Published online before print March 4, 2004, doi: 10.1161/01.RES.0000124920.09738.26
© 2004 American Heart Association, Inc.
Molecular Medicine |
Glucose Enhances Human Macrophage LOX-1 Expression
Role for LOX-1 in Glucose-Induced Macrophage Foam Cell Formation
From the Department of Biomedical Sciences (L.L.) and Medicine (G.R.), University of Montreal, Centre Hospitalier de l’Université de Montréal (CHUM) Research Centre, Notre-Dame Hospital, Montreal, Quebec, Canada, and the Department of Bioscience (T.S.), National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka, Japan.
Correspondence to Dr Geneviève Renier, CHUM Research Centre, Notre-Dame Hospital, J.-A. De Seve Pavilion, Door Y 3622, 1560 Sherbrooke St E, Montreal, Quebec H2L 4M1, Canada. E-mail genevieve.renier{at}umontreal.ca
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Abstract |
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Lectin-like oxidized LDL receptor-1 (LOX-1) is a newly identified receptor for oxidized LDL that is expressed by vascular cells. LOX-1 is upregulated in aortas of diabetic rats and thus may contribute to the pathogenesis of human diabetic atherosclerosis. In this study, we examined the regulation of human monocyte-derived macrophage (MDM) LOX-1 expression by high glucose and the role of LOX-1 in glucose-induced foam cell formation. Incubation of human MDMs with glucose (5.6 to 30 mmol/L) enhanced, in a dose- and time-dependent manner, LOX-1 gene and protein expression. Induction of LOX-1 gene expression by high glucose was abolished by antioxidants, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), nuclear factor-
B (NF-B), and activated protein-1 (AP-1) inhibitors. In human MDMs cultured with high glucose, increased expression of PKCß2 and enhanced phosphorylation of extracellular signal-regulated protein kinase 1/2 was observed. Activation of these kinases was inhibited by the antioxidant N-acetyl-L-cysteine (NAC) and by the PKCß inhibitor LY379196. High glucose also enhanced the binding of nuclear proteins extracted from human MDMs to the NF-B and AP-1 regulatory elements of the LOX-1 gene promoter. This effect was abrogated by NAC and PKC/MAPK inhibitors. Finally, high glucose induced human macrophage-derived foam cell formation through a LOX-1–dependent pathway. Overall, these results demonstrate that high glucose concentrations enhance LOX-1 expression in human MDMs and that this effect is associated with foam cell formation. Pilot data showing that MDMs of patients with type 2 diabetes overexpress LOX-1 support the relevance of this work to human diabetic atherosclerosis.
Key Words: macrophages • lectin-like oxidized low-density lipoprotein receptor-1 • glucose • diabetes • foam cell formation
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Introduction |
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The prevalence, incidence, and mortality from all forms of cardiovascular diseases are increased in patients with diabetes.1 Among the cardiovascular risk factors documented in diabetes, hyperglycemia appears as an independent risk factor for diabetic macrovascular complications.2 Mechanisms through which hyperglycemia may promote the development of diabetic cardiovascular disease include glycoxidation and lipoxidation, increased oxidative stress, and protein kinase C (PKC) activation.3–8 One of the earliest events in atherogenesis is the accumulation of oxidized LDL (oxLDL) in the intima and the subsequent uptake of this modified lipoprotein by macrophages, leading to foam cell formation.9 One limiting factor for oxLDL uptake by endothelial cells is lectin-like oxLDL receptor-1 (LOX-1), a newly identified vascular receptor for oxLDL.10–12 Accumulating evidence indicates a key role for LOX-1 in atherogenesis. First, uptake of oxLDL by endothelial cells through LOX-1 induces endothelial dysfunction. Second, the two main LOX-1 ligands, oxLDL and advanced glycation end products (AGE), are implicated in the pathogenesis of atherosclerosis.5,9 Third, expression of LOX-1 by vascular cells, including endothelial cells and macrophages, is enhanced by proatherogenic factors.13–18 Finally, LOX-1 is expressed in vivo in the aortas of animals with proatherogenic settings16,19,20 and is upregulated in early human atherosclerotic lesions.21
LOX-1 expression is increased in the endothelium and aortas of diabetic rats16 and thus may play a role in atherogenesis associated with diabetes. Evidence that AGE induce LOX-1 expression in cultured endothelial cells16 and macrophages22 supports a primary role for these products in modulating vascular LOX-1 expression in diabetes.
