Peripheral Arterial Disease Subjects Research Articles

Effects of acute whole-body and lower limb heating on hemodynamic responses to walking in patients with peripheral artery disease

Background: The exercise pressor reflex is exaggerated in patients with peripheral artery disease (PAD). There has been speculation regarding cardiovascular benefits of heating therapy for patients with PAD. The impact of acute leg heating and whole-body heating on hemodynamic responses during walking remains incompletely understood. The purpose of this study was to evaluate the effects of acute whole-body and lower leg heating on hemodynamic responses to walking in both patients with PAD and healthy control subjects. Hypothesis: We hypothesized that both acute whole-body and lower leg heating would attenuate the pressor response to walking in patients with PAD. Methods: 10 PAD patients (68 ± 7 yrs; Male/Female, N = 7/3) and 8 age-matched healthy control subjects (64 ± 5 yrs, Male/Female, N = 5/3) participated in this study. The treadmill walking test followed the Gardner protocol, involving a constant speed of 2 mph with a 2%-grade increment every 2 minutes. The study encompassed 3 trials/visits: 1) non-heating (control), 2) post leg heating, and 3) post whole-body heating. The order of these visits was randomized. During the leg heating visit, the subjects' lower limbs (below the knees) were immersed in warm water (40-42℃) for 30 minutes. Whole-body heating was administered by outfitting the subjects in a tube-lined heating suit until their internal temperature increased by 0.3℃ (~ 45 min). Hemodynamic responses were assessed by comparing the average systolic blood pressure (SBP) and heart rate (HR) during the last 3 stages of treadmill walking with baseline values. Results: Resting SBP and HR did not exhibit significant differences between groups (all P > 0.05). During the control visit, the increase in SBP due to treadmill walking was significantly (P = 0.04) higher in patients with PAD than in healthy subjects. No group differences were observed in the increase in HR by treadmill walking. The responses in SBP and HR to treadmill walking, following leg heating, were comparable to those observed in the control visit for both groups (all P > 0.05). Compared to the control visit, whole-body heating significantly attenuated the SBP (ΔSBP%, 32.9 ± 13.5 vs. 21.3 ± 9.5 %, P = 0.02) and HR responses (ΔHR%, 61.6 ± 24.3 vs. 42.7 ± 15.6 %, P = 0.03) to treadmill walking in PAD. However, in healthy subjects after whole-body heating, these responses were not different from the control visit (both P > 0.05). Neither whole-body nor leg heating had any impact on the walking distance in patients with PAD. Discussion and Conclusion: Acute whole-body heating significantly attenuated the pressor response to walking in patients with PAD, while lower leg heating exhibited no such impact. These findings suggest potential benefits of acute whole-body heating for patients with PAD. We speculate that the comprehensive systemic adjustments by whole-body heat exposure play a pivotal role in improving the exaggerated pressor response to exercise in PAD. Conversely, local lower leg heating, as observed in our study, seems insuffcient to change the hemodynamic response to exercise in patients with PAD. This study was supported by NIH R01 HL141198 and R01 HL144781. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 544: Evaluating The Diagnostic Ability Of Six Different Artificial Neural Networks From The Subcellular Microenvironment To The Clinical Manifestation

Peripheral arterial disease (PAD) is affecting a large number of patients in the USA and worldwide. The natural history of the disease leads to detrimental clinical implications including walking disability, pain and eventually limb loss. The clinical management of the disease is largely based on clinical criteria (e.g. pain, tissue loss). We have prepared muscle biopsy specimens from patients with PAD versus control subjects and utilizing electron-microscopy we captured in the subcellular level the mitochondrial architecture changes. Using the acquired data as input we tested the ability of six structurally different artificial neural networks (ANNs) to identify PAD patients. For the study we acquired human tissue biopsies from the gastrocnemius muscle. The specimens were processed and cut as ultrathin sections (70 nm) for analysis using a JEM1010 transmission electron microscope. The images were used to generate an image preprocessing database of 992 images and subsequently a custom python-based code was developed with Keras - TensorFlow platforms in order to perform the ANN analysis. We implementing six different deep artificial neural network architectures namely, VGG-19 , ResNet50 , Densenet201 , InceptionV3 , InceptionsResnetV2 and EfficientNetB7.The diagnostic accuracy was found to be respectively: VGG-19 0.81%, ResNet50 0.75%, Densenet201 0.81%, InceptionV3 0.79%, InceptionsResnetV2 0.78% and EfficientNetB7 0.87%.This study presents data for the ability of simple and more advanced ANNs to perform diagnostic predictions based on electron microscopy images of intracellular organelles. The ability to take advantage of applied artificial intelligence technologies and serve this information for clinical decision making could be a pathway for improved patient care. The presented work established insight in some of the currently available deep artificial neural networks, applied in a daily clinical problem and their ability to harness biological information. Our goal is to stimulate the development of higher computational capacity artificial intelligence systems in order to assist clinicians and support improved guidelines geared towards individualized precision medicine.

Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Identifies Abnormal Calf Muscle-Specific Energetics in Peripheral Artery Disease.

Peripheral artery disease (PAD) results in exercise-induced ischemia in leg muscles. 31Phosphorus (P) magnetic resonance spectroscopy demonstrates prolonged phosphocreatine recovery time constant after exercise in PAD but has low signal to noise, low spatial resolution, and requires multinuclear hardware. Chemical exchange saturation transfer (CEST) is a quantitative magnetic resonance imaging method for imaging substrate (CEST asymmetry [CESTasym]) concentration by muscle group. We hypothesized that kinetics measured by CEST could distinguish between patients with PAD and controls. Patients with PAD and age-matched normal subjects were imaged at 3T with a transmit-receive coil around the calf. Four CEST mages were acquired over 24-second intervals. The subjects then performed plantar flexion exercise on a magnetic resonance imaging-compatible ergometer until calf exhaustion. Twenty-five CEST images were obtained at end exercise. Regions of interest were drawn around individual muscle groups, and (CESTasym) decay times were fitted by exponential curve to CEST values. In 10 patients and 11 controls, 31P spectra were obtained 20 minutes later after repeat exercise. Five patients and 5 controls returned at a mean of 1±1 days later for repeat CEST studies. Thirty-five patients with PAD (31 male, age 66±8 years) and 29 controls (11 male, age 63±8 years) were imaged with CEST. The CESTasym decay times for the whole calf (341±332 versus 153±72 seconds; P<0.03) as well as for the gastrocnemius and posterior tibialis were longer in patients with PAD. Agreement between CESTasym decay and phosphocreatine recovery time constant was good. CEST is a magnetic resonance imaging method that can distinguish energetics in patients with PAD from age-matched normal subjects on a per muscle group basis. CEST agrees reasonably well with the gold standard 31P magnetic resonance spectroscopy. Moreover, CEST has higher spatial resolution, creates an image, and does not require multinuclear hardware and thus may be more suitable for clinical studies in PAD.

Novel and robust auxiliary indicators to ankle-brachial index using multi-site pulse arrival time and detrended fluctuation analysis for peripheral arterial disease assessment

The ankle-brachial index (ABI) test is a common tool used for screening peripheral arterial disease (PAD), but its accuracy has been doubted especially in patients with incompressible arteries attributed to calcification. Advanced wearable sensing technologies have potential to offer novel solutions for PAD assessment. This study investigated several novel indicators using the Detrended Fluctuation Analysis (DFA) on beat-to-beat RR interval (RRI) and pulse arrival time (PAT) from the four limbs of 7 young healthy and 7 elderly PAD subjects in supine and sitting postures. The auxiliary value of these indicators in addition to ABI for PAD assessment was also studied using a binary logistic regression model. The results showed that the short-scale DFA coefficients (α1) of RRI, α1 of PATs of fingers and α1_ratio of PAT (the ratio of α1 of PAT between the ipsilateral finger and toe) in supine posture, were significantly different between the PAD and healthy groups (1.26 ± 0.24 vs 0.76 ± 0.21, 1.03 ± 0.30 vs 0.68 ± 0.19, and 0.84 ± 0.20 vs 1.42 ± 0.47, respectively, p < 0.05), and the α1 of RRI and α1_ratio of PAT were not significantly influenced by postural change. Moreover, using ABI together with the supine α1_ratio of PAT was more sensitive in detecting PAD (sensitivity = 92.9%) than ABI alone (78.6%) with the same specificity of 100%. The short-scale DFA coefficients of multi-site PAT in supine posture, which can be measured by wearable devices, is an auxiliary to ABI for detecting PAD.

Abstract 12812: Creatine Chemical Exchange Transfer MRI Demonstrates Abnormal Calf Muscle Energetics in Peripheral Arterial Disease

