Upregulation of Endothelial Receptor for Oxidized LDL (LOX-1) by Oxidized LDL and Implications in Apoptosis of Human Coronary Artery Endothelial Cells
Evidence From Use of Antisense LOX-1 mRNA and Chemical Inhibitors
Abstract—A specific lectinlike endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL–induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 μg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL–mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL–mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL–mediated apoptosis of HCAECs. Nuclear factor (NF)-κB was markedly activated in ox-LDL–treated HCAECs. The critical role of NF-κB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL–mediated NF-κB activation. In this process, an NF-κB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL–mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL–induced apoptosis is mediated by the action of LOX-1. In this process, NF-κB activation may play an important role as a signal transduction mechanism.
- Received July 13, 1999.
- Accepted December 1, 1999.
Vascular endothelial cells in culture1 and in vivo2 internalize and degrade oxidized LDL (ox-LDL) through a putative receptor-mediated pathway that does not seem to involve the classic macrophage scavenger receptor. The endothelial receptor for ox-LDL (LOX-1) is a membrane protein that belongs structurally to the C-type selectin family and is expressed in vivo in vascular endothelium and in vascular-rich organs.3 Because endothelial uptake of ox-LDL is important in the genesis of atherosclerosis, understanding of the role and regulation of LOX-1 may be of immense clinical significance.
Recent studies show that cytokine tumor necrosis factor (TNF)-α4 and fluid shear stress5 markedly upregulate LOX-1 gene expression in endothelial cells. Another study6 has shown that LOX-1 expression is dramatically increased in hypertensive rats. A more recent study from our laboratory7 has demonstrated that angiotensin II upregulates LOX-1 expression as well as the uptake of ox-LDL in human coronary artery endothelial cells (HCAECs).
It is widely appreciated that LDL, especially its oxidatively modified form (ox-LDL), is a critical factor in atherogenesis. Endothelial dysfunction elicited by ox-LDL has been identified in the course of atherogenesis and its complications.8 LDL is oxidized in vascular endothelial cells to a highly injurious product that results in characteristic cell dysfunction in large arteries and resistant vessels. Endothelial dysfunction (ie, loss of vasodilation, vasoconstriction, thrombosis, and inflammation) occurs before and throughout the development of atherosclerosis and particularly during plaque rupture. Ox-LDL appears to induce this cellular dysfunction in a time- and concentration-dependent manner.9 Recent studies show that apoptosis, which is a common accompaniment of atherosclerosis, is induced by ox-LDL in cultured vascular smooth muscle cells,10 monocytes/macrophages,11 and human endothelial cells.12 The mechanisms of ox-LDL–mediated apoptosis, particularly in endothelial cells, and of its relation with LOX-1 have not been defined.
Nuclear factor (NF)-κB, a transcription factor, regulates the transcription of a variety of cellular genes, including injury response and growth control.13 Activation of NF-κB is inhibited by antioxidant compounds, such as N-acetyl-l-cysteine, pyrrolidine dithiocarbamate,14 and vitamin E.15 It has thus been proposed that NF-κB is primarily an oxidative stress–responsive transcription factor.14 15 Previous studies have demonstrated that NF-κB is activated in accelerated and in advanced atherosclerosis.16 Recent studies show that NF-κB activation plays a critical role in apoptosis17 18 and that ox-LDL induces the activation of NF-κB in fibroblasts and in endothelial and smooth muscle cells.19
Accordingly, we hypothesized that (1) ox-LDL upregulates its own receptor, LOX-1; (2) ox-LDL–mediated apoptosis of cultured HCAECs is associated with the action of LOX-1; and (3) NF-κB activation plays an important role in this process. The present study was conducted to examine these hypotheses.
The initial batch of HCAECs was purchased from Clonetics Corp. The endothelial cells were pure, on the basis of morphology and staining for factor VIII and acetylated LDL. These cells were 100% negative for α-actin smooth muscle expression. Microvascular endothelial growth medium consisted of 500 mL of endothelial cell basal medium, 5 ng of human recombinant epidermal growth factor, 5 mg of hydrocortisone, 25 mg of gentamycin, 25 μg of amphotericin B, 6 mg of bovine brain extract, and 25 mL of FBS.
