factors (CSF) (15), and a monocyte chemotactic protein (MCP- 1) (16). Since the results of in vitro studies can often be

Transcription

1 Rapd Publcaton Mnmally Modfed Low Densty Lpoproten Is Bologcally Actve In Vvo n Mce Feng Lao,* Judth A. Berlner,* Margarete Mehraban,* Mahamad Navab,* Lnda L. Demer,* Aldons J. Luss,* and Alan M. Fogelman* *Dvson ofcardology, Department ofmedcne, Department ofmcrobology and Molecular Genetcs, and tdepartment ofpathology, Unversty ofcalforna School ofmedcne, Los Angeles, Calforna Abstract Mnmally modfed low densty lpoproten (MM-LDL), derved by mld ron oxdaton or prolonged storage at 4 C, has been shown to nduce certan nflammatory responses n vascular cells n tssue culture. These nclude nducton of monocyte (but not neutrophl) adherence to endothelal cells (EC), nducton of EC producton of colony stmulatng factors (CSF), and nducton of EC and smooth muscle cell producton of monocyte chemotactc proten (MCP-1). To test for bologc actvty n vvo, mcrogram quanttes of MM-LDL were njected nto mce, sera were assayed for CSF actvty, and tssues were subjected to Northern analyss. After njecton of MM-LDL, CSF actvty ncreased 7-26-fold but remaned - near control levels after njecton of natve LDL. Essentally all of the nduced CSF actvty was due to macrophage CSF as judged by antbody nhbton. Injecton of MM-LDL nto a mouse stran (C3H/ HeJ) that s resstant to bacteral LPS gave smlar results, ndcatng that the nducton of CSF was not due to contamnatng LPS and suggestng that there are dfferences n the pathways by whch LPS and MM-LDL trgger cytokne producton. In addton, after njecton of MM-LDL, mrna for JE, the mouse homologue of MCP-1, was markedly nduced n varous tssues, but was not nduced after njecton of natve LDL. We conclude, therefore, that MM-LDL s bologcally actve n vvo and may contrbute to the early stages of atheroscleross by actng as an nflammatory agent. (J. Cln. Invest : ) Key words: atheroscleross * monocytes - monocyte colony stmulatng factors * JE/monocyte chemotactc proten- * oxdzed low densty lpoproten Introducton The earlest event observed n expermental atherogeness s the retenton of apolpoproten B-contanng lpoprotens n the extracellular matrx of the subendothelal space (1-3). There s an ncreasng body ofevdence that oxdatve modfcaton of LDL may play a sgnfcant role n atherogeness (4-12). Address correspondence to Dr. Feng Lao, Dvson ofcardology, Department of Medcne, Room CHS, UCLA School ofmedcne, Los Angeles, CA Receved for publcaton 19 September 1990 and n revsedform 20 March J. Cln. Invest. The Amercan Socety for Clncal Investgaton, Inc /91/06/2253/05 $2.00 Volume 87, June 1991, Most studes have focused on hghly oxdzed LDL whch s no longer recognzed by the LDL receptor but s recognzed by the scavenger (acetyl-ldl) receptor and has been postulated to play a role n foam cell formaton (13). We (J. A. Berlner) hypotheszed that f LDL retenton was the frst step n leson development ntally there would be relatvely few cells present n the subendothelal space to oxdze LDL to the extent that t would be recognzed by the scavenger receptor. Therefore we studed the bologc consequences of mnmally oxdzng LDL. We found that ths LDL was not suffcently modfed to be recognzed by the scavenger receptor and that t appeared to nteract normally wth the LDL receptor (14). However, n vtro ths mnmally modfed LDL (MM-LDL)' was bologcally qute dfferent from natve LDL. As a result of the oxdaton of lpd(s) n MM-LDL t nduced the bndng of monocytes but not neutrophls to cultured endothelal cells (EC) (14). It also nduced EC