Cholesterol Accumulation in Aortic Smooth Muscle Cells Exposed to Low Density Lipoproteins. Contribution of Free Cholesterol Transfer

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1 Chlesterl Accumulatin in Artic Smth Muscle Cells Expsed t Lw Density Lipprteins Cntributin f Free Chlesterl Transfer J. Peter Sltte, Alan Chait, and Edwin L. Bierman Incubatin f cultured arterial smth muscle cells with large cncentratins f lw density lipprteins (LDL) resulted in a net increase in cell chlesterl and chlesteryl ester mass that was dependent n LDL cncentratin and time f incubatin. Use f an inhibitr f acyl-caxhlesterl acyl-transferase (ACAT) reduced the accumulatin f chlesteryl ester mass by 40% (range 25% t 50%), suggesting that a significant prprtin f the chlesteryl ester mass that accumulated frm LDL did s withut being hydrlyzed and re-esterified. Quiescent arterial smth muscle cells expsed fr 48 hurs t 0.5 mg/ml f 125 I-LDL accumulated 115 nml ttal sterl/mg cell prtein. Hwever, these cells tk up and degraded nly 21 /ig f 12S I-LDL prtein, which cntains 64 nml ttal chlesterl. Hence, nly abut 60% f the increase in cell-assciated chlesterl mass was accunted fr by LDL particle uptake and degradatin. Further, when cells were incubated with 3 H chlesteryl linleyl ether-labeled LDL, the net increase f ttal cell chlesterl was 81 nml/mg cell prtein. Hwever, nly 49 nml f ttal chlesterl was taken up by LDL particle uptake, as calculated frm the uptake f the 3 H chlesteryl linleyl ether tracer. It thus appears that abut 40% f the accumulated chlesterl mass was derived independent f LDL particle uptake, suggesting the pssibility f transfer f free chlesterl frm the surface f LDL t the cell surface. The ccurrence f chlesterl surface transfer was independently verified by the measurement f the uptake and cellular distributin f LDL-derived free 3 H-chlesterl. A substantial fractin f the accumulated cell chlesterl mass (apprximately 40%) was derived frm surface transfer f LDL free chlesterl. These findings suggest that mechanisms ther than the classical LDL receptr pathway are likely t play imprtant rles in chlesterl accumulatin and fam cell frmatin in arterial smth muscle cells. (Arterisclersis 8: , Nvember/December 1988) The accumulatin f chlesterl and chlesteryl esters in arterial wall cells is an early event during athergenesis. 1 The mechanisms respnsible fr chlesterl accumulatin in cells are nt fully understd. While mst cells have n their surface a specific receptr fr aplipprtein (ap) B and ap E-cntaining lipprteins [the lw density lipprtein (LDL) receptr 23 ], the activity f this receptr is inversely prprtinal t the chlesterl level in cells. 45 Thus, when the cell chlesterl level is increased, the activity f the LDL-receptr is decreased. Hence, it is unlikely that LDL receptr-mediated uptake f LDL can accunt slely fr the bserved chlesterl Frm the Divisin f Metablism, Endcrinlgy, and Nutritin; Department f Medicine, University f Washingtn, Seattle, Washingtn. This research was supprted by NIH Grants HL 18645, HL 30086, and DK J. Peter Sltte was supprted by a Research Fellwship frm the American Heart Assciatin, Washingtn Affiliate. His present address is the Department f Bichemistry and Pharmacy, ABO AKADEMI, SF Turku, Finland. Alan Chait is an Established Investigatr f the American Heart Assciatin. Address fr crrespndence: Edwin L. Bierman, M.D., Divisin f Metablism, Endcrinlgy, and Nutritin; Department f Medicine RG-26, University f Washingtn, Seattle, WA Received April 7, 1988; revisin accepted June 6, Olga Stein, M.D., kindly acted as Guest Editr fr this manuscript. accumulatin in cells expsed t high cncentratins f ap B- r ap E-cntaining lipprteins. 6-9 Macrphages have scavenger receptrs that bind and take up several mdified frms f lipprteins, leading t massive chlesterl accumulatin and fam cell frmatin. 10 Hwever, arterial smth muscle cells lack scavenger receptrs. 10 Thus, receptr-independent mechanisms are likely t cntribute t the bservable accumulatin f chlesterl mass in arterial smth muscle cells expsed t high levels f LDL. Accumulatin f chlesterl by LDL receptr-independent mechanisms culd, in part, be explained by surface transfer f free chlesterl frm lipprteins t cells fllwed by subsequent esterificatin. The plausibility f such a transfer and esterificatin prcess has been demnstrated in cell culture studies by using a variety f different cell types and extracellular chlesterl dnr particles In this study, the ability f arterial smth muscle cells t accumulate chlesterl after expsure t LDL at cncentratins sufficient t express the LDL receptr was examined. Further, the hypthesis that surface transfer f unesterified chlesterl between LDL and arterial smth muscle cells can cntribute t a mass accumulatin f chlesterl in the cells was tested directly. Arterial smth muscle cells frm the nnhuman primate, Macaca nemestrina, were expsed t high cncentratins f LDL labeled 750

