• Open Access

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging

Authors


Amy L. Sindler, PhD, University of Colorado, 354 UCB, Boulder, CO 80309, USA. Tel.: +303 492 3010; fax: +303 492 6778; e-mail: amy.sindler@colorado.edu

Summary

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26–28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L−1 in drinking water) restored nitrite levels to or above young (4–6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100–300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Introduction

Cardiovascular diseases (CVD) remain the leading cause of death in modern societies (Lloyd-Jones et al., 2010). Advancing age is the major risk factor for CVD (Lakatta, 2002; Lakatta & Levy, 2003). Most CVD are linked to disorders of arteries (Lloyd-Jones et al., 2010), and it is now recognized that aging increases risk of CVD in large part by causing arterial dysfunction, which then leads to clinical vascular and cardiac diseases (Lakatta & Levy, 2003).

Many changes to arteries likely contribute to the increased risk of CVD with aging. One of these is the development of vascular endothelial dysfunction, as most commonly indicated by impaired endothelium-dependent dilation (EDD) (Celermajer et al., 1994; Taddei et al., 1995; Lakatta & Levy, 2003). Impaired EDD with aging is mediated by excessive superoxide, at least in part as a result of increases in the oxidant enzyme nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) (Zalba et al., 2000; Bedard & Krause, 2007; Donato et al., 2007; Durrant et al., 2009). Increased superoxide reduces the bioavailability of nitric oxide (NO) both by reacting with NO to form peroxynitrite and by oxidizing tetrahydrobiopterin (BH4), an essential co-factor for NO production by endothelial nitric oxide synthase (eNOS) (Cosentino et al., 1998; Taddei et al., 2001; Landmesser et al., 2003; Blackwell et al., 2004; Shi et al., 2004). Another key vascular change with aging is the stiffening of large elastic arteries (Lakatta & Levy, 2003), possibly linked in part to the development of vascular endothelial dysfunction (Fitch et al., 2001; Wilkinson et al., 2004). Large elastic artery stiffness has emerged as a major independent risk factor for age-associated CVD and a key therapeutic target (Lakatta & Levy, 2003; Nilsson et al., 2009; Mitchell et al., 2010; Vlachopoulos et al., 2010).

Oxidative stress and inflammation are believed to be important mechanisms contributing to these effects of aging on arteries (Brandes et al., 2005; Donato et al., 2007; Csiszar et al., 2008; Donato et al., 2008; Ungvari et al., 2010). Arterial oxidative stress develops with aging as a consequence of excessive production of superoxide by NADPH oxidase or other mechanisms (eNOS uncoupling, mitochondrial dysfunction) (Vasquez-Vivar et al., 1998; Ungvari et al., 2008; Durrant et al., 2009; Rippe et al., 2010) and possibly via selective reductions in antioxidant enzymes, including superoxide dismutase (SOD) (Sun et al., 2004; Rippe et al., 2010). As a result of oxidative stress, injury, or other causes, vascular inflammation also develops with aging, as indicated by increases in the expression of pro-inflammatory cytokines such as interleukins 1 and 6 (IL-1, IL6), interferon γ (INFγ), and tumor necrosis factor α (TNFα) (Csiszar et al., 2008; Donato et al., 2008; Rippe et al., 2010; Ungvari et al., 2010).

Given the above, treatments that improve or reverse vascular endothelial dysfunction and large elastic artery stiffness in middle/older age may prevent much of the increase in CVD risk with aging (Lakatta & Levy, 2003; Lonn et al., 2010; Seals et al., 2010). In this context, the nitrite anion (inorganic nitrite) would seem to hold promise. Once considered an inert byproduct of NO metabolism, nitrite now is recognized as a physiologically important storage form of NO (Lundberg & Weitzberg, 2008). Nitrite is a cytoprotective molecule considered to have broad therapeutic potential for the prevention and treatment of CVD (Calvert & Lefer, 2010; Lundberg & Weitzberg, 2008). Sodium nitrite administration protects against ischemia/reperfusion injury (Bryan et al., 2007, 2008; Gonzalez et al., 2008) and improves vascular endothelial dysfunction in hypercholesterolemic mice (Stokes et al., 2009). However, the efficacy of sodium nitrite for treating arterial aging is entirely unknown.

