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(Circulation Research. 2002;90:1159.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Aging-Induced Phenotypic Changes and Oxidative Stress Impair Coronary Arteriolar Function

Anna Csiszar, Zoltan Ungvari, John G. Edwards, Pawel Kaminski, Michael S. Wolin, Akos Koller, Gabor Kaley

From the Department of Physiology, New York Medical College, Valhalla, NY.

Correspondence to Gabor Kaley, PhD, Dept of Physiology, New York Medical College, Valhalla, NY 10595. E-mail gabor_kaley@ nymc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract— We aimed to elucidate the possible role of phenotypic alterations and oxidative stress in age-related endothelial dysfunction of coronary arterioles. Arterioles were isolated from the hearts of young adult (Y, 14 weeks) and aged (A, 80 weeks) male Sprague-Dawley rats. For videomicroscopy, pressure-induced tone of Y and A arterioles and their passive diameter did not differ significantly. In A, arterioles L-NAME (a NO synthase blocker)–sensitive flow-induced dilations were significantly impaired (Y: 41±8% versus A: 3±2%), which could be augmented by superoxide dismutase (SOD) or Tiron (but not L-arginine or the TXA₂ receptor antagonist SQ29,548). For lucigenin chemiluminescence, O₂^·- generation was significantly greater in A than Y vessels and could be inhibited with SOD and diphenyliodonium. NADH-driven O₂^·- generation was also greater in A vessels. Both endothelial and smooth muscle cells of A vessels produced O₂^·- (shown with ethidium bromide fluorescence). For Western blotting, expression of eNOS and COX-1 was decreased in A compared with Y arterioles, whereas expressions of COX-2, Cu/Zn-SOD, Mn-SOD, xanthine oxidase, and the NAD(P)H oxidase subunits p47^phox, p67^phox, Mox-1, and p22^phox did not differ. Aged arterioles showed an increased expression of iNOS, confined to the endothelium. Decreased eNOS mRNA and increased iNOS mRNA expression in A vessels was shown by quantitative RT-PCR. In vivo formation of peroxynitrite was evidenced by Western blotting, and immunohistochemistry showing increased 3-nitrotyrosine content in A vessels. Thus, aging induces changes in the phenotype of coronary arterioles that could contribute to the development of oxidative stress, which impairs NO-mediated dilations.

Key Words: arteriole • endothelium • superoxide • reactive oxygen species • free radical scavenger

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Numerous studies suggest that aging is an important risk factor for the development of ischemic heart disease. This may be due to an age-related increase in coronary vascular resistance,¹ leading to a reduction in myocardial blood flow and flow reserve.^1,2 Studies utilizing pharmacological probes, such as acetylcholine (ACh), suggest that aging is associated with endothelial dysfunction in humans^3,4 and in laboratory animals.^5,6 One of the most important mechanisms that contribute to the local regulation of myocardial blood flow is the flow (shear stress)–induced NO-mediated dilation of small coronary arteries and arterioles⁷; however, its age-related alterations have not yet been elucidated. Previous studies demonstrated that pathophysiological conditions that are associated with an increased risk of coronary heart disease, such as hypercholesterolemia, diabetes, hypertension, and hyperhomocysteinemia, are characterized by decreased NO synthesis/release and a significant impairment of flow-induced dilation of arterioles.^{8– 10}

Thus, it can be hypothesized that aging may also impair NO synthesis/release in the coronary endothelium by decreasing availability of the eNOS substrate L-arginine¹¹ or by decreasing the activity of eNOS.¹² Also, there may be an increased breakdown of NO due to an augmented arteriolar production of superoxide (O₂^·-) anions¹³ or a loss of antioxidant capacity, which normally provides protection against reactive oxygen species (oxidative stress, reviewed in Beckman and Ames¹⁴). In addition, age-related dysfunction in some conduit vessels may involve an enhanced synthesis of thromboxane A₂ (TXA₂),⁵ suggesting that the underlying mechanisms associated with vascular aging are multifactorial with significant anatomic heterogeneity.⁶ Recent studies showing that aging significantly alters regulation of gene expression by hormonal and growth factors¹⁵ raise the possibility that complex phenotypic changes affecting expression of eNOS⁶ and/or a shift in the expression of pro- and antioxidant enzymes^6,12,13,16 may elicit age-related decreases in NO bioavailability.

