Ashley E. Walker

25-Hydroxyvitamin D Deficiency Is Associated With Inflammation-Linked Vascular Endothelial Dysfunction in Middle-Aged and Older Adults

We tested the hypothesis that vascular endothelial function, assessed by endothelium-dependent dilation, is related to serum vitamin D status among middle-aged and older adults without clinical disease, and that this is linked to inflammation. Brachial artery flow-mediated dilation, a measure of endothelium-dependent dilation, was lower (P<0.01) in vitamin D–insufficient (3.7±0.2%; serum 25-hydroxyvitamin D [25(OH)D]: 20 to 29 ng/mL; 62±1 years of age; n=31; mean± SE) and vitamin D–deficient (3.2±0.3%; 25(OH)D: <20 ng/mL; 63±2 years of age; n=22) versus vitamin D–sufficient (4.6±0.4%; 25(OH)D: >29 ng/mL; 61±1 years of age; n=22) subjects, whereas endothelium-independent dilation (brachial dilation to sublingual nitroglycerine) did not differ (P=0.45). Among all subjects, brachial flow-mediated dilation was positively related to serum 25(OH)D (%&Dgr;: r=0.35; P<0.01) but not 1,25-dihydroxyvitamin D (r=−0.06; P=0.61), the active form of vitamin D. Vascular endothelial cell expression of the proinflammatory transcription factor nuclear factor &kgr;B was greater in deficient versus sufficient subjects (0.59±0.07 versus 0.44±0.05; P<0.05), and inhibition of nuclear factor &kgr;B (4 days oral salsalate) improved flow-mediated dilation to a greater extent in subjects with lower versus higher 25(OH)D (+3.7±0.6 versus +2.0±0.2%; P<0.05). Endothelial cell expression of the downstream proinflammatory cytokine interleukin-6 also was higher in deficient versus sufficient subjects (0.67±0.08 versus 0.47±0.05; P<0.01) and inversely related to serum 25(OH)D level (r=−0.62; P<0.01), whereas vitamin D receptor and 1-&agr; hydroxylase, the 25(OH)D to 1,25-dihydroxyvitamin D converting enzyme, were lower (P<0.05). Inadequate serum 25(OH)D is associated with vascular endothelial dysfunction among healthy middle-aged/older adults, and this is mediated in part by nuclear factor &kgr;B–related inflammation. Reduced vitamin D receptor and 1-&agr; hydroxylase may be molecular mechanisms linking vitamin D insufficiency to endothelial dysfunction.

Habitual exercise and vascular ageing.

Age is the major risk factor for cardiovascular diseases (CVD) and this is attributable in part to stiffening of large elastic arteries and development of vascular endothelial dysfunction (e.g. impaired endothelium‐dependent dilatation, EDD). In contrast, regular aerobic exercise is associated with reduced risk of CVD. Endurance exercise‐trained middle‐aged/older adults demonstrate lower large elastic artery stiffness and greater EDD than their sedentary peers. With daily brisk walking, previously sedentary middle‐aged/older adults show reduced stiffness and improved EDD. The mechanisms underlying the effects of regular aerobic exercise on large elastic artery stiffness with ageing are largely unknown, but are likely to include changes to the composition of the arterial wall. Enhanced EDD in older adults who exercise is mediated by increased nitric oxide (NO) bioavailability associated with reduced oxidative stress. Arteries from old rodents that perform regular aerobic exercise demonstrate increased expression and activity of endothelial NO synthase, reduced oxidative damage associated with reduced expression and activity of the oxidant enzyme NADPH oxidase, and increased activity of the antioxidant enzyme superoxide dismutase. Aerobic exercise also may protect arteries with ageing by increasing resistance to the effects of other CVD risk factors like LDL‐cholesterol. Habitual aerobic exercise is an effective strategy to combat arterial ageing.

Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy

The phosphodiesterase 5A inhibitor tadalafil restores normal blood flow regulation in exercising skeletal muscle of patients with Becker muscular dystrophy. A Shot in the Arm for Muscular Dystrophy Becker muscular dystrophy (BMD), characterized by progressive skeletal muscle wasting, is caused by mutations in the muscle protein dystrophin. Preclinical research in the dystrophin-deficient mdx mouse model of a related disease Duchenne muscular dystrophy shows that inhibitors of phosphodiesterase 5 (PDE5)—which boosts guanosine 3′,5′-monophosphate (cGMP), the downstream target of nitric oxide (NO) in vascular smooth muscle—alleviate some features of the dystrophic phenotype including vasospasm of skeletal muscle microvessels that can lead to muscle injury and fatigue. The challenge is to determine whether these compelling results in mice can be translated to benefit human patients with muscular dystrophy. In a new study, Martin et al. assessed exercise-induced attenuation of reflex sympathetic vasoconstriction in the muscles of 10 patients with BMD and 7 age-matched healthy male controls. This is a protective mechanism that optimizes perfusion of skeletal muscle to meet the metabolic demands of exercise. Reflex vasoconstriction was induced by simulated orthostatic stress and was measured as the decrease in forearm muscle oxygenation using near-infrared spectroscopy. The authors took the measurements when the forearm muscles were rested or lightly exercised in the form of a rhythmic handgrip. First, the investigators showed that exercise-induced attenuation of reflex vasoconstriction was defective in 9 of 10 patients with BMD in whom the common dystrophin mutations disrupt targeting of neuronal NO synthase (nNOS) to the muscle sarcolemma (the response was preserved in one patient in whom nNOS was localized to the muscle sarcolemma). Then, in a double-blind randomized placebo-controlled crossover trial, the authors showed that normal blood flow regulation was fully restored in eight of nine patients by a single oral 20-mg dose of the drug tadalafil, a specific PDE5 inhibitor. These findings support an essential role for sarcolemmal nNOS in modulating sympathetic vasoconstriction in exercising human skeletal muscles and implicate PDE5 inhibition as a putative therapeutic strategy for treating BMD. Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is no treatment. Like Duchenne muscular dystrophy (DMD), BMD is caused by mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the muscle sarcolemma. Among these is neuronal nitric oxide synthase (nNOSμ), which requires certain spectrin-like repeats in dystrophin’s rod domain and the adaptor protein α-syntrophin to be targeted to the sarcolemma. When healthy skeletal muscle is subjected to exercise, sarcolemmal nNOSμ-derived NO attenuates local α-adrenergic vasoconstriction, thereby optimizing perfusion of muscle. We found previously that this protective mechanism is defective—causing functional muscle ischemia—in dystrophin-deficient muscles of the mdx mouse (a model of DMD) and of children with DMD, in whom nNOSμ is mislocalized to the cytosol instead of the sarcolemma. We report that this protective mechanism also is defective in men with BMD in whom the most common dystrophin mutations disrupt sarcolemmal targeting of nNOSμ. In these men, the vasoconstrictor response, measured as a decrease in muscle oxygenation, to reflex sympathetic activation is not appropriately attenuated during exercise of the dystrophic muscles. In a randomized placebo-controlled crossover trial, we show that functional muscle ischemia is alleviated and normal blood flow regulation is fully restored in the muscles of men with BMD by boosting NO-cGMP (guanosine 3′,5′-monophosphate) signaling with a single dose of the drug tadalafil, a phosphodiesterase 5A inhibitor. These results further support an essential role for sarcolemmal nNOSμ in the normal modulation of sympathetic vasoconstriction in exercising human skeletal muscle and implicate the NO-cGMP pathway as a putative new target for treating BMD.

Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

Background— Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results— We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning–based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. Conclusions— By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Life-Long Caloric Restriction Reduces Oxidative Stress and Preserves Nitric Oxide Bioavailability and Function in Arteries of Old Mice

Aging impairs arterial function through oxidative stress and diminished nitric oxide (NO) bioavailability. Life‐long caloric restriction (CR) reduces oxidative stress, but its impact on arterial aging is incompletely understood. We tested the hypothesis that life‐long CR attenuates key features of arterial aging. Blood pressure, pulse wave velocity (PWV, arterial stiffness), carotid artery wall thickness and endothelium‐dependent dilation (EDD; endothelial function) were assessed in young (Y: 5–7 month), old ad libitum (Old AL: 30–31 month) and life‐long 40% CR old (30–31 month) B6D2F1 mice. Blood pressure was elevated with aging (P < 0.05) and was blunted by CR (P < 0.05 vs. Old AL). PWV was 27% greater in old vs. young AL‐fed mice (P < 0.05), and CR prevented this increase (P < 0.05 vs. Old AL). Carotid wall thickness was greater with age (P < 0.05), and CR reduced this by 30%. CR effects were associated with amelioration of age‐related changes in aortic collagen and elastin. Nitrotyrosine, a marker of cellular oxidative stress, and superoxide production were greater in old AL vs. young (P < 0.05) and CR attenuated these increase. Carotid artery EDD was impaired with age (P < 0.05); CR prevented this by enhancing NO and reducing superoxide‐dependent suppression of EDD (Both P < 0.05 vs. Old AL). This was associated with a blunted age‐related increase in NADPH oxidase activity and p67 expression, with increases in superoxide dismutase (SOD), total SOD, and catalase activities (All P < 0.05 Old CR vs. Old AL). Lastly, CR normalized age‐related changes in the critical nutrient‐sensing pathways SIRT‐1 and mTOR (P < 0.05 vs. Old AL). Our findings demonstrate that CR is an effective strategy for attenuation of arterial aging.

Regular aerobic exercise protects against impaired fasting plasma glucose-associated vascular endothelial dysfunction with aging.

In the present study, we tested the hypothesis that age-associated vascular endothelial dysfunction is exacerbated by IFG (impaired fasting plasma glucose) and that regular aerobic exercise prevents this effect. Data were analysed from a cohort of 131 non-smoking men and women without overt clinical disease. Compared with young adult controls (age=24±1 years, n=29; values are means±S.E.M.), brachial artery FMD (flow-mediated dilation), a measure of conduit artery EDD (endothelium-dependent dilation), was 33% lower [7.93±0.33 against 5.27±0.37%Δ (% change), P<0.05] in MA/O (middle-aged/older) adults with NFG (normal fasting plasma glucose) (≤99 mg/dl, 62±1 years, n=35). In MA/O adults with IFG (100-125 mg/dl, 64±1 years, n=28), FMD was 30% lower (3.37±0.35%Δ) than in their peers with NFG and 58% lower than young controls (P<0.05). Brachial artery FMD was greater (6.38±0.35%Δ) in MA/O adults with NFG who regularly performed aerobic exercise (>45 min/day for ≥5 days/week, 62±1 years, n=23) compared with their non-exercising peers and only slightly less than young controls (P<0.05). Most importantly, FMD was completely preserved in MA/O adults with IFG who regularly performed aerobic exercise (6.99±0.69%Δ, 65±1 years, n=16). In the pooled sample, fasting plasma glucose was inversely related to FMD (r=-0.42, P<0.01) and was the strongest independent predictor of FMD (R(2)=0.32). Group differences in FMD were not affected by other subject characteristics or brachial artery properties, including brachial artery dilation to sublingual NTG (nitroglycerine, i.e. endothelium-independent dilation). IFG exacerbates age-associated vascular endothelial dysfunction and this adverse effect is completely prevented in MA/O adults who regularly perform aerobic exercise.

Fenofibrate Improves Vascular Endothelial Function by Reducing Oxidative Stress While Increasing Endothelial Nitric Oxide Synthase in Healthy Normolipidemic Older Adults

