© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Aspirin inhibits ox-LDL-mediated LOX-1 expression and metalloproteinase-1 in human coronary endothelial cells
Department of Internal Medicine, Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, 4301 West Markham St., #532, Little Rock, AR 72205-7199, United States
* Corresponding author. Tel.: +1 501 296 1401; fax: +1 501 686 6180. Email address: MehtaJL{at}uams.edu
Received 8 April 2004; revised 29 June 2004; accepted 2 July 2004
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Background: Aspirin is thought to exert salutary effects in vascular disease states by inhibiting platelet aggregation. Endothelial activation, accumulation of oxidized low-density lipoprotein (ox-LDL) and intense inflammation also characterize atherosclerotic plaque in acute myocardial ischemia. Ox-LDL induces expression of lectin-like receptors (LOX-1) on endothelial cells and leads to the expression of matrix metalloproteinases (MMPs), which destabilize the atherosclerotic plaque. We hypothesized that aspirin may interfere with LOX-1 expression and subsequent MMP activation.
Methods and results: Cultured human coronary artery endothelial cells (HCAECs) were incubated with aspirin (1–5 mM), sodium salicylate (5 mM) or the cyclo-oxygenase inhibitor indomethacin (0.25 mM) before treatment with ox-LDL. Aspirin, in a dose- and time-dependent fashion, reduced ox-LDL-mediated LOX-1 expression (P<0.01). Ox-LDL also increased MMP-1 expression and activity, and treatment of HCAECs with aspirin decreased this effect (P<0.01). Ox-LDL also enhanced the activity of p38MAPK in HCAECs, and aspirin blocked this effect of ox-LDL (P<0.01). Treatment of HCAECs with salicylate, but not indomethacin, resulted in a suppression of LOX-1 expression, an effect similar to that of aspirin. Importantly, both aspirin and salicylate, but not indomethacin, decreased superoxide anion generation in ox-LDL-treated HCAECs (P<0.05).
Conclusion: These observations suggest that aspirin inhibits ox-LDL-mediated LOX-1 expression and interferes with the effects of ox-LDL in intracellular signaling (p38MAPK activation) and subsequent MMP-1 activity. These novel effects of aspirin may complement its platelet inhibitory effect in acute myocardial ischemia.
KEYWORDS Aspirin; Endothelial cells; Lectin-like ox-LDL receptor (LOX-1); MAP kinase; Metalloproteinases; Oxidation
This article is referred to in the Editorial by G. Kojda (pages 192–194) in this issue.
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Acetylsalicylic acid (aspirin) has been used in the primary and secondary prevention of cardiovascular disease for the past two decades. Aspirin decreases the evolution of vascular events by 20–25% in patients with a variety of vascular diseases [1]. This drug is now a cornerstone of therapy in patients with acute coronary syndromes. The American Heart Association/American College of Cardiology Task Force recommends immediate administration in patients with suspected acute myocardial infarction or unstable angina [2].
The pathogenesis of acute coronary syndrome is generally believed to involve rupture and/or hemorrhage into a vulnerable atherosclerotic plaque [3,4]. Following the disruption of endothelial lining, platelets adhere to subendothelial surface, and intense platelet aggregation and deposition of interspersed fibrin bands and inflammatory cells takes place [3,4].
Rupture of the atherosclerotic plaque is probably a result of intense inflammatory response, and release of collagen degrading metalloproteinases (MMPs) [4–6]. The atherosclerotic site vulnerable to rupture exhibits enhanced oxidant activity, manifested by deposition of oxidatively modified low-density lipoproteins (ox-LDL) [7] and reduced nitric oxide (NO) synthesis [4]; both these phenomena lead to some degree of vasospasm in acute coronary syndromes [3].
Aspirin is believed to be effective primarily by inhibiting platelet aggregation, but inhibition of inflammation [8], smooth muscle cell proliferation [9] and oxidative process [10,11] also contribute to aspirin's effects. Stimulation of apoptosis [12] and NO synthesis [13,14] by aspirin may also relate to its overall salutary effect.
A lectin-like receptor for ox-LDL, termed LOX-1, has recently been described in endothelial cells [15–19]. This receptor is activated by shear stress, endothelin, angiotensin II and ox-LDL. Activation of this receptor initiates intracellular signaling pathways leading to endothelial activation, dysfunction and apoptosis [18]. LOX-1 activation also stimulates MMP-1 (collagenase) synthesis and release [19] and platelet activation [15]. Recent studies in animal and human tissues indicate that LOX-1 is upregulated in atherosclerotic lesions [20,21] and is involved in the reduction in protein kinase B (PKB/Akt), a key signaling pathway in endothelial constitutive NO synthase (cNOS) expression [22].