Fifth generation HCAECs7 12 were incubated with ox-LDL (40 μg/mL) for 24 hours to study the expression of LOX-1 as well as NF-κB activity. Several groups of HCAECs were incubated with different concentrations of ox-LDL (10 to 100 μg/mL) for 24 hours to observe apoptosis. HCAECs were preincubated with antisense to LOX-1 mRNA (antisense LOX-1) or sense LOX-1 (0.5 μmol/L) for 48 hours. HCAECs were then incubated with ox-LDL (40 μg/mL) to determine the expression of LOX-1. HCAECs incubated with antisense LOX-1 or sense LOX-1 were also used to examine ox-LDL–mediated apoptosis and NF-κB activation. Parallel groups of HCAECs were incubated with 2 different chemical inhibitors of LOX-1 (250 μg/mL polyinosinic acid or 250 μg/mL carrageenan) or NF-κB inhibitor (25 μg/mL caffeic acid phenethyl ester [CAPE]) for 1 hour and then exposed to ox-LDL for 24 hours to observe NF-κB activity and apoptosis of HCAECs. The concentration of these inhibitors was based on previous studies.20 21
Preparation of Antisense LOX-1
Antisense phosphorothioate oligonucleotides (ODNs) and sense phosphorothioate ODNs (as controls) directed to the 5′-coding sequence of the human LOX-1 mRNA were designed and manufactured by Biognostik GmbH. The antisense LOX-1 was synthesized as a 16-mer product (8 bases) targeted at 5′-CAG TTA AAT GAG GCC G-3′ of the LOX-1 sequence. The corresponding control (sense) was 16-mer (8 bases) targeted at 5′-ACC TAC GTG ACT ACG T-3′. Hereafter, the antisense and sense to LOX-1 mRNA will be referred to as antisense LOX-1 and sense LOX-1, respectively. Logarithmically growing endothelial cells were transfected by adding ODNs into culture medium according to the instructions of the manufacturer. Recent experiments from our laboratory demonstrated that the uptake of antisense LOX-1 was maximal, with 0.5 μmol/L after incubation for 48 hours. Hence, the present study describes the use of a 0.5 μmol/L concentration of antisense ODNs.
Preparation of Lipoproteins
Native LDL and ox-LDL were prepared as described earlier.12 In brief, human native LDL was isolated from human blood plasma by discontinuous centrifugation. LDL was oxidized by exposure to CuSO4 (5 μmol/L free Cu2+ concentration) in PBS at 37°C for 24 hours. The thiobarbituric acid–reactive substance content of ox-LDL was 18.2±0.28 versus 0.56±0.16 nmol per 100 μg protein in the native LDL preparation (P<0.01). LDL and ox-LDL were kept in 50 mmol/L Tris-HCl, 0.15 mol/L NaCl, and 2 mmol/L EDTA at pH 7.4 and were used within 10 days of preparation.
