Arq Bras Cardiol Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia XcWq9tmtphptBBLQDHTFYfC S0066-782X2015000200011 00011 S0066-782X2015005040210 10.5935/abc.20140210 Review Article Microparticles as Potential Biomarkers of Cardiovascular Disease França Carolina Nunes 1 2 Izar Maria Cristina de Oliveira 1 Amaral Jônatas Bussador do 1 Tegani Daniela Melo 1 Fonseca Francisco Antonio Helfenstein 1 Universidade Federal de São Paulo São Paulo SP Brazil Universidade Federal de São Paulo – UNIFESP – UNISA, SP, São Paulo – Brazil Universidade de Santo Amaro São Paulo SP Brazil Universidade de Santo Amaro – UNISA, SP, São Paulo – Brazil Mailing Address: Carolina Nunes França, Rua Pedro de Toledo, 276, Vila Clementino, Postal code 04039030, São Paulo, SP- Brazil. E-mail: carolufscar24@gmail.com; cnfranca@unisa.br

Author contributions

Acquisition of data: França CN. Writing of the manuscript: França CN, Amaral JB, Tegani DM. Critical revision of the manuscript for intellectual content: Izar MCO, Fonseca FAH. Figure Confection:Amaral JB.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 02 2015 27 01 2015 104 2 169 174 10 09 2014 19 09 2014 21 10 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Primary prevention of cardiovascular disease is a choice of great relevance because of its impact on health. Some biomarkers, such as microparticles derived from different cell populations, have been considered useful in the assessment of cardiovascular disease. Microparticles are released by the membrane structures of different cell types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered physiological) and in patients with different pathologies. Many studies have suggested an association between microparticles and different pathological conditions, mainly the relationship with the development of cardiovascular diseases. Moreover, the effects of different lipid-lowering therapies have been described in regard to measurement of microparticles. The studies are still controversial regarding the levels of microparticles that can be considered pathological. In addition, the methodologies used still vary, suggesting the need for standardization of the different protocols applied, aiming at using microparticles as biomarkers in clinical practice.

 Cardiovascular Diseasesv Biomarkers, Pharmacological / analysis Cell-Derived Microparticles Atherosclerosis / prevention & control Introduction

Microparticles (MP) are defined as a population of vesicles derived from different cell types (Table 1) after activation or apoptosis, measuring from 50 nm to 1000 nm, and containing cell material, such as proteins, mRNA and lipoproteins, which are fundamental to the identification of those vesicles by use of different techniques, such as flow cytometry1. All blood cells produce MP, the greatest amount being released by platelets, platelet MP (PMP), corresponding to 70%-90% of the total amount of MP in the plasma of healthy individuals2-4.

Antigens on the surface of microparticles derived from platelets, endothelium and monocytes
Microparticles Surface antigens References
PMP CD31, CD41, CD42, CD61, CD62P, CD63 3 , 8 , 10 , 39 , 58 , 62
EMP CD31, CD51, CD54, CD62E,CD105, CD106, CD144, CD146, E-selectina, VE-caderina 8 , 9 , 23 , 34 , 36 , 39 , 42 , 43 , 58 - 61
MMP CD14, CD54 28 , 29 , 31

PMP: platelet microparticles; EMP: endothelial microparticles; MMP: monocyte microparticles 62

Until the 1990s, no biological importance had been given to MP, which were considered inert particles resulting from cell destruction or only markers of apoptosis. In 1996, however, Raposo et al.5 suggested that MP played an important role in adaptative immune response. Since then, several studies have shown the importance of MP as vectors of intracellular exchange of biological information, by use of identification, characterization and quantification of MP in several situations, such as obesity, diabetes mellitus, infarction, depression, cancer, HIV and renal failure.

As the atherosclerotic process develops, monocytes accumulate lipoproteins and change into cholesterol-rich macrophages, which undergo apoptosis, releasing a high amount of lipids to the extracellular medium, causing a vicious cycle of inflammation, oxidative stress, apoptosis of endothelial cells or endothelial erosion (Figure 1). This culminates in atherothrombotic outcomes, such as myocardial infarction or ischemic cerebral vascular accident, which result from the contact of inner plaque substances with blood, producing immediate coagulation and consequent total and sudden obstruction of the vessel6.

Representação ilustrativa de algumas interações celulares no decorrer da formação do ateroma.

Healthy individuals and those with different diseases have MP in their plasma7. Inflammatory stimuli to the release of MP include Gram-negative bacterial lipopolysaccharides (LPS) and cytokines, such as tumor necrosis factor alpha (TNF-α), interleukine 6 (IL-6) and interleukine 1 (IL-1β). After activation, the cells change their asymmetrical conformation, exposing phosphatidylserine, an aminophospholipid responsible for high procoagulant capacity8.

 Microparticles derived from different cell populations Platelet microparticles

The PMP are the most abundant in human plasma4,9. Several studies have shown the relationship between those PMP and blood coagulation10, inflammatory processes11, thrombosis and tumor progression12, as well as the interaction between leukocytes and endothelial cells13.

However, some studies have suggested that PMP have an important role in tissue regeneration, because they are strongly associated with angiogenesis14. Some authors have shown that, in vitro, PMP promote budding of aortic rings by activating the signaling pathways of PI3-kinase and Extracellular Signal- Regulated Kinase (ERK)13, in addition to promoting postischemic revascularization15.

 Endothelial microparticles

Endothelial MP (EMP) represent a smaller population of MP in plasma, but have been associated with cardiovascular disease, mainly endothelial dysfunction8. Similarly to PMP, some studies have suggested a relationship between EMP and angiogenesis16, tumor growth17 and increased oxidative stress18.

Some studies have shown the importance of EMP in the proliferation and differentiation of endothelial progenitor cells, which are essential for vascular regeneration19, indicating a possible protective function related to vascular regeneration, repair and protection20.

