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* Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Rome, Italy; and
Department of Internal Medicine, University of Rome "Tor Vergata," Rome, Italy
1Correspondence: Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza," Policlinico Umberto I-00185, Rome, Italy. E-mail: francesco.violi{at}uniroma1.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: X-linked chronic granulomatous disease • ROS • gp91phox activation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Accordingly, administration of vitamin E, a well-known antioxidant, to patients undergoing carotid atherectomy demonstrated a significant inhibition of LDL uptake by human atherosclerotic plaque (2)
. Several cells, including endothelial, macrophage, and smooth muscle cells, are likely to play a role in generating oxLDL, as suggested by in vitro studies showing that, under appropriate conditions, these cells convert native LDL to a modified form that is rapidly degraded by macrophages (3)
. Cell-mediated modification of LDL is characterized by lipid peroxidation with extensive breakdown of cholesterol, phospholipids, and apolipoprotein B-100 (Apo B-100) (4)
. Among circulating cells, platelets may represent another potential stimulus for oxLDL generation. Resting and stimulated platelets (5
, 6)
produce reactive oxidant species (ROS), such as superoxide anion (O2–). This has an important role in stimulating platelet function under some conditions such as anoxia-reoxygenation, and also in favoring platelet recruitment (6
7
8)
. Further support for the idea of production of ROS by stimulated platelets has been provided by experiments showing that platelets express four subunits of NADPH oxidase (8)
, one of the most important cellular producers of O2– (9)
. The relevant role of this enzymatic pathway in generating ROS by activated platelets has been provided by experiments showing that platelets from patients with X-linked chronic granulomatous disease (X-CGD) due to hereditary deficiency of gp91phox, the central core of NADPH oxidase, had almost complete suppression of O2– (9)
. X-CGD is a rare (1:200.000 live births) primary immunodeficiency affecting the innate immunological system; X-CGD is characterized by life-threatening bacterial and fungal infections (10)
. The disease is caused by mutations in any of the four genes encoding subunits of the O2– generating NADPH oxidase, resulting in defective O2– generation and intracellular killing (11)
. Based on these data showing that platelets represent an alternative source of ROS formation, we speculated that they could help to modify LDL, and in turn determine enhanced LDL uptake by macrophages via an oxidative stress-mediated mechanism. Therefore, the first aim of the study was to assess whether, upon stimulation, platelets elicit human LDL oxidation, and in turn enhance LDL uptake by human macrophages. The second aim was to investigate the mechanism through which platelets oxidize LDL. For this purpose, we studied the oxidation of LDL using platelets taken from patients with gp91phox hereditary deficiency. Here we report for the first time that platelets modify LDL via gp91phox-mediated O2– generation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients’ description
We performed a cross-sectional study comparing LDL oxidation elicited by platelets taken from 10 patients with hypercholesterolemia (males 5, females 5, age 44 years) and 10 sex- and age-matched normocholesterolemic subjects (males 5, females 5, age 45 years). Patients and controls were recruited from the same geographic area and followed a typical Mediterranean diet. None of the patients had clinical evidence of cardiovascular disease (as shown by clinical history, physical examination or ECG), diabetes mellitus, or hypertension. Patients with hypercholesterolemia had not taken any lipid-lowering agents or antiplatelet drugs in the previous 30 days. LDL cholesterol was 191 ± 13 mg/dl in patients and 110 ± 12 mg/dl in controls.
X-CGD, an inherited disorder characterized by an absence or deficiency of phagocyte-NADPH oxidase activity, was diagnosed in two male patients (33 and 38 years of age) by demonstrating the absence or manifest deficiency of oxidase activity in stimulated neutrophils (12)
. Blood samples were taken from each patient with X-CGD on three separate occasions.
