key: cord-0314743-sutirysr authors: Frey, Kathrin; Goetze, Sandra; Rohrer, Lucia; von Eckardstein, Arnold; Wollscheid, Bernd title: Decoding functional high-density lipoprotein particle surfaceome interactions date: 2022-03-22 journal: bioRxiv DOI: 10.1101/2022.03.22.485140 sha: 7b3f8e54955e6a39a07ba500c6e2a75e03a7a280 doc_id: 314743 cord_uid: sutirysr High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport in the human body and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL’s journey throughout the body, its function is mediated through interactions with cell surface receptors on different cell types. Using four different cellular model systems HDL is interacting with, we comparatively analyzed their surfaceomes to define the HDL receptome. Surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAEC) revealed the main currently known HDL-receptor scavenger receptor B1 (SCRB1), as well as 154 shared cell surface receptors representing potential HDL interaction receptor candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF modulated surfaceome of HAEC using auto-CSC technology. VEGF-A treatment led to a remodeling of the surfaceome of HAEC cells including the previously reported higher surfaceome abundance of SCRB1 upon treatment. 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using HATRIC-based ligand receptor capturing (HATRIC-LRC) technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK), which we show is directly contributing to endothelial HDL binding and uptake. Subsequent proximity ligation assays (PLA) demonstrate furthermore the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence functional HDL binding and uptake. List of human gene and protein names discussed in the paper ABCA1: Phospholipid-transporting ATPase ABCA1 (ABCA1) ABCA7: Phospholipid-transporting ATPase ABCA7 (ABCA7) ABCG1: ATB-binding cassette G1 (ABCG1) ACVR2A: Activin receptor type-2A (AVR2A) APOA1: Apolipoprotein A1 (APOA1) APOB: Apolipoprotein B (APOB) APOC3: Apolipoprotein C-III (APOC3) APOH: Beta-2-glycoprotein 1 (APOH) APOM: Apolipoprotein M (APOM) ATP5MF: Ecto-F1-ATPase (ATPK) CD4: T-cell surface glycoprotein CD4 (CD4) CD14: Monocyte differentiation antigen CD14 (CD14) CD36: platelet glycoprotein 4 (CD36) EGFR: Epidermal growth factor receptor (EGFR) ITGAM: Integrin alpha-M (ITAM) ITGAV: Integrin alpha-V (ITAV) KDR: Vascular endothelial growth factor receptor 2 (VGFR2) KIT: Mast/stem cell growth factor receptor Kit (KIT) MEGF8: Multiple epidermal growth factor-like domains protein 8 (MEGF8) MEGF10: Multiple epidermal growth factor-like domains protein 10 (MEGF10) MERTK: tyrosine-protein kinase Mer (MERTK) MRC2: C-type mannose receptor 2 (MRC2) MSR1: Macrophage scavenger receptor types I and II (MSRE) PCDHAC1: Protocadherin alpha-C1 (PCDC1) PLTP: Phospholipid transfer protein (PLTP) S1PR1: sphingosine 1-phosphate receptor 1 (S1PR1) S1PR2: sphingosine 1-phosphate receptor 2 (S1PR2) S1PR3: sphingosine 1-phosphate receptor 3 (S1PR3) SCARA3: Scavenger receptor class A member 3 (SCAR3) SCARB1: Scavenger receptor B1 (SCRB1) SLC8A3: Sodium/calcium exchanger 2 (NAC2) TF: Transferrin (TRFE) TFRC: Transferrin receptor protein 1 (TFR1) VEGFA: vascular endothelial growth factor A (VEGF-A) Significance statement A molecular understanding of the high-density lipoprotein (HDL) receptome would provide the rational basis for modulating reverse cholesterol transport. Here we used cellular model systems mimicking tissues involved in HDL-trafficking and reverse cholesterol transport to define the HDL receptome. The receptor tyrosine-protein kinase Mer (MERTK) was identified as a novel mediator of HDL binding and uptake in human aortic endothelial cells. Endothelial MERTK resides within the surfaceome proximal to SCRB1 representing a potential co-receptor. The molecular nanoscale organization of the cell-specific surfaceome may influence cellular HDL interactions and functionality. PLTP: Phospholipid transfer protein (PLTP) S1PR1: sphingosine 1-phosphate receptor 1 (S1PR1) S1PR2: sphingosine 1-phosphate receptor 2 (S1PR2) S1PR3: sphingosine 1-phosphate receptor 3 (S1PR3) A molecular understanding of the high-density lipoprotein (HDL) receptome would provide the rational basis for modulating reverse cholesterol transport. Here we used cellular model systems mimicking tissues involved in HDL-trafficking and reverse cholesterol transport to define the HDL receptome. The receptor tyrosine-protein kinase Mer (MERTK) was identified as a novel mediator of HDL binding and uptake in human aortic endothelial cells. Endothelial MERTK resides within the surfaceome proximal to SCRB1 representing a potential co-receptor. The molecular nanoscale organization