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CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells
2003; Elsevier BV; Volume: 1; Issue: 2 Linguagem: Inglês
10.1046/j.1538-7836.2003.00065.x
ISSN1538-7933
AutoresT. Holm, J Damås, Kirsten B. Holven, Ingvild Nordøy, Frank Brosstad, Thor Ueland, T. Währe, John Kjekshus, Stig S. Frøland, Hans Geir Eiken, Nils Olav Solum, Lars Gullestad, Marit S. Nenseter, Pål Aukrust,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoSummaryCXC-chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC-chemokines in the inflammatory interactions between oxidized (ox-) low-density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without ‘traditional’ risk factors and 15 carefully matched controls. Our main findings were: (a) ox-LDL stimulated the release of the CXC-chemokines interleukin (IL)-8, ENA-78 and GRO-α from PBMC, particularly in CAD. (b) In platelets, ox-LDL induced release of ENA-78 and, when combined with SFLLRN, also of GRO-α, with significantly higher response in CAD. (c) Platelet-rich plasma, especially when costimulated with ox-LDL, enhanced the release of IL-8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL-8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox-LDL, platelets and PBMC in CAD patients involving CXC-chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients. CXC-chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC-chemokines in the inflammatory interactions between oxidized (ox-) low-density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without ‘traditional’ risk factors and 15 carefully matched controls. Our main findings were: (a) ox-LDL stimulated the release of the CXC-chemokines interleukin (IL)-8, ENA-78 and GRO-α from PBMC, particularly in CAD. (b) In platelets, ox-LDL induced release of ENA-78 and, when combined with SFLLRN, also of GRO-α, with significantly higher response in CAD. (c) Platelet-rich plasma, especially when costimulated with ox-LDL, enhanced the release of IL-8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL-8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox-LDL, platelets and PBMC in CAD patients involving CXC-chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients. An important pathogenic event in atherogenesis is increased recruitment of leukocytes into arterial subendothelium [1Ross R. Atherosclerosis – an inflammatory disease.N Engl J Med. 1999; 340: 115-26Crossref PubMed Scopus (19170) Google Scholar, 2Ross R. The pathogenesis of atheroscleosis: a perspective for the 1990s.Nature. 1993; 362: 801-9Crossref PubMed Scopus (9962) Google Scholar]. The ability of these cells to produce cytokines, proteolytic enzymes and growth factors may play a critical role in the progression of atherosclerosis and notably, oxidized low density lipoprotein (ox-LDL) may in itself endorse such an inflammatory response [3Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity.Arterioscler Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar]. Also platelets have received increasing attention as contributors to atherogenesis, not only by promoting thrombosis, but also for their role in inflammation [1Ross R. Atherosclerosis – an inflammatory disease.N Engl J Med. 1999; 340: 115-26Crossref PubMed Scopus (19170) Google Scholar]. Thus, platelets may, upon activation, release chemokines and other inflammatory mediators, contributing to an enhanced inflammatory response in coronary artery disease (CAD) [4Theilmeier G. Lenaerts T. Remacle C. Collen D. Vermylen J. Hoylaerts M.F. Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress.Blood. 1999; 94: 2725-34Crossref PubMed Google Scholar, 5Henn V. Slupsky J.R. Grafe M. Anagnostopoulos I. Forster R. Muller-Berghaus G. Kroczek R.A. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.Nature. 1998; 391: 591-4Crossref PubMed Scopus (1737) Google Scholar, 6Aukrust P. Müller F. Ueland T. Berget T. Aaser E. Brunsvig A. Solum N.O. Forfang K. Frøland S.S. Gullestad L. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes.Circulation. 1999; 100: 614-20Crossref PubMed Scopus (482) Google Scholar]. Chemokines are a family of cytokines characterized by their ability to cause directed migration of leukocytes into inflamed tissues [7Adams D.H. Lloyd A.R. Chemokines: leucocyte recruitment and activation cytokines.Lancet. 