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.

Diet-induced obesity augments ischemic myopathy and functional decline in a murine model of peripheral artery disease.

Peripheral artery disease (PAD) causes an ischemic myopathy contributing to patient disability and mortality. Most preclinical models to date use young, healthy rodents with limited translatability to human disease. Although PAD incidence increases with age, and obesity is a common comorbidity, the pathophysiologic association between these risk factors and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of age, diet-induced obesity and chronic hindlimb ischemia (HLI) on (1) mobility, (2) muscle contractility, and markers of muscle (3) mitochondrial content and function, (4) oxidative stress and inflammation, (5) proteolysis, and (6) cytoskeletal damage and fibrosis. Following 16-weeks of high-fat, high-sucrose, or low-fat, low-sucrose feeding, HLI was induced in 18-month-old C57BL/6J mice via the surgical ligation of the left femoral artery at 2 locations. Animals were euthanized 4-weeks post-ligation. Results indicate mice with and without obesity shared certain myopathic changes in response to chronic HLI, including impaired muscle contractility, altered mitochondrial electron transport chain complex content and function, and compromised antioxidant defense mechanisms. However, the extent of mitochondrial dysfunction and oxidative stress was significantly greater in obese ischemic muscle compared to non-obese ischemic muscle. Moreover, functional impediments, such as delayed post-surgical recovery of limb function and reduced 6-minute walking distance, as well as accelerated intramuscular protein breakdown, inflammation, cytoskeletal damage, and fibrosis were only evident in mice with obesity. As these features are consistent with human PAD myopathy, our model could be a valuable tool to test new therapeutics.

MicroRNAs in peripheral artery disease: potential biomarkers and pathophysiological mechanisms.

Peripheral artery disease (PAD) is a disease of atherosclerosis in the lower extremities. PAD carries a massive burden worldwide, while diagnosis and treatment options are often lacking. One of the key points of research in recent years is the involvement of microRNAs (miRNAs), which are short 20-25 nucleotide single-stranded RNAs that can act as negative regulators of post-transcriptional gene expression. Many of these miRNAs have been discovered to be misregulated in PAD patients, suggesting a potential utility as biomarkers for PAD diagnosis. miRNAs have also been shown to play an important role in many different pathophysiological aspects involved in the initiation and progression of the disease including angiogenesis, hypoxia, inflammation, as well as other cellular functions like cell proliferation and migration. The research on miRNAs in PAD has the potential to lead to a whole new class of diagnostic tools and treatments.

High-Fat Diet Augments the Effect of Alcohol on Skeletal Muscle Mitochondrial Dysfunction in Mice.

Previous studies have shown that chronic heavy alcohol consumption and consumption of a high-fat (HF) diet can independently contribute to skeletal muscle oxidative stress and mitochondrial dysfunction, yet the concurrent effect of these risk factors remains unclear. We aimed to assess the effect of alcohol and different dietary compositions on mitochondrial activity and oxidative stress markers. Male and female mice were randomized to an alcohol (EtOH)-free HF diet, a HF + EtOH diet, or a low-Fat (LF) + EtOH diet for 6 weeks. At the end of the study, electron transport chain complex activity and expression as well as antioxidant activity and expression, were measured in skeletal muscles. Complex I and III activity were diminished in muscles of mice fed a HF + EtOH diet relative to the EtOH-free HF diet. Lipid peroxidation was elevated, and antioxidant activity was diminished, in muscles of mice fed a HF + EtOH diet as well. Consumption of a HF diet may exacerbate the negative effects of alcohol on skeletal muscle mitochondrial health and oxidative stress.

Skeletal muscle MiR-210 expression is associated with mitochondrial function in peripheral artery disease patients.

Previous studies have demonstrated that circulating microRNA (miR)-210 levels are elevated in peripheral artery disease (PAD) patients. MiR-210 is known to be a negative regulator of mitochondrial respiration; however, the relationship between miR-210 and mitochondrial function has yet to be studied in PAD. We aimed to compare skeletal muscle miR-210 expression of PAD patients to non-PAD controls (CON) and to examine the relationship between miR-210 expression and mitochondrial function. Skeletal muscle biopsies from CON (n = 20), intermittent claudication (IC) patients (n = 20), and critical limb ischemia (CLI) patients (n = 20) were analyzed by high-resolution respirometry to measure mitochondrial respiration of permeabilized fibers. Samples were also analyzed for miR-210 expression by real-time PCR. MiR-210 expression was significantly elevated in IC and CLI muscle compared to CON (P = 0.008 and P < 0.001, respectively). Mitochondrial respiration of electron transport chain (ETC) Complexes II (P = 0.001) and IV (P < 0.001) were significantly reduced in IC patients. Further, CLI patients demonstrated significant reductions in respiration during Complexes I (state 2: P = 0.04, state 3: P = 0.003), combined I and II (P < 0.001), II (P < 0.001), and IV (P < 0.001). The expression of the miR-210 targets, cytochrome c oxidase assembly factor heme A: farnesyltransferase (COX10), and iron-sulfur cluster assembly enzyme (ISCU) were down-regulated in PAD muscle. MiR-210 may play a role in the cellular adaptation to hypoxia and may be involved in the metabolic myopathy associated 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.