We have recently observed that aspirin blocks the expression of LOX-1 and subsequently MMP-1 synthesis and activity. This report describes these studies.
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2. Materials and methods |
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2.1 Cell culture
The methodology for culture of human coronary artery endothelial cells (HCAECs) has been described earlier [16–19]. 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. Fourth generation 70% confluent cells with were used in this study.
2.2 Study design
HCAECs were incubated with ox-LDL (80 µg/ml) for 24 h. Other groups of cells were pretreated with different concentrations (0.5–5 mM) of aspirin (Sigma, St. Louis, MO) for 1–24 h. In parallel experiments, HCAECS were pretreated with sodium salicylate (5 mM) or the cyclo-oxygenase inhibitor indomathacin (0.25 mM). Concentrations of different reagents and the duration of incubation were chosen based on previous studies [9–16].
To explore the molecular basis of the action of LOX-1, we studied p38MAPK signaling pathway in the HCAECs. For this purpose, HCAECs were used to measure p38MAPK protein and its phosphorylation. Aliquots of HCAECs were also used for measurement of MMP-1 protein and its activity.
2.3 Preparation of lipoproteins
Native LDL and ox-LDL were prepared as described earlier [15–17]. TBARS content of ox-LDL and native LDL was 15.2±0.53 and 0.79±0.26 nmol/100 µg protein, respectively (P<0.01). Ox-LDL was extensively dialyzed against tris-saline. Ox-LDL was kept in 50 µM Tris–HCl, 0.15 M NaCl and 2 µM EDTA at pH 7.4, and was used within 10 days of preparation. The level of endotoxin was measured by the E-Toxate kit (Sigma) and found to be consistently less than 0.005 EU/ml (lowest detection limit).
2.4 RT-PCR for LOX-1 mRNA expression
The method for LOX-1 mRNA expression was same as described earlier [16]. In brief, 1.5 µl of the reverse-transcripted material of each sample of total RNA was amplified with Taq DNA polymerase (Promega, Madison, WI) using a primer pair specific to human endothelial receptor (forward primer, 5'-TTACTCTCCATGGTGGTGCC-3', reverse primer, 5'-AGCTTCTTCTGCTTGTTGCC-3'). PCR product was 193 base pairs. For PCR, 35 cycles were used at 94 °C for 40 s, 55 °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. Human β-actin was amplified as a reference for quantitation of LOX-1 mRNA. Relative intensity of bands of interest were analyzed by NSF-300G scanner (Microtek) and scan analysis software (Biosoft) and expressed as the ratio to β-actin mRNA band.
The method for LOX-1 protein measurement by Western analysis was same as described earlier [16]. The primary antibody to LOX-1 was a gift of Dr. T. Sawamura, Osaka, Japan. The second antibody was purchased from Amersham Life Science, Arlington Heights, IL.
2.5 Western analysis for MMP-1
For MMP-1 protein determination, HCAEC lysates from each experiment (40 µg per lane) were separated by SDS-PAGE, and transferred to nitrocellulose membranes. After incubation in blocking solution (4% non-fat milk, Sigma), membranes were incubated with 1:1000 dilution primary antibody to human MMP-1 (Oncogene) for overnight at 4 °C. Membranes were washed and incubated with 1:2000 dilution second antibody (Amersham) for 1 h. Relative intensities of protein bands were analyzed by Scan-gel-it software.
2.6 Collagenase (MMP-1) activity assay
Collagenase zymography was carried out according to the method described by Guarda et al. [23]. Briefly, the conditioned culture medium was collected from the dishes and 10 µl of the medium was subjected to electrophoresis in SDS polyacrylamide gel containing 0.1% gelatin under non-reducing conditions. The gels were soaked in 2.5% Triton-X100 for 60 min and then washed with water for 60 min to remove SDS. The gels were then incubated in a developing buffer containing 50 mM Tris, pH 7.4, 5 mM CaCl2, and 0.02% sodium azide for 18 h at 37 °C. After the incubation the gels were stained Coomassie blue and photographed.
2.7 Measurement of P38MAPK protein and phosphorylation
HCAEC lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, the membranes were incubated with 1:1000 dilution antibodies (Calbiochem, CA) that detect P38 component of MAPK and its phosphorylated form. Thereafter, the membrane was stripped and reprobed with the MAPK antibody [17].
