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Cysteine Protease Cathepsin F Is Expressed in Human Atherosclerotic Lesions, Is Secreted by Cultured Macrophages, and Modifies Low Density Lipoprotein Particles in Vitro

2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês

10.1074/jbc.m310814200

ISSN

1083-351X

Autores

Katariina Öörni, Mia Sneck, Dieter Brömme, Markku O. Pentikäinen, Ken A. Lindstedt, Mikko I. Mäyränpää, Helena Aitio, Petri T. Kovanen,

Tópico(s)

Lipoproteins and Cardiovascular Health

Resumo

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis. During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis. During atherogenesis, lipid droplets accumulate extracellularly within the inner layer of the arterial wall, the intima. Initially, the droplets accumulate subendothelially (1Guyton J.R. Klemp K.F. Black B.L. Bocan T.M.A. Eur. Heart. J. 1990; 11: 20-28Crossref PubMed Google Scholar). These droplets, which appear to be derived from low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; apo, apolipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4,6-diamidino-2-phenylindole; BSA, bovine serum albumin; TTBS, Tris-buffered saline plus Tween 20; DTT, dithiothreitol; GM-CSF, granulocytemacrophage colony-stimulating factor; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. particles, are entrapped by the arterial extracellular matrix, especially by its proteoglycans (2Öörni K. Pentikäinen M.O. Ala-Korpela M. Kovanen P.T. J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). In the intima, the proteoglycans form an organized tight network (3Wight T.N. Fuster V. Ross R. Topol E.J. Atherosclerosis and Coronary Artery Disease. Lippincott-Raven Publishers, Philadelphia1996: 421-440Google Scholar) that has the potential to bind apolipoprotein (apo) B-100-containing lipoproteins, notably LDL particles (4Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 5Hurt-Camejo E. Olsson U. Wiklund O. Bondjers G. Camejo G. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1011-1017Crossref PubMed Scopus (137) Google Scholar, 6Camejo G. Hurt-Camejo E. Wiklund O. Bondjers G. Atherosclerosis. 1998; 139: 205-222Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 7Williams K.J. Tabas I. Curr. Opin. Lipidol. 1998; 9: 471-474Crossref PubMed Scopus (300) Google Scholar). Binding of LDL by proteoglycans increases their residence time in the arterial intima and renders the particles more susceptible to various types of modifications, which leads to increased binding strength and accumulation of the LDL-derived cholesterol in the arterial intima (2Öörni K. Pentikäinen M.O. Ala-Korpela M. Kovanen P.T. J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). The importance of the initial LDL-proteoglycan interaction has been directly assessed with the use of transgenic mice expressing proteoglycan binding-deficient human apoB-100 (8Borén J. Olin K. Lee I. Chait A. Wight T.N. Innerarity T.L. J. Clin. Invest. 1998; 101: 2658-2664Crossref PubMed Scopus (231) Google Scholar). Thus, despite the accompanying hypercholesterolemia, the binding-deficient LDL caused delayed atherosclerosis as compared with that in control mice expressing normal human apoB-100 (9Skalen K. Gustafsson M. Rydberg E.K. Hulten L.M. Wiklund O. Innerarity T.L. Boren J. Nature. 2002; 417: 750-754Crossref PubMed Scopus (735) Google Scholar). The apoB-100 in the LDL particles isolated from the human atherosclerotic arterial intima is fragmented to variable degrees (10Clevidence B.A. Morton R.E. West G. Dusek D.M. Hoff H.F. Arteriosclerosis. 1984; 4: 196-207Crossref PubMed Google Scholar, 11Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar, 12Hoff H.F. O'Neil J. Arterioscler. Thromb. 1991; 11: 1209-1222Crossref PubMed Google Scholar, 13Steinbrecher U.P. Lougheed M. Arterioscler. Thromb. 