On the basis of these results and given the key role for macrophages as precursors of foam cells in the vascular wall,9 the present study was aimed at investigating the regulation of human macrophage LOX-1 expression by hyperglycemia and the role of this receptor in glucose-induced macrophage foam cell transformation.
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Materials and Methods |
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Reagents
See the online data supplement, available at http://circres. ahajournals.org, for details about reagents.
Cell Culture
Freshly isolated human monocytes23 or THP-1 monocytes were differentiated into macrophages in vitro and treated with high glucose (see the online data supplement for details).
Analysis of mRNA Expression
Northern Blot Analysis
LOX-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in THP-1 monocyte-derived macrophages (MDMs) (10x106) was analyzed by hybridization with [32P] dCTP-labeled human LOX-1 and GAPDH cDNA probes (see the online data supplement for details).
Polymerase Chain Reaction Analysis
Total RNA for use in the polymerase chain reaction (PCR) reaction was extracted from human MDMs (2x106/mL) by an improvement of the acid-phenol technique of Chomczynski and Sacchi.24 cDNA was synthesized from RNA and amplified by synthetic primers specific for human LOX-1 and GAPDH (see the online data supplement for details).
Western Blot
LOX-1, mitogen-activated protein kinase (MAPK), and PKC-ß2 expression in human MDMs was analyzed by Western blot analysis using specific antibodies (see the online data supplement for details).
DNA Binding Assay
Nuclear proteins were isolated from THP-1 MDMs, and their binding to consensus sequences of the LOX-1 promoter nuclear factor-B (NF-B) and activated protein-1 (AP-1)–enhancing elements was assessed by DNA retardation electrophoretic mobility shift assay25,26 (see the online data supplement for details).
DNA Probes
See the online data supplement for details about DNA probes.
Uptake of Dil-oxLDL by Human MDMs
Native LDL (density, 1.019 to 1.063) was isolated from plasma obtained from healthy donors by sequential ultracentrifugation27 and extensively dialyzed for 24 hours at 4°C against 5 mmol/L Tris/50 nmol/L NaCl to remove EDTA. Oxidation of LDL was performed by incubating native LDL (2 mg of protein per mL) at 37°C for 20 hours in serum-free RPMI 1640 containing 7.5 µg/mL CuSO4. Oxidation of LDL was monitored by measuring the amount of thiobarbituric acid–reactive substances and by electrophoretic mobility on agarose gel. OxLDL was labeled with Dil as described previously.28 Uptake of DiI-oxLDL by human MDMs was assessed by fluorescence microscopy and determination of fluorescence at 520/564 nm (see the online data supplement for details). Results were normalized to total cell protein concentrations.29
Quantification of Cytosolic AGE in MDMs
The total AGE content present in the cytosolic extracts of glucose-treated MDMs was determined by competitive ELISA. Results were expressed as B/Bo (see the online data supplement for details).
Patients
The study group comprised 7 patients with type 2 diabetes and 12 healthy control subjects. The patients with diabetes were recruited from the Notre-Dame Hospital outpatient clinic and gave written consent to participate in this study. The patients had a mean (±SEM) age of 65±3 years, body mass index of 32±2 kg/m2, fasting glucose of 9.4±1.2 mmol/L, triglyceride level of 3.26±1.21 mmol/L, LDL cholesterol level of 3.08±0.31 mmol/L, and serum glycohemoglobin of 0.072±0.006. All patients were treated with glyburide and metformin. None of the patients was primarily insulin dependent. One patient was hypertensive and was treated with enalapril, and one had macroangiopathy and microalbuminuria. Control subjects were recruited from the hospital staff and relatives. They had a mean (±SEM) age of 38±4 years, body mass index of 23±1 kg/m2, fasting glucose of 5.0±0.1 mmol/L, triglyceride level of 1.30±0.20 mmol/L, and LDL cholesterol level of 3.50±0.40 mmol/L. Subjects who had infectious or inflammatory conditions or cardiac, renal, or pulmonary decompensated diseases or who were treated with antiinflammatory or antioxidant drugs were excluded from the study.
