In Vivo Catabolism of Biologically Modified LDL

Transcription

1 In Viv Catablism f Bilgically Mdified LDL J. Fred Nagelkerke, Luis Havekes, Victr W.M. van Hinsbergh, and The J.C. van Berkel Incubatin f human lw density llpprtein (LDL) at 37 C in the presence f human umbilical vein endthellal (EC) caused a time-dependent shift in the charge and density f LDL. The physical changes f the human LDL ccurred parallel with an increase In Its clearance frm the serum and uptake in the liver when injected int rats. The serum decay f the EC-mdified LDL (44 hurs incubatin) was 20 times faster than fr cntrl LDL. EC-mdified LDL, cleared frm the bld, was quantitatively recvered in the liver. Islatin f the different liver cell types (parenchymal, Kupffer, and endthellal ) after In viv injectin f 125 l-ec-mdified LDL shwed that apprximately 30 times mre radiactivity was assciated with the endthelial than with the parenchymal (per milligram f cell prtein). In vitr experiments indicated that EC-mdlfled-LDL was prcessed by the rat liver endthelial via a high affinity, saturable pathway related t the pathway by which these prcessed acetyl-ldl. We cncluded that, if EC-mdified LDL Is generated In viv, the liver, and In particular the endthelial cell, frms the majr prtectin system against the ccurrence f athergenic particles in the bld. (Arterisclersis 4: , May/June 1984) The mechanism by which lw density lipprtein (LDL) is cleared frm the bld circulatin has been a subject f intensive research fr the last decade. The best knwn mechanism is the classical LDL-receptr pathway, initially described by Gldstein and Brwn. 1 Hwever, in viv studies indicate that the LDL clearance frm the bld is nt quantitatively mediated by the classical receptr. 2 Therefre, ther mechanisms have been prpsed, generally called "nnreceptr" pathways. This name was based upn the bservatin that LDL, in which the recgnitin site fr the classical LDL receptr is blcked, is nevertheless cleared frm the circulatin, althugh at a lwer rate. 2 One such additinal clearance pathway, perating via a different receptr site, is the J.F. Nagelkerke and T.J.C. van Berkel are frm the Department f Bichemistry I, Medical Faculty, Erasmus University, Rtterdam; L. Havekes and V.W.M. van Hinsbergh are frm the Gaubius Institute TNO, Leyden, The Netherlands. J. F. Nagelkerke was the recipient f FUNGO Grant T.J.C. van Berkel is an Established Investigatr f the Dutch Heart Fundatin. Requests fr reprints: Dr. J.F. Nagelkerke, Department f Bichemistry I, Medical Faculty, Erasmus University, P.O. Bx 1738, 3000 DR Rtterdam, The Netherlands. Received September 7, 1983; revisin accepted January 25, scavenger pathway. Its existence was prpsed n the basis f the findings f Brwn et al. 3 that accumulatin f chlesterl esters in macrphages, bserved in patients with increased LDL levels, culd nly be prvked in vitr by incubatin f macrphages with acetylated lw density lipprtein (acetyl-ldl), while native LDL was ineffective. The scavenger pathway is predminantly present in cell types belnging t the mnnuclear phagcyte system. 4 Furthermre, the receptr site is present in the fam cell, 5 which is thught t be derived frm belnging t this system. Recently we demnstrated 6 that human acetyl- LDL is almst cmpletely cleared frm the circulatin by the liver within 3 minutes after injectin int rats. Mrever, it was demnstrated that the rat liver endthelial cell is by far the mst active liver cell type in the uptake f acetyl-ldl. In vitr studies indicated that a high affinity saturable receptr site fr acetyl- LDL is present n the liver endthelial cell at a relatively high cncentratin. In viv acetylatin f LDL is, hwever, dubtful and, therefre, the physilgical imprtance f the scavenger pathway was unclear. Recently a mre relevant bilgical mdificatin f LDL, leading tward in vitr recgnitin by the scavenger receptr n macrphages, was reprted by Henriksen et al. 78 The mdificatin was achieved by incubating the LDL with rabbit artic r human umbilical vein end-

