Oxidized Low Density Lipoproteins Induce mrna Expression and Release of Endothelin

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1 1191 Oxidized Low Density Lipoproteins Indue mrna Expression and Release of Endothelin From Human and Porine Endothelium Chantal M. Boulanger, Felix C. Tanner, Marie-Lue Bea, Alfred W.A. Hahn, Annik Werner, and Thomas F. Lusher Downloaded from by on September 3, 218 Experiments were designed to examine the effet of oxidized low density lipoproteins (Ox-LDLs) on the expression and the release of endothelin from ultured endothelial ells and intat blood vessels. Ox-LDLs (3-3 jtg/ml), but not native low density lipoproteins (2,ug/ml), stimulated the expression of preproendothelin mrna in porine and human endothelial ells, leading to a time- and onentrationdependent release of the peptide into the ulture medium. The Ox-LDL-stimulated release of endothelin was mimiked by aetylated low density lipoprotein and abolished by downregulation of protein kinase C by phorbol ester. In the intat porine aorta, Ox-LDLs, but not native low density lipoproteins, also inreased the release of peptide in an endothelium- and onentration-dependent manner. The maximal effet was observed at a onentration of 1,g/ml. Inubation of the intat porine aorta with the savenger reeptor antagonist dextran sulfate dereased the formation of endothelin evoked by Ox-LDLs. The Ox-LDL-stimulated prodution of the peptide was further augmented in the presene of thrombin (4 units/ml) and was unaffeted by nitri oxide-generating ompound 3-morpholinosydnonimine (1` M). These results suggest that Ox-LDL may be an endogenous mediator of the augmented release of endothelin observed in hyperlipidemia and atheroslerosis. The inreased prodution of the peptide ould ontribute to vasospasti events and may promote vasular smooth musle proliferation and progression of atherosleroti vasular disease. (Cirulation Researh 1992;7: ) KEY WoRDs * thrombin * native low density lipoprotein * ultured endothelial ells * intat porine aorta * dextran sulfate * phorbol esters * aetylated low density lipoprotein * GMP T he endothelium partiipates in the regulation of vasular tone by releasing relaxing fators, suh as nitri oxide, or ontrating substanes, suh as the peptide endothelin.,2 Endothelial ells are primary targets for ardiovasular risk fators suh as low density lipoproteins (LDLs).3,4 Plasma LDLs an be oxidatively modified by the ells of the blood vessel wall,4-6 and oxidized LDLs (Ox-LDLs) have been found to aumulate in early plaques and atherosleroti vasular lesions, where they may profoundly affet the endothelial funtion.7-1 Atherosleroti arteries are more prone to vasospasti events than are normal blood vessels.11-'3 One feature of this disease is the impairment of the relaxations mediated by the endothelium This endothelial From the Department of Researh, Laboratories of Vasular Researh (C.M.B., F.C.T., M.-L.B., A.W., T.F.L.) and Hypertension (A.W.A.H.), and the Department of Mediine (T.F.L.), Divisions of Clinial Pharmaology and Cardiology, University Hospital, Basel, Switzerland. Supported by the Swiss National Researh Foundation (grant and SCORE grant ), the Swiss Cardiology Foundation, the Helmut Horten Foundation, the Stanley Thomas Johnson Foundation, the Rohe Researh Foundation, the Foundation for Nutrition Researh, and the Institute Lipha for Medial Researh. Address for orrespondene: Thomas F. Lusher, MD, Division of Clinial Pharmaology, University Hospital, CH-431 Basel, Switzerland. Reeived September 3, 1991; aepted February 1, dysfuntion16-2 appears to be partially due to the inhibition of the formation of nitri oxide by Ox-LDL, whih aumulates within human atherosleroti plaques.4-6 However, an enhaned prodution of the potent vasoonstritor peptide endothelin2 ould also partiipate, partiularly sine nitri oxide inhibits its release from intat blood vessels.21 Indeed, the