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• W2156107349 abstract "Serum amyloid A (SAA) circulates bound to HDL3 during the acute-phase response (APR), and recent evidence suggests that elevated levels of SAA may be a risk factor for cardiovascular disease. In this study, SAA-HDL was produced in vivo during the APR and without the APR by injection of an adenoviral vector expressing human SAA-1. SAA-HDL was also produced in vitro by incubating mouse HDL with recombinant mouse SAA and by SAA-expressing cultured hepatoma cells. Whether produced in vivo or in vitro, SAA-HDL floated at a density corresponding to that of human HDL3 (d 1.12 g/ml) separate from other apolipoproteins, including apolipoprotein A-I (apoA-I; d 1.10 g/ml) when either apoA-I or apolipoprotein E (apoE) was present. In the absence of both apoA-I and apoE, SAA was found in VLDL and LDL, with low levels in the HDL and the lipid-poor fractions suggesting that other HDL apolipoproteins are incapable of facilitating the formation of SAA-HDL.We conclude that SAA does not exist in plasma as a lipid-free protein. In the presence of HDL-associated apoA-I or apoE, SAA circulates as SAA-HDL with a density corresponding to that of human HDL3. In the absence of both apoA-I and apoE, SAA-HDL is not formed and SAA associates with any available lipoprotein. Serum amyloid A (SAA) circulates bound to HDL3 during the acute-phase response (APR), and recent evidence suggests that elevated levels of SAA may be a risk factor for cardiovascular disease. In this study, SAA-HDL was produced in vivo during the APR and without the APR by injection of an adenoviral vector expressing human SAA-1. SAA-HDL was also produced in vitro by incubating mouse HDL with recombinant mouse SAA and by SAA-expressing cultured hepatoma cells. Whether produced in vivo or in vitro, SAA-HDL floated at a density corresponding to that of human HDL3 (d 1.12 g/ml) separate from other apolipoproteins, including apolipoprotein A-I (apoA-I; d 1.10 g/ml) when either apoA-I or apolipoprotein E (apoE) was present. In the absence of both apoA-I and apoE, SAA was found in VLDL and LDL, with low levels in the HDL and the lipid-poor fractions suggesting that other HDL apolipoproteins are incapable of facilitating the formation of SAA-HDL. We conclude that SAA does not exist in plasma as a lipid-free protein. In the presence of HDL-associated apoA-I or apoE, SAA circulates as SAA-HDL with a density corresponding to that of human HDL3. In the absence of both apoA-I and apoE, SAA-HDL is not formed and SAA associates with any available lipoprotein. Atherosclerosis is now recognized as a chronic inflammatory response in which a vast number of mediators of the inflammatory process are superimposed upon hyperlipidemia (1Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar, 2Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation are partners in crime.Nat. Med. 2002; 8: 1211-1217Crossref PubMed Scopus (592) Google Scholar). Two proteins of the innate acute inflammatory response are the focus of intensive research, C-reactive protein and serum amyloid A (SAA). These proteins have been shown to exhibit higher risk relationships with cardiovascular disease than serum cholesterol (3Delanghe J.R. Langlois M.R. De Bacquer D. Mak R. Capel P. Van Renterghem L. De Backer G. Discriminative value of serum amyloid A and other acute-phase proteins for coronary heart disease.Atherosclerosis. 2002; 160: 471-476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 4Hoffmeister A. Rothenbacher D. Bazner U. Frohlich M. Brenner H. Hombach V. Koenig W. Role of novel markers of inflammation in patients with stable coronary heart disease.Am. J. Cardiol. 2001; 87: 262-266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Whether these proteins have any causal relationship with cardiovascular diseases or are simply markers of the inflammatory process is not known. This study focuses on SAA, a plasma protein that increases by greater than 1,000-fold within 24 to 48 h after the induction of the acute-phase response (APR) by infectious and noninfectious inflammatory processes. A highly conserved family of proteins, SAA is present in all vertebrates from fish to human (5Uhlar C.M. Whitehead A.S. Serum amyloid A, the major vertebrate acute-phase reactant.Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (893) Google Scholar, 6Ureili-Shoval S. Linke R.P. Matzner Y. Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states.Curr. Opin. Hematol. 2000; 7: 64-69Crossref PubMed Scopus (371) Google Scholar, 7Sipe J.D. Serum amyloid A: from fibril to function. Current status.Amyloid. 2000; 7: 10-12Crossref PubMed Scopus (26) Google Scholar). Despite years of study, the biological significance of its dramatic increase during the APR remains unclear. SAA may play a role in both amyloidogenesis and atherogenesis. Its role in amyloidogenesis is better defined, SAA being the precursor of the AA protein deposited in secondary amyloidosis. Its role in atherogenesis is circumstantial, although evidence is accumulating that it may be more than an innocent bystander and may affect processes and functions related to atherogenesis. For example, SAA circulates in plasma bound to HDL3, and when incubated with HDL in vitro, can displace apolipoprotein A-I (apoA-I) (8Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar, 9Cabana V.G. Lukens J.R. Rice K.S. Hawkins T.J. Getz G.S. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease.J. Lipid Res. 1999; 37: 2662-2674Abstract Full Text PDF Google Scholar), which may result in decreased HDL levels. We have shown also that SAA can displace the antioxidant enzyme paraoxonase from HDL (10Cabana V.G. Reardon C.A. Feng N. Neath S. Lukens J. Getz G.S. Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response.J. Lipid Res. 2003; 44: 780-792Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and that this may contribute to the reduction in the antioxidant properties of HDL after the acute phase (11Van Lenten B.J. Hama S.Y. de Beer F.C. Stafforini D.M. McIntyre T.M. Prescott S.M. La Du B.N. Fogelman A.M. Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.J. Clin. Invest. 1995; 96: 2758-2767Crossref PubMed Scopus (706) Google Scholar). Thus, although basal HDL may protect against atherosclerosis, this activity of HDL may be either attenuated or reversed when it contains SAA. SAA may function in cholesterol transport (12Kisilevsky R. Tam S.P. Acute phase serum amyloid A, cholesterol metabolism, and cardiovascular disease.Pediatr. Pathol. Mol. Med. 2002; 21: 291-305Crossref PubMed Scopus (43) Google Scholar) in an isoform-specific manner (13Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). It apparently binds cholesterol and could alter HDL-mediated cholesterol efflux (14Liang J-S. Sipe J.D. Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux.J. Lipid Res. 1995; 36: 37-47Abstract Full Text PDF PubMed Google Scholar, 15Banka C.L. Yuan T. de Beer M.C. Kindy M. Curtiss L.K. de Beer F.C. Serum amyloid A (SAA): influence on HDL-mediated cellular cholesterol efflux.J. Lipid Res. 1995; 36: 1058-1065Abstract Full Text PDF PubMed Google Scholar), thereby affecting the transport of cholesterol from the tissues back to the liver for excretion. SAA mRNA has been detected in cells of the human vessel wall involved in atherogenesis, such as endothelial cells, smooth muscle cells, macrophage-derived foam cells, and adventitial macrophages (16Meek R.L. Urieli-Shoval S. Benditt E.P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function.Proc. Natl. Acad. Sci. USA. 1994; 91: 3186-3190Crossref PubMed Scopus (276) Google Scholar). This expression pattern has been observed also in the aortic lesions of C57BL/6 and apoA-II transgenic mice fed an atherogenic diet and in apoE gene knockout mice fed a chow diet (17Qiao J.H. Xie P.Z. Fishbein M.C. Kreuzer J. Drake T.A. Demer L.L. Lusis A.J. Pathology of atheromatous lesions in inbred and genetically engineered mice. Genetic determination of arterial calcification.Arterioscler. Thromb. 1994; 14: 1480-1497Crossref PubMed Scopus (222) Google Scholar). Among endothelial cells, those lining the lumen of atherosclerotic vessels express the highest level of SAA mRNA, suggesting a role for SAA in the pathogenesis or regression of atherosclerotic plaques (16Meek R.L. Urieli-Shoval S. Benditt E.P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function.Proc. Natl. Acad. Sci. USA. 1994; 91: 3186-3190Crossref PubMed Scopus (276) Google Scholar). SAA induces the migration, adhesion, and tissue infiltration of monocytes (18Badolato R. Johnston J.A. Wang J.M. McVicar D. Xu L.L. Oppenheim J.J. Kelvin D.J. Serum amyloid A induces calcium mobilization and chemotaxis of human monocytes by activating a pertussis toxin-sensitive signaling pathway.J. Immunol. 1995; 155: 4004-4010PubMed Google Scholar), cells strongly implicated in the pathogenesis of atherosclerosis. Atherosclerosis-susceptible strains of mice on an atherogenic diet accumulate lipid oxidation products, activate the nuclear factor-κB transcription factor involved in the inflammatory response, and activate inflammatory genes. The induction of inflammatory genes, including SAA, is associated with susceptibility to aortic lesion formation (19Liao F. Andaligi A. Lusis A.J. Fogelman A.M. Genetic control of the inflammatory response induced by oxidized lipids.Am. J. Cardiol. 1995; 23: 65B-66BAbstract Full Text PDF Scopus (21) Google Scholar). A high-fat, high-cholesterol diet induces the synthesis of the acute phase (but not the constitutive) SAA isoforms (20Liao F. Lusis A.J. Berliner J.A. Fogelman A.M. Kindy M. de Beer M.C. de Beer F.C. Serum amyloid A protein family. Differential induction by oxidized lipids in mouse strains.Arterioscler. Thromb. 1994; 14: 1475-1479Crossref PubMed Google Scholar). Its presence in cells of the atherosclerotic plaque and its several possible functions in the atherogenic process indicate that more extensive study of these functions is called for. SAA exists in the plasma bound to HDL3. We have previously shown that HDL particles containing only SAA exist in the plasma of normal and apoA-I-deficient mice after the induction of the APR by bacterial lipopolysaccharide (LPS) (21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). In this report, we have further analyzed the formation of SAA-containing lipoproteins in the presence of either apoA-I or apoE in the following systems: 1) in vivo during the APR induced by LPS injection; 2) in vivo without the APR by injection of adenoviruses expressing human SAA1 (adv-huSAA1); 3) in vitro by incubation of non-APR sera with recombinant SAA; and 4) in cultured hepatoma-derived cells infected with adv-huSAA1. We have also analyzed the formation of SAA-containing lipoproteins in vivo and in vitro in the absence of both apoA-I and apoE. In all of the systems in which either apoA-I or apoE was present, the SAA was found mainly in particles with a density comparable to that of human HDL3. However, in mice deficient in both apoE and apoA-I genes (EA−/−) that lack HDL, SAA associated almost exclusively with VLDL and intermediate density lipoprotein (IDL) and/or LDL. We conclude that apoA-I and apoE play an important role in the formation of SAA-HDL. LPS Escherichia coli serotype 0127:B8, tribromoethyl alcohol, tertiary amyl alcohol, horseradish peroxidase-coupled antibodies, and protease inhibitors were purchased from Sigma Chemical (St. Louis, MO). Tris-glycine 4–20% polyacrylamide gels were from Invitrogen (Carlsbad, CA). Immobilon-P was purchased from Millipore (Bedford, MA), and broad-range nonstained (6,500–200,000 Da) or prestained Kaleidoscope (7,600–216,000 Da) standards were obtained from Bio-Rad (Richmond, CA). The enhanced chemiluminescence (ECL) kit was supplied by Amersham Corporation (Arlington Heights, IL). All cell culture media reagents were from Gibco (Gaithersburg, MD). Kits for enzymatic measurement of cholesterol and phospholipids were purchased from Roche Diagnostics (Indianapolis, IN) and Wako Chemicals (Osaka, Japan), respectively. Mice deficient in both the apoE gene (apoE−/−) and the recombination activating gene 2 (RAG2−/−) gene (RE−/−) were produced by mating the respective strains at the animal facilities of the University of Chicago as described (22Reardon C.A. Blachowicz L. White T. Cabana V. Wang Y. Lukens J. Bluestone J. Getz G.S. Effect of immune deficiency on lipoproteins and atherosclerosis in male apolipoprotein E-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1011-1016Crossref PubMed Scopus (164) Google Scholar). C57BL/6 mice purchased from Jackson Laboratories (Bar Harbor, ME) were used as controls. Mice deficient in both the apoA-I and apoE genes (EA−/− mice) were bred and maintained in the animal facilities of the University of Chicago as described (10Cabana V.G. Reardon C.A. Feng N. Neath S. Lukens J. Getz G.S. Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response.J. Lipid Res. 2003; 44: 780-792Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The mice were housed in a specific-pathogen-free environment in a temperature-controlled room with a 12-h light-dark cycle. All of the mice were fed mouse chow diet, and food and water were available at all times. During injections and bleedings, the mice were anesthetized by intraperitoneal injection (17.5 μl/g body weight) of a sterile Avertin solution [2.5% in saline prepared from a stock solution of equal tribromoethyl alcohol and tertiary amyl alcohol (w/v)]. The APR was induced in vivo by intraperitoneal injection of 50 μg of bacterial LPS per mouse. After 24 h, the mice were killed by cardiac puncture under anesthesia. Blood was collected without EDTA, and the sera separated by centrifugation were stored at 4°C in the presence of 1 mM PMSF (in ethanol) and anti-bacterial agents (per milliliter of plasma: 0.5 μg of gentamycin sulfate, 50 μg of NaN3, and 1 μg of chloramphenicol) and used within 2 weeks. Sera were used because some of the mice used in this study were also used for the study of paraoxonase (10Cabana V.G. Reardon C.A. Feng N. Neath S. Lukens J. Getz G.S. Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response.J. Lipid Res. 2003; 44: 780-792Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), which is irreversibly inactivated by EDTA. All procedures performed on the mice were in accordance with National Institutes of Health and institutional guidelines. The construction of a replication-defective adv-huSAA1 was described previously (23Hosoai H. Webb N.R. Glick J. Purdom M.S. de Beer F.C. Rader D.J. Expression of serum amyloid A protein in the absence of the acute phase response is not sufficient to reduce HDL cholesterol or apoA-I levels in mice.J. Lipid Res. 1999; 40: 648-653Abstract Full Text Full Text PDF PubMed Google Scholar). Infectious titers of adenovirus preparations were determined by plaque assay using HEK-293 cells as described (24Graham F.L. Prevec L. Methods for construction of adenovirus vectors.Mol. Biotechnol. 1995; 3: 207-220Crossref PubMed Scopus (437) Google Scholar). Mice were injected via the retro-orbital sinus with various amounts of the virus [expressed as plaque-forming units (pfu)]. Blood was collected at various time points by retro-orbital or cardiac puncture and processed as described above. Two hundred fifty microliters of sera from RE−/− mice was incubated with recombinant mouse SAA (r-moSAA) at a ratio of 600 μg of SAA per milliliter of serum, the ratio usually found during the APR. The r-moSAA was isolated from the pGEX bacterial system according to procedures detailed previously (21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). We have shown that in vitro this recombinant SAA formed fibrils similar to prion proteins as analyzed by atomic force microscopy (25Xu, S., V. G. Cabana, G. S. Getz, and M. F. Arnsdorf. 2000. Scanning force microscopy of amyloid fibers. The Biophysical Society Meeting, New Orleans, LA, February 12–16, 2000.Google Scholar). After a 1 h incubation at room temperature, the incubation mixture was subjected to density gradient centrifugation as described below. The fractions were collected, and the distributions of apoA-I and SAA were analyzed as described below. The rat hepatoma-derived McA RH7777 cells were obtained from the American Type Culture Collection (ATCC No. CRL 1601; Rockville, MD). The cells were maintained in high-glucose (4.5 g/l) DMEM with 10% FBS, 5% horse serum, and 1% each of glutamine, penicillin, and streptomycin (henceforth referred to as growth medium). Subconfluent cells were trypsinized, and 1.5 × 106 cells were replated in T-75 culture flasks and grown for 48 h in growth medium. After washing twice with growth medium, adv-huSAA1 with predetermined multiplicity of infection in serum-free medium was added and incubated in serum-free medium for 1 h. The virus media were removed, and the cells were washed twice with serum-free medium and then grown for 48 h in DMEM with 1% lipoprotein-deficient serum isolated as described (26Reardon C.A. Blachowicz L. Watson K.M. Barr E. Getz G.S. Association of human apolipoprotein E with lipoproteins secreted by transfected McA RH7777 cells.J. Lipid Res. 1998; 39: 1372-1381Abstract Full Text Full Text PDF PubMed Google Scholar) and supplemented with 1% each of glutamine, penicillin, and streptomycin. The cells were separated by centrifugation, and the supernatant culture media were harvested in solution containing a protease cocktail (0.1% aprotinin, 1 mM PMSF, 2 mM EDTA, and 0.02% NaN3), concentrated by centrifugal filtration (Centriprep; Amicon, Beverly, MA), and the lipoprotein fractions were isolated by density gradient centrifugation as described below. The density distribution of apolipoproteins was analyzed by equilibrium density gradient centrifugation according to procedures described previously (27Cabana V.G. Siegel J.N. Sabesin S.M. Effects of the acute phase response on the concentration and density distribution of plasma lipids and apolipoproteins.J. Lipid Res. 1989; 30: 39-49Abstract Full Text PDF PubMed Google Scholar). In this procedure, 250 μl of serum or 2 ml of concentrated culture supernatant was fractionated on 3–20% NaBr gradients by centrifugation for 66 h at 38,000 rpm in a SW41 Ti rotor. After centrifugation, 30 0.4 ml fractions were collected using a gradient fractionator with UV monitor (ISCO, Lincoln, NE) and pump assembly (Brandel). The refractive index of each of the fractions was assessed as an indicator of the density based on the refractive index of salt solutions of known concentration, density, and refractive index. The fractions were dialyzed against Tris-buffered saline (10 mM Tris, 150 mM NaCl, 0.01% EDTA, and 20 mM NaN3, pH 7.4) and used for analyses. One microliter of plasma or 2–5 μl of fraction from the density gradient centrifugation was loaded per lane on a 4–20% polyacrylamide Tris-glycine gel in a sample buffer containing 5% β-mercaptoethanol. The gels were either stained with Coomassie blue or used for electrotransfer of the protein bands to Immobilon-P membrane for Western immunoblotting using polyclonal antibodies to the respective proteins as described (9Cabana V.G. Lukens J.R. Rice K.S. Hawkins T.J. Getz G.S. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease.J. Lipid Res. 1999; 37: 2662-2674Abstract Full Text PDF Google Scholar). The approximate proportion of apolipoproteins present in a given lipoprotein fraction was assessed by scanning densitometry of the protein bands developed on a photographic film and quantitated (Advanced Quantifier; BioImage) or quantitated using a chemiluminescence imager (Alpha Innotech, San Leandro, CA). Where indicated, apoA-I was quantitated by radial immunodiffusion (28Albers J.J. Wahl P.W. Cabana V.G. Hazzard W.R. Hoover J.J. Quantitation of apolipoprotein A-I of human plasma high density lipoprotein.Metabolism. 1976; 25: 633-644Abstract Full Text PDF PubMed Scopus (238) Google Scholar) after denaturation of the protein with 1% Triton X-100, using antibody against mouse apoA-I. Total cholesterol was quantified enzymatically (Roche Diagnostics). Phospholipids were quantified by an enzymatic-colorimetric assay of choline-containing phospholipids (Wako Chemicals). All lipoprotein analyses were performed by methods standardized against Centers for Disease Control and Prevention-furnished standards. Protein was quantified according to the procedure of Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with SDS to disrupt the lipid micelles (30Markwell M.A. Hass S.M. Beiber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5333) Google Scholar) using BSA as a standard. In published studies, we have shown that the induction of the APR by LPS in wild-type C57BL/6 mice changes the HDL profile from a monodisperse peak (peak at 1.10 g/ml) on density gradients into two separate peaks (peaks at 1.09 and 1.12 g/ml) within 24 h of injection of LPS (9Cabana V.G. Lukens J.R. Rice K.S. Hawkins T.J. Getz G.S. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease.J. Lipid Res. 1999; 37: 2662-2674Abstract Full Text PDF Google Scholar, 21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). We also showed that SAA-HDL (d 1.12 g/ml) essentially devoid of other apolipoproteins can be produced in wild-type C57BL/6 mice and in the absence of apoA-I in apoA-I-deficient (apoA-I−/−) mice after the induction of the APR (21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). The production of SAA-HDL in apoA-I−/− mice showed that apoA-I is not required for the production of these particles. In the current study, we further examined the role of apoA-I and apoE in the production of SAA-HDL initially using immunoincompetent double gene knockout RE−/− mice. These mice are deficient in both apoE and RAG2. ApoE−/− mice develop atherosclerosis spontaneously on chow diet and are used as models for the development of atherosclerosis. RAG2−/− mice lack mature T and B cells. This immunodeficiency allows the sequential injections of replication-defective adenoviruses carrying the SAA gene to maintain a high SAA level without the complication of a host-adaptive immune response related to the APR or the viral vector. Our results show that even in the absence of mature T and B cells, RE−/− mice were able to mount a characteristic rapid increase of SAA in plasma after the injection of LPS. This is expected, as the induction of SAA is a part of the innate acute inflammatory response. Before the injection of LPS, a single HDL peak was present at d 1.10 g/ml as detected by density gradient ultracentrifugal flotation of plasma (Fig. 1A, fraction 20). Twenty-four hours after LPS injection (Fig. 1B), two HDL peaks were present: one at d 1.09 g/ml (fraction 19) and a second at the region equivalent to human HDL3 (fractions 22–23; d 1.12 g/ml). This is similar to the results obtained upon induction of the APR in C57BL/6 mice (21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). Western immunoblotting of the fractions for mouse apoA-I and SAA followed by densitometric scanning of the protein bands showed that before the injection of LPS, apoA-I had a monodisperse distribution that peaked at d 1.10 g/ml (Fig. 2A). Twenty-four hours after LPS injection, apoA-I had a broader distribution, with its peak shifted to lighter density at 1.08 g/ml. SAA floated separately from apoA-I, with a peak at d 1.12 g/ml, suggesting that they are on separate particles. We have previously shown that ∼90% of the SAA is found in the density of HDL3 in C57BL/6 and apoA-I−/− mice (9Cabana V.G. Lukens J.R. Rice K.S. Hawkins T.J. Getz G.S. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease.J. Lipid Res. 1999; 37: 2662-2674Abstract Full Text PDF Google Scholar, 21Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar) (Fig. 2B, Table 1). In contrast, in the RE−/− mice, which have high levels of VLDL and IDL/LDL (22Reardon C.A. Blachowicz L. White T. Cabana V. Wang Y. Lukens J. Bluestone J. Getz G.S. Effect of immune deficiency on lipoproteins and atherosclerosis in male apolipoprotein E-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1011-1016Crossref PubMed Scopus (164) Google Scholar), 14.1 ± 6.5% of SAA was in the VLDL/IDL/LDL fractions (Fig. 2B, Table 1). Most of the SAA (69.7 ± 1.5%) was still found in the fraction corresponding to human HDL3 (d 1.12 g/ml), with less than 15% in the lipid-poor fractions (fractions 28–30).TABLE 1Distribution of SAA in vivo in three mouse strainsSAAMouse StrainVLDLIDL/LDLHDLLipid-Poor% of totalapoA-I−/−aAverage, n = 2.0086.913.1RE−/−bMean ± SD, n = 6.,cSimilar distributions were observed in apoE−/− mice.10.1 ± 4.04.4 ± 2.569.7 ± 1.513.0 ± 3.0EA−/−bMean ± SD, n = 6.25.0 ± 5.041.5 ± 10.919.0 ± 5.314.0 ± 5.2apoA-I, apolipoprotein A-I; apoA-I−/− , mice deficient in the apoA-I gene; EA−/− , mice deficient in both the apoA-I and apolipoprotein E (apoE) genes; IDL, intermediate density lipoprotein; RE−/− , mice deficient in both the apoE gene and the recombination activation gene 2; SAA, serum amyloid A.a Average, n = 2.b Mean ± SD, n = 6.c Similar distributions were observed in apoE−/− mice. Open table in a new tab apoA-I, apolipoprotein A-I; apoA-I−/− , mice deficient in the apoA-I gene; EA−/− , mice deficient in both the apoA-I and apolipoprotein E (apoE) genes; IDL, intermediate density lipoprotein; RE−/− , mice deficient in both the apoE gene and the recombination activation gene 2; SAA, serum amyloid A. Production of SAA-HDL in the absence of the APR was induced in RE−/− mice by the injection of adenoviruses carrying a single copy of the adv-huSAA1 gene (5 × 108 pfu/mouse). Three days after injection, the level of adv-huSAA1 was ∼50 μg/ml (Fig. 3A). This is substantially lower than the mouse SAA level achieved after the induction of the APR by LPS injection. The levels progressively decreased thereafter. Because the level of SAA decreased progressively after a single dose of adv-huSAA1, injections of 5 × 108 pfu of adv-huSAA1 per mouse were repeated every 21 days. After each injection of the adenovirus, huSAA levels in the plasma increased 3 days postinjection, and although the level decreased by 21 days after each injection, the levels were always higher than that at 21 days after the previous injection (Fig. 3B). Although endogenous mouse SAA increased modestly with each viral injection (Fig. 3C), these levels were low compared with the 24 h post-LPS injection level, showing that a full APR was not a significant complication of the adenoviral injections. Analysis of the RE−/− plasma at 3 days after injection revealed huSAA and endogenous apoA-I floating at separate density peaks; the SAA floated at d 1.12 g/ml, and apoA-I peaked at d 1.10 g/ml (Fig. 4A). At this low level of SAA, greater than 90% of the SAA was found in HDL (Fig. 4B), even in the presence of high VLDL and LDL/IDL in these animals. Similar distributions of huSAA were obtained after adenoviral injections into apoA-I−/− and C57BL/6 mice (data not shown). With a 10-fold higher dose of adv-huSAA1 (50 × 108 pfu), higher levels of huSAA were produced, with ∼15% of the SAA found in VLDL and <10% in the lipid-poor fraction (data not shown). This distribution is almost identica" @default.
• W2156107349 created "2016-06-24" @default.
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• W2156107349 date "2004-02-01" @default.
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• W2156107349 title "Influence of apoA-I and apoE on the formation of serum amyloid A-containing lipoproteins in vivo and in vitro" @default.
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