LDL modification by activated platelets
Oxidation of LDL was carried out by incubating LDL (50 µg protein/ml) at 37°C for 30 min with platelets (5x108/ml) taken from healthy subjects (HS), hypercholesterolemic patients (HC), or X-CGD patients and stimulated with collagen (6 µg/ml). Collagen was selected as agonist because it elicits much more production of ROS than other agonists, such as ADP or thrombin (13)
. To assess the role of PLA2 and cyclooxygenase upon LDL oxidation, AACOCF3 (14 µM) or ASA (100 µM), selective inhibitors of PLA2 and cyclooxygenase 1 (COX-1), respectively, were incubated for 10 min at 37°C with LDL-treated platelets before agonist stimulation.
Samples were centrifuged for 3 min at 37°C and the supernatant was treated to measure the conjugated dienes, LPC formation, vitamin E consumption, LDL electrophoretic mobility, degradation of Apo B-100, and uptake of oxLDL (see below).
Conjugated dienes
The standard oxidation assay was performed using a Perkin Elmer Lambda 4B UV/VIS spectrometer (Norwalk, CT, USA).
Measurement of the 234 nm absorption was read at intervals of 2 min for a period of 2 h, as described, and expressed as micromoles of conjugated dienes (14)
.
Phospholipid analysis
The lipid extracts by Folch et al. (15)
were separated on silica gel 60 plates (Merck) with a solvent system of toluene/diethyl ether/ethanol (105: 30: 3, v/v/v). The zones on silica gel corresponding to phospholipids (PLs) were scraped off, extracted with chloroform/methanol (2:1, v/v), and further developed by high-performance thin layer chromatography (HPTLC) with a solvent system of chloroform/methanol/acetic acid/water (50:37.5:3.5: 2, v/v/v/v).
Areas containing individual phospholipids were identified by comigration of standards and were visualized by staining with molybdenum blue reagent (16)
. Relative quantification of individual lipid classes was performed using the "NIH IMAGE 1.63" and expressed as µg/50 mg protein/ml of LDL.
Vitamin E estimation
Samples (100 µl) were supplemented with tocopheryl acetate (internal standard), deproteinized by the addition of ethanol, and extracted with hexane. Phase separation was achieved by centrifugation. The collected upper phase was evaporated and analyzed by HPLC (17)
. Vitamin E was expressed as 1 µg/50 µg protein/ml of LDL.
Agarose gel electrophoresis
Electrophoretic mobility was measured in barbital/sodium barbital, pH 8.0 on 1% agarose gel SPE (Beckman Coulter, Fullerton, CA, USA) (18)
and expressed as percentage of mobility compared with native LDL.
SDS-polyacrylamide gradient gel electrophoresis
The protein interface was removed during phospholipid extraction. The protein band was then dissolved in sample buffer containing 3% SDS, 10% glycerol, and 5% 2-mercapto-ethanol by incubating in boiling water for 3–5 min. Vertical gel electrophoresis was performed according to the method of Laemmli (19)
, using a 3–14% gradient acrylamide gel (20)
.
Cholesteryl esters content within human macrophages
Human peripheral blood mononuclear cells (monocytes and lymphocytes) were isolated from whole blood as described (21)
. To isolate monocytes, we used MS Columns according to the manufacturer’s instructions (MACS® by Miltenyi Biotech GmbH). Monocytes (2x106) (97% of purity) were cultured in DMEM containing 20% FCS for 24 h. After 24 h each dish was washed with 2 ml of DMEM without serum to remove nonadherent cells. Adherent cells received 1 ml of DMEM containing 50 µg/ml modified LDL (see above) supplemented with [1-14C] oleic acid (0.5 µCi/ml) bound to albumin (2 mg/ml) (22)
. Monocyte neutral lipids were extracted as described (15)
and separated by HPTLC on silica gel 60 plates, with hexane/diethyl ether/acetic acid (70:30:1 v/v/v) as the developing system. Quantitative analyses of the cholesteryl esters were performed by autoradiography using BIOMAX MR-1 film, quantified using "NIH IMAGE 1.63," and by scraping off the area corresponding to cholesteryl oleate and measuring the radioactivity by liquid scintillation counting. For each method, the amount of cholesteryl esters was expressed as nanomoles of cholesteryl [1-14C] oleate formed per 50 mg of total cell protein (cholesteryl [1-14C] oleate standard was 0.1 mmol).
