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Cardiovascular Research 2001 52(1):130-135; doi:10.1016/S0008-6363(01)00371-6
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Statins inhibit oxidized-LDL-mediated LOX-1 expression, uptake of oxidized-LDL and reduction in PKB phosphorylation

D.Y Li, H.J Chen and J.L Mehta*

Departments of Medicine and Physiology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Health Care System, Little Rock, AR, USA

* Corresponding author. Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, 4301 West Markham St., Mail Slot 532, Little Rock, AR 72205-7199, USA. Tel.: +1-501-296-1401; fax: +1-501-686-6180 mehtajl{at}uams.edu

Received 16 February 2001; accepted 28 May 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objectives: LOX-1, a lectin-like receptor on endothelial cells, facilitates the uptake of oxidized-LDL. Expression of LOX-1 is involved in the pathobiological effects of oxidized-LDL in endothelial cells, including apoptosis, suppression of cNOS activity and cell adhesion. Recent studies show that intracellular signal protein kinase B (PKB) is involved in the regulation of cNOS. Further, HMG CoA reductase inhibitors (statins) may affect LOX-1 expression. In this study, we examined the modulation of LOX-1 expression and PKB activity in response to oxidized-LDL by two different statins (simvastatin and atorvastatin). Methods and results: Cultured human coronary artery endothelial cells (HCAECs) were used in this study. Oxidized-LDL (40 µg/ml) was found to upregulate the expression of LOX-1 (mRNA and protein), enhance [125I]-ox-LDL uptake and to reduce the phosphorylation of PKB (p-PKB). Two different statins, simvastatin and atorvastatin (each 1 and 10 µM), upregulated the activity of PKB and decreased LOX-1 expression and [125I]-ox-LDL uptake. A high concentration of statins (10 µM) gave a more potent effect than the low concentration (1 µM). The effects of the two different statins were similar. Conclusions: These observations show that statins decrease LOX-1 expression, a novel oxidized-LDL endothelial receptor, and uptake of oxidized-LDL in HCAECs. The effect of statins on LOX-1 expression is associated with an increase in PKB activity in HCAECs.

KEYWORDS Endothelial function; Lipoproteins; Protein kinases; Statins

This article is referred to in the Editorial by S.J. Miller (pages 5–7) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Vascular endothelial cells internalize and degrade oxidized low-density lipoprotein (ox-LDL) through a unique receptor-mediated pathway, independent of the classic macrophage scavenger receptor [1,2]. This lectin-like receptor for ox-LDL (LOX-1) may mediate many of the biological effects of ox-LDL in endothelial cells [3–6]. Studies from our [4,7] and other [8,9] laboratories show that the expression of LOX-1 gene is upregulated by ox-LDL, angiotensin II, inflammatory cytokines (such as TNF{alpha}) and shear stress. Other studies have demonstrated that LOX-1 expression is upregulated in atherosclerotic tissues [10,11].

Recent studies have shown that protein kinase B (PKB), the cellular homologue of v-Akt, is a key signaling component downstream of phosphatidylinositide (PI) 3-kinase [12]. PKB activation appears to be critically important in the expression of constitutive nitric oxide synthase (cNOS). It has been recently proposed that treatment of endothelial cells with a variety of growth factors in a PI-3 kinase dependent fashion, stimulates cNOS activity [13,14]. Since ox-LDL downregulates cNOS expression [15], we proposed that changes in PKB activity may play an important role in modulating this major effect of ox-LDL.

The development of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) has been a major milestone in the primary and secondary prevention of coronary heart disease. These agents, besides lowering total and low-density lipoprotein cholesterol, have a multitude of other effects, which may have a bearing on the cardioprotective effect of these agents [16]. Kureishi et al. [17] have recently suggested endothelial cell PKB as a new biological target for the action of statins. These agents, as a class, modulate the paracrine and vasodilatory functions of endothelium in hypercholesterolemia [18]. In a variety of animal models, these agents limit the development of atherosclerosis [19,20].

