Transcriptomic Profiling Identifies Ferroptosis-Related Gene Signatures in Ischemic Muscle Satellite Cells Affected by Peripheral Artery Disease-Brief Report.

BACKGROUND: We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. METHODS: Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. RESULTS: Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. CONCLUSIONS: This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.

A biomechanical perspective on walking in patients with peripheral artery disease.

The most common symptom of peripheral artery disease (PAD) is intermittent claudication, which consists of debilitating leg pain during walking. In clinical settings, the presence of PAD is often noninvasively evaluated using the ankle-brachial index and imaging of the arterial supply. Furthermore, various questionnaires and functional tests are commonly used to measure the severity and negative effect of PAD on quality of life. However, these evaluations only provide information on vascular insufficiency and severity of the disease, but not regarding the complex mechanisms underlying walking impairments in patients with PAD. Biomechanical analyses using motion capture and ground reaction force measurements can provide insight into the underlying mechanisms to walking impairments in PAD. This review analyzes the application of biomechanics tools to identify gait impairments and their clinical implications on rehabilitation of patients with PAD. A total of 18 published journal articles focused on gait biomechanics in patients with PAD were studied. This narriative review shows that the gait of patients with PAD is impaired from the first steps that a patient takes and deteriorates further after the onset of claudication leg pain. These results point toward impaired muscle function across the ankle, knee, and hip joints during walking. Gait analysis helps understand the mechanisms operating in PAD and could also facilitate earlier diagnosis, better treatment, and slower progression of PAD.

Impaired microcirculatory function, mitochondrial respiration, and oxygen utilization in skeletal muscle of claudicating patients with peripheral artery disease.

Peripheral artery disease (PAD) is an atherosclerotic disease that impairs blood flow and muscle function in the lower limbs. A skeletal muscle myopathy characterized by mitochondrial dysfunction and oxidative damage is present in PAD; however, the underlying mechanisms are not well established. We investigated the impact of chronic ischemia on skeletal muscle microcirculatory function and its association with leg skeletal muscle mitochondrial function and oxygen delivery and utilization capacity in PAD. Gastrocnemius samples and arterioles were harvested from patients with PAD (n = 10) and age-matched controls (Con, n = 11). Endothelium-dependent and independent vasodilation was assessed in response to flow (30 µL·min-1), acetylcholine, and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiration was quantified by high-resolution respirometry, microvascular oxygen delivery, and utilization capacity (tissue oxygenation index, TOI) were assessed by near-infrared spectroscopy. Vasodilation was attenuated in PAD (P < 0.05) in response to acetylcholine (Con: 71.1 ± 11.1%, PAD: 45.7 ± 18.1%) and flow (Con: 46.6 ± 20.1%, PAD: 29.3 ± 10.5%) but not SNP (P = 0.30). Complex I + II state 3 respiration (P < 0.01) and TOI recovery rate were impaired in PAD (P < 0.05). Both flow and acetylcholine-mediated vasodilation were positively associated with complex I + II state 3 respiration (r = 0.5 and r = 0.5, respectively, P < 0.05). Flow-mediated vasodilation and complex I + II state 3 respiration were positively associated with TOI recovery rate (r = 0.8 and r = 0.7, respectively, P < 0.05). These findings suggest that chronic ischemia attenuates skeletal muscle arteriole endothelial function, which may be a key mediator for mitochondrial and microcirculatory dysfunction in the PAD leg skeletal muscle. Targeting microvascular dysfunction may be an effective strategy to prevent and/or reverse disease progression in PAD.NEW & NOTEWORTHY Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity is compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration and oxygen delivery and utilization capacity in claudicating patients with PAD.

Machine Learning-Based Peripheral Artery Disease Identification Using Laboratory-Based Gait Data.

