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In JoVE (1)
Other Publications (18)
- Medicine and Science in Sports and Exercise
- Medicine and Science in Sports and Exercise
- American Heart Journal
- Free Radical Biology & Medicine
- Dynamic Medicine : DM
- Free Radical Biology & Medicine
- Ultrasound in Medicine & Biology
- Nitric Oxide : Biology and Chemistry / Official Journal of the Nitric Oxide Society
- British Journal of Pharmacology
- Free Radical Biology & Medicine
- Thrombosis Research
- BMC Cancer
- Arteriosclerosis, Thrombosis, and Vascular Biology
- Biomaterials
- Vascular Medicine (London, England)
- Journal of Applied Physiology (Bethesda, Md. : 1985)
- Journal of Applied Physiology (Bethesda, Md. : 1985)
- Journal of Applied Physiology (Bethesda, Md. : 1985)
Articles by Jason D. Allen in JoVE
JoVE Bioengineering
Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.
Other articles by Jason D. Allen on PubMed
Stability and Reproducibility of Brachial Artery Flow-mediated Dilation
Brachial artery flow-mediated dilation (BAFMD) is a noninvasive technique, which has been suggested as a potential means of identifying patients with early atherosclerosis and therefore has enormous clinical appeal. Despite this, the stability and reproducibility of this technique are not yet clear. Therefore, the purpose of this study was to establish the stability and reproducibility of BAFMD after 5 min of forearm occlusion and to produce power calculations to aid in clinical trial design.
Time Course of Improved Flow-mediated Dilation After Short-term Exercise Training
The purpose was to determine the influence of a unilateral localized short-term handgrip training protocol on brachial artery flow-mediated dilation (BAFMD) and to examine the time course of such changes.
Regional and Whole-body Markers of Nitric Oxide Production Following Hyperemic Stimuli
The measurement of nitric oxide (NO) bioavailability is of great clinical interest in the assessment of vascular health. However, NO is rapidly oxidized to form nitrite and nitrate and thus its direct detection in biological systems is difficult. Venous plasma nitrite (nM concentrations) has been shown to be a marker of forearm NO production following pharmacological stimulation of the endothelium utilizing acetylcholine (Ach). In the present study, we demonstrate, within 15 apparently healthy subjects (34.1 +/- 7.3 years), that reactive hyperemia of the forearm, a physiological endothelial stimulus, results in a 52.5% increase in mean plasma nitrite concentrations (415 +/- 64.0 to 634 +/- 57.1 nM, P = 0.015). However, plasma nitrite is readily oxidized to nitrate within plasma, and thus its utility as a marker of NO production within the clinical setting may be limited. Alternatively, NOx (predominantly nitrate) is relatively stable in plasma (microM concentrations), but is produced by sources other than the vasculature and has been shown to be unsuitable as a measure of localized NO production. We reasoned that the principle source of NOx generation during exercise is NO production and thus have examined the change in NOx following treadmill exercise stress. In this study, 12 apparently healthy subjects showed an increase (from baseline) in venous NOx at peak effort and during recovery (12 +/- 9.1 and 17 +/- 15.3 microM respectively, P < 0.05). In contrast, 10 subjects with cardiovascular disease showed no significant increases. Additionally, a correlation between VO(2peak) and the change in circulating NOx (r(2) = 0.4585, P < or = 0.01) indicated the subjects who could exercise hardest also produced the most NO.
Biphasic Responses of the Brachial Artery Diameter Following Forearm Occlusion: a Blunted Response in the Elderly
The purpose was to examine the temporal response of the brachial artery diameter following 5 minutes of forearm occlusion in young men. A secondary objective was to compare the main features of the temporal pattern between young and old.