RT-PCR for LOX-1 mRNA Expression in HCAECs
Total RNA (1 μg) extracted from cultured HCAECs was reverse-transcribed with oligo dT (Promega) and M-MLV reverse transcriptase (Promega) at 37°C for 1 hour. Two microliters of the reverse-transcribed material was amplified with Taq DNA polymerase (Promega) by using a primer pair specific to human endothelial receptor (sense primer, 5′-TTACTCTCCATGGTGGTGCC-3′; antisense primer, 5′-AGCTTCTTCTGCTTGTTGCC-3′). The polymerase chain reaction (PCR) product was 193 bp. For PCR, 35 cycles were used at 94°C for 40 seconds, 55°C for 1 minute, and 72°C for 1 minute. The reverse transcription (RT)-PCR–amplified samples were visualized on 1.5% agarose gels by using ethidium bromide. β-Actin was amplified as a reference for quantification of LOX-1 mRNA. The signal intensity of each LOX-1 mRNA band was normalized by that of β-actin. The RT-PCR product for LOX-1 was sequenced. The sequence of RT-PCR product for LOX-1 in HCAECs was the same as the previously published sequence for LOX-1.3 7
Western Blot Analysis for LOX-1 in HCAECs
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% nonfat milk, Sigma Chemical Co), membranes were incubated with 1:100 dilution primary antibody (monoclonal antibody to human LOX-1 was a gift of Dr Sawamura, National Cardiovascular Center Research Unit, Osaka, Japan) overnight at 4°C. Membranes were washed and then incubated with secondary antibody (1:2000 dilution, Amersham Life Sciences) for 1 hour, the membranes were detected with the ECL system (Amersham Life Sciences), and relative intensities of protein bands were analyzed by an MSF-300G Scanner (Microtek Laboratory).7
Determination of Apoptosis
In Situ TUNEL and Propidium Iodide Staining
To detect DNA fragmentation in situ, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) was performed as previously described.22 Briefly, the cells plated on slides were fixed with 4% methanol-free formaldehyde, pH 7.4, for 25 minutes at 4°C and washed with PBS. The slides were incubated with 0.2% Triton X-100 for 5 minutes on ice to increase cell permeability and were equilibrated with terminal deoxynucleotidyl transferase (TDT) buffer (including 30 mmol/L Tris-HCl, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) for 10 minutes at room temperature. The slides were covered with 0.3 U/μL TDT and 0.04 nmol/μL fluorescein-12-dUTP (Promega) in TDT buffer for 60 minutes at 37°C. The slides were immersed in 2× SSC buffer for 15 minutes at room temperature and then washed with PBS to remove unincorporated fluorescein-dUTP. The slides were immersed in 1 μg/mL of propidium iodide in PBS for 15 minutes at room temperature and washed with deionized water. The slides were viewed under a fluorescence microscope with green fluorescence set at 520 nm and red fluorescence (of propidium iodide) set at >620 nm. The negative controls were cells without TDT enzyme. The positive controls were samples pretreated with DNase I.
DNA Fragmentation Gel Electrophoresis (DNA Laddering)
HCAECs were removed from culture dishes, washed twice with PBS, and pelleted by centrifugation. Cell pellets were then treated for 10 minutes with lysis buffer (1% NP-40 in 20 mmol/L EDTA and 50 mmol/L Tris-HCl, pH 7.5). After centrifugation for 5 minutes at 1600g, the supernatant was collected, and the extraction was repeated with the same amount of lysis buffer. The supernatants were brought to 1% SDS and treated for 2 hours with RNase A (final concentration 5 μg/μL) at 56°C, followed by digestion with proteinase K (final concentration 2.5 μg/μL) for 2 hours at 37°C. After addition of 1/2 vol of 10 mol/L ammonium acetate, the DNA was precipitated with 2.5 vol of absolute ethanol. DNA was recovered by centrifugation at 12 000g for 10 minutes and dissolved in gel loading buffer. DNA was separated by electrophoresis in 1.6% agarose gel with ethidium bromide.22
Preparation of Nuclear Extracts
Nuclear extracts were prepared as described previously.22 Briefly, the cells were washed with 1 mL PBS and resuspended in 100 μL hypotonic buffer (mmol/L: HEPES 10, pH 7.3, KCl 10, MgCl2 1.5, dithiothreitol [DTT] 1, and phenylmethylsulfonyl fluoride [PMSF] 1). After centrifugation, cells were lysed by resuspension in 300 μL lysis buffer (10 mmol/L HEPES, pH 7.3, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.4% Nonidet P-40, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin). After a 10-minute incubation at 4°C, nuclei were collected by centrifugation for 1 minute at 8000g, and the pellets were washed once in 1 mL of 20 mmol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 20 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin). The isolated nuclei were resuspended in 100 μL of 484 mmol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 484 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin). Nuclear proteins were extracted by incubation on ice for 30 minutes. After centrifugation for 15 minutes at 8000g, the supernatant containing nuclear proteins was transferred to a precooled microcentrifuge tube, and nuclear protein concentration was quantified.