of the cell-specific surfaceome may influence cellular HDL interactions and functionality. High-density lipoprotein (HDL) is the term given a complex mixture of particles of different sizes, shapes, densities, and compositions. HDL particles contain about 300 proteins (1), a similarly large number of lipid species, and non-coding RNAs (2) . Low plasma levels of HDL containing cholesterol are associated with an increased risk of mortality and other morbidities including atherosclerotic cardiovascular diseases, diabetes, chronic kidney disease, infections, and autoimmune diseases (3) . The causal role of HDL in the pathogenesis of these diseases is controversial in part due to the particle's structural and functional complexity (4) . The classical function of HDL is the delivery of excess cholesterol from peripheral tissues, via lipid-laden macrophages (foam cells), to the liver for biliary excretion. This reverse cholesterol transport (RCT) involves interactions between HDL and cells of various types. For example with endothelial cells to travel between intravascular and extravascular compartments (5), with lipid-laden macrophages (foam cells) for the induction of cholesterol efflux, and lastly with hepatocytes for either selective uptake of cholesterol or holoparticle uptake before excretion (6) . Additionally, HDL is involved in many other signaling events unrelated to RCT such as regulation of the endothelial barrier integrity, angiogenesis, vasoreactivity, and inflammation (5). The molecular mechanisms involved in the binding of HDL to cells that result in particle or lipid uptake and/or signaling are poorly understood, partially because the inventory of HDL receptors appears to be still incomplete. SCRB1 is currently the only confirmed HDL receptor (7) . Binding of HDL to SCRBI mediates the selective uptake of lipids into hepatocytes as well as steroidogenic cells and facilitates cholesterol efflux from macrophages (7, 8) . In endothelial cells, SCRB1 limits holoparticle uptake and mediates several signaling functions of HDL such as stimulation of nitric oxide production, proliferation, migration, and progenitor cell differentiation, inhibition of apoptosis, as well as suppression of adhesion molecule expression and, hence, leukocyte diapedesis (5) . Given the multiplicity of signaling events that appear to be mediated by HDL, it is conceivable that HDL's functionality is mediated by multiple receptor and intracellular adapter proteins (9) . It was recently demonstrated that S1PR1 transiently interacts with SCRB1 to trigger calcium flux and S1PR1 internalization (10) . Calcium flux through S1PR1 is triggered by binding of the ligand S1P, which is enriched on HDL particles carrying APOM (11) . Binding of HDL's core protein component APOA1 to ABCA1 and ecto-F1-ATPase (ATPK) elicits cholesterol efflux and the generation of ADP. The latter activates purinergic receptors to trigger HDL holoparticle uptake into hepatocytes and endothelial cells by an as yet unknown pathway (12, 13) . Finally, platelet glycoprotein CD36, which shares high sequence similarity with SCRB1, reportedly binds HDL specifically on hepatocytes (14) . However, with the exception of SCRB1, which mediates selective lipid uptake, the interactions of HDL with these receptor proteins do not lead to specific HDL-mediated functionalities, suggesting the involvement of other co-receptors and receptor synapses. To better understand the dynamic interplay between surfaceome-residing receptor neighborhoods and HDL functionality, we set out to characterize the HDL receptome using a combination of chemoproteomic technologies including the automated Cell Surface Capturing (auto-CSC) (15) and HATRIC-based ligandreceptor capturing (LRC) (16) . Both proteotyping technologies enable the mass-spectrometric-based identification and quantitation of N-linked glycosylated receptors at the cellular surface. While auto-CSC technology enables the identification of the acute N-glycosylated surfaceome, HATRIC-LRC technology enables the identification of an unknown receptor for a known ligand such as HDL via a trifunctional crosslinker based strategy. To establish a cell surface atlas of potential HDL-interacting proteins, we applied auto-CSC to model systems mimicking tissues relevant for RCT and frequently used in HDL research. Tissuespecific receptor neighborhoods are considered hubs that translate information from the extracellular environment to the cell interior (17) . Such signaling hubs are highly dynamic (18) and are affected by external stimuli that in turn can influence ligand-receptor interactions (17) . We investigated the dynamic behavior of such receptor neighborhoods by