Title: Secretory Sphingomyelinase, a Product of the Acid Sphingomyelinase Gene, Can Hydrolyze Atherogenic Lipoproteins at Neutral pH
Abstract: The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase)in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitroSM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation. The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase)in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitroSM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation. A critical event in early atherogenesis is the subendothelial retention of atherogenic lipoproteins, including LDL 1The abbreviations used are: LDL, low-density lipoprotein; apo, apolipoprotein; L-SMase, lysosomal sphingomyelinase; PC, phosphatidylcholine; SM, sphingomyelin; SMase, sphingomyelinase; sPLA2, nonpancreatic secretory phospholipase A2; S-SMase; secretory sphingomyelinase; TBARS, thiobarbituric acid-reactive substances; DAG, diacylglycerol; LDLr0, LDL receptor-deficient; E0, apolipoprotein E-deficient. (1Schwenke D.C. Carew T.E. Arteriosclerosis. 1989; 9: 895-907Crossref PubMed Google Scholar, 2Nievelstein P.F.E.M. Fogelman A.M. Mottino G. Frank J.S. Arterioscler. Thromb. 1991; 11: 1795-1805Crossref PubMed Google Scholar), lipoprotein(a) (3Kreuzer J. Lloyd M.B. Bok D. Fless G.M. Scanu A.M. Lusis A.J. Haberland M.E. Chem. Phys. Lipids. 1994; 67/68: 175-190Crossref Scopus (41) Google Scholar), and triglyceride-rich lipoproteins (4Rapp 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). Retained lipoproteins likely trigger a series of biological responses, such as endothelial changes and recruitment of macrophages to the arterial wall, that are central to the initiation and progression of atherosclerosis (5Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar). Subendothelial lipoproteins are exposed to several modifying enzymes, including lipases (6Ylä-Herttuala S. Lipton B.A. Rosenfeld M.E. Goldberg I.J. Steinberg D. Witztum J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10143-10147Crossref PubMed Scopus (174) Google Scholar, 7Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (282) Google Scholar, 8Hurt-Camejo E. Andersen S. Standal R. Rosengren B. Sartipy P. Stadberg E. Johansen B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 300-309Crossref PubMed Scopus (168) Google Scholar), oxidizing enzymes (9Leeuwenburgh C. Rasmussen J.E. Hsu F.F. Mueller D.M. Pennathur S. Heinecke J.W. J. Biol. Chem. 1997; 272: 3520-3526Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), and proteases (10Kaartinen M. Penttila A. Kovanen P.T. Arterioscler. Thromb. 1994; 14: 966-972Crossref PubMed Google Scholar). The actions of these and other unknown factors lead to the several prominent lipoprotein modifications observed in vivo, including oxidation (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), enrichment with the phospholipid sphingomyelin (SM) (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, 12Daugherty A. Zweifel B.S. Sobel B.E. Schonfeld G. Arteriosclerosis. 1988; 8: 768-777Crossref PubMed Google Scholar, 13Hoff H.F. Lewis L.A. Handbook of Electrophoresis. 3. CRC Press, Inc., Boca Raton, FL1983: 133-165Google Scholar), and self-aggregation (2Nievelstein P.F.E.M. Fogelman A.M. Mottino G. Frank J.S. Arterioscler. Thromb. 1991; 11: 1795-1805Crossref PubMed Google Scholar, 14Hoff H.F. Morton R.E. Ann. N. Y. Acad. Sci. 1985; 454: 183-194Crossref PubMed Scopus (66) Google Scholar, 15Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Crossref PubMed Scopus (213) Google Scholar). Lipoprotein aggregation is likely to be important in atherogenesis for at least two reasons. First, processes that promote lipoprotein aggregation before or during retention dramatically increase the amount of lipoprotein retained (16Tabas I. Li Y. Brocia R.W. Xu S.W. Swenson T.L. Williams K.J. J. Biol. Chem. 1993; 268: 20419-20432Abstract Full Text PDF PubMed Google Scholar). Second, aggregated LDL, but not unaggregated LDL, is a potent inducer of macrophage foam cell formation (17Hoff H.F. O'Neill J. Pepin J.M. Cole T.B. Eur. Heart J. 1990; 11: 105-115Crossref PubMed Google Scholar, 18Khoo J.C. Miller E. McLoughlin P. Steinberg D. Arteriosclerosis. 