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• W1993900511 abstract "In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a−)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a−), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3–4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5–2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis. In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a−)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a−), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3–4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5–2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis. lipoprotein(a) low density lipoprotein apolipoprotein(a) Kringle V plasminogen activator inhibitor interleukin 5,6-dichlorobenzimidazole ε-aminocaproic acid diisopropylfluorophosphate dithioerythritol kallikrein inactivator apo(a)-free LDL derived from parental Lp(a) phosphate buffered saline glyceraldehyde-3-phosphate-dehydrogenase enzyme-linked immunosorbent assay phosphate-buffered saline polymerase chain reaction Lipoprotein(a) (Lp(a))1represents a low density lipoprotein (LDL) variant in which apoB100 is linked by a single disulfide bond to apolipoprotein(a) (apo(a)), a multikringle structure shown to have a high degree of homology with plasminogen (1McLean J.W. Tomlinson J.E. Kuang W. Eaton D.L. Chen E.Y. Fless G.M. Scanu A.M. Lawn R.M. Nature. 1987; 330: 132-137Crossref PubMed Scopus (1597) Google Scholar, 2Scanu A.M. Edelstein C. Biochim. Biophys. Acta. 1995; 1256: 1-12Crossref PubMed Scopus (61) Google Scholar). Lp(a) has been associated with an increased risk for coronary heart (3Rhoads G.G. Dahlen G. Berg K. Morton N.E. Dannenberg A.L. J. Am. Med. Assoc. 1986; 256: 2540-2544Crossref PubMed Scopus (668) Google Scholar, 4Budde T. Fechtrup C. Bosenberg E. Vielhauer C. Enbergs A. Schulte H. Assmann G. Breithardt G. Arterioscler. Thromb. 1994; 14: 1730-1736Crossref PubMed Google Scholar), cerebrovascular (5Bolger C. Phillips J. Gilligan S. Zourob T. Farrell M. Croake D. Collins P. Bouchierhayes D. Neurosurgery. 1995; 37: 241-245Crossref PubMed Scopus (13) Google Scholar, 6Shintani S. Kikuchi S. Hamaguchi H. Shiigai T. Stroke. 1993; 24: 965-969Crossref PubMed Scopus (121) Google Scholar, 7Nagayama M. Shinohara Y. Nagayama T. Stroke. 1994; 25: 74-78Crossref PubMed Scopus (95) Google Scholar), and peripheral vascular disease (8Groves P. Rees A. Bishop A. Morgan R. Ruttley M. Lewis N. Lane I. Hall R. Br. Heart J. 1993; 69: 26-30Crossref PubMed Scopus (12) Google Scholar, 9Valentine R.J. Grayburn P.A. Vega G.L. Grundy S.M. Arch. Intern. Med. 1994; 154: 801-806Crossref PubMed Scopus (107) Google Scholar, 10Bandello F. Vigano'D'Angelo S. Parlavecchia M. Tavola A. Della Valle P. Brancato R. D'Angelo A. Thromb. Haemostasis. 1994; 72: 39-43Crossref PubMed Scopus (65) Google Scholar) by still poorly defined mechanisms. Whether the whole Lp(a) particle is required for the pathogenicity is unclear. The potential contribution by the LDL moiety of Lp(a) to the cardiovascular risk has received relatively limited attention, although based on the information available on authentic LDL, it is likely to be dependent on LDL particle size and type and extent of modifications due to oxidative, lipolytic, or proteolytic events. On the contrary, free apo(a), either derived from parent Lp(a) or as a recombinant, has been reported to be an active component of Lp(a) in many cellular systems and in binding to members of the vascular extracellular matrix. In an endothelial cell system, native Lp(a), via apo(a), has been shown to stimulate the production of adhesion molecules such as intercellular adhesion molecule (11Takami S. Yamashita S. Kihara S. Ishigami M. Takemura K. Kume N. Kita T. Matsuzawa Y. Circulation. 1998; 97: 721-728Crossref PubMed Scopus (99) Google Scholar), vascular cell adhesion molecule-1 (12Allen S. Khan S. Tam S. Koschinsky M. Taylor P. Yacoub M. FASEB J. 1998; 12: 1765-1776Crossref PubMed Scopus (88) Google Scholar), and E-selectin (12Allen S. Khan S. Tam S. Koschinsky M. Taylor P. Yacoub M. FASEB J. 1998; 12: 1765-1776Crossref PubMed Scopus (88) Google Scholar), as well as endothelin-1 (13Berge K.E. Djurovic S. Muller H.J. Alestrom P. Berg K. Clin. Genet. 1997; 52: 314-325Crossref PubMed Scopus (10) Google Scholar) and I-309, a potent chemoattractant for monocytes (14Haque N.S. Zhang X. French D.L. Li J. Poon M. Fallon J.T. Gabel B.R. Taubman M.B. Koschinsky M. Harpel P.C. Circulation. 