to produce colony stmulatng factors (CSF) (15), and a monocyte chemotactc proten (MCP- 1) (16). Snce the results of n vtro studes can often be dfferent from those n vvo, we sought to further test the bologc actvty of MM-LDL n vvo. We chose the mouse model for ths n vvo work because of the convenence of workng wth these anmals and the avalablty ofmouse strans that are known to be genetcally susceptble or resstant to the development of atheroscleross and to cytokne nducton by bacteral LPS (17-19). Methods Reagents. Ant-mouse macrophage CSF (M-CSF) antbody, rased aganst M-CSF purfed from the condtoned medum of L cells, was a generous gft from Dr. E. R. Stanley, Albert Ensten College of Medcne, Bronx, NY. Lpopolysaccharde prepared from Eschercha col :B4 was purchased from Lst Bologcal Laboratores, Inc., Campbell, CA. All other reagents were from prevously descrbed sources (14-16). Mce. BALB/cHS, C3H/HS, and C57BL/6HS female mce, 3-6 mo of age, were purchased from Harlan Sprague Dawley, Inc., Indanapols, IN, and C3H/HeJ and C57BL/6J female mce, 3-6 mo ofage, were purchased from Jackson Laboratores, Bar Harbor, ME. The test substances were njected nto the tal vens 4 or 5 h before kllng unless otherwse ndcated. Lpoprotens. Human LDL was solated as prevously descrbed (14) from the serum of normal donors after an overnght fast and after 1. Abbrevatons used n ths paper: CSF, colony stmulatng factors; EC, endothelal cells; JE, the mouse homologue ofthe gene for MCP- 1; MCP-1, monocyte chemotactc proten; M-CSF, macrophage CSF; MM-LDL, mnmally modfed LDL; TBARS, thobarbturc acdreactve substances. Mnmally Modfed Low Densty Lpoproten In Vvo 2253

2 obtanng wrtten permsson. MM-LDL was prepared as prevously descrbed by ether cold storage or ron oxdaton ( 14) and contaned nmol of thobarbturc acd reactve substances (TBARS) as malondaldehyde equvalents per mg cholesterol, and < 4 pg ofcontamnatng LPS per Ag ofldl proten. MM-LDL prepared by cold storage was kept sterle n plastc tubes at 4VC for 6-11 mo n phosphate buffered 0.15 M NaCI contanng 0.01% EDTA. MM-LDL was also prepared by ron oxdaton by dalyzng natve LDL aganst 9M4M FeSO4 n phosphate buffer, ph 7.2, for 72 h at 4VC as descrbed by Kosug and colleagues (20). Each preparaton of MM-LDL was tested n vtro for ts ablty to nduce monocyte bndng to EC as descrbed prevously (14) or for the nducton ofmrna for JE (the mouse homologue ofthe gene for MCP-I) n mouse L cells by Northern analyss as descrbed below. Not all preparatons ofldl that were stored under these condtons became bologcally actve. Only a mnorty (on the order of one n fve) ofthe preparatons demonstrated bologc actvty even though almost all ofthe preparatons demonstrated a small ncrease n TBARS (2-4 nmol of TBARS as malondaldehyde equvalents per mg cholesterol) characterstc of MM-LDL. Smlarly, actvty after ron oxdaton vared from preparaton to preparaton as a functon ofthe tme of exposure to FeSO4. Therefore, the optmum tme for exposng natve LDL to FeSO4 must be determned emprcally for each LDL preparaton n order to produce bologcally actve MM-LDL. As prevously reported (14) the bologcally actve component n MM-LDL appears to be a polar lpd(s) presumably formed by oxdaton of unsaturated lpd contaned n natve LDL. Wth contnued oxdaton (.e., contnued exposure to FeSO4 beyond the tme requred to nduce bologc actvty) the nduced bologc actvty was lost, as would be expected f the bologcally actve agent n MM-LDL were an oxdzed polar lpd. Between one n four and one n two preparatons can be expected to show bologc actvty after exposure to FeSO4 for