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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 (SIRT 1), 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.sub.2][O.sub.2]-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular [H.sub.2][O.sub.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. vasoprotection; histone deacetylase; sirtuin 1 doi: 10.1152/ajpheart.00375.2009.

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1[alpha], nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRTl-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. vasoprotection; histone deacetylase; endothelial dysfunction; diabetes; obesity

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.sup.*-.sub.2] and [H.sub.2][O.sub.2], endothelial function, mitochondrial ROS generation, and inflammatory gene expression in cultured aortic segments. In P. leucopus aortas, steady-state endothelial [O.sup.*-.sub.2] and [H.sub.2][O.sub.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. senescence; comparative biology; vascular disease; atherosclerosis

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial [O.sub.2.sup.x-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress. senescence; vascular disease; atherosclerosis; [Prop1.sup.df/df] mice

There is increasing evidence that TGF-[beta] family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased [[O.sub.2].sup.*-] and [H.sub.2][O.sub.2] generation, induced NF-[kappa]B activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased [[O.sub.2].sup.*-] production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation. systemic; oxidative stress; endothelial dysfunction; pulmonary

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-[kappa]B 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 anti-aging action in mammals and may be especially beneficial in pathophysiological conditions associated with accelerated vascular aging. tobacco; polyphenol; stroke; inflammation; apoptosis; vascular aging; sirtuin 1

Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating agerelated alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-[gamma]/coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells. vascular senescence

The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known [maximum lifespan potential (MLSP): >28 yr] and is a unique model of successful aging showing attenuated declines in most physiological function. This study addresses age-related changes in endothelial function and production of reactive oxygen species in NMR arteries and vessels of shorter-living Fischer 344 rats (MLSP: ~3 yr). Rats exhibit a significant age-dependent decline in acetylcholine-induced responses in carotid arteries over a 2-yr age range. In contrast, over a 10-yr age range nitric oxide (NO)-mediated relaxation responses to acetylcholine and to the NO donor S-nitrosopencillamine (SNAP) were unaltered in NMRs. Cellular superoxide anion ([O.sub.2.sup.x-]) and [H.sub.2][O.sub.2] production significantly increased with age in rat arteries, whereas they did not change substantially with age in NMR vessels. Indicators of apoptotic cell death (DNA fragmentation rate, caspase 3/7 activity) were significantly enhanced (~250-300%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a ~50% increase in apoptotic cell death. In the hearts of NMRs (2 to 26 yr old), expression of endothelial NO synthase, antioxidant enzymes (Cu,Zn-SOD, Mn-SOD, catalase, and glutathione peroxidase), the NAD(P)H oxidase subunit gp[91.sup.phox], and mitochondrial proteins (COX-IV, ATP synthase, and porin, an indicator of mitochondrial mass) did not change significantly with age. Thus long-living NMRs can maintain a youthful vascular function and cellular oxidant-antioxidant phenotype relatively longer and are better protected against aging-induced oxidative stress than shorter-living rats. senescence; comparative biology; vascular disease; atherosclerosis; oxidative stress

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-[kappa]B activity (assessed by luciferase reporter gene assay and NF-[kappa]B binding assay), which was attenuated by scavenging [H.sub.2][O.sub.2]. Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-[kappa]B. In endothelial cells of aged vessels there was an increased production of [H.sub.2][O.sub.2] (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial [H.sub.2][O.sub.2] generation, which was associated with activation of NF-[kappa]B. In aged vessels inhibition of NF-[kappa]B (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-[kappa]B activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial [H.sub.2][O.sub.2] production and/or directly inhibiting NF-[kappa]B novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects. inflammation; endothelial cell; senescence; aging; resveratrol doi:10.1152/ajpheart.01346.2006

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-[alpha] elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment ([10.sup.-6]-[10.sup.-4] mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular [H.sub.2][O.sub.2] levels and [H.sub.2][O.sub.2]-mediated apoptotic cell death induced by oxidative stressors (exogenous [H.sub.2][O.sub.2], paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heine oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged [H.sub.2][O.sub.2] in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging [H.sub.2][O.sub.2] and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects. endothelial cell; comet assay; caloric restriction mimetics; apoptosis; polyphenol; heme oxygenase antioxidant doi:10.1152/ajpheart.01258.2006.