irulating levels of endothelin are inreased in patients with hyperlipidemia or atheroslerosis.22'23 Hene, this study was designed to investigate the effet of Ox-LDL, as ompared with that of native LDL (Nat-LDL) on the expression and release of the peptide by both ultured endothelial ells and intat blood vessels. Materials and Methods Blood Vessels Aortas were obtained from farm pigs killed at the nearby slaughterhouse and plaed in modified Krebs- Ringer solution (ontrol solution) of the following omposition (mm): NaCl 118.3, KCI 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25., alium EDTA.26, and gluose 11.1 (ph 7.4). The blood vessels were leaned of onnetive tissue and opened longitudinally. Great are was taken to preserve the intimal surfae. In some experiments, the endothelial layer was rubbed. Cell Culture Porine aorti endothelial ells (PAECs) and human aorti endothelial ells (HAECs) were obtained as

2 Downloaded from by on September 3, Cirulation Researh Vol 7, No 6 June 1992 previously desribed.24 Aortas were plaed and washed several times in sterile, old Earle's balaned salt solution ontaining 3 units/ml peniillin and streptomyin. PAECs, obtained by mehanial sraping with a razor blade of the intimal layer, were ultured in RPMI 164 medium supplemented with 1% fetal alf serum, 2 mm glutamine, 2,ug/ml heparin, 1 units/ml peniillin, and 1 units/ml streptomyin. HAECs, obtained intraoperatively from the aorta of a 41-year-old male organ donor patient in the same manner as desribed for PAECs, were ultured in the same medium as PAECs and supplemented with 2% serum and 15 j,g/ml endothelial ell growth fator. Identifiation of HAECs was onfirmed by the presene of fator VIII-related antigen. PAECs and HAECs were used from passages 6 to 12 and from passages 11 to 15 respetively. Confluent ultures were rendered quiesent by inubation in endothelial ell growth fator-free and serum-free medium supplemented with.5% bovine serum albumin and were exposed either to vehiles, Ox-LDLs, Nat- LDLs, or aetylated LDLs (A-LDLs). Preliminary experiments have shown that Ox-LDLs up to 3 ug/ml did not modify the release of [3H]adenine from ultured PAECs.25 Indeed, the [3H]adenine ontent was 14 ±3% of the ontrol value in ells exposed for 48 hours to Ox-LDLs (3,ug/ml), demonstrating that Ox-LDLs did not exert any major ytotoxi effet on endothelial ells in the experimental onditions used. Preparation of LDLs LDLs were isolated from human plasma olleted in EDTA (1` M) and butylated hydroxytoluene (BHT, 1` M) using sequential ultraentrifugation with density adjustments by potassium bromide.26 The isolated LDLs were dialyzed against phosphate-buffered saline (PBS) in the presene of EDTA and BHT and then sterilized by filtration (filter pore size,.45,um; Gelman Sienes In., Ann Arbor, Mih.). Protein onentration was determined as desribed using bovine serum albumin as a standard.27 LDL samples were stored at 4 C in the dark and used within 2 weeks. LDLs prepared under these onditions are referred to as Nat-LDLs. Before oxidation, LDLs were dialyzed against PBS to remove EDTA and BHT. Then they were oxidized at a onentration of 2,ug/ml by exposure to 5,uM CuCl2 for 24 hours at 37C.28 The entire proedure was performed with sterile agents under sterile onditions to prevent any baterial ontamination. The extent of lipid peroxidation was estimated as for thiobarbituri aidreative substanes.28,29 Tetramethoxypropane was used as a standard, and results are expressed as nanomoles of malondialdehyde equivalents per milliliter of the diluted solution. The average degree of oxidation subsequently inreased from values below.2 to 4.3±.4 nmol malondialdehyde equivalents per 1,ug proteins for Nat-LDL and Ox-LDL, respetively (n=6 preparations). Northern Blot Analysis Quiesent HAECs and PAECs were exposed to sterile Ox-LDLs (1 gg/ml) or Nat-LDLs (2,ug/ml with 1-5 M BHT) in RPMI 164 