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.     

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.     

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.     

Resveratrol increases vascular oxidative stress resistance

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(-6)-10(-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(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(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 heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(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.     

Age-related alterations in NOS and oxidative stress in mesenteric arteries from male and female rats.

Epidemiological evidence suggests that advancing age affects the cardiovascular system of men and women differently. The purpose of this study was to determine whether the effects of aging on nitric oxide synthase (NOS), oxidative stress, and vascular function are different in males and females. Mesenteric arteries from young (3 mo) and old (24 mo) male and female Fischer 344/Brown Norway rats were studied. Western blot analysis and NOS activity were performed on the homogenized mesenteric arterial bed separated into cytosolic and membrane-associated fractions. Plasma 8-isoprostane measurements assessed oxidative stress. Vascular reactivity was determined by using a wire myograph in the absence and presence of a NOS inhibitor, N(omega)-nitro-l-arginine, to examine endothelial function and basal and stimulated nitric oxide release. In additional arteries, reactivity was performed in the presence of polyethylene glycol-SOD to assess the impact of superoxide on vascular function. Among females, aging was associated with a decline in membrane-associated NOS activity and membrane-associated NOS III protein expression. Advancing age in males was associated with increased cytosolic NOS III protein expression. Among both males and females, advancing age resulted in increased oxidative stress. Vascular function was maintained with age in arteries from both males and females, and there was no difference in either basal or stimulated nitric oxide release with age. Despite sex-specific effects of advancing age on the NOS system and increases in markers of oxidative stress, vascular function is maintained in mesenteric arteries from aged Fischer 344/Brown Norway rats. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.     

NADPH oxidases are in part responsible for increased cardiovascular superoxide production during aging

The aim of our study was to examine in rats, age-related differences in myocardial ischemic recovery and to determine the possible relationship with modification of cardiac and vascular oxidative stress. Isolated perfused hearts from young (2 months), adult (6 months), and old (21 months) Wistar rats were subjected to a ischemia–reperfusion sequence. Vascular histomorphological analyses were performed and NADPH oxidase was studied. The expression of angiotensin AT₁ receptors was evaluated using immunostaining. During the preischemic period, but also after ischemia, an aged-related decrease in myocardial functional parameters was observed, and was associated with an increased release of reactive oxygen species. In aortas, the activity and expression of NADPH oxidase increased with age according to the ESR, fluorescence microscopy, and immunohistochemistry; the NADPH oxidase involved was localized in endothelial cells. We found an age-related increase in the expression of endothelial angiotensin AT₁. Our study suggests that myocardial function and adaptation to ischemia–reperfusion declined during aging and are related to a higher level of oxidative stress. Endothelial NADPH oxidase is a major contributor to age-related cardiovascular deterioration. One of the regulators of vascular NADPH oxidase activity, the renin–angiotensin system, may be involved in the modulation of vascular superoxide production during the aging process.     

Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.     

Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF‐α is CEACAM1‐dependently upregulated in the aging vasculature. Vice versa, TNF‐α induces CEACAM1 expression. This results in a feed‐forward loop in the aging vasculature that maintains a chronic pro‐inflammatory milieu. Furthermore, we demonstrate that age‐associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age‐dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR‐2 signaling. Consequently, aging‐related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis.     

Loss of adaptation to oxidative stress as a mechanism for aortic damage in aging rats

Cells are armed with a vast repertoire of antioxidant defence mechanisms to prevent the accumulation of oxidative damage. The cellular adaptive response is an important antioxidant mechanism against physiological and pathophysiological oxidative alterations in a cell's microenvironment. The aim of this paper was to study, in the rat aorta, whether this adaptive response and the inflammation associated with oxidative stress were expressed throughout the aging process. We examined the rat aorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12, 18 and 24 months of age were used. Superoxide anion (O2(-)) generation; levels of two antioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels of prostaglandin E2 (PGE2), an inflammatory marker, were measured. The results for rats at different ages were compared with those for 3 months of age. A balance between production of O2(-) and SOD activity was found in the aorta of rats from 1.5 to 12 months old. Oxidative stress was present in the aorta of old animals (18-24 months), due to a failure in the mechanisms of adaptation to oxidative stress. The observed increase in PGE2 levels in these rats reflected an inflammatory response. All together suggest that vascular oxidative stress and the inflammatory process observed in the old groups of rats could be closely related to vascular aging. Our results also remark the importance of the adaptative response to oxidative stress.     

Aldose Reductase and AGE–RAGE pathways: central roles in the pathogenesis of vascular dysfunction in aging rats

Aging is inevitably accompanied by gradual and irreversible innate endothelial dysfunction. In this study, we tested the hypothesis that accentuation of glucose metabolism via the aldose reductase (AR) pathway contributes to age‐related vascular dysfunction. AR protein and activity levels were significantly increased in aged vs. young aortic homogenates from Fischer 344 rats. Immunostaining revealed that the principal site of increased AR protein was the aortic endothelium as well as smooth muscle cells. Studies revealed that endothelial‐dependent relaxation (EDR) in response to acetylcholine was impaired in aged rats compared to young rats and that treatment with the AR inhibitor (ARI) zopolrestat significantly improved EDR in aged rats. Methylglyoxal (MG), a key precursor of advanced glycation endproducts (AGEs), was significantly increased in the aortas of aged rats vs. young rats. Consistent with central roles for AR in generation of MG in aging, ARI treatment significantly reduced MG levels in aged rat aorta to those in young rats. Treatment of aged rats with soluble(s) RAGE, a soluble form of the chief signal transduction receptor for AGEs, RAGE, significantly improved EDR in aged rats, thus establishing the contribution of age‐related increases in AGEs to endothelial dysfunction. These findings reveal that significant increases in AR expression and activity in aged rat vasculature linked to endothelial dysfunction may be mitigated, at least in part, via ARI and that aging‐linked increased flux via AR generates AGEs; species which transduce endothelial injury consequent to their interaction with RAGE. These data demonstrate for the first time that AR mediates aging‐related vascular dysfunction, at least in part, via RAGE.     

