Impaired hemodynamic response to exercise in patients with peripheral artery disease: evidence of a link to inflammation and oxidative stress

Abstract

An exaggerated mean arterial blood pressure (MAP) response to exercise in patients with peripheral artery disease (PAD), likely driven by inflammation and oxidative stress and, perhaps, required to achieve an adequate blood flow response, is well described. However, the blood flow response to exercise in patients with PAD actually remains equivocal. Therefore, eight patients with PAD and eight healthy controls completed 3 min of plantar flexion exercise at both an absolute work rate (WR) (2.7 W, to evaluate blood flow) and a relative intensity (40%WR_max, to evaluate MAP). The exercise-induced change in popliteal artery blood flow (BF, Ultrasound Doppler), MAP (Finapress), and vascular conductance (VC) were quantified. In addition, resting markers of inflammation and oxidative stress were measured in plasma and muscle biopsies. Exercise-induced ΔBF, assessed at 2.7 W, was lower in PAD compared with controls (PAD: 251 ± 150 vs. Controls: 545 ± 187 mL/min, P < 0.001), whereas ΔMAP, assessed at 40%WR_max, was greater for PAD (PAD: 23 ± 14 vs. Controls: 11 ± 6 mmHg, P = 0.028). The exercise-induced ΔVC was lower for PAD during both the absolute WR (PAD: 1.9 ± 1.6 vs. Controls: 4.7 ± 1.9 mL/min/mmHg) and relative intensity exercise (PAD: 1.9 ± 1.8 vs. Controls: 5.0 ± 2.2 mL/min/mmHg) trials (both, P < 0.01). Inflammatory and oxidative stress markers, including plasma interleukin-6 and muscle protein carbonyls, were elevated in PAD (both, P < 0.05), and significantly correlated with the hemodynamic changes during exercise (r = −0.57 to −0.78, P < 0.05). Thus, despite an exaggerated ΔMAP response, patients with PAD exhibit an impaired exercise-induced ΔBF and ΔVC, and both inflammation and oxidative stress likely play a role in this attenuated hemodynamic response.

INTRODUCTION

Peripheral arterial disease (PAD) is an atherosclerotic disease that predominantly affects the lower limbs causing plaque development and, ultimately, accelerating functional decline (1), putting those who suffer from this disease at a markedly increased risk of cardiovascular morbidity and mortality (2, 3). In addition, patients with PAD demonstrate an exaggerated blood pressure response to exercise compared with healthy controls (4, 5), which further exacerbates the risk of adverse cardiovascular events such as myocardial infarction and stroke during physical activity (4, 6, 7). The typical blood pressure response to exercise is a fundamental component of the complex hemodynamic adjustments required to increase oxygen delivery (i.e., blood flow) to the active skeletal muscle. However, the blood flow response to exercise in PAD is equivocal, with reports of both reduced (8–10) and conserved (11–13) exercising blood flow compared with healthy control participants. Traditionally, impaired blood flow during exercise, and, thus, a reduced oxygen delivery, creating a mismatch between delivery and utilization, is thought to influence the exercise pressor response primarily through metaboreceptor activation (14–16). Indeed, this reflex was used to explain the preserved exercising blood flow in several studies of patients with PAD compared with control participants (11, 12). Recent evidence suggests that other chemical stimuli, outside of the scarcity of oxygen and subsequent metabolic byproducts, such as inflammatory cytokines and oxidative stress, can also activate muscle metaboreceptors and trigger an inappropriate cardiovascular response to exercise (17), particularly in PAD (18, 19). Therefore, developing a better understanding of the mechanisms contributing to the impaired hemodynamic response to exercise in these patients is vital to better characterize this disease and develop therapeutics to improve quality of life in this population.

