Judy M. Muller-Delp

Effects of ageing and exercise training on endothelium‐dependent vasodilatation and structure of rat skeletal muscle arterioles

Ageing reduces endothelium‐dependent vasodilatation in humans and animals, and in humans, exercise training reverses the ageing‐associated reduction in endothelium‐dependent vasodilatation. The purpose of this study was to determine the mechanism(s) by which 10–12 weeks of treadmill exercise enhances endothelium‐dependent vasodilatation in muscles of differing fibre composition from young and old rats. Three‐ and 22‐month‐old male Fischer 344 rats were assigned to young sedentary, young exercise‐trained, old sedentary, or old exercise‐trained groups. Arterioles were isolated from the soleus and gastrocnemius muscles; luminal diameter changes were determined in response to the endothelium‐dependent vasodilator acetylcholine (ACh, 10−9–10−4 mol l−1) alone and in the presence of the nitric oxide synthase (NOS) inhibitor l‐NAME (10−5 mol l−1) or the combination of l‐NAME and the cyclooxygenase inhibitor indomethacin (10−5 mol l−1). Training ameliorated the ageing‐induced reduction in endothelium‐dependent vasodilatation in soleus muscle arterioles. Treatment with l‐NAME alone and in combination with indomethacin abolished differences in ACh vasodilatation occurring with ageing and training. Expression of endothelial NOS (eNOS) mRNA in soleus arterioles was unaltered by ageing, whereas eNOS protein was increased with age; training elevated both eNOS mRNA and protein. In gastrocnemius muscle arterioles, ageing did not alter maximal vasodilatation, but ageing and training increased maximal arteriolar diameter. These results demonstrate that ageing‐induced reductions and training‐induced enhancement of endothelial vasodilatation both occur through the nitric oxide signalling mechanism in highly oxidative skeletal muscle, but ageing and training do not appear to act on the same portion of the signalling cascade.

Effects of ageing and exercise training on eNOS uncoupling in skeletal muscle resistance arterioles

Reduced availability of tetrahydrobiopterin (BH4) contributes to the age‐related decline of nitric oxide (NO)‐mediated vasodilatation of soleus muscle arterioles. Depending on availability of substrate and/or necessary co‐factors, endothelial nitric oxide synthase (eNOS) can generate NO and/or superoxide (O2−). We evaluated the effects of age and chronic exercise on flow‐induced vasodilatation and levels of NO and O2− in soleus muscle arterioles. Young (3 months) and old (22 months) male rats were exercise trained or remained sedentary (SED) for 10 weeks. Flow‐stimulated NO and O2−, as well as BH4 and l‐arginine content, were determined in soleus muscle arterioles. Flow‐induced vasodilatation was assessed under control conditions and during the blockade of O2− and/or hydrogen peroxide. Exercise training enhanced flow‐induced vasodilatation in arterioles from young and old rats. Old age reduced, and exercise training restored, BH4 content and flow‐stimulated NO availability. Flow‐stimulated, eNOS‐derived O2− levels were higher in arterioles from old SED compared to those from young SED rats. Exercise training increased flow‐stimulated eNOS‐derived O2− levels in arterioles from young but not old rats. O2− scavenging with Tempol reduced flow‐induced vasodilatation from all groups except young SED rats. Addition of catalase to Tempol‐treated arterioles eliminated flow‐induced vasodilatation in arterioles from all groups. Catalase reduced flow‐induced vasodilatation from all groups. In Tempol‐treated arterioles, flow‐induced vasodilatation was restored by deferoxamine, an iron chelator. These data indicate that uncoupling of eNOS contributes to the age‐related decline in flow‐induced vasodilatation; however, reactive oxygen species are required for flow‐induced vasodilatation in soleus muscle arterioles from young and old rats.

Ageing diminishes endothelium-dependent vasodilatation and tetrahydrobiopterin content in rat skeletal muscle arterioles.

