Cholesterol-Induced Membrane Microvesicles: Novel Contributors to Atherothrombosis
Ming-Lin Liu, M.D., Ph.D., Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Thomas Jefferson University, Philadelphia, PA, E-
Microvesicles in Cardiovascular Diseases and Atherothrombosis
Microvesicles (MV), also known as microparticles, are small membranous structures that are released from cells upon activation or during apoptosis [1-5]. Many cell types have been shown to release MVs, including circulating platelets, leukocytes, and erythrocytes, as well as cells of the vascular wall, mainly endothelium, macrophages, and smooth muscle cells [1-5]. MVs can be detected by flow cytometry, electromicroscopy, or ELISA with Annexin V-coated plates . Of these methods, flow cytometry has been the most commonly used, owing to its wide availability and its capacity for individually characterizing large numbers of MVs. By flow cytometry, MVs are generally identified based on size (forward scatter) and surface exposure of phosphatidylserine (PS; specifically binding with fluorescently labeled annexin V). PS is a membrane phospholipid that is generally confined to the inner leaflet of native plasma membranes, but becomes exposed on membrane MVs during vesiculation . MVs can serve as mediators of intercellular crosstalk or communication and induce a variety of cellular responses, including activation, apoptosis, thrombosis, and inflammation [3,4,6,7], owing to their content of biologically active lipids and proteins.
Increased levels of MVs in the circulation have been reported in cardiovascular disease (CVD), hyperlipidemia, hypertension, diabetes, the metabolic syndrome, and end-stage renal disease [3,4]. High circulating levels of MVs have been suggested to be a potential marker for CVD [3,4], and circulating leukocyte-derived MVs predict subclinical atherosclerosis burden in asymptomatic subjects . In addition, MVs have also been detected within human atherosclerotic plaques , where leukocyte- or monocyte/macrophage-derived MVs account for over 52% of the MVs . A potential pathogenic role for MVs in the development and progression of atherosclerosis, however, remains understudied. The local stimuli that provoke MV generation within atheromata have not been defined, and their local, pro-atherogenic effects are only now beginning to be explored. Several reports indicate that MVs may activate endothelial cells, thereby promoting leukocyte/monocyte adhesion in vitro , as well as impairing NO release and endothelium-dependent relaxation of mouse aortic rings ex vivo [12,13].
Cholesterol Loading of Human Monocyte/Macrophages Induces the Release of Biologically Active Microvesicles
Monocyte/macrophages accumulate unesterified cholesterol (UC) in vivo during atherogenesis  presumably owing to their uptake of lipoproteins that have been retained [15,16] and modified [17-19] within the arterial wall. Cholesterol loading of primary human monocyte-derived macrophages (MDM) in vitro was reported to stimulate the expression of tissue factor (TF) . Increased TF expression was found in atherosclerotic vessels from cholesterol-fed rabbits  in human lipid-rich plaque macrophages  and in monocytes from hypercholesterolemic patients , although none of these studies examined microvesicles.
In this context, we sought a pathophysiologically plausible process for stimulating the release of MVs within human atheromata. We recently reported that unesterified cholesterol (UC) enrichment of human monocyte/macrophages, a process known to occur in lesions , induces the release of biologically-active TF-positive MVs . To avoid potentially confounding effects from engagement of cell-surface receptors, we delivered the unesterified cholesterol as a water-soluble complex with methyl-?-cyclodextrin (UC/MCD; 1:6 molar ratio of UC:MCD; Sigma) . Beginning with human THP-1 monocytic cells, we observed UC-induced MV release as early as 2 hours after addition of UC/MCD, and the effect persisted for the entire 20-hour period we examined. By 20 hours, the total MV count from UC-enriched human THP-1 monocytes was nearly three times the value from untreated control cells. In contrast, media supplemented with the same, low concentration of MCD (0.15 mM), but not complexed to cholesterol, failed to measurably affect THP-1 MV generation over time.
In our system, we found that cholesterol loading of THP-1 monocytes caused large increases in the cellular release of MVs that contained TF, as measured either by flow cytometry of the cell culture suspension using fluorescently labeled anti-TF antibodies, or by TF ELISA of cell-culture supernatants. Importantly, UC loading of THP-1 monocytes caused a remarkable 10-fold increase in the TF procoagulant activity of THP-1 MVs, consistent with the increase we observed in TF mass as well as possible TF de-encryption .