Inflammation plays a critical role in the development of atherosclerosis and the progression of PAD (20). Indeed, circulating markers of inflammation relate directly to the clinical severity of PAD assessed by ankle-brachial index (ABI), brachial artery flow-mediated dilation, and maximal treadmill walking speed (21–23). Furthermore, Copp et al. (18) documented evidence of a direct link between inflammation and the exercise pressor response. In this interventional animal study, an intra-arterial infusion of interleukin-6 in healthy rats increased the exercise pressor response to both static and intermittent isometric contractions. In addition, the infusion of an interleukin-6 inhibitor into the circulation of rats with chronic femoral artery ligations (an animal model of PAD) reduced the exercise pressor response (18). A subsequent clinical study documented that systemic administration of ketorolac, a nonselective inhibitor of cyclooxygenase, attenuated the exercise pressor response in patients with PAD (24). Together, these studies implicate an important role for inflammation in the etiology of PAD in general, but also importantly, in the exaggerated blood pressure response to exercise in patients with PAD. However, neither of the latter two studies characterized the role of inflammation to the peripheral blood flow response in PAD.

Oxidative stress also contributes significantly to the progression of PAD and is inextricably linked to the inflammatory process (25). Much of the research, to date, investigating oxidative stress in PAD has concentrated on myopathy, and particularly, mitochondrial dysfunction (26, 27). The role of oxidative stress in vascular dysfunction is well understood and has been examined across the spectrum of health and disease (28–33). However, the contribution of oxidative stress to the hemodynamic responses to exercise in patients with PAD has received only minimal attention. As in the investigations evaluating inflammation, human and preclinical animal studies have revealed a role of oxidative stress in the exaggerated blood pressure response in PAD (5, 19), although, again, the peripheral hemodynamics during exercise were not characterized in these studies.

Therefore, although an exaggerated blood pressure response to exercise in patients with PAD compared with healthy controls is well documented, the blood flow response to exercise is poorly understood in this population. Furthermore, to guide future therapeutics, the potential role of both inflammation and oxidative stress in the blood flow response to exercise in PAD deserves further investigation. Thus, this study sought to test the following hypotheses: 1) patients with PAD would exhibit an exaggerated blood pressure response, but a markedly attenuated increase in exercising blood flow compared with healthy control participants, and 2) endogenous levels of inflammation and oxidative stress would be associated with the exercise-induced changes in blood flow, mean arterial pressure (MAP), and vascular conductance (VC).

METHODS

Ethical Approval

The protocol was approved and written informed consent was obtained according to the University of Utah and Salt Lake City Veterans Affairs Medical Center Institutional Review Board requirements. These procedures adhered to the Declaration of Helsinki. All data collection took place at the Utah Vascular Research Laboratory located at the Veterans Affairs Salt Lake City Geriatric, Research, Education, and Clinical Center.

Participants

Eight patients (2 females, 6 males) with mild PAD, classified as Fontaine Stage II (symptomatic intermittent claudication, ABI < 0.90, and no lower limb revascularization procedures) volunteered for and enrolled in this study. Eight healthy control subjects matched for age, sex, and physical activity participated in this study. Participant characteristics are presented in Table 1. All participants reported to the laboratory in a fasted state (>8 h) and refrained from caffeine and strenuous activity before any assessments (>24 h). The current study utilizes a subset of participants included in previous investigations that evaluated in vivo and ex vivo mitochondrial function in PAD (8, 34).

Table 1.

Participant characteristics

	Patients with Peripheral Artery Disease	Healthy Control Participants	P
Sex (female/male)	2/6	2/6	—
Age, yr	64 ± 9	60 ± 10	0.23
Height, cm	167 ± 8	173 ± 8	0.19
Weight, kg	77 ± 14	85 ± 22	0.51
Body mass index, kg/m2	27.6 ± 4.0	28.2 ± 5.5	0.81
Mean arterial pressure, mmHg	100 ± 14	106 ± 10	0.34
Ankle brachial index	0.67 ± 0.11	1.16 ± 0.07	<0.001
Smoker (former/current)	4/3	1/0	—
Steps, number/day	5,496 ± 4217	6,872 ± 3467	0.49
Maximal work rate, W	5.1 ± 1.6	10.6 ± 3.1	<0.001

Data are means ± SD; n = 8 in each group. Bolded P values indicate significant differences.