Mezentnev et al.21 have assessed in vitro different patterns of angiogenesis (cell division rate, capillary formation and apoptosis of endothelial cells), comparing physiological levels of EMP present in healthy individuals (between 103 and 104 EMP/mL)22,23 and pathological concentrations (present in individuals with cardiovascular disease, 105 EMP/mL).24-26 Those authors have reported that pathological levels of EMP affected all parameters associated with angiogenesis in a directly proportional manner to the concentration of EMP. Those same authors had previously shown that 105 EMP/mL impaired endothelium-dependent relaxation, which was not seen with 104 EMP/mL27.

 Monocyte microparticles

Similarly to PMP, the MP originated from monocytes, monocyte MP (MPM), can contain procoagulant substances and be related to endothelial dysfunction28 and sepsis29. The study by Wang et al.30 has shown that MPM can activate endothelial cells, because MPM contain IL-1β, which enhances the inflammatory process.

Hoyer et al.31 have assessed the role of MPM in vascular inflammation and reported that the treatment of ApoE -/- mice with MPM promoted the formation of atherosclerotic plaque in the mice and increased the accumulation of macrophages in the vascular wall. Those authors have suggested an important interaction between MPM and inflammatory cells in the atherosclerotic disease of ApoE -/- mice.

Microparticles and coronary disease

Several studies have suggested a direct relationship between the increase in MP and development of coronary disease. Augustine et al.32, assessing patients undergoing dobutamine stress echocardiography, have reported an elevation in MP derived from different cell types (platelets, erythrocytes and endothelial cells) immediately after the test followed by a rapid MP clearance from the circulation during the next hour in response to cardiac stress. Those authors have suggested that the release of MP is a protective mechanism to clear cell stress in those patients.

Sarlon-Bartoli et al.33 have measured the plasma levels of leukocyte-derived MP (LMP) in 42 individuals with carotid artery stenosis greater than 70%. They have shown that patients with unstable plaque had increased levels of the CD11bCD66b+ and CD15+ LMP, suggesting that even less frequently found subpopulations of MP in plasma, as compared to PMP, can provide important information regarding clinical studies on atherosclerotic plaque vulnerability in patients with high-grade carotid stenosis.

Morel et al.34 have assessed the levels of LMP and EMP within occluded coronary arteries of ST-segment elevation myocardial infarction patients treated with primary angioplasty and have compared them with the levels of MP in peripheral blood. Those authors have reported an increase in MP within arteries, indicating the importance of those vesicles in the development of coronary atherothrombosis.

Faille et al.35 have measured CD11b+ MP (monocyte marker) in patients with acute coronary syndrome with no ST-segment elevation on the electrocardiogram, aiming at assessing whether the quantification of those MP could contribute to the identification of patients at higher risk for a recurring cardiovascular event within one month after coronary stent implantation. A smaller amount of CD11b+ MP was found in individuals with recurring cardiovascular event as compared to that in patients with no complications, suggesting greater capture of those MP in sites of atherosclerotic lesions.

Jeanneteau et al.36 have assessed in rats and humans the role of MP in the mechanism of remote ischemic conditioning (RIC), which has been described as an infarction-related cardioprotective strategy. No differences were found in the total number of MP in the group of animals undergoing RIC as compared to the control group. After phenotypic characterization of MP, elevations in the endothelial and Annexin V+ (apoptotic) subpopulations were observed in the RIC group. Similarly, elevations in EMP and Annexin V+ MP were found in the group of individuals submitted to RIC.

Porto et al.37 have assessed the concentrations of MP in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention, and the relationship of those vesicles with microvascular obstruction (defined by multiple angiography and electrocardiography). The main finding was that the MP subpopulations assessed (PMP and EMP) showed higher levels within the coronary arteries as compared to those in aortic blood. In addition, a greater release of both MP subpopulations was observed in the impaired coronary artery than in ascending aorta, indicating local MP production. Those authors have suggested that their findings can support the hypothesis that MP act as active elements in the embolization and pathophysiology of microvascular obstruction.

Kaabi et al.38 have assessed the relationship between the levels of MP and treatment of stable coronary artery disease patients with external counterpulsation (ECP). That therapy has been considered effective and safe for patients with refractory angina pectoris. Those authors have found an increase in PMP after ECP therapy, and no difference in EMP and MPM levels.

Willians et al.11 have assessed platelet activation and depression levels in coronary artery disease patients, because depression, even mild, is an independent predictor of increased mortality after myocardial infarction. Those authors have reported that patients with moderate depression and high levels of TNF-α, IL-6 and PCR also released more PMP, indicating that a pro-inflammatory component could change platelet function in those patients.

Bernal-Mizrachi et al.39 have shown that, depending on the cell stimulus (activation or apoptosis), different surface proteins are expressed. Those authors have conducted a study analyzing two subpopulations of EMP (CD31+/CD42- and CD51+) in coronary artery disease patients and have reported that CD31+/CD42- EMP were more frequently expressed in acute events (myocardial infarction and unstable angina), and CD51+ EMP were released in similar amounts both in acute and chronic events (stable angina).

 Microparticles and diabetes mellitus

Some studies have shown higher concentrations of PMP related to diabetes mellitus. Ogata et al.40 have assessed the levels of PMP in 92 patients with diabetic retinopathy. Those authors have reported increased release of PMP in those patients as compared to that in healthy individuals, and the increase was higher the more severe the retinopathy. In atherothrombosis, MP are related to the release of cytokines by leukocytes and endothelial cells, monocyte recruitment to the atherosclerotic plaque, smooth muscle cell proliferation, angiogenesis and increased oxidative stress. In addition, MP can be signalers of cell homeostasis, promoting balance between cell stimulus, proliferation and apoptosis12.

Lumsden et al.41 have assessed patients with type 2 diabetes mellitus after acute coronary syndrome (six months prior to event), who had reduced levels of EMP and no PMP changes. Those authors have suggested that those unexpected findings, which disagree with most studies in the literature, can result from concomitant medications used by patients42-44.