2 BIOLOGICALLY MODIFIED LDL Nagelkerke et al. 257 thelial (EC-mdified LDL). The purpse f this study was t investigate the behavir f EC-mdified LDL in viv and t crrelate this with receptr studies in vitr. In additin, the in viv and in vitr uptake and degradatin rates f EC-mdified LDL were cmpared with thse f acetyl-ldl. Methds Islatin and Culture f Umbilical Vein Endthelial Cells Human umbilical crds were kept after delivery in ice-cld crd buffer (140 mm NaCI, 4 mm KCI, 11 mm D-glucse, 10 mm Hepes, ph 7.3, 100 lu/ml penicillin, 0.10 mg/ml streptmycin). They were cllected nce a day frm the hspital. Endthelial were islated frm veins as described by van Hinsbergh et al. 9 Briefly, the vein was rinsed and incubated fr 15 minutes at 37 C with 0.1% cllagenase in M-199 medium at 37 C. The cllected venus were centrifuged (5 minutes 200 g) and suspended in M-199 medium cntaining 20% human serum and 15 mm Hepes buffer. They were seeded rather densely (1-5 x 10 4 /cm 2 ) in six 2 cm 2 wells cated with a crude fibrnectin slutin. The cating was perfrmed at 37 C fr 30 minutes. The fibrnectin slutin was remved by aspiratin immediately befre the were seeded. The were cultured at 37 C in M-199 medium supplemented with 20% pled human serum (nt inactivated), 15 mm Hepes buffer, 100 lu/ml penicillin and 0.1 mg/ml streptmycin under 5% CO 2 in air. The medium (0.2 ml/cm 2 ) was refreshed every 2 r 3 days. At cnfluency, the were used, r released with trypsin/ EDTA and passaged with a 1:3 split rati t btain subcultures. Lw Density Lipprtelns LDL was islated frm freshly prepared human serum by density gradient ultracentrifugatin accrding t the methd f Redgrave et al. 10 fllwed by tube slicing. SDS-plyacrylamide gel electrphresis shwed nly the presence f ap B. The LDL was immediately used fr idinatin by the 125 l-idine mnchlride methd described by Bilheimer et al. 11 After idinatin, LDL was dialyzed against phsphate-buffered saline (PBS), withut EDTA, fr 4 hurs (4 x 500 vlumes). Thereafter, it was stabilized by the additin f 10 mg f bvine serum albumin (BSA) per ml and further dialyzed vernight against 500 vlumes f PBS at 4 C. The specific activity ranged frm 80 t 150 cpm/ng f LDL prtein. The acid-sluble (nnidine) fractin was less than 0.1 %. This 125 l-labelled LDL was used fr endthelial cell mdificatin within 2 days f strage at 4 C. Endthelial Cell-Mdified LDL EC-mdified LDL was prepared by incubating cnfluent endthelial cell cultures at 37 C with medium M-199 supplemented with 10 g/l BSA, instead f 20% human serum, and with 100 fig f LDL prtein per ml medium. After the indicated time intervals, the medium was aspirated and stred at 4 C. The mrphlgy f the was then examined by phase cntrast light micrscpy. At all time intervals, n mrphlgical changes culd be detected. Incubatins f LDL in the same medium at 37 C but withut were used as cntrls. EC-mdified LDL and cntrl LDL preparatins were, withut prir islatin frm the culture media, subjected t agarse electrphresis accrding t the methd f Demacker. 12 After electrphresis, the agarse plate was dried by a stream f ht air and subjected t autradigraphy. Simultaneusly, 100 ix\ f the culture media were subjected t density gradient ultracentrifugatin accrding t the methd f Redgrave et al. 10 After 14 hurs f ultracentrifugatin (4 C, mean RCF is 200,000 g) density fractins f 0.5 ml were cllected using the methd described by Grt et al. 13 The fractins were measured fr density using a DMA 602 M densitmeter and cunted fr radiactivity. The acid sluble (nnidine) fractins f the EC-mdified and cntrl LDL samples were measured as described by Bierman et al. 14 Acetylatin f LDL LDL was acetylated with acetic anhydride as described by Basu et al. 15 Rat Liver Cells Thrughut this study 3-mnth-ld male Wistar R1 rats (Centraal Prefdiezen Bedzyf, Rtterdam, The Netherlands) were used. Rat liver endthelial, Kupffer, and parenchymal were prepared as previusly nted. 6 After injectin f the indicated lipprtein preparatins, the were islated with either prnase r cllagenase by a lw temperature prcedure. Earlier studies 8 shwed that in viv endcytsed material was nt degraded during the applied islatin prcedures, which led t a reliable determinatin f the cntributin f each cell type t ttal liver uptake in viv. Fr in vitr studies were islated with cllagenase at 37 C as described earlier. 6 ' 16 We fund that this prcedure gives the best preservatin f lipprtein receptr activity. Freshly islated endthelial were incubated at 37 C in Hams F-10 medium cntaining 2% BSA and the indicated amunts f lipprteins. Incubatins were carried ut in silicnized Srvall tubes. At the indicated times, 1 ml samples were withdrawn, transferred t plastic Eppendrff tubes, and centrifuged fr 2 minutes at 600 g. The cell pellets were suspended in 1 ml f medium, cntaining 50 mm Tris-HCI (ph 7.4), 0.15 M NaCI and 2 mg BSA; incubated fr 5 minutes at 4 C; and centrifuged again. This washing prcedure was repeated twice. The last washing was perfrmed with similar medium withut BSA fr a reliable cell prtein determinatin.