Although the cardiovascular morbidity and mortality induced by cigarette smoking exceed those attributable to lung cancer, the molecular basis of smoking-induced vascular injury remains unclear. To test the link between cigarette smoke, oxidative stress, and vascular inflammation, rats were exposed to the smoke of five cigarettes per day (for 1 wk). Also, isolated arteries were exposed to cigarette smoke extract (CSE; 0 to 40 [micro]g/ml, for 6 h) in organoid culture. We found that smoking impaired acetylcholine-induced relaxations of carotid arteries, which could be improved by the NAD(P)H oxidase inhibitor apocynin. Lucigenin chemiluminescence measurements showed that both smoking and in vitro CSE exposure significantly increased vascular 02' production. Dihydroethidine staining showed that increased [O.sub.2].sub.*.sup.-] generation was present both in endothelial and smooth muscle cells. CSE also increased vascular [H.sub.2][O.sup.2] production (dichlorofluorescein fluorescence). Vascular mRNA expression of the proinflammatory cytokines IL-1[beta], IL-6, and TNF-[beta] and that of inducible nitric oxide synthase was significantly increased by both smoking and CSE exposure, which could be prevented by inhibition of NAD(P)H oxidase (diphenyleneiodonium and apocynin) or scavenging of [H.sub.2]]O.sub.2]. In cultured endothelial cells, CSE elicited NF-[kappa]B activation and increased monocyte adhesiveness, which were prevented by apocynin and catalase. Thus we propose that water-soluble components of cigarette smoke (which are likely to be present in the bloodstream in vivo in smokers) activate the vascular NAD(P)H oxidase. NAD(P)H oxidase-derived [H.sub.2][O.sub.2] activates NF-[kappa]B, leading to proinflammatory alterations in vascular phenotype, which likely promotes development of atherosclerosis, especially if other risk factors are also present. tobacco; oxidative stress; stroke; plaque development; cytokine; arteriosclerosis; endothelial dysfunction

Vascular aging is characterized by decreased nitric oxide (NO) bioavailability, oxidative stress, and enhanced apoptotic cell death. We hypothesized that interspecies comparative assesment of vascular function among rodents with disparate longevity may offer insight into the mechanisms determining successful vascular aging. We focused on four rodents that show approximately an order of magnitude range in maximum longevity (ML). The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known (ML > 28 yr), Damara mole rats (DMRs, Cryptomys damarensis; ML ~ 16 yr) and guinea pigs (GPs, Cavia porcellus; ML ~ 6 yr) have intermediate longevity, whereas laboratory mice are short living (ML ~ 3.5 yr). We compared interspecies differences in endothelial function, [O.sub.2.sup.-] and [H.sub.2][O.sub.2] production, and resistance to apoptotic stimuli in blood vessels. Sensitivity to acetylcholine-induced, NO-mediated relaxation was smaller in carotid arteries from NMRs, GPs, and DMRs than in mouse vessels. Measurements of production of [O.sub.2.sup.-]. (lucigenin chemiluminescence and ethidium bromide fluorescence) and [H.sub.2][O.sub.2] (dichlorofluorescein fluorescence) showed that free radical production in vascular endothelial and smooth muscle cells is comparable in vessels of the three longer-living species and in arteries of shorter-living mice. In mouse arteries, [H.sub.2][O.sub.2] (from [10.sup.-6] to [10.sup.-3] mol/l) and heat exposure (42[degrees]C for 15-45 min) enhanced apoptotic cell death, as indicated by an increased DNA fragmentation rate and increased caspase 3/7 activity. In NMR vessels, only the highest doses of [H.sub.2][O.sub.2] enhanced apoptotic cell death, whereas heat exposure did not increase DNA fragmentation rate. Interspecies comparison showed there is a negative correlation between [H.sub.2][O.sub.2]-induced apoptotic cell death and ML. Thus endothelial vasodilator function and vascular production of reactive oxygen species do not correlate with maximal lifespan, whereas increased lifespan potential is associated with an increased vascular resistance to proapoptotic stimuli. senescence; comparative biology; vascular disease; atherosclerosis;

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its cardioprotective effects are not completely understood. Because TNF-[alpha]-induced endothelial activation and vascular inflammation play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits TNF-[alpha]-induced signal transduction in human coronary arterial endothelial cells (HCAECs). We found that TNF-[alpha] significantly increased adhesiveness of the monocytic THP-1 cells to HCAECs, an effect that could be inhibited by pretreatment with resveratrol and the NF-[kappa]B inhibitor pyrrolidine dithiocarbamate. Previously, we found that TNF-[alpha] activates NAD(P)H oxidases, and our recent data showed that TNF-[alpha]-induced endothelial activation was prevented by the NAD(P)H oxidase inhibitor apocynin or catalase plus SOD. Resveratrol also inhibited [H.sub.2][O.sub.2]-induced monocyte adhesiveness. Using a reporter gene assay, we found that, in HCAECs, TNF-[alpha] significantly increased NF-[kappa]B activity, which could be inhibited by resveratrol (>50% inhibition at [10.sup.-6] mol/l) and pyrrolidine dithiocarbamate. Resveratrol also inhibited TNF-[alpha]-induced, NF-[kappa]B-driven luciferase expression in rat aortas electroporated with the reporter gene construct. In TNF-[alpha]-treated HCAECs, resveratrol (in the submicromolar range) significantly attenuated expression of NF-[kappa]B-dependent inflammatory markers inducible nitric oxide synthase, IL-6, bone morphogenetic protein-2, ICAM-1, and VCAM. Thus resveratrol at nutritionally relevant concentrations inhibits TNF-[alpha]-induced NF-[kappa]B activation and inflammatory gene expression and attenuates monocyte adhesiveness to HCAECs. We propose that these anti-inflammatory actions of resveratrol are responsible, at least in part, for its cardioprotective effects. stilbene; atheroprotection; phytoestrogen; vascular aging; inflammation