To test the hypothesis that aging is associated with impaired NO-mediation of flow-induced dilation due to an increased production of superoxide (which scavenges NO) or TXA₂ (which may counteract the effect of NO), we characterized in isolated coronary arterioles age-related alterations in flow-induced dilation, O₂^·- production, peroxynitrite generation,¹⁷ and expression of eNOS and the pro- and antioxidant enzymes known to be involved in the modulation of flow-induced arteriolar dilation (SOD and COX isoforms, NAD(P)H oxidases, xanthine oxidase, iNOS).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Fourteen- to sixteen-week-old (young, Y) and 74- to 82-week-old (aged, A) male Sprague-Dawley rats (n=36; Harlan Sprague-Dawley, Indianapolis, Ind) were used. Protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conformed to the current guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.

Functional Studies on Isolated Arterioles
Intramural arteriolar branches of the septal artery were isolated from the hearts of Y and A rats. Changes in diameter of pressurized (at 80 mm Hg) Y and A arterioles in response to step increases in intraluminal flow (from 0 to 60 µL/min) were measured with videomicroscopy,^8,9,18,19 before and after incubation with N-nitro-L-arginine-methyl-ester (L-NAME, 3x 10^-4 mol/L, for 20 minutes, an inhibitor of NO synthesis). Responses to acetylcholine (ACh), the voltage-operated Ca²⁺ channel inhibitor verapamil, and the NO donor sodium nitroprusside (SNP) were also obtained. In separate experiments, responses of arterioles to flow and ACh (10^-6 mol/L) were measured; the arterioles were also incubated with L-arginine (10^{- 4} mol/L, the substrate of eNOS), SQ29,548 (10^{- 6} mol/L, a TXA₂ receptor antagonist), or the superoxide scavengers Tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid, 10 mmol/L)²⁰ or superoxide dismutase (SOD, 120 U/mL),^18,20 and responses were reassessed.

Measurement of Vascular Superoxide Level
Vascular O₂^·- production was assessed from vascular samples similar to those used for biochemical studies, by the lucigenin chemiluminescence method, ¹³ according to the modified protocol of Mohazzab et al.²⁰ In separate experiments, O₂^·- production of aged vessels was determined in the absence and presence of rotenone (10^-5 mol/L), aminoguanidine (10^-4 mol/L), indomethacin (10^-5 mol/L, a COX inhibitor), allopurinol (10^-4 mol/L, an inhibitor of xanthine oxidase), diphenyliodonium (DPI, 10^-4 mol/L, an inhibitor of flavoprotein-containing oxidases, including NAD(P)H oxidases), or SOD (200 U/mL). In other experiments, superoxide production by aortic segments (4 mm) from young and aged rats was also measured.

Xanthine Oxidase Assay and NADH Oxidase Assay
Pooled coronary vessels of Y and A rats were homogenized in liquid nitrogen and lysed in buffer containing protease inhibitors. Xanthine oxidase and NADH oxidase activity were measured with the lucigenin assay^13,20,21 in 20 µL of the particulate fraction of the homogenate after addition of 10^-4 mol/L xanthine and NADH, respectively. Protein content was measured in an aliquot of the homogenate by the Lowry method.

Ethidium Bromide Fluorescence
Hydroethidine, an oxidative fluorescent dye, was used to localize superoxide production in situ according to the modified protocol of Lund et al²² and Rey et al.²³ This method provides sensitive detection of O₂^·- levels in situ.²⁴ The number of fluorescent ethidium bromide (EB)–stained nuclei was counted per arterial cross sections in unfixed frozen sections of the left ventricle after hydroethidine (2x10^-6 mol/L) exposure. The sections were also counterstained with hematoxylin and immunostained for -smooth muscle actin. Samples exposed to hydroethidine in the presence of Tiron (10 mmol/L) served as control.