3 es ARTERIOSCLEROSIS VOL Degradatin f the lipprteins was measured accrding t the methd f Bierman et al.14 T 0.5 ml f the first supernatant, 0.2 ml f 35% trichlracetic acid was added, fllwed by incubatin fr 15 minutes at 37 C; subsequently the mixture was centrifuged fr 2 minutes at 15,000 g. T 0.5 ml f the supernatants, 5 fi\ f 40% ptassium idide and 25 fi\ f 30% hydrgen perxide were added. After 5 minutes at rm temperature, 0.8 ml chlrfrm was added and the mixture was shaken fr anther 5 minutes. After centrifugatin fr 2 minutes at 15,000 g, 0.4 ml f the aqueus phase (cntaining idinated degradatin prducts) and 0.5 ml f the chlrfrm phase (cntaining free idine) were sampled. Radiactivity was cunted in a LKB-Wallace ultragamma cunter. The viability f the befre and after incubatin was mre than 95% as judged by the Trypan blue exclusin test. The purity f the rat liver endthelial cell preparatins was checked by a light micrscpe as described earlier6 and was always mre than 95% pure. Prtein Determinatin Prtein determinatin was accrding t the methd f Lwry et al.17 using BSA as a standard. Chemicals 4, N 3, MAY/JUNE 1984 Results Characterizatin f the LDL Preparatins Human LDL incubated with human umbilical vein endthelial fr increasing perids f time shwed an increasing electrphretic mbility n agarse gels (Figure 1). This effect was already bserved after 9 hurs f incubatin. The mbility increased further with prlnged incubatin times withut reaching a plateau during the time studied. Nte that LDL incubated fr 44 hurs at 37 C in the absence f als migrated slightly faster than LDL that was nt incubated. Density gradient ultracentrifugatin demnstrated that after 44 hurs f incubatin, the buyant density had shifted frm the nrmal LDL range tward a mean density f d = g/ml (Figure 2). The incubatin f LDL in the absence f did nt lead t a significant shift in buyant density. The acid sluble (nnidine) radiactivity in all lipprtein fractins was less than 1 % f the acid-precipitable radiactivity (Table 1). The mdificatin was a result f a direct interactin between LDL and the umbilical vein endthelial. LDL that was incubated fr 44 hurs in medium in which endthelial were previusly incubated fr 44 hurs was nt significantly altered (data nt shwn). In Viv Studies Type I cllagenase and BSA, Fractin V, were frm Sigma Cmpany, St. Luis, Missuri; B-grade prnase was btained frm Calbichem-Behring Crpratin, La Jlla, Califrnia; metrizamide was frm Nyegaard and Cmpany, A/S, Osl, Nrway; Ham's F-10 was frm Gibc-Eurpe, Hfddrp, The Netherlands; and 12SI (carrier-free) in NaOH was frm New England Nuclear, Dreieich, West Germany. frnt - Serum decay and liver uptake after intravenus injectin int rats were determined with the same lipprtein preparatins that are characterized in Figures 1 and 2 and Table 1. The LDL preparatins were injected int the recipient animals withut prir islatin frm the media. Figure 3 shws the serum decay f the 125l-labeled EC-mdified and cntrl lipprteins after injectin int rats. (Rutinely, a lipprtein preparatin cntaining /*g f apprtein, specific activity dpm/ng apprtein was injected.) There was a strng effect f prlnged incubatin f the LDL with the umbilical vein endthelial n the clearance frm serum. Als the LDL that was incubated fr 44 hurs at 37 C in the absence f was cleared slightly faster frm the circulatin than LDL that was nt incubated. This Table 1. Water-Sluble (Nnidine) Radiactivity In the Lipprtein Fractins start cntrl Incubatin time (hrs) Presence f umbilical vein endthelial Absence f umbilical vein endthelial hurs Figure 1. Agarse electrphresis f 125l-labeled ECmdified LDL and 125l-labeled cntrl LDL. After incubatin in the presence r absence f at 37 C fr the indicated time, 2 ix\ f medium was subjected t agarse electrphresis accrding t the methd f Demacker et al. 12 After 3 hurs f electrphresis, the agarse plate was dried by ht air and subjected t autradigraphy fr 18 hurs. Acid-precipitable and acid-sluble (nnidine) radiactivity was determined as in reference 14. Values are expressed as percentage f the acid-precipitable radiactivity.

