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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences logoLink to The Journals of Gerontology Series A: Biological Sciences and Medical Sciences
. 2011 Feb 8;66A(4):409–418. doi: 10.1093/gerona/glq233

Salicylate Treatment Improves Age-Associated Vascular Endothelial Dysfunction: Potential Role of Nuclear Factor κB and Forkhead Box O Phosphorylation

Lisa A Lesniewski 1,✉, Jessica R Durrant 1, Melanie L Connell 1, Brian J Folian 1, Anthony J Donato 1, Douglas R Seals 1
PMCID: PMC3055281  PMID: 21303813

Abstract

We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction.

Keywords: EDD, NADPH oxidase, SOD

AGING is the major risk factor for the development of cardiovascular diseases (CVDs), largely by adversely affecting the function and health of arteries (1). A key change in arteries that contributes to increased risk of CVD with aging is the development of endothelial dysfunction (1), most commonly established as impaired endothelium-dependent dilation (EDD) (2–5). Impaired EDD with aging is caused primarily by a reduction in the bioavailability of the endothelium-derived dilator molecule, nitric oxide (NO), which is produced by the enzyme endothelial NO synthase (eNOS) (3,6). This decrease in NO bioavailability is, in turn, mediated by an increase in bioactivity of reactive oxygen species such as superoxide anion (3,5,7,8).

Aging also is considered a state of chronic low-grade inflammation generally characterized by increased circulating levels of proinflammatory cytokines and acute phase proteins (8–11), with accompanying vascular inflammation (8,11–14). It is possible that inflammation suppresses EDD by stimulating oxidant enzyme systems to produce excessive amounts of superoxide (15). However, the specific cellular and molecular events that contribute to the development of oxidative stress, inflammation, reduced NO bioavailability, and impaired EDD with aging are incompletely understood.

One mechanism that could play an important role is tonic activation of I kappa B kinase (IKK). Upon activation via phosphorylation, IKK has biologically important downstream effects on multiple signaling pathways and contributes to the reciprocal regulation of the nuclear transcription factors nuclear factor kappa B (NFκB) and forkhead box O (FoxO). IKK-associated phosphorylation of IκB leads to NFκB activation and nuclear translocation (16,17) with consequent increases in production of proinflammatory cytokines (16,18) and superoxide, the latter via increased transcription and expression of the oxidant enzyme, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (19–21). In contrast, IKK phosphorylation of FoxO results in its nuclear exclusion and transcriptional inactivation (22,23), which may contribute to oxidative stress through reduced expression of antioxidant enzymes such as manganese superoxide dismutase (MnSOD) (24,25). IKK activation in arteries may increase with age (14). However, its role in mediating vascular endothelial dysfunction with aging via these potential mechanisms is unknown.

Here we test the hypothesis that short-term high-dose salicylate treatment will reduce age-associated arterial IKK activation and that this will, in turn, reduce arterial oxidative stress and inflammation and improve EDD via increases in eNOS expression/activity and/or decreases in superoxide-related suppression of NO bioavailability. We further hypothesized that this salicylate-mediated decrease in IKK bioactivity will be concomitant with reduced activation of NFκB and increased FoxO activation, with consequent decreases in NADPH oxidase and increases in MnSOD expression. To test these hypotheses, we studied young and old B6D2F1 mice under control conditions, and old mice after short-term, high-dose in vivo treatment with sodium salicylate, an inhibitor of IKK.