In the present study, we hypothesized that short-term oral sodium nitrite treatment would improve or reverse vascular endothelial dysfunction and large elastic artery stiffness in old mice while having no effects in young mice. As a secondary aim, we sought to gain initial insight into the mechanisms by which nitrite-induced improvements in function in the old mice might be mediated, particularly those related to reduced oxidative stress and inflammation.

Results

Animal characteristics

Characteristics of the groups are shown in Table 1. Body mass and heart mass were greater, and gastrocnemius muscle mass was smaller in the old compared with the young groups (P < 0.05). Carotid artery baseline diameter was greater in the old animals (P < 0.05), and sodium nitrite treatment had no effect on these characteristics. Carotid artery preconstriction to phenylephrine was not different between groups.

Table 1.   Animal characteristics
 YCOCYNON
  1. Values are mean ± SEM.

  2. *< 0.05 vs. YC.

  3. YC, young control; OC, old control; YN, young nitrite; ON, old nitrite.

Body mass (g)23 ± 135 ± 1*23 ± 134 ± 1*
Heart mass (mg)136 ± 9194 ± 8*137 ± 1189 ± 9*
Gastrocnemius mass (mg)195 ± 9156 ± 14*191 ± 13146 ± 7*
Carotid artery lumen diameter (μm)389 ± 11421 ± 8*387 ± 10410 ± 13*

Sodium nitrite treatment restores NO-mediated EDD in old mice

Endothelium-dependent dilation to acetylcholine (ACh) was lower in old compared with young control mice (P < 0.05) as a result of a smaller NO dilatory influence, as indicated by a smaller reduction in EDD in the presence vs. absence of the NO inhibitor N-G-nitro-l-arginine methyl ester (l-NAME) (Fig. 1A,B). Nitrite treatment restored EDD to ACh in old mice by restoring NO-mediated dilation, but had no effect in the young animals (Fig. 1A,B). There were no differences in NO-mediated EDD among the young control and young and old nitrite-treated animals. Endothelium-independent dilation to sodium nitroprusside did not differ among the groups (Fig. 1C). TEMPOL, a SOD mimetic (Fig. 2A), apocynin, a NADPH oxidase inhibitor (Fig. 2B), and sepiapterin, a precursor of BH4 (Fig. 2C), each restored EDD to ACh in old control animals, while not affecting responses in young control or old sodium nitrite-treated animals. Endothelial nitric oxide synthase protein in the aorta was lower in the old vs. young control animals (Table 2), and this was not influenced by sodium nitrite treatment. Sodium nitrite supplementation increased eNOS expression in young mice. These data demonstrate that short-term sodium nitrite treatment restores NO-mediated EDD in old mice via a superoxide/NADPH oxidase/BH4-related mechanism and not by affecting vascular smooth muscle sensitivity to NO or eNOS protein.

Figure 1.

 Endothelium-dependent, nitric oxide (NO)-dependent and endothelium-independent dilation. (A) Dose-responses to the endothelium-dependent dilator acetylcholine (ACh) in the absence and presence of the endothelial NO synthase inhibitor N-G-nitro-l-arginine methyl ester (l-NAME) in young and old control (YC and OC) and nitrite-supplemented (YN and ON) mice. (B) NO-dependent dilation (Max DilationACh − Max DilationACh + l-NAME). (C) Dose-responses to the endothelium-independent dilator sodium nitroprusside (SNP). Values are mean ± SEM. (n = 7 per group). *< 0.05 vs. YC.

Figure 2.

 Superoxide-, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), and tetrahydrobiopterin-dependent modulation of endothelium dependent dilation. (A) Maximal dilation of carotid arteries to acetylcholine (ACh) and to ACh + TEMPOL, a superoxide dismutase mimetic. (B) Maximal dilation of carotid arteries to ACh and to ACh + apocynin, a NADPH oxidase inhibitor. (C) Maximal dilation of carotid arteries to ACh and to ACh + sepiapterin, an exogenous tetrahydrobiopterin donor. Values are mean ± SEM. (n = 6–9 per group) *< 0.05 vs. YC.

