High-Density Lipoprotein Transport Through Aortic Endothelial Cells Involves Scavenger Receptor BI and ATP-Binding Cassette Transporter G1
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 ≈30% 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 homeostasis
- endothelial cells
- vascular endothelial function
- vascular permeability
Plasma concentrations of high-density lipoprotein (HDL) 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.1–4 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 for albumin.8 Recently, we have shown that ECs bind, internalize, and transport lipid-free apoA-I in a specific manner. The transcellular transport of apoA-I is temperature-dependent, involves the ATP-binding cassette transporter (ABC)A1, and generates a lipidated particle.9,10
In the plasma, the majority of apoA-I is lipid-bound and assembled in spherical HDL. We hypothesized that spherical HDL crosses the endothelium similar to lipid-free apoA-I and therefore investigated binding, uptake, and transport of HDL by cultured aortic ECs and the involvement of the ATP-binding cassette transporters ABCA1 and ABCG1, as well as the HDL receptor scavenger receptor (SR)-BI.
Materials and Methods
Lipoproteins and Cells
Aortic ECs were isolated from bovine aorta and cultured in DMEM (Sigma) supplemented with 5% FCS (medium A) as described previously.10
Plasma HDL (1.063<d<1.21 g/mL) were isolated from fresh normolipidemic human plasma of blood donors by sequential ultracentrifugation as described previously.9,10 HDL was iodinated with Na125I by the McFarlane monochloride procedure as modified for lipoproteins.11 Specific activities of approximately 300 to 700 cpm/ng protein were obtained.
Liposome preparations are described in the expanded Materials and Methods section, available in the online data supplement at http://circres.ahajournals.org.
Interactions of 125I-HDL With Endothelial Cells
Binding, cell association, degradation, internalization, and transport of 125I-HDL were assayed, in principle, as described previously for 125I-apoA-I10 (see the online data supplement).
Small Interfering RNA Transfection
ECs were transfected with 30 nmol/L BLOCK-iT fluorescent oligo and 100 nmol/L Stealth small interfering (si)RNA (Invitrogen) or 100 nmol/L siRNA siGenome ON-TARGETplus SMARTpool duplex (Dharmacon) against either SR-BI, ABCG1, or scrambled as described in the online data supplement.
RNA quantitative PCR was performed as described in the online data supplement.
Western blotting and gel filtration were performed as described in the online data supplement.
HDL Interaction With Aortic Endothelial Cells
At first, the interaction of HDL with ECs was characterized as binding at 4°C, cell association at 37°C, internalization, degradation, and transport. The binding capacity of ECs for HDL was analyzed by using 125I-HDL. Specific binding was calculated by subtracting from the values of the total binding those of the nonspecific binding, which is measured in the presence of a 40-fold excess of unlabeled HDL (Figure 1A). Binding was largely competed with a 40-fold excess of unlabeled HDL and to a much lesser degree but still significantly with LDL but not at all with excess of lipid-free apoA-I and BSA (Figure 1C). Apolipoprotein-free liposomes of different lipid composition did not compete for HDL binding, but adding apoA-I into the liposomes partially restored the competition capacity (Figure 1D). These data argue for a specific receptor-mediated binding of HDL to ECs. ECs bound 125I-HDL in a saturable manner. Scatchard plot analysis of the binding curve revealed multiple binding sites (Figure 1B), with Bmax of 7.3±0.4 ng/mg for the high-affinity and Bmax of 73.7±9.1 ng/mg for the low-affinity binding, respectively. Moreover, the Kd values were 1.2±0.4 μg/mL for high-affinity and 32.1±10.9 μg/mL for low-affinity binding (Figure 1B).
At 37°C, cell association was almost completely competed with a 40-fold excess of unlabeled HDL and partially competed with LDL but not at all with an excess of apoA-I or BSA (Figure 2A). The 125I-HDL-specific cell association Bmax values estimated from the Scatchard plot (data not shown) were 55.7±12.9 ng/mg for the high-affinity binding and 102±26 ng/mg for the low-affinity binding. In addition, the Kd values of HDL cell association (37°C) were similar to the Kd of binding (4°C) and amounted to 2.4±1.2 and 43.9±3.3 μg/mL for the high- and low-affinity sites, respectively.