METHODS

Animals

B6D2F1 mice were obtained from the National Institute on Aging rodent colony. All mice were housed in an animal care facility at the University of Colorado at Boulder on a 12:12 light:dark cycle. Thirteen young (4–7 months) and 15 old (29–32 months, age at approximately 50% survival) male B6D2F1 mice were fed normal rodent chow ad libitum and housed in standard mouse cages. A second cohort of old mice (n = 14) was fed ad libitum a diet containing 1.2 g/kg sodium salicylate (Sigma–Aldrich, St. Louis, MO) for 5 days prior to sacrifice. This was calculated to provide approximately 200 mg/kg body weight/day to a 30-g mouse eating 5 g/day (26). Reductions in inflammatory signaling via inhibition of NFκB can be achieved through oral administration of high-dose salicylates, aspirin-like compounds that inhibit the activator of NFκB, IKKβ (27–29). However, because aspirin is known to be a cyclooxygenase (COX) inhibitor, and the products of the COX enzyme are well-known vasoactive agents, it should also be noted that unlike aspirin, salicylates lack the acetyl group required for the inhibition the enzymes, COX-1 and COX-2 (30–32). Indeed, in our hands, short-term high-dose salsalate, a United States Federal Drug Administration (FDA)-approved salicylate, reduced total nuclear expression of NFκB in endothelial cells isolated from human veins but failed to reduce endothelial cell expression of COX-1 or COX-2 or plasma 6-keto prostanglandin F1α, a marker of prostacyclin metabolism (33). Thus, the mechanism of action of salicylate is distinct from that of aspirin and neither modifies nor inhibits the activity of the COX enzymes (31). Food intake was monitored daily for the 5 days prior to sacrifice, and actual sodium salicylate intake was calculated. All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 2010) and were approved by the University of Colorado at Boulder Animal Care and Use Committee.

Carotid Artery Endothelial Function

Mice (n = 7–14 per group) were euthanized via exsanguinations by cardiac puncture while under isoflurane anesthesia. Right and left carotid arteries were excised, and EDD to acetylcholine (ACh: 1 × 10−9 to 1 × 10−4 mol/L) in the absence or presence of the NO synthase inhibitor, G-nitro-L-arginine-methyl ester (L-NAME, 0.1 mmol/L, 30 min), and/or the superoxide dismutase (SOD) mimetic, TEMPOL (1 mmol/L, 60 min), was assessed as previously described (34). Carotid artery endothelium independent dilation to sodium nitroprusside (SNP: 1 × 10−10 to 1 × 10−4 mol/L) also was determined (5). Vessel segments were imaged and diameters measured by MyoView software (DMT, Inc., Atlanta, GA). All dose–response data are presented as a percent of possible dilation after phenylephrine (2 μmol/L) preconstriction. Sensitivity was defined as the concentration of vasoactive agent (ACh, SNP) that yielded 50% of the maximal response (IC50). NO bioavailability was defined as the difference in maximal dilation in response to ACh and in the presence versus absence of L-NAME.

Arterial Protein Expression

Arterial protein expression was determined by Western blot in thoracic aorta as described previously by our laboratory (5,34) and others (35,36). Briefly, the thoracic aorta was excised, cleared of surrounding tissues, whole artery lysates were prepared, and Western blots performed as previously described (5) using the following primary antibodies, IKKβ (1:500, 87 kDa; Santa Cruz Biotechnology, Santa Cruz, CA), ser177/181-phosphorylated IKKβ (1:500, 87 kDa; Cell Signaling, Boston, MA), nitrotyrosine (1:1000, 25, 55, 160 kDa; Abcam, Cambridge, MA), eNOS (1:1000; 140 kDa; BD Biosciences, San Jose, CA), inhibitor of NFκB α (IκBα, 1:1000, 41 kDa; Santa Cruz Biotechnology), ser32-phosphorylated IκBα (1:1000, 40 kDa; Cell Signaling), ser1177-phosphorylated eNOS (1:1000, 140 kDa; Cell Signaling, Danvers, MA), p65 NFκB (1:500, 65 kDa; Santa Cruz Biotechnology), ser536-phosphorylated p65 NFκB (1:1000, 65 kDa; Cell Signaling), p50 NFκB (1:100, 50 kDa; Santa Cruz Biotechnology), ser337-phosphorylated p50 NFκB (1:200, 50 kDa; Santa Cruz Biotechnology), p67phox-NADPH oxidase (1:1000, 67 kDa; BD Biosciences), FoxO3a (1:500, 82–97 kDa; Cell Signaling), ser253-phosphorylated FoxO3a (1:1000, 97 kD; Cell Signaling), MnSOD (1:2000, 25 kDa; Stressgen, Ann Arbor, MI), catalase (1:2500, 65 kD; Abcam, Cambridge, MA), the appropriate horse radish peroxidase-conjugated secondary antibody (Jackson Immunological, West Grove, PA), and Supersignal ECL (Pierce, Rockford, IL). Bands were visualized using a digital acquisition system (ChemiDoc-It, UVP, Upland, CA) and quantified using ImageJ software (NIH, Bethesda, MD). To account for differences in protein loading, expression of proteins of interest were normalized to glyceraldehyde-3-phosphate dehydrogenase (1:1000, 37 kDa; Cell Signaling). At least two to four samples per group for all three experimental groups were loaded into all gels, and direct comparisons between groups were made within each gel. The mean of the young control group within a given blot was used to normalize the data for all groups allowing data to be combined from multiple blots. Representative blots show bands from all groups from the same blot and exposure, and normalizer protein was assessed on the same blot after stripping.

