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Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-[Lepr.sup.db]) mice and type 2 diabetic ([Lepr.sup.db]) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in [Lepr.sup.db] mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O2 production by inhibiting NAD(P)H oxidase activity and gp91phox mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in [Lepr.sup.db] mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in [Lepr.sup.db] mice. Furthermore, TNF-[alpha] mRNA and protein expression, as well as NF-[kappa]B activation, were reduced in resveratrol-treated [Lepr.sup.db] mice. Both [Lepr.sup.db] mice null for TNF-[alpha] ([db.sup.TNF-] /[db.sup.TNF-mice]) and [Lepr.sup.db] mice treated with the NF-[kappa]B inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-[Lepr.sup.db] mice treated with TNF-[alpha] showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-[alpha]-induced NF-[kappa]B activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes. cardiac diabetic complication; antioxidants and free radicals; anti-inflammation; nitric oxide; magnetic resonance spectroscopy doi: 10.1152/ajpheart.00489.2010.

The role of circulating, systemic TGF-[beta] levels in endothelial function is not clear. TGF-[[beta].sub.1] may cause endothelial dysfunction in apolipoprotein E-deficient ([apoE.sup.-/-]) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-too-old control (C57B1/6), [apoE.sup.-/-], TGF-[[beta].sub.1]-overexpressing (TGF[[beta].sub.1]), and crossbred [apoE.sup.-/-] x TGF[[beta].sub.1] mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-too-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-[[beta].sub.1] levels (ELISA) were elevated in TGF[[beta].sub.1] mice without downregulation of TGF-[beta]-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in [apoE.sup.-/-] x TGF[[beta].sub.1] mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the [apoE.sup.-/-] x TGF[beta] vs. C57B1/6 mice. Our results demonstrate that elevated circulating TGF-[[beta].sub.1] causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in [apoE.sup.-/-] mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGF[[beta].sub.1]. aorta; oxidative stress; nicotinamide adenine dinucleotide phosphate; transforming growth factor-[beta]; apolipoprotein E-deficient mice doi: 10.1152/ajpheart.01042.2009.

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-E2-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 oxido-reductase 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.sup.+/+] 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.sup.-/-] 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. endothelial cell; gracilis; resveratrol; nuclear factor-Ez-related factor-2 doi: 10.1152/ajpheart.00260.2010.

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

Targeting cannabinoid-2 ([CB.sub.2]) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which [CB.sub.2] activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of [CB.sub.2]-receptor activation on TNF-[alpha]-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-[alpha] induced NF-[kappa]B and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF-[alpha] were attenuated by [CB.sub.2] agonists. [CB.sub.2] agonists also decreased the TNF-[alpha]- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. [CB.sub.1] and [CB.sub.2] receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF-[alpha]-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting [CB.sub.2] receptors on endothelial cells may offer a novel approach in the treatment of these pathologies. endothelial activation; inflammation; RhoA; adhesion molecules

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

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising novel approach for the treatment of various inflammatory disorders. In this study, we have investigated the age-associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (FAA[H.sup.-/-]) mice and their wild-type (FAA[H.sup.+/+]) littermates. Additionally, we have explored the effects of anandamide on TNF-[alpha]-induced ICAM-1 and VCAM-1 expression and monocyte-endothelial adhesion in human coronary artery endothelial cells (HCAECs). There was no difference in the cardiac function (measured by the pressure-volume conductance catheter system) between 2- to 3-mo-old (young) FAA[H.sup.-/-] and FAA[H.sup.+/+] mice. In contrast, the aging-associated decline in cardiac function and increased myocardial gene expression of TNF-[alpha], gp91phox, matrix metalloproteinase (MMP)-2, MMP-9, caspase-3 and caspase-9, myocardial inducible nitric oxide synthase protein expression, nitrotymsine formation, poly (ADP-ribose)polymerase cleavage and caspase3/9 activity, observed in 28- to 31-mo-old (aging) FAA[H.sup.+/+] mice, were largely attenuated in knockouts. There was no difference in the myocardial cannabinoid C[B.sub.1] and C[B.sub.2] receptor gene expression between young and aging FAA[H.sup.-/-] and FAA[H.sup.+/+] mice. Anandamide dose dependently attenuated the TNF-[alpha]-induced ICAM-1 and VCAM-1 expression, NF-[kappa]B activation in HCAECs, and the adhesion of monocytes to HCAECs in a C[B.sub.1]-and C[B.sub.2]-dependent manner. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis. cardiac function; anandamide; pressure-volume relationship; endocannabinoids

