|Year : 2014 | Volume
| Issue : 3 | Page : 143-148
Evaluation of the procoagulant potential of endothelial microparticles CD144 (VE-Cadherin) positive in coronary syndrome patients
Amany H Mansour1, Azaa M Abd Elbaky1, Shahir Kamal2, M Noura3, Abdulrahman Alshaikh MD 4
1 Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Cardiology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
4 Department of Emergency Hospitals, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||05-May-2014|
|Date of Acceptance||13-Oct-2014|
|Date of Web Publication||31-Dec-2014|
Mansoura University Hospital, Algomhouria Street, Mansoura 35516
Source of Support: None, Conflict of Interest: None
Background Apoptotic microparticles are responsible for almost all tissue factor activity of the plaque lipid core. We hypothesized that elevated levels of procoagulant microparticles could also circulate in the peripheral blood of patients with recent clinical signs of plaque disruption and thrombosis. The present study included 60 acute coronary syndrome (ACS) adult patients. Group I included 30 patients with diabetes mellitus who presented with ACS. Group II included 30 nondiabetic patients complaining of ACS and 25 healthy individuals as controls. ACS patients were further classified according to laboratory and radiological findings (troponin test and ECG) as follows: group A included 16 ST-segment elevation myocardial infarction (STEMI) patients, group B included 19 non-ST-segment elevation myocardial infarction (NSTEMI) patients, and group C included 25 patients with unstable angina. Traditional laboratory investigations and special laboratory assessments of CD144 fluorescein isothiocyanate by flow cytometry were performed.
Results The present study found highly elevated CD144 percentages in diabetic ACS patients compared with healthy controls (P≤0.0001(, and highly elevated creatine kinase-MB (CK-MB), fasting sugar, total cholesterol, triglyceride, HDL, and LDL (P = 0.0001, 0.0001, 0.0002, 0.0002, 0.0001, and 0.0001, respectively). In contrast, nondiabetic ACS patients had significantly elevated CD144, CK-MB, total cholesterol, triglyceride, HDL, and LDL (P = 0.0001, 0.0001, 0.0001, 0.0021, 0.0001, and 0.0021, respectively), whereas fasting sugar and HbA1c did not change significantly. However, the patients in group B (NSTEMI) had significantly elevated CD144% in comparison with patients with unstable angina (group C) ( P = 0.05), but patients with group A (STEMI) had significantly elevated CK-MB compared with patients with unstable angina (group C) (P = 0.02).
Conclusion The high levels of circulating microparticles of endothelial origin are increased in diabetic patients with coronary artery disease, suggesting an important role for endothelial injury in the prediction of ACS. Hyperglycemia in ACS is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes.
Keywords: acute coronary syndrome, endothelial microparticles, platelets microparticles
|How to cite this article:|
Mansour AH, Abd Elbaky AM, Kamal S, Noura M, Alshaikh A. Evaluation of the procoagulant potential of endothelial microparticles CD144 (VE-Cadherin) positive in coronary syndrome patients. Egypt J Haematol 2014;39:143-8
|How to cite this URL:|
Mansour AH, Abd Elbaky AM, Kamal S, Noura M, Alshaikh A. Evaluation of the procoagulant potential of endothelial microparticles CD144 (VE-Cadherin) positive in coronary syndrome patients. Egypt J Haematol [serial online] 2014 [cited 2018 May 23];39:143-8. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/143/148244
| Introduction|| |
Cardiovascular disease is a major global cause of mortality in the developed countries. Intravascular thrombogenesis, the main pathogenic mechanism of coronary artery disease (CAD), is influenced by a complex interplay of procoagulant, anticoagulant, fibrinolytic, endothelial damage/dysfunction, and inflammatory processes  . Chest pain (or discomfort) is the main complaint of patients found to have ischemic heart disease  . The majority of acute CAD are precipitated by vascular occlusion caused by atherosclerotic plaque disruption, platelet aggregation, platelet adhesion, and the resulting intravascular thrombosis. Systems that are involved in maintaining the integrity and patency of the vasculature including endothelial and platelet function, coagulation, and fibrinolysis are impaired in diabetes, thereby shifting the balance to favor thrombus formation. The endothelium can shed microparticles (MP) as a result of cell activation or apoptosis. MP were defined as small vesicular structures with a heterogeneous diameter (from 0.1 to 1 μm), resulting from the remodeling of membrane phospholipids and expressing phosphatidylserine and antigens representative of their parent cells  . Endothelial dysfunction (ED) plays an important role in the pathogenesis and clinical expression of atherosclerosis. It has been linked to type II diabetes mellitus (DM) and insulin resistance states such as obesity in experimental and clinical studies  . This reflects a number of abnormalities that include loss of bioavailable nitric oxide, increased production of vasoconstrictors, and disturbed regulation of inflammation, thrombosis, and cell growth in the vascular wall  .