Enzyme-Linked Imunosorbent Assays

The concentrations of proinflammatory cytokines, interleukin-1β, interleukin-6, interferon γ, and tumor necrosis factor α were determined in aortic whole-cell lysates by multiplex enzyme-linked immunosorbent assay (Searchlight Mouse Inflammatory Cytokine kit; Aushon Biosystems, Billerica, MA) according to manufacturer instructions.

Enzyme Activity

NADPH oxidase activity was determined in aortic lysates (10-μg protein) using the Amplex Red Xanthine/Xanthine Oxidase Assay kit (Invitrogen, Carlsbad, CA) according to manufacturer instructions with NADPH (200 μmol/L per reaction) as the reaction substrate. Total SOD activity was determined in aortic lysates (1-μg protein) using the SOD Activity Assay kit (Cayman Chemical, Ann Arbor, MI) according to manufacturer instructions. Data are expressed relative to the mean of the young control group to account for interassay variability.

Statistics

For animal and vessel characteristics, Western blotting, enzyme-linked immunosorbent assays, maximal vasodilation, and sensitivity, group differences were determined by one-way analysis of variance. For dose responses to ACh and SNP, group differences were determined by repeated-measures analysis of variance. Least-squares difference post hoc tests were used where appropriate. Data are presented as mean ± SEM. Significance was set at p < .05.

RESULTS

Average salicylate intake of old-treated mice was 163 ± 14 mg/kg/day. Neither body mass (young: 35.7 ± 1.1 g; old: 34.0 ± 1.1 g; old salicylate treated: 35.1 ± 1.2 g) nor daily food intake (young: 4.8 ± 0.1 g/day; old: 5.0 ± 0.1 g/day; old salicylate treated: 4.5 ± 0.3 g/day) differed among the groups.

Aging Was Associated With Increased Aortic Phosphorylation of IKKβ and Decreased Expression of IκBα

Total IKKβ expression did not differ among the groups. Phosphorylated IKKβ was 82% greater in old compared with young control mice (p < .05), and this age-associated increase was abolished in old salicylate-treated mice (Figure 1A). Consistent with this, old control mice demonstrated a reduction in total IκBα expression (p < .05) that was reversed by salicylate treatment (p < .05), whereas phosphorylation of IκBα was unaffected by either aging or salicylate (Figure 1B).

Figure 1.

Figure 1.

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Aortic expression of total (left) and phosphorylated (right) inhibitor of nuclear factor κB (NFκB) kinase (IKK) (n = 7–12 per group) (A) and the inhibitor of NFκB α (IκBα) (n = 7–9 per group) (B) in young, old, and old salicylate (Sal)-treated mice with representative Western blots. Protein expression was normalized to own glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as a ratio of young control mean. Values are mean ± SEM. *p < .05 versus young, †p < .05 versus old.

IKK Activation Contributes to a Prooxidant/Proinflammatory Arterial Phenotype With Aging

Nitrotyrosine abundance (p < .01, Figure 2A), a marker of cellular oxidative stress, was 95% greater in aorta of old compared with young control mice. In contrast, nitrotyrosine was not different between young mice and salicylate-treated old mice. Aortic concentrations of proinflammatory cytokines were greater in old compared with young control mice (all p < .05, Figure 2B). Salicylate treatment reduced the concentrations of interleukin-6 (p < .05), interferon γ (p = .01), and tumor necrosis factor α (p < .05) and tended to decrease interleukin-1β (p = .06) in old mice (Figure 2B).

Figure 2.

Figure 2.