The present study was designed to examine the following hypotheses: (a) ox-LDL via action of its receptor LOX-1 decreases PKB activity; (b) statins increase PKB activity, and subsequently inhibit LOX-1 expression and ox-LDL uptake in cultured human coronary artery endothelial cells (HCAECs).


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
The methodology for culture of HCAECs has been described earlier [4,5]. The initial batch of HCAECs was purchased from Clonetics (San Diego, CA). The endothelial cells were pure based on morphology and staining for factor VIII-related antigen and acetylated LDL. These cells were 100% negative for alpha-actin smooth muscle expression.

2.2 Study design
HCAECs were incubated with ox-LDL (40 µg/ml) for 24 h to determine the expression of LOX-1 mRNA and protein. We have earlier shown that ox-LDL upregulates the expression of LOX-1 [4,21]. To determine the effects of two most commonly used statins on the expression of LOX-1, parallel batches of HCAECs were pretreated with simvastatin (Merck) or atorvastatin (Pfizer) (each 1 and 10 µM) for 30 min and then the cells were exposed to ox-LDL. In order to examine the significance of the upregulation of LOX-1 expression, uptake of [125I]-ox-LDL by HCAECs was studied. HCAECs were pretreated with simvastatin or atorvastatin (1 and 10 µM) and then exposed to ox-LDL for 24 h. Thereafter, [125I]-ox-LDL (10 µg/ml) was added into each dish to observe binding of LOX-1. To explore the signal transduction mechanism of LOX-1 expression, we studied PKB activity. HCAECs were pretreated with simvastatin (10 µM), atorvastatin (10 µM) and then exposed to ox-LDL (40 µg/ml) without or with the PKB inhibitor LY294002 (10 µM) for 24 h. Thereafter, the harvested cells were used to examine PKB activity. In preliminary studies, we found that 0.1–10 µM of simvastatin or atorvastatin had no effect on cell function, whereas 100 µM of simvastatin or atorvastatin caused cell injury determined by LDH release. Therefore, we used 1 and 10 µM of simvastatin and atorvastatin in the present study. The concentration of the other reagents and the duration of incubation were based on previous studies [4,5,21].

2.3 Preparation of lipoproteins
Native LDL and ox-LDL were prepared as described earlier [5]. The TBARS content of ox-LDL was 18.2±0.28 versus 0.56±0.16-nmoles/100 µg protein in the native-LDL preparation (P<0.01). Ox-LDL was extensively dialyzed against Tris–saline. Ox-LDL was kept in 50 mM Tris–HCl, 0.15 M NaCl and 2 mM EDTA at pH 7.4, and was used within 10 days of preparation. [125I]-Labeled ox-LDL was purchased from Biomedical Technologies (Stoughton, MA).

2.4 Semiquantitative RT-PCR for LOX-1 mRNA
Total RNA (1 µg) extracted from cultured HCAECs was reverse transcripted with Oligo dT (Promega) and M-MLV reverse transcriptase (Promega) at 37°C for 1 h. One point five (1.5) µl of the reverse-transcripted material was amplified with Taq DNA polymerase (Promega) using a primer pair specific to human LOX-1 (sense primer, 5'-TTACTCTCCATGGTGGTGCC-3', antisense primer, 5'-AGCTTCTTCTGCTTGTTGCC-3'). PCR product was 193 base pairs. For PCR, 30 cycles were used at 94°C for 40s, 55°C for 1 min, and 72°C for 1 min. In same experiments, human β-actin was amplified with equal efficiency as a reference for quantitation of LOX-1 mRNA. A primer pair of human β-actin was used (sense primer, 5'-TCGAATTCTGGAGAAGAGCTATGAGCTGCCG-3', antisense primer, 5'-TCGGATCCGTGCCACCAGACAGCACTGTGTTG-3'). PCR product was 201 base pairs. For PCR, 30 cycles were used at 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min. The RT-PCR amplified samples were visualized on 1.5% agarose gels using ethidium bromide. Each LOX-1 mRNA band was normalized with a band of the relative internal reference β-actin mRNA. Relative intensity of band of interest was analyzed by NSF-300G scanner (Microtek) and scan analysis software (Biosoft) and expressed as the ratio to β-actin mRNA band [4,5,21].

