Immune mechanisms have been suggested to play a key role in

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1 Antibodies to Oxidized LDL in Relation to Caotid Atheoscleosis, Cell Adhesion Molecules, and Phospholipase A 2 Johannes Hulthe, Olov Wiklund, Eva Hut-Camejo, Göan Bondjes Abstact The ole of the humoal immune esponse to oxidized low density lipopotein () in atheogenesis is unclea and available studies ae contadictoy. The aims of the pesent study wee (1) to compae antibody tites to modified LDL in a goup of patients with hypecholesteolemia (n 102) with those in matched contols (n 102), (2) to analyze whethe these tites wee elated to atheoscleosis development as measued by ultasound, and (3) to analyze whethe these tites wee elated to soluble cell adhesion molecules and secetoy type II phospholipase A 2 in plasma. The esults showed that male patients with hypecholesteolemia had lowe immunoglobulin G () tites compaed with those in healthy contols. In the contol goup, thee was an invese coelation between intima-media thickness of the caotid atey bulb and tites against and malondialdehyde-ldl ( 0.35, P 0.001; and 0.31, P 0.003, espectively). In the patient goup, howeve, only weak associations wee seen. tites wee positively associated with soluble intecellula adhesion molecule-1, soluble E-selectin, and secetoy type II phospholipase A 2. Taken togethe, the esults of this study suppot the concept that the humoal immune esponse against may be potective in ealy atheoscleosis. The patten, howeve, is complex, and the ole of the immune esponse may diffe in diffeent patient goups as well as at diffeent stages of the disease. (Ateioscle Thomb Vasc Biol. 2001;21: ) Key Wods: antibodies atheoscleosis intima-media thickness inflammation Downloaded fom by on Decembe 26, 2018 Immune mechanisms have been suggested to play a key ole in atheoscleosis development. 1 Seveal lines of evidence suppot the concept that oxidized (Ox)-LDL may be a key antigen in this pocess. T-cell clones esponsive to have been isolated fom human lesions. 2 Futhemoe, in seveal studies, antibodies against epitopes of have been found in both human and abbit plasma 3 and in atheoscleotic lesions. 4,5 A elationship between ciculating antibodies against and atheoscleotic disease has, howeve, not been unequivocally shown. Thee ae case-contol studies suggesting an elevated antibody tite against in patients with vaious manifestations of atheoscleotic disease. 6 8 High tites of antibodies have also been found to be independent pedictos of the pogession of caotid atheoscleosis. 9 In othe ecent studies, howeve, no such elationships have been found between atheoscleotic disease and antibody tites. 10,11 Soluble foms of cell adhesion molecules (vascula cell adhesion molecule-1 [VCAM-1], intecellula adhesion molecule-1 [ICAM-1], and E-selectin) can be detected in seum, and these molecules have also ecently been detected in seveal components of human atheoma 12 ; in addition, coss-sectional and pospective data suggest that soluble foms of these poteins ae elevated among patients with divese manifestations of atheoscleosis. 13,14 Anothe possible make fo atheoscleosis development is the seceted goup IIa phospholipase A 2 (snppla 2 ), which has been descibed in association with local and systemic inflammation. 15 snppla 2 has been shown to stimulate oxidation of LDL by lipoxygenase. 16 snppla 2 has been found in human atheoscleotic lesions 17 and has also ecently been found to be coelated with coonay atey disease and to pedict coonay events. 18 Development of the B-mode ultasound technique has made it possible to noninvasively study the atheoscleotic pocess. Intima-media thickness (IMT) of the caotid atey has been used as a noninvasive indicato fo the atheoscleotic pocess in the coonay ateies. 