treating human endothelial cells with VEGF-A, a known mediator of SCRB1 translocation to the cell surface (19) , which led to major changes in the potential HDLreceptor interaction landscape. Finally, we sought to identify endothelial HDL-receptors in a HATRIC-LRC, which enables direct ligand-receptor capture via a trifunctional linker. The HATRIC-LRC experiments led to the discovery of tyrosine-protein kinase MERTK, a TAM receptor family member involved in the maintenance of vascular cell homeostasis (20) , as a modulator of HDL binding and uptake. To better understand HDL-cell interactions, the underlying surfaceome must be defined. Employing auto-CSC (15), we set out to qualitatively and quantitatively characterize the cellular surfaceomes of model systems mimicking tissues relevant for RCT and frequently used in HDL research ( Fig. 1A and B) : (i) the human endothelial somatic hybrid EA.hy926 cells, (ii) primary human aortic endothelial cells (HAECs), (iii) the human hepatocyte cell line HEPG2, and (iv-vi) the human monocyte THP1 cells before and after activation with phorbol 12-myristate 13-acetate (PMA) and after differentiation into foam cells upon treatment with acetylated LDL (acLDL) (Fig. S1, Table S1 ). Based on the scaled rank of all quantified glycosylated proteins over all samples, the endothelial cells EA.hy926 cells and HAECs clustered in the principal component analysis and the heatmap dendrogram ( Fig. 2A and B , Table S2 ). Although closely related, we observed surfaceome remodeling during the differentiation of THP1 monocytes into THP1 macrophages and upon transformation into lipid laden foam cells ( Fig. S2 and S3) . As previously described, quantitative surface receptor differences induced by stimulation of THP1 with PMA include increased abundances of the activin receptor AVR2A, monocyte differentiation antigen CD14, the integrin ITAM, and the receptor tyrosine kinase MERTK, as well as the decreased abundance of T cell surface glycoprotein CD4, growth factor receptor KIT, and mannose receptor MRC2 (Fig. S4) (21) . In addition, the abundance of ABCA1 on the cellular surface increased during the differentiation of THP1 cells induced by treatment with acLDL, whereas the abundance of SCRB1 decreased upon PMA stimulation but increased again upon differentiation into foam cells. The oxidized LDL (oxLDL) receptor CD36 and the macrophage scavenger receptors MSRE1 and MSRE2 were slightly enriched on foam cells compared to THP1 monocytes and THP1 macrophages. In reverse cholesterol transport, HDL crosses the endothelial barrier and takes up free cholesterol from lipid-laden macrophages in the intima of blood vessels. HDL then travels via the lymph to the liver to deliver cholesterol for biliary excretion. B) THP1 monocytes, PMA-activated THP1 macrophages, acLDL-treated THP1 foam cells, HEPG2 hepatocytes, human aortic endothelial HAECs, and EA.hy926 microvascular endothelial cells were used as cellular model systems to investigate the potential HDL-interacting cellular surfaceome. (1) Cells were first mildly oxidized to (2a) biotinylate the surfaceome for auto-CSC or (2b) to tag receptors proximal to HDL using HATRIC-LRC. (3a and 3b) For auto-CSC, proteins were digested and biotinylated peptides were enriched and (3b) for HATRIC-LRC proteins were on-bead digested (4) on an automated liquid handling system. (5) Peptides were identified and quantified by mass spectrometry. We identified 419, 496, 580, and 497 surfaceome proteins on EA.hy926, HEPG2, foam cells, and HAECs, respectively ( Fig. 2C, Table S3 ). Of these, 155 proteins were detected on the surfaces of all four of these cell types, and 40 were identified exclusively on HAECs and EA.hy926 cells (Fig. 2C , purple bar). One of the proteins identified only on endothelial cells was S1PR1, which is known to mediate several effects of HDL on endothelial cells including barrier integrity, nitric oxide production, and suppression of leukocyte adhesion (22) . The main HDL receptor SCRB1 was identified on all four cell types and most abundantly on HEPG2 cells ( Fig. 2D) . Other receptors involved in cholesterol-or lipoprotein-related biological processes were also differentially abundant on the different surfaceomes. ABCA1 and CD36, for instance, were predominantly detected on foam cells and HEPG2 cells but were less abundant or were not detected on the endothelial cell lines. Furthermore, we detected apolipoprotein APOB on both hepatocytes and foam cells and apolipoprotein APOM and glycoprotein APOH on hepatocytes. Hepatocytes are known to synthesize and secrete apolipoproteins (23) , whereas APOB on foam cells is most likely derived from the acLDL treatment. PLTP, which