FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2115519661> ?p ?o ?g. }

• W2115519661 endingPage "39881" @default.
• W2115519661 startingPage "39874" @default.
• W2115519661 abstract "Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(–)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(–) remain poorly described. Here we demonstrate that LDL(–) increases tumor necrosis factor α (TNFα)–induced inflammatory responses through NFκB and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(–) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFα alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor α (PPARα) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein ≫ LDL > high density lipoprotein). The PPARα activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPARα to an extent proportional to its LDL(–) content. As compared with LDL(–) alone, LPL-treated LDL(–) increased PPARα activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(–) suppressed NFκB and AP-1 activation, increasing expression of the PPARα target gene IκBα, although only in the genetic presence of PPARα and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(–) releases hydroxy-octadecadienoic acids (HODEs), potent PPARα activators. These findings suggest LPL-mediated PPARα activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(–). Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(–)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(–) remain poorly described. Here we demonstrate that LDL(–) increases tumor necrosis factor α (TNFα)–induced inflammatory responses through NFκB and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(–) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFα alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor α (PPARα) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein ≫ LDL > high density lipoprotein). The PPARα activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPARα to an extent proportional to its LDL(–) content. As compared with LDL(–) alone, LPL-treated LDL(–) increased PPARα activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(–) suppressed NFκB and AP-1 activation, increasing expression of the PPARα target gene IκBα, although only in the genetic presence of PPARα and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(–) releases hydroxy-octadecadienoic acids (HODEs), potent PPARα activators. These findings suggest LPL-mediated PPARα activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(–). Extensive data links low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; AP-1, adaptor protein 1; BAEC, bovine aortic endothelial cell; EC, endothelial cell; ELISA, enzyme-linked immunosorbent assay; EMSA, electromobility shift assay; HBLDL, LDL modified during hemoglobin-mediated plasma oxidation; HDL, high density lipoprotein; HODE, hydroxy-octadecadienoic acid; HpODE, hydroperoxy-octadecadienoic acid; HPLC, high pressure liquid chromatography; LBD, ligand binding domain; LDL(–), electronegative LDL; LDLR, LDR receptor; LPL, lipoprotein lipase; MS, mass spectrometry; NFκB, nuclear factor κB; oxLDL, oxidized LDL; PPAR, peroxisome proliferation-activated receptor; PPRE, peroxisome proliferator response element; TNFα, tumor necrosis factor α; VCAM1, vascular cell adhesion molecule-1; VLDL, very low density lipoprotein.1The abbreviations used are: LDL, low density lipoprotein; AP-1, adaptor protein 1; BAEC, bovine aortic endothelial cell; EC, endothelial cell; ELISA, enzyme-linked immunosorbent assay; EMSA, electromobility shift assay; HBLDL, LDL modified during hemoglobin-mediated plasma oxidation; HDL, high density lipoprotein; HODE, hydroxy-octadecadienoic acid; HpODE, hydroperoxy-octadecadienoic acid; HPLC, high pressure liquid chromatography; LBD, ligand binding domain; LDL(–), electronegative LDL; LDLR, LDR receptor; LPL, lipoprotein lipase; MS, mass spectrometry; NFκB, nuclear factor κB; oxLDL, oxidized LDL; PPAR, peroxisome proliferation-activated receptor; PPRE, peroxisome proliferator response element; TNFα, tumor necrosis factor α; VCAM1, vascular cell adhesion molecule-1; VLDL, very low density lipoprotein. to atherosclerosis (1Steinberg D. Circulation. 