Annemarie Silver

Direct Evidence of Endothelial Oxidative Stress With Aging in Humans: Relation to Impaired Endothelium-Dependent Dilation and Upregulation of Nuclear Factor-κB

Aging is associated with impaired vascular endothelial function, as indicated in part by reduced endothelium-dependent dilation (EDD). Decreased EDD with aging is thought to be related to vascular endothelial cell oxidative stress, but direct evidence is lacking. We studied 95 healthy men: 51 young (23±1 years) and 44 older (63±1 years). EDD (brachial artery flow-mediated dilation) was ≈50% lower in older versus young men (3.9±0.3% versus 7.6±0.3%, P<0.01; n=42 older/n=51 young). Abundance of nitrotyrosine (quantitative immunofluorescence), an oxidatively modified amino acid and marker of oxidative stress, was higher in endothelial cells (ECs) obtained from the brachial artery (1.25±0.12 versus 0.61±0.11 nitrotyrosine intensity/human umbilical vein EC [HUVEC] intensity, P=0.01; n=11 older/n=11 young) and antecubital veins (0.55±0.04 versus 0.34±0.03, P<0.05; n=19 older/n=17 young) of older men. Flow-mediated dilation was inversely related to arterial EC nitrotyrosine expression (r=−0.62, P=0.01; n=22). In venous samples, EC expression of the oxidant enzyme NAD(P)H oxidase-p47phox was higher in older men (0.71±0.05 versus 0.57±0.05 NAD[P]H oxidase-p47phox intensity/HUVEC intensity, P<0.05; n=19 older/n=18 young), whereas xanthine oxidase and the antioxidant enzymes cytosolic and mitochondrial superoxide dismutase and catalase were not different between groups. Nuclear factor-&kgr;B p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-&kgr;B, was elevated in both arterial (0.73±0.07 versus 0.53±0.05 NF-&kgr;B p65 intensity/HUVEC intensity, P<0.05; n=9 older/n=12 young) and venous (0.65±0.07 versus 0.34±0.05, P<0.01; n=13 older/n=15 young) EC samples of older men and correlated with nitrotyrosine expression (r=0.51, P<0.05 n=16). These results provide direct support for the hypothesis that endothelial oxidative stress develops with aging in healthy men and is related to reductions in EDD. Increased expression of NAD(P)H oxidase and nuclear factor-&kgr;B may contribute to endothelial oxidative stress with aging in humans.

Overweight and Obese Humans Demonstrate Increased Vascular Endothelial NAD(P)H Oxidase-p47phox Expression and Evidence of Endothelial Oxidative Stress

Background— Obesity may alter vascular endothelial cell protein expression (VECPE) of molecules that influence susceptibility to atherosclerosis. Methods and Results— Quantitative immunofluorescence was performed on vascular endothelial cells collected from 108 men and women free of clinical disease who varied widely in adiposity (body mass index 18.4 to 36.7 kg/m2; total body fat 5.8 to 55.0 kg; waist circumference: 63.0 to 122.9 cm). All 3 expressions of adiposity were positively associated with VECPE of the oxidant enzyme subunit NAD(P)H oxidase-p47phox (part correlation coefficient [rpart] 0.22 to 0.24, all P<0.05) and the antioxidant enzyme catalase (rpart=0.71 to 0.75, all P<0.001). Total body fat was positively associated with VECPE of nitrotyrosine (rpart=0.36, P=0.003), a marker of protein oxidation, and, in men, with Ser1177-phosphorylated endothelial nitric oxide synthase (rpart=0.46, P=0.02), an activated form of endothelial nitric oxide synthase. Overweight/obese subjects (body mass index ≥25 kg/m2) had 35% to 130% higher VECPE of NAD(P)H oxidase-p47phox, nitrotyrosine, catalase, and the cytosolic antioxidant CuZn superoxide dismutase (all P<0.05), as well as a 56% greater VECPE of the potent local vasoconstrictor endothelin-1 (P=0.05) than normal-weight subjects (body mass index <25 kg/m2). Nuclear factor-&kgr;B protein expression was ≈60% to 100% greater in the most obese adults than in the leanest adults (P≤0.01). These relations were independent of sex but were selectively reduced after accounting for the influence of plasma C-reactive protein, fasting glucose-insulin metabolism, or serum triglycerides. Conclusions— Compared with their normal-weight peers, overweight and obese adults demonstrate increased vascular endothelial expression of NAD(P)H oxidase-p47phox and evidence of endothelial oxidative stress, with selective compensatory upregulation of antioxidant enzymes and Ser1177-phosphorylated endothelial nitric oxide synthase. Endothelin-1 and nuclear factor-&kgr;B protein expression also appear to be elevated in obese compared with lean adults. These findings may provide novel insight into the molecular mechanisms linking obesity to increased risk of clinical atherosclerotic diseases in humans.