2000; 102: 786-792Crossref PubMed Scopus (85) Google Scholar). Native Lp(a) has also been reported to enhance endothelial plasminogen activator inhibitor-1 expression (15Etingin O.R. Hajjar D.P. Hajjar A.K. Harpel P.C. Nachman R.L. J. Biol. Chem. 1991; 266: 2459-2465Abstract Full Text PDF PubMed Google Scholar, 16Grenett H.E. Benza R.I. Fless G.M. Li X.-N. Davis G.C. Booyse F.M. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1803-1809Crossref PubMed Scopus (29) Google Scholar), although those data have not been corroborated by other studies (13Berge K.E. Djurovic S. Muller H.J. Alestrom P. Berg K. Clin. Genet. 1997; 52: 314-325Crossref PubMed Scopus (10) Google Scholar, 17Levin E.G. Miles L.A. Fless G.M. Scanu A.M. Baynham P. Curtiss L.K. Plow E.F. Arterioscler. Thromb. 1994; 14: 438-442Crossref PubMed Google Scholar). Moreover, in a vascular smooth muscle cell system, native Lp(a), and particularly apo(a), was shown to inhibit the proteolytic activation of transforming factor β via a decrease in cell surface generation of plasmin, resulting in increased vascular smooth muscle cells proliferation (18Grainger D.L. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Nature. 1994; 370: 460-462Crossref PubMed Scopus (341) Google Scholar). There is also evidence that the proteolytic fragment of apo(a), namely F2 which corresponds to the C-terminal domain of apo(a), may exhibit pro-inflammatory properties in that it binds in vitro to the members of the vascular extracellular matrix (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, 20Klezovitch O. Edelstein C. Zhu L. Scanu A.M. J. Biol. Chem. 1998; 273: 23856-23865Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), is present in vivo in unstable atheromatous carotid plaques (21Fortunato J.E. Bassiouny H.S. Song R.H. Kocharian H. Glagov S. Edelstein C. Scanu A.M. J. Vasc. Surg. 2000; 32: 555-563Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and stimulates the production of monocyte chemoattractant I-309 in cultured endothelial cells (14Haque N.S. Zhang X. French D.L. Li J. Poon M. Fallon J.T. Gabel B.R. Taubman M.B. Koschinsky M. Harpel P.C. Circulation. 2000; 102: 786-792Crossref PubMed Scopus (85) Google Scholar). Macrophages play a pivotal role in atherosclerosis as cellular components of the underlying chronic inflammatory process. These cells, derived from blood monocytes, are virtually absent in the normal artery but are abundant in unstable plaques, where they exhibit an increased expression of pro-inflammatory elements that contribute to the progression of the atherosclerotic lesion (22Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar). At this time, it is unclear whether Lp(a) in its native form has pro-inflammatory properties. In the current study we tested this hypothesis by examining the effect of Lp(a) and some of its derivatives on the production of inflammatory mediators, using as a model system human THP-1 macrophages obtained by phorbol ester stimulation of THP-1 monocytes. This cell line is highly differentiated and, upon stimulation with phorbol ester, is known to acquire properties similar to those of human monocyte-derived macrophages (23Tsuchiya S. Kobayashi Y. Goto Y. Okumura H. Nakae S. Konno T. Tada K. Cancer Res. 1982; 42: 1530-1536PubMed Google Scholar, 24Via D.P. Pons L. Dennison D.K. Fanslow A.E. Bernini F. J. Lipid Res. 1989; 30: 1515-1524Abstract Full Text PDF PubMed Google Scholar). We show here that in the chosen cell system, Lp(a), Lp(a)-derived apo(a), and its C-terminal domain, all cause, although to a different degree, an increased production of interleukin (IL)-8, a potent pro-inflammatory chemokine. We also show that this effect is exhibited by neither the LDL isolated from parent Lp(a) nor by authentic LDL isolated from the plasma that served as a source of Lp(a). All the tissue culture reagents, cholera toxin, and pertussis toxin were obtained from Life Technologies, Inc., and were of low endotoxin grade. Phorbol 12-myristate 13-acetate, 5,6-dichlorobenzimidazole (DRB), ε-aminocaproic acid (EACA), diisopropyl fluorophosphate (DFP), EDTA, dithioerythritol (DTE), β-mercaptoethanol, SDS, porcine pancreatic elastase (EC 1.4.21.36), trehalose, formamide, and formaldehyde were purchased from Sigma. Kallikrein