Circulating CD45⁺/CD3⁺ lymphocyte-derived microparticles map lipid-rich atherosclerotic plaques in familial hypercholesterolaemia patients

Summary

Circulating microparticles (cMPs) seem to play important roles in vascular function. Beyond markers of activated cells, cMPs may have potential paracrine functions and influence atherosclerosis. Here, our objective was to characterise a) the abundance and phenotype of cMPs in stable statin-treated heterozygous familial hypercholesterolaemia (FH) patients exposed to life-long hypercholesterolaemia and b) the principal phenotype associated to lipid-rich atherosclerotic plaques in hFHpatients with significant atherosclerotic plaque burden. An age/gender/ treatment-matched group of adult-onset non-FH hypercholesterolaemic patients (n=37/group) was comparatively analysed. cMPs were characterised by flow cytometry using annexin-V and cell surface-specific antibodies. Our study shows that LLT-FH patients had higher overall cMP-numbers (p<0.005) than LLT-non-FH patients. Endothelial cellshed cMPs were also significantly higher in FH (p<0.0005). Within the leukocyte-derived cMP-subpopulations, FH-patients had significantly higher lymphocyte- and monocyte-derived cMP-numbers as well as cMPs carrying leukocyte-activation markers. Normalisation of cMPs by LDL levels did not affect cMP number or phenotype, indicating that the proinflammatory effect was derived from chronic vascular damage. Levels of AV⁺-total, CD45⁺-pan-leukocyte and CD45⁺/CD3⁺-lymphocyte-derived cMPs were significantly higher in FH-patients with subclinical lipid-rich atherosclerotic plaques than fibrous plaques. Levels of CD45⁺/CD3⁺-lymphocyte-MPs above 20,000/ml could differentiate between FH-patients with lipidic or non-lipidic plaques (area under the ROC curve of 0.803, 95%CI: 0.641–0.965, p=0.008). In summary, in this snapshot cross-sectional study cMP concentration and phenotype in FH differed markedly from non-FH hypercholesterolaemia. Patients with life-long high LDL exposure have higher endothelial activation and higher proinflammatory profile, even under current state-of-the-art LLT. cMPs carrying lymphocyte-epitopes appear as markers of lipid-rich atherosclerotic plaques in FH.

Keywords

Atherosclerosis - circulating microparticles - endothelial dysfunction - hypercholesterolaemia - inflammation