1997; 95: 1062-1071Crossref PubMed Scopus (702) Google Scholar, 2Scandinavian Simvastin Survival Study GroupLancet. 1994; 344: 1383-1389PubMed Google Scholar). This occurs in part through the induction of early atherogenic inflammatory responses, including the expression of adhesion molecules like VCAM1 by endothelial cells (ECs) (3Li H. Cybulsky M.I. Gimbrone Jr., M.A. Libby P. Arterioscler.Thromb. 1993; 13: 197-204Crossref PubMed Google Scholar, 4Glass C.K. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2597) Google Scholar). Consistent with this, increased dietary cholesterol rapidly induces atherosclerosis in animal models, with changes in VCAM1 expression seen within 2 weeks (3Li H. Cybulsky M.I. Gimbrone Jr., M.A. Libby P. Arterioscler.Thromb. 1993; 13: 197-204Crossref PubMed Google Scholar). LDL, a major carrier of cholesterol, circulates in several forms in vivo (5Sevanian A. Hwang J. Hodis H. Cazzolato G. Avogaro P. Bittolo-Bon G. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 784-793Crossref PubMed Scopus (143) Google Scholar, 6Sanchez-Quesada J. Benitez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. J. Lipid Res. 2002; 43: 699-705Abstract Full Text Full Text PDF PubMed Google Scholar). Most LDL pathogenicity becomes manifest after LDL oxidation (1Steinberg D. Circulation. 1997; 95: 1062-1071Crossref PubMed Scopus (702) Google Scholar). For example, oxidized LDL (oxLDL), but not native LDL, induces endothelial VCAM1 expression in the presence of TNFα (7Khan B. Parthasarathy S. Alexander R. Medford R. J. Clin. Invest. 1995; 95: 1262-1270Crossref PubMed Scopus (409) Google Scholar).More recently, a form of native LDL containing intermediately modified subfractions with higher electronegative charge, referred to as LDL(–), has been identified and characterized (8Cazzolato G. Avogaro P. Bittolo-Bon G. Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (99) Google Scholar, 9Hodis H. Kramsch D. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar). Several lines of evidence implicate LDL(–) as a particularly pro-atherogenic particle. Like oxLDL, LDL(–) has pro-inflammatory effects, e.g. inducing the chemokine monocyte chemotactic protein-1 and cytokine interleukin-8 (10Sanchez-Quesada J. Camacho M. Anton R. Benitez S. Vila L. Ordonez-Llanos J. Atherosclerosis. 2003; 166: 261-270Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), both of which are NFκB- and AP-1-regulated (11Roebuck K. Carpenter L. Lakshminarayanan V. Page S. Moy J. Thomas L. J.Leukoc.Biol. 1999; 65: 291-298Crossref PubMed Scopus (209) Google Scholar). LDL(–) is a significant component of more atherogenic small dense LDL subfractions (5Sevanian A. Hwang J. Hodis H. Cazzolato G. Avogaro P. Bittolo-Bon G. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 784-793Crossref PubMed Scopus (143) Google Scholar, 6Sanchez-Quesada J. Benitez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. J. Lipid Res. 2002; 43: 699-705Abstract Full Text Full Text PDF PubMed Google Scholar). LDL(–) also appears to be enriched in conditions characterized by accelerated atherosclerosis, namely familial hypercholesterolemia (10Sanchez-Quesada J. Camacho M. Anton R. Benitez S. Vila L. Ordonez-Llanos J. Atherosclerosis. 2003; 166: 261-270Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), diabetes (12Sanchez-Quesada J. Perez A. Caixas A. Ordonmez-Llanos J. Carreras G. Payes A. Gonzalez-Sastre F. de Leiva A. Diabetologia. 1996; 39: 1469-1476Crossref PubMed Scopus (67) Google Scholar), and hemodialysis (13Ziouzenkova O. Asatryan L. Akmal M. Tetta C. Wratten M. Loseto-Wich G. Jurgens G. Heinecke J. Sevanian A. J. Biol. Chem. 1999; 274: 18916-18924Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).Despite some similarities, native LDL, oxLDL, and LDL(–) have distinct characteristics that likely determine their biologic effects (14Ziouzenkova O. Sevanian A. Blood Purif. 