Increased availability of angiotensin AT 1 receptors leads to sustained arterial constriction to angiotensin II in diabetes - role for Rho-kinase activation.

BACKGROUND AND PURPOSE: Antagonists of angiotensin AT(1) receptors elicit beneficial vascular effects in diabetes mellitus. We hypothesized that diabetes induces sustained availability of AT(1) receptors, causing enhanced arterial constriction to angiotensin II. EXPERIMENTAL APPROACH: To assess functional availability of AT(1) receptors, constrictions to successive applications of angiotensin II were measured in isolated skeletal muscle resistance arteries (∼150 µm) of Zucker diabetic fatty (ZDF) rats and of their controls (+/Fa), exposed acutely to high glucose concentrations (HG, 25 mM, 1 h). AT(1) receptors on cell membrane surface were measured by immunofluorescence. KEY RESULTS: Angiotensin II-induced constrictions to first applications were greater in arteries of ZDF rats (maximum: 82 ± 3% original diameter) than in those from +/Fa rats (61 ± 5%). Constrictions to repeated angiotensin II administration were decreased in +/Fa arteries (20 ± 6%), but were maintained in ZDF arteries (67 ± 4%) and in +/Fa arteries vessels exposed to HG (65 ± 6%). In ZDF arteries and in HG-exposed +/Fa arteries, Rho-kinase activities were enhanced. The Rho-kinase inhibitor, Y27632 inhibited sustained constrictions to angiotensin II in ZDF arteries and in +/Fa arteries exposed to HG. Levels of surface AT(1) receptors on cultured vascular smooth muscle cells (VSMCs) were decreased by angiotensin II but were maintained in VSMCs exposed to HG. In VSMCs exposed to HG and treated with Y27632, angiotensin II decreased surface AT(1) receptors. CONCLUSIONS AND IMPLICATIONS: In diabetes, elevated glucose concentrations activate Rho-kinase which inhibits internalization or facilitates recycling of AT(1) receptors, leading to increased functional availability of AT(1) receptors and sustained angiotensin II-induced arterial constriction.

Role of NAD(P)H oxidase in superoxide generation and endothelial dysfunction in Goto-Kakizaki (GK) rats as a model of nonobese NIDDM.

BACKGROUND: Cardiovascular disease is the leading cause of mortality in diabetics, and it has a complex etiology that operates on several levels. Endothelial dysfunction and increased generation of reactive oxygen species are believed to be an underlying cause of vascular dysfunction and coronary artery disease in diabetes. This impairment is likely the result of decreased bioavailability of nitric oxide (NO) within the vasculature. However, it is unclear whether hyperglycemia per se stimulates NADPH oxidase-derived superoxide generation in vascular tissue. METHODS AND RESULTS: This study focused on whether NADPH oxidase-derived superoxide is elevated in vasculature tissue evoking endothelial/smooth muscle dysfunction in the hyperglycemic (169+/-4 mg%) Goto-Kakizaki (GK) rat. By dihydroethidine fluorescence staining, we determined that aorta superoxide levels were significantly elevated in 9 month-old GK compared with age matched Wistar (GK; 195+/-6%, Wistar; 100+/-3.5%). Consistent with these findings, 10(-6) mol/L acetylcholine-induced relaxation of the carotid artery was significantly reduced in GK rats compared with age matched Wistar (GK; 41+/-7%, Wistar; 100+/-5%) and measurements in the aorta showed a similar trend (p = .08). In contrast, relaxation to the NO donor SNAP was unaltered in GK compared to Wistar. Endothelial dysfunction was reversed by lowering of superoxide with apocynin, a specific Nox inhibitor. CONCLUSIONS: The major findings from this study are that chronic hyperglycemia induces significant vascular dysfunction in both the aorta and small arteries. Hyperglycemic induced increases in NAD(P)H oxidase activity that did not come from an increase in the expression of the NAD(P)H oxidase subunits, but more likely as a result of chronic activation via intracellular signaling pathways.

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.

Resveratrol prevents monocrotaline-induced pulmonary hypertension in rats.

Proliferation of pulmonary arterial smooth muscle cells, endothelial dysfunction, oxidative stress, and inflammation promotes the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exerts antioxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteries remain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonary hypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment (25 mg/kg per day, PO, from day 1 postmonocrotaline) attenuated right ventricular systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (tumor necrosis factor-alpha, interleukin 1beta, interleukin 6, and platelet-derived growth factor-alpha/beta), and limited leukocyte infiltration in the lung. Resveratrol also inhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol increased expression of endothelial NO synthase, decreased oxidative stress, and improved endothelial function in small pulmonary arteries. Pulmonary hypertension was associated with an upregulation of NAD(P)H oxidase in small pulmonary arteries, which was significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory, antioxidant, and antiproliferative effects in the pulmonary arteries, which may contribute to the prevention of pulmonary hypertension.

