Author + information
- Received June 19, 2002
- Revision received August 29, 2002
- Accepted September 26, 2002
- Published online February 5, 2003.
- Luciano Cominacini, MD*,* (, )
- Anna Fratta Pasini, MD*,
- Ulisse Garbin, MD*,
- Antonio Pastorino, BSc*,
- Anna Rigoni, MD*,
- Cristina Nava, MD*,
- Anna Davoli, BSc*,
- Vincenzo Lo Cascio, MD* and
- Tatsuya Sawamura, MD†
- ↵*Reprint requests and correspondence:
Dr. Luciano Cominacini, Dipartimento di Scienze Biomediche e Chirurgiche (Medicina D), Università di Verona, Ospedale Policlinico, 37134 Verona, Italy.
Objectives To address the potential role of the endothelial lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the thrombotic system, in this study we first examined whether platelet interaction with LOX-1 generated reactive oxygen species (ROS) and superoxide (O2·−) and then investigated the relationship between the intracellular production of O2·− and the availability of nitric oxide (NO).
Background Oxidative inactivation of NO is regarded as an important cause of its decreased biologic activity which may favor platelet-dependent arterial thrombosis.
Methods Bovine aortic endothelial cells (BAECs) and Chinese hamster ovary-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were incubated at different times with human platelets. The ROS, O2·−, and NO were measured in cells by flow cytometry.
Results The incubation of BAECs and BLOX-1-CHO cells with human platelets induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·− (p from <0.01 to <0.001). The increase in intracellular concentration of O2·− was followed by a dose-dependent reduction in basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001). The increase in O2·− and the reduction of NO were inhibited by the presence of vitamin C and anti-LOX-1 monoclonal antibody (p < 0.001).
Conclusions The results of this study show that one of the pathophysiologic consequences of platelet binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2·−.
Basic science in context
Focus on the endothelial cell response to activated platelets
Endothelial dysfunction is important in the pathogenesis of atherosclerosis and acute coronary syndromes. Multiple risk factors contribute to endothelial injury, such as dyslipidemia, platelet activation, oxidant stress, inflammation, hypertension, and diabetes. Although therapy aimed at activating factors is effective (e.g., platelet inhibition for the treatment and prevention of acute coronary syndromes), complementary approaches are needed to protect the target cell and prevent endothelial cell dysfunction. This study examines a novel mechanism for platelet-vascular endothelial interactions to induce endothelial cell damage.
Oxidized low-density lipoprotein (ox-LDL) damages endothelial cells and impairs the release of nitric oxide (NO), a key regulator of normal endothelial function. Oxidized low-density lipoprotein binds to endothelial cells by scavenger receptors, including lectin-like ox-LDL receptor-1 (LOX-1), a recently described receptor for ox-LDL. This study demonstrates that activated human platelets also target LOX-1. Similar to ox-LDL, activated platelets induce a LOX-1–mediated increase in reactive oxygen species and superoxide that decrease NO availability in endothelial cells. This dual role makes LOX-1 a novel therapeutic target for preventing endothelial dysfunction induced by ox-LDL or by activated platelets.
—Wilbur Y. W. LewUniversity of California at San DiegoVA San Diego Healthcare SystemLa Jolla, California
Nitric oxide was originally discovered as a vasodilator product of the endothelium (1–3). It is known that there is decreased NO availability in the early stages of atherosclerosis (4)and in the presence of vascular risk factors (5)which contribute to the impairment of endothelium-dependent relaxation (6,7). Nitric oxide possesses many anti-atherogenic properties (8–11). Oxidative inactivation of NO is regarded as an important cause of its decreased biologic activity (12). In this context, it has recently been shown that the binding of ox-LDL to an endothelial scavenger receptor called LOX-1 activates the transcription factor, nuclear factor kappa-B, through an increased production of reactive oxygen species (ROS) (13)and superoxide (O2·−) (14). The LOX-1 is highly expressed in vascular endothelial cells also in humans (15), and many pathologic changes—including monocytes adhesion via up-regulation of monocyte chemotactic protein-1 expression (16), and induction of apoptosis (17)—have been reported to be correlated to the activation of this receptor.
Very recently it has been demonstrated that LOX-1 is also involved in the platelet-endothelium interaction through its binding to phosphatidylserine (PS) and other negatively charged phospholipids (18,19)that are mainly exposed on the surface of platelets on activation (20). Notably, the binding of platelets to LOX-1 enhanced the release of endothelin-1 from endothelial cells, supporting the induction of endothelial dysfunction, which would in turn promote the atherogenic process (18).
