Published online before print October 7, 2004, doi: 10.1161/01.RES.0000147309.54227.42
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2004 American Heart Association, Inc.
Molecular Medicine |
C-Reactive Protein Enhances LOX-1 Expression in Human Aortic Endothelial Cells
Relevance of LOX-1 to C-Reactive Protein–Induced Endothelial Dysfunction
From Department of Biomedical Sciences (L.L.) and Medicine (N.R., 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 East, Montreal, Quebec H2L 4M1, Canada. E-mail genevieve.renier{at}umontreal.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C-reactive protein (CRP), a characteristic inflammatory marker, is a powerful predictor of cardiovascular events. Recent data suggest that CRP may also promote atherogenesis through inducing endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is a newly identified endothelial receptor for oxLDL that plays a pivotal role in oxLDL-induced endothelial dysfunction. Whether CRP may regulate endothelial LOX-1 and induce endothelial dysfunction through this receptor is unknown. In the present study, we studied the in vitro effect of CRP on LOX-1 expression in human aortic endothelial cells (HAECs) and the role of LOX-1 in CRP-induced human monocyte adhesion to endothelium and oxLDL uptake by endothelial cells. Incubation of HAECs with CRP enhanced, in a dose- and time-dependent manner, LOX-1 mRNA and protein levels. Induction of LOX-1 protein was already present at 5 µg/mL CRP and reached a maximum at 25 µg/mL. This effect was reduced by antibodies against CD32/CD64, endothelin-1 (ET-1) and interleukin-6 (IL-6). The extent of stimulation of LOX-1 achieved by CRP was comparable to that elicited by high glucose and IL-6 and remained unchanged in presence of these factors. Finally, CRP increased, through LOX-1, both human monocyte adhesion to endothelial cells and oxLDL uptake by these cells. We conclude that CRP enhances endothelial LOX-1 expression and propose a new mechanism by which CRP may promote endothelial dysfunction, that of inducing LOX-1.
Key Words: C-reactive protein • endothelium • lectin-like oxidized low-density lipoprotein receptor-1 • inflammation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Atherosclerosis is an inflammatory process that takes place in the arterial wall and is accompanied by a systemic response. Since the concept of an inflammatory soil of atherosclerosis has been validated,1–3 serum markers of inflammation have been identified as risk markers for cardiovascular disease. Among these, highly sensitive C-reactive protein (CRP) has been proven to be the strongest predictor of cardiovascular events.4–5 Besides being a risk marker, CRP may further play a pivotal role in promoting atherogenesis. Arguing for this hypothesis, it has been shown that CRP increases the release of inflammatory cytokines,6 enhances the binding of monocytes to endothelium,7 and favors macrophage foam cell formation.8 CRP also decreases endothelial nitric oxide synthase activation while increasing the expression of endothelial cell adhesion molecules, chemokines, endothelin-1 (ET-1), and plasminogen activator inhibitor-1.9–13
Unregulated uptake of oxidized low-density lipoprotein (oxLDL) by vascular cells is a crucial step in atherogenesis. Endothelial lectin-like oxidized LDL receptor-1 (LOX-1) is the major receptor of oxLDL,14 and accumulating evidences indicate that oxLDL uptake through this receptor induces endothelial dysfunction. oxLDL binding to endothelial LOX-1 generates superoxide anions, decreases nitric oxide production, and activates nuclear factor-
B (NF-B).15–16 Furthermore, inhibition of LOX-1 reduces oxLDL-mediated upregulation of monocyte chemoattractant protein-1 (MCP-1) and monocyte adhesion to endothelial cells.17 Endothelial LOX-1 expression is induced by various pro-inflammatory cytokines, such as tumor necrosis factor-
(TNF-)18 and transforming growth factor-ß (TGF-ß),19 as well as by pro-atherogenic factors, such as oxLDL and advanced glycation end products in vitro.20 This receptor is expressed in the aortas of hypertensive,21 diabetic,20 and hyperlipidemic22 animals and is upregulated in early human atherosclerotic lesions.23 In the present study, we hypothesized that CRP-induced endothelial dysfunction may be mediated in part via LOX-1. Thus, we tested the effect of CRP on endothelial LOX-1 expression and the role of this receptor in CRP-induced monocyte adhesion and oxLDL uptake by endothelial cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Reagents
Phenylmethylsulfonyl fluoride, Hanks balanced salt solution, penicillin–streptomycin, glycine, sodium dodecyl sulfate (SDS), and Trizol reagent were obtained from Invitrogen Life Technologies (Burlington, Ontario, Canada). Human aortic endothelial cells (HAECs), endothelial growth culture medium (EGM), and EGM bullet kit were obtained from Cedarlane Laboratories Limited (Hornby, Ontario, Canada). D-Glucose, bovine serum albumin fraction V, dianisinine dihydrochloride, hexadecyltrimethylamine ammonium bromide (HTAB), isopropanol, and E-TOXATE kit were purchased from Sigma. END-X B15 endotoxin removal affinity resin kit was obtained from Seikagaku America (Falmouth, Mass). Ficoll and horseradish peroxidase-conjugated anti-mouse IgG were obtained from Amersham Biosciences. Monoclonal antibody against ß-actin was bought from Santa Cruz Biotechnology (Santa Cruz, Calif). Recombinant human interleukin-6 (IL-6), human neutralizing antibodies against IgG1 (10 µg/mL), intercellular adhesion molecule-1 (ICAM-1) (20 µg/mL), vascular cell adhesion molecule-1 (VCAM-1) (20 µg/mL), E-selectin (20 µg/mL), IL-6 (5 µg/mL), and ET-1 (0.1 µg/mL) were purchased from R&D Systems (Minneapolis, Minn). Monoclonal antibody to human CD32 (5 µg/mL) was purchased from MEDICORP (Montreal, QC, Canada). Monoclonal antibodies to human LOX-1 (20 µg/mL) and human CD64 (5 µg/mL) were kindly provided by Dr Sawamura (National Cardiovascular Center Research Unit, Osaka, Japan) and Dr Sarfati (CHUM Research Center, Montreal, Canada), respectively. Recombinant human CRP, BAY11–7085, and actinomycin D were obtained from Calbiochem (La Jolla, Calif). 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was bought from Molecular Probes.