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Nitrotyrosine abundance (A) and proinflammatory cytokine concentrations (B) (all n = 8–13 per group) in aortas from young, old, and old salicylate (Sal)-treated mice with representative Western blot (subsequently left). Protein expression was normalized to own glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as a ratio of young control mean. *p < .05 versus young, †p < .05 versus old. IL-1β, interleukin-1β; IL-6, interleukin-6; IFNg, interferon gamma; and TNFα, tumor necrosis factor alpha.

Salicylate Treatment Improves Age-Associated Endothelial Dysfunction by Increasing NO Bioavailability via Decreases in Superoxide Bioactivity and Increased eNOS Phosphorylation

Carotid artery EDD was impaired in old compared with young (p < .01) control mice, but was preserved in old mice after salicylate treatment (Figure 3A). Sensitivity (IC50) to ACh did not differ among the groups (Table 1).

Figure 3.

Figure 3.

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Carotid artery endothelium-dependent dilation (EDD) in the absence or presence of G-nitro-L-arginine-methyl ester (L-NAME) (n = 6–10 per group) (A), aortic total (left) and phosphorylated (right) endothelial nitric oxide synthase (eNOS, n = 10–12 per group) (B) with representative Western blot, carotid artery EDD in the absence or presence of L-NAME after pretreatment with the superoxide dismutase mimetic (TEMPOL) (C) (n = 6–10 per group), and carotid artery endothelium-independent dilation (n = 6–10 per group) in young, old, and old salicylate (Sal)-treated mice (D). Protein expression was normalized to own glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as a ratio of young control mean. Values are mean ± SEM. *p < .05 versus young, †p < .05 versus old.

Table 1.

Sensitivity (IC50) to ACh Alone and in the Presence of a Superoxide Dismutase Mimetic (TEMPOL) and to SNP

Young Old Old Sal
ACh
 Alone (× 10−9 M) 4.5 ± 0.4 8.6 ± 2.4 11.9 ± 6.4
 TEMPOL (× 10−9 M) 8.5 ± 2.4 5.1 ± 0.7 3.9 ± 0.9
SNP (× 10−9 M) 6.9 ± 2.5 5.9 ± 2.5 3.6 ± 1.2
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Note: Ach = acetylcholine; SNP = sodium nitroprusside.

The NO inhibitor L-NAME reduced carotid artery EDD in all groups (all P<0.01, Figure 3A). NO bioavailability was lower in old compared with young control mice (26 ± 7 % vs. 50 ± 8%, P=0.05), but was restored in old mice treated with salicylate (52 ± 10%, P<0.05).

eNOS protein expression was unchanged with aging or inhibition of IKK. However, aging resulted in a 59% decrease ser1177-phosphorylation of eNOS (p < .01) that was ameliorated by salicylate treatment (p < .01, Figure 3B).

The SOD mimetic TEMPOL restored EDD (Figure 3C) and increased NO bioavailability (to 47 ± 8% vs. 26 ± 7 %, p < .05) in old control mice, but did not affect sensitivity to ACh (Table 1). TEMPOL had no effect on EDD (Figure 3C), sensitivity to ACh (Table 1), or NO bioavailability (49 ± 7% and 46 ± 7%, respectively) in either young control mice or old salicylate-treated mice.

Endothelium-independent dilation (Figure 3D) and sensitivity to SNP (Table 1) did not differ among the groups.

Salicylate Reduces Age-Associated Phosphorylation of Aortic NFκB and Increased Expression/Activity of NADPH Oxidase

Total expression of NFκB p65 in the aorta did not differ among the groups (Figure 4A). Aortic NFκB activation, as indicated by phosphorylation of the p65 subunit of NFκB, was 110% greater in old compared with young control mice (p < .01, Figure 4A) but did not differ between young and IKK-inhibited old mice. No differences were found with aging or salicylate treatment for either total expression or phosphorylation of the p50 subunit of NFκB (data not shown, n = 5 per group).

Figure 4.

Figure 4.

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Expression of total (left) and phosphorylated (p-) (right) nuclear factor κB p65 subunit (A) (n = 11–13 per group), NADPH oxidase p67 subunit expression (B) with representative Western blots in aortas from young, old, and old salicylate (Sal)-treated mice (all n = 10–14 per group), and NADPH oxidase activity (C) (n = 9–15). Protein expression was normalized to own glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as a ratio of young control mean. Values are mean ± SEM. *p < .05 versus young, †p < .05 versus old.

NADPH oxidase expression was approximately 120% greater in old compared with young control mice (p < .01, Figure 4B), an effect that was abolished in salicylate-treated old mice. NADPH oxidase activity also was higher in aorta from old compared with young animals (p < .01), and this difference was abolished by inhibition of IKK (p < .05, Figure 4C).

Salicylate Treatment Activates FoxO3a and Increases Expression of MnSOD in Old Mice

Total expression of FoxO3a in the aorta tended to be lower in old compared with young control mice (p = .09) and was increased in old mice after salicylate treatment (p < .05, Figure 5A). Phosphorylation (inactivation) of FoxO3a was 57% greater in old compared with young control mice (p < .01), whereas salicylate-treated old mice were similar to young (Figure 5A). Consistent with these effects on FoxO3a activation, aortic MnSOD expression was lower in old compared with young control mice (p < .01) but was restored in salicylate-treated old mice (Figure 5B). Similar trends were observed with catalase but were not statistically significant (p = .07–.1, data not shown). Total SOD activity was not different between young and old mice but was increased in old mice after inhibition of IKK (p < .01, Figure 5C).

Figure 5.

Figure 5.

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Expression of total (left) and phosphorylated (p-) (right) forkhead box O3a (FoxO3a) (A) (n = 7–10 per group), manganese superoxide dismutase (MnSOD) expression (B) with representative Western blots in aortas from young, old, and old salicylate (Sal)-treated mice (n = 9–14 per group), and SOD activity (C) (n = 6–9). Protein expression was normalized to own glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as a ratio of young control mean. Values are mean ± SEM. *p < .05 versus young, †p < .05 versus old.

DISCUSSION

Utilizing high-dose sodium salicylate as an IKK inhibitor, here we show that an increase in IKK activation with aging is associated with a reduction in the ser1177-phosphorylation of eNOS and superoxide-mediated suppression of NO bioavailability and vascular endothelial dysfunction in B6D2F1 mice. We also provide evidence that IKK activation may produce these effects in part via changes in the phosphorylation/activation of the transcription factors NFκB and FoxO, with consequent increases in the expression of the oxidant enzyme, NADPH oxidase, and decreases in the antioxidant enzyme, MnSOD.

IKK, Vascular Endothelial Dysfunction, and Impaired NO Bioavailability With Aging

Our data confirm an earlier report in rats (14) that phosphorylation (activation) of IKKβ is increased in aorta with aging and show that this occurs in the absence of an increase in total IKKβ expression. We also demonstrate that the IKKβ activation in aorta of old mice is associated with reduced total expression IκBα, the cytosolic protein that inhibits NFκB translocation to the nucleus where it stimulates proinflammatory gene transcription. These results are consistent with recent observations from our laboratory in vascular endothelial cells obtained from young and older humans (11). Most importantly, we show that short-term treatment with sodium salicylate normalized aortic IKKβ activity in old mice, as indicated by a reduction in aortic ser177/181-phosphorylated IKKβ to levels observed in young controls and that this was associated with a marked increase in expression of IκBα.

Our results extend previous biochemical observations on IKKβ (14) by demonstrating that the increase in IKK activity with aging may play a major role in the development of vascular endothelial dysfunction. This conclusion is supported by the finding that short-term high-dose sodium treatment with sodium salicylate inhibited IKK phosphorylation and improved EDD in old mice to levels not significantly different from young animals.

This effect of salicylate was not caused by reductions in vascular smooth muscle sensitivity to NO because endothelium-independent dilation to SNP was not altered with aging or salicylate treatment. Rather, our data show that salicylate-sensitive IKK activation suppressed EDD in old mice by reducing NO bioavailability, as indicated by the absence of age-related differences in EDD after inhibition of NO production with L-NAME.

Oxidative Stress

Our findings suggest that IKK activation reduces NO bioavailability and impairs EDD in old mice largely via the development of oxidative stress linked to superoxide production. This conclusion is supported by two main observations. First, sodium salicylate treatment in old mice markedly reduced the age-associated increase in aortic staining of nitrotyrosine, a cellular footprint of oxidative stress (37). Second, ex vivo administration of TEMPOL, a mimetic of the antioxidant enzyme SOD, selectively improved carotid artery EDD in old control mice, restoring function to levels observed in young mice and salicylate-treated old animals. Salicylate sensitive, IKK-associated oxidative stress caused impaired EDD with aging by reducing NO bioavailability because the improvement in EDD with TEMPOL in old control mice was abolished in the presence of L-NAME.

eNOS Phosphorylation

Although not observed in all models of arterial aging (38–40), recently we established that eNOS protein is unchanged, but that ser1177-phosphorylation of eNOS is reduced in aorta of old B6D2F1 mice (34). In the present study, we demonstrate that salicylate treatment restores ser1177-phosphorylation of eNOS in old mice to levels observed in young mice. Whether this is a primary effect of IKK on eNOS activity or is secondary to other actions of salicylate remains to be elucidated. Nevertheless, our results suggest that increased IKK signaling may contribute to reductions in NO bioavailability with aging in B6D2F1 mice in part by suppressing eNOS activity.

NFκB, Proinflammatory Cytokines, NADPH Oxidase, and MnSOD

DNA binding activity and nuclear localization of NFκB are increased in arteries and vascular endothelial cells with aging (8,11,13,14) and contribute to increases in expression of proinflammatory cytokines (11,12,14,41,42). Unlike the p50 subunit, p65 (also termed RelA) along with Rel-B and c-Rel, make up a class of NFκB subunits that contain amino acids sequences essential for its transcriptional activity. Translocation of the p50 and p52 subunits alone cannot induce NFκB transcriptional activity and may actually act as suppressors when present as homo- or heterodimers in vivo (43). Here we demonstrate that despite no changes in either the total expression or phosphorylation of the p50 subunit, phosphorylation of the p65 subunit was markedly increased in old control mice. Treatment with sodium salicylate normalized this activated state of NFκB by reducing phosphorylation of the p65 subunit of NFκB in old mice. Consistent with this, high-dose salicylate treatment also reduced expression of inflammatory cytokines in the aorta of old mice to levels not different from young mice.

NFκB transcribes more than 150 genes, including the oxidative stress-modulating genes NADPH oxidase and MnSOD (18,20,21). In the present study, expression of the p67 subunit of the oxidant enzyme NADPH oxidase was markedly increased, whereas expression of MnSOD, the mitochondrial isoform of the antioxidant enzyme SOD, was lower in old compared with young control mice. Both of these changes with aging were reversed after salicylate treatment suggesting that they are indeed dependent on IKK activation in old animals. In addition to the increase in NADPH oxidase expression, the IKK-mediated increase in the proinflammatory cytokine tumor necrosis factor α may contribute to the observed increase in NADPH oxidase activity and development of oxidative stress in aorta of old mice because tumor necrosis factor alpha induces NADPH oxidase activity (44). Finally, these observations indicate that although increased NFκB activation may contribute to the increase in NADPH oxidase expression/activity, it does not explain the observed reduction in aortic MnSOD expression with aging.

FoxO3a Activation and MnSOD

Although typically associated with NFκB activation, IKKβ also phosphorylates downstream FoxO transcription factors, modulating their transcriptional activity. Unlike NFκB, however, phosphorylation by IKK and other signaling kinases (eg, protein kinase B) results in nuclear export and inactivation of FoxO (22,24). Nuclear FoxO can transcribe gene targets related to oxidative stress resistance such as MnSOD and catalase (24,45). Here we demonstrate for the first time that increased IKKβ activity with advancing age is concomitant with an increase in the phosphorylation/inactivation of FoxO3a in aortas of mice and that this may be a mechanism underlying age-related decreases in MnSOD expression. We also found that salicylate treatment increased SOD activity in aorta of old mice, suggesting that salicylate also may have contributed to improvements in EDD by boosting antioxidant bioactivity. This could be a direct effect of salicylate-dependent inhibition of IKK on SOD activity or secondary to nitrosylation of SOD proteins given that salicylate also reduced aortic nitrotyrosine (46).