2000; 18: 169-176Crossref PubMed Scopus (27) Google Scholar). Most fundamentally, these particles have unique compositional profiles. For example, LDL(–) contains fewer lipid peroxidation products than oxLDL, but more than native LDL (9Hodis H. Kramsch D. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar, 14Ziouzenkova O. Sevanian A. Blood Purif. 2000; 18: 169-176Crossref PubMed Scopus (27) Google Scholar). These forms of LDL are also cleared from the circulation in different ways, potentially contributing to their unique roles in atherosclerosis. In contrast to both native LDL and LDL(–), which are taken up through the LDL receptor (LDLR) (15Avogaro P. Bon G. Cazzolato G. Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google Scholar), oxLDL is removed after binding to scavenger or Fc receptors (1Steinberg D. Circulation. 1997; 95: 1062-1071Crossref PubMed Scopus (702) Google Scholar). Hydrolytic pathways for LDL particles also differ. For example, lipoprotein lipase (LPL), the predominant enzyme in triglyceride-rich lipoprotein (TRL) metabolism, can hydrolyze mildly oxidized LDL forms like LDL(–) (16Wang X. Greilberger J. Levak-Frank S. Zimmermann R. Zechner R. Jurgens G. Biochem. J. 1999; 343: 347-353Crossref PubMed Scopus (15) Google Scholar), although this ability may be limited as suggested by the higher content of both triglycerides (8Cazzolato G. Avogaro P. Bittolo-Bon G. Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (99) Google Scholar) and the LPL inhibitor Apo CIII (17Vedie B. Jeunemaitre X. Megnien J.L. Myara I. Trebeden H. Simon A. Moatti N. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1780-1789Crossref PubMed Scopus (25) Google Scholar) in LDL(–) as compared with native LDL fractions.Recently, we reported LPL enzymatic action as a mechanism for generating endogenous peroxisome proliferator-activated receptor α (PPARα) ligands (18Ziouzenkova O. Perrey S. Asatryan L. Hwang J. MacNaul K.L. Moller D.E. Rader D.J. Sevanian A. Zechner R. Hoefler G. Plutzky J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2730-2735Crossref PubMed Scopus (195) Google Scholar). This LPL/PPARα pathway replicated synthetic PPARα agonist effects, e.g. decreasing cytokine-induced VCAM1 expression (18Ziouzenkova O. Perrey S. Asatryan L. Hwang J. MacNaul K.L. Moller D.E. Rader D.J. Sevanian A. Zechner R. Hoefler G. Plutzky J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2730-2735Crossref PubMed Scopus (195) Google Scholar) in vitro and in vivo. The PPARα activation through LPL varied depending on the lipoprotein substrate; it was greatest with VLDL, less with LDL, and minimal with HDL, a series corresponding to triglyceride content (18Ziouzenkova O. Perrey S. Asatryan L. Hwang J. MacNaul K.L. Moller D.E. Rader D.J. Sevanian A. Zechner R. Hoefler G. Plutzky J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2730-2735Crossref PubMed Scopus (195) Google Scholar). Although this pattern and the absolute requirement for intact enzymatic catalysis for LPL-mediated PPARα activation suggests fatty acid release accounted for the responses seen, LPL-treated LDL activated PPARα to a disproportionate extent. This suggested LPL might release different PPARα mediators from LDL as compared with VLDL. If so, this indicates that the transcriptional responses to LDL might vary depending on LPL action, LDL particle composition, or its mechanism of uptake.To pursue this hypothesis, we tested cellular responses, including PPARα activation, to different forms of native and oxidized LDL with and without LPL treatment. In the presence of TNFα, LDL(–) uptake by the LDLR induced VCAM1 through NFκB and AP-1 activation, a previously unreported pathogenic LDL(–) effect. In contrast, LPL treatment of LDL(–) reversed this response, decreasing VCAM1 expression in a PPARα-dependent manner. Further studies reveal that LPL hydrolysis of LDL(–) generated oxidized linoleic acid (hydroxy-octadecadienoic acid, or HODE) in concentrations likely to account for the PPARα activation and the subsequent anti-inflammatory effects.EXPERIMENTAL PROCEDURESReagents—All reagents were purchased from Sigma-Aldrich unless otherwise indicated. All media were obtained from BioWhittaker (Walkersville, Maryland) and contained fungizone/penicillin/streptomycin. Human and murine TNFα were purchased from R&D Systems (Minneapolis, Minnesota). Fenofibric acid was a generous gift from Laboratories Fournier (Daix, France).Cell Culture—Human ECs, isolated from a saphenous vein and cultured in M199 medium (supplemented with endothelial cell growth factor (ECGF) and 5% fetal calf serum) as before (19Marx N. Sukhova G. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (543) Google Scholar), were all passage 3 to 5. PPARα+/+ (129S3/SvImJ) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). PPARα–/– mice were a generous gift from F. Gonzalez (National Institutes of Health) (20Lee S. Gonzalez F. Ann. N. Y. Acad. Sci. 1996; 804: 524-529Crossref PubMed Scopus (26) Google Scholar). Murine ECs from 1-month-old PPARα+/+ and PPARα–/– mouse hearts were isolated using double selection with intercellular adhesion molecule 2 (ICAM-2) and platelet endothelial cell adhesion molecule 1 (PECAM-1) antibodies (BD PharMingen) bound to Dynabeads (Dynal, Lake Success, New York) as before (21Marelli-Berg F.M. Peek E. Lidington E.A. Stauss H.J. Lechler R.I. J. Immunol. Methods. 2000; 244: 205-215Crossref PubMed Scopus (166) Google Scholar).LDL Isolation—Lipoproteins were isolated using gradient ultracentrifugation of human plasma pooled from at least six healthy donors (9Hodis H. Kramsch D. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar). Plasma of healthy donors was subjected to hemoglobin-mediated oxidation as described (13Ziouzenkova O. Asatryan L. Akmal M. Tetta C. Wratten M. Loseto-Wich G. Jurgens G. Heinecke J. Sevanian A. J. Biol. Chem. 1999; 274: 18916-18924Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The specific form of LDL that is formed by this type of oxidation is referred to as HBLDL. Anion exchange chromatography was utilized to purify the LDL(–) fraction and measure LDL(–) content in LDL modified during hemoglobin-mediated plasma oxidation (9Hodis H. Kramsch D. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar).RNA Analysis—Total cell RNA was isolated using RNeasy kit (Qiagen, Valencia, California), separated in 1% agarose gel, and transferred to Hydrobond membrane (Amersham Biosciences). Northern blotting was performed using cDNA probes obtained from American Type Culture Collection (Manassas, VA).Western Blotting—Western blotting was performed on proteins extracted from cells harvested in phosphate-buffered saline lysis buffer containing freshly added 1 mm phenylmethylsulfonyl fluoride at 4 °C. Nuclear extracts were isolated as before (19Marx N. Sukhova G. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (543) Google Scholar). Protein concentration was determined using the bicinchoninic acid kit (Pierce). Electrophoresis was performed on 12% polyacrylamide gels under reducing conditions (β-mercaptoethanol). Proteins were transferred onto Immobilon-P membranes using semi-dry transfer (45min, 16V). Nonspecific binding sites were blocked for 1 h with 5% delipidated milk in TBST (20 mm Tris, 55 mm NaCl, and 0.1% Tween 20). Monoclonal antibody against PPARα was a gift from Dr. J. Woods (1:500, Merck), polyclonal antibody against IκBα (1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000) was from Biodesign (Saco, Maine). Signals were visualized by chemiluminescence (PerkinElmer Life Sciences) after incubation with secondary horseradish peroxidase-conjugated antibody.Electromobility Shift Assay (EMSA)—Nuclear extracts were analyzed by EMSA (19Marx N. Sukhova G. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (543) Google Scholar). Oligonucleotide sequences for NFκB, AP-1, and PPRE were purchased from Santa Cruz Biotechnology. Supershift was performed using antibodies for PPARα, PPARγ (Biomol, Plymouth Meeting, PA), p50, and p65 (Santa Cruz Biotechnology). Nonspecific binding was blocked with 0.5 μg/μl bovine serum albumin and 0.1 μg/μl poly(dI·dC).Enzyme-linked Immunosorbent Assay (ELISA)—ELISA was performed in 96-well plates on confluent human umbilical vein endothelial cell (HUVEC) monolayers (19Marx N. Sukhova G. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (543) Google Scholar). Treated cells were kept on ice for 10 min, washed with cold phosphate-buffered saline, incubated with human VCAM1 monoclonal antibodies (gift from Dr. M. Gimbrone), and visualized using alkaline phosphate secondary antibody (450 nm).Flow Cytometry—Flow cytometry was performed using confluent mouse ECs obtained from heart of PPARα+/+ and PPARα–/– mice. Cells were washed in phosphate-buffered saline, harvested by trypsinization, and incubated (1 h at 4 °C) with fluorescein isothiocyanate-conjugated anti-mouse VCAM1 antibody (BD PharMingen). The EC culture purity was examined using anti-mouse phosphatidylethanolamine-conjugated platelet endothelial cell adhesion molecule 1 (BD PharMingen). Subsequently, washed cells were analyzed in a BD Biosciences FACScan™ flow cytometer using CELLQuest™ software. At least 20,000 viable cells per condition were analyzed.HPLC-MS—A stock standard solution containing hydroperoxy-octadecadienoic (HpODE) and hydroxy-octadecadienoic acids was prepared by diluting and combining solutions of standard mixtures obtained from Cayman Chemical (Ann Arbor, MI). Samples were extracted by the Folch method, reconstituted in isopropanol, and filtered (0.2-μm nylon). The samples and stock standard were serially diluted with 50:50 acetonitrile/H2O.The separation was performed using a Waters 2690 LC with photo-diode array (Waters 996) and time-of-flight mass spectrometry (TOF-MS) detection (Micromass LCT). The column used was a Luna C18 (2Scandinavian Simvastin Survival Study GroupLancet. 1994; 344: 1383-1389PubMed Google Scholar) 50 × 2 mm, 5 μm from Phenomenex (Torrance, CA). Mobile phases for the isocratic separation were 50% A, 4 mm ammonium acetate (Sigma-Aldrich) in H2O, and 50% B, acetonitrile (Sigma-Aldrich) flowing at 0.5 ml/min. The separation was performed at 30 °C with a total run time of 5 min. UV absorption was acquired from 200–400 nm. MS was performed using electrospray ionization operating in negative ionization mode. The ionization parameters were as follows: capillary voltage, 3200V; sample cone, 37V; extraction cone, 4V; desolvation temperature, 300 °C; source temperature, 120 °C; and ion scanning m/z range, 100–1000. Extracted ion chromatograms were constructed for HODE and HpODE using m/z values of 295 and 293, respectively. When the signal-to-noise ratio was sufficient, samples were quantified using an external calibration curve. When the signal-to-noise ratio was too low, only semi-quantitative estimates were made.Transfection—Transfection was carried out in 24-well plates at 2.3 × 104 primary bovine aortic ECs (BAECs) (passage 3–6) per well using FuGENE (F. Hoffmann-La Roche). Cells were transfected 3 h after replating in Dulbecco's modified Eagle's medium containing 1% of delipidated fetal calf serum (for PPAR ligand binding domain (LBD) studies) or 1% Nutridoma SP (F. Hoffmann-La Roche) for VCAM1 promoter studies. Transfected cells were treated with the indicated compounds for at least 10 h. Constructs were generous gifts from T. Willson (GlaxoSmithKline; LBD/yeast Gal4), T. Collins (Children's Hospital, Boston, MA; VCAM1 promoter), H. Hobbs (University of Texas Southwestern, LDLR expression vector), and D. Rader (University of Pennsylvania; LPL and LPL mutant expression vectors). The catalytically inactive LPL mutant has a two-base pair difference (AGC/GCC) that replaces the active site serine 132 with alanine (18Ziouzenkova O. Perrey S. Asatryan L. Hwang J. MacNaul K.L. Moller D.E. Rader D.J. Sevanian A. Zechner R. Hoefler G. Plutzky J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2730-2735Crossref PubMed Scopus (195) Google Scholar). PCMX-β-galactosidase expression vector was used for transfection control. Luciferase (BC Pharmingen) and β-galactosidase activity utilizing chlorophenol red-β-d-galactopyranoside as substrate (F. Hoffmann-La Roche) were measured according to manufacturer's protocols.Scintillation Proximity Assay (SPA)—A scintillation proximity assay was carried out using human full-length cDNA for PPARα, PPARδ, and PPARγ2 that were subcloned into the pGEX-KT expression vector (22Berger J.P. Petro A.E. Macnaul K.L. Kelly L.J. Zhang B.B. Richards K. Elbrecht A. Johnson B.A. Zhou G. Doebber T.W. Biswas C. Parikh M. Sharma N. Tanen M.R. Thompson G.M. Ventre J. Adams A.D. Mosley R. Surwit R.S. Moller D.E. Mol. Endocrinol. 