Detection of Coronary Arteriole Protein Expression With Western Blotting
We have modified a Western blot protocol enabling us to use single arterioles, identical to those used for functional studies. A detailed description and validation of the method can be found in the expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org. Expressions of eNOS, COX-1, COX-2, Mn-SOD and Cu, Zn-SOD, iNOS, the NAD(P)H oxidase subunits p22^phox, p47^phox, p67^phox and Mox-1, and xanthine oxidase were analyzed in coronary vessels isolated from the same Y and A rats that were used for functional studies. Anti–ß-actin was used to normalize for loading variations.

RNA Isolation and Real-Time RT-PCR
A quantitative reverse transcription–PCR technique was used to determine relative expression levels of eNOS mRNA in coronary arterioles of A and Y rats. Total RNA from single coronary arterioles was isolated and was reverse transcribed. PCR reactions were performed using a real-time fluorescent determination in the Roche Molecular Biochemicals LightCycler System. The housekeeping gene ß-actin was used for internal normalization.

Detection of Coronary 3-Nitrotyrosine Content With Western Blotting
3-Nitrotyrosine content in single coronary arterioles was quantified with Western blotting. Isolated coronary arterioles of control rats incubated with authentic ONOO^- (10 µmol/L and 10 mmol/L) served as positive controls.

Immunohistochemical Detection of 3-Nitrotyrosine
Frozen sections from the interventricular septum and the anterior wall of the left ventricle were labeled for 3-nitrotyrosine, stained with Vector-DAB, and counterstained with hematoxylin.

Localization of iNOS With Dual Immunofluorescence Staining and Immunohistochemistry
Immunolabeling was carried out with primary anti-iNOS and anti–-smooth muscle actin antibody (to visualize smooth muscle) or anti-CD31 antibody (to stain endothelium). Separate tissue sections were immunolabeled for iNOS, stained with Vector-ALP substrate, and counterstained with hematoxylin.

Measurement of Serum Nitrite/Nitrate (NOx)
NOx content was measured in serum samples of Y and A rats as described previously.²⁵

Data Analysis
Arteriolar dilations were expressed as percentage of the maximal dilation in the absence of extracellular Ca²⁺ at 80 mm Hg intraluminal pressure. Lucigenin chemiluminescence data were normalized to the respective control mean values. Densitometric ratios were expressed as a percentage of the control mean value. Data are mean±SEM Statistical analyses of data were performed by Student’s t test or by 2-way ANOVA followed by the Tukey post hoc test, as appropriate. A value of P<0.05 was considered statistically significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolated coronary arterioles developed active tone in response to intraluminal pressure of 80 mm Hg without the use of any vasoactive agent (Y: 31±2%, A: 33±4%; NS). The passive diameters of Y and A arterioles in the absence of extracellular Ca²⁺ were 200±11 and 229±16 µm (NS), respectively (at 80 mm Hg).

Flow- and Agonist-Induced Dilations of Coronary Arterioles
Step increases in intraluminal flow (from 0 to 60 µL/min, corresponding to a shear stress of 20 dyn/cm²) elicited marked dilations of coronary arterioles of Y rats, which were significantly inhibited by L-NAME (Figure 1A). In contrast, in arterioles of aged rats, responses to flow were greatly diminished and were unaffected by L-NAME (Figure 1B).

View larger version (32K): [in this window] [in a new window]   Figure 1. Flow-induced dilation of coronary arterioles of young adult (A) and aged (B) rats as a function of intraluminal flow in the presence and absence of the NO synthase inhibitor L-NAME. C, Flow-induced responses of coronary arterioles of aged rats in the absence and presence of L-arginine (ARG), SQ29,548, superoxide dismutase (SOD), or Tiron. D, Time course of dilations of coronary arterioles from aged rats in response to an increase in intraluminal flow (from 0 to 60 µL/min) before and after administration of SOD or SOD+L-NAME. E and F, Arteriolar dilations to acetylcholine (ACh, E) and sodium nitroprusside (SNP, F) (n=6 to 7 for each groups, 1 vessel from each animal was used). Data are mean±SEM; *P< 0.05.