Vascular Endothelial Estrogen Receptor α Is Modulated by Estrogen Status and Related to Endothelial Function and Endothelial Nitric Oxide Synthase in Healthy Women

CONTEXT AND OBJECTIVE Estrogen receptor alpha (ER alpha), a potent transcription factor expressed in vascular endothelial cells, plays a key role in regulating vascular function and health. We determined whether vascular endothelial cell expression of ER alpha is influenced by estrogen status and is related to vascular endothelial function in healthy women. METHODS ER alpha protein expression was measured (quantitative immunofluorescence) in endothelial cells from peripheral veins of 16 healthy, premenopausal women during the early follicular (EF) and late follicular (LF) phases of the menstrual cycle and 17 estrogen-deficient postmenopausal women. Endothelial-dependent dilation (EDD; brachial artery flow-mediated dilation) and endothelial nitric oxide synthase (eNOS) expression and activation were also measured in a subgroup of women. RESULTS In premenopausal women (n = 10), ER alpha expression was 30% lower (P < 0.001) during the EF (low estrogen) compared with the LF (high estrogen) phase of the menstrual cycle. In postmenopausal women, ER alpha expression was 33% lower (P < 0.001) compared with the LF phase of the menstrual cycle in premenopausal women. ER alpha expression was strongly related (r = 0.67; P < 0.001) to EDD, which was reduced in postmenopausal women. ER alpha abundance was positively related to expression of eNOS (r = 0.54; P = 0.009; n = 21) and ser1177 phosphorylated eNOS (r = 0.59; P = 0.006; n = 20). CONCLUSIONS These results provide the first evidence that expression of ER alpha in vascular endothelial cells is modulated by estrogen status and may be a key determinant of vascular endothelial function in healthy pre- and postmenopausal women. ER alpha expression may influence vascular endothelial function in women by affecting protein levels and activation of eNOS.

Habitually exercising older men do not demonstrate age-associated vascular endothelial oxidative stress

We tested the hypothesis that older men who perform habitual aerobic exercise do not demonstrate age‐associated vascular endothelial oxidative stress compared with their sedentary peers. Older exercising men (n = 13, 62 ± 2 years) had higher (P < 0.05) physical activity (79 ± 7 vs. 30 ± 6 MET hours per week) and maximal exercise oxygen consumption (42 ± 1 vs. 29 ± 1 mL kg−1 per minute) vs. sedentary men (n = 28, 63 ± 1 years). Brachial artery flow‐mediated dilation (FMD), a measure of vascular endothelial function, was greater (P < 0.05) in the exercising vs. sedentary older men (6.3 ± 0.5 vs. 4.9 ± 0.4%Δ) and not different than young controls (n = 20, 25 ± 1 years, 7.1 ± 0.5%Δ). In vascular endothelial cells sampled from the brachial artery, nitrotyrosine, a marker of oxidative stress, was 51% lower in the exercising vs. sedentary older men (0.38 ± 0.06 vs. 0.77 ± 0.10 AU). This was associated with lower endothelial expression of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox subunit, 0.33 ± 0.05 vs. 0.61 ± 0.09 AU) and the redox‐sensitive transcription factor nuclear factor kappa B (NFκB) (p65 subunit, 0.36 ± 0.05 vs. 0.72 ± 0.09 AU). Expression of the antioxidant enzyme manganese superoxide dismutase (SOD) (0.57 ± 0.13 vs. 0.30 ± 0.04 AU) and activity of endothelium‐bound extracellular SOD were greater (6.4 ± 0.5 vs. 5.0 ± 0.6 U mL−1 per minute) in the exercising men (both P < 0.05), but differences no longer were significant after correcting for adiposity and circulating metabolic factors. Overall, values for the young controls differed with those for the sedentary, but not the exercising older men. Older men who exercise regularly do not demonstrate vascular endothelial oxidative stress, and this may be a key molecular mechanism underlying their reduced risk of cardiovascular diseases.