Anti-oxidative and anti-inflammatory vasoprotective effects of caloric restriction in aging: role of circulating factors and SIRT1.

Endothelial dysfunction, oxidative stress and inflammation are associated with vascular aging and promote the development of cardiovascular disease. Caloric restriction (CR) mitigates conditions associated with aging, but its effects on vascular dysfunction during aging remain poorly defined. To determine whether CR exerts vasoprotective effects in aging, aortas of ad libitum (AL) fed young and aged and CR-aged F344 rats were compared. Aging in AL-rats was associated with impaired acetylcholine-induced relaxation, vascular oxidative stress and increased NF-kappaB activity. Lifelong CR significantly improved endothelial function, attenuated vascular ROS production, inhibited NF-kappaB activity and down-regulated inflammatory genes. To elucidate the role of circulating factors in mediation of the vasoprotective effects of CR, we determined whether sera obtained from CR animals can confer anti-oxidant and anti-inflammatory effects in cultured coronary arterial endothelial cells (CAECs), mimicking the effects of CR. In CAECs cultured in the presence of AL serum TNFalpha elicited oxidative stress, NF-kappaB activation and inflammatory gene expression. By contrast, treatment of CAECs with CR serum attenuated TNFalpha-induced ROS generation and prevented NF-kappaB activation and induction of inflammatory genes. siRNA knockdown of SIRT1 mitigated the anti-oxidant and anti-inflammatory effects of CR serum. CR exerts anti-oxidant and anti-inflammatory vascular effects, which are likely mediated by circulating factors, in part, via a SIRT1-dependent pathway.

Resveratrol induces mitochondrial biogenesis in endothelial cells.

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-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-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.

Oxidative-nitrosative stress in a rabbit pup model of germinal matrix hemorrhage: role of NAD(P)H oxidase.

BACKGROUND AND PURPOSE: Germinal matrix hemorrhage-intraventricular hemorrhage is the most common neurological problem of premature infants. Despite this, mechanisms of brain injury from intraventricular hemorrhage are elusive. We hypothesized that germinal matrix hemorrhage-intraventricular hemorrhage, by induction of NAD(P)H oxidases, might cause oxidative/nitrosative stress contributing to brain injuries and that NAD(P)H oxidase inhibition could offer neuroprotection. METHODS: To test this hypothesis, we exploited our rabbit pup model of glycerol-induced germinal matrix hemorrhage-intraventricular hemorrhage. We delivered rabbit pups prematurely (E29) by cesarean section and administered intraperitoneal glycerol at 2 hours postnatal age. Free-radical adducts, including nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine as well as O(2)(.-) and H(2)O(2) levels were measured in the forebrain. To determine the source of free-radical generation, we used inhibitors for NAD(P)H oxidase (apocynin), xanthine oxidase (allopurinol), cyclo-oxygenase-2 (indomethacin), or nitric oxide synthases (L-NAME). Intraventricular hemorrhage pups were treated with apocynin and cell death was compared between apocynin-treated and vehicle-treated pups. RESULTS: Nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine levels were higher in pups with intraventricular hemorrhage than controls. Likewise, O(2)(.-) and H(2)O(2) levels were significantly greater in both the periventricular area and cerebral cortex of pups with intraventricular hemorrhage than controls. In pups with intraventricular hemorrhage, reactive oxygen species production was more in the periventricular area than in the cortex. Apocynin, but not allopurinol, indomethacin, or nitric oxide synthases, inhibited reactive oxygen species generation. Importantly, apocynin reduced cell death in pups with intraventricular hemorrhage. CONCLUSIONS: Activation of NAD(P)H oxidase was the predominant mechanism of free-radical generation in pups with intraventricular hemorrhage. NAD(P)H oxidase inhibition by apocynin might suppress reactive oxygen species production and confer neuroprotection in premature infants with intraventricular hemorrhage.

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.

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice.

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 O2(-) and H2O2 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 O2(-) and H2O2 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 O2(-) and H2O2 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.

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.

Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells.

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(2)(*-) and H(2)O(2) generation, induced NF-kappaB 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(2)(*-) 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.

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.

Dysregulation of mitochondrial biogenesis in vascular endothelial and smooth muscle cells of aged rats.

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 age-related 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.