Total Nitrogen Oxide Following Exercise Testing Reflects Endothelial Function and Discriminates Health Status
Nitric oxide (NO) bioavailability is important in vascular health, but unsuitable as a clinical measure due to biological oxidation. Total nitrogen oxides (NO(x)) are stable but background nitrate levels make it difficult to detect disease-based variation. We investigated the clinical discriminatory value of NO(x) as it relates to exercise capability (VO(2peak)) and brachial artery reactivity (BAR, an NO-dependent measure of endothelial health), in healthy (H), increased risk (RF), and known cardiovascular disease (CVD) subjects. BAR was measured using forearm occlusion/hyperemia stimulus. Subjects performed a maximal graded exercise test (GXT). Blood at rest, exercise termination, and 10 min into recovery was mixed equally with 0.1 M NaOH at 4 degrees C, filtered, and stored at -70 degrees C. NO(x) was measured by chemiluminescence. Seven of the RF group then exercise-trained for 6 months prior to retesting. The H group (n = 12) was younger, had higher VO(2peak), HDL levels, and baseline NO(x) values than the RF (n = 15) and CVD (n = 10) groups. NO(x) increased from baseline to recovery in the H group only (75.85 +/- 19.04 microM vs 97.76 +/- 31.93 microM; P
Acoustic Radiation Force Impulse Imaging for Noninvasive Characterization of Carotid Artery Atherosclerotic Plaques: a Feasibility Study
Atherosclerotic disease in the carotid artery is a risk factor for stroke. The susceptibility of atherosclerotic plaque to rupture, however, is challenging to determine by any imaging method. In this study, acoustic radiation force impulse (ARFI) imaging is applied to atherosclerotic disease in the carotid artery to determine the feasibility of using ARFI to noninvasively characterize carotid plaques. ARFI imaging is a useful method for characterizing the local mechanical properties of tissue and is complementary to B-mode imaging. ARFI imaging can readily distinguish between stiff and soft regions of tissue. High-resolution images of both homogeneous and heterogeneous plaques were observed. Homogeneous plaques were indistinguishable in stiffness from vascular tissue. However, they showed thicknesses much greater than normal vascular tissue. In heterogeneous plaques, large and small soft regions were observed, with the smallest observed soft region having a diameter of 0.5 mm. A stiff cap was observed covering the large soft tissue region, with the cap thickness ranging from 0.7-1.3 mm.
Plasma Nitrite Response and Arterial Reactivity Differentiate Vascular Health and Performance
NO is crucial for endothelial function and vascular health. Plasma nitrite (NO(2)(-)) is the main oxidation product of NO and has been shown to reflect changes in eNOS activity. We hypothesized that plasma NO(2)(-) response to physical exercise stress along with physiological endothelial function would be reduced with increasing severity of vascular disease. Subject groups were: (a) risk factors but no vascular disease (RF); (b) Type 2 diabetes with no vascular disease (DM); (c) diagnosed peripheral arterial disease (PAD); and (d) DM+PAD. Venous blood was drawn at rest and 10min following maximal exercise. Plasma samples were analyzed by reductive chemiluminescence. Brachial diameters were imaged prior to, during and following 5min of forearm occlusion (BAFMD). There were no differences in resting plasma NO(2)(-) or BA diameters between groups. The PAD groups had lower age adjusted BAFMD responses (p0.05). Within group analysis revealed an increase in NO(2)(-) in the RF group (+39.3%), no change in the DM (-15.51%), and a decrease in the PAD (-44.20%) and PAD+DM (-39.95%). This was maintained after adjusting for age and VO(2peak) (p0.05). DeltaNO(2)(-) and BAFMD were the strongest independent predictors of VO(2peak) in multivariate linear regression. These findings suggest DeltaNO(2)(-) discriminates severity of cardiovascular disease risk, is related to endothelial function and predicts exercise capacity.
Nitrite, NO and Hypoxic Vasodilation
The ability to deliver oxygen and other nutrients to working tissues at a rate acutely matched to demand is the quintessential function of the cardiovascular system. Thus, an understanding of the biochemical mechanisms involved in hypoxic vasodilation remains a major goal in vascular biology. Nitric oxide, its metabolites, and oxidation status are recognized as playing important roles in this process. Previous work examining how nitrite can be converted to bioactive nitric oxide (NO) under hypoxic conditions has focused on the role of the red blood cell and haemoglobin. In a recent issue of the British Journal of Pharmacology, Pinder et al. demonstrate that plasma nitrite, in the absence of haemoglobin, is capable of increasing the maximal dilation of rabbit aortic rings under hypoxic conditions. Furthermore, they demonstrate that this relaxation can occur with or without the endothelium. This observation, even if it is only a small proportion of the relaxant activity of nitrite, highlights how NO metabolites may be involved in a variety of mechanisms of vessel control.