with.2% bovine serum albumin for 1-1 hours. The medium was removed, and ell layers were washed twie with PBS before lysis in guanidinium- isothioyanate buffer, whih was added diretly onto the ulture dishes. Total RNA was olleted on CsC12 gradients as desribed before.3,31 For Northern analysis, 9,ug (HAECs) to 2 ug (PAECs) of total RNA was eletrophoresed through a 1.2% agarose gel ontaining 2.2 M formaldehyde at a onstant voltage of 5 V for 6-8 hours in MOPS buffer. The gel was vaublotted onto HYbond N membranes (Amersham Corp., Arlington Heights, Ill.) with 2x standard saline itrate as transfer buffer. Blotted RNA was fixed to the membranes by ultraviolet irradiation at 32 nm for 3 minutes. Blots were hybridized to a random-primed DNA probe speifi for endothelin (1.3 kb, gift of Dr. J. Powell, Hoffmann-La-Rohe, Basel, Switzerland)3'31'32 aording to the method of Churh and Gilbert,33 washed at high stringeny (.1x standard saline itrate/.1% sodium dodeyl sulfate at 65 C), and then exposed to Kodak X-Omat films at -7 C overnight using one intensifying sreen. To assess for variabilities in sample RNA, blots were rehybridized to a 1.5-kb DNA probe speifi for major histoompatibility omplex (MHC) lass I antigens (lone pmf 48)34 and exposed for 3-6 hours to X-Omat film. Densitometri analysis of hybridization signals was performed by sanning (525 nm) autoradiographs; the given arbitrary optial density (OD) units were obtained by normalization of signals with respet to the OD values obtained for the MHC internal ontrol (MHC OD value of unstimulated ells was arbitrarily taken as 1%). Measurement of Endothelin Confluent and quiesent endothelial ells were inubated in 1 ml RPMI 164 supplemented with.2% bovine serum albumin and exposed to Ox-LDLs, A- LDLs (Biomedial Tehnologies In., Stoughton, Mass.), or Nat-LDLs for up to 24 hours. The inubation medium was entrifuged at 25g to remove nonadherent ells and was used for the determination of endothelin by radioimmunoassay (Peninsula Laboratories, Merseyside, UK). The detetion limit of the assay was 1 pg, and ross-reativity of the antibody was 1% for endothelin-1 (porine, human), 7% for endothelin-2 and -3, and 35% for big endothelin (porine, human). Intat porine aortas with endothelium (1-m2 intimal surfae) were inubated at 37 C in 3 ml ontrol solution ontaining.1% bovine serum albumin and aerated with a mixture of 95% 2-5% CO2 as previously reported.21 Endothelin was determined by radioimmunoassay in the inubation medium as desribed above. Previous work has shown that the release of endothelin from intat porine aorta was endothelium dependent.2' Measurement of GMP Confluent and quiesent endothelial ells (35-mm Petri dishes, seond passage) were washed three times and inubated in ontrol solution ontaining indomethain (1` M) and isobutylmethylxanthine (1-4 M) for 45 minutes at 37 C as previously reported.35 Cells were then stimulated either with 3-morpholinosydnonimine (SIN-1, 1` M) or vehile. Preliminary experiments have shown that maximal effet of the ompound was observed after 2 minutes of stimulation, in agreement with Shini and Vanhoutte.36 Inubation medium was removed after 2 minutes of stimulation, and intraellular

3 Boulanger et al Ox-LDLs Indue Endothelin Expression and Release PA E C hours a +Ox-LDL StIRS * + Nat-LDL, 4 l! _a MHC time, hours HAEC hours a +Ox-LDL # Vl U e -C +Nat-LDL & -W * m I*' I MHC 2 3 time, hours Downloaded from by on September 3, 218 GMP was extrated with 5 gl old ethanol/in HCl (1%). Samples were lyophilized and resuspended in 25 gl sodium aetate buffer (.5 mm, ph 6.2), and GMP ontent was determined after aetylation by radioimmunoassay (Amersham-Rahn, Zuirih, Switzerland). Drugs All tissue ulture material and hemials were from GIBCO, Basel, Switzerland, and AMIMED, Basel, Switzerland, with the exeption of fetal alf serum (Boehringer, Mannheim, FRG). Lipopolysaharide (from Salmonella typhosa) and