inactivator (KI) was purchased from Calbiochem. Nuclease-free water and the RNase-free plasticware were from Ambion, Inc. (Austin, TX). Human Glu-plasminogen was purchased from Enzyme Research Laboratories (South Bend, IN). Rabbit affinity-purified antibodies to Lp(a), apo(a), and LDL and anti-KV monoclonal antibody were prepared as described previously (25Fless G.M. Snyder M.L. Scanu A.M. J. Lipid Res. 1989; 30: 651-662Abstract Full Text PDF PubMed Google Scholar). Anti-Lp(a) and anti-apo(a) did not react against LDL and plasminogen, and anti-LDL was unreactive to apo(a). The plasma from a healthy donor with a single apo(a) isoform of 289 kDa (26Edelstein C. Mandala M. Pfaffinger D. Scanu A.M. Biochemistry. 1995; 34: 16483-16492Crossref PubMed Scopus (42) Google Scholar) was obtained by plasmapheresis performed in the Blood Bank of the University of Chicago. The steps for Lp(a) and LDL isolation were carried out immediately after blood drawing using the procedure outlined below. To prevent lipoprotein degradation, the plasma was adjusted with 0.15% EDTA, 0.01% sodium azide, 10,000 units/liter KI, and 1 mm phenylmethylsulfonyl fluoride. Lp(a) was isolated by sequential ultracentrifugation and lysine-Sepharose chromatography as described previously (27Fless G.M. Snyder M.L. Furbee J.W.J. Garcia-Hedo M.T. Mora R. Biochemistry. 1994; 33: 13492-13501Crossref PubMed Scopus (41) Google Scholar). The purity of the product was assessed by electrophoresis on precast 1% agarose gels (Ciba-Corning, Palo Alto, CA) and Western blots of 4% SDS-polyacrylamide gel electrophoresis (NOVEX, San Diego, CA), utilizing anti-Lp(a) and anti-LDL. The LDL preparations used in this study were isolated from the same donor used for the Lp(a) preparation, at d = 1.030–1.050 g/ml by sequential flotation as described previously (28Schumaker V.N. Puppione D.L. Methods Enzymol. 1986; 128: 155-170Crossref PubMed Scopus (467) Google Scholar) and assessed to have no apo(a) by electrophoresis and Western blot criteria. Lp(a−),i.e. apo(a)-free LDL, was isolated from Lp(a) as reported previously (26Edelstein C. Mandala M. Pfaffinger D. Scanu A.M. Biochemistry. 1995; 34: 16483-16492Crossref PubMed Scopus (42) Google Scholar). The protein concentrations of Lp(a), Lp(a−), and LDL were determined by either a sandwich ELISA as described previously (25Fless G.M. Snyder M.L. Scanu A.M. J. Lipid Res. 1989; 30: 651-662Abstract Full Text PDF PubMed Google Scholar) or by the Bio-Rad DC protein assay. The purified lipoproteins showed no evidence of oxidation as determined by measuring the amounts of thiobarbituric acid-reactive substances and diene formation (29Sattler W. Kostner G.M. Waeg G. Esterbauer H. Biochim. Biophys. Acta. 1991; 1081: 65-74Crossref PubMed Scopus (124) Google Scholar). The endotoxin content in lipoprotein preparations was analyzed using theLimulus amoebocyte lysate assay (Sigma) and was estimated to be less than 0.3 pg/μg Lp(a) protein and 0.2 pg/μg LDL protein. Apo(a) was isolated from Lp(a) under mild reductive conditions in the presence of 1.5–2 mm DTE as described by Edelstein et al. (26Edelstein C. Mandala M. Pfaffinger D. Scanu A.M. Biochemistry. 1995; 34: 16483-16492Crossref PubMed Scopus (42) Google Scholar). The final preparation of apo(a) was assessed for purity by Western blot with an anti-apo(a) and stored in 10 mm phosphate buffer containing 1 mm EDTA, 0.02% sodium azide, and 125 mmtrehalose at −80 °C. The concentration of apo(a) was determined either by ELISA or using an extinction coefficient (ε278= 1.31 ml mg−1 cm−1) established previously for apo(a) (30Edelstein C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The endotoxin content in apo(a) preparations was estimated to be less than 0.25 pg/μg apo(a). The structure of apo(a) fragments obtained by limited proteolysis using either human leukocyte or pancreatic elastase is outlined in Fig. 1. Lp(a) was digested with a purified preparation of human leukocyte elastase (1 μg of enzyme per 3 mg of Lp(a) protein) for 30 min at 37 °C, and the reaction was terminated by the addition of DFP to a final concentration of 5 mm. Apo(a) fragments containing KIV-9, retained its linkage to apoB100 via the disulfide bond, forming a miniLp(a) particle. The digest was centrifuged in 30% sucrose solutions, d 1.127 g/ml containing 100 mm EACA at 15 °C, 412,160 ×g. The floating