Plasma Nitrite Flux Predicts Exercise Performance in Peripheral Arterial Disease After 3months of Exercise Training
Plasma nitrite is a major oxidation product of nitric oxide. It has also recently been suggested to perform an endocrine-like function as a nitric oxide donor in hypoxic tissues, allowing vasodilation. Exercise performance is limited in peripheral arterial disease because of an inadequate blood supply to working tissues. We hypothesized that exercise training in peripheral arterial disease subjects will improve "plasma nitrite flux" and endothelial function, to accompany increased exercise performance. Peripheral arterial disease subjects were tested at baseline and after 3 months supervised or home exercise training. Venous blood (arm) was drawn at rest and 10 min after a maximal graded treadmill test. Samples were added to heparin and centrifuged and plasma was snap-frozen for analysis by reductive chemiluminescence. Brachial artery endothelial function was measured in response to a hyperemic stimulus (flow-mediated dilation). At 3 months the peripheral arterial disease-supervised exercise group showed increases in claudication onset pain time (+138 s, p< or =0.05), peak walking time (+260 s, p< or =0.01), VO(2peak) (1.3 ml/kg/min, p< or =0.05), brachial artery flow-mediated dilation (+2%, p< or =0.05), and plasma nitrite flux (+33% p< or =0.05). There were no changes in the peripheral arterial disease-home exercise group. The change in plasma nitrite flux predicted the change in claudication onset pain (r(2)=0.59, p< or =0.01). These findings suggest that changes in plasma nitrite are related to endothelial function and predict exercise performance in peripheral arterial disease.
The Effect of Aspirin on Endothelial Progenitor Cell Biology: Preliminary Investigation of Novel Properties
Atherosclerosis develops in an environment of endothelial injury and inflammation. Circulating endothelial progenitor cells (EPCs) are required for vascular repair and restoration of normal endothelial function. We tested the hypothesis that the nonselective cyclooxygenase (COX) inhibitor aspirin (ASA) exerts an effect on circulating EPCs.
Rationale and Design of the Exercise Intensity Trial (EXCITE): A Randomized Trial Comparing the Effects of Moderate Versus Moderate to High-intensity Aerobic Training in Women with Operable Breast Cancer
The Exercise Intensity Trial (EXcITe) is a randomized trial to compare the efficacy of supervised moderate-intensity aerobic training to moderate to high-intensity aerobic training, relative to attention control, on aerobic capacity, physiologic mechanisms, patient-reported outcomes, and biomarkers in women with operable breast cancer following the completion of definitive adjuvant therapy.
Angiogenesis in Skeletal Muscle Precede Improvements in Peak Oxygen Uptake in Peripheral Artery Disease Patients
Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, causing claudication and exercise intolerance. The mechanism(s) by which exercise training improves functional capacity is not understood. This study tested the hypothesis that in PAD patients who undergo supervised exercise training, increases in capillary density (CD) in calf muscle take place before improvements in peak oxygen uptake (VO(2)).
The Biocompatibility of Titanium Cardiovascular Devices Seeded with Autologous Blood-derived Endothelial Progenitor Cells: EPC-seeded Antithrombotic Ti Implants
Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.
The Development and Potential of Acoustic Radiation Force Impulse (ARFI) Imaging for Carotid Artery Plaque Characterization
Stroke is the third leading cause of death and long-term disability in the USA. Currently, surgical intervention decisions in asymptomatic patients are based upon the degree of carotid artery stenosis. While there is a clear benefit of endarterectomy for patients with severe (> 70%) stenosis, in those with high/moderate (50-69%) stenosis the evidence is less clear. Evidence suggests ischemic stroke is associated less with calcified and fibrous plaques than with those containing softer tissue, especially when accompanied by a thin fibrous cap. A reliable mechanism for the identification of individuals with atherosclerotic plaques which confer the highest risk for stroke is fundamental to the selection of patients for vascular interventions. Acoustic radiation force impulse (ARFI) imaging is a new ultrasonic-based imaging method that characterizes the mechanical properties of tissue by measuring displacement resulting from the application of acoustic radiation force. These displacements provide information about the local stiffness of tissue and can differentiate between soft and hard areas. Because arterial walls, soft tissue, atheromas, and calcifications have a wide range in their stiffness properties, they represent excellent candidates for ARFI imaging. We present information from early phantom experiments and excised human limb studies to in vivo carotid artery scans and provide evidence for the ability of ARFI to provide high-quality images which highlight mechanical differences in tissue stiffness not readily apparent in matched B-mode images. This allows ARFI to identify soft from hard plaques and differentiate characteristics associated with plaque vulnerability or stability.