2003; 17: 662-676Crossref PubMed Scopus (299) Google Scholar). The 3H2-labeled known synthetic PPAR agonists used were nTZD3 and nTZD4 with relative Kd values as follows: nTZD3, PPARγ 2.5 nm PPARα 5 nm; and nTZD4, PPARδ 1 nm (22Berger J.P. Petro A.E. Macnaul K.L. Kelly L.J. Zhang B.B. Richards K. Elbrecht A. Johnson B.A. Zhou G. Doebber T.W. Biswas C. Parikh M. Sharma N. Tanen M.R. Thompson G.M. Ventre J. Adams A.D. Mosley R. Surwit R.S. Moller D.E. Mol. Endocrinol. 2003; 17: 662-676Crossref PubMed Scopus (299) Google Scholar). Results are expressed as percent inhibition with a calculated inhibitory constant (Kis ).RESULTSLPL Decreases LDL(–)-mediated VCAM-1 Induction in a PPARα-dependent Manner—We compared the effect of native LDL and LDL(–) on TNFα-induced VCAM1 expression in human ECs. As expected, TNFα induced VCAM1 protein in ECs (8-fold, set as 100% induction) on ELISA (Fig. 1A). Although concomitant treatment with native LDL led to only a modest further increase in VCAM1 (20%), LDL(–) augmented VCAM1 levels by 70% relative to TNFα alone (14-fold as compared with basal untreated levels). The presence of LPL inhibited LDL(–)-mediated VCAM1 induction in a dose-dependent fashion (Fig. 1A). Similar effects were evident on Northern blotting (data not shown) and activation of the human VCAM1 promoter (Fig. 1B). Although LDL(–) treatment induced the VCAM1 promoter 120–180% in bovine aortic EC transfections, this same stimulation in the presence of LPL repressed this response by 60–80% as compared, in both cases, with TNFα alone (Fig. 1B). These effects of LPL/LDL(–) on TNFα-induced VCAM1 expression equaled those seen with synthetic PPARα ligands (Wy14163 or fenofibric acid, Fig. 1B).To test whether treatment of LDL(–) reduced VCAM1 expression in a PPARα-dependent manner, VCAM1 responses were examined using FACS analysis of PPARα+/+ and PPARα–/– microvascular ECs (Fig. 1C). Both TNFα and TNFα/LDL(–) stimulation of PPARα+/+ ECs markedly increased VCAM1 content on the EC surface. LPL-treated LDL(–) repressed this TNFα induction in the presence, but not the genetic absence, of PPARα with effects replicating those seen with the synthetic PPARα agonist WY14643. These data suggest that LPL action on LDL(–) limits inflammation through a PPARα mechanism.LPL Treatment of LDL(–) Decreases NFκB Binding—We and others have shown that PPARα ligands decrease cytokine-mediated VCAM1 expression through effects on NFκB signaling (19Marx N. Sukhova G. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (543) Google Scholar, 23Jackson S.M. Parhami F. Xi X.P. Berliner J.A. Hsueh W.A. Law R.E. Demer L.L. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2094-2104Crossref PubMed Scopus (350) Google Scholar). Mechanisms involved reportedly include direct PPARα interaction with p65 and PPARα-dependent expression of IκBα, sequestering the inactive p65/p50 complex in the cytoplasm (24Delerive P. Gervois P. Fruchart J.C. Staels B. J. Biol. Chem. 2000; 275: 36703-36707Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). To explore how LPL negatively regulates LDL(–)/TNFα induction of the VCAM1 promoter, we performed an EMSA of EC nuclear extracts using NFκB and PPRE binding sites. As expected, TNFα induced NFκB binding, a response augmented in the presence of LDL(–) (Fig. 2A). In the presence of LPL, however, LDL(–) significantly decreased NFκB binding; Wy14163 had similar effects. This decrease in NFκB binding was paralleled by increased IκBα levels from the very same ECs (Fig. 2B). Similarly, the increase in AP-1 binding induced by LDL(–)/TNFα was decreased after LPL treatment (data not shown). In parallel with decreased NFκB binding, both LPL/LDL(–) and WY14643 markedly increased