Table 2.   Protein expression in aorta
 YCOCYNON
  1. Values are mean ± SEM.

  2. *< 0.05 vs. YC.

  3. MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; ecSOD, extracellular superoxide dismutase; eNOS, endothelial nitric oxide synthase; YC, young control; OC, old control; YN, young nitrite; ON, old nitrite.

MnSOD1.0 ± 0.10.7 ± 0.1*0.2 ± 0.05*0.7 ± 0.1*
CuZnSOD1.0 ± 0.31.2 ± 0.11.2 ± 0.21.1 ± 0.2
ecSOD1.0 ± 0.11.1 ± 0.11.0 ± 0.11.0 ± 0.2
eNOS1.0 ± 0.10.7 ± 0.1*1.5 ± 0.1*0.6 ± 0.1*

Sodium nitrite treatment normalizes aortic pulse wave velocity in old mice

Large elastic artery stiffness, as determined by aortic pulse wave velocity, was ∼70% greater in old compared with young control mice (P < 0.05) (Fig. 3). Sodium nitrite treatment reversed the age-associated increase in aortic pulse wave velocity in old mice. These observations indicate that short-term treatment with sodium nitrite ameliorates age-associated stiffening of the aorta.

Figure 3.

 Large elastic artery stiffness. Aortic pulse wave velocity in young and old control (YC and OC) and young and old nitrite-supplemented (YN and ON) mice. Values are mean ± SEM. (n = 5 per group) *< 0.05 vs. YC.

Sodium nitrite treatment reduces vascular oxidative stress and inflammation in old mice

Nitrotyrosine, a cellular marker of oxidative stress, was ∼100% greater in the aorta of old compared with young control mice (P < 0.05) (Fig. 4A) and was associated with similarly greater aortic superoxide production, and an ∼50% reduction in aortic SOD activity (P < 0.05) (Fig. 4B,C). Nicotinamide adenine dinucleotide phosphate-oxidase subunit p67 protein expression was ∼300% greater in the aorta of old compared with young control mice (P < 0.05) (Fig. 4D). Nitrite treatment in old animals reduced aortic nitrotyrosine to levels observed in young control mice, and this was associated with complete reversal of the age-associated changes in superoxide production, SOD activity, and aortic p67 expression. Protein expression of manganese SOD (MnSOD) in aorta was decreased in old control mice (P < 0.05), and this was unaltered by nitrite supplementation; young nitrite-supplemented mice had reduced MnSOD (P < 0.05) (Table 2). Protein expression of the inflammatory cytokines IL-1β, IL6, INFγ, and TNFα was increased in aorta of old compared with young mice. Short-term nitrite treatment reduced aortic inflammatory cytokines selectively in old mice, normalizing the expression to levels observed in young controls (P < 0.05, Fig. 5). These observations indicate that short-term sodium nitrite treatment ameliorates arterial oxidative stress (by normalizing superoxide production and SOD activity) and inflammation with aging.

Figure 4.

 Aortic oxidative stress, superoxide production, and oxidant/antioxidant enzymes. (A) Nitrotyrosine in aorta of young and old control (YC and OC) and young and old nitrite-supplemented (YN and ON) mice. Data are expressed relative to GAPDH and normalized to YC mean value. Representative Western blot images below. (B) Mean electron paramagnetic resonance signal for superoxide from aortic rings. (C) Aortic superoxide dismutase enzymatic activity. (D) Protein expression of p67 subunit of nicotinamide adenine dinucleotide phosphate-oxidase in aorta of young and old control (YC and OC) and young and old nitrite-supplemented (YN and ON) mice. Data are expressed relative to GAPDH and normalized to YC mean value. Representative Western blot images below. Values are mean ± SEM. (n = 6–9 per group) *< 0.05 vs. YC.

Figure 5.

 Aortic inflammatory cytokines. Expression of inflammatory cytokines IL-1β, IL-6, IFNγ and TNFα in aorta from young and old control (YC and OC) and young and old nitrite-supplemented (YN and ON) mice. Values are mean ± SEM. (n = 5–8 per group) *< 0.05 vs. YC.