Peripheral artery disease (PAD) manifests from atherosclerosis, which limits blood flow to the legs and causes changes in muscle structure and function, and in gait performance. PAD is underdiagnosed, which delays treatment and worsens clinical outcomes. To overcome this challenge, the purpose of this study is to develop machine learning (ML) models that distinguish individuals with and without PAD. This is the first step to using ML to identify those with PAD risk early. We built ML models based on previously acquired overground walking biomechanics data from patients with PAD and healthy controls. Gait signatures were characterized using ankle, knee, and hip joint angles, torques, and powers, as well as ground reaction forces (GRF). ML was able to classify those with and without PAD using Neural Networks or Random Forest algorithms with 89% accuracy (0.64 Matthew's Correlation Coefficient) using all laboratory-based gait variables. Moreover, models using only GRF variables provided up to 87% accuracy (0.64 Matthew's Correlation Coefficient). These results indicate that ML models can classify those with and without PAD using gait signatures with acceptable performance. Results also show that an ML gait signature model that uses GRF features delivers the most informative data for PAD classification.

Patient Compliance With Wearing Lower Limb Assistive Devices: A Scoping Review.

OBJECTIVE: The aim of this scoping review was to identify information on compliance with wearing orthoses and other supportive devices, to discuss the barriers to adherence, and to suggest strategies for improvement based on these findings. METHODS: Online databases of PubMed, Web of Science, and the Cochrane Library were searched for articles about patients' compliance with regard to lower limb assistive devices. In addition, a methodological quality control process was conducted. Studies were included if in the English language and related to compliance and adherence to the lower limb assistive device. Exclusion was based on first reading the abstract and then the full manuscript confirming content was not related to orthotic devices and compliance. RESULTS: Twelve studies were included. The data revealed between 6% and 80% of patients were not using a prescribed device. Barriers to the use of the orthotic device included medical, functional, device properties and lack of proper fit. Strategies for improved compliance included better communication between patient and clinician, patient education, and improved comfort and device esthetics. CONCLUSIONS: Individualized orthotic adjustments, rehabilitation, and patient education were promising for increasing adherence. Despite positive aspects of improvements in gait, balance in elderly, and a sense of security produced by using assistive devices, compliance remains less than ideal due to barriers. As compliance in recent studies has not improved, continued work in this area is essential to realize the benefits of technological advances in orthotic and assistive devices.

Claudicating patients with peripheral artery disease have meaningful improvement in walking speed after supervised exercise therapy.

OBJECTIVE: Supervised exercise therapy (SET) is a first-line treatment for patients with peripheral artery disease (PAD). The efficacy of SET is most commonly expressed by significant statistical improvement of parameters that do not clarify how each individual patient will benefit from SET. This study examined the minimal clinically important difference (MCID) in walking speed in claudicating patients with PAD after SET. METHODS: A total of 63 patients with PAD-related claudication (Fontaine stage II PAD) participated in a 6-month SET program. Self-selected walking speed was measured before and after SET. Distribution and anchor-based approaches were used to estimate the MCID for small and substantial improvement. The ability to walk one block and the ability to climb one flight of stairs questions were chosen as anchor questions from the Medical Outcomes Study 36-item Short Form questionnaire. Receiver operating characteristics curve analyses were performed to detect the threshold for MCID in walking speed after treatment. RESULTS: The distribution-based method estimated 0.03 m/s as a small improvement and 0.08 m/s as a substantial improvement after SET. Small and substantial improvements according to the anchor question walking one block were 0.05 m/s and 0.15 m/s, respectively. For the climbing one flight of stairs anchor question, 0.10 m/s was a small improvement. Receiver operating characteristics curve analyses identified an increase of 0.04 m/s and 0.03 m/s for improvement based on walking one block and climbing one flight of stairs, respectively. CONCLUSIONS: We report our findings for the MCID for walking speed among claudicating patients receiving SET. Claudicating patients who increase walking speed of 0.03 m/s or greater are more likely to experience a meaningful improvement in walking impairment than those who do not. The MCID reported in this study can serve as a benchmark for clinicians to develop goals and interpret clinically meaningful progress in the care of claudicating patients with PAD.

Acute mitochondrial antioxidant intake improves endothelial function, antioxidant enzyme activity, and exercise tolerance in patients with peripheral artery disease.