Circulating endothelial cell MPs (EMPs) can be identified and measured by the detection of antigens constitutively expressed by mature endothelial cells (ECs) [e.g. CD31 (platelet-derived growth factor), CD105 (endoglin), CD144 (vascular endothelium cadherin), CD146, etc  . There are strong associations between high values of EMPs (CD31+, CD51+, and CD144+) and various vascular structural and functional abnormalities. Annexin V+/CD31+EMPs correlate with vasodilatory ED and have been shown to have potential as a marker of coronary endothelial function, independent of classic cardiovascular risk factors , . Furthermore, therapeutic improvement in endothelial function has been associated with a reduction in circulating EMP levels  . EMP are also known to be elevated in conditions associated with endothelial injury, as in acute coronary syndromes (ACS)  . Patients with different cardiovascular risk factors have significant upregulation of EMPs before the development of CAD  . For example, various EMP types (CD144+, CD31+, CD51+, and annexin V+) are increased in diabetic patients, and appear to be good predictors of CAD  . Diabetes not only increases the risk of myocardial infarction (MI) but also increases the mortality associated with the acute event. Hyperglycemia in ACS is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes. Acute hyperglycemia occurs in up to 50% of all ST-segment elevation myocardial infarctions (STEMI)  , whereas patients with diabetes represent ~25% of patients with STEMI. When glucose tolerance testing is performed, 65% of patients with MI and a negative history of diabetes can be diagnosed with diabetes or impaired glucose tolerance  . The aim of this study is to assess the levels of procoagulant MP CD144 in diabetic and nondiabetic ACS patients and to evaluate its role in the prediction of acute coronary events in these patients.
| Patients and methods|| |
The present study included 60 ACS adult patients (46 men and 14 women) recruited from the Cardiology Department of Internal Medicine Hospital, Mansoura University. Twenty-five healthy individuals matched for age, sex, and BMI were included as healthy controls. They were classified according to the presence or absence of DM into the following groups: group I included 30 patients (22 men and eight women) with DM who presented with ACS. Their ages ranged from 35 to 70 years. Group II included 30 nondiabetic patients (24 men and six women) complaining of ACS. Their ages ranged from 40 to 70 years. The group of healthy controls included 25 healthy individuals (18 men and seven women).Their ages ranged from 38 to 65 years. ACS patients were further classified according to laboratory and radiological findings (troponin test and ECG) into the following groups: group A included 16 STEMI patients, group B included 19 non-ST-segment elevation myocardial infarction (NSTEMI) patients, and group C included 25 unstable angina patients. A written consent was signed by every participant.
All study participants were subjected to a full assessment of history, clinical examination, radiological, and laboratory investigations including the following:
- Complete blood count.
- Liver function tests including aspartate aminotransferase, alanine aminotransferase, serum albumin, and bilirubin levels.
- Kidney function tests including serum creatinine.
- Fasting blood glucose and glycated hemoglobin (HbA1c).