2.5 Western analysis for LOX-1 protein
HCAEC lysates from each experiment (30 µg per lane) were separated by 10% SDS–PAGE and transferred to nitrocellulose membranes. After incubation in blocking solution (4% non-fat milk, Sigma, St. Louis, MO), membranes were incubated with 1:1000 dilution primary antibody (monoclonal antibody to LOX-1, gifted by professor T. Sawamura, Osaka, Japan) for overnight at 4°C. Membranes were washed and then incubated with 1:2000 dilution second antibody (Amersham) for 1 h, and the membranes were detected with the ECL system, and relative intensities of protein bands were analyzed by MSF-300G Scanner [4,5,21].

2.6 Binding of [125I]-ox-LDL
Details of [125I]-ox-LDL binding and uptake by HCAECs have been recently published [7]. In brief, cells from different experimental groups were prechilled for 30 min in Hepes buffer, pH 7.4. [125I]-ox-LDL was added to each dish in a final concentration of 10 µg/ml. Incubation was carried out at 4°C for 2 h. Cells were washed three times on ice with 150 nM NaCl, 50 mM Tris, 2 mM EDTA pH 7.4, containing 2 mg/ml BSA. Cells were then rinsed with cold saline without BSA. Cells were lysed at room temperature in 0.5 M NaOH solution. An aliquot of the cell lysate was counted to determine the amount of cell bound [125I]-ox-LDL.

2.7 Determination of PKB phosphorylation
HCAEC lysates from different experimental groups were separated by 10% SDS–PAGE and transferred to nitrocellulose membranes. After incubation in blocking solution (4% non-fat milk), the membranes were incubated overnight at 4°C with 1:800 dilution rabbit polyclonal phospho-specific antibody to PKB (p-PKB) that detects p-PKB only when catalytically activated by phosphorylation at Ser473 (New England Biolabs, Beverly, MA). Membranes were washed and then incubated with 1:2000 dilution second antibody (Amersham) for 1 h, and the membranes were detected with the ECL system. Thereafter, protein on the membrane was stripped and reprobed with PKB antibody (New England Biolabs, Beverly, MA) and relative intensities of protein bands were analyzed by MSF-300G Scanner [21].

2.8 Data analysis
All data represent mean of at least six independently performed experiments. Data are presented as mean±S.D. 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 P-value ≤0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 OX-LDL-induced expression of LOX-1 and regulation of PKB
In previous studies [4,21], we demonstrated that ox-LDL upregulates its own receptor LOX-1 in a concentration (10–60 µg/ml)-dependent fashion. In the present study, we again confirmed that ox-LDL (40 µg/ml) markedly increased the expression of LOX-1 mRNA and protein (P<0.01 vs. control group, n=6). Incubation of HCAECs with ox-LDL did not affect PKB protein levels, but caused a decrease in activated PKB, determined by phosphorylation of PKB (p-PKB) (P<0.01 vs. control, n=6) (Fig. 1).


Figure 1
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  		Fig. 1 Effect of ox-LDL on LOX-1 expression and modulation of PKB activity. Treatment of HCAECs with ox-LDL (40 µg/ml) increased the expression of LOX-1 (mRNA and protein). Treatment of HCAECs with ox-LDL markedly decreased PKB activity, (p-PKB) without affecting PKB protein. Left panel is representative of LOX-1 expression from 6 independent experiments. Right panel is representative of PKB activity and PKB protein from 6 independent experiments.