Peripheral artery disease (PAD) is a manifestation of atherosclerosis in the leg arteries, which causes claudication. This may be in part due to vascular mitochondrial dysfunction and excessive reactive oxygen species (ROS) production. A mitochondrial-targeted antioxidant (MitoQ) has been shown to improve vascular mitochondrial function that, in turn, led to improved vascular function in older adults and animal models. However, the roles of vascular mitochondria in vascular function including endothelial function and arterial stiffness in patients with PAD are unknown; therefore, with the use of acute MitoQ intake, this study examined the roles of vascular mitochondria in endothelial function, arterial stiffness, exercise tolerance, and skeletal muscle function in patients with PAD. Eleven patients with PAD received either MitoQ or placebo in a randomized crossover design. At each visit, blood samples, brachial and popliteal artery flow-mediated dilation (FMD), peripheral and central pulse-wave velocity (PWV), blood pressure (BP), maximal walking capacity, time to claudication (COT), and oxygen utility capacity were measured pre- and-post-MitoQ and placebo. There were significant group by time interactions (P < 0.05) for brachial and popliteal FMD that both increased by Δ2.6 and Δ3.3%, respectively, and increases superoxide dismutase (Δ0.03 U/mL), maximal walking time (Δ73.8 s), maximal walking distance (Δ49.3 m), and COT (Δ44.2 s). There were no changes in resting heart rate, BP, malondialdehyde, total antioxidant capacity, PWV, or oxygen utility capacity (P > 0.05). MitoQ intake may be an effective strategy for targeting the vascular mitochondrial environment, which may be useful for restoring endothelial function, leg pain, and walking time in patients with PAD.NEW & NOTEWORTHY The results of this study reveal for the first time that acute oral intake of mitochondrial-targeted antioxidant (MitoQ, 80 mg) is effective for improving vascular endothelial function and superoxide dismutase in patients with peripheral artery disease (PAD). Acute MitoQ intake is also effective for improving maximal walking capacity and delaying the onset of claudication in patients with PAD. These findings suggest that the acute oral intake of MitoQ-mediated improvements in vascular mitochondria play a pivotal role for improving endothelial function, the redox environment, and skeletal muscle performance in PAD.

Supervised walking exercise therapy improves gait biomechanics in patients with peripheral artery disease.

OBJECTIVE: In patients with peripheral artery disease (PAD), supervised exercise therapy is a first line of treatment because it increases maximum walking distances comparable with surgical revascularization therapy. Little is known regarding gait biomechanics after supervised exercise therapy. This study characterized the effects of supervised exercise therapy on gait biomechanics and walking distances in claudicating patients with PAD. METHODS: Forty-seven claudicating patients with PAD underwent gait analysis before and immediately after 6 months of supervised exercise therapy. Exercise sessions consisted of a 5-minute warmup of mild walking and stretching of upper and lower leg muscles, 50 minutes of intermittent treadmill walking, and 5 minutes of cooldown (similar to warmup) three times per week. Measurements included self-perceived ambulatory limitations measured by questionnaire, the ankle-brachial index (ABI), walking distance measures, maximal plantar flexor strength measured by isometric dynamometry, and overground gait biomechanics trials performed before and after the onset of claudication pain. Paired t-tests were used to test for differences in quality of life, walking distances, ABI, and maximal strength. A two-factor repeated measures analysis of variance determined differences for intervention and condition for gait biomechanics dependent variables. RESULTS: After supervised exercise therapy, quality of life, walking distances, and maximal plantar flexor strength improved, although the ABI did not significantly change. Several gait biomechanics parameters improved after the intervention, including torque and power generation at the ankle and hip. Similar to previous studies, the onset of claudication pain led to a worsening gait or a gait that was less like healthy individuals with a pain-free gait. CONCLUSIONS: Six months of supervised exercise therapy produced increases in walking distances and quality of life that are consistent with concurrent improvements in muscle strength and gait biomechanics. These improvements occurred even though the ABI did not improve. Future work should examine the benefits of supervised exercise therapy used in combination with other available treatments for PAD.

A low-cost, wireless near-infrared spectroscopy device detects the presence of lower extremity atherosclerosis as measured by computed tomographic angiography and characterizes walking impairment in peripheral artery disease.