Next, the cellular distribution of HDL was analyzed. Cell surface biotinylation experiments recovered ≈30% of the total cell-associated 125I-HDL in intracellular compartments (Figure 2B). In contrast, at 4°C almost all 125I-HDL bound to ECs was recovered on the surface, as expected. Internalization of HDL by ECs was further investigated by confocal fluorescence microscopy. ECs were incubated with FITC-HDL together with Alexa 594-transferrin. After 10 minutes of incubation, vesicles containing fluorescent HDL were partially colocalized with Alexa 594-transferrin (Figure 2C), confirming that HDL is internalized by ECs. The degradation of 125I-HDL after 4 hours was measured as the release of radiolabeled degraded amino acids into the medium in the presence or absence of excess unlabeled HDL. The specific degradation was <5% of the specific cell association (data not shown).
Transport of HDL Through a Monolayer of Endothelial Cells
In the vessel wall, the ECs are polarized. To separately analyze the binding affinity of both apical and basolateral sides, we cultured the cells on porous membrane inserts to form a confluent and presumably polarized cell layer. The distribution of the HDL-binding sites in these cells was studied by adding the label either into the apical compartment or into the basolateral compartment. HDL bound specifically to the apical side, but no specific binding to the basolateral side of the cell layer was obtained (Figure 3A). To analyze the accessibility of the cell layer on the basolateral side, as well as the intactness of the barrier function, we measured the permeability coefficient of 3H-inulin, which does not cross cell membranes and hence represents a paracellular transport marker. The tracer was added to the apical chamber, and the filtered radioactivity was measured in the basolateral compartment and vice versa. The permeability coefficients for 3H-inulin across the EC layer were calculated over a time period of 1 hour and were similar for both directions, namely 3.06±0.22×10−5 cm/sec and 3.25±0.03×10−5 cm/sec for apical-to-basolateral and basolateral-to-apical transports, respectively. This indicates that the membrane did not hinder inulin diffusion from the basolateral to the apical compartment (Figure 3B). Furthermore, we assessed the influence of 30 μg/mL HDL in the donor compartment on the permeability of 3H-inulin. The permeability coefficients of 3H-inulin in the presence of HDL were similar, as described above, namely 3.07±0.2×10−5 cm/sec and 3.31±0.02×10−5 cm/sec for apical-to-basolateral and basolateral-to-apical transports, respectively, and not significantly different from the permeability coefficients of 3H-inulin alone (Figure 3B). The data indicate that HDL does not change the barrier function of the monolayer.
With this model, we addressed the question of whether ECs transport HDL through the cell layer. At first, the transport direction of HDL in the insert was analyzed by adding the label in the presence of a 40-fold excess and in the absence of excess cold HDL into either the apical or the basolateral compartments of the cell culture system (Figure 3C). The appearance of 125I-HDL in the opposite compartment was measured. We recorded specific apical-to-basolateral transport but no basolateral-to-apical transport (Figure 3C), whereas inulin permeability was similar in both directions (Figure 3B). This suggests that HDL is transported transcellularly through ECs from apical to basolateral. SDS-PAGE analysis of the radioactive material recovered in the basolateral compartment revealed that the protein moiety of HDL remained intact during transendothelial transport (Figure 4A). Interestingly, gel filtration analysis of the transported material showed a reduced size of the transported particle (Figure 4B). The Stokes diameter of the material harvested in the basolateral compartment after translocation through the EC layer was 9.8±0.03 nm in contrast to 10.6±0.05 nm of the starting material or in contrast to 6.16±0.1 nm for lipid-free apoA-I (Table I in the online data supplement). By contrast, HDL that was filtered through the cell-free porous membrane did not change its size (Online Table I).
The transport capacity of the cells was further investigated after adding the tracer into the apical chamber in the presence or absence of excess cold HDL at different concentrations. The specific transport was calculated by subtracting nonspecific transport from total transport (Figure 5A). The partial competition of 25I-HDL transport through the cell layer suggests the occurrence of a specific transport. To further analyze the transport, the experiment was repeated at 4°C, a temperature that prevents internalization. The transport was almost abolished at this reduced temperature (Figure 5B), which supports the notion that HDL is transported transcellularly.