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

Pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (Epi), were employed to examine whether changes in NADP(H) redox regulates contractile force in endothelium-removed bovine coronary arteries (BCAs). 6-AN (0.01-5 mM) or Epi (1-500 [micro]M) elicited dose-dependent relaxation in BCAs contracted with 30 mM KCl, 0.1 [micro]M U-44619, and endothelin-1 but not with phorbol 12,13-dibutyrate, a protein kinase C activator that causes [Ca.sup.2+]-independent contraction. Relaxation to PPP inhibition was associated with oxidation of NADPH and glutathione (GSH). Relaxation to 6-AN was not mediated by [H.sub.2][O.sub.2], because it was not altered by hypoxia or the peroxide scavenger ebselen (100 [micro]M). The thiol reductant DTT (3 mM) attenuated the relaxation to 6-AN and Epi by 30-40%. Inhibition of glycolysis of mitochondrial electron transport did not elicit relaxation in BCAs contracted with 30 mM KCl, suggesting these pathways may not be involved in relaxation elicited by PPP inhibition. High doses of [K.sup.+] channel blockers [e.g., TEA (10 mM) and 4-aminopyridine (10 mM)] only partially inhibited the relaxation to 6-AN. On the basis of changes in the fura-2 fluorescence ratio, 6-AN and Epi appeared to markedly reduce intracellular [Ca.sup.2+]. Thus PPP inhibition oxidizes NADPH and GSH and appears to activate a novel coordination of redox-controlled relaxing mechanisms in BCAs mediated primarily through decreasing intracellular C[a.sup.2+] calcium; redox signaling; vasodilator mechanisms

In skeletal muscle arterioles, the pathway leading to non-nitric oxide (NO), non-prostaglandin-mediated endothelium-derived hyperpolarizing factor (EDHF)-type dilations is not well characterized. To elucidate some of the steps in this process, simultaneous changes in endothelial intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and the diameter of rat gracilis muscle arterioles (~60 [micro]m) to acetylcholine (ACh) were measured by fura 2 microfluorimetry (in the absence of NO and prostaglandins). ACh elicited rapid increases in endothelial [[[Ca.sup.2+]].sub.i] (101 [+ or -] 7%), followed by substantial dilations (73 [+ or -] 2%, coupling time: 1.3 [+ or -] 0.2 s) that were prevented by endothelial loading of an intracellular [Ca.sup.2+] chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Arteriolar dilations to ACh were also inhibited by intraluminal administration of the [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channel blockers charybdotoxin plus apamin or by palmitoleic acid, an uncoupler of myoendothelial gap junctions without affecting changes in endothelial [[[Ca.sup.2+]].sub.i]. The presence of large conductance [K.sub.Ca] channels on arteriolar endothelial cells was demonstrated with immunohistochemisty. We propose that in skeletal muscle arterioles, EDHF-type mediation is evoked by an increase in endothelial [[[Ca.sup.2+]].sub.i], which by activating endothelial [K.sub.Ca] channels elicits hyperpolarization that is conducted via myoendothelial gap junctions to the smooth muscle resulting in decreases in [[[Ca.sup.2+]].sub.i] and consequently dilation. endothelium-dependent hyperpolarizing factor; arteriolar endothelium; potassium channels; charybdotoxin