lipoprotein fraction (top) containing miniLp(a)s, and the sedimenting fraction (bottom) containing F1, F6, and F7 were collected separately and dialyzed against phosphate-buffered saline (PBS). The bottom fraction was subjected to a lysine-Sepharose affinity chromatography. The fraction eluted in PBS (unbound) contained F1 and F7 and was further fractionated by molecular sieve chromatography with Superdex 200; F1, due to a higher molecular weight, eluted first followed by F7. The fraction eluted from lysine-Sepharose with 100 mm EACA (bound) contained only F6. The miniLp(a)s in the top d 1.12 g/ml fraction consist of four species of miniLp(a) particles each containing apoB100 disulfide linked to individual apo(a) fragments, namely F2, F3, F4, and F5. The fragments were obtained free of apoB100 by mild reduction with DTE (1.5 mm) in the presence of 100 mm EACA and separated from the floating LDL fraction (Lp(a−) by ultracentrifugation. The sedimenting fraction (bottom) containing F2, F3, F4, and F5 was fractionated by lysine-Sepharose chromatography using a gradient of EACA from 0 to 100 mm. The order of elution with increasing EACA molarity was F4, F5, F2, and F3. The purity of the fragments was assessed by SDS-polyacrylamide gel electrophoresis on Coomassie-stained gels and by Western blot analysis using polyclonal monospecific rabbit anti-apo(a) and anti-apoB100 antibodies as well as monoclonal anti-KV antibodies. In addition, N-terminal sequence analysis of the first 20 amino acids confirmed the purity and identity of each fragment. When the studies involved only F1 and F2, these fragments were prepared by digestion of apo(a) with pancreatic elastase as described previously by Edelstein et al. (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar). Briefly, apo(a) in 50 mm Tris-HCl, 100 mm NaCl, pH 8.0, KI (200 units/ml) was digested with porcine pancreatic elastase at a molar ratio of 25:1 (protein:enzyme) at 22 °C for 2 h, and the reaction was terminated by the addition of 5 mm DFP with further incubation for 20 min. The digest was applied to a lysine-Sepharose affinity column that was then washed sequentially with 3 column volumes of PBS, 500 mm NaCl, and 200 mm EACA. Fragment F1 eluted with PBS and F2 with EACA. The PBS and EACA fractions containing these fragments were each pooled, dialyzed against 10 mm phosphate buffer, pH 7.4, containing 1 mm EDTA, 0.02% sodium azide, and concentrated using Centriprep membranes (Amicon Corp., Beverly, MA). Human monocytic leukemia cell line, THP-1, was purchased from the American Type Culture Collection. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 μg/ml), gentamicin (50 μg/ml), and 50 μm mercaptoethanol at 37 °C, 5% CO2. To prepare THP-1 macrophages, monocytes were plated in 6-well plates at the density of 1·106 cells/ml (2 ml per well) and incubated in the complete growth medium in the presence of 100 nm phorbol 12-myristate 13-acetate. After 72 h, the cells were washed once with RPMI 1640 serum-free medium and incubated with a new aliquot of the serum-free medium for 16 h. At this point, the medium was replaced with a fresh serum-free medium containing the indicated amounts of Lp(a), Lp(a−), LDL, apo(a), or the apo(a) fragments and incubated with THP-1 macrophages for the indicated times. In some experiments, cells were preincubated for 30 min with cholera or pertussis toxins (1 μg/ml) prior to the addition of apo(a). At the end of incubation, the cells were immediately processed for the isolation of RNA as described below. The supernatants from each well were collected, centrifuged to eliminate debris, and either used for determination of the concentration of IL-8 or frozen at −20 °C until further analysis. At the concentrations of apo(a) and lipoproteins used in our studies, cell viability was >95% as assessed by trypan blue exclusion. All the experiments were conducted in duplicate and were repeated at least twice. Total cellular RNA was isolated using the TRIZOL reagent (Life Technologies, Inc.). Quality of the RNA preparations was verified by 1% denatured formaldehyde agarose gel electrophoresis as described by Sambrook et al. (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Microarray analysis of total RNA samples isolated from both control and treated cells, 5-μg aliquots, was performed using the human inflammatory response cytokines GE array kit