Endothelial cell aging
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1 Endothelial cell aging Molekulare Präventivmedizin Molecular preventive medicine Judith (Jojo) Haendeler, PhD Leibniz Institut fuer Umweltmedizinische Forschung (IUF) Molecular Cell & Aging Research
2 atherosclerosis endothelial cell apoptosis Aging Disturbed angiogenesis endothelial cell migration Reduced organ functions disturbed redox balance increased apoptosis sensitivity reduced bioavailable NO reduced migratory capacity
3 Young Old Aging and apoptosis sensitivity correlate in vivo DAPI TUNEL Apoptosis and Endothelial Cell Density in Aorta and Femoral Artery Apoptosis, % of endothelial cells Young Old Endothelium n Mean ± SEM n Mean ± SEM P Thoratic aorta ± ± 0.6 <0.05 Abdominal aorta ± ± 1.3 <0.05 Femoral artery ± ± 0.8 <0.01 K. Asai et al., 2000
4 Increased apoptosis sensitivity during endothelial cell aging Apoptosis (%) Control oxldl TNF Passage Number: Population Doubling: 0 P2 P8 P9 P10 P11 P12 P13 P14 P (Hoffmann/Haendeler et al., 2001)
5 Which factors contribute to endothelial cell aging? disturbed mitochondrial! of Reduction NO/ROS! balance damage telomerase Passage 9-10 Passage /telomeres Passage 11-13! Contribute to the development of atherosclerosis
6 -gglaktosidase positive cells (%) -Galaktosidase positive cells (%) NO, antioxidants and statins delay replicative senescence control SNAP (NO-donor) control NAC Statin passage Passage (Vasa et al., 2000) (Haendeler et al., 2004)
7 NO, statins and antioxidants Delay endothelial cell aging Prevent age related increase in apoptosis sensitivity Mechanisms?
8 Cellular antioxidants TRX-TRXR system GSH-GSSG system SOD, catalase etc. Cellular oxidants NADPH oxidases Respiratory chain enzymes etc. ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS ROS
9 How can NO and/or statins influence the redox balance? transcriptional/protein expression protein modification S-nitros(yl)ation protein SH+ NO protein SNO
10 Thioredoxin is a cytosolic intracellular disulfide reductase and important for regulating the cellular redox - status. Thioredoxin knockout is embryonal lethal. (Matsui et al., 1996 Dev. Biol.) Trx-1 TRX Protein (SH) 2 Reduction and oxidation of proteins and reduction of ROS (Holmgren, 1989) Protein S SH SH TRX -S S--Sprotein X Binding to active cysteines thereby modulation of protein function (Saitoh et al., 1998) TRX Nucleus ARE Translocation into the nucleus: binding and regulation of transcription factors (Schroeder et. al. 2007) 69 SH potential S-nitrosylation site
11 HS HS ON-S Posttranslational modification of Trx-1 TRX active cysteines C C C In mice: increased cardioprotection and anti-apoptotic capacity 73 C S-SG Trx In cells and plants: reduction of oxidoreductase activity In cells: increased oxidoreductase activity and anti-apoptotic capacity (Modified from Haendeler, 2006)
12 Trx activity ( OD340nm/ T*mg protein) Effect of statins on Trx-1 activity Enzymeactivity 100% Trx Trxwt TRXwt TRX C69S SH SH S-NO O -. O O % Trx TrxC69S 0.01 SH SH O -. 2 O -. 2 O -. O O -. 2 OH AT (Haendeler et al., 2004)
13 Conclusion: Trx-1 SH SH S- NO S-nitros(yl)ation of thioredoxin leads to an increase in thioredoxin activity, to a reduction of intracellular ROS and to a reduction of endothelial cell apoptosis Is there a role for Trx-1 in endothelial cell aging?
14 apoptosis (%) Role of Trx-1 in aged EC Young EC Aged EC (Altschmied & Haendeler, 2009) - Trx-1 - actin young aged EV EV/TNF Trx-1wt Trx-1wt/TNF Trx-1 overexpression rescued aged endothelial cells towards increased apoptosis sensitivity: Implication of Trx-1 loss for endothelial cells?
15 Trx-1 interacts with γ-actin I IP with anti-trx-1 Mass spectrometry 97 kda 66 kda IgG γ-actin 45 kda 30 kda IgG Trx-1 20 kda 14 kda
16 Trx-1 interacts with γ-actin II IP with anti-γ-actin Trx-1 Immunostaining (endogenous) actin 52 kda 38 kda IgG γ-actin 24 kda IgG DAPI merge 17 kda 12 kda Trx-1
17 Trx-1 prevents H 2 O 2 -induced actin stress fiber formation Trx-1 overexpressing cell Xpress - Trx-1 actin stress fiber formation Trx-1 overexpressing cells Xpress - Trx-1 +1h 200µM H 2 O 2
18 Focal Adhesion Kinase (FAK) is directly linked to H 2 O 2 -induced actin stress fiber formation physiological conditions oxidative stress Integrin Receptor Integrin Receptor FAK P Tyr397 FAK P Tyr397 H 2 O 2 Ras Ras PF (FAKi) actin fiber formation actin stress fiber formation
19 PF573228, a FAK-Inhibitor (FAKi), inhibits H 2 O 2 -induced actin stress fiber formation 7h control or 40nM FAKi 0h -6h 1h 1h control or 200µM H 2 O 2 control H 2 O 2 FAKi/H 2 O 2
20 Experimental procedure to elucidate the influence of H 2 O 2 -induced actin stress fiber formation on cellular events Pre-incubation with FAKi (FAKi/H 2 O 2 ) -6h 0h 24h control or 40nM FAKi 18h 0h 18h control or 200µM H 2 O 2 18h Post-incubation with FAKi (H 2 O 2 /FAKi) 1h 17h control or 40nM FAKi 18h 0h 1h 18h control or 200µM H 2 O 2 18h
21 H 2 O 2 -induced actin stress fiber formation during pre- and post-incubation Pre-incubation with FAKi con H 2 O 2 FAKi FAKi/H 2 O 2 FAKi/H 2 O 2 H 2 O 2 /FAKi Post-incubation with FAKi con H 2 O 2 FAKi H 2 O 2 /FAKi
22 oxidative stress via the aspartyl protease Cathepsin D Trx-1 degradation Trx-1 (overexpression, exogenous) aging/apoptosis
23 relative Trx-1 level relative Trx-1 level Trx-1 levels during pre- and post-incubation pre (no stress fibers) post (actin stress fibers) * ** * con H 2 O 2 FAKi FAKi + H 2 O con H 2 O 2 FAKi H 2 O 2 + FAKi Trx-1 Trx-1 GAPDH GAPDH con H 2 O 2 FAKi FAKi + H 2 O 2 con H 2 O 2 FAKi H 2 O 2 + FAKi * p<0,05 vs. con ** p<0,05 vs. H 2 O 2 Blocking phosphorylation of FAK prior to H 2 O 2 inhibits Trx-1 degradation in contrast to post-incubation with FAKi
24 relative apoptosis relative apoptosis Apoptosis during pre- and post-incubation pre (no actin stress fibers) post (actin stress fibers) * ** * * 1 1 con H 2 O 2 FAKi FAKi + H 2 O 2 con H 2 O 2 FAKi H 2 O 2 + FAKi * p<0,05 vs. con ** p<0,05 vs. H 2 O 2 Blocking phosphorylation of FAK prior to H 2 O 2 inhibits apoptosis induction in contrast to post-incubation with FAKi
25 Summary nucleus endothelial cell Trx-1 γ-actin Oxidative stress FAK P nucleus Apoptosis/Aging
26 TERT Team Sascha Jakob Nicole Büchner Kerstin Kunze Anika Rühl Cooperations G. Saretzki T. von Zglinicki Newcastle, UK U. Laufs Homburg Saar U. Brandt Frankfurt J. Krutmann IUF Trx-1 Team Yogi Altschmied Tim C. Zschauer Philip Czypiorski Diane Schmiegelt Kerstin Kunze Cooperations J. Sadoshima Newark, USA B.C. Berk J. Miano Rochester, USA R. Popp Frankfurt p27/caffeine Team Sascha Jakob Tim C. Zschauer Diane Schmiegelt Marius Beylebens Cooperations I. Spyridopoulos Newcastle, UK R. Popp Frankfurt C. Weber Aachen V. Andrés Valencia, Spanien J. Schrader HHU Düsseldorf T. Rassaf M. Kelm HHU Düsseldorf GRHL-3 Team Yogi Altschmied Maga Lukosz Arne Mlynek Anika Rühl Christopher Güttler Cooperations C. Winkler Singapore J. Miano Rochester, USA H. Sültmann Heidelberg A. Gödecke HHU Düsseldorf Molekulare Präventivmedizin DFG GRK 1033 SFB 728 GRK 1089 SFB 612 Molecular preventive medicine
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28 oxidative stress (fold passage 2) Role of ROS during endothelial cell aging 4,5 4 3,5 TERT activity ROS senescence 20 Telomere length Passage , , , passage
29 mtdna (arbitary units) Loss of intact mitochondrial DNA during endothelial cell aging passage M H 2 O kb 6 kb Intact mitochondrial DNA * *
30 DCF fluorescence S-Nitrosylation of TRX regulates ROS NO 325 S-nitrosylation of Cys Thioredoxin Activity Reactive oxygen Species 175 vector TRXwt TRX (C69S) TRX (C32S/C35S) (Nature Cell Biology, 4, 2002)
31 H 2 O 2 -induced actin bundle formation during pre- and post-incubation Pre-incubation with FAKi con H 2 O 2 FAKi FAKi/H 2 O 2 Post-incubation with FAKi con H 2 O 2 FAKi H 2 O 2 /FAKi Blocking phosphorylation of FAK prior to H 2 O 2 inhibits actin bundle formation in contrast to post-incubation with FAKi indicating that once actin bundle formation is initiated it is not reversible
32 apoptosis (fold control) NO prevents apoptosis in vivo mice serum wild type enos (-/-)
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