Western Blot for NF-κB
Monoclonal antibody to the P65 subunit of NF-κB from mouse to mouse hybrid cells was purchased from Boehringer-Mannheim. The antibody recognizes an epitope overlapping the nuclear location signal of the P65 subunit and, therefore, selectively binds the activated form of NF-κB. Equal amounts of protein (15 μg) from the nuclear extract of each group were separated by 7.5% SDS-PAGE and transferred to nitrocellulose filters (Sigma). The membrane was blocked by incubating the membrane for 1 hour in Tris-saline buffer (pH 7.4) containing 3% nonfat milk (Sigma) and incubating the membrane in the same buffer containing 7.5 μg/mL of monoclonal antibody to the P65 subunit of NF-κB. Anti-mouse alkaline phosphatase–conjugated antibody was used as a secondary antibody at 1: 3000 dilution. The blot was used for color development on the membrane. Sites of antigen localization turn a dark purple color as a result of alkaline phosphatase activity. Relative intensities of bands of interest were analyzed by use of an MSF-300G Scanner (Microtek Laboratory).7 12 22
All data represent the mean of duplicate samples from 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 value of P<0.05 was considered significant.
Regulation of LOX-1 by Ox-LDL
A previous study from our laboratory showed that LOX-1 expression is upregulated by ox-LDL in a concentration-dependent manner, with maximal effect at 40-μg/mL.20 Therefore, a concentration of only 40 μg/mL ox-LDL was used in the present study. In keeping with the previous report, incubation of HCAECs with ox-LDL (40 μg/mL) for 24 hours increased LOX-1 mRNA (RT-PCR) and protein expression (Western analysis), whereas native LDL had no effect on the regulation of LOX-1. This upregulation of LOX-1 by ox-LDL was blocked by pretreatment of HCAECs with antisense LOX-1, but not with sense LOX-1. Notably, basal LOX-1 mRNA and protein were also suppressed by antisense LOX-1, but not by sense LOX-1 (Figure 1⇓).
Ox-LDL and Apoptosis in HCAECs
Because a small number of cells normally die during culture or are damaged during processing, 1% to 5% (3.8±1.6%) of control cells stained positive on TUNEL staining. Cultured HCAECs incubated with ox-LDL (10 to 100 μg/mL) for 24 hours showed a concentration-dependent increase in the number of apoptotic cells (from 6.8±3.0% to 48.4±4.2%). Ox-LDL also caused a time-dependent (6- to 24-hour) increase in the number of apoptotic cells (Figure 2⇓). The proapoptotic effect of ox-LDL was also confirmed by DNA fragmentation on gel electrophoresis (see below).
LOX-1 Inhibition and Apoptosis in HCAECs
Pretreatment of cells with antisense LOX-1 markedly decreased the number of apoptotic cells in response to ox-LDL (P<0.01). In contrast, sense LOX-1 had no effect on the degree of apoptosis. These effects of antisense LOX-1 were confirmed by TUNEL staining (Figure 3⇓) and gel electrophoresis (Figure 4⇓).
The presence of 2 different chemical inhibitors of LOX-1, polyinosinic acid and carrageenan, in the HCAEC culture medium before the cells were exposed to ox-LDL also reduced the number of apoptotic cells in response to ox-LDL (P<0.01). These observations were confirmed by TUNEL staining as well as by DNA laddering (Figure 4⇑).
LOX-1 Inhibition and NF-κB Activation and Apoptosis in HCAECs
Cultured HCAECs in normal conditions did not show NF-κB activation. Treatment of cells with ox-LDL, on the other hand, markedly enhanced the activation of NF-κB. Pretreatment of cells with antisense LOX-1 for 48 hours before the cells were exposed to ox-LDL significantly inhibited ox-LDL–mediated activation of NF-κB (Figure 5⇓). The chemical inhibitors of LOX-1, polyinosinic acid and carrageenan, also inhibited the ox-LDL–mediated activation of NF-κB (P<0.05, Figure 5⇓).