Dietary Nitrate Supplementation Enhances Exercise Performance in Peripheral Arterial Disease
Peripheral arterial disease (PAD) results in a failure to adequately supply blood and oxygen (O(2)) to working tissues and presents as claudication pain during walking. Nitric oxide (NO) bioavailability is essential for vascular health and function. Plasma nitrite (NO(2)(-)) is a marker of vascular NO production but may also be a protected circulating "source" that can be converted to NO during hypoxic conditions, possibly aiding perfusion. We hypothesized that dietary supplementation of inorganic nitrate in the form of beetroot (BR) juice would increase plasma NO(2)(-) concentration, increase exercise tolerance, and decrease gastrocnemius fractional O(2) extraction, compared with placebo (PL). This was a randomized, open-label, crossover study. At each visit, subjects (n = 8) underwent resting blood draws, followed by consumption of 500 ml BR or PL and subsequent blood draws prior to, during, and following a maximal cardiopulmonary exercise (CPX) test. Gastrocnemius oxygenation during the CPX was measured by near-infrared spectroscopy. There were no changes from rest for [NO(2)(-)] (152 ± 72 nM) following PL. BR increased plasma [NO(2)(-)] after 3 h (943 ± 826 nM; P ≤ 0.01). Subjects walked 18% longer before the onset of claudication pain (183 ± 84 s vs. 215 ± 99 s; P ≤ 0.01) and had a 17% longer peak walking time (467 ± 223 s vs. 533 ± 233 s; P ≤ 0.05) following BR vs. PL. Gastrocnemius tissue fractional O(2) extraction was lower during exercise following BR (7.3 ± 6.2 vs. 10.4 ± 6.1 arbitrary units; P ≤ 0.01). Diastolic blood pressure was lower in the BR group at rest and during CPX testing (P ≤ 0.05). These findings support the hypothesis that NO(2)(-)-related NO signaling increases peripheral tissue oxygenation in areas of hypoxia and increases exercise tolerance in PAD.
Relationship Between Leg Muscle Capillary Density and Peak Hyperemic Blood Flow with Endurance Capacity in Peripheral Artery Disease
The aim of this study was to determine if skeletal muscle capillary density is lower in patients with peripheral artery disease (PAD) and if capillary density relates to functional limitations. PAD patients with intermittent claudication (IC) have a decreased exercise tolerance due to exercise-induced muscle ischemia. Despite the apparent role diminished arterial flow has in this population, the degree of walking pain and functional limitation is not entirely explained by altered hemodynamics of the affected limbs. We hypothesized that skeletal muscle capillary density is lower in PAD and is related to the functional impairment observed in this population. Sixty-four patients with PAD and 56 controls underwent cardiopulmonary exercise testing and a gastrocnemius muscle biopsy. A subset of these patients (48 PAD and 47 controls) underwent peak hyperemic flow testing via plethysmography. Capillary density in PAD patients was lower compared with controls (P < 0.001). After adjustment for several baseline demographic imbalances the model relating capillary density to peak oxygen consumption (Vo(2)) remained significant (P < 0.001). In PAD subjects, capillary density correlated with peak Vo(2), peak walking time (PWT), and claudication onset time (COT). Peak hyperemic blood flow related to peak Vo(2) in both PAD and control subjects. PAD is associated with lower capillary density, and capillary density is related to the functional impairment as defined by a reduced peak Vo(2), PWT, and COT. These findings suggest that alterations in microcirculation may contribute to functional impairment capacity in PAD.