- Lipid profile including [total cholesterol, triglyceride (TG), HDL, LDL].
- Cardiac markers including creatine kinase-MB (CK-MB) and troponin.
- Measurement of thrombin generation: plasma levels of the thrombin-antithrombin complex (TAT) were measured using commercially available enzyme-linked immunosorbent assay kits (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL, USA).
- CD144 MP were determined by flow cytometric analysis.
Flow cytometry analysis was carried out using the kit supplied by Dako (Copenhagen, Denmark) using Coulter  . Briefly, 10 μl of fluorescein isothiocyanate-conjugated monoclonal mouse antihuman CD144 antibodies were added and mixed with 100 μl of a fresh EDTA blood sample and was incubated in the dark for 30 min. A lysing reagent was added to lyse the red cells and separate the mononuclear cells. The cells were washed twice with PBS containing 2% BSA. The supernatant was removed and the cells were suspended in an appropriate medium. Negative controls were included in each run to determine the cut-off value between the negative and the positive population of cells. Analysis on the flow cytometer was carried out within 1 h using forward and side scatter. Gating on mononuclear cell population was used for analysis of samples. Mononuclear cells were separated from bone marrow samples of the controls and the patients by Ficoll Hypaque density gradient centrifugation (Sigma Chemicals, St Louis, Missouri, USA). The separated cells were washed twice using PBS and the count in the suspension was adjusted to 5 × 10 6 /ml. Data were displayed on two histograms: histogram I: two-parameter histograms (FS vs. SS). Histogram II: usually displayed as the mean fluorescence intensity value of CD144.
After an overnight fast, 7 ml venous was obtained from every participant. They were divided into 3 ml on EDTA (1 mg/ml) for complete blood count and HbA1c, CD144, and TAT, and 4 ml into plain tubes, and serum samples were separated for basic laboratory investigations.
Results were collected, tabulated, and analyzed statistically using the statistical package SPSS, version 16 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were obtained. Data were presented as mean, SD, and SEM. One-way analysis of variance is a test of significance used for comparison between three or more normally distributed groups with quantitative variables. Independent-sample t-test is a test of significance used for comparison between two normally distributed groups with quantitative variables. The Mann-Whitney test (nonparametric test) is a test of significance used for comparison between two non-normally distributed groups with quantitative variable. The level of significance was set at a P-value of up to 0.05.
| Results|| |
Demographic and some clinical data are presented in [Table 1].
|Table 1 Differences in age, sex, presence of hypertension, and smoking in all the groups studied|
Click here to view
[Table 2] shows a significant increase in the mean level of endothelial MP CD144 in ACS versus control participants (P = 0.001). The increased MPs level paralleled the increased levels of C-reactive protein (CRP) and TAT (markers of thrombin generation) among patients compared with the healthy participants. Increased levels of CRP and TAT (markers of thrombin generation) were found among patients compared with the healthy participants.
|Table 2 Comparison between CD144, CK-MB, TAT, and CRP in the groups studied compared with the healthy controls|
Click here to view
Comparison between the three main groups showed the following: CD144, creatine phosphokinase (CPK), CK-MB, total cholesterol, TG, and LDL-cholesterol were significantly increased in groups I and II in comparison with the healthy controls. Fasting plasma glucose and HbA1C were significantly higher in group I in comparison with the healthy controls. Significant differences in CD144, fasting blood sugar, HbA1c, and TG were detected on comparing groups I and II ([Table 3] and [Figure 1]).
| Discussion|| |
ACS is a term that includes all clinical syndromes compatible with acute myocardial ischemia resulting in an imbalance between myocardial oxygen demand and supply. In contrast to stable angina, an ACS results primarily from reduced myocardial blood flow secondary to an occlusive or a partially occlusive coronary artery thrombus.