Vascular endothelial dysfunction develops with aging, as indicated by impaired endothelium-dependent dilation, and is related to increased cardiovascular disease risk. We hypothesized that short-term treatment with fenofibrate, a lipid-lowering agent with potential pleiotropic effects, would improve endothelium-dependent dilation in middle-aged and older normolipidemic adults by reducing oxidative stress. Brachial artery flow-mediated dilation, a measure of endothelium-dependent dilation, was assessed in 22 healthy adults aged 50 to 77 years before and after 7 days of fenofibrate (145 mg/d; n=12) or placebo (n=10). Brachial flow-mediated dilation was unchanged with placebo, but improved after 2 and 7 days of fenofibrate (5.1±0.7 versus 2 days: 6.0±0.7 and 7 days: 6.4±0.6%&dgr;; both P<0.005). The improvements in flow-mediated dilation after 7 days remained significant (P<0.05) after accounting for modest changes in plasma total and low-density lipoprotein cholesterol. Endothelium-independent dilation was not affected by fenofibrate or placebo (P>0.05). Intravenous infusion of the antioxidant vitamin C improved brachial flow-mediated dilation at baseline in both groups and during placebo treatment (P<0.05), but not after 2 and 7 days of fenofibrate (P>0.05). Fenofibrate treatment also reduced plasma-oxidized low-density lipoprotein, a systemic marker of oxidative stress, compared with placebo (P<0.05). In vascular endothelial cells sampled from peripheral veins of the subjects, endothelial nitric oxide synthase protein expression was unchanged with placebo and after 2 days of fenofibrate, but was increased after 7 days of fenofibrate (0.54±0.03 versus 2 days: 0.52±0.04 and 7 days: 0.76±0.11 intensity/human umbilical vein endothelial cell control; P<0.05, 7 days). Short-term treatment with fenofibrate improves vascular endothelial function in healthy normolipidemic middle-aged and older adults by reducing oxidative stress and induces an increase in endothelial nitric oxide synthase.

Prevention of age-related endothelial dysfunction by habitual aerobic exercise in healthy humans: possible role of nuclear factor κB.

Habitual aerobic exercise prevents age-related impairments in endothelium-dependent dilation (EDD). We have hypothesized that the pro-inflammatory transcription factor nuclear factor κB (NF-κB) impairs EDD with sedentary aging, and habitual aerobic exercise prevents this age-related suppression of EDD by NF-κB. To test this hypothesis, we have inhibited NF-κB signalling via oral salsalate administration in healthy older aerobic exercise-trained adults (OT, n=14, 58 ± 2 years), older non-exercising adults (ON, n=16, 61 ± 1 years) and young non-exercising controls (YN, n=8, 23 ± 1 years). Salsalate reduced endothelial cell expression of NF-κB p65 by ~25% in ON (P<0.05) but did not significantly change expression in OT or YN (P>0.05). EDD, assessed by brachial artery flow-mediated dilation (FMD), was improved by salsalate in ON (4.0 ± 0.7% compared with 6.8 ± 0.7%, placebo compared with salsalate, P<0.001) but did not change with salsalate in OT or YN (OT: 7.2 ± 0.7% compared with 7.7 ± 0.6%; YN: 7.6 ± 0.9% compared with 8.1 ± 0.8%; placebo compared with salsalate, P>0.05). Endothelium-independent dilation was not affected by salsalate in any group (P>0.05). In ON, vitamin C infusion improved FMD by ~30% during placebo (P<0.001) but had no affect during salsalate (P>0.05). In OT and YN, vitamin C infusion did not affect FMD during either placebo or salsalate (P>0.05). Salsalate reduced endothelial cell nitrotyrosine content by ~25% and NADPH oxidase p47phox expression by ~30% in ON (P<0.05) but had no effect in OT or YN (P>0.05). Our results suggest that endothelial NF-κB signalling is associated with oxidative stress-related impairment of EDD in healthy non-exercising but not aerobically exercising older adults. This may be a key mechanism by which regular aerobic exercise preserves endothelial function and reduces cardiovascular risk with aging.

Vascular Endothelial Function Is Related to White Blood Cell Count and Myeloperoxidase Among Healthy Middle-Aged and Older Adults

Endothelium-dependent dilation (EDD) is impaired with aging, but there is significant variability among healthy middle-aged and older adults. We tested the hypothesis that EDD is related to white blood cell (WBC) count in healthy men and women aged 55 to 75 years (n=48) who have a WBC count within the clinically normal range. The peak forearm blood flow response to intrabrachial artery infusion of acetylcholine was inversely related to WBC count (r=−0.38; P=0.004) and was 34% smaller in subjects with higher versus lower WBC count (more versus less than the median of 5.0×109 cells per liter; P=0.001). Vascular smooth muscle responsiveness to NO (peak forearm blood flow response to sodium nitroprusside) was inversely related to WBC count (r=−0.30; P=0.02) but did not fully explain the associations with EDD. Inhibition of NO with NG-monomethyl-l-arginine reduced EDD in subjects with lower (−56%; P=0.01) but not higher WBC count. Tetrahydrobiopterin selectively improved EDD in subjects with higher WBC count (+35%; P=0.01) by increasing NO bioavailability. EDD was related (P<0.05) to neutrophil, eosinophil, and monocyte but not lymphocyte or basophil counts. Myeloperoxidase, which is secreted by neutrophils and monocytes, consumes NO and produces molecules that oxidize tetrahydrobiopterin, was inversely related to EDD (r=−0.35; P=0.02), and was 42% higher in subjects with a higher WBC count (P=0.02). No other factors contributed to the relation between EDD and WBC count. Among healthy middle-aged and older adults, impaired EDD is related to higher neutrophil, eosinophil, and monocyte-based WBC count mediated by reduced responsiveness to NO and increased myeloperoxidase-associated reductions in tetrahydrobiopterin and NO bioavailability.

TNF-α impairs endothelial function in adipose tissue resistance arteries of mice with diet-induced obesity

We tested the hypothesis that high fat (HF) feeding results in endothelial dysfunction in resistance arteries of epididymal white adipose tissue (eWAT) and is mediated by adipose tissue inflammation. When compared with normal chow (NC)-fed mice (n = 17), HF-fed male B6D2F1 mice were glucose intolerant and insulin resistant as assessed by glucose tolerance test (area under the curve; HF, 18,174 ± 1,889 vs. NC, 15,814 ± 666 mg·dl(-1)·min(-1); P < 0.05) and the homeostatic model assessment (HF, 64.1 ± 4.3 vs. NC, 85.7 ± 6.4; P = 0.05). HF diet-induced metabolic dysfunction was concomitant with a proinflammatory eWAT phenotype characterized by greater macrophage infiltration (HF, 3.9 ± 0.8 vs. NC, 0.8 ± 0.4%; P = 0.01) and TNF-α (HF, 22.6 ± 4.3 vs. NC, 11.4 ± 2.5 pg/dl; P < 0.05) and was associated with resistance artery dysfunction, evidenced by impaired endothelium-dependent dilation (EDD) (maximal dilation; HF, 49.2 ± 10.7 vs. NC, 92.4 ± 1.4%; P < 0.01). Inhibition of nitric oxide (NO) synthase by N(ω)-nitro-L-arginine methyl ester (L-NAME) reduced dilation in NC (28.9 ± 6.3%; P < 0.01)- and tended to reduce dilation in HF (29.8 ± 9.9%; P = 0.07)-fed mice, eliminating the differences in eWAT artery EDD between NC- and HF-fed mice, indicative of reduced NO bioavailability in eWAT resistance arteries after HF feeding. In vitro treatment of excised eWAT arteries with recombinant TNF-α (rTNF) impaired EDD (P < 0.01) in NC (59.7 ± 10.9%)- but not HF (59.0 ± 9.3%)-fed mice. L-NAME reduced EDD in rTNF-treated arteries from both NC (21.9 ± 6.4%)- and HF (29.1 ± 9.2%)-fed mice (both P < 0.01). In vitro treatment of arteries with a neutralizing antibody against TNF-α (abTNF) improved EDD in HF (88.2 ± 4.6%; P = 0.05)-fed mice but was without effect on maximal dilation in NC (89.0 ± 5.1%)-fed mice. L-NAME reduced EDD in abTNF-treated arteries from both NC (25.4 ± 7.5%)- and HF (27.1 ± 16.8%)-fed mice (both P < 0.01). These results demonstrate that inflammation in the visceral adipose tissue resulting from diet-induced obesity impairs endothelial function and NO bioavailability in the associated resistance arteries. This dysfunction may have important implications for adipose tissue blood flow and appropriate tissue function.