Sodium nitrite treatment restores the reductions in nitrite concentrations in plasma, large elastic arteries and heart with aging

To obtain sufficient tissue to determine nitrite concentrations in large elastic arteries, carotid arteries and aorta were pooled from each group of mice (Fig. 6). Nitrite concentrations in the large elastic arteries, heart, and plasma were lower in old compared with young control animals (P < 0.05). In the old mice, nitrite treatment restored nitrite concentrations to levels either not different from (elastic arteries and heart) or well above (plasma) those observed in young control mice (P < 0.05). Nitrite supplementation also increased nitrite levels in young mice (P < 0.05). These results show that nitrite concentrations in large elastic arteries, heart, and plasma decrease with aging and that short-term sodium nitrite treatment restores nitrite concentrations in old mice.

Figure 6.

 Circulating and tissue nitrite concentrations from plasma, pooled large elastic arteries, and heart. Nitrite concentrations in plasma, large elastic arteries (pooled aortic and carotid arteries), and heart in young and old control (YC and OC) and young and old nitrite-supplemented (YN and ON) mice. Values are mean ± SEM. (n = 8 animals per group) *< 0.05 vs. YC.

Discussion

The key finding of this study was that 3 weeks of sodium nitrite treatment in old C57BL6 mice ameliorated carotid artery endothelial dysfunction and reduced large elastic artery stiffness to a level not different from young mice. Age is the major risk factor for CVD, and vascular endothelial dysfunction and large elastic stiffening are believed to explain much of the increase in CVD risk with aging (Lakatta, 2002; Vita & Keaney, 2002; Mitchell et al., 2010). Our preclinical findings, therefore, suggest that sodium nitrite has intriguing translational potential as a treatment for the prevention of age-associated CVD in humans. The present results also provide novel insight into the mechanisms by which sodium nitrite reverses arterial aging, which include restoring NO and BH4 bioavailability, suppressing superoxide-dependent oxidative stress by reducing NADPH oxidase and increasing SOD activity, and reducing inflammation. Finally, our results indicate that aging is associated with reduced concentrations of nitrite in large elastic arteries, the heart, and plasma, providing additional rationale for therapies that enhance circulating and tissue nitrite stores.

Vascular endothelial dysfunction

The present results are consistent with previous reports from our laboratory (Durrant et al., 2009; Lesniewski et al., 2009; Rippe et al., 2010) and others (Muller-Delp et al., 2002; Soucy et al., 2006) that vascular endothelial dysfunction develops with aging, as indicated by impaired EDD. The latter is mediated by reduced NO bioavailability (Luscher & Barton, 1997; Spier et al., 2004; Sindler et al., 2009) as a result of NADPH oxidase-associated increases in superoxide (Hamilton et al., 2002; Rippe et al., 2010) and inadequate BH4 bioactivity (Cosentino et al., 1998; Blackwell et al., 2004; Eskurza et al., 2005; Delp et al., 2008).

Recently it was shown that 3 weeks of sodium nitrite treatment reverses impaired EDD to ACh in cremaster muscle arterioles of hypercholesterolemic mice (Stokes et al., 2009). In the present study, we show that in a model of primary arterial aging, short-term treatment with sodium nitrite completely restores EDD in old mice to levels observed in young animals. Importantly, our findings show for the first time that sodium nitrite treatment restores EDD in a setting of impaired baseline function by restoring NO bioavailability (i.e., the NO-component of EDD) to normal control levels. Endothelial nitric oxide synthase protein in the aorta was reduced with aging, but was not influenced by sodium nitrite treatment in the older mice, consistent with earlier findings in myocardial tissue (Bryan et al., 2007).

The fact that administration of TEMPOL, a SOD mimetic, restored EDD in old control mice, while having no effect in young animals or old sodium nitrite-treated mice, supports the idea that a reduction in superoxide was responsible for the normalization of endothelial function by sodium nitrite in old mice. That apocynin, an inhibitor of NADPH oxidase, selectively restored EDD in old control mice further suggests that the effects of sodium nitrite in old animals was mediated by reduced NADPH oxide production of superoxide. Lastly, sepiapterin, an exogenous donor of BH4, restored EDD in old control, but had no effect in old nitrite-treated mice. This suggests that sodium nitrite may enhance NO bioavailability also by preserving BH4, a critical co-factor for eNOS-dependent NO production, as shown recently in the liver of hypercholesterolemic mice (Stokes et al., 2009).