To address the potential role of LOX-1 in the thrombotic system, in this study we first examined whether platelet interaction with endothelial cells generated ROS and O2·− in endothelial cells. Then we investigated the relationship between the intracellular production of ROS, and in particular of O2·−, and the intracellular concentration of NO in endothelial cells exposed to platelets.
Platelets were isolated by using the standard method of Baenziger and Majerus (21). Blood samples were obtained from healthy graduate student donors who were medication-free and non-smokers (with informed consent).
Bovine aortic endothelial cells (BAECs) were isolated and cultured as previously described (22). Chinese hamster ovary-K1 (CHO-K1) cells stably expressing bovine LOX-1 (BLOX-1-CHO) were obtained by transfecting a bovine LOX-1 expression vector, designated pBLOX-1 (1 μg), into CHO-K1 cells by the calcium phosphate transfection method as described (15). The CHO-K1 cells and BLOX-1-CHO cells were cultured as previously described (22). Cell survival was monitored according to the method of Landegren (23).
ROS and O2·− measurement
Intracellular ROS and O2·− levels were measured as described (13,14)by following the oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and hydroethidine (HE) by flow cytometry (Coulter Corporation, Hialeah, Florida). The BAECs were incubated in Dulbecco’s modified Eagle medium (Sigma, St. Louis, Missouri) containing 10% fetal bovine serum and 10 μmol/l DCFH-DA or 1μmol/l HE for 20 min. The density of washed platelets was first adjusted to 1 × 108/ml in Hepes-Tyrode’s buffer. Then different amounts of non-activated and activated platelets (ranging from 1.5 × 107/ml to 4.5 × 107/ml) were added to the BAEC medium for 5 to 30 min at 37°C in the presence of 5 mmol/l arginine and 3 μmol/l tetrahydrobiopterin to prevent O2·− formation by endothelial nitric oxide synthase (eNOS) (24). Platelets were stimulated with different amounts of thrombin (Sigma) (from 0.03 to 1 U/ml) (activated platelets). After 15 min, hirudin (Calbiochem, San Diego, California) was added to a final concentration of 1 U/ml to inactivate thrombin. To exclude any direct effect of thrombin and hirudin on ROS and O2·− generated by the cells, thrombin (1 U/ml) and hirudin (1 U/ml) were also added to the control (BAECs without addition of platelets). Finally, to exclude the interference of biologically highly active compounds secreted by platelets on ROS and O2·− in BAECs, the effects of platelet releasate (obtained by removing the supernatant after centrifugating 4.5 × 107/ml thrombin-activated platelets), and of some antagonists (preincubated for 30 min before the addition of 4.5 × 107/ml thrombin-activated platelets) such as SQ29548 (1 μmol/l), a thromboxane A2receptor antagonist (Cayman, Ann Arbor, Michigan), ketanserin (5 μmol/l), a serotonin receptor antagonist (Alexis, San Diego, California), and yohimbine (140 nmol/l), an alpha2-receptor antagonist (Sigma) were evaluated. The adenosine diphosphate (ADP) scavengers apyrase (Sigma) (1 U/ml) and phosphoenolpyruvate (PEP)-pyruvate kinase (PK) (Alexis) (PEP = 0.28 mmol/l, PK = 3 U/ml) were also preincubated for 5 min before the addition of thrombin-activated platelets (4.5 × 107/ml).
To test the response specificity, vitamin C (5 μmol/l) and LOX-1 antibody (Ab) (30 μg/ml) (15)were incubated with BAECs, CHO-K1, and BLOX-1-CHO cells under the experimental conditions specified previously. To compare with other antibodies or antagonists possibly involved in platelet–endothelium interaction, anti-CD41a (Pharmingen, San Diego, California), anti-CD40L (Pharmingen), anti-CD11a (Pharmingen), anti-CD62 (Santa Cruz, Santa Cruz, California), anti-CD31 (Santa Cruz), and anti-von Willebrand factor (Santa Cruz) antibodies, sialyl Lewis X oligosaccharide (Calbiochem), and annexin V (Pharmingen) were also preincubated with the medium.
The effect of PS (incubated as liposomes) on O2·− generation by BAECs, CHO-K1, and BLOX-1-CHO cells was also evaluated. The PS liposomes were obtained as previously described (25). To determine which oxidative systems contribute to the release of O2·− after platelet exposure, BAECs were also preincubated with L- and D-N-monomethyl arginine (L- and D-NMMA) (1 mmol/l), L- and D-N-arginine (L-and D-NAME) (1 mmol/l), aspirin (100 μmol/l), oxypurinol (200 μmol/l), rotenone (1 μmol/l), and diphenyleneiodonium (DPI) (100 μmol/l).