Protocol

The methods used herein have been described in detail previously (8, 34) and are, therefore, described in brief later. All participants were familiarized with the dynamic plantar flexion exercise before completing an incremental test to determine their peak work rate using 1-min stages with 0.5- to 1.0-W increments at a cadence of 60 contractions per min. On a separate day, participants performed exercise for 3 min at both an absolute work rate (2.7 W) and a relative exercise intensity (40% peak power), while maintaining 60 contractions per minute. With the expectation that peak work rate would differ between groups, the absolute work rate and relative exercise intensity plantar flexion trials were performed to facilitate the most appropriate comparison of the blood flow (absolute work rate, 2.7 W) and MAP (relative exercise intensity, 40% peak power) responses to exercise between patients with PAD and healthy controls.

Measurements

Popliteal artery blood velocity and vessel diameter measurements were performed in the exercising leg using a Logiq 7 Doppler ultrasound system (General Electric Medical Systems, Milwaukee, WI) operating in duplex mode with both imaging and pulse wave frequencies optimized in accordance with recently published guidelines for Doppler ultrasound assessment of resistance vessel function (35). The popliteal artery was insonated in the popliteal fossa, proximal to the branching of the medial inferior genicular artery. All blood velocity measurements were obtained with the probe appropriately positioned to maintain an insonation angel of 60°. The sample volume was maximized according to vessel size and was centered within the vessel. Blood flow (mL/min) was calculated as: [V_mean × π (vessel diameter/2)² × 60]. MAP was measured with a Finometer (Finapres Medical Systems, Amsterdam, The Netherlands) placed on the left middle finger and adjusted for height difference from heart level. Finometer data were recorded at 1,000 Hz and saved for offline analysis. VC (mL/min/mmHg) was calculated as blood flow divided by MAP.

On a day preceding the exercise studies, venous blood samples were drawn from the antecubital fossa and muscle biopsies were taken to assess inflammation and oxidative stress in both blood and muscle. The specifics of these methods have been described, in depth, elsewhere (34). Briefly, both groups had a percutaneous needle biopsy performed under sterile conditions, whereby a sample was obtained from the medial gastrocnemius muscle ∼10 cm distal to the tibial tuberosity at a depth of ∼1 cm. Biopsies were obtained from the most symptomatic leg in the patients with PAD and from the right leg of the healthy control subjects.

Whole blood samples were centrifuged to allow the collection of plasma and stored at −80°C until analysis for systemic blood-borne markers of inflammation and oxidative stress. Specifically, the proinflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-6 were determined by solid-phase sandwich ELISA (R&D Systems, Minneapolis, MN), whereas plasma protein oxidation by protein carbonyl levels was assessed by colorimetric ELISA assay (R&D Systems) with each performed according to the manufacturer’s recommended protocols.

Skeletal muscle protein carbonyl content was assessed following treatment with 2.4-dinitrophenylhydraziine (DNPH) (36). Briefly, two 200-µL samples were transferred to two 2-mL plastic microtubes, a sample tube, and a control tube. The sample tube then received 800 µL of DNPH and the control tube received 800 µL of 2.5 M hydrochlorides. Both tubes were incubated for 1 h at room temperature while protected from light and were vortexed every 15 min. Then 1 mL of 20% trichloroacetic acid was added and the tubes were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was discarded, and the pellets were washed three times with 1 mL ethanol-ethyl acetate (1:1) to remove free reagent. The precipitated protein was dissolved in 500 µL of guanidine hydrochloride via vortexing and centrifuged at 10,000 g for an additional 10 min to separate any insoluble material. Protein carbonyl content was determined spectrophotometrically at 360 nm and calculated using a molar absorption coefficient of 22,000 M⁻¹·cm⁻¹.

The relative abundance of the target proteins in skeletal muscle tissue samples for interleukin-6 and 4-hydroxynonenal were determined by immunoblotting, with the protein abundance of each normalized to β-actin that served as a loading control. Briefly, muscle samples were homogenized 1:12 (weight/volume) in an ice-cold buffer containing 5 mM Tris buffer (pH 7.5) and 5 mM ethylenediaminetetraacetate (pH 8) with a protease inhibitor cocktail. The homogenate was centrifuged at 1,500 g for 10 min at 4°C. After centrifugation, the supernatant was collected and the Bradford assay technique was used to determine protein concentration. Proteins from the supernatant were separated via polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and incubated with primary (1:1,000 dilution) and secondary antibodies (1:5,000 dilution) specific to the proteins of interest (anti-interleukin 6: ab6672, Abcam; 4-hydroxynonenal: ab46545, Abcam; β-actin: ab8227, Abcam). Representative Western blot images are presented in Supplemental Fig. S1 (see, https://doi.org/10.6084/m9.figshare.21111646.v1). Furthermore, the entire gel is presented for 4-hydroxynonenal in Supplemental Fig. S2 (see, https://doi.org/10.6084/m9.figshare.21171424.v1).

Data and Statistical Analysis

The exercise-induced changes in blood flow, MAP, and VC were calculated as the difference between an average of the last 60 s of exercise and a 2 min average at rest. All statistical analyses and figure production were performed using a commercially available software package (SigmaPlot 14.0, Systat Software, San Jose, CA). Comparisons of descriptive variables, resting measurements, and exercise-induced changes between groups were performed with unpaired Student’s t tests or Mann–Whitney Rank Sum tests when a Shapiro–Wilk normality test was failed. Of note, appropriately, data interpretation between groups for exercise-induced blood flow and MAP was restricted to the absolute work rate (2.7 W) and relative exercise intensity (40% peak power) trials, respectively, although, for completeness, all data and statistics are presented. In contrast, data interpretation between groups for exercise-induced VC was performed for both exercise trials as VC incorporates both blood flow and MAP. The assessment of relationships between variables was determined using Pearson’s product-moment correlation analysis. Data are presented as means ± standard deviation. Significance was accepted at P < 0.05.

RESULTS

Resting Hemodynamics and Markers of Inflammation and Oxidative Stress

As documented in Table 1, resting MAP was not different between the patients with PAD and healthy controls. Similarly, resting blood flow (PAD: 114 ± 82 vs. Control: 134 ± 78 mL/min; P = 0.44) and VC (PAD: 1.2 ± 1.0 vs. Control: 1.3 ± 0.8 mL/min/mmHg; P = 0.72) in the popliteal artery were not different.

The assessments of inflammation and oxidative stress in blood and tissue, at rest, are presented in Table 2. Not all participants in each group completed the required procedures to assess these variables, or had samples that were successfully analyzed, which resulted in 11 biopsies (PAD: 6, Control: 5) and 14 blood draws (PAD: 6, Control: 8). The PAD group had significantly elevated levels of the plasma inflammatory cytokines TNFα and interleukin-6 (both, P < 0.01), as well as intramuscular interleukin-6 (P < 0.01). Furthermore, the intramuscular markers of oxidative stress, protein carbonyl and 4-hydroxynonenal, were elevated in the PAD group (both, P < 0.01).

Table 2.

Participant blood panels, inflammation, and oxidative stress markers

	Patients with Peripheral Artery Disease	Healthy Control Participants	P
Glucose, mg/dL	110 ± 32	88 ± 18	0.08
Total cholesterol, mg/dL	174 ± 32	195 ± 30	0.21
High-density lipoprotein, mg/dL	42 ± 7	54 ± 12	0.03
Low-density lipoprotein, mg/dL	102 ± 36	129 ± 21	0.11
Triglycerides, mg/dL	154 ± 30	107 ± 19	0.19
White blood cells, M/μL	8.0 ± 1.7	6.6 ± 2.3	0.19
Red blood cells, M/μL	4.5 ± 0.3	4.9 ± 0.2	<0.01
Hemoglobin, g/dL	14.3 ± 1.2	15.0 ± 0.9	0.21
Hematocrit, %	43 ± 4	45 ± 2	0.19
Intramuscular markers	n = 6	n = 5
Protein carbonyl, nM/mg	3.5 ± 0.4	2.2 ± 0.3	<0.001
4-Hydroxynonenal, AU	0.21 ± 0.01	0.13 ± 0.06	<0.01
Interleukin-6, AU	0.17 ± 0.03	0.09 ± 0.02	<0.01
Plasma markers	n = 6	n = 8
Tumor necrosis factor α, pg/mL	2.1 ± 1.5	0.9 ± 0.2	<0.01
Protein carbonyl, nM/mg	0.03 ± 0.04	0.04 ± 0.03	0.43
Interleukin-6, pg/mL	4.5 ± 1.3	2.3 ± 1.2	<0.01

Data are means ± SD; n = 8 for each variable, except for the intramuscular and plasma markers, which are noted above the columns. Bolded P values indicate significant differences.

Exercising Hemodynamic Responses

The individual and group mean Δblood flow, ΔMAP, and ΔVC responses to exercise at 2.7 W are presented in Fig. 1. The PAD group exhibited a significantly attenuated Δblood flow (Fig. 1A, P < 0.001), an elevated ΔMAP response (Fig. 1B, P < 0.01), and an attenuated ΔVC (Fig. 1C, P < 0.01) compared with the healthy controls. There was a negative correlation between Δblood flow and ΔMAP during 2.7 W exercise (r = −0.68, P < 0.01). The individual and group mean Δblood flow, ΔMAP, and ΔVC responses to exercise at 40% peak power are presented in Fig. 2. During the 40% peak power intensity, the PAD group displayed a significantly attenuated Δblood flow (Fig. 2A, P < 0.001), elevated MAP response (Fig. 2B, P = 0.028), and an attenuated ΔVC (Fig. 2C, P < 0.01) compared with the healthy controls.

Figure 1.

Hemodynamic responses to absolute work rate dynamic plantarflexion exercise. Individual and group changes in popliteal artery blood flow (A), mean arterial pressure (B), and vascular conductance (C) in patients with peripheral artery disease (PAD, open symbols), and healthy controls (closed symbols) during exercise at an absolute work rate of 2.7 W. Dashed line in the box plots represents the mean and the solid line represents the median. Upper and lower limits of the box plot represent the 75th and 25th percentile, respectively. *Significantly different from PAD (P < 0.05). Of note, in B, the significant difference between patients with PAD and controls was maintained with the uppermost patient data point removed (P < 0.01).

Figure 2.

Hemodynamic responses to relative intensity dynamic plantarflexion exercise. Individual and group changes in popliteal artery blood flow (A), mean arterial pressure (B), and vascular conductance (C) in patients with peripheral artery disease (PAD, open symbols) and healthy controls (closed symbols) during exercise at relative intensity of 40% peak power. The dashed line in box plots represents the mean and the solid line represents the median. Upper and lower limits of the box plot represent the 75th and 25th percentile, respectively. *Significantly different from PAD (P < 0.05). Of note, in B, the significant difference between patients with PAD and controls was maintained with the uppermost patient data point removed (P = 0.034).

Correlations between Hemodynamics and Markers of Inflammation and Oxidative Stress

The correlations between Δblood flow during the absolute work rate trial (2.7 W) and ΔMAP during the relative exercise intensity trial (40% peak power) are presented in Table 3. ΔVC was negatively correlated with plasma interleukin-6 (Fig. 3, A and C) and intramuscular protein carbonyl (Fig. 3, B and D) during both the absolute work rate (2.7 W) and relative exercise intensity trials (40% peak power) (all, P < 0.01). ΔVC was negatively correlated with intramuscular interleukin-6 and TNFα during both the absolute work rate (2.7 W) (both r = −0.62, P < 0.05) and relative exercise intensity trials (40% peak power) (r = −0.54 and -0.53, respectively; both P < 0.05). In addition, ABI was correlated with peak work rate during the incremental exercise test (r = 0.85, P < 0.001) and several of the hemodynamic responses to both the absolute work rate (2.7 W) and relative exercise intensity trials (40% peak power). Specifically, ABI was positively correlated with Δblood flow (r = 0.64) and ΔVC (r = 0.63) during both the absolute work rate (2.7 W) and relative exercise intensity trials (40% peak power) (all, P < 0.01). ABI was also associated with ΔMAP during the relative exercise intensity trial (40% peak power) (r = −0.55, P = 0.03).

Table 3.

Pearson’s correlation coefficients for exercise-induced changes in blood flow and mean arterial pressure during plantar flexion exercise with resting intramuscular and plasma markers of inflammation and oxidative stress in patients with peripheral artery disease and well-matched controls

ΔBlood Flow, mL/min	2.7 W Correlation Coefficient	P
Protein carbonyl, im	−0.78	<0.01
4-Hydroxynonenal, im	−0.67	0.03
Interleukin-6, im	−0.69	<0.01
Plasma interleukin-6	−0.72	0.01
Plasma TNFα	−0.57	0.04

ΔMAP, mmHg	40% Peak Correlation Coefficient	P
Protein carbonyl, im	0.6	0.04
Plasma interleukin-6	0.57	0.04
Plasma TNFα	0.82	<0.01

im, intramuscular; MAP, mean arterial pressure; TNFα, tumor necrosis factor-α.

Figure 3.

Relationships between the exercise-induced change in vascular conductance and markers of inflammation and oxidative stress. Vascular conductance was related to plasma interleukin-6 (a systemic marker of inflammation; A and C) and intramuscular protein carbonyl (indicative of intramuscular oxidative stress; B and D) during both the absolute work rate trial (2.7 W; left) and the relative intensity exercise trial (40% peak power; right). Individual data represent both patients with peripheral artery disease (open circles) and healthy controls (filled circles). The solid lines represent the overall correlations (coefficients and P values inset).

DISCUSSION

The main novel finding of this investigation, in agreement with our primary hypothesis, was that patients with PAD exhibited an attenuated exercise-induced increase in blood flow and VC compared with healthy controls, despite an exaggerated MAP response. Furthermore, in line with our second hypothesis, in general, endogenous levels of inflammation and oxidative stress, assessed in both blood and muscle, were strongly related to the exercise-induced changes in blood flow, MAP, and VC during exercise across all participants. Specifically, inflammation and oxidative stress were negatively related to the exercise-induced increase in blood flow and vasodilation, assessed by VC, and positively related to the exercise-induced increase in MAP. In addition, the most common clinical diagnostic assessment of PAD, ABI, positively correlated with the exercise-induced Δblood flow and ΔVC response and negatively correlated with the ΔMAP response. Therefore, these findings support the tenet that both inflammation and oxidative stress may play an important role in the abnormal exercise-induced hemodynamic response exhibited by patients with PAD and highlight potential pathways to target for therapeutic treatment to improve exercise tolerance and quality of life in this population.

Hemodynamic Responses to Exercise

The exercise-induced change in blood flow and VC were lower in the patients with PAD compared with the age- and activity-matched healthy controls during both the absolute WR trial, and, although hard to interpret for blood flow due to the different WRs, the relative intensity exercise trial. This is in agreement with the findings of some (9, 10, 37), but not all (11–13, 38), studies that have quantified the exercise-induced change in blood flow in both patients with PAD and healthy controls. This apparent disagreement is surprising given the widespread attribution of hemodynamic dysfunction to patients with PAD, however, this dysfunction is largely based on attenuated reactive hyperemia in response to occlusion in PAD (37–41). The discrepancy in the aforementioned exercise-induced responses could be a consequence of methodological differences in the assessment of blood flow or the exercise protocols utilized for the comparisons. It should be noted that the current patients were relatively active and able to maintain daily physical activity levels in line with the age-matched healthy controls. This suggests that despite a moderate level of PAD, as classified by their lower ABI, and an apparently dramatically attenuated capacity to increase blood flow in response to exercise, daily functional capacity was not severely limited.

In this investigation, the exercise-induced change in blood flow, during the absolute WR trial (2.7 W), was negatively related to the change in MAP, which is in agreement with previous work that indicated oxygen delivery plays an important role in the exercise pressor response (14–16). This observation, however, disagrees with recent work that suggested the exaggerated MAP in this population was requisite to achieve an appropriate blood flow response to exercise (11, 12). An alternative explanation for this difference is that the level of exercising blood flow achieved by the patients in the present study was bolstered by the elevated MAP response but was still inadequate to match that achieved by the control participants. Future work could usefully use methodologies to attempt to tease apart this reflex in these patients. The absolute work rate performed during this trial was a greater proportion of the patients’ peak work rate compared with controls. Thus, it is reasonable to assume that a greater central command was required in the patients during the effort, in addition to the greater metabolic stress and attenuated O₂ delivery to the exercising muscle, and this contributed to this increased MAP response. Importantly, when matched for relative exercise intensity (40% peak power), which is necessary to appropriately compare blood pressure responses across populations with different exercise capacities, the exercise-induced change in MAP was still greater for the patients, suggesting that there is some inherent stimulus in the patients that contributes to the exaggerated increase in MAP, independent of differences in central command. Recent evidence from animal models of PAD suggests that an increased afferent sensitivity in this population contributes to the exaggerated pressor response (42), potentially due to upregulation of P2X or ASIC3 receptors (43, 44).

Inflammation and PAD

In this investigation, exercise-induced changes in blood flow, VC, and MAP were associated with several markers of inflammation, such as interleukin-6 and TNFα. Previously, markers of inflammation have been related to clinical risk markers such as ABI, brachial artery flow-mediated dilation, and maximal treadmill walking speed (21–23) in patients with PAD. To our knowledge, this study is the first to document a relationship between these inflammatory cytokines and exercise-induced blood flow and VC. To date, the most compelling evidence of a direct role of inflammatory cytokines on the MAP response in PAD was reported by Copp et al. (18) and Xing et al. (45) in a rodent model using femoral artery ligation. These studies reported that inhibition of interleukin-6 (18) and TNFα (45) reduced the MAP response to muscle contraction. However, blood flow was only measured in the former study, and these authors reported that the direct modulation of interleukin-6 had no effect on blood flow during muscle contractions (18). The present investigation found that both the blood flow and MAP responses to exercise were related to level of endogenous inflammation. Further research is required to determine if altering the levels of these inflammatory markers will alter the blood flow, MAP, and VC responses to exercise in patients with PAD. Previous work suggests that proinflammatory cytokines, such as TNFα, impair endothelial function (46, 47) through interactions with nitric oxide signaling pathways (48, 49). Furthermore, it has also been reported that inhibiting TNFα can improve endothelial function in a number of cardiovascular diseases (47, 49–51) and, particularly relevant to the current study, reduce the exercise pressor reflex in the femoral artery ligation rat model of PAD (45). Together, these findings indicate that endogenous inflammation may be a therapeutic target to improve the peripheral hemodynamic responses in patients with PAD. The negative relationships, observed in the present investigation, between inflammatory markers and the exercise-induced change in VC suggest that the higher levels of these cytokines may be linked to an attenuated capacity to vasodilate during exercise.

Oxidative Stress and PAD

In this investigation, intramuscular 4-hydroxynonenal was negatively related to the exercise-induced increase in blood flow and intramuscular protein carbonyl was negatively related to both blood flow and VC and positively related to MAP. Increased levels of 4-hydroxynonenal and protein carbonyls are evidence of irreversible lipid and protein oxidation, respectively (52, 53), and may be common in patients with PAD (26, 34, 54). These markers of oxidative stress have been associated with vascular and endothelial dysfunction (55–57) and could be the cause of the attenuated increase in VC in the patients with PAD observed in the present investigation. The deleterious role of oxidative stress in vascular dysfunction has been explored extensively across the health and disease spectrum (28–33), but patients with PAD have received little attention despite the important role of oxidative stress in the development and progression of this disease (25). To the best of our knowledge, the current report of significant relationships between intramuscular markers of oxidative stress and hemodynamic dysfunction in PAD is novel. These elationships may have important clinical relevance to the exaggerated MAP response in these patients due to the link between blood flow and MAP during exercise (14–16). Previous investigations, which administered antioxidant treatments, revealed that the exaggerated MAP response in PAD could be attenuated by aiming to minimize oxidative stress (5, 19), but these studies did not quantify the blood flow response. This highlights an important question for future research to determine whether these improvements in the exercise-induced MAP response in patients with PAD were due to augmented blood flow and oxygen delivery, the direct alteration in the exercise pressor response (i.e., changes in Group III/IV afferent activation), or some currently unrecognized effect or combination of effects. Based upon our understanding of these interactions, it is likely that this pro-oxidative state leads to an accumulation of oxidative stress and inflammation, which promotes a heightened sensitivity or activation of skeletal muscle afferents (42, 58, 59), but there are likely other mechanisms involved.

Experimental Considerations

There are, of course, limitations when interpreting the results of this investigation. There may have been a self-selection bias in those participants who volunteered for the study, in that the patients with PAD may have had relatively better health than subjects who did not volunteer. However, because the group comprised patients with mild-to-moderate PAD and the controls were well matched for various descriptive characteristics (importantly daily physical activity), this investigation expands the current understanding of the hemodynamic response to exercise in patients with PAD and the potential role of inflammation and oxidative stress. The number of current and past smokers was considerably higher in the patients with PAD, as smoking is a leading risk factor for developing the disease. Smoking is understood to induce systemic inflammation through heightened epigenetic activation of inflammatory pathways (60); this could have contributed to associations observed herein between inflammatory mediators and hemodynamic adjustments. Future investigation in patients with PAD and smokers without clinical signs of PAD is certainly warranted to further dissect how the disease, per se, and smoking individually contribute to the alterations in peripheral hemodynamics. This study did not include an intervention to change the endogenous levels of inflammation or oxidative stress to determine if this would alter the blood flow, MAP, or VC responses to exercise. Because of this limitation, cause and effect cannot be attributed to inflammation and oxidative stress in these exercise-induced responses. However, a recent investigation utilizing a mitochondrial-targeted antioxidant in patients with PAD revealed improvements in both endothelial function and exercise tolerance (61), supporting a role for oxidative stress in the differences, reported herein, between the exercise-induced hemodynamic responses to exercise in the patients with PAD compared with healthy controls.

Perspectives and Significance

Patients with PAD exhibited an attenuated exercise-induced increase in blood flow and VC compared with healthy controls, despite an exaggerated MAP response. In general, endogenous levels of inflammation and oxidative stress, assessed in both blood and muscle, were strongly related to the exercise-induced changes in blood flow, MAP, and VC during exercise across all participants. Furthermore, the most common clinical diagnostic assessment of PAD, ABI, was strongly associated with the exercise-induced changes in the primary physiological measurements quantified in this investigation. Although these associations do not indicate causation, these findings support the tenet that both inflammation and oxidative stress may play an important role in the exaggerated blood pressure response and the impaired oxygen delivery and utilization evident in PAD and highlight potential pathways to target for therapeutic treatment to improve exercise tolerance and quality of life in this population.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.21111646.v1.

Supplemental Fig. S2: https://doi.org/10.6084/m9.figshare.21171424.v1.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants T32HL007576 (to J. C. Craig), T32HL139451 (to J. C. Craig), K99HL125756 (to G. Layec), P01HL091830 (to R. R. Richardson), and R01HL142603 (to J. D. Trinity); the Flight Attendant Medical Research Institute; Veterans Administration Rehabilitation Research and Development Service Awards E6910-R (to R. S. Richardson), E1697-R (to R. S. Richardson), E1433-P (to R. S. Richardson), E9275-L (to R. S. Richardson), and I01CX001999 (to J. D. Trinity), and Small Projects in Rehabilitation Research Award E1572P (to R. S. Richardson).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.C.C., C.R.H., G.L., R.S.R., and J.D.T. conceived and designed research; C.R.H., G.L., O.S.K., and J.D.T. performed experiments; J.C.C. and O.S.K. analyzed data; J.C.C., R.S.R., and J.D.T. interpreted results of experiments; J.C.C. prepared figures; J.C.C. drafted manuscript; J.C.C., C.R.H., G.L., O.S.K., R.S.R., and J.D.T. edited and revised manuscript; J.C.C., C.R.H., G.L., O.S.K., R.S.R., and J.D.T. approved final version of manuscript.