Peripheral artery disease (PAD) is a cardiovascular disease manifesting from systemic atherosclerosis, which blocks the leg arteries and results in insufficient blood flow to the lower extremities. Limb ischemia from PAD is the most common disorder treated within the vascular surgery service at the Omaha Veterans' Affairs Medical Center. PAD also accounts for one-third of the operations performed in the VA Medical Centers nationwide. This project aims to establish the feasibility and acceptability of a muscle oxygen-guided supervised exercise program for patients with PAD. The investigators will determine the potential benefits of using this intervention over standard supervised exercise therapy. This modified intervention may enable patients to increase overall physical activity without the negative impacts on muscle structure and function. Increasing physical activity will decrease cardiovascular morbidity and mortality. If proven beneficial, the findings will lead to an improved exercise program that directly benefits veterans nationwide.
The long-term goal of this research is to improve mobility, functional independence, and quality of life in patients with peripheral artery disease (PAD) by developing conservative interventions. PAD is a manifestation of atherosclerosis, which produces blockages in the leg arteries, and results in insufficient blood flow to the legs. PAD results in significant gait deficits and an overall sedentary lifestyle. The investigators' prior work showed that the biochemical (oxidative stress and mitochondrial dysfunction) and histological (myofiber degeneration and fibrosis) characteristics of PAD muscles are altered compared to controls. These myopathic changes are due to cycles of ischemia and reperfusion, and they correlate to functional limitations (walking distance and calf muscle strength). Limb ischemia from PAD is the most common disorder treated within the vascular surgery service of the Omaha Veterans Affairs Medical Center. The most effective treatment for increasing the distances that patients with PAD can walk is supervised exercise therapy (SET). However, SET uses pain onset to guide exercise intensity and rest cycles. These repeated bouts of ischemia/reperfusion could cause further pathological damage to muscle myofibers and oxidative processes. Such adverse effects of ischemia may explain why not all patients improve with SET. Preventing cycles of ischemia-reperfusion during SET can improve walking performance without further muscle biochemistry damage in patients with PAD. The investigators propose using hemodynamic responses to modulate exercise intensity and prevent ischemia during SET. Muscle oxygen-guided supervised exercise therapy (M-SET) can prevent ischemia onset and thus preserve muscle mass and function during exercise therapy in patients with PAD. The aims are: Aim 1: To determine the exercise outcomes of an M-SET versus a standard SET session in patients with PAD. Aim 2: To determine the acute effect of M-SET versus standard SET on oxidative stress and inflammation in patients with PAD. Aim 3: To determine the effect of 12 weeks of M-SET versus standard SET on exercise outcomes, muscle biochemistry, and subject-reported preference in patients with PAD. The investigators will measure exercise outcomes during one session each of M-SET (using StO2 on the calf to modulate exercise) and SET, in 12 patients with PAD using a cross-over design. Exercise outcomes will be assessed by the total distance patients walked during the session, metabolic cost, metabolic equivalents of the task, physical activity enjoyment, and rate of perceived exertion. In Aim 2, oxidative stress will be measured using peripheral blood mononuclear cells before and after each exercise session (SET and M-SET) in all patients. During the standard SET session, subjects will walk until claudication pain becomes severe and the participant needs to stop. Then subjects will rest until claudication pain subsides. Afterward, subjects will walk again, repeating the cycle for up to 50 minutes. M-SET will use an StO2 threshold that is 15% lower level than baseline StO2 levels. Subjects will stop walking once they reach the threshold and rest until StO2 levels return to baseline. Then, subjects will begin walking again, repeating this cycle for 50 total minutes (including walking and rest). Patients from Aims 1 and 2 will be randomly assigned to either a 12-week M-SET or standard SET (6 per group) program. Training will be performed three times weekly as described above. Subjects will be evaluated before and after the intervention to capture initial and absolute walking distances and 7-day physical activity. Muscle biochemistry will be quantified using mitochondrial function, mitochondrial oxidative dynamics, and reactive oxygen species production before and after training. Results will support a full clinical trial and rehabilitation recommendations.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
NONE
Enrollment
12
Subjects will be enrolled in a 12-week intervention program of either modified supervised exercise therapy or standard supervised exercise therapy. The program will be 3 sessions/week for 12 weeks. Each session will consist of 50 minutes plus 5 minutes warm up and cool down.