is known to mediate the transfer of phospholipids and free cholesterol to HDL (24) , was identified on all four RCT-relevant cellular model systems but was only slightly above the lower limit of quantification on HEPG2 cells and HAECs. The different characteristics of the surfaceome landscapes of these models suggest tissue-specific encoding of HDL functionality through receptor neighborhoods. The cellular surfaceome is not a static organization of receptors and lipids. It is continually exposed to extracellular stimuli and therefore reacts to and adapts to environmental changes. Upon HDL uptake by HAECs, VEGF-A triggers the translocation of SCRB1 from an intracellular pool to the plasma membrane (19) . As VEGF-A might also affect the availability of additional co-receptors of HDL on the cellular surface, we assessed surfaceome changes on HAECs treated with VEGF-A. Although SCRB1 was the most prominently affected protein, we observed a significant quantitative reorganization of 165 additional cell-surface receptors (Fig. 3A, Table S4 ). Like SCRB1, MERTK was upregulated in the VEGF-A treated condition. In contrast, PLTP, S1PR1, and S1PR2 were downregulated. Unexpectedly, the decoration of the cellular surface with the VEGF-A receptor VGFR2 was not affected by either presence or absence of its ligand. To globally assess the functional processes of up-or downregulated protein groups, we performed a gene ontology (GO) biological processes and molecular function enrichment analysis (Tables S5 and S6 ). This analysis revealed that there were 23 significantly enriched GO terms for the group of upregulated protein and 12 significantly enriched terms in the group of downregulated proteins. The most enriched GO terms, according to the family-wise error rate, were G protein-coupled receptor binding, which was enriched in the group of downregulated proteins, and virus receptor activity, which was enriched in the group of upregulated proteins (Fig. 3B) . The proteins associated with the most terms were the integrin ITAV (15 terms), SCRB1 (13 terms), and EGFR (13 terms). About 80% of all proteins were specifically associated with three or less GO terms. This analysis showed that VEGF-A not only influences the surface abundance of SCRB1 on HAECs but also quantitatively modulates a large fraction of the cellular surfaceome landscape, which presumably has functional implications. Table S4 for details). Labeled are proteins belonging to the top GO term of each protein group (for upregulated proteins: virus receptor activity; for down-regulated proteins: G protein-coupled receptor binding) as well as MERTK, which was associated with apoptotic cell clearance, regulation of phagocytosis, and regulation of vesicle-mediated transport. B) GO analysis of proteins in HAECs up-and downregulated by VEGF-A treatment. The top 15 terms are labeled based on family-wise error rate (FWER). Non-significant terms with a p value > 0.01 and cellular component terms were excluded from the plot. The size of the dot corresponds to the log.FWER (i.e., smaller FWER corresponds to larger dots). To identify novel direct interactors of HDL on the cellular surface, we performed a HATRIC-LRC experiment on EA.hy926 cells as previously described (16) . EA.hy926 cells were incubated with either lipid-free APOA1, a minimal artificial HDL particle (rHDL) reconstituted from APOA1 and palmitoylphopshatidylcholine (POPC) in a 1:80 molar ratio, or native HDL as ligands. Although lipid-free APOA1 still binds to SCRB1 on human endothelial cells, it does so with a much lower affinity than does APOA1 formulated in a lipid (25) . Both rHDL and native HDL are fully functional and strongly bind to SCRB1. The native HDL is more complex than rHDL (Tabel S7 and (1) and presumably mediates more ligand-receptor interactions at the cellular surface. As a negative control we included TRFE in our HATRIC-LRC experiment; TRFE is the ligand for the transferrin receptor TFR1, a receptor that has not been shown to be involved in HDL signaling. As expected, in our TRFE control, TRFE and its receptor TFR1 were highly enriched in comparison to APOA1, rHDL, and native HDL conditions (Fig. 4A, Table S8 ). In cells treated with APOA1, rHDL, and native HDL conditions, we identified HDL proteins that were significantly enriched compared to the TRFE control (Fig. 4A, Table S8 ). Enrichment increased as the ligand complexity increased. These HDL derived proteins were most likely enriched due to the labeling of the ligand (i.e., HDL) itself. With lipid-free APOA1 as a ligand, we also identified APOC3, most likely an artifact of the APOA1 purification. We did not identify ABCA1 under any condition, most likely because