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.

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).

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.

Elastin-Derived Peptides Promote Abdominal Aortic Aneurysm Formation by Modulating M1/M2 Macrophage Polarization.

Abdominal aortic aneurysm is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix degradation. Damage to elastin in the extracellular matrix results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Proinflammatory M1 macrophages initially are recruited to sites of injury, but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. Abdominal aortic aneurysm tissue reveals a high M1/M2 ratio in which proinflammatory cells and their associated markers dominate. In the current study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57BL/6 mice, Ab-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and proinflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2-polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a proinflammatory environment in aortic tissue by inducing M1 polarization, and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation.

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.

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.

Abnormal myofiber morphology and limb dysfunction in claudication.

BACKGROUND: Peripheral artery disease (PAD), which affects an estimated 27 million people in Europe and North America, is caused by atherosclerotic plaques that limit blood flow to the legs. Chronic, repeated ischemia in the lower leg muscles of PAD patients is associated with loss of normal myofiber morphology and myofiber degradation. In this study, we tested the hypothesis that myofiber morphometrics of PAD calf muscle are significantly different from normal calf muscle and correlate with reduced calf muscle strength and walking performance. METHODS: Gastrocnemius biopsies were collected from 154 PAD patients (Fontaine stage II) and 85 control subjects. Morphometric parameters of gastrocnemius fibers were determined and evaluated for associations with walking distances and calf muscle strength. RESULTS: Compared with control myofibers, PAD myofiber cross-sectional area, major and minor axes, equivalent diameter, perimeter, solidity, and density were significantly decreased (P < 0.005), whereas roundness was significantly increased (P < 0.005). Myofiber morphometric parameters correlated with walking distances and calf muscle strength. Multiple regression analyses demonstrated myofiber cross-sectional area, roundness, and solidity as the best predictors of calf muscle strength and 6-min walking distance, whereas cross-sectional area was the main predictor of maximum walking distance. CONCLUSIONS: Myofiber morphometrics of PAD gastrocnemius differ significantly from those of control muscle and predict calf muscle strength and walking distances of the PAD patients. Morphometric parameters of gastrocnemius myofibers may serve as objective criteria for diagnosis, staging, and treatment of PAD.

Lower limb revascularization leads to faster walking but with less efficient mechanics in claudicating patients.

Peripheral artery disease (PAD) is characterized by reduced blood flow to the extremities due to atherosclerosis. Studies report impaired gait mechanics in patients with lower extremity PAD. We hypothesized that revascularization surgery would improve gait mechanics when quantified by net lower limb joint work across the stance phase of walking. We performed gait analyses in 35 patients with PAD and 35 healthy, older adults. Patients with PAD performed a walking protocol prior to and six months following revascularization surgery. Healthy adults only took part in a single walking session. Lower limb joint powers were calculated using inverse dynamics and were integrated across early, middle, and late stance phases to determine the work performed during each phase (J kg-1). The work mechanical ratio between positive-producing and negative-producing phases of stance was calculated for each lower-limb joint. Self-selected walking speed significantly increased from 1.13 ± 0.2 ms-1 to 1.26 ± 0.18 ms-1 in patients following revascularization (p < 0.001). We observed a significant decrease in positive late stance work (p < 0.001) in conjunction with more negative work during early stance (p < 0.001) in patients following revascularization. Revascularization surgery led to faster walking without an increase in the ankle joint's mechanical ratio. Our results suggest faster walking was achieved via work done at the hip rather than the ankle. These findings suggest that additional therapies that facilitate the restoration of muscle, tissue, and nervous system damage caused by years of having reduced blood flow to the limbs might still be beneficial following revascularization.

Oxidative damage in the gastrocnemius predicts long-term survival in patients with peripheral artery disease.

Patients with peripheral artery disease (PAD) have increased mortality rates and a myopathy in their affected legs which is characterized by increased oxidative damage, reduced antioxidant enzymatic activity and defective mitochondrial bioenergetics. This study evaluated the hypothesis that increased levels of oxidative damage in gastrocnemius biopsies from patients with PAD predict long-term mortality rates. Oxidative damage was quantified as carbonyl adducts in myofibers of the gastrocnemius of PAD patients. The oxidative stress data were grouped into tertiles and the 5-year, all-cause mortality for each tertile was determined by Kaplan-Meier curves and compared by the Modified Peto test. A Cox-regression model was used to control the effects of clinical characteristics. Results were adjusted for age, sex, race, body mass index, ankle-brachial index, smoking, physical activity, and comorbidities. Of the 240 study participants, 99 died during a mean follow up of 37.8 months. Patients in the highest tertile of oxidative damage demonstrated the highest 5-year mortality rate. The mortality hazard ratios (HR) from the Cox analysis were statistically significant for oxidative damage (lowest vs middle tertile; HR = 6.33; p = 0.0001 and lowest vs highest; HR = 8.37; p < 0.0001). Survival analysis of a contemporaneous population of PAD patients identifies abundance of carbonyl adducts in myofibers of their gastrocnemius as a predictor of mortality rate independently of ankle-brachial index, disease stage and other clinical and myopathy-related covariates.