Together, our data are consistent with the possibility that short-term sodium nitrite treatment ameliorates vascular endothelial dysfunction in old mice by restoring NO bioavailability as a result of reduced NADPH oxidase superoxide production and enhanced bioactivity of BH4.

Large elastic artery stiffness

Our finding that aortic pulse wave velocity was greater in old compared with young control mice is consistent with previous reports in both animals and humans that large elastic arteries stiffen with advancing age in the absence of disease (Reddy et al., 2003; Eskurza et al., 2004; Sutton-Tyrrell et al., 2005; Soucy et al., 2006). The present results extend these earlier findings by showing that 3 weeks of sodium nitrite therapy initiated in old age reduces aortic pulse wave velocity to levels that are no longer significantly different from those of young control and nitrite-treated mice. Nitrite treatment had no effect on aortic pulse wave velocity in young mice, indicating that treatment selectively reduced large elastic artery stiffness in old animals. Our findings in old mice are clinically important because recently it was shown that aortic pulse wave velocity is a major independent risk factor for incident CV events and all-cause mortality in older adults (Mitchell et al., 2010; Vlachopoulos et al., 2010). This provides experimental support for the idea that sodium nitrite treatment holds promise for reducing CVD and all-cause deaths in middle-aged and older humans.

Oxidant stress and inflammation

The present findings are consistent with previous work from our laboratory (Lesniewski et al., 2009; Rippe et al., 2010) and others (van der Loo et al., 2000; Csiszar et al., 2002; Yang et al., 2009) showing increased arterial oxidative stress with aging as indicated by a marked increase in aortic nitrotyrosine staining in old compared with young control mice. Nitrotyrosine is produced by nitration of tyrosine residues on proteins primarily by peroxynitrite, which is formed when superoxide reacts with NO (Radi, 2004). The present findings also confirm recent results from our laboratory (Rippe et al., 2010) showing that this increase in arterial oxidative stress with aging in mice is associated with both increased superoxide production, as measured directly by spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and reduced activity of SOD, an important antioxidant enzyme.

Our findings here suggest that sodium nitrite treatment completely reversed the increase in arterial oxidative stress with aging, as indicated by a reduction in aortic nitrotyrosine abundance in nitrite-treated old animals to levels observed in young control mice. An earlier report found that nitrite inhibits myeloperoxidase-mediated modification of low-density lipoprotein (Carr & Frei, 2001), consistent with an antioxidant effect. Nitrite treatment appeared to reduce arterial oxidative stress in old mice, at least in part, by normalizing arterial superoxide production. Indeed, to our knowledge this is the first evidence that short-term sodium nitrite administration reduces superoxide formation in arteries, which is consistent with previous observations following ischemia/reperfusion injury in the heart (Dezfulian et al., 2009). Moreover, our results show that nitrite treatment restored SOD activity in aorta of old animals and this also may have contributed to reductions in arterial superoxide bioavailability and oxidative stress in this group. The effect appeared to be the result of an increase in activity of the enzyme and not because of the changes in expression, as protein concentrations of the SOD isoforms were unaffected by sodium nitrite treatment in the old animals.

In this study, we also show that sodium nitrite treatment reduced the expression of several pro-inflammatory cytokines in aorta of old mice to concentrations observed in young controls. This is consistent with the results of a previous study in hypercholesterolemic mice, in which sodium nitrite treatment lowered plasma C-reactive protein and reduced leukocyte adhesion and infiltration through venular endothelium (Stokes et al., 2009). Taken together, our results provide the first evidence of a potent combined antioxidant and anti-inflammatory effect of sodium nitrite treatment on arteries.