• References

• 1 Jy W, Horstman LL, Jimenez JJ. et al. Measuring circulating cell-derived micro-particles. J Thromb Haemost 2004; 2: 1842-1851.
• 2 Boulanger CM. Microparticles, vascular function and hypertension. Curr Opin Nephrol Hypertens 2010; 19: 177-180.
• 3 Berckmans RJ, Nieuwland R, Boing AN. et al. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 2001; 85: 639-646.
• 4 Diehl P, Aleker M, Helbing T. et al. Increased platelet, leukocyte and endothelial microparticles predict enhanced coagulation and vascular inflammation in pulmonary hypertension. J Thromb Thrombolysis 2010; 31: 173-179.
• 5 Koga H, Sugiyama S, Kugiyama K. et al. Elevated levels of VE-cadherin-positive endothelial microparticles in patients with type 2 diabetes mellitus and coronary artery disease. J Am Coll Cardiol 2005; 45: 1622-1630.
• 6 Tan KT, Tayebjee MH, Lim HS. et al. Clinically apparent atherosclerotic disease in diabetes is associated with an increase in platelet microparticle levels. Diabet Med 2005; 22: 1657-1662.
• 7 Amabile N, Guerin AP, Tedgui A. et al. Predictive value of circulating endothe-lial microparticles for cardiovascular mortality in end-stage renal failure: a pilot study. Nephrol Dial Transplant 2011; 27: 1873-1880.
• 8 Bulut D, Tuns H, Mugge A. CD31+/Annexin V+ microparticles in healthy offsprings of patients with coronary artery disease. Eur J Clin Invest 2009; 39: 17-22.
• 9 Chirinos JA, Heresi GA, Velasquez H. et al. Elevation of endothelial micropar-ticles, platelets, and leukocyte activation in patients with venous thromboem-bolism. J Am Coll Cardiol 2005; 45: 1467-1471.
• 10 Mallat Z, Benamer H, Hugel B. et al. Elevated levels of shed membrane micro-particles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 2000; 101: 841-843.
• 11 Sinning JM, Losch J, Walenta K. et al. Circulating CD31+/Annexin V+ micro-particles correlate with cardiovascular outcomes. Eur Heart J 2010; 32: 2034-2041.
• 12 Pirro M, Schillaci G, Paltriccia R. et al. Increased ratio of CD31+/CD42- micro-particles to endothelial progenitors as a novel marker of atherosclerosis in hy-percholesterolaemia. Arterioscler Thromb Vasc Biol 2006; 26: 2530-2535.
• 13 Chironi G, Simon A, Hugel B. et al. Circulating leukocyte-derived micropar-ticles predict subclinical atherosclerosis burden in asymptomatic subjects. Arte-rioscler Thromb Vasc Biol 2006; 26: 2775-2780.
• 14 Sarlon-Bartoli G, Bennis Y, Lacroix R. et al. Plasmatic level of leukocyte-derived microparticles is associated with unstable plaque in asymptomatic patients with high-grade carotid stenosis. J Am Coll Cardiol. 2013 epub ahead of print.
• 15 Gill PJ, Harnden A, Karpe F. Familial hypercholesterolaemia. Br Med J 2012; 344: e3228
• 16 Neefjes LA, Ten Kate GJ, Alexia R. et al. Accelerated subclinical coronary atherosclerosis in patients with familial hypercholesterolaemia. Atherosclerosis 2011; 219: 721-727.
• 17 Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352: 1685-1695.
• 18 Yuan G, Wang J, Hegele RA. Heterozygous familial hypercholesterolaemia: an underrecognized cause of early cardiovascular disease. Cmaj 2006; 174: 1124-1129.
• 19 Sjouke B, Kusters DM, Kastelein JJ. et al. Familial hypercholesterolaemia: present and future management. Curr Cardiol Rep 2011; 13: 527-536.
• 20 Caballero P, Alonso R, Rosado P. et al. Detection of subclinical atherosclerosis in familial hypercholesterolaemia using non-invasive imaging modalities. Atherosclerosis 2012; 222: 468-472.
• 21 Mata N, Alonso R, Badimon L. et al. Clinical characteristics and evaluation of LDL-cholesterol treatment of the Spanish Familial Hypercholesterolaemia Longitudinal Cohort Study (SAFEHEART). Lipids Health Dis 2011; 10: 94.
• 22 Wierzbicki AS, Humphries SE, Minhas R. Familial hypercholesterolaemia: summary of NICE guidance. Br Med J 2008; 337: a1095
• 23 Graham I, Atar D, Borch-Johnsen K. et al. European guidelines on cardiovascular disease prevention in clinical practice: executive summary: Fourth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (Constituted by representatives of nine societies and by invited experts). Eur Heart J 2007; 28: 2375-2414.
• 24 Pijlman AH, Huijgen R, Verhagen SN. et al. Evaluation of cholesterol lowering treatment of patients with familial hypercholesterolaemia: a large cross-sectional study in The Netherlands. Atherosclerosis 2010; 209: 189-194.
• 25 Gehi AK, Ali S, Na B. et al. Self-reported medication adherence and cardiovascular events in patients with stable coronary heart disease: the heart and soul study. Arch Intern Med 2007; 167: 1798-1803.
• 26 Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499-502.
• 27 Mozas P, Castillo S, Tejedor D. et al. Molecular characterisation of familial hy-percholesterolaemia in Spain: identification of 39 novel and 77 recurrent mutations in LDLR. Hum Mutat 2004; 24: 187.
• 28 Nieuwland R, Berckmans RJ, McGregor S. et al. Cellular origin and procoagu-lant properties of microparticles in meningococcal sepsis. Blood 2000; 95: 930-935.
• 29 Rank A, Liebhardt S, Zwirner J. et al. Circulating microparticles in patients with benign and malignant ovarian tumors. Anticancer Res 2012; 32: 2009-2014.
• 30 Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost 2011; 105: 396-408.
• 31 Corti R, Fuster V. Imaging of atherosclerosis: magnetic resonance imaging. Eur Heart J 2011; 32: 1709-1719b.
• 32 Vilades Medel D, Leta Petracca R, Carreras Costa F. et al. Coronary computed tomographic angiographic findings in asymptomatic patients with heterozygous familial hypercholesterolaemia and null allele low-density lipoprotein receptor mutations. Am J Cardiol 2013; 111: 955-961.