2 FREE CHOLESTEROL TRANSFER FROM LDL TO SMC Sltte et al. 751 either with 125 I in the prtein miety, with 3 H-chlesterl in the particle surface, r with 3 H-chlesteryl linleyl ether ( 3 H-CLE) in the lipid cre f the particle. The extent t which the cellular free and esterified chlesterl cntent increased and the degree t which the accumulatin f chlesterl mass in the cells culd be accunted fr by LDL particle uptake, by exchange f LDL free 3 H-chlesterl, and by LDL chlesteryl ester uptake was determined. Methds Materials 3 H-chlesterl (55 Ci/mml) was btained frm Du Pnt-New England Nuclear (Bstn, MA) and prved t be greater than 98% pure. 3 H-chlesteryl linleyl ether was synthesized frm 3 H-chlesterl and linleyl alchl (Sigma, St. Luis, MO), as described by Halperin and Gatt. 20 The cmpund [3-decyldimethylsilyl-A/-2- (4-methylphenyl)-1-phenylethyl prpanamide], an inhibitr f the enzyme acyl-ca:chlesterl acyltransferase (ACAT), was generusly prvided by Sandz Incrprated (East Hanver, NJ). Cell culture media and supplements were btained frm Gibc (Grand Island, NY). Chlesterl xidase was btained frm Calbichem (San Dieg, CA). Cell Culture Arterial smth muscle cells were derived frm intimamedial explants f mnkey (Macaca nemestrina) thracic artery. 21 The cells were cultivated in Dulbecc's mdified Eagle's medium (DMEM) with 10% calf serum. Fr experiments, cells were seeded either in 16 mm diameter cell culture wells r in 35 mm diameter cell culture dishes. The grwth medium was replaced with fresh medium every third day (0.5 ml grwth medium/ well r 1.0 ml/dish), and the cells became cnfluent within 5 t 7 days. Then cells were kept fr 3 days in DMEM supplemented with 0.2% calf serum t render them quiescent. The cell number ceased t increase after 2 t 3 days in this lw-serum medium. Lipprteins LDL (d=1.019 t g/ml) were prepared frm human plasma (EDTA 4 mm) by vertical rtr ultracentrifugatin 22 fllwed by standard sequential gradient ultracentrifugatin. 5 The LDL stck slutin (abut 11 t 15 mg/ml) was stred under N 2 in the dark at 4 C until used within 2 weeks f preparatin. Lipprteindeficient serum was prepared by ultracentrifugatin at d=1.25 g/ml. 23 Radi-idinatin f LDL was perfrmed with the idine mnchlride methd as mdified fr lipprteins. 24 The 125 I-LDL stck slutin (apprximately 5 mg prtein/ml) had a specific activity f apprximately 200 t 300 cpm/ng prtein and was diluted with unlabeled LDL (frm the same batch) befre the experiments. Fresh batches f LDL were prepared every 2 weeks, s that different experiments were usually perfrmed with different LDL preparatins. LDL labeled with trace amunts f 3 H-chlesterl was prepared as fllws: 100 ^Ci 3 H-chlesterl (in ethanl) was dried nt the wall f a sterile plastic tube. Then 30 mg LDL (prtein) was added t the tube, and chlesterl exchange was allwed t prceed fr 48 hurs at 4 C. The resultant 3 H-chlesterl LDL was passed thrugh a 0.22 jum sterile filter befre use and had a specific activity f apprximately 400 t 500 cpm/nml unesterified chlesterl. Less than 0.1% f the label in LDL was fund as 3 H-chlesteryl ester. Labeling f LDL with trace amunts f 3 H-chlesterl did nt alter the lipid cmpsitin f the lipprtein particles (data nt shwn). LDL labeled with trace amunts f 3 H-chlesteryl linleyl ether was prepared as described by Stein et al. 25 The final specific activity was 10 t 15 cpm/nml LDL chlesteryl ester, assuming cmplete mixing f the 3 H-chlesteryl linleyl ether with chlesteryl esters f the LDL particle cre. Incubatin Prcedure Cnfluent and quiescent smth muscle cells were incubated fr 48 hurs (unless therwise indicated) in DMEM with 0.2% calf serum supplemented with LDL (0.5 mg prtein/ml, unless therwise indicated). The LDL used was native LDL, r native LDL labeled with either 125 I, 3 H-chlesterl, r 3 H-chlesteryl linleyl ether fr measurement f particle uptake, exchange f unesterified chlesterl, r LDL chlesteryl ester uptake, respectively. Sme cells were als expsed t 5 ^.g/ml f the ACAT-inhibitr cmpund (added frm an ethanlic stck slutin, final ethanl cncentratin <0.1 % vl/vl). Cntrl cells run in parallel received 0.2% calf serum alne, with apprpriate amunts f ethanl. The cmplete incubatin medium als cntained 0.1 fim butylated hydrxytluene as anti-xidant. When cells were expsed t LDL fr lnger than 48 hurs, the incubatin medium was replaced every 48 hurs. At the cmpletin f the experiments, the media were remved (and saved when necessary), and the cell mnlayers were washed five times with phsphate-buffered saline (PBS, ph 7.4). The cells were stred at -20 C until further prcessed within 4 days. Cellular uptake f either 3 H-chlesterl r 3 H-chlesteryl linleyl ether, and cellular frmatin f 3 H-chlesteryl ester was determined frm the neutral lipid extract, as described under lipid analysis. Uptake and Degradatin f 125 I-LOW Density Lipprtein T determine the extent f lipprtein degradatin, cnfluent and quiescent mnkey arterial smth muscle cells were incubated with 0.5 mg/ml 125 I-LDL (final specific activity 10 t 20 cpm/ng LDL prtein) in DMEM cntaining 0.2% calf serum fr 48 hurs. At the cmpletin f the incubatin, a sample f the incubatin medium was taken fr measurement f 125 I-LDL degradatin prducts. 26 Degradatin f 125 I-LDL was measured in parallel in cell-free dishes, and was subtracted frm ttal degradatin t give a measure f LDL that was degraded by cells. Degradatin f 12S I-LDL in the absence f cells was usually less than 2% f the ttal amunt degraded by cells. After cllectin f incubatin media, cell mnlayers were washed five times with PBS, lipids were extracted, and the prteins were digested int 0.1 N NaOH. Aliquts were taken fr cunting f cellular 125 l-radiactivity (cellretained, undegraded 125 I-LDL), and fr prtein determinatin 27 using bvine serum albumin as standard.