Identification of Proteins Modulating Transendothelial HDL Transport
To identify proteins that regulate HDL translocation, a candidate-based approach was chosen. SR-BI, ABCG1, and ABCA1 were considered as candidate receptors mediating binding, internalization, and, ultimately, the transport of HDL though ECs. All candidate genes are expressed in ECs. To evaluate their role in HDL binding, we suppressed SR-BI, ABCG1, and ABCA1 expression in ECs individually by the use of RNA interference. SR-BI, ABCA1, and ABCG1 transcriptions were reduced by 80% in cells transfected with SR-BI-, ABCA1-, and ABCG1-specific siRNA, respectively (Online Figure I). By contrast, no significant SR-BI mRNA reduction was observed in cells transfected with the ABCA1-specific siRNA, ABCG1-specific siRNA, or noncoding siRNA (Online Figure I). Similar results were observed for ABCA1- and ABCG1-specific silent cells (Online Figure I). On the protein level, the targeted protein expression was found significantly and specifically downregulated by each gene-specific siRNA (Figure 6A and 6B). The reduction on the protein level was less pronounced compared with corresponding mRNAs, probably because of the stability of the preformed protein.
Knock down of SR-BI reduced both cellular binding (4°C) and cell association (37°C) of HDL by 50% and 35% to 40%, respectively (Figure 6C and 6D). Suppression of ABCG1 reduced both cellular binding and association of HDL by 35% and 35% to 40% (Figure 6C and 6D). Interestingly the partial competition of 125I-HDL binding by LDL (Figure 1C) was completely lost in the SR-BI knockdown cells but not in the ABCG1 knockdown cells, suggesting that SR-BI but not ABCG1 binds LDL in addition to HDL (Figure 6E). Interestingly, and in contrast to SR-BI or ABCG1 silencing, suppression of ABCA1 did not significantly reduce HDL binding and association by ECs. However, the binding of apoA-I (the typical interacting partner of ABCA1) was significantly reduced when ABCA1 expression was diminished (data not shown).9 Thus, the role of ABCA1 in HDL cell internalization or HDL transport was not further studied. We next investigated the participation of SR-BI and ABCG1 in HDL internalization. After diminishing SR-BI or ABCG1 expression, the internalization of HDL was similarly reduced by approximately 40% to 45%, respectively, as compared to control (Figure 7E). We reproduced all findings described above by using additional sets of siRNAs (Online Figure II).
The finding that both SR-BI and ABCG1 participate in HDL cell binding, association, and internalization prompted us to cotransfect the cells with siRNAs against both genes simultaneously. Cosuppression of SR-BI and ABCG1 with specific siRNA resulted in a reduced expression of both genes by 80% on the RNA level (Online Figure I). The protein expression levels of both genes were significantly reduced, as assessed by Western blotting (Figure 7A and 7B). HDL binding and association with SR-BI and ABCG1 cosuppressed cells were reduced by ≈30%, ie, to a similar degree as found in cells with only ABCG1 knockdown (Figure 7C and 7D). In the cotransfected cells, the internalization was reduced by 20% (Figure 7E). In general, the cotransfected cells revealed large variations both within and between experiments.
Finally, the implication of SR-BI and ABCG1 in HDL transport through the ECs was analyzed. HDL transport was reduced by 35% to 40% and 50% after silencing SR-BI or ABCG1, respectively (Figure 8A). Interestingly, whereas silencing of SR-BI had a stronger impact on HDL binding, ABCG1 knockdown had a more pronounced effect in HDL transport. Cosuppression of both genes together reduced transendothelial HDL transport by 30%, ie, to a similar extent as found in cells with a single knockdown of either SR-BI or ABCG1. Moreover, the integrity of the monolayer was verified after the specific siRNA treatment by analyzing the permeability of 3H-inulin (Figure 8B). The permeability values for all transfected cells were similar, namely 2.97×10−5, 2.90×10−5, and 2.69×10−5 cm/sec for EC layers with knock downs of SR-BI, ABCG1, and SR-BI+ABCG1, respectively. These numbers are not significantly higher than in control (ie, 2.61×10−5 cm/sec). Addition of 30 μg/mL HDL slightly reduced the permeability coefficients of 3H-inulin to values of 2.88×10−5, 2.67×10−5, and 2.66×10−5 cm/sec. Also, these values are not significantly different from the permeability coefficients of 3H-inulin alone (Figure 8B). This indicates that the transcellular HDL translocation is modulated by both SR-BI and ABCG1.