2.8 Determination of superoxide anion generation in HCAECs
Superoxide anion generation in HCAECs was measured with a superoxide anion-sensitive chemiluminescent probe coelenterazine [24]. Briefly, HCAECs which were pretreated ox-LDL and vehicle or aspirin, sodium salicylate or indomethacin were suspended in Krebs–Ringer buffer (pH 7.4) containing 10 µM coelenterazine. The chemiluminescence of coelenterazine was then detected with the use of a scintillation counter (LS 7000; Beckman, Irvine, CA) in out-of-coincidence mode with a single active photomultiplier tube. The data on superoxide anion generation was expressed as counts per minute per milligram (cpm/mg) protein.
2.9 Data analysis
All data represent mean of five separately performed experiments. Data are presented as mean±S.D. Statistical significance was determined in multiple comparisons among different groups of data in which ANOVA and the F test indicated the presence of significant differences. A P value 
0.05 was considered significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1 Modulation of LOX-1 expression by aspirin
As shown earlier [10–18], ox-LDL increased LOX-1 expression (protein and mRNA). Treatment of HCAECs with aspirin (5 mM) resulted in suppression of ox-LDL-mediated increase in LOX-1 expression. The decrease in LOX-1 expression became evident at 1 h. of incubation and was maximal at 6–12 h of incubation, although the effect was still present at 24 h (Fig. 1). The suppressive effect of aspirin on ox-LDL-mediated upregulation of LOX-1 protein was seen with 0.5 mM concentration (incubation time 24 h), and was maximal with 5 mM concentration (Fig. 2A). Aspirin alone had no effect on basal LOX-1 expression. In subsequent experiments, HCAECs were exposed to 5 mM aspirin for 24 h. LOX-1 expression was also suppressed by sodium salicylate, but not by indomethacin (Fig. 2B).
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3.2 Intracellular signaling
Ox-LDL had no effect on p38MAPK expression, but it markedly increased its phosphorylation (P<0.01). In parallel experiments, aspirin decreased ox-LDL-induced p38MAPK phosphorylation without affecting its protein levels (Fig. 3A). As control, treatment of HCAECs with the specific p38MAPK inhibitor SB203580 decreased ox-LDL-induced p38MAPK activation without affecting its protein levels (Fig. 3B).
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3.3 Inhibition of MMP-1 Protein expression and activity by aspirin
Treatment of HCAECs with ox-LDL resulted in an increase in MMP-1 protein expression and activity (P<0.01 vs. control). Pretreatment of cells with aspirin decreased ox-LDL-mediated MMP-1 protein expression, as well as MMP-1 activity (both P<0.01 vs. ox-LDL alone, n=5) (Fig. 3C). Aspirin had no effect on MMP-1 protein expression/activity in the absence of ox-LDL.
The role of p38MAPK in signaling the effect of ox-LDL on MMP-1 expression was evident in experiments with the specific p38MAPK inhibitor SB203580. As shown in Fig. 3D, pretreatment of cells with SB203580 markedly reduced MMP-1 protein expression and activity.
3.4 Antioxidant effect of aspirin
Next, we examined as to which component of aspirin decreases LOX-1 expression. We measured superoxide anion generation in HCAECs, and found that ox-LDL increased superoxide anion generation. HCAECs were incubated with aspirin for 24 h. Aspirin decreased superoxide anion generation in a concentration-dependent fashion (P<0.01). Sodium salicylate, but not indomethacin, also significantly attenuated the increase in superoxide anion generation in response to ox-LDL (P<0.05 vs. ox-LDL treatment alone, n=5) (Fig. 4). In control experiments, the solvent for aspirin ethyl alcohol (0.1%) had no effect on superoxide anion generation.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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This study shows that aspirin decreases the expression of LOX-1 (RNA and protein) in HCAECs that have been exposed to ox-LDL. This effect of aspirin is qualitatively similar to that of the salicylate moiety. Previous studies [25,26] have shown that superoxide anion upregulates LOX-1 expression. It is not surprising, therefore, that both sodium salicylate and aspirin, which inhibited superoxide anion generation, also decreased LOX-1 expression. Since another cyclo-oxygenase inhibitor indomethacin did not affect either superoxide anion generation or the LOX-1 expression, it is unlikely that the effect of aspirin was mediated by inhibition of cyclo-oxygenase in the endothelial cells. These observations suggest that it is the salicylate component of aspirin that is responsible for the antioxidant effect of aspirin and subsequent inhibition of ox-LDL-induced LOX-1 expression.
