Ceramide activation of RhoA/Rho kinase impairs actin polymerization during aggregated LDL catabolism
2017; Elsevier BV; Volume: 58; Issue: 10 Linguagem: Inglês
10.1194/jlr.m076398
ISSN1539-7262
AutoresRajesh Kumar Singh, Abigail S. Haka, Alexandria Brumfield, Inna Grosheva, Priya Bhardwaj, Harvey F. Chin, Yuquan Xiong, Timothy Hla, Frederick R. Maxfield,
Tópico(s)Caveolin-1 and cellular processes
ResumoMacrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation. Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation. A critical initiating event in atherogenesis is the progressive deposition of LDL in the arterial wall (1.Tabas I. Williams K.J. Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications.Circulation. 2007; 116: 1832-1844Crossref PubMed Scopus (977) Google Scholar). This LDL becomes modified, aggregated, and retained. Macrophages encountering such deposits are unable to use standard phagocytic or endocytic mechanisms to catabolize this aggregated LDL (agLDL). Instead, they form an intimate contact with the agLDL, a lysosomal synapse (LS) (2.Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar, 4.Singh R.K. Barbosa-Lorenzi V.C. Lund F.W. Grosheva I. Maxfield F.R. Haka A.S. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.J. Cell Sci. 2016; 129: 1072-1082Crossref PubMed Scopus (25) Google Scholar). The LS is characterized by local actin polymerization (3.Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar), exocytosis of lysosomal contents (2.Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar), and regions of low pH at macrophage contact sites with agLDL (2.Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar). We have shown previously that actin polymerization is important for formation of the LS, because it drives macrophage plasma membrane contact with agLDL (4.Singh R.K. Barbosa-Lorenzi V.C. Lund F.W. Grosheva I. Maxfield F.R. Haka A.S. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.J. Cell Sci. 2016; 129: 1072-1082Crossref PubMed Scopus (25) Google Scholar). Exocytosis of lysosomal contents into the LS allows delivery of hydrolytic enzymes, such as lysosomal acid lipase and acid SMase residing in the lysosomes, which promote catabolism of agLDL and generation of catabolites such as free cholesterol (3.Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar, 4.Singh R.K. Barbosa-Lorenzi V.C. Lund F.W. Grosheva I. Maxfield F.R. Haka A.S. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.J. Cell Sci. 2016; 129: 1072-1082Crossref PubMed Scopus (25) Google Scholar). This free cholesterol promotes macrophage actin polymerization, likely through activation of Rac/Cdc42 GTPases (3.Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar). Free cholesterol at the LS that is subsequently internalized by the macrophage promotes foam cell formation (3.Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar). We have postulated that release of free cholesterol from macrophages into the extracellular space during agLDL catabolism may be a precursor for the formation of extracellular cholesterol crystals. Apart from free cholesterol, other metabolites are also produced in the microenvironment of the plaque. In the atherosclerotic plaque, lesional agLDL is known to be rich in ceramide, and it contains 10- to 50-fold-higher content of ceramide when compared with plasma LDL (5.Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (269) Google Scholar). Ceramides are usually found within cellular membranes, and studies have shown that they can act as potent signaling mediators regulating processes such as cell differentiation and proliferation (6.Sharma K. Shi Y. The yins and yangs of ceramide.Cell Res. 1999; 9: 1-10Crossref PubMed Scopus (19) Google Scholar). Ceramide can be generated by acid SMase that exists in two forms, a lysosomal acid SMase (L-SMase) that requires a low pH for activity (7.Callahan J.W. Jones C.S. Davidson D.J. Shankaran P. The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups.J. Neurosci. Res. 1983; 10: 151-163Crossref PubMed Scopus (36) Google Scholar) and a secretory acid SMase (S-SMase), which is not localized to the lysosome and can function at neutral pH (8.Schissel S.L. Jiang X. Tweedie-Hardman J. Jeong T. Camejo E.H. Najib J. Rapp J.H. Williams K.J. Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development.J. Biol. Chem. 1998; 273: 2738-2746Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). S-SMase has been reported to be responsible for conversion of sphingomyelin to ceramide in LDL, whereas L-SMase has been implicated in cellular signaling and apoptosis (9.Jenkins R.W. Canals D. Hannun Y.A. Roles and regulation of secretory and lysosomal acid sphingomyelinase.Cell. Signal. 