Cholesterol enrichment of human monocyte/macrophages induces surface exposure of phosphatidylserine and the release of biologically-active tissue factor-positive microvesicles

Abstract

Objective: Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages.

Methods and results: Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.

Conclusions: Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis.

Figure 1

Cholesterol enrichment of monocytic cells increases total MV generation. Suspensions of THP-1 monocytes were treated for 0 to 20 hours at 37°C with UC, LPS, both, or neither (Control), as indicated, and then samples were analyzed by flow cytometry. A, Representative dot-plots of forward scatter (FSC, indicating size) vs annexin V-PE staining (indicating PS exposure) for suspensions of control and UC-enriched cells. The squares enclose the population of PS-positive MVs. The ovals indicate cellular populations with the same size range and PE intensity as control THP-1 cells at t=0 hours, whereas the circles identify a population of cells that appeared over time, particularly with cholesterol enrichment. B, Quantitative counts of PS-positive MVs generated over time under the four experimental conditions. P<0.01 by ANOVA. *P<0.05, †P<0.01, and ‡P<0.001 vs the control group by the Student-Newman-Keuls test.

Figure 2

Cholesterol enrichment of monocytic cells increases the generation of biologically active TF-positive MVs. Suspensions of THP-1 monocytes were treated as in Figure 1. A, Quantitative counts at 4 hours of PS-positive MVs that were also TF-positive. B, TF procoagulant activity of MV-containing supernatants in an assay using exogenous factor VIIa and collagen-activated platelets. P<0.01 by ANOVA. *P<0.05, †P<0.01, and ‡P<0.001 vs the control group by the Student-Newman-Keuls test.

Figure 3

Cholesterol enrichment of primary human monocyte-derived macrophages increases the generation of total as well as biologically-active TF-positive MV. Adherent primary human MDMs in 6-well plates were treated without (Control) or with UC for 20 hours. A, Total concentrations of PS-positive MVs in the conditioned medium, as assessed by flow cytometry. B, TF procoagulant activity of MV-containing culture supernatants. ‡P<0.001 vs the control group by Student unpaired t test.

Figure 4

Apoptosis during cholesterol-induced MV generation. THP-1 monocytes were incubated at 37°C with or without UC, LPS, or combinations, as indicated, then analyzed by flow cytometry. Displayed are representative histograms of TUNEL staining after 0 hours (red), 2 hours (black), 4 hours (green), and 20 hours (blue). Each histogram includes approximately 1000 cells. Percentages indicate the proportion of cells that were TUNEL positive at 20 hours.

Figure 5

Role for apoptosis in cholesterol-induced MV generation. THP-1 monocytic cells were incubated at 37°C without or with UC, Z-DEVD-FMK (a caspase-3 inhibitor), or combinations, as indicated, then analyzed by flow cytometry. Displayed are quantitative counts of PS-positive MVs over time after the indicated treatments. P<0.01 by ANOVA. Symbols labeled with different lowercase letters are statistically different by the Student- Newman-Keuls test (P<0.05).