The quantification of circulating ECs could indicate the presence of endothelial injury, and is a simple method of evaluation of endothelial-related physiologic and pathophysiologic states. Flow cytometry is a method of choice for the quantification of ECs. Numerous attempts have been made to standardize flow cytometric protocols, but results from these studies show a high degree of variability among centers  .
Koga et al.  reported that endothelial MP and platelets MP work synergistically to achieve maximal procoagulant activity. Consistent with this finding, the present study proved that the increased level of inflammatory markers such as CRP and the hemostatic marker of thrombin generation such as TAT pass paralleled increased EMP levels. CD144 are not only markers for endothelial injury/activation and but also contribute toward the pathogenesis of vascular injury observed in ACS patients. This finding is consistent with other literature that suggests that coronary diseases lead to abnormalities of endothelial function and platelet activation  . Also, the existing literature observed variable levels of MPs between patients. These results are in agreement with those of Mallat et al.  , who reported that differences in the levels of MP reflect differences in medications, however, because heparin has been reported either to have no effect or to decrease the generation of MP from thrombin-activated platelets. This observation suggests that the level of circulating MP could be useful as an indicator of the persistence or recurrence of thrombus and therefore as a prognostic marker of the recurrence of ischemic events  .
Evidence on the pathophysiological links between EMP formation and vascular dysfunction/damage has been provided by studies on hypertension, both systemic and pulmonary. Hypertension causes increased shear stress and direct mechanical damage of arteries. EMPs appear to be very sensitive to hemodynamic changes in hypertension, and their numbers are increased even in mild hypertension and increase further in proportion to an increase in blood pressure, even in the presence of multiple risk factors .
Moreover, EMP generation is associated with abnormalities of arterial elastic properties in hypertension and persistent arterial elastic abnormalities correlate with EMPs even after blood pressure normalization  , and our study confirms this as all hypertensive patients from group I and group II had mildly elevated CD144%.
The current study aimed to assess CD144+EMP in diabetic versus nondiabetic ACS patients. Hyperglycemia, hyperlipidemia with reduced HDL-cholesterol as well as hypertension are among the traditional risk factors for CAD. They have all been associated independently with increased cardiovascular events and are associated with ED  . Platelet count was found to be reduced in diabetic acute coronary patients in the current study. The decreased platelet count may result from its consumption in the process of thrombogenesis in ACS  . CD144+ EMP might participate in ACS through its procoagulant role. Phosphatidylserine expressed on EMP can bind to coagulation factors and promote their activation. Moreover, EMP harbor tissue factor, the initiator of the extrinsic coagulation pathway leading to thrombin generation. In this respect, Combes et al.  reported a reduction in the clotting time of normal plasma incubated with increasing amount of EMP released in vitro.
The procoagulant activity of EMP was confirmed by the fact that EMP from activated cells triggered TF-dependent thrombin formation in vitro and thrombus formation in vivo  .
Moreover, Montalescot et al.  found elevated EMP in infarction-related arteries than in the peripheral arteries and suggested that they might participate in ACS through its procoagulant role.
Although the cellular mechanism(s) of the role of EMP in atherosclerosis and coronary heart disease (CHD) largely remain unclear, it may increase reactive oxygen species and enhance cellular apoptosis. However, the proangiogenic role of EMP may have a deleterious effect in many diseases including cancer spread, proliferative diabetic retinopathy, and atherosclerotic plaque destabilization by promoting intraplaque neovascularization  .
ED and hence elevation in EMP (CD144) could be caused by various processes including enhanced oxidative stress. DM is typified by an increased tendency for oxidative stress and elevated levels of oxidized LDL, which represent the nidus for atherosclerosis and CHD. In addition, overexpression of growth factors is among the key players, and is indicated as a link between DM and proliferation of EC and smooth muscle cells that are active participants in plaque maturation  .
However, the diabetic state is associated with prothrombotic tendency as well as platelet activation and aggregation, which may be the cause for the low platelet count in diabetic ACS in the present study  .