Determination of Cell Viability
Cell viability after treatment with the different agents under study was assessed by trypan blue exclusion and was consistently found to be >90%.
Statistical Analysis
Values are expressed as mean±SEM. Data were analyzed by one-way ANOVA followed by the Tukey test. P<0.05 was considered statistically significant.
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Results |
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Effect of D-Glucose on Human MDM LOX-1 mRNA Expression
Incubation of human MDMs for 24 to 72 hours with 5.6 or 30 mmol/L D-glucose increased, in a time-dependent manner, macrophage LOX-1 gene expression. Maximal effect was observed from 48 to 72 hours (Figure 1Aa). Under these experimental conditions, no modulation of the mRNA expression of GAPDH, used as internal control, was observed (Figure 1Ab). LOX-1 mRNA levels, normalized to the levels of GAPDH mRNA, are shown in Figure 1Ac. The stimulatory effect of D-glucose on human MDM LOX-1 mRNA expression was dose-dependent, with maximal effect occurring between 20 and 30 mmol/L glucose (Figure 1Ba). Under these experimental conditions, no modulation of the mRNA expression of GAPDH was observed (Figure 1Bb). LOX-1 mRNA levels, normalized to the levels of GAPDH mRNA, are shown in Figure 1Bc. Incubation of human MDMs with L-glucose or mannitol (30 mmol/L) did not enhance LOX-1 mRNA expression (LOX-1 mRNA expression [% of control values]: L-glucose, 98±5; mannitol, 107±6).
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Effect of D-Glucose on Human MDM LOX-1 Protein Expression
Treatment of human MDMs with 30 mmol/L D-glucose enhanced LOX-1 protein expression in these cells. This effect was observed from 72 to 96 hours (Figure 2Aa). Under these experimental conditions, no modulation of ß-actin, used as internal control, was observed (Figure 2Ab). LOX-1 protein levels normalized to the levels of ß-actin protein are illustrated in Figure 2Ac. Incubation of human MDMs for 72 hours with increasing D-glucose concentrations (5.6 to 30 mmol/L) enhanced, in a dose-dependent manner, LOX-1 protein expression in these cells (Figure 2Ba). LOX-1 protein levels normalized to the levels of ß-actin (Figure 2Bb) are illustrated in Figure 2Bc. No stimulatory effect of mannitol (30 mmol/L) on macrophage LOX-1 protein expression was observed (LOX-1 protein expression [% of control values]: mannitol, 109±9).
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Role of AGE in the Induction of MDM LOX-1 by High Glucose
To evaluate whether intracellular AGE formation may play a role in the induction of MDM LOX-1 expression by high glucose, the levels of cytosolic glycated proteins present in MDMs exposed to high glucose for 24 to 48 hours were determined. Regardless of the glucose concentrations used, levels of glycated proteins in MDMs consistently fell below the minimum concentration of AGE detected by this assay, ie, <0.25 ng AGE/µg protein (B/Bo: glucose at 24 hours [in mmol/L], 5.6: 2.5±0.7; 10: 3.9±1.3; 20: 3.4±0.8; 30: 3.9±0.7; glucose at 48 hours [in mmol/L], 5.6: 3.7±1.7; 10: 2.9±1.2; 20: 3.3±0.3; 30: 2.3±0.5). Although nonglycated BSA (50 ng/mL), used as negative control, failed to inhibit antiserum binding (B/Bo, 1.1), competition for antibody binding was observed with methylglyoxal- and glucose-derived AGE-BSA (50 ng/mL) used as positive controls (B/Bo, 0.53 and 0.57, respectively).
