Recombinant anti-pc IgG antibodies reduce intimal thickening and vascular inflammation in a mouse model for accelerated atherosclerosis
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1 Recombinant anti-pc IgG antibodies reduce intimal thickening and vascular inflammation in a mouse model for accelerated atherosclerosis Mark M. Ewing Departments of Cardiology and Surgery Leiden University Medical Center, Leiden, The Netherlands
2 Restenosis Restenosis is the result of accelerated atherosclerosis and vascular inflammation, elicited by de-endothelization and plaque injury during PCI, exposing endogenous ligands Ross, N Engl J Med. 1999;340(2):
3 Immunogenic auto-antigens elicit vascular inflammation Oxidation-specific epitopes in the vascular wall are generated OxLDL cholesterol uptake by PRRs activates macrophages Phosphorylcholine (PC) is an important immunogenic antigen Binder et al. Nat Med. 2002;8(11): Binder et al. J Lipid Res 2005; 46:
4 Natural antibodies against PC part of innate immunity Natural IgM anti-pc (T15/E06) antibodies are suggested to prevent atherogenesis by blocking oxldl uptake by macrophages Rose et al. Nat Med. 2003;9(6):
5 PC as therapeutic target against atherosclerosis in mice Atherosclerotic lesion reduction in mice by plasma anti-pc antibodies after immunization: Actively with PC-containing pneumococci (Binder et al., Nat Med, 2003) Passively with IgM anti-pc (Faria-Neto et al, Atherosclerosis, 2006) Actively with PC-KLH (keyhole limpet hemocyanin) (Caligiuri et al, JACC, 2007) Unlike IgM, IgG anti-pc antibodies can be produced in large quantities, allowing clinical application to prevent restenosis after PCI
6 Caidahl et al, submitted Protective role of anti-pc antibodies in CVD: clinical data ELSA study: inverse relation between anti-pc titers and carotid intima thickness (Su et al. Atherosclerosis, 2006) Göteborg study: low levels of anti-pc predict poor outcome in ACS patients (Caidahl et al. submitted) Anti-PC 1st quartile Anti-PC 2nd quartile Anti-PC 3rd quartile Anti-PC 4th quartile Caidahl et al. Submitted
7 Anti-PC antibodies block MΦ adhesion to injured cells Injured and apoptotic cells contain PC, activating endothelial cells and stimulating MΦ adhesion Since IgM anti-pc (E06) prevents MΦ adhesion, we tested recombinant anti-pc IgG (bearing E06/T15) against restenosis after PCI Recombinant hu-igg anti-pc (T15) Murine T15/E06 Human IgG1 Chang et al. J Exp Med. 2004;200(11):
8 In vivo immunization of recombinant anti-pc IgG1 (T15) ApoE3*Leiden transgenic mice: Human-like lipoprotein profile by attenuated (V)LDL clearance Develop diet-induced atherosclerosis Femoral arterial cuff placement gives vascular injury and leads to restenosis development Immunization with recombinant anti-pc IgG1 and control AB Non-constrictive cuff Femoral artery Lardenoye et al. Circ Res. 2000;87(3):
9 % leukocytes of total cells % macrophages of total cells Significantly reduced early leukocyte recruitment Plasma human anti-pc IgG titer reached 1208 ±110 U/ml after 3d (>> 100 μg/ml) No foam cells present 3d after surgery Control Hu anti-strep A2 IgG1 Anti-PC IgG Control Hu anti-strep A2 IgG1 Anti-PC IgG 0 Cells in medial layer Attached cells to endothelium 0 Cells in medial layer Attached cells to endothelium
10 Intimal thickening (μm²) Anti-PC IgG (T15) reduces intimal thickening Control Hu anti-strep A2 IgG1 0 Control Hu anti-strep A2 IgG1 Anti-PC IgG Anti-PC IgG
11 Lumenstenosis (%) Anti-PC IgG (T15) reduces lumenstenosis and intima/media ratio Intima / media ratio Lumenstenosis (%) Intima / media ratio Control Hu anti-strep A2 IgG1 Anti-PC IgG 0.0 Control Hu anti-strep A2 IgG1 Anti-PC IgG
12 SMC MΦ Anti-PC IgG (T15) affects vascular composition Control Hu anti-strepa2 IgG1 Anti-PC IgG Arrows indicate internal elastic laminae
13 % MΦ from total intimal layer area % MΦ from total medial layer area Anti-PC IgG (T15) affects macrophage presence Intimal layer composition Medial layer composition Control Hu anti-strep A2 IgG1 Anti-PC IgG 0 Control Hu anti-strep A2 IgG1 Anti-PC IgG
14 Polyclonal IgM anti-pc inhibits oxldl-uptake by MΦ apc IgM (50 μg/ml) apc IgM (200 μg/ml) unb. IgM apc IgM macro Unb. IgM apc IgM macro
15 Polyclonal IgG anti-pc does not inhibit oxldl-uptake by MΦ apc IgG (50 μg/ml) apc IgG (200 μg/ml ) unb. IgG apc IgG macro unb. IgG apc IgG macro In vivo concentration recombinant hu anti-pc IgG >> 100 μg/ml
16 Recombinant anti-pc T15 IgG1 Recombinant apc IgG1/IgG4 apc IgG4 apc IgG1 + oxldl macro Murine T15/E06 Human IgG1/4 Both anti-pc IgG4 and IgG1 + anti-cd32/cd64 gave similar results as IgG1 alone: Fc-γ receptors not involved Recombinant anti-pc IgG1 does not prevent Cu-oxLDL uptake
17 Conclusions Anti-PC antibodies clinically interesting with high therapeutic potential Recombinant anti-pc IgG reduced leukocyte adhesion and is therapeutically effective against restenosis by reducing vascular inflammation Unlike IgM anti-pc (T15, E06), this did not inhibit oxldl-uptake by MΦ in vitro Mechanism of action remains to be further investigated
18 Contributors MM Ewing JC Karper MR de Vries JW Jukema PHA Quax K Pettersson M. Nordzell J Frostegård H Lettesjö D Sexton
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RECOMBINANT HUMAN ACTIVATED PROTEIN C IN THE TREATMENT OF SEVERE SEPSIS ANAESTHESIA TUTORIAL OF THE WEEK 133 11 TH MAY 2009 Dr Richard Eve, Royal Cornwall Hospital, Cornwall, UK Correspondence to richardlloydeve@gmail.com