BACKGROUND: Patients with peripheral artery disease (PAD) who experience intermittent claudication report a range of symptoms. Patients with symptoms other than classically described intermittent claudication may be at the highest risk for functional decline and mobility loss. Therefore, technologies allowing for characterization of PAD severity are desirable. Near-infrared spectroscopy (NIRS) allows for measurements of muscle heme oxygen saturation (StO2) during exercise. We hypothesized lower extremities affected by PAD would exhibit distinct NIRS profiles as measured by a low-cost, wireless NIRS device and that NIRS during exercise predicts walking limitation. METHODS: We recruited 40 patients with PAD and 10 control participants. All patients with PAD completed a computed tomographic angiography, 6-minute walk test, and a standardized treadmill test. Controls completed a 540-second treadmill test for comparison. StO2 measurements were continuously taken from the gastrocnemius during exercise. Variables were analyzed by Fischer's exact, χ2, Wilcoxon rank-sum, and Kruskal-Wallis tests as appropriate. Correlations were assessed by partial Spearman correlation coefficients adjusted for occlusive disease pattern. RESULTS: Patients with PAD experienced claudication onset at a median of 108 seconds with a median peak walking time of 288 seconds. The baseline StO2 was similar between PAD and control. The StO2 of PAD and control participants dropped below baseline at a median of 1 and 104 seconds of exercise, respectively (P < .0001). Patients with PAD reached minimum StO2 earlier than control participants (119 seconds vs 522 seconds, respectively; P < .001) and experienced a greater change in StO2 at 1 minute of exercise (-73.2% vs 8.3%; P < .0001) and a greater decrease at minimum exercise StO2 (-83.4% vs -16.1%; P < .0001). For patients with PAD, peak walking time, and 6-minute walking distance correlated with percent change in StO2 at 1 minute of exercise (r = -0.76 and -0.67, respectively; P < .001) and time to minimum StO2 (r = 0.79 and 0.70, respectively; P < .0001). CONCLUSIONS: In this initial evaluation of a novel, low-cost NIRS device, lower extremities affected by PAD exhibited characteristic changes in calf muscle StO2, which differentiated them from healthy controls and were strongly correlated with walking impairment. These findings confirm and expand on previous work demonstrating the potential clinical value of NIRS devices and the need for further research investigating the ability of low-cost NIRS technology to evaluate, diagnose, and monitor treatment response in PAD.

Collateral Development and Arteriogenesis in Hindlimbs of Swine After Ligation of Arterial Inflow.

BACKGROUND: Development of collateral vasculature is key in compensating for arterial occlusions in patients with peripheral artery disease (PAD). We aimed to examine the development of collateral pathways after ligation of native vessels in a porcine model of PAD. METHODS: Right hindlimb ischemia was induced in domestic swine (n = 11) using two versions of arterial ligation. Version 1 (n = 6) consisted of ligation with division of the right external iliac, profunda femoral, and superficial femoral arteries. Version 2 (n = 5) consisted of the ligation of version 1 with additional ligation with division of the right internal iliac artery. Development of collateral pathways was evaluated with standard angiography before arterial ligation and at termination (30 days later). Relative luminal diameter of the arteries supplying the ischemic right hind limb were determined by two-dimensional angiography. RESULTS: The dominant collateral pathway that developed after version 1 ligation connected the right internal iliac artery to the right profunda femoral and then to the right superficial femoral and popliteal artery. Mean luminal diameter of the right internal iliac artery at termination increased by 38% compared with baseline. Two codominant collateral pathways developed in version 2 ligation: (i) from the left profunda femoral artery to the reconstituted right profunda femoral artery and (ii) from the common internal iliac trunk and the left internal iliac artery to the reconstituted right internal iliac artery, which then supplied the right profunda femoral and then the right superficial femoral and popliteal artery. The mean diameter of the left profunda and the left internal iliac artery increased at termination by 26% and 21%, respectively (P < 0.05). CONCLUSIONS: Two versions of hindlimb ischemia induction (right ilio-femoral artery ligation with and without right internal iliac artery ligation) in swine produced differing collateral pathways, along with changes to the diameter of the inflow vessels (i.e., arteriogenesis).

IL-1ß (Interleukin-1ß) and TNF-α (Tumor Necrosis Factor-α) Impact Abdominal Aortic Aneurysm Formation by Differential Effects on Macrophage Polarization.