Introduction: Creatine chemical exchange saturation transfer (CrCEST) is a novel MRI technique utilizing radiofrequency pulses to monitor creatine concentrations at high spatial resolution. The study utilized CrCEST kinetics to compare post-exercise creatine decay in peripheral arterial disease (PAD) patients to normal subjects and to validate against phosphocreatine (PCr) recovery by 31 phosphorus (P) MR spectroscopy (MRS). Methods: 23 subjects with PAD (claudication and ankle-brachial index&lt;0.9) and 28 healthy subjects were enrolled. All underwent 3T MRI (Siemens Prisma) using a knee coil around the calf. Pre- and post-exercise images were obtained around plantar flexion ergometry (Ergospect) until exhaustion. Regions of interests were drawn around major muscle groups. Creatine decay times were obtained by fitting an exponential curve to the CrCEST values. 31 P spectra were obtained 20 minutes later at peak exercise using a surface-coil localized, free induction decay acquisition with 25 signal averages at a repetition time of 550s with calculation of PCr recovery time constant. Results: 23 PAD subjects (mean ABI 0.66 ± 0.11, age 64 ± 8, 17% females) had prolonged median overall calf muscle creatine decay time of 286 seconds (s) (Interquartile Range (IQR) 134 to 370) versus 152 s (IQR 114 to 193) in controls (age 63 ± 9, 70% females), p = 0.009. The gastrocnemius muscle demonstrated a creatine decay time of 316 s in PAD (IQR 189 to 510) versus 181 s in controls (IQR 102 to 236), p = 0.003 and the posterior tibialis muscle, 186 s (IQR 119 to 653) versus 125 s (IQR 72 to 221), p = 0.013. 8 PAD patients and 6 controls underwent CrCEST and 31 P MRS which were compared over the entire calf. Bland-Altman analysis (Figure 1B) yielded a mean difference of 71s (SD 109). Conclusions: Imaging with CrCEST demonstrates prolonged creatine kinetics in PAD patients compared to normal subjects with high spatial resolution and good agreement with 31 P MRS, although PCr recovery appears faster.

Investigation of Roll-Over Characteristics in Healthy Old Individuals and Patients With Peripheral Artery Disease

Atherosclerotic blockages in the leg arteries, cause leg pain with ambulation (called claudication), impair gait and substantially reduce the walking ability of patients with peripheral artery disease (PAD). Ankle-foot orthoses are being developed and applied so patients can walk more and with less pain. Roll-over shape (ROS) is a potential design objective for such assistive devices. In the proposed work, we study roll-over characteristics in patients with PAD and healthy older subjects. Gait data of ten healthy older individuals (Age: 72.8 ± 5.5 years) and twenty patients with PAD (Age: 64.1 ± 6.6 years) were collected at self-selected walking speed. In patients with PAD, gait data were collected before the onset of pain and after claudication pain was induced. To generate ROS, the center of pressure data was transformed to the shank-based coordinate system and circular arcs were fit using an optimization program in MATLAB. Independent t-tests with Bonferroni corrections were used to separately compare roll-over radius differences (p<0.05) between healthy older to both walking conditions in patients with PAD. The mean roll-over radius was not significantly different between healthy older vs PAD pain-free (p=0.468) or PAD pain-induced (p=0.289) walking conditions. Our results indicate invariance of ROS radius in patients with PAD, which is consistent with previous literature showing general invariance of ROS in healthy young individuals. Previous biomechanical studies show gait kinematics and kinetics are more affected by PAD than by age. Future studies should focus on the potential adaptive mechanisms in patients with PAD achieving invariant ROS.

Association of apolipoprotein levels with peripheral arterial disease: a meta-analysis of literature studies.

Lower limb peripheral artery disease (PAD) is a leading cause of atherosclerotic cardiovascular disease (ASCVD). Discordant data are available on the association between apolipoprotein and PAD. We performed a meta-analyses on the association between apolipoprotein (apo)B, apoA-I, and apoB/apoA-I ratio with PAD. PubMed, Web of Science, Scopus databases were systematically searched. Studies providing data about apoB, apoA-I, apoB/apoA-I ratio in PAD subjects and non-PAD controls were included. Differences between PAD and non-PAD subjects were expressed as mean difference (MD) with pertinent 95% confidence intervals (95%CI). Twenty-two studies were included. Peripheral artery disease subjects showed higher apoB (MD: 12.5 mg/dL, 95%CI: 2.14, 22.87) and lower apoA-I levels (MD: -7.11 mg/dL, 95%CI: -11.94, -2.28) than non-PAD controls. Accordingly, ApoB/ApoA-I ratio resulted higher in PAD subjects than non-PAD controls (MD: 0.11, 95% CI: 0.00, 0.21). Non-HDL-C showed a direct association with the difference in apoB (z-value: 4.72, P < 0.001) and an inverse association with the difference of apoA-I (z-value: -2.43, P = 0.015) between PAD subjects and non-PAD controls. An increasing BMI was associated with an increasing difference in apoA-I values between PAD subjects and non-PAD controls (z-value: 1.98, P = 0.047). Our meta-analysis suggests that PAD subjects exhibit increased apoB and reduced apoA-I levels, accompanied by an increased apoB/apoA-I ratio as compared with non-PAD controls.

Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

Peripheral Artery Disease in Vulnerable Patient Populations: Outcomes of Orbital Atherectomy in Native Americans Compared to Non-Native Americans. A Single-Center Experience in Rural Oklahoma

Background/purposeAlthough the incidence of peripheral artery disease (PAD) and amputations is higher in Native Americans (NA) than Caucasians, the study of revascularization NA is limited, resulting in their under representation in clinical studies. Orbital atherectomy (OA) is widely utilized for endovascular revascularization of significantly calcified peripheral arteries and has been shown to improve limb salvage rates. Methods/materialsA cohort of 74 consecutive PAD subjects undergoing OA treatment was retrospectively analyzed via Kaplan Meier (KM) and Propensity Score Matched (PSM) analysis. ResultsA significant proportion of the subjects were NA (16.2%). Compared to the non-NA, the NA had higher numerical baseline rates of wounds, dialysis, chronic kidney disease (CKD), and critical limb ischemia, but were numerically less likely to smoke and had similar rates of diabetes. There were very high rates of severe calcification (100% vs. 87%) and pre-procedure diameter stenosis (99% vs. 95%) in both groups. The NA and non-NA had good angiographic outcomes, resulting in low rates of post-procedure residual diameter stenosis (10% vs. 11%). Lastly, KM analysis indicated high freedom from amputation in both groups at 1 year (89% vs. 95%), as well as in the PSM subjects (89% vs. 100%). ConclusionsDespite numerically higher rates of co-morbidities at baseline (e.g., CKD, dialysis, and presence of non-healing wounds), the NA underwent successful revascularization with OA, resulting in high freedom from amputation at 1-year. Given the small sample size of NA, these results may not be generalizable—thus, larger studies on NA are warranted.

Associations of Peripheral Artery Disease With Calf Skeletal Muscle Mitochondrial DNA Heteroplasmy.

BackgroundPatients with peripheral artery disease (PAD) undergo frequent episodes of ischemia‐reperfusion in lower extremity muscles that may negatively affect mitochondrial health and are associated with impaired mobility. We hypothesized that skeletal muscle from PAD patients will show high mitochondrial DNA heteroplasmy, especially in regions more susceptible to oxidative damage, such as the displacement loop, and that the degree of heteroplasmy will be correlated with the severity of ischemia and mobility impairment.Methods and ResultsMitochondrial mutations and deletions and their relative abundance were identified by targeted mitochondrial DNA sequencing in biopsy specimens of gastrocnemius muscle from 33 PAD (ankle brachial index <0.9) and 9 non‐PAD (ankle brachial index >0.9) subjects aged ≥60 years. The probability of heteroplasmy per DNA base was significantly higher for PAD subjects than non‐PAD within each region. In adjusted models, PAD was associated with higher heteroplasmy than non‐PAD (P=0.003), but the association was limited to microheteroplasmy, that is heteroplasmy found in 1% to 5% of all mitochondrial genomes (P=0.004). Heteroplasmy in the displacement loop and coding regions were significantly higher for PAD than non‐PAD subjects after adjustment for age, sex, race, and diabetes mellitus (P=0.037 and 0.004, respectively). Low mitochondrial damage, defined by both low mitochondrial DNA copy number and low microheteroplasmy, was associated with better walking performance.ConclusionsPeople with PAD have higher “low frequency” heteroplasmy in gastrocnemius muscle compared with people without PAD. Among people with PAD, those who had evidence of least mitochondrial damage, had better walking performance than those with more mitochondrial damage.RegistrationURL: http://www.clinicaltrials.gov. Unique identifier: NCT02246660.

P948Rapid and automated risk stratification by determination of aortic stiffness in healthy and cardiovascular diseased

Abstract Background and aims Aortic stiffness is an independent predictor of cardiovascular morbidity and mortality and thus simple, rapid and preferably automated techniques are indispensableto persue a global approach in risk statification. A novel oscillometric based carotido-femoral pulse-wave velocity (cfPWV) was investigated, regarding diagnostic accuracy, sensitivity and specificity and with emphasis on costs, training curves and procedural time effort. Methods In a single-center crossover study, we evaluated subjects free of known cardiovascular disease (CVD) and CVD subjects and a subgroup with peripheral artery disease (PAD) undergoing ankle-brachial index (ABI) and PWV measurements using oscillometry (BoSo, Bosch + Sohn, Germany) compared to tonometry (SphymoCor, Atcor Australia). Pearson's correlation analysis was used to assess the relationship of PWV measurements for both methods. Moreover, examination times and costs were compared. Results A total of 174 study subjects underwent assessment of oscillometric and tonometric PWV measurements. CVD-free subjects (n=57) were younger (60.4±15.6 vs. 67±12.9 years, p=0.003) compared to CVD subjects (n=117). PWV measurements showed significant correlations in CVD-free subjects (r=0.797, p&lt;0.001), in CVD subjects (r=0.817, p&lt;0.001) and the subgroup of PAD subjects (r=0.807, p&lt;0.001). Sensitivity, specificity, positive predictive value and negative predictive value of the oscillometric measurement for detecting arterial stiffness reached 93%, 84%, 86%, and 92%. The examination time of the oscillometric method was shorter compared to tonometry (4.4±0.5 vs. 9.2±0.8 min, p&lt;0.001) in line with reduced costs. Conclusion Using a simple and rapid automated oscillometric method, we provide good diagnostic accuracy for the determination of aortic stiffness through PWV, both in CVD-free and in CVD subjects. This might help in terms of cost-effectiveness and simplification in daily practice to screen for cardiovascular morbidity and for vascular damage in atherosclerosis.