Omaha VA Nebraska-Western Iowa Health Care System, Omaha, NE
Omaha, Nebraska, United States
RECRUITINGDistance walked
Total distance walked during the exercise session (standard or M-SET) will be determined using the total time of walking multiplied by the walking speed.
Time frame: Before and after 12 week exercise program
Energy expenditure
Energy expenditure57 will be assessed via oxygen consumption as measured by a metabolic cart (K5, Cosmed USA, Inc., Chicago, IL). Subjects will stand quietly for five minutes to collect resting energy expenditure. Standing steady state energy expenditure will be calculated for each subject by plotting VO2 and assigning a sliding best fit line over a two-minute window. Steady-state oxygen consumption in ml/kg/min will be calculated during the exercise session. Energy cost of transport will be calculated by subtracting the energy consumption values at standing from walking to achieve net metabolic cost for each bout of exercise.
Time frame: Before and after 12 weeks after the exercise program
Reactive oxygen species
Quantification of reactive oxygen species (ROS) related markers and free radicals: Electron paramagnetic resonance (EPR), which has been known as a gold standard for free radical assessment, will be used to quantify free radicals such as superoxide as we have previously performed (1-3). Plasma samples will be used for measuring ROS-related markers such as TAC, SOD, GSH:GSSG, ONOO- and MDA with commercially available and previously validated ELISA Assay Kits (CAT#s ab65329, ab65354, ab138881, ab233469, and ab233471 Abcam, UK) according to the manufacturer's instructions60. The average intra-assay variability measured as the coefficient of variation for each sample was 2.9%, 3.8%, and 3.3% for SOD, TAC, and MDA, respectively.
Time frame: Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise
Protein expression of ROS-related enzymes
Protein expression of ROS-related enzymes: Enzymes related to ROS will be quantified with western blots of isolated PBMCs analyzed for catalase, 4 hydroxynonenal (4HNE), and glutathione peroxidase 1 (GPX) (4). All samples will be homogenized in RIPA buffer (Boston Bioproducts) and 100X protease inhibitor (Sigma Aldrich). A 150-volt electrical potential will be applied to the gel for 1 hr to separate proteins based on mass. A 250-volt electrical potential will then be applied to the gel for 7 min to transfer protein bands to transfer paper with TurboTransfer (Biorad). Primary antibodies for catalase, 4HNE, and GPX will be recombinant anti-catalase antibody (ab209211), anti-4 hyroxynonenal antibody (ab243070), and recombinant anti-glutathione peroxidase 1 antibody (ab108429), respectively.
Time frame: Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise
Markers of systemic inflammation
Markers of systemic inflammation: Standard ELISA kits will be used to test blood samples for interleukin 6, tumor necrosis factor-a, vascular cell adhesion protein 1, and vascular endothelial growth factor-a (Cat#s, ab178013, ab285312, ab223591, ab119566). Oxidized low density lipoproteins (LDL) will be measured with an oxidized LDL assay kit (ab242302). All measurements will be made per validated manufacturer specifications.
Time frame: Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise
Initial and absolute walking distances
Initial and absolute walking distances will be determined from the progressive-load treadmill test. Subjects will walk on a treadmill that starts at 0% grade and 2.0 mph. Every two minutes, the grade will be increased by 2% up to a maximum of 15% grade, and the speed held constant throughout the test. Initial claudication distance is the distance walked prior to claudication pain onset. Absolute claudication distance is the total distance walked before subjects had to stop walking due to claudication pain.
Time frame: Before and after 12 week supervised exercise program
Comprehensive mitochondrial respiratory complex function
Comprehensive mitochondrial respiratory complex function (complex I, II, III, IV, and V) will be completed with previously validated high resolution respirometry with complex agonists/antagonists. Skeletal muscle tissue will be manually teased and permeabilized with 15 l of digitonin for 30 min. Samples will then be rinsed twice in 2 ml of MIRO5 for 10-min. 3-5 mg (dry weight) of tissue will then be isolated and placed in the high-resolution respirometer with 2 ml of MIRO5 solution. The order and quantity of each agonist and antagonist will be applied as follows: 5 l malate and 10 l glutamate (complex I agonist), 20 l adenosine diphosphate (complex V agonist), 20 l succinate (complex II agonist), 1 l rotenone (complex I antagonist), 1 l oligomycin (complex V antagonist), 1 l antimycin A (complex III antagonist), and 5 l ascorbate + 5 l TMPD (complex IV agonist).
