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Cardiovascular Research 2006 69(1):263-271; doi:10.1016/j.cardiores.2005.08.013
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Copyright © 2005, European Society of Cardiology

Oxidized LDL receptor LOX-1 is involved in neointimal hyperplasia after balloon arterial injury in a rat model

Jun-ichi Hinagataa,b, Makoto Kakutania, Takao Fujiic, Takahiko Narukod, Nobutaka Inouea, Yoshiko Fujitaa, Jawahar L. Mehtae, Makiko Uedac and Tatsuya Sawamuraa,b,*

aDepartment of Vascular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan
bDepartment of Molecular Pathophysiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
cDepartment of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan
dDepartment of Cardiology, Osaka City General Hospital, Osaka 534-0021, Japan
eDivision of Cardiovascular Medicine, University of Arkansas Medical School, Little Rock, AR, United States

*Corresponding author. Department of Vascular Physiology, National Cardiovascular Center research Institute, 5-7-1, Fujishirodai, Suita, Osaka, 565-8565, Japan. Tel.: +81 6 6833 5012x2518; fax: +81 6 6835 5329. Email address: t-sawamura{at}umin.ac.jp

Received 29 October 2004; revised 14 August 2005; accepted 16 August 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Objective: LOX-1 is a multi-ligand receptor originally identified as the endothelial oxidized LDL receptor. LOX-1 expression is also induced in smooth muscle cells in response to proinflammatory and oxidative stimuli. Here, we report on the role of LOX-1 in intimal hyperplasia, in which proinflammatory and oxidative stimuli are increased.

Methods and results: Left common carotid artery of rat was injured by a balloon catheter. The expression of LOX-1 was significantly increased within 24 h after the balloon injury and peaked at day 7. LOX-1 expression was observed predominantly in medial smooth muscle cells until day 3, and then shifted to predominantly intimal smooth muscle cells. At day 14, the expression was concentrated in the regenerated endothelial cells. To examine the contributory role of LOX-1 in the growth of intimal smooth muscle cells, rats were administered anti-LOX1 antibody intravenously every 3 days after balloon injury. Anti-LOX-1 antibody administration effectively suppressed intimal hyperplasia, oxidative stress, and leukocyte infiltration compared with control IgG. These findings suggest the importance of LOX-1 expression in the pathogenesis of neointimal formation in conjunction with oxidative stress and leukocyte infiltration.

Conclusion: The LOX-1 expressed in smooth muscle cells is involved in intimal hyperplasia in a rat model of balloon injury. Manipulation of LOX-1 activity is a novel potential therapeutic target to prevent restenosis after angioplasty.

KEYWORDS Restenosis; Neointimal formation; LOX-1; Smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Restenosis is a major complication after coronary angioplasty that occurs in 30% to 50% of patients within 6 months after the procedure [1]. Although the use of a stent significantly reduces the rate of restenosis, often the interventional procedures need to be repeated because of recurrent restenosis [2]. The major cause of restenosis is neointimal hyperplasia, which results from an excessive proliferative response of vascular smooth muscle cells (VSMC) to mechanical injury. This proliferative process includes VSMC activation, migration from media to the intima, and intimal growth [2,3].

There is a great deal of evidence to show the process of VSMC growth involves oxidative stress and inflammation related to the accumulation of leukocytes and platelets [4–8]. After injury to the vascular surface, attachment of platelets to the injured surface area and their activation/aggregation occur almost immediately, and the migration of leukocytes follows. The platelets and leukocytes distributed in the injured blood vessel release reactive oxygen species and enhance oxidative stress on the vessel wall. These cells also release cytokines that induce the migration and proliferation of VSMCs. Mechanical stress to the vessel wall further contributes to the oxidative stress in VSMCs.