1997; 349: 490-5Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar]. Several reports suggest that chemokines [e.g. interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1] play a pathogenic role in atherosclerosis by activating and recruiting inflammatory cells into the atherosclerotic lesions [8Boisvert W.A. Curtiss L.K. Terkeltaub R.A. Interleukin-8 and its receptor CXCR2 in atherosclerosis.Immunol Res. 2000; 21: 129-37Crossref PubMed Scopus (122) Google Scholar, 9Terkeltaub R. Boisvert W.A. Curtiss L.K. Chemokines and atherosclerosis.Cur Opin Lipidol. 1998; 9: 397-405Crossref PubMed Scopus (144) Google Scholar, 10Boisvert W.A. Santiago R. Curtiss L.K. Terkeltaub R.A. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-63Crossref PubMed Scopus (436) Google Scholar]. However, except for some reports on IL-8 [3Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity.Arterioscler Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar], few studies have examining the effect of ox-LDL on CXC-chemokines, and the literature is virtually devoid of data on the effect of ox-LDL on CXC-chemokine release from platelets. Several patients with normal LDL levels develop CAD suggesting that other risk factors may be involved. One possibility is that such individuals may have an enhanced inflammatory response to LDL. Based on the pathogenic role of platelets, leukocytes and their interactions, possibly involving CXC-chemokines, we hypothesized that CAD patients could have an increased CXC-chemokine response to ox-LDL upon stimulation of platelets and peripheral blood mononuclear cells (PBMC), resulting in enhanced inflammatory interactions between these cells. Herein this hypothesis was investigated by different experimental approaches in 15 male patients with verified CAD and 15 age-matched men with normal coronary angiograms, all with serum cholesterol 70% narrowing of luminal diameter) and 15 had normal coronary angiograms (controls) (Table 1). All individuals received aspirin (160 mg once daily) and all had serum levels of total cholesterol <5.0 mmol L−1. The exclusion criteria included previous acute coronary syndromes, left ventricular ejection fraction <50%, statin treatment, significant concomitant disease, cigarette smoking or extensive use of alcohol.Table 1Clinical characteristics of patients with coronary artery disease (CAD) and control subjectsCAD (n = 15)Controls (n = 15)Age (years)57 ± 655 ± 7Body mass index (kg m−2)24.4 ± 0.723.1 ± 0.7Left ventricular ejection fraction (%)78 ± 580 ± 6Mean blood pressure (mmHg)96 ± 1.493 ± 1.9Creatinine (mmol L−1)86 ± 482 ± 4Total cholesterol (mmol L−1)4.3 ± 0.24.5 ± 0.3LDL-cholesterol (mmol L−1)2.9 ± 0.22.8 ± 0.2HDL-cholesterol (mmol L−1)1.4 ± 0.11.3 ± 0.2Triglycerides (mmol L−1)1.2 ± 0.21.3 ± 0.2Smokers (n)00Medication Asprin (n)1515 Calcium antagonist (n)32 Betablocker (n)32 Statins (n)00Values are mean ±SEM. Open table in a new tab Values are mean ±SEM. Plasma from healthy blood donors, collected in lipopolysaccharide (LPS)-free heparin, was stored in 0.6% sucrose at −80 °C. LDL was isolated by sequential ultracentrifugation for 5 h at 10 °C. The final preparations were dialyzed extensively against phosphate-buffered saline (PBS) without EDTA at 4 °C. LDL (300 µg mL−1) in PBS was oxidized in the presence of freshly prepared 5 µm CuSO4 (final concentration) at 37 °C. The oxidation was terminated by placing the tubes on ice and adding 200 µm EDTA and 40 µm butylated hydroxy toluene (final concentrations). The degree of oxidation of the lipid residues was determined by measuring formation of lipid peroxides by a commercial available kit from Kamiya Biochemical Co (Thousand Oaks, CA, USA). Oxidation of the proteins was measured as altered relative electrophoretic mobility (REM) of oxidized LDL in Paragon lipoprotein electrophoresis agarose gels (Beckman Instruments Inc.). The ox-LDL samples contained 503 ± 173 nmol lipid peroxides per mg LDL protein, and REM was 4.1 ± 0.6. LDL was stored under N2 and used within 2 weeks. PBMC, obtained from heparinized blood by Isopaque–Ficoll gradient centrifugation [11Aukrust P. Müller F. Frøland S.S. Enhanced generation of reactive oxygen species in monocytes from patients with common variable immunodeficiency.Clin Exp Immunol. 