Intracellular NO was measured in the presence and absence of endothelial activation with bradykinin (100 nmol/l) (Sigma), as previously described (14), using the fluorescent indicator 4,5 diaminofluorescein diacetate (DAF-2 DA) in flow cytometry (Coulter Electronics GmBH). Confluent BAECs were incubated in Krebs Ringer Phosphate buffer containing 10 μmol/l DAF-2 DA for 10 min at 37°C and with 100 nmol/l bradykinin (Sigma) in the presence of 5 mmol/l arginine and 3 μmol/l tetrahydrobiopterin. The NO production was also monitored by following levels of nitrite in the supernatant of stimulated BAECs and activated platelets (4.5 × 107/ml) and thereafter in the supernatant of stimulated BAECs incubated with activated platelets. Concentrations of nitrite were determined by a fluorimetric assay (26).
Different amounts of non-activated and thrombin-activated platelets (ranging from 1.5 × 107/ml to 4.5 × 107/ml) were added to BAECs for 10 min. To exclude any direct effect of thrombin and hirudin on NO generated by the cells, thrombin (1 U/ml) and hirudin (1 U/ml) were also added to the control (BAECs without addition of platelets). Finally, to exclude the interference of biologically highly active compounds secreted by platelets on NO in BAECs, the effects of platelet releasate and of the same antagonists and ADP scavengers specified previously were evaluated.
To ascertain whether the effect of platelets on intracellular NO concentration was dependent on O2·− production and to test the response specificity, vitamin C and LOX-1 Ab (15)were also used under the experimental conditions specified earlier.
ENOS activity measurement
The effect of platelets on eNOS metabolism of 3H arginine to 3H citrulline was determined as described previously (14,27).
Statistical analysis was performed by one- or two-way analysis of variance (ANOVA) for repeated measures followed by post hoc Tukey test for multiple comparisons using the “SYSTAT” program and statistical software manual (SYSTAT Inc., Evanston, Illinois) for Macintosh. Statistical significance was inferred at p values <0.05.
In our experimental conditions, the incubation of BAECs with increasing amounts of platelets for 10 min induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·− (p from <0.01 to <0.001) (Fig. 1A). We also performed some time-course experiments of O2·− formation in BAECs incubated with platelets activated with different amounts of thrombin. By two-way ANOVA with repeated measures, there was a significant effect for the thrombin concentrations (p < 0.001) and for the times of incubation (p < 0.001), with a significant concentrations/times interaction (p < 0.001) on O2·− generation. As shown in Figure 1B, from the evaluation of the time-course of O2·− formation in BAECs using post-ANOVA Tukey test for multiple comparisons, it was clear that the effect of platelets was already present after 2 min of incubation even with submaximal doses of thrombin.
To exclude the interference of biologically highly active compounds secreted by thrombin-activated platelets on ROS and O2·− generated by BAECs, the effects of thrombin-activated platelet releasate and of specific antagonists were analyzed. The incubation of thrombin-activated platelet releasate with BAECs did not significantly affect intracellular concentration of ROS and O2·−. Similarly the preincubation of BAECs with SQ29548, ketanserin, yohimbine, and with the ADP scavengers apyrase and PEP-PK did not modify intracellular ROS and O2·− concentration induced by activated platelets in BAECs.
To test the response specificity and to verify if O2·− increase was dependent on platelets binding to LOX-1, vitamin C and LOX-1 Ab were incubated with BAECs, CHO-K1, and BLOX-1-CHO cells. As shown in Figure 2, the O2·− concentration was markedly reduced in BAECs and BLOX-1-CHO cells preincubated with vitamin C (p < 0.001) and LOX-1 Ab (p < 0.001), whereas control CHO-K1 cells were not affected. Besides LOX-1 Ab, anti-CD41a slightly inhibited the production of O2·−, and the simultaneous application of anti-LOX-1 and anti-CD41a showed an additive effect (p < 0.001). Finally, annexin V significantly reduced the production of O2·− induced by thrombin-activated platelets (p < 0.001) (Fig. 3). Annexin V also decreased the generation of O2·− induced by non-stimulated platelets (3 × 107/ml) by 14 ± 1.3% (p < 0.05).
