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Background and Aims. Atherosclerosis is an inflammatory disease with long-lasting activation of innate immunity and monocytes are the main blood cellular effectors. We aimed to investigate monocyte phenotype (subset fraction and marker expression) at different stages of coronary atherosclerosis in stable coronary artery disease (CAD) patients. Methods. 73 patients with chronic coronary syndrome were evaluated by CT coronary angiography (CTCA) and classified by maximal diameter stenosis of major vessels into three groups of CAD severity: CAD1 (no CAD/minimal CAD, n°=30), CAD2 (non-obstructive CAD, n°=21), and CAD3 (obstructive CAD, n°=22). Flow cytometry for CD14, CD16, and CCR2 was used to quantify Mon1, Mon2, and Mon3 subsets. Expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, and CXCR4 was also measured. Adhesion molecules and cytokines were quantified by ELISA. Results. Total cell count and fraction of Mon2 were higher in CAD2 and CAD3 compared to CAD1. By multivariate regression analysis, Mon2 cell fraction and Mon2 expression of CX3CR1, CD18, and CD16 showed a statistically significant and independent increase, parallel to stenosis severity, from CAD1 to CAD2 and CAD3 groups. A similar trend was also present for CX3CR1 and HLA-DR expressions on total monocyte population. A less calcified plaque composition was associated to a higher Mon2 expression of CD16 and higher TNF-α levels. IL-10 levels were lower at greater stenosis severity, while the IFN-γ/IL-10 ratio, a marker of a systemic pro-inflammatory imbalance, was directly correlated to stenosis degree and number of noncalcified plaques. Conclusions. The results of this study suggest that a specific pattern of inflammation-correlated monocyte marker expression is associated to higher stenosis severity and less calcified lesions in stable CAD. The clinical trial Identifier is NCT04448691.

URL: http://www.clinicaltrials.gov. Unique identifier: NCT01725178.

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Objective: Inflammation and immunity activation play a key role in atherosclerosis (ATS) onset and progression. Aim of this study was to investigate the relationships between phenotype of circulating monocytes and coronary artery disease (CAD) development in a histologically well-characterized swine model of ATS. Methods:Blood samples were obtained from 6 animals at baseline and after 16 weeks high fat cholesterolenriched diet. Flow cytometry monocyte identification was performed (CD172a marker). Adhesion (CD18a, CD11a, CD11R3, CD49d, CD29), differentiation (CD14) and activation receptors (SLA-DR, CD16, CD163) were quantified as percentage of positivity (%) and Relative Fluorescence Intensity (RFI). Lipid parameters (LDL, oxLDL, HDL) and soluble endothelial ICAM-1 were measured and histologic quantitative assessment of coronary ATS was performed. Results:Flow cytometry analysis demonstrated a significant post-diet decrease of CD14 RFI and an increment of % SLA-DR. Pre-diet values of ICAM-1 and % SLA-DR correlated reciprocally (P=0.0191) and with several CAD severity indexes (P≤0.02). Positive correlations between RFI changes of CD29 (P=0.0213) and CD18a (P=0.0341) and morphometric indexes of coronary ATS were found. Post-diet RFI values of CD29, CD18a and CD16 were also closely related to morphometric parameters (P<0.03). A cumulative post-diet tendency to increase of CD14 low / CD163 high monocyte fraction (45.07 ± 2.27 vs. 40.14 ± 3.16) and a tight correlation between changes of this monocyte subset and corresponding HDL variations (P=0.0100) were also observed. Conclusions:Blood monocyte orientation towards a macrophage-like phenotype, similar to a HDL-induced maturation, and a close association between markers changes and severity of diet induced coronary ATS could provide new insights into plaque growth and progression in CAD.

Introduction Non-familial ascending thoracic aorta dilation and aneurysms (TAAs) are silent diseases in elderly patients. Histopathology revealed that functionally polarized infiltrating CD4 + T-cells play a key role in aortic wall weakening. Objective To evaluate the possible associations between phenotype and cytokine production of circulating CD4 + T-lymphocytes and the presence of TAA in patients with aortic valve disease (AVD). Methods We studied blood samples from 10 patients with TAA and 10 patients with AVD. Flow cytometry was used to quantify: a) CD4 + T-lymphocytes surface expression of CD25, CD28, and chemokine receptors (CCR5, CXCR3, CX3CR1); b) fractions of in vitro stimulated CD4 + T-cells producing cytokines (interferon gamma [IFN-γ], interleukin [IL]-17A, IL-21, IL-10); c) CD4 + CD25 high FoxP3 + regulatory T-cells (Treg) fraction. Enzyme-linked immunosorbent assays (ELISA) were performed for cytokines (IFN-γ, IL-6, IL-10, IL-17A, IL-23, transforming growth factor beta [TGF-β]) and chemokines (RANTES, CX3CL1). Results The total CD4 + CD28 ± CD4 + /CX3CR1 + T-cells fraction was higher ( P =0.0323) in AVD (20.452±4.673) than in TAA patients (8.633±2.030). The frequency ratio of CD4 + T-lymphocytes producing IFN-γ vs . IL-17A+IL-21 cytokine-producing CD4+ T-cells was higher ( P =0.0239) in AVD (2.102±0.272) than in TAA (1.365±0.123) patients. The sum of CD4 + CD28 ± CD4 + /CX3CR1 + T-cells correlated positively with values of the previous cytokine ratio ( P =0.0002, R=0.732). The ratio of CD4 + CD28 ± CD4 + /CX3CR1 + T-cells vs . Treg was higher ( P =0.0008) in AVD (20.859±3.393) than in TAA (6.367±1.277) patients. Conclusion Our results show that the presence of TAA in subjects with AVD is associated with imbalance between phenotypic and cytokine-producing subsets of circulating CD4+ T-lymphocytes, prevalently oriented towards a pro-fibrotic and IFN-γ counteracting effect to functional polarization.

INTRODUCTION: Ascending thoracic aortic aneurysm (TAA) is a multi-factorial process in which histological modifications and immune-mediated inflammation are closely associated. The predominant role of a Th1-mediated response in influencing aortic wall remodeling, dilation, and aneurysm formation has been suggested by previous studies. Recently, the importance of chemokine receptors for Th1 cells recruitment into vascular inflammatory sites, as well as of the balance between pro- and anti-inflammatory T-cell subsets in influencing the severity of coronary artery disease, have been described.MATERIAL AND METHODS: We evaluated activation markers and chemokine receptors expression on peripheral T-cell and NK cell subsets of subject with aortic valve disease associated with ascending TAA (ascending aortic diameter > 4 cm) and undergoing elective surgery for TAA (Group A), in comparison with patients with aortic valve disease without TAA (ascending aortic diameter < 4 cm) (Group B). Peripheral blood samples from the two groups were also compared for intracellular T-lymphocyte cytokine production, frequency of regulatory T cells (Treg) and soluble levels of cytokine and chemokines. The aortic size index (ASI) was considered a parameter able to reflect aortic pathophysiological modifications leading to aortic dilation.RESULTS: The results demonstrated correlations between ASI values and CCR5 expression on CD3+, CD3+/CD8+, CD4+ and CD4+/CD28- T-cell subsets. In Group A the expression of CCR5 was higher on CD3+/CD8+, CD4+ and CD4+/CD28- T-cell subsets, when compared with Group B. CD4+ and CD4+/CD28- T-cells in Group A showed also a higher expression for the co-stimulatory molecule CD28 and the activation marker CD25, respectively. An increased expression of CXCR3 was found on CD4+, CD3+/CD8+ and CD3+/TCR+ T-cell subsets in Group A. A higher circulating fraction of NK cells, together with a higher NK cell positivity for CX3CR1, were observed in aneurysmatic patients. Intracellular cytokine analysis demonstrated a higher fraction of CD3+/CD4+ T-cells producing IL-17A and IL-10 in Group A, together with a higher intracellular content for IL-21. Finally, a higher soluble level of fractalkine (CX3CL1) has been detected in aneurysm group.CONCLUSION: Results indicate a higher activation state, migratory capacity and cytotoxic potential of peripheral blood NK and T-cell subsets in patients with aortic valve disease associated with ascending TAA, when compared with patients affected by aortic valve disease alone. These findings, together with the observed higher polarization towards a Th17 in patients with aortic aneurysm could suggest the involvement of autoimmune mechanisms leading to cellular loss, inflammation and fibrosis during ascending aortic wall dilation and aneurysmatic progression.Journal of Universal College of Medical Sciences Vol. 3, No. 1, 2015: 11-20