19 The aims of the pesent study wee (1) to compae antibody tites to modified LDL in a goup of patients with hypecholesteolemia (n 102) with those in matched contols (n 102); (2) to analyze whethe these tites wee elated to atheoscleosis development, as measued by ultasound; and (3) to analyze whethe these tites wee elated to othe inflammatoy makes of possible inteest in atheoscleosis development, eg, soluble cell adhesion molecules and snppla 2, in plasma. Methods Study Goups Study Goup With Pimay Hypecholesteolemia Subjects with pimay hypecholesteolemia (n 105) wee ecuited fom the sceening of subjects fom the geneal population pefomed at the Wallenbeg Laboatoy (n 50) and also fom patients Received Decembe 13, 1999; evision accepted Septembe 29, Fom the Wallenbeg Laboatoy fo Cadiovascula Reseach, Sahlgenska Univesity Hospital, Götebog Univesity, Gothenbug, Sweden. Coespondence to Johannes Hulthe, MD, PhD, Wallenbeg Laboatoy, Fack 16, Sahlgenska Univesity Hospital, S Gothenbug, Sweden. Johannes.Hulthe@wlab.wall.gu.se 2001 Ameican Heat Association, Inc. Ateioscle Thomb Vasc Biol. is available at 269

2 270 Ateioscle Thomb Vasc Biol. Febuay 2001 Downloaded fom by on Decembe 26, 2018 at the Lipid Clinic of the Sahlgenska Univesity Hospital (n 55). Inclusion citeia wee willingness to paticipate, 20 to 70 yeas of age, seum cholesteol 6.5 mmol/l, LDL cholesteol 5.0 mmol/l, and seum tiglyceides 4.5 mmol/l. Futhemoe, the patients had to fulfill at least 1 of the following ultasound citeia: (1) a maximum IMT in the common caotid atey 1.0 mm o (2) a measuable plaque in the caotid atey, abitaily defined as a 50% incease in IMT compaed with that of neighboing sites. Lipids and ultasound vaiables wee measued twice befoe inclusion, and subjects had to fulfill inclusion citeia at both occasions. Because of technical easons, 3 patients had no seum samples available; thus, the total numbe of patients included fo the pesent epot was 102. The study goup has peviously been well descibed. 20 Study Goup of Contol Subjects Fo each patient, 1 contol subject was ecuited fom the same sceening examination as mentioned above. Contol subjects had no symptoms of o teatment fo cadiovascula disease. All contol subjects had seum cholesteol levels 6.5 mmol/l at the initial sceening examination. Contol subjects wee matched fo sex, age, height, weight, body mass index (BMI), and smoking habits. 20 The potocol was appoved by the ethics committee of Götebog Univesity, and infomed consent was obtained fom all subjects. Anthopometic Data, Seum Lipids, Blood Pessue, IMT, and Plaque Occuence in the Patient and Contol Goups Patients and contols wee well matched accoding to age, body height, body weight, and BMI. As expected, patients had highe seum cholesteol, LDL cholesteol, and seum tiglyceide levels compaed with contols (Table 1). IMT in the caotid atey bulb was significantly thicke in male patients compaed with contols: mm (95% confidence inteval [CI], 1.13 to 1.31] and mm (95% CI, 0.93 to 1.12), espectively, as well as in female patients compaed with contols: mm (95% CI, 1.24 to 1.51) and mm (95% CI, 0.85 to 0.97), espectively. The fequencies of modeate to lage plaques in male patients and contols wee 44.2% and 15.4%, espectively, and in female patients and contols, 52% and 2%, espectively. 20 Antibody Tites Against Modified Lipopoteins Detemination of Antibody Tites Against Modified Lipopoteins Antibody tites wee detemined with a solid-phase ELISA, as ealie descibed. 21 Antibody tite was defined as absobance (patient seum postcoat)/(intenal antibody tite standad seum postcoat). Fo, the postcoated wells gave no absobance; theefoe, this coection was made only fo. Intenal Antibody Tite Standad Used On each plate, 2 diffeent intenal standad seum samples wee epeatedly tested. The absobances fo these 2 samples, named the intenal contol sample (ICS) and the intenal standad sample (ISS), wee used to calculate thei atio (ie, ICS/ISS), which was used as the intenal antibody tite, the vaiability has peviously been shown to be satisfactoy. 