Andrej Podlutsky

Oxidative stress and accelerated vascular aging: Implications for cigarette smoking

Cigarette smoking is the major cause of preventable morbidity and mortality in the United States and constitutes a major risk factor for atherosclerotic vascular disease, including coronary artery disease and stroke. Increasing evidence supports the hypothesis that oxidative stress and inflammation provide the pathophysiological link between cigarette smoking and CAD. Previous studies have shown that cigarette smoke activates leukocytes to release reactive oxygen and nitrogen species (ROS/RNS) and secrete pro-inflammatory cytokines, increases the adherence of monocytes to the endothelium and elicits airway inflammation. Here we present an overview of the direct effects of water-soluble cigarette smoke constituents on endothelial function, vascular ROS production and inflammatory gene expression. The potential pathogenetic role of peroxynitrite formation, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation in cardiovascular complications in smokers are also discussed.

Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms

An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H2O2) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, medium‐sized mammals, birds). During succinate oxidation, H2O2 production rates were generally lower in the longer‐lived species; the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, white‐footed mouse, naked mole‐rat, Damara mole‐rat, guinea pig, baboon, little brown bat, Brazilian free‐tailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H2O2 production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H2O2 production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H2O2 production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms.

Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations

The dietary polyphenolic compound resveratrol, by activating the protein deacetylase enzyme silent information regulator 2/sirtuin 1 (SIRT1), prolongs life span in evolutionarily distant organisms and may mimic the cytoprotective effects of dietary restriction. The present study was designed to elucidate the effects of resveratrol on cigarette smoke-induced vascular oxidative stress and inflammation, which is a clinically highly relevant model of accelerated vascular aging. Cigarette smoke exposure of rats impaired the acetylcholine-induced relaxation of carotid arteries, which could be prevented by resveratrol treatment. Smoking and in vitro treatment with cigarette smoke extract (CSE) increased reactive oxygen species production in rat arteries and cultured coronary arterial endothelial cells (CAECs), respectively, which was attenuated by resveratrol treatment. The smoking-induced upregulation of inflammatory markers (ICAM-1, inducible nitric oxide synthase, IL-6, and TNF-alpha) in rat arteries was also abrogated by resveratrol treatment. Resveratrol also inhibited CSE-induced NF-kappaB activation and inflammatory gene expression in CAECs. In CAECs, the aforementioned protective effects of resveratrol were abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicked the effects of resveratrol. Resveratrol treatment of rats protected aortic endothelial cells against cigarette smoking-induced apoptotic cell death. Resveratrol also exerted antiapoptotic effects in CSE-treated CAECs, which could be abrogated by knockdown of SIRT1. Resveratrol treatment also attenuated CSE-induced DNA damage in CAECs (comet assay). Thus resveratrol and SIRT1 exert antioxidant, anti-inflammatory, and antiapoptotic effects, which protect the endothelial cells against the adverse effects of cigarette smoking-induced oxidative stress. The vasoprotective effects of resveratrol will likely contribute to its antiaging action in mammals and may be especially beneficial in pathophysiological conditions associated with accelerated vascular aging.

The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis

Altered structure, and hence function, of cellular macromolecules caused by oxidation can contribute to loss of physiological function with age. Here, we tested whether the lifespan of bats, which generally live far longer than predicted by their size, could be explained by reduced protein damage relative to short‐lived mice. We show significantly lower protein oxidation (carbonylation) in Mexican free‐tailed bats (Tadarida brasiliensis) relative to mice, and a trend for lower oxidation in samples from cave myotis bats (Myotis velifer) relative to mice. Both species of bat show in vivo and in vitro resistance to protein oxidation under conditions of acute oxidative stress. These bat species also show low levels of protein ubiquitination in total protein lysates along with reduced proteasome activity, suggesting diminished protein damage and removal in bats. Lastly, we show that bat‐derived protein fractions are resistant to urea‐induced protein unfolding relative to the level of unfolding detected in fractions from mice. Together, these data suggest that long lifespan in some bat species might be regulated by very efficient maintenance of protein homeostasis.—Salmon, A. B., Leonard, S., Masamsetti, V., Pierce, A., Podlutsky, A. J., Podlutskaya, N., Richardson, A., Austad, S. N., Chaudhuri, A. R. The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis. FASEB J. 23, 2317–2326 (2009)

Oxidative stress in vascular senescence: Lessons from successfully aging species

Cardiovascular disease is a main cause of morbidity and a leading cause of death of elderly Americans. Studies identifying the pathophysiological mechanisms underlying cardiovascular aging hold promise to develop treatments to delay/prevent coronary artery disease and stroke in the elderly. Evidence supporting the roles of oxidative stress and inflammation in the cardiovascular aging process is presented in detail in this review. Mammalian lifespan ranges hundred-fold and we propose that long-living species may be useful models for successful cardiovascular aging in humans. Comparative studies exploiting the large differences in maximum lifespan potential and cardiovascular aging patterns may be particularly relevant. Comparisons of mechanisms related to oxidative stress, oxidative stress resistance and redox signaling between long-living species and shorter-living ones may elucidate key mechanisms for delaying cardiovascular aging. We discuss the potential use of three long-lived but mouse-sized mammalian species, the naked mole-rat (Heterocephalus glaber), the white-footed mouse (Peromyscus leucopus) and the little brown bat (Myotis lucifugus) to test predictions of the oxidative stress theory of aging and elucidate mechanisms by which cardiovascular aging can be delayed.