2009; 21: 836-846Crossref PubMed Scopus (224) Google Scholar). The major pathway of generation of ceramide is through activation of L-SMase, which can cleave sphingomyelin to generate ceramide (10.Bismuth J. Lin P. Yao Q. Chen C. Ceramide: a common pathway for atherosclerosis?.Atherosclerosis. 2008; 196: 497-504Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Generation of ceramide through activation of L-SMase occurs in response to ionizing radiation, UV exposure and TNFα treatment, and can be thought of as a stress signal, inducing cell apoptosis under these conditions (11.Kolesnick R.N. Kronke M. Regulation of ceramide production and apoptosis.Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (728) Google Scholar, 12.Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). Defective function of acid SMase in humans results in Niemann-Pick disease types A and B (13.Schneider P.B. Kennedy E.P. Sphingomyelinase in normal human spleens and in spleens from subjects with Niemann-Pick disease.J. Lipid Res. 1967; 8: 202-209Abstract Full Text PDF PubMed Google Scholar), and interestingly, absence of acid SMase in mice results in the inability to signal apoptosis (14.Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar). Much attention has been paid to the role of ceramide in atherosclerosis, particularly in the vascular system, where it functions as a critical second messenger in many atherosclerotic processes (10.Bismuth J. Lin P. Yao Q. Chen C. Ceramide: a common pathway for atherosclerosis?.Atherosclerosis. 2008; 196: 497-504Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, few studies have focused on the role of ceramide in macrophage-specific modulation of atherogenic processes. This is surprising because macrophage-derived SMase has been shown to induce partial digestion and aggregation of LDL (15.Jeong T. Schissel S.L. Tabas I. Pownall H.J. Tall A.R. Jiang X. Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase.J. Clin. Invest. 1998; 101: 905-912Crossref PubMed Scopus (120) Google Scholar). Furthermore, when ceramide is utilized in studies, a short-chain (C2-ceramide) analog of ceramide is often used because it is more water soluble than are longer-chain, biologically relevant ceramides. In this study, we used both long-chain C16-ceramide (as well as C2-ceramide) and bone marrow-derived macrophages cultured from sphingosine kinase 2 KO (SK2KO) mice, found previously to contain elevated levels of longer-chain ceramides (16.Xiong Y. Lee H.J. Mariko B. Lu Y.C. Dannenberg A.J. Haka A.S. Maxfield F.R. Camerer E. Proia R.L. Hla T. Sphingosine kinases are not required for inflammatory responses in macrophages.J. Biol. Chem. 2013; 288 ([Erratum. 2016 J. Biol. Chem. 291: 11465.]): 32563-32573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We used these strategies to replicate elevated levels of ceramide that macrophages experience in the microenvironment of the plaque and to investigate the role of ceramide in agLDL catabolism and foam cell formation. We confirm that macrophages accumulate ceramide in atherosclerotic plaques. By taking a microscopy approach, we show that this ceramide can inhibit actin polymerization specifically at the LS and foam cell formation in response to agLDL in a RhoA/Rho kinase-dependent manner. We also show that ceramide from agLDL can be taken up by macrophages and can regulate macrophage agLDL catabolism. These data highlight a novel regulatory pathway that macrophages use to modulate agLDL catabolism and provide a new context in which to view ceramide signaling during foam cell formation and atherogenesis. J774a.1 macrophages and RAW264.7 macrophages (American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere (5% CO2) at 37°C and used at low passage numbers. Cells were confirmed to be contamination free. Bone marrow-derived macrophages (BMMs) were cultured as follows. Bone marrow was isolated from female mice ages 6–13 weeks of age. We flushed sterilized femurs and tibias from wild-type (C57BL/6), Sphk1flox/flox Sphk2−/− (SK2KO), Sphk1−/−Sphk2−/− [Sphingosine kinase 1 (SK1)/2KO], S1pr1−/− [Sphingosine-1-phosphate 1 (S1P1) KO], and S1pr2−/− (S1P2 KO) mice on a C57BL/6 background [as has been described previously (16.Xiong Y. Lee H.J. Mariko B. Lu Y.C. Dannenberg A.J. Haka A.S. Maxfield F.R. Camerer E. Proia R.L. Hla T. Sphingosine kinases are not required for inflammatory responses in macrophages.J. Biol. Chem. 2013; 288 ([Erratum. 