of its relatively low abundance on the cellular surface of EA.hy926 cells (Fig. 2D) . Eleven proteins were identified on cells treated with reconstituted minimal HDL and with native HDL as ligands. Nine are annotated as cell-surface proteins including MERTK and the scavenger receptors SCRB1 and SCAR3. In the rHDL HATRIC-LRC condition, we identified the phospholipid-transporting ATPase ABCA7 and the protocadherin PCDC1 as potential HDL co-receptors. ABCA7 was previously reported to bind APOA1 to mediate phospholipid efflux from cells (26) . In the LRC-HATRIC comparison of native HDL to TRFE as ligand, we identified two surface proteins that are potentially part of the HDL-receptome, the sodium/calcium exchanger NAC2 and multiple epidermal growth factor-like domains protein 8 (MEGF8). Another MEGF family member and scavenger receptor, MEGF10, was shown to bind the complement protein C1Q, which in turn was shown to associate with HDL (27, 28) . Whether MEGF8 also acts as a scavenger receptor remains to be determined. In addition to the significant enrichment in HATRIC-LRC experiments with both rHDL and native HDL (Fig. 4A, Table S8 ), MERTK was also upregulated together with SCRB1 upon VEGF-A treatment of HAECs (Fig. 3A, Table S4 ). MERTK is a receptor tyrosine kinase that regulates many physiological processes including cell survival, migration, differentiation, and phagocytosis of apoptotic cells (20) . It was also shown to mediate efferocytosis in atherosclerotic lesions (29) . Therefore, we selected MERTK as a potential HDL receptor candidate for follow-up validation experiments to confirm its role as HDL-(co)receptor. Using proximity ligation assay (PLA), we confirmed that SCRB1 and MERTK localize in the same neighborhood on the surface of both EA.hy926 cells and HAECs (Fig. 4B) . To show the functional relevance of MERTK in the context of HDL binding and uptake we suppressed MERTK expression using agents that mediate RNA interference ( Fig. S5 and S6) . HDL binding and uptake were significantly reduced in both endothelial cell types after MERTK depletion to an extent similar to that observed after SCRB1 silencing ( Fig. 4C and D) . Ligand binding to the MERTK receptor induces autophosphorylation of the protein on its intracellular domain providing a docking site for downstream signaling molecules (30) . However, when we measured the level of MERTK phosphorylation before and after HDL binding in endothelial cells, we did not detect a difference in the phosphorylation state of the receptor (Fig. S7) . Thus, our data indicate that both SCRB1 and MERTK can modulate HDL binding and uptake and that phosphorylation of MERTK is not likely necessary for this activity. HDL, a multimolecular complex of proteins and lipids, exerts a broad spectrum of functions in different cell types. It is conceivable that the molecular mode of action of HDL with cellular surface proteins is many-tomany rather than one-to-one or many-to-one. We therefore set out to characterize the cellular interaction space of HDL using chemoproteomic technologies. Our cell surface protein atlas provides a first step toward understanding of what we demonstrate are many-to-many interactions. We identified around 500 cell surface proteins in each of the four cellular models of RCT: EA.hy926 cells, HAECs, HEPG2 cells, and foam cells resulting from differentiation of THP1 cells. Of these, only 155 surface proteins were shared by all four cell types. The cellular surfaceome differences imply different functionalities, in line with known tissuespecific roles of HDL. Of the total 1054 proteins characterized across the four RCT relevant cell types, more than 30% were not annotated in Surfy, an in silico surfaceome resource (31) , which highlights the importance of our experimental cell surface protein atlas. By auto-CSC we also captured proteins that are not linked directly to the plasma membrane via transmembrane domains or glycosylphosphatidylinositol anchors but that were associated through interaction with other components of the plasma membrane. Some of these proteins are of high relevance for HDL formation and remodeling. For instance, we identified PLTP on all four cell types. HDL-particle-bound PLTP transfers phospholipids from triglyceride-rich particles to HDL and remodels lipid-poor and protein-rich HDL3 into lipid-rich and protein-poor HDL2 (32) . Cell-bound PLTP within atherosclerotic plaques may serve as a bridging protein to mediate association of HDL with the extracellular matrix (33) . The auto-CSC technology only captures cell surface proteins carrying extracellular Nglycosylation motifs, which comprises the majority of the cellular surfaceome: According to