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. Author manuscript; available in PMC: 2014 Apr 15.
Published in final edited form as: Exp Physiol. 2008 Nov 7;94(3):305–310. doi: 10.1113/expphysiol.2008.043315

Signaling processes in endothelial aging in relation to chronic oxidative stress and their potential therapeutic implications

Bernd van der Loo 1, Stefan Schildknecht 2, Rebecca Zee 3, Markus M Bachschmid 3
PMCID: PMC3987961  NIHMSID: NIHMS129012  PMID: 18996949

Abstract

Aging is an important risk factor for the development of cardiovascular diseases. Vascular aging 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 posttranslational oxidative modification of intracellular regulatory proteins.

Mitochondria are a major source of age-associated superoxide (•O2–) 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 (mtDNA) 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 aging process.

Introduction

Cardiovascular diseases have a higher incidence with increasing age, even in the absence of established risk factors. This suggests that aging per se alters vascular function. Vascular aging 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 finely balanced to ensure vascular homeostasis. However, with increasing age, this equilibrium cannot be preserved any longer. An age-associated enhanced •O2– 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 “oxidative-stress theory” of vascular aging. It is obvious that ROS play a pivotal role in endothelial cell redox signalling. Knowledge about their sources, the signalling cascades they modify, as well as the compensatory mechanisms will enhance our knowledge of the vascular aging process. The particular understanding about how redox systems are regulated in the elderly may also help to identify new targets to treat aging, besides smoking, diabetes 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 inflammation, exogenous noxes and aging. For the description of endothelial alterations several terms and definitions 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 inflammation. Locally invading pathogens can trigger the release of cytokines by tissue macrophages, thus activating the endothelium and guiding immune cells to the site of inflammation. This involves the opening of the endothelial cell barrier, reduction of the release of repellent molecules, expression of adhesion molecules and finally the controlled diapedesis of immune cells. This process is a fully reversible physiological reaction to inflammation or injury, but chronic exposure to ROS, as in the aging 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 type I and II. The first 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 •NO and prostaglandin I2 (PGI2) production by a novel mechanism involving ONOO– formation from •NO and a still unknown source of •O2– radicals (Ullrich and Bachschmid, 2000). ONOO– then causes tyrosine nitration and concomitant inhibition of prostaglandin synthase (PGIS) (Ullrich and Bachschmid, 2000). This leads to accumulation of PGH2, which is able to activate the TxA2/PGH2 receptor both mediating smooth muscle contraction and activation of platelets and white cells.

A second stage (ECA type II) becomes apparent after 1h and involves induction and expression of early genes like the adhesion molecules ICAM or VCAM, proinflammatory cytokines as well as regulatory enzymes such as NOS-2 and COX-2. As a consequence white cells can tightly adhere and migrate into tissues, •NO and prostaglandins are formed in excess and cytokines orchestrate the inflammatory 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 inflammation.

Free radicals and vascular aging

•NO is one of the most important mediators of the cardiovascular system and has been extensively studied in the past. Pathophysiologically, endothelial dysfunction is described as a paradoxical response of the vasculature to acetylcholine, reacting with constriction instead of relaxation. Normally, acetylcholine triggers the specific endothelium-derived release of •NO leading to vascular relaxation. Studies on aging in animal models and humans have proven that the bioavailability of •NO is continuously diminished and, in parallel, endothelium-derived superoxide (•O2–) increases (van der Loo et al. 2000; Krause 2007).

•O2– is a highly efficient scavenger for •NO causing formation of the very reactive and deleterious ONOO– in a near diffusion-controlled reaction (Beckman and Koppenol, 1996). At high concentrations (micromolar range) peroxynitrite oxidizes unspecifically any biological macromolecule such as DNA, proteins and lipids. At lower concentrations (nanomolar) it interferes with important vascular signalling pathways (Xie et al. 2006) such as •NO and prostaglandin signalling, calcium homeostasis, MAP kinase cascade, NFκB and AP-1. Thus an increase in •O2– 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 significant accumulation of nitrotyrosine in the mitochondria, suggesting primarily a mitochondrial source of •O2– (van der Loo et al. 2000). However, other potential sources of •O2– production, such as subunits of the NADPH oxidase (Krause 2007), xanthine oxidase, and an uncoupled eNOS (NOS III) 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 increasing electron transfer to molecular oxygen.

The role of mitochondria

Under physiological conditions, approximately 0.1% of •O2– consumed by mitochondria is reduced to •O2– (Fridovich 2004).

Age-associated mitochondrial dysfunction implies increased generation of •O2– and peroxynitrite by misdirection of electrons from the respiratory chain into ROS production (van der Loo and Lüscher, 2000), 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 DNA (mtDNA) damage (Finkel and Holbrook, 2000) (figure 1). MtDNA is considered to be far more susceptible to oxidative stress than nuclear DNA because

  1. of the proximity of mtDNA to the source of free oxygen radicals,

  2. protective mitochondrial histone proteins are absent (Larsen et al., 2005), and

  3. mitochondria lack efficient DNA repair mechanisms (Larsen et al. 2005).