1994; 97: 232-8Crossref PubMed Scopus (32) Google Scholar], were incubated in 24-well trays (Costar; 106/mL) in medium alone [RPMI 1649 with 2 mmol L−1l-glutamine and 25 mmol L−1 HEPES buffer (Gibco, Paisley, UK) supplemented with 10% heat-inactivated autolog serum] or with stimulants [ox- or native (n-) LDL, final concentration 100 µg mL−1]. Cell-free supernatants were harvested after 20 h and stored at −80 °C. Preparation and stimulation of citrated PRP was performed as previously described [6Aukrust P. Müller F. Ueland T. Berget T. Aaser E. Brunsvig A. Solum N.O. Forfang K. Frøland S.S. Gullestad L. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes.Circulation. 1999; 100: 614-20Crossref PubMed Scopus (482) Google Scholar, 12Holme P.A. Müller F. Solum N.O. Brosstad F. Frøland S.S. Aukrust P. Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.FASEB J. 1998; 12: 79-89Crossref PubMed Scopus (143) Google Scholar]. Briefly, PRP was incubated by gently tilting for 30 min at room temperature after addition of the thrombin receptor agonist SFLLRN (final concentration 10 µm) or Tris-buffered saline (TS) only (unstimulated sample). In some experiments, ox- or n-LDL (final concentration 100 µg mL−1) were added to PRP 5 min before start of incubation with SFLLRN or TS. At baseline and after 30 min, PRP were centrifuged at 10 000 g for 10 min and platelet-free plasma (PFP) and platelet pellets (with TS) were stored separately at −80 °C. Platelet pellets were lyzed by freezing and thawing three times, and chemokine levels were analyzed in the lysates. The increase in chemokine levels (ng per 108 platelets) was expressed as the concentration in PFP (or in platelet pellets) at the end of the experiments minus the concentration at baseline. In some experiments the expression of CD63 on platelet surface was quantified by flow cytometry [12Holme P.A. Müller F. Solum N.O. Brosstad F. Frøland S.S. Aukrust P. Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.FASEB J. 1998; 12: 79-89Crossref PubMed Scopus (143) Google Scholar]. Platelet aggregation and the release of ATP from these cells were measured in PFP and PRP by a Chrono-Log platelet Aggregometer (Chrono-log Cooperation) [13Knofler R. Weissbach G. Kuhlisch E. Release of adenosine triphosphate by adenosine diphosphate in whole blood and in erythrocyte suspensions.Am J Hematol. 1997; 56: 259-65Crossref PubMed Scopus (15) Google Scholar]. PBMC, PRP and PFP from the same individuals were obtained and prepared as described above and kept at 37 °C. PBMC were resuspended in unstimulated or SFLLRN stimulated (see above) PRP or PFP (3 × 106 PBMC mL−1 plasma). After 30 min, the mixtures were transferred to 96-well trays (Costar). After culturing for 20 h, cell-free supernatants were harvested and stored at −80 °C. In some experiments ox-LDL or n-LDL was added to PRP and PFP 5 min before PBMC and platelets were coincubated. Endotoxin levels in all media, buffers and stimulants were <10 pg mL−1 (Limulus amebocyte test). ENA-78, GROα and regulated on activation normally T cell expressed and secreted (RANTES) were measured by EIAs (R & D Systems, Minneapolis, MN, USA). IL-8 was measured by an EIA obtained from CLB. PBMC pellets were stored in liquid nitrogen. Total RNA was extracted from frozen cells using RNeasy columns (Qiagen) and stored in RNA storage solution (Ambion) at −80 °C. Chemokine multiprobe (hCK5) and reagents for in vitro transcription and RPA was purchased from Pharmingen (RiboQuant). The RPA was used for the detection and quantification of IL-8 mRNA as previously described [14Damås J.K. Eiken H.G. Øie E. Bjerkeli V. Yndestad A. Ueland T. Tønnessen T. Geiran O.R. Aass H. Simonsen S. Christensen G. Frøland S.S. Attramadal H. Gullestad L. Aukrust P. Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure.Cardiovasc Res. 2000; 47: 778-87Crossref PubMed Scopus (188) Google Scholar]. The mRNA signal was normalized to the signal from the control gene GAPDH. Differences between groups were compared with Mann–Whitney rank sum test for unpaired data. In the paired situation, Wilcoxon's signed-rank test for paired data was performed. Correlations between variables were tested using Spearman's rank test. Data are given as mean ± SEM if not otherwise stated, P-values are two-sided and considered significant when <0.05. The two groups were comparable with respect to demographic, clinical and hemodynamic variables (Table 1). There were no differences in platelet and leukocyte counts or leukocyte differential counts between controls and CAD patients. In a separate experiment in PBMC from two CAD patients, we found a dose-dependent increase in IL-8, ENA-78 and GROα after ox-LDL stimulation with the highest response at a concentration of 100 µg mL−1 of ox-LDL, and this dosage was used in further experiments. As shown in Fig. 1, n-LDL and in particular ox-LDL, markedly enhanced the release of IL-8, ENA-78 and GROα from PBMC in both CAD patients (n = 15) and controls (n = 15), but notably, this inflammatory response to ox-LDL was significantly higher in CAD patients. We have shown that human platelets may release ENA-78 and GROα, but not IL-8, upon activation [15Damås J.K. Gullestad L. Ueland T. Solum N.O. Simonsen S. Frøland S.S. Aukrust P. CXC-chemokines, a new group of cytokines in congestive heart failure – possible role of platelets and monocytes.Cardiovasc Res. 2000; 45: 428-36Crossref PubMed Scopus (127) Google Scholar]. We therefore next examined the release of ENA-78 and GROα in unstimulated and SFLLRN, ox- and n-LDL stimulated platelets from 10 of the CAD patients and 10 of the controls. Several findings were revealed (Fig. 2). (a) ox-LDL induced ENA-78 release, with significantly higher levels in CAD patients. Ox-LDL had no effect on GROα, but because of very low concentrations comparing ENA-78 even after SFLLRN stimulation, it could be hard to detect any increase in GROα after ox-LDL stimulation alone by the method used (detection limit 7.0 pg mL−1). (b) ox-LDL markedly enhanced the SFLLRN-stimulated release of GROα with particularly enhancing effect in CAD patients. As for ENA-78, only a modest additive effect was seen between ox-LDL and SFLLRN. (c) The significant increase in ENA-78 after ox-LDL and in GROα after ox-LDL + SFLLRN stimulation in CAD patients comparing controls was accompanied by a more marked decrease in levels of these chemokines in platelet pellets (ENA-78: 2078 ± 248 pg per 108 platelets vs. 1469 ± 132 pg per 108 platelets, P < 0.05; GROα: 31.4 ± 2.6 pg per 108 platelets vs. 17.5 ± 1.8 pg per 108 platelets, P < 0.05; decrease in platelet pellets after stimulation, CAD patients and controls, respectively). (d) n-LDL stimulation did not induce release of chemokines from platelets. We also examined if the effect of ox-LDL on platelets was specific for the release of ENA-78 and GROα in five CAD patients and five controls. These experiments revealed (data not shown): (a) ox-LDL stimulated the release of the CC-chemokine RANTES from α-granules. (b) ox-LDL enhanced the expression of CD63, a marker of lysosome secretion, and the combined stimulation of ox-LDL and SFLLRN showed additive effects. (c) ox-LDL induced an increase in platelet aggregation and released ATP, a marker of dense body release, and the combined stimulation of ox-LDL and SFLLRN showed synergistic effects on these parameters. A similar pattern was seen in CAD and controls. It could be argued that the effect of ox-LDL on platelets is related to platelet agonists (e.g. thrombin or ADP) in the lipid preparation. However, the effect of ox-LDL on platelets was tested in four ox-LDL preparations with similar pattern in all preparations. Moreover, and most importantly, in contrast to ox-LDL, n-LDL prepared from the same blood donors, had no effect on platelet activation. We next examined if platelets activated by SFLLRN or ox-LDL or both could modulate the release of IL-8 from PBMC in 10 of the CAD patients and 10 of the controls (see methods). While PRP had no effect on IL-8 in PBMC from the control group, it markedly enhanced the release of IL-8 from these cells in CAD patients (Fig. 3). Moreover, when PBMC were incubated