Other studies have reported that the increase in the expression of adhesion molecules is associated with the activation of monocytes, which can bind to endothelial cells in vessel walls, leading to diabetic retinopathy progression. Those data suggest that measuring the levels of MPM can be a useful biomarker of diabetic retinopathy progression45.

 Microparticles in endothelial dysfunction and dyslipidemia

Leroyer et al.46 have shown that MP originated from macrophages (CD40+) can promote angiogenesis within the plaque, suggesting that these MP can determine plaque vulnerability. However, studies assessing the relationship between MP and angiogenesis have proved contraversial and inconclusive, because some MP have been reported to be other studies have reported that MP can both stimulate and able to stimulate angiogenesis (PMP, for example)13,47, while inhibit angiogenesis, depending on cell origin48-50.

Some studies have shown that individuals with metabolic syndrome have increased levels of MP as compared to those of healthy individuals, and that MP are related to endothelial dysfunction, due to decreased eNOS expression and increased release of reactive oxygen species51.

The first study to show the direct effect of MP on vascular function has been developed by Boulanger et al.52 Those authors have assessed whether the MP present in peripheral blood of patients with non-ischemic syndrome and after acute myocardial infarction would influence the endothelium-dependent response in aortic rings of rats. The MP of post-infarction individuals have been reported to reduce acetylcholine‑induced vascular relaxation (by influencing the nitric oxide pathway), suggesting that MP could contribute to the endothelial dysfunction observed after the acute event.

Diehl et al.53 have analyzed different subpopulations of MP in individuals with pulmonary hypertension, and have reported increased levels of LMP, EMP and PMP, indicating higher inflammatory and procoagulant activity, which can be related to thromboembolic complications and endothelial dysfunction in those patients.

The improvement in endothelial function promoted by calcium channel blockers has been well described in the literature. Nomura et al.54 have shown a reduction in EMP in patients with type 2 diabetes mellitus after treatment with the calcium channel blocker, nifedipine. Similar result has been reported by the same group55 with patients with type 2 diabetes mellitus and hypertension after treatment with benidipine, belonging to the same drug class. The effect of the renin-angiotensin system blocker valsartan on the levels of MPM in individuals with type 2 diabetes mellitus has also been assessed by the same group56, who has reported that the drug inhibited the release of MPM. Those results have suggested that the renin-angiotensin system blocker can contribute to the treatment of atherosclerosis.

 Microparticles and lipid-lowering therapies

New strategies that can either inhibit MP functions or provide greater clearance have been searched. Several studies have assessd the effect of different lipid-lowering strategies on the amount of MP released by different cell types57,58.

Pinheiro et al.59 have assessed the effect of the antiplatelet drug clopidogrel in association or not with rosuvastatin (40 mg) on the levels of EMP and PMP in patients with stable coronary disease on statins for at least three months. Those authors have identified an increase in the levels of PMP after suspension of rosuvastatina and maintenance of only clopidogrel for four weeks and a tendency towards greater release of EMP in those patients. They have suggested that an increase in the apoptosis of platelets occurred, and that rosuvastatin might have a protective effect on the endothelium when associated with clopidogrel. In a similar study, França et al.60 have assessed the influence of atorvastatin (80 mg) in association or not with clopidogrel in patients with stable coronary disease. Those authors have suggested higher vascular stability promoted by atorvastatin after identifying an inverse relationship between the plasma concentration of atorvastatin and the levels of PMP.

Another study61 has assessed the effect of the treatment with vitamin C for five days on the levels of MP in patients with diabetes, dyslipidemia, or at least two risk factors for post‑infarction cardiovascular disease. A reduction in the amount of EMP and PMP was observed, which has been associated with the reduction in oxidative stress caused by vitamin C.

Thus, the studies have shown that MP can be useful markers not only to assess cardiovascular disease, but also cancer10,17, sepsis22 and other illnesses. However, the MP levels considered physiological and pathological are still controversial. Although flow cytometry is considered a reference for the identification and phenotypic characterization of MP, the studies have used several methods (time of centrifugation and incubation with antibodies, different markers), which makes the comparison between publications in the literature difficult.

In conclusion, the search for new biomarkers that might be related to cardiovascular disease has increased the interest in MP derived from different cells, especially platelets. However, studies are still controversial, and further research on standardization of more sensitive techniques to obtain, characterize and quantify those vesicles is required, so that the findings can be applied to clinical practice.

Sources of Funding

There were no external funding sources for this study.

Study Association

This study is not associated with any thesis or dissertation work.