Interestingly, experimental evidence in C. 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 life-long damage to mtDNA and age-associated vascular dysfunction are not yet fully understood to date.

Figure 1. Mitochondrial reactive oxygen and nitrogen species formation.

Figure 1

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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 (O2), producing water (H2O). Complex 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 •O2– formation, which is sequentially detoxified 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 NO synthase isoform, from nitrite reduction at complex III or diffuses from the outside into the mitochondria. •NO combines with mitochondrial •O2– 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,Zn SOD can translocate into the mitochondrial intermembrane space, is then folded and loaded with Zn and Cu in order to shield the cytosol from •O2–.

Abbreviations: •NO, nitric oxide; ONOO–, peroxynitrite; •O2–, 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, IMS, intermembrane space.

Antioxidant systems and compensatory mechanisms

Based upon the oxidative stress hypothesis of vascular aging, 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, α-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 sufficiently high levels of antioxidant vitamin E may be built up from a normal diet in an attempt to counterbalance age-associated oxidative stress and that this may represent a self-regulatory protective adaptation.

Under physiological conditions antioxidant enzymes prevent the detrimental effects of •O2–. Apart from mitochondrial MnSOD (Sod 2), EC-SOD (Sod 3) is the main scavenger of •O2– in the extracellular space and Cu,ZnSOD (Sod 1) in the cytosol, respectively. 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 •O2– 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 aging

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 and Guarente, 2005). These mechanisms include most importantly the control of glucose metabolism in hepatocytes by modulation (deacetylation) of PGC-1α (PPAR (peroxisome proliferator-activated receptor) γ coactivator 1) (Rodgers et al. 2005) and suppression of PPARγ -controlled genes involved in the regulation of fat tissue (Picard et al. 2004). At least seven isoforms exerting diverse functions exist in humans. SIRT1, by far the best investigated of the homologue so far, 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 and Lecour, 2007). Resveratrol has been shown to be able to activate SIRT1 (but not other sirtuin homologs) 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 aging process. Treatment of mice with resveratrol leads to an increase in mitochondrial size, enzymatic activity, mitochondrial DNA (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-1α, with SIRT1 deacetylating and hereby activating PGC-1α (Lagouge et al. 2006). A recent study in mice on a high-fat diet moderately over-expressing SIRT1 proved that SIRT1 can lower lipid-induced inflammation along with an improved glucose tolerance. In part these effects are mediated by down-modulation of NFkB resulting in reduced activation of proinflammatory cytokines such as TNFα and IL-6 and an increased expression of MnSOD (Pfluger et al. 2007). The effects of a natural compound on life span will certainly lead to the development of more potent analogs of resveratrol for in vivo use in the future (Koo and 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). Also in a model of stress-induced premature senescence (SIPS) by treating human umbilical vein endothelial cells with hydrogen peroxide, the selective PDE3 inhibitor cilostazol reduced the senescent cell phenotype by upregulation of SIRT1 (Ota et al. 2008). These findings 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 beneficial for the aged cardiovascular system in order to restore endothelial function by activating eNOS and by reducing mitochondrial •O2– formation via induction of MnSOD expression.

A mechanistic concept of age-associated mitochondrial dysfunction

Mitochondrial proofreading-deficient polymerase γ (Polγ) knock-in mice have been shown to accumulate mtDNA mutations and deletions, resulting in premature aging (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. MnSOD, 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 and M. Bachschmid, unpublished data). MnSOD 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 finger. This suggests that sirtuins are sensitive to ROS and can lose their ability to maintain mtDNA into 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 Polγ proofreading-deficient mice exhibiting the features of premature aging (Trifunovic et al. 2004; Kujoth et al. 2005) (figure 2).

Figure 2. Cytosolic control of mitochondrial ROS formation and effects of sirtuins.

Figure 2

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Various signaling cascades can affect mitochondrial physiology, mediate mitochondrial radical formation or are connected to apoptotic signaling. Oxidative stress activates PKCβ leading to serine-phosphorylation of p66Shc, which subsequently translocates to the mitochondrial intermembrane space and associates with the 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 and p21Ras. Both molecules are assumed to sequester antiapoptotic proteins of the Bcl-family, 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. ROS may inactivate SIRT1 by oxidation of an essential structural Zn-finger that may result in p53 accumulation and mitochondrial translocation. Another example is TNF-signaling involving 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, protein-DNA complexes, and increase the mtDNA mutation rate. Similar to organization of nuclear DNA, mitochondrial sirtuins may regulate nucleoid plasticity.

Abbreviations: SIRT, sirtuin, Ac, acetylation; ROS, reactive oxygen species; MnSOD, manganese superoxide dismutase; Gpx, glutathione peroxidase; mTFA, mitochondrial transcription factor A.

If this concept holds true, the potential therapeutic implications to combat the loss of protection of mtDNA against ROS may have an enormous impact.

Acknowledgements

This work was supported by the Whitaker Cardiovascular Institute and NIH grants R01 AG027080-04.

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