thrombin were from Sigma Chemial Co., St. Louis, Mo. SIN-1 was a kind gift from Dr. V. Balteaux, Hoehst Laboratory, Paris. Unless otherwise indiated, all other hemials were purhased from Fluka, Buhs, Switzerland; Sigma, and Bio-Rad, Glattbrugg, Switzerland. Statistial Analysis Results are expressed as mean±sem; in experiments involving ultured endothelial ells (yli nuleotide and endothelin measurements), data were obtained from at least three different dishes and were observed with two other ultures. In experiments perforrmed with intat blood vessels, n refers to the number of animals from whih the tissues were obtained. In experiments involving densitometri analysis of Northern blots, data are expressed as mean ± SEM of perent inrease in OD arbitrary value as ompared with unstimulated ells (n =3). Statistial omparisons were made using Student's paired t test or Sheffe's test for multiple omparison. Values of p<.5 were onsidered statistially signifiant FIGURE 1. Effet of oxidized low density lipoprotein (Ox-LDL) and native low density lipoprotein (Nat-LDL) on the expression ofpreproendothelin mrna in ultured porine aorti endothelial ells (PAECs, top panel) and human aorti endothelial ells (4AECs, bottom panel). Confluent and quiesent endothelial ells were exposed either to sterile Ox-LDLs (1 pglml) or sterile Nat-LDLs (2 gg/ml). Total RNA was extrated, and Northern blot analysis was performed. Blots were hybridized with a random-primed DNA probe speifi for endothelin (1.3 kb) first and then with a human l.5-kb DNA probe speifi for major histoompatibility omplex (MHC) lass I antigens to orret for possible variability in the amount of RNA fixed to the membrane. The left side of both panels represents a typial Northern blot hybridized with the endothelin and MHC probes. Densitometri analysis of Northern blot autoradiographs with normalization for ontrol MHC hybridization (as perent inrease in arbitrary optial density [ODJ units) is plotted against time on the right side of the figure (n =3). Ox-LDLs but not Nat-LDLs stimulate the expression of endothelin mrna in both PAECs and HAECs. Results Expression of Endothelin mrna Ox-LDLs (1 gg/ml) inreased endothelin transription both in PAECs (Figure 1, top panel) and in HAECs (Figure 1, bottom panel). In PAECs, the inrease in mrna expression ould already be observed after 2 hours and was maintained for the following 6 hours of stimulation (Figure 1). In IAECs, the effet of Ox-LDLs (1 gig/ml) was deteted after 3 hours of stimulation, with a maximum after 4 hours (Figure 1). In ontrast, Nat-LDLs (2 gg/ml) failed to stimulate preproendothelin mrna within a omparable time period, both in PAECs and HAECs (Figure 1). Release of Endothelin From Cultured Endothelial Cells Ox-LDLs stimulated the release of endothelin from PAECs as ompared with ontrol onditions (Figure 2A). This effet was onentration dependent in the range of 3-3,ug/ml; onentrations lower than 3 gg/ml (data not shown) did not augment the release of the peptide as ompared with that ourring in unstimulated ells. The effet of Ox-LDLs was also time dependent (-24 hours); after 24 hours, the prodution rate of the peptide was similar in stimulated and ontrol ells (ontrol ells, 98±4 pg/16 ells per hour; OxLDLs [3 gig/ml], 17±6 pg/16 ells per hour) (Figure 2A). Ox-LDLs also aused a 2.3-fold inrease of the release of endothelin from HAECs. Under the same experimental onditions, lipopolysaharide (1 ng/ml, 4 hours of inubation) did not augment the release of