Cells
Commercially available HAECs (passage 3) were grown to confluence in EGM under recommended conditions. The EGM was supplemented with 2% fetal bovine serum (FBS) containing 0.01 µg/mL human epidermal growth factor, 0.1% gentamicin sulfate amphotericin-B, 1 µg/mL hydrocortisone, and 12 µg/mL bovine brain extract protein content. Cells were used at passages 3 to 5. Confluent HAECs, cultured in medium containing 2% FBS, were exposed for different time periods to various concentrations of CRP. Endotoxin was removed from CRP using END-X B15 endotoxin removal affinity resin kit at 4°C overnight. After removal of endotoxin, CRP contained <0.03 EU/mL endotoxin, as detected by E-TOXATE kit. In some experiments, cells were pretreated with antibodies against human CD32, CD64, ET-1, or IL-6, or coincubated with CRP in the presence or absence of glucose (30 mmol/L) or IL-6.
Human monocytes were isolated as previously described.24 Briefly, peripheral blood mononuclear cells were isolated from healthy control subjects by density centrifugation using Ficoll, allowed to aggregate in the presence of fetal calf serum, then further purified by the rosetting technique. After density centrifugation, highly purified monocytes (85% to 90%) were recovered. Human monocyte purity was assessed by flow cytometry (FACScan; Becton Dickinson) using phycoerythrin-conjugated anti-CD14 monoclonal antibody (Becton Dickinson).
Analysis of mRNA Expression
Expression of the LOX-1 gene in human HAECs cultured in 6-well plates (FALCON) was measured by polymerase chain reaction (PCR) technique. Total RNA for use in the PCR reaction was extracted from cells by an improvement of the acid–phenol technique of Chomczynski.25 Briefly, cells were lysed with TRIzol reagent and chloroform was added to the solution. After centrifugation, the RNA present in the aqueous phase was precipitated and resuspended in diethyl pyrocarbonate water. cDNA was synthesized from RNA by incubating total cellular RNA with 0.1 µg oligodT (Pharmacia) for 5 minutes at 98°C, then by incubating the mixture with reverse transcription buffer for 1 hour at 37°C. The cDNA obtained was amplified by using 0.8 µmol/L of two synthetic primers specific for human LOX-1 (5'-TTACTCTCCATGGTGGTGCC-3') (5'-AGCTTCTTCTDCTTGTTGCC-3') and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-CCCTTCATTGACCTCAACTACATGG-3') (5'-AGTCTTCTGGGTGGCAGTGATGG-3'), used as internal standard in the PCR reaction mixture. A 193-base pair human LOX-1 cDNA fragment and a 456-base pair human GAPDH cDNA fragment were amplified enzymatically by 30 and 25 repeated cycles, respectively. An aliquot of each reaction mixture was then subjected to electrophoresis on 1% agarose gel containing ethidium bromide. The intensity of the bands was measured by an image analysis scanning system (Alpha Imager 2000; Packard Instrument Company). Titrating the cDNA samples ensured that the signal lies on the exponential part of the standard curve.
Western Blot
HAEC protein extracts (12 µg) were applied to 10% SDS-PAGE and transferred to a nitrocellulose membrane using a Bio-Rad transfer blotting system at 100 V for 1 hour. Nonspecific binding was blocked with 5% bovine serum albumin for 1 hour at room temperature. After washing with PBS–Tween 0.1%, blots were incubated overnight at 4°C with anti-LOX-1 or anti-ß-actin antibodies. After further wash, membranes were incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated donkey anti-mouse IgG (1/5000). Antigen detection was performed with an enhanced chemiluminescence detection system (Amersham).