Effect of caregiver gender, age, and feedback prompts on chest compression rate and depth

BACKGROUND Quality of chest compressions (CC) is an important determinant of resuscitation outcome for cardiac arrest patients. PURPOSE To characterize the quality of CC performed by hospital personnel, evaluate for predictors of CC performance, and determine the effects of audiovisual feedback on CC performance. METHODS Seven hundred and fifty four individuals participated in a CPR quality improvement challenge at 30 US hospitals. Participants performed 2min of CC on a manikin with an accelerometer-based system for measuring both rate (CC/min) and depth (in.) of CC (AED Plus:ZOLL Medical). Real-time audiovisual feedback was disabled. A subset of participants performed a second trial of CC with the audiovisual feedback prompts activated. RESULTS Mean depth of CC was below AHA minimum guidelines (<1.5in.) for 34% (1.30+/-0.14in.) and above maximum guidelines (>2.0in.) for 12% of participants (2.20+/-0.22in.). Depth of CC was greater for male vs. female (p<0.001) and younger vs. older (p=0.009) but did not differ between ACLS, BCLS, and non-certified participants (p=0.6). Predictors of CC depth included CC rate (r(part)=-0.34, p<0.0001), gender (r(part)=0.13, p=0.001), and age (r(part)=-0.09, p=0.02). Mean depth of CC increased, mean rate decreased, and variance in CC depth and rate declined when feedback was used (p< or =0.0001 vs. without feedback). The percentage of CC performed within AHA guidelines (1.5-2in.) improved from 15 to 78% with feedback. CONCLUSIONS The quality of CC performed by personnel at US hospitals as judged by their performance on a manikin is often suboptimal. Quality of CC can be improved with use of CPR feedback technologies.

Habitual aerobic exercise is associated with smaller femoral artery intima–media thickness with age in healthy men and women:

Background Femoral artery intima–media thickness (IMT), an independent predictor of atherosclerotic disease risk, increases with age in sedentary adults. It is not known whether regular aerobic exercise modulates femoral IMT with ageing. Methods and results Study 1: Femoral IMT was measured in 173 sedentary, moderately active, and endurance-trained young (20–39 years), middle-aged (40–59 years) and older (60–79 years) men. IMT increased with age in all activity groups (P<0.001). However, IMT was 20–27% smaller in age-matched, endurance-trained compared with sedentary men (P<0.001), and the age-associated increase in IMT was 33% smaller in endurance-trained compared with sedentary men (+0.32 versus +0.45 mm). There was a trend for the IMT to be smaller in moderately active compared with sedentary older men, and the age-associated increase in IMT was 37% smaller in moderately active than sedentary men (+0.28 mm). Study 2: Among 74 premenopausal and postmenopausal sedentary or endurance-trained women, IMT was higher (P<0.001) in postmenopausal compared with premenopausal women regardless of activity status. However, IMT was 15% smaller in endurance-trained compared with sedentary postmenopausal women (P<0.001), and the premenopausal to postmenopausal difference in IMT was approximately 45% smaller in endurance-trained compared with sedentary women (+0.13 versus +0.23 mm). Conclusions Femoral IMT increases with age even in habitually exercising adults. However, the age-associated increase and absolute level of IMT are smaller in middle-aged and older adults who perform regular aerobic-endurance exercise, and may contribute to their lower incidence of atherosclerotic disease.

Protein Expression in Vascular Endothelial Cells Obtained from Human Peripheral Arteries and Veins

Studying molecular mechanisms of vascular endothelial function in humans is difficult in part because of limited access to arteries. Access to peripheral veins is more practical. We determined if differences in protein expression of endothelial cells (EC) collected from a peripheral artery are reflected in measurements made on EC obtained from peripheral veins. EC were collected from the brachial artery and an antecubital vein of 106 healthy adults (60 men and 46 women, age 18–77 years). Quantitative immunofluorescence was used to measure protein expression of endothelial nitric oxide synthase (eNOS), Ser-1177 phosphorylated eNOS, manganese superoxide dismutase, nitrotyrosine, xanthine oxidase and nuclear factor-κB p65. Protein expression in EC obtained from brachial artery and antecubital vein sampling was moderately to strongly related (r = 0.59–0.81, all p < 0.0001, mean r = 0.70). Moreover, differences between subgroups in the lowest and highest tertiles of protein expression in EC obtained from arterial samples were consistently reflected in EC obtained from venous collections. These findings indicate that interindividual and group differences in expression of several proteins involved in nitric oxide production, oxidant production, antioxidant defense and inflammatory signaling in EC obtained from brachial artery sampling are consistently reflected in EC obtained from venous samples. Thus, EC collected from peripheral veins may provide a useful surrogate for EC obtained from arteries for measurements of EC protein expression in humans.