21 Standad deviations (SDs) fo the mean value of the atio ICS/ISS (ie, the intenal antibody tite standad used) fom all plates wee 0.06 and 0.05 fo tites against and malondialdehyde (MDA)-LDL, espectively, and 0.05 and 0.08 fo tites against and, espectively, when ealie pedefined citeia wee used. 21 Ultasonogaphy Examination was pefomed with an ultasound scanne (Acuson 128) with a 7-MHz linea tansduce apetue of 38 mm, as descibed ealie. 22,23 The ight caotid atey was scanned at the level of the bifucation, and images fo IMT measuements wee ecoded fom the fa wall of the common caotid atey and the caotid atey bulb. The images wee measued in an automated analyzing system. 24 As descibed befoe, a subjective semiquantitative scale was used to gade the size of plaques into gades 1, 2, and 3, whee gade 1 TABLE 1. Anthopometic Data, Seum Lipids, and Blood Pessue in Patients and Contols Contol Goup Patients Males/females, n/n 52/50 52/50 Age, y Male Female BMI, kg/m 2 Male Female Seum cholesteol, mmol/l Male Female Seum tiglyceides, mmol/l Male Female HDL cholesteol, mmol/l Male Female LDL cholesteol, mmol/l Male Female SBP, mm Hg Male Female DBP, mm Hg Male Female SBP indicates systolic blood pessue; DBP, diastolic blood pessue. Values shown ae mean SD. coesponds to 1 o moe small plaques (less than 10 mm 2 ) and gade 3 coesponds to lage plaques causing a hemodynamic change in blood flow. 23,24 Repoducibility studies of blinded eeading of plaque occuence in 53 male subjects showed that plaque size was assessed in the same way on both occasions in 95% of the cases. Quantification of Type II snppla 2 by ELISA Levels of snppla 2 in human sea wee measued by enzyme-linked captue antibody immunoassay. 25 The antibodies used wee a monoclonal antibody against human snppla 2 (1 g/ L) fom Cayman Chemicals and a polyclonal antibody ( faction) against human ecombinant snppla 2 that was poduced in ou laboatoy and demonstated no coss-eactivity with type V PLA 2 (Edwad A. Dennis, pesonal communication) o actin. 26 ELISA plates wee ead at 405 nm (Specta MAX Plus micoplate spectophotomete system, Molecula Devices). Puified human ecombinant snppla 26 2 was used to geneate a standad cuve (62.5 to 2000 pg/50 L pe well). The between-assay vaiation was detemined by compaing the same blood samples (n 15) on 3 diffeent days. The coefficient of vaiation was found to ange between 8.6% and 9.6%. Cell Adhesion Molecules Ciculating soluble VCAM-1, E-selectin, and ICAM-1 levels wee detemined by commecially available ELISA kits and standads (R&D Systems Euope Ltd). The between-assay vaiation was detemined by compaing the same blood samples (n 41) on 2 diffeent plates. The coefficient of vaiation was found to be 3.3%, 5.1%, and 4.8% fo VCAM-1, E-selectin, and ICAM-1 levels, espectively.

3 Hulthe et al Antibodies to and Caotid Atheoscleosis 271 TABLE 2. Antibody Tites Against and in Patients and Contols Antibody Contol Goup Patients Mean Diffeence 95% CI fo Diffeence P Value fo Diffeence Males/females, n/n 52/50 52/50 Male to Female to Male to Female to Male to Female to Male to Female to Values shown fo tites ae mean SD. Downloaded fom by on Decembe 26, 2018 Biochemical Analysis Blood samples fo seum cholesteol, seum tiglyceides, and lipopotein factions wee dawn afte a fasting peiod of 10 to 12 hous. Cholesteol and tiglyceide levels wee detemined by fully enzymatic techniques. 