OBJECTIVE: Abdominal aortic aneurysms are inflammatory in nature and are associated with some risk factors that also lead to atherosclerotic occlusive disease, most notably smoking. The purpose of our study was to identify differential cytokine expression in patients with abdominal aortic aneurysm and those with atherosclerotic occlusive disease. Based on this analysis, we further explored and compared the mechanism of action of IL (interleukin)-1ß versus TNF-α (tumor necrosis factor-α) in abdominal aortic aneurysm formation. APPROACH AND RESULTS: IL-1ß was differentially expressed in human plasma with lower levels detected in patients with abdominal aortic aneurysm compared with matched atherosclerotic controls. We further explored its mechanism of action using a murine model and cell culture. Genetic deletion of IL-1ß and IL-1R did not inhibit aneurysm formation or decrease MMP (matrix metalloproteinase) expression. The effects of IL-1ß deletion on M1 macrophage polarization were compared with another proinflammatory cytokine, TNF-α. Bone marrow-derived macrophages from IL-1ß-/- and TNF-α-/- mice were polarized to an M1 phenotype. TNF-α deletion, but not IL-1ß deletion, inhibited M1 macrophage polarization. Infusion of M1 polarized TNF-α-/- macrophages inhibited aortic diameter growth; no inhibitory effect was seen in mice infused with M1 polarized IL-1ß-/- macrophages. CONCLUSIONS: Although IL-1ß is a proinflammatory cytokine, its effects on aneurysm formation and macrophage polarization differ from TNF-α. The differential effects of IL-1ß and TNF-α inhibition are related to M1/M2 macrophage polarization and this may account for the differences in clinical efficacy of IL-1ß and TNF-α antibody therapies in management of inflammatory diseases.

Quantification of Daily Physical Activity and Sedentary Behavior of Claudicating Patients.

BACKGROUND: Claudication is the most common manifestation of peripheral artery disease (PAD), producing significant ambulatory compromise. Limited information exists on the routine physical activity of claudicating patients. Our objective was to record the intensity/time profiles of physical activity and the timing and duration of sedentary behavior of a sample of community-dwelling claudicating patients. METHODS: Forty-four claudicating patients referred to our vascular clinic were recruited. Physical activity was recorded using the ActiGraph GT1M activity monitor. The Actigraph monitor is a lightweight instrument designed to measure human movement through changes in acceleration, measured as counts over 1-minute time periods. Data from 7 consecutive days were used for the calculations. We processed the data using the ActiLife software program. RESULTS: The average daily activity of the claudicating patients shows a steady increase beginning approximately 05:30 AM until a peak plateau from approximately 10:00 AM to 01:30 PM followed by a steady decrease until approximately 09:30 PM, when a sustained period of inactivity begins. The average claudicating patient takes 3586 steps per day at an average intensity of 1.77 metabolic equivalents of task (METs, a physiological measure expressing the energy cost of physical activities). Average physical activity intensity and peak intensity fluctuate very little during the day, and they rarely exceed the level of light activity (light = <3 METs maximum effort, such as casual walking or light housework). During awake time, approximately 7 hours are spent in sedentary behaviors (<1.5 METs), and sedentary time is spread throughout the day mostly in short intervals between periods of low-energy activity. CONCLUSIONS: Our study objectively demonstrates the reduced physical activity of claudicating patients and documents physical activity/duration profiles throughout the day. The intensity of the physical activity of the average claudicating patient fluctuates very little during the day and rarely exceeds a light intensity level. Claudicating patients spend approximately half of their awake time in sedentary behavior and when they walk they do it in short bursts followed by several minutes of rest. We anticipate that changes in routine physical activity/duration profiles of patients with PAD will provide relevant, sensitive, and direct measures of the effectiveness of therapeutic interventions.

Gait Mechanics Differences Between Healthy Controls and Patients With Peripheral Artery Disease After Adjusting for Gait Velocity Stride Length and Step Width.