Aging-Induced Dysregulation of Dicer1-Dependent MicroRNA Expression Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells

Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

Longevity is associated with increased vascular resistance to high glucose-induced oxidative stress and inflammatory gene expression in Peromyscus leucopus

Vascular aging is characterized by increased oxidative stress and proinflammatory phenotypic alterations. Metabolic stress, such as hyperglycemia in diabetes, is known to increase the production of ROS and promote inflammatory gene expression, accelerating vascular aging. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower steady-state production of ROS and/or superior resistance to the prooxidant effects of metabolic stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), which show a more than twofold difference in maximum lifespan potential (8.2 and 3.5 yr, respectively). We compared interspecies differences in steady-state and high glucose (HG; 30 mmol/l)-induced production of O(2)(*-) and H(2)O(2), endothelial function, mitochondrial ROS generation, and inflammatory gene expression in cultured aortic segments. In P. leucopus aortas, steady-state endothelial O(2)(*-) and H(2)O(2) production and ROS generation by mitochondria were less than in M. musculus vessels. Furthermore, vessels of P. leucopus were more resistant to the prooxidant effects of HG. Primary fibroblasts from P. leucopus also exhibited less steady-state and HG-induced ROS production than M. musculus cells. In M. musculus arteries, HG elicited significant upregulation of inflammatory markers (TNF-alpha, IL-6, ICAM-1, VCAM, and monocyte chemoattractant protein-1). In contrast, the proinflammatory effects of HG were blunted in P. leucopus vessels. Thus, increased life span potential in P. leucopus is associated with decreased cellular ROS generation and increased resistance to prooxidant and proinflammatory effects of metabolic stress, which accord with predictions of the oxidative stress hypothesis of aging.

Testing the Oxidative Stress Hypothesis of Aging in Primate Fibroblasts: Is There a Correlation Between Species Longevity and Cellular ROS Production?

The present study was conducted to test predictions of the oxidative stress theory of aging assessing reactive oxygen species production and oxidative stress resistance in cultured fibroblasts from 13 primate species ranging in body size from 0.25 to 120 kg and in longevity from 20 to 90 years. We assessed both basal and stress-induced reactive oxygen species production in fibroblasts from five great apes (human, chimpanzee, bonobo, gorilla, and orangutan), four Old World monkeys (baboon, rhesus and crested black macaques, and patas monkey), three New World monkeys (common marmoset, red-bellied tamarin, and woolly monkey), and one lemur (ring-tailed lemur). Measurements of cellular MitoSox fluorescence, an indicator of mitochondrial superoxide (O2(·-)) generation, showed an inverse correlation between longevity and steady state or metabolic stress-induced mitochondrial O2(·-) production, but this correlation was lost when the effects of body mass were removed, and the data were analyzed using phylogenetically independent contrasts. Fibroblasts from longer-lived primate species also exhibited superior resistance to H(2)O(2)-induced apoptotic cell death than cells from shorter-living primates. After correction for body mass and lack of phylogenetic independence, this correlation, although still discernible, fell short of significance by regression analysis. Thus, increased longevity in this sample of primates is not causally associated with low cellular reactive oxygen species generation, but further studies are warranted to test the association between increased cellular resistance to oxidative stressor and primate longevity.

Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity

Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2-8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16 (INK4a) ), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

Free Radical Production, Antioxidant Capacity, and Oxidative Stress Response Signatures in Fibroblasts From Lewis Dwarf Rats: Effects of Life Span-Extending Peripubertal GH Treatment

The discovery that in invertebrates, disruption of the insulin/insulin-like growth factor (IGF)-1 pathway extends life span and increases resistance to oxidative injury led to the hypothesis that IGF-1 signaling may play a role in regulating cellular reactive oxygen species production, oxidative stress resistance, and consequentially, organismal life span in mammals. However, previous studies testing this hypothesis in rodent models of IGF-1 deficiency yielded controversial results. The Lewis dwarf rat is a useful model of human growth hormone (GH)/IGF-1 deficiency as it mimics many of the pathophysiological alterations present in human GH/IGF-1-deficient patients as well as elderly individuals. Peripubertal treatment of Lewis dwarf rats with GH results in a significant extension of life span. The present study was designed to test the role of the GH/IGF-1 axis in regulating cellular oxidative stress and oxidative stress resistance, utilizing primary fibroblasts derived from control rats, Lewis dwarf rats and GH-replete dwarf rats. Measurements of cellular dihydroethidium and C-H(2)DCFDA fluorescence showed that cellular O(2)(·-) and peroxide production were similar in each group. Fibroblasts from control and Lewis dwarf rats exhibited similar antioxidant capacities and comparable sensitivity to H(2)O(2), rotenone, high glucose, tunicamycin, thapsigargin, paraquat, and mitomycin, which cause apoptosis through increasing oxidative stress, mitochondrial damage, ATP depletion, and/or by damaging DNA, lipids and proteins. Fibroblasts from GH-replete rats exhibited significantly increased antioxidant capacities and superior resistance to H(2)O(2), rotenone and bacterial lipopolysaccharide-induced cell death compared with cells from Lewis dwarf rats, whereas their sensitivity to the other stressors investigated was not statistically different. Thus, low circulating IGF-1 levels present in vivo in Lewis dwarf rats do not elicit long-lasting alterations in cellular reactive oxygen species generation and oxidative stress resistance, whereas life span-extending peripubertal GH treatment resulted in increased antioxidant capacity and increased resistance to cellular injury caused by some, but not all, oxidative stressors.