LOX-1 is a multiligand receptor, originally identified by our laboratory as the major OxLDL receptor in endothelial cells [9]. The expression of LOX-1 in endothelial cells is markedly increased in vitro by cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}) and transforming growth factor-β1(TGF-β1) [10,11], and also is induced in vivo in hypertension, diabetes, and hyperlipidemia in animal models [12–14]. Ligand binding to LOX-1 induces superoxide generation, which is accompanied by a reduction of nitric oxide (NO) in endothelial cells. This is followed by the activation of transcription factors and the induction of the expression of adhesion molecules and chemokines [15–17]. These data suggest that LOX-1 over-expression promotes endothelial dysfunction and leads to pathological changes of blood vessels [18]. Recent studies have demonstrated that LOX-1 recognizes aged/apoptotic cells, activated platelets, and leukocytes [19–21]. As a leukocyte-adhesion molecule, LOX-1 is actually involved in inflammation, and an anti-LOX-1 antibody has been shown to suppress endotoxin-and zymosan-induced inflammation in an animal model [21,22]. Therefore, LOX-1 appears to be an important component of inflammation.

In addition to endothelial cells, LOX-1 is expressed in smooth muscle cells, macrophages, and platelets [23–25]. When the endothelium is injured and removed, as in the case of balloon induced injury, the LOX-1 expressed in smooth muscle cells and platelets could play an important pathophysiological role in events occurring in the vessel wall. To investigate this hypothesis, we examined whether LOX-1 is involved in the process of neointimal hyperplasia after balloon arterial injury.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
2.1 Preparation of lipoproteins
LDL (1.019–1.063 g/mL) was isolated by sequential ultracentrifugation from healthy human plasma as described previously [9]. LDL was oxidatively modified by exposing to 7.5 µM CuSO4 for 16 h at 37 °C at the protein concentration of 3 mg/ml in phosphate-buffered saline (PBS). The degree of oxidation was estimated by measuring the amount of thiobarbituric acid-reactive substances (TBARS) and the relative electrophoretic mobility (REM) in agarose gel compared with native LDL. TBARS and REM values of OxLDL were 10.7 nmol/mg protein and 3.25, respectively. Labeling of OxLDL with 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes) was performed as described [9].

2.2 Cells
Rat LOX-1 cDNA [26] was subcloned into a mammalian expression vector pME18s. The plasmid was co-transfected with pSV2bsr carrying blasticidin S resistant gene into CHO-K1 cells as described [9]. CHO cells stably expressing rat LOX-1 (rat LOX-1-CHO) was selected and maintained under Ham's F12/10% fetal bovine serum and 10 µg/mL blasticidin S. Wild-type CHO-K1 cells were maintained under Ham–s F12/10% fetal bovine serum.

2.3 Anti-LOX-1 antibody
Anti-LOX-1 monoclonal antibody was generated by immunizing mice with cells stably expressing bovine LOX-1 (bLOX-1-CHO). Hybridomas which produce antibody, JTX20, reactive to both bLOX-1 CHO and rat-LOX-1 CHO were selected for this purpose.

2.4 Immunofluorescent staining of culture cells
Culture cells were washed three times with PBS and fixed with 4% (v/v) paraformaldehyde in PBS for 10 min. Nonspecific reactions were blocked by incubating cells with 10% non-immune horse serum in PBS. Then, the cells were incubated with 5 µg/mL anti-LOX-1 antibody (JTX20) or non-immune mouse IgG in PBS containing 1%(w/v) BSA, and subsequently with biotinylated horse anti-mouse IgG (Vector Laboratories) in PBS containing 1%(v/v) horse serum according to the manufacturer–s instruction. The cells were then incubated with streptavidin–fluorescein conjugate (Vector Laboratories) and subjected to observation with fluorescence microscopy.

2.5 Internalization of DiI-OxLDL by rat LOX-1-CHO
Rat LOX-1-CHO cells were incubated with 3 µg/mL DiI-OxLDL in Ham's F12, 10% newborn calf serum (NBCS, Gibco BRL) for 3 h at 37 °C. To observe the effects of antibody, anti-LOX-1 antibody or non-immune mouse IgG were added to the cells 5 min before the addition of DiI-OxLDL. The cells were washed three times with PBS to remove unbound DiI-OxLDL, and lysed in isopropanol. The fluorescence intensity of the cell lysate was determined by a fluorescence spectrophotometer (excitation: 530 nm, emission: 590 nm, Spectro Fluor, Tecan).