3 752 ARTERIOSCLEROSIS VOL 8, N 6, NOVEMBER/DECEMBER 1988 Oxidatin f Cell Surface 3 H-Chlesterl T measure the extent f chlesterl exchange between LDL and the cell surface (i.e., plasma membrane), chlesterl xidase was used t prbe the distributin f chlesterl between the plasma membrane and intracellular membranes Befre the experiments, cells were pretreated fr 24 hurs with either DMEM with 0.2% calf serum r the same medium supplemented with unlabeled LDL (0.5 mg prtein/ml). Cells in 35 mm dishes were then incubated with 3 H-chlesterl-labeled LDL (0.5 mg prtein/ ml) fr up t 4 hurs. Cells were then washed three times with PBS at 4 C and incubated with dextran sulphate (4 mg/ml in PBS) fr 10 minutes at 4 C t dissciate LDL frm the LDL receptr. 23 After three further washes with PBS, the cells were fixed with glutaraldehyde (1 % vl/vl in PBS) fr 10 minutes at 4 C. Excess glutaraldehyde was remved with three additinal washes, and 1 ml PBS at 37 C was added t each dish tgether with 1 U f chlesterl xidase. The cells were expsed t the chlesterl xidase fr exactly 10 minutes at 37 C. Cells were then washed nce with ice-cld PBS and immediately frzen (-20 C). Lipid Analysis Cellular neutral lipids were extracted with hexane/ isprpanl (3:2 vl/vl) 30 cntaining butylated hydrxytluene (50 pm) as an anti-xidant. Chlesterl, 3 H-chlesterl, 3 H-chlestenne, chlesteryl esters, and 3 H-chlesteryl esters were islated n either Silica Gel H (fr chlesterl mass analysis) r Silica Gel G (fr scintillatin cunting) thin-layer chrmatgraphy plates (Analtech, Newark, NJ) and were eluted with hexane/diethyl ether/acetic acid (130:40:1.5) (vl/vl/vl). Sterl mass was determined frm extracts f the apprpriate spts n the thin-layer chrmatgram by the chlesterl xidase methd. 31 The radiactivity in the free and esterified chlesterl spts was determined by cunting an aliqut f the chrmatgram extracts. Results Cellular Uptake f Chlesterl frm Lw Density Lipprtein and Accumulatin f Chlesterl Mass Cultured cells expsed t high levels f LDL have been shwn t accumulate free and esterified chlesterl mass T determine the extent f chlesterl and chlesteryl ester accumulatin in cultured arterial smth muscle cells, cnfluent and quiescent cells were expsed t varying amunts f LDL fr 4 days. An accumulatin f bth free and esterified chlesterl in cells expsed t LDL was bserved (Figure 1). The extent f chlesteryl ester accumulatin was prprtinal t the cncentratin f LDL in the incubatin medium, whereas the increase in cellular free chlesterl mass was maximal at 50 ^9 prtein/ml and did nt increase further with higher LDL cncentratins. An LDL cncentratin f 0.5 mg LDL prtein/ml was used in subsequent studies, since it resulted in marked elevatins f cellular chlesterl cntent LDL Cncentratin (mg/ml) Figure 1. Accumulatin f free and esterified chlesterl in smth muscle cells expsed t increasing amunts f lw density lipprtein (LDL). Cnfluent and quiescent cells in 35 mm diameter cell culture dishes were incubated fr 4 days with increasing amunts f LDL in Dulbecc's mdified Eagle medium (DMEM) with 0.2% calf serum. The cntent f free chlesterl (FC) () and chlesteryl ester (A) mass in cells was determined frm the neutral lipid extract, as described in Methds. Values are expressed as nml chlesterl (r equivalent) per milligram f cell prtein (mean±sd f triplicate dishes frm tw separate experiments). The percent-cntent f chlesteryl ester ( ) was calculated frm mass values f free and esterified chlesterl, CE=[CE/(CE+FC)]100. Cntributin f Endgenus Esterificatin t Cellular Accumulatin f Chlesteryl Ester Mass in Cells Expsed t Lw Density Lipprtein T determine the rle f endgenus esterificatin f chlesterl by ACAT t the bservable accumulatin f chlesteryl ester mass in smth muscle cells expsed t LDL, cells were incubated with 3 H-chlesterl LDL and the uptake and esterificatin f 3 H-chlesterl was determined in cells treated with r withut the ACAT-inhibitr, Sandz cmpund Cells were expsed t 0.5 mg/ml f 3 H-chlesterl-LDL fr up t 96 hurs in the presence r absence f cmpund at 5 ^g/ml. The arterial smth muscle cells incrprated LDL-derived free 3 H-chlesterl readily, and at the end f the 96-hur incubatin perid, mre than 90% f the cellular free chlesterl had been exchanged with 3 H-chlesterl frm LDL (Figure 2). Cells that were nt expsed t cmpund had esterified abut 13% f the incrprated 3 H-chlesterl at the end f the 96-hur incubatin (Figure 2A). Cells treated with 5 ptg/ml f cmpund incrprated LDL-derived 3 H-chlesterl t the same extent as untreated cells, but the subsequent esterificatin f 3 H-chlesterl was cmpletely blcked (Figure 2B). These results cnfirm that cmpund at 5 /xg/ml als is a ptent inhibitr f ACAT in mnkey arterial smth muscle cells. In additin t uptake and esterificatin f LDL-derived 3 H-chlesterl, the smth muscle cells als gained sterl mass during the 24- t 96-hur incubatin with 0.5 mg/ml f 3 H-chlesterl LDL. Whereas the cellular level f free chlesterl increased nly during the first 24 hurs and then remained steady at abut 110 nml/mg cell prtein,