perods up to 72 h. Moreover, n testng these preparatons n vtro we found that as prevously descrbed (14) one can nduce resstance n cultured cells to MM-LDL and, therefore, the preparatons ofmm-ldl must be tested aganst cells that have not been nduced to be resstant. Each preparaton of MM-LDL used n these studes was found to be actve n vtro before testng n vvo. Serum CSF assay. Blood was obtaned from the mce by heart puncture under Halothane anesthesa. CSF actvty was determned by mouse bone marrow cell colony formaton assay as prevously descrbed (21). Serum CSF actvty was expressed as CFU per ml of serum. One CFU s defned as the amount of CSF stmulatng formaton of one colony per I0O plated mouse bone marrow cells (wthn the lnear range of the assay). RNA analyss. A 445-bp JE cdna contanng the complete codng sequence was obtaned by polymerase chan reacton amplfcaton of cdna syntheszed from total NIH 3T3 cell RNA. Densty arrested cell cultures grown n DME supplemented wth 10% fetal bovne serum were transferred to fresh DME supplemented wth 5% platelet-poor plasma, free of platelet derved growth factor (PDGF), for 16 h. Cells were then treated wth 0.1 ng/ml rpdgf (BB-homodmer, Genzyme Corp., Cambrdge, MA) for 2 h (22) before RNA extracton (23). The cdna synthess was performed usng olgo(dt) prmer followed by polymerase chan reacton amplfcaton for 30 cycles wth Taq DNA polymerase (Perkn-Elmer/Cetus, Emeryvlle, CA) usng a denaturaton temperature of 95 C (1 mn), an annealng temperature of 55 C (1 mn), and an extenson temperature of72 C (3 mn). The amplfcaton buffer contaned 10 mm Trs-HCl, ph 8.3, 50 mm KCI, 1.5 mm MgC12, 0.01% (wt/vol) gelatn, and 200MM each datp, dttp, dgtp, and dctp. Reactons were performed n a IOO-Ml vol contanng 100 pmol ofthe two prmer olgonucleotdes. The synthetc olgomers used for the amplfcaton were based on the JE nucleotde sequence determned by Kawahara and Deuel (24): (5') 5'ATGCAGGTCCCTGT- CATGCTTCTGGGC3' and (3') 5'GTTCACTGTCACACTGGT- CACTCCTAC 3'. The resultng double stranded cdna ofthe expected sze of 445 bp was gel purfed and labeled wth 32P to a sp act of cpm/,ug usng random prmng. Northern blot analyss was performed as prevously descrbed (16). In addton, the dentty of the gel-purfed cdna was confrmed to be JE by sequence analyss followng subclonng of the fragment nto a PUC 18 vector. Results Inducton of M-CSF actvty n serum after njecton of MM- LDL. Wthn 5 h after njecton of MM-LDL nto BALB/cHS or nto C57BL/6HS mce there was a concentraton-dependent ncrease n serum CSF actvty whch was not seen after the njecton of natve LDL. In addton, njecton of LPS n concentratons that were determned to be the maxmum that could have contamnated the MM-LDL preparatons faled to nduce CSF actvty (Fg.- 1). As shown n Fg. 2, CSF actvty peaked 9 h after njecton ofmm-ldl and was declnng by - 20 h. Experments wth neutralzng antbody (Table I) ndcated that almost all of the nduced CSF actvty was due to M-CSF. As shown n Fg. 3, BALB/cHS mce readly nduced CSF actvty when njected wth LPS (at a concentraton 2,000- fold greater than that determned to be the maxmum LPS that could have contamnated the MM-LDL) whereas the C3H/ HeJ mce that are genetcally resstant to LPS showed poor nducton of CSF at even hgher concentratons. In contrast CSF nducton was equal n the two strans after njecton of MM-LDL. These results provde defntve proof that the bologc actvty of MM-LDL was not due to contamnatng LPS. Moreover, they ndcate that there are dfferences n the pathways by whch LPS and MM-LDL nduce cytoknes. The MM- LDL used for the experments n Fgs. 1-3 and n Table I were prepared by cold storage. Fg. 4 demonstrates that MM-LDL prepared by ron oxdaton also gave smlar results. MM-LDL nduces JE mrna. The mouse homologue of the human MCP-l gene s known as JE (25). As shown n Fg Fgure 1. Serum CSF BALB/HS E actvty after the njecton of LDL, MM-LDL, or LPS. BALB/cHS and 2000 C57BL/6HS mce were njected ntravenously v Ioo wth LDL (*), MM- LDL (prepared by cold storage and contanng nmol of TBARS as Am911 malondaldehyde equvalents per mg cholesterol) (o), or LPS (*) 5 h C57BUAHS before kllng. The concentratons shown on the abscssa are the concentraton of MM-LDL A or LDL n mouse blood mmedately after njecton assumng a blood co I vol of 2 ml. The concentraton of LPS ( pg/ml) was determned as the maxmum level that could have been present n the MM-LDL at a blood concentraton of 100 Mg/ml MM-LDL. Nether MM-LDL nor LPS had CSF actvty when added to the mouse bone marrow cell cultures (data not shown). The data shown are from the sera of two mce wth each serum assayed n duplcate to gve a total of four assays for each data pont. The values are the mean± 1 SD of these assays Lao, Berlner, Mehraban, Navab, Demer, Luss, and Fogelman

3 E z 1200 :L 0 0 F. 4% HOURS: Fgure 2. Tme course of the nducton of serum CSF actvty after njecton of MM-LDL. BALB/cHS were njected ntravenously wth MM-LDL (prepared by cold storage of LDL from a dfferent donor from that descrbed n Fg. 1 but also contanng 3 nmol of TBARS as malondaldehyde equvalents per mg cholesterol) to gve a blood concentraton of 100 jg/ml MM-LDL mmedately after njecton (assumng a blood vol of 2 ml). Blood was collected at the tme ponts ndcated on the abscssa and CSF actvty n the sera was determned. Four mce were used for each tme pont and ther sera was assayed n duplcate. The values shown are the mean± I SD of these assays. j Fgure 3. Inducton of serum CSF actvty after BALB/cHS njecton of LPS or l10 - MM-LDL nto BALB/ chs and C3H/HeJ C3H mce. Blood was col- 500 son vlected 5 h after njecton of LPS or MM-LDL (prepared by cold stor age of LDL from a do o*20 *04L {,,9010 nor dfferent from those 2000 used n the experments of Fgs. 1 and 2 and F. 5W * * T contanng 2 nmol of XALWBCHS TBARS as malondaldehyde equvalents per 1W00 mg cholesterol) nto 5 C3H/F.J BALB/cHS (o) or C3H/ SW son,hej (*) -.1mce. The concentratons shown on the abscssa are those mmedately after njec- LPS (bhnl) ton assumng a blood vol of 2 ml. Each data pont represents serum taken from two mce and assayed n duplcate. The values are the mean± I SD of these assays. 5, there was a dramatc ncrease n JE mrna n lver after the njecton of MM-LDL (prepared by mld ron oxdaton) wthout a sgnfcant change n tubuln mrna. In contrast, njecton of the same LDL (but wthout ron oxdaton) dd not nduce mrna for JE (Fg. 5). Smlar results were seen after njecton of MM-LDL prepared by cold storage and there was also nducton of JE mrna n heart, lung, spleen, and kdney n all strans tested ncludng C57BL/6HS, BALB/cHS, and C3H/HS (data not shown). 9 h after the njecton, mrna levels for JE had returned to baselne (data not shown). Dscusson In varous anmal models ofatheroscleross cholesterol-feedng results n the rapd retenton of apolpoproten B-contanng lpoprotens n the subendothelal space (1-3) followed by the Table I. MM-LDL Induces M-CSF Prencubaton Percentage of orgnal actvty None 100.0±1.1 Ant-M-CSF 17.9±10.5 Irrelevant IgG 83.6±3.7 Duplcate BALB/cHS mce were njected wth MM-LDL (prepared by cold storage and contanng 3 nmol of TBARS as malondaldehyde equvalents per mg cholesterol) to gve a blood concentraton mmedately after njecton of 50,g/ml (assumng a blood vol of 2 ml). Blood was collected 5 h later and the sera were prencubated for 20 mn wthout addtons, or wth ant-m-csf antbody, or wth an rrelevant IgG before addton of the sera to the mouse bone marrow cell cultures. The sera were assayed n trplcate or quadruplcate and the data shown are the mean± I SD of the assays from both mce. adherence ofmonocytes to the endothelum at stes ofpredlecton such as the orfces of the ntercostal arteres (26-29). Enhanced monocyte progentor cell prolferaton occurs n the bone marrow of cholesterol-fed swne and ths s accompaned by ncreased ablty ofthe sera from these swne to nduce monocytc colones n bone marrow cell cultures (30). Our recent E U. 3000, o 1000 U- 0 CON LDL MM-LDL Fgure 4. Serum CSF actvty n control mce or after njecton of LDL or MM-LDL. C57BL/6J mce were ether not njected (CON) or were njected wth LDL from a donor dfferent from those used n the experments descrbed above and contanng < 2 nmol of TBARS as malondaldehyde equvalents per mg cholesterol (LDL) or the same LDL exposed to FeSO4 as descrbed n Methods and subsequently contanng 3.7 nmol of TBARS as malondaldehyde equvalents per mg cholesterol (MM-LDL) 3-6 h before kllng to gve a blood concentraton of 100 yg/ml of LDL or MM-LDL mmedately after njecton (assumng a blood vol of 2 ml). The sera from three control mce, three mce njected wth LDL, and two mce njected wth MM-LDL were assayed n duplcate for CSF actvty. The values are the mean± 1 SD of these assays. Mnmally Modfed Low Densty Lpoproten In Vvo 2255

4 Award from the Amercan Heart Assocaton, Greater Los Angeles Afflate. JE Fgure 5. Inducton of mrna for JE after njecton of LDL or MM-LDL nto C57BL/6J mce. RNA was extracted from the lvers of two of the mce descrbed n Fg. 4 after njecton wth LDL (lanes I and 2) or from both ofthe mce njected wth MM-LDL (lanes 3 and 4) and subjected to Northern blot analyss usng probes for JE and a-tubuln. fndngs that n vtro MM-LDL nduced the bndng of monocytes but not neutrophls to cultured EC (14), nduced EC to produce CSF (15) and MCP- 1 (16), together wth the fndngs reported here that MM-LDL nduced smlar events n vvo, suggest that the early cellular events n atherogeness could be due, n part, to the formaton of MM-LDL. As the monocytes mgrate nto the subendothelal space and convert nto macrophages further modfcaton of LDL could occur as a result of the release of metaboltes from the macrophages (1 1, 31) nto the mcroenvronment. The resultng hghly modfed LDL would then be taken up by the scavenger receptor, producng foam cells. There are genetc dfferences n susceptblty of nbred strans of mce to the det-nduced development of the early events of atherogeness (monocyte adheson to the endothelum, monocyte chemotaxs nto the subendothelal space, monocyte converson nto macrophages, and monocyte foam cell formaton) (17, 18). C57BL/6 mce are hghly susceptble whle BALB/c and C3H are relatvely resstant (17, 18). Snce we found that all of these strans readly nduced M-CSF n ther sera and mrna for JE n ther tssues after njecton of MM-LDL, t s possble that the vared susceptblty to det-nduced development ofthese early events of atherogeness s due to dfferences n the formaton of oxdatvely modfed LDL. The nbred strans ofmce that are resstant to the development of these early events of atherogeness have sgnfcantly hgher levels ofhdl compared to the strans that are susceptble (18). Evdence from several laboratores suggests that HDL s a potent antoxdant that s capable of preventng the formaton of oxdatvely modfed