Effect of High Glucose on Tumor Necrosis Factor-
–Induced MDM LOX-1 Expression
One pathophysiological stimulus relevant to atherosclerosis in diabetes is tumor necrosis factor- (TNF-).30,31 Because this cytokine stimulates LOX-1 expression in vascular cells13,17 and is released by monocytic cells in response to high glucose and AGE,32–35 we determined the modulatory effect of TNF- on human MDM LOX-1 expression under normal and high glucose conditions. As shown in Figures 3A and 3B, TNF-–treated human MDMs cultured under normoglycemic conditions express similar levels of LOX-1 gene and protein expression as high glucose-treated cells. Levels of LOX-1 expression elicited by this cytokine did not differ when human MDMs were cultured in high glucose conditions. The effect of TNF- alone on LOX-1 protein expression was blocked by an anti–TNF- antibody (Figure 3B).
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Signaling Pathways Involved in Glucose-Induced Human MDM LOX-1 Gene Expression
To identify the signaling pathways involved in the stimulatory effect of high glucose on LOX-1 gene expression, human MDMs were pretreated for 2 hours with PKC, MAPK, tyrosine kinase, NF-B, or AP-1 inhibitors before exposure to glucose. As shown in Figure 4A, the pan-specific PKC inhibitor calphostin C (0.1 µg/mL) and the PKCß inhibitor LY379196 (30 nmol/L) totally abrogated glucose-induced macrophage LOX-1 gene expression. A similar effect was observed when the cells were preincubated with the MAPK inhibitor PD98059 (50 µmol/L), the AP-1 inhibitor curcumin (10 µmol/L), or the NF-B inhibitor BAY 11-7085 (40 µmol/L)36 (Figure 4A). In contrast, tyrosine kinase inhibitors did not affect this parameter (data not shown). Under these experimental conditions, no modulation of the mRNA expression of GAPDH was observed (Figure 4Ab). LOX-1 mRNA levels, normalized to the levels of GAPDH mRNA, are presented in Figure 4Ac. Because diabetes and high glucose induce increased oxidative stress,37 we next determined the role of oxidative stress in the regulation of LOX-1 gene expression by glucose. As shown in Figure 4B, preincubation of human MDMs with various antioxidants, including N-acetyl-l-cysteine (NAC) (10 mmol/L), vitamin E (50 µmol/L), vitamin C (10 µmol/L), and DMSO (0.5%), totally prevented the stimulatory effect of high glucose on LOX-1 gene expression. Involvement of these signaling events was confirmed in THP-1 MDMs by demonstrating that PKC and MAPK inhibitors as well as antioxidants abolished glucose-induced LOX-1 mRNA expression in these cells (Figures 4C and 4D). Having established the relevance of THP-1 cells to human MDMs, we next assessed using these cells the sequential events leading to glucose-induced PKC/MAPK activation. As shown in Figure 5, treatment of THP-1 cells for 48 hours with high glucose induced PKCß2 (Figure 5A) and extracellular signal-regulated protein kinase (ERK) 1/2 (Figure 5B) activation, as assessed by Western blot analysis. Glucose-induced activation of these kinases was reduced by NAC (Figures 5A and 5B). Furthermore, ERK1/2 activation in glucose-treated macrophages was inhibited by LY379196 (Figure 5B), thereby identifying MAPK as downstream targets of PKC.
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Effect of High Glucose Concentrations on the Binding of Nuclear Proteins to the Regulatory NF-B and AP-1 Sequences of the LOX-1 Gene Promoter
Exposure of THP-1 MDMs to a high glucose environment increased the binding of nuclear proteins to the NF-B (Figure 6) and AP-1 (Figure 7) consensus sequences of the human LOX-1 promoter. These binding complexes were specifically competed in the presence of a 1000-molar excess of the unlabeled NF-B or AP-1 oligonucleotides and were significantly decreased by BAY 11-7085 (Figure 6) or curcumin (Figure 7). Nuclear protein binding was additionally inhibited in the presence of antibodies against p50 and p65 (Figure 6) or c-fos and c-Jun (Figure 7). In contrast, irrelevant antibodies or competitors did not alter glucose-induced NF-B and AP-1 activation, thus confirming the specificity of the inhibition documented in these electrophoretic mobility shift assays (Figures 6 and 7
). Preincubation of THP-1 cells with NAC, PKC, and MAPK inhibitors also suppresses the nuclear binding to the NF-B and AP-1 sequences of the LOX-1 gene promoter (Figures 6 and 7 ).