Testing hypotheses of aging in long-lived mice of the genus Peromyscus: association between longevity and mitochondrial stress resistance, ROS detoxification pathways, and DNA repair efficiency.

In the present review we discuss the potential use of two long-lived mice of the genus Peromyscus--the white-footed mouse (P. leucopus) and the deer mouse (P. maniculatus) maximum lifespan potential approximately 8 years for both--to test predictions of theories about aging from the oxidative stress theory, mitochondrial theory and inflammatory theory. Previous studies have shown that P. leucopus cells exhibit superior antioxidant defense mechanisms and lower cellular production of reactive oxygen species (ROS) than do cells of the house mouse, Mus musculus (maximum lifespan approximately 3.5 years). We present new data showing that mitochondria in P. leucopus cells produce substantially less ROS than mitochondria in M. musculus cells, and that P. leucopus mitochondria exhibit superior stress resistance to those of M. musculus. We also provide evidence that components of the DNA repair system (e.g., pathways involved in repair of DNA damage induced by gamma-irradiation) are likely to be more efficient in P. leucopus than in M. musculus. We propose that mitochondrial stress resistance, ROS detoxification pathways and more efficient DNA repair contribute to the previously documented resistance of P. leucopus cells toward oxidative stress-induced apoptosis. The link between these three pathways and species longevity is discussed.

Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity.

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.

Vascular aging in the longest-living rodent, the naked mole rat.

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: approximately 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(2)(*-)) and H(2)O(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 ( approximately 250-300%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a approximately 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 gp91(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.

Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries.

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-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(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(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.

Downregulation of bone morphogenetic protein 4 expression in coronary arterial endothelial cells: role of shear stress and the cAMP/protein kinase A pathway.

OBJECTIVE: Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor beta family member cytokine that exerts proinflammatory effects on the endothelium and is likely to play a role in atherogenesis. Recent studies suggested that atheroprotective levels of shear stress control endothelial BMP-4 expression; however, the underlying mechanisms remained unknown. METHODS AND RESULTS: We found that shear stress downregulated BMP-4 expression in human and rat coronary arterial endothelial cells (CAECs) as well as in cultured mesenteric arterioles, although it had no effect on the expression of BMP-2, a related cytokine. In human coronary arterial endothelial cells, 8-bromo-cAMP, the adenylate cyclase activator forskolin, or a cAMP-dependent protein kinase (PKA) activator effectively decreased BMP-4 expression, mimicking the effects of shear stress. Indeed, shear stress induced the nuclear translocation of PKA-c, and inhibition of PKA attenuated the effects of shear stress and forskolin on BMP-4 expression. RNA decay assay and BMP-4 promoter-driven luciferase reporter gene assay showed that cAMP regulates BMP-4 expression at the transcriptional level. CONCLUSIONS: Laminar shear stress and the cAMP/PKA pathway are important negative regulators of BMP-4 expression in the vascular endothelium. Because BMP-4 elicits endothelial activation and dysfunction, hypertension, and vascular calcification, inhibition of BMP-4 expression by shear stress and the cAMP/PKA pathway is likely to exert antiatherogenic and vasculoprotective effects.

Vasculoprotective effects of anti-tumor necrosis factor-alpha treatment in aging.

Vascular aging is associated with dysregulation of tumor necrosis factor (TNF)-alpha expression. TNF-alpha is a master regulator of vascular proatherogenic phenotypic changes, and it has been linked to endothelial dysfunction and apoptosis. To test the hypothesis that anti-TNF-alpha treatment exerts vasculoprotective effects in aging, aged (29 months old) F344 rats were treated with etanercept (1 mg/kg/week for 4 weeks), which binds and inactivates TNF-alpha. In aged carotid arteries, relaxations to acetylcholine were decreased, and endothelial O2* production was increased (as shown by dihydroethidine fluorescence measurements). Etanercept treatment significantly improved responses to acetylcholine and decreased vascular NAD(P)H oxidase activity and expression. In aged carotid and coronary arteries, there were increases in DNA fragmentation rate and caspase 3/7 activity (indicating an increased rate of apoptotic cell death), which were attenuated by etanercept treatment. In aged vessels, there was an up-regulation of inflammatory markers, including inducible nitric-oxide synthase and intercellular adhesion molecule-1, which was decreased by etanercept treatment. In carotid arteries of young animals, recombinant TNF-alpha elicited endothelial dysfunction, oxidative stress, and increased apoptosis and proinflammatory gene expression, mimicking many of the symptoms of vascular aging. Thus, we propose that anti-TNF-alpha treatment exerts anti-aging vasculoprotective effects.