Phytochemicals as Therapeutic Interventions in Peripheral Artery Disease.

Peripheral artery disease (PAD) affects over 200 million people worldwide, resulting in significant morbidity and mortality, yet treatment options remain limited. Among the manifestations of PAD is a severe functional disability and decline, which is thought to be the result of different pathophysiological mechanisms including oxidative stress, skeletal muscle pathology, and reduced nitric oxide bioavailability. Thus, compounds that target these mechanisms may have a therapeutic effect on walking performance in PAD patients. Phytochemicals produced by plants have been widely studied for their potential health effects and role in various diseases including cardiovascular disease and cancer. In this review, we focus on PAD and discuss the evidence related to the clinical utility of different phytochemicals. We discuss phytochemical research in preclinical models of PAD, and we highlight the results of the available clinical trials that have assessed the effects of these compounds on PAD patient functional outcomes.

The Nitric Oxide System in Peripheral Artery Disease: Connection with Oxidative Stress and Biopterins.

Peripheral artery disease (PAD) pathophysiology extends beyond hemodynamics to include other operating mechanisms, including endothelial dysfunction. Oxidative stress may be linked to endothelial dysfunction by reducing nitric oxide (NO) bioavailability. We aimed to investigate whether the NO system and its regulators are altered in the setting of PAD and to assess the relationship between NO bioavailability and oxidative stress. Sera from 35 patients with intermittent claudication (IC), 26 patients with critical limb ischemia (CLI), and 35 non-PAD controls were analyzed to determine levels of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), nitrate/nitrite (nitric oxides, or NOx), arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and the oxidative stress markers 8-Oxo-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), advanced glycation end products (AGEs), and protein carbonyls. NOx was significantly lower in IC and CLI patients compared to controls in association with elevated oxidative stress, with the greatest NOx reductions observed in CLI. Compared with controls, IC and CLI patients had reduced BH4, elevated BH2, and a reduced BH4/BH2 ratio. SDMA, the arginine/SDMA ratio, and the arginine/ADMA ratio were significantly higher in CLI patients. The NO system and its regulators are significantly compromised in PAD. This dysregulation appears to be driven by increased oxidative stress and worsens as the disease progresses from claudication to CLI.

Altered Metabolomic Profile in Patients with Peripheral Artery Disease.

Peripheral artery disease (PAD) is a common atherosclerotic disease characterized by narrowed or blocked arteries in the lower extremities. Circulating serum biomarkers can provide significant insight regarding the disease progression. Here, we explore the metabolomics signatures associated with different stages of PAD and investigate potential mechanisms of the disease. We compared the serum metabolites of a cohort of 26 PAD patients presenting with claudication and 26 PAD patients presenting with critical limb ischemia (CLI) to those of 26 non-PAD controls. A difference between the metabolite profiles of PAD patients from non-PAD controls was observed for several amino acids, acylcarnitines, ceramides, and cholesteryl esters. Furthermore, our data demonstrate that patients with CLI possess an altered metabolomic signature different from that of both claudicants and non-PAD controls. These findings provide new insight into the pathophysiology of PAD and may help develop future diagnostic procedures and therapies for PAD patients.

Resistance Training, Antioxidant Status, and Antioxidant Supplementation.

Resistance training is known to promote the generation of reactive oxygen species. Although this can likely upregulate the natural, endogenous antioxidant defense systems, high amounts of reactive oxygen species can cause skeletal muscle damage, fatigue, and impair recovery. To prevent these, antioxidant supplements are commonly consumed along with exercise. Recently, it has been shown that these reactive oxygen species are important for the cellular adaptation process, acting as redox signaling molecules. However, most of the research regarding antioxidant status and antioxidant supplementation with exercise has focused on endurance training. In this review, the authors discuss the evidence for resistance training modulating the antioxidant status. They also highlight the effects of combining antioxidant supplementation with resistance training on training-induced skeletal muscle adaptations.

Oxidative Stress and Arterial Dysfunction in Peripheral Artery Disease.

Peripheral artery disease (PAD) is an atherosclerotic disease characterized by a narrowing of the arteries in the lower extremities. Disease manifestations are the result of more than just reduced blood flow, and include endothelial dysfunction, arterial stiffness, and inflammation. Growing evidence suggests that these factors lead to functional impairment and decline in PAD patients. Oxidative stress also plays an important role in the disease, and a growing amount of data suggest a link between arterial dysfunction and oxidative stress. In this review, we present the current evidence for the involvement of endothelial dysfunction, arterial stiffness, and inflammation in the pathophysiology of PAD. We also discuss the links between these factors and oxidative stress, with a focus on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2)-derived reactive oxygen species (ROS) and decreased nitric oxide (NO) bioavailability. Finally, the potential therapeutic role of NOX2 antioxidants for improving arterial function and functional status in PAD patients is explored.