Ageing reduces endothelium‐dependent vasodilatation through an endothelial nitric oxide synthase (NOS) signalling pathway. The purpose of this study was to determine whether arginase activity diminishes endothelium‐dependent vasodilatation in skeletal muscle arterioles from old rats, and whether NOS substrate (l‐arginine) and cofactor (tetrahydrobiopterin; BH4) concentrations are reduced. First‐order arterioles were isolated from the soleus muscle of young (6 months old) and old (24 months old) male Fischer 344 rats. In vitro changes in luminal diameter in response to stepwise increases in flow were determined in the presence of the NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−5 mol l−1), the arginase inhibitor Nω‐hydroxy‐nor‐l‐arginine (NOHA, 5 × 10−4 mol l−1), exogenous l‐arginine (3 × 10−3 mol l−1) or the precursor for BH4 synthesis sepiapterin (1 μmol l−1). Arteriolar l‐arginine and BH4 content were determined via HPLC. Ageing decreased flow‐mediated vasodilatation by 52%, and this difference was abolished with NOS inhibition. Neither inhibition of arginase activity nor addition of exogenous l‐arginine had any effect on flow‐mediated vasodilatation; arteriolar l‐arginine content was also not different between age groups. BH4 content was lower in arterioles from old rats (94 ± 8 fmol (mg tissue)−1) relative to controls (234 ± 21 fmol (mg tissue)−1), and sepiapterin elevated flow‐mediated vasodilatation in arterioles from old rats. These results demonstrate that the impairment of endothelium‐dependent vasodilatation induced by old age is due to an altered nitric oxide signalling mechanism in skeletal muscle arterioles, but is not the result of increased arginase activity and limited l‐arginine substrate. Rather, the age‐related deficit in flow‐mediated vasodilatation appears to be the result, in part, of limited BH4 bioavailability.

Aging-Induced Adaptations of Microvascular Reactivity

Control of blood flow to skeletal muscle depends on the vasomotor tone present in the resistance vasculature. Although muscle blood flow has been shown to decline with advancing age, our knowledge of how alterations of reactivity of the resistance vasculature contribute to reduced delivery or altered distribution of blood in the aged is limited. Recent work has demonstrated that age alters the reactivity of resistance arteries and arterioles from skeletal muscle, and that impairment of both vasodilator and vasoconstrictor responses occurs with advancing age. The alterations in cellular mechanisms that contribute to age‐related impairment of vasoreactive responses encompass both the vascular endothelium and smooth muscle, and differ in muscles of varying function and fiber type. Current research suggests that some degree of age‐induced endothelial dysfunction occurs in resistance arteries and arterioles from most skeletal muscle; however, the severity of endothelial impairment appears greater in resistance arteries and arterioles from highly oxidative locomotory muscles. Age‐related impairment of vasoconstrictor responses to metabolites and endogenous constrictor agents has also been documented. These age‐related reductions in vasoreactivity that occur in the skeletal muscle resistance vasculature may contribute to inadequate delivery or distribution of blood flow during exercise and ultimately be a factor in loss of exercise capacity that occurs with advancing age.

Aging impairs flow-induced dilation in coronary arterioles: role of NO and H2O2

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.

Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats

The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 +/- 4 vs. old: 34 +/- 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by approximately 160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME), declined with age. l-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, l-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age.

Age impairs Flk-1 signaling and NO-mediated vasodilation in coronary arterioles

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P<or=0.05); however, ACh-induced vasodilation was preserved with age. NG-nitro-L-arginine methyl ester (L-NAME) (1x10(-5) M) eliminated vasodilation to flow, VEGF, and ACh, indicating dependence of these responses on NO. SU-1498, an inhibitor of vascular endothelial growth factor receptor 2 (VEGFR, also known as Flk-1), abolished age-related differences in flow-induced vasodilation. Flow-stimulated phosphorylation of Flk-1 in coronary arterioles from young but not old rats and Flk-1 protein was reduced in coronary arterioles from old rats compared with those from young rats. Flow stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) in coronary arterioles from both young and old rats. VEGF induced phosphorylation of both protein kinase B (Akt) and eNOS in coronary arterioles. VEGF-induced phosphorylation of Akt, but not eNOS, was significantly reduced in arterioles from old rats compared with arterioles from young rats. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase, eliminated age-related differences in both flow- and VEGF-induced vasodilation. These results indicate that impairment of Flk-1/PI3-kinase signaling contributes to the reduction of flow-induced vasodilation in coronary arterioles with advancing age.

Spaceflight-induced alterations in cerebral artery vasoconstrictor, mechanical, and structural properties: implications for elevated cerebral perfusion and intracranial pressure

