BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells

Abstract

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis. Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis. It is believed that the initiation of atherosclerosis occurs from the subendothelial retention and accumulation of cholesterol-rich, apolipoprotein B100 (apoB-100) containing particles, primarily low density lipoprotein (LDL) (1Tabas I. Williams K.J. Borén J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: Update and therapeutic implications.Circulation. 2007; 116 (17938300): 1832-184410.1161/CIRCULATIONAHA.106.676890Crossref PubMed Scopus (871) Google Scholar). The phenomenon of LDL permeating the vascular endothelium has been studied for decades, and classic EM studies have shown that this occurs by transcytosis of LDL, independent of the LDL receptor (LDLR) (2Vasile E. Simionescu M. Simionescu N. Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ.J. Cell Biol. 1983; 96 (6853599): 1677-168910.1083/jcb.96.6.1677Crossref PubMed Scopus (237) Google Scholar). Despite the potential importance of LDL transcytosis in atherosclerotic vascular disease, the mechanisms and pathways responsible for this phenomenon have not been explored until relatively recently. Work from our group has shown that a member of the TGF-β type 1 receptor family, activin-like kinase 1 (ALK-1), can serve as a binding protein and potential receptor for the uptake and transcytosis of LDL and very low density lipoprotein, but not oxidized LDL or high density lipoprotein across the vascular endothelium (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar). Interestingly, LDL binding to ALK-1 does promote transcytosis but does not evoke canonical ALK-1–dependent signaling. In contrast, the high affinity ligands, bone morphogenetic protein-9 (BMP-9) and BMP-10, bind to ALK-1 and several TGF-β type 2 receptors to induce the phosphorylation of SMAD1/5/8 and subsequent recruitment of SMAD4 to regulate gene expression (4Goumans M.J. Zwijsen A. Ten Dijke P. Bailly S. Bone morphogenetic proteins in vascular homeostasis and disease.Cold Spring Harb. Perspect. Biol. 2018; 10 (28348038)a03198910.1101/cshperspect.a031989Crossref PubMed Scopus (60) Google Scholar). Although purified BMP-9 does not compete for LDL binding to purified ALK-1, implying different binding sites for BMP-9 versus LDL on ALK-1, if and how BMP-9 regulates ALK-1 internalization, and impact of this on LDL uptake and transcytosis has not been systematically studied. Because fluctuations in BMP-9 plasma levels occur (5Bidart M. Ricard N. Levet S. Samson M. Mallet C. David L. Subileau M. Tillet E. Feige J.J. Bailly S. BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain.Cell Mol. Life Sci. 2012; 69 (21710321): 313-32410.1007/s00018-011-0751-1Crossref PubMed Scopus (100) Google Scholar, 6David L. Mallet C. Keramidas M. Lamandé N. Gasc J.M. Dupuis-Girod S. Plauchu H. Feige J.J. Bailly S. Bone morphogenetic protein-9 is a circulating vascular quiescence factor.Circ. Res. 2008; 102 (18309101): 914-92210.1161/CIRCRESAHA.107.165530Crossref PubMed Scopus (280) Google Scholar) and LDL and very low density lipoprotein levels can exceed the capacity for LDL receptor hepatic clearance during hyperlipidemia, it is feasible that elevated levels LDL may impact the magnitude or duration of BMP-9 function in the vasculature. Thus, the goal of the present study was to examine the relationship between BMP-9 and LDL as ligands for ALK-1, to discern the pathways mediating their internalization and to interrogate the actions of BMP-9 on LDL transcytosis in endothelial cells. BMP-9 and -10 are the cognate ligands for the type 1 receptor, ALK-1; however, little is known about the fate of the receptor after binding BMP-9 or -10. Initially, we assessed if BMP-9 and -10 could induce internalization and endocytosis of ALK-1. HUVECs were surface labeled with a cell-impermeable biotin analog and treated with BMP-9 followed by Western blot analysis of biotin-labeled plasma membrane (PM) proteins. As seen in Fig. 1A, BMP-9 treatment for 30 min did not change the levels of total ALK-1 in cell lysates (input, bottom panel) but dose-dependently