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Role of LOX-1 in Mediating Glucose-Induced Human MDM Foam Cell Formation
To evaluate whether increased expression of LOX-1 by high glucose resulted in enhanced uptake of oxLDL by human MDMs, these cells were treated for 48 hours with 5.6 or 30 mmol/L glucose, and then incubation was pursued for an additional 24-hour period in the presence of saturating amounts (20 µg/mL) of antibodies to CD36, SR-A, LOX-1, or IgG1. At the end of the incubation period, cells were exposed for 3 hours to DiI-oxLDL (80 µg/mL) in the presence or absence of excess unlabeled oxLDL. Incubation of human MDMs with high glucose in the presence of anti-CD36 and anti-SR-A antibodies led to enhanced uptake of oxLDL by these cells, as assessed by fluorescence microscopy (Figure 8A) and measurement of extracted DiI-oxLDL (Figure 8B). This effect was abrogated by incubating human MDMs with excess unlabeled oxLDL or with anti–LOX-1 antibody. In contrast, exposure of these cells to anti-IgG1 did not affect glucose-induced MDM foam cell formation (Figures 8A and 8B).
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Levels of LOX-1 mRNA in MDMs of Patients With Diabetes
MDMs of patients with type 2 diabetes demonstrated a significant increase in LOX-1 mRNA levels compared with those isolated from control subjects (LOX-1 mRNA levels [%], controls: 100±7; patients with diabetes, 169±25; P<0.01).
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Discussion |
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Despite the recent evidence linking experimental diabetes with increased vascular LOX-1 expression,16 only few studies have examined the regulation of LOX-1 by metabolic factors dysregulated in diabetes. The present study demonstrates for the first time that high glucose increases human macrophage LOX-1 expression, both at gene and protein levels. These results together with our preliminary observations that MDMs of diabetic patients exhibit increased LOX-1 gene expression suggest a role of hyperglycemia in the regulation of vascular LOX-1 in human diabetes. In macrophages that do express multiple scavenger receptors,38 >50% of the uptake of oxLDL seems to occur via CD36,39 whereas SR-A shares the rest with several other scavenger receptors, including LOX-1. Because high glucose enhances macrophage CD36 expression,40 it is tempting to postulate that upregulation of macrophage scavenger receptors in response to glucose may play a role in the pathogenesis of atherosclerosis in human diabetes.
It has been previously shown that AGE enhance LOX-1 mRNA expression in cultured aortic endothelial cells and human macrophages.16,22 On the basis of the time course and concentration of glucose required to modulate macrophage LOX-1 expression, we speculated that generation of AGE might be responsible for LOX-1 induction in glucose-treated MDMs. However, arguing against this hypothesis, we did not ascertain the presence of AGE in these cells over the time course required to modulate LOX-1 gene expression. Considering the short incubation period of macrophages with high glucose, lack of intracellular AGE detection may be related to this in vitro variable. Alternatively, characteristics relating to the sensitivity of the ELISA and the specificity of the anti–AGE-RNAse antiserum used in this assay41 may account for these negative results.
Interestingly, we found that the extent of stimulation of macrophage LOX-1 expression achieved by glucose was comparable to that elicited by TNF- and that these two stimuli did not synergize for macrophage LOX-1 induction. Because glucose and AGE stimulate TNF- secretion,32–35 one possible explanation for this observation is that induction of LOX-1 by glucose involves TNF-. However, this hypothesis is not supported by our finding that immunoneutralization of TNF- does not affect glucose-induced LOX-1 expression. Alternatively, glucose and TNF- may regulate macrophage LOX-1 through one major and possibly identical pathway. Like TNF-, glucose is a well-known activator of NF-B and AP-142–46 and may therefore induce, through the activation of these factors, the transcription of the LOX-1 gene. Consistent with this, we found that glucose increases the LOX-1 mRNA levels in macrophages and enhances the binding of nuclear proteins to the NF-B and AP-1 regulatory sequences of the LOX-1 promoter.47 Although final proof for a role for NF-B and AP-1 as functional responsive elements involved in the transcriptional activation of the LOX-1 gene would require promoter-reporter gene assays, these data support a role for these transcriptional factors in the regulation of LOX-1 gene expression by glucose.