Statistical analysis
Continuous variables are reported as means ± SD. For each gp91 phox-deficient patient, data reported represent the mean ± SD of three separate experiments. Comparisons between groups were carried out by nonparametric tests (Wilcoxon test for related samples and Mann-Whitney U test for independent samples).
A value of P < 0.05 was considered statistically significant. All analyses were carried out with Statistical Packages for the Social Sciences 13.0 software (SPSS Inc., Chicago, IL, USA).
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RESULTS |
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The rate of conjugated dienes was 17.32 µM ± 1.46 in HC and 14.38 µM ± 0.581 in HS (n=10, P<0.005) (Fig. 1
). Vitamin E consumption was 0.271 µg ± 0.057 in HC and 0.400 µg ± 0.08 in HS (n=10, P=0.001) (Fig. 2
). The content of cholesteryl esters in monocytes was 287 nmol ± 18.5 in HC and 234 nmol ± 28.49 in HS (n=10, P=0.038) (Fig. 3
).
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In vitro analysis of platelet-induced LDL oxidation
Experiments were conducted by measuring LDL oxidation induced by platelets taken from HS or X-CGD patients. Several assays were used to measure LDL oxidation: formation of conjugated dienes, consumption of vitamin E in LDL, content of cholesteryl esters in monocytes, formation of LPC, LDL electrophoretic mobility, and Apo B-100 degradation.
Conjugated dienes
As shown in Fig. 1
, we compared the rate of conjugated dienes produced by LDL treated with collagen-stimulated platelets from HS vs. LDL treated with unstimulated platelets. The results showed a maximum increase of conjugated dienes production at 30 min in LDL treated with collagen-stimulated platelets compared to LDL treated with unstimulated platelets (14.38 µM±0.581 vs. 6.27 µM±1.09, respectively, n=10, P<0.005). Incubation of LDL with collagen-stimulated platelets supplemented with AACOCF3, an inhibitor of phospholipase A2 (PLA2), resulted in a significant reduction of conjugated dienes compared to LDL treated with collagen-stimulated platelets (7.59 µM±0.681 vs. 14.38 µM±0.581, respectively, n=10, P<0.005); treatment with ASA resulted in a partial but significant reduction of conjugated dienes compared to LDL treated with collagen-stimulated platelets (12.1 µM±0.438 vs. 14.38 µM±0.581, respectively, n=10, P=0.0013) (Fig. 1)
.
Conjugated dienes were also measured in platelets from patients with X-CGD. LDL treated with collagen-stimulated platelets showed a marked reduction of conjugated dienes compared to LDL treated with collagen-stimulated platelets from HS (8.45 µM±1.43 vs. 14.38 µM±0.581, respectively, n=6, P<0.0001) (Fig. 1)
.
Loss of vitamin E in LDL
Analysis of vitamin E in LDL treated with unstimulated platelets from HS showed a weak but significant decrease of vitamin E compared with native LDL (0.74 µg±0.10 vs. 0.95 µg±0.13, respectively, n=10, P<0.005) (Fig. 2)
; a more marked decrease of vitamin E was observed in LDL treated with collagen-stimulated platelets vs. LDL incubated with unstimulated platelets (0.4 µg±0.08 vs. 0.74 µg±0.10, respectively, n=10, P<0.005) (Fig. 2)
. Preincubation of platelets with the inhibitor of PLA2 prevented vitamin E loss compared to LDL treated with collagen-stimulated platelets alone (0.70 µg/mg±0.05 vs. 0.40 µg±0.08, respectively, n=10, P<0.005) (Fig. 2)
; preincubation with ASA partially prevented vitamin E loss compared to LDL treated with collagen-stimulated platelets alone (0.46 µg/mg±0.017 vs. 0.40 µg±0.08, respectively, n=10, P=0.04) (Fig. 2)
. No change in vitamin E content was observed in LDL treated with collagen-stimulated platelets from X-CGD compared with native LDL (0.84 µg±0.04 vs. 0.95 µg±0.13, respectively, n=6, P=0.06) (Fig. 2)
.