from Super Array, Inc. (Bethesda, MD) according to the manufacturer's instructions. This array is composed of 23 genes involved in inflammatory response including a variety of cytokines, growth factors, and interleukins such as IL-1α, -1β, -2, -4, -5, -6, -8, -10, -12A, -12B, -16, -17, and -18. It also includes two housekeeping genes, β-actin and glyceraldehyde-3-phosphate-dehydrogenase (G3PDH). The relative mRNA level of each gene was normalized against the levels of both housekeeping genes and expressed as a ratio of sample to control. For Northern blot analysis, 10 μg of total RNA were fractionated by 1% denatured formaldehyde agarose gel electrophoresis and blotted onto Zeta Probe Nylon membrane (Bio-Rad) by capillary transfer according to the manufacturer's instructions. After cross-linking with UV irradiation (Stratalinker model 2400, Stratagene, La Jolla, CA), the membranes were hybridized with a human radiolabeled IL-8 probe (289 base pairs), stripped, and subsequently hybridized with a human radiolabeled G3PDH probe (983 base pairs). For preparation of the hybridization probes, total RNA isolated from THP-1 cells was subjected to the reverse transcriptase-PCR by using the Super Script One-step kit from Life Technologies, Inc., and the corresponding PCR primers were from CLONTECH (Palo Alto, CA): IL-8, forward 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and reverse 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′; G3PDH, forward 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and reverse 5′-CATGTGGGCCATGAGGTCCACCAC-3′. Total RNA, 100-ng aliquot, was converted to cDNA according to the kit's instructions, and the PCR was conducted thereafter in the same reaction tube as follows: denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, and primer extension at 72 °C for 2 min, total of 30 cycles. The resulting PCR products were analyzed by agarose gel electrophoresis, purified by QIAEX II gel extraction kit (Qiagen, Valencia, CA), and subsequently labeled with [α-32P]dCTP (Amersham Pharmacia Biotech) using a Prime It II kit (Stratagene, La Jolla, CA). Hybridization and washes were performed as recommended by the manufacturer of the membrane (Bio-Rad). After autoradiography, the signals were quantified by densitometric analysis (ImageQuant software version 3.3, Molecular Dynamics, Sunnyvale, CA), and the relative mRNA level of IL-8 was normalized against that of G3PDH. The amount of IL-8 released in the media was assessed by ELISA (BIOSOURCE International, Camarillo, CA) according to the manufacturer's instructions. All of the measurements were conducted in triplicate. In this preliminary work we used apo(a) because according to our previous studies (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, 20Klezovitch O. Edelstein C. Zhu L. Scanu A.M. J. Biol. Chem. 1998; 273: 23856-23865Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) it is the active component of Lp(a). Human THP-1 monocytes and THP-1 macrophages were incubated in the presence and absence of apo(a), 220 nm, for 24 h at 37 °C, and the gene expression in these cells was analyzed by the human inflammatory response cytokines microarray (Super Array, Inc., Bethesda, MD). Among the 23 genes examined, a 6-fold stimulation was observed for IL-8 mRNA in the apo(a)-treated THP-1 macrophages as compared with the untreated cells. This apo(a) effect on IL-8 was not observed in the THP-1 monocytes. Based on these results, we set out to identify in more detail the elements of Lp(a) responsible for the IL-8 stimulation. These products were obtained from the same donor. We first incubated THP-1 macrophages with various concentrations of Lp(a) and then evaluated the level of IL-8 expression by both Northern blot analysis and ELISA. As shown in Fig. 2, treatment of the THP-1 macrophages with this lipoprotein resulted in a dose-dependent induction of both IL-8 mRNA level (up to 12-fold) and the secretion of IL-8 protein (up to 6-fold) into the culture medium. Apo(a) also caused a dose-dependent increase in the production of IL-8 mRNA (up to 32-fold) and protein (up to 22-fold), indicating a greater stimulating efficiency compared with its parent Lp(a). Because bacterial endotoxin is a potent inducer of IL-8 expression in macrophages (32Wilson L. Butcher C.J. Kellie S. FEBS Lett. 1993; 325: 295-298Crossref PubMed Scopus (32) Google Scholar, 33Schmidt J. Fleissner S. Heimann-Weitschat I. Lindstaet R. Pomberg B. Werner U. Szelenyi I. Immunopharmacology. 1994; 27: 173-179Crossref PubMed Scopus (57) Google Scholar), we determined the endotoxin content of Lp(a) and apo(a) by using the Limulus amoebocyte lysate assay. The amount of endotoxin in Lp(a) and apo(a) was extremely small (less than 0.3 pg/μg for Lp(a) protein and 0.25 pg/μg for apo(a)), a value that is in the same order of magnitude of that reported for the LDL preparations used by other investigators (34Wang N. Tabas I. Winchester R. Ravalli S. Rabbani L.E. Tall A. J. Biol. Chem. 1996; 271: 8837-8842Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 35Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Arterioscler. Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar) in this cell system. Neither Lp(a−) (Fig. 2) nor LDL (endotoxin 0.2 pg/μg of LDL protein) had an effect on IL-8 production. Taken together, these results indicate that the increased production of IL-8 by THP-1 macrophages induced by Lp(a) was due to apo(a) and was not endotoxin-related. To define the region on apo(a) responsible for the effect on IL-8 in THP-1 macrophages, we used in our assay the two main proteolytic fragments of apo(a), F1 and F2, along with a full-length apo(a). We first established that apo(a), upon incubation with THP-1 macrophages, remained intact as assessed by Western blot analysis of the immunoreactive apo(a) present in the culture medium (data not shown). F2 also remained intact and was 1.5–2-fold more potent than apo(a) both in terms of IL-8 RNA and protein induction (Fig. 3). In contrast, F1 exhibited only a limited activity. To define the region in F2 responsible for the IL-8 induction, we studied non-overlapping apo(a) fragments of F2 obtained by elastase digestion, namely F5, F6, and F7 (see Fig. 1). Of them, F7, located in the C-terminal portion of F2, stimulated IL-8 production more efficiently than F5 and F6 (Fig. 3). However, none of those fragments was individually as potent as the whole F2. Of note, plasminogen exhibited only a minimal effect regarding IL-8 induction (Fig. 3). The evidence that F2 studied as a fragment is the domain responsible for the action of apo(a) on the production of IL-8 in THP-1 macrophages, prompted us to determine whether this also applies to F2 as a part of apo(a). To this end, we exposed apo(a) to different concentrations of a monoclonal antibody specific for KV, prior to the incubation with the cells. As shown in Fig. 4, this antibody caused a concentration-dependent inhibition of the IL-8 mRNA production. In turn, no inhibition was observed when an irrelevant mouse IgG was used (data not shown). For this purpose, we performed experiments using DRB, an inhibitor of RNA polymerase II. THP-1 macrophages were cultured in either the presence or absence of apo(a) for 20 h and then exposed to DRB for various time intervals. As shown in Fig. 5, following the arrest of transcription, the rate of decay of IL-8 mRNA in both apo(a)-treated and untreated cells was similar indicating that apo(a) had no effect on the degradation and stability of IL-8 mRNA, suggesting an induction of expression at the transcriptional level. These experiments were carried out to determine whether the G-proteins were required for the action of apo(a) on IL-8 production. For this purpose, THP-1 macrophages were incubated with apo(a) in either the presence or absence of either cholera toxin (inhibitor of stimulatory G protein, Gs) or pertussis toxin (inhibitor of inhibitory G protein, Gi). Cholera toxin totally inhibited the apo(a)-mediated accumulation of IL-8 mRNA contrary to the lack of effect by the pertussis toxin (Fig. 6). These results suggest that the stimulatory G protein signal transduction pathway might have been responsible for the apo(a)-mediated effect. In the present study we have shown that in THP-1 macrophages, Lp(a) under low endotoxin conditions is an inducer of IL-8, a major pro-inflammatory chemokine (36Boisvert W.A. Curtiss L.K. Terkeltaub R.A. Immunol. Res. 2000; 21: 129-137Crossref PubMed Scopus (122) Google Scholar). We have also shown that this effect is at both the mRNA and protein levels and that apo(a) is responsible for the action of Lp(a). This stimulation was macrophage-specific because no effect was elicited when apo(a) was incubated with the unstimulated THP-1 monocytes. In inducing IL-8, apo(a) was markedly more efficient than parent Lp(a) possibly due to the masking of the apo(a)-active site(s) by the LDL moiety. In this vein, we have shown previously that in vitro apo(a) is more efficient than parent Lp(a) in binding to lysine-Sepharose (26Edelstein C. Mandala M. Pfaffinger D. Scanu A.M. Biochemistry. 1995; 34: 16483-16492Crossref PubMed Scopus (42) Google Scholar), fibrinogen (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, 37Klezovitch O. Edelstein C. Scanu A.M. J. Clin. Invest. 1996; 98: 185-191Crossref PubMed Scopus (39) Google Scholar), fibronectin (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar), and decorin (20Klezovitch O. Edelstein C. Zhu L. Scanu A.M. J. Biol. Chem. 1998; 273: 23856-23865Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The importance of apo(a) in the Lp(a) action was also supported by the finding that both Lp(a−) and authentic LDL from the same subject had little or no effect on the production of IL-8 by THP-1 macrophages even at very high lipoprotein concentrations. Of note, our finding regarding authentic LDL is in agreement with the previous observations by Wang et al. (34Wang N. Tabas I. Winchester R. Ravalli S. Rabbani L.E. Tall A. J. Biol. Chem. 1996; 271: 8837-8842Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) in THP-1 macrophages and Terkeltaub et al. (35Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Arterioscler. Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar) in THP-1 monocytes. In both studies, LDL needed to undergo either oxidative (34Wang N. Tabas I. Winchester R. Ravalli S. Rabbani L.E. Tall A. J. Biol. Chem. 1996; 271: 8837-8842Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,35Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Arterioscler. Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar), acetylation (34Wang N. Tabas I. Winchester R. Ravalli S. Rabbani L.E. Tall A. J. Biol. Chem. 1996; 271: 8837-8842Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), or phospholipase A2-induced modification (35Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Arterioscler. Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar) to stimulate production of IL-8. Of note, in the studies by Terkeltaub et al. (35Terkeltaub R. Banka C.L. Solan J. Santoro D. Brand K. Curtiss L.K. Arterioscler. Thromb. 1994; 14: 47-53Crossref PubMed Google Scholar) the stimulatory effect of oxidized LDL was mediated by oxidized lipid end products. In turn, our studies with unmodified Lp(a) demonstrated that the action on IL-8 was protein- and not lipid-dependent pointing at important functional differences between LDL and Lp(a) when studied in their native state. By using proteolytic derivatives of apo(a), we also provided evidence that F2 was the domain responsible for the IL-8 stimulatory effect and that this effect was 1.5–2-fold higher than that exhibited by intact apo(a). There are two possible explanations for these findings. First, F2, as an isolated fragment, may assume a conformation that is different from that in intact apo(a). Second, isolated F1, although in our system exhibited little activity on IL-8, may exhibit some hindering action on F2 when a component of full-length apo(a) was used. Irrespective of mechanisms, however, it is apparent that apo(a) and some of its fragments can differ in their functional expression and that this difference should be taken into account when studying Lp(a) at the tissue level. In this context, we have reported previously (21Fortunato J.E. Bassiouny H.S. Song R.H. Kocharian H. Glagov S. Edelstein C. Scanu A.M. J. Vasc. Surg. 2000; 32: 555-563Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) that F2 is abundant in surgical segments of unstable human carotid plaques and co-localized with MMP-2 and MMP-9 in areas enriched in macrophages. Both MMPs cleave apo(a). Moreover, MMP-9 is the major MMP produced by macrophages and thus a potential contributor to apo(a) fragmentation. In our experimental system we observed no fragmentation of apo(a) upon its incubation with cultured macrophages, suggesting that the secreted MMPs were not adequately activated to cause proteolysis. Based on the finding on F2, we may surmise that if apo(a) fragmentation were to occur either in vitro or in vivo, the pro-inflammatory action of apo(a) will be maintained or even