LDL n vtro (32-35). Ifths s also true n vvo, some of the genetc dfferences n susceptblty of nbred strans of mce to the development of these early events of atherogeness may be due to dfferences n ther HDL levels on the hgh fat det and the resultng dfferences n ther ablty to resst oxdatve modfcaton of LDL. Acknowledgments We thank Dr. E. R. Stanley for hs generous gft of antbody to mouse M-CSF. Ths research was supported n part by Natonal Insttutes ofhealth Grants HL-30568, RR865, the Amercan Heart Assocaton, Greater Los Angeles Afflate, the Laubsch, Rachel Israel Berro, and Mlt Grey Funds. A. J. Luss was an Establshed Investgator of the Amercan Heart Assocaton. L. L. Demer s the recpent of a Clncan Scentst References 1. Schwenke, D. C., and T. E. Carew Intaton of atherosclerotc lesons n cholesterol-fed rabbts I. Focal ncreases n arteral LDL concentraton precede development of fatty streak lesons. Arteroscleross. 9: Schwenke, D. C., and T. E. Carew Intaton of atherosclerotc lesons n cholesterol-fed rabbts. II. Selectve retenton of LDL vs. selectve ncreases n LDL permeablty n susceptble stes of arteres. Arteroscleross. 9: Frank, J. S., and A. M. Fogelman Ultrastructure of the ntma n WHHL and cholesterol-fed rabbt aortas prepared by ultra-rapd freezng and freeze-etchng. J. Lpd Res. 30: Stenberg, D., S. Parthasarathy, T. E. Carew, J. C. Khoo, and J. L. Wtztum Beyond cholesterol. Modfcatons of low-densty lpoproten that ncrease ts atherogencty. N. Eng. J. Med. 320: Stenbrecher, U., S. Parthasarathy, D. S. Leake, J. L. Wtztum, and D. Stenberg Modfcaton of low densty lpoproten by endothelal cells nvolves lpd peroxdaton and degradaton of low densty lpoproten phospholpds. Proc. Natl. Acad. Sc. USA. 83: Morel, D. W., P. E. DCorleto, and G. M. Chsolm Endothelal and smooth muscle cells alter low densty lpoproten n vtro by free radcal oxdaton. Arteroscleross. 4: Kta, T., Y. Nagano, M. Yokode, K. Ish, N. Kume, A. Ooshuma, H. Yoshda, and C. Kawa Probucol prevents the progresson ofatheroscleross n WHHL rabbt. Proc. Natl. Acad. Sc. USA. 84: Carew, T. E., D. C. Schwenke, and D. Stenberg Antatherogenc effect of probucol unrelated to the hypocholesterolemc effect: evdence that antoxdants n vvo can selectvely nhbt LDL degradaton n macrophage rch fatty streaks and slow progresson of atheroscleross n the WHHL rabbt. Proc. Nal. Acad. Sc. USA. 84: Chsolm, G. M., and D. W. Morel Lpoproten oxdaton and cytotoxcty: effect of probucol on streptozotocn-treated rats. Am. J. Cardol. 62:20B-26B. 10. Haberland, M. E., D. Fong, and L. Cheng Malondaldehyde altered proten occurs n atheroma ofwhhl rabbts. Scence (Wash. DC). 241: Palnsk, W., M. E. Rosenfeld, S. Yla-Herttuala, G. C. Gurtner, S. S. Socher, S. W. Butler, S. Parthasarathy, T. E. Carew, D. Stenberg, and J. L. Wtztum LDL undergoes oxdatve modfcaton n vvo. Proc. Natl. Acad. Sc. USA. 86: Boyd, H. C., A. M. Gown, G. Wolfbauer, and A. Chat Drect evdence for a proten recognzed by a monoclonal antbody aganst oxdatvely modfed LDL n atherosclerotc lesons from a Watanabe hertable hyperlpdemc rabbt. Am. J. Pathol. 135: Brown, M. S., and J. L. Goldsten Scavengng for receptors. Nature (Lond.). 343: Berlner, J. A., M. C. Terrto, A. Sevanan, S. Ramn, J. A. Km, B. Bamshad, M. Esterson, and A. M. Fogelman Mnmally modfed low densty lpoproten stmulates monocyte endothelal nteractons. J. Cln. Invest. 85: Rajavashsth, T. B., A. Andalb, M. C. Terrto, J. A. Berlner, M. Navab, A. M. Fogelman, and A. J. Luss Inducton ofendothelal cell expresson of granulocyte and macrophage colony-stmulatng factors by modfed low-densty lpoprotens. Nature (Lond.). 