Patients with peripheral artery disease (PAD) experience significant leg dysfunction. The effects of PAD on gait include shortened steps, slower walking velocity, and altered gait kinematics and kinetics, which may confound joint torques and power measurements. Spatiotemporal parameters, joint torques and powers were calculated and compared between 20 patients with PAD and 20 healthy controls using independent t-tests. Separate ANCOVA models were used to evaluate group differences after independently adjusting for gait velocity, stride length and step width. Compared to healthy controls, patients with PAD exhibited reduced peak extensor and flexor torques at the knee, and hip. After adjusting for all covariates combined, differences between groups remained for ankle power generation in late stance, and knee flexor torque. Reduced walking velocity observed in subjects affected by PAD was closely connected with reductions in joint torques and powers during gait. Gait differences remained, at the knee and ankle, after adjusting for the combined effect of spatiotemporal parameters. Improving muscle function through exercise or with the use of assistive devices needs to be a key tool in the development of interventions that aim to enhance the ability of PAD patients to restore spatiotemporal gait parameters.

Muscle strength and control characteristics are altered by peripheral artery disease.

OBJECTIVE: Peripheral artery disease (PAD), a common manifestation of atherosclerosis, is characterized by lower leg ischemia and myopathy in association with leg dysfunction. Patients with PAD have impaired gait from the first step they take with consistent defects in the movement around the ankle joint, especially in plantar flexion. Our goal was to develop muscle strength profiles to better understand the problems in motor control responsible for the walking impairment in patients with PAD. METHODS: Ninety-four claudicating PAD patients performed maximal isometric plantar flexion contractions lasting 10 seconds in two conditions: pain free (patient is well rested and has no claudication symptoms) and pain induced (patient has walked and has claudication symptoms). Sixteen matched healthy controls performed the pain-free condition only. Torque curves were analyzed for dependent variables of muscle strength and motor control. Independent t-tests were used to compare variables between groups, and dependent t-tests determined differences between conditions. RESULTS: Patients with PAD had significantly reduced peak torque and area under the curve compared with controls. Measures of control differed between PAD conditions only. Load rate and linear region duration were greater in the pain condition. Time to peak torque was shorter in the pain condition. CONCLUSIONS: This study conclusively demonstrates that the plantar flexor muscles of the PAD patient at baseline and without pain are weaker in patients with PAD compared with controls. With the onset of claudication pain, patients with PAD exhibit altered muscle control strategies and further strength deficits are manifest compared to baseline levels. The myopathy of PAD legs appears to have a central role in the functional deterioration of the calf muscles, as it is evident both before and after onset of ischemic pain.

An endovascular model of ischemic myopathy from peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is a significant age-related medical condition with limited pharmacologic options. Severe PAD, termed critical limb ischemia, can lead to amputation. Skeletal muscle is the end organ most affected by PAD, leading to ischemic myopathy and debility of the patient. Currently, there are not any therapeutics to treat ischemic myopathy, and proposed biologic agents have not been optimized owing to a lack of preclinical models of PAD. Because a large animal model of ischemic myopathy may be useful in defining the optimal dosing and delivery regimens, the objective was to create and to characterize a swine model of ischemic myopathy that mimics patients with severe PAD. METHODS: Yorkshire swine (N = 8) underwent acute right hindlimb ischemia by endovascular occlusion of the external iliac artery. The effect of ischemia on limb function, perfusion, and degree of ischemic myopathy was quantified by weekly gait analysis, arteriography, hindlimb blood pressures, femoral artery duplex ultrasound scans, and histologic examination. Animals were terminated at 5 (n = 5) and 6 (n = 3) weeks postoperatively. Ossabaw swine (N = 8) fed a high-fat diet were used as a model of metabolic syndrome for comparison of arteriogenic recovery and validation of ischemic myopathy. RESULTS: There was persistent ischemia in the right hindlimb, and occlusion pressures were significantly depressed compared with the untreated left hindlimb out to 6 weeks (systolic blood pressure, 31 ± 21 vs 83 ± 15 mm Hg, respectively; P = .0007). The blood pressure reduction resulted in a significant increase of ischemic myopathy in the gastrocnemius muscle in the treated limb. Gait analysis revealed a functional deficit of the right hindlimb immediately after occlusion that improved rapidly during the first 2 weeks. Peak systolic velocity values in the right common femoral artery were severely diminished throughout the entire study (P < .001), and the hemodynamic environment after occlusion was characterized by low and oscillatory wall shear stress. Finally, the internal iliac artery on the side of the ischemic limb underwent significant arteriogenic remodeling (1.8× baseline) in the Yorkshire but not in the Ossabaw swine model. CONCLUSIONS: This model uses endovascular technology to produce the first durable large animal model of ischemic myopathy. Acutely (first 2 weeks), this model is associated with impaired gait but no tissue loss. Chronically (2-6 weeks), this model delivers persistent ischemia, resulting in ischemic myopathy similar to that seen in PAD patients. This model may be of use for testing novel therapeutics including biologic therapies for promoting neovascularization and arteriogenesis.