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Porto I Biasucci LM de Maria GL Leone AM Niccoli G Burzotta F et al Intracoronary microparticles and microvascular obstruction in patiens with ST elevation myocardial infarction undergoing primary percutaneous intervention Eur Heart J 2012 33 23 2928 2938 Kaabi AA, Traupe T, Stutz M, Buchs N, Heller M. Cause or effect of atherogenesis: compositional alterations of microparticles from CAD patients undergoing external counterpulsation therapy. Plos One. 2012;7(10):e46822. Kaabi AA Traupe T Stutz M Buchs N Heller M Cause or effect of atherogenesis: compositional alterations of microparticles from CAD patients undergoing external counterpulsation therapy Plos One 2012 7 10 e46822 Bernal-Mizrachi L, Jy W, Jimenez JJ, Pastor J, Mauro LM, Horstman LL, et al. High levels of circulating endothelial microparticles in patients with acute coronary syndromes. Am Heart J. 2003;145(6):962-70. Bernal-Mizrachi L Jy W Jimenez JJ Pastor J Mauro LM Horstman LL et al High levels of circulating endothelial microparticles in patients with acute coronary syndromes Am Heart J 2003 145 6 962 970 Ogata N, Imaizumi M, Nomura S, Shozu A, Arichi M, Matsuoka M, et al. Increased levels of platelet-derived microparticles in patients with diabetic retinopathy. Diab Res Clin Pract. 2005;68(3):193-201. Ogata N Imaizumi M Nomura S Shozu A Arichi M Matsuoka M et al Increased levels of platelet-derived microparticles in patients with diabetic retinopathy Diab Res Clin Pract 2005 68 3 193 201 Lumsden NG, Andrews KL, Bobadilla M, Moore XL, Sampson AK, Shaw JA, et al. Endothelial dysfunction in patients with type 2 diabetes post acute coronary syndrome. Diab Vasc Dis Res. 2013;10(4):368-74. Lumsden NG Andrews KL Bobadilla M Moore XL Sampson AK Shaw JA et al Endothelial dysfunction in patients with type 2 diabetes post acute coronary syndrome Diab Vasc Dis Res 2013 10 4 368 374 Tramontano AF, Lyubarova R, Tsiakos J, Palaia T, Deleon JR, Ragolia L. Circulating endothelial microparticles in diabetes mellitus. Mediators Inflamm. 2010;2010:250476. Tramontano AF Lyubarova R Tsiakos J Palaia T Deleon JR Ragolia L Circulating endothelial microparticles in diabetes mellitus Mediators Inflamm 2010 2010 250476 Koga H, Sugiyama S, Kugiyama K, Watanabe K, Fukushima H, Tanaka T, 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(10):1622-30. Koga H Sugiyama S Kugiyama K Watanabe K Fukushima H Tanaka T 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 10 1622 1630 Feng B, Chen Y, Luo Y, Chen M, Li X, Ni Y. Circulating level of microparticles and their correlation with arterial elasticity and endothelium-dependent dilation in patients with type 2 diabetes mellitus. Atherosclerosis. 2010;208(1):264-9. Feng B Chen Y Luo Y Chen M Li X Ni Y Circulating level of microparticles and their correlation with arterial elasticity and endothelium-dependent dilation in patients with type 2 diabetes mellitus Atherosclerosis 2010 208 1 264 269 Ogata N, Nomura S, Shouzu A, Imaizumi M, Arichi M, Matsumura M. Elevation of monocyte-derived microparticles in patients with diabetic retinopathy. Diabetes Res Clin Pract. 2006;73(3):241-8. Ogata N Nomura S Shouzu A Imaizumi M Arichi M Matsumura M Elevation of monocyte-derived microparticles in patients with diabetic retinopathy Diabetes Res Clin Pract 2006 73 3 241 248 Leroyer AS, Rautou PE, Silvestre JS, Castier Y, Leseche G, Devue C, et al. CD40 ligand- microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization. J Am Coll Cardiol. 2008;52(16):1302-11. Leroyer AS Rautou PE Silvestre JS Castier Y Leseche G Devue C et al CD40 ligand- microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization J Am Coll Cardiol 2008 52 16 1302 1311 Brill A, Elinav H, Varon D. Differential role of platelet granular mediators in angiogenesis. Cardiovasc Res. 2004;63(2):226-35. Brill A Elinav H Varon D Differential role of platelet granular mediators in angiogenesis Cardiovasc Res 2004 63 2 226 235 Mostefai HA, Agouni A, Carusio N, Mastronardi ML, Heymes C, Henrion D, et al. Phosphatidylinositol 3-kinase and xanthine oxidase regulate nitric oxide and reactive oxygen species productions by apoptotic lymphocyte microparticles in endothelial cells. J Immunol. 2008;180(7):5028-35. Mostefai HA Agouni A Carusio N Mastronardi ML Heymes C Henrion D et al Phosphatidylinositol 3-kinase and xanthine oxidase regulate nitric oxide and reactive oxygen species productions by apoptotic lymphocyte microparticles in endothelial cells J Immunol 2008 180 7 5028 5035 Agouni A, Mostefai HA, Porro C, Carusio N, Favre J, Richard V, et al. Sonic hedgehog carried by microparticles corrects endothelial injury through nitric oxide release. FASEB J. 2007;21(11):2735-41. Agouni A Mostefai HA Porro C Carusio N Favre J Richard V et al Sonic hedgehog carried by microparticles corrects endothelial injury through nitric oxide release FASEB J 2007 21 11 2735 2741 Yang C, Mwaikambo BR, Zhu T, Gagnon C, Lafleur J, Seshadri S, et al. Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGFinduced pathways. Am J Physiol Regul Integr Comp Physiol. 2008;294(2):R467-76. Yang C Mwaikambo BR Zhu T Gagnon C Lafleur J Seshadri S et al Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGFinduced pathways Am J Physiol Regul Integr Comp Physiol 2008 294 2 R467 R476 Martinez MC, Tesse A, Zobairi F, Andriantsitohaina R. Shed membrane microparticles from circulating and vascular cells in regulating vascular function. Am J Physiol Heart Circ Physiol. 2005;288(3):H1004-9. Martinez MC Tesse A Zobairi F Andriantsitohaina R Shed membrane microparticles from circulating and vascular cells in regulating vascular function Am J Physiol Heart Circ Physiol 2005 288 3 H1004 H1009 Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, et al. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation. 2001;104(22):2649-52. Boulanger CM Scoazec A Ebrahimian T Henry P Mathieu E Tedgui A et al Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction Circulation 2001 104 22 2649 2652 Diehl P, Aleker M, Helbing T, Sossong V, Germann M, Sorichter S, et al. Increased platelet, leukocyte and endothelial microparticles predict enhanced coagulation and vascular inflammation in pulmonary hypertension. J Thromb Thrombolysis. 2011;31(2):173-9. Diehl P Aleker M Helbing T Sossong V Germann M Sorichter S et al Increased platelet, leukocyte and endothelial microparticles predict enhanced coagulation and vascular inflammation in pulmonary hypertension J Thromb Thrombolysis 2011 31 2 173 179 Nomura S, Inami N, Kimura Y, Omoto S, Shouzu A, Nishikawa M, et al. Effect of nifedipine on adiponectin in hypertensive patients with type 2 diabetes mellitus. J Hum Hypertens. 2007;21(1):38-44. Nomura S Inami N Kimura Y Omoto S Shouzu A Nishikawa M et al Effect of nifedipine on adiponectin in hypertensive patients with type 2 diabetes mellitus J Hum Hypertens 2007 21 1 38 44 Nomura S, Shouzu A, Omoto S, Nishikawa M, Iwasaka T. Benidipine improves oxidized LDLdependent monocyte and endothelial dysfunction in hypertensive patients with type 2 diabetes mellitus. J Hum Hypertens. 2005;19(7):551-7. Nomura S Shouzu A Omoto S Nishikawa M Iwasaka T Benidipine improves oxidized LDLdependent monocyte and endothelial dysfunction in hypertensive patients with type 2 diabetes mellitus J Hum Hypertens 2005 19 7 551 557 Nomura S, Shouzu A, Omoto S, Nishikawa M, Fukuhara S, Iwasaka T. Effect of valsartan on monocyte/endothelial cell activation markers and adiponectin in hypertensive patients with type 2 diabetes mellitus. Thromb Res. 2006;117(4):385-92. Nomura S Shouzu A Omoto S Nishikawa M Fukuhara S Iwasaka T Effect of valsartan on monocyte/endothelial cell activation markers and adiponectin in hypertensive patients with type 2 diabetes mellitus Thromb Res 2006 117 4 385 392 Camargo LM, França CN, Izar MC, Bianco HT, Lins LS, Barbosa SP, et al. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy. Braz J Med Biol Res. 2014;47(5):432-7. Camargo LM França CN Izar MC Bianco HT Lins LS Barbosa SP et al Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy Braz J Med Biol Res 2014 47 5 432 437 Lins LC, França CN, Fonseca FA, Barbosa SP, Matos LN, Aguirre AC, et al. Effects of ezetimibe on endothelial progenitor cells and microparticles in high-risk patients. Cell Biochem Biophys. 2014;70(1):687-96. Lins LC França CN Fonseca FA Barbosa SP Matos LN Aguirre AC et al Effects of ezetimibe on endothelial progenitor cells and microparticles in high-risk patients Cell Biochem Biophys 2014 70 1 687 696 Pinheiro LF, França CN, Izar MC, Barbosa SP, Bianco HT, Kasmas SH, et al. Pharmacokinetic interactions between clopidogrel and rosuvastatin: effects on vascular protection in subjects with coronary heart disease. Int J Cardiol. 2012;158(1):125-9. Pinheiro LF França CN Izar MC Barbosa SP Bianco HT Kasmas SH et al Pharmacokinetic interactions between clopidogrel and rosuvastatin: effects on vascular protection in subjects with coronary heart disease Int J Cardiol 2012 158 1 125 129 França CN, Pinheiro LF, Izar MC, Brunialti MK, Salomão R, Bianco HT, et al. Endothelial progenitor cell mobilization and platelet microparticle release are influenced by clopidogrel plasma levels in stable coronary artery disease. Circ J. 2012;76(3):729-36. França CN Pinheiro LF Izar MC Brunialti MK Salomão R Bianco HT et al Endothelial progenitor cell mobilization and platelet microparticle release are influenced by clopidogrel plasma levels in stable coronary artery disease Circ J 2012 76 3 729 736 Morel O, Jesel L, Hugel B, Douchet MP, Zupan M, Chauvin M, et al. Protective effects of vitamin C on endothelium damage and platelet activation during myocardial infarction in patients with sustained generation of circulating microparticles. J Thromb Haemost. 2003;1(1):171-7. Morel O Jesel L Hugel B Douchet MP Zupan M Chauvin M et al Protective effects of vitamin C on endothelium damage and platelet activation during myocardial infarction in patients with sustained generation of circulating microparticles J Thromb Haemost 2003 1 1 171 177 Montoro-Garcia S, Shantsila E, Hernández-Romero D, Jover E, Valdés M, Marín F, et al. Small-size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P-selectin. Br J Haematol. 2014;166(4):571-80. Montoro-Garcia S Shantsila E Hernández-Romero D Jover E Valdés M Marín F et al Small-size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P-selectin Br J Haematol 2014 166 4 571 580 Artigo de Revisão Micropartículas como Possíveis Biomarcadores da Doença Cardiovascular França Carolina Nunes 1 2 Izar Maria Cristina de Oliveira 1 Amaral Jônatas Bussador do 1 Tegani Daniela Melo 1 Fonseca Francisco Antonio Helfenstein 1 Universidade Federal de São Paulo São Paulo SP Brasil Universidade Federal de São Paulo – UNIFESP – UNISA, SP, São Paulo – Brasil Universidade de Santo Amaro São Paulo SP Brasil Universidade de Santo Amaro – UNISA, SP, São Paulo – Brasil Correspondência: Carolina Nunes França, Rua Pedro de Toledo, 276, Vila Clementino, CEP 04039030, São Paulo, SP- Brasil. E-mail: carolufscar24@gmail.com; cnfranca@unisa.br

Contribuição dos autores

Obtenção de dados: França CN. Redação do manuscrito: França CN, Amaral JB, Tegani DM. Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Izar MCO, Fonseca FAH. Confecção de figura: Amaral JB.

Potencial conflito de interesse

Declaro não haver conflito de interesses pertinentes.

A prevenção primária da doença cardiovascular constitui uma opção de grande relevância pelos seus impactos na saúde. Alguns biomarcadores têm sido considerados úteis na avaliação da doença cardiovascular, dentre eles micropartículas originadas de diferentes populações de células. Micropartículas são estruturas liberadas pela membrana de diferentes tipos celulares após ativação ou apoptose, presentes tanto no plasma de indivíduos saudáveis (níveis considerados fisiológicos) quanto em portadores de diferentes doenças. Muitos estudos têm sugerido uma associação entre micropartículas e diferentes condições patológicas, destacando-se a relação com o desenvolvimento das doenças cardiovasculares. Além disso, têm sido descritos os efeitos de diferentes terapias hipolipemiantes na mensuração de micropartículas. Os estudos ainda são controversos quanto aos níveis de micropartículas que possam ser considerados patológicos, e os métodos utilizados ainda são variados, o que sugere a necessidade da padronização dos diferentes protocolos utilizados, visando à utilização de micropartículas como biomarcadores úteis na prática clínica.

 Doenças cardiovasculares Biomarcadores Farmacológicos / análise Micropartículas Derivadas de Células Aterosclerose / prevenção & controle Introdução

As micropartículas (MP) são definidas como uma população de vesículas originadas de diferentes tipos celulares (Tabela 1) após ativação ou apoptose, possuindo um tamanho entre 50 nm e 1000 nm e contendo material celular, como proteínas, mRNA e lipoproteínas, que são fundamentais para a identificação dessas vesículas por diferentes técnicas, como citometria de fluxo1. Todas as células sanguíneas produzem MP, e as plaquetas liberam a maior quantidade de MP plaquetárias (MPP), correspondendo a 70%-90% do total de MP no plasma de indivíduos saudáveis2-4.

Antígenos presentes na superfície de micropartículas plaquetárias, endoteliais e monocíticas
Micropartículas Antígenos de superfície Referências
MPP CD31, CD41, CD42, CD61, CD62P, CD63 3 , 8 , 10 , 39 , 58 , 62
MPE CD31, CD51, CD54, CD62E,CD105, CD106, CD144, CD146, E-selectina, VE-caderina 8 , 9 , 23 , 34 , 36 , 39 , 42 , 43 , 58 - 61
MPM CD14, CD54 28 , 29 , 31

MPP: micropartículas plaquetárias; MPE: micropartículas endoteliais; MPM: micropartículas monocíticas62

Até a década de 1990, nenhuma importância biológica foi dada às MP, e elas eram consideradas como partículas inertes resultantes de destruição celular ou apenas marcadores de apoptose. Porém, em 1996, Raposo e col.5 sugeriram a importância das MP na resposta imune adaptativa. A partir disso, vários trabalhos têm mostrado a importância dessas vesículas como vetores de troca intracelular de informação biológica, por meio de identificação, caracterização e quantificação das MP em várias situações, como obesidade, diabetes, infarto, depressão, câncer, HIV e insuficiência renal.

Com o desenvolvimento do processo aterosclerótico, os monócitos passam a acumular lipoproteínas, transformam-se em macrófagos ricos em colesterol, que sofrem apoptose, liberando alta quantidade de lípides no meio extracelular, ocasionando um ciclo vicioso de inflamação, estresse oxidativo, apoptose de células endoteliais ou erosão endotelial (Figura 1), culminando com os desfechos aterotrombóticos, como infarto do miocárdio ou acidente vascular isquêmico, que ocorrem em razão do contato das substâncias do interior da placa com o sangue, produzindo imediata coagulação e, consequentemente, obstrução total e súbita do vaso6.

Representação ilustrativa de algumas interações celulares no decorrer da formação do ateroma.

Tanto indivíduos saudáveis quanto portadores de diferentes enfermidades possuem MP no plasma7. Estímulos inflamatórios para a liberação incluem lipopolissacarídeo (LPS) de bactérias Gram-negativas e citocinas, como fator de necrose tumoral alfa (TNF-α), interleucina 6 (IL-6) e interleucina 1 (IL-1β). Após ativação, as células mudam sua conformação assimétrica, expondo a fosfatidilserina, um aminofosfolipídeo responsável por alta capacidade pró-coagulante8.

 Micropartículas de diferentes populações celulares Micropartículas plaquetárias

As MPP são as mais abundantes no plasma humano4,9. Há vários trabalhos mostrando a relação entre essas MP e coagulação sanguínea10, processos inflamatórios11, trombose e progressão de tumores12 e interação entre leucócitos e células endoteliais13.

Por outro lado, estudos sugerem que MPP podem ter importante relação na regeneração tecidual, por estarem fortemente associadas à angiogênese14. Alguns autores mostraram que as MPP in vitro promovem o brotamento de anéis aórticos por meio da ativação das vias de sinalização da PI3-cinase e Extracellular Signal-Regulated Kinase (ERK)13 e promovem a neovascularização pós-isquemia15.

 Micropartículas endoteliais

As MP endoteliais (MPE) representam uma menor população de MP presentes no plasma, porém têm sido associadas à doença cardiovascular, especialmente à disfunção endotelial8. Assim como ocorre com as MPP, estudos sugerem haver relação entre MPE e angiogênese16, crescimento tumoral17 e aumento de estresse oxidativo18.

Há trabalhos mostrando a importância das MPE na proliferação e diferenciação de células progenitoras endoteliais, que são fundamentais na regeneração vascular19, indicando uma possível função protetora relacionada a regeneração, reparo e proteção vascular20.

Mezentnev e col.21 avaliaram in vitro diferentes padrões de angiogênese (razão de divisão celular, formação de capilares e apoptose de células endoteliais), comparando níveis fisiológicos de MPE presentes em indivíduos saudáveis (entre 103 e 104 MPE/mL, conforme literatura)22,23 e concentrações patológicas (presentes em portadores de doença cardiovascular, 105 MPE/mL)24-26. Observou-se que níveis patológicos de MPE afetaram todos os parâmetros associados à angiogênese de maneira diretamente proporcional à concentração de MPE. Os mesmos autores haviam demonstrado anos antes que 105 MPE/mL prejudicaram o relaxamento dependente do endotélio, o que não foi visto com 104 MPE/mL27.

 Micropartículas monocíticas

Assim como as MPP, MP originadas de monócitos (MPM) podem conter substâncias pró-coagulantes e estar relacionadas à disfunção endotelial28 e à sepse29. Estudo desenvolvido por Wang e cols.30 mostrou que MPM são capazes de ativar células endoteliais, por conterem IL-1β que aumenta o processo inflamatório.