Plasma and tissue nitrite concentrations

The present data are the first to show that nitrite concentrations are reduced with aging in large elastic arteries (−44% vs. young controls), the heart (−43%), and plasma (−46%). Reductions in nitrite in plasma and the heart have been documented previously in other mouse models of low NO bioavailability including hypercholesterolemia and eNOS deficiency (Bryan et al., 2008; Stokes et al., 2009). In the present study, nitrite supplementation in the drinking water increased arterial, cardiac, and plasma nitrite concentrations in old mice to levels not different from (arterial and heart) or even greater than (plasma) young control mice. These findings are consistent with the results of earlier studies of nitrite supplementation in states in which baseline nitrite concentrations are reduced (Bryan et al., 2008; Stokes et al., 2009). We did not observe an increase in tissue nitrite above normal control concentrations reported previously in hypercholesterolemic mice (Stokes et al., 2009), but rather only in plasma levels in our old mice. Overall, our results demonstrate that short-term nitrite therapy in old mice restores (arterial and cardiac) or enhances above normal (plasma) nitrite concentrations compared with levels observed in young controls.

Conclusions

The results of this study show for the first time that nitrite concentrations are reduced with aging in the circulation and cardiovascular tissues, and that short-term sodium nitrite therapy ameliorates vascular endothelial dysfunction and de-stiffens large elastic arteries, two clinically significant expressions of arterial aging, in old C57BL6 mice. Our findings also provide evidence that the improvements in vascular endothelial function in old mice treated with sodium nitrite are mediated by increased NO bioavailability as a result of reduced NADPH oxidase-dependent superoxide bioavailability and enhanced BH4 bioactivity. Moreover, our data provide the first evidence for antioxidant and anti-inflammatory influences of sodium nitrite on arterial tissue. Overall, these findings from a preclinical model establish an experimental basis for translational research aimed at determining the efficacy of nitrite therapy for reversing arterial aging and reducing the risk of age-associated CVD in humans.

Experimental procedures

Animals

Young (4–6 months) and old (26–28 months; ∼50% survival) male C57BL6 mice were obtained from the National Institute on Aging rodent colony and were fed normal rodent chow ad libitum. After an acclimation period of 2 weeks, the young and old mice were divided into two subgroups: control animals continued on regular drinking water and the other animals that had nitrite-supplemented (50 mg L−1) drinking water for 3 weeks. All mice were housed in an animal care facility at the University of Colorado at Boulder on a 12:12 h light-dark cycle. All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals (NIH publication n. 85–23, revised 1996) and were approved by the UCB Animal Care and Use Committee.

Carotid artery vasodilatory responses

Endothelium-dependent dilation and endothelium-independent dilation were determined ex vivo in isolated carotid arteries as previously described (Durrant et al., 2009; Lesniewski et al., 2009; Rippe et al., 2010). Briefly, mice were anesthetized using isoflurane and euthanized by exsanguination via cardiac puncture. The carotid arteries were carefully excised, cannulated onto glass micropipettes, and secured with nylon (11-0) suture in myograph chambers (DMT Inc., Ann Arbor, MI, USA) containing buffered physiological saline solutions. The arteries were pressurized to 50 mmHg at 37 °C and were allowed to equilibrate for 1 h. After submaximal preconstriction with phenylephrine (2 μm), increases in luminal diameter in response to acetylcholine (ACh: 1 × 10−9 − 1 × 10−4 m) with and without co-administration of the NO synthase inhibitor l-NAME, (0.1 mm, 30 min incubation), or the SOD mimetic, TEMPOL, (1 mm, 60 min incubation), were determined. Endothelium-dependent dilation also was determined in the presence of the NADPH oxidase inhibitor, apocynin, (1 mm, 60 min incubation) and the exogenous BH4 donor, sepiapterin, (1 mm, 60 min incubation). Endothelium-independent dilation was determined by vasodilation in response to sodium nitroprusside (SNP: 1 × 10−10 − 1 × 10−4 m).

All dose–response data are presented on a percent basis. Preconstriction was calculated as a percentage of maximal diameter according to the following formula:

image

Because of differences in maximal carotid artery diameter between young and old animals, vasodilator responses were recorded as actual diameters expressed as a percentage of maximal response according to the following formula:

image

Where Dm is maximal inner diameter at 50 mmHg, Ds is the steady-state inner diameter recorded after the addition of drug, and Db is the steady-state inner diameter following preconstriction before the first addition of drug.