Adhesion Assay
Confluent HAECs cultured in 96-well plates (FALCON) were exposed for 15 hours to 25 µg/mL CRP, then treated for 1 hour in the presence of antibodies to IgG1 (10 µg/mL) or LOX-1. HAECs were then washed twice with Hanks balanced salt solution and incubated for 2 hours with freshly purified human monocytes (280 000 cells/well) resuspended in serum-free RPMI medium. At the end of this incubation period, nonadherent monocytes were removed by washing the cells with PBS (pH 6.0). Monocyte adhesion to HAECs was quantified by measuring monocyte myeloperoxidase (MPO) activity.26
Determination of Endothelial Cell-Associated Adhesion Molecule Expression
Endothelial cell surface expression of ICAM-1, VCAM-1, and E-selectin was determined by the cellular enzyme-linked immunosorbent assay method. HAECs cultured in 96-well plates (FALCON) were treated with CRP for 15 hours, and then washed with PBS. To block nonspecific binding, endothelial cells were treated for 1 hour at room temperature with PBS–3% bovine serum albumin. Twenty µg/mL of monoclonal antibodies against ICAM-1, VCAM-1, E-selectin, and control IgG1 were then added to the cells for 2 hours at 37°C. After washing, endothelial cells were incubated for 90 minutes with horseradish-conjugated anti-mouse IgG (1/1000) (Bio-Rad). The peroxidase substrate, o-phenylnediamine dihydrochloride, was then added to the cells. The reaction was stopped by addition of 50 µL of H2SO4 (0.5 mol/L) and the optical density was read at 490 nm.
Uptake of DiI–oxLDL
Native LDL (density, 1.019 to 1.063) was isolated from plasma obtained from healthy donors by sequential ultracentrifugation using potassium bromide for density adjustment.27 Native LDL was 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 protein/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 (
10 nmol malondialdehyde equivalent/mg protein) and by electrophoretic mobility on agarose gel (data not shown). Labeling of oxLDL with DiI was performed as described previously.28 To examine cellular uptake of oxLDL, HAECs were seated in 8-well culture slides (FALCON) and incubated for 3 hours in medium containing 5% of lipoprotein-deficient serum with DiI-labeled oxLDL (80 µg/mL) in the presence or absence of a 500-fold excess of unlabeled oxLDL. At the end of the incubation period, cells were washed, mounted on coverslips with mounting medium, and examined by fluorescence microscopy. To measure amounts of DiI–oxLDL accumulated in cells, HAECs were seated in 12-well plates receiving the same treatment as mentioned. At the end of the incubation period, DiI was extracted by isopropanol and the fluorescence was determined at 520/564 nm. Results were normalized to total cell protein concentrations.29
Cell Viability
Cell viability after treatment with CRP was assessed by trypan blue exclusion and determination of total cell number. It was consistently found to be higher than 95%.
Statistical Analysis
All values were expressed as the mean±SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey test. P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effect of CRP on Endothelial LOX-1 mRNA Expression
Incubation of HAECs for 6 hours with 25 µg/mL CRP enhanced LOX-1 mRNA levels. This effect was sustained up to 24 hours (Figure 1A-a). LOX-1 mRNA levels, normalized to the levels of GAPDH mRNA (Figure 1A-b), are presented in Figure 1A-c. The stimulatory effect of CRP on LOX-1 was already present at a concentration of 5 µg/mL and was maximal with CRP concentrations ranging between 10 and 25 µg/mL (Figure 1B-a). LOX-1 mRNA levels, normalized to the levels of GAPDH mRNA (Figure 1B-b), are presented in Figure 1B-c. Preincubation of the cells with actinomycin D (5 µg/mL) for 1 hour before exposure of the cells to CRP prevented this effect (Figure 1C). Interestingly, an increase in LOX-1 mRNA expression was also observed when cells were treated for 7 days with 5 µg/mL CRP (LOX-1 gene expression [% of control values]: CRP (5 µg/mL),143±8; P<0.05 versus controls).
|
Effect of CRP on Endothelial LOX-1 Protein Expression
Treatment of HAECs for 15 hours with 25 µg/mL CRP significantly increased LOX-1 protein levels. This effect was sustained up to 48 hours (Figure 2 A-a). LOX-1 protein levels normalized to the levels of ß-actin (Figure 2A-b) are illustrated in Figure 2A-c. CRP-induced LOX-1 protein expression was dose-dependent, with maximal effect being observed between 10 and 25 µg/mL CRP (Figure 2B-a). LOX-1 protein levels normalized to the levels of ß-actin (Figure 2B-b) are illustrated in Figure 2B-c. Because CRP binds on vascular cells to Fc
RI/CD64 and FcRIIa/CD32 receptors, we next determined whether the CRP effect on LOX-1 is receptor-mediated. HAECs were pre-incubated in the presence of antibodies to CD32 and/or CD64 before treatment with CRP. We found that CRP effect on LOX-1 was reduced, in part, by pre-incubating HAECs with anti-CD32 and anti-CD64 antibodies, and that coincubation of the endothelial cells with both antibodies totally abrogated this effect (Figure 2C). Treatment of endothelial cells with these antibodies did not influence basal LOX-1 expression (LOX-1 protein expression [% of control values]: CD32 antibody, 85±12; CD64 antibody, 102±9; CD32 +CD64 antibodies, 107±8).