Hoyer e cols.31 avaliaram o papel das MPM na inflamação vascular e encontraram que o tratamento de camundongos ApoE -/- com MPM promoveu a formação de placa aterosclerótica nos animais e aumentou o acúmulo de macrófagos na parede vascular. Os autores sugeriram uma importante interação entre MPM e células inflamatórias na doença aterosclerótica nos camundongos ApoE -/-.

 Micropartículas e doença coronariana

Muitos estudos sugerem uma relação direta entre aumento de MP e desenvolvimento de doença coronariana. Augustine e cols.32 avaliaram pacientes expostos a estresse cardíaco durante ecocardiografia com dobutamina e encontraram aumento de MP de diferentes tipos celulares (plaquetas, hemácias e células endoteliais) seguido por rápido clearance em resposta ao estresse cardíaco. Os autores sugeriram que a liberação de MP seria um mecanismo protetor para remover o estresse celular nesses pacientes.

Sarlon-Bartoli e cols.33 mensuraram níveis de MP de origem leucocitária (MPL) no plasma de 42 indivíduos que apresentaram estenose da artéria carótida acima de 70%. Eles mostraram que pacientes com placa instável tiveram aumento nos níveis de MPL CD11bCD66b+ e CD15+, sugerindo que mesmo subpopulações de MP menos frequentes no plasma (em comparação às MPP) podem agregar importantes informações no que concerne aos estudos clínicos analisando vulnerabilidade da placa aterosclerótica em pacientes com elevado grau de estenose carotídea.

Estudo desenvolvido por Morel e cols.34 avaliou os níveis de MPL e MPE dentro das artérias coronarianas ocluídas de pacientes após infarto com elevação do segmento ST, tratados por angioplastia primária, e comparou com as quantificações dessas MP no sangue periférico. Os autores encontraram aumento das MP no interior das artérias, mostrando a importância dessas vesículas no desenvolvimento da aterotrombose coronariana.

Faille e cols.35 mensuraram MPs CD11b+ (marcador de monócitos) em pacientes com síndrome coronariana aguda sem elevação do segmento ST no eletrocardiogama. O intuito foi verificar se a quantificação dessas MP poderia contribuir para a identificação de pacientes com maior risco de um evento cardiovascular recorrente em um mês após o implante do stent coronariano. Foi encontrada menor quantidade de MP CD11b+ em indivíduos com evento cardiovascular recorrente do que em pacientes que não tinham complicações, sugerindo que haja uma maior captura dessas MP nos sítios de lesões ateroscleróticas.

Jeanneteau e cols.36 avaliaram em ratos e humanos o papel das MP no mecanismo de condicionamento isquêmico remoto (CIR), que é descrito como uma estratégia de cardioproteção relacionada ao infarto. Não foram encontradas diferenças no número total de MP no grupo de animais submetidos ao CIR comparado ao grupo controle. Após caracterização fenotípica das MP, observaram-se elevações nas subpopulações endotelial e anexina V+ (apoptóticas) no grupo CIR. Assim como no modelo animal, houve aumento de MPE e MP Anexina V+ no grupo de indivíduos submetidos ao CIR.

Porto e cols.37 avaliaram as concentrações de MP em pacientes pós-infarto com elevação do segmento ST submetidos a intervenção coronariana percutânea primária, e a relação dessas vesículas com a obstrução microvascular (definida por angiografia múltipla e eletrocardiografia). Os principais achados foram que as subpopulações de MP avaliadas (MPP e MPE) tiveram níveis mais elevados no interior das coronárias se comparadas ao sangue aórtico. Além disso, houve maior liberação das duas subpopulações de MP na artéria coronariana comprometida do que na aorta ascendente, indicando uma produção local de MP. Os autores sugerem que os dados encontrados possam reforçar a hipótese de que as MP atuem como elementos ativos na embolização e na fisiopatologia da obstrução microvascular.

Kaabi e cols.38 avaliaram a relação entre o tratamento de pacientes portadores de doença arterial coronariana estável por contrapulsação externa de alta pressão (CEAP) e níveis de MP. Essa terapia tem sido considerada efetiva e segura para pacientes com angina de peito refratária. Os autores encontraram aumento de MPP após tratamento por CEAP, sem diferenças nos níveis de MPE e MPM.

Williams e cols.11 avaliaram ativação plaquetária e níveis de depressão em portadores de doença arterial coronariana, uma vez que a depressão, mesmo em condições leves, é um preditor independente de maior mortalidade após infarto do miocárdio. Os autores encontraram que pacientes portadores de moderada depressão e altos níveis de TNF-α, IL-6 e PCR possuíam, paralelamente, maior liberação de MPP, indicando que um componente pró-inflamatório foi capaz de alterar a função plaquetária nesses pacientes.

Bernal-Mizrachi e cols.39 mostraram que, de acordo com o estímulo que a célula recebe (ativação ou apoptose), ocorre a expressão de diferentes proteínas de superfície. Os autores desenvolveram um estudo em que eles analisaram duas subpopulações de MPE (CD31+/CD42- e CD51+) em indivíduos com doença arterial coronariana e encontraram que as MPE CD31+/CD42- foram mais expressas em eventos agudos (infarto do miocárdio e angina instável), enquanto as MPE CD51+ foram liberadas em quantidades semelhantes tanto em eventos agudos quanto crônicos (angina estável).

 Micropartículas e diabetes melito

Alguns trabalhos mostram maiores concentrações de MPP relacionadas ao diabetes. Ogata e cols.40 avaliaram os níveis de MPP em 92 portadores de retinopatia diabética. Os autores encontraram maior liberação de MPP nesses pacientes comparados a indivíduos saudáveis, e o aumento foi tanto maior quanto mais grave a retinopatia. Na aterotrombose, MP estão relacionadas com a liberação de citocinas pelos leucócitos e células endoteliais, o recrutamento de monócitos para a placa aterosclerótica, a proliferação de células musculares lisas, a angiogênese e o aumento do estresse oxidativo. Por outro lado, MP podem atuar como sinalizadores para a homeostase celular, promovendo um balanço entre estímulo celular, proliferação e apoptose12.