Cardiovascular long-term outcome and prophylactic treatment patterns in peripheral arterial disease in a population-based cohort.

This study evaluates 10-year follow-up data on associated comorbidity, mortality, and pharmacological treatment patterns for men and women with different stages of peripheral arterial disease (PAD) in a population-based setting. This was a prospective observational population-based cohort study, based on physical examinations and questionnaires at baseline supplemented with national register data between 2005 and 2015. Subjects were placed in subgroups defined by ankle-brachial index levels and reported symptoms; asymptomatic PAD (APAD), intermittent claudication (IC), severe limb ischaemia (SLI), or references (Ref). Cox proportional hazards regression models were used for analysis with adjustments for sex and baseline age and comorbidity. The cohort consisted of 5080 subjects (45% males). At baseline, APAD, IC, and SLI were prevalent in 559 (11%), 320 (6.3%), and 78 (1.5%) subjects, respectively. A significant increased risk for cardiovascular (CV) death, even when adjusted for age and baseline morbidity, were noted in all PAD stages as compared with reference group with a small difference between APAD and IC, an adjusted hazard ratio 1.80 (confidence interval 1.45-2.22) and 1.95 (1.50-2.53), respectively. Only about 60% of PAD subjects received medical prophylactic treatment as recommended in guidelines. Peripheral arterial disease subjects had significantly increased CV morbidity and mortality risks, especially males. Asymptomatic PAD subjects confer similar risk for CV events as symptomatic patients. Our findings motivate enhanced preventive efforts of all PAD stages, including in asymptomatic disease.

Diabetes and smoking are more important for prognosis of patients with peripheral arterial disease than some genetic polymorphisms.

Background: The role of genetic polymorphisms in peripheral arterial disease (PAD) is incompletely understood. We tested whether selected single nucleotide polymorphisms (SNPs) were associated with PAD and with adverse events in an observational study cohort. Also, the role of diabetes and smoking was studied. Patients and methods: 742 patients with PAD and 713 age- and gender-matched control subjects were subjected to yearly physical and laboratory investigations and were managed for 5 years according to the European guidelines on cardiovascular disease prevention. The occurrence of all-cause death, cardiovascular death, non-fatal myocardial infarction, ischemic stroke or critical limb ischemia (major events) and revascularization procedures (minor events) was recorded. In 655 patients with PAD and 612 control subjects the following SNPs were determined: rs1466408, rs13428968 and rs12803 of NR4A2 gene, rs10499563 of IL6 gene, rs668 and rs12953 of PECAM1 gene, and rs10861032 of Chr12 locus. Results: The distribution of selected SNPs did not differ between patients with PAD and control subjects, and neither between subjects with or without major adverse events. In contrast, diabetes and smoking affected survival and event-free survival. Among patients with PAD, diabetes doubled the hazard ratio (HR) for cardiovascular death and smoking doubled the HR for death or major event. The 5-year survival of diabetics with PAD was 0.80 (CI 0.75-0.85) and of non-diabetics with PAD 0.87 (CI 0.84-0.90), p = 0.045. The 5-year survival of active smokers with PA D was 0.80 (CI 0.75-0.62), of former smokers 0.83 (CI 0.79-0.88), and of never-smokers 0.89 (CI 0.86-0.93), p = 0.024. Conclusions: SNPs of NR4A2, IL6, PECAM1 and Chr12 were not associated with PAD or with major adverse events. However, diabetes and smoking were associated with worse survival and event-free survival in patients with PAD.

Microparticles Carrying Sonic Hedgehog Are Increased in Humans with Peripheral Artery Disease.