1992; 12: 608-625Crossref PubMed Google Scholar, 14Tailleux A. Torpier G. Caron B. Fruchart J.-C. Fievet C. J. Lipid Res. 1993; 34: 719-728Abstract Full Text PDF PubMed Google Scholar, 15Rapp J.H. Lespine A. Hamilton R.L. Colyvas N. Chaumeton A.H. Tweedie-Hardman J. Kotite L. Kunitake S.T. Havel R.J. Kane J.P. Arterioscler. Thromb. 1994; 14: 1767-1774Crossref PubMed Google Scholar). Moreover, when compared with LDL in plasma, arterial lipid droplets have a reduced protein content and contain no immunoreactive apoB-100 (16Kruth H.S. Subcell. Biochem. 1997; 28: 319-362Crossref PubMed Scopus (30) Google Scholar), and LDL particles, when deposited in human atherosclerotic lesions, lose their apoB-100 immunoreactivity (17Kruth H.S. Shekhonin B. Atherosclerosis. 1994; 105: 227-234Abstract Full Text PDF PubMed Scopus (19) Google Scholar), suggesting that apoB-100 in the arterial intima is subjected to proteolytic degradation. Indeed, proteolysis of apoB-100 in vitro has been shown to induce fusion of LDL particles into lipid droplets that resemble those found in atherosclerotic lesions (18Kovanen P.T. Kokkonen J.O. J. Biol. Chem. 1991; 266: 4430-4436Abstract Full Text PDF PubMed Google Scholar, 19Paananen K. Kovanen P.T. J. Biol. Chem. 1994; 269: 2023-2031Abstract Full Text PDF PubMed Google Scholar, 20Paananen K. Saarinen J. Annila A. Kovanen P.T. J. Biol. Chem. 1995; 270: 12257-12262Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Piha M. Lindstedt L. Kovanen P.T. Biochemistry. 1995; 34: 10120-10129Crossref PubMed Scopus (66) Google Scholar). On the basis of the above findings, we have proposed that proteolytic modification of LDL particles may be one mechanism leading to LDL fusion and the appearance of the typical extracellular lipid droplets in the arterial intima (2Öörni K. Pentikäinen M.O. Ala-Korpela M. Kovanen P.T. J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). However, only certain neutral proteases have been shown to be able to trigger aggregation and fusion of LDL particles, these proteases having in common the ability to cause extensive cleavage of apoB-100, i.e. degradation into small peptide fragments, some of which are released from the LDL particles (21Piha M. Lindstedt L. Kovanen P.T. Biochemistry. 1995; 34: 10120-10129Crossref PubMed Scopus (66) Google Scholar). Cultured monocyte-derived macrophages (22Reddy V.Y. Zhang Q.Y. Weiss S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3849-3853Crossref PubMed Scopus (279) Google Scholar, 23Punturieri A. Filippov S. Allen E. Caras I. Murray R. Reddy V. Weiss S.J. J. Exp. Med. 2001; 192: 789-799Crossref Scopus (190) Google Scholar) and smooth muscle cells, when stimulated with proinflammatory cytokines (24Sukhova G.K. Shi G.P. Simon D.I. Chapman H.A. Libby P. J. Clin. Invest. 1998; 102: 576-583Crossref PubMed Scopus (565) Google Scholar), have been shown to secrete lysosomal papain-like cysteine proteases. Normally, these cysteine proteases play a major role in intracellular protein degradation and turnover in lysosomes, but they are also capable of degrading proteins extracellularly (25Turk B. Turk D. Turk V. Biochim. Biophys. Acta. 2000; 1477: 98-111Crossref PubMed Scopus (695) Google Scholar). By degrading the components of the arterial extracellular matrix, the secreted lysosomal cysteine proteases could contribute to the development of atherosclerotic lesions. Indeed, when human arteries were examined for the presence of two cysteine proteases, cathepsins S and K, normal arterial segments were found to contain little or none, whereas atherosclerotic lesions contained abundant immunoreactive cathepsins S and K (24Sukhova G.K. Shi G.P. Simon D.I. Chapman H.A. Libby P. J. Clin. Invest. 