binding to a canonical PPRE (Fig. 2C). This response was much greater than that seen with LDL(–), TNFα, or their combination (Fig. 2C). PPRE binding involved PPARα as indicated by the supershift in the presence of PPARα but not the PPARγ antibody. These effects appear to be due to increases in PPARα activators and not PPARα itself, given the lack of significant differences in nuclear PPARα protein levels after treating cells with LDL(–), LPL, or their combination (Fig. 2D). Together, these data suggest that LPL treatment of LDL(–) exerts its effects through direct interaction of LDL-derived components with NFκB and AP-1 in a manner similar to that of synthetic PPARα ligands.Fig. 2LPL treated LDL(–) inhibits NFκB activation and increases IκBα expression while inducing PPRE binding. Confluent human ECs were pretreated with LPL (200 units/ml, 4 h) in the presence or absence of LDL(–) (10 μg/ml) and then stimulated with human TNFα (10 ng/ml, 1.5 h). Fenofibric acid (Feno; 100 μm) pre-treatment was used for comparison. Nuclear extracts were analyzed by EMSA using 5 μg of nuclear extracts and 100 ng of radiolabeled NFκB (A), AP-1 (data not shown), and PPRE (C) sequences (Santa Cruz Biotechnology). Supershift analysis was performed to confirm the identity of PPARα and PPARγ as well as p65 and p50 (data not shown) using specific antibodies. Cytosolic fractions (25 μg protein) of the same cells were analyzed for IκBα expression (B), whereas nuclear fractions (25 μg protein) were studied for PPARα expression (D) using Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression confirmed equal loading. Data shown are representative of one experiment from three with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)LPL Lipolysis of LDL(–) Generates PPARα Ligands—We further examined PPARα ligand generation as a result of LPL treatment of LDL(–). The yeast Gal4/PPAR LBD hybrid assay is classically used to screen for PPARα ligand formation (25Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2712) Google Scholar). LDL(–) stimulation led to a modest 3-fold PPARα LBD activation; in the presence of LPL and LDL(–), PPARα activation increased 30-fold (Fig. 3A). In addition to its catalytic activity, LPL can also promote the uptake of lipoproteins like LDL through non-enzymatic bridging of lipoproteins to receptors like the LDLR (26Merkel M. Eckel R.H. Goldberg I.J. J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). We investigated the contribution of LPL bridging to the effects reported above. Transient transfection of the LDLR into EC did not alter PPARα LBD activation by LDL(–)/LPL. In contrast, LDL(–) treatment of LDLR-transfected ECs markedly increased VCAM1 promoter activity (Fig. 3B).Fig. 3Divergent cellular responses to LDL(–) depend on mechanisms of lipoprotein uptake. LPL-mediated hydrolysis of LDL(–) activates PPARα LBD independent of LDLR expression. BAECs were co-transfected with expression constructs for PPARα-LBD, the luciferase response pUASx4-TK-luc, and β-galactosidase. Cells were also" @default.
• W2115519661 created "2016-06-24" @default.
• W2115519661 creator A5004343492 @default.
• W2115519661 creator A5017593302 @default.
• W2115519661 creator A5020442736 @default.
• W2115519661 creator A5025426780 @default.
• W2115519661 creator A5037617286 @default.
• W2115519661 creator A5038033830 @default.
• W2115519661 creator A5049638521 @default.
• W2115519661 creator A5051351787 @default.
• W2115519661 creator A5063639681 @default.
• W2115519661 creator A5064529739 @default.
• W2115519661 creator A5064907948 @default.
• W2115519661 date "2003-10-01" @default.
• W2115519661 modified "2023-10-13" @default.
• W2115519661 title "Dual Roles for Lipolysis and Oxidation in Peroxisome Proliferation-Activator Receptor Responses to Electronegative Low Density Lipoprotein" @default.
• W2115519661 cites W1505047300 @default.
• W2115519661 cites W1650965741 @default.
• W2115519661 cites W1671834350 @default.
• W2115519661 cites W1879201657 @default.
• W2115519661 cites W1933491090 @default.
• W2115519661 cites W1997745709 @default.
• W2115519661 cites W1998287845 @default.
• W2115519661 cites W1999975112 @default.
• W2115519661 cites W2005674716 @default.
• W2115519661 cites W2012896945 @default.
• W2115519661 cites W2024059969 @default.
• W2115519661 cites W2037019145 @default.
• W2115519661 cites W2038610374 @default.
• W2115519661 cites W2044136517 @default.
• W2115519661 cites W2045773814 @default.
• W2115519661 cites W2049647259 @default.
• W2115519661 cites W2050776161 @default.
• W2115519661 cites W2050904588 @default.
• W2115519661 cites W2057132562 @default.
• W2115519661 cites W2058232747 @default.
• W2115519661 cites W2069537563 @default.
• W2115519661 cites W2087426673 @default.
• W2115519661 cites W2087647782 @default.
• W2115519661 cites W2092059547 @default.
• W2115519661 cites W2096832025 @default.
• W2115519661 cites W2103100606 @default.
• W2115519661 cites W2106809354 @default.
• W2115519661 cites W2120871101 @default.
• W2115519661 cites W2128263037 @default.
• W2115519661 cites W2131222777 @default.
• W2115519661 cites W2143340286 @default.
• W2115519661 cites W2148326332 @default.
• W2115519661 cites W2153054296 @default.
• W2115519661 cites W2159083249 @default.
• W2115519661 cites W2159090847 @default.
• W2115519661 cites W2160271044 @default.
• W2115519661 cites W2188633998 @default.
• W2115519661 cites W2343447675 @default.
• W2115519661 doi "https://doi.org/10.1074/jbc.m306786200" @default.
• W2115519661 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4231711" @default.
• W2115519661 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12878589" @default.
• W2115519661 hasPublicationYear "2003" @default.
• W2115519661 type Work @default.
• W2115519661 sameAs 2115519661 @default.
• W2115519661 citedByCount "58" @default.
• W2115519661 countsByYear W21155196612012 @default.
• W2115519661 countsByYear W21155196612013 @default.
• W2115519661 countsByYear W21155196612014 @default.
• W2115519661 countsByYear W21155196612017 @default.
• W2115519661 countsByYear W21155196612019 @default.
• W2115519661 countsByYear W21155196612020 @default.
• W2115519661 countsByYear W21155196612021 @default.
• W2115519661 countsByYear W21155196612022 @default.
• W2115519661 countsByYear W21155196612023 @default.
• W2115519661 crossrefType "journal-article" @default.
• W2115519661 hasAuthorship W2115519661A5004343492 @default.
• W2115519661 hasAuthorship W2115519661A5017593302 @default.
• W2115519661 hasAuthorship W2115519661A5020442736 @default.
• W2115519661 hasAuthorship W2115519661A5025426780 @default.
• W2115519661 hasAuthorship W2115519661A5037617286 @default.
• W2115519661 hasAuthorship W2115519661A5038033830 @default.
• W2115519661 hasAuthorship W2115519661A5049638521 @default.
• W2115519661 hasAuthorship W2115519661A5051351787 @default.
• W2115519661 hasAuthorship W2115519661A5063639681 @default.
• W2115519661 hasAuthorship W2115519661A5064529739 @default.
• W2115519661 hasAuthorship W2115519661A5064907948 @default.
• W2115519661 hasBestOaLocation W21155196611 @default.
• W2115519661 hasConcept C124952713 @default.
• W2115519661 hasConcept C127078168 @default.
• W2115519661 hasConcept C134018914 @default.
• W2115519661 hasConcept C140985366 @default.
• W2115519661 hasConcept C142362112 @default.
• W2115519661 hasConcept C170493617 @default.
• W2115519661 hasConcept C171089720 @default.
• W2115519661 hasConcept C185592680 @default.
• W2115519661 hasConcept C2780980858 @default.
• W2115519661 hasConcept C55493867 @default.
• W2115519661 hasConcept C86803240 @default.
• W2115519661 hasConcept C88045685 @default.
• W2115519661 hasConcept C95444343 @default.
• W2115519661 hasConceptScore W2115519661C124952713 @default.
• W2115519661 hasConceptScore W2115519661C127078168 @default.

• next