4 1194 Cirulation Researh Vol 7, No 6 June 1992 A en C) 11 CD CD U) B 2 24 Tirne (hours) A "E u n=8._ X 4 - * B N E ), CL 2 - o - ontrol oxidized - LDL (,ug / ml) * Ipi-T * 2S T Downloaded from by on September 3, 218 ) (D -1._ CD._ T1 ontrol Ox-LDL A-LDL Nat-LDL 3,g ml 3 gg ml 2 gg ml FIGURE 2. Prodution of endothelin by ultured porine aorti endothelial ells. Panel A: Time ourse of the release over 24 hours of inubation with oxidized low density lipoproteins (Ox-LDLs, 3-3 pg/ml). Panel B: Bar graph showing effet of aetylated low density lipoprotein (A-LDL) and native low density lipoprotein (Nat-LDL) on theprodution of immunoreative (ir) endothelin after 4 hours of inubation. Ox-LDLs (1 tig/ml) did not have any signifiant effet. Endothelin released in the inubation medium was determined by radioimmunoassay. *Signifiant differene ompared with ontrol. the peptide from ultured PAECs (ontrol ells, ; treated ells, 1.48±.9 pg/16 ells; n=4). Release of Endothelin From Intat Blood Vessels In the intat porine aorta with endothelium, Ox- LDLs indued a onentration-dependent release of endothelin, with a maximal effet at 1,g/ml after 5 hours of inubation (:2.7-fold inrease, Figure 3A). No detetable amounts of the peptide were observed in the ontrol D.S. Ox-LDL Ox-LDL D.S FIGURE 3. Bar graphs showing stimulation by oxidized low density lipoprotein (Ox-LDL) of the release of immunoreative (ir) endothelin from intat porine aorta with endothelium. Panel A: Effet of inreasing onentrations of Ox- LDLs (1-3 g/ml). Panel B: Inhibition by dextran sulfate (D.S., 4 pg/ml; moleular weight, 5,) of the release of the peptide evoked by Ox-LDLs (1 pgiml). *Signifiant differene ompared with ontrol; #signifiant differene ompared with Ox-LDL effet. inubating medium of preparations without endothelium (n=4). Cellular Mehanism ofation Savenger reeptor. In ultured PAECs, the effet indued by Ox-LDLs was also mimiked by A-LDLs (3,ug/ml) but not by Nat-LDLs (2,ug/ml, in the presene of 1-5 M BHT) (Figure 2B). As in ultured endothelial ells, Nat-LDLs (2,g/ml, with 1-5 M BHT) did not inrease the release of endothelin from intat porine aorta (ontrol ells, 3±3 pg/m2; Nat- LDLs, 26±6 pg/m2; n=5). Inubation of intat porine aorta with the savenger reeptor antagonist dextran sulfate (4 ug/ml; moleular weight, 5,) prevented the release of endothelin indued by Ox-LDLs (1 1 g/ml) (Figure 3B). Dextran sulfate by itself did not have any signifiant effet on the spontaneous prodution of the peptide (Figure 3B). Protein kinase C. Chroni treatment of PAECs with phorbol 12-myristate 13-aetate (1 nm, 48 hours) and onsequent downregulation of protein kinase C mark-

5 Boulanger et al Ox-LDLs Indue Endothelin Expression and Release 1195 Downloaded from by on September 3, 218 A C4 E es CL.E f 1 B CD n=6 NE 6- -2'- *-+ ontrol thrombin ox-ldl thrombin TI Tr- I_'_ ox-ldl TF thrombin thrombin Ox-LDL Ox-LDL SIN - 1 SIN - 1 FIGURE 4. Bar graphs showing release of immunoreative (ir) endothelin from intat porine aorta. Panel A: Effet of thrombin (4 units/ml) and oxidized low density lipoproteins (Ox-LDLs, 1 pg/ml). Panel B: Differential effet of 3-morpholinosydnonimine (SIN-i, 1-5 M) on the prodution of endothelin evoked by thrombin (4 units/ml) and Ox-LDLs (1 pg/ml). *Signifiant differene ompared with ontrol; +, signifiant differene ompared with thrombin effet. edly redued the basal prodution of endothelin (from to ng/16 ells after 24 hours) and the (from ability of Ox-LDLs (1 gg/ml) to stimulate it 4.9±.7 to 1.±.1 ng/16 ells after 24 hours). Effet of nitri oxide. When tissues were exposed simultaneously to maximal onentrations of Ox-LDLs (1,g/ml) and thrombin (4 units/ml),34 their effets on the release of endothelin from intat porine aorta were additive (Figure 4A). As previously reported,37 the thrombin-indued release of the peptide was inhibited by SIN-1 (1` M), a nitri oxide-generating ompound (Figure 4B). In ontrast, the release of endothelin evoked by Ox-LDLs (1,g/ml) was not affeted by SIN-1 (1-5 M) (Figure 4B). Inubation of endothelial ells for 5 hours with Ox-LDLs (1 gg/ml) did not alter the 83-fold inrease of intraellular GMP evoked by SIN-1 (1-5 M) (ontrol ells, 1.23±.21 pg GMP/16 ells after 2 minutes; Ox-LDLs, 1.7±.18 pg GMP/16 ells after 2 minutes; n=3). As shown with SIN-1, 8-bromo-GMP (1`3 M) did