Zoltan Ungvari

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Aging-Induced Phenotypic Changes and Oxidative Stress Impair Coronary Arteriolar Function

We aimed to elucidate the possible role of phenotypic alterations and oxidative stress in age-related endothelial dysfunction of coronary arterioles. Arterioles were isolated from the hearts of young adult (Y, 14 weeks) and aged (A, 80 weeks) male Sprague-Dawley rats. For videomicroscopy, pressure-induced tone of Y and A arterioles and their passive diameter did not differ significantly. In A, arterioles L-NAME (a NO synthase blocker)–sensitive flow-induced dilations were significantly impaired (Y: 41±8% versus A: 3±2%), which could be augmented by superoxide dismutase (SOD) or Tiron (but not l-arginine or the TXA2 receptor antagonist SQ29,548). For lucigenin chemiluminescence, O2·− generation was significantly greater in A than Y vessels and could be inhibited with SOD and diphenyliodonium. NADH-driven O2·− generation was also greater in A vessels. Both endothelial and smooth muscle cells of A vessels produced O2·− (shown with ethidium bromide fluorescence). For Western blotting, expression of eNOS and COX-1 was decreased in A compared with Y arterioles, whereas expressions of COX-2, Cu/Zn-SOD, Mn-SOD, xanthine oxidase, and the NAD(P)H oxidase subunits p47phox, p67phox, Mox-1, and p22phox did not differ. Aged arterioles showed an increased expression of iNOS, confined to the endothelium. Decreased eNOS mRNA and increased iNOS mRNA expression in A vessels was shown by quantitative RT-PCR. In vivo formation of peroxynitrite was evidenced by Western blotting, and immunohistochemistry showing increased 3-nitrotyrosine content in A vessels. Thus, aging induces changes in the phenotype of coronary arterioles that could contribute to the development of oxidative stress, which impairs NO-mediated dilations.

Mechanisms of Vascular Aging: New Perspectives

This review focuses on molecular, cellular, and functional changes that occur in the vasculature during aging; explores the links between mitochondrial oxidative stress, inflammation, and development of vascular disease in the elderly patients; and provides a landscape of molecular mechanisms involved in cellular oxidative stress resistance, which could be targeted for the prevention or amelioration of unsuccessful vascular aging. Practical interventions for prevention of age-associated vascular dysfunction and disease in old age are considered here based on emerging knowledge of the effects of anti-inflammatory treatments, regular exercise, dietary interventions, and caloric restriction mimetics.

Inflammation and endothelial dysfunction during aging: role of NF-κB

One of the major conceptual advances in our understanding of the pathogenesis of age-associated cardiovascular diseases has been the insight that age-related oxidative stress may promote vascular inflammation even in the absence of traditional risk factors associated with atherogenesis (e.g., hypertension or metabolic diseases). In the present review we summarize recent experimental data suggesting that mitochondrial production of reactive oxygen species, innate immunity, the local TNF-alpha-converting enzyme (TACE)-TNF-alpha, and the renin-angiotensin system may underlie NF-kappaB induction and endothelial activation in aged arteries. The theme that emerges from this review is that multiple proinflammatory pathways converge on NF-kappaB in the aged arterial wall, and that the transcriptional activity of NF-kappaB is regulated by multiple nuclear factors during aging, including nuclear enzymes poly(ADP-ribose) polymerase (PARP-1) and SIRT-1. We also discuss the possibility that nucleophosmin (NPM or nuclear phosphoprotein B23), a known modulator of the cellular oxidative stress response, may also regulate NF-kappaB activity in endothelial cells.

Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. Resveratrol was also shown to confer vasoprotection in animal models of type 2 diabetes and aging. However, the mechanisms by which resveratrol exerts its antioxidative vasculoprotective effects are not completely understood. Using a nuclear factor-E(2)-related factor-2 (Nrf2)/antioxidant response element-driven luciferase reporter gene assay, we found that in cultured coronary arterial endothelial cells, resveratrol, in a dose-dependent manner, significantly increases transcriptional activity of Nrf2. Accordingly, resveratrol significantly upregulates the expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1, gamma-glutamylcysteine synthetase, and heme oxygenase-1. Resveratrol treatment also significantly attenuated high glucose (30 mM)-induced mitochondrial and cellular oxidative stress (assessed by flow cytometry using MitoSox and dihydroethidine staining). The aforementioned effects of resveratrol were significantly attenuated by the small interfering RNA downregulation of Nrf2 or the overexpression of Kelch-like erythroid cell-derived protein 1, which inactivates Nrf2. To test the effects of resveratrol in vivo, we used mice fed a high-fat diet (HFD), which exhibit increased vascular oxidative stress associated with an impaired endothelial function. In HFD-fed Nrf2(+/+) mice, resveratrol treatment attenuates oxidative stress (assessed by the Amplex red assay), improves acetylcholine-induced vasodilation, and inhibits apoptosis (assessed by measuring caspase-3 activity and DNA fragmentation) in branches of the femoral artery. In contrast, the aforementioned endothelial protective effects of resveratrol were diminished in HFD-fed Nrf2(-/-) mice. Taken together, our results indicate that resveratrol both in vitro and in vivo confers endothelial protective effects which are mediated by the activation of Nrf2.