2016 J. Biol. Chem. 291: 11465.]): 32563-32573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17.Michaud J. Im D.S. Hla T. Inhibitory role of sphingosine 1-phosphate receptor 2 in macrophage recruitment during inflammation.J. Immunol. 2010; 184: 1475-1483Crossref PubMed Scopus (107) Google Scholar, 18.Blaho V.A. Galvani S. Engelbrecht E. Liu C. Swendeman S.L. Kono M. Proia R.L. Steinman L. Han M.H. Hla T. HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation.Nature. 2015; 523: 342-346Crossref PubMed Scopus (158) Google Scholar)] and from ASM−/− (A-SMaseKO) and wild-type control [as has been described previously (19.Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar)], and cells were differentiated for 7 days by culture in DMEM supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin, supplemented with 20% L-929 cell-conditioned media in a humidified atmosphere (5% CO2) at 37°C. Control and KO mice were housed in the same facility, in a pathogen-free environment at Weill Cornell Medical College, and used in accordance with protocols approved by the Institutional Animal Care and Utilization Committees. AlexaFluor546 (Alexa546), LipidTOX Green, LipidTOX Deep Red, Alexa488-phalloidin, Alexa647-phalloidin, and BODIPY-FL-C5-ceramide were purchased from Invitrogen. C16-ceramide (d18:1/16:0) and C2-ceramide (d18:1/2:0) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Monoclonal anti-ceramide antibody (clone MID 15B4) and desipramine were purchased from Sigma-Aldrich (St. Louis, MO). Y-27632 was purchased from Tocris Bioscience (Bristol, UK). Rho inhibitor I was purchased from Cytoskeleton, Inc. (Denver, CO). Phospho(Thr18/Ser19)-Myosin Light Chain 2 antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-RhoA and anti-calnexin antibodies were purchased from Abcam (Cambridge, MA). pcDNA3-enhanced green fluorescent protein (EGFP) was a gift from Doug Golenbock (Addene plasmid #13031). pcDNA3-EGFP-RhoAwt [WTRhoA-green fluorescent protein (GFP)] and pcDNA3-EGFP-RhoAQ63L (CARhoA-GFP) were a gift from Gary Bokoch (Addgene plasmid #12965 and #12968). ApoE−/− mice were obtained from Jackson Laboratories and placed on a high-fat diet (21% milk fat, 0.15% cholesterol; Harlan Teklad) for 13 weeks. Mice were euthanized and perfused with PBS, and aortas were taken for sectioning. Aortas were fixed overnight in 3% paraformaldehyde at 4°C. Fixed aortas were placed in a solution of 30% sucrose in PBS and stored at 4°C overnight. Aortas were then gently agitated in embedding media (1:2 ratio of 30% sucrose in PBS in OCT medium) and then frozen in the same media using 2-methylbutane and liquid nitrogen. Samples were then cut into 8 μm sections using a Cryostat, mounted onto glass slides and coverslips attached using Vectorshield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). After blocking with 10% goat serum for 1 h, macrophages were identified using a rabbit polyclonal antibody for F4/80 (Abcam ab100790, Cambridge, MA) at 1:300 dilution overnight at 4°C and AlexaFluor546 anti-rat secondary antibody (Invitrogen) at 1:400 dilution for 2 h at room temperature. Ceramide was stained using a mouse monoclonal antibody (clone MID 15B4) at 1:100 dilution overnight at 4°C and AlexaFluor488 anti-mouse secondary antibody (Invitrogen) at 1:400 dilution for 2 h at room temperature. All antibody labeling was carried out in PBS containing 3% goat serum. Images were acquired with a Zeiss LSM 510 laser-scanning confocal microscope (Thornwood, NY) using a 63 × 1.4 NA objective. Human LDL was prepared from donor plasma as has been described previously (20.Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6480) Google Scholar). AcLDL was purchased from Alfa Aesar (Haverhill, MA). LDL was labeled using succinimidyl esters of Alexa546. LDL was aggregated by vigorous vortexing for 30 s (21.Buton X. Mamdouh Z. Ghosh R. Du H. Kuriakose G. Beatini N. Grabowski G.A. Maxfield F.R. Tabas I. Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL).J. Biol. Chem. 1999; 274: 32112-32121Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). BODIPY-FL-C5-ceramide was incorporated by incubating 10 μM concentration complexed with BSA (1:1) in 1 mg/ml Alexa546-LDL at room temperature with slow rotation for 1 h prior to aggregation. The pellet was washed once with serum-free DMEM prior to incubation with cells. For imaging, cells were plated on poly-D-lysine-coated glass-coverslip-bottom dishes. Images were acquired with a Zeiss LSM510 or LSM880 laser-scanning confocal microscope using a 40× Air, 0.8 NA, or 40× Oil, 1.3 NA, objective respectively. For actin measurements, z-stacks were obtained with a step size of 0.98 μm. All data were analyzed with MetaMorph image analysis software (Molecular Devices, Dowingtown, PA). All measurements were made using MetaMorph software. Cell area was measured by outlining cells. Elongation index was calculated by measuring the maximum cell length and dividing by maximum cell width. For ceramide immunostaining, BMMs were fixed with 3% (w/v) paraformaldehyde in PBS for 20 min at room temperature, washed extensively with PBS, and subsequently blocked/permeabilized for 1 h at room temperature using 10% goat serum by 0.05% (w/v) saponin in PBS. Cells were then stained with anti-ceramide antibody (1:100 dilution) overnight at 4°C. Cells were washed extensively in PBS prior to staining using Alexa488-anti-mouse secondary antibody (1:400) for 30 min at room temperature. All staining was performed in 3% goat serum by 0.05% (w/v) saponin in PBS. Cells were washed extensively with PBS prior to imaging. For foam cell formation, BMMs were treated for 24 h with Alexa546-agLDL, fixed with 3% (w/v) paraformaldehyde in PBS for 20 min at room temperature, washed with PBS, and then stained using LipidTOX Green or LipidTOX Deep Red (at 1:1,000 dilution in PBS) for 15 min at room temperature followed by extensive washing in PBS prior to imaging. Cells were left untreated or pretreated where indicated prior to treatment with Alexa546-agLDL for 1 h. Cells were fixed with 3% (w/v) paraformaldehyde in PBS for 20 min at room temperature and stained for filamentous actin (F-actin) using 0.02 U/ml of Alexa488-phalloidin or Alexa647-phalloidin in 0.5% (w/v) saponin in PBS for 1 h. Cells were washed extensively with PBS and then imaged. Plasmids obtained from agar stab cultures were purified using plasmid DNA maxiprep kits (Qiagen) according to manufacturer's instructions. RAW264.7 macrophages were transiently transfected using Fugene HD reagent (Promega, Madison, WI). Two micrograms plasmid and 6 µl Fugene reagent were added to 100 µl serum-free DMEM, gently mixed and then incubated at room temperature for 15 min. Eight hundred microliters complete DMEM culture media was added to this, mixed gently, and added to macrophages on coverslip dishes. Cells were then cultured for 24 h prior to agLDL treatment. WT and SK2KO BMMs were transfected with siRNA using the Amaxa nucleofector I device (Lonza, Basel, Switzerland) using Amaxa Cell Line Nucleofector Kit T (Lonza). Four–5 × 106 cells were resuspended in 100 µl nucleofector solution. Control scrambled all-stars negative siRNA (Qiagen) or a pool of four different siRNA sequences targeting RhoA (Qiagen) were added to a final concentration of 2 µM. Cells were added to cuvettes and nucleofected using program T-20. Cells were transferred to prewarmed culture media and plated into tripartition petri dishes and cultured for 48 h. Cell solutions were trypsinized and plated into coverslip microscopy dishes for microscopy experiments or six-well plates overnight for assessment of knockdown efficiency. Typically, RhoA protein levels 72 h posttransfection were reduced ≥60% by RhoA siRNA in comparison with scrambled siRNA control. Actin polymerization was assessed, as has been described previously (3.Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar, 4.Singh R.K. Barbosa-Lorenzi V.C. Lund F.W. Grosheva I. Maxfield F.R. Haka A.S. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.J. Cell Sci. 2016; 129: 1072-1082Crossref PubMed Scopus (25) Google Scholar). In brief, z-stacks were acquired and a binary mask was created for each slice in the z-stack using Alexa546-agLDL signal intensity. This binary mask was applied to the Alexa488-phalloidin image, and the integrated Alexa488-phalloidin fluorescence colocalized with Alexa546-agLDL per z-slice was obtained. These values were summed for the entire stack to obtain the total integrated Alexa488-fluoresence colocalized with Alexa546-agLDL per z-stack. This was then divided by the number of cells in the field. For assessment of neutral lipid content, images were thresholded to exclude any fluorescence not associated with lipid droplets. Then the integrated LipidTOX Green fluorescence per field was quantified and divided by the number of cells in the field. For quantification of the extracellular agLDL/field, we outlined cells and measured the total integrated fluorescence of agLDL within the cell boundaries, then measured the total integrated fluorescence of agLDL per field and subtracted the intracellular fluorescence from the total fluorescence to obtain extracellular agLDL fluorescence per field. We then expressed this as a percentage of total agLDL per field. For pairwise comparisons, Student's two-tailed t test was performed. For comparisons of more than two groups, a one-way ANOVA followed by Bonferroni correction was performed. All statistical comparisons were performed using Excel software. Lesional LDL becomes enriched in ceramide during atherosclerosis (5.Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (269) Google Scholar). To determine whether macrophages come into contact with such ceramide, we took aortic sections from hyperlipidemic ApoE−/− mice and immunostained them for F4/80 to detect macrophages, and for ceramide using a well-characterized anti-ceramide antibody that has been used successfully for staining mouse tissue sections (22.Chen X. Talati M. Fessel J.P. Hemnes A.R. Gladson S. French J. Shay S. Trammell A. Phillips J.A. Hamid R. et al.Estrogen metabolite 16alpha-hydroxyestrone exacerbates bone morphogenetic protein receptor type II-associated pulmonary arterial hypertension through microRNA-29-mediated modulation of cellular metabolism.Circulation. 2016; 133: 82-97Crossref PubMed Scopus (70) Google Scholar). We found that lesional macrophages contact ceramide, particularly in regions close to necrotic cores (Fig. 1A, arrows, and Fig. 1C). We also observed macrophages that were accumulating ceramide (Fig. 1A, arrowheads and inset). Sections stained with secondary antibodies alone showed little background staining (Fig. 1B, D). A proportion of ceramide in the plaque is likely to be lesional aggregated LDL, which is known to be enriched in ceramide and accumulate in the subendothelium (intima) of the plaque (5.Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (269) Google Scholar, 23.Rocha V.Z. Libby P. Obesity, inflammation, and atherosclerosis.Nat. Rev. Cardiol. 2009; 6: 399-409Crossref PubMed Scopus (651) Google Scholar). When macrophages catabolize and internalize this LDL, they accumulate the accompanying ceramide. SK2KO macrophages contain increased levels of ceramide (16.Xiong Y. Lee H.J. Mariko B. Lu Y.C. Dannenberg A.J. Haka A.S. Maxfield F.R. Camerer E. Proia R.L. Hla T. Sphingosine kinases are not required for inflammatory responses in macrophages.J. Biol. Chem. 2013; 288 ([Erratum. 2016 J. Biol. Chem. 291: 11465.]): 32563-32573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Consistent with this, we found that SK2KO BMMs stained more intensely for ceramide than did WT BMMs (Fig. 2A, B). Quantification of this showed a 65% increase in ceramide staining in SK2KO compared with WT BMMs (Fig. 2C). We have used SK2KO macrophages to investigate the role of ceramide in the response of BMMs to agLDL. As is discussed below, we found that other changes in the SK2KO BMMs such as decreased S1P or SK1 upregulation cannot explain differences in the response of SK2KO BMMs to agLDL. We treated WT and SK2KO macrophages with agLDL to assess the effect of SK2 KO on actin polymerization. WT macrophages treated with agLDL displayed robust actin polymerization at the LS (Fig. 2D, arrow), but SK2KO BMMs displayed a 40% reduction in actin polymerization in response to agLDL at the LS (Fig. 2E, arrow, and Fig. 2F). By taking a microscopy approach, we were able to pinpoint changes in actin polymerization in SK2KO BMM, specifically the LS, indicating that these are local and not global phenomena. SK1 has previously been observed to be significantly upregulated in SK2KO BMMs (16.Xiong Y. Lee H.J. Mariko B. Lu Y.C. Dannenberg A.J. Haka A.S. Maxfield F.R. Camerer E. Proia R.L. Hla T. Sphingosine kinases are not required for inflammatory responses in macrophages.J. Biol. Chem. 2013; 288 ([Erratum. 2016 J. Biol. Chem. 291: 11465.]): 32563-32573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To assess the role of upregulated SK1 expression in impairment of actin polymerization in response to agLDL observed in SK2KO BMMs, we cultured BMMs deficient in both SK1 and SK2 and analyzed actin polymerization in response to agLDL. SK1 and SK2KO BMMs displayed a similar level of reduction in actin polymerization as did SK2KO BMMs specifically at the LS, suggesting that deficiency of SK2 underlies impaired actin polymerization (supplemental Fig. S1A, arrows, S1B). The primary function of sphingosine kinases is to phosphorylate sphingosine to generate S1P. S1P can bind a family of S1P receptors (S1P1-5) and can mediate actin reorganization through binding of S1P to receptors S1P1 and S1P2 (24.Formigli L. Meacci E. Sassoli C. Chellini F
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