Administration of the eNOS substrate L-arginine or the presence of SQ29,548 (Figure 1C) did not restore flow-induced arteriolar dilations,⁸ excluding the possibility that decreased availability of L-arginine¹¹ or an increased PGH₂/TXA₂ synthesis⁵ affects coronary arteriolar responses in aging. In contrast, administration of the free radical scavengers SOD or Tiron significantly augmented dilations of A arterioles to step increases in intraluminal flow (Figures 1C and 1D). In the presence of SOD, administration of L-NAME diminished flow-induced dilation of A arterioles (Figure 1D). SOD, Tiron, L-arginine, or SQ29548 had no significant effect on responses of Y arterioles.

We also demonstrated that ACh-induced dilation of aged coronary arterioles is impaired (Figure 1E), extending results of previous studies on vessels from other vascular beds.^5,6 Dilations to the NO donor SNP (Figure 1F)^5,6 and the Ca²⁺ antagonist verapamil (at 10^-6 mol/L, Y: 75±8%, A: 81±9%; NS, not shown), as well as the passive mechanical properties of arterioles of Y and A rats were similar.

Vascular Superoxide Production
Under basal conditions, levels of O₂^·- produced by coronary vessels of A rats were significantly higher than by vessels of Y rats (Figure 2A). O₂^·- level in A vessels was significantly decreased by administration of DPI or SOD, whereas it was unaffected by rotenone, aminoguanidine, indomethacin, or allopurinol (Figure 2B). In aortic segments of A rats, there was also a significantly increased O₂^·- production (Y: 100±11%, A: 239±16%), confirming results of previous studies.^13,17

View larger version (73K): [in this window] [in a new window]   Figure 2. A, O2·- generation as determined by lucigenin chemiluminescence in coronary vessels of young and aged rats. Data are normalized to the mean value of the young group. *P<0.05 (n=7). B, O2·- generation in coronary vessels of aged rats under control conditions and in the presence of rotenone (ROT, 10-5 mol/L), aminoguanidine (AG, 10-4 mol/L), indomethacin (INDO, 10-5 mol/L), allopurinol (ALLO, 10-4 mol/L), DPI (10- 4 mol/L), or SOD (200 U/mL). Data are normalized to the mean value of the time control group. *P< 0.05 (n=4 to 7). C, Xanthine- (10-4 mol/L) and NADH- (10-4 mol/L) driven O2·- production in coronary vessels of young (n=6) and aged (n=6) rats. *P<0.05. D, Representative fluorescent photomicrographs of aged (D, left) and young (E, left) coronary vessel sections labeled with the dye dihydroethidium, which produces a red fluorescence when oxidized to ethidium bromide (EB) by O2·-. Brightfield photomicrographs of the same vessel sections (hematoxylin staining, D and E, right). F through I, Overlaying (H) of EB-stained fluorescent (F) and hematoxylin-stained brightfield (G) images of an aged vessel or EB-stained fluorescent images with images of the same vessel section stained for smooth muscle -actin (I, green) shows that increased O2·- levels are present both in the endothelium and the smooth muscle of aged vessels. Similar findings were observed in 3 separate experiments. Scale bars=10 µ m.

Xanthine Oxidase and NADH Oxidase Assays
Xanthine-driven O₂^{· -} production by coronary vessel homogenates from A and Y rats did not differ significantly (Figure 2C). NADH-driven O₂^·- production by vascular samples from A rats was significantly increased as compared with Y controls (Figure 2C). Both xanthine- and NADH-induced increases in lucigenin chemiluminescence could be abolished by addition of SOD.