In general, transendothelial transport of proteins occurs by paracellular and transcellular pathways. We have previously demonstrated that aortic ECs bind, internalize, and resecrete apoA-I in a competed and temperature-dependent manner.9,10 Furthermore, we demonstrated that ABCA1 but not SR-BI modulates this process.9 In the present study, we extend these findings by showing that ECs also bind, internalize, and transport mature HDL, however, by characteristics that are distinct from those of transendothelial apoA-I transport. Most importantly, SR-BI and ABCG1 but not ABCA1 are rate-limiting for HDL transport.
The presence of different pathways for the transendothelial transport of apoA-I and HDL parallels the need of at least 2 distinct molecules interacting with cells of the arterial wall and other extravascular compartments. Lipid-free apoA-I dissociates from mature HDL as a result of HDL remodeling by lipid transfer proteins and lipases.12–14 Lipid-free apoA-I is important to mediate lipid efflux from macrophage foam cells via the ABCA1 pathway.15 Although this lipid-efflux pathway generates a ligand for ABCG1-mediated lipid efflux, there appears to be additional need for preformed cholesterol acceptors, namely HDL.16 In addition, after lipid loading of HDL from macrophage foam cells, it must leave the intimal space into the blood stream to deliver the acquired lipids to the liver. Also, this step requires the passage through the endothelium.
Consistent with previously published data,17,18 we showed here that ECs specifically bind and associate HDL. Nonspecific binding of HDL to the cell surface can be ruled out because HDL binding was not competed by apolipoprotein-free liposomes but by apoA-I-containing liposomes, suggesting that HDL binding to ECs is protein-mediated (Figure 1D). Interestingly, HDL binding and association are not competed by apoA-I, whereas HDL competes for apoA-I binding and association. At first sight, these contradictory findings are well supported by the fact that lipid-free apoA-I is a poor ligand for SR-BI and ABCG1.15,19 Furthermore, structural analysis of lipid-free apoA-I and apoA-I in HDL particles revealed different folding patterns20 and consequently different functional domains. The partial competition of HDL binding and association by LDL can be explained by the involvement of SR-BI in HDL binding because LDL is also a ligand of this receptor, although with lower affinity than HDL.21 This is further supported by the failure of LDL to compete for HDL binding in SR-BI knockdown cells (Figure 6E).
Interestingly, the binding of HDL to the polarized ECs appears to specifically occur on the apical site of the cells, which is in good agreement with our postulated transport of HDL from the blood into the arterial wall. Because the cells are cultivated on a membrane, one could argue that binding of HDL to the basolateral site is spatially hindered. However, the diffusion of inulin from the apical to basolateral and the basolateral to apical site did not differ significantly, arguing against spatial hindrance. However, the specific binding of HDL to the apical site of aortic ECs raises the question on how HDL particles leave the arterial wall after they have been loaded with cholesterol to complete reverse cholesterol transport. This process likely occurs via vasa vasorum, which grow from the adventitia into the thickening intima. ECs of these microvessels may well differ in their HDL-binding properties from ECs of the macrovasculature such as aorta, which have been used in our experiments.
By both cell surface biotinylation and fluorescent microscopy, we demonstrated that ECs internalize HDL. Like Wüstner et al,22 in hepatocytes, we found FITC-labeled HDL partially colocalized with Alexa 594-conjugated transferrin. However, it is important to note that not all cells internalize HDL, even if they express ABCG1 and SR-BI.16 For example, we have previously demonstrated that cholesterol efflux from RAW macrophages is coupled with internalization of apoA-I but not HDL. Importantly, the majority of the HDL internalized by ECs is not degraded but resecreted as intact proteins.