Sonic hedgehog (Shh) is a prototypical angiogenic agent with a crucial role in the regulation of angiogenesis. Experimental studies have shown that Shh is upregulated in response to ischemia. Also, Shh may be found on the surface of circulating microparticles (MPs) and MPs bearing Shh (Shh + MPs) have shown the ability to contribute to reparative neovascularization after ischemic injury in mice. The goal of this study was to test the hypothesis that, in humans with peripheral artery disease (PAD), there is increased number of circulating Shh + MPs. This was done by assessing the number of Shh + MPs in plasma of patients with PAD and control subjects without PAD. We found significantly higher number of Shh + MPs in plasma of subjects with PAD, compared to controls, while the global number of MPs—produced either by endothelial cells, platelets, leukocytes, and erythrocytes—was not different between PAD patients and controls. We also found a significant association between the number of Shh + MPs and the number of collateral vessels in the ischemic limbs of PAD patients. Interestingly, the concentration of Shh protein unbound to MPs—which was measured in MP-depleted plasma—was not different between subjects with PAD and the controls, indicating that, in the setting of PAD, the call for Shh recapitulation does not lead to secretion of protein into the blood but to binding of the protein to the membrane of MPs. These findings provide novel information on Shh signaling during ischemia in humans, with potentially important biological and clinical implications.

Innovative Multi-Site Photoplethysmography Analysis for Quantifying Pulse Amplitude and Timing Variability Characteristics in Peripheral Arterial Disease.

Photoplethysmography (PPG) is a simple-to-perform vascular optics measurement technique that can detect blood volume changes in the microvascular bed of tissue. Beat-to-beat analysis of the PPG waveform enables the study of the variability of pulse features, such as the amplitude and the pulse arrival time (PAT), and when quantified in the time and frequency domains, has considerable potential to shed light on perfusion changes associated with peripheral arterial disease (PAD). In this pilot study, innovative multi-site bilateral finger and toe PPG recordings from 43 healthy control subjects and 31 PAD subjects were compared (recordings each at least five minutes, collected in a warm temperature-controlled room). Beat-to-beat normalized amplitude variability and PAT variability were then quantified in the time-domain using two simple statistical measures and in the frequency-domain bilaterally using magnitude squared coherence (MSC). Significantly reduced normalized amplitude variability (healthy control 0.0384 (interquartile range 0.0217–0.0744) vs. PAD 0.0160 (0.0080–0.0338) (p < 0.0001)) and significantly increased PAT variability (healthy control 0.0063 (0.0052–0.0086) vs. PAD 0.0093 (0.0078–0.0144) (p < 0.0001)) was demonstrated for the toe site in PAD using the time-domain analysis. Frequency-domain analysis demonstrated significantly lower MSC values across a range of frequency bands for PAD patients. These changes suggest a loss of right-to-left body side coherence and cardiovascular control in PAD. This study has also demonstrated the feasibility of using these measurement and analysis methods in studies investigating multi-site PPG variability for a wide range of cardiac and vascular patient groups.

Beet the Best?

A primary goal of therapy for patients with peripheral artery disease (PAD) and intermittent claudication is increased ambulatory function. Supervised exercise rehabilitation was recently shown to confer superior walking benefits to pharmacological or surgical interventions. Increases in plasma inorganic nitrite, via oral nitrate, have been shown to increase exercise performance in both human and animal models, especially in hypoxic conditions. To determine whether a 36-session exercise rehabilitation program while consuming oral inorganic nitrate (4.2 mmol concentrated beetroot juice) would produce superior benefits over exercise plus placebo in pain-free walking and markers of increased skeletal muscle perfusion in patients with PAD and intermittent claudication. This was a randomized, double-blind, per-protocol study design. After the 12-week protocol, claudication onset time on a maximal treadmill test increased by 59.2±57.3 s for the exercise plus placebo group (n=13) and by 180.3±46.6 s for the exercise plus beetroot juice group (n=11; P≤0.05). This produced a between treatment medium to large standardized effect size (Cohen d) of 0.62 (95% CI, -0.23 to +1.44). The data for 6-minute walk distance showed a similar pattern with increases of 24.6±12.1 and 53.3±19.6 m ( P≤0.05) in the exercise plus placebo and exercise plus beetroot juice groups, respectively. Measures of gastrocnemius perfusion, including ankle-brachial index, peak reactive hyperemic blood flow, and tissue deoxygenation characteristics, during exercise (assessed my near-infrared spectroscopy) all changed significantly for the exercise plus beetroot juice group with moderate-to-large effect sizes over exercise plus placebo changes. Although it is premature to speculate on overall clinical utility of a nitrate-based therapy for PAD, this early pilot study evidence is encouraging. Specifically, our data suggests that increasing plasma nitrite before exercise may allow PAD subjects to train with less pain, at higher workloads for longer durations at each training session, thereby maximizing the beneficial peripheral vascular and skeletal muscle adaptations. URL: http://www.clinicaltrials.gov . Unique identifier: NCT01684930 and NCT01785524.