NO-dependent dilation was determined from the maximal EDD in the absence or presence of l-NAME according to the following formula:

image

In vivo aortic pulse wave velocity

Aortic pulse wave velocity was measured as described previously (Kim et al., 2009). Mice were anesthetized with 2% isoflurane and placed supine on a heating board with legs secured to ECG electrodes. Aortic velocity was measured with Doppler probes at the transverse aortic arch and abdominal aorta. Pre-ejection time, the time between the R-wave of the ECG to foot of the Doppler signal, was determined for each site. Aortic pulse wave velocity was calculated by dividing the distance between the transverse and abdominal probes by the difference in the thoracic and abdominal pre-ejection times.

Arterial superoxide production

Production of superoxide was measured by EPR spectrometry using the spin probe 1-hydroxy-3-methoxycarbonly-2,2,5,5-tetramethylpyrrolidine (CMH; Alexis Biochemicals, San Diego, CA, USA) as previously described (Rippe et al., 2010). Two-millimeter aortic rings were incubated for 60 min at 37 °C in 200 μL of Krebs-HEPES buffer containing 0.55 mm CMH and analyzed immediately on an MS300 X-band EPR spectrometer (Magnettech, Berlin, Germany).

Arterial protein expression and enzyme activities

Aortas were used as a surrogate large elastic artery to provide sufficient tissue for analysis of protein expression by Western blot and enzyme activity as described previously (Durrant et al., 2009; Lesniewski et al., 2009; Rippe et al., 2010). Aortas were excised, cleared of surrounding tissues, and frozen in liquid nitrogen before storage at −80 °C. The tissue was pulverized over liquid nitrogen and homogenized in ice-cold Radioimmunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors [Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA) and 0.01% phosphatase inhibitor cocktail (Sigma, St Louis, MO, USA)]. Ten micrograms of protein was loaded on 4–12% polyacrylamide gels, separated by electrophoresis, and transferred onto nitrocellulose membranes for Western blot analysis. Antibodies for Western blot analysis included anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH 1:1000; Cell Signaling), anti-nitrotyrosine (1:100; Abcam, Cambridge, MA, USA), anti-p67 phox (1:1000; Cell Signaling, Danvers, MA, USA), anti-MnSOD, anti-CuZn (1:2000; Stressgen, Ann Arbor, MI, USA), anti-ecSOD (1:500; Sigma), and anti-endothelial NO synthase (eNOS 1:500; BD Biosciences, San Jose, CA, USA). Total SOD activity in aortic lysates (1 μg protein) was determined using the SOD Activity Assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Concentrations of the pro-inflammatory cytokines IL-1β, IL-6, IFNγ and TNF-α were determined in aortic whole cell lysates by multiplex ELISA (Searchlight Mouse Inflammatory Cytokine Kit; Aushon Biosystems; Billerica, MA, USA) as previously described (Rippe et al., 2010).

Analysis of nitrite in large elastic arteries, heart and plasma

Nitrite analysis procedures have been described in detail (Bryan et al., 2007, 2008; Elrod et al., 2008). Nitrite concentrations were quantified by ion chromatography (ENO20 Analyzer; Eicom, San Diego, CA, USA). Plasma was obtained by centrifugation at 800 g for 10 min.

Statistics

Results are presented as mean ± SEM. Statistical analysis was performed with spss 17.0 software (IBM, Somers, NY, USA). For the ex vivo vasodilatory dose response, group differences were determined by repeated measures anova. A two-way anova was used to analyze stiffness. For maximal dilation, protein expression, enzyme activities, superoxide production, and animal characteristics comparisons between groups were made using anova. Significance was determined using P < 0.05.

Funding

This work is supported by the National Institutes of Health (AG013038, HL098481-01, AG000279, HL007822, HL092141 and HL093579) and the American Diabetes Association (7-09-BS-26).

Author contributions

Amy L. Sindler, Bradley S. Fleenor, David J. Lefer, and Douglas R. Seals contributed to the conception, experimental design, and interpretation of the data. Amy L. Sindler and Melanie L. Zigler collected most of the data; David J. Lefer and John W. Calvert were responsible for the nitrite measures in arteries and tissue; Kurt D. Marshal assisted with the collection and analysis of the Western blot data. The manuscript was largely written by Douglas R. Seals and Amy L. Sindler; however, all authors were involved in the writing and in the final approval of the paper.

Ancillary