The presence of CAPE, an inhibitor of NF-κB, in the cell culture medium before the cells were exposed to ox-LDL markedly reduced the number of apoptotic cells in response to ox-LDL (P<0.01), but alone, it had no effect on the magnitude of apoptosis (Figure 6⇓).
We have recently identified high-affinity LOX-1 in cultured HCAECs by RT-PCR, Western blot, and radioligand binding.7 20 Native LDL does not bind to this receptor. We have also demonstrated that angiotensin II enhances LOX-1 expression, uptake of ox-LDL, and injury to HCAECs.7 In the present study, we confirm that ox-LDL upregulates the expression of its own endothelial receptor, LOX-1. Additionally, we show that ox-LDL induces apoptosis of HCAECs via action of LOX-1. The critical role of LOX-1 in the genesis of apoptosis became evident in that a highly specific antisense to LOX-1 mRNA as well as 2 different chemical LOX-1 inhibitors attenuated the proapoptotic effect of ox-LDL. Last, these studies indicate that NF-κB activation is an important signal transduction mechanism in LOX-1–mediated apoptosis, because the antisense as well as the chemical inhibitors of LOX-1 prevented the ox-LDL–mediated activation of NF-κB. The NF-κB inhibitor, CAPE, also significantly attenuated the ox-LDL–induced apoptosis of HCAECs.
Regulation of LOX-1
Inflammatory stimuli, such as TNF-α and phorbol 12-myristate 13-acetate, increase LOX-1 expression in a time- and dose-dependent fashion in cultured bovine aortic endothelial cells. Upregulation of the expression of LOX-1 by TNF-α and phorbol 12-myristate 13-acetate in bovine endothelial cells is associated with an increase in the uptake of ox-LDL.4 Fluid shear stress, which activates endothelium, also upregulates LOX-1 expression by enhancing intracellular calcium mobilization.5 In an in vivo study, Nagase et al6 found that LOX-1 mRNA is markedly upregulated in the aorta and vein of hypertensive rats, which may be the basis of impaired endothelium-dependent vasodilation in these rats. In a recent study,7 we demonstrated that LOX-1 expression is upregulated by angiotensin II via activation of the Ang II type 1 receptor and that the upregulation of LOX-1 is associated with an increased uptake of ox-LDL by HCAECs and induction of cell injury. In the present study, we confirm that ox-LDL upregulates its own receptor LOX-1 mRNA and protein expression. These observations may be the basis of the increased uptake of ox-LDL and endothelial activation and dysfunction as plasma LDL levels rise.
LOX-1 and Apoptosis
Nishio and Watanabe10 have suggested that ox-LDL, but not native LDL, induces apoptosis in cultured rabbit vascular smooth muscle cells. Yang et al11 have also shown that ox-LDL induces apoptosis of human monocytes and macrophages. Recent work from our laboratory12 has shown that ox-LDL markedly augments hypoxia/reoxygenation-mediated apoptosis of cultured HCAECs. Ox-LDL decreases endogenous superoxide dismutase activity and constitutive nitric oxide synthase activity and increases free radical generation beyond that caused by hypoxia/reoxygenation. In the present study, we demonstrate that ox-LDL, per se, induces apoptosis of cultured HCAECs in a concentration- and time-dependent fashion. The importance of LOX-1 expression became obvious in our experiments in which a specific antisense to LOX-1 mRNA decreased ox-LDL–mediated apoptosis of HCAECs by 75%. In other experiments, we found that 2 different nonspecific chemical inhibitors of LOX-1, polyinosinic acid and carrageenan, also decreased ox-LDL–mediated apoptosis of HCAECs by 62% and 72%, respectively. Notably these 2 LOX-1 inhibitors prevent [125I]ox-LDL binding to LOX-1 by 60% to 90%.4 20
Apoptosis in the present study was documented by 2 independent methods, TUNEL stating and gel electrophoresis. The results of the 2 methods were similar and complementary. We have shown previously that apoptosis in HCAECs in response to ox-LDL, as measured by TUNEL staining and DNA laddering, is associated with characteristic changes in markers of apoptosis, such as bcl-2 and Fas proteins.12
LOX-1 and NF-κB Signal Transduction
The mechanism of ox-LDL–induced apoptosis continues to be discussed. Nishio and Watanabe10 have shown that oxysterols induce apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Dimmeler et al23 have shown that ox-LDL induces apoptosis of endothelial cells through activation of CPP32-like proteases. Another study24 has shown that ox-LDL increases cellular calcium to trigger apoptosis. Recent work12 from our laboratory has shown that ox-LDL changes the expression of certain genes associated with apoptosis in endothelial cells, such as bcl-2 and Fas. Concurrently, ox-LDL activates protein kinase C and protein tyrosine K, and inhibitors of protein kinase C and protein tyrosine K markedly reduce ox-LDL–mediated apoptosis.