enhanced by some of its fragments. As we reported previously (19Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, 30Edelstein C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 38Edelstein C. Shapiro S.D. Klezovitch O. Scanu A.M. J. Biol. Chem. 1999; 274: 10019-10023Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), the two major fragments, F1 and F2, are in vitro products of a limited proteolysis by either elastase or MMPs. However, under conditions of a more extensive digestion, smaller apo(a) fragments are generated (30Edelstein C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig. 1). The use of them in our current study permitted us to identify the region in apo(a) comprising KV and the protease domain predominantly responsible for the IL-8 effect. Of interest, this region has been suggested to be involved in the binding of apo(a) to fibronectin. 2O. Klezovitch and A. M. Scanu, unpublished observations. This region is structurally very similar but not identical to its counterpart in plasminogen that we found to have a very limited effect on IL-8 production, indicating that even subtle structural divergence might have been responsible for the difference in action between the two proteins. Of the 13 interleukins examined, IL-8 was the one that was stimulated by apo(a) in our cell system. IL-8 is a known pro-inflammatory chemokine that is expressed in macrophage-rich areas of human coronary atheromas as assessed by both immunohistochemical techniques andin situ hybridization studies (34Wang N. Tabas I. Winchester R. Ravalli S. Rabbani L.E. Tall A. J. Biol. Chem. 1996; 271: 8837-8842Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The pathogenicity of this chemokine is mostly attributed to its chemotactic activity toward neutrophils (39Baggiolini M. Walz A. Kunkel S.L. J. Clin. Invest. 1989; 84: 1045-1049Crossref PubMed Scopus (1632) Google Scholar), T-cells (40Larsen C.G. Anderson A.O. Appella E. Oppenheim J.J. Matsushima K. Science. 1989; 243: 1464-1466Crossref PubMed Scopus (914) Google Scholar), monocytes (41Gersztein R.E. Garcia-Zepeda E.A. Lim Y.-C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rozenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar), smooth muscle (42Yue T.L. McKenna P.J. Gu J.L. Feuerstein G.Z. Eur. J. Pharmacol. 1993; 240: 81-84Crossref PubMed Scopus (55) Google Scholar, 43Yue T.L. Wang X. Sung C.P. Olson B. Mckenna P.J. Gu J.L. Feuerstein G.Z. Circ. Res. 1994; 75: 1-7Crossref PubMed Google Scholar), and endothelial cells (44Koch A.E. Polverini P.J. Kunkel S.L. Harlow L.A. DiPietro L.A. Elner V.M. Elner S.G. Strieter R.M. Science. 1992; 268: 447-448Google Scholar), as well as to its mitotic (43Yue T.L. Wang X. Sung C.P. Olson B. Mckenna P.J. Gu J.L. Feuerstein G.Z. Circ. Res. 1994; 75: 1-7Crossref PubMed Google Scholar) and angiogenic properties (44Koch A.E. Polverini P.J. Kunkel S.L. Harlow L.A. DiPietro L.A. Elner V.M. Elner S.G. Strieter R.M. Science. 1992; 268: 447-448Google Scholar). IL-8 is also a long-lived chemokine resistant to proteolytic degradation compared with the other cytokines produced by macrophages (45Colditz I.G. Zwahlen R.D. Biggiolini M. J. Leukocyte Biol. 1990; 48: 129-137Crossref PubMed Scopus (73) Google Scholar). Thus, we may speculate that the apo(a)-dependent increased production and secretion of IL-8 by activated macrophages and its subsequent accumulation in the vascular extracellular matrix may contribute to the progression of the chronic inflammation process in atheromas. The mechanism by which apo(a)/fragments stimulate IL-8 production remains to be established. Our current data with DRB, an inhibitor of transcription, suggest that apo(a) stimulated the synthesis of IL-8 mRNA rather than affected its degradation. Moreover, the results of our toxin studies favor an involvement of the G protein signal transduction pathway in the Lp(a)/apo(a) action. These promising leads and the potential pleiotropic effects of Lp(a) justify further mechanistic studies utilizing larger scale microarray systems comprising a variety of genes relevant to the general areas of inflammation and atherosclerosis." @default.
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• W1993900511 title "Stimulation of Interleukin-8 Production in Human THP-1 Macrophages by Apolipoprotein(a)" @default.
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