Analysis of ischemic muscle in patients with peripheral artery disease using X-ray spectroscopy.

BACKGROUND: Peripheral artery disease (PAD) is a vascular disease caused by atherosclerosis, resulting in decreased blood flow to the lower extremities. The ankle-brachial index (ABI) is a standard PAD diagnostic test but only identifies reduced blood flow based on blood pressure differences. The early signs of PAD manifest themselves not only at a clinical level but also at an elemental and biochemical level. However, the biochemical and elemental alterations to PAD muscle are not well understood. The objective of this study was to compare fundamental changes in intracellular elemental compositions between control, claudicating, and critical limb ischemia muscle tissue. MATERIALS AND METHODS: Gastrocnemius biopsies from three subjects including one control (ABI ≥ 0.9), one claudicating (0.4 ≤ ABI < 0.9), and one critical limb ischemia patient (ABI < 0.4) were evaluated using a scanning electron microscope and energy dispersive X-ray spectroscopy to quantify differences in elemental compositions. Spectra were collected for five myofibers per specimen. An analysis of variance was performed to identify significant differences in muscle elemental compositions. RESULTS: This study revealed that intracellular magnesium and calcium were lower in PAD compared with control myofibers, whereas sulfur was higher. Magnesium and calcium are antagonistic, meaning, if magnesium concentrations go down calcium concentrations should go up. However, our findings do not support this antagonism in PAD. Our analysis found decreases in sodium and potassium, in PAD myofibers. CONCLUSIONS: These findings may provide insight into the pathologic mechanisms that may operate in ischemic muscle and aid in the development of specialized preventive and rehabilitative treatment plans for PAD patients.

Contained Rupture of an Abdominal Aortic Aneurysm With Extensive Vertebral Body and Retroperitoneal Space Destruction.

Chronic contained rupture of an abdominal aortic aneurysm with vertebral body erosion most commonly presents with symptoms of low back pain. Although not well known, vertebral body erosion or destruction may be seen in up to 25% of patients with sealed or contained rupture of an abdominal aortic aneurysm. This appearance on cross-sectional imaging may mimic a malignant or infectious process. Although these cases can present a diagnostic challenge, published cases of chronic contained rupture of an abdominal aortic aneurysm with vertebral body erosion demonstrate clinical and imaging similarities that, when recognized, can assist in diagnosis.

Transforming growth factor-beta 1 produced by vascular smooth muscle cells predicts fibrosis in the gastrocnemius of patients with peripheral artery disease.

BACKGROUND: Lower leg ischemia, myopathy, and limb dysfunction are distinguishing features of peripheral artery disease (PAD). The myopathy of PAD is characterized by myofiber degeneration in association with extracellular matrix expansion, and increased expression of transforming growth factor-beta 1 (TGF-ß1; a pro-fibrotic cytokine). In this study, we evaluated cellular expression of TGF-ß1 in gastrocnemius of control (CTRL) and PAD patients and its relationship to deposited collagen, fibroblast accumulation and limb hemodynamics. METHODS: Gastrocnemius biopsies were collected from PAD patients with claudication (PAD-II; N = 25) and tissue loss (PAD-IV; N = 20) and from CTRL patients (N = 20). TGF-ß1 in slide-mounted specimens was labeled with fluorescent antibodies and analyzed by quantitative wide-field, fluorescence microscopy. We evaluated co-localization of TGF-ß1 with vascular smooth muscle cells (SMC) (high molecular weight caldesmon), fibroblasts (TE-7 antigen), macrophages (CD163), T cells (CD3) and endothelial cells (CD31). Collagen was stained with Masson Trichrome and collagen density was determined by quantitative bright-field microscopy with multi-spectral imaging. RESULTS: Collagen density increased from CTRL to PAD-II to PAD-IV specimens (all differences p < 0.05) and was prominent around microvessels. TGF-ß1 expression increased with advancing disease (all differences p < 0.05), correlated with collagen density across all specimens (r = 0.864; p < 0.001), associated with fibroblast accumulation, and was observed exclusively in SMC. TGF-ß1 expression inversely correlated with ankle-brachial index across PAD patients (r = -0.698; p < 0.001). CONCLUSIONS: Our findings support a progressive fibrosis in the gastrocnemius of PAD patients that is caused by elevated TGF-ß1 production in the SMC of microvessels in response to tissue hypoxia.