4 BIOLOGICALLY MODIFIED LDL Nagelkerke et al , cntrl, 0 hr 100- cntrl, 23 hr 10, cntrl, 44 hr 50- E D. O ~ 10, ta, 9 hr (0 IT, 23 hr Density (g/ml) I- Figure 2. Density gradient ultracentrifugatin f labeled EC-mdified LDL and cntrl LDL. After incubatin in the presence r absence f at 37 C fr the indicated time, 100 ix\ f medium was mixed with 4 ml KBr salt slutin (density 1.21 g/ml) and subjected t density gradient ultracentrifugatin accrding t the methd f Redgrave et al. 10 After 14 hurs f ultracentrifugatin (RCF 200,000 g, 4 C) the density fractins (0.5 ml) were cllected using the apparatus described by Grt et al. 13 cnnected t a fractin cllectr. Each fractin was measured fr density and cunted fr radiactivity. In the figure nly the fractins with densities between 1.00 and 1.12 g/ml are shwn. clearance is, hwever, much slwer than that f LDL incubated fr 44 hurs in the presence f. T determine the kinetics f the liver assciatin in viv, the '^-labeled lipprteins (40-60 ^g apprtein) were injected int rats. At different time intervals after injectin, a liver lbule was tied ff and excised. After weighing the lbule and cunting its radiactivity, the ttal liver uptake was extraplated using the assumptin that 3.75% f the ttal bdy weight is cntributed by the liver. 18 Figure 4 shws that the uptake f the EC-mdified LDL by the liver increased with prlnged incubatin f the LDL with the endthelial. Als the peak values f the lipprtein cntent in the liver shifted t shrter time intervals after injectin time (min) Figure 3. Serum decay f 125 l-labeled lipprtems after injectin int rats. Bld samples were drawn as indicated. The samples were centrifuged fr 2 minutes at 20,000 g and radiactivity was cunted in the supernatants. The values are expressed as percentages f the injected dse. LDL was incubated with human umbilical vein endthelial fr 9 (), 23 ( ) r 44 ( ) hurs r in the absence f fr 0 (A) r 44 (A) hurs. Three different batches f LDL were mdified fr different time perids. The results shwn are frm ne batch. Experiments with the ther tw batches gave similar results. Determinatin f the cell types respnsible fr the liver uptake was perfrmed by islating the varius cell types 10 minutes after the in viv injectin f the 125 l-labelled lipprteins (40 t 60 fig apprtein) int rats (Figure 5). It appeared that the liver endthelial and parenchymal were mainly respnsible fr the increased liver assciatin f EC-mdified LDL. The relative cntributin f these cell types t the uptake f EC-mdified LDL appeared t be similar t that bserved earlier fr chemically mdified LDL (acetyl-ldl). 6 Apprximately 30 times mre ECmdified LDL became assciated with the endthelial than with the parenchymal (per mg cell prtein). Taking int accunt the cntributin f each cell type t the ttal liver prtein, 18 the ttal rat liver

5 260 ARTERIOSCLEROSIS VOL 4, N 3, MAY/JUNE 1984 endthelial cell ppulatin was the majr site fr the liver uptake f EC-mdified LDL, similar t acetyl- LDL (Table 2). Figure 5 als shws that LDL incubated 44 hurs in the absence f was taken up mre than the nnincubated LDL. Hwever, the presence f umbilical vein endthelial during incubatin resulted in an uptake by endthelial and parenchymal that was abut 10 times as high. T determine the cellular metablism f EC-mdified LDL assciated with the liver endthelial in viv, we warmed the, islated at 8 C, t 37 C. At different time intervals thereafter, we drew a sample and determined the cell-bund and excreted acidsluble (nnidine) degradatin prducts. Figure 6 shws that the radiactivity initially bund t the appeared as acid-sluble (nnidine) radiactivity in the medium, indicating the degradatin f the apprtein. T cmpare the intracellular prcessing rate f EC-mdified LDL with acetyl-ldl, we repeated the experiment. This time acetyl-ldl was injected. Onehalf f the acetyl-ldl, initially cell-assciated, was Whle liver rat X c 5 a. ~l Parenchymal time (min) 120 Figure 4. Liver uptake f 125 l-labeled lipprteins after injectin int rats. After injectin, lbules were tied ff and excised at the indicated times. Values are expressed as percentages f the injected dses. The lbules were nt perfused; liver values here include the amunt f lipprteins present in the entrapped bld (apprx. 