Leukocyte Phenotype Is Altered in Peripheral Arterial Disease

Peripheral arterial disease (PAD) is characterized by low-grade systemic inflammation. Monocytes and monocyte-derived macrophages (MDMs) play a central role in vascular inflammation and its resolution. We hypothesize that impaired resolution in PAD contributes to adverse clinical outcomes. In a cross-sectional study, we profiled serum cytokines, phagocytic activity of leukocytes, monocyte cell surface markers, and gene expression of MDM from healthy individuals (n ≥ 10) and patients with stable claudication (n ≥ 10). Leukocyte phagocytosis of fluorescently labeled Escherichia coli and monocyte surface markers were determined by flow cytometry. MDMs were cultured from peripheral blood monocytes isolated by density gradient centrifugation. MDM cytokine production and gene expression, before and after stimulation with lipopolysaccharide, were determined by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. Patients with PAD had elevated serum high-sensitivity C-reactive protein (3.7 vs 0.6 mg/L; P = .004) and interleukin (IL) 6 (5.1 vs 1.1 pg/mL; P = .01) and trended toward higher levels of monocyte chemoattractant protein 1 and lower adiponectin compared with healthy individuals. Circulating monocytes and polymorphonuclear cells (PMNs) from PAD patients had reduced phagocytic activity for E. coli (monocytes, >30% [P < .001]; PMNs, >25% [P < .01]). Flow cytometry demonstrated a higher proportion of the proinflammatory intermediate monocyte subset (CD14++16+, 1.8-fold; P = .04) in PAD subjects. MDMs from PAD patients retain their intrinsic inflammatory program, producing more IL-6 (>4-fold; P = .03) and IL-1β (>10-fold; P = .04) and demonstrating increased expression of M1 genes (tumor necrosis factor α, monocyte chemoattractant protein 1, CXCL10) and decreased expression of M2 genes (CCL17, MRC1) vs healthy individuals (Table). Clinically stable PAD patients have elevated serum inflammatory markers compared with healthy individuals. Circulating monocytes and PMNs in patients with PAD have reduced phagocytic activity and a greater proportion of the proinflammatory intermediate monocyte subset. MDMs from PAD patients preserve their elevated inflammatory state in culture. These data demonstrate a heightened inflammatory and impaired resolution phenotype in PAD that has potential implications for disease progression and response to interventions.TableMonocyte-derived macrophage (MDM) gene expression of the peripheral arterial disease (PAD) cohort vs healthy individualsGeneMl or M2PAD patients relative to healthy individualsExpression fold changeP valueTNF-α (veh)Ml2.71.02aTNF-α (LPS)0.81.33MCP-1 (veh)Ml1.92.05aMCP-1 (LPS)1.91.01aiNOS (veh)Ml1.34.26iNOS (LPS)1.04.94CXCL10 (veh)Ml7.87.04aCXCL10 (LPS)1.8129IL-10 (veh)M21.50.19IL-10 (LPS)2.72.009aCCL17 (veh)M20.48.09CCL17 (LPS)022.14MRC1 (veh)M20.52.09MRC1 (LPS)0.30.01aCCL17, Chemokine (C-C motif) ligand 17; CXCL10, C-X-C motif chemokine 10; IL-10, interleukin 10; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; MRC1, mannose receptor C type 1; TNF-α, tumor necrosis factor α; veh, vehicle.Expression fold change was calculated as 2ˆ(–ΔΔCT) and represents the fold change in the average expression of the target gene within the PAD cohort (n = 10) vs the average expression of the target gene within the cohort of healthy individuals (n = 10), normalized to the housekeeping gene (HPRT). MDMs were stimulated with lipopolysaccharide to mimic the occurrence of an acute inflammatory event or vehicle for 24 hours before assessment of gene expression.aP < .05 by unpaired Student t-test. Open table in a new tab

Epidemiology of peripheral artery disease in Europe: VAS Educational Paper.

This paper reviews the current epidemiological data on peripheral artery disease (PAD) in Europe. Cardiovascular disease (CVD) burden is presented and PAD prevalence is discussed, as compared to coronary (CAD) and cerebrovascular (CeVD) diseases, in European Union (EU) countries and European continent countries. The article reviews PAD clinical manifestations, PAD diagnosis, risk factors for PAD incidence and progression, PAD prognosis and financial implications. CVD mortality is higher in women than in men and in European continent countries, as compared to EU countries. While CAD and CeVD have a lower prevalence in EU countries, as compared to the whole continent, PAD and other CVDs still have a higher prevalence in EU countries. PAD prevalence vary widely between countries, increases sharply with aging and has a relation with ethnicity. CVD comorbidities are high in PAD subjects and particularly high in those with critical limb ischemia. PAD prognosis is related to CAD and CeVD mortality, that are particularly high in critical limb ischemia. This review promotes the need for global awareness on PAD burden, stimulates PAD screening and emphasizes the importance of early diagnosis and treatment of PAD and associated cardiovascular comorbidities.