Hydroethidine Fluorescence
Representative fluorescent photomicrographs of EB-stained aged (Figure 2D, left) and young (Figure 2E, left) coronary vessel sections are shown in Figure 2 (brightfield photomicrographs of the same vessel sections: Figures 2D and 2E, right). The relative number of EB-positive nuclei in arterioles and arteries was significantly greater in sections of A hearts than in sections of Y hearts. Overlaying (Figure 2H) of EB-stained fluorescent (Figure 2F) and hematoxylin-stained brightfield (Figure 2G) images of aged vessels or EB-stained fluorescent images with images of the same vessel sections stained for smooth muscle -actin (Figure 2I, green) showed that increased O₂^·- levels are present both in the endothelium and the smooth muscle of aged vessels. Incubation of tissue sections with Tiron (10 mmol/L) prevented EB staining of nuclei.

Detection of Coronary 3-Nitrotyrosine Content With Western Blotting and Immunohistochemistry
Aging was associated with an increased prevalence of nitrated tyrosine residues of proteins in coronary vessels, whereas 3-nitrotyrosine immunoreactivity was weak or absent in coronary vessels of young rats (Figure 3A, left). In Y arteries pretreated with authentic ONOO^- (Figure 3A, lanes 1 and 2), there was a strong 3-nitrotyrosine immunoreactivity. The summary data of the total background-corrected band densities normalized to the vascular ß -actin content (Figure 3A, right) show that 3-nitrotyrosine content of coronary vessels of A rats is significantly increased, compared with vessels of Y rats.

View larger version (99K): [in this window] [in a new window]   Figure 3. A, Western blot analysis of protein 3-nitrotyrosine content in coronary arterioles of young adult (lanes 3 to 7) and aged (lanes 8 to 12) rats. Coronary arterioles of control rats incubated with 1 µmol/L (lane 1) or 10 mmol/L (lane 2) authentic ONOO- served as positive controls. Anti–ß-actin was used to normalize for loading variations. Bar graphs are summary data of normalized densitometric ratios. *P<0.05. B through F, In coronary arteries and arterioles of aged rats, immunostaining for 3-nitrotyrosine (brown reaction product, DAB staining) was localized both to the endothelium (arrows) and the media/adventitia (arrowheads). Consecutive sections served as negative controls (G, antigen absorption; H, omission of the primary antibody). Hematoxylin counterstaining. Scale bars=10 µ m.

In intramural coronary arterioles and arteries of A rats, immunostaining for 3-nitrotyrosine was localized both to the endothelium (Figures 3B through 3F, arrows) and the media/adventitia (arrowheads). In control experiments on consecutive tissue sections, in the absence of the primary antibody or after absorption of the primary antibody with the homologous antigen for 3-nitrotyrosine (Figures 3G and 3H), there was no evidence of nonspecific immunostaining.

Changes in Protein Expression
Arteriolar protein expressions were assessed in single vessels by Western blotting and normalized to arteriolar ß-actin content. Original Western blots in Figures 4 through 6 are representative of 3 to 4 separate experiments (each vessel was isolated from a different animal). The bar graphs show summary data of the total background-corrected band densities normalized to the arteriolar ß-actin content. ß-Actin expression did not change significantly with aging. Figure 4 shows that in arterioles of A rats, expression of eNOS was significantly decreased compared with arterioles of Y rats. Arteriolar expressions of Cu/Zn-SOD and Mn-SOD were not affected significantly by aging (Figure 5A). Expression of COX-1 (Figure 5B) was significantly decreased in A arterioles, whereas expressions of COX-2 (Figure 5B) and xanthine oxidase (Figure 5C) were unaltered. Expression of the NAD(P)H oxidase subunits p22^phox and mox-1, p47^phox and p67^phox (Figure 6) did not differ significantly between Y and A arterioles, whereas expression of iNOS was significantly greater in A than in Y arterioles (Figure 7A).