The PS liposomes (at concentrations of 10 and 20 μmol/l total lipids/106cells) determined a dose-dependent increase in O2·− production in BAECs (mean fluorescence intensity [MFI] from 0.3 ± 0.029 to 1.5 ± 0.13 and to 2.7 ± 0.23, p < 0.001, n = 10) and BLOX-1-CHO cells (MFI from 0.24 ± 0.020 to 1.8 ± 0.17 and to 3.1 ± 0.29, p < 0.001, n = 10). The PS liposomes produced no effect on CHO-K1 cells.
Anti-CD40L, antiCD11a, anti-CD62, anti-CD31, anti-VWF, sialyl Lewis X oligosaccharide, and control immunoglobulin G (IgG) did not affect O2·− generation.
The incubation of BAECs with increasing amounts of platelets for 10 min in the presence of DAF-2 DA dose-dependently reduced basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001) (Fig. 4). Similarly, concentrations of nitrite in the supernatant of stimulated BAECs dropped from 635 ± 25 pmol/well to 211 ± 12 pmol/well (p < 0.001, n = 12) after the addition of activated platelets (4.5 × 107/ml), the contribution of activated platelets alone being 124 ± 8 pmol/4.5 × 107platelets.
The incubation of platelet releasate with BAECs did not significantly affect basal and stimulated concentration of intracellular NO and supernatant nitrite. Similarly, the preincubation of BAECs with SQ29548, ketanserin, yohimbine, and with the ADP scavengers apyrase and PEP-PK did not interfere with the modifications of basal and stimulated intracellular NO and supernatant nitrite induced by activated platelets.
To test if the reduction of intracellular NO concentration induced by platelets was dependent on O2·− generation, we preincubated BAECs with vitamin C and LOX-1 Ab. Figure 5shows that the preincubation of BAECs with vitamin C and LOX-1 Ab significantly counteracted the effect of activated platelets on basal and stimulated generation of NO (p from <0.01 to <0.001).
The effect of platelets on endothelial cell eNOS activity was examined by the 3H citrulline assay. Activated platelets (4.5 × 107/ml) did not significantly modify the ability of eNOS to metabolize L-arginine to L-citrulline (BAECs = 76.6 ± 10.4 pmol citrulline/mg protein/min, BAECs + platelets = 72 ± 9.9 pmol citrulline/mg protein/min, p = NS).
As for the oxidative system involved, we found that aspirin, L-NAME, L-NMMA, D-NAME, D-NMMA, oxypurinol, and rotenone did not modify O2·− concentration in endothelial cells after platelet incubation, whereas DPI drastically reduced the intracellular amount of O2·− (MFI from 8.20 ± 0.88 to 0.34 ± 0.04, p < 0.001, n = 10).
Lectin-like ox-LDL receptor-1 is a type II membrane protein with a C-type lectin-like structure at the C terminus (15). The expression of LOX-1 in endothelial cells in vitro and in vivo is highly regulated (22). Besides ox-LDL, LOX-1 binds aged/apoptotic cells (19)and platelets (18). The binding of platelets to LOX-1 has recently been shown to increase the release of endothelin from endothelial cells supporting the induction of endothelial dysfunction (18).
In this study, we demonstrated for the first time that the binding of platelets to LOX-1 reduces the intracellular concentration of NO in cultured BAECs. This decrease was associated with a contemporary fall of nitrite in the supernatant, indicating that the reduction in intracellular NO concentrations also reflects the extracellular NO concentration. Taken together, these results suggest a role of platelets in the selective impairment of endothelium-derived relaxation which is characteristic of the early stages of atherosclerosis (6,7).
The rapid decrease in NO induced by platelets was paralleled by a specular fast increase in ROS and O2·− formation. The increased cellular production of ROS and O2·− was prevented by preincubating BAECs with vitamin C, an antioxidant known to work as a radical scavenger and with LOX-1 Ab. Furthermore, the incubation of platelets with BLOX-1 CHO cells determined a time- and dose-dependent significant increase in ROS and O2·− formation, which once again were abolished by vitamin C and LOX-1 Ab. As for the effect of LOX-1 Ab, our results agree with those of Kakutani et al. (18), who demonstrated that the preincubation of BAECs or BLOX-1 CHO cells with LOX-1 Ab greatly reduced the binding of platelets to LOX-1. To compare with other molecules possibly involved in platelet-endothelium interaction, antibodies or antagonists for these molecules were introduced into the medium. Besides LOX-1 Ab, anti-CD41a inhibited the O2·− formation approximately 25%, indicating that glycoprotein IIb/IIIa-mediated binding is also involved in the platelet–endothelium interaction. The simultaneous addition of LOX-1 Ab and anti-CD41 antibody showed an additive effect on O2·−, indicating, as previously shown (18), that LOX-1 and glycoprotein IIb/IIIa work independently on platelet-endothelium interaction. Anti-CD40L, anti-CD11a, anti-CD62, anti-CD31, anti-VWF, sialyl Lewis X oligosaccharide, and control IgG did not affect the O2·− production induced by platelets.