Peripheral artery disease causes consistent gait irregularities regardless of the location of leg claudication pain.

BACKGROUND: The most common symptom of peripheral artery disease (PAD) is intermittent claudication that involves the calf, thigh, and/or buttock muscles. How the specific location of this leg pain is related to altered gait, however, is unknown. OBJECTIVES: We hypothesized that because the location of claudication symptoms uniquely affects different leg muscle groups in people with PAD, this would produce distinctive walking patterns. METHODS: A total of 105 participants with PAD and 35 age-matched older volunteers without PAD (CTRL) were recruited. Participants completed walking impairment questionnaires (WIQ), Gardner-Skinner progressive treadmill tests, the six-minute walk test, and we performed an advanced evaluation of the biomechanics of their overground walking. Participants with PAD were categorized into 4 groups according to their stated pain location(s): calf only (C, n = 43); thigh and calf (TC, n = 18); buttock and calf (BC, n = 15); or buttock, thigh, and calf (BTC, n = 29). Outcomes were compared between CTRL, C, TC, BC and BTC groups using a one-way ANOVA with post-hoc comparisons to identify and assess statistically significant differences. RESULTS: There were no significant differences between CTRL, C, TC, BC and BTC groups in distances walked or walking speed when either pain-free or experiencing claudication pain. Each participant with PAD had significantly dysfunctional biomechanical gait parameters, even when pain-free, when compared to CTRL (pain-free) walking data. During pain-free walking, out of the 18 gait parameters evaluated, we only identified significant differences in hip power generation during push-off (in C and TC groups) and in knee power absorption during weight acceptance (in TC and BC groups). There were no between-group differences in gait parameters while people with PAD were walking with claudication pain. CONCLUSIONS: Our data demonstrate that PAD affects the ischemic lower extremities in a diffuse manner irrespective of the location of claudication symptoms. DATABASE REGISTRATION: ClinicalTrials.gov NCT01970332.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle.

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre (+), FoxO1,3,4 (L/L) , referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

Oxidative damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease.

Peripheral arterial disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in arteries supplying the legs, affects an estimated 27 million people in Europe and North America. Increased production of reactive oxygen species by dysfunctional mitochondria in leg muscles of PAD patients is viewed as a key mechanism of initiation and progression of the disease. Previous studies demonstrated increased oxidative damage in homogenates of biopsy specimens from PAD gastrocnemius compared to controls, but did not address myofiber-specific damage. In this study, we investigated oxidative damage to myofibers as a possible cause of the myopathy of PAD. To achieve this, we developed and validated fluorescence microscopy procedures for quantitative analysis of carbonyl groups and 4-hydroxy-2-nonenal (HNE) adducts in myofibers of biopsy specimens from human gastrocnemius. PAD and control specimens were evaluated for differences in 1) myofiber content of these two forms of oxidative damage and 2) myofiber cross-sectional area. Furthermore, oxidative damage to PAD myofibers was tested for associations with clinical stage of disease, degree of ischemia in the affected leg, and myofiber cross-sectional area. Carbonyl groups and HNE adducts were increased 30% (p < 0.0001) and 40% (p < 0.0001), respectively, in the myofibers of PAD (N = 34) compared to control (N = 21) patients. Mean cross-sectional area of PAD myofibers was reduced 29.3% compared to controls (p < 0.0003). Both forms of oxidative damage increased with clinical stage of disease, blood flow limitation in the ischemic leg, and reduced myofiber cross-sectional area. The data establish oxidative damage to myofibers as a possible cause of PAD myopathy.

Morphometric analysis of gastrocnemius muscle biopsies from patients with peripheral arterial disease: objective grading of muscle degeneration.