Because of the cytotoxicity of vesicular transport inhibitors such as N-ethylenimide, filipin, or dansylcadaverine, it was not possible to further study the nature of HDL translocation. For lipid-free apoA-I, we demonstrated by the use of siRNA technology that ABCA1 is a rate-limiting factor in the transport through ECs.9 Therefore, we also used siRNA to analyze which proteins are mediating the specific interactions of HDL with ECs. In agreement with others,23–25 we found that the best candidates were SR-BI, ABCA1, and ABCG1 expressed in ECs of bovine and human origin.26 Their expression was successfully diminished by RNA interference (Figure 5A). As previously published, reduction of ABCA1 expression reduced neither HDL binding nor cell association because lipid-free apoA-I, rather than lipidated apoA-I or HDL, interacts with this transporter.9 Knock down of both SR-BI and ABCG1 had a significant inhibitory effect on HDL-binding cell association and internalization. In addition, the transport of HDL was significantly impaired by suppression of either SR-BI or ABCG1. Interestingly, the transport in ABCG1-diminished cells was even more impaired than by knock down of SR-BI (Figure 8A). These differences may reflect that SR-BI, which is known to bind HDL by protein-protein interactions,27 is a receptor in the proper sense, whereas ABCG1 is a lipid transporter for which the direct protein-protein interaction with HDL has not been proven. It is possible that ABCG1 only indirectly mediates cellular HDL binding by rearranging lipid domains in the plasma membranes that interact with HDL. This is in agreement with a recent publication by Terasaka et al26 describing a nonredundant role of ABCG1 for the regulation of endothelium-dependent vasoreactivity in mice fed a high-cholesterol diet.
The stronger inhibitory effect of SR-BI knockdown on HDL binding than on internalization or transport may reflect that this receptor mediates fluxes of lipids from HDL into cells or from cells to HDL without holoparticle uptake and may explain the reduced particles size of the transported material. However, it is also important to highlight that the siRNA approach used cannot distinguish SR-BI from its splice variant SR-BII, which has been described as an endocytic receptor.28 The lower impact of SR-BI knockdown on HDL cell association and transport than binding indicates that HDL binding on the cell surface is a rate-limiting step in the transendothelial transport of HDL but also the coexistence of downstream effectors of this transport. In fact, the interaction of HDL with SR-BI was previously shown to promote endothelial repair by entailing several intracellular G protein-coupled signaling pathways, such as activation of src, phosphatidylinositol 3-kinase, or mitogen-activated protein kinases.29 It is therefore tempting to speculate that one or several of these SR-BI mediated signaling pathways modulate endocytosis and intracellular transport of HDL (ie, transendothelial transport of HDL). In this regard, it is important to mention that HDL and sphingosine-1-phosphate, which in plasma is predominantly carried by HDL, as well as SR-BI, were previously shown to elicit several signaling effects in ECs,30,31 which favor closure of interendothelial junctions and hence restrict the paracellular pathway of transendothelial transport.32–35
As yet, ABCG1 is only known as an important regulator of lipid efflux from macrophages to HDL. However, Terasaka et al26 reported previously that ABCG1, unlike ABCA1, regulates the activity of endothelial nitric oxide synthase and thereby endothelium-dependent vasorelaxation in mice. The authors provided evidence that this regulatory effect of ABCG1 on nitric oxide production involves cholesterol and oxysterol efflux. Our data show that ABCG1 in ECs participates in HDL internalization and transport (Figures 7 and 8⇑). Furthermore, the lower impact of ABCG1 suppression on HDL binding than on HDL internalization and transport supports the concept that ABCG1 does not directly bind HDL (Figure 6). Also, in the context of the work of Terasaka et al,26 this suggests that ABCG1 modifies the lipid distribution of the plasma membrane and thereby the organization of integral plasma membrane proteins including those involved in endocytosis, such as caveolin or clathrin.
The finding that both SR-BI and ABCG1 participate in HDL binding and cell association prompted us to silence both genes simultaneously to determine whether the 2 proteins interact cooperatively or independently in the binding, internalization, and transport of HDL. Interestingly, the cosuppression of SR-BI and ABCG1 did not decrease HDL binding and cell association more severely than the single knockdowns, although on both the mRNA and protein level, the joint knockdowns were equally as efficient as the single knockdowns. Thus, it appears that SR-BI and ABCG1 cooperate in the modulation of HDL transport rather than act in parallel.
In summary, aortic ECs cultured on porous inserts bind, internalize, and translocate HDL particles from the apical to the basolateral compartment in a specific and temperature-dependent manner, without degrading the protein moiety but reducing the size of the particle. This process is modulated by SR-BI and ABCG1, but not ABCA1, which was previously found to modulate apoA-I transcytosis. Further studies are needed to evaluate the precise function of the identified proteins.
We thank Silvija Radosavljevic for technical assistance.
Sources of Funding
This work is supported by grants from the Swiss National Research Foundation (3100A0-100693/1 and 3100A0-116404/1), as well as by the European Union (LSHM-C-2006-037631).
Original received November 2, 2008; revision received March 28, 2009; accepted April 2, 2009.
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