayashi H, Suzuki F, Yan ZQ, Sirsjo A, Putnam CW, and Hansson GK. The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque. J Vasc Surg 30: 36–50, 1999.[CrossRef][ISI][Medline]
  21. Ito S. Nitric oxide in the kidney. Curr Opin Nephrol Hypertens 4: 23–30, 1995.[CrossRef][Medline]
  22. Jankowski J, van der Giet M, Jankowski V, Schmidt S, Hemeier M, Mahn B, Giebing G, Tölle M, Luftmann H, Schlüter H, Zidek W, and Tepel M. Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. J Clin Invest 112: 256–264, 2003.[CrossRef][ISI][Medline]
  23. Jessup W and Dean RT. Autoinhibition of murine macrophage-mediated oxidation of low-density lipoprotein by nitric oxide synthesis. Atherosclerosis 101: 145–155, 1993.[CrossRef][ISI][Medline]
  24. Jessup W and Kritharides L. Metabolism of oxidized LDL by macrophages. Curr Opin Lipidol 11: 473–481, 2000.[CrossRef][ISI][Medline]
  25. Jouni ZE, Winzerling JJ, and McNamara DJ. 1,25-Dihydroxyvitamin D3-induced HL-60 macrophages: regulation of cholesterol and LDL metabolism. Atherosclerosis 117: 125–138, 1995.[CrossRef][ISI][Medline]
  26. Kataoka H, Kume N, Miyamoto S, Minami M, Moriwaki H, Murase T, Sawamura T, Masaki T, Hashimoto N, and Kita T. Expression of lectinlike oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions. Circulation 99: 3110–3117, 1999.[Abstract/Free Full Text]
  27. Kelm M. The L-arginine-nitric oxide pathway in hypertension. Curr Hypertens Rep 5: 80–86, 2003.[ISI][Medline]
  28. Kielstein JT, Bode-Boger SM, Frolich JC, Ritz E, Haller H, and Fliser D. Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation 107: 1891–1895, 2003.[Abstract/Free Full Text]
  29. Kielstein JT, Bode-Boger SM, Klein G, Graf S, Haller H, and Fliser D. Endogenous nitric oxide synthase inhibitors and renal perfusion in patients with heart failure. Eur J Clin Invest 33: 370–375, 2003.[CrossRef][ISI][Medline]
  30. Kimura C, Oike M, Ohnaka K, Nose Y, and Ito Y. Constitutive nitric oxide production in bovine aortic and brain microvascular endothelial cells: a comparative study. J Physiol 554: 721–730, 2004. First published November 14, 2003; 10.1113/jphysiol.2003.057059.[Abstract/Free Full Text]
  31. Kita T, Kume N, Minami M, Hayashida K, Murayama T, Sano H, Moriwaki H, Kataoka H, Nishi E, Horiuchi H, Arai H, and Yokode M. Role of oxidized LDL in atherosclerosis. Ann NY Acad Sci 947: 199–206, 2001.[Abstract/Free Full Text]
  32. Kita T, Kume N, Yokode M, Ishii K, Arai H, Horiuchi H, Moriwaki H, Minami M, Kataoka H, and Wakatsuki Y. Oxidized-LDL and atherosclerosis. Role of LOX-1. Ann NY Acad Sci 902: 95–102, 2000.[Abstract/Free Full Text]
  33. Klinge JM, Topf HG, Trusen B, Rauh M, Rascher W, and Dotsch J. Endothelial cells play an important role in the antiaggregatory effect of nitric oxide. Crit Care Med 31: 2010–2014, 2003.[CrossRef][ISI][Medline]
  34. Kockx MM, Cromheeke KM, Knaapen MW, Bosmans JM, De Meyer GR, Herman AG, and Bult H. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol 23: 440–446, 2003.[Abstract/Free Full Text]
  35. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, and Krieger M. Type I macrophage scavenger receptor contains alpha-helical and collagen-like coiled coils. Nature 343: 531–535, 1990.[CrossRef][Medline]
  36. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, and Nagano T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70: 2446–2453, 1998.[Medline]
  37. Kosaka S, Takahashi S, Masamura K, Kanehara H, Sakai J, Tohda G, Okada E, Oida K, Iwasaki T, Hattori H, Kodama T, Yamamoto T, and Miyamori I. Evidence of macrophage foam cell formation by very low-density lipoprotein receptor: interferon-gamma inhibition of very low-density lipoprotein receptor expression and foam cell formation in macrophages. Circulation 103: 1142–1147, 2001.[Abstract/Free Full Text]
  38. Kruth HS, Huang W, Ishii I, and Zhang WY. Macrophage foam cell formation with native low-density lipoprotein. J Biol Chem 277: 34573–34580, 2002.[Abstract/Free Full Text]
  39. Kuhlencordt PJ, Chen J, Han F, Astern J, and Huang PL. Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice. Circulation 103: 3099–3104, 2001.[Abstract/Free Full Text]