Expression of the pro-apoptotic gene gadd153/chop is elevated in liver with aging and sensitizes cells to oxidant injury

Aging is generally accompanied by reduced tolerance to oxidative stress and altered responsiveness to proliferative signals. We have shown that hepatocytes derived from aged rats (24-26 months) exhibit greater sensitivity to H(2)O(2) treatment and reduced proliferation following epidermal growth factor (EGF) treatment than cells of young adult rats (5-6 months). Here we examined the effects of aging and calorie restriction (CR) on expression of the oxidative stress-inducible and pro-apoptotic gene gadd153 (chop) in these hepatocytes, and we investigated its influence on sensitivity to oxidants. We show that aging was associated with elevated expression of gadd153, both basally and in response to H(2)O(2) treatment. CR, which attenuates age-associated declines in stress tolerance, prevented the age-related increase in gadd153 expression. EGF treatment also resulted in gadd153 induction in old cells. This effect was absent in young cells and in old cells of CR rats. gadd153 induction by EGF was reactive oxygen species-dependent and correlated with heightened sensitivity to subsequent H(2)O(2) treatment, suggesting that elevated Gadd153 contributes to the greater sensitivity of EGF-pretreated old cells to oxidative stress. Additional support for this hypothesis was provided by experiments with Rat1 fibroblasts in which conditional expression of Gadd153 conferred increased sensitivity to H(2)O(2). We propose a model whereby the diminished ability of old hepatocytes to overcome an EGF-triggered reactive oxygen species load leads to induction of the proapoptotic gene gadd153, which, in turn, sensitizes the cells to oxidant injury. Our findings point to gadd153 expression levels as an important factor in liver aging.     

Nrf2-dependent induction of proteasome and Pa28αβ regulator are required for adaptation to oxidative stress

The ability to adapt to acute oxidative stress (e.g. H(2)O(2), peroxynitrite, menadione, and paraquat) through transient alterations in gene expression is an important component of cellular defense mechanisms. We show that such adaptation includes Nrf2-dependent increases in cellular capacity to degrade oxidized proteins that are attributable to increased expression of the 20 S proteasome and the Pa28αβ (11 S) proteasome regulator. Increased cellular levels of Nrf2, translocation of Nrf2 from the cytoplasm to the nucleus, and increased binding of Nrf2 to antioxidant response elements (AREs) or electrophile response elements (EpREs) in the 5'-untranslated region of the proteasome β5 subunit gene (demonstrated by chromatin immunoprecipitation (or ChIP) assay) are shown to be necessary requirements for increased proteasome/Pa28αβ levels, and for maximal increases in proteolytic capacity and stress resistance; Nrf2 siRNA and the Nrf2 inhibitor retinoic acid both block these adaptive changes and the Nrf2 inducers DL-sulforaphane, lipoic acid, and curcumin all replicate them without oxidant exposure. The immunoproteasome is also induced during oxidative stress adaptation, contributing to overall capacity to degrade oxidized proteins and stress resistance. Two of the three immunoproteasome subunit genes, however, contain no ARE/EpRE elements, and Nrf2 inducers, inhibitors, and siRNA all have minimal effects on immunoproteasome expression during adaptation to oxidative stress. Thus, immunoproteasome appears to be (at most) minimally regulated by the Nrf2 signal transduction pathway.     

Increased expression of endothelin-1 and inducible nitric oxide synthase isoform II in aging arteries in vivo: implications for atherosclerosis

We here report that aging increases expression of endothelin-1 and NO synthases in the vasculature and kidney of normotensive rats in vivo. Expression of preproendothelin-1 mRNA was quantified by RT-PCR and in situ hybridization, and endothelin-1 protein was determined by radioimmunoassay/HPLC. Vascular mRNA expression of NO synthase isoforms II and III was analyzed by RT-PCR. In young animals, vascular endothelin-1 protein was differentially expressed (aorta < renal artery < carotid artery) and increased with aging in all vascular beds (P < 0.05). In the intact aorta of aged rats, mRNA expression of preproendothelin-1, "inducible" NO synthase II, and endothelial cell NO synthase III gene was up-regulated (P < 0.05). Moreover, preproendothelin-1 mRNA expression increased in glomeruli and tubulointerstitial cells (P < 0.05). To our knowledge this is the first study demonstrating local vascular up-regulation of the trophic factor endothelin under physiological conditions. Activation of vascular endothelin and NO synthases may be important, pressure-independent factors contributing to structural and functional abnormalities of age-dependent diseases, including atherosclerosis.