|
Effect of IL-6 and Glucose on CRP-Induced Endothelial LOX-1 Protein Expression
Because diabetes is associated with inflammatory processes with an increase in the levels of CRP30 and pro-inflammatory cytokines,31,32 we studied the effect of CRP on LOX-1 expression in IL-6- and high glucose-treated HAECs. Treatment of HAECs with IL-6 (0 to 200 ng/mL) resulted in a dose-dependent increase in LOX-1 protein expression, with maximal effect at 100 to 200 ng/mL (Figure 3 A). CRP did not potentiate this effect (Figure 3B). CRP-induced endothelial LOX-1 expression was reduced by ET-1 and IL-6 inhibition (Figure 3B) and coincubation of HAECs with anti-ET-1 and IL-6 antibodies totally suppressed this effect (Figure 3B). Remarkably, HAECs cultured with CRP (25 µg/mL for 15 hours) showed a 3-fold increase in IL-6 secretion (data not shown). As reported earlier,33 incubation of HAECs in a high-glucose environment led to increased endothelial LOX-1 protein expression (Figure 3B). Addition of CRP did not further increase LOX-1 expression under hyperglycemic conditions (Figure 3B). Pretreatment of HAECs with the NF-B inhibitor, BAY11–7085 (10 µmol/L), totally abrogated the independent and combined effects of glucose and CRP on LOX-1 expression (LOX-1 protein expression [% of control values]: glucose,193.1±13; BAY+glucose, 94±10; CRP, 160±10; BAY+CRP, 110±21; glucose+CRP, 165±11; BAY+glucose+CRP, 106±10).
|
CRP Enhances Monocyte Binding to HAECs: Role of LOX-1
Treatment of HAECs with CRP (25 µg/mL) or lipopolysaccharide (10 ng/mL), used in these experiments as positive control, increased monocyte adhesion to these cells (Figure 4). Anti-LOX-1 antibody suppressed this effect, whereas anti-IgG1 antibody was ineffective (Figure 4). HAECs cultured with CRP for 15 hours in medium containing 2% FBS did not show induction of ICAM-1, VCAM-1, or E-selectin expression (adhesion molecule protein expression [% over control values]: ICAM-1, 115±7; VCAM-1, 97±5; E-selectin, 90±9). Intriguingly, similar negative results were obtained when CRP was added for 6 or 24 hours to HAECs cultured in medium containing 10% or 20% FBS (data not shown). Incubation of HAECs with antibodies to ICAM-1, VCAM-1, and E-selectin failed to affect CRP-induced monocyte adhesion (Figure 4). Treatment of endothelial cells with these antibodies did not influence basal monocyte adhesion (monocyte adhesion [% of control values]: VCAM-1+ICAM-1+E-selectin antibodies, 116±11; LOX-1 antibody, 102±13).
|
CRP Stimulates oxLDL Uptake in Endothelial Cells Via LOX-1
To evaluate whether induction of LOX-1 by CRP results in enhanced uptake of oxLDL by endothelial cells, HAECs were treated for 14 hours with CRP (25 µg/mL), then incubation was pursued for 1 hour in the presence of saturating amounts (20 µg/mL) of antibodies to 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 HAECs with CRP led to enhanced uptake of oxLDL by these cells as assessed by fluorescence microscopy (Figure 5 A) and measurement of extracted DiI–oxLDL (Figure 5B). This effect was abrogated by incubating HAECs with anti-LOX-1 antibody or with excess unlabeled oxLDL. In contrast, exposure of these cells to anti-IgG1 did not affect CRP-induced oxLDL uptake by endothelial cells (Figure 5A and 5B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Accumulating evidences indicate that chronic low-grade inflammation is a major pathogenic component of endothelial dysfunction.1 Consistent with this concept, elevated serum CRP levels have been found to be associated with blunted endothelium-dependent vasodilation in vivo,34 and a direct proinflammatory effect of CRP on endothelial cells has been documented in vitro.9–13 The present study, which demonstrates that CRP enhances the expression of LOX-1, a limiting factor for oxLDL uptake by endothelial cells, further stresses the potential key role of CRP in endothelial dysfunction. Induction of LOX-1 by CRP is receptor-mediated and appears to be exerted at the transcriptional level, as reflected by the parallel increase in LOX-1 gene and protein expression and the inhibitory effect of actinomycin D on CRP-induced LOX-1 gene expression. Transcriptional activation of the LOX-1 gene by CRP may theoretically involve NF-