View larger version (27K): [in this window] [in a new window]   Figure 4. A, Western blot analysis of eNOS protein expression in single coronary arterioles of young adult (lane 1 to 7) and aged (lane 8 to 13) rats. Anti–ß-actin was used to normalize for loading variations. Bar graphs are summary data of normalized densitometric ratios. B, Calculated eNOS:ß -actin mRNA ratios in young and aged coronary arterioles (n=7 for each group, 1 vessel from each animal was used). Quantification of mRNA expression was performed by real-time PCR with the LightCycler System. Data are mean± SEM; *P< 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 5. Western blot analysis of Cu, Zn-SOD, Mn-SOD (A), COX-1, COX-2 (B), and xanthine oxidase (XO, C) protein expression in single coronary arterioles of young adult and aged rats. Anti–ß-actin was used to normalize for loading variations. Bar graphs are summary data of normalized densitometric ratios (n=5 to 7 for each group, 1 vessel from each animal was used). Data are mean±SEM.

View larger version (44K): [in this window] [in a new window]   Figure 6. A, Western blot analysis of expression of the membrane-bound NAD(P)H oxidase subunits p22phox (A) and Mox-1 (B) and the cytoplasmic subunits p47phox (C) and p67phox (D) in single coronary arterioles of young adult and aged rats. Anti–ß -actin was used to normalize for loading variations. Bar graphs are summary data of normalized densitometric ratios (n=5 to 7 for each group, 1 vessel from each animal was used). Data are mean± SEM.

View larger version (52K): [in this window] [in a new window]   Figure 7. A, Western blot analysis of iNOS protein expression in single coronary arterioles of young adult and aged rats. Lysate of cytokine-stimulated endothelial cells was used as positive control (lane 1). Anti–ß-actin was used to normalize for loading variations. Bar graphs are summary data of normalized densitometric ratios. B, Calculated iNOS:ß-actin mRNA ratios in young and aged coronary arterioles (n=7). Quantification of mRNA expression was performed by real-time PCR with the LightCycler System. Data are mean± SEM; *P<0.05. C, In coronary arteries and arterioles of aged rats, immunofluorescent staining for iNOS (red) was confined to the endothelium (arrow), whereas the smooth muscle (-smooth muscle actin staining, green) was free from immunoreactivity (40x). D, Colocalization of iNOS with the endothelial cell marker CD31 in coronary vessel of aged rat (40x). E, G through J, In coronary vessels of aged rats, immunostaining for iNOS (red reaction product, alkaline phosphatase staining) was confined to the endothelium, whereas the media was free from immunoreactivity. K, Lack of immunoreactivity for iNOS in the endothelium of coronary artery of young rat. F and L, Consecutive sections served as negative controls (omission of the primary antibody). Hematoxylin counterstaining. Scale bars=10 µm (E through H, J), 50 µm (I), and 20 µm (K and L).

Changes in eNOS mRNA Expression
Expression of eNOS mRNA in A coronary arterioles was significantly decreased (Figure 4B), whereas expression of iNOS mRNA was significantly increased (Figure 7B), compared with arterioles of Y rats.

Localization of iNOS Expression With Dual Immunofluorescence
In intramural coronary arterioles and arteries of A rats, immunostaining for iNOS (red) did not overlap with smooth muscle -actin staining (green, Figure 7C) and was colocalized with CD31 in the endothelium (green, Figure 7D). Immunohistochemistry also showed that in coronary vessels of A rats immunostaining for iNOS was predominantly localized to the endothelium (Figures 7E and 7G through 7J), whereas the vascular endothelium of Y rats (Figure 7K) was free of immunoreactivity. In control experiments on consecutive tissue sections in the absence of the primary antibody (Figures 7F and 7L), there was no evidence for nonspecific immunostaining.

Measurement of Serum NOx
Serum NOx concentration in Y and A rats was 5.9±0.9x 10^-6 mol/L and 3.5±0.3x 10^-6 mol/L, respectively (P<0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the new findings of the present study is that in arterioles isolated from rats with a biological age corresponding to that of 65- to 70-year-old humans,²⁶ increases in intraluminal flow did not elicit substantial NO-mediated dilation (Figure 1B), a response that is present in arterioles of young adult animals⁷ with a biological age corresponding to that of 18- to 20-year-old humans.²⁶ Our findings have important clinical implications because healthy elderly humans of similar age frequently have significant coronary endothelial dysfunction, as demonstrated in vivo by vasomotor responses to pharmacological probes.^3,4 An age-related decreased vascular release of NO is also supported by microelectrode measurements in the aorta²⁷ and a significant decrease in serum level and urinary excretion¹¹ of the NO metabolites, nitrate and nitrite. However, the mechanisms responsible for reduced NO release are less clear.