Time frame: Before and after 12 week supervised exercise program
Mitochondrial dynamics
Specific proteins we will assess are Fis-1 and DRP-1 (fission), Mfn-1, Mfn-2 and OPA-1 (fusion), and UPC-1 and UCP-2 (dynamics). We will perform the same Western Blot technique mentioned in Aim #262. Primary antibodies for Fis-1, DRP-1, Mfn-1, Mfn-2, OPA-1, UPC-1, and UCP-2 will be recombinant anti- TTC11/FIS1 (ab156865), recombinant anti-DRP1 (ab184247), anti-mitofusin 1 antibody-N-terminal (ab191853), recombinant anti-mitofusin 2 (ab205236), recombinant anti-OPA1 (ab157457), anti-UCP1 (ab155117), and anti-UCP2 (ab247184), respectively.
Time frame: Before and after 12 week supervised exercise program
Mitochondrial reactive oxygen species production
Mitochondrial reactive oxygen species production will be assessed with fluorescent imaging of skeletal muscle biopsy samples treated with H2DCFDA (D399, ThermoFisher), which is validated to detect ROS within cells. The validated mitochondrial targeted antioxidant MitoTempo (SML0737, Sigma-Aldrich) will be used to confirm the presence of mitochondrial ROS. EPR will be used to quantify free radicals such as superoxide.
Time frame: Before and after 12 week supervised exercise program
Metabolic equivalents of task
Metabolic equivalents of task (MET) will be used to quantify exercise dose accumulated over the course of the SET or M-SET session. The MET value (measured by metabolic cart) will be multiplied by the number of minutes exercised during each bout. Session MET/min will be summed to quantify total dose (Exercise intensity).
Time frame: Immediate
Physical activity enjoyment scale
Physical activity enjoyment scale is an 18-item measure developed to assess the extent to which an individual enjoys a given physical activity. Ratings span from 1(most enjoyment) to 7 (least enjoyment) and the questionnaire has been extensively validated1. The scale will be given at the end of each exercise session.
Time frame: Immediate
Rating of perceived exertion
Rating of perceived exertion will be recorded using the Borg Scale, which ranges from 6 (no exertion) to 20 (maximal exertion) at the end of exercise session.
Time frame: Immediate
Physical activity average steps/day
Physical activity will be measured with an accelerometer (Actigraph GT3X, Actigraph, FL, USA) placed on subjects' hip for seven days after the intervention but prior to the assessment visit. Average steps/day will be calculated. Accelerometers will be given to patients at their last exercise session prior to the assessment data collection.
Time frame: Before and after 12 week supervised exercise program
Muscle strength
Muscle strength will be measured with an isokinetic dynamometer (Biodex 4 Medical Systems, Inc. USA). Peak isokinetic strength (torque) of the ankle and hip of the more symptomatic leg will be measured. Subjects will perform one trial of five repetitions at 60 degrees/second to assess strength.
Time frame: Before and after 12 week supervised exercise program
Acceptability
In each intervention group, patients will be interviewed after completing the 12 week intervention. Interviews will provide the potential feasibility of exercise by identifying acceptability (satisfaction, intent to continue participation), perceived demand, and practicality (positive/negative effects).
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Time frame: Before and after 12 week supervised exercise program
Maximum cadence/day
Physical activity will be measured with an accelerometer (Actigraph GT3X, Actigraph, FL, USA) placed on subjects' hip for seven days after the intervention but prior to the assessment visit. Accelerometers will be given to patients at their last exercise session prior to the assessment data collection. Cadence is the number of steps per minute and the maximum cadence in a 24 hour period will be recorded as the maximum cadence/day.
Time frame: Before and after 12 week supervised exercise program
Muscle endurance
Muscle endurance will be measured with an isokinetic dynamometer (Biodex 4 Medical Systems, Inc. USA). Peak isokinetic torque of the ankle and hip of the more symptomatic leg will be measured. Subjects will perform one trial of 15 repetitions at 120 degrees/second to assess endurance.
Time frame: Before and after 12 weeks supervised exercise program.