B. Supporting this possibility, a NF-B–responsive element has been located in the promoter of the LOX-1 gene,35 and increased transcriptional activity of this factor has been documented in CRP-treated endothelial cells.36 CRP predicts incident type 2 diabetes37,38 and is increased in subjects with diabetes.30 Thus, hyperglycemia, in states of high CRP, may exaggerate the deleterious effects of CRP on endothelial cell activation. In contrast to previous observations showing that high glucose potentiates the proatherogenic effects of CRP in endothelial cells,13,39 we found that induction of LOX-1 by CRP remained unchanged by hyperglycemia, suggesting that CRP and glucose may operate through common molecular mechanisms to induce LOX-1. Because glucose induces endothelial LOX-1 through NF-B activation,33 and because CRP activates this transcription factor in endothelial cells,36 NF-B may represent a common signal mediator of CRP and glucose effect on LOX-1. Our data, which show that inhibition of NF-B prevents LOX-1 induction elicited by these two agents, support this possibility. CRP and glucose are well-known activators of ET-1 release by endothelial cells,11,40 and this peptide mediates the proinflammatory effects of CRP.11 Interestingly, ET-1 is also a LOX-1 stimulatory factor,41 and thus may mediate CRP effect on LOX-1. In support of this possibility, our data demonstrate that inhibition of ET-1 attenuates CRP effect on LOX-1. ET-1 is one of the upstream activators of IL-6 secretion,42 and recent data suggest that CRP may stimulate the release of this cytokine by endothelial cells.11 Our observation that CRP exerts a direct stimulatory effect on IL-6 secretion by cultured HAECs, and our findings that IL-6 induces endothelial LOX-1 and that inhibition of IL-6 reduces CRP-induced LOX-1 expression strongly support a role of IL-6 as one mediator of CRP effect on LOX-1. Our finding that attenuation of CRP-induced LOX-1 expression was greater during coincubation with anti-ET-1 and IL-6 antibodies support the concept that CRP induces LOX-1 via stimulating the production of ET-1 and IL-6 concurrently.
One major pathophysiological event associated with endothelial dysfunction is increased monocyte adhesion to endothelial cells. Recent studies have demonstrated that CRP increases monocyte–endothelium interaction7,43 by inducing endothelial and monocyte adhesion molecules.7–9,11,44,45 Our findings that CRP increases the expression of LOX-1, a well-identified cell adhesion molecule,46 and that blockade of LOX-1 abolished CRP-induced monocyte adhesion, suggest a new role for LOX-1, that of mediating the stimulatory effect of CRP on monocyte adhesion. The similar 1.5-fold increase in adhesion of mononuclear leukocytes documented in CRP-treated endothelial cells and CHO-K1 cells expressing human LOX-147 further stress this possibility.
In contrast to previous studies showing that CRP increases the expression of E-selectin, ICAM-1, and VCAM-1 in endothelial cells,8,11,44,45 we did not observe an upregulation of these antigens in CRP-treated HAECs, regardless of the concentrations of serum present in the culture medium or the time incubation periods with CRP. These results differ from previous findings that CRP-induced expression of adhesion molecules is serum- and time-dependent.8,45 Although the reasons for these discrepancies remain unknown, some experimental conditions specifically used in our work, such as the use of heat-inactivated serum, very low-endotoxin CRP, and cell enzyme-linked immunosorbent assay for adhesion molecule detection, might provide an explanation for these apparent conflicting results.
Endothelial cells do express different types of modified LDL scavenger receptors, including SR-A, SR-B, CD36, SREC, and LOX-1. Although it has long been suggested that endothelial cells internalize and degrade modified LDL, previous studies have suggested that this process occurs through a receptor-mediated pathway that does not involve classic scavenger receptors.48 These data and the recent finding that endothelial cells internalize and degrade oxLDL via the LOX-1 receptor14 support the notion that LOX-1 is the major receptor for oxLDL expressed in endothelial cells, presumably mediating the majority of the uptake of oxLDL by these cells. Our results showing that CRP enhances, through LOX-1, the uptake of oxLDL by endothelial cells suggest that CRP may trigger the toxic effect of oxLDL on vascular endothelium.
In conclusion, the present study demonstrates that CRP, at concentrations applicable to both acute and low-grade chronic inflammation, increases endothelial LOX-1 expression. Whether this potentially pro-atherogenic effect of CRP has clinical relevance remains to be evaluated.
|
Acknowledgments |
|---|
This study was supported by a grant from the Association Diabète Québec.
|
Footnotes |
|---|
Original received March 31, 2004; revision received August 24, 2004; accepted September 28. 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999; 138: S419–S420.[CrossRef][Medline] [Order article via Infotrieve]
2. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.[CrossRef][Medline] [Order article via Infotrieve]
3. Fan J, Watanabe T. Inflammatory reactions in the pathogenesis of atherosclerosis. J Atheroscler Thromb. 2003; 10: 63–71.[Medline] [Order article via Infotrieve]
4. Shah SH, Newby LK. C-reactive protein: a novel marker of cardiovascular risk. Cardiol Rev. 2003; 11: 169–179.[CrossRef][Medline] [Order article via Infotrieve]
5. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000; 342: 836–843.