4 FREE CHOLESTEROL TRANSFER FROM LDL TO SMC Sltte et al. 753 cells. The remainder f the accumulated chlesteryl esters must have been derived frm uptake and retentin f LDL chlesteryl esters withut lyssmal hydrlysis. Chlesteryl Ester Mass Accumulatin in Cells Treated with 3 H-Chlesteryl Linleyl Ether-cntaining Lw Density Lipprtein 3 H-chlesteryl linleyl ether is a nndegradable marker fr chlesteryl esters. Uptake f this tracer is representative f chlesteryl ester uptake, since it is nt degraded but, instead, accumulates in cells Radilabeled chlesteryl linleyl ether has been used t accurately measure the internalizatin f lipprtein-derived chlesteryl esters Cnfluent and quiescent smth muscle cells were expsed fr 48 hurs t 0.5 mg prtein/ ml f LDL cntaining trace amunts f 3 H-chlesteryl linleyl ether ( 3 H-CLE-LDL), and the subsequent cellular accumulatin f 3 H-CLE-LDL tracer and sterl mass was determined. Under these incubatin cnditins, smth muscle cells internalized 49 and 47 nml ttal LDLderived sterl/mg cell prtein when incubated in the absence r presence f cmpund (Table 2). These values are calculated frm the internalizatin f the nndegradable 3 H-CLE-tracer, assuming that uptake f 3 H-CLE-LDL was accmpanied by LDL-particle uptake. The actual net increase in cellular ttal sterl mass was 81 and 89 nml/mg cell prtein in untreated and cm- " a. CP E a> O O " Ec E 100 a> t 0) 6 75 c Incubatin Time ( hr) 96 Figure 2. Time-curse f accumulatin f chlesterl mass and 3 H-chlesterl in cells expsed t 3 H-chlesterl-labeled lw density lipprtein (LDL). Cnfluent and quiescent smth muscle cells in 16 mm diameter wells were incubated fr 24,48, 72, r 96 hurs with 0.5 mg prtein/ml f 3 H-chlesterl-labeled LDL in Dulbecc's mdified Eagle medium (DMEM) with 0.2% calf serum. The accumulatin f free (O) and esterified chlesterl (A) mass, f 3 H-chlesterl ( ), and the frmatin f 3 H-chlesteryl esters (A) was determined frm the neutral lipid extract, as described in Methds. In additin, cells were simultaneusly incubated withut (A) r with 5 ^g/ml f cmpund (B), an inhibitr f acyl-ca:chlesterl acyl-transferase (ACAT). Values are means±sd f six wells frm ne representative experiment. chlesteryl ester mass cntinued t increase during the incubatin perid (Figure 2A). The accumulatin f chlesteryl ester mass in cells treated with cmpund was abut ne-third lwer (Figure 2B) than the level fund in untreated cells (Figure 2A). In general, the accumulatin f chlesteryl ester mass in cells expsed t the ACAT inhibitr was abut 40% [average frm five separate experiments (range 25% t 50%)] less than in cntrl cells. Hence it appears that a maximum f 50% f the accumulated chlesteryl ester mass was derived frm endgenus synthesis f chlesteryl esters, i.e., was due t the ACATreactin that culd be blcked by cmpund Exgenus chlesterl available t the ACAT reactin culd be derived frm uptake and degradatin f LDL, r frm surface transfer f free chlesterl between LDL and Cellular Uptake and Degradatin f 125 I-LOW Density Lipprtein Cnfluent and quiescent smth muscle cells were incubated in DMEM cntaining (0.2% calf serum) with r withut 0.5 mg prtein/ml f 125 I-LDL fr 48 hurs. During incubatin with LDL, cntrl cells internalized and degraded 20.9 /xg LDL prtein per milligram f cell prtein. Cells treated with cmpund internalized and degraded 20.8 /xg LDL prtein per milligram f cell prtein (Table 1). Based n the lipid cmpsitin f the 125 I-LDL (2.1 nml chlesteryl ester and 1.0 nml unesterified chlesterl//^g LDL prtein), it can be calculated that these cells internalized a maximum f 64.0 nml (cntrls) and 63.7 nml (cells treated with cmpund ) ttal sterl/mg cell prtein, respectively (ttal sterl=sum f LDL free and esterified chlesterl). Hwever, the increase in cell sterl mass (ttal sterl in LDL-treated cells minus ttal sterl in cells grwn in 0.2% calf serum nly) was 115 nml/mg cell prtein fr cntrl cells and 96.4 nml/mg in cells treated with cmpund Hence, a substantial amunt (abut 44% and 34% fr cntrl cells and treated cells, respectively) f the accumulated cell sterl mass culd nt be accunted fr by LDL particle uptake and degradatin. Als, whereas cells incubated with the ACAT inhibitr internalized and degraded equal amunts f 125 I-LDL cmpared t cntrl cells, they accumulated nly ne-half t three-quarters the chlesteryl ester mass f untreated cells (Table 1). The accumulatin f unesterified chlesterl was abut the same in the tw treatment grups. T further examine the relatinship between LDL lipid that was internalized and the actual mass f sterl that accumulated intracellularly, the uptake f the chlesteryl ester miety f LDL was traced with 3 H-chlesteryl linleyl ether.