not modify the release of endothelin evoked by Ox-LDLs (14+8 and 17±1 pg/m2 in the absene and presene of 8-bromo-GMP, respetively; n =6). Disussion LDLs are a well-known risk fator for atheroslero- SiS.3,4 Plasma LDLs may be oxidized in vivo by either endothelial ells, marophages, or vasular smooth musle ells and aumulate in human early plaques as well as fully developed atherosleroti lesions.4-6 Inreased irulating levels of endothelin have been observed in hyperlipidemia and in atheroslerosis.22'23 The present study demonstrates that Ox-LDLs, but not Nat-LDLs, stimulate the expression and the release of endothelin from ultured and native endothelial ells of intat blood vessels. Therefore, it provides evidene for Ox- LDL as a potential endogenous mediator of the inreased release of endothelin in hyperlipidemia and atherosleroti diseases. The stimulation of the expression of endothelin mrna and the subsequent release of the peptide were observed both in PAECs and HAECs, emphasizing the importane of these results in humans. The release of endothelin indued by Ox-LDL most likely involves ativation of the endothelial savenger reeptor Indeed, the prodution of the peptide indued by Ox-LDL ould be reprodued by A-LDL, another ligand of the savenger reeptor,38-41 whereas Nat-LDL, whih interats with its speifi reeptor,42 failed to stimulate the release of endothelin. The involvement of the savenger reeptor in the effet of Ox-LDL is further supported by the fat that the savenger reeptor antagonist dextran sulfate28 totally abolished the release of endothelin indued by Ox- LDL, whereas the substane by itself was without effet. Ativation of the endothelial savenger reeptor by Ox-LDL has also been involved in the inhibition of the release of endothelium-derived nitri oxide by Ox-LDL in intat porine oronary arteries.'7,18 The effets of Ox-LDL are unlikely to be influened by the presene of endotoxin ontaminating the preparations of Ox-LDLs, sine the lipoproteins were prepared under sterile onditions and beause a high onentration of lipopolysaharide did not stimulate the release of endothelin from ultured ells. Indeed, the stimulating effet of endotoxin on the release of endothelin from ultured endothelial ells requires the presene of serum.43 Both the basal prodution of endothelin and that stimulated by Ox-LDL must involve ativation of protein kinase C in endothelial ells, sine they were markedly redued by downregulation of the enzyme by phorbol ester. In this regard, Ox-LDL shares a similar pathway with other agonists releasing endothelin, suh as angiotensin II in ultured vasular smooth musle ells and thrombin in ultured endothelial ells.31"44 The prodution of endothelin is regulated both by ativating and inhibitory mehanisms. Indeed, previous work has shown that the thrombin-indued release of the peptide in the intat porine aorta is redued by the onomitant prodution of endothelium-derived nitri oxide34 as well as by exogenous nitri oxide-generating ompounds suh as SIN-1 or nitroglyerin.37,45 In ontrast to thrombin, however, the release of endothelin evoked by Ox-LDLs was insensitive to SIN-1. One possible explanation ould be that Ox-LDLs savenge and inativate free nitri oxide46 or that the modified lipoproteins interfere with the ativation of soluble guanylate ylase by nitri oxide, as it was observed with

