Cardiology, CardioVascular Center University Hospital Zurich Zurich, Switzerland
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1 Modification of by the lipoxidation product malondialdehyde leads to LOX-1 dependent activation of endothelial PKCbeta-2 and adverse endothelial effects of in patients with coronary artery disease Christian Besler, Kathrin Heinrich, Lucia Rohrer, Meliana Riwanto, Angeliki Chroni, Aldons J. Lusis, Arnold von Eckardstein, Alan M. Fogelman, Thomas F. Lüscher, Ulf Landmesser Cardiology, CardioVascular Center University Hospital Zurich Zurich, Switzerland
2 Conflict of interest statement Nothing to declare
3 Reduced Cholesterol is associated with increased cardiovascular risk despite intense statin therapy Barter P et al., N Engl J Med 27; 357: 131-1
4 : Proposed anti-atherogenic effects VLDL/ LDL Bile LDL-R FC Liver 1. -mediated promotion of RCT (reverse cholesterol transport) CE TG CE SR-BI PLTP CETP A-I FC CE Mature LCAT A-I FC Nascent ABCG1 ABCA1 Macrophage 2. Direct -mediated endothelial-protective effects SR-BI? Antioxidant Effects Anti-inflam. Effects Endothelial NO Production Anti-thromb. Effects Endothelial Regeneration Besler C et al. & Landmesser U; Curr Pharm Des 21; 16(13):148-93
5 Assocation of cholesterol efflux capacity of with carotid IMT and angiographic CAD Assocation between cholesterol efflux capacity and carotid IMT (beta coefficients) Assocation between cholesterol efflux capacity and angiographic CAD (odds ratios) Khera A et al., N Engl J Med 211;364(2):127-35
6 Aim of the present study To characterize the effects of isolated from patients with an acute coronary syndrome and stable coronary disease as compared to from healthy subjects on endothelial NO production, anti-inflammatory effects and endothelial repair To examine potential mechanisms leading to impaired endothelial-protective effects of in patients with coronary disease
7 Experimental setup Patients with acute coronary syndrome (n=25) Patients with stable coronary disease (n=25) control subjects (n=25) Endothelial Function (Endothelial cell NO production and vasoreactivity) ESR spectroscopy Organ chamber Isolation of (by sequential ultracentrifugation) Vascular effects Effects on Re- Endothelialization Carotid artery injury model in nude mice Anti-oxidant effects (Endothelial cell superoxide production) ESR spectroscopy Anti-inflammatory effects (Endothelial cell inflammatory activation) Monocyte adhesion VCAM-1 expression
8 Characteristics of the study population
9 Effect of from patients with CAD on endothelial nitric oxide production production [in % of buffer-treated cells] P<.1 P<.1 D Endothelial nitric oxide stable CAD ACS AU -5 Arbitrary Units -5 Arbitrary Units Magnetic field (G) Magnetic field (G) Magnetic field (G) Besler C et al. & Landmesser U, J Clin Invest 211; 121(7):
10 VCAM-1 expression [in % of TNF a -stimulated cells] Effects of on TNFα-stimulated endothelial VCAM-1 expression and endothelial monocyte adhesion Endothelial monocyte adhesion [number of monocytes per high power field] P<.1 15 P<.1 P<.1 35 P< VCAM1 scad ACS 5 scad ACS GAPDH Besler C et al. & Landmesser U, J Clin Invest 211; 121(7):
11 Reendothelialized area [in %] Effect of on endothelial repair after carotid artery injury in nude mice n.s. n.s. P= mm PBS scad ACS PBS scad ACS Besler C et al. & Landmesser U, J Clin Invest 211; 121(7):
12 enos-activating [in % of buffer-treated cells] Ser1177 phosphorylation Effect of on enos phosphorylation at serine residue 1177 in endothelial cells scad ACS penos (Ser1177) total enos penos (Ser1177) scad 5 total enos penos (Ser1177) ACS Time (min) total enos
13 enos-inhibiting [in % of buffer-treated cells] Thr495 phosphorylation Effect of on enos phosphorylation at threonine residue 495 in endothelial cells 3 2 scad ACS penos (Thr495) total enos penos (Thr495) scad 1 total enos penos (Thr495) ACS Time (min) total enos
14 PKCßII-activating Ser-66 phosphorylation [in % of buffer-treated cells] Effects of on activation of protein kinase Cβ-II PKCßII membrane translocation [in % of buffer-treated cells] P<.25 P<.1 P< P<.1 4 P<.25 n.s ppkcßii (Ser66) total PKCßII Basal CAD ACS IB: PKCßII membrane cytosol Basal scad ACS
15 enos activating Ser1177 phosphorylation [in % of buffer-treated cells] Role of PKCβ-II for altered effects of on enos phosphorylation enos inhibiting Thr495 phosphorylation [in % of buffer-treated cells] P=n.s. P<.1 P=n.s. P<.25 2 P=n.s. P<.25 2 P=n.s. P< CAD LY CGP penos (Ser1177) total enos CAD LY CGP penos (Thr495) total enos
16 Role of PKCβ-2 for altered effects of on endothelial NO production production [in % of buffer-treated cells] P=n.s. P<.1 P=n.s. P<.1 D Endothelial nitric oxide CAD LY CGP
17 PKCβ-2 activating Ser66 phosphorylation [in % of buffer-treated cells] Relevance of endothelial LOX-1 for stimulation of PKCβ-2 phosphorylation by from patients with coronary disease [in % of buffer-treated cells] P< ppkcβ-2 (Ser66) + buffer n.s. CAD + isotype + anti-lox1 control D Endothelial NO production n.s. + Isotype ctrl. + anti- LOX1 P<.5 CAD + Isotype ctrl. + anti- LOX1 total PKCβ-2
18 Protein-bound MDA content of [nmol/mg protein] Protein-bound MDA content and MDA-lysine adducts in MDA-lysine adducts in as detected by mass spectrometry [in % of healthy subjects] P<.1 P< P< scad ACS CAD
19 Role of MDA for altered effects of on endothelial NO production and PKCβ-2 activation Effect of on endothelial nitric oxide production [in % of buffer-treated ] PKCbeta-2 activating Ser66 phosphorylation [in % of buffer-treated cells] n.s. 15 P<.25 P< * * MDA / apoa-i [mol/mol] ppkcbeta-2 (Ser66) Basal +MDA +MDA +anti LOX-1 Total PKCbeta-2
20 -associated paraoxonase-1 activity and content Paraoxonase-1 activity Paraoxonase-1 content P<.5 P<.5 Paraoxonase Paraoxonase activity [in % of ] P<.5 CAD ACS Paraoxonase-1 content [in % of ] 2 1 P<.5 CAD ACS
21 Role of PON-1 for the effects of on endothelial PKCβ-2 activation PKCßII-activating Ser-66 phosphorylation [in % of buffer-treated cells] P<.25 2 P<.25 P<.25 1 ppkcßii (Ser66) Basal + HQ + EDTA HQ EDTA total PKCßII
22 [in % of buffer-treated cells] Role of PON-1 for the effects of on endothelial NO production [in % of buffer-treated cells] D Endothelial NO production PBS P<.25 P<.25 HQ + EDTA D Endothelial NO production Wild type P<.1 PON-1 -/-
23 Summary and Conclusion The effects of on endothelial NO production, endothelial inflammatory activation and endothelial repair are impaired in patients with stable CAD and ACS In contrast to from healthy subjects, from patients with CAD activates endothelial PKCβ-2 and inhibits enosactivating pathways Furthermore, the present study indicates that impaired associated PON-1 activity and an accumulation of the advanced lipoxidation end product malondialdehyde (MDA) represents an important mechanism underlying stimulation of PKCβ-2 by from patients with CAD These findings may have important implications for designing -targeted therapies, since only raising with vasoprotective properties can be expected to exert beneficial effects in patients with cardiovascular disease
24 Thank you
25 Effects of the CETP-inhibitor torcetrapib on cardiovascular outcome in patients at high risk for coronary events (ILLUMINATE) All-cause mortality Major cardiovascular events despite an increase in -cholesterol levels by 72% and a decrease in LDL-cholesterol levels by 25% Barter P et al., N Engl J Med 27; 357:219-22
26 Effect of protein kinase Cβ on endothelial nitric oxide bioavailiability Protein kinase Cβ Akt-activating serine 473 phosphorylation enos-activating serine 1177 phosphorylation + enos-inhibitory threonine 495 phosphorylation Endothelial NO bioavailability Naruse K et al.; Diabetes. 26; 55(3):691-8 Payne GA et al.; Am J Physiol Heart Circ Physiol. 29;291(1):H46-5
27 Effects of increasing concentrations of on NO production in endothelial cells production [in % of buffer-treated cells] D Endothelial nitric oxide P<.5 3 scad ACS m g/ml 5 m g/ml 1 m g/ml
![Reendothelialized area [in %] Effect of on](/docs-images/83/88969041/images/28-0.jpg "endothelial repair after carotid artery injury in")
28 Reendothelialized area [in %] Effect of on endothelial repair after carotid artery injury in enos-deficient mice n.s mm enos-/- - buffer control enos-/- + enos-/- - buffer control enos-/- +
29 Total [ 14 C] cholesterol efflux [% of total cholesterol] Total cholesterol efflux capacity of from healthy subjects and patients with CAD n.s. 4 n.s scad ACS
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