9% f the serum value based upn 3 H-albumin measurements). LDL was incubated with human umbilical vein endthelial fr 9 (), 22 ( ) r 44 ( ) hurs r in the absence f fr 0 (A) r 44 (A) hurs. Table 2. Relative Cntributin f the Different Liver Cell Types t the Ttal Uptake f EC-Mdified LDL and Acetyl-LDL by Rat Liver Cell type Parenchymal (%) Endthelial (%) Kupffer (%) EC-mdified 1_DL incubatin time (hrs) Acetyl- LDL The percentage was calculated by multiplying the values frm Figure 4 with the amunt f prtein that each cell type cntributes t ttal liver prtein. The values are expressed as a percentage f the ttal injected dse O) V) V u 0) a> c ai L. a Endthelial 1 1 Kupffer cntrl u acetyl- LDL Figure 5. Cellular distributin f 125 l-labeled LDL in liver after intravenus injectin int rats. Ten minutes later perfusin was started by cannulatin f the vena prta. The perfusin and cell islatin was perfrmed at 8 C t prevent degradatin f endcytsed LDL. 6 Eight minutes after perfusin began a lbule was tied ff and excised; then different cell types were islated and separated. Values are expressed as percentages f the injected dse per mg liver r cell prtein. Frm each lipprtein fractin tested tw independent ttal liver uptake values were btained. The averages are shwn at the tp (left t right): 1.55 ± 0.15; 5.95 ± 1.15; 3.7 ± 0.1; ± 0.35; 22.3 ± 4.4; 39.3 ± 2.25.

6 BIOLOGICALLY MODIFIED LDL Nagelkerke et al. 261 degraded within 10 minutes, althugh it tk 30 t 40 minutes f incubatin at 37 C befre ne-half f the cell-assciated EC-mdified LDL was degraded. Nature f the Recgnitin Site The time curse f in vitr cell-assciatin f the EC-mdified LDL, (incubated fr 44 hurs) with islated liver endthelial, indicates a linear increase with time up t 2 hurs (Figure 7 A). In the insert the results f a similar experiment with acetyl- LDL as a substrate are shwn. Here cell assciatin was mre rapid and reached a plateau after 30 minutes f incubatin. The degradatin rate f EC-mdified LDL shwed a lag-phase f 30 minutes befre TCA-sluble (nnidine) degradatin prducts were detected (Figure 7 B). In the insert the degradatin rate f acetyl-ldl is shwn, with degradatin detectable between 10 and 30 minutes. Additin f the lyssmtrpic agent chlrquine (50 fim) led t a virtually cmplete blckade f the degradatin f ECmdified LDL, suggesting the invlvement f the lyssmes (Figure 7 B). The slwer interactin f EC-mdified LDL with the endthelial as cmpared with acetyl LDL at 10 /Ltg/ml culd be due t the lwered affinity f ECmdified LDL fr the receptr site, a lwered maximal cell binding, r t differences in intracellular prcessing. With increasing cncentratins f 125 I-ECmdified LDL, the saturatin f the cell-assciatin (Figure 8 A) and f the degradatin (Figure 8 B) indicates a high affinity assciatin that is half-maximal at /ig f EC-mdified LDL apprtein. T test whether the EC-mdified LDL binds t the same site as acetyl-ldl n the rat liver endthelial cell, we perfrmed a cmpetitin experiment. Ends -i i. a Q. O a re I 10 1 time i 30 (min) i 45 Figure 6. Time curse f intracellular prcessing f ECmdified LDL and acetyl-ldl by islated liver endthelial after in viv injectin f lipprteins. Rat liver endthelial were islated frm rats injected with /xg 125 l-labeled lipprteins 10 minutes befre starting liver perfusin. The islated pure endthelial were resuspended in buffer at 37 C. Samples were drawn at indicated times, the were centrifuged, and the amunt f cell-assciated and acid-sluble radiactivity in the supernatant was determined. Clsed symbls represent cellassciated radiactivity; pen symbls, acid-sluble (nnidine) radiactivity. Triangles indicate acetyl-ldl, circles indicate LDL cincubated with umbilical vein endthelial fr 44 hurs. Values are per mg/cell prtein. The 100% value represents the amunt f cell-bund radiactivity frm the freshly prepared. D) C at 01 0) T S 1000" a a r en c time (min) 120 Figure 7. In vitr time curse f cell assciatin (A) and degradatin (B) f EC-mdified LDL (44 hurs incubatin) by islated rat liver endthelial. Islated were incubated at 37 C with 20 ^g/ml EC-mdified LDL. Values are the ng applipprtein cell-assciated (A) r degraded (acid-sluble radiactivity) (B) per mg cell prtein. Cells were incubated in the absence (circles) r presence (squares) f 50 /xm chlrquine. Inserts shw the time curse f in vitr assciatin (A) and degradatin (B) f acetyl-ldl by rat liver endthelial in vitr. Data is the same as in the main figures.