1998; 102: 576-583Crossref PubMed Scopus (565) Google Scholar). Moreover, cystatin C, a natural extra-cellular cysteine protease inhibitor, was found to be down-regulated in the lesions (26Shi G.P. Sukhova G.K. Grubb A. Ducharme A. Rhode L.H. Lee R.T. Ridker P.M. Libby P. Chapman H.A. J. Clin. Invest. 1999; 104: 1191-1197Crossref PubMed Scopus (413) Google Scholar). In addition, atherosclerotic mouse models have provided further support for the view that cysteine proteases play a role in the pathobiology of the arterial wall. Thus, the expression of cathepsins B, L, and S, was found to be increased in apoE-deficient mice (27Jormsjo S. Wuttge D.M. Sirsjo A. Whatling C. Hamsten A. Stemme S. Eriksson P. Am. J. Pathol. 2002; 161: 939-945Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and deficiency of cathepsin S was shown to reduce atherosclerosis in LDL receptor-deficient mice (28Sukhova G.K. Zhang Y. Pan J.H. Wada Y. Yamamoto T. Naito M. Kodama T. Tsimikas S. Witztum J.L. Lu M.L. Sakara Y. Chin M.T. Libby P. Shi G.P. J. Clin. Invest. 2003; 111: 897-906Crossref PubMed Scopus (320) Google Scholar). We have now examined the possible role of a newly described lysosomal cysteine protease, cathepsin F (29Wang B. Shi G.P. Yao P.M. Li Z. Chapman H.A. Brömme D. J. Biol. Chem. 1998; 273: 32000-32008Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), in human atherosclerosis and examined the ability of cathepsins to generate lipid droplets from LDL particles. We have tested the effects of human recombinant cathepsins F, S, and K on LDL particles, notably on the degradation of apoB-100, on the aggregation and fusion of LDL particles, and on the retention of LDL particles by human aortic proteoglycans in vitro. We have also followed the expression and secretion of cathepsin F in cultured human monocyte-derived macrophages. Finally, we have also looked for the presence of cathepsin F in normal and atherosclerotic human coronary arteries and compared its localization with that of cathepsins S and K. Materials—Bovine serum albumin, diaminobenzidine, dermatan sulfate, heparan sulfate, and hyaluronan were purchased from Sigma. Chondroitin 4-sulfate and chondroitin 6-sulfate were obtained from Seikagaku Kogyo (Tokyo, Japan). [1,2-3H]Cholesteryl linoleate, t-Butoxycarbonyl-l-[35S]methionine N-hydroxysuccinimidyl ester (the 35S labeling reagent), HiTrap SP columns, protein A-Sepharose, and the nucleotides were from Amersham Biosciences (Uppsala, Sweden). Cystatin C was from Calbiochem (San Diego, CA). Vectastain ABC kits and methyl green were from Vector Laboratories (Burlingame, CA), and anti-human CD68 (PG-M1), anti-human macrophage (HAM56), anti-muscle actin (HHF35) and horseradish peroxidase-conjugated anti-mouse IgG (P0447) antibodies were obtained from Dako (Glostrup, Denmark). A rabbit polyclonal antibody against human recombinant cathepsin F was generated (mature cathepsin F protein produced in Escherichia coli). In addition, a rabbit polyclonal anti-cathepsin F antibody (SC-13987) from Santa Cruz Biotechnology (Santa Cruz, CA) and a mouse monoclonal anti-cathepsin F antibody from Novocastra Laboratories (United Kingdom) were used. Anti-cystatin C antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). Alexa-conjugated isotype-specific goat anti-mouse IgG antibodies and DAPI were from Molecular Probes (Leiden, The Netherlands). Microtiter plates (Combiplate 8, Enhanced Binding) were from Labsystems (Helsinki, Finland), Falcon 12-well cell culturing plates from Becton Dickinson (NJ) and 25-cm2 cell culturing bottles from Nalge Nunc International. Dulbecco's modified Eagle's medium was purchased from BioWhittaker Europe (Verviers, Belgium), nitrocellulose filters (Transblot Transfer Medium) from Bio-Rad, and Vivaspin concentrators from Vivascience (Hannover, Germany). l-Glutamine, macrophage-SFM medium, Moloney murine leukemia virus reverse transcriptase kit, nonessential amino acids, penicillin-streptomycin, random