As for the mechanism of platelet-endothelium interaction, the results we obtained with annexin V, a PS binding protein (28), are consistent with the hypothesis that PS may be one of the major determinants of platelet binding to LOX-1. Phosphatidylserine in fact is an efficient ligand for LOX-1 (19), and although PS binding sites have also been demonstrated in non-activated platelets (20), they are mainly exposed on the surface of activated platelets (19,20). In addition, the results of this study show that PS dose-dependently increased O2·− production both in BAECs and BLOX-1-CHO cells. Even if these results should be considered preliminary, another conclusion of this study is that PS not only may be one of the major determinants of platelet binding to LOX-1, but may also trigger a train of events leading to O2·− generation.
Taken together, these data show that the incubation of platelets with BAECs is associated with an increased intracellular production of ROS and O2·− and that the platelet ligation to LOX-1 plays a crucial role in intracellular ROS and O2·− generation. The results of this study also demonstrate that the decrease of intracellular NO concentration was prevented by preincubating BAECs with vitamin C and with LOX-1 Ab. The data support the conclusion that the incubation of platelets with BAECs is associated with a receptor-dependent, abnormally increased intracellular production of ROS, and in particular of O2·−. The NO decrease therefore may be secondary to O2·− formation, which is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (12). In our experimental conditions, platelets did not significantly alter the ability of eNOS to metabolize L-arginine to L-citrulline. Because the conversion of 3H arginine into 3H citrulline, under apparent maximum velocity conditions (14,27), is a measure of eNOS levels, the results of this study show that the binding of platelets to LOX-1 did not alter, at least quantitatively, the ability to produce NO.
As for the oxidative system involved in ROS and O2·− generation, the results of this study clearly show that only DPI, a non-selective inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (29), reduced O2·− generation in BAECs stimulated by platelets. Because specific inhibitors of cyclooxygenase, eNOS, xanthine oxidase, and mitochondrial nicotinamide adenine dinucleotide dehydrogenase did not reduce O2·− generation in BAECs stimulated by platelets, another conclusion of this study is that the increase of O2·− release after platelet exposure may be related to the increased activity of endothelial NADPH oxidase.
The decrease in intracellular NO concentration as a result of O2·− generation may also have implications in vivo. For example, the impaired endothelium-dependent vasodilation is associated with an augmented susceptibility of atherosclerotic blood vessels to the development of vasospasm in vivo (6), the inappropriate constriction leading to ischemic manifestations (30). Increased oxidative stress within the vascular wall may facilitate platelet activation (31,32). The O2·− produced by the ligation of activated platelets to LOX-1 may finally inactivate NO in a chemical reaction during which peroxynitrite is formed. Furthermore, a deficiency of NO has been shown to be associated with arterial thrombosis in animal models (33)and in individuals with acute coronary syndromes (34). The decrease in NO availability induced by the binding of platelets to LOX-1 may, therefore, in turn further increase platelet activation, induce adhesion and aggregation, and finally favor platelet-dependent arterial thrombosis.
In conclusion, the results of this study show that one of the pathophysiologic consequences of platelets binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2·−.
☆ Supported in part by grants from the ministry of Education, Culture, Sports, Science, and Technology of Japan; the ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation.
- bovine aortic endothelial cells
- BLOX-1-CHO cells
- CHO-K1 cells stably expressing bovine LOX-1
- CHO-K1 cells
- Chinese hamster ovary-K1 cells
- DAF-2 DA
- 4, 5 diaminofluorescein diacetate
- 2′,7′-dichlorofluorescin diacetate
- endothelial nitric oxide synthase
- lectin-like ox-LDL receptor-1
- LOX-1 Ab
- anti-LOX-1 monoclonal antibody
- mean fluorescence intensity
- nitric oxide
- oxidized low-density lipoprotein
- pyruvate kinase
- reactive oxygen species
- Received June 19, 2002.
- Revision received August 29, 2002.
- Accepted September 26, 2002.
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