The association between chest compression release velocity and outcomes from out-of-hospital cardiac arrest

BACKGROUND Previous studies have demonstrated significant relationships between cardiopulmonary resuscitation (CPR) quality metrics and survival to hospital discharge from out-of-hospital cardiac arrest (OHCA). Recently, it has been suggested that a new metric, chest compression release velocity (CCRV), may be associated with improved survival from OHCA. METHODS AND RESULTS We performed a retrospective review of all treated adult OHCA occurring over a two year period beginning January 1, 2012. CPR metrics were abstracted from accelerometer measurements during each resuscitation. Multivariable regression analysis was used to examine the impact of CCRV on survival to hospital discharge. Secondary outcome measures were the impact of CCRV on return of spontaneous circulation (ROSC) and neurologically intact survival (MRS ≤ 3). Among 1800 treated OHCA, 1137 met inclusion criteria. The median (IQR) age was 71.6 (60.6, 82.3) with 724 (64%) being male. The median (IQR) CCRV (mm/s) amongst 96 survivors was 334.5 (300.0, 383.2) compared to 304.0 (262.6, 354.1) in 1041 non survivors (p < 0.001). When adjusted for Utstein variables, the odds of survival to hospital discharge for each 10 mm/s increase in CCRV was 1.02 (95% CI: 0.98, 1.06). Similarly the odds of ROSC and neurologically intact survival were 1.01 (95% CI: 0.99, 1.03) and 1.02 (95% CI: 0.98, 1.06), respectively. CONCLUSIONS When adjusted for Utstein variables, CCRV was not significantly associated with outcomes from OHCA. Further research in other EMS systems is required to clarify the potential impact of this variable on OHCA survival.

A New Defibrillator Mode to Reduce Chest Compression Interruptions for Health Care Professionals and Lay Rescuers: A Pilot Study in Manikins

Abstract Background. Chest compression interruptions are detrimental during the resuscitation of cardiac arrest patients, especially immediately prior to shock delivery. Objective. To evaluate the effect of use of a new defibrillator technology, which filters compression-induced artifact and provides reliable rhythm analysis with automatic defibrillator charging during chest compressions, on preshock chest compression interruption. Methods. Thirty subjects (20 basic life support [[BLS]]; 10 advanced life support [[ALS]]) worked in pairs to perform two randomly ordered simulated cardiac resuscitations with the defibrillator operating in either standard mode (ALS == manual; BLS == automated external defibrillator [[AED]]) or the new Analysis and Charging during CPR (AC-CPR) mode. During each resuscitation simulation, rescuers switched roles as chest compressor and defibrillator operator every two segments of CPR (one segment == 2 minutes of chest compressions, rhythm analysis, and shock delivery, if appropriate), for eight total segments. The participants rested ≥30 minutes between trials and received brief AC-CPR training (BLS == 30 seconds; ALS == 5 minutes). Heart rate and perceived exertion were measured with pulse oximetry and the Borg scale, respectively. Results. Mean (± standard deviation) preshock chest compression pause time was considerably shorter in each CPR segment with AC-CPR versus standard defibrillator operation (2.13 ± 0.99 sec vs. 10.93 ± 1.33, p < 0.0001), demonstrating effective use of AC-CPR with minimal training. Despite reduced chest compression interruption with AC-CPR, rescuer fatigue and perceived exertion did not differ in any CPR segment with standard defibrillator operation versus AC-CPR (p == 0.2–1.0). Conclusions. Preshock pause time is reduced by 80%% utilizing a novel technology that employs automated analysis and charging during chest compression. Although chest compression pause time is reduced with the use of the new technology, participants do not excessively fatigue.

Cardiac rhythm analysis during ongoing cardiopulmonary resuscitation using the Analysis During Compressions with Fast Reconfirmation technology

BACKGROUND Pauses in chest compressions (CCs) have a negative association with survival from cardiac arrest. Electrocardiographic (ECG) rhythm analysis and defibrillator charging are significant contributors to CC pauses. OBJECTIVE Accuracy of the Analysis During Compressions with Fast Reconfirmation (ADC-FR) algorithm, which features automated rhythm analysis and charging during CCs to reduce CC pauses, was retrospectively determined in a large database of ECGs from 2701 patients with out-of-hospital cardiac arrest. METHODS The ADC-FR algorithm generated a total of 7264 advisories, of which 3575 were randomly assigned to a development data set and 3689 to a test data set. With ADC-FR, a high-pass digital filter is used to remove CC artifacts, while the underlying ECG rhythm is automatically interpreted. When CCs are paused at the end of the 2-minute cardiopulmonary resuscitation interval, a 3-second reconfirmation analysis is performed using the artifact-free ECG to confirm the shock/no-shock advisory. The sensitivity and specificity of the ADC-FR algorithm in correctly identifying shockable/nonshockable rhythms during CCs were calculated. RESULTS In both data sets, the accuracy of the ADC-FR algorithm for each ECG rhythm exceeded the recommended performance goals, which apply to a standard artifact-free ECG analysis. Sensitivity and specificity were 97% and 99%, respectively, for the development data set and 95% and 99% for the test data set. CONCLUSION The ADC-FR algorithm is highly accurate in discriminating shockable and nonshockable rhythms and can be used to reduce CC pauses.