Our finding, that scavenging of superoxide with SOD and Tiron augmented flow-induced NO-mediated dilation in coronary arterioles of aged rats (Figures 1C and 1D), suggests that in older vessels, increases in flow do elicit NO synthesis and release, but elevated levels of O₂^{· -} react with NO to decrease its bioavailability. This is likely to be the case in human aging because ACh-induced increases in brachial blood flow in elderly patients can be significantly augmented by administration of the antioxidant vitamin C.²⁸ To further support this hypothesis in the present study, increased vascular O₂^·- production was evidenced by showing increased lucigenin chemiluminescence in aged coronary vessels (Figure 3A). We also identified NAD(P)H oxidase(s) as major sources of vascular O₂^·- generation in aging because increased lucigenin chemiluminescence of aged vessels could be substantially inhibited by DPI (as well as by SOD), but not by rotenone or inhibitors of iNOS, COX, and xanthine oxidase (Figure 2B). A primary role for increased NAD(P)H oxidase activity in aging is further supported by the findings that in aged coronary vessels NADH-driven (but not xanthine-driven) O₂^·- generation was substantially enhanced (Figure 2C). Similar findings have also been reported recently on aged rat aorta and carotid artery.¹³ Localization of EB-stained nuclei suggests that in aging both endothelial and smooth muscle cells generate substantial amounts of O₂^·- (Figures 2H and 2I). This finding also accords with results of recent studies showing that NAD(P)H oxidases are expressed and active both in vascular endothelium^20,29 and smooth muscle.³⁰ Importantly, an increased activity of vascular NAD(P)H oxidase(s) has been shown recently to be associated with several pathophysiological conditions with increased risk for coronary heart disease (eg, hypercholesterolemia, hypertension, and diabetes).^13,29,31

It is known that interaction of superoxide with NO results in the formation of peroxynitrite (ONOO^-).³² Because in coronary vessels of A rats there is an increased protein 3-nitrotyrosine content (a biomarker of in vivo production of ONOO^-32,33; Figure 3A), we assume that in aging the increased levels of O₂^·- in vivo scavenge endothelium-derived NO, resulting in increased formation of ONOO^-. 3-Nitrotyrosine immunoreactivity was present both in the endothelium and smooth muscle of coronary arteries and arterioles of aged rats (Figures 3B through 3E), consistent with the idea that NO released from the endothelium to flow reacts with O₂^·- generated in both endothelial and smooth muscle cells (Figures 2D through 2K). A recent study also demonstrated enhanced 3-nitrotyrosine staining in aged rat aortas.¹⁷ Because flow-induced dilation of aged arterioles could be improved by scavenging O₂^·-, it is likely that ONOO^- did not inactivate eNOS, yet ONOO^--induced protein modification (eg, tyrosine nitration or cysteine oxidation) is likely to affect the function of other enzymes,^17,33–35 further promoting coronary vessel dysfunction in aging.

We also sought to characterize age-related changes in arteriolar expression of some of the pro- and antioxidant enzymes that are likely to be involved in the maintenance of normal flow-induced arteriolar dilation. In coronary arterioles of aged rats, expression of eNOS protein was significantly decreased (Figure 4A). Because levels of eNOS mRNA were also decreased in A arterioles, as determined by QRT-PCR (Figure 4B), transcription of the eNOS gene is likely to be downregulated in aging. Similar age-related decreases in eNOS mRNA and/or protein expression have also been reported in some other, but not all,¹⁷ vascular beds.³⁶ Interestingly, a recent study also reported a time-dependent decline in eNOS expression in cultured endothelial cells in vitro,³⁷ suggesting that this decline may contribute to the development of aging-induced oxidative stress promoting endothelial apoptosis.³⁷

