Silvia Montoro-García

Immunophenotypic characterization of human monocyte subsets: possible implications for cardiovascular disease pathophysiology

Summary.  Objectives: Monocytes include several subsets with different and sometimes divergent roles in immunity, atherogenesis and reparative processes. Objectives: We aimed to perform detailed immunophenotypic and functional characterization of human monocyte subsets. Patients/methods: Analysis of surface markers of blood and bone marrow monocyte subsets and functional characterization of blood monocyte subsets in healthy volunteers was performed using flow cytometry. Results: In the present study, we show the presence of three subsets which could be unequivocally distinguished by surface expression of CD14, CD16 and CCR2 as CD14+CD16−CCR2+(Mon1), CD14+CD16+CCR2+(Mon2) and CD14lowCD16+CCR2−(Mon3) subsets. In comparison with the classic Mon1, the Mon2 subset had the highest expression of Tie2, CXCR4, CD163, CD115, receptors to inter‐cellular adhesion molecule‐1 (ICAM‐1), vascular endothelial growth factor (VEGF), and the highest surface levels of apolipoprotein B and ferritin. In contrast, Mon3 had maximal expression of VCAM‐1 receptors and CD204. The Mon2 and Mon3 subsets had significantly lower activity of the NFκB pathway than Mon1. Mon1 and Mon2 had similar phagocytic activity, which was significantly higher compared with Mon3. All three subsets were present in bone marrow, although the relative proportion of Mon2 in bone marrow was about 2.5‐fold higher compared with that seen in blood. Significant differences in cytokine production in response to endotoxin stimulation were observed between the three monocyte subsets. Conclusion: Given their immunophenotypic similarity, the newly characterized Mon2 population may represent the previously reported pluripotent progenitor/pro‐angiogenic monocytes.

Circulating microparticles: new insights into the biochemical basis of microparticle release and activity

Circulating microparticles released from various cell types are present in healthy individuals and the number and composition of their membrane vary in different disorders. Long considered to be cellular debris, microparticles have been recently identified as regulatory vectors of intercellular cross-talk. Indeed, circulating microparticles represent a heterogeneous mixture of spheroids of diverse surface membrane glycoproteins and lipids, with diverse cytoplasm components, the pattern of which depends on the type of stimulation and pathophysiology of parental cells. Despite extensive research into the procoagulant and proinflammatory properties of microparticles, there are few data that can provide information on the mechanism(s) of their formation and biological effects. Although several mechanisms of microparticle release have been suggested, the precise order of the events associated with key features of microparticle formation, transmembrane phosphatidylserine redistribution and cytoskeleton disruption remain to be clarified. In this review, we provide an overview of the molecular mechanisms involved in microparticle formation, as well as the diverse physiological and pathological roles they are able to undertake. Understanding the mechanism(s) governing microparticle release processes may be critical to understanding their precise role in various pathophysiological processes and thus indicate new potential routes to therapy.

Small-size circulating microparticles in acute coronary syndromes: relevance to fibrinolytic status, reparative markers and outcomes.

