Luke D. Tapp

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.

The CD14++CD16+ monocyte subset and monocyte-platelet interactions in patients with ST-elevation myocardial infarction.

Summary.  Aim:  Monocytes contribute to both myocardial damage and repair by virtue of subset heterogeneity. The dynamics and functional characteristics of the three human monocyte subsets, including the unique CD14++CD16+ subset, and their contributions to monocyte platelet aggregates (MPAs) following ST‐elevation myocardial infarction (STEMI) are unknown. We aimed to examine dynamic changes and relation to left ventricular ejection fraction (LVEF) of the three human monocyte subsets and their aggregates with platelets following STEMI.

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.

CD14++CD16+ monocytes in patients with acute ischaemic heart failure

Monocytes play important roles in inflammation, angiogenesis and tissue repair and may contribute to the pathophysiology of heart failure (HF).

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.

Increased Formation of Monocyte-Platelet Aggregates in Ischemic Heart Failure

Background— Cross-talk between monocytes and platelets is reflected by the formation of monocyte-platelet aggregates (MPAs). It is not known whether MPAs are affected in heart failure (HF), and we examined differences in patients with acute HF (AHF), stable HF (SHF), stable coronary artery disease (CAD) without HF, and healthy controls (HCs). Methods and Results— MPAs were analyzed by flow cytometry for the 3 monocyte subsets (CD14++CD16-CCR2+ [Mon1], CD14++CD16+CCR2+ [Mon2] and CD14+CD16++CCR2– [Mon3]) in patients with AHF (n=51), SHF (n=42), stable CAD (n=44), and HCs (n=40). Counts of total MPA and MPAs associated with Mon1 and Mon2 were significantly higher in AHF compared with SHF, CAD, and HCs (P<0.001 for all). The proportion of Mon1 aggregated with platelets was increased in AHF compared with SHF (P=0.033), CAD (P<0.001), and HCs (P<0.001). A higher percentage of Mon3 aggregated with platelets was also seen in AHF compared with SHF (P=0.012) and HCs (P<0.001) but not compared with CAD (P=0.647). MPAs associated with Mon2 were significantly lower in patients who experienced adverse clinical outcomes of death or rehospitalization compared with those who remained free of events (P=0.03). Mon2 count remained an independent negative predictor of combined death and rehospitalization after adjustment for age, left ventricular ejection fraction, creatinine, and brain natriuretic peptide (hazard ratio, 0.58 [95% CI, 0.34–0.98]; P=0.043). Conclusions— MPA formation in patients with both acute and stable HF is increased and seems to be confined to monocytes from Mon1 and Mon2 subsets. MPAs associated with Mon2 seem to be negatively predictive of a worse prognosis in AHF.

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.

Ethnic Differences in Macrovascular and Microvascular Function in Systolic Heart Failure

Background— Endothelial dysfunction is implicated in the pathophysiological features of heart failure (HF), and ethnic differences in the presentation of cardiovascular disease are evident, with an excess seen among South Asians (SAs). However, data on ethnic differences in endothelial function in HF are limited. Methods and Results— In a cross-sectional study, we recruited 128 subjects with systolic HF: 50 SAs, 50 whites, and 28 African Caribbeans (ACs). In addition, SAs with systolic HF were compared with 40 SAs with coronary artery disease without HF (“disease controls”) and 40 SA healthy controls. Macrovascular endothelial function was assessed by measurement of flow-mediated dilation (FMD) in response to hyperemia, arterial stiffness was assessed by the pulse-wave velocity, and microvascular endothelial function was assessed by forearm laser Doppler flowmetry. CD144-expressing endothelial microparticles were measured by flow cytometry. When compared with disease controls and healthy controls, SAs with HF had an impaired microvascular response to acetylcholine (P=0.001) and reduced FMD (P<0.001). In comparing ethnic groups, SAs with HF had an impaired response to acetylcholine (123±95.5%) compared with whites (258±156%) and ACs (286±173%, P<0.001 for both). Whites had a higher FMD (8.49±4.63%) than SAs (4.76±4.78%, P<0.001) and ACs (4.55±3.56%, P=0.01). No difference in endothelial-independent response was observed between study groups or in pulse-wave velocity. Ethnicity remained associated with microvascular endothelial function even after adjustment for age, presence of hypertension and diabetes mellitus, blood pressure, and glucose levels (P=0.003). There were no differences in numbers of endothelial microparticles. Conclusions— The SAs with HF have impaired microvascular and macrovascular endothelial function but preserved arterial elastic properties. Significant ethnic differences in endothelial function are evident in subjects with HF, with ethnicity being associated with microvascular endothelial dysfunction in this disorder.

Fibrinolytic status in acute coronary syndromes: Evidence of differences in relation to clinical features and pathophysiological pathways

Limited data are available on the role of innate fibrinolysis in acute coronary syndromes (ACS). In the present study we evaluated the dynamic alterations of fibrinolytic markers in patients presenting with ACS. Tissue-type-(tPA) and urokinase type-(uPA) plasminogen activators, plasminogen activator inhibitor (PAI-1) antigen and activity and thrombin activatable fibrinolysis inhibitor (TAFI) were analysed in 50 patients with ST elevation myocardial infarction (STEMI), 47 non-STEMI patients (NSTEMI), 40 patients with stable coronary artery disease (CAD) and 39 controls. The parameters were measured on day 1 and days 3, 7 and 30. Counts of monocyte subsets, monocyte-platelet aggregates and plasma inflammatory cytokines were assessed on admission. On day 1, TAFI was higher in NSTEMI vs. STEMI (p<0.001) while PAI-1 activity was higher in STEMI (p<0.001). In STEMI, uPA activity levels was low on day 1 but significantly increased on day 30 (p<0.001). TAFI levels were increased in NSTEMI on day 1 and gradually reduced by day 30 (p<0.05). In STEMI, TAFI levels peaked at day 7 (p<0.05) and dropped significantly by day 30 (p<0.05). CD14++CD16+ monocytes were independently associated with PAI-1 activity in ACS (p=0.03). Monocyte-platelet aggregates rather than platelet-free monocytes were an independent determinant of tPA, PAI-1 antigen and TAFI on a multivariate analysis (p<0.05). There are significant differences in fibrinolytic activity between patients with STEMI and NSTEMI. These changes could reflect the role of these factors in post-MI myocardial healing. Monocyte-platelet interactions are independently associated with the regulation of the fibrinolytic status in ACS.