Evidence indicates that cerebral blood flow is both increased and diminished in astronauts on return to Earth. Data from ground‐based animal models simulating the effects of microgravity have shown that decrements in cerebral perfusion are associated with enhanced vasoconstriction and structural remodeling of cerebral arteries. Based on these results, the purpose of this study was to test the hypothesis that 13 d of spaceflight [Space Transportation System (STS)‐135 shuttle mission] enhances myogenic vasoconstriction, increases medial wall thickness, and elicits no change in the mechanical properties of mouse cerebral arteries. Basilar and posterior communicating arteries (PCAs) were isolated from 9‐wk‐old female C57BL/6 mice for in vitro vascular and mechanical testing. Contrary to that hypothesized, myogenic vasoconstrictor responses were lower and vascular distensibility greater in arteries from spaceflight group (SF) mice (n=7) relative to ground‐based control group (GC) mice (n=12). Basilar artery maximal diameter was greater in SF mice (SF: 236±9 μm and GC: 215±5 μm) with no difference in medial wall thickness (SF: 12.4±1.6 μm; GC: 12.2±1.2 μm). Stiffness of the PCA, as characterized via nanoindentation, was lower in SF mice (SF: 3.4±0.3 N/m; GC: 5.4±0.8 N/m). Collectively, spaceflight‐induced reductions in myogenic vasoconstriction and stiffness and increases in maximal diameter of cerebral arteries signify that elevations in brain blood flow may occur during spaceflight. Such changes in cerebral vascular control of perfusion could contribute to increases in intracranial pressure and an associated impairment of visual acuity in astronauts during spaceflight.—Taylor, C. R., Hanna, M., Behnke, B. J., Stabley, J. N., McCullough, D. J., Davis III, R. T., Ghosh, P., Papadopoulos, A., Muller‐Delp, J. M., Delp, M. D. Spaceflight‐induced alterations in cerebral artery vasoconstrictor, mechanical, and structural properties: implications for elevated cerebral perfusion and intracranial pressure. FASEB J. 27, 2282–2292 (2013). www.fasebj.org

Age and exercise training alter signaling through reactive oxygen species in the endothelium of skeletal muscle arterioles

Exercise training ameliorates age-related impairments in endothelium-dependent vasodilation in skeletal muscle arterioles. Additionally, exercise training is associated with increased superoxide production. The purpose of this study was to determine the role of superoxide and superoxide-derived reactive oxygen species (ROS) signaling in mediating endothelium-dependent vasodilation of soleus muscle resistance arterioles from young and old, sedentary and exercise-trained rats. Young (3 mo) and old (22 mo) male rats were either exercise trained or remained sedentary for 10 wk. To determine the impact of ROS signaling on endothelium-dependent vasodilation, responses to acetylcholine were studied under control conditions and during the scavenging of superoxide and/or hydrogen peroxide. To determine the impact of NADPH oxidase-derived ROS, endothelium-dependent vasodilation was determined following NADPH oxidase inhibition. Reactivity to superoxide and hydrogen peroxide was also determined. Tempol, a scavenger of superoxide, and inhibitors of NADPH oxidase reduced endothelium-dependent vasodilation in all groups. Similarly, treatment with catalase and simultaneous treatment with tempol and catalase reduced endothelium-dependent vasodilation in all groups. Decomposition of peroxynitrite also reduced endothelium-dependent vasodilation. Aging had no effect on arteriolar protein content of SOD-1, catalase, or glutathione peroxidase-1; however, exercise training increased protein content of SOD-1 in young and old rats, catalase in young rats, and glutathione peroxidase-1 in old rats. These data indicate that ROS signaling is necessary for endothelium-dependent vasodilation in soleus muscle arterioles, and that exercise training-induced enhancement of endothelial function occurs, in part, through an increase in ROS signaling.

Increased nitric oxide-mediated vasodilation of bone resistance arteries is associated with increased trabecular bone volume after endurance training in rats

Old age-associated osteoporosis is related to diminished bone blood flow and impaired nitric oxide (NO)-mediated vasodilation of the bone vasculature. Endurance exercise training restores the age-associated reduction of vasodilation in numerous vascular beds, as well as improving bone properties. The purpose of this study was to determine whether functional improvements in the bone vasculature are associated with increased bone properties after an endurance training intervention. Young adult (4-6 months) and old (24-26 months) male Fischer-344 rats remained sedentary or were trained (15 m/min walking, 15 degrees incline, 5 days/week, 10-12 weeks). Endothelium-dependent vasodilation of the femoral principal nutrient artery (PNA) was assessed in vitro using acetylcholine (ACh) and inhibitors of NO synthase (NOS) and cyclooxygenase (COX). PNA endothelium-dependent vasodilation was greater after training by 16% in young and by 24% in old animals. The NOS-mediated contribution to endothelium-dependent vasodilation was enhanced by 77% after training in old rats. Distal femur trabecular bone volume (BV/TV, %) was lower with old age in sedentary animals (young: 27+/-2%, old: 23+/-1%; P<0.05). Exercise-induced elevations in bone and marrow blood flow and the NOS signaling pathway were associated with greater BV/TV (young trained: 34+/-2%, old trained: 26+/-1%; P<0.05) relative to sedentary groups. These data demonstrate that training-induced increases in bone properties are associated with enhanced endothelium-dependent vasodilation through a NOS signaling pathway in the bone vasculature.