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257 inhibition of atherosclerosis in mice

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257 inhibition of atherosclerosis in mice

  1. 1. Inhibition of Atherosclerosis in ApoE-Inhibition of Atherosclerosis in ApoE- Null Mice by Immunization withNull Mice by Immunization with ApoB-100 Peptide SequencesApoB-100 Peptide Sequences Gunilla Nordin FredriksonGunilla Nordin Fredrikson andand Jan NilssonJan Nilsson
  2. 2. The early atherosclerotic plaque - theThe early atherosclerotic plaque - the inflammatory responseinflammatory response LDL oxidation and aggregation LDL VCAM-1
  3. 3. The atherosclerotic plaque – immuneThe atherosclerotic plaque – immune responses against oxidized LDLresponses against oxidized LDL LDL Oxidized LDL T cell Macrophage MHC II T cell receptor Oxidized LDL peptide
  4. 4. Evidence for immune response againstEvidence for immune response against oxidized LDLoxidized LDL Antibodies against oxidized LDL in humanAntibodies against oxidized LDL in human serum (Palinski et al PNAS 1989)serum (Palinski et al PNAS 1989) Activation of peripheral T cells by oxidizedActivation of peripheral T cells by oxidized LDL (Frostegård et al ATVB 1992)LDL (Frostegård et al ATVB 1992) Activation of T cells from humanActivation of T cells from human atherosclerotic plaques by oxidized LDLatherosclerotic plaques by oxidized LDL (Stemme et al PNAS 1995)(Stemme et al PNAS 1995)
  5. 5. Why does oxidized LDL becomeWhy does oxidized LDL become immunogenic?immunogenic? Fragmentation of apo B-100Fragmentation of apo B-100 Oxidation of phospholipidsOxidation of phospholipids Formation of covalent aldehyde (MDA and 4-Formation of covalent aldehyde (MDA and 4- HNE) and apo B-100 amino acid (lysine andHNE) and apo B-100 amino acid (lysine and histidine) adductshistidine) adducts
  6. 6. How does immunization with oxidizedHow does immunization with oxidized LDL affect atherosclerosis?LDL affect atherosclerosis? Reduced development of atherosclerosis inReduced development of atherosclerosis in rabbits (Palinski et al 1995, Ameli et al 1996)rabbits (Palinski et al 1995, Ameli et al 1996) Reduced development of atherosclerosis in apoReduced development of atherosclerosis in apo E knockout mice (George et al 1998, FreigangE knockout mice (George et al 1998, Freigang et al 1998, Zhou et al 2001)et al 1998, Zhou et al 2001) Reduced intimal plaque development afterReduced intimal plaque development after vascular injury in rabbits (Nilsson et al 1997)vascular injury in rabbits (Nilsson et al 1997)
  7. 7. Evidence for antibody /B cell –Evidence for antibody /B cell – mediated athero-protective immunitymediated athero-protective immunity IgG treatment inhibits atherosclerosis in apo EIgG treatment inhibits atherosclerosis in apo E null mice (Nicoletti et al JCI, 1998)null mice (Nicoletti et al JCI, 1998) B cell substitution inhibits atherosclerosis inB cell substitution inhibits atherosclerosis in spleen-ectomized apo E null mice (Caliguri etspleen-ectomized apo E null mice (Caliguri et al, JCI 2002)al, JCI 2002) B cell substitution inhibits injury-inducedB cell substitution inhibits injury-induced plaque formation in lymphocyte deficient (Rag-plaque formation in lymphocyte deficient (Rag- 1) mice (Dimayuga et al, ATVB 2002)1) mice (Dimayuga et al, ATVB 2002)
  8. 8. Aims of the projectAims of the project Identify the structures in oxidized LDLIdentify the structures in oxidized LDL recognized immune responses in manrecognized immune responses in man Develop ELISAs for analyses of antibodiesDevelop ELISAs for analyses of antibodies against these structuresagainst these structures Determine the relation between these immuneDetermine the relation between these immune responses and cardiovascular diseaseresponses and cardiovascular disease Develop an immunization therapyDevelop an immunization therapy
  9. 9. Development of ELISAs for analyses ofDevelopment of ELISAs for analyses of antibodies against native and MDA-antibodies against native and MDA- modified apo B-100 peptide sequencesmodified apo B-100 peptide sequences 302 peptides, corresponding to the complete302 peptides, corresponding to the complete amino acid sequence of Apo B-100amino acid sequence of Apo B-100 Each peptide consists of 20 amino acids with 5Each peptide consists of 20 amino acids with 5 amino acids overlapamino acids overlap Native or MDA-modified peptides used forNative or MDA-modified peptides used for coatingcoating Screening of the ELISA library with pooledScreening of the ELISA library with pooled healthy control plasmahealthy control plasma
  10. 10. Antibody binding to native and MDA-Antibody binding to native and MDA- modified apo B-100 peptide sequencesmodified apo B-100 peptide sequences Antibody binding to over 100 different sites inAntibody binding to over 100 different sites in apo B-100 identifiedapo B-100 identified IgM binds to more sites than IgGIgM binds to more sites than IgG No difference in binding to hydrophobic andNo difference in binding to hydrophobic and hydrophilic sequenceshydrophilic sequences High covariation in binding to native and MDAHigh covariation in binding to native and MDA sequencessequences
  11. 11. Antibodies against apoB-100 peptides andAntibodies against apoB-100 peptides and CHD –clinical charcteristics of the studyCHD –clinical charcteristics of the study groupgroup Subjects Cases Controls Number 78 149 Age (years) 61 (49 –67) 61 (49 –67) Male sex (%) 68 69 Current smokers (%) 27 27 Diabetes mellitus (%) 19 11 Hypertension(%) 64 64 Total cholesterol (mmol/L) 6.25 (3.47 – 8.24) 6.00 (4.08 – 9.90) LDL-cholesterol (mmol/L) 4.4 (1.7 – 6.2) 4.0 (1.6 – 7.6) HDL-cholesterol (mmol/L) 1.1 (0.6 – 2.5) 1.2 (0.6 –2.9) Triglycerides (mmol/L) 1.5 (0.5 – 10.0) 1.2 (0.4 – 7.3) Carotid ultrasonography Common carotid intima-media thickness (mm) 0.81 (0.36 – 1.67) 0.82 (0.47 – 1.58) Carotid plaques, (%) 64 40
  12. 12. IgM antibodies against MDA-modifiedIgM antibodies against MDA-modified apoB-100 peptides decrease more rapidlyapoB-100 peptides decrease more rapidly with age in CHD caseswith age in CHD cases CHD casesCHD cases ControlsControls Age 70605040IgMagainstMDApeptide32(abs405nm) 2,0 1,5 1,0 ,5 0,0 Rsq= 0,0261 Age 70605040 IgMagainstMDApeptide32(abs405nm) 2,0 1,5 1,0 ,5 0,0 Rsq = 0,3074
  13. 13. Inverse correlation between IgM antibodiesInverse correlation between IgM antibodies against MDA-modified apoB-100 peptidesagainst MDA-modified apoB-100 peptides and oxidized LDL levels in CHD casesand oxidized LDL levels in CHD cases Age 70605040 IgMagainstMDApeptide32(abs405nm) 2,0 1,5 1,0 ,5 0,0 Rsq = 0,3074 Age (years) 70605040 OxidizedLDL-cholesterol(U/L) 200 180 160 140 120 100 80 60 40 20 0 Rsq = 0,1984 IgM abIgM ab oxLDLoxLDL
  14. 14. Immunization of apo E null mice withImmunization of apo E null mice with apo B-100 peptide sequencesapo B-100 peptide sequences Immunization with 100 µg peptides at 6Immunization with 100 µg peptides at 6 and 9 weeks of ageand 9 weeks of age High cholesterol diet at 10 weeks of ageHigh cholesterol diet at 10 weeks of age Atherosclerosis assessed by Oil Red OAtherosclerosis assessed by Oil Red O staining of the descending aorta at 25staining of the descending aorta at 25 weeks of ageweeks of age
  15. 15. Lipids, body weight and serum amyloid A inLipids, body weight and serum amyloid A in immunized apo E null miceimmunized apo E null mice at the age of 25 weeks.at the age of 25 weeks. PeptidesPeptides ControlsControls ((nn = 10)= 10) ((nn = 10)= 10) Plasma-Cholesterol,Plasma-Cholesterol, (mg/mL)(mg/mL) 4.38±1.584.38±1.58 4.57±1.334.57±1.33 HDL-Cholesterol,HDL-Cholesterol, (mg/mL)(mg/mL) 0.23±0.040.23±0.04 0.18±0.120.18±0.12 Triglycerides,Triglycerides, (mg/mL)(mg/mL) 0.76±0.130.76±0.13 0.80±0.120.80±0.12 Body weight,Body weight, (g)(g) 36.2±4.236.2±4.2 38.4±3.038.4±3.0 SAA,SAA, (µg/mL)(µg/mL) 227±67227±67 252±94252±94
  16. 16. Reduced plaque area in descending aorta of apoEReduced plaque area in descending aorta of apoE null mice immunzed with Apo B-100 peptidenull mice immunzed with Apo B-100 peptide sequencessequences 0.0 0.1 0.2 0.3 0.4 0.5 Controls Peptides ***
  17. 17. Increased collagen content in subvalvular plaquesIncreased collagen content in subvalvular plaques of apoE null mice immunized with Apo B-100of apoE null mice immunized with Apo B-100 peptide sequencespeptide sequences 0 10 20 30 40 50 *** Controls Peptides Trichromestainedareain subvalvularplaques(%)
  18. 18. Activation of athero-protective immuneActivation of athero-protective immune responses against MDA-apo B-100responses against MDA-apo B-100 fragmentsfragments Immunization with oxidized LDL has previouslyImmunization with oxidized LDL has previously been found to reduce atherosclerosis in animalsbeen found to reduce atherosclerosis in animals Human oxidized LDL antibodies were found toHuman oxidized LDL antibodies were found to bind to specific MDA-modified peptidebind to specific MDA-modified peptide sequences in apo B-100sequences in apo B-100 Antibodies to these sequences correlated to ageAntibodies to these sequences correlated to age and oxidized LDL in plasmaand oxidized LDL in plasma Immunization with these MDA apo B-100Immunization with these MDA apo B-100 peptide sequences inhibited atherosclerosis inpeptide sequences inhibited atherosclerosis in apo E null miceapo E null mice The findings suggest the possibility to develop aThe findings suggest the possibility to develop a vaccine-based therapy for atherosclerosisvaccine-based therapy for atherosclerosis
  19. 19. Department of Medicine, UMAS, Lund University Gunilla Nordin Fredrikson Jan Nilsson Ragnar Alm Mikko Ares Göran Berglund Paul Dimayuga Bo Hedblad Isabel Goncalves Marie Lindholm Alexandru Schiopu Ingrid Söderberg Division of Cardiology, Cedars-Sinai Medical Center, UCLA Bojan Cercek Prediman K Shah Kuang-Yuh Chyu

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