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) Key Words: arteriole Ⅲ endothelium Ⅲ superoxide Ⅲ reactive oxygen species Ⅲ free radical scavenger N umerous 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 NOmediated dilation of small coronary arteries and arterioles 7 ; 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-arginine 11 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 (O 2 ·Ϫ ) anions 13 or a loss of antioxidant capacity, which normally provides protection against reactive oxygen species (oxidative stress, reviewed in Beckman and Ames 14 ). In addition, age-related dysfunction in some conduit vessels may involve an enhanced synthesis of thromboxane A 2 (TXA 2 ), 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 factors 15 raise the possibility that complex phenotypic changes affecting expression of eNOS 6 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 TXA 2 (which may counteract the effect of NO), we characterized in isolated coronary arterioles age-related alterations in flow-induced dilation, O 2 ·Ϫ production, peroxynitrite generation, 17 and expression of eNOS and the pro-and antioxi-

This review focuses on molecular, cellular, and functional changes that occur in the vasculature during aging; explores the links between mitochondrial oxidative stress, inflammation, and development of vascular disease in the elderly patients; and provides a landscape of molecular mechanisms involved in cellular oxidative stress resistance, which could be targeted for the prevention or amelioration of unsuccessful vascular aging. Practical interventions for prevention of age-associated vascular dysfunction and disease in old age are considered here based on emerging knowledge of the effects of anti-inflammatory treatments, regular exercise, dietary interventions, and caloric restriction mimetics.

We have studied the effect of changes in shear stress on diameter of isolated arterioles of rat cremaster muscle. The steady-state active diameter of arterioles at a constant perfusion pressure (60 mm Hg) was 80 +/- 1.2 microns. The vessels' passive diameter (Ca(2+)-free solution) was 156 +/- 1.8 microns. Changes in shear stress were induced either by an increase in flow (velocity) or by an increase in viscosity of the perfusion solution. At a constant perfusion pressure, the stepwise increase in perfusion flow (0-80 microliters/min in 10-microliters/min steps) elicited, with a delay of approximately 20 seconds, a gradual increase in diameter up to 46%. At a constant 20-microliters/min flow rate, increases in viscosity of the perfusate (2%, 4%, and 6% dextran [molecular weight, 77,800]) caused a gradual vasodilation up to 22%. Varying flow and viscosity of the perfusate simultaneously resulted in an upward shift of the flow-diameter curve. Both flow- and viscosity-induced dilations were eliminated by the removal of the endothelium of arterioles (by air) or were inhibited by indomethacin (10(-5) M). The efficacy and specificity of these inhibitory treatments were assessed with vasoactive agents whose action, with regard to endothelial mediation, has been determined previously. The arteriolar dilation maintained calculated wall shear stress close to control values during increases in flow and/or viscosity of the perfusate, but when the dilation was inhibited by removal of the endothelium or by indomethacin, wall shear stress increased significantly in a cumulative manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial nitric oxide synthase (eNOS) uncoupling is a mechanism that leads to endothelial dysfunction. Previously, we reported that shear stress-induced release of nitric oxide in vessels of aged rats was significantly reduced and was accompanied by increased production of superoxide (18,27). In the present study, we investigated the influence of aging on eNOS uncoupling. Mesenteric arteries were isolated from young (3 mo) and aged (24 mo) C57 BL/6J mice. The expression of eNOS protein in young vs. aged mice was not significantly different. However, the aged mice had remarkable increases in the ratio of eNOS monomers to dimers and N -nitro-L-arginine methyl ester-inhibitable superoxide formation. The level of nitrotyrosine in the total protein and precipitated eNOS of aged vessels was increased compared with that in young vessels. HPLC analysis indicated a reduced level of tetrahydrobiopterin (BH4), an essential cofactor for eNOS, in the mesenteric arteries of aged mice. Quantitative PCR results implied that the diminished BH4 may result from the decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, enzymes involved in BH4 biosynthesis. When isolated and cannulated second-order mesenteric arteries (ϳ150 m) from aged mice were treated with sepiapterin, acetylcholine-induced, endothelium-dependent vasodilation improved significantly, which was accompanied by stabilization of the eNOS dimer. These data suggest that eNOS uncoupling and increased nitrosylation of eNOS, decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, and subsequent reduced BH4 bioavailability may be important contributors of endothelial dysfunction in aged vessels. tetrahydrobiopterin; nitrotyrosine; superoxide A NUMBER OF STUDIES REPORTED that, in various diseases, such as atherosclerosis and diabetes, the function of endothelial nitric oxide (NO) synthase (eNOS) is altered, and that it produces superoxide instead of NO. This alteration is referred to as "eNOS uncoupling" and is linked to an increased monomerization of the enzyme (15, 32). When endothelial cells were treated with peroxynitrite, a product of NO and superoxide, the reduction in eNOS activity was associated with disruption of eNOS dimers (33). On the other hand, tetrahydrobiopterin (BH4) has been identified as a critical cofactor for the production of NO. Two molecules of BH4 bind to each eNOS dimer and facilitate electron transfer for L-arginine oxidation. When BH4 is limited, by decreasing its synthesis or increasing its oxidation, eNOS becomes uncoupled, and superoxide is produced (20,21). It has also been demonstrated that BH4 preserves eNOS dimerization and improves endothelial function (6). Thus BH4 availability is essential for normal endothelial function.Aging of blood vessels is a risk factor for the development of cardiovascular diseases. However, the cellular and molecular mechanisms underlying the aging of blood vessels remain unclear. Our laboratory has demonstrated previously that, in mesenteric arteries of aged rats, flow-ind...

We have studied the mechanisms responsible for the mediation of flow (shear stress)-induced dilation of isolated arterioles of rat gracilis muscle. Active diameter of arterioles at a constant perfusion pressure (PP, 80 mmHg) was approximately 92 microns, while their passive diameter (Ca(2+)-free solution) was approximately 165 microns. At a constant PP the stepwise increase in flow of the perfusion solution (PS, 0-60 microliters/min in 10-microliters/min steps) elicited a gradual increase in diameter up to approximately 140 microns. Flow-induced dilations were eliminated by the removal of the endothelium of arterioles (by air). Dilations were significantly reduced by the cyclooxygenase blocker, indomethacin (Indo, 10(-5) M), by the nitric oxide synthase blocker, N omega-nitro-L-arginine (L-NNA, 10(-4) M), or by the endothelium-derived relaxing factor inhibitor, oxyhemoglobin (Hb, 10(-5) M), as indicated by the significant changes in the slope of the regression lines of the flow-diameter curves. For example, during administration of the inhibitors, dilation to 60 microliters/min perfusate flow was reduced by 41.1, 54.3, and 39.3%, respectively. Combined application of Indo and L-NNA almost completely eliminated flow-induced dilation. Arteriolar dilation maintained calculated wall shear stress close to control values (approximately 30 dyn/cm2 at 60 microliters/min) despite increases in flow, but when the dilation was inhibited by removal of the endothelium or by the combined administration of Indo and L-NNA, wall shear stress was greatly increased as a function of increases in flow of the PS (approximately 125 dyn/cm2).(ABSTRACT TRUNCATED AT 250 WORDS)