Abstract-Cholesterol efflux from macrophage foam cells is a rate-limiting step in reverse cholesterol transport. In this process cholesterol acceptors like high-density lipoproteins (HDL) and apolipoprotein (apo)A-I must cross the endothelium to get access to the donor cells in the arterial intima. Previously, we have shown that apoA-I passes a monolayer of aortic endothelial cells (ECs) from the apical to the basolateral side by transcytosis, which is modulated by the ATP-binding cassette transporter (ABC)A1. Here, we analyzed the interaction of mature HDL with ECs. ECs bind HDL in a specific and saturable manner. Both cell surface biotinylation experiments and immunofluorescence microscopy of HDL recovered Ϸ30% of the cell-associated HDL intracellularly. Cultivated on inserts ECs bind, internalize, and translocate HDL from the apical to the basolateral compartment in a specific and temperature-dependent manner. The size of the translocated particle was reduced, but its protein moiety remained intact. Using RNA interference, we investigated the impact of SR-BI, ABCA1, and ABCG1 on binding, internalization, and transcytosis of HDL by ECs. HDL binding was reduced by 50% and 30% after silencing of SR-BI and ABCG1, respectively, but not at all after diminishing ABCA1 expression. Knock down of SR-BI and, even more so, ABCG1 reduced HDL transcytosis but did not affect inulin permeability. Cosilencing of both proteins did not further reduce HDL binding, internalization, or transport. In conclusion, ECs transcytose HDL by mechanisms that involve either SR-BI or ABCG1 but not ABCA1. cholesterol show an inverse association with the incidence of coronary artery disease. The cardioprotective effect of HDL and its major apolipoprotein (apo)A-I are, in part, related to the ability to promote the reverse transport of cholesterol from macrophage foam cells in the arterial intima to the liver for excretion into the bile. [1][2][3][4] An early step in the reverse transport of cholesterol is the transfer of excess cholesterol from the lipid-laden macrophages to HDLs. Importantly, the loading of cellular cholesterol to HDL does not take place in the plasma compartment but in the subendothelial space of arteries. 1 Consequently, HDLs must cross the endothelium to get into close proximity to the cholesterol donor cells. This passage is not well understood. 5 The endothelium lines the vasculature as a single layer of endothelial cells (ECs). As a semipermeable barrier, it regulates the flux of liquid, solutes, and cells between blood and interstitial space. Two principal pathways are known for transendothelial macromolecule translocation, the transcellular transport, including transcytosis, and the paracellular transfer between adjacent cells. 6,7 The paracellular pathway is formed by gaps between ECs, but regulated adherence and tight junctions restrict and control the free passage of macromolecules larger than 6 nm. 7 Endothelial transcytosis, which is defined as vesicle-mediated transport of proteins, has been best investigated ...
The precision and accuracy of this five-pod 3D photosystem suggests its suitability for clinical applications, particularly anthropometric studies. Three-hundred-and-sixty degree surface-contour mapping of the craniofacial region within milliseconds is particularly useful in paediatric patients. Proper patient positioning is essential for high-quality imaging.
Objective-In the reverse cholesterol transport pathway, high-density lipoprotein (HDL) passes the endothelial cell barrier by mechanisms involving the scavenger receptor class B type I and the ATP-binding cassette G1. However, little is known on how inflammation influences this transendothelial transport. Approach and Results-On stimulation with interleukin-6, cultivated primary endothelial cells showed increased binding and transport of 125 I-HDL without changing the expression of scavenger receptor class B type I and ATP-binding cassette G1. Therefore, we analyzed the involvement of endothelial lipase (EL), a known HDL-binding protein expressed by endothelial cells. Here, we show an increased EL expression after interleukin-6 stimulation. Moreover, using pharmacological inhibitors or RNA interference against EL, we demonstrated its participation in HDL binding and transport through the endothelium. Furthermore, adenovirus-mediated transfection of endothelial cells with either catalytically active or nonactive EL revealed that EL facilitates the endothelial binding and transport by both bridging and lipolysis of HDL. EL was also found responsible for the reduction of HDL particle size occurring during the specific transport through a monolayer of endothelial cells. Finally, pharmacological inhibition of EL reversed the inducing effect of interleukin-6 on HDL binding and transport.Conclusions-Interleukin-6 stimulates the translocation of HDL through the endothelium, the first step in reverse cholesterol transport pathway, by enhancing EL expression. In addition, we demonstrated the role of EL in the transendothelial transport of HDL. [13][14][15][16] Moreover, several groups demonstrated the upregulation of EL on inflammatory stimulation both in vitro and in vivo. [17][18][19][20] In this study, we provide evidence that EL participates in endothelial binding, cell association, and transport of HDL. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results IL-6 Induces HDL Binding, Cell Association, and TransportTo investigate whether the interaction of HDL with endothelial cells is changed by the inflammatory cytokine IL-6, we stimulated aortic endothelial cells with IL-6. The interaction of HDL with the endothelial cells was characterized as binding at 4°C, cell association and transport at 37°C. IL-6 induced HDL binding to endothelial cells in a dose-and timedependent manner ( Figure IA and IB in the online-only Data Supplement). HDL binding was significantly increased after incubation with 1-or 10-ng/mL IL-6. Because of the maximal effects seen, all subsequent experiments were performed on cells that were stimulated without 0-or 10-ng/mL IL-6 for 24 hours. After 24 hours of IL-6 stimulation, specific binding was induced by 200±67% compared with not stimulated cells (100±28%; Figure 1A). Conversely, the specific cellularassociated HDL and the transported HDL were also increased by 144±21% and 178±39%, respectively, compared with not stimulated cells (100±17%; Figure 1B a...
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