Regulation of LOX-1 gene expression is redox sensitive.14 Therefore, reactive oxygen species (ROS) generated by glucose in vascular cells6,7,46 may represent key intermediates in the regulation of LOX-1 gene expression by this metabolic factor. Evidence linking glucose-induced oxidative stress with activation of PKC and MAPK in vascular cells6–8,48,49 additionally supports a role of these kinases in the control of LOX-1 expression by hyperglycemia. In line with these hypotheses, we found that antioxidants and PKC/MAPK inhibitors abolish glucose-induced macrophage LOX-1 mRNA levels, thus implicating ROS and kinases as signaling molecules in this effect. Our findings that antioxidants suppressed glucose-induced PKC/MAPK activation and that PKC inhibition abolished glucose-induced MAPK activation support the hypothesis that glucose-induced kinase activation involves oxidative stress and that MAPKs act in this signaling cascade as intermediate molecules transducing signals from PKC to macrophage LOX-1. Convincing data also indicate a role for oxidative stress and kinases in NF-B and AP-1 activation.7,42,46,49–51 In accordance with these results, we found that antioxidants as well as PKC/MAPK inhibitors block glucose-induced NF-B and AP-1 activation, thus identifying these transcriptional factors as downstream ROS and kinase targets. Taken together, these results indicate that increased production of intracellular ROS and activation of PKC/MAPK pathways are initial signaling events in the regulation of LOX-1 gene by glucose that are required for subsequent activation of NF-B and AP-1.
Accumulation of cholesterol-loaded foam cells in the arterial intima is a hallmark and key event of early atherogenesis. Evidence that incubation of macrophages in high glucose conditions leads to increased intracellular accumulation of cholesterol ester52 suggests a role for hyperglycemia in foam cell formation. Like other scavenger receptors, LOX-1 is highly expressed in macrophages present in human atherosclerotic lesions21 and thus may play a role in macrophage foam cell formation. The present study demonstrates for the first time that increased LOX-1 surface expression in glucose-treated macrophages is associated with enhanced uptake of oxLDL by these cells, suggesting thereby a new role for LOX-1, that of mediating glucose-induced foam cell formation. Importantly, such a role for LOX-1 in foam cell formation was only evident after functional blockade of CD36. It is widely believed that much of the oxLDL uptake by human macrophages occurs via CD36.39 Although the quantitative contribution of CD36 in glucose-induced foam cell formation is unknown, it has been shown that glucose-induced macrophage CD36 expression correlates with a 10-fold increase in CD36-mediated oxLDL uptake,40 thus suggesting a major role of this receptor in glucose-induced foam cell formation. In the present study, we reported a 2-fold increase in non-CD36/non–SR-A–mediated oxLDL uptake in glucose-treated macrophages that was only partly reduced by an anti-LOX-1 antibody. Although these results demonstrate a role of LOX-1 in glucose-induced foam cell formation, they do not argue for a major contribution of LOX-1 in this process. Consistent with this idea, one recent study failed to demonstrate a key role of LOX-1 in the progression of macrophages to foam cells in vitro.53 Nevertheless, extrapolation of in vitro results to the in vivo situation is hazardous, and additional studies are needed to assess the functional significance of increased LOX-1 expression on foam cell formation in vivo.
In summary, the present study demonstrates that high glucose enhances human MDM LOX-1 expression in vitro and that this effect is associated with foam cell formation. Our preliminary results showing increased MDM LOX-1 expression in human diabetes support the relevance of this work to the human setting.
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Acknowledgments |
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This study was supported by a grant from the Association Diabète Québec. We thank Dr O. Serri (CHUM, Notre-Dame Hospital, Montreal, Quebec, Canada) for the referral of the patients with diabetes.
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Footnotes |
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Original received March 5, 2003; resubmission received September 30, 2003; revised resubmission received February 19, 2004; accepted February 20, 2004.
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