reduced ALK-1 levels, but not VE-cadherin (VECAD), in PM (top panel and quantified in Fig. 1B). As little as 100 pg/ml of BMP-9 triggered internalization of ∼30% of ALK-1 whereas 100 ng/ml reduced ALK-1 on the cell surface by greater than 90%. BMP-9 (10 ng/ml)–mediated internalization of ALK-1 occurred rapidly (within 1 min) and maximal internalization was achieved after 60–120 min of BMP-9 treatment (Fig. 1C and quantified in Fig. 1D). Several EC plasma membrane proteins were quantified and BMP-9 (10 ng/ml) did not affect the levels of VECAD, VEGFR2, or CD31 (Fig. S1A and quantified in Fig. S1B). Similar data were obtained using BMP-10 as a ligand for ALK-1; however, BMP-10 was less efficient (Fig. S1, C and D). Moreover, neither TGF-β1 (10 ng/ml) nor VEGF-A (10 ng/ml), ligands that activate endothelial cell signaling, affected ALK-1 internalization (Fig. S1, E and F). Thus, ALK-1 is selectively and efficiently internalized by specific ligands, BMP-9 and -10, in endothelial cells. Next, we sought to determine which endocytic pathway is responsible for BMP-9–mediated ALK-1 internalization. Ligand-induced endocytosis in endothelial cells can be mediated through several pathways including clathrin (clathrin heavy chain; CHC) or caveolin (CAV-1) dependent entry, both of which utilze dynamin-2 (DNM2) as the intracellular GTPase critical for membrane fission. Thus, HUVECs were treated with siRNAs to reduce levels of CAV-1, DNM2, and CHC (Fig. S2, A and B for siRNA validation) and BMP-9–mediated ALK-1 internalization from the PM assessed. As seen in Fig. 2A (and quantified in Fig. 2B), BMP-9 (0.5 ng/ml for 30 min) induced the internalization of ALK-1, an effect attenuated by knockdown of CAV-1 and DNM2, but not CHC. Interestingly, silencing of CAV-1 enhanced the basal levels of ALK-1 in PM. To examine this using a different method, the amount of internalized ALK-1 (versus ALK-1 remaining on the cell surface) was quantified after cleavage of biotin on the cell surface. Indeed, knockdown of DNM2 and CAV-1, but not CHC, reduced BMP-9 stimulated ALK-1 internalization (Fig. 2C and quantified in Fig. 2D). Next, we took a genetic approach to examine if the loss of CAV-1 affected ALK-1 endocytosis. Mouse lung endothelial cells (mLEC) were isolated from WT and CAV-1 knockout (CAV-1KO) mice and BMP-9–mediated ALK-1 internalization assessed. As seen in Fig. 2E, the loss of CAV-1 reduced BMP-9–mediated ALK-1 endocytosis. Thus, BMP-9 mediates ALK-1 internalization, in part, via a CAV-1–dependent pathway. To investigate the relationship between BMP-9–induced ALK-1 internalization and signaling, BMP-9 stimulated SMAD1/5 phosphorylation and SMAD-dependent gene expression was measured. Depletion of CAV-1 (Fig. 3A and quantified in Fig. 3B) reduced BMP-9–stimulated SMAD1/5 phosphorylation, whereas depletion of CHC did not impact signaling (Fig. 3, C and D). Next, a panel of BMP-9–inducible genes were quantified in HUVEC after either CAV-1 or ALK-1 silencing. As seen in Fig. 3E, BMP-9 (0.5 ng/ml) induced expression of canonical SMAD1/5-dependent genes, TMEM100, ID1, and SMAD6, and this induction was reduced by CAV-1 depletion and abrogated by ALK-1 silencing. BMP-9 did not affect the expression of ALK-2 or CAV-1. Similar results were obtained with different concentrations of BMP-9 (Fig. S3, A–C). Thus, CAV-1 is critical for BMP-9–stimulated ALK-1 endocytosis, signaling, and gene expression. To explore the spatial connection between CAV-1 and ALK-1, confocal microscopy was employed and endogenous CAV-1 and ALK-1 colocalize in HUVECs (Fig. 4A). To examine if BMP-9 impacts recruitment of BMPR2, a type 2 receptor critical for ALK-1–dependent signaling to SMAD1/5 (7David L. Mallet C. Mazerbourg S. Feige J.J. Bailly S. Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells.Blood. 