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.

Abnormal accumulation of desmin in gastrocnemius myofibers of patients with peripheral artery disease: associations with altered myofiber morphology and density, mitochondrial dysfunction and impaired limb function.

Patients with peripheral artery disease (PAD) develop a myopathy in their ischemic lower extremities, which is characterized by myofiber degeneration, mitochondrial dysfunction and impaired limb function. Desmin, a protein of the cytoskeleton, is central to maintenance of the structure, shape and function of the myofiber and its organelles, especially the mitochondria, and to translation of sarcomere contraction into muscle contraction. In this study, we investigated the hypothesis that disruption of the desmin network occurs in gastrocnemius myofibers of PAD patients and correlates with altered myofiber morphology, mitochondrial dysfunction, and impaired limb function. Using fluorescence microscopy, we evaluated desmin organization and quantified myofiber content in the gastrocnemius of PAD and control patients. Desmin was highly disorganized in PAD but not control muscles and myofiber content was increased significantly in PAD compared to control muscles. By qPCR, we found that desmin gene transcripts were increased in the gastrocnemius of PAD patients as compared with control patients. Increased desmin and desmin gene transcripts in PAD muscles correlated with altered myofiber morphology, decreased mitochondrial respiration, reduced calf muscle strength and decreased walking performance. In conclusion, our studies identified disruption of the desmin system in gastrocnemius myofibers as an index of the myopathy and limitation of muscle function in patients with PAD.

Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective.

BACKGROUND: Peripheral artery disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in the arteries supplying the legs, affects approximately 5% of Americans. We have previously, demonstrated that a myopathy characterized by myofiber oxidative damage and degeneration is central to PAD pathophysiology. OBJECTIVES: In this study, we hypothesized that increased oxidative damage in the myofibers of the gastrocnemius of PAD patients is myofiber-type selective and correlates with reduced myofiber size. METHODS: Needle biopsies were taken from the gastrocnemius of 53 PAD patients (28 with early PAD and 25 with advanced PAD) and 25 controls. Carbonyl groups (marker of oxidative damage), were quantified in myofibers of slide-mounted tissue, by quantitative fluorescence microscopy. Myofiber cross-sectional area was determined from sarcolemma labeled with wheat germ agglutinin. The tissues were also labeled for myosin I and II, permitting quantification of oxidative damage to and relative frequency of the different myofiber Types (Type I, Type II and mixed Type I/II myofibers). We compared PAD patients in early (N=28) vs. advanced (N=25) disease stage for selective, myofiber oxidative damage and altered morphometrics. RESULTS: The carbonyl content of gastrocnemius myofibers was higher in PAD patients compared to control subjects, for all three myofiber types (p<0.05). In PAD patients carbonyl content was higher (p<0.05) in Type II and I/II fibers compared to Type I fibers. Furthermore, the relative frequency and cross-sectional area of Type II fibers were lower, while the relative frequencies and cross-sectional area of Type I and Type I/II fibers were higher, in PAD compared to control gastrocnemius (p<0.05). Lastly, the type II-selective oxidative damage increased and myofiber size decreased as the disease progressed from the early to advanced stage. CONCLUSIONS: Our data confirm increased myofiber oxidative damage and reduced myofiber size in PAD gastrocnemius and demonstrate that the damage is selective for type II myofibers and is worse in the most advanced stage 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.

Parallel changes in neuronal AT1R and GRK5 expression following exercise training in heart failure.

Although exercise training (ExT) is an important therapeutic strategy for improving quality of life in patients with chronic heart failure (CHF), the central mechanisms by which ExT is beneficial are not well understood. The angiotensin II type 1 receptor (AT1R) plays a pivotal role in the development of CHF and is upregulated in a number of tissues owing, in part, to transcription factor nuclear factor kappa B (NF-κB). In addition, AT1R is marked for internalization and recycling via G-protein-coupled receptor kinase (GRK) phosphorylation. Because previous studies have shown that the beneficial effects of ExT in CHF rely on a reduction in angiotensin II, we hypothesized ExT would decrease AT1R, GRK5, and NF-κB protein expression in the paraventricular nucleus and rostral ventrolateral medulla of CHF rats. Following infarction by coronary artery ligation, animals were exercised 4 weeks postsurgery on a treadmill at a final speed of 25 miles per minute for 60 minutes, 5 days per week for 6 weeks. Western blot analysis of paraventricular nucleus and rostral ventrolateral medulla micropunches revealed an upregulation of AT1R, GRK5, and NF-κB in the infarcted group that was reversed by ExT. Furthermore, the relative expression of phosphorylated AT1R and AT1R/GRK5 physical association was increased in the CHF sedentary group and reversed by ExT. Overexpression of GRK5 in cultured CATH.a neurons blunted angiotensin II-mediated upregulation of AT1R and NF-κB; conversely, silencing of GRK5 exacerbated angiotensin II-mediated AT1R and NF-κB upregulation. Taken together, increased GRK5 may regulate AT1R expression in CHF, and ExT mitigates AT1R and its pathway components.