Accumulation of cholesteryl esters in monocytes
Monocytes accumulate large amounts of cholesteryl ester when incubated with human LDL that has been modified by oxidation. This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of oxLDL (23)
.
Monocyte incubation with LDL and collagen-stimulated platelets caused a 2-fold increase in the concentration of cholesteryl 14C oleate compared to LDL incubated with unstimulated platelets (234 nmol±28.49 vs. 110 nmol±13.62, n=10, P<0.005) (Fig. 3)
. Such an increase in etherified cholesterol was significantly inhibited in the presence of the inhibitor of PLA2 (130 nmol±16.2 vs. 234 nmol±28.49, n=10, P<0.005); a partial but significant inhibition was observed with ASA (186.6 nmol±18.2 vs. 234 nmol±28.49, n=10, P=0.001) (Fig. 3)
. Conversely, a weak accumulation of cholesteryl 14C oleate was observed in monocytes incubated with LDL treated with collagen-stimulated platelets from X-CGD patients compared to monocytes incubated with LDL and collagen-stimulated platelets from HS (150.6 nmol±16.28 vs. 234 nmol±28.49, respectively, n=6, P<0.0001) (Fig. 3)
.
Identification of lysophosphatidylcholine
No difference in LPC content was observed between native LDL and LDL treated with unstimulated platelets from HS (0.277 µg±0.036 vs. 0.281 µg±0.034, respectively, n=10, P=0.8) (Fig. 4
). An enhanced formation of LPC was detected in LDL treated with collagen-stimulated platelets from HS compared to LDL treated with unstimulated platelets (5.23 µg±0.82 vs. 0.281 µg±0.034, respectively, n=10, P<0.005) (Fig. 4)
. In samples to which the inhibitor of PLA2 was added, LPC formation was reduced compared to LDL treated with collagen-stimulated platelets alone (1.45 µg±0.36 of LDL vs. 5.23±0.82 µg, respectively, n=10, P<0.005); a less marked reduction was obtained by treating samples with ASA (3.12 µg±0.42 of LDL vs. 5.23±0.82 µg, respectively, n=10, P=0.001) (Fig. 4)
. Conversely, LPC was not detected in LDL treated with collagen-stimulated platelets from X-CGD patients compared to LDL treated with collagen-stimulated platelets from HS (0.33 µg±0.05 vs. 5.23±0.82 µg, respectively, n=6, P<0.0001) (Fig. 4)
.
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LDL electrophoresis
We observed a greater mobility in LDL treated with collagen-stimulated platelets from HS compared to LDL treated with unstimulated platelets from HS (64.84±3.16 vs. 21.68±2.81, P<0.001) (Fig. 5
). This agrees with other reports (16
, 17)
showing that oxidation renders the LDL more negatively charged and increases anodic electrophoretic mobility on agarose gel. Preincubation of collagen-stimulated platelets with the inhibitor of PLA2 mitigated electrophoresis mobility of LDL (28.76±3.87 vs. 64.84±3.16, P<0.001); pretreatment with ASA only partially reduced the electrophoresis mobility of LDL (41.23±4.57 vs. 64.84±3.16, P=0.001) (Fig. 5)
.
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Conversely, LDL treated with collagen-stimulated platelets from X-CGD patients did not show changes of electrophoretic mobility (4.28±1.15) (Fig. 5)
.