Increased Superoxide Production in Coronary Arteries in Hyperhomocysteinemia Role of Tumor Necrosis Factor-α, NAD(P)H Oxidase, and Inducible Nitric Oxide Synthase

Objective—In coronary arteries, hyperhomocysteinemia (HHcy, a known risk factor for coronary heart disease) impairs flow-induced dilations, which can be reversed by superoxide dismutase (SOD). To evidence increased O2.− generation and elucidate its source, we characterized changes in activity (lucigenin chemiluminescence, hydroethidine staining) and expression of arterial pro- and antioxidant systems (Western blotting, immunohistochemistry, cDNA microarray, reverse-transcription polymerase chain reaction) in the coronary arteries of rats by using methionine diet-induced HHcy. Methods and Results—The increased generation of O2.− by HHcy coronary arteries was inhibited by SOD, diphenyleneiodonium, apocynin, and apocynin plus amino guanidine but was unaffected by allopurinol and rotenone. Also, diphenyleneiodonium-sensitive NADPH-driven O2.− generation was increased in HHcy vessels. In HHcy arteries expression of the smooth muscle-confined NAD(P)H oxidase subunit nox1 and that of iNOS was increased. Expression of p67phox, p22phox, and p47phox subunits and that of endothelial nitric oxide synthase, Cu,Zn-SOD, Mn-SOD, extracellular SOD (mRNA), and xanthine oxidase was unchanged. Microarray analysis showed increased expression of tumor necrosis factor (TNF)-&agr; (confirmed by reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry) that was localized in smooth muscle. In vitro incubation (18 hours) of HHcy arteries with anti-TNF-&agr; antibody decreased O2.− production, whereas incubation of control vessels with TNF-&agr; increased O2.− generation and nox1 expression. Conclusions—In coronary arteries, HHcy increases TNF-&agr; expression, which enhances oxidative stress through upregulating a nox1-based NAD(P)H oxidase and inducible nitric oxide synthase. Thus, TNF-&agr; induces a proinflammatory vascular phenotype in HHcy that potentially contributes to the development of coronary atherosclerosis.

Potent Metalloporphyrin Peroxynitrite Decomposition Catalyst Protects Against the Development of Doxorubicin-Induced Cardiac Dysfunction

Background—Increased oxidative stress and dysregulation of nitric oxide have been implicated in the cardiotoxicity of doxorubicin (DOX), a commonly used antitumor agent. Peroxynitrite is a reactive oxidant produced from nitric oxide and superoxide in various forms of cardiac injury. Using a novel metalloporphyrinic peroxynitrite decomposition catalyst, FP15, and nitric oxide synthase inhibitors or knockout mice, we now delineate the pathogenetic role of peroxynitrite in rodent models of DOX-induced cardiac dysfunction. Methods and Results—Mice received a single injection of DOX (25 mg/kg IP). Five days after DOX administration, left ventricular performance was significantly depressed, and high mortality was noted. Treatment with FP15 and an inducible nitric oxide synthase inhibitor, aminoguanidine, reduced DOX-induced mortality and improved cardiac function. Genetic deletion of the inducible nitric oxide synthase gene was also accompanied by better preservation of cardiac performance. In contrast, inhibition of the endothelial isoform of nitric oxide synthase with N-nitro-l-arginine methyl ester increased DOX-induced mortality. FP15 reduced the DOX-induced increase in serum LDH and creatine kinase activities. Furthermore, FP15 prevented the DOX-induced increase in lipid peroxidation, nitrotyrosine formation, and metalloproteinase activation in the heart but not NAD(P)H-driven superoxide generation. Peroxynitrite neutralization did not interfere with the antitumor effect of DOX. FP15 also decreased ischemic injury in rats and improved cardiac function and survival of mice in a chronic model of DOX-induced cardiotoxicity. Conclusions—Thus, peroxynitrite plays a key role in the pathogenesis of DOX-induced cardiac failure. Targeting peroxynitrite formation may represent a new cardioprotective strategy after DOX exposure or in other conditions associated with peroxynitrite formation, including myocardial ischemia/reperfusion injury.

Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells.

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted H(2)O(2)-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular H(2)O(2) levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Are sirtuins viable targets for improving healthspan and lifespan

Although the increased lifespan of our populations illustrates the success of modern medicine, the risk of developing many diseases increases exponentially with old age. Caloric restriction is known to retard ageing and delay functional decline as well as the onset of disease in most organisms. Studies have implicated the sirtuins (SIRT1–SIRT7) as mediators of key effects of caloric restriction during ageing. Two unrelated molecules that have been shown to increase SIRT1 activity in some settings, resveratrol and SRT1720, are excellent protectors against metabolic stress in mammals, making SIRT1 a potentially appealing target for therapeutic interventions. This Review covers the current status and controversies surrounding the potential of sirtuins as novel pharmacological targets, with a focus on SIRT1.

Aging-induced proinflammatory shift in cytokine expression profile in coronary arteries

The phenotypic and functional changes of coronary arteries with aging promote ischemic heart disease. We hypothesized that these alterations reflect an aging‐induced proinflammatory shift in vascular regulatory mechanisms. Thus, in isolated coronary arteries of young (3‐month‐old) and aged (25‐month‐old) male Fischer 344 rats the expression of 96 cytokines, chemokines, and their receptors were screened by a cDNA‐based microarray technique. In aged vessels expressions of tumor necrosis factor (TNF)‐α (3.3x), interleukin (IL)‐1β (3.0x), IL‐6 (2.9x), IL‐6Rα (2.8x) and IL‐17 (6.1x) genes were significantly increased over young vessels. Quantitative reverse transcriptase‐polymerase chain reaction confirmed these results. Western blotting demonstrated that protein expressions of TNF‐α, IL‐1β, IL‐6, and IL‐17 were also significantly increased in vessels of aged rats compared with those of young rats. Immunofluorescent double labeling showed that in aged vessels IL‐1β and IL‐6 are predominantly localized in the endothelium, whereas TNF‐α and IL‐17 are localized in smooth muscle. Thus, a proinflammatory shift in the profile of vascular cytokine expression may contribute to the aging‐induced phenotypic changes in coronary arteries, promoting the development of ischemic heart disease in the elderly.