5 754 ARTERIOSCLEROSIS VOL 8, N 6, NOVEMBER/DECEMBER 1988 Table 1. Cmparisn f Sterl Mass Accumulatin with LDL Uptake in Mnkey Artic Smth Muscle Cells Incubated with 125 I-LDL Additin Nne LDL LDL FC 97.2 ± ± ±23.3 Cell sterl mass* EC 6.2 ± ± ±3.1 TC ± ± ±26.4 FC Net sterl increase* EC TC I-LDL uptake (/*g/mg) 20.9 ± ±2.8 Sterl uptake based n 125 I-LDL uptakef EC TC 'Values are nml sterl eqv./mg prtein. t 125 l-ldl uptake=degradatin+cell-retained 125 I-LDL, expressed as /j.g LDL-prtein/mg cell prtein. 125 I-LDL was added t a final cncentratin f 0.5 mg prtein/ml. Cntrl cells received DMEM alne. Cmpund was added t a final cncentratin f 5 jug/ml. Values fr sterl mass uptake frm LDL were calculated based n the mass f chlesterl and chlesteryl esters that wuld be incrprated int cells fllwing 125 I-LDL uptake (degradatin+cell=assciated 125 l-activity; LDL cntained abut 2.1 nml f esterified chlesterl per /*g LDL-prtein and 1.0 nml unesterified chlesterl per ^g prtein). LDL=lw density lipprtein, FC=free (unesterified) chlesterl, EC=esterified chlesterl, TC=ttal chlesterl. Values are means ±SD f six wells frm tw separate experiments. Table 2. Cmparisn f Sterl Mass Accumulatin with 3 H-chlesteryl Linleyl Ether Incrpratin in Mnkey Artic Smth Muscle Cells Incubated with 3 H-Chlesteryl Linleyl Ether-labeled Lw Density Lipprteins Additin Nne FC 71.0 ±1.0 Sterl uptake based Cell sterl mass* Net sterl increase* n 3 H-CLE uptaket EC 1.3 ±0.3 TC 72.3 ±1.3 LDL ± ± ± ± ±1.6 LDL ± ± ± ± ±10.1 'Values are nml sterl eqv./mg prtein. t 3 H-CLE= 3 H-chlesteryl linleyl ether. Cells were incubated fr 48 hurs in DMEM (0.2% calf serum) with r withut 0.5 mg prtein/ml f 3 H-CLE-LDL (32.1 cpm//xg LDL-prtein). Sme cells als received Cmpund , added t a final cncentratin f 5 /tg/ml. The uptake f 3 H-CLE was calculated frm the cell-assciated activity f 3 H-CLE. Values fr chlesterl uptake frm LDL (sum f LDL free and esterified chlesterl) were calculated assuming that 3 H-CLE uptake was accmpanied by LDL particle uptake. Values are means±sd f six wells frm a representative experiment. pund treated cells, respectively (Table 2). Thus, abut 39% and 47% (cntrl cells and treated cells, respectively) f the accumulated ttal sterl mass culd nt be accunted fr by the calculated internalizatin f LDL particles. Since LDL-derived free chlesterl is readily exchangeable with cell chlesterl (Figure 2), it appears that the discrepancy in the amunts f actual cell sterl accumulatin and the amunt f LDL sterl uptake after LDL particle internalizatin culd, in part, be due t additinal surface transfer f LDL free chlesterl t the cells. T further test fr this pssibility, the distributin f LDLderived free 3 H-chlesterl between the cell surface and intracellular pls was determined. Distributin f LDL Free 3 H-Chlesterl between Chlesterl Oxidase-Susceptible and -Resistant Pls Chlesterl xidase has successfully been used t prbe the lcalizatin f cell chlesterl between the cell surface FC EC TC EC 43.8 ± ± ± ±6.2 TC r plasma membrane (xidase-susceptible) and intracellular cmpartments (xidase-resistant) This apprach was used t examine the distributin f LDL-derived free 3 H-chlesterl between the cell surface (the site f exchange with lipprteins) and the intracellular cmpartments (i.e., the endsmes and lyssmes where LDL is trapped after internalizatin). Cnfluent and quiescent smth muscle cells that had been pretreated fr 48 hurs with either DMEM (0.2% calf serum) alne r the same medium cntaining 0.5 mg/ml f LDL prtein (t dwn-regulate the LDL receptr) were expsed t 0.5 mg prtein/ml f 3 H-chlesterl-labeled LDL fr up t 4 hurs. The subsequent uptake f 3 H-chlesterl and the xidase susceptibility f the tracer was determined. Cells grwn in DMEM with 0.2% calf serum internalized 14 nml 3 H-chlesterl per milligram f cell prtein during the 4-hur incubatin (Figure 3A). Cells pretreated with 0.5 mg/ml f LDL internalized nly 9 nml 3 H-chlesterl per milligram f cell prtein, reflecting the dwn-regulatin f the activity f the LDL receptr after a 48-hur pretreatment with LDL. After