2.6 Animal experiments
The animal experiments in the present study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.

2.7 Clearance of anti-LOX-1 antibody after a bolus intravenous injection
Rats (n=3) were injected anti-LOX-1 antibody through tail vein at a dose of 10 mg/kg per rat. Blood (~0.1 mL) were collected from the tail vein at the indicated time points. To determine the plasma concentration of anti-LOX-1 antibody, sandwich enzyme immunoassay was performed by using anti-mouse IgG-Fc (Sigma) and horseradish peroxidase-conjugated anti-mouse immunoglobulin kappa chain (Southern biotechnology Associates, Inc.).

2.8 Arterial injury model
Male Sprague–Dawley rats weighing ~300 g were obtained from Japan SLC Inc. (Hamamatsu, Japan). Rats were anesthetized with pentobarbital (50 mg/kg, i.p.). The left common carotid artery was exposed under a surgical microscope. A deflated 2F balloon catheter (Fogarty, E-060-2F, Baxter) was inserted from external carotid artery and advanced to the aortic arch. An inflated balloon with 0.03 ml air was pull back toward the external carotid artery to denude the endothelium in common carotid artery. This process was repeated three times. Then, deflated catheter was pulled out, and the external carotid artery was ligated. At the indicated time points, the animals were sacrificed by injecting excess amount of anesthetic. The carotid arteries of the rats were perfused with phosphate-buffered saline (PBS), isolated, and excised into ~10 mm length (from the 5 mm proximal point from the internal–external branch), and then, snap-frozen in liquid nitrogen for RNA preparation, or in OCT compound (Miles Laboratories) chilled by isopentan-dry ice for immunohistochemistry.

2.9 Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from rat carotid arteries with Trizol reagent (Gibco BRL) according to manufacturer's manual. Total RNA (1 µg/mL per sample) was reverse transcribed with oligo (dT)12–18 by SuperScript II reverse transcriptase (GIBCO BRL) at 42 °C for 1h. Five percent of the reaction was subjected to PCR using a primer pair specific to rat LOX-1 cDNA (forward primer: 5'-actcttcaggtccttgtccaca-3', reverse primer: 5'-gttgggatttttccgatgtaat-3') in 35 thermal cycles of 94 °C for 40 s, 55 °C for 1 min, and 74 °C for 1 min to amplify 218 bp-product. GAPDH cDNA was amplified as internal control with forward primer: 5'-gcgcctggtcaccagggctgctt-3', and reverse primer: 5'-tgccgaagtggtcgtggatgacct-3') in 30 thermal cycles of 94 °C for 40 s, 60 °C for 1 min, and 74 °C for 1 min, to amplify 465 bp. The RT-PCR products were separated in 4% NuSieve 3:1 agarose gel (FMC bioproducts) and visualized with ethidium bromide. The relative intensity of the bands was quantified using Scion Image software.

2.10 Immunohistochemistry
Left common carotid arteries were isolated and snap-frozen at 3, 7 and 14 days after balloon injury. The samples were sectioned serially at 6-µm thickness and fixed in acetone. Every first section was stained with hematoxylin-eosin; the other sections were used for immunohistochemical staining. The cellular components were analyzed by use of monoclonal antibodies against von Willebrand factor (1:1000, Dako A/S), myeloperoxidase (1:500, Abcam, Cambridge, UK), and LOX-1 (1 µg/mL). Sections were incubated at 4 °C overnight, and then subjected to a three-step staining procedure using the streptavidin-biotin complex method. Visualization was performed by horseradish peroxidase-based colorimetric reaction with 3-amino-9-ethyl-carbazole (10 min, room temperature), and the sections were faintly counterstained with hematoxylin.

2.11 Analyses of the effects of anti-LOX-1 antibody on the neointimal hyperplasia
Anti-LOX-1 antibody (10 mg/kg) or nonimmune mouse IgG (10 mg/kg) were administered intravenously to a subset of rats (n=12) immediately after balloon injury and every 3 days thereafter. Fourteen days after the surgery, the carotid artery was isolated as described above. The carotid artery was fixed in 4%(w/v) formaldehyde and embedded in paraffin for histological analyses. Six 5 µm-thick sections from each carotid artery in interval of 2 mm were stained by the elastica van Gieson methods. Neointimal and medial areas were quantified with Scion Image software.

2.12 Detection of in situ generation of reactive oxygen species (ROS)
To detect in situ generation of ROS in the carotid artery specimens, fluorescence microphotography with dihydroethidium was performed as previously described [27,28]. Briefly, unfixed frozen samples were cut into 6 µm-thick sections and placed on glass slides. The slides were washed with PBS twice before loading dihydroethdium. Dihydroethidium (DHE, 10 µM) was applied to each tissue section, and then the sections were incubated in a light-protected chamber for 40 min. After washing with PBS, the image of dihydroethidium was obtained by a laser scanning confocal imaging system. Generation of ROS was demonstrated by red fluorescence labeling.

2.13 Statistics
All data are presented as mean ± SEM. Difference of means was analyzed by two-tailed, unpaired Student's t test. Differences with P value less than 0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1 A neutralizing antibody against rat LOX-1
An anti-LOX-1 monoclonal antibody (JTX20) [29] that cross-reacts with rat LOX-1 was screened from a number of anti-bovine LOX-1 monoclonal antibodies [29]. The immunostaining of the CHO cell line expressing rat LOX-1 reveals the specificity of this antibody towards rat LOX-1 (Fig. 1). JTX20 has exhibited potent neutralizing activity in various in vitro experiments; i.e., OxLDL binding and its biological responses, as well as the binding of platelets, bacteria, and advanced glycation endproducts [15,17,20,29–32]. The epitope of the antibody involves the C-terminus of LOX-1, where the amino acid sequences are well conserved among the bovine, human, rat, porcine, rabbit, and rat [29] versions. JTX20 exhibited neutralizing activity against the internalization of fluorescently labeled OxLDL via rat LOX-1 (Fig. 2). Rat-LOX-1-CHO cells significantly incorporated DiI-OxLDL after incubation with 3 µg/ml DiI-OxLDL at 37 °C for 3 h. Anti-LOX-1 antibody blocked the uptake in a dose-dependent manner, while control IgG did not suppress the internalization even at 30 µg/mL. The IC50 value was approximately 1 µg/mL.


Figure 1
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  		Fig. 1 Specificity of anti-LOX-1 antibody to rat LOX-1. Anti-LOX-1 antibody recognized CHO cells expressing rat LOX-1 (A) but not wild-type CHO cells (B). Non-immune mouse IgG did not recognize CHO cells expressing rat LOX-1 (C).

 

Figure 2
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  		Fig. 2 Anti-LOX-1 antibody inhibits the binding and internalization of DiI-OxLDL. CHO cells stably expressing rat LOX-1 were incubated with indicated concentrations of anti-LOX-1 antibody (JTX20, bullet), nonimmune IgG ({circ}) and 3 µg/ml DiI-OxLDL in Ham's F12 with 10% NBCS for 3 h. The fluorescence intensity of DiI-OxLDL internalized by cells is expressed as a percentage of that in the absence of the antibody. Values are mean ± SEM of three experiments in duplicate determinations.

 
3.2 Effects of balloon injury on LOX-1 gene expression
Next, we analyzed the expression of LOX-1 after balloon injury, utilizing RT-PCR. As shown in Fig. 3, LOX-1 mRNA expression was markedly upregulated as early as 1 day after balloon injury in the injured left common carotid artery. The LOX-1 gene expression level was elevated 4-fold in the injured carotid artery in comparison with the uninjured artery (P<0.01). The genetic expression was induced over the entire 2-week observation after balloon injury, while it remained at the basal level in the control contra-lateral right common carotid artery (data not shown).


Figure 3
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  		Fig. 3 (A) RT-PCR analysis of the expression of LOX-1 gene after balloon injury. Control displays mRNA level of LOX-1 before injury. (B) Densitometric analysis of the electrophoretic bands of the RT-PCR. Values are mean ± SEM of the results (n=4 for control and day 1; n=5 for day 3, 7, and 14). *P<0.01 vs. control.

 
3.3 Localization of LOX-1 expressing cells in balloon-injured artery
The localization of LOX-1 expression in the carotid artery was determined by the immunostaining of serial sections with anti-rat LOX-1 antibody. In the control uninjured artery, LOX-1 protein was not detected (Fig. 4A). One day after balloon injury, LOX-1 was markedly expressed in medial smooth muscle cells (Fig. 4B and F). The expression of LOX-1 in smooth muscle cells gradually shifted from the media to the intima (Fig. 4C, D and E). In a section of the carotid artery 14 days after injury, while the LOX-1 expression was still evident in smooth muscle cells, a powerful expression of LOX-1 became observable in luminal endothelial cells (Fig. 4E and G). The localization of LOX-1 in the regenerated endothelial cells was confirmed by the staining of endothelial cells in the serial section with anti-von Willebrand factor antibody (Fig. 5) In the intact right common carotid artery, the expression of LOX-1 was not detectable under the present immunostaining conditions (data not shown).


Figure 4
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  		Fig. 4 Immunohistochemical detection of LOX-1 expression after balloon injury. Samples were collected before (A), and 1 (B and F), 3 (C), 7 (D), 14 day(s) (E and G) after balloon injury.

 

Figure 5
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  		Fig. 5 Detection of regenerated endothelial cells 14 days after balloon injury. Endothelial cells were detected by anti-von Willebrand factor antibody. In the adjacent section, LOX-1 expression in the regenerated endothelial cells was observed as well as intimal smooth muscle cells.

 
3.4 Effects of anti-LOX-1 antibody on intimal thickening
To examine the clearance of the anti-LOX-1 antibody in plasma, we administered 10 mg/kg of anti-LOX-1 antibody by bolus intravenous injection and determined the level of the antibody in the serum. The half-life of anti-LOX-1 antibody was determined to be approximately 24 h in the rat, which is shorter than the time determined by reports for mouse IgG [33–35] (Fig. 6). This might be due to the distribution of the antibody to LOX-1 expressed in vascular endothelial cells. In the dose administered to the rat, the serum anti-LOX-1 antibody level was maintained at >50 µg/ml over three days, which is sufficient to suppress the activity of rat LOX-1. Therefore, in order to suppress the increased activity of LOX-1 after balloon injury in a sustained manner, we administrated 10 mg/kg of anti-LOX-1 antibody every 3 days until the rats were sacrificed after 14 days.


Figure 6
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  		Fig. 6 Clearance of anti-LOX-1 antibody after single bolus intravenous injection (10 mg/kg). Data are mean ± SEM (n=3).

 
Progressive intimal thickening in the injured left common carotid artery was observed 14 days after balloon injury in the control IgG-treated group. In contrast, the thickening was significantly suppressed in the injured left common carotid artery of rats treated with the anti-LOX-1 antibody (Fig. 7, A and B). Quantitative analyses of the thickened area showed that the neointimal area was reduced by 29.4%, and the intima to media ratio was reduced by 27.8% in the anti-LOX-1 antibody-treated group as compared with the control IgG-treated group (Fig. 7, C and D).


Figure 7
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  		Fig. 7 Effects of the anti-LOX-1 antibody on neointimal formation in rat carotid arteries. Elastica van Gieson-staining of the cross section of injured artery treated with control IgG (A), and anti-LOX-1 antibody (B) are shown. (C) Intimal and medial area of the arteries at 14 days after injury. (D) Intimal/media ratio of the injured arteries. Data are mean ± SEM from 12 rats. *P<0.01 vs. control IgG.

 
To clarify the mechanisms of the effects of the anti-LOX-1 antibody, we further analyzed the specimens 7 days after balloon injury. Analysis of in situ ROS generation detected by DHE revealed that the ROS level generated from the specimens of the anti-LOX-1-treated rat was markedly reduced compared with IgG-treated rats (Fig. 8). Furthermore, the leukocyte infiltration detected by immunostaining of myeloperoxidase was decreased in the injured artery of the anti-LOX-1 treated rat (Fig. 9).


Figure 8
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  		Fig. 8 Detection of in situ generation of reactive oxygen species (ROS). ROS generation were monitored by the oxidation of DHE on the sections from the injured arteries at day 7 treated with antiLOX-1 antibody or IgG.

 

Figure 9
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  		Fig. 9 Immunohistochemical detection of myeloperoxidase. Injured arteries at day 7 treated with antiLOX-1 antibody or IgG were stained with anti-myeloperoxidase antibody.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
LOX-1 has been shown to be expressed in endothelial cells, macrophages, smooth muscle cells, and activated platelets [23–25,36]. LOX-1 expression can be induced by proinflammatory stimuli and oxidative stress, such as TNF-{alpha}, TGF-β, angiotensin II and reactive oxygen species [10,11,36–38]. These factors are thought to be important in neointimal hyperplasia after balloon injury [8,39–41]. In the present study, we demonstrated that balloon catheter injury induces LOX-1 expression and this enhances neointima formation. LOX-1 expression was induced in smooth muscle cells immediately after balloon injury. The localization of LOX-1 expression progressed with the pathological phases of neointima formation. In the early and intermediate phases, LOX-1 expression was localized in the medial and intimal smooth muscle cells, respectively, and in the advanced stages in the newly generated endothelial cells. The removal of endothelial cells appears to be involved in the induction of LOX-1 in VSMCs as these cells were exposed to proinflammatory cytokines and oxidative stress. Removal of endothelial cells leading to the decrease in endothelium-derived nitric oxide might also up-regulate the expression of LOX-1 in VSMCs, as reported in endothelial cells [42]. In addition, mechanical stimulation of VSMCs exposed to the shear stress of the flowing blood may also be involved, since fluid shear force has been shown to induce LOX-1 expression [43].

After mechanical injury/stimulation by the balloon catheter, cells in the arterial wall change their phenotype to the activated state [2]. In this sense, LOX-1 might be one of the immediate early genes that are induced on the stimulation of VSMCs. However, the significant suppression by anti-LOX-1 antibody of intimal hyperplasia suggests that LOX-1 might not be merely an inducible gene, but rather, might also be involved in the initiation and progression of the neointimal hyperplasia after balloon injury. It has been shown that the binding of ligands to LOX-1 induces oxidative stress, producing the superoxide anion. Oxidative stress, in turn, induces the expression of LOX-1 [38], forming a positive feedback loop [44], which may contribute to the vicious cycle which characterizes the pathogenesis of intimal hyperplasia.

It has been reported that a potent anti-oxidative drug, probucol, inhibits neointimal formation in normocholesterolemic rabbits and porcine [45,46]. In the Multivitamins and Probucol (MVP) clinical trial, probucol was shown effective in reducing the rate of restenosis after balloon coronary angioplasty, although the cholesterol level was decreased in the probucol-treated group [47]. Furthermore, Muscoli et al. reported that a synthetic superoxide dismutase mimetic suppressed neointimal formation after balloon injury and suppressed the expression of LOX-1 in the developing lesion, although the evidence for the involvement of LOX-1 was indirect [48]. In the present study, blocking LOX-1 function with an anti-LOX-1 antibody actually reduced oxidative stress in the injured vascular wall. These findings support the importance of oxidative stress in neointimal formation in relationship with LOX-1.

As a multi-ligand receptor LOX-1 binds oxidized LDL, activated platelets, leukocytes, apoptotic cells and possibly other, as yet unidentified ligands. It is likely that LOX-1 interacts with these ligands on injured smooth muscle cells and increases the oxidative stress in the lesion which promotes intimal thickening. As a leukocyte adhesion molecule, LOX-1 has been shown to be involved in inflammation [21,22], one of the crucial factors in the pathogenesis of intimal hyperplasia. It is noteworthy that inflammation further enhances oxidative stress in the growing lesion. LOX-1 expression and activation may promote intimal thickening by promoting leukocyte attachment and the initiation of inflammation and oxidative stress. Actually, in the present study, the decrease in the infiltration of leukocytes as the result of treatment with the anti-LOX-1 antibody suggests the importance of the function of LOX-1 for leukocyte-adhesion.

In the final phase of pathological changes in the balloon-injured artery, marked expression of LOX-1 in the regenerated endothelium was observed two weeks after balloon injury. It has been reported that the regenerated endothelial cells exhibit an increased uptake of modified LDL and a reduced production of NO [49]. These findings may result from the up-regulated expression of LOX-1 in the regenerated endothelial cells.

In conclusion, we observed that the administration of an anti-LOX-1 antibody suppressed the neointimal thickening which occurs after balloon injury. Although the observation period in the present study was limited to 14 days, the findings suggest that manipulation of LOX-1 activity holds promise as a useful strategy to suppress restenosis after coronary intervention with a balloon catheter.


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
This work was supported in part by the grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labor and Welfare of Japan, the Organization for Pharmaceutical Safety and Research. We thank Pacific Edit for reviewing the manuscript prior to publication.


   Notes
 
Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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