Over two-thirds of individuals aged 65 and older are obese or overweight in the United States. Epidemiological data show an association between the degree of adiposity and cognitive dysfunction in the elderly. In this review, the pathophysiological roles of microvascular mechanisms, including impaired endothelial function and neurovascular coupling responses, microvascular rarefaction, and blood-brain barrier disruption in the genesis of cognitive impairment in geriatric obesity are considered. The potential contribution of adipose-derived factors and fundamental cellular and molecular mechanisms of senescence to exacerbated obesity-induced cerebromicrovascular impairment and cognitive decline in aging are discussed. [ABSTRACT FROM AUTHOR]

Age-related alterations in endothelium and the resulting vascular dysfunction critically contribute to a range of pathological conditions associated with old age. To develop therapies rationally that improve vascular health and thereby increase health span and life span in older adults, it will be essential to understand the cellular and molecular mechanisms contributing to vascular aging. Preclinical studies in model organisms demonstrate that NAD+ availability decreases with age in multiple tissues and that supplemental NAD+ precursors can ameliorate many age-related cellular impairments. Here, we provide a comprehensive overview of NAD+-dependent pathways [including the NAD+-using silent information regulator- 2-like enzymes and poly(ADP-ribose) polymerase enzymes] and the potential consequences of endothelial NAD+ deficiency in vascular aging. The multifaceted vasoprotective effects of treatments that reverse the age-related decline in cellular NAD+ levels, as well as their potential limitations, are discussed. The preventive and therapeutic potential of NAD+ intermediates as effective, clinically relevant interventions in older adults at risk for ischemic heart disease, vascular cognitive impairment, and other common geriatric conditions and diseases that involve vascular pathologies (e.g., sarcopenia, frailty) are critically discussed. We propose that NAD+ precursors [e.g., nicotinamide (Nam) riboside, Nam mononucleotide, niacin] should be considered as critical components of combination therapies to slow the vascular aging process and increase cardiovascular health span. [ABSTRACT FROM AUTHOR]

In cultured endothelial cells, [[Ca.sup.2+]]-dependent and -independent activation of nitric oxide (NO) synthesis to agonists and flow/wall shear stress (WSS) has been demonstrated. However, the presence and function of these pathways are less well known in microvessels that can be exposed to a high level of WSS. We hypothesized that the role of changes in endothelial intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) is different in agonist- and WSS-induced release of NO. Thus changes in endothelial [[[Ca.sup.2+]].sub.i] and diameter of intact pressurized (~100 [micro]m at 80 mmHg) gracilis skeletal muscle arterioles of rats were measured by fluorescent video-microscopy. Acetylcholine (ACh) and increases in WSS (by increasing intraluminal flow) elicited dilations (maximum 91 [+ or -] 2% and 34 [+ or -] 4%) that could be inhibited by [N.sup.[Omega]]-nitro-L-arginine methyl ester (L-NAME), a NO synthase blocker. In diameter-clamped arterioles, ACh caused substantial increases in the endothelial calcium fluorescence ratio ([ER.sub.Ca], maximum 43 [+ or -] 5%), which was significantly greater than changes in [ER.sub.Ca] (maximum ~10%) to increases in WSS. The [[Ca.sup.2+]] ionophore A-23187 also substantially increased [ER.sub.Ca] (maximum 38 [+ or -] 5%) and elicited significant L-NAME-sensitive arteriolar dilations (maximum 45 [+ or -] 7%). Intraluminal administration of the tyrosine kinase inhibitor genistein had no effect on dilations induced by ACh or the NO donor sodium nitroprusside, whereas it eliminated WSS-induced dilations. Collectively, our data suggest that, in endothelium of skeletal muscle arterioles, NO synthesis is activated by shear stress without a substantial increase in [[[Ca.sup.2+]].sub.i], most likely by activation of tyrosine kinase pathways, whereas NO release by ACh and A-23187 is associated with substantial increases in [[[Ca.sup.2+]]i.sub.]. fura 2; endothelial calcium fluorescence ratio; calcium-independent pathways; tyrosine kinase; endothelium-derived hyperpolarizing factor

Impairment of moment-tomoment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS-/-) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS-/- mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia. [ABSTRACT FROM AUTHOR]