7 262 ARTERIOSCLEROSIS VOL 4, N 3, MAY/JUNE 1984 T3 0) u G IOOO , thelial were incubated fr 2 hurs with l-ec-mdified LDL and increasing amunts f unlabeled acetyl-ldl. Figure 9 shws that the binding and degradatin f EC-mdified LDL is abut 80% inhibited at equimlar amunts f bth lipprteins. The insert shws a similar cmpetitin experiment between labeled and unlabeled acetyl-ldl. Cmparisn f the insert and the main figure shw that unlabeled acetyl-ldl cmpetes mre effectively with labeled EC-mdified LDL than with labeled acetyl-ldl. A cmpetitin experiment using labeled ECmdified LDL and native LDL shwed that a 30-fld excess f native LDL had n effect n either assciatin r degradatin f EC-mdified LDL by endthelial (data nt shwn). u-s _ B I-EC-mdified LDL (pg/ml) Figure 8. Cell assciatin (A) and degradatin (B) f 12^-mdified LDL (44 hurs incubatin) by rat liver endthelial as a functin f the aplipprtein cncentratin. Values are expressed as ng aplipprtein per mg cell prtein. Incubatin time was 2 hurs at 37 C unlabeled acetyl-ldl (yg/ml) Figure 9. The effect f increasing cncentratins f unlabeled acetyl-ldl n the cell assciatin ( ) and degradatin () f 125 l-labeled EC-mdified LDL by rat liver endthelial. The cncentratin f 125 l-ec-mdifled LDL was 10 jug/ml aplipprtein. Cells were incubated fr 2 hurs at 37 C. Values are given as percentages f the cntrl incubated in the absence f unlabeled acetyl-ldl. Insert shws a similar experiment fr 125 l-acetyl-ldl and unlabeled acetyl-ldl. Data is the same as in the main figure. A = assciatin, A = degradatin. Discussin This study was undertaken t determine the in viv behavir f bilgically mdified LDL. Recent studies by Henriksen et al. 78 indicated that incubatin f LDL with endthelial induces changes in structure that resulted in recgnitin by macrphages and subsequent degradatin at three t five times the rate f unmdified LDL. This bilgically mdified LDL may be pathphysilgically significant. Earlier studies with macrphages indicated that the uptake and degradatin f EC-mdified LDL is exerted by a pathway that is shared t sme extent by acetyl-ldl. The present study shws that in viv EC-mdified LDL was prcessed at similar cellular sites as acetyl- LDL. EC-mdified LDL was rapidly cleared frm the bld and recvered quantitatively in the liver (Figures 3 and 4) at 10 minutes after injectin f the lipprteins. At lnger time intervals after injectin, the uptake f the disappeared EC-mdified LDL was n lnger quantitative, prbably because idinelabeled degradatin prducts f EC-mdified LDL left the liver. This explanatin is supprted by the data n the in vitr degradatin f in viv recgnized EC-mdified LDL. Endthelial that were islated 10 minutes after injectin f EC-mdified LDL, actively degraded the cell-assciated radiactivity t acid-sluble prducts, which were subsequently released frm the. The cntributin f the different cell types t ttal liver uptake was similar t that bserved with acetyl- LDL. The rat liver endthelial cell was apprximately 30 times mre active per milligram f cell prtein than the parenchymal cell. Prlnged cincubatin f umbilical vein endthelial with LDL strengthened the acetyl-ldl character f the EC-mdified LDL, i.e., a faster serum decay and increased liver uptake (up t 20-fld), althugh the clearance and liver uptake rate f the chemically generated acetyl- LDL was never reached. In the case f acetyl-ldl 80% f the injected dse is present in the liver, after 10 minutes, indicating a high capacity f the liver acetyl-ldl receptr. Because similar cncentratins f EC-mdified LDL and acetyl-ldl were injected, the lwer uptake f EC-mdified LDL as cmpared