2007; 109 (17068149): 1953-196110.1182/blood-2006-07-034124Crossref PubMed Scopus (473) Google Scholar), HUVECs were treated with BMP-9 for 10 min and immunocomplex formation examined by immunoprecipitation of ALK-1. As seen in Fig. 4B, precipitation of ALK-1 resulted in the co-association of CAV-1 (in the absence and presence of BMP-9 treatment), and this complex was not found in control precipitates with an isotype control Ab. Interestingly, precipitation of ALK-1 under BMP-9 stimulated conditions resulted in the co-association of CAV-1 and BMPR2 with the complex. Detergent-free isolation of CAV-1/lipid raft–enriched domains from HUVECs showed that ALK-1 is exclusively located in buoyant, CAV-1–enriched membranes (Fig. 4C; fractions 4 and 5), whereas BMPR2 is in both light and heavy membranes (fractions 9–12) similar to that seen with endothelial nitric oxide synthase (eNOS). Previous work uncovered ALK-1 as receptor for native LDL transcytosis in endothelial cells. The binding occurs through apoB-100 on the LDL particle, is independent of BMP-9 binding, and does not directly activate SMAD1/5 signaling (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar). Thus, we examined if LDL-mediated internalization of ALK-1 affected BMP-9 signaling to SMAD1/5. Preincubation of HUVEC in lipoprotein-depleted serum (LPDS) containing increasing concentrations of LDL resulted in dose-dependent suppression of BMP-9–mediated SMAD1/5 activation (Fig. 5A and quantified in Fig. 5B). Incubation with LDL blunted the increase in SMAD1/5 phosphorylation in response to different concentrations of BMP-9 (Fig. 5C and quantified in Fig. 5D), and this occurred in a time-dependent manner (Fig. 5E and quantified in Fig. 5F). Accordingly, the reduction in SMAD1/5 phosphorylation triggered by LDL-blunted BMP-9–induced gene expression of TMEM100 and ID1, but not SMAD6 (Fig. 5G). Next, we tested if LDL could serve as a nonclassical ligand for ALK-1. Treatment of HUVEC with LDL (2.5–100 μg/ml for 60 min) did not result in SMAD1/5 phosphorylation whereas 0.5 ng/ml of BMP-9 robustly induced SMAD1/5 phosphorylation (Fig. S4A). These data are consistent with previous work showing that loss of function or gain of function mutants of the kinase domain of ALK-1 equally internalized LDL (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar). Interestingly, LDL time-dependently reduced ALK-1 levels on the plasma membrane in HUVECs replete with serum (Fig. 6A) or in cells in LPDS (Fig. S4B and quantified in Fig. S4C). LDLR is a high affinity receptor for LDL on the endothelium that mediates the uptake and distribution of LDL-derived cholesterol to cells, whereas ALK-1 and serve as receptors for LDL transcytosis (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar, L. X. E. S. M. P. K. A. L. C. endothelial cell LDL transcytosis via to promote PubMed Scopus Google Scholar, Sugiyama M.G. C. P. M. C. B. et an role for in LDL Res. PubMed Scopus (64) Google Scholar), but not for the of LDL cholesterol to intracellular membranes X. C. transcytosis of in 2018; PubMed Scopus Google Scholar). Thus, we the internalization of LDL the time-dependent internalization of both LDLR and ALK-1, but not (Fig. and quantified in Fig. BMP-9 treatment reduced ALK-1 protein levels in the PM LDLR or levels (Fig. and quantified in Fig. The of effect of BMP-9 on LDLR levels was by in cells (Fig. and To examine the pathway of internalization of ALK-1, cells were treated with siRNAs CAV-1, DNM2, and CHC. As seen in Fig. and quantified in Fig. of CAV-1 and DNM2, but not CHC, reduced LDL-mediated ALK-1 endocytosis. Previous work has shown that ALK-1, in part, mediates LDL transcytosis across the the LDLR receptor is and transcytosis occurs independent of the LDLR via ALK-1 or X. C. transcytosis of in 2018; PubMed Scopus Google Scholar). Thus, we EC to the of BMP-9 versus ALK-1 silencing on LDL transcytosis (Fig. arterial were treated with BMP-9 (10 ng/ml for 60 min) to reduce ALK-1 on the plasma membrane followed by transcytosis by total microscopy BMP-9 treatment reduces LDL and this effect is not reduced in cells of ALK-1 (Fig. Thus, BMP-9–induced endocytosis of ALK-1 LDL-mediated transcytosis in of BMP-9 and LDL could ALK-1 internalization, which is the to signaling or transcytosis (Fig. The goal of this study was to explore the potential crosstalk between BMP-9 and LDL as proteins that bind to ALK-1. we a BMP-9–mediated internalization pathway for ALK-1 that is for SMAD1/5 phosphorylation and subsequent gene expression. BMP-9–mediated ALK-1 endocytosis both CAV-1 and and of CAV-1 reduces BMP-9–stimulated ALK-1 internalization, SMAD1/5 phosphorylation, and gene expression. Interestingly, LDL treatment of EC promotes ALK-1 internalization and reduces BMP-9–stimulated SMAD1/5 treatment of EC with BMP-9 reduces LDL transcytosis in an ALK-1–dependent implying that the levels of BMP-9 may regulate LDL transcytosis and the of The of BMP-9 and -10 in the have on their in during and the to genetic showing that loss of function in ALK-1, or its to In mice and the loss of ALK-1 reduces endothelial cell to activation of signaling pathways vascular (4Goumans M.J. Zwijsen A. Ten Dijke P. Bailly S. Bone morphogenetic proteins in vascular homeostasis and disease.Cold Spring Harb. Perspect. Biol. 2018; 10 (28348038)a03198910.1101/cshperspect.a031989Crossref PubMed Scopus (60) Google Scholar, H. of the ALK1 ligands, BMP9 and 2016; PubMed Scopus Google Scholar). In the of BMP-9 or has been shown to in of L. A. Mallet C. L. A. C. R. J. A. A. L. Feige J.J. M. et BMP-9 Res. PubMed Scopus Google Scholar, L. X. M. S. M. B. J.M. et of endothelial with BMP9 arterial PubMed Scopus Google Scholar). In with ALK-1 levels are in A. E. K. kinase receptor 1 (ALK1) in atherosclerotic and vascular Res. 2007; PubMed Scopus Google Scholar) and of signaling in reduces the of atherosclerotic M. R. E. of bone morphogenetic protein signaling reduces vascular and Biol. 2012; PubMed Scopus Google Scholar, X. C. of bone morphogenetic proteins atherosclerosis and vascular Res. PubMed Scopus Google Scholar). Previous work by our group has shown of ALK-1 reduces LDL uptake in mice the LDLR (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar). Because BMP-9 and -10 are high affinity ligands for ALK-1 and their levels with (5Bidart M. Ricard N. Levet S. Samson M. Mallet C. David L. Subileau M. Tillet E. Feige J.J. Bailly S. BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain.Cell Mol. Life Sci. 2012; 69 (21710321): 313-32410.1007/s00018-011-0751-1Crossref PubMed Scopus (100) Google Scholar), it is feasible that the elevated levels of LDL during may bind to ALK-1, in its transcytosis and subendothelial retention of Interestingly, in the present we that CAV-1 is for both LDL and BMP-9–mediated ALK-1 endocytosis. is it is that the loss of CAV-1 in the endothelium reduces LDL and strongly atherosclerosis C. J. C. A. a critical role of endothelial caveolin-1 during the of 10 PubMed Scopus Google Scholar, Lee H. Park of caveolin-1 Biol. PubMed Scopus Google Scholar, C.M. Zhang X. C. N. Sugiyama M.G. B. X. S. J.R. H. et regulates by low-density lipoprotein transcytosis and vascular of endothelial nitric oxide synthase PubMed Scopus Google Scholar). The loss of CAV-1 reduces LDL uptake in isolated and in and reduces levels in the as using several endogenous and LDL C. J. C. A. a critical role of endothelial caveolin-1 during the of 10 PubMed Scopus Google Scholar, C.M. Zhang X. C. N. Sugiyama M.G. B. X. S. J.R. H. et regulates by low-density lipoprotein transcytosis and vascular of endothelial nitric oxide synthase PubMed Scopus Google Scholar). Previous work has shown that ALK-1 can be in C. and with the type receptor ALK1 in endothelial Res. 2008; PubMed Scopus Google Scholar). we and that ALK-1 can be in complex with CAV-1 and the type 2 receptor, the loss of CAV-1 but does not BMP-9 signaling, that CAV-1 regulates ALK-1 signaling the of the plasma The work a role for the endocytic in signaling in is a showing that the clathrin 2 is for the function of during vascular H. B. Lee M.Y. M. A. function of bone morphogenetic protein signaling is mediated by 2012; PubMed Scopus Google Scholar). of ALK-1 endocytosis by CAV-1 or in our study from pathways that on of BMP-9 signaling by and protein (4Goumans M.J. Zwijsen A. Ten Dijke P. Bailly S. Bone morphogenetic proteins in vascular homeostasis and disease.Cold Spring Harb. Perspect. Biol. 2018; 10 (28348038)a03198910.1101/cshperspect.a031989Crossref PubMed Scopus (60) Google Scholar). In the present we that treatment with BMP-9 to promote extensive ALK-1 internalization reduces LDL transcytosis to levels to the loss of ALK-1, implying that elevated BMP-9 levels may be and that the loss of ALK-1 in reduce LDL transcytosis. have identified the lipoprotein receptor as an LDL transcytosis L. X. E. S. M. P. K. A. L. C. endothelial cell LDL transcytosis via to promote PubMed Scopus Google Scholar, Sugiyama M.G. C. P. M. C. B. et an role for in LDL Res. PubMed Scopus (64) Google however, treatment with BMP-9 effect on internalization, are pathways that LDL transcytosis. Thus, our data are consistent with BMP-9 and LDL using the pathway to ALK-1 internalization. the ligand are BMP-9–mediated internalization is critical for signaling to whereas LDL-mediated internalization mediates and mechanisms of receptor internalization are not BMP-9 is mainly produced in by hepatocytes and cells, and of it is in the active form in the BMP-9 and signaling and has been as a quiescence in mice L. Mallet C. Keramidas M. Lamandé N. Gasc J.M. Dupuis-Girod S. Plauchu H. Feige J.J. Bailly S. Bone morphogenetic protein-9 is a circulating vascular quiescence factor.Circ. Res. 2008; 102 (18309101): 914-92210.1161/CIRCRESAHA.107.165530Crossref PubMed Scopus (280) Google Scholar, B. C. E. R. A. M. A. ALK1 signaling by with the 2012; PubMed Scopus Google Scholar). In in the endothelium to the actions of BMP-9 N. B. Ola R. B. A. B. E. M. H. A. mediates Cell Biol. 2016; PubMed Scopus Google Scholar) and may be to ALK-1 internalization to the of LDL transcytosis. to the actions of BMP-9 and -10 LDL binding to ALK-1 of this pathway in HUVECs were obtained from the of and were from isolation was (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google mice were using and for mice after were to endothelial cells. and from WT or were for of endothelial cells were in with serum and in a with LDL and were obtained from BMP-9 and was from was to and the of In for surface after the on with a 1 cells were with a and in immunoprecipitation the endocytosis of the the biotin-labeled cells were treated with BMP-9 or LDL for in with the biotin-labeled receptors remaining on the cell surface were using 1 and min on protein was were and was fractions were after the a The were several in to Detergent-free isolation was as M. between signaling mechanisms in Sci. S. A. PubMed Scopus Google Scholar). In HUVECs were to in with cells were with 1 with a of and was the bottom of and to by the of 2 of in 0.5 A to was of and 4 of both in containing and for in a fractions and a were from the and with and of were by and protein distribution assessed by or were on with containing 100 10 1 1 1 and 2 and protein was followed by to was with the LDLR ALK-1 DNM2 Cell BMPR2 CAV-1 were on the were on using As in our previous study (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar), HUVECs were with for 10 min in in for 1 The cells were with ALK-1 and CAV-1 with a of in for were using a and with were on a confocal The surface LDLR was using (3Kraehling J.R. Chidlow J.H. Rajagopal C. Sugiyama M.G. Fowler J.W. Lee M.Y. Zhang X. Ramírez C.M. Park E.J. Tao B. Chen K. Kuruvilla L. Larriveé B. Folta-Stogniew E. Ola R. et al.Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.Nat. Commun. 2016; 7 (27869117)1351610.1038/ncomms13516Crossref PubMed Scopus (64) Google Scholar). HUVECs were from using in were and labeled with or as 1 the cells were with for 30 were by and using In from cells or were isolated using the 0.5 was with the were in using the were obtained from were using the LDL transcytosis by was by cells were in a cell and treated with in for 10 min to binding internalization. were with to LDL and was were on the cell for 2 min of the were after with and microscopy of the basal membrane was to microscopy was on an with and and and with were between of were of LDL transcytosis was and has been in S. Sugiyama M.G. Lee LDL transcytosis by endothelial cells via and receptor type Biol. 2018; PubMed Scopus Google Scholar). were with an or with for A of was analysis was with data are in the and with low density lipoprotein LDL receptor endothelial cell plasma membrane VE-cadherin endothelial cell vascular endothelial clathrin heavy lung endothelial cells lipoprotein-depleted serum arterial EC total microscopy analysis of

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