with ox-LDL and PFP, there was surprisingly no significant effect on IL-8 release comparing incubation with PFP alone in either CAD patients or controls (Fig. 3). In contrast, the combination of ox-LDL and PRP further enhanced the release of IL-8 comparing PRP alone with particularly enhancing effects in CAD patients (Fig. 3). Actually, in CAD patients the IL-8-inducing effect of PRP + ox-LDL was almost similar to that of PRP + SFLLRN and SFLLRN + ox-LDL suggesting that a plateau was reached. The present study suggests that ox-LDL in combination with autologous serum or PRP may enhance the release of IL-8 from PBMC, particularly in CAD patients. To examine the potential in vivo relevance of these findings, we analyzed the gene expression of IL-8 in freshly isolated PBMC from CAD patients and controls. As shown in Fig. 4, RPA documented markedly increased IL-8 mRNA expression in vivo in PBMC from CAD patients (∼20-fold increase) comparing very low levels in controls. It is well known that individuals with normal lipid profile may develop CAD, suggesting that other pathogenic mechanisms than those associated with ‘traditional’ risk factors may exist. Herein, non-smoking CAD patients with body mass index and cholesterol levels within normal limits, without the use of statins, were compared with a carefully matched control group referred to our hospital for evaluation of chest discomfort, but with normal coronary angiograms. Despite these similarities, CAD patients had increased gene expression of IL-8 in freshly isolated PBMC. Moreover, platelets from these CAD patients markedly enhanced the release of IL-8 from PBMC comparing platelets from the control group. Finally, our findings also suggest that the increased levels of CXC-chemokines in these CAD patients with no ‘traditional’ risk factors at least partly may reflect an augmented inflammatory response to ox-LDL in platelets and PBMC. We believe that the enhanced CXC-chemokine response in CAD patients may reflect important pathogenic processes. Thus, increased IL-8 expression is found in human coronary atheroma, mediating chemoattractant and mitogenic effects on T cells and smooth muscle cells [8Boisvert W.A. Curtiss L.K. Terkeltaub R.A. Interleukin-8 and its receptor CXCR2 in atherosclerosis.Immunol Res. 2000; 21: 129-37Crossref PubMed Scopus (122) Google Scholar]. Moreover, intimal macrophages with enhanced expression of the receptor for GROα, ENA-78 and IL-8 (i.e. CXCR2) have been reported in advanced murine and human atherosclerotic lesions [8Boisvert W.A. Curtiss L.K. Terkeltaub R.A. Interleukin-8 and its receptor CXCR2 in atherosclerosis.Immunol Res. 2000; 21: 129-37Crossref PubMed Scopus (122) Google Scholar, 10Boisvert W.A. Santiago R. Curtiss L.K. Terkeltaub R.A. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-63Crossref PubMed Scopus (436) Google Scholar]. Also, knock-out mice lacking CXCR2 have significantly reduced progression of atherosclerosis [10Boisvert W.A. Santiago R. Curtiss L.K. Terkeltaub R.A. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-63Crossref PubMed Scopus (436) Google Scholar] further underscoring the pathogenic role of CXC-chemokine in this disorder. Oxidative modification of LDL seems to play a key role in the lipid-mediated inflammation in atherosclerosis [1Ross R. Atherosclerosis – an inflammatory disease.N Engl J Med. 1999; 340: 115-26Crossref PubMed Scopus (19170) Google Scholar, 2Ross R. The pathogenesis of atheroscleosis: a perspective for the 1990s.Nature. 1993; 362: 801-9Crossref PubMed Scopus (9962) Google Scholar]. Thus, ox-LDL may induce an inflammatory response in various cells such as monocytes [3Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity.Arterioscler Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar, 16Janabi M. Yamashita S. Hirano K. Sakai N. Hiraoka H. Matsumoto K. Zhang Z. Nozaki S. Matsuzawa Y. Oxidized LDL-induced NF-κB activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients.Arterioscler Thromb Vasc Biol. 2000; 20: 1953-60Crossref PubMed Scopus (162) Google Scholar, 17Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Role for peroxisome proliferator-activated receptor α in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.Circ Res. 2000; 87: 516-21Crossref PubMed Scopus (271) Google Scholar, 18Sindermann J.R. Schmidt A. Breithardt G. Upregulation of the interleukin-8 system in hypercholesterolemic patients. Does inhibition of the mevalonate pathway lower interleukin-8 levels in the vessel wall?.Atherosclerosis. 