Therapeutic potential of sustained-release sodium nitrite for critical limb ischemia in the setting of metabolic syndrome.

Nitrite is a storage reservoir of nitric oxide that is readily reduced to nitric oxide under pathological conditions. Previous studies have demonstrated that nitrite levels are significantly reduced in cardiovascular disease states, including peripheral vascular disease. We investigated the cytoprotective and proangiogenic actions of a novel, sustained-release formulation of nitrite (SR-nitrite) in a clinically relevant in vivo swine model of critical limb ischemia (CLI) involving central obesity and metabolic syndrome. CLI was induced in obese Ossabaw swine (n = 18) by unilateral external iliac artery deployment of a full cross-sectional vessel occlusion device positioned within an endovascular expanded polytetrafluoroethylene-lined nitinol stent-graft. At post-CLI day 14, pigs were randomized to placebo (n = 9) or SR-nitrite (80 mg, n = 9) twice daily by mouth for 21 days. SR-nitrite therapy increased nitrite, nitrate, and S-nitrosothiol in plasma and ischemic skeletal muscle. Oxidative stress was reduced in ischemic limb tissue of SR-nitrite- compared with placebo-treated pigs. Ischemic limb tissue levels of proangiogenic growth factors were increased following SR-nitrite therapy compared with placebo. Despite the increases in cytoprotective and angiogenic signals with SR-nitrite therapy, new arterial vessel formation and enhancement of blood flow to the ischemic limb were not different from placebo. Our data clearly demonstrate cytoprotective and proangiogenic signaling in ischemic tissues following SR-nitrite therapy in a very severe model of CLI. Further studies evaluating longer-duration nitrite therapy and/or additional nitrite dosing strategies are warranted to more fully evaluate the therapeutic potential of nitrite therapy in peripheral vascular disease.

Age and disease-related geometric and structural remodeling of the carotid artery.

BACKGROUND: Carotid artery geometry has been suggested as a risk factor for atherosclerotic carotid artery disease (ACD). Although normal aging and development of disease can both lead to geometric changes in the artery, whether geometric changes in a given artery actually predispose to disease or are just a consequence of remodeling during aging is unclear. We investigated carotid artery geometric changes with aging to identify geometric features associated with the presence of ACD. METHODS: Carotid artery geometry was quantified by measuring carotid artery diameter, tortuosity, and bifurcation angle using three-dimensional reconstructions of thin-section computed tomography angiography scans in 15 healthy individuals (average age, 43 ± 18 years; range, 15-64 years). The same geometric features were measured in 17 patients (68 ± 10 years old) with unilateral ACD. Geometric features associated with presence of ACD were determined by using the nondiseased contralateral carotid artery as an intrinsic control. Elastin-stained carotid arteries were analyzed to assess age-related structural changes in 12 deceased individuals. RESULTS: Increases were noted in bulb diameter (0.64 mm), bifurcation angle (10°), and tortuosity of the common carotid (CCA; 0.03) and internal carotid arteries (ICA; 0.04) for every decade of life. Density and continuity of circumferential and longitudinal elastin in the CCA and ICA decreased with age. Compared with normal carotid arteries, those with ACD demonstrated larger bulb diameters (P = .001) but smaller bifurcation angles (P = .001). CCA tortuosity (P = .038) increased in ACD arteries compared with normal carotid arteries, but ICA tortuosity was decreased (P = .026). CONCLUSIONS: With increasing age, bulb diameter, tortuosity, and bifurcation angle increases in carotid arteries. These geometric changes may be related to degradation and fragmentation of intramural elastin. Arteries with atherosclerotic occlusive disease demonstrate decreased ICA tortuosity and smaller bifurcation angles compared with nondiseased carotid arteries.