6. Ballou SP, Lozanski G. Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein. Cytokine. 1992; 4: 361–368.[CrossRef][Medline] [Order article via Infotrieve]
7. Woollard KJ, Phillips DC, Griffiths HR. Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells. Clin Exp Immunol. 2002; 130: 256–262.[CrossRef][Medline] [Order article via Infotrieve]
8. Fu T, Borensztajn J. Macrophage uptake of low-density lipoprotein bound to aggregated C-reactive protein: possible mechanism of foam-cell formation in atherosclerotic lesions. Biochem J. 2002; 366: 195–201.[Medline] [Order article via Infotrieve]
9. Pasceri V, Willerson JT, Yeh ET. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation. 2000; 102: 2165–2168.
10. Pasceri V, Cheng JS, Willerson JT, Yeh ET, Chang J. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. Circulation. 2001; 103: 2531–2534.
11. Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA. Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation. 2002; 105: 1890–1896.
12. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, Dhillon B, Weisel RD, Li RK, Mickle DA, Stewart DJ. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation. 2002; 106: 913–919.
13. Devaraj S, Xu DY, Jialal I. C-reactive protein increases plasminogen activator inhibitor-1 expression and activity in human aortic endothelial cells: implications for the metabolic syndrome and atherothrombosis. Circulation. 2003; 107: 398–404.
14. Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, Tanaka T, Miwa S, Katsura Y, Kita T, Masaki T. An endothelial receptor for oxidized low-density lipoprotein. Nature. 1997; 386: 73–74.[CrossRef][Medline] [Order article via Infotrieve]
15. Cominacini L, Pasini AF, Garbin U, Davoli A, Tosetti ML, Campagnola M, Rigoni A, Pastorino AM, Lo Cascio V, Sawamura T. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-B through an increased production of intracellular reactive oxygen species. J Biol Chem. 2000; 275: 12633–12638.
16. Cominacini L, Rigoni A, Pasini AF, Garbin U, Davoli A, Campagnola M, Pastorino AM, Lo Cascio U, Sawamura T. The binding of oxidized low-density lipoprotein (oxLDL) to ox-LDL receptor-a in endothelial cells reduces the intracellular concentration of nitric oxide through an increased production of superoxide. J Biol Chem. 2001; 276: 13750–13755.
17. Li D, Mehta J. L. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells. Circulation. 2000; 101: 2889–2895.
18. Kume N, Murase T, Moriwaki H, Aoyama T, Sawamura T, Masaki T, Kita T. Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res. 1998; 83: 322–327.
19. Draude G, Lorenz R. L. TGF-ß 1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages. Am J Physiol Heart Circ Physiol. 2000; 278: H1042–H1048.
20. Chen M, Nagase M, Fujita T, Narumiya S, Masaki T, Sawamura T. Diabetes enhances lectin-like oxidized LDL receptor-1 (LOX-1) expression in the vascular endothelium: possible role of LOX-1 ligand and AGE. Biochem Biophys Res Commun. 2001; 287: 962–968.[CrossRef][Medline] [Order article via Infotrieve]
21. Nagase M, Hirose S, Sawamura T, Masaki T, Fujita T. Enhanced expression of endothelial oxidized low-density lipoprotein receptor (LOX-1) in hypertensive rats. Biochem Biophys Res Commun. 1997; 237: 496–498.[CrossRef][Medline] [Order article via Infotrieve]
22. Chen M, Kakutani M, Minami M, Kataoka H, Kume N, Narumiya S, Kita T, Sawamura T. Increased expression of lectin-like oxidized low density lipoprotein receptor-1 in initial atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits. Arterioscler Thromb Vasc Biol. 2000; 20: 1107–1115.
23. Kataoka H, Kume N, Miyamoto S, Minami M, Moriwaki H, Murase T, Sawamura T, Masaki T, Hashimoto N, Kita T. Expression of lectin-like oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions. Circulation. 1999; 99: 3110–3117.
24. Mentzer SJ, Guyre PM, Burakoff SJ, Faller DV. Spontaneous aggregation as a mechanism for human monocyte purification. Cell Immunol. 1986; 101: 312–319.[CrossRef][Medline] [Order article via Infotrieve]
25. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162: 56–159.