2000; 150: 443-4Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar] and platelets [19Essler M. Retzer M. Bauer M. Zangl K.J. Tigyi G. Siess W. Stimulation of platelets and endothelial cells by mildly oxidized LDL proceeds through activation of lysophosphatidic acid receptors and the Rho/Rho-kinase pathway. Inhibition by lovastatin.Ann N Y Acad Sci. 2000; 905: 282-6Crossref PubMed Scopus (36) Google Scholar, 20Siess W. Zangl K.J. Essler M. Bauer M. Brandl R. Corrinth C. Bittman R. Tigyi G. Aepfelbacher M. Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions.Proc Natl Acad Sci USA. 1999; 96: 6931-6Crossref PubMed Scopus (369) Google Scholar, 21Maschberger P. Bauer M. Baumann-Siemons J. Kangl K.J. Negrescu E.V. Reininger A.J. Siess W. Mildly oxidized low density lipoprotein rapidly stimulates via activation of the lysophosphatidic acid receptor Src family and Syk tyrosine kinases and Ca2+ influx in human platelets.J Biol Chem. 2000; 275: 19159-66Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Chen M. Kakutani M. Naruko T. Ueda M. Narumiya S. Masaki T. Sawamura T. Activation-dependent surface expression of LOX-1 in human platelets.Biochem Biophys Res Commun. 2001; 282: 153-8Crossref PubMed Scopus (136) Google Scholar], and herein we show that ox-LDL also induces the release of CXC-chemokines from these cells. Several receptors for modified LDL are identified including CD36 and the lectin-like oxidized LDL receptor-1, LOX-1 [16Janabi M. Yamashita S. Hirano K. Sakai N. Hiraoka H. Matsumoto K. Zhang Z. Nozaki S. Matsuzawa Y. Oxidized LDL-induced NF-κB activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients.Arterioscler Thromb Vasc Biol. 2000; 20: 1953-60Crossref PubMed Scopus (162) Google Scholar, 17Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Role for peroxisome proliferator-activated receptor α in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.Circ Res. 2000; 87: 516-21Crossref PubMed Scopus (271) Google Scholar, 19Essler M. Retzer M. Bauer M. Zangl K.J. Tigyi G. Siess W. Stimulation of platelets and endothelial cells by mildly oxidized LDL proceeds through activation of lysophosphatidic acid receptors and the Rho/Rho-kinase pathway. Inhibition by lovastatin.Ann N Y Acad Sci. 2000; 905: 282-6Crossref PubMed Scopus (36) Google Scholar, 20Siess W. Zangl K.J. Essler M. Bauer M. Brandl R. Corrinth C. Bittman R. Tigyi G. Aepfelbacher M. Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions.Proc Natl Acad Sci USA. 1999; 96: 6931-6Crossref PubMed Scopus (369) Google Scholar, 23Li D. Mehta J.L. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells.Circulation. 2000; 101: 2889-95Crossref PubMed Scopus (374) Google Scholar], and these receptors seems also to mediate the cytokine-modifying effect of ox-LDL on PBMC [16Janabi M. Yamashita S. Hirano K. Sakai N. Hiraoka H. Matsumoto K. Zhang Z. Nozaki S. Matsuzawa Y. Oxidized LDL-induced NF-κB activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients.Arterioscler Thromb Vasc Biol. 2000; 20: 1953-60Crossref PubMed Scopus (162) Google Scholar, 23Li D. Mehta J.L. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells.Circulation. 2000; 101: 2889-95Crossref PubMed Scopus (374) Google Scholar]. Moreover, modification of the protein rather than the lipid moiety could be of importance for the ox-LDL-mediated effect on platelets involving interaction with LOX-1 and lysophosphatidic acid receptors [19Essler M. Retzer M. Bauer M. Zangl K.J. Tigyi G. Siess W. Stimulation of platelets and endothelial cells by mildly oxidized LDL proceeds through activation of lysophosphatidic acid receptors and the Rho/Rho-kinase pathway. Inhibition by lovastatin.Ann N Y Acad Sci. 2000; 905: 282-6Crossref PubMed Scopus (36) Google Scholar, 22Chen M. Kakutani M. Naruko T. Ueda M. Narumiya S. Masaki T. Sawamura T. Activation-dependent surface expression of LOX-1 in human platelets.Biochem Biophys Res Commun. 