primers, RPMI 1640, sodium pyruvate, and Ultrapure Agarose were from Invitrogen (Paisley, Scotland). Taq polymerase, RNase inhibitor, and the lactate dehydrogenase kit were from Roche (Basel, Switzerland), RNeasy minikit and DNase from Qiagen (Hilden, Germany), and human GM-CSF (Leucomax) from Schering-Plough. Cholesteryl ester transfer protein was a kind gift from Drs. Christian Ehnholm and Matti Jauhiainen at the National Public Health Institute, Helsinki, Finland. Preparation and Labeling of LDL—Human LDL (d = 1.019–1.050 g/ml) was isolated from the plasma of fasting healthy volunteers by sequential ultracentrifugation in the presence of 3 mm EDTA (30Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar, 31Radding C.M. Steinberg D. J. Clin. Invest. 1960; 39: 1560-1569Crossref PubMed Scopus (170) Google Scholar). 35S-Bolton-Hunter-LDL was prepared by labeling the protein component of the lipoproteins with a 35S labeling reagent by the Bolton-Hunter procedure (32Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2398) Google Scholar), as described previously (18Kovanen P.T. Kokkonen J.O. J. Biol. Chem. 1991; 266: 4430-4436Abstract Full Text PDF PubMed Google Scholar). [3H]Cholesteryl linoleate-LDL was prepared by incubating a mixture of LDL and cholesteryl ester transfer protein with solid dispersions of [3H]cholesteryl linoleate on Celite, as described (21Piha M. Lindstedt L. Kovanen P.T. Biochemistry. 1995; 34: 10120-10129Crossref PubMed Scopus (66) Google Scholar). In each experiment, the labeled lipoproteins were mixed with unlabeled lipoproteins. The amounts of LDL are expressed in terms of their protein concentrations, which were determined by the method of Lowry et al. (33Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as standard. Cathepsins F, S, and K—Human cathepsin F was produced by the Pichia expression system and purified using a HiTrap SP column, as previously described (29Wang B. Shi G.P. Yao P.M. Li Z. Chapman H.A. Brömme D. J. Biol. Chem. 1998; 273: 32000-32008Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Human cathepsins K and S were expressed as Pichia pastoris (34Linnevers C.J. McGrath M.E. Armstrong R. Mistry F.R. Barnes M.G. Klaus J.L. Palmer J.T. Katz B.A. Bromme D. Protein Sci. 1997; 6: 919-921Crossref PubMed Scopus (107) Google Scholar) and in Sf9 cells using the baculovirus expression system (35Brömme D. Bonneau P.R. Lachance P. Wiederanders B. Kirschke H. Peters C. Thomas D.Y. Storer A.C. Vernet T. J. Biol. Chem. 1993; 268: 4832-4838Abstract Full Text PDF PubMed Google Scholar), respectively. Molar concentrations of active cathepsins K and S were obtained by titration with E64 (36Barrett A.J. Kirschke H. Brown M.A. Kirschke H. Knight G.C. Tamai M. Hanada K. Biochem. J. 1982; 201: 189-198Crossref PubMed Scopus (920) Google Scholar), and that of cathepsin F with the irreversible inhibitor, LHVS using the same method as described for E64 (kindly provided by Celera Corp, South San Francisco, CA). Treatment of LDL with Cathepsins F, S, and K—LDL (0.5 mg/ml) was incubated with 20–100 nm human recombinant cathepsin F, S, or K in buffer A (20 mm MES, 150 mm NaCl, 2.5 mm EDTA, 1 mm DTT, pH 6.0) at 37 °C for the times indicated. When the effect of pH in LDL degradation was studied, the incubations were carried out in either buffer A, 20 mm PIPES, 150 mm NaCl, 2.5 mm EDTA, 1 mm DTT, pH 6.5 or 7.0, or 20 mm HEPES, 150 mm NaCl, 2.5 mm EDTA, 1 mm DTT, pH 7.5. In some experiments, the degradation assays were carried out in the presence of various glycosaminoglycans or proteoglycans. In control samples, LDL was incubated in the absence of proteolytic enzymes. Analysis of Proteolyzed LDL—The degree of proteolytic degradation was determined by measuring the amount of trichloroacetic acid-soluble radioactivity produced (20Paananen K. Saarinen J. Annila A. Kovanen P.T. J. Biol. Chem. 1995; 270: 12257-12262Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The degree of aggregation and/or fusion of proteolyzed [3H]cholesteryl linoleate-labeled LDL was determined by rate zonal ultracentrifugation (37Polacek D. Byrne M.E. Scanu A.M. J. Lipid Res. 1988; 29: 797-808Abstract Full Text PDF PubMed Google Scholar), as described previously (38Hakala J.K. Öörni K. Pentikäinen M.O. Hurt-Camejo E. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar). Briefly, a linear NaBr gradient (d = 1.006–1.10 g/ml) was layered on top of 50-μl samples of modified [3H]cholesteryl linoleate-LDL in 250 μl of 40% NaBr (w/v) and centrifuged at 33,000 rpm in a SW 40 Ti rotor (Beckman) for 1 h at 20 °C. The gradient was then divided into 500-μl fractions, and the radioactivities were determined using a scintillation counter 1H NMR Spectroscopy—For 1H NMR spectroscopy measurements, LDL samples were prepared at 1 mg/ml concentration. The samples were incubated at 37 °C in the NMR spectrometer in buffer A in the presence and absence (control LDL) of 100 nm cathepsin F, K, or S during data acquisition for 24 h. During the initial 3 h, a spectrum was recorded every 10 min, and subsequently once every 1 h. The spectral width was set to 7008 Hz, comprising 32,000 points yielding a free induction decay of 2.34 s. The recycle delay was 6.4 s. Data were zero-filled eight times and Fourier-transformed. Sodium 3-trimethylsilyl[2,2,3,3-D4]propionate (8 mm) and MnSO4 (0.6 mm), in 99.8% D2O, in a thin coaxial capillary were used as an external chemical shift reference. All the spectra were obtained with a 600-MHz Varian Inova NMR spectrometer at the Institute for Biotechnology NMR Laboratory (Helsinki, Finland). Electron Microscopy of LDL—For thin-section transmission electron microscopy, LDL samples were cast in agarose, and then fixed (39Pentikäinen M.O. Lehtonen E.M.P. Kovanen P.T. J. Lipid Res. 1996; 37: 2638-2649Abstract Full Text PDF PubMed Google Scholar), and stained with the osmium-tannic acid-paraphenylenediamine technique (40Guyton J.R. Klemp K.F. J. Histochem. Cytochem. 1988; 36: 1319-1328Crossref PubMed Scopus (72) Google Scholar). For negative staining electron microscopy, samples (3 μl) were dried on carbon-coated grids, after which 3 μl of 1% potassium phosphotungstate, pH 7.4, was added and also dried on the grids (41Forte T. Nichols A.V. Adv. Lipid Res. 1972; 10: 1-41Crossref PubMed Google Scholar). The samples were viewed and photographed in a JEOL 1200EX electron microscope at the Institute for Biotechnology, Department of Electron Microscopy (Helsinki, Finland). Preparation and Characterization of Aortic Proteoglycans—Proteoglycans from the intima media of human aortas obtained at autopsy within 24 h of accidental death were prepared essentially by the method of Hurt-Camejo et al. (42Hurt-Camejo E. Camejo G. Rosengren B. López F. Wiklund O. Bondjers G. J. Lipid Res. 1990; 31: 1387-1398Abstract Full Text PDF PubMed Google Scholar), as described previously (43Öörni K. Pentikäinen M.O. Annila A. Kovanen P.T. J. Biol. Chem. 1997; 272: 21303-21311Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Glycosaminoglycans were determined by the method of Bartold and Page (44Bartold P.M. Page R.C. Anal. Biochem. 1985; 150: 320-324Crossref PubMed Scopus (59) Google Scholar), and the amounts of the proteoglycans are expressed in terms of their glycosaminoglycan contents. Binding of LDL to Proteoglycans in a Microtiter Well Assay—The wells in polystyrene 96-well plates were coated with human aortic proteoglycans (50 μg/ml) or with BSA (5 mg/ml) and blocked as described (38Hakala J.K. Öörni K. Pentikäinen M.O. Hurt-Camejo E. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar). 