Lumsden e cols.41 avaliaram portadores de diabetes tipo 2 após síndrome coronariana aguda (seis meses antes do evento), que tiveram reduzidos níveis de MPE sem alterações em MPP. Os autores sugerem que esses resultados inesperados, uma vez que estão em discordância com a maioria dos trabalhos da literatura, podem ser resultantes de medicações concomitantes utilizadas pelos pacientes42-44.

Outros trabalhos indicaram que o aumento na expressão de moléculas de adesão está associado à ativação de monócitos, que podem se ligar a células endoteliais presentes na parede dos vasos, levando a progressão da retinopatia diabética. Esses dados sugerem que a mensuração dos níveis de MPM seja um biomarcador útil da progressão da retinopatia diabética45.

 Micropartículas na disfunção endotelial e na dislipidemia

Leroyer e cols.46 mostraram que MP originadas de macrófagos (CD40+) podem promover angiogênese no interior da placa, sugerindo que essas MP possam ser determinantes na vulnerabilidade da placa. Porém, estudos avaliando a relação entre MP e angiogênese têm-se mostrado contraditórios e inconclusivos, uma vez que alguns afirmam que certas MP podem estimular angiogênese (MPP, por exemplo)13,47, bem como outros estudos afirmam que MP podem tanto estimular quanto inibir a angiogênese, dependendo da origem celular48-50.

Alguns estudos mostraram que portadores de síndrome metabólica possuem aumento nos níveis de MP comparados a indivíduos saudáveis e que elas estão relacionadas à disfunção endotelial, por diminuição na expressão de eNOS e aumento da liberação de espécies reativas de oxigênio51.

O primeiro trabalho a demonstrar o efeito direto das MP na função vascular foi desenvolvido por Boulanger e cols.52 Os autores avaliaram se MP presentes no sangue periférico de pacientes com síndrome não isquêmica e pós-infarto agudo do miocárdio influenciariam a resposta dependente do endotélio em anéis aórticos de ratos. Observou-se que MP de indivíduos pós-infarto reduziram o relaxamento vascular induzido por acetilcolina (por influenciar a via do óxido nítrico), sugerindo que as MP poderiam contribuir com a disfunção endotelial observada após o evento agudo.

Diehl e cols.53 analisaram diferentes subpopulações de MP em portadores de hipertensão pulmonar, tendo encontrado níveis aumentados de MPL, MPE e MPP, o que indica maior atividade inflamatória e pró-coagulante, que pode estar relacionada a complicações tromboembólicas e disfunção endotelial nesses pacientes.

Já está bem descrita na literatura a melhora da função endotelial promovida pelos bloqueadores de canais de cálcio. Nomura e cols.54 mostraram redução de MPE em portadores de diabetes tipo 2 após tratamento com o bloqueador de canal de cálcio nifedipino. Resultado similar foi obtido pelo mesmo grupo55 com portadores de diabetes tipo 2 e hipertensão após tratamento com benidipina, pertencente à mesma classe farmacológica. O efeito do bloqueador do sistema renina-angiotensina (BRA) valsartana nos títulos de MPM em indivíduos portadores de diabetes melito tipo 2 também foi avaliado pelo mesmo grupo56, que observou que o fármaco inibiu a liberação de MPM. Esses resultados sugerem que BRA possa contribuir no tratamento da aterosclerose.

 Micropartículas e terapias hipolipemiantes

Têm-se buscado novas estratégias que possam inibir as funções das MP ou que proporcionem um maior clearance. Muitos estudos têm avaliado o efeito de diferentes estratégias hipolipemiantes na quantidade de MP liberadas por diferentes tipos celulares57,58.

Pinheiro e cols.59 avaliaram o efeito do antiplaquetário clopidogrel associado ou não à rosuvastatina (40 mg) nos níveis de MPE e MPP em pacientes coronarianos estáveis em uso de estatinas há pelo menos três meses. Os autores identificaram um aumento nos níveis de MPP após a suspensão por quatro semanas da rosuvastatina (mantendo‑se apenas o clopidogrel) e uma tendência à maior liberação de MPE nesses pacientes, sugerindo que tenha ocorrido um aumento da apoptose das plaquetas, e também que a rosuvastatina possa ter um efeito protetor sobre o endotélio quando em associação com o clopidogrel. França e cols.60 analisaram em estudo similar a influência da atorvastatina (80 mg) em associação ou não com o clopidogrel, também em coronarianos estáveis. Os autores sugeriram uma maior estabilidade vascular promovida pela atorvastatina após identificarem uma relação inversa entre a concentração plasmática de atorvastatina e os níveis de MPP.

Nesse sentido, os estudos mostram que as MP podem ser marcadores úteis não só na avaliação da doença cardiovascular, estando também associadas a câncer10,17, sepse22 e outras enfermidades. Porém, ainda há muita controvérsia sobre níveis considerados fisiológicos e patológicos. Embora a citometria de fluxo seja considerada padrão de referência para identificação e caracterização fenotípica de MP, os estudos utilizam-se de métodos bastante variados (tempo de centrifugação e de incubação com os anticorpos, diferentes marcadores), o que ainda dificulta a comparação entre as publicações existentes na literatura.

Em conclusão, a pesquisa por novos biomarcadores que possam estar relacionados com a doença cardiovascular tem despertado o interesse pelo estudo de MP de diferentes origens celulares, especialmente plaquetárias. Porém, os trabalhos ainda são controversos, sendo necessários estudos buscando a padronização de técnicas mais sensíveis para obtenção, caracterização e quantificação dessas vesículas para que, futuramente, os dados possam ser transpostos para a prática clínica.

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