344: Cushng, S. D., J. A. Berlner, A. J. Valente, M. C. Terrto, M. Navab, F. Parham, R. Gerrty, C. J. Schwartz, and A. M. Fogelman Mnmally modfed low densty lpoproten nduces monocyte chemotactc proten 1 n human endothelal cells and smooth muscle cells. Proc. Natl. Acad. Sc. USA. 87: Pagen, B., A. Morrow, C. Brandon, D. Mtchell, and P. A. Holmes Varaton n susceptblty to atheroscleross among nbred strans ofmce. Atheroscleross. 57: Pagen, B., D. Mtchell, K. Reue, A. Morrow, A. J. Luss, and R. C. LeBoeuf Ath-l, a gene determnng atheroscleross susceptblty and hgh densty lpoproten levels n mce. Proc. Natl. Acad. Sc. USA. 84: Rosenstrech, D. L Genetc control of endotoxn response: C3H/ HeJ mce. In Handbook of Endotoxn. Vol. 3. L. J. Berry, edtor. Elsever/North- Holland Bomedcal Press. New York Kosug, K., D. W. Morel, P. E. D Corleto, and G. M. Chsholm Toxcty of oxdzed LDL to cultured fbroblasts s selectve for S phase ofthe cell cycle. J. Cell Physol. 130: Luss, A. J., D. H. Quon, and D. W. Golde Purfcaton and characterzaton of a human T-lymphocyte-derved granulocyte-macrophage colonystmulatng factor. Blood. 57: Scher, C. D., W. J. Pledger, P. Martn, H. Antonades, and C. D. Stles Transformng vruses drectly reduce the cellular growth requrement for a platelet derved growth factor. J. Cell. Physol. 97: Lao, Berlner, Mehraban, Navab, Demer, Luss, and Fogelman

5 23. Chomczynsk, P., and N. Sacch Sngle-step method ofrna solaton by acd guandnum thocyanate-phenol-chloroform extracton. Anal. Bochem. 162: Kawahara, R. S., and T. F. Deuel Platelet-derved growth factor-nducble gene JE s a member ofa famly ofsmall nducble genes related to platelet factor 4. J. Bol. Chem. 264: Rollns, B. J., P. Ster, T. Ernst, and G. G. Wong The human homolog of the JE gene encodes a monocyte secretory proten. Mol. Cell. Bol. 9: Gerrty, R. G., H. K. Nato, M. Rchardson, and C. J. Schwartz Detary nduced atherogeness n swne. Am. J. Pathol. 95: Gerrty, R. G The role ofthe monocyte n atherogeness. I. Transton of blood-borne monocytes nto foam cells n fatty lesons. Am. J. Pathol. 103: Faggotto, A., R. Ross, and L. Harker Studes of hypercholesterolema n the nonhuman prmate. I. Changes that lead to fatty streak formaton. Arteroscleross. 4: Gerrty, R. G., J. Goss, and L. Soby Control of monocyte recrutment by chemotactc factor(s) n leson-prone areas ofswne aorta. Arteroscleross. 5: Averll, L. E., R. C. Meagher, and R. G. Gerrty Enhanced monocyte progentor cell prolferaton n bone marrow of hyperlpdemc swne. Am. J. Pathol. 135: Fogelman, A. M., I. Shechter, J. Seager, M. Hokom, J. S. Chld, and P. A. Edwards Malondaldehyde alteraton of low densty lpoprotens leads to cholesteryl ester accumulaton n human monocyte-macrophages. Proc. Natl. Acad. Sc. USA. 77: Hessler, J. R., A. L. Robertson, Jr., and G. M. Chsolm LDL-nduced cytotoxcty and ts nhbton by HDL n human vascular smooth muscle and endothelal cells n culture. Atheroscleross. 32: Ohta, T., K. Takata, S. Horuch, Y. Morno, and I. Matsuda Protectve effect of lpoprotens contanng apoproten A-I on Cu2+-catalyzed oxdaton of human low densty lpoproten. FEBS (Fed. Eur. Bochem. Soc.) Lett. 257: Parthasarathy, S., J. Barnett, and L. G. Fong Hgh-densty lpoproten nhbts the oxdatve modfcaton of low-densty lpoproten. Bochm. Bophys. Acta. 1044: Navab, M., G. P. Hough, J. A. Berlner, L. A. Ross, A. J. Valente, and A. M. Fogelman Modfcaton of LDL by serum contanng cocultures of human artery wall cells nduces monocyte chemotactc proten (MCP- 1). Arteroscleross. 10:759a (Abstr.) Mnmally Modfed Low Densty Lpoproten In Vvo 2257