In concert with previous studies [19], we found that ox-LDL increased MMP-1 protein levels and its activity. We observed that aspirin inhibited the ox-LDL-mediated increase in MMP-1 protein and activity in HCAECs. MMP-1 activity has been ascribed to the degradation of collagen in the atherosclerotic plaque [5,6]. Atherosclerotic areas, particularly those that are prone to rupture, are associated with an increased formation of ROS [27,28], modulation of enzyme systems, including NADH/NADPH oxidase [27], release of inflammatory cytokines [4,28], activation of neurohormonal factors [27,29] and a loss of constitutive NO synthase expression and activity [4,14]. Recent studies have suggested a link between ROS and MMP-1 activity [30,31]. Increased synthesis and activity of MMP-1 in the ox-LDL-rich plaque [5,6] could be the basis of weakening of the fibrous cap and rupture of atherosclerotic plaque, resulting in the syndrome of acute myocardial ischemia [4]. The beneficial effect of aspirin in acute myocardial ischemia could be related to the inhibition of MMP-1 protein and activity, as identified in the present study.
We examined the role of p38MAPK as a target for the action of aspirin in ox-LDL-mediated HCAECs since our previous studies have suggested that ox-LDL enhances p38MAPK phosphorylation [18]. Indeed, we observed that aspirin rapidly decreased p38MAPK phosphorylation in ox-LDL-treated endothelial cells (Fig. 3). Aspirin may also influence other components of MAPK, but those were not examined. The role of p38MAPK in signaling ox-LDL's effect on MMP-1 expression became evident from studies in which pretreatment of cells with the specific p38MAPK inhibitor SB203580 blocked the increase in MMP-1 expression/activity in response to ox-LDL.
Yuan et al. [32] have suggested that both aspirin and salicylate enhance phosphorylation of PKB/Akt. Wu et al. [10] have confirmed the antioxidant properties of aspirin in vascular tissues from normotensive and hypertensive rats. In rat vascular smooth muscle cells, Detmer et al. [33] showed that aspirin reduces TNF-
and IL-6 mRNA after LPS stimulation by about 50%. Recently, Voisard et al. [34] have shown that aspirin in 5 mM concentration decreases the adherence of monocytes or CD4 (+) lymphocytes to HCAECs, chemotaxis of monocytes, and proliferation of cultured human coronary artery smooth muscle cells. Aspirin and sodium salicylate similarly inhibit vascular smooth muscle cell proliferation by cell cycle arrest at the G1-S phase [9]. We have recently shown that aspirin decreases DNA and protein synthesis by upregulating P53 expression [35]. Other studies show that both aspirin and sodium salicylate enhance NO production in vascular smooth muscle and endothelial cells [13,14]. Bellosillo et al. [11] have shown that aspirin and salicylate induce apoptosis of chronic lymphocytic leukemia cells by activation of caspase by cyclo-oxygenase-independent mechanisms.
We recognize that large concentrations were required to show the unique effects of aspirin and salicylate in this study. These concentrations are often necessary to demonstrate and amplify the effect of different compounds in in vitro experiments. Almost all investigators who examined the platelet-dependent or platelet-independent effects of aspirin have used concentrations exceeding 1 mM, and often exceeding 10 mM [9–14,33–36].
Taken together, these observations indicate that aspirin has a multitude of cyclo-oxygenase-independent effects. These effects most likely reflect the effect of salicylate moiety in aspirin. Wu et al. [10] have recently shown that aspirin reduces basal superoxide generation and NAD(P)H oxidase activity in rat aortic rings. Oberle et al. [37] showed that aspirin can increase ferritin synthesis in bovine endothelial cells in a time- and concentration-dependent manner. The cytoprotective effect of aspirin in their studies was not evident when other non-steroidal anti-inflammatory agents such as indomethacin and diclofenac were used. It is possible that similar mechanisms are operative in the effects of aspirin on HCAECs. We suggest that inhibition of oxygen free radicals may attenuate several key intracellular signals, including p38MAPK, which lead to a decrease in MMP-1 protein expression and activity. Coupled with the inhibitory effect on inflammatory cytokines and redox-sensitive transcription factors, such as NF-
B [33,36], the effects of aspirin on LOX-1 expression, generation of superoxide anions, activation of p38MAPK and enhanced MMP-1 activity provide a roadmap of the salutary effect of aspirin in vascular disease states.
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Notes |
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Time for primary review 11 days
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