We report the application of fluorescent pegylated nanoparticles for the visualization of fluid-phase pinocytosis in vivo. Previously, no suitable method existed for in vivo detection of this important endocytic process that, as we have shown here, functions in atherosclerotic lesion macrophages. The previous use of fluorescently labeled dextran to monitor fluid-phase pinocytosis has been found to be unsuitable because this probe can be internalized by receptor-mediated uptake in cells such as macrophages expressing the mannose receptor (16
). As discussed below, fluid-phase pinocytosis can mediate macrophage cholesterol accumulation, an important process in the development of atherosclerotic lesions. Furthermore, fluid-phase pinocytosis has been shown to function in vitro in dendritic cell and macrophage uptake of soluble antigens that are then processed for antigen presentation to T cells (9
). The techniques we report here make it possible now to study fluid-phase pinocytosis in vivo as it occurs in atherosclerosis and during the process of antigen presentation. Also, fluid-phase pinocytosis by other cell types in vivo can now be discovered and investigated.
Learning how macrophages accumulate cholesterol and transform into foam cells within atherosclerotic plaques has been a major goal of research directed at understanding the pathogenesis of atherosclerosis. The widely accepted hypothesis for foam cell formation in atherosclerotic lesions involves receptor-mediated endocytosis of blood-derived LDL that has entered the vessel wall and become modified presumably by oxidation or aggregation (3
). We have recently shown that modification of LDL is not necessary for its uptake and degradation by macrophages. Human monocyte–derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase pinocytosis, constitutively or after activation with PMA, depending on the macrophage phenotype (5
). We believe pinocytosis of fluid-phase rather than bound LDL to be a novel mechanism of lipoprotein-induced macrophage cholesterol accumulation. The current study has shown that this fluid phase–mediated mechanism of macrophage lipoprotein uptake could also occur in vivo in atherosclerotic plaques.
We encountered an unexpected technical problem with the use of pegylated nanoparticles to probe fluid pinocytosis in vivo. Because the nanoparticles do not bind to cell contents or matrix and cannot be fixed in place (by design, the nanoparticles lack reactive functional groups), the particles are not well retained in tissue sections that are rinsed. To overcome this problem, it was optimal to image the fluorescent nanoparticles in DAPI-stained frozen sections without rinsing, fixation, immunostaining, or mounting medium. Then further staining could be carried out, imaged, and superimposed on the nanoparticle image.
The culture model used for the research reported here is characterized by human monocyte–derived macrophages, differentiated in the presence of M-CSF, that constitutively take up large amounts of native LDL by fluid-phase pinocytosis (7
). We showed that these macrophages can also take up by fluid-phase pinocytosis fluorescent nanoparticles similar in size to native LDL. DiI-LDL particles are LDL labeled with the fluorescent lipophilic dye DiI, which diffuses into the hydrophobic portion of LDL without affecting lipoprotein receptor binding (18
). Thus, as expected from our previous studies with native LDL (5
), DiI-LDL, like the pegylated nanoparticles, showed nonsaturable fluid-phase uptake by macrophages. In contrast, DiI-AcLDL showed saturable receptor-mediated uptake by macrophages, consistent with its known interaction with macrophage scavenger receptors (21
After injection of AngioSPARK and quantum dots into Apoe-knockout mice, both types of fluorescent nanoparticles could be localized within aortic arch atherosclerotic lesions that develop in these hypercholesterolemic mice. Macrophages within the atherosclerotic lesions accumulated the nanoparticles. We hypothesized that the mechanism underlying their uptake was fluid-phase pinocytosis, since this process was shown to occur in vitro with nanoparticles treated with plasma from Apoe-knockout mice that were then incubated with bone marrow–derived macrophages from Apoe-knockout mice. Two findings made it unlikely that the fluorescent nanoparticles were taken up by monocytes in the circulation and carried into the vessel wall. Rare CD68-labeled monocytes that were attached to the luminal surface of the vessel wall did not show accumulation of quantum dots. Moreover, when we exposed atherosclerotic aortas removed from Apoe-knockout mice to quantum dots in vitro rather than by injecting the quantum dots into mice, quantum dots also accumulated within atherosclerotic lesion macrophages. This shows that the quantum dots could enter the vessel wall and be taken up by macrophages already present within atherosclerotic lesions.
Our results showed that pinocytosis persists even in lipid-filled macrophage foam cells, because macrophages that showed extensive lipid deposits also accumulated substantial amounts of quantum dots (Figure ). Thus, macrophage lipid accumulation does not downregulate fluid-phase pinocytosis, consistent with our in vitro findings that LDL-induced macrophage cholesterol accumulation does not downregulate macrophage pinocytosis (7
). Unregulated uptake of LDL could eventually lead to toxic levels of cholesterol accumulation within macrophages, contributing to macrophage death, since excess unesterified cholesterol accumulation is one factor shown to trigger macrophage apoptosis (23
Previously, it was shown that a minimum of one-third of LDL catabolism in animals and humans occurs via a receptor-independent route, most prominently in the spleen, liver, small intestine, and kidney (24
). These same tissues show the highest rates of fluid-phase pinocytosis (25
), consistent with the suggestion that receptor-independent catabolism of plasma LDL is due to fluid-phase pinocytosis (26
). Also, consistent with the hypothesis that receptor-independent catabolism of plasma LDL is due to fluid-phase pinocytosis is the finding that receptor-independent catabolism of plasma LDL is a linear function of the plasma LDL concentration (24
). Suppression of macrophage function results in decreased receptor-independent catabolism of LDL and raised LDL levels, suggesting that receptor-independent uptake of LDL is mediated in part by macrophages (26
). Furthermore, a recent study (27
) has shown enhanced fluid-phase (i.e., bulk-phase) uptake of LDL cholesterol in those tissues infiltrated by macrophages in a mouse model of Niemann-Pick type C disease, a cholesterol storage disorder caused by a defect in cellular trafficking of cholesterol.
Our findings here show substantial fluid-phase pinocytosis by macrophages in atherosclerotic plaques. Thus, not only is macrophage fluid-phase pinocytosis of LDL a mechanism for foam cell formation that we have shown occurs with cultured macrophages (5
), but it is also a plausible mechanism to explain foam cell formation of atherosclerotic plaque macrophages. Monocyte differentiation into plaque macrophages is well established. Also, M-CSF is present in plaques (29
), and we have shown that this factor differentiates monocytes into a macrophage phenotype showing constitutive pinocytosis (7
). LDL is present within atherosclerotic plaques at levels that are about twice the blood concentrations of LDL (31
). These levels are greater than 1 mg/ml (i.e.,
2 μM) LDL, and we showed these LDL levels can produce substantial macrophage cholesterol accumulation in human monocyte–derived macrophages (5
). Fluid-phase pinocytosis of LDL produces macrophage LDL uptake that occurs in a linear relationship with LDL concentration. Thus, fluid-phase pinocytosis is an uptake mechanism that can explain why plasma cholesterol concentration is an indicator of risk for the development of coronary artery disease (34
). Also, fluid-phase pinocytosis of LDL can explain why macrophage foam cell formation occurs even in the absence of the scavenger receptors that mediate uptake of oxidized LDL (36
). These findings indicate that future studies aimed at limiting macrophage cholesterol accumulation in atherosclerotic plaques should consider fluid-phase pinocytosis of LDL as a potentially important target pathway.