• 33 Working Group on Vascular Biology. 55th Annual Scientific and Standardisation Committee Meeting. ISTH Scientific Subcommittee Minutes; Boston, USA: 2009
• 34 Biro E, Akkerman JW, Hoek FJ. et al. The phospholipid composition and cholesterol content of platelet-derived microparticles: a comparison with platelet membrane fractions. J Thromb Haemost 2005; 3: 2754-2763.
• 35 Lacroix R, Robert S, Poncelet P. et al. Standardisation of platelet-derived micro-particle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost 2010; 8: 2571-2574.
• 36 Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC. et al. Cell-derived micro-particles generated in patients during cardiopulmonary bypass are highly pro-coagulant. Circulation 1997; 96: 3534-3541.
• 37 Hazen SL. Neutrophils, hypercholesterolaemia, and atherogenesis. Circulation 2010; 122: 1786-1788.
• 38 Dignat-George F, Boulanger CM. The many faces of endothelial microparticles. Arterioscler Thromb Vasc Biol 2011; 31: 27-33.
• 39 Alonso R, Mata P, De Andres R. et al. Sustained long-term improvement of arterial endothelial function in heterozygous familial hypercholesterolaemia patients treated with simvastatin. Atherosclerosis 2001; 157: 423-429.
• 40 Nozaki T, Sugiyama S, Koga H. et al. Significance of a multiple biomarkers strategy including endothelial dysfunction to improve risk stratification for cardiovascular events in patients at high risk for coronary heart disease. J Am Coll Cardiol 2009; 54: 601-608.
• 41 Huber J, Vales A, Mitulovic G. et al. Oxidized membrane vesicles and blebs from apoptotic cells contain biologically active oxidized phospholipids that induce monocyte-endothelial interactions. Arterioscler Thromb Vasc Biol 2002; 22: 101-107.
• 42 Hartvigsen K, Chou MY, Hansen LF. et al. The role of innate immunity in athe- rogenesis. J Lipid Res 2009; 50 Suppl S388-393.
• 43 Wang JG, Williams JC, Davis BK. et al. Monocytic microparticles activate en-dothelial cells in an IL-1beta-dependent manner. Blood 2011; 118: 2366-2374.
• 44 Leroyer AS, Isobe H, Leseche G. et al. Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 2007; 49: 772-777.
• 45 Rautou PE, Leroyer AS, Ramkhelawon B. et al. Microparticles from human atherosclerotic plaques promote endothelial ICAM-1-dependent monocyte adhesion and transendothelial migration. Circ Res 2011; 108: 335-343.
• 46 Scanu A, Molnarfi N, Brandt KJ. et al. Stimulated T cells generate micropar-ticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins. J Leukoc Biol 2008; 83: 921-927.
• 47 Martin S, Tesse A, Hugel B. et al. Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 2004; 109: 1653-1659.
• 48 Hoyer FF, Giesen MK, Nunes Franca C. et al. Monocytic microparticles promote atherogenesis by modulating inflammatory cells in mice. J Cell Mol Med 2012; 16: 2777-2788.
• 49 Becker L, Gharib SA, Irwin AD. et al. A macrophage sterol-responsive network linked to atherogenesis. Cell Metab 2010; 11: 125-135.
• 50 Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res 2010; 107: 1047-1057.
• 51 Liu ML, Reilly MP, Casasanto P. et al. Cholesterol enrichment of human mono-cyte/macrophages induces surface exposure of phosphatidylserine and the release of biologically-active tissue factor-positive microvesicles. Arterioscler Thromb Vasc Biol 2007; 27: 430-435.
• 52 Llorente-Cortes V, Otero-Vinas M, Camino-Lopez S. et al. Aggregated low-density lipoprotein uptake induces membrane tissue factor procoagulant activity and microparticle release in human vascular smooth muscle cells. Circulation 2004; 110: 452-459.
• 53 Owens 3rd AP, Passam FH, Antoniak S. et al. Monocyte tissue factor-dependent activation of coagulation in hypercholesterolaemic mice and monkeys is inhibited by simvastatin. J Clin Invest 2012; 122: 558-568.
• 54 Suades R, Padro T, Alonso R. et al. Lipid-lowering therapy with statins reduces microparticle shedding from endothelium, platelets and inflammatory cells indicating protection against cell activation. Thromb Haemost 2013; 110: 366-377.
• 55 Ridker PM, Danielson E, Fonseca FA. et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 2008; 359: 2195-2207.
• 56 Perez-Pujol S, Marker PH, Key NS. Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry A 2007; 71: 38-45.
• 57 Burger D, Schock S, Thompson CS. et al. Microparticles: biomarkers and beyond. Clin Sci 2013; 124: 423-441.
• 58 Nielsen CT, Ostergaard O, Johnsen C. et al. Distinct features of circulating microparticles and their relationship to clinical manifestations in systemic lupus erythematosus. Arthritis Rheum 2011; 63: 3067-3077.
• 59 Xu Y, Mintz GS, Tam A. et al. Prevalence, distribution, predictors, and outcomes of patients with calcified nodules in native coronary arteries: a 3-vessel intravas-cular ultrasound analysis from Providing Regional Observations to Study Predictors of Events in the Coronary Tree (PROSPECT). Circulation 2012; 126: 537-545.
• 60 Beckman JA, Ganz J, Creager MA. et al. Relationship of clinical presentation and calcification of culprit coronary artery stenoses. Arterioscler Thromb Vasc Biol 2001; 21: 1618-1622.
• 61 Suades R, Padro T, Vilahur G. et al. Circulating and platelet-derived micropar-ticles in human blood enhance thrombosis on atherosclerotic plaques. Thromb Haemost 2012; 108: 1208-1219.
• 62 Mause SF. Platelet microparticles: reinforcing the hegemony of platelets in athe-rothrombosis. Thromb Haemost 2012; 109: 5-6.
• 63 Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeutic options. Nat Med 2011; 17: 1410-1422.