BACKGROUND Recent evidence suggests that circulating microparticles (MPs) contribute to inflammation, coagulation and vascular injury. Majority of MPs have usually not been included into prior analyses due their small size and limited resolution of conventional equipment. Our aim was to assess levels of MPs of different cellular origin sized below 0.5 μm polystyrene beads, denoted as small-size microparticles (sMP), their relation to markers of cardiovascular repair and their impact on prognosis in patients with acute coronary syndromes (ACS). METHODS In a cross-sectional study, we initially compared levels of sMP between patients with ST-segment elevation myocardial infarction (STEMI, n = 50), non-STEMI (n = 47), stable coronary artery disease (CAD, n = 40) and healthy individuals (HC, n = 40). In a separate study, the prognostic value of sMP was assessed in patients with non-STEMI (n = 160). Annexin V-binding sMP (sAMP), platelet CD42b(+) sMPs (sPMP), endothelial CD144(+) sMP (sEMP) and monocyte CD14(+) sMP (sMMP) were quantified using high resolution flow cytometry. Endothelial progenitor cells (EPCs) and monocyte expression of scavenger receptors was quantified by flow cytometry. Fibrinolytic factors were measured by ELISA. RESULTS Counts of sAMP and sEMP were lower in STEMI after PCI (p < 0.001 and p = 0.025, respectively) but not in non-STEMI vs. CAD. sAMP was positively correlated with EPCs in non-STEMI (p < 0.001). Likewise, plasminogen activators negatively correlated with sAMP in non-STEMI and STEMI (p = 0.02 and p = 0.002, respectively). In non-STEMI patients, sEMP and sMMP were independently predictive for future admissions related to heart failure (p = 0.034 and 0.013, respectively) and sPMP for major bleedings (p = 0.002). The sAMP/EPCs ratio was higher in patients (before PCI) compared to STEMI patients. CONCLUSIONS Small-size MPs could be potentially implicated in the modulation of the post-ACS reparative response to injury, with prognostic implications. Besides, the sAMP/EPCs ratio could reflect a change in the apoptotic/reparative potential, being a putative indicator for vascular repair.

Role of microRNAs in cardiac remodelling: New insights and future perspectives

Cardiac remodelling is a key process in the progression of cardiovascular disease, implemented in myocardial infarction, valvular heart disease, myocarditis, dilated cardiomyopathy, atrial fibrillation and heart failure. Fibroblasts, extracellular matrix proteins, coronary vasculature, cardiac myocytes and ionic channels are all involved in this remodelling process. MicroRNAs (miRNAs) represent a sizable sub-group of small non-coding RNAs, which degrade or inhibit the translation of their target mRNAs, thus regulating gene expression and play an important role in a wide range of biologic processes. Recent studies have reported that miRNAs are aberrantly expressed in the cardiovascular system under some pathological conditions. Indeed, in vitro and in vivo models have revealed that miRNAs are essential for cardiac development and remodelling. Clinically, there is increasing evidence of the potential diagnostic role of miRNAs as potential diagnostic biomarkers and they may represent a novel therapeutic target in several cardiovascular disorders. This paper provides an overview of the impact of several miRNAs in electrical and structural remodelling of the cardiac tissue, and the diagnostic and therapeutic potential of miRNA in cardiovascular disease.

Circulating microparticles: challenges and perspectives of flow cytometric assessment

Circulating blood microparticles are likely to play a significant role as messengers of biological information. Their accurate quantification and characterisation is challenging and needs to be carefully designed with preferable usage of fresh minimally-processed blood samples. Utilisation of flow cytometers specifically designed for analysis of small-size particles is likely to provide considerable methodological advantages and should be the preferable option. This viewpoint manuscript provides a critical summary of the key methodological aspects of microparticle analysis.

Monocyte subsets in coronary artery disease and their associations with markers of inflammation and fibrinolysis

AIMS The multiple roles of monocytes in atherogenesis, including inflammation, angiogenesis and repair are attributed to the existence of different monocyte sub-populations. Scarce data are available on changes in phenotype and functional status of human monocyte subsets in patients with coronary artery disease (CAD), especially when monocytes are evaluated as three distinct subsets. METHODS AND RESULTS Surface expression of receptors implicated in inflammation, repair and activation status (intracellular IKKβ) of monocyte subsets was assessed by flow cytometry in 53 patients with CAD and compared to 50 age- and sex-matched healthy controls. Monocyte subsets were defined as CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR2+ (Mon2), and CD14+CD16++CCR2- (Mon3). Plasma levels of inflammatory cytokines (FACSArray) and fibrinolytic factors (ELISA) were measured in CAD. CAD was associated with reduced expression of CD14 on Mon1 (p = 0.02) and Mon3 (p = 0.036), higher expression of IL6 receptor on Mon1 (p = 0.025) and Mon2 (p = 0.015), CXCR4 on Mon1 (p = 0.035) and Mon3 (p = 0.003), and CD34 on all subsets (all p < 0.007). Monocyte CD163 expression correlated negatively with interleukin (IL)-6 levels (p < 0.01 for all subsets). Expression of vascular endothelial growth factor receptor-1 correlated positively with plasminogen activator inhibitor (PAI)-1 antigen levels (r = 0.47, p = 0.006). In vitro, monocyte subsets derived from CAD patients showed significantly altered responses to endotoxin stimulation compared to monocytes from healthy controls. CONCLUSIONS There is a complex interplay between phenotype and activity of monocytes and plasma cytokines and fibrinolytic factors. These findings support the presence of unique roles for the three human monocyte subsets in atherogenesis and CAD pathogenesis.

An innovative flow cytometric approach for small-size platelet microparticles: Influence of calcium

Microparticles (MPs) are small submicron membrane-derived vesicles shed from a variety of cells and they have been implicated in different disorders. Accordingly, understanding of physiological characteristics of MPs and improvement of methods of their quantification are important for further advance in the field. Although flow cytometry is the most widely applied technique for MP analysis, it is limited by lack of adequate standardisation. Annexin V (AnV), which binds surface phosphatidylserine (PS) with high affinity, has been long regarded as a marker of MPs, but AnV binding is Ca2+-dependent and it is unclear how [Ca2+] concentrations could affect AnV binding to MPs and its enumeration. MPs from citrated and heparinised plasma were labelled with AnV, anti-CD42b and quantified using an Apogee A50 flow cytometer. The small-size MP gate was defined with the use of size beads (from 0.1 to 0.5 μm) and confirmed with an in vitro assessment of platelet stimulation. Biotinylated anti-CD42b antibodies were then bound to streptavidin conjugated with different fluorochromes, leading to an amplified signal of platelet MPs (PMPs). Moderate increase of [Ca2+] concentrations in the annexin V staining buffer allows initial plasma recalcification and more accurate MP quantification in citrated plasma. Thrombin stimulation of platelet-free plasma containing only MPs did not produce any changes in the concentration of AnV+ MPs, but decreased the anti-CD42b binding. The results also indicate that prolonged storage and thrombin induce the release of AnV+ MPs whereas PS exposure in pre-existent MPs is not affected by thrombin. In conclusion, we present a sensitive protocol for the analysis of circulating and in vitro induced small-size PMPs that might contribute to future cardiovascular and clinical research.

The effects of exercise and diurnal variation on monocyte subsets and monocyte‐platelet aggregates

Eur J Clin Invest 2012

TLR4 expression on monocyte subsets in myocardial infarction

Monocyte toll‐like receptor 4 (TLR4) has been implicated in the pathogenesis of atherosclerosis with increased levels in myocardial infarction. The aim of this study was to assess the numbers of TLR4+ monocytes in each monocyte subset in MI, the expression of TLR4 and association with markers of monocyte activation, inflammation, myocardial damage and postmyocardial infarction (MI) cardiac contractility.

Small‐size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P‐selectin

This study aimed to examine the mechanisms of cellular activation by small‐size platelet microparticles (sPMP) and to present the performance of high‐resolution flow cytometry for the analysis of subcellular entities from different origins. Plasma counts of sPMP were analysed in coronary artery disease patients (n = 40) and healthy controls (n = 40). The effect of sPMP and platelet debris (PD) in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry. New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Addition of sPMP resulted in platelet degranulation and P‐selectin redistribution to the membrane (P = 0·019) in a dose and time‐dependent manner. Blood clotting time decreased after addition of sPMP (P = 0·005), but was not affected by PD. Blocking P‐selectin (CD62P) in sPMP markedly reverted the effect on thrombus kinetics (P = 0·035). Exposure to sPMP stimulated monocyte expression of intercellular adhesion molecule‐1 (P < 0·03) and decreased monocyte interleukin‐6 receptor density (P < 0·01). These results implicate sPMP as a direct source of downstream platelet and monocyte activation. In pathological coronary artery disease conditions, higher levels of sPMP favour a prothrombotic state, partly through P‐selectin expression.