35S/3H-LDL (10 μg) was incubated with or without recombinant cathepsins F, K, or S (20 nm) in reaction buffer (buffer A containing 1% BSA) for 16 h at 37 °C. The supernatants were removed, and proteolysis was measured by determining the amounts of trichloroacetic acid-soluble radioactivity in the supernatants. The wells were washed three times with 250 μl of buffer A containing 50 mm NaCl, and the radioactivity bound to the wells was measured. Specific binding to the proteoglycans was calculated by subtracting the amount of LDL bound to the BSA-coated wells from the amount of LDL bound to the proteoglycan-coated wells. Preparation of Macrophage Monolayers—Human monocytes were isolated from buffy coats (kind gifts from the Finnish Red Cross Blood Transfusion Center, Helsinki, Finland) by centrifugation in Ficoll-Paque gradient as described (45Saren P. Welgus H.G. Kovanen P.T. J. Immunol. 1996; 157: 4159-4165PubMed Google Scholar). Washed cells were suspended in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin, counted, and seeded in bottles (25 × 106 cells/25 cm2) for Western blotting or in 12-well plates (4 × 106 cells/well) for mRNA analysis. After 1 h, nonadherent cells were removed and the medium was replaced with macrophage-SFM supplemented with penicillin-streptomycin and GM-CSF (11 ng/ml). mRNA Analysis—Monocyte-macrophages were cultured for up to 15 days, and at various time points the total RNA was isolated using an RNeasy minikit (Qiagen) in the presence of DNase. For the isolation, the cells from three different donors were pooled. Total RNA was reverse transcribed into cDNA using a Moloney murine leukemia virus reverse transcriptase kit (Invitrogen) in the presence of an RNase inhibitor. The cDNA obtained was further amplified by PCR using specific oligonucleotides for cathepsin F: 5′-TCA GTG ATC TCA CAG AGG AGG (sense) and 5′-TAG TCA TCC TCT GTC TCC AGC (anti-sense) and conditions were 40 cycles, Tm 58 °C. GAPDH-PCR was used for quality control. Primers for GAPDH were 5′-ACC ACA GTC CAT GCC ATC AC (sense) and 5′-TCC ACC ACC CTG TTG CTG TA (anti-sense). Conditions used were 25 cycles, Tm 58 °C. The PCR products were separated on a 1.4% agarose gel, stained with ethidium bromide, and quantified with a Gel Doc 2000 gel documentation system. Western Blot Analysis of Monocyte-Macrophage Media and Lysates— Monocyte-macrophages were cultured for up to 13 days, and at various time points the medium was replaced with RPMI 1640 supplemented with penicillin-streptomycin and l-glutamine (2 mm). The cells were further cultured for 2 days, after which the media were collected. Lactate dehydrogenase activity in the media and in the cells was measured from parallel incubations using a commercial kit. The level of lactate dehydrogenase activity in the media varied between 5 and 10% of the total cellular activity and did not increase during the 15-day culture period. For each sample, media from three different donors were pooled. Nonadherent cells were removed from the media by centrifugation, protease inhibitors (1 mm PMSF, 2 mm benzamidine, 5 mm EDTA) were added, and the samples were concentrated into 1/20 using Vivaspin concentrators. 20 μl of reducing SDS-PAGE buffer (0.25 m Tris-HCl, pH 6.8, 4% SDS, 0.002% bromphenol blue, 40% glycerol, 1% β-mercaptoethanol) was added to 20 μl of the sample. Whole cell lysates were prepared by lysing the cells with reducing SDS-PAGE buffer. For each sample, cells from three different donors were pooled. Proteins were separated by SDS-PAGE using 4–20% gradient gels, after which the samples were transferred to nitrocellulose filters. The filters were blocked by incubation in 3% BSA in 10 mm Tris-HCl, pH 7.4, containing 0.15 m NaCl, and 0.1% Tween 20 (TTBS) for 1 h. Cathepsin F was detected using anti-human cathepsin F monoclonal antibody (1:50 in 1% BSA-TTBS) and horseradish peroxidase-conjugated anti-mouse antibody (1:2000 in 1% BSA-TTBS). The bands were detected by using a commercial enhanced chemiluminescence kit (Amersham Biosciences). Detection of Active Cathepsin F in Macrophage-conditioned Media— Monocyte-derived macrophages were cultured for 13 days as described above. The cells were then cultured for 2 days in the absence and presence of cystatin C (15 μg/ml) in serum-free RPMI supplemented with penicillin-streptomycin and l-glutamine, after which the media were collected as described above. The media were pre-precipitated with 50 μl of protein A-Sepharose for 1 h at 4 °C, after which the media were first incubated with 2 μl of anti-cystatin C antibody for 1 h at 4 °C and then with 50 μl of protein A-Sepharose for 2 h at 4 °C. The bound proteins were eluted with 20 μl of reducing SDS-PAGE sample buffer, loaded into a 4–20% SDS-polyacrylamide gel, and immunoblotted with cathepsin F monoclonal antibody as described above. Immunohistochemistry—Coronary samples were obtained, with permission from the Ethical Committee of Helsinki University Central Hospital, from hearts discarded during heart transplantation. The samples were fixed in 10% formalin and embedded in paraffin, using standard procedures. Paraffin (8 μm) sections were cut, and the sections were deparaffinized, rehydrated, microwaved at high power for 2 × 5 min in 10 mm citrate buffer (pH 6.0), and immunostained with ABC Elite kits from Vector Laboratories according to the instructions from the manufacturer, using diaminobenzidine as the peroxidase substrate. The sections were then counterstained with methyl green, dehydrated, and mounted with Permount. The sections were also double immunostained for cathepsin F and various cell type markers by incubating the samples first with a combination of a monoclonal anti-cathepsin F (IgG2a) antibody and monoclonal cell type markers (IgG1) followed by fluorescently labeled isotype-specific secondary antibodies, and in these samples the nuclei were stained with DAPI. The samples were viewed and photographed with a Nikon E600 fluorescence microscope equipped with a cooled CCD camera (Spot RT, Diagnostic Instruments). The primary antibodies used were rabbit anti-human cathepsin F anti-serum (29Wang B. Shi G.P. Yao P.M. Li Z. Chapman H.A. Brömme D. J. Biol. Chem. 1998; 273: 32000-32008Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) (1:200 dilution), commercial rabbit anti-human cathepsin F antibody (2 μg/ml), commercial mouse-anti human cathepsin F monoclonal antibody (1:50), mouse anti-human CD68 antibody (7 μg/ml), mouse anti human-macrophage (HAM-56) antibody (0.7 μg/ml), mouse anti-CD31 antibody (10 μg/ml) for endothelial cell, mouse anti-CD43 (2.7 μg/ml) for T lymphocytes, and mouse anti-muscle actin (1 μg/ml) for smooth muscle cells. In the controls, the primary antibodies were omitted or replaced with similar concentrations of nonimmune rabbit serum or nonimmune mouse IgG1 or IgG2a. As a further control, immunostaining was conducted on methanol-fixed frozen sections of human coronary arteries. Cathepsins F, K, and S have a slightly acidic pH optimum (∼pH 6) and although cathepsins F and K are rapidly inactivated at neutral pH, cathepsin S is stable also at neutral pH (29Wang B. Shi G.P. Yao P.M. Li Z. Chapman H.A. Brömme D. J. Biol. Chem. 1998; 273: 32000-32008Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 35Brömme D. Bonneau P.R. Lachance P. Wiederanders B. Kirschke H. Peters C. Thomas D.Y. Storer A.C. Vernet T. J. Biol. Chem. 1993; 268: 4832-4838Abstract Full Text PDF PubMed Google Scholar, 47Bromme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Here, we first examined the ability of cathepsins F, K, and S to degrade apoB-100 in LDL particles at pH range 6.0–7.5. The degree of proteoly

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