6 1196 Cirulation Researh Vol 7, No 6 June 1992 Downloaded from by on September 3, 218 purified enzyme.47 These explanations are unlikely, however, sine the aumulation of GMP indued by SIN-1 by endothelial ells36 was not impaired in the presene of Ox-LDLs. Thus, in ontrast to the response to thrombin, the Ox-LDL-stimulated release of endothelin is not modulated by exogenously added nitri oxide. The absene of a GMP-dependent regulation of the endothelin release indued by Ox-LDLs annot be explained by a diret ytotoxi effet of the modified lipoproteins on endothelial ells under the experimental onditions used. Indeed, Ox-LDLs (up to 3,tg/ml) did not modify [3H]adenine release as ompared with ontrol ells.24 Moreover, the rate of endothelin release measured after 24 hours of stimulation with Ox-LDLs was not different from the basal rate, demonstrating that the endothelial ells kept their onstitutive release of the peptide even after 24 hours' exposure to Ox-LDLs. An inreased prodution of the peptide may have important vasular effets. Indeed, the peptide auses potent and protrated ontrations in most blood vessels.2'48,49 In addition, threshold and low onentrations of endothelin potentiate the ontration indued by norepinephrine or serotonin in human blood vessels.5 This may be of partiular importane at sites of platelet ativation with release of serotonin5 and thromboxane A Other substanes formed or released under these onditions, suh as thrombin and transforming growth fator-,l,53,54 would further augment the prodution of endothelin already inreased by Ox-LDLs. Indeed, the stimulatory effet of thrombin on vasular endothelin prodution (whih may be partiularly relevant in myoardial infartion55) was fully maintained even in the presene of Ox-LDLs. Hene, loally inreased levels of endothelin may upregulate the responsiveness of the blood vessel wall to vasoonstritor stimuli and may explain that vasospasti events preferentially our at the site of early atherosleroti lesions.11-13,56 Inhibition of the release of endotheliumderived relaxing fator by Ox-LDLs17 may also ontribute to vasospasti events, sine a loally dereased prodution of nitri oxide would further augment endothelin prodution34 as well as inrease the sensitivity of blood vessel to vasoonstritor stimuli. The Ox-LDL-stimulated release of endothelin may be of partiular relevane in aging, whih is aompanied by aumulation of oxidized proteins,57 inrease plasma levels of endothelin,58 and vasular hyperreativity.59 In addition to induing vasoonstrition, endothelin may failitate loal proliferation of the vasular smooth musle ells. The peptide evokes intraellular events known to be linked to proliferative responses suh as alkalinization and expression of -fos and -my protoonogenes in ultured vasular smooth musle and mesangial ells.6-62 Endothelin also stimulates the prodution of autorine growth fators by inreasing the expression of platelet-derived growth fator mrna transript in vasular smooth musle ells.31 Thus, the release of endothelin from the intat endothelium indued by Ox-LDLs trapped in the vessel wall may ontribute to the formation of advaned atherosleroti lesions ontaining proliferative smooth musle ells (i.e., the fibrous ape). An inreased release of endothelin indued by Ox-LDLs ould also explain why proliferation at sites in atherosleroti lesions where the endothelium remains intat.3 Referenes 1. Furhgott RF, Vanhoutte PM: Endothelium-derived relaxing and ontrating fators. Faseb J 1989;3: Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yasaki Y, Goto K, Masaki T: A novel potent vasoonstritor peptide produed by vasular endothelial ells. Nature 1988;333: Ross R: The pathogenesis of atheroslerosis: An update. N Engi J Med 1986;314: Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witzum JL: Modifiations of low density lipoprotein that inrease its atherogeniity. N Engl J Med 1989;32: Haberland ME, Fong D, Cheng L: Malondialdehyde-altered protein ours in atheroma of Watanabe heritable hyperlipidemi rabbits. Siene 1988;241: Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witzum JL, Steinberg DJ: Evidene for the presene of oxidatively modified low-density lipoproteins in atherosleroti lesions of rabbit and man. J Clin Invest 1989;84: Jaobs M, Plane F, Brukdorfer KR: Native and oxidized lowdensity lipoproteins have different inhibitory effets on endothelium-derived relaxing fator in the rabbit aorta. 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