1988; 8: 348-358Crossref PubMed Google Scholar, 19Suits A.G. Chait A. Aviram M. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2713-2717Crossref PubMed Scopus (175) Google Scholar, 20Xu X.X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar). Although the mechanism of lipoprotein aggregation in lesions is not known, several studies from our laboratories suggest a role for the enzyme sphingomyelinase (SMase). First, LDL treated with bacterial SMase forms lesion-like self-aggregates (20Xu X.X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar) due to enrichment in ceramide (7Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (282) Google Scholar), the major product of SM hydrolysis; furthermore, these aggregates potently induce macrophage foam cell formation in vitro (16Tabas I. Li Y. Brocia R.W. Xu S.W. Swenson T.L. Williams K.J. J. Biol. Chem. 1993; 268: 20419-20432Abstract Full Text PDF PubMed Google Scholar, 20Xu X.X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar). Second, aggregated LDL from human atherosclerotic lesions, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an extracellular SMase, and LDL retained in rabbit aortic strips ex vivo is hydrolyzed by an extracellular, cation-dependent SMase (7Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (282) Google Scholar). Third, and most important, we have found that several cell types present in atherosclerotic lesions, namely macrophages (21Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar) and endothelial cells, 2Marathe, S., Schissel, S. L., Schuchman, E., Yellin, M., Beatini, N., Minzer, R., Williams, K. J., and Tabas, I. (1998) J. Biol. Chem. 273, in press. secrete a Zn2+-activated SMase (S-SMase). The cellular origins, secretion, and cation dependence make S-SMase a leading candidate for the arterial wall SMase that acts on retained lipoproteins. Nonetheless, two major issues regarding the relevance of S-SMase to atherogenesis needed to be addressed. First, mammalian SMases are much more selective than bacterial SMases in terms of the milieu in which the SM is presented to the enzyme (22Spence M.W. Adv. Lipid Res. 1993; 26: 3-23PubMed Google Scholar). 3S. L. Schissel and I. Tabas, unpublished data. Second, studies on the molecular origin of S-SMase have revealed that it is a product of the same gene, the acid SMase (ASM) gene, that gives rise to lysosomal SMase (L-SMase) (21Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Not surprisingly, therefore, S-SMase shares with L-SMase a sharp acid pH optimum when assayed under standardin vitro conditions using detergent-solubilized SM-micelles as a substrate (21Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 23Spence M.W. Byers D.M. St C. Palmer F.B. Cook H.W. J. Biol. Chem. 1989; 264: 5358-5363Abstract Full Text PDF PubMed Google Scholar). Although it is possible that acidic enzymes are active in advanced atherosclerotic lesions, where local pockets of acidity may occur (24Menkin V. Am. J. Pathol. 1934; 10: 193-210PubMed Google Scholar, 25Smith E.B. Adv. Exp. Med. Biol. 1979; 115: 245-297Crossref Google Scholar, 26Maroudas A. Weinberg P.D. Parker K.H. Winlove C.P. Biophys. Chem. 1988; 32: 257-270Crossref PubMed Scopus (65) Google Scholar, 27Tapper H. Sundler R. Biochem. J. 1992; 281: 245-250Crossref PubMed Scopus (42) Google Scholar, 28Silver I.A. Murrills R.J. Etherington D.J. Exp. Cell Res. 1988; 175: 266-276Crossref PubMed Scopus (761) Google Scholar), a role for such enzymes in pre-lesional or early lesional events would require activity at neutral pH with physiologic substrates. In this context, the goal of the current study was to test whether S-SMase can hydrolyze LDL-SM, particularly at neutral pH. Herein, we show that S-SMase can hydrolyze and aggregate native LDL at acid but not neutral pH. LDL modified by several means that have been shown to occur or might occur during atherogenesis, however, is an excellent substrate for S-SMase at pH 7.4. Most importantly, LDL extracted from human atherosclerotic lesions is efficiently hydrolyzed by S-SMase at neutral pH, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. Our results support a role for S-SMase in the subendothelial hydrolysis of LDL-SM, perhaps leading to lipoprotein aggregation and lesion initiation and progression. sn-1,2-Diacylglycerol kinase (fromEscherichia coli) was purchased from Calbiochem. Cardiolipin and 1,2-dioleoylglycerol were purchased from Avanti Polar Lipids (Alabaster, AL). [9,10-3H]Palmitic acid and [γ-32P]ATP were obtained from NEN Life Science Products. Tissue culture media and reagents were purchased from Life Technologies, Inc., and fetal bovine serum was from Gemini Bioproducts (Calabasas, CA). Human native apoCIII (29Clavey V. Lestavel-Delattre S. Copin C. Bard J.M. Fruchart J.C. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 963-971Crossref PubMed Scopus (168) Google Scholar), human recombinant nonpancreatic soluble PLA2 (sPLA2) (30Sartipy P. Johansen B. Camejo G. Rosengren B. Bondjers G. Hurt-Camejo E. J. Biol. Chem. 1996; 271: 26307-26314Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and partially purified phospholipid transfer protein (31Tollefson J.H. Ravnik S. Albers J.J. J. Lipid Res. 1988; 29: 1593-1602Abstract Full Text PDF PubMed Google Scholar) were prepared as described previously. Soybean lipoxygenase and all other reagents were from Sigma. LDL receptor-deficient (LDLr0) (32Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1314) Google Scholar) and apolipoprotein E-deficient (E0) mice (33Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1925) Google Scholar, 34Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1875) Google Scholar) were purchased from Jackson Laboratories and crossed into the C57BL/6J background. LDL receptor-deficient mice expressing a human apoCIII transgene (LDLr0/CIII) were derived as described by Masucci-Magoulas et al. (35Masucci-Magoulas L. Goldberg I.J. Bisgaier C.L. Serajuddin H. Francone O.L. Breslow J.L. Tall A.R. Science. 1997; 275: 391-394Crossref PubMed Scopus (123) Google Scholar). Human and murine LDL (density, 1.020–1.063 g/ml) were isolated from fresh plasma by preparative ultracentrifugation as described previously (36Havel R.J. Eder H. Bragdon J. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6714) Google Scholar). LDL (5 mg/ml) was oxidized by dialysis against 150 mm NaCl, 6 μm FeSO4, 0.04% azide for 36 h at room temperature followed by addition of EDTA (1 mm) and BHT (150 μm) and then dialysis against 150 mmNaCl, 0.3 mm EDTA (37Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1047) Google Scholar). Alternatively, LDL (1 mg) was incubated with 275 units soybean lipoxygenase/ml and 60 μg of linoleic acid/ml in 50 mm Tris-HCl, pH 7.4, 0.04% azide for 24 h at 37 °C (38Dzeletovic S. Babiker A. Lund E. Diczfalusy U. Chem. Phys. Lipids. 1995; 78: 119-128Crossref PubMed Scopus (73) Google Scholar); LDL was re-isolated using a G-200 gel filtration column and then concentrated using a Centricon 30 (molecular weight cut-off = 30,000) ultrafiltration device. For modification by sPLA2, LDL (5 mg/ml) was incubated with 15 μg of pure human recombinant sPLA2/ml in 0.12 m Tris-HCl, pH 8.0, 12 mm CaCl2, 0.1 mm EDTA, 10 μm butylated hydroxytoluene for 14 h at 37 °C; control and modified LDL (50 μg protein) were then treated directly with S-SMase as described below. Acetyl-LDL was prepared by acetylation of LDL with acetic anhydride (39Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (2076) Google Scholar). LDL was extracted from abdominal aortic aneurysm plaque material as described previously (4Rapp 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). Briefly, aortic plaque was removed from individuals as part of the standard reconstructive surgery for abdominal aortic aneurysms at the San Francisco Veterans Affairs Medical Center. Plaque material, which ranged in weight from 2 to 12 g, was obtained in the operating room and immediately placed into ice-cold 7 mm citrate buffer, pH 7.4, containing 15 mm NaCl, 3 mmEDTA, 0.5 mm butylhydroxytoluene, 1 mmphenylmethylsulfonyl fluoride, 1.5 mg aprotinin/ml, 2 mmbenzamidine, and 0.08 mg of gentamicin sulfate/ml. Blood and adherent thrombus were removed by blotting with absorbent gauze, scrubbing with a small brush, and sharp dissection as necessary. Loosely retained lipoproteins were extracted by mincing the plaque into 0.5–1.0-mm2 pieces and incubating them overnight on a Labquake shaker at 4 °C in a non-denaturing buffer (0.1m citrate, pH 7.4, with 1 mg of EDTA/ml, 0.3 mg of benzamidine/ml, 0.08 mg of gentamicin sulfate/ml, 10 μg of aprotinin/ml, 10 μg of Trolox (an anti-oxidant)/ml, and 20 μg of phenylmethylsulfonyl fluoride/ml). The extracted material was cleared of particulate matter by centrifuging at 800 × g for 10 min, and 1.019 < d < 1.063-g/ml lipoproteins were isolated by sequential potassium bromide density ultracentrifugation (4Rapp 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, 36Havel R.J. Eder H. Bragdon J. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6714) Google Scholar). [N-palmitoyl-9,10-3H]SM was synthesized as follows (7Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (282) Google Scholar, 40Sripada P.K. Maulik P.R. Hamilton J.A. Shipley G.G. J. Lipid Res. 1987; 28: 710-718Abstract Full Text PDF PubMed Google Scholar, 41Ahmad T.Y. Sparrow J.T. Morrisett J.D. J. Lipid Res. 1985; 26: 1160-1165Abstract Full Text PDF PubMed Google Scholar): [9,10-3H]palmitic acid (25 mCi, 450 nmol) was stirred for 12 h at room temperature with an equimolar equivalent of N-hydroxysuccinimide and with 3 m eq of 1,3-dicyclohexylcarbodiimide inN, N-dimethylformamide. The reaction was run under dry argon in the dark. Sphingosylphosphorylcholine (300 nmol) and N, N-diisopropylethylamine (10 μl) were then added, and the reaction was stirred another 12 h at room temperature. The reaction was stopped by evaporating the N, N-dimethylformamide under a stream of N2. [N-palmitoyl-9,10-3H]SM was purified by preparative thin layer chromatography of the reaction products three consecutive times in chloroform:methanol (95:5) and then twice in chloroform:methanol:acetic acid:water (50:25:8:4). Greater than 95% of the [N-palmitoyl-9,10-3H]SM was converted to [N-palmitoyl-9,10-3H]ceramide after treatment with 10 milliunits of SMase/ml (Bacillus cereus) for 1 h at 37 °C, as assayed by TLC, indicating a pure, functional substrate. Plasma LDL was labeled with [N-palmitoyl-9,10-3H]SM as follows (7Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (282) Google Scholar). ∼3.5 mCi (63 nmol) of [N-palmitoyl-9,10-3H]SM and 13 nmol of phosphatidylcholine (PC) were mixed in chloroform, and the solvent was removed first under a stream of nitrogen and then by lyophilization. The dried lipids were resuspended in 1 ml of 150 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 7.5, and, to prepare [3H]SM/PC liposomes, sonicated for three 50-s pulses at 4 °C using a tapered microtip on a Branson 450 sonicator (setting 3). The liposomes were then incubated with 30 mg (by protein mass) of LDL, 50 μg of partially purified phospholipid transfer protein, 100 units of penicillin, and 100 μg of streptomycin for 18 h at 37 °C under argon. LDL was then separated from the liposomes after phospholipid transfer by centrifuging the mixture at density = 1.006 g/ml for 8 h at 35,000 rpm in a Beckman 50.3 rotor; the supernatant containing the liposomes was removed, and the LDL band at the bottom of the tube was harvested. The LDL solution was mixed with buffer containing 150 mm NaCl, 0.3 mm EDTA, pH 7.4, and centrifuged as before. This wash procedure was performed a total of four times, resulting in the removal of 95% of the