27,28 HDL was detemined afte pecipitation of apolipopotein (apo) B containing lipopoteins with MnCl 2 and dextan sulfate. LDL cholesteol was calculated as descibed by Fiedewald et al. 29 All lipid analyses wee pefomed at the Wallenbeg Laboatoy. Statistical Analysis All statistical analyses wee pefomed with SPSS 8.0 fo Windows. The Mann-Whitney test was used when compaing mean values of antibody tites in patients and contols. Futhemoe, a t-distibuted vaiable was used to calculate 95% CIs fo diffeences. Compaisons between goups fo anthopometic and ultasound data wee not fomally tested fo significance. Only 95% CIs fo mean values ae pesented because patients and contols had been selected and matched on the basis of anthopometic and ultasound vaiables. Mantel s test fo linea association was used to test the elationship between antibody tites and and plaque occuence and size in the caotid atey. Patial coelation (adjusted fo sex) coefficients wee used to investigate the elationships between antibody tites against Ox- LDL, IMT, cell adhesion molecules, and snppla 2 in the patient and contol goups. Pobability values 0.05 (2-sided) wee egaded as statistically significant. Results Antibody Tites Against Modified Lipopoteins in Patients With Hypecholesteolemia and Contols Male patients with hypecholesteolemia had significantly lowe tites against and compaed with contol subjects. No othe significant diffeences wee obseved (Table 2). When patients and contols wee analyzed togethe, women had significantly highe mean values fo tites against and compaed with men (P and 0.010, espectively). Patial Coelations Between Antibody Tites, Cell Adhesion Molecules, and snppla 2 in Patients With Hypecholesteolemia and Contols When adjusted fo sex, tites against both and in the patient goup wee significantly associated with ICAM, E-selectin, and snppla 2. In addition, tites against wee also associated with VCAM in the patient goup (Table 3). Othewise thee wee no significant elationships obseved in the patient and contol goups (Table 3). IMT in Relation to Antibody Tites in the Patient and Contol Goups When adjusted fo sex, no significant elationships wee obseved in the patient goup between IMT of the common caotid atey o the caotid atey bulb and antibodies (both and ) against and, espectively TABLE 3. Patial Coelation Coefficients, Adjusted fo Sex, Regading the Relations Between Antibody Tites Against, Cell Adhesion Molecules, and snppla 2 Patients (n 102) ICAM 0.30* 0.26* VCAM * E-selectin 0.27* PLA * 0.28* Contols (n 102) ICAM VCAM E-selectin PLA *P 0.01, P 0.05.

4 272 Ateioscle Thomb Vasc Biol. Febuay 2001 TABLE 4. Patial Coelation Coefficients, Adjusted fo Sex, Regading the Relations Between Antibody Tites Against and Intima-Media Thickness Downloaded fom by on Decembe 26, 2018 Patients (n 102) IMT CCA IMT bulb Contols (n 102) IMT CCA IMT bulb * 0.31* CCA indicates common caotid atey. *P (Table 4). Howeve, in the contol goup, common caotid atey IMT tended to be negatively associated with tites against ( 0.19, P 0.055). Futhemoe, IMT of the caotid atey bulb was significantly and negatively associated with tites against both and MDA- LDL ( 0.35, P 0.001; and 0.31, P 0.003, espectively). tites in the contol goup wee not significantly associated with IMT (Table 4). Plaque Occuence and Size in Relation to Antibody Tites Against Modified Lipopoteins in the Patient and Contol Goups Fo men in the patient goup, deceasing tites against wee significantly associated with plaque occuence and size in the caotid atey (P 0.041). Fo women in the patient goup, inceasing tites against wee significantly associated with plaque occuence and size in the caotid atey (P 0.045). Othewise no significant elationships wee found between antibody tites and plaque occuence and size in the patient goup (Table 5). In the contol goup, thee wee no significant elationships between tites and plaque occuence and size (Table 5). Howeve, deceasing tites against wee significantly associated with plaque occuence and size fo men in the contol goup (the Figue). Deceasing tites against wee also significantly associated with plaque occuence and size in both men and women in the contol goup (P and P 0.010, espectively; Table 5). Discussion The main finding