 
3.2 Statins and the expression of LOX-1
To examine the effect of statins on LOX-1 expression, cells were pretreated with statins before exposure to ox-LDL. As shown in Fig. 2, pretreatment of HCAECs with simvastatin or atorvastatin (1 and 10 µM) markedly decreased ox-LDL-induced upregulation of LOX-1 protein and mRNA (all P<0.05). High concentration of simvastatin and atorvastatin (10 µM) was more potent than the low concentration (1 µM) (P<0.05). Furthermore, the effect of the two statins appeared quantitatively similar. In parallel experiments, incubation of HCAECs with simvastatin or atorvastatin (10 µM) alone did not affect the expression of LOX-1 in cultured HCAECs.


Figure 2
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  		Fig. 2 Effects of statins on LOX-1 expression. Pretreatment of HCAECs with simvastatin or atorvastatin decreased ox-LDL-induced upregulation of LOX-1. The high concentration of simvastatin or atorvastatin (10 µM) gave a more potent effect than the low concentration (1 µM). LOX-1 mRNA band density was normalized by β-actin and expressed as the ratio of LOX-1 mRNA/β-actin mRNA. LOX-1 protein band density was expressed as the ratio of LOX-1 protein to the expression in control. The top panel is representative of 6 independent experiments. The bottom panel is the summary of data (mean±S.D.) from these 6 experiments.

 
3.3 Statins and binding of [125I]-ox-LDL to HCAECs
As shown in Fig. 3, incubation of HCAECs with ox-LDL increased the binding of [125I]-ox-LDL to HCAECs. To determine the significance of LOX-1 expression in [125I]-ox-LDL binding, HCAECs were treated with statins followed by incubation with ox-LDL. Pretreatment of HCAECs with simvastatin or atorvastatin markedly decreased [125I]-ox-LDL binding to HCAECs compared with ox-LDL alone (all P<0.05 vs. ox-LDL alone). High concentration of statins (10 µM) had a more potent inhibitory effect on [125I]-ox-LDL binding than the low concentration (1 µM) (P<0.05). In parallel experiments, incubation of HCAECs with simvastatin or atorvastatin (10 µM) alone did not affect the binding of [125I]-ox-LDL to HCAECs.


Figure 3
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  		Fig. 3 Ox-LDL and [125I]-ox-LDL binding and modulation by statins. Incubation of HCAECs with ox-LDL increased binding of [125I]-ox-LDL to HCAECs. Pretreatment of HCAECs with simvastatin or atorvastatin decreased [125I]-ox-LDL binding to HCAECs. The high concentration of statins gave a more potent effect than the low concentration. These data are the summary (mean±S.D.) of 6 independent experiments.

 
3.4 Statins and PKB intracellular signal transduction
As shown in Fig. 4, incubation of HCAECs with ox-LDL caused a decrease in p-PKB. Pretreatment of HCAECs with simvastatin or atorvastatin markedly increased PKB activity compared to ox-LDL alone (P<0.01). However, the effects of these two statins on PKB activity were reversed by treatment of cells with LY294002 (10 µM), an upstream inhibitor of PKB.


Figure 4
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  		Fig. 4 Inhibition of PKB activity (p-PKB) by ox-LDL and its modulation by statins. Ox-LDL decreased p-PKB determined by Western analysis. Pre-treatment of HCAECs with simvastatin or atorvastatin inhibited the effects of ox-LDL on p-PKB. The concentration of statins was 10 µM in these experiments. In addition, pretreatment of HCAECs with the PKB inhibitor LY294002 (10 µM) reversed the effect of statins. Note that ox-LDL or statins had no effect on PKB protein levels. The top panel is representative of 6 independent experiments. The bottom panel is the summary of data (mean±S.D.) from these 6 experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this study in human coronary endothelial cells, we found that ox-LDL decreases PKB activity via LOX-1 action. Furthermore, we found that two different statins (simvastatin and atorvastatin) markedly attenuated ox-LDL-induced expression of LOX-1, binding of [125I]-ox-LDL, and reversed the reduction in PKB activity.

