Aging impairs flow-induced dilation in coronary arterioles: role of NO and H(2)O(2)

Abstract

Aging contributes significantly to the development of cardiovascular disease and is associated with elevated production of reactive oxygen species (ROS). The beneficial effects of nitric oxide (NO)-mediated vasodilation are quickly abolished in the presence of ROS, and this effect may be augmented with aging. We previously demonstrated an age-induced impairment of flow-induced dilation in rat coronary arterioles. Therefore, the purpose of this study was to determine the effects of O(2)(-) scavenging, as well as removal of H(2)O(2), the byproduct of O(2)(-) scavenging, on flow-mediated dilation in coronary resistance arterioles of young (4 mo) and old (24 mo) male Fischer 344 rats. Flow increased NO and H(2)O(2) production as evidenced by enhanced diaminofluorescein and dichlorodihydrofluorescein fluorescence, respectively, whereas aging reduced flow-induced NO and H(2)O(2) production. Endothelium-dependent vasodilation was evaluated by increasing intraluminal flow (5-60 nl/s) before and after treatment with the superoxide dismutase mimetic Tempol (100 muM), the H(2)O(2) scavenger catalase (100 U/ml), or Tempol plus catalase. Catalase reduced flow-induced dilation in both groups, whereas Tempol and Tempol plus catalase diminished vasodilation in young but not old rats. Tempol plus deferoxamine (100 muM), an inhibitor of hydroxyl radical formation, reversed Tempol-mediated impairment of flow-induced vasodilation in young rats and improved flow-induced vasodilation in old rats compared with control. Immunoblot analysis revealed increases in endogenous superoxide dismutase, catalase, and nitrotyrosine protein levels with aging. Collectively, these data indicate that NO- and H(2)O(2)-mediated flow-induced signaling decline with age in coronary arterioles and that elevated hydroxyl radical formation contributes to the age-related impairment of flow-induced vasodilation.

Fig. 1.

Flow-induced dilation in coronary arterioles from young (Y) and old (O) male rats. Aging reduces flow-mediated vasodilation in coronary arterioles from Fischer 344 rats. *P < 0.05, significant difference from young rats.

Fig. 2.

Flow-induced nitric oxide (NO) production in coronary arterioles as measured by 4-amino-5-methylamino-2′2′-difluorofluorescein diacetate (DAF) fluorescence. A: representative images of vessels from young and old rats at flow rates of 0 (flow 0), 35, and 50 nl/s. B: increasing flow rates enhanced DAF fluorescence in coronary arterioles from both young (n = 11) and old (n = 9) rats. NO production in response to flow declined with age, as evidenced by reduced DAF intensity. Values are means ± SE. *P < 0.05, significant difference from flow 0. #P < 0.05, significant difference from young rats at respective flow rate.

Fig. 3.

Flow-induced increases in DAF fluorescence were abolished by both N^G-nitro-l-arginine methyl ester (l-NAME) and catalase. Representative images of coronary arterioles from young and old rats are shown at flow rates of 0 and 50 nl/s after treatment with l-NAME (A) or catalase (B). In the presence of l-NAME (C) or catalase (D), DAF fluorescence intensity did not change with increasing flow over time. Arrows in C and D indicate stepwise increases in flow to 7.5, 35, and 50 nl/s, respectively. Values are means ± SE; n = 4 young and 4 old rats per group (8 total).

Fig. 4.

Flow-induced H₂O₂ production in coronary arterioles as measured by 5(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCF) fluorescence. A: representative images of vessels from young and old rats at flow rates of 0 and 50 nl/s. B: DCF fluorescence increased in response to flow (50 nl/s) in coronary arterioles from both young and old rats; however, flow-induced H₂O₂ production declined with age. Catalase blunted DCF fluorescence in arterioles from both age groups. Values are means ± SE; n = 6 rats per group. *P < 0.05 significant difference from young rats. #P < 0.05, significant difference from control.

Fig. 5.

Effect of H₂O₂ on coronary arterioles from young and old rats. H₂O₂ dilated coronary arterioles concentration dependently. EC₅₀ values differed significantly between young (EC₅₀ = 2.5 ± 0.8 mM) and old rats (EC₅₀ = 1.0 ± 0.1 mM). Values are means ± SE. *P < 0.05.

Fig. 6.

Flow-induced dilation in coronary arterioles following treatment with reactive oxygen species (ROS) scavengers. A: Tempol significantly inhibited flow-mediated dilation in vessels from young rats but had no effect in vessels from old animals. B: treatment with catalase blunted flow to a greater extent in arterioles from young vs. old rats, consistent with direct H₂O₂ measures shown in Fig. 4. C: Tempol plus catalase (Tem + Cat) inhibited the flow response in vessels from young, but not old, rats. Similar to treatment with Tempol alone, the effect of additional Tem + Cat resulted in no net change in the flow response. Values are means ± SE. *P < 0.05, significant difference from control.

Fig. 7.

Effect of deferoxamine on flow-induced vasodilation in coronary arterioles from young and old rats. A: treatment with deferoxamine (Tem + Def) reversed Tempol-induced reduction of flow in vessels from young rats and augmented flow-induced dilation above control levels in vessels from old rats. B: deferoxamine alone similarly enhanced the flow response in old rats but did not alter flow-induced dilation in vessels from young animals. Values are means ± SE. *P < 0.05, significant difference from deferoxamine.

Fig. 8.

Immunoblot analysis of catalase, nitrotyrosine (NT), and superoxide dismutase (SOD) protein expression in coronary arterioles from young and old rats. A and B: aging increased protein levels of nitrotyrosine, a marker of oxidative stress. Catalase and SOD protein levels also increased with advancing age. C: real-time PCR analysis revealed an increase in SOD mRNA with aging in coronary arterioles. Values are means ± SE; n = 4 rats per group. *P < 0.05, significant difference from young rats.

Fig. 9.

H₂O₂ and O₂⁻ regulatory pathways involved in flow-mediated vasodilation. With aging, elevated Fe³⁺ leads to increased OH⁻ production and diminished vasodilation. A dramatic age-related increase in SOD also may contribute to reduced flow-induced dilation with aging as the endogenous catalase system is overwhelmed and more H₂O₂ is shunted to formation of OH⁻. GC, guanylate cyclase.