Aging was shown to decrease the activity/expression of superoxide dismutases, which may impair elimination of O₂^·- in certain organs, such as the liver, of aged rats (reviewed by Matsuo³⁸). Because in coronary arterioles the expression of neither Cu, Zn-SOD, nor Mn-SOD (which are the major SOD isoforms in the cytoplasm and the mitochondria, respectively, whereas the function of extracellular SOD may be somewhat less pronounced in the rat³⁹) were affected by aging (Figure 5A), it is likely that aging-induced oxidative stress in these vessels is not due to a decreased presence of SOD. Previous studies also revealed important tissue- and species-dependent differences in SOD expression and demonstrated no significant changes in SOD expression in aged rat hearts.³⁸ Yet, one cannot exclude the possibility that vascular SOD activity declines with age due to posttranslational protein modification⁴⁰ or that vascular antioxidant systems distinct from SOD are altered by the aging process.³⁸

The findings that expression of COX-1 decreased, whereas that of COX-2 did not change significantly with aging (Figure 5B), together with the lack of an effect of SQ29,548 and indomethacin on flow-induced responses and superoxide production (Figures 2A and 3B, respectively), suggest that in coronary arterioles endothelial dysfunction is unlikely to be due to COX-related production of oxygen free radicals and/or TXA₂.⁵ Arteriolar expression of xanthine oxidase (Figure 5C), similar to xanthine oxidase activity (Figures 2B and 2C), was also unchanged in aging.

Because lucigenin chemiluminescence measurements showed that increased NAD(P)H oxidase activity contributes importantly to the development of oxidative stress in aged coronary vessels (Figures 2B and 2C), we investigated the expression of the 2 membrane-bound and the 2 cytosolic subunits of NAD(P)H oxidases known to be involved in superoxide production. Although in coronary arterioles of aged rats expression of the membrane-associated subunits (p22^phox, which is essential for the oxidase activity in endothelial and smooth muscle cells²⁹ and Mox-1,²⁹ an analog of the leukocyte-type gp91^phox subunit) tended to increase, this change did not reach statistical significance (Figures 6A and 6B). Interestingly, a previous study using semiquantitative immunohistochemical analysis showed an age-dependent upregulation of p22^phox in the rat aorta.¹³ Aging also did not seem to affect the arteriolar expressions of p47^phoxand p67^phox (Figures 6C and 6D), cytosolic subunits that are known to regulate the function of the membrane oxidase complex. Nevertheless, it is known that activity of NAD(P)H oxidases is regulated by nontranslational mechanisms (eg, phosphorylation by protein kinases). Recent studies demonstrated that vascular NAD(P)H oxidase activity is regulated by cytokines, including TNF-,²¹ the plasma level⁴¹ and vascular expression⁴² (see also the online data supplement) of which is increased in aging.

One could speculate that increased vascular production of cytokines⁴² and/or an increased NF-B–dependent sensitivity of vascular cells to cytokine stimulation⁴³ may also be responsible for the increased iNOS expression found in aged arterioles (Figure 7A), aorta,¹² and other vascular tissues.⁴⁴ QRT-PCR analysis also showed increased levels of iNOS mRNA in arterioles of aged rats (Figure 7B). Because expression of iNOS was confined predominantly to the endothelium (Figures 7C through 7J), it seems likely that basal NO production by iNOS may contribute to the increased endothelial ONOO^- formation (Figure 3).

In conclusion, flow-induced NO-mediated dilation of coronary arterioles is significantly diminished in aged rats due to increased O₂^·- production. We propose that a shift in arteriolar phenotype, including decreased expression of eNOS, increased expression of iNOS, and increased activity of NAD(P)H oxidases, underlies these changes and contributes to ONOO^- generation and the impairment of the vasoactive function of the coronary endothelium in aging, which could limit or reduce myocardial perfusion thereby promoting ischemic heart disease.

	Acknowledgments

 
This study was supported by grants from NIH PO 43023, HL-59417 and HL-46813, AHA NY State Aff. Inc 00500849T, 0020144T, and 0120166T.

Received January 17, 2002; revision received April 22, 2002; accepted April 22, 2002.

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