SDS-polyacrylamide gradient gel electrophoresis
The ApoB-100 becomes progressively altered during oxidation; its loss of reactive amino groups and fragmentation to smaller peptides can be used as an index of oxidative modification (19)
. A partial degradation of ApoB-100 was detected in LDL treated with unstimulated platelets from HS (Fig. 6
). Conversely, in LDL treated with collagen-stimulated platelets, fragmentation of ApoB-100 was observed (Fig. 6)
. With the loss of ApoB-100, the appearance of a variety of Coomassie blue-stained bands of lower molecular weight was evident. ApoB-100 electrophoresis was also investigated in LDL treated with collagen-stimulated platelets along with an inhibitor of PLA2. These experiments demonstrated a less marked degradation of ApoB-100 compared to LDL treated with collagen-stimulated platelets alone (Fig. 6)
. Apo-B 100 degradation was only partially prevented by LDL treated with collagen-stimulated platelets to which ASA was added (Fig. 6)
.
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The electrophoresis of ApoB-100 was repeated using platelets from patients with X-CGD and showed only a partial fragmentation of ApoB-100 (Fig. 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, the vitamin E content of LDL coincubated with collagen-activated platelet was significantly lower than that found in LDL incubated with unstimulated platelets. Finally, LDL electrophoresis showed that platelet stimulation by collagen increased LDL mobility and determined degradation of ApoB-100, suggesting that LDL incubated with activated platelets underwent oxidation of protein moiety.
An earlier study (25)
proved that in other cell lines, such as smooth muscle cells, O2– was able to give formation of oxLDL. Thus, incubation of LDL with smooth muscle cells resulted in LDL modification as demonstrated by an increase in thiobarbituric acid-reactive substances (TBARS) assay content and electrophoretic mobility. Modification of LDL was prevented by superoxide dismutase, suggesting that O2– production by smooth muscle cells had a key role in LDL modification.
As previous studies have shown that, upon stimulation, platelets produce O2– and that NADPH oxidase activation has a crucial role in agonist-induced platelet O2– generation (8
, 9)
, we investigated whether O2– generated by platelets had a role in the formation of oxLDL. For this purpose, we measured the oxidation of LDL using platelets taken from patients with gp91 phox hereditary deficiency, a human knockout model characterized by almost complete suppression of platelet O2– production (9)
. The novel finding of our study was that, as opposed to normal platelets, those from patients with gp91 phox deficiency weakly modified LDL. Therefore, compared to experiments performed with normal platelets, formation of conjugated dienes was markedly reduced, LDL mobility was unaffected, and Apo B-100 underwent only partial degradation. This result suggests that O2– platelet production is crucial for LDL modification. Consistent with this finding was the result concerning the content of vitamin E. Thus, whereas the level of vitamin E dropped in LDL to which collagen-stimulated platelets from HS were added, no change in vitamin E was detected in experiments performed with platelets from patients with gp91 phox hereditary deficiency.
Taken together, these data demonstrated that upon stimulation with collagen, platelets are able to oxidize LDL via an NADPH-oxidase dependent mechanism, and suggested that the NADPH oxidase subunit gp91phox has a crucial role in LDL modification.
Further studies were performed to analyze the intracellular signaling through which agonist-induced platelet activation enhanced the formation of ROS. We previously demonstrated that, as for other cell lines (13)
, platelet O2– formation is mediated by arachidonic acid-induced NADPH oxidase activation (26)
. This also seems to occur in our experimental model, where platelets supplemented with an inhibitor of PLA2 induced less oxidation of LDL, suggesting a role for arachidonic acid pathway in stimulating NADPH oxidase and, in turn, eliciting LDL oxidation. This phenomenon would be only partially attributable to a COX-1 pathway, as ASA less markedly inhibited platelet-induced LDL oxidation than the PLA2 inhibitor. These data suggest that a COX-1-independent pathway is primarily responsible for platelet-induced LDL oxidation.