We also examined primary human monocyte-derived macrophages (MDMs). UC enrichment of these cells likewise caused large, statistically significant increases in total and TF-positive MV release compared to control . Moreover, MV-containing cell culture supernatants from cholesterol-enriched primary human MDMs showed the same, striking 10-fold increase in TF procoagulant activity over control. These results suggest that cholesterol-induced release of pro-coagulant MVs from human monocyte/macrophages could contribute to the general prothrombotic state in hypercholesterolemia [23,24] and to the prothrombotic interior of lipid-rich, vulnerable plaques [9,16,25].
To explore the mechanism by which UC stimulates biologically active MV release, we found cholesterol enrichment caused a majority of THP-1 monocytes to promptly display strong staining for PS on their surface by 2 hours, and to gradually shrink in size over time. In contrast, our data showed no significant changes in either cell-surface exposure of PS or cellular size when THP-1 cells were incubated in unsupplemented serum-free medium or when exposed to LPS, a known stimulus for microparticle release. Since cell-surface exposure of PS often indicates early-stage apoptosis, we examined a definitive marker of late-stage apoptosis, namely, DNA fragmentation by terminal deoxynucleotidyl transferase FITC-dUTP nick end labeling of the cells (TUNEL). At the 20-hour time point, the proportions of TUNEL-positive cells were only 1-2% of control cells incubated in unsupplemented medium, but rose to approximately 30% of cholesterol enriched THP-1 monocytes. Similarly, the proportions of primary human MDMs that were TUNEL-positive were only 1% in serum-free medium but rose to approximately 11% after 20 hours of cholesterol enrichment. Thus, similar to prior literature , we saw significant cholesterol-induced apoptosis. Interestingly, our human monocyte/macrophages underwent UC-induced apoptosis even in the absence of other manipulations (cf. [26-28]), indicating an unexpectedly robust response in these cells. Addition of caspase-3 inhibitor (Z-DEVD-FMK) during cholesterol enrichment of THP-1 monocytes delayed cell-surface exposure of PS by 2-4 hours, and decreased the percentage of TUNEL-positive cells at 20 hours from 28 ± 2.8% to 12.5 ± 2.2%. Importantly, the caspase-3 inhibitor significantly decreased the number of PS-positive MVs released during UC-enrichment over time, although not entirely to the level of unenriched control cells. These results indicated that at least a portion of the cholesterol-induced MV generation in our system arises from apoptotic blebbing.
Summary and Conclusion
Taken together, our results directly link cellular cholesterol enrichment, a known consequence of hyperlipidemia and arterial retention of lipoproteins [15,16] with the enhanced generation of biologically active monocyte/macrophage-derived MVs, at least in part through induction of apoptosis. Related effects may occur with other cell types, though independent of apoptosis . The effects of TF-positive monocyte/macrophage-derived MVs and their potential contributions to atherothrombosis have been investigated by us  and others [9,30].
Importantly, however, we found that over 80% of the MVs in our study  and in prior literature  were PS-positive but TF-negative. We speculate that these particles may be biologically active as well, for example, by enhancing clot propagation [3,25], causing apoptosis of nearby cells , blocking PS receptors that could otherwise promote phagocytosis of apoptotic bodies , and altering gene expression via their display of PS . In addition, we and others found that apoptotic MVs from monocyte/macrophages (our unpublished data) or endothelial cells  may contain oxidatively modified phospholipids and thereby contribute to the presence of oxidized epitopes in atherosclerotic lesions , but without necessarily having to invoke LDL oxidation per se.
Of particular interest, we recently found that injection of cholesterol-induced monocyte MVs into rats provoked substantial leukocyte rolling and adherence to post-capillary venules in vivo, indicative of endothelial activation (Liu, M-L., Scalia, R., Williams, K.J., unpublished data, cited in ).
The biological effects of MVs will be determined by their protein and lipid composition, which may vary depending on their cell of origin and the type of stimulus involved in their formation . A crucial advantage of our cell system in the generation of biologically active MVs is that we use a pathophysiologically plausible stimulus, cholesterol enrichment, to generate biologically active MVs. We have identified that cholesterol-induced monocyte/macrophage MVs carry PS, TF, as well as modified phospholipids and that these MVs are highly pro-thrombotic in vitro and can activate endothelium in vivo. Therefore, cholesterol-induced MVs may be novel contributors to atherothrombosis.
The author thanks Dr. Kevin J. Williams, M.D. for his helpful support and advice. Our work here received support from NIH (USA) grants HL73898 and HL56984.
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