in the pesent study was the association between subclinical atheoscleosis and low antibody tites Scatteplots showing the elationship between tites against and plaque occuence and size in male contol subjects (left panel) and female contol subjects (ight panel), espectively. against o. Male patients with hypecholesteolemia and signs of ealy atheoscleosis had lowe tites compaed with those in healthy contols. Futhemoe, in the contol goup, thee was a significant invese coelation between IMT of the caotid atey bulb and tites against and ( 0.35, P 0.001; and 0.31, P 0.003, espectively). Thee was also a highly significant invese coelation between plaque occuence and size and tites in the contol goup. In the patient goup, only weak associations wee seen. tites against both and wee significantly and positively associated with ICAM, E-selectin, and snppla 2 levels in the patient goup but not in the contol goup. The above-mentioned obsevations between subclinical atheoscleosis and antibody tites ae confusing but inteesting. So fa, the geneal hypothesis has been that high antibody tites to pedispose to atheoscleosis. 6 8 On the othe hand, thee ae ecent epots indicating that immunization of expeimental animals with, leading to damatically inceased levels, may inhibit the pogession of atheoscleosis In this context, the ole of humoal immune eactions in atheogenesis is not clea. 33 The potective ole of the humoal immune esponse is suppoted by a ecent study by Nicoletti et al. 34 In that, study a spleen-associated immune esponse could potect against atheoscleosis in apoe-deficient mice. B cells can poduce without T-cell help, and the high tite of to in the pesent study may eflect T-cell independent B-cell activation. In geneal, it may be hypothesized that the physiological ole of antibodies against and elated compounds is TABLE 5. P Values fo Tend Between Antibody Tites and Plaque Occuence and Size in the Caotid Atey Plaque occuence and size Male patients (negative) Female patients (positive) Male contols (negative) (negative) Female contols (negative)

5 Hulthe et al Antibodies to and Caotid Atheoscleosis 273 Downloaded fom by on Decembe 26, 2018 to tigge emoval of the compounds fom the ciculation and possibly also fom the ateial wall. Accodingly, Shoji et al 35 ecently showed that thee was a negative coelation between antibody tites to and plasma levels of. Also in clinical studies thee ae epots suppoting an association between low antibody tites and inceased atheoscleosis. We have epoted ealie on patients with familial hypecholesteolemia that those with a pevious myocadial infaction had lowe tites compaed not only with patients without a pevious myocadial infaction but also with contols. 21 A low antibody tite has also been epoted in subjects with bodeline hypetension. 36 Anothe explanation fo the invese elationship between antibody tites to and atheoscleosis could be the fomation of soluble antigen-antibody complexes. Lopes- Viella et al 37 ecently showed that thee was a significant negative coelation between fee antibodies and immune complexes in subjects with insulin-dependent diabetes mellitus. Hence, the possibility that immune complex fomation may affect antibody tites, as detemined in the pesent study, should be consideed. The above-mentioned explanations fo the invese elationship between atheoscleosis and antibody tites (eg, emoval of antigen fom the ciculation and complex fomation) do not explain why thee was a elationship between tites (but not tites) and atheoscleosis in the contol goup (but not in the patient goup). We find methodological inconsistencies less likely as an explanation fo the vaying esults, since we in ealie studies have shown that the measuement of subclinical atheoscleosis as well as the detemination of antibody tites have good epoducibility and specificity. 21,38 The discepancies may instead indicate diffeences in the chaacte of atheoscleotic disease between patients and contols. The patient goup was chaacteized by hypecholesteolemia and a high pevalence of sub-clinical atheoscleosis. On the othe hand the contol goup was nomocholesteolemic and had a low pevalence of sub-clinical atheoscleosis. In this context it may be suggested that the humoal immune esponse play a diffeent ole in diffeent stages of the development of atheoscleosis as well as in the pesence of vaious othe isk factos. In the above mentioned study by Shoji et al 35 the subjects wee all healthy. Also, the animals immunized with modified LDL wee fee fom significant atheoscleosis at the time of immunization. Futhemoe, Fukumoto et al 39 ecently showed a negative elationship between common caotid IMT and antibody tites to in a healthy population. The esults fom the pesent study ae in line with these latte findings. Because the composition of immune-competent cells in atheoscleotic plaques is quite diffeent between ealy and advanced lesions, 33 the biological ole of immune esponses against pobably diffes in vaious stages of atheogenesis. This might explain why we found an invese coelation in the contol goup but not in the patient goup. Anothe diffeence between the patient and contol goups that could have affected the esults is that some of the patients had been on lipid-loweing theapy. The dugs wee withdawn befoe inclusion to the study, but one may hypothesize that teatment could have long-tem effects on antibody tite fomation. Futhemoe, it is not known whethe the withdawal of lipid-loweing medication may lead to an acute incease in antigen (ie, LDL cholesteol) fomation, which in the pesent study might have caused shot-tem changes in antibody tites. In the contol goup, was associated with subclinical atheoscleosis in the caotid bulb only but not with IMT in the common caotid atey. Atheoscleotic changes ae fist seen in the caotid bulb, and subclinical atheoscleosis in this egion, as measued by ultasound, has also peviously been shown to be associated with coonay atheoscleosis, as measued by coonay angiogaphy. 40 It is theefoe not supisingly to find a elationship between atheoscleotic changes in the caotid bulb and antibody tites in this goup of healthy subjects. tites against and wee positively coelated with cell adhesion molecule levels and snppla 2 levels in patients but not in contols. Again, this could be due to diffeent esponses in diffeent stages of the atheoscleotic pocess. The ole of these inflammation makes and whethe they ae egulated by the immune esponse o nonspecific inflammatoy stimulation in atheoscleosis cannot be concluded fom the pesent study. Howeve, inflammation may tigge LDL oxidation. Futhemoe, the degadation of LDL by snppla 2 may make the lipopotein paticles moe sensitive to oxidation. These factos may theefoe paticipate in tiggeing the immune esponse against. Taken togethe, the findings of this study suppot the concept that the humoal immune esponse against may be potective in ealy atheoscleosis. The patten, howeve, is complex, and the ole of the immune esponse may be diffeent in diffeent patient goups as well as at diffeent stages of the disease. Acknowledgments This study was suppoted by gants fom the Swedish Heat-Lung Foundation, the Swedish Medical Reseach Council (pojects 10880, 4531), Doctos Educational Ageement gants fo clinical eseach, and Asta Hässle, Mölndal, Sweden. We thank laboatoy technologists Aia Lidell, Anna Födén, and Caoline Schmidt fo excellent eseach assistance. Refeences 1. Hansson GK. Immune esponses in atheoscleosis. In: Hansson GK, Libby P, eds. Immune Functions of the Vessel Wall. Amstedam, Nethelands: Hawood Academic Publishes; Stemme S, Fabe B, Holm J, Bondjes G, Witztum JL, Hansson GK. T lymphocytes fom human atheoscleotic plaques ecognize oxidized low density lipopotein. Poc Natl Acad Sci U S A. 1995;92: Palinski W, Rosenfeld ME, Ylä-Hettuala S, Gutne GC, Soche SS, Butle SW, Pathasaathy S, Caew TE, Steinbeg D, Witztum JL. Low density lipopotein undegoes oxidative modification in vivo. Poc Natl Acad Sci U S A. 1989;86: Ylä-Hettuala S, Palinski W, Butle SW, Picad S, Steinbeg D, Witztum JL. Rabbit and human atheoscleotic lesions contain that ecognizes epitopes of oxidized LDL. Ateioscle Thomb. 1994;14: Rosenfeld ME, Palinski W, Ylä-Hettuala S, Butle S, Witztum JL. Distibution of oxidation specific lipid potein adducts and apolipopotein B in atheoscleotic lesions of vaying seveity fom WHHL abbits. Ateioscleosis. 1990;10: Maggi E, Machesi E, Ravetta V, Matignoni A, Finadi G, Bellomo G. Pesence of autoantibodies against oxidatively modified low-density lipopotein in essential hypetension: a biochemical signatue of an enhanced in vivo low-density lipopotein oxidation. J Hypetens. 1995; 13: Begmak C, Wu R, de Faie U, Lefvet AK, Swedenbog J. Patients with ealy-onset peipheal vascula disease have inceased levels of autoan-

6 274 Ateioscle Thomb Vasc Biol. Febuay 2001 Downloaded fom by on Decembe 26, 2018 tibodies against oxidized LDL. Ateioscle Thomb Vasc Biol. 1995;15: Lehtimäki T, Lehtinen S, Solakivi T, Nikkilä M, Jaakkola O, Jokela H, Ylä-Hettuala S, Luoma JS, Koivula T, Nikkae T. Autoantibodies against oxidized low density lipopotein in patients with angiogaphically veified coonay atey disease. Ateioscle Thomb Vasc Biol. 1999; 19: Salonen JT, Ylä-Hettuala S, Yamamoto R, Butle S, Kopela H, Salonen R, Nyssönen K, Palinski W, Witztum JL. Autoantibodies against oxidized LDL and pogession of caotid atheoscleosis. Lancet. 1992;339: Uusitupa M, Niskanen L, Luoma J, Vilja P, Mecui M, Rauamaa R, Ylä-Hettuala S. Autoantibodies against oxidized LDL do not pedict atheoscleotic vascula disease in non-insulin-dependent diabetes mellitus. Ateioscle Thomb Vasc Biol. 1996;16: Van de Vije L, Steyge R, Poppel G, Boe J, Kuijssen D, Seidell J, Pincen H. Autoantibodies against in subjects with sevee and mino atheoscleosis and healthy population contols. Atheoscleosis. 1996;122: Poston RN, Haskad DO, Couche JR, Gall NP, Johnson-Tidey RR. Expession of intecellula adhesion molecule-1 in atheoscleotic plaques. Am J Pathol. 1992;140: Ridke PM, Hennekens CH, Roitman-Johnson B, Stampfe MJ, Allen J. Plasma concentations of soluble intecellula adhesion molecule-1 and isks of futue myocadial infaction in appaently healthy men. Lancet. 1998;351: Hwang SJ, Ballantyne CM, Shaett AR, Smith LC, Davis CE, Gotto AM, Boewinkle E. Ciculating adhesion molecules VCAM-1, ICAM-1 and E-selectin in caotid atheoscleosis and incident coonay heat disease cases. Ciculation. 1997;96: Fanson R, Dobov R, Weiss J, Elsbach P, Weglicki W. Isolation and chaacteization of a phospholipase A 2 fom an inflammatoy exudate. J Lipid Res. 1978;19: Mangin EL, Kugiyama K, Nguy JH, Dens SA, Heny PD. Effects of lysolipids and oxidatively modified low density lipopotein on endothelium-dependent elaxation of abbit aota. Cic Res. 1993;72: Hut-Camejo E, Andeson S, Standal R, Rosengen B, Satipy P, Stadbeh E, Johansen B. Localization of nonpanceatic secetoy phospholipase A 2 in nomal and atheoscleotic ateies. Ateioscle Thomb Vasc Biol. 1997;17: Kugiyama K, Ota Y, Takazoe K, Moiyama Y, Kawano H, Miyao Y, Sakamoto T, Soejima H, Ogawa H, Doi H, Suiyama S, Yasue H. Ciculating levels of secetoy type II phospholipase A 2 pedict coonay events in patients with coonay atey disease. Ciculation. 1999;100: Gobbee DE, Bots ML. Caotid atey intima-media thickness as an indicato of genealized atheoscleosis. J Inten Med. 1994;236: Hulthe J, Wiklund O, Bondjes G, Wikstand J. LDL paticle size in elation to intima-media thickness and plaque occuence in the caotid and femoal ateies in patients with hypecholesteolemia. J Inten Med. 2000;248: Hulthe J, Wikstand J, Lidell A, Wendelhag I, Hansson GK, Wiklund O. Antibody tites against oxidized LDL ae not elevated in patients with familial hypecholesteolemia. Ateioscle Thomb Vasc Biol. 1998;18: Wendelhag I, Gustavsson T, Suukula M, Beglund G, Wikstand J. Ultasound measuement of wall thickness in the caotid atey: fundamental pinciples and desciption of a computeized analysing system. Clin Physiol. 1991;11: Wendelhag I, Wiklund O, Wikstand J. Atheoscleotic changes in the femoal and caotid ateies in familial hypecholesteolemia: ultasonogaphic assessment of intima-media thickness and plaque occuence. Ateioscle Thomb. 1993;13: Wendelhag I, Liang Q, Gustavsson T, Wikstand J. A new automated computeized analyzing system simplifies eadings and educes the vaiability in ultasound measuement of intima-media thickness. Stoke. 1997;28: Peilot H, Rosengen B, Bondjes G, Hut-Camejo E. Intefeon-gamma induces secetoy goup IIA phospholipase A2 in human ateial smooth muscle cells: involvement of cell diffeentiation, STAT-3 activation, and modulation by othe cytokines. J Biol Chem. 2000;28: Satipy P, Johansen B, Camejo G, Rosengen G, Bondjes G, Hut- Camejo E. Binding of human phospholipase A 2 type II to poteoglycans: diffeential effect of glycosaminoglycans on enzyme activity. J Biol Chem. 1997;271: Klose S, Bone K. Enzymatische Bestimmung des Gesamtcholesteins mit dem Geine Selective Analyze (GSA-II). J Clin Chem Clin Biochem. 1978;15: Wahlefeld AW. Tiglyceides: detemination afte enzymatic hydolysis. In: Bemeye HU, ed. Methods of Enzymatic Analysis, 2nd English ed. New Yok, NY: Academic Pes Inc; 1974: Fiedewald WT, Levy RI, Fedickson DS. Estimation of the concentation of low density lipopotein in plasma, without use of the pepaative ultacentifuge. Clin Chem. 1972;18: Palinski W, Mille E, Witztum JL. Immunization of low density lipopotein (LDL) ecepto-deficient abbits with homologous malondialdehyde modified LDL educes atheogenesis. Poc Natl Acad Sci U S A. 1995;92: Ameli S, Hultgådh-Nilsson A, Regnstöm J, Calaa F, Yano J, Cecek B, Shah PK, Nilsson J. Effect of immunization with homologous LDL and oxidized LDL on ealy atheoscleosis in hypecholesteolemic abbits. Ateioscle Thomb Vasc Biol. 1996;16: Nilsson J, Calaa F, Regnstöm J, Hultgadh-Nilsson A, Ameli S, Cecek B, Shah PK. Immunization with homologous oxidized low density lipopotein educes neointimal fomation afte balloon injuy in hypecholesteolemic abbits. J Am Coll Cadiol. 1997;30: Hansson GK. Cell-mediated immunity in atheoscleosis. Cu Opin Lipidol. 1997;8: Nicoletti A, Caligui G, Hansson GK. Reduction of atheoscleosis by adoptive tansfe of potective immunity in apoe knockout mice. Eu Heat J. 1999;20(suppl):607. Abstact. 35. Shoji T, Nishizawa Y, Fukumoto M, Shimamua K, Kimua J, Kanda H, Emoto M, Kawagishi T, Moii H. Invese elationship between ciculating oxidized low density lipopotein (oxldl) and anti-oxldl antibody levels in healthy subjects. Atheoscleosis. 2000;148: Ruihua W, de Faie U, Lemne C, Witztum J, Fostegåd J. Autoantibodies to OxLDL ae deceased in individuals with bodeline hypetension. Hypetension. 1999;33: Lopes-Viella MF, Viella G, Ochad TJ, Koskinen S, Evans RW, Becke DJ, Foest K. Antibodies to oxidized LDL and LDL-containing immune complexes as isk factos fo coonay atey disease in diabetes mellitus. Clin Immunol. 1999;90: Schmidt C, Wendelhag I. How can the vaiability in ultasound measuement of intima-media thickness be educed? studies of inteobseve vaiability in caotid and femoal ateies. Clin Physiol. 1999;1: Fukumoto M, Shoji T, Emoto M, Kawagishi T, Okuno Y, Nishizawa Y. Antibodies against oxidized LDL and caotid atey intima-media thickness in a healthy population. Ateioscle Thomb Vasc Biol. 2000; 20: Hulthe J, Wikstand J, Emanuelsson H, Wiklund O, de Feyte PJ, Wendelhag I. Atheoscleotic changes in the caotid atey bulb as measued by B-mode ultasound ae associated with the extent of coonay atheoscleosis. Stoke. 1997;28: