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Walking Training Improves Systemic and Local Pathophysiological Processes in Intermittent Claudication

Open ArchivePublished:April 16, 2021DOI:https://doi.org/10.1016/j.ejvs.2021.02.022

      Objective

      This study examined the impact of submaximal walking training (WT) on local and systemic nitric oxide (NO) bioavailability, inflammation, and oxidative stress in patients with intermittent claudication (IC).

      Methods

      The study employed a randomised, controlled, parallel group design and was performed in a single centre. Thirty-two men with IC were randomly allocated to two groups: WT (n = 16, two sessions/week, 15 cycles of two minutes walking at an intensity corresponding to the heart rate obtained at the pain threshold interspersed by two minutes of upright rest) and control (CO, n = 16, two sessions/week, 30 minutes of stretching). NO bioavailability (blood NO and muscle nitric oxide synthase [eNOS]), redox homeostasis (catalase [CAT], superoxide dismutase [SOD], lipid peroxidation [LPO] measured in blood and muscle), and inflammation (interleukin-6 [IL-6], C-reactive protein [CRP], tumour necrosis factor α [TNF-α], intercellular adhesion molecules [ICAM], vascular adhesion molecules [VCAM] measured in blood and muscle) were assessed at baseline and after 12 weeks.

      Results

      WT statistically significantly increased blood NO, muscle eNOS, blood SOD and CAT, and muscle SOD and abolished the increase in circulating and muscle LPO observed in the CO group. WT decreased blood CRP, ICAM, and VCAM and muscle IL-6 and CRP and eliminated the increase in blood TNF-α and muscle TNF-α, ICAM and VCAM observed in the CO group.

      Conclusion

      WT at an intensity of pain threshold improved NO bioavailability and decreased systemic and local oxidative stress and inflammation in patients with IC. The proposed WT protocol provides physiological adaptations that may contribute to cardiovascular health in these patients.

      Keywords

      Walking training near maximal intermittent claudication (IC) symptoms is considered the best treatment for increasing walking capacity in patients with peripheral artery disease (PAD) and IC. However, ischaemia and reperfusion induced by maximal walking produce oxidative stress and inflammation during and after the effort, which may contribute to PAD progression. This study showed that a submaximal walking protocol at an intensity of pain threshold besides increasing walking capacity is effective in ameliorating not only systemic but also local pathophysiological processes associated to PAD, which provides support for the use of submaximal WT protocols in clinical practice.

      Introduction

      Peripheral artery disease (PAD) affects more than 200 million people worldwide.
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      with therapeutic interventions encouraged to ameliorate the abovementioned pathophysiological processes. Walking training (WT) is considered the best treatment to improve walking capacity in symptomatic PAD
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      ,
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      with walking to near maximal IC symptoms producing greatest improvements.
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      Plasma nitrite flux predicts exercise performance in peripheral arterial disease after 3months of exercise training.
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      oxidative stress,
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      • Bukowska-Strakova K.
      • Nizankowski R.
      • et al.
      Exercise training in intermittent claudication: effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells.
      ,
      • Gardner A.W.
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      Changes in vascular and inflammatory biomarkers after exercise rehabilitation in patients with symptomatic peripheral artery disease.
      and inflammation
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      • Bukowska-Strakova K.
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      • et al.
      Exercise training in intermittent claudication: effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells.
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      Changes in vascular and inflammatory biomarkers after exercise rehabilitation in patients with symptomatic peripheral artery disease.
      • Saetre T.
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      • Lyberg T.
      • Stranden E.
      • Jorgensen J.J.
      • Sundhagen J.O.
      • et al.
      Supervised exercise training reduces plasma levels of the endothelial inflammatory markers E-selectin and ICAM-I in patients with peripheral arterial disease.
      • Delaney C.L.
      • Spark J.I.
      A randomised controlled trial of two supervised exercise regimens and their impact on inflammatory burden in patients with intermittent claudication.
      • Schlager O.
      • Hammer A.
      • Giurgea A.
      • Schuhfried O.
      • Fialka-Moser V.
      • Gschwandtner M.
      • et al.
      Impact of exercise training on inflammation and platelet activation in patients with intermittent claudication.
      • Januszek R.
      • Mika P.
      • Konik A.
      • Petriczek T.
      • Nowobilski R.
      • Nizankowski R.
      Effect of treadmill training on endothelial function and walking abilities in patients with peripheral arterial disease.
      in IC have provided controversial results with many of them showing no benefits,
      • Nowak W.N.
      • Mika P.
      • Nowobilski R.
      • Kusinska K.
      • Bukowska-Strakova K.
      • Nizankowski R.
      • et al.
      Exercise training in intermittent claudication: effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells.
      ,
      • Delaney C.L.
      • Spark J.I.
      A randomised controlled trial of two supervised exercise regimens and their impact on inflammatory burden in patients with intermittent claudication.
      ,
      • Schlager O.
      • Hammer A.
      • Giurgea A.
      • Schuhfried O.
      • Fialka-Moser V.
      • Gschwandtner M.
      • et al.
      Impact of exercise training on inflammation and platelet activation in patients with intermittent claudication.
      ,
      • Leicht A.S.
      • Crowther R.G.
      • Golledge J.
      Influence of peripheral arterial disease and supervised walking on heart rate variability.
      which may be explained, at least in part, by the training protocols involving maximal ischaemia.
      Walking until maximal ischaemia increases oxidative stress and inflammation during and after the effort,
      • Turton E.P.
      • Coughlin P.A.
      • Kester R.C.
      • Scott D.J.
      Exercise training reduces the acute inflammatory response associated with claudication.
      which may contribute to the progression of the disease,
      • Wildman R.P.
      • Muntner P.
      • Chen J.
      • Sutton-Tyrrell K.
      • He J.
      Relation of inflammation to peripheral arterial disease in the national health and nutrition examination survey, 1999–2002.
      blunting the chronic benefits of WT. On the other hand, recent guidelines from the American College of Cardiology, American Heart Association, and Society for Vascular Surgery
      • Gerhard-Herman M.D.
      • Gornik H.L.
      • Barrett C.
      • Barshes N.R.
      • Corriere M.A.
      • Drachman D.E.
      • et al.
      2016 AHA/ACC Guideline on the Management of Patients With Lower Extremity Peripheral Artery Disease: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines.
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American Heart Association.
      • Conte M.S.
      • Pomposelli F.B.
      • Clair D.G.
      • Geraghty P.J.
      • McKinsey J.F.
      • et al.
      Society for Vascular Surgery Lower Extremity Guidelines Writing Group
      Society for Vascular Surgery practice guidelines for atherosclerotic occlusive disease of the lower extremities: management of asymptomatic disease and claudication.
      point out that pain free and low intensity walking protocols that avoid moderate to maximum claudication ischaemia and pain can improve walking ability and functional status, being recommended for these patients. Thus, some useful and reproducible submaximal protocols for patients with PAD and IC have been created and tested.
      • Chehuen M.
      • Cucato G.G.
      • Carvalho C.R.F.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      • Manfredini F.
      • Conconi F.
      • Malagoni A.M.
      • Manfredini R.
      • Mascoli F.
      • Liboni A.
      • et al.
      Speed rather than distance: a novel graded treadmill test to assess claudication.
      • Manfredini F.
      • Malagoni A.M.
      • Mascoli F.
      • Mandini S.
      • Taddia M.C.
      • Basaglia N.
      • et al.
      Training rather than walking: the test in -train out program for home-based rehabilitation in peripheral arteriopathy.
      Among them, the present authors have developed a WT protocol based on the heart rate (HR) of pain threshold that is a reproducible physiological marker.
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      This training protocol has been shown to evoke low pain and produce a notable physiological stimulus above the anaerobic threshold during its execution.
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      Additionally, it has improved cardiovascular function and autonomic regulation in patients with IC after a period of training.
      • Chehuen M.
      • Cucato G.G.
      • Carvalho C.R.F.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      However, despite these benefits, little is known about its effects on important factors for vascular health, such as NO bioavailability, oxidative stress, and inflammation.
      Previous studies examining the effects of WT on biomarkers of inflammation and oxidative stress in patients with IC
      • Allen J.D.
      • Stabler T.
      • Kenjale A.
      • Ham K.L.
      • Robbins J.L.
      • Duscha B.D.
      • et al.
      Plasma nitrite flux predicts exercise performance in peripheral arterial disease after 3months of exercise training.
      ,
      • Saetre T.
      • Enoksen E.
      • Lyberg T.
      • Stranden E.
      • Jorgensen J.J.
      • Sundhagen J.O.
      • et al.
      Supervised exercise training reduces plasma levels of the endothelial inflammatory markers E-selectin and ICAM-I in patients with peripheral arterial disease.
      • Delaney C.L.
      • Spark J.I.
      A randomised controlled trial of two supervised exercise regimens and their impact on inflammatory burden in patients with intermittent claudication.
      • Schlager O.
      • Hammer A.
      • Giurgea A.
      • Schuhfried O.
      • Fialka-Moser V.
      • Gschwandtner M.
      • et al.
      Impact of exercise training on inflammation and platelet activation in patients with intermittent claudication.
      • Januszek R.
      • Mika P.
      • Konik A.
      • Petriczek T.
      • Nowobilski R.
      • Nizankowski R.
      Effect of treadmill training on endothelial function and walking abilities in patients with peripheral arterial disease.
      ,
      • Turton E.P.
      • Coughlin P.A.
      • Kester R.C.
      • Scott D.J.
      Exercise training reduces the acute inflammatory response associated with claudication.
      have shown the systemic impact of WT. However, such analyses do not reflect the direct effects of WT on the source of these processes, the disease affected muscle.
      • Turrens J.F.
      • Beconi M.
      • Barilla J.
      • Chavez U.B.
      • McCord J.M.
      Mitochondrial generation of oxygen radicals during reoxygenation of ischemic tissues.
      Thus, simultaneous examination of the systemic and local effects of submaximal WT on inflammatory, oxidative stress, and NO bioavailability biomarkers may provide a more comprehensive understanding of the effects of WT on patients with IC management. Therefore, this study sought to determine whether a submaximal WT could improve systemic and local pathophysiological processes associated with PAD in patients with IC.

      Materials and Methods

      The study’s protocol followed the Declaration of Helsinki, was registered at the Brazilian Clinical Trials database (http://www.ensaiosclinicos.gov.br, RBR-3pq58k), and was approved by an Ethics Committee of Human Research at the University of São Paulo (process 667.382). Written informed consent was obtained prior to participation.

      Participants

      Patients with PAD and IC symptoms enrolled at a tertiary centre specialising in treatment of vascular disease were invited to participate. Patients were included if they met the following criteria: (i) male; (ii) aged ≥ 50 years; (iii) diagnosed with Fontaine stage II and Rutherford stages 1–3 PAD; (iv) ankle brachial index (ABI) at rest ≤ .90 in at least one lower limb; (v) absence of non-compressible arteries; (vi) resting systolic and diastolic blood pressure (SBP and DBP) lower than 160 and 105 mmHg, respectively; (vii) absence of revascularisation surgery or angioplasty in the last year; (viii) not receiving β blocker, non-dihydropyridine calcium channel antagonist, anticoagulant clopidogrel, and insulin; (ix) ability to walk at least two minutes at 3.2 km/h on a treadmill; (x) ability to undertake an incremental treadmill test limited by symptoms of IC; (xi) absence of myocardial ischaemia or complex dysrhythmias during a treadmill test. Patients were excluded if they: (i) presented with diabetes with clinical autonomic neuropathy; (ii) presented other medical comorbidities (such as cardiomyopathies, neurodegenerative conditions, and others) that prevent exercise execution; and iii) changed medication during the study.

      Study design

      This randomised, controlled, parallel group study was performed at a single centre. Patients who fulfilled the study criteria after preliminary evaluations underwent the experimental protocol in which the study’s outcomes were evaluated at baseline and after 12 weeks of intervention. Participants were randomly assigned, via a computer random number generator (https://www.randomizer.org), into two groups: control (CO) and walking training (WT).

      Preliminary procedures

      Patients were interviewed to identify age, presence of cardiovascular disease and risk factors, comorbid conditions, and current medication. ABI was evaluated as previously described.
      • Hirsch A.T.
      • Haskal Z.J.
      • Hertzer N.R.
      • Bakal C.W.
      • Creager M.A.
      • Halperin J.L.
      • et al.
      ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation.
      Body mass and height were assessed to calculate body mass index. Auscultatory BP was measured in triplicate after five minutes of seated rest in two visits and the mean value was calculated. Diabetic patients (n = 9, 28.1%) underwent the American Diabetes Association’s battery for detecting cardiovascular autonomic neuropathy.
      American Diabetes Association
      10. Microvascular Complications and Foot Care: Standards of Medical Care in Diabetes-2018.
      Afterwards, the patients underwent a graded maximal walking test on a treadmill (Imbrasport, ATL, Porto Alegre, Brazil) with the speed set at 3.2 km/h and grade increased by 2% every two minutes until maximal leg pain.
      • Gardner A.W.
      • Skinner J.S.
      • Cantwell B.W.
      • Smith L.K.
      Progressive vs single-stage treadmill tests for evaluation of claudication.
      Patients were monitored with a 12 lead ECG (Welch Allyn, Inc., Cardio Perfect MD, New York, USA). Heart rate (HR) at the pain threshold was recorded when the patients first reported claudication pain. This index has been previously shown to have high reliability and good agreement (ICC = 0.92; SEM = 3.2 bpm; SDD = 8.8 bpm).
      • da Rocha Chehuen M.
      • Cucato G.G.
      • Saes G.F.
      • Costa L.A.
      • Leicht A.S.
      • Ritti-Dias R.M.
      • et al.
      Reproducibility of anaerobic and pain thresholds in male patients with intermittent claudication.
      Claudication onset distance (COD) and total walking distance (TWD) were also recorded.

      Experimental protocol

      For all evaluations, patients were instructed to maintain similar routines in the prior 24 hours and to avoid physical exercise for the previous 48 hours, alcoholic beverages for 24 hours, and smoking on the day of the sessions. Patients had to take their medication as normal and attend to the laboratory in a fasted state. Laboratory temperature was kept between 20°C and 22°C.
      Patients arrived at 7:00 a.m. and received a standardised meal (two cereal bars and 50 mL of juice). Then, a catheter was inserted into an antecubital vein and kept patent with saline. They then rested in the supine position until 8:00 a.m., when assessments were initiated. HR and auscultatory blood pressure were measured in triplicate and the mean value was calculated. Afterwards, venous blood and muscle biopsy samples were collected.
      Patients were informed about their allocated group after the initial evaluation, while researchers who assessed the main outcomes (biomarkers analyses) of the study were blinded to the group allocation.

      Outcomes

      The primary outcomes were blood and muscle inflammatory and oxidative stress biomarkers. Secondary outcomes were walking capacity and cardiovascular function. (For the full list of biomarkers evaluated and their definitions, please see the Measurements section.)

      Measurements

      Cardiovascular function

      HR was determined from ECG (EMG System do Brazil, EMG 030110/00B, Brazil). Auscultatory SBP/DBP was measured using a mercury sphygmomanometer (Unitec, São Paulo, Brazil). Rate product pressure (RPP) was calculated as HR × SBP.

      Blood analysis

      Blood samples (16 mL) were collected into standard EDTA treated vacutainer tubes, centrifuged and plasma aliquots stored at −80°C. Enzyme linked immunosorbent assays (ELISA) were used to evaluate plasma concentrations of C reactive protein (CRP), tumour necrosis factor-α (TNF-α), vascular cell adhesion molecule (VCAM), intercellular adhesion molecules (ICAM), and interleukin-6 (IL-6), according to the manufacturer’s instructions (Cayman Chemical, USA and R&D Systems, USA). Plasma concentrations of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (LPO) were analysed using specific kits (Cayman Chemical, USA). Plasma NO was analysed by the chemiluminescence method with a specific analyser (Sievers Nitric Oxide Analyzer NOA 280, USA).

      Muscle biopsy

      Tissue samples (∼70 – 100 mg) from the gastrocnemius muscle of the leg with the lowest ABI were removed by biopsy following Bergstrom’s technique.
      • Bergstrom J.
      Percutaneous needle biopsy of skeletal muscle in physiological and clinical research.
      The samples were cleaned (blood and excessive connective tissue removed), immediately frozen in liquid nitrogen, and stored at −80°C.
      The mRNA levels of inflammatory biomarkers (IL-6, CRP, TNF-α, VCAM, ICAM) and endothelial nitric oxide synthase (eNOS) were assessed with real time PCR (SYBRGreen PCR Master Mix and ABI PRISM 7500 Sequence Detection System, Applied Biosystems). Cyclophilin mRNA levels were used as a reference gene expression level. Relative quantities of target gene expressions were compared after normalisation to the reference gene value (ΔCT). Changes in mRNA expression were calculated using the differences in ΔCT between the target gene expressions and cyclophilin (ΔΔCT) and the equation 2-ΔΔCT. Results are expressed as a percentage of the value from the baseline assessment of the CO group.
      For SOD, CAT, and LPO analyses, muscle biopsies were homogenised (1:4 wt/vol) in potassium phosphate buffered saline (50 mM, pH 7.8), centrifuged and stored at 4°C. SOD maximal activity was analysed based on the inhibition of xanthine/xanthine oxidase driven cytochrome C and expressed as U/mg. For CAT activity analysis, the rate of hydrogen peroxide (H2O2) decomposition was assessed. LPO was evaluated as an index of skeletal muscle oxidative injury following the ferrous oxidation xylenol orange technique (FOX2).

      Interventions

      The WT group followed a protocol already used in this population.
      • Chehuen M.
      • Cucato G.G.
      • Carvalho C.R.F.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      ,
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      Patients from this group executed two supervised training sessions per week. Each session consisted of 15 bouts of two minutes walking on a treadmill interspersed by two minutes of upright rest, resulting in a 60 minute total session with 30 minutes of active walking. During walking bouts, intensity was adjusted to maintain HR at the HR obtained at the pain threshold
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      evaluated in the graded maximal test executed in the preliminary procedures. A range of 4 bpm above or below of the HR obtained at this threshold was used to determine the upper and lower limits for training. Thus, during training sessions, treadmill speed was set at 3.2 km/h and the slope was adjusted when necessary to keep HR (measured by a thorax monitor Polar A3, Helsinki, Finland) within the desired range (i.e. in each two minute walking bout, if HR during walking was below the lower HR limit, the slope was increased in the next bout, and if HR was above the upper limit, slope was decreased in the next bout).
      In this population, the inclusion of a CO group that had to move for supervised sessions was essential to ensure that improvements obtained by the WT group could not be attributed to the active commuting necessary to get to the training sessions. Stretching was chosen as the control intervention because it is an alternative mode of physical activity that has been shown not to modify walking capacity or cardiovascular function and regulation in patients with IC.
      • Chehuen M.
      • Cucato G.G.
      • Carvalho C.R.F.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      ,
      • Cucato G.G.
      • Chehuen Mda R.
      • Costa L.A.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Saxton J.M.
      • et al.
      Exercise prescription using the heart of claudication pain onset in patients with intermittent claudication.
      Thus, the CO group performed stretching classes for 30 minutes, twice a week. In each class, approximately 20 stretching exercises were performed for all body segments. Each exercise was executed two to three times with passive technique and maximal stretch maintained for 20 seconds.

      Statistical analyses

      The required sample sizes were calculated based on a pilot study that showed differences between the groups of: CRP = 500 pg/mL; TNF-α = 0.29 pg/mL; ICAM = 25 ng/mL; VCAM = 85 ng/mL; LPO = 6.40 μM; NO = 1.78 μM; and CAT = 4.91 nmol.min−1.mL−1. Considering a power of 80% and an alpha error of 5%, the minimum sample size required was calculated to be 30 subjects.
      Normality of data distribution and homogeneity of variance were evaluated, respectively, by Shapiro–Wilk and Levene tests. Baseline group characteristics were compared by t or chi square tests. The effects of training were assessed via mixed two way (group vs. phase) ANOVAs (Statsoft, Statistic for Windows 4.3, Oklahoma, USA) and Newman–Keuls post-hoc tests were used when necessary. A p value ≤ .05 was considered to be statistically significant and data were presented as mean ± SE.

      Results

      The study flowchart is presented in Fig. 1. Seventy-eight patients were contacted, 54 provided informed consent, 35 undertook the baseline assessments, and 32 completed the study (16 in each group), which represents a loss of 40.7% of the sample that had signed the informed consent. The baseline characteristics were similar between the groups (Table 1).
      Figure 1
      Figure 1Study flowchart for enrolment, randomisation, interventions, and follow up of 32 patients with intermittent claudication (IC) studied for the impact of submaximal walking training (WT) on local and systemic nitric oxide bioavailability, inflammation, and oxidative stress. CO = control; SBP = systolic blood pressure; DBP = diastolic blood pressure; ABI = ankle brachial index; ECG = electrocardiogram.
      Table 1Baseline characteristics of patients in the control (CO) and walking training (WT) groups of 32 patients with intermittent claudication
      CO (n = 16)WT (n = 16)p value
      Age – y69 ± 366 ± 2.36
      Body mass index – kg/m225.4 ± 0.925.7 ± 0.8.79
      Diagnosis of peripheral artery disease
       Ankle brachial index0.62 ± 0.040.64 ± 0.03.67
       Claudication onset distance – m231 ± 22212 ± 20.51
       Total walking distance – m593 ± 88572 ± 47.83
      Cardiovascular measures
       Systolic blood pressure – mmHg137 ± 5136 ± 2.78
       Diastolic blood pressure – mmHg79 ± 283 ± 2.21
       Heart rate – bpm75 ± 370 ± 2.15
      Comorbidities
      Diabetes, hypertension, dyslipidaemia, chronic obstructive pulmonary disease, heart disease, and stroke defined by previous diagnosis.
       Obesity
      Obesity defined as body mass index ≥ 30 kg/m2.
      1 (6.2)1 (6.2).99
       Hypertension12 (75.0)11 (68.8).69
       Diabetes mellitus5 (31.2)4 (25.0).69
       Dyslipidaemia14 (87.5)16 (100.0).14
       Current smoker4 (25.0)6 (37.5).45
       Alcohol intake4 (25.0)6 (37.5).43
       Chronic obstructive pulmonary disease1 (6.2)0 (0.0).31
       Heart disease/stroke3 (18.7)3 (18.7).99
       Atrial fibrillation0 (0.0)0 (0.0)
       Valve disorder0 (0.0)0 (0.0)
      Drug therapy
       Aspirin15 (93.8)15 (93.8).99
       Statin14 (87.5)16 (100.0).14
       Oral hypoglycaemic5 (31.2)4 (25.0).69
      Antihypertensive agent
      Angiotensin converting enzyme inhibitor6 (37.5)9 (56.2).48
      Diuretic6 (37.5)2 (12.5).10
      Dihydropyridine calcium channel antagonist1 (6.2)5 (31.2).078
      Angiotensin receptor antagonists3 (18.7)0 (0.0).074
      Data are presented as mean ± standard error or n (%).
      Diabetes, hypertension, dyslipidaemia, chronic obstructive pulmonary disease, heart disease, and stroke defined by previous diagnosis.
      Obesity defined as body mass index ≥ 30 kg/m2.
      WT increased COD (212 ± 15 vs. 372 ± 48 m, p = .004) and TWD (572 ± 35 vs. 947 ± 61 m, p < .001), while no change was observed in the CO group (COD = 231 ± 16 vs. 191 ± 47 m, and TWD = 593 ± 73 vs. 633 ± 75 m; p = .54 and p = .48, respectively).
      Statistically significant interactions were identified for SBP, HR, and RPP (Table 2). These variables were similar between the groups at baseline and notably decreased only after WT.
      Table 2Cardiovascular variables measured at baseline and after 12 weeks for the control (CO) and walking training (WT) groups of 32 patients with intermittent claudication
      Cardiovascular functionCO (n = 16)WT (n = 16)p value
      Baseline12 weeksBaseline12 weeks
      Systolic BP – mmHg136 ± 4141 ± 5137 ± 3126 ± 3
      Different from baseline within the group (p ≤ .05).
      Different from CO at the same study phase (p ≤ .05).
      <.001
      Diastolic BP – mmHg75 ± 276 ± 279 ± 277 ± 2.082
      HR – bpm69 ± 269 ± 366 ± 262 ± 2
      Different from baseline within the group (p ≤ .05).
      Different from CO at the same study phase (p ≤ .05).
      .038
      RPP – bpm.mmHg9 293 ± 3569 684 ± 5128 998 ± 3577 798 ± 365
      Different from baseline within the group (p ≤ .05).
      Different from CO at the same study phase (p ≤ .05).
      <.001
      Data are presented as mean ± standard error. BP = blood pressure; HR = heart rate; RPP = rate pressure product.
      Different from baseline within the group (p ≤ .05).
      Different from CO at the same study phase (p ≤ .05).
      Systemic biomarkers are shown in Fig. 2. Statistically significant interactions were identified for plasma NO (p = .001), SOD (p = .009), CAT (p = .004), LPO (p = .030), CRP (p = .007), TNF-α (p = .005), ICAM (p = .008), and VCAM (p < .001), showing that WT had a statistically significant effect different from CO on these variables. Baseline levels of these variables were similar between the groups. WT statistically significantly increased NO, SOD, and CAT, and prevented the increase of LPO observed in the CO group. Additionally, WT statistically significantly decreased CRP, ICAM, and VCAM, and prevented the increase in TNF-α observed in the CO group.
      Figure 2
      Figure 2Blood biomarkers of nitric oxide (NO) bioavailability, oxidative stress, and inflammation measured at baseline and after 12 weeks in the control (CO; n = 16) and walking training (WT; n = 16) groups of patients with intermittent claudication. (A) nitric oxide (NO); (B) superoxide dismutase (SOD); (C) catalase (CAT); (D) lipid peroxidation (LPO); (E) interleukin 6 (IL-6); (F) C reactive protein (CRP); (G) tumour necrosis factor-α (TNF-α); (H) intercellular adhesion molecule (ICAM); and (I) vascular cell adhesion protein (VCAM). Different from baseline within the group (p ≤ .05). Different from the CO at the same study phase (p ≤ .05).
      Local muscle biomarkers are shown in Fig. 3. Statistically significant interactions were identified for muscle eNOS (p = .050), CAT (p = .036), LPO (p = .035), IL-6 (p = .043), CRP (p = .026), TNF-α (p = .017), ICAM (p = .008), and VCAM (p = .006), showing that WT had a statistically significant effect different from CO on these variables. Baseline levels of these variables were similar between groups. WT statistically significantly increased eNOS and CAT and prevented the increase in LPO observed in the CO group. Additionally, WT statistically significantly decreased IL-6 and CRP, and prevented the increase in TNF-α, ICAM, and VCAM observed in the CO group.
      Figure 3
      Figure 3Muscle biomarkers of nitric oxide bioavailability, oxidative stress, and inflammation measured at baseline and after 12 weeks in the control (CO; n = 16) and walking training (WT; n = 16) groups of patients with intermittent claudication. (A) endothelial nitric oxide synthase (eNOS); (B) superoxide dismutase (SOD); (C) catalase (CAT); (D) lipid peroxidation (LPO); (E) interleukin 6 (IL-6); (F) C reactive protein (CRP); (G) tumour necrosis factor-α (TNF-α); (H) intercellular adhesion molecule (ICAM); (I) vascular cell adhesion protein (VCAM). Different from baseline within the group (p ≤ .05). Different from the CO at the same study phase (p ≤ .05).

      Discussion

      The findings of this study were that, in IC patients, 12 weeks of submaximal WT: i) increased walking capacity (COD, TWD); ii) ameliorated cardiovascular function (decreased SBP, HR, and RPP; iii) increased systemic and local NO bioavailability; iv) decreased systemic and local oxidative stress (blunted the increase in LPO observed in CO); and v) decreased systemic and local inflammation (decreased blood CRP, ICAM, and VCAM; decreased muscle IL-6 and CRP; and blunted the increase in blood TNF-α and muscle TNF-α, ICAM, and VCAM observed in the CO group).
      As expected, the employed submaximal WT protocol improved walking capacity and cardiovascular function.
      • Chehuen M.
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      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      COD and TWD improvements (75.5 and 65.5%, respectively) were similar to those obtained with supervised maximal WT protocols.
      • Treat-Jacobson D.
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      • Collins T.C.
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      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American Heart Association.
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      Furthermore, the decrease of 10 ± 2 mmHg in SBP obtained in this study may have clinical impact as a reduction of 5 mmHg is associated with a decreased incidence of cardiovascular events.
      • Grenet G.
      • Le H.H.
      • Bejan-Angoulvant T.
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      Association between difference in blood pressure reduction and risk of cardiovascular events in a type 2 diabetes population: A meta-regression analysis.
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      • Pathak A.
      • Dimitriadis K.
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      • et al.
      European Society of Hypertension position paper on renal denervation 2018.
      Similarly, studies using other walking protocols based on the individual pain threshold speed
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      also reported positive claudication, functional and haemodynamic adaptation,
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      corroborating the applicability of protocols that avoid maximum ischaemia for managing patients with IC.
      The novelty of the present investigation was the benefits of WT on NO bioavailability, oxidative stress, and inflammation. The increases in plasma NO and muscle eNOS suggest a global effect of WT, enhancing whole body NO bioavailability. Systemic NO response is similar to that in previous studies.
      • Allen J.D.
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      • Kenjale A.
      • Ham K.L.
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      Plasma nitrite flux predicts exercise performance in peripheral arterial disease after 3months of exercise training.
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      Increased endogenous nitric oxide production induced by physical exercise in peripheral arterial occlusive disease patients.
      However, the present findings extended this knowledge to eNOS mRNA levels within the muscle affected by PAD. It is possible that increased local shear stress within active muscles during walking increased eNOS gene expression, leading to NO production and release to the blood.
      • Erkens R.
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      ,
      • Green D.J.
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      These responses may improve endothelial function, contributing to an improvement in walking capacity
      • Chehuen M.
      • Cucato G.G.
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      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      and a better cardiovascular prognosis.
      • Godo S.
      • Shimokawa H.
      Endothelial functions.
      WT also increased systemic and local antioxidant defence as demonstrated by increases in blood SOD and CAT activities and muscle CAT activity. These increases may have prevented the increase in oxidative stress observed in the CO group (i.e., increase in LPO), reflecting a beneficial effect of WT on redox homeostasis. Previous studies reported controversial results regarding the effects of WT on oxidative stress biomarkers assessed in the blood.
      • Nowak W.N.
      • Mika P.
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      • Kusinska K.
      • Bukowska-Strakova K.
      • Nizankowski R.
      • et al.
      Exercise training in intermittent claudication: effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells.
      ,
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      Changes in vascular and inflammatory biomarkers after exercise rehabilitation in patients with symptomatic peripheral artery disease.
      ,
      • Schlager O.
      • Giurgea A.
      • Schuhfried O.
      • Seidinger D.
      • Hammer A.
      • Groger M.
      • et al.
      Exercise training increases endothelial progenitor cells and decreases asymmetric dimethylarginine in peripheral arterial disease: a randomized controlled trial.
      Thus, the present results expand on this knowledge by demonstrating that submaximal WT was able to increase antioxidant defence and prevent increased oxidative stress not only in blood but also in the muscle. It is interesting to note that although CAT activity increased in both blood and muscle, muscle SOD activity did not increase. Increased SOD activity after training was observed mainly in muscles predominantly composed of oxidative fibres.
      • Powers S.K.
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      • Lawler J.
      • Ji L.L.
      • Martin D.
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      • et al.
      Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle.
      As patients with IC have reduced oxidative fibres within the affected muscles,
      • Koutakis P.
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      • Shostrom V.K.
      • Papoutsi E.
      • Ha D.M.
      • et al.
      Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective.
      this characteristic may explain, at least in part, the absence of increase in muscle SOD activity after WT. The mechanisms by which WT increased antioxidant response were not investigated, but it has been suggested that the oxidative stress generated during walking may activate pathways that lead to chronic adaptations.
      • Yavari A.
      • Javadi M.
      • Mirmiran P.
      • Bahadoran Z.
      Exercise-induced oxidative stress and dietary antioxidants.
      A reduction in systemic inflammation with WT also has been reported previously,
      • Nowak W.N.
      • Mika P.
      • Nowobilski R.
      • Kusinska K.
      • Bukowska-Strakova K.
      • Nizankowski R.
      • et al.
      Exercise training in intermittent claudication: effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells.
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      Changes in vascular and inflammatory biomarkers after exercise rehabilitation in patients with symptomatic peripheral artery disease.
      • Saetre T.
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      • Stranden E.
      • Jorgensen J.J.
      • Sundhagen J.O.
      • et al.
      Supervised exercise training reduces plasma levels of the endothelial inflammatory markers E-selectin and ICAM-I in patients with peripheral arterial disease.
      ,
      • Januszek R.
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      • Konik A.
      • Petriczek T.
      • Nowobilski R.
      • Nizankowski R.
      Effect of treadmill training on endothelial function and walking abilities in patients with peripheral arterial disease.
      ,
      • Turton E.P.
      • Coughlin P.A.
      • Kester R.C.
      • Scott D.J.
      Exercise training reduces the acute inflammatory response associated with claudication.
      and the present study expands this finding to the muscle affected by the disease, the potential major source of inflammation in IC.
      • Turrens J.F.
      • Beconi M.
      • Barilla J.
      • Chavez U.B.
      • McCord J.M.
      Mitochondrial generation of oxygen radicals during reoxygenation of ischemic tissues.
      The mechanisms responsible for this anti-inflammatory adaptation were beyond the scope of this study. However, a reduction in circulating immune cells related to inflammatory mediators may be involved, as aerobic training was reported to decrease the number of circulating monocytes and to decrease TNF-α production.
      • Barry J.C.
      • Simtchouk S.
      • Durrer C.
      • Jung M.E.
      • Little J.P.
      Short-term exercise training alters leukocyte chemokine receptors in obese adults.
      ,
      • Abd El-Kader S.M.
      • Al-Shreef F.M.
      Inflammatory cytokines and immune system modulation by aerobic versus resisted exercise training for elderly.
      Potentially, WT may inhibit cytokine release, as muscle contractions increase IL-10 from regulatory T cells,
      • Shill D.D.
      • Polley K.R.
      • Willingham T.B.
      • Call J.A.
      • Murrow J.R.
      • McCully K.K.
      • et al.
      Experimental intermittent ischemia augments exercise-induced inflammatory cytokine production.
      which inhibits the expression of pro-inflammatory cytokines.
      • Saraiva M.
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      The regulation of IL-10 production by immune cells.
      Further, the improvement of walking capacity may promote fewer episodes of ischaemia and reperfusion throughout the day, resulting in less local inflammation.
      • Turton E.P.
      • Coughlin P.A.
      • Kester R.C.
      • Scott D.J.
      Exercise training reduces the acute inflammatory response associated with claudication.
      Finally, the improvement in oxidative stress may also impact decreasing inflammation.
      • Siti H.N.
      • Kamisah Y.
      • Kamsiah J.
      The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review).
      The effects of WT on NO bioavailability, oxidative stress, and inflammation may have important clinical implications for IC patients. Increased NO bioavailability may improve endothelial function, blood flow, and vasodilatory capacity,
      • Chehuen M.
      • Cucato G.G.
      • Carvalho C.R.F.
      • Ritti-Dias R.M.
      • Wolosker N.
      • Leicht A.S.
      • et al.
      Walking training at the heart rate of pain threshold improves cardiovascular function and autonomic regulation in intermittent claudication: A randomized controlled trial.
      contributing to increase walking capacity, mobility, and independent living. As endothelial dysfunction,
      • Jud P.
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      • Verheyen N.
      • Meinitzer A.
      • Gary T.
      • Brodmann M.
      • et al.
      Homoarginine/ADMA ratio and homoarginine/SDMA ratio as independent predictors of cardiovascular mortality and cardiovascular events in lower extremity arterial disease.
      oxidative stress,
      • Wilson A.M.
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      • Weatherby C.
      • Harada R.K.
      • Ng M.K.
      • Nair N.
      • et al.
      Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease.
      and inflammation
      • Wildman R.P.
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      • He J.
      Relation of inflammation to peripheral arterial disease in the national health and nutrition examination survey, 1999–2002.
      are associated with morbimortality and disease progression in IC, their improvement may reduce cardiovascular risk and ameliorate prognosis. Thus, the present study provided novel evidence (systemic and local) about the beneficial effects of a submaximal WT on key factors associated with PAD progression and cardiovascular risk (a complete hypothetic model can be seen in the Supplementary material).
      The main practical implication of the present results is to support the use of submaximal WT protocols in patients with PAD and IC. Maximal WT (i.e., near maximal claudication symptoms) has been consistently recommended
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American Heart Association.
      ,
      • Conte M.S.
      • Pomposelli F.B.
      • Clair D.G.
      • Geraghty P.J.
      • McKinsey J.F.
      • et al.
      Society for Vascular Surgery Lower Extremity Guidelines Writing Group
      Society for Vascular Surgery practice guidelines for atherosclerotic occlusive disease of the lower extremities: management of asymptomatic disease and claudication.
      for clinical practice because of the walking capacity improvement obtained.
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American Heart Association.
      However, high levels of pain, ischaemia, and reperfusion induced by maximal effort increase oxidative stress and inflammation during and after walking,
      • Allen J.D.
      • Stabler T.
      • Kenjale A.
      • Ham K.L.
      • Robbins J.L.
      • Duscha B.D.
      • et al.
      Plasma nitrite flux predicts exercise performance in peripheral arterial disease after 3months of exercise training.
      ,
      • Turton E.P.
      • Coughlin P.A.
      • Kester R.C.
      • Scott D.J.
      Exercise training reduces the acute inflammatory response associated with claudication.
      which may favour the atherosclerotic process, contributing to PAD progress. By showing that a submaximal WT protocol can increase walking capacity and improve oxidative stress and inflammation, the present results support its use in the clinical treatment of PAD and IC as suggested by the recent guidelines from the Society for Vascular Surgery/American College of Cardiology/American Heart Association.
      • Gerhard-Herman M.D.
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      • Barrett C.
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      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American Heart Association.
      • Conte M.S.
      • Pomposelli F.B.
      • Clair D.G.
      • Geraghty P.J.
      • McKinsey J.F.
      • et al.
      Society for Vascular Surgery Lower Extremity Guidelines Writing Group
      Society for Vascular Surgery practice guidelines for atherosclerotic occlusive disease of the lower extremities: management of asymptomatic disease and claudication.
      Future studies, however, should compare directly maximal and submaximal WT protocols effects on these outcomes.
      This study has limitations that should be acknowledged. It included only men with specific disease characteristics, which limits extrapolation of the results. There is evidence that women with PAD and IC present distinct walking capacity,
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      • Khurana A.
      • Ritti-Dias R.M.
      • Blevins S.M.
      Gender differences in daily ambulatory activity patterns in patients with intermittent claudication.
      ,
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      • Sosnowska D.
      • Casanegra A.I.
      • Ungvari Z.
      • et al.
      Gender and racial differences in endothelial oxidative stress and inflammation in patients with symptomatic peripheral artery disease.
      biomarkers,
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      • Sosnowska D.
      • Casanegra A.I.
      • Ungvari Z.
      • et al.
      Gender and racial differences in endothelial oxidative stress and inflammation in patients with symptomatic peripheral artery disease.
      and cardiovascular variables,
      • Correia M.A.
      • de Sousa A.S.A.
      • Andrade-Lima A.
      • Germano-Soares A.H.
      • Zerati A.E.
      • Puech-Leao P.
      • et al.
      Functional and cardiovascular measurements in patients with peripheral artery disease: comparison between men and women.
      and respond differently to training
      • Gardner A.W.
      • Parker D.E.
      • Montgomery P.S.
      Predictors of improved walking after a supervised walking exercise program in men and women with peripheral artery disease.
      ,
      • Gommans L.N.
      • Scheltinga M.R.
      • van Sambeek M.R.
      • Maas A.H.
      • Bendermacher B.L.
      • Teijink J.A.
      Gender differences following supervised exercise therapy in patients with intermittent claudication.
      than men. Thus, future studies should examine the impact of the proposed WT protocol in women and compare this with men’s responses. Additionally, patients at other stages of the disease may present less or more difficult to perform WT, which may produce different results that should be addressed in the future. Patients receiving medications that directly affected cardiac autonomic regulation were excluded because of the possibility that these drugs blunt adaptations to exercise,
      • Mert K.U.
      • Sener E.
      • Yilmaz A.S.
      • Mert G.O.
      • Yetmis F.
      • Dural M.
      • et al.
      The association of exaggerated hypertensive response to exercise and beta-blockers use in hypertensives.
      ,
      • Gomides R.S.
      • Costa L.A.
      • Souza D.R.
      • Queiroz A.C.
      • Fernandes J.R.
      • Ortega K.C.
      • et al.
      Atenolol blunts blood pressure increase during dynamic resistance exercise in hypertensives.
      which could minimise biomarker responses to training. As many patients use these medications, future studies should broaden their examination to include such patients and check whether they present similar adaptations to WT. Several muscle biomarkers were measured by mRNA expression that despite being widely used and representing gene transcription, may or may not be an indicative of functional protein. Finally, although the sample size was adequate for the whole group comparisons, it did not allow stratifications within groups, which could strength the discussion. Futures studies are encouraged to use sample sizes that allow group stratification.
      In conclusion, 12 weeks of WT at an intensity of pain threshold improved NO bioavailability and decreased systemic and local oxidative stress and inflammation in patients with PAD and IC.

      Acknowledgements

      The authors thank all volunteers for participating.

      Conflict of interest

      None.

      Funding

      Fundação de Amparo à Pesquisa do Estado de São Paulo (process number 2015/13800–0 ); Conselho Nacional de Desenvolvimento Científico e Tecnológico (process number 442507/2014–03 , 304436/2018–6 ); and Coordenadoria de Aprimoramento Pessoal de Nível Superior (CAPES-PROEX; process number 001 ).

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:
      • Hypothetical model of beneficial effects of a submaximal walking training on key factors associated with progression of peripheral artery disease and cardiovascular risk

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