unreacted [3H]SM/PC liposomes. All lipoproteins were stored under argon at 4 °C and were used within 2 weeks of preparation. [3H]SM-emulsions with a lipid composition similar to human LDL were prepared as follows: 5.4 mg of cholesteryl oleate, 0.48 mg of triolein, 1.08 mg of free cholesterol, 2.04 mg of phosphatidylcholine, 0.96 mg of sphingomyelin, and 50 μCi of [N-palmitoyl-9,10-3H]sphingomyelin were all added in chloroform to a sonication vial, and the solvent was completely evaporated by exposure to a stream of nitrogen, followed by the high vacuum of a lyophilizer. The dried lipids were resuspended in 3 ml of buffer containing 150 mm NaCl, 0.3 mmEDTA, pH 7.4, and sonicated under a stream of argon at 40 °C until translucent (approximately 90 min). The sonicated material was then centrifuged twice at 15,000 × g to pellet any titanium shed from the sonication probe. SM-rich emulsions were prepared exactly as above except that 1.2 mg of phosphatidylcholine and 1.8 mg of SM were used, and sonication time was increased to 120 min. [3H]SM-emulsions were enriched with apoCIII based on the method of Ahmad et al. (42Ahmad T.Y. Beaudet A.L. Sparrow J.T. Morrisett J.D. Biochemistry. 1986; 25: 4415-4420Crossref PubMed Scopus (9) Google Scholar). Briefly, [3H]SM-emulsions (0.4 ml; 182 nmol of SM) were incubated with 200 μg of apoCIII (22.5 nmol) for 2 h at 40 °C. A portion of the emulsions was re-isolated from free apoCIII using ultrafiltration (29Clavey V. Lestavel-Delattre S. Copin C. Bard J.M. Fruchart J.C. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 963-971Crossref PubMed Scopus (168) Google Scholar) as follows: the crude emulsion/apoCIII mixture was diluted to 2 ml with buffer containing 150 mm NaCl, 0.3 mm EDTA, pH 7.4, and ultrafiltered and concentrated to 0.2 ml using a Centricon 30 (molecular weight cut-off = 30,000); the concentrated emulsions were then diluted to 2 ml, and the process was repeated 5 times. [3H]SM-emulsions run through the same enrichment and re-isolation protocols in the absence of apoCIII served as the control for the experiments in Fig. 5 A. Ceramide was measured from LDL lipid extracts using the method described by Schneider and Kennedy (43Schneider E.G. Kennedy E.P. Biochim. Biophys. Acta. 1976; 441: 201-212Crossref PubMed Scopus (39) Google Scholar) and adapted by Preiss et al. (44Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). In this method, diacylglycerol (DAG) kinase phosphorylates ceramide and DAG using [γ-32P]ATP. For ceramide measurement, the lipids were first incubated with 0.1 n KOH in methanol for 1 h at 37 °C, which hydrolyzes DAG but not ceramide. The extracted lipids were dried under nitrogen and then solubilized in 5 mmcardiolipin, 7.5% octyl-β-glucopyranoside, and 1 mmdiethylenetriaminepentaacetic acid by bath sonication. This solution was then added to reaction buffer (50 mm imidazole HCl, pH 6.6, 50 mm NaCl, 12.5 mm MgCl2, 1 mm EGTA) containing sn-1,2-DAG kinase (0.7 units/ml). The reaction was initiated by the addition of [γ-32P]ATP (final concentration = 10 mm). After incubation at room temperature for 60 min, the reaction was stopped by lipid extraction with chloroform:methanol:HCl (100:100:1, v/v/v) and 10 mm EDTA. Ceramide 1-[32P]phosphate in the organic phase was separated by TLC using chloroform:methanol:acetic acid (65:15:5, v/v/v) and visualized with autoradiography and identified by comparing with standards. The spots corresponding to ceramide 1-[32P]phosphate were scraped and counted, and the mass was calculated by comparison with a ceramide standard curve. To rule out changes in the DAG kinase enzyme itself (cf. Ref. 45Watts J.D. Gu M. Polverino A.J. Patterson S.D. Aebersold R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7292-7296Crossref PubMed Scopus (123) Google Scholar), C2:0 ceramide was added to some of the lipid extracts and shown not to undergo increased phosphorylation in samples in which LDL ceramide was found to be elevated. Lipid extracts (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44558) Google Scholar) of lipoproteins were chromatographed by TLC using chloroform:methanol:acetic acid:H2O (50:25:8:4, v/v/v/v). Individual phospholipid subclasses were visualized by iodine vapor staining, and the SM and PC spots were identified by comparison with standards. The spots were scraped, extracted twice with chloroform:methanol (2:1), and assayed for phosphate content by the method of Bartlett (47Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). LDL lipid peroxides were measured using the method of El-Saadani et al. (48El-Saadani M. Esterbauer H. El-Sayed M. Goher M. Nassar A.Y. Jurgens G. J. Lipid Res. 1989; 30: 627-630Abstract Full Text PDF PubMed Google Scholar). LDL (50–150 μg of protein), in a volume of no more than 100 μl, was added to 1 ml of color reagent (0.2 m KH2PO4, 0.12 m KI, 0.15 mm NaN3, 2 g of Triton X-100/liter, 0.1 g of benzalkonium chloride/liter, 10 μm ammonium molybdate, 20 μm BHT, 25 μm EDTA, pH 6.2) and incubated in the dark for 30 min at room temperature; light absorbance at 365 nm was then measured and lipid peroxides were quantified by comparison with a H2O2 standard curve. Thiobarbituric acid-reactive substances (TBARS) were measured using a standard method (49Puhl H. Waeg G. Esterbauer H. Methods Enzymol. 1994; 233: 425-441Crossref PubMed Scopus (283) Google Scholar). Briefly, LDL (100 μl, 100–200 μg of protein) was mixed with 1 ml of 20% trichloroacetic acid and incubated on ice for 30 min. Following precipitation, 1 ml of 1% thiobarbituric acid was added, and the samples were heated at 95 °C for 45 min. After cooling, the samples were centrifuged at 1000 × g for 20 min, and the light absorbance at 532 nm was measured. TBARS were quantified by comparison with a malonaldehyde standard curve prepared using tetramethoxypropane. LDL electrophoretic mobility was assayed by loading 30 μg of LDL protein onto a polyacrylamide 0.75–27% gradient gel (Lipogel; Zaxis, Hudson, OH) and electrophoresing in 0.1m Tris base, 0.1 m boric acid, 20 mm EDTA (upper chamber, pH 8.7; lower chamber, pH 8.3) for 12 h at 100 V. The gel was then stained with Sudan black and the bands visualized by counter-staining with methanol:acetic acid:water (10:7:83, v/v/v). The source of S-SMase was serum-free conditioned medium from DG44 Chinese hamster ovary cells stably transfected with the human acid SMase cDNA (21Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 50Schuchman E.H. Suchi M. Takahashi T. Sandhoff K. Desnick R.J. J. Biol. Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar). Our previous work demonstrated that S-SMase is the only detectable SMase secreted into the culture medium (21Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Cells were plated in 100-mm dishes and cultured for 48 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 292 μg of glutamine/ml, 100 units penicillin/ml, and 100 μg of streptomycin/ml. The cells were then changed to low protein serum-free media for 12 h, washed 3 times with phosphate-buffered saline, and finally incubated for 18 h in fresh serum-free media (6 ml per 100-mm dish). The “18-h conditioned medium” was then collected, centrifuged at 1000 × g to pellet any cells, and, except where indicated, ZnCl2 (final concentration = 100 μm) was added to fully activate and stabilize S-SMase; this S-SMase-containing conditioned medium was then used fresh to treat LDL and lipid emulsions. The standard incubation mixture consisted of up to 50 μl of sample (LDL or emulsions), 25 μl of S-SMase-containing conditioned media (see above), and a volume of assay buffer (0.1 m Tris-HCl, pH 7.4, 0.04% azide; or, where indicated, 0.1 m sodium acetate, pH 5.5, 0.04% azide) to bring the final volume to 200 μl. The reactions were incubated at 37 °C for no longer than 16 h and then extracted by the method of Bligh and Dyer (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44558) Google Scholar). For the samples containing [N