CD144+ EMP were reported to be lower in STEMI after percutaneous coronary intervention, but not in NSTEMI and stable CAD. Moreover, CD144 and monocyte MP were independently predictive for future admission related to heart failure. Small-size MP including CD144 MP could be potentially implicated in the modulation of a post-ACS reparative response to injury with prognostic implications. CD144+ EMP is correlated to CK-MB in diabetic ACS patients. This finding is in agreement with that of Holmes et al.  , who reported that CD144+ EMP and platelet MP are correlated with myocardium at risk and infarct size, especially in STEMI.
Of particular interest, the present study found a significant increase in CD144+ EMP in smoker and hypertensive patients compared with nonsmokers and nonhypertensive ACS patients, respectively. These findings could be explained by a cumulative effect of traditional risk factors in the same patients. Evidence on the pathophysiological links between EMP formation and vascular dysfunction/damage has been provided by studies on hypertension, both systemic and pulmonary. Hypertension causes increased shear stress and direct mechanical damage on arteries. EMPs appear to be very sensitive to hemodynamic changes in hypertension, and their numbers are increased even in mild hypertension and increase further in proportion to blood pressure elevation, even in the presence of multiple risk factors  . Circulating EMP levels were associated with the presence of cardiometabolic risk factors, particularly dyslipidemia  .
| Conclusion|| |
The high levels of circulating MP of endothelial origin are increased in diabetic patients with CAD, suggesting an important role for endothelial injury in the prediction of ACS. Hyperglycemia in ACS is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Naghavi M, Libby P, Falk E, Casscells SW, Litovsky S, Rumberger J, et al
. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part I. Circulation
Gupta M, Tabas JA, Kohn MA. Presenting complaint among patients with myocardial infarction who present to an urban, public hospital emergency department. Ann Emerg Med
Daleke DL. Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res
Lüscher TF, Creager MA, Beckman JA, Cosentino F. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part II. Circulation
Habib SS. Cardiovascular disease in diabetes: an enigma of dyslipidemia, thrombosis and inflammation. Basic Res J Med Clin Sci
Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res
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
Bulut D, Maier K, Bulut-Streich N, Börgel J, Hanefeld C, Mügge A. Circulating endothelial microparticles correlate inversely with endothelial function in patients with ischemic left ventricular dysfunction. J Card Fail
Boulanger CM, Amabile N, Tedgui A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension
Bernard S, Loffroy R, Sérusclat A, Boussel L, Bonnefoy E, Thévenon C, et al
. Increased levels of endothelial microparticles CD144 (VE-Cadherin) positives in type 2 diabetic patients with coronary noncalcified plaques evaluated by multidetector computed tomography (MDCT). Atherosclerosis
Shet AS, Aras O, Gupta K, Hass MJ, Rausch DJ, Saba N, et al
. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood
Libby P. The molecular mechanisms of the thrombotic complications of atherosclerosis. J Intern Med
Wahab NN, Cowden EA, Pearce NJ, Gardner MJ, Merry H, Cox JLICONS Investigators. Is blood glucose an independent predictor of mortality in acute myocardial infarction in the thrombolytic era? J Am Coll Cardiol
Norhammar A, Tenerz A, Nilsson G, Hamsten A, Efendíc S, Rydén L, Malmberg K. Glucose metabolism in patients with acute myocardial infarction and no previous diagnosis of diabetes mellitus: a prospective study. Lancet
Garcia S, Chirinos J, Jimenez J, Del Carpio Muñoz F, Canoniero M, Jy W, et al