To the best of our knowledge, there is only one report showing that stimulated platelets modify LDL and enhance LDL accumulation within macrophages. Fogelman et al. (22)
demonstrated that malondialdehyde (MDA) produced by thrombin-stimulated platelets reacted with LDL, forming MDA-LDL; platelet-modified LDL was taken up by monocyte-macrophages, suggesting that platelets contribute to the accumulation of cholesterol within atherosclerotic plaques (27)
. Our paper extends these findings because it demonstrated that LDL uptake by monocytes occurred with a mechanism involving platelet O2– formation. This hypothesis was supported by experiments with platelets from X-CGD that elicit much less LDL uptake by monocytes compared with normal platelets.
A previous study showed that upon incubation with endothelial cells, LDL uptake by macrophages was also associated with phospholipids degradation. As much as 40% of the LDL PC was converted to lysophosphatydilcholine by a PLA2-like activity (28)
. Inhibition of PLA2 reduced phospholipid as well as LDL degradation, suggesting that phospholipid breakdown has a crucial role in inducing changes leading to LDL modification (28)
. In our experimental model, we showed that LDL modification mediated by collagen-stimulated platelets induced extensive hydrolysis of LDL PC to LPC. The role of phospholipid breakdown in generating LDL modification was also evident in our system where incubation of platelets with an inhibitor of PLA2 resulted in inhibition of LDL modification. Additional study is necessary to establish whether phospholipid degradation is dependent on a phospholipase associated with platelets and/or with LDL.
Atherosclerosis is characterized by an inflammatory process that is mediated by resident and nonresident cells of the arterial wall. Among the nonresident cells, the role of platelets as inflammatory cells contributing to the atherosclerotic process has attracted the attention of many researchers. Recently three independent studies focused on activation of platelets as a mechanism implicated in the formation of atherosclerotic lesion (29)
. Burger et al. (30)
demonstrated that in Apo E-deficient animals platelets contribute to atherosclerotic lesion via up-regulation of P-selectin. Massberg et al. (31)
showed that the early stage of atherosclerotic lesion is characterized by platelet adhesion to the endothelium: in particular, prolonged blockage of platelet adhesion by antibodies against GP1b alpha resulted in a decrease of atherosclerotic lesion. Finally, Huo et al. (32)
demonstrated that repeated infusions of activated platelets induced atherosclerosis in Apo E-deficient animals with a mechanism involving platelet expression of P-selectin. These data indicate that, once recruited into atherosclerotic lesion, platelets generate a variety of inflammatory molecules that contribute to lesion progression.
The present study provides further insight into the inflammatory property of platelets, as it shows that they modify LDL via a free radical-mediated mechanism and enhance LDL uptake by human macrophages. This effect could be particularly relevant in the early stage of atherosclerosis, where platelets, upon adhesion to the endothelium, could release oxygen free radicals, help to oxidize LDL present in the subintima layer, and eventually promote LDL accumulation within atherosclerotic plaque. Oxidative modification of LDL and LDL uptake by macrophages were more marked with platelets taken from hypercholesterolemic patients compared with platelets from healthy subjects. These preliminary data would indicate that in patients at risk of atherosclerotic disease, platelets could contribute to atherosclerotic progression by an oxidative stress mechanism.
This study has some limitations. Even if platelet expression of gp91phox was a prerequisite for the formation of oxLDL, we cannot exclude the possibility that other free radical-generating pathways may contribute to LDL modification. Furthermore, the intrinsic mechanism through which arachidonic acid activates platelet NADPH oxidase was not investigated. Studies are necessary to explore the other intracellular pathways involved in the activation of such enzymes.
In conclusion, we provide evidence that platelets modify LDL via an oxidative stress-mediated mechanism involving gp91phox activation, and we suggest that this may represent a novel pathway through which platelets contribute to the progression of atherosclerotic disease.
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ACKNOWLEDGMENTS |
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Received for publication July 13, 2006. Accepted for publication October 11, 2006.
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REFERENCES |
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generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J. Biol. Chem. 275,32357-32362This article has been cited by other articles:
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