NF-κB is an oncogene protein that regulates transcription of a variety of cellular genes, including immune and inflammatory response and growth control.13 NF-κB is present in cytosol as a heterodimer composed of NF-κB1 (P50) and Rel (P65) subunits bound to an inhibitor protein, I-κB. After activation, NF-κB translocates from the cytosol to the nucleus of the cell, binds to specific DNA sequences, and initiates transcription. Maziere et al19 have shown that ox-LDL activates NF-κB in fibroblasts and endothelial and smooth muscle cells and causes cell injury. Collins25 has suggested oxidative activation of endothelial cell transcription factors, especially NF-κB, as a mechanism for changing endothelial cell phenotype and for initiating atherosclerotic lesions. Hernan dez-Presa et al16 have also demonstrated NF-κB activation in early atherosclerosis. Other studies have shown a critical role of NF-κB activation in myocardial26 and endothelial cell17 apoptosis. Some studies have also shown NF-κB as a proapoptotic signal in human endothelial cells.27 It is possible that NF-κB activation plays different roles depending on the stimulus and conditions for cell injury. In the present study, we demonstrate that ox-LDL induces NF-κB activation. The role of LOX-1 in this process appears to be important, because antisense LOX-1 and chemical LOX-1 inhibitors significantly reduced NF-κB activation in cultured HCAECs. Importantly, we found that CAPE, an inhibitor of NF-κB,21 markedly attenuated ox-LDL–induced apoptosis of HCAECs. Natarajan et al21 demonstrated that CAPE completely blocks the activation of NF-κB induced by TNF-α but that it has no effect on other transcription factors, such as activator protein-1, Oct-1, and TFIID. Although a few studies show that CAPE also induces apoptosis in fibroblast cells,28 we did not find evidence for a proapoptotic effect of CAPE. We propose that ox-LDL via LOX-1 triggers a suicide pathway leading to apoptosis in HCAECs and that this pathway is closely linked with the activation of NF-κB. It is possible that ox-LDL–induced apoptosis may involve other signal transduction pathways, such as the activation of CPP32-like protease and protein kinase C, because the NF-κB inhibitor did not completely block the effect of ox-LDL.
In summary, ox-LDL upregulates the expression of its own receptor in HCAECs. Ox-LDL also induces apoptosis in HCAECs via LOX-1 in concert with upregulation of its receptor, and last, NF-κB activation plays an important role as a signal transduction mechanism in this process. The confirmation of these concepts was obtained with the use of a specific antisense to LOX-1 mRNA and 2 different chemical blockers of LOX-1, polyinosinic acid and carrageenan, as well as CAPE, a powerful NF-κB inhibitor. These observations may have important implications with regard to the propagation of endothelial cell injury in the presence of ox-LDL.
This study was supported by a Merit Review Award from the VA Central Office and an award from the Department of Defense, Washington, DC.
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