predictions based on Surfy, less than 5% of cell surface proteins do not contain an extracellular N-glycosylation motif (31) . Nevertheless, non-N-glycosylated proteins such as ATPK or ABCG1 also contribute to the uptake of HDL by hepatic and endothelial cells (12, 13, 25) . The analysis of such proteins with respect to HDL functionality will require different technological approaches. Extrinsic factors can trigger changes of the cellular surfaceome and, thereby, HDL-related functionalities. For example, we demonstrated that there is extensive, quantitative remodeling of the endothelial cell surface proteome upon VEGF-A treatment. Corroborating previous findings of our lab (19) , SCRB1 was one of the most VEGF-A-responsive proteins (19) . Interestingly, the majority of receptors upregulated together with SCRB1 upon VEGF-A treatment are associated with gene ontology traits of host entry and virus receptors. VEGF-A treatment promotes vaccinia host entry via activation of the AKT pathway (34), the same mechanism that was also shown to be of relevance for HDL cell endocytosis (19) . HDL particles share structural similarities with lipid-coated viruses in terms of size, mixed protein and lipid cell surface composition, and the cargo of RNA. During infection, viruses and HDL might both co-opt endocytic mechanisms and pathways. Of note, the scavenger receptor family is known to be targeted by different viral particles (27) . In particular, SCRB1, the main HDL receptor, is also involved in hepatitis C virus entry into cells (35) , and, as recently shown, SARS-CoV-2 entry is HDL dependent (36) . VEGF-A treatment of HAECs decreased the abundances of several cell surface proteins including sphingosine-1-phosphate receptors S1PR1 and S1PR2. This finding contrasts the previous report that VEGF-A induces the mRNA expression of S1PR1 (37). We found that S1PR3 was enriched in the VEGF-A treated cells. Both S1PR1 and S1PR3 were previously shown to mediate the effects of HDL on endothelial barrier integrity, nitric oxide production, and leukocyte diapedesis (22) . As VEGF-A stimulation promotes angiogenesis, cell proliferation, and migration (38, 39) , we assume that the ligands S1P and VEGF-A balance each other, to secure endothelial integrity and functionality. Using HATRIC-LRC, we set out to identify the cell surface proteins that interact with HDL on human endothelial cells. We identified MERTK as a novel co-receptor critical for HDL binding and uptake. MERTK was present on the surfaces of all investigated cell types. In macrophages, MERTK was reported to influence atherosclerosis progression through apoptotic cell clearance (40) . In THP1 cells, MERTK mitigates MSRE abundance upon interaction with protein S resulting in decreased acLDL uptake (41) . Both MERTK and HDL mitigate the inflammatory response triggered by lipid-laden macrophages (42) . These findings support a MERTK-dependent link between lipid metabolism and inflammation (20) . Endothelial MERTK has been reported to contribute to the maintenance of endothelial barrier function in human lung microvascular endothelial cells (43) . Furthermore, like HDL, MERTK inhibits neutrophil trans-endothelial migration in vitro (43) . Finally, MERTK facilitates cellular entry of filoviruses (44) , again supporting the hypothesis that viruses and HDL share cellular entry routes. Ligand binding to the MERTK receptor induces its autophosphorylation, but we did not detect a difference in the phosphorylation state of the receptor in the presence of HDL. This might be an indication that HDLmediated functionalities function via pathways that do not involve intracellular MERTK phosphorylation. MERTK may modulate HDL binding and uptake through extracellular interactions with HDL and co-receptors such as SCRB1. We thank Silvija Radosavljevic for her help with HDL, APOA1 and LDL isolation and lipidated APOA1 preparation. We thank Anika Koetemann for her help with establishing the ligand receptor capturing protocol and Audrey van Drogen for assistance with the western blot experiments. We thank J. R. Wyatt for text editing. This work was supported by ETH grant 30 If not mentioned otherwise, experiments and data analysis was performed by KF and SG. HDL-binding and uptake assays were performed by LR. AVE and LR provided expertise and critical feedback at all stages of the project. KF, SG and BW conceived the project and designed experiments. KF and SG wrote the first and subsequent versions of the paper. All authors critically read and revised the manuscript. HAECs (Cell Applications Inc., 304-05a) were grown in Human Endothelial Cell Growth Medium, All-in-one ready-to-use (LONZA, Clonetics CC-3156). LDL (1.019