8 BIOLOGICALLY MODIFIED LDL Nagelkerke et al. 263 with acetyl-ldl culd nly be the result f a slwer assciatin rate. These findings can be explained by the in vitr cell-assciatin data. The dse respnse curves fr EC-mdified LDL and acetyl-ldl were similarly dependent n the substrate cncentratin; half-maximal cell-assciatin and degradatin ccurred at abut ^g apprtein/ml fr bth mdified frms f LDL. Hwever, the maximal amunt that became cell-assciated was clearly different: 20,000 ng/mg cell prtein fr acetyl-ldl 6 and 7500 ng/mg cell prtein fr EC-mdified LDL at 2 hurs after incubatin. This results in a higher assciatin rate fr acetyl-ldl than fr EC-mdified LDL at cmparable cncentratins f EC-mdified LDL and acetyl-ldl. This difference in maximal cell assciatin rate is reflected in the lnger time needed fr EC-mdified LDL t reach a steady-state level (determined by the cell-assciatin rate versus the degradatin rate). The cmpetitin experiments indicated that acetyl- LDL was a mre efficient cmpetitr fr 125 I-ECmdified LDL than fr 125 l-acetyl-ldl. With this mdel, ne shuld expect that cncentratins f ECmdified LDL higher than acetyl-ldl shuld be necessary t cmpete fr 125 l-acetyl-ldl cell-assciatin, a finding presented earlier by Henriksen et al. 7 In cnclusin, it is evident that EC-mdified LDL binds t the receptr n endthelial which als bind acetyl-ldl. Als the in viv assciatin f ECmdified LDL with parenchymal can be explained by the presence f an acetyl-ldl binding site n these as demnstrated earlier. 19 It shuld be nted that we have injected human lipprteins int rats, which culd be a ptential prblem due t the species difference. Hwever, it was reprted 20 that upn acetylatin, bth rat and human LDL were prcessed in a similar fashin after injectin int rats. During incubatin f LDL with umbilical vein endthelial, tw physical characteristics (charge and density) f the LDL particle change. Which, if either, f these changes induces the enhanced serum decay and liver uptake is nt clear at the mment. Mdificatin f the LDL by incubatin with umbilical vein endthelial is a cntinuus prcess. 8 The electrphretic mbility and buyant density gradually increases and there is a gradual increase in serum decay and liver uptake. Besides the changes induced by incubatin f LDL with umbilical vein endthelial, we als fund that incubatin f LDL fr 44 hurs at 37 C in the absence f slightly altered its behavir. The electrphretic mbility and serum decay, as cmpared with native LDL were increased. The in viv assciatin was enhanced, but in cntrast with ECmdified LDL, the live Kupffer were mstly respnsible fr the increased liver assciatin f the 37 C incubated LDL. Mdified lipprteins are cnsidered ptentially athergenic because uptake f these particles can lead t accumulatin f chlesterl esters in f the mnnuclear phagcyte system. 4 The uptake f these abnrmal lipprteins by macrphages in viv might explain the frmatin f fam in the arterial wall. 5 If EC-mdified LDL is generated in viv and enters the general circulatin, it wuld very rapidly be remved by the liver. Therefre the liver, and in particular the liver endthelial cell, frms an imprtant prtectin system against these pathphysilgical lipprteins. It is pssible that a delicate balance between frmatin f these particles and the capacity f the liver t entrap them determines their pathlgical actin. In this view, a small disturbance f the balance culd give rise t prlnged circulatin f these particles, thereby allwing the slw frmatin f athersclertic lesins. Acknwledgments We thank Ken Bart fr technical assistance, Cecile Hansn fr typing, and Willem C. Hulsmann fr critical reading f the manuscript. References 