26. Wang J, Beekhuizen H, van Furth R. Surface molecules involved in the adherence of recombinant interferon-gamma (rIFN-gamma)-stimulated human monocytes to vascular endothelium cells. Clin Exp Immunol. 1994; 95: 263–269.[Medline] [Order article via Infotrieve]
27. Hatch FT. Practical methods for plasma lipoprotein analysis. Adv Lipid Res. 1968; 6: 1–68.[Medline] [Order article via Infotrieve]
28. Stephan ZF, Yirachek EC. Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL. J Lipid Res. 1993; 34: 325–330.[Abstract]
29. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248–254.[CrossRef][Medline] [Order article via Infotrieve]
30. Rodriguez-Moran M, Guerrero-Romero F. Increased levels of C-reactive protein in noncontrolled type II diabetic subjects. J Diabetes Complications. 1999; 13: 211–215.[CrossRef][Medline] [Order article via Infotrieve]
31. Theuma P, Fonseca VA. Inflammation and emerging risk factors in diabetes mellitus and atherosclerosis. Curr Diab Rep. 2003; 3: 248–254.[Medline] [Order article via Infotrieve]
32. Dandona P. Endothelium, inflammation, and diabetes. Curr Diab Rep. 2002; 2: 311–315.[Medline] [Order article via Infotrieve]
33. Li L, Sawamura T, Renier G. Glucose enhances endothelial LOX-1 expression: role for LOX-1 in glucose-induced human monocyte adhesion to endothelium. Diabetes. 2003; 52: 1843–1850.
34. Fichtlscherer S, Rosenberger G, Walter DH, Breuer S, Dimmeler S, Zeiher AM. Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease. Circulation. 2000; 102: 1000–1006.
35. Nagase M, Abe J, Takahashi K, Ando J, Hirose S, Fujita T. Genomic organization and regulation of expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) gene. J Biol Chem. 1998; 273: 33702–33707.
36. Verma S, Badiwala MV, Weisel RD, Li SH, Wang CH, Fedak PW, Li RK, Mickle DA. C-reactive protein activates the nuclear factor-kappaB signal transduction pathway in saphenous vein endothelial cells: implications for atherosclerosis and restenosis. J Thorac Cardiovasc Surg. 2003; 126: 1886–1891.
37. Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM. C-reactive protein, interleukin 6, and the risk of developing type II diabetes mellitus. JAMA. 2001; 286: 327–334.
38. Freeman DJ, Norrie J, Caslake MJ Gaw A, Ford L, Lowe GD, O’Reilly DS, Packard CJ, Sattar N, West of Scotland Coronary Prevention Study. C-reactive protein is an independent predictor of risk for the development of diabetes in the West of Scotland Coronary Prevention Study. Diabetes. 2002; 51: 1596–1600.
39. Verma S, Wang CH, Weisel RD, Badiwala MV, Li SH, Fedak PW, Li RK, Mickle DA. Hyperglycemia potentiates the proatherogenic effects of C-reactive protein: reversal with rosiglitazone. J Mol Cell Cardiol. 2003; 35: 417–419.[CrossRef][Medline] [Order article via Infotrieve]
40. Park JY, Takahara N, Gabriele A Chou E, Naruse K, Suzuma K, Yamauchi T, Ha SW, Meier M, Rhodes CJ, King GL. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes. 2000; 49: 1239–1248.[Abstract]
41. Morawietz H, Duerrschmidt N, Niemann B Galle J, Sawamura T, Holtz J. Induction of the oxLDL receptor LOX-1 by endothelin-1 in human endothelial cells. Biochem Biophys Res Commun. 2001; 284: 961–965.[CrossRef][Medline] [Order article via Infotrieve]
42. Browatzki M, Schmidt J, Kubler W Kranzhofer R. Endothelin-1 induces interleukin-6 release via activation of the transcription factor NF-B in human vascular smooth muscle cells. Basic Res Cardiol. 2000; 95: 98–105.[CrossRef][Medline]
[Order article via Infotrieve]
43. Torzewski M, Rist C, Mortensen RF, Zwaka TP, Bienek M, Waltenberger J, Koening W, Schmitz G, Hombach V, Torzewski J. C-reactive protein in the arterial intima: role of C-reactive protein receptor-dependent monocyte recruitment in atherosclerosis. Arterioscler Thromb Vasc Biol. 2000; 20: 2094–2099.
44. Blann AD, Lip GY. Effects of C-reactive protein on the release of von Willebrand factor, E-selectin, thrombomodulin and intercellular adhesion molecule-1 from human umbilical vein endothelial cells. Blood Coagul Fibrinolysis. 2003; 14: 335–340.[CrossRef][Medline] [Order article via Infotrieve]
45. Wadham C, Albanese N, Roberts J, Wang L, Bagley CJ, Gamble JR, Rye K-A, Barter PJ, Vadas MA, Xia P. High-density lipoproteins neutralize C-reactive protein proinflammatory activity. Circulation. 2004; 109: 2115–2121.
46. Honjo M, Nakamura K, Yamashiro K, Kiryu J, Tanibara H, McEvoy LM, Honda Y, Butcher EC, Masaki T, Sawamura T. Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation. Proc Natl Acad Sci U S A. 2003; 100: 1274–1279.
47. Hayashida K, Kume N, Minami M, 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 Letters. 2002; 511: 133–138.[CrossRef][Medline] [Order article via Infotrieve]
48. Kume N, Arai H, Kawai C, Kita T. Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein. Biochim Biophys Acta. 1991; 1091: 63–67.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  H. H. Shih, S. Zhang, W. Cao, A. Hahn, J. Wang, J. E. Paulsen, and D. C. Harnish CRP is a novel ligand for the oxidized LDL receptor LOX-1 Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1643 - H1650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Devaraj, U. Singh, and I. Jialal The Evolving Role of C-Reactive Protein in Atherothrombosis Clin. Chem., February 1, 2009; 55(2): 229 - 238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Ponnoth, A. Nadeem, and S. J. Mustafa Adenosine-mediated alteration of vascular reactivity and inflammation in a murine model of asthma Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2158 - H2165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Vidula, L. Tian, K. Liu, M. H. Criqui, L. Ferrucci, W. H. Pearce, P. Greenland, D. Green, J. Tan, D. B. Garside, et al. Biomarkers of Inflammation and Thrombosis as Predictors of Near-Term Mortality in Patients with Peripheral Arterial Disease: A Cohort Study Ann Intern Med, January 15, 2008; 148(2): 85 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Haugen, I. P. Casserly, J. G. Regensteiner, and W. R. Hiatt Risk assessment in the patient with established peripheral arterial disease Vascular Medicine, November 1, 2007; 12(4): 343 - 350. [Abstract] [PDF]
|
|
|
|
 |
|
 H. Teoh, A. Quan, and S. Verma Does C-reactive protein predict saphenous vein graft patency? J. Thorac. Cardiovasc. Surg., August 1, 2007; 134(2): 277 - 279. [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Lorenz, P. Karbstein, H. S. Markus, and M. Sitzer High-Sensitivity C-Reactive Protein Is Not Associated With Carotid Intima-Media Progression: The Carotid Atherosclerosis Progression Study Stroke, June 1, 2007; 38(6): 1774 - 1779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Ramzy, V. Rao, L. C. Tumiati, N. Xu, R. Sheshgiri, J. Jackman, D. H. Delgado, and H. J. Ross Endothelin-1 accentuates the proatherosclerotic effects associated with C-reactive protein J. Thorac. Cardiovasc. Surg., May 1, 2007; 133(5): 1137 - 1146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Bisoendial, J. J. P. Kastelein, S. L. M. Peters, J. H. M. Levels, R. Birjmohun, J. I. Rotmans, D. Hartman, J. C. M. Meijers, M. Levi, and E. S. G. Stroes Effects of CRP infusion on endothelial function and coagulation in normocholesterolemic and hypercholesterolemic subjects J. Lipid Res., April 1, 2007; 48(4): 952 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Fujii, S.-H. Li, P. E. Szmitko, P. W.M. Fedak, and S. Verma C-Reactive Protein Alters Antioxidant Defenses and Promotes Apoptosis in Endothelial Progenitor Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2476 - 2482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Paffen and M. P.M. deMaat C-reactive protein in atherosclerosis: A causal factor? Cardiovasc Res, July 1, 2006; 71(1): 30 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Scirica, D. A. Morrow, S. Verma, S. Devaraj, I. Jialal, B. M. Scirica, D. A. Morrow, S. Verma, S. Devaraj, and I. Jialal The Verdict Is Still Out Circulation, May 2, 2006; 113(17): 2128 - 2151. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Davis, G. Valacchi, E. Pagnin, Q. Shao, H. B. Gross, L. Calo, and W. Yokoyama Walnuts Reduce Aortic ET-1 mRNA Levels in Hamsters Fed a High-Fat, Atherogenic Diet J. Nutr., February 1, 2006; 136(2): 428 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Crimmins and C. E. Finch Infection, inflammation, height, and longevity PNAS, January 10, 2006; 103(2): 498 - 503. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashida, N. Kume, T. Murase, M. Minami, D. Nakagawa, T. Inada, M. Tanaka, A. Ueda, G. Kominami, H. Kambara, et al. Serum Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Levels Are Elevated in Acute Coronary Syndrome: A Novel Marker for Early Diagnosis Circulation, August 9, 2005; 112(6): 812 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Liu, S. Wang, A. Deb, K. A. Nath, Z. S. Katusic, J. P. McConnell, and N. M. Caplice Proapoptotic, Antimigratory, Antiproliferative, and Antiangiogenic Effects of Commercial C-Reactive Protein on Various Human Endothelial Cell Types In Vitro: Implications of Contaminating Presence of Sodium Azide in Commercial Preparation Circ. Res., July 22, 2005; 97(2): 135 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Devaraj, T. W. Du Clos, and I. Jialal Binding and Internalization of C-Reactive Protein by Fcgamma Receptors on Human Aortic Endothelial Cells Mediates Biological Effects Arterioscler. Thromb. Vasc. Biol., July 1, 2005; 25(7): 1359 - 1363. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||