. Phenotypic assessment of endothelial microparticles in patients with heart failure and after heart transplantation: switch from cell activation to apoptosis. J Heart Lung Transplant
Mobarrez F, Antovic J, Egberg N, Hansson M, Jörneskog G, Hultenby K, Wallén H. A multicolor flow cytometric assay for measurement of platelet-derived microparticles. Thromb Res
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
Ahn YS, Jy W, Jimenez JJ, Horstman LL. More on: cellular microparticles: what are they bad or good for? J Thromb Haemost
Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freyssinet JM, Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation
Jones WS, Annex BH. Growth factors for therapeutic angiogenesis in peripheral arterial disease. Curr Opin Cardiol
Preston RA, Jy W, Jimenez JJ, Mauro LM, Horstman LL, Valle M, et al
. Effects of severe hypertension on endothelial and platelet microparticles. Hypertension
Wang JG, Manly D, Kirchhofer D, Pawlinski R, Mackman N. Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice. J Thromb Haemost
Lukasik M, Rozalski M, Luzak B, Michalak S, Kozubski W, Watala C. Platelet activation and reactivity in the convalescent phase of ischaemic stroke. Thromb Haemost
Morel O, Pereira B, Averous G, Faure A, Jesel L, Germain P, et al
. Increased levels of procoagulant tissue factor-bearing microparticles within the occluded coronary artery of patients with ST-segment elevation myocardial infarction: role of endothelial damage and leukocyte activation. Atherosclerosis
Combes V, Simon AC, Grau GE, Arnoux D, Camoin L, Sabatier F, et al
. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest
Abid Hussein MN, Böing AN, Biró E, Hoek FJ, Vogel GM, Meuleman DG, et al
. Phospholipid composition of in vitro endothelial microparticles and their in vivo thrombogenic properties. Thromb Res
Montalescot G, Cayla G, Collet JP, Elhadad S, Beygui F, Le Breton H, et al
. ABOARD Investigators Immediate vs delayed intervention for acute coronary syndromes: a randomized clinical trial. JAMA
Leroyer AS, Rautou PE, Silvestre JS, Castier Y, Leseche G, Devue C, et al
. CD40ligand+ microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization. J Am Coll Cardiol
Wackers FJ, Young LH, Inzucchi SE, Chyun DA, Davey JA, Barrett EJ, et al
. Detection of Ischemia in Asymptomatic Diabetics Investigators Detection of silent myocardial ischemia in asymptomatic diabetic subjects: the DIAD study. Diabetes Care
Libby P. Mechanisms of acute coronary syndromes and their implications for therapy. N Engl J Med 2013; 368
Holmes DR Jr, Lerman A, Moreno PR, King SB 3rd, Sharma SK. Diagnosis and management of STEMI arising from plaque erosion. JACC Cardiovasc Imaging
Simak J, Gelderman MP, Yu H, Wright V, Baird AE. Circulating endothelial microparticles in acute ischemic stroke: a link to severity, lesion volume and outcome. J Thromb Haemost
Amabile N, Cheng S, Renard JM, Larson MG, Ghorbani A, McCabe E, et al
. Association of circulating endothelial microparticles with cardiometabolic risk factors in the Framingham Heart Study. Eur Heart J
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Niveles circulantes de MCP-1, VEGF-A, sICAM-1, sVCAM-1, sE-selectina y sVE-cadherina: su relación con componentes del síndrome metabólico en población joven
| ||Iris Paola Guzmán-Guzmán,Oscar Zaragoza-García,Amalia Vences-Velázquez,Natividad Castro-Alarcón,José Francisco Muñoz-Valle,Isela Parra-Rojas |
| ||Medicina Clínica. 2016; |
|[Pubmed] | [DOI]|
||Circulating levels of MCP-1, VEGF-A, sICAM-1, sVCAM-1, sE-selectin and sVE-cadherin: Relationship with components of metabolic syndrome in young population
| ||Iris Paola Guzmán-Guzmán,Oscar Zaragoza-García,Amalia Vences-Velázquez,Natividad Castro-Alarcón,José Francisco Muñoz-Valle,Isela Parra-Rojas |
| ||Medicina Clínica (English Edition). 2016; |
|[Pubmed] | [DOI]|