Abstract 262: Circulating Exosomes from PAD Patients Modulate Vascular Repair and Inflammation

Objectives: Peripheral arterial disease (PAD) is a chronic disease characterized by inflammation. Recent work suggests that circulating exosomes may contribute to vascular injury and remodeling. We hypothesize that exosomes from PAD subjects negatively modulate vascular repair via miRNA and bioactive lipid mediators (LM). Methods: Exosomes (particle size 30-100nm) were isolated from plasma of healthy (n=6) and PAD (n=6) subjects. Exosome miRNA was isolated and assessed by qPCR. Targeted metabolo-lipidomics was performed by liquid-chromatography-tandem mass spectrometry. VSMC and EC migration were assessed via scratch assay. Monocyte-derived macrophage gene expression after exposure to exosomes was assessed via RT-qPCR. Results: Compared to healthy subjects, exosomes from PAD subjects contained lower levels of pro-angiogenic miR-126 and miR-210 (25.2±6.4 vs 8.3±1.7, p&lt;0.05 and 0.29±0.07 vs 0.08±0.02, p&lt;0.05, respectively). Exosomes contained arachidonic acid, eicosapentaenoic acid and docosapentanoic acid, as well as both pro-inflammatory and pro-resolving bioactive LMs and their pathway markers, including prostaglandins, leukotrienes, lipoxins, resolvins (D- and E-series) and maresins. By principle component analysis, exosome LM profiles differed significantly between healthy and PAD subjects. Exosomes from PAD subjects increased VSMC migration (1.5±.09-fold vs 1.0±.09-fold wound closure, p&lt;0.005) and decreased EC migration (1.5±.04-fold vs. 1.8±.06-fold wound closure, p&lt;0.005) compared to healthy controls. Both PAD and healthy exosomes increased MDM expression of pro-inflammatory genes TNF-α and MCP-1. Conclusion: Plasma-derived exosomes from PAD patients contain an altered profile of vascular-active miRNA and LMs and confer effects on VSMCs and ECs that may impair vessel remodeling. We describe the first known evidence that plasma exosomes contain pro-resolving LMs. Collectively these data suggest that circulating exosome-based signaling may modulate vascular inflammation and repair in PAD patients.

Abstract 136: Near-Infrared Spectroscopy for Diagnosis and Characterization of Peripheral Artery Disease Severity

Background: Peripheral artery disease (PAD) frequently is undertreated. ABI is the standard for diagnosis of PAD but is limited in its ability to predict functional status and risk of disease progression. Previous work with Near-Infrared Spectroscopy (NIRS) has demonstrated impaired oxygen utilization in PAD muscle and may allow for improved diagnosis and stratification of disease severity. We hypothesize that exercise produces characteristic changes in lower-extremity muscle oxygenated heme percent (StO 2 ) which differentiates PAD from control and predicts PAD severity better than ABI. Methods: We recruited 31 PAD subjects with intermittent claudication (IC) (ABI &lt; 0.9) and 9 controls (ABI ≥ 0.9 and no IC). All subjects completed a Gardner maximal treadmill test. PAD subjects walked until IC was prohibitive (peak walking time, PWT) and controls walked for 540 seconds. StO 2 measurements were taken from the lateral gastrocnemius with the wireless MOXY NIRS monitor. StO 2 was documented at baseline, 60s, claudication onset time (COT), and PWT. For each subject, StO 2 values were expressed as percent of baseline to allow for comparison across subjects. Data were analyzed by student’s t-test and linear regression. Results: Mean baseline StO 2 values were 46±11% and 58±17%, respectively, for PAD and control subjects. Among controls, StO 2 dropped below baseline at 140±101s whereas PAD subjects dropped below baseline at 6±10s (P&lt;0.001). PAD StO 2 s at 60s, COT, and PWT were compared to control StO 2 s at the corresponding time points. PAD patients’ StO 2 at 60s, COT, and PWT were 65.2±37.9% (n=31), 66.4±35.5% (n=31), and 73.2±26.0% (n=25) lower than controls, respectively (P &lt; 0.001). PAD subjects were separated into tertiles of PWT. Relative to baseline, StO 2 decreased 84.2±18.4% (n=11) in the lowest tertile compared to 29.8±30.6% (n=10) in the highest tertile (P&lt;0.05). Percent decrease in StO 2 at 60s correlated to PWT (R 2 =0.56) in contrast to ABI (R 2 =0.018). Conclusion: During exercise, PAD patients exhibited a characteristic change in StO 2 . In contrast to ABI, StO 2 predicted PAD-related exercise limitation. These data suggest NIRS in conjunction with traditional methods could be used to guide diagnostic and treatment decisions.