The aim of this study was to determine whether persistently high levels of interleukin-6 (IL-6) or soluble vascular adhesion molecule–1 (sVCAM-1) are associated with faster functional decline compared to fluctuating or persistently low biomarker levels in 255 participants with peripheral arterial disease. Participants underwent baseline and ≥2 annual follow-up measures of IL-6 and sVCAM-1. Participants were categorized as follows: category 1, annual levels of IL-6 (or sVCAM-1) were in the lowest tertile for ≥3 study visits; category 3, annual levels of IL-6 (or sVCAM-1) were in the highest tertile for ≥3 visits. Category 2 levels of IL-6 (or sVCAM-1) did not meet criteria for group 1 or 3. Six-minute walking distance, fastest paced 4-m walking velocity, and the Short Physical Performance Battery were measured annually. Results were adjusted for age, gender, race, co-morbidities, statin use, physical activity, the ankle-brachial index, and other confounders. Across IL-6 categories, average annual decreases in 6-minute walking distance were −21.4 feet in category 1, −49.2 feet in category 2, and −76.8 feet in category 3 (p for trend = 0.013), and average annual decreases in Short Physical Performance Battery score were −0.18, −0.45, and −0.62, respectively (p for trend = 0.022). Similar associations of IL-6 categories with decrease in fastest paced walking velocity were observed (p for trend = 0.034). There were no significant associations of sVCAM-1 categories with functional decline. In conclusion, in participants with peripheral arterial disease, persistently high IL-6 levels are associated with faster functional decline compared to those with fluctuating or persistently low IL-6 levels.
Persistently high levels of inflammation may promote functional decline in patients with lower-extremity peripheral arterial disease (PAD) if persistent inflammation impairs skeletal muscle function or promotes more rapid progression of lower-extremity atherosclerosis. We studied associations of persistently high levels of interleukin-6 (IL-6) and soluble vascular cellular adhesion molecule–1 (sVCAM-1) with average annual functional decline in participants with PAD.
Methods
The protocol received institutional review board approval from Northwestern University Feinberg School of Medicine and Catholic Health Partners Hospitals. Participants gave informed consent. Participants were men and women with PAD in the Walking and Leg Circulation Study (WALCS) II who had blood drawn during ≥3 of their 4 annual WALCS II visits.
The WALCS II cohort consisted of 368 participants with PAD completing their fourth annual follow-up visit for the original WALCS cohort and 402 newly identified participants with PAD. Participants with PAD were aged ≥59 years at baseline and were identified from among consecutive patients in the noninvasive vascular laboratories at Chicago-area hospitals. Baseline data were collected from November 2002 to May 2004. Follow-up data were collected annually through October 31, 2009.
PAD was defined as an ankle-brachial index <0.90 at baseline. Patients with recent major surgery were excluded. At the time of enrollment for WALCS and WALCS II, patients with dementia, nursing home residents, wheelchair-bound patients, and patients with foot or leg amputations or critical limb ischemia were excluded. Non-English-speaking patients were excluded because investigators were not fluent in non-English languages.
Participants rested supine for 5 minutes before the ankle-