2001; 282: 153-8Crossref PubMed Scopus (136) Google Scholar, 24Volf I. Bielek E. Moeslinger T. Koller F. Koller E. Modification of protein moiety of human low density lipoprotein by hypochlorite generates strong platelet agonist.Arterioscler Thromb Vasc Biol. 2000; 20: 2011-8Crossref PubMed Scopus (13) Google Scholar]. Whatever the mechanisms, an inflammatory response to ox-LDL may be of pathogenic importance. If such a CXC-chemokine response also exists within the atherosclerotic lesions, it may contribute to further activation and recruitment of leukocytes into the vessel wall, promoting additional inflammatory interactions between ox-LDL, platelets and PBMC. Recent reports describe the induction of chemokine expression in monocytes by binding of thrombin-stimulated platelets [15Damås J.K. Gullestad L. Ueland T. Solum N.O. Simonsen S. Frøland S.S. Aukrust P. CXC-chemokines, a new group of cytokines in congestive heart failure – possible role of platelets and monocytes.Cardiovasc Res. 2000; 45: 428-36Crossref PubMed Scopus (127) Google Scholar, 25Weyrich A.S. Elstad M.R. McEver R.P. McIntyre T.M. Moore K.L. Morrissey J.H. Prescott S.M. Zimmerman G.A. Activated platelets signal chemokine synthesis by human monocytes.J Clin Invest. 1996; 97: 1525-34Crossref PubMed Scopus (540) Google Scholar], and increased leukocyte–platelet interaction is found in patients with acute myocardial infarction [26Neumann F.J. Zohlnhofer D. Fakhoury L. Ott I. Gawaz M. Schomig A. Effect of glycoprotein IIb/IIIa receptor blockade on platelet–leukocyte interaction and surface expression of the leukocyte integrin Mac-1 in acute myocardial infarction.J Am Coll Cardiol. 1999; 34: 1420-6Crossref PubMed Scopus (225) Google Scholar], possibly contributing to the inflammatory response in this disorder. Herein we show that ox-LDL-stimulated platelets may increase IL-8 production in PBMC. Moreover, we found that this inflammatory interaction between platelets and PBMC was markedly enhanced in CAD patients. In fact, while PRP alone had no stimulatory effect on IL-8 levels in PBMC from control subjects, PRP induced a pronounced increase in IL-8 in CAD patients. Finally, while ox-LDL induced IL-8 in PBMC when incubated in autologous serum or PRP, no response was seen when ox-LDL and PBMC was incubated in PFP, suggesting that platelet-derived factors, such as P-selectin, RANTES and ENA-78 [12Holme P.A. Müller F. Solum N.O. Brosstad F. Frøland S.S. Aukrust P. Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.FASEB J. 1998; 12: 79-89Crossref PubMed Scopus (143) Google Scholar, 27Johnsen C.R. Chapman S.M. Dong Z.M. Ordovas J.M. Mayadas T.N. Herz J. Hynes R.O. Schaefer E.J. Wagner D.D. Absence of P-selectin delays fatty streak formation in mice.Circulation. 2000; 99: 1037-43Google Scholar] may contribute to the ox-LDL mediated inflammatory interactions between on these cells. Although limited, e.g. few patients were studied and a high concentration of ox-LDL was used, the present study suggests that inflammatory interactions between ox-LDL, platelets and PBMC may contribute to atherogenesis in CAD patients without ‘traditional’ risk factors and possible also other CAD patients. Moreover, our findings further underscore the pathogenic role of platelets in CAD possibly involving CXC-chemokine related mechanisms. All authors have contributed significantly to the design, analysis and writing of the study. In addition TH, SSF, NOS, FB, MN, JK and PA initiated the study; TH, JK, TW and LG were responsible for recruitment and clinical characteristics; MN and KH performed the isolation and oxidation of LDL; TH, JKD, TW, IN, KH and HGE performed the immunologic analysis; TU and JKD performed the ribonuclease-protection-assay; TH, JKD, TW, NOS and FB performed the platelet analysis; TH and PA performed the statistical analysis and TH, NOS, MN, SSF and PA were main responsible for writing of the manuscript. We thank Bodil Lunden and Anne Brunsvig for excellent laboratory assistance. This work was supported by a grant from the Norwegian Health and Rehabilitation Foundation.
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