Peripheral arterial disease (PAD), which affects ~10 million Americans, is characterized by atherosclerosis of the noncoronary arteries. PAD produces a progressive accumulation of ischemic injury to the legs, manifested as a gradual degradation of gastrocnemius histology. In this study, we evaluated the hypothesis that quantitative morphological parameters of gastrocnemius myofibers change in a consistent manner during the progression of PAD, provide an objective grading of muscle degeneration in the ischemic limb, and correlate to a clinical stage of PAD. Biopsies were collected with a Bergström needle from PAD patients with claudication (n = 18) and critical limb ischemia (CLI; n = 19) and control patients (n = 19). Myofiber sarcolemmas and myosin heavy chains were labeled for fluorescence detection and quantitative analysis of morphometric variables, including area, roundness, perimeter, equivalent diameter, major and minor axes, solidity, and fiber density. The muscle specimens were separated into training and validation data sets for development of a discriminant model for categorizing muscle samples on the basis of disease severity. The parameters for this model included standard deviation of roundness, standard deviation of solidity of myofibers, and fiber density. For the validation data set, the discriminant model accurately identified control (80.0% accuracy), claudicating (77.7% accuracy), and CLI (88.8% accuracy) patients, with an overall classification accuracy of 82.1%. Myofiber morphometry provided a discriminant model that establishes a correlation between PAD progression and advancing muscle degeneration. This model effectively separated PAD and control patients and provided a grading of muscle degeneration within clinical stages of PAD.

Gastrocnemius mitochondrial respiration: are there any differences between men and women?

INTRODUCTION: Work on human and mouse skeletal muscle by our group and others has demonstrated that aging and age-related degenerative diseases are associated with mitochondrial dysfunction, which may be more prevalent in males. There have been, however, no studies that specifically examine the influence of male or female sex on human skeletal muscle mitochondrial respiration. The purpose of this study was to compare mitochondrial respiration in the gastrocnemius of adult men and women. METHODS: Gastrocnemius muscle was obtained from male (n = 19) and female (n = 11) human subjects with healthy lower-extremity musculoskeletal and arterial systems and normal ambulatory function. All patients were undergoing operations for the treatment of varicose veins in their legs. Mitochondrial respiration was determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Complex I-, II-, III-, and IV-dependent respiration was measured individually and normalized to muscle weight, total protein content, and citrate synthase (CS, index of mitochondrial content). RESULTS: Male and female patients had no evidence of musculoskeletal or arterial disease and did not differ with regard to age, race, body mass index, or other clinical characteristics. Complex I-, II-, III-, and IV-dependent respiration normalized to muscle weight, total protein content, and CS did not statistically differ for males compared with females. CONCLUSIONS: Our study evaluates, for the first time, gastrocnemius mitochondrial respiration of adult men and women who have healthy musculoskeletal and arterial systems and normal ambulatory function. Our data demonstrate there are no differences in the respiration of gastrocnemius mitochondria between men and women.

Loss of neuronal integrity during progressive HIV-1 infection of humanized mice.

Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.

Muscle forces and power are significantly reduced during walking in patients with peripheral artery disease.

Patients with peripheral artery disease (PAD) have significantly reduced lower extremity muscle strength compared with healthy individuals as measured during isolated, single plane joint motion by isometric and isokinetic strength dynamometers. Alterations to the force contribution of muscles during walking caused by PAD are not well understood. Therefore, this study used simulations with PAD biomechanics data to understand lower extremity muscle functions in patients with PAD during walking and to compare that with healthy older individuals. A total of 12 patients with PAD and 10 age-matched healthy older controls walked across a 10-meter pathway with reflective markers on their lower limbs. Marker coordinates and ground reaction forces were recorded and exported to OpenSim software to perform gait simulations. Walking velocity, joint angles, muscle force, muscle power, and metabolic rate were calculated and compared between patients with PAD and healthy older controls. Our results suggest that patients with PAD walked slower with less hip extension during propulsion. Significant force and power reductions were observed in knee extensors during weight acceptance and in plantar flexors and hip flexors during propulsion in patients with PAD. The estimated metabolic rate of walking during stance was not different between patients with PAD and controls. This study is the first to analyze lower limb muscular responses during walking in patients with PAD using the OpenSim simulation software. The simulation results of this study identified important information about alterations to muscle force and power during walking in those with PAD.

Endothelial cell-derived pro-fibrotic factors increase TGF-ß1 expression by smooth muscle cells in response to cycles of hypoxia-hyperoxia.

BACKGROUND: The vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R. METHODS: Primary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs. FINDINGS: The mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-ß1, TGF-ß pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-ß1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-ß1 levels. INTERPRETATION: In an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-ß1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R. FUNDING: National Institute on Aging at the National Institutes of Health grant number R01AG064420. RESEARCH IN CONTEXT: Evidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-ß1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-ß1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-ß1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.