1. Gldstein JL, Brwn MS. Lw density lipprtein pathway and its relatin t athersclersis. Annu Rev Bichem 1977; 46: Mahley RW, Welsgraber KH, Melchlr QW, Innerarrty TL, Hlcmbe KS. Inhibitin f receptr-mediated clearance f lyslne and arginine-mdified lipprteins frm the plasma f rats and mnkeys. Prc Natl Acad Sd USA 1980;77: Brwn MS, Gldstein JL, Krieger M, H YK, Andersn RGW. Reversible accumulatin f chlesteryl esters in macrphages incubated with acetylated lipprteins. J Cell Bil 1979:82: Brwn MS, Basu SK, Falck JR, H YK, Gldstein JL. The scavenger cell pathway fr lipprtein degradatin. Specificity f the binding site that mediates the uptake f negativelycharged LDL by macrphages. J Supraml Struct 1980; 13: Pitas RE, Innerarrty TL, Mahley RW. Fam in explants f athersclertic rabbit artas have receptrs fr p-very lw density lipprteins and mdified lw density lipprteins. Arterisclersis 1983;3: Nagelkerke JF, Bart KP, van Berkel TJC. In viv and in vitr uptake and degradatin f acetylated lw density lipprtein by rat liver endthelial, Kupffer and parenchymal. J Bil Chem 1983;258: Henriksen T, Mahney EM, Steinberg D. Enhanced macrphage degradatin f lw density lipprtein previusly incubated with cultured endthelial : recgnitin by receptrs fr acetylated lw density lipprtein. Prc Natl Acad Sd USA 1981;78: Henriksen T, Mahney EM, Steinberg D. Enhanced macrphage degradatin f bilgically mdified lw density lipprtein. Arterisclersis 1983;3: van Hlnsbergh VWM, Havkes L, Emels JJ, van Crven E, Scheffer M. Metablism f lw density lipprtein (LDL) and acetylated LDL by subcnfluent and cnfluent endthelial frm human umbilical crd arteries and veins. Arterisclersis 1983:3: Redgrave TG, Rberts DCK, West CE. Separatin f plasma lipprteins by density gradient ultracentrifugatin. Anal Bichem 1975;65:42^9 11. Bllhelmer DW, Elsenberg S, Levy Rl. The metablism f very lw density lipprteins. I: Preliminary in vitr and in viv bservatins. 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9 264 ARTERIOSCLEROSIS VOL 4, N 3, MAY/JUNE Demacker PNM. Hyperlipprteinemia a study n diagnstic methds. [Dissertatin]. Nijmegen, The Netherlands: University f Nijmegen, Grt PHE, Scheek LM, Havekes L, van Nrt WL, van't Hft FM. A ne-step separatin f human serum high density lipprteins 2 and 3 by rate-znal density gradient ultracentrifugatin In a swinging bucket rtr. J Upid Res 1982; 23: Blerman EL, Stein O, Stein Y. Lipprtein uptake and metablism by rat artic smth muscle in tissue-culture. Circ Res 1974;35: Basu SK, Gldstein JL, Andersn RGW, Brwn MS. Degradatin f catinized lw density lipprtein and regulatin f chlesterl metablism in hmzygus familial hyperchlesterlemia fibrblasts. Prc Natl Acad Sci USA 1976; 73: van Berkel TJC, Nagelkerke JF, Kruljt JK. The effect f Ca 2+ tnfluperazine n the prcessing f human acetylated lw density lipprtein by nn-parenchymal liver. FEBS Lett 1981;132: Lwry OH, Rsebrugh NJ, Farr AL, Randall RJ. Prtein measurement with the Flin phenl reagent. J Bil Chem 1951;93: Bluln A, Blender RP, Welbel ER. Distributin f rganelles and membranes between hepatcytes and nn-hepatcytes in the rat liver parenchyma. J Cell Bil 1977;72: Van Berkel TJC, Nagelkerke JF, Harkes L, Kruljt JK. Prcessing f acetylated human lw-density lipprtein by parenchymal and nn-parenchymal liver. Bichem J 1982;2O8: Mahley RW, Welsgraber KH, Innerarlty TL, Wlndmueller HG. Accelerated clearance f lw-density and high-density lipprteins and retarded clearance f E apprtein-cntaining lipprteins frm the plasma f rats after mdificatin f lysine residues. Prc Natl Acad Sci USA 1979;76: Index Terms: rat liver endthelial cell mdified LDL umbilical vein endthelial cell