4.1 Ox-LDL and LOX-1
Accumulating evidence indicates that ox-LDL is a key component in atherogenesis [22]. LOX-1, a novel receptor for ox-LDL, found predominantly on endothelial cells, has a different biochemical structure from the scavenger receptor [3]. Several studies [3,7,21] have demonstrated that the uptake of ox-LDL by endothelial cells is mediated by LOX-1. Ox-LDL also upregulates the expression of LOX-1 mRNA and protein in a concentration-dependent fashion [4]. Ox-LDL uptake induces endothelial activation, dysfunction and loss of integrity, and alterations in cell secretory function [5,23,24]. These changes in endothelial biology provide a basis for leukocyte, monocyte and platelet deposition on the vascular intima, which is an early event in atherosclerosis [24]. Many of these effects of ox-LDL are mediated by LOX-1 activation [21]. It is of note that the expression of LOX-1 is markedly increased in rabbit [9] and human [10] atherosclerotic tissues.

4.2 Statins and LOX-1
Statins are used widely for the treatment of hypercholesterolemia. Clinical trials in patients with and without coronary heart disease have consistently shown that statins reduce the progression of coronary atherosclerosis [25] as well as the risk of major coronary events [26–28]. Proposed mechanisms for the beneficial effect of statins include favorable effects on plasma lipoproteins, endothelial function, plaque architecture and stability, thrombosis, and inflammation. Mechanisms independent of LDL lowering may also play an important role in the clinical benefits conferred by these drugs [21]. Recent studies have shown that statins protect against ox-LDL-induced upregulation of endothelin gene expression and the downregulation of cNOS gene expression [29,30]. In the present study, we show that two different commonly used statins simvastatin and atorvastatin inhibit the expression of LOX-1 elicited by ox-LDL in HCAECs. A decrease in LOX-1 expression resulted in a reduction in binding (uptake) of [125I]-ox-LDL in HCAECs. A high concentration (10 µM) of these statins was more potent than the low concentration (1 µM) in this regard. These findings indicate that statins may directly protect vascular endothelium against the adverse effect of ox-LDL.

4.3 Signal transduction mechanism of ox-LDL action and the role of statins therein
Experimental studies have shown that ox-LDL injures endothelial cells via activation of a variety of signal transduction pathways [31,32]. PKB is a member of the second-messenger family that regulates the sub-family of protein kinases implicated in signaling downstream of tyrosine kinases and PI 3-kinase. PKB is activated by phosphorylation in response to mitogens and survival factors [13,14]. Recent studies [14,33] show that the activation of PKB is an independent signal transduction pathway in the expression of cNOS gene in endothelial cells. In the present study, we found that incubation of HCAECs with ox-LDL markedly decreased the activity of PKB, and pre-treatment of cells with statins blocked this effect of ox-LDL. Furthermore, we demonstrated that LY294002, an upstream (PI 3-kinase) inhibitor of PKB, prevented the effect of statins on PKB activity. This finding indicates that ox-LDL modulates PKB activity through PI 3-kinase-PKB pathway. Statins, which block HMG-CoA reductase, have earlier been shown to increase cNOS gene expression in endothelial cells [34,35].

In this context, the present study HCAECs provides evidence that ox-LDL upregulates expression of LOX-1 associated with a decrease in PKB activity. Simvastatin and atorvastatin inhibit LOX-1 expression through an increase in PKB activity and prevent ox-LDL uptake by HCAECs. These observations indicate that statins modulate endothelial secretory function and signal transduction. These findings further support the clinically beneficial effect of statins on cardiovascular diseases without high cholesterol levels.

Time for primary review 27 days.


   Acknowledgements
 
Supported by a Scientist Development Award from the American Heart Association National Center, a Merit Review Award from the VA Central Office and a contract with the Department of Defense.


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

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