6 FREE CHOLESTEROL TRANSFER FROM LDL TO SMC Sltte et al. 755 E 15 B 1 10 O ne c0) V) (V O E c a> D 10 V, 200 a> " _c O 100 Q Incubatin Time (hr) Figure 3. Distributin f 3 H-chlesterl between the cell surface and intracellular rganelles during shrt-term expsures f cells t 3 H-chlesterl-labeled lw density lipprteins (LDL). Cnfluent and quiescent smth muscle cells in 35 mm diameter cell culture dishes were pretreated fr 48 hurs with either Dulbecc's mdified Eagle medium (DMEM) with 0.2% calf serum (A and C) r DMEM (0.2% calf serum) supplemented with 0.5 mg prtein/ml f unlabeled LDL (B and D). Cells were then washed twice with 2 ml phsphate-buffered saline (PBS), and incubated fr 0, 1, 2, r 4 hurs with 0.5 mg prtein/ ml f 3 H-chlesterl-labeled LDL. After the apprpriate incubatins, surface-bund LDL was dissciated with dextran sulphate (4 mg/ml in PBS), cells were fixed (1% glutaraldehyde in PBS), and treated with 1 U/ml f chlesterl xidase, as detailed under Methds. The amunt f cell-assciated ttal 3 H-sterl () (sum f 3 H-chlesterl, 3 H-chlestenne, and 3 H-chlesteryl ester); 3 H-chlestenne ( ); and 3 H-chlesteryl ester (A) was determined frm the neutral lipid extract. The amunt f cell 3 H-chlesterl xidized t 3 H-chlestenne () was calculated as the percent 3 H-chlestenne ver the ttal cell 3 H sterl. Values are means±sd f triplicate dishes frm ne representative experiment. expsure f cells t 3 H-chlesterl-LDL fr 1 hur, abut 40% t 45% f the cell-assciated 3 H-chlesterl-label was susceptible t xidatin by chlesterl xidase, whether r nt the cells had been pretreated with LDL (Figures 3A and 3B). With time, the surface-assciated amunt f 3 H-chlesterl decreased, albeit slightly mre slwly in cells with a dwn-regulated LDL receptr activity (pretreated with LDL). Thus, it appears that the exchange f 3 H-chlesterl between LDL and the cell surface (xidasesusceptible) was nt influenced by the state f the LDL receptr regulatin, whereas the internalizatin (xidaseresistant) f 3 H-chlesterl LDL clearly was. In bth states f LDL receptr activity, 3 H-chlesterl frm LDL prgressively accumulated in cell chlesteryl esters t a cmparable degree (Figures 3C and 3D). Discussin Accumulatin f bth unesterified and esterified chlesterl in cells f the arterial wall is an early event in athergenesis. 1 Bth arterial smth muscle cells and mncyte-derived macrphages are fund in early lesins as lipid-laden fam cells. 38 Whereas macrphages can internalize mdified frms f lipprteins by the scavengerreceptr, 10 the activity f which des nt respnd t changes in cell chlesterl cntent, arterial smth muscle cells internalize LDL by the classical LDL receptr pathway. 2 Since the activity f the LDL receptr is regulated in respnse t changes in cellular chlesterl hmestasis, 2539 this pathway is likely t be dwnregulated when the extracellular level f lipprteinchlesterl is high. Arterial smth muscle cells are, therefre, likely t accumulate chlesterl and becme fam cells by LDL receptr-independent mechanisms. In this study, arterial smth muscle cells were rendered quiescent t limit the use f cell chlesterl fr membrane synthesis during cell divisin. When incubated with LDL frm nrmlipidemic dnrs, the cells accumulated chlesteryl esters in a time- and cncentratin-dependent fashin, althugh nt t the same extent as has been reprted fr cultured macrphages incubated with chemically mdified frms f LDL. 10

7 756 ARTERIOSCLEROSIS VOL 8, N 6, NOVEMBER/DECEMBER 1988 One majr fcus f the present study was t elucidate the ptential mechanisms by which arterial smth muscle cells accumulate chlesterl, with particular reference t the rle f chlesterl surface transfer between LDL and cells. By cmparing the accumulatin f chlesterl mass in cells n ne hand, and the uptake and degradatin f 125 I-LDL n the ther, it was fund that cells expsed t high cncentratins f 125 I-LDL (0.5 mg LDL prtein/ml, crrespnding t 1.55 mml/l ttal sterl) accumulated mre chlesterl mass (bth free and esterified) than culd be accunted fr by 125 I-LDL particle uptake alne (sum f 125 l-degradatin prducts and cellassciated 125 l-activity). Under the experimental cnditins used, abut 60% f the accumulated chlesterl mass was derived frm 125 I-LDL particle uptake, whereas the rest (i.e., 40%) derived frm LDL, but was taken up int cells independent f LDL particle uptake r aplipprtein uptake and degradatin. Additinal evidence fr chlesterl uptake int cells independent f LDL particle uptake was btained frm studies where the cellular metablism f 3 H-chlesteryl linleate ether-labeled LDL was examined. When the uptake f LDL-derived 3 H-chlesteryl linleyl ether was quantified, it was fund that nly abut 60% f the accumulated chlesterl mass in the cells culd be accunted fr by uptake f LDL particles. The calculatin f this percentage was based n the assumptin that uptake f 3 H-chlesteryl linleyl ether was accmpanied by LDL particle uptake. Recently it has been shwn that chlesteryl esters in high density lipprtein can enter cells independent f particle uptake Ptential mechanisms fr selective uptake f chlesteryl esters independent f particle uptake include preferential transfer f lipid int cells r lipid release during retrendcytsis f lipprteins If analgus mechanisms led t the cellular uptake f 3 H-chlesteryl linleyl ether frm LDL bserved in the present study, then the prprtin f chlesterl that accumulated independent f LDL particle uptake wuld be even greater than that calculated. Use f an inhibitr f ACAT reduced the accumulatin f chlesteryl ester mass by 40%, suggesting that a significant prprtin f the chlesteryl ester mass that accumulated frm LDL did s withut being hydrlyzed and reesterified. It is cnceivable that LDL chlesteryl ester delivered t cells by such mechanisms culd accumulate withut underging lyssmal hydrlysis and withut dwnregulating the LDL receptr, leading t fam cell frmatin. Since mre chlesterl mass accumulates in cells expsed t LDL than can be accunted fr by LDL particle uptake, additinal mechanisms that prmte transfer f LDL chlesterl t cells independent f LDL particle r aplipprtein uptake must exist. The mst plausible prcess invlves the surface transfer f unesterified chlesterl frm LDL particles t cell membranes. Chlesterl in lipprteins can exchange readily with chlesterl in ther lipprtein particles 4445 and with chlesterl in cells such as adipcytes 46 and hepatcytes If the chlesterl-t-phsphlipid mlar rati in the dnr particles (e.g., LDL) exceeds that in the acceptr structure (e.g., cell membranes) the exchange prcess will lead t a net transfer f chlesterl between dnr and acceptr t equilibrate the apparent cncentratin gradient This mechanism culd lead t a net transfer f free chlesterl frm LDL t the cell surface and subsequent incrpratin intracellularly. Delivery f free chlesterl t cells withut degradatin f LDL als culd ccur during retrendcytsis Cnsistent with previus studies, incubatin f smth muscle cells with LDL cntaining trace amunts f free 3 H-chlesterl led t a rapid cellular uptake f the 3 H-chlesterl frm LDL Whereas sme f the cellassciated 3 H-chlesterl was derived frm LDL particle uptake, mst f it was transferred frm LDL t cells by surface transfer independent f particle uptake. The cellassciated 3 H-chlesterl was distributed evenly between the cell surface (chlesterl xidase-susceptible) and the cell interir (xidase-resistant) after the first hur f expsure. With time, hwever, the fractin f 3 H-chlesterl in the xidase-susceptible pl diminished, whereas the fractin f 3 H-chlesterl in the xidase-resistant pl increased. The state f LDL receptr activity affected the distributin f 3 H-chlesterl between the xidase susceptible and resistant pls. With "nrmal" LDL receptr activity and cellular intemalizatin f LDL, a large fractin f the LDL-derived 3 H-chlesterl was resistant t xidatin by chlesterl xidase, suggesting it was lcated mainly inside the cells (mst likely in endsmes and lyssmes). Hwever, in cells pretreated with LDL t dwn-regulate LDL receptr activity and thus decrease the rate f receptr-mediated LDL particle intemalizatin, a substantially smaller fractin f the cell assciated 3 H-chlesterl was xidase-resistant, presumably in the cell interir. The results f this study shw that when cells are expsed t large amunts f LDL, the accumulatin f cell chlesterl cannt be ttally accunted fr by LDL particle uptake (either receptr-mediated r nnspecific uptake). It appears that surface transfer f LDL free chlesterl int cells cntributes significantly t the cellular sterl mass accumulatin seen in this in vitr mdel. It als is likely that surface transfer f free chlesterl frm LDL t vascular cells plays a significant rle fr chlesterl accumulatin in viv. It has been reprted that lipprteins (bth very lw density lipprtein and LDL) frm patients with increased risk fr athersclersis shw a cnsistent increase in their free chlesterl cntent The surface film f these particles appears t be almst cmpletely saturated with chlesterl Of interest in this regard, an increased plasma unesterified chlesterl-t-phsphlipid rati has been assciated with athersclersis in humans 55 and atherma develpment in mice. 56 Surface transfer f free chlesterl between lipprteins and cells is a physicchemical prcess that is nt subject t the same kind f metablic regulatin seen with the LDL receptr pathway, but instead is dependent n the saturatin f chlesterl in lipprtein particles. Therefre, surface transfer f chlesterl is likely t be f majr imprtance as a mechanism fr chlesterl accumulatin in arterial cells f patients with a high chlesterl saturatin in lipprteins (such as thse with nninsulin-dependent diabetes 52 ) and with high circulating cncentratins f LDL.

8 FREE CHOLESTEROL TRANSFER FROM LDL TO SMC Sltte et al. 757 Acknwledgments We thank Maria Culala, Lucy Suzuki, Karen Tittle, Thmas Jhnsn, and Weiling King fr expert technical assistance during varius parts f this study. The secretarial assistance f J Casterline and Janetta Shepard is gratefully appreciated. References 1. Smith EB, Slater PB. The micrdissectin f large athersclertic plaques t give mrphlgically and tpgraphically defined fractins fr analysis. Part 1. The lipids in the islated fractins. Athersclersis 1972;15: Gldstein JL, Brwn MS. The lw density lipprtein pathway and its relatin t athersclersis. Annu Rev Bichem 1977;46: Mahley RW, Innerarlty TL. Lipprtein receptrs and chlesterl hmestasis. Bichim Biphys Acta 1983; 737: Brwn MS, Gldstein JL. Regulatin f the lw density lipprtein receptr in human fibrblasts. Cell 1975; 6: Oram JF, Albers JJ, Bierman EL. Rapid regulatin f the activity f the lw density lipprtein receptr f cultured human fibrblasts. 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Med Bil 1986; 64: Phillips MC, Jhnsn WJ, Rthblat GH. Mechanisms and cnsequences f cellular chlesterl exchange and transfer. Bichin Biphys Acta 1987;906: Shireman RB, Remsen JF. Uptake f ph] chlesterl frm lw density lipprteins by cultured human fibrblasts. Bichim Biphys Acta 1982;711: Halperin G, Gatt S. The synthesis f chlesteryl alkyl ethers. Sterids 1980:35: Rss R. Smth muscle cell. II. Grwth f smth muscle cells in culture and frmatin f elastic fibers. J Cell Bil 1971 ;50: Chung BH, Wilkinsn T, Geer JC, Segrest JP. Preparative and quantitative islatin f plasma lipprteins: rapid, single discntinuus density gradient ultracentrifugatin in a vertical rtr. J Lipid Res 1980;21: Gldstein JL, Basu SK, Brwn MS. Receptr-mediated endcytsis f lw-density lipprteins in cultured cells. Methds Enzyml 1983;98: Bllhelmer DW, Elsenberg S, Levy Rl. Metablism f very lw density lipprtein. Bichim Biphys Acta 1972; 260: Stein Y, Halperin G, Stein Y. The fate f chlesteryl linleyl ester and chlesterl linleate in the intact rat after injectins f bilgically labeled human lw density lipprtein. Bichim Biphys Acta 1981;663: Bierman EL, Stein O, Stein Y. Lipprtein uptake and metablism by rat artic smth muscle cells in tissue culture. Circ Res 1974;35: Lwry OH, Rsebrugh NJ, Farr AL, Randall RJ. Prtein measurement with Flin phenl-reagent. J Bil Chem 1951; 193: Lange Y, Rams BV. Analysis f the distributin f chlesterl in intact cells. J Bil Chem 1983:258: Sltte JP, Oram JF, Bierman EL. Binding f high density lipprteins t cell receptrs prmtes translcatin f chlesterl frm intracellular membranes t the cell surface. J Bil Chem 1987;262: Brwn MS, H YK, Gldstein JL. The chlesteryl ester cycle in macrphage fam cells. Cntinual hydrlysis and re-esterificatin f cytplasmic chlesteryl esters. J Bil Chem 1980:255: Heider JG, Byett RL. The picmle determinatin f free and esterified chlesterl in cells in culture. 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10 Chlesterl accumulatin in artic smth muscle cells expsed t lw density lipprteins. Cntributin f free chlesterl transfer. J P Sltte, A Chait and E L Bierman Arteriscler Thrmb Vasc Bil. 1988;8: di: /01.ATV Arterisclersis, Thrmbsis, and Vascular Bilgy is published by the American Heart Assciatin, 7272 Greenville Avenue, Dallas, TX Cpyright 1988 American Heart Assciatin, Inc. All rights reserved. Print ISSN: Online ISSN: The nline versin f this article, alng with updated infrmatin and services, is lcated n the Wrld Wide Web at: Permissins: Requests fr permissins t reprduce figures, tables, r prtins f articles riginally published in Arterisclersis, Thrmbsis, and Vascular Bilgy can be btained via RightsLink, a service f the Cpyright Clearance Center, nt the Editrial Office. Once the nline versin f the published article fr which permissin is being requested is lcated, click Request Permissins in the middle clumn f the Web page under Services. Further infrmatin abut this prcess is available in the Permissins and Rights Questin and Answerdcument. Reprints: Infrmatin abut reprints can be fund nline at: Subscriptins: Infrmatin abut subscribing t Arterisclersis, Thrmbsis, and Vascular Bilgy is nline at: