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Experimental Physiology Symposium Report

Signalling processes in endothelial ageing in relation to chronic oxidative stress and their potential therapeutic implications in humans
Bernd van der Loo1 , Stefan Schildknecht2 , Rebecca Zee3 and Markus M. Bachschmid3
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Clinic of Cardiology, Cardiovascular Centre, Department of Medicine, University Hospital Zurich, Switzerland Department of Biology, Faculty of Sciences, University of Konstanz, Germany 3 Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA

Ageing is an important risk factor for the development of cardiovascular diseases. Vascular ageing is mainly characterized by endothelial dysfunction, an alteration of endothelium-dependent signalling processes and vascular remodelling. The underlying mechanisms comprise increased production of reactive oxygen species (ROS), inactivation of nitric oxide (NO) and subsequent formation of peroxynitrite (ONOO ). Elevated ONOO may exhibit new messenger functions by post-translational oxidative modication of intracellular regulatory proteins. Mitochondria are a major source of age-associated superoxide formation, as electrons are misdirected from the respiratory chain. Manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, is an integral part of the nucleoids and may protect mitochondrial DNA from ROS. A model linking NO, mitochondria, MnSOD and its acetylation/deacetylation by sirtuins (NAD+ -dependent class III histone deacetylases) may be the basis for a potentially new powerful therapeutic intervention in the ageing process.
(Received 13 August 2008; accepted after revision 5 November 2008; rst published online 7 November 2008) Corresponding author B. van der Loo: Clinic of Cardiology, Cardiovascular Centre, Department of Medicine, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. Email: bernd.vanderloo@usz.ch

Cardiovascular diseases have a higher incidence with increasing age, even in the absence of established risk factors. This suggests that ageing per se alters vascular function. Vascular ageing is mainly characterized by endothelial dysfunction (van der Loo et al. 2000), associated with decreased endothelium-dependent relaxations with increasing age (Tschudi et al. 1996). The endothelium exerts a multimodal regulation of vascular tone, structure and function by the release of vasoactive substances, which, under physiological conditions, are nely balanced to ensure vascular homeostasis. However, with increasing age, this equilibrium cannot be preserved any longer. An age-associated enhanced superoxide (O 2 ) production in the vasculature (Oudot et al. 2006; van der Loo et al. 2000), mainly derived from the endothelium, occurs and is the basis for the oxidativestress theory of vascular ageing. It is obvious that reactive oxygen species (ROS) play a pivotal role in endothelial cell redox signalling. Knowledge about their sources and the signalling cascades they modify, as well as the compensatory mechanisms, will enhance our
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knowledge of the vascular ageing process. The particular understanding about how redox systems are regulated in the elderly may also help to identify new targets to treat ageing, which is, besides smoking, diabetes, hypertension and obesity, a major cardiovascular risk factor.
Endothelial dysfunction versus endothelial cell activation

The endothelium controls homeostatic conditions by the release of potent endothelium-derived autacoids. Endothelial cell properties and the signalling processes are altered in a variety of pathophysiological conditions, such as inammation, exogenous noxious substances and ageing. For the description of endothelial alterations, several terms and denitions have been used, such as endothelial dysfunction and endothelial cell activation (Cines et al. 1998). The latter is based on the fact that the endothelium is an integral part of the innate immune system, controlling adhesion and transcytosis of leukocytes and monocytes to the site of inammation.
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Locally invading pathogens can trigger the release of cytokines by tissue macrophages, thus activating the endothelium and guiding immune cells to the site of inammation. This involves the opening of the endothelial cell barrier, reduction of the release of repellent molecules, expression of adhesion molecules and nally the controlled diapedesis of immune cells. This process is a fully reversible physiological reaction to inammation or injury, but chronic exposure to ROS, as in the ageing process, may lead to irreversible changes and damage of the endothelial cell layer. At the molecular level, two subsequent strictly timed phases can be distinguished and are called endothelial cell activation (ECA) type I and type II. The rst phase (ECA type I), in which endothelial cells retract from each other, express P-selectin (Prescott et al. 2001) and release the von Willebrand factor, is entirely independent of de novo protein synthesis and essentially completed within 1 h. We suggested an inhibition of nitric oxide (NO) and prostaglandin I 2 production by a novel mechanism involving peroxynitrite (ONOO ) formation from NO and a still unknown source of O 2 radicals (Ullrich & Bachschmid, 2000). Peroxynitrite then causes tyrosine nitration and concomitant inhibition of prostaglandin synthase (PGIS; Ullrich & Bachschmid, 2000). This leads to accumulation of prostaglandin H 2 , which is able to activate the thromboxane A 2 /prostaglandin H 2 receptor, mediating both smooth muscle contraction and activation of platelets and white cells. A second stage (ECA type II) becomes apparent after 1 h and involves induction and expression of early genes, such as the adhesion molecules intracellular adhesion molecule or vascular cell adhesion molecule and proinammatory cytokines, as well as regulatory enzymes such as nitric oxide synthase 2 (NOS 2) and cyclo-oxygenase 2 (COX2). As a consequence, white cells can tightly adhere and migrate into tissues, NO and prostaglandins are formed in excess, and cytokines orchestrate the inammatory response. It is crucial to understand the molecular mechanisms behind endothelial cell activation in order to develop pharmacological strategies to prevent this process or, more importantly, to induce resolution and reversal of inammation.

relaxation. Studies on ageing in animal models and humans have proven that the bioavailability of NO is continuously diminished and, in parallel, endotheliumderived O 2 increases (van der Loo et al. 2000; Krause, 2007). Superoxide is a highly efcient scavenger for NO, causing formation of the very reactive and deleterious ONOO in a near diffusion-controlled reaction (Beckman & Koppenol, 1996). At high concentrations (micromolar range), peroxynitrite oxidizes unspecically any biological macromolecule, such as DNA, proteins and lipids. At lower concentrations (nanomolar), it interferes with important vascular signalling pathways, such as NO and prostaglandin signalling, calcium homeostasis, mitogenactivated protein kinase cascade, nuclear factor B (NFB) and activator protein 1 (Xie et al. 2006). Thus an increase in O 2 and subsequent peroxynitrite formation reduces the bioavailability of NO and leads to endothelial dysfunction, one of the major causes for the progression of vascular disease and senescence. When studying the distribution of 3-nitrotyrosine residues, a typical end-product of the reaction of ONOO with biological compounds, by using immunogold labelling and electron microscopy, we found the most signicant accumulation of nitrotyrosine in the mitochondria, suggesting primarily a mitochondrial source of O 2 (van der Loo et al. 2000). However, other potential sources of O 2 production, such as subunits of the NADPH oxidase (Krause, 2007), xanthine oxidase, and an uncoupled endothelial (e)NOS (NOS3) have to be considered. Both upregulation of eNOS (van der Loo et al. 2000) and reduced levels of enzyme expression (Cernadas et al. 1998) have been observed with increasing age. Our own data demonstrating a steep increase both in expression and in activity of eNOS support the concept that eNOS may, as an oxidase, become part of a redox system that increases electron transfer to molecular oxygen.

The role of mitochondria

Free radicals and vascular ageing

Nitric oxide is one of the most important mediators of the cardiovascular system and has been extensively studied. Pathophysiologically, endothelial dysfunction is described as a paradoxical response of the vasculature to acetylcholine, reacting with constriction instead of relaxation. Normally, acetylcholine triggers the specic endothelium-derived release of NO, leading to vascular

Under physiological conditions, approximately 0.1% of O 2 consumed by mitochondria is reduced to O 2 (Fridovich, 2004). Age-associated mitochondrial dysfunction implies increased generation of O 2 and peroxynitrite by misdirection of electrons from the respiratory chain into ROS production due to a decline of mitochondrial function (van der Loo et al. 2000), such as inactivation of aconitase, reduced ATP synthesis and enhanced permeability transition pore opening, leading to apoptosis. This initiates a vicious cycle of increased (nuclear and mitochondrial) DNA damage, leading again to more ROS generation, entailing further mitochondrial
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DNA (mtDNA) damage (Finkel & Holbrook, 2000; Fig. 1). Mitochondrial DNA is considered to be far more susceptible to oxidative stress than nuclear DNA because of the proximity of mtDNA to the source of free oxygen radicals, because protective mitochondrial histone proteins are absent (Larsen et al. 2005) and because mitochondria lack efcient DNA repair mechanisms (Larsen et al. 2005). Interestingly, experimental evidence in Caenorhabditis elegans has also been provided for a direct link between mitochondrial ROS generation and nuclear (genomic) DNA damage (Hartman et al. 2004). However, the exact signalling pathways determining the molecular basis for the link between lifelong damage to mtDNA and age-associated vascular dysfunction are not yet fully understood.

Antioxidant systems and compensatory mechanisms

Based upon the oxidative stress hypothesis of vascular ageing, naturally occurring antioxidants such as vitamins would seem to be attractive candidates to prevent ROS production. Vitamin E is an important electron source for the reduction of ONOO (Lee et al. 1998). However, in aged rats fed a normal diet and not susceptible to atherosclerosis, -tocopherol, the biologically most active form of vitamin E, was found to be markedly increased in both plasma and major organs. The highest, up to 70-fold, increase was found in the aortic wall (van der Loo et al. 2002). This suggests that sufciently high levels of antioxidant vitamin E may be built up from a normal diet in an attempt to counterbalance

Figure 1. Formation of mitochondrial reactive oxygen and nitrogen species Abbreviations: NO, nitric oxide; ONOO , peroxynitrite; O 2 , superoxide; TOM, translocase of the outer membrane; MnSOD, manganese superoxide dismutase; GSH, reduced glutathione; GSSG, oxidized glutathione; CCS, copper chaperone for Cu,Zn superoxide dismutase; Q, ubiquinone or coenzyme Q10; and IMS, intermembrane space. The mitochondrial respiratory chain continuously shuttles complex I or II-derived electrons via ubiquinone, complex III and cytochrome c to complex IV (cytochrome c oxidase). The terminal complex IV transfers electrons to molecular oxygen (O 2 ), producing water (H 2 O). Complexes I, III and IV use the energy from the electrons to create the electrochemical gradient by pumping protons across the inner mitochondrial membrane for ATP synthesis at complex V (oxidative phosphorylation). Electron leaks at complex I, III and ubiquinone lead to O 2 formation, which is sequentially detoxied by MnSOD, glutathione peroxidase and mitochondrial peroxiredoxins or glutaredoxin. Changes in the mitochondrial GSH/GSSG ratio can cause protein glutathiolation, which in the case of complex I results in inhibition. Mitochodrial NO may be derived from a mitochondrial nitric oxide synthase isoform, from nitrite reduction at complex III or may diffuse from the outside into the mitochondria. Nitric oxide combines with mitochondrial O 2 to form the highly reactive peroxynitrite, which may nitrate and inactivate MnSOD, cause mtDNA lesions or diffuse into the cytosol. Oxidative mtDNA lesions promote mutation and dysfunction of respiratory chain components, thus enhancing free radical formation and mitochondrial dysfunction. In order to compensate for an age-dependent increase in mitochondrial superoxide formation, newly synthesized and unfolded Cu,ZnSOD can translocate into the mitochondrial intermembrane space, where it is then folded and loaded with Zn and Cu in order to shield the cytosol from O 2 .
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age-associated oxidative stress and that this may represent a self-regulatory protective adaptation. In physiological conditions, antioxidant enzymes prevent the detrimental effects of O 2 . Apart from mitochondrial manganese superoxide dismutase (MnSOD; Sod 2), extracellular superoxide dismutase (EC-SOD; Sod 3) is the main scavenger of O 2 in the extracellular space and Cu,Zn superoxide dismutase (Cu,ZnSOD; Sod 1) in the cytosol, respectively. Copper/zinc superoxide dismutase (Cu,ZnSOD) was recently shown to lose its membrane and caveolae association and to relocate to the mitochondria in an age-dependent manner (van der Loo et al. 2006). Unlike MnSOD, constitutively expressed in mitochondria, Cu,ZnSOD is not inactivated by ONOO -mediated tyrosine nitration as a function of age. The process may be regulated by age-dependent elevated O 2 levels of copper chaperone for superoxide dismutase in the

intermembrane space, resulting in retention of Cu,ZnSOD in the mitochondria. Thus, the enzyme becomes part of a compensatory defense against compromised mitochondrial function.
Sirtuins in vascular ageing

Sirtuins (SIRT, silent information regulator two) are a class of NAD+ -dependent protein deacetylases and are known to be able to increase lifespan by mechanisms similar to calorie restriction (Bordone & Guarente, 2005). These mechanisms include, most importantly, the control of glucose metabolism in hepatocytes by modulation (deacetylation) of PGC1 [PPAR (peroxisome proliferator-activated receptor) coactivator 1; Rodgers et al. 2005] and suppression of PPAR -controlled genes involved in the regulation of fat tissue (Picard et al. 2004). At least seven isoforms,

Figure 2. Cytosolic control of mitochondrial ROS formation and effects of sirtuins Abbreviations: SIRT, sirtuin, Ac, acetylation; ROS, reactive oxygen species; MnSOD, manganese superoxide dismutase; Gpx, glutathione peroxidase; and mTFA, mitochondrial transcription factor A. Various signalling cascades can affect mitochondrial physiology, mediate mitochondrial radical formation or are connected to apoptotic signalling. Oxidative stress activates protein kinase C , leading to serine-phosphorylation of the adaptor protein p66Shc , which subsequently translocates to the mitochondrial intermembrane space and associates with the translocator inner membrane-outer membrane (TIM-TOM) l protein import complex. Proapoptotic stimuli release p66Shc from the complex and, via interaction with cytochrome c, hydrogen peroxide is formed, which triggers the permeability transition pore and initiates apoptosis. Mitochondrial translocation has also been reported for p53 a transcription factor promoting senescence and p21Ras a signaling molecule involved in cell cycle regulation. Both molecules are assumed to sequester antiapoptotic proteins of the Bcl-mammalian gene family regulating mitochondrial membrane permeabilization thus promoting free radical formation and apoptosis. Furthermore, the stability of p53 is regulated by lysine-acetylation, which competes with ubiquitinylation and subsequent proteasomal degradation. Reactive oxygen species may inactivate SIRT1 by oxidation of an essential structural Zn-nger that may result in p53 accumulation and mitochondrial translocation. Another example is tumour necrosis factor signalling, which involves the ceramide pathway that increases mitochondrial superoxide formation at complex III. Mitochondria also respond very sensitively to oxidative stimuli or metabolic cues with enhanced electron leakage and free radical formation. This may alter the composition of mitochondrial nucleoids and protein DNA complexes, and increase the mtDNA mutation rate. Similar to organization of nuclear DNA, mitochondrial sirtuins may regulate nucleoid plasticity.
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exerting diverse functions, exist in humans. By far the best investigated of the homologues so far is SIRT1, which deacetylates p53, a well-known transcription factor promoting senescence, thereby limiting premature cellular senescence (Langley et al. 2002). Resveratrol is one of the polyphenols found in red wine and responsible for cardiovascular protective effects (Opie & Lecour, 2007). Resveratrol has been shown to be able to activate SIRT1 (but not other sirtuin homologues) by promoting a conformational change in the enzyme (Borra et al. 2005). Resveratrol can extend lifespan in yeast and in mice on a high-calorie diet (Baur et al. 2006), indicating a potentially new powerful intervention in the ageing process. Treatment of mice with resveratrol leads to an increase in mitochondrial size, enzymatic activity, mtDNA content and enrichment of genes involved in mitochondrial biogenesis in skeletal muscle (Lagouge et al. 2006). These effects on mitochondrial function are mediated by SIRT1 and PGC-1, with SIRT1 deacetylating and thereby activating PGC-1 (Lagouge et al. 2006). A recent study in mice on a high-fat diet moderately overexpressing SIRT1 proved that SIRT1 can lower lipid-induced inammation and improve glucose tolerance. In part, these effects are mediated by downmodulation of NFB, resulting in reduced activation of proinammatory cytokines such as tumour necrosis factor and interleukin-6 and an increased expression of MnSOD (Puger et al. 2007). The effects of a natural compound on lifespan will certainly lead to the development of more potent analogues of resveratrol for in vivo use in the future (Koo & Montminy, 2006). Interestingly, calorie restriction was also recently shown to induce eNOS expression in various mouse tissues, accompanied by a promotion of mitochondrial biogenesis, increased mtDNA content and SIRT1 expression, dependent on eNOS-derived NO (Nisoli et al. 2005). Furthermore, in a model (treatment of human umbilical vein endothelial cells with hydrogen peroxide) of stress-induced premature senescence, the selective phosphodiesterase 3 inhibitor cilostazol reduced the senescent cell phenotype by upregulation of SIRT1 (Ota et al. 2008). These ndings may lead to a recently proposed model linking calorie restriction, NO, SIRT1 and activation of mitochondria (Guarente, 2005). Therefore, the development of SIRT1 activators may potentially be highly benecial for the aged cardiovascular system in order to restore endothelial function by activating eNOS and by reducing mitochondrial O 2 formation via induction of MnSOD expression.
A mechanistic concept of age-associated mitochondrial dysfunction

mtDNA mutations and deletions, resulting in premature ageing (Trifunovic et al. 2004; Kujoth et al. 2005). Protective mechanisms must exist within the nucleoids to shield mtDNA from ROS produced by the nearby electron transport chain. Manganese superoxide dismutase, an antioxidant enzyme localized in the mitochondria, has been demonstrated to be an integral component of the nucleoids, where it can protect mtDNA from ROS (J. Kienhoefer & M. Bachschmid, unpublished data). Manganese superoxide dismutase can be acetylated, which may modulate its association to mtDNA (Kim et al. 2006), in analogy to the control of nuclear chromatin plasticity by histone acetylation/deacetylation. Mitochondria harbour three sirtuin isoforms which, by deacetylation, may increase the association of MnSOD and other protective proteins with mtDNA and may regulate replication and transcription of mtDNA by modulating the composition and density of the nucleoids. Peroxynitrite can inactivate SIRT1, presumably via disruption of the zinc nger (Min et al. 2001). This suggests that sirtuins are sensitive to ROS and can lose their ability to maintain mtDNA in a more protected form. Finally, with an increasing rate of mutations in mtDNA, this will lead to a malfunctioning respiratory chain and enhanced ROS generation within mitochondria, similar to the effects seen in Pol proofreading-decient mice exhibiting the features of premature ageing (Trifunovic et al. 2004; Kujoth et al. 2005; Fig. 2). If this concept holds true, the potential therapeutic implications to combat the loss of protection of mtDNA against ROS may have an enormous impact. References
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