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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:816.)
© 2003 American Heart Association, Inc.

Vascular Biology

LOX-1, an Oxidized LDL Endothelial Receptor, Induces CD40/CD40L Signaling in Human Coronary Artery Endothelial Cells

Dayuan Li; Ling Liu; Hongjiang Chen; Tatsuya Sawamura; Jawahar L. Mehta

From the Departments of Medicine and Physiology and Biophysics (D.L., L.L., J.L.M.), University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, and the Department of Bioscience (T.S.), National Cardiovascular Center Research Institute, Osaka, Japan.

Correspondence to J.L. Mehta, MD, PhD, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W Markham St, #532, Little Rock, AR 72205. E-mail mehtajl{at}uams.edu

	Abstract

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Background— Despite increasing appreciation that atherogenesis involves participation of inflammatory cells, information on mediators of communication between different constituents of atherosclerotic plaque remain incomplete. We examined the role of LOX-1, a receptor for oxidized (ox) LDL, in the expression of CD40/CD40L in cultured human coronary artery endothelial cells (HCAECs).

Methods and Results— We observed that ox-LDL increased the expression of CD40 and CD40L in a concentration (10 to 80 µg/mL)- and time (1 to 24 hours)- dependent manner. These effects of ox-LDL were mediated by activation of LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92) prevented the expression of CD40 and CD40L in response to ox-LDL (P<0.01). In parallel experiments, HCAECs were incubated with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, and the cells were then exposed to ox-LDL. Both LOX-1 antibody and the PKC inhibitor inhibited PKC activation in response to ox-LDL (P<0.01). The PKC inhibitor also blocked the effects of ox-LDL on the expression of CD40 and CD40L (P<0.01). In additional experiments, we found that it is the PKC, but not PKCß and PKC, isoform that mediated ox-LDL–induced CD40 and CD40L upregulation. Further experiments showed that upregulation of CD40 mediated induction of proinflammatory genes, because CD40 antibody markedly reduced ox-LDL–induced TNF- generation and P-selectin expression, whereas nonspecific mouse IgG had no effect.

Conclusions— These findings indicate that ox-LDL through its receptor LOX-1 triggers the CD40/CD40L signaling pathway that activates the inflammatory reaction in HCAECs. These observations provide novel insight into ox-LDL–mediated inflammation in atherosclerosis.

Key Words: CD40/CD40L • endothelial cells • LOX-1 • oxidized LDL • protein kinase

	Introduction

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Endothelial dysfunction elicited by oxidized (ox) LDL plays a critical role in the pathogenesis of atherosclerosis.¹ Ox-LDL changes the secretory activities of the endothelium and causes it to become dysfunctional.² Ox-LDL inhibits the expression of constitutive endothelial nitric oxide synthase,³ induces expression of adhesion molecules on the endothelium, and facilitates inflammatory cells to adhere to the intima.⁴

Scavenger receptors on macrophages and smooth muscle cells are believed to mediate the biologic effect of ox-LDL.⁵ Recent studies show that LOX-1, a novel lectinlike receptor for ox-LDL, facilitates the uptake of ox-LDL and mediates several of its biologic effects.^6,7 LOX-1 mediates ox-LDL–induced apoptosis in endothelial cells⁸ and phagocytosis of aged and apoptotic cells.⁹ Expression of the LOX-1 gene is upregulated by ox-LDL, angiotensin II, inflammatory cytokines such as tumor necrosis factor (TNF)-, and shear stress.^7,10–12 Recent studies show that LOX-1 expression is upregulated in atherosclerotic tissues from rabbits and humans.^13,14

Recently, a critical role for CD40/CD40L signaling in atherosclerosis has been reported.^15–17 In atherosclerotic regions, CD40 and CD40L are present on vascular endothelial cells, smooth muscle cells, macrophages, and T lymphocytes.^16,17 In vitro stimulation of CD40/CD40L signaling in atheroma-derived cells¹⁸ results in the activation of proatherogenic pathways, such as the synthesis of chemokines,¹⁸ cytokines,¹⁸ matrix metalloproteinases,^17,19 and leukocyte adhesion molecules.^20–22 A recent study showed an important role for CD40/CD40L interactions in the progression of atherosclerosis in apolipoprotein E–deficient mice.¹⁵ In another study, administration of antibody to CD40L, when given early in the development of atherosclerosis, was shown to inhibit atherosclerotic lesion initiation in LDL receptor–knockout mice.¹⁶

Both ox-LDL and CD40 have been identified to colocalize in atherosclerotic plaques. In the present study, we investigated the role of LOX-1 in CD40 and CD40L gene expression and the related intracellular mechanism in human coronary artery endothelial cells (HCAECs).

	Methods

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Cell Culture
The methodology for culture of HCAECs has been described earlier.^7,8 The initial batch of HCAECs was purchased from Clonetics Corporation (San Diego, Calif). The endothelial cells were pure, based on their morphology and staining for factor VIII–related antigen and acetylated LDL. These cells were 100% negative for -actin smooth muscle expression.

Study Design
HCAECs were incubated with ox-LDL (20 to 80 µg/mL) for 1 to 24 hours to determine the expression of CD40 and CD40L (mRNA and protein). The concentration and time point for maximal effect of ox-LDL were used in subsequent experiments. To examine the receptor specificity of ox-LDL action, the HCAECs were pretreated with human LOX-1–blocking antibody (JTX92, 10 µg/mL) and then exposed to ox-LDL. The details of preparation of antibody and its specificity have been presented earlier.²³ The harvested cells were used to measure expression of CD40 and CD40L and protein kinase C (PKC) activity.

To explore the molecular basis of the action of LOX-1, we studied the PKC signaling pathway. For this purpose, HCAECs were pretreated with the PKC inhibitor bisindolylmaleimide I (1 µmol/L) for 30 minutes, and then the cells were exposed to ox-LDL. The harvested cells were used to measure CD40 and CD40L expression and PKC activity.

To further explore the roles of the PKC isoforms (, ß, and subunits) in this process, HCAECs were pretreated with the inhibitors of PKC (Ro-32[hyphen]0432, 20 nmol/L), PKCß (hispidin, 4 µmol/L), or PKC (Ro-31[hyphen]7549, 0.4 µmol/L) for 30 minutes, and then the cells were exposed to ox-LDL. The harvested cells were used to measure CD40 and CD40L expression and PKC activity. To determine whether or not ox-LDL activates CD40 transcription, HCAECs were incubated with ox-LDL. HCAECs were harvested to isolate nuclei. Nuclei were then extracted from cells treated or not with ox-LDL, and then nuclear run-on experiments were performed.

To examine the specific role of upregulation of CD40, HCAECs were pretreated with CD40-blocking antibody (10 µg/mL) or a nonspecific mouse IgG (10 µg/mL) and then exposed to ox-LDL. The harvested supernatant was used to measure TNF-, as determined by ELISA. The harvested cells were used to measure expression of P-selectin. Concentrations of all reagents and the duration of incubation were chosen on the basis of previous studies.^7,8,23–25

Preparation of Lipoproteins
Native LDL and ox-LDL were prepared as described earlier.^7,8 The thiobarbituric acid–reactive substances content of ox-LDL and native LDL was 15.2±0.28 and 0.56±0.16 nmol/100 µg protein, respectively (P<0.01). Ox-LDL was extensively dialyzed against Tris-saline. Ox-LDL was kept in 50 mmol/L Tris-HCl, 0.15 mol/L NaCl, and 2 mmol/L EDTA at pH 7.4 and was used within 10 days of preparation. I¹²⁵-labeled ox-LDL was purchased from Biomedical Technologies Inc. The level of endotoxin was measured by the E-Toxate kit (Sigma) and found to be consistently <0.005 EU/mL (lowest detection limit).

Semiquantitative RT-PCR and Northern Blot Assay
CD40L mRNA was examined by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Total RNA (1 µg) extracted from cultured HCAECs was reverse-transcripted with oligo dT (Promega) and Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Promega) at 37°C for 1 hour. A small amount (1.5 µL) of the reverse-transcribed material was amplified with Taq DNA polymerase (Promega) by using specific primers for human CD40L.²⁶ The products of PCR-amplified samples were visualized on 1.5% agarose gels with ethidium bromide staining. The CD40L mRNA band density was semiquantified by UN-SCAN-IT gel software. Each specific mRNA band was normalized against the ß-actin mRNA band.

CD40 mRNA was examined by Northern blot analysis. Total RNA (15 µg) from HCAECs was fractionated on a formaldehyde-denatured 1.2% agarose gel and transferred to a nylon membrane filter. The filter was hybridized overnight with a ³²P-labeled human CD40 probe (10⁶ counts per minute/mL) in a solution containing 50% formamide at 42°C. The CD40 probe was made by RT-PCR²⁷ and labeled by the random-primer labeling method. The membrane was washed twice in 2x standard saline citrate (SSC) containing 0.1% (wt/vol) sodium dodecyl sulfate (SDS) at room temperature, followed by washes in 0.2x SSC containing 0.1% (wt/vol) SDS at 50°C for 15 minutes. Kodak film was exposed to the filter with an intensifying screen at -80°C. The band density was semiquantified by UN-SCAN-IT gel software. CD40 mRNA expression was standardized against that of the human housekeeping gene ubiqilin.¹²

Western Blot Analysis
HCAEC lysates from each experiment (40 µg per lane) were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After incubation in blocking solution (4% non-at milk, Sigma), the membranes were incubated with a 1:1000 dilution of primary antibody (monoclonal antibody to CD40, CD40L, or P-selectin; Santa Cruz) overnight at 4°C. Membranes were washed and then incubated with a 1:2000 dilution of a second antibody (Amersham) for 1 hour, the membranes were detected with an enhanced chemiluminescence system, and relative intensities of the protein bands were analyzed by UN-SCAN-IT gel software.^7,8,23

Measurement of PKC Activity
Cells (5 to 10 x 10⁶ cells/100-mm dish) from different groups were washed twice with phosphate-buffered saline and scraped into 1 mL membrane-bound PKC extraction buffer containing (in mmol/L) 25 Tris-HCl (pH 7.4), 0.5 EDTA, 0.5 EGTA, 0.0% Triton X-100, 10 ß-glycerophosphatase, 0.5 1,4-(2-aminoethyl)benzenesulfonyl fluoride, 1 µg/mL leupeptin, and 1 µg/mL aprotinin. The lysate was homogenized and centrifuged at 14 000g at 4°C for 30 minutes, and the supernatant was used to measure PKC activity. An assay system (Promega) was used to determine PKC activity as described previously.²⁸ The reaction for each sample was performed separately in the presence of phospholipids (activated PKC reaction) and in the absence of phospholipids (control reaction). Results were expressed as picomoles of phosphate per minute per microgram of protein.

ELISA for TNF- Measurement
TNF- secreted by endothelial cells was quantitated with an ELISA kit (Oncogene). Two hundred microliters of conditioned medium was added to each well of the plate coated with monoclonal anti-human TNF- antibodies, and incubation was carried out at room temperature for 2 hours with shaking. After incubation, the plate was washed 4 times with washing buffer, and secondary antibodies conjugated with horseradish peroxidase were added for a 1-hour incubation at room temperature; the plate was then washed, which was followed by addition of tetramethylbenzidine. The plate was incubated in the dark at room temperature for 30 minutes, and the reaction was stopped by addition of 2.5N H₂SO₄. The absorbance at 450 nm was measured by a spectrophotometric plate reader. A standard curve was obtained with purified TNF- as the antigen supplied with the kit. The expression of TNF- was normalized by the protein amount in each experiment.

Nuclear Run-On Assay
A nuclear run-on transcription assay was performed as described.²⁴ Endothelial cells treated with or without ox-LDL were dislodged from culture dishes by scraping with a rubber policeman. The cells were then centrifuged and lysed with a lysis buffer containing 10 mmol/L Tris-HCl, 10 mmol/L NaCl, 3 mmol/L MgCl₂, and 0.5% NP-40. The nuclei were spun down at 500g at 4°C, and the supernatant was removed. The nuclear pellet was resuspended in a buffer containing 50 mmol/L Tris-HCl, pH 8.3, 40% glycerol, 5 mmol/L MgCl₂, and 0.1 mmol/L EDTA. Nuclei were incubated in buffer containing 5 mmol/L Tris-HCl, pH 8.0; 2.5 mmol/L MgCl₂; 150 mmol/L KCl; 1 mmol/L each ATP, GTP, CTP, and [³²P]UTP (100 µCi, Amersham); and 12.5 mmol/L dithiothreitol for 40 minutes at 37°C with shaking. Radiolabeled transcripts were isolated with phenol and chloroform and dissolved in DNAse- and RNase-free water.

Two microgram of CD40 and ubiquilin cDNA was boiled for 5 minutes, chilled quickly on ice, and then applied to a nylon membrane mounted in a dot-blot apparatus. The immobilized cDNAs were hybridized with the radiolabeled transcripts obtained from the nuclear run-on reaction for 48 hours at 45°C in buffer containing 50 mmol/L PIPES, 500 mmol/L NaCl, 33% formamide, 0.1% SDS, 2 mmol/L EDTA, and 5 mg/mL salmon sperm DNA. The nylon membrane was washed twice with 1x SSC with 0.1% SDS at 50°C for 30 minutes each. The dried membrane was exposed to x-ray film.

Data Analysis
All data represent the mean of 3 to 6 independently performed experiments. Data are presented as mean±SD. Statistical significance was determined in multiple comparisons among independent groups of data in which ANOVA and the F test indicated the presence of significant differences. A probability value 0.05 was considered significant.

	Results

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Ox-LDL and CD40/CD40L Gene Expression
Incubation of HCAECs with ox-LDL (20 to 80 µg/mL) increased the expression of CD40 (mRNA and protein) in a concentration- and time-dependent manner (Figure 1). Incubation of HCAECs with ox-LDL (20 to 80 µg/mL) also increased the expression of CD40L (mRNA and protein) in a concentration- and time-dependent manner (Figure 2). To determine whether or not ox-LDL activates CD40 transcription, nuclear run-on analysis was performed. We observed higher CD40 hybridization signals after using transcription products from nuclei of ox-LDL–treated HCAECs compared with those from nuclei of control cells, indicating that ox-LDL induces CD40 gene transcriptional activation (Figure 3).

View larger version (75K): [in this window] [in a new window]   Figure 1. Ox-LDL and CD40 expression. Incubation of HCAECs with ox-LDL (20 to 80 µg/mL) for 24 hours increased expression of CD40 mRNA and protein in a concentration-dependent manner (top). Upregulation of CD40 (mRNA and protein) by ox-LDL (80 µg/mL) occurred in a time-dependent manner (bottom). CD40 protein band density was normalized by its control. CD40 mRNA band density (determined by Northern blot) was normalized by housekeeping gene ubiquilin. Left panel is representative of 6 separate experiments. Right panel is the summary of data (mean±SD) from these 6 experiments.

View larger version (72K): [in this window] [in a new window]   Figure 2. Ox-LDL and CD40L expression. Incubation of HCAECs with ox-LDL (20 to 80 µg/mL) for 24 hours increased expression of CD40L mRNA and protein in a concentration-dependent manner (top). Upregulation of CD40L (mRNA and protein) by ox-LDL (80 µg/mL) occurred in a time-dependent manner (bottom). CD40L protein band density was normalized by its control. CD40L mRNA band density (determined by RT-PCR) was normalized by the housekeeping gene ß-actin. Left panel is representative of 6 separate experiments. Right panel is the summary of data (mean±SD) from these 6 experiments.

View larger version (23K): [in this window] [in a new window]   Figure 3. Ox-LDL–induced CD40 expression. HCAECs were treated without or with 80 µg/mL ox-LDL for 24 hours. Nuclear run-on analysis showed higher CD40 hybridization signals from nuclei of ox-LDL–treated HCAECs compared with those from nuclei of control cells.

Role of LOX-1 in Expression of CD40
Incubation of HCAECs with ox-LDL (80 µg/mL) markedly increased the expression of CD40. In contrast, native LDL (80 µg/mL) did not affect the expression of CD40. Pretreatment of HCAECs with LOX-1–blocking antibody (10 µg/mL) for 30 minutes before exposure to ox-LDL reduced the ox-LDL–mediated upregulation of CD40 (P<0.01). LOX-1 antibody alone did not affect the expression of CD40 (Figure 4).

View larger version (31K): [in this window] [in a new window]   Figure 4. Mechanism of LOX-1–mediated CD40 expression. Incubation of HCAECs with ox-LDL (80 µg/mL) markedly increased expression of CD40. In contrast, native LDL (80 µg/mL) did not affect expression of CD40. Pretreatment of HCAECs with LOX-1 antibody (Ab) reduced upregulation of CD40 in response to ox-LDL. LOX-1 antibody alone did not affect expression of CD40.

Activation of Ox-LDL-Induced PKC
To determine the mechanism of CD40 and CD40L expression elicited by ox-LDL, we examined the PKC pathway. Ox-LDL increased PKC activity in HCAECs (P<0.01 vs control). Ox-LDL–induced PKC activation was inhibited by pretreatment of HCAECs with the LOX-1 antibody or with the total PKC inhibitor (both P<0.01). Notably, pretreatment of cells with the LOX-1 antibody or the PKC inhibitor alone did not affect PKC activity (Figure 5A).

View larger version (40K): [in this window] [in a new window]   Figure 5. Ox-LDL, LOX-1, and PKC activation. Treatment of HCAECs with ox-LDL (80 µg/mL) for 24 hours caused PKC activation (P<0.01 vs control, n=6). Ox-LDL–induced PKC activation was inhibited by pretreatment of HCAECs with LOX-1 antibody (Ab; 10 µg/mL) as well as the PKC inhibitor bisindolylmaleimide I (1 µmol/L) (both P<0.01, n=6). Pretreatment of cells with LOX-1 antibody or PKC inhibitor alone did not significantly affect PKC activity (A). Further experiments showed that pretreatment of HCAECs with the inhibitor of PKC (Ro-32[hyphen]0432, 20 nmol/L), PKCß (hispidin, 4 µmol/L), or PKC (Ro-31[hyphen]7549, 0.4 µmol/L) for 30 minutes inhibited ox-LDL–mediated activation of PKC (Figure 5B).

To further define the role of PKC isoforms in ox-LDL–induced CD40 and CD40L expression, HCAECs were pretreated with different inhibitors of PKC isoforms (, ß, or ) and exposed to ox-LDL. We found that all PKC subunit inhibitors (, ß, and ) inhibited ox-LDL–induced activation of PKC (Figure 5B) but that only PKC inhibitor reduced ox-LDL–induced CD40 and CD40L expression.

Activation of PKC Subunits and Expression of CD40 and CD40L in Response to Ox-LDL
Along with the inhibition of PKC activity by its global inhibitor, treatment of HCAECs with the inhibitor markedly reduced ox-LDL–induced CD40/CD40L expression. As a control, the PKC inhibitor by itself did not affect the expression of CD40/CD40L (Figure 6,left). To further define the role of PKC isoforms in ox-LDL–induced CD40/CD40L expression, HCAECs were pretreated with different inhibitors of PKC isoforms (, ß, or ) and exposed to ox-LDL. Consistent with previous observations, ox-LDL increased the activation of PKC and the expression of CD40 and CD40L in HCAECs. Importantly, we found that PKC activation played a critical role in the expression of CD40/CD40L, because PKC inhibitor Ro-32[hyphen]0432 reduced ox-LDL–induced PKC activation as well as CD40/CD40L expression. The other PKC subunit (ß and ) inhibitors did not affect ox-LDL–induced CD40 and CD40L expression (Figure 6, right).

View larger version (28K): [in this window] [in a new window]   Figure 6. PKC activation and CD40 and CD40L expression. Ox-LDL upregulated CD40 and CD40L protein expression (P<0.01 vs control). Pretreatment of HCAECs with the PKC inhibitor bisindolylmaleimide I (1 µmol/L) for 30 minutes inhibited ox-LDL–mediated CD40 and CD40L upregulation (both P<0.01 vs ox-LDL, n=6; left). Furthermore, PKC inhibitor (Ro-32[hyphen]0432, 20 nmol/L) inhibited ox-LDL–induced CD40 and CD40L expression (P<0.01 vs ox-LDL), whereas, PKCß (hispidin, 4 µmol/L), and PKC (Ro-31[hyphen]7549, 0.4 µmol/L) inhibitors had no effect on CD40 and CD40L expression (right).

Significance of CD40 Upregulation Elicited by Ox-LDL in HCAECs
To determine the pathophysiological significance of CD40 upregulation elicited by ox-LDL, we examined the induction of proinflammatory genes. Ox-LDL increased TNF- release and P-selectin expression in HCAECs (P<0.01 vs control). Ox-LDL–induced TNF- release and P-selectin expression were inhibited by pretreatment of HCAECs with the CD40 antibody (P<0.01). Notably, pretreatment of cells with the nonspecific mouse IgG antibody had no effect (Figure 7).

View larger version (20K): [in this window] [in a new window]   Figure 7. Significance of CD40 upregulation in response to ox-LDL. To determine the significance of CD40 upregulation elicited by ox-LDL, HCAECs were pretreated with CD40 antibody (Ab; 10 µg/mL) or nonspecific mouse IgG (10 µg/mL) and then exposed to ox-LDL. Ox-LDL increased TNF- release and P-selectin expression in HCAECs (P<0.01 vs control). Effects of ox-LDL on TNF- and P-selectin were inhibited by pretreatment of HCAECs with human CD40 antibody (both P<0.01). Notably, pretreatment of cells with the nonspecific mouse IgG antibody had no effect.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows that ox-LDL upregulates the expression of CD40/CD40L in HCAECs. Importantly, we found that upregulation of CD40/CD40L by ox-LDL is mediated by LOX-1, because inhibition of LOX-1 action by a specific blocking antibody reduced the upregulation of CD40 elicited by ox-LDL. Furthermore, we observed that it is the activation of PKC, but not the ß and isoforms, that plays a critical role in this process, because pretreatment of cells with the PKC inhibitor markedly reduced the expression of CD40/CD40L in response to ox-LDL. Lastly, CD40 upregulation increases activation of inflammatory signaling, because CD40 antibody decreased TNF- release and P-selectin expression in response to ox-LDL in HCAECs.

Role of Ox-LDL in the Inflammatory Signal (CD40/CD40L)
There is ample evidence that ox-LDL plays an important role in atherosclerosis and associated inflammatory reactions. Ox-LDL increases free radical generation²⁹; release of the cytokines TNF- and interleukin-6,³⁰ leukocyte chemokines (monocyte chemoattractant protein [MCP]-1 and interleukin-8),³¹ and angiotensin II³²; and the expression of adhesion molecules (intercellular adhesion molecule [ICAM]-1, vascular cell adhesion molecule [VCAM]-1, and E-selectin)³³; all of these contribute to the inflammatory process and initiate and/or accelerate atherosclerosis. There is emerging evidence that CD40/CD40L activation is a critical inflammatory signal in atherosclerosis. Ox-LDL may trigger CD40/CD40L signaling and initiate and augment progression of atherosclerosis.

In the present study, we demonstrate that ox-LDL upregulates the gene expression of CD40 and CD40L in cultured HCAECs. The effects of ox-LDL on CD40 and CD40L expression are concentration and time dependent. Importantly, we found that CD40 upregulation increases induction of proinflammatory genes, because CD40 antibody markedly reduced ox-LDL–induced TNF- release and P-selectin expression in coronary endothelial cells. Other studies also showed that activation of the CD40 and CD40L pathway in endothelial cells results in the upregulation of ICAM-1, VCAM-1, and E-selectin,^20–22 which are associated with macrophage recruitment in the plaque, expansion of the lipid core, inhibition of collagen synthesis, and degradation of the fibrous cap in vivo.³⁴ A recent study has shown that inhibition of CD40L/CD40 signal in mice with advanced atherosclerotic lesions results in the development of a lipid-poor, collagen-rich, stable-plaque phenotype.³⁵ The present study provides the first direct link between ox-LDL and the CD40/CD40L signaling pathway. These observations provide a clue as to how ox-LDL promotes activation of endothelial cells and initiation of inflammatory pathways.

Role of LOX-1 in CD40 Expression
Many studies^6,36 have shown that the pathological effects of ox-LDL are mediated by its receptors. LOX-1,⁶ found predominantly on endothelial cells, has a different biochemical structure from the scavenger receptor.³⁶ Several investigators^6–11 have demonstrated that the uptake of ox-LDL by endothelial cells is mediated by LOX-1. Studies from our laboratory⁸ showed that LOX-1 mediates ox-LDL–induced apoptosis in HCAECs. LOX-1 mediates ox-LDL–induced expression of adhesion molecules (such as ICAM-1, VCAM-1, P-selectin, and MCP-1) and monocyte adhesion to endothelial cells.^37,38

In the present study, we show that ox-LDL upregulates the expression of CD40 through its own endothelial receptor, LOX-1. The confirmatory evidence for the role of LOX-1 came from experiments in which a specific LOX-1–blocking antibody decreased the expression of CD40 in response to ox-LDL. Taken together, these findings suggest that ox-LDL through its receptor LOX-1 activates the CD40 signal pathway, leading to inflammatory reaction and endothelial injury.

Mechanisms of Ox-LDL-Mediated CD40 and CD40L Expression
Experimental work has shown that ox-LDL causes injury to endothelial cells through activation of PKC²⁸ and mitogen-activated protein kinase (MAPK).³⁹ Ox-LDL also activates nuclear factor (NF)-B as well as activator protein-1 in several cell lines.^40,41 A recent study from our laboratory⁴² showed that LOX-1 activation is associated with a decrease in PKB activity. Another study⁸ showed that LOX-1–mediated apoptosis in endothelial cells and cardiac myocytes involves activation of MAPKs and NF-B. In the present study, we demonstrate that activation of PKC plays a critical role in ox-LDL–induced gene expression of CD40/CD40L, because the PKC inhibitor markedly reduced CD40/CD40L expression elicited by ox-LDL. The LOX-1 antibody blocked PKC activation, indicating that activation of PKC is located downstream of LOX-1 activation. Additional experiments defined that the activation of PKC plays a critical role in the expression of CD40/CD40L elicited by ox-LDL, because the PKC inhibitor Ro-32[hyphen]0432 inhibited ox-LDL–induced PKC activation as well as CD40/CD40L expression. In contrast, other PKC subunits (ß and ) did not affect ox-LDL–induced CD40/CD40L expression.

Summary
This study shows that ox-LDL induces gene expression of CD40/CD40L in adult HCAECs. The effect of ox-LDL is mediated through its newly described receptor LOX-1. In this process, activation of PKC plays an important role. These observations indicate that inhibition of LOX-1 may be effective in blocking inflammation-related vascular injury.

	Acknowledgments

 
This study was supported by a scientist development grant from the American Heart Association and a merit review award from the Veterans Affairs Central Office.

Received December 30, 2002; accepted March 3, 2003.

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