  40. Kume N and Kita T. Roles of lectin-like oxidized LDL receptor-1 and its soluble forms in atherogenesis. Curr Opin Lipidol 12: 419–423, 2001.[CrossRef][ISI][Medline]
  41. Kume N, Moriwaki H, Kataoka H, Minami M, Murase T, Sawamura T, Masaki T, and Kita T. Inducible expression of LOX-1, a novel receptor for oxidized LDL, in macrophages and vascular smooth muscle cells. Ann NY Acad Sci 902: 323–327, 2000.[Abstract/Free Full Text]
  42. Li AC and Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med 8: 1235–1242, 2002.[CrossRef][ISI][Medline]
  43. London GM and Drueke TB. Atherosclerosis and arteriosclerosis in chronic renal failure. Kidney Int 51: 1678–1695, 1997.[ISI][Medline]
  44. Lougheed M, Moore ED, Scriven DR, and Steinbrecher UP. Uptake of oxidized LDL by macrophages differs from that of acetyl LDL and leads to expansion of an acidic endolysosomal compartment. Arterioscler Thromb Vasc Biol 19: 1881–1890, 1999.[Abstract/Free Full Text]
  45. MacAllister RJ and Vallance P. Endogenous inhibitors of nitric oxide synthesis: how important are they? Exp Nephrol 6: 195–199, 1998.[CrossRef][ISI][Medline]
  46. MacAllister RJ, Whitley GS, and Vallance P. Effects of guanidino and uremic compounds on nitric oxide pathways. Kidney Int 45: 737–742, 1994.[ISI][Medline]
  47. Mehta JL and Li D. Identification, regulation and function of a novel lectin-like oxidized low-density lipoprotein receptor. J Am Coll Cardiol 39: 1429–1435, 2002.[Abstract/Free Full Text]
  48. Mehta JL, Li DY, Chen HJ, Joseph J, and Romeo F. Inhibition of LOX-1 by statins may relate to upregulation of eNOS. Biochem Biophys Res Commun 289: 857–861, 2001.[CrossRef][ISI][Medline]
  49. Morawietz H, Rueckschloss U, Niemann B, Duerrschmidt N, Galle J, Hakim K, Zerkowski HR, Sawamura T, and Holtz J. Angiotensin II induces LOX-1, the human endothelial receptor for oxidized low-density lipoprotein. Circulation 100: 899–902, 1999.[Abstract/Free Full Text]
  50. Moriwaki H, Kume N, Kataoka H, Murase T, Nishi E, Sawamura T, Masaki T, and Kita T. Expression of lectin-like oxidized low-density lipoprotein receptor-1 in human and murine macrophages: upregulated expression by TNF-alpha. FEBS Lett 440: 29–32, 1998.[CrossRef][ISI][Medline]
  51. Moriwaki H, Kume N, Sawamura T, Aoyama T, Hoshikawa H, Ochi H, Nishi E, Masaki T, and Kita T. Ligand specificity of LOX-1, a novel endothelial receptor for oxidized low-density lipoprotein. Arterioscler Thromb Vasc Biol 18: 1541–1547, 1998.[Abstract/Free Full Text]
  52. Nicholson AC, Han J, Febbraio M, Silversterin RL, and Hajjar DP. Role of CD36, the macrophage class B scavenger receptor, in atherosclerosis. Ann NY Acad Sci 947: 224–228, 2001.[Abstract/Free Full Text]
  53. Rattazzi M, Puato M, Faggin E, Bertipaglia B, Grego F, and Pauletto P. New markers of accelerated atherosclerosis in end-stage renal disease. J Nephrol 16: 11–20, 2003.[CrossRef][ISI][Medline]
  54. Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, Tanaka T, Miwa S, Katsura Y, Kita T, and Masaki T. An endothelial receptor for oxidized low-density lipoprotein. Nature 386: 73–77, 1997.[CrossRef][Medline]
  55. Vainio S and Ikonen E. Macrophage cholesterol transport: a critical player in foam cell formation. Ann Med 35: 146–155, 2003.[CrossRef][ISI][Medline]
  56. Vallance P. Importance of asymmetrical dimethylarginine in cardiovascular risk. Lancet 358: 2096–2097, 2001.[CrossRef][ISI][Medline]
  57. Vallance P, Leone A, Calver A, Collier J, and Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339: 572–575, 1992.[CrossRef][ISI][Medline]
  58. Yang F, von Knethen A, and Brune B. Modulation of nitric oxide-evoked apoptosis by the p53-downstream target p21WAF1/CIP1. J Leukoc Biol 68: 916–922, 2000.[Abstract/Free Full Text]
  59. Yates MT, Lambert LE, Whitten JP, McDonald I, Mano M, Ku G, and Mao SJ. A protective role for nitric oxide in the oxidative modification of low-density lipoproteins by mouse macrophages. FEBS Lett 309: 135–138, 1992.[CrossRef][ISI][Medline]
  60. Zingg JM, Ricciarelli R, and Azzi A. Scavenger receptors and modified lipoproteins: fatal attractions? IUBMB Life 49: 397–403, 2000.[CrossRef][ISI][Medline]



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