Clinical Relevance

NFκB activation and its inhibition by salicylates/aspirin is implicated in a number of age-associated events and functions including skeletal muscle atrophy, immunosenescence, and longevity, although there appear to be species- and sex-specific effects (47–51). It is possible that interventions that favorably modulate NFκB signaling with aging may reduce age-associated morbidities and/or increase lifespan. The present study extends our understanding of this issue by demonstrating that high-dose salicylate treatment initiated at older age reduces IKK activation and improves vascular endothelial dysfunction, a key antecedent of CVD and common comorbidity of aging, in old mice.

The dose of salicylate employed in the present study was more than 50-fold greater than the dose of aspirin recommended for prevention of CVD/events in humans (ie, 81 mg) but less than half the aspirin dose described to induce ulcers in mice (52). Whereas chronic low-dose aspirin typically is used prophylactically in humans for its COX-dependent antithrombotic effects, salicylates, metabolites of aspirin, are much less effective COX inhibitors (53). Therefore, we chose to use high-dose, short-term salicylate treatment to take advantage of its reported inhibitory effects on IκB/IKK signaling (27), while limiting effects on COX. This approach allowed us to test the hypothesis that IKK activation contributes to age-associated vascular endothelial dysfunction, perhaps via modulation of NFκB and FoxO activity, whereas short-term inhibition of IKK with salicylate may improve this age-associated dysfunction. We recognize that the dose of salicylate used here is not likely sustainable for chronic treatment. As such, future investigations should determine the efficacy of long-term, low-dose treatment with salicylates in old mice and humans, consistent with recent trials in patients with type 2 diabetes (31).

Nevertheless, the IKK-associated vascular endothelial dysfunction with aging shown here may have important implications for increases in CVD risk with aging in humans. We recently showed that 4 days of treatment with the FDA-approved drug salsalate improved brachial artery flow-mediated dilation, a measure of vascular endothelial function, and reduced NFκB and NADPH oxidase in vascular endothelial cells in overweight and obese middle-aged/older adults with chronic inflammation and features of the metabolic syndrome (33). The results of the present investigation suggest that tonic activation of IKK in arteries with primary aging could “partner” with conventional risk factors to facilitate the development of CVD in middle-aged and older adults. Together, these observations highlight excessive IKK signaling as a potentially important therapeutic target to prevent arterial endothelial aging and age-associated CVD. Accordingly, lifestyle and pharmacological strategies aimed at the prevention and treatment of increased activation of IKK with aging should be an important goal of future research.

Limitations

We recognize the complex signaling interactions among IKK, NFκB, FoxO, NADPH oxidase, and MnSOD. We also acknowledge that because IKK inhibition acts on both NFκB and FoxO, interpretation of our results in the context of these downstream signaling events is limited. However, because the available NFκB inhibitors (MG132, BAY11-7085, etc.) all act at the level of the upstream kinases IκB and IKK (24,54), and no specific FoxO inhibitors are available, inhibition of IKK presently is the only viable pharmacological option to assess the roles of these transcription factors. We chose to use high-dose salicylate as our IKK inhibitor because of its potential for translation to future clinical studies.

Conclusions

The present findings are the first to demonstrate that increased phosphorylation and activation of IKKβ play a key role in the development of vascular endothelial dysfunction with aging by reducing NO bioavailability via superoxide-mediated oxidative stress. Our results also provide novel evidence that age-associated activation of IKKβ may produce oxidative stress by excessive downstream phosphorylation of the nuclear transcription factors NFκB and FoxO, which, in turn, may induce the expression and activity of NADPH oxidase and downregulate MnSOD expression, respectively. The present findings also show that increased phosphorylation and activation of IKKβ is an important mechanism in mediating arterial inflammation with aging, characterized by elevated concentrations of proinflammatory cytokines. Finally, these observations provide further support for the therapeutic use of salicylates in the prevention and treatment of age-associated dysfunction and disorders, including arterial aging.

FUNDING

National Institute on Aging at the National Institutes of Health (AG013038, AG029337, AG000279, AG033196 and AG033755).

CONFLICT OF INTEREST

There are no conflicts of interest to disclose.

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