Monocyte and macrophage subtypes as paired cell biomarkers for coronary artery disease

Abstract

New findings: What is the central question of this study? Are circulating monocyte markers correlated with their derived macrophage polarization patterns and coronary artery disease severity? What is the main finding and its importance? There was an inverse relationship between circulating CD16⁺ monocytes (high) and M2 macrophages (low) that marked coronary disease severity, and the differences in polarization of macrophages were seen despite a week of cell culture ex vivo. This study highlights the importance, and potential prognostic implications, of circulating monocyte and descendant macrophage phenotypes in coronary artery disease.

Abstract: Monocytes and macrophages are central to atherosclerosis, but how they combine to mark progression of human coronary artery disease (CAD) is unclear. We tested whether patients' monocyte subtypes paired with their derived macrophage profiles were correlated with extent of CAD. Peripheral blood was collected from 40 patients undergoing cardiac catheterization, and patients were categorized as having no significant CAD, single vessel disease or multivessel disease according to the number of affected coronary arteries. Mononuclear cells were measured for the monocyte markers CD14 and CD16 by flow cytometry, and separate monocytes were cultured into macrophages over 7 days and measured for the polarization markers CD86 and CD206. At baseline, patients with a greater CAD burden were older, with higher rates of statin, β-blocker and antiplatelet drug use, whereas other characteristics were similar across the spectrum of coronary disease. CD16⁺ (both intermediate and non-classical) monocytes were elevated in patients with single vessel and multivessel disease compared with those without significant CAD (P < 0.05), whereas regulatory M2 macrophages (CD206⁺ ) were decreased in patients with single vessel and multivessel disease (P < 0.001). An inverse relationship between paired CD16⁺ monocytes and M2 macrophages marked CAD severity. On multivariable linear regression, CAD severity was associated, along with age and traditional cardiovascular risk factors, with CD16⁺ monocytes (directly) and M2 macrophages (inversely). Circulating monocytes may influence downstream polarization of lesional macrophages, and these measures of monocyte and macrophage subtypes hold potential as biomarkers in CAD.

Keywords: cell-based biomarkers; coronary artery disease; macrophage; monocyte.

Figure 1:. Monocytes subtypes as a function of CAD burden.

Percentages represent each monocyte subpopulation as a percentage of the total monocyte population. (A) Classical monocytes are the majority of circulating monocytes. (B) Although a minority population, non-classical monocytes were relatively enriched in patients with CAD. (C) Intermediate monocytes also showed this difference with CAD burden (D), as did total CD16⁺ monocytes (intermediate and non-classical combined) and the ratio of CD16⁺ to classical monocytes (E). Data are presented as mean ± SEM. For each graph, p value by ANOVA is reported, and if this is < 0.05, post-hoc Dunnett’s test is used to report p values for the comparison to “no CAD” (shown just below the error bars).

Figure 2:. Profiles of cultured macrophage from patients with CAD.

(A) Of the macrophages cultured from all patients with or without CAD, most had the M1 surface marker CD86, without significant differences between clinical presentations. (B) The minority population of M2 macrophages (CD206⁺) was suppressed in patients with CAD. (C) The M1:M2 macrophage ratio rises according to CAD severity. Data are presented as mean ± SEM. (D) M2 macrophages plotted against CD16⁺ monocytes show an inverse relationship that tracks with CAD burden. For A-C, p value by ANOVA is reported, and if this is < 0.05, post-hoc Dunnett’s test is used to report p values for the comparison to “no CAD” (shown just below the error bars). For D, the correlation coefficient r and the p value are reported for a linear fit to all data points combined.

Figure 3:. Myeloid-derived foam cell formation, response to pooled human serum, and association with CAD severity in multivariable linear regression.

(A) After generation of blood-derived macrophages (MΦs) from six CAD and three non-CAD patients, these cells were incubated with DiI-labeled oxLDL for six hours and subjected to quantified fluorescence microscopy. At each dose of oxLDL, there was no difference in uptake between CAD and non-CAD macrophages. An average of 358 cells were analyzed per CAD status per oxLDL dose. (B) Pooled serum from CAD patients, when incubated for 48 hours with macrophages from non-CAD patients, had no impact on the M1/M2 profile of those macrophages. Sera from non-CAD patients likewise did not increase M2 content of CAD patients’ macrophages. Nine patients were analyzed (3 non-CAD, 6 CAD) and pooled sera contained the serum from 3 CAD and non-CAD patients, respectively. (C) Four variables had significant association with CAD severity score on univariate linear analysis [CD16⁺ monocyte percentage, M2 macrophage percentage, age, and total number of cardiovascular risk factors, see Table 2 for details]. When these four variables were analyzed through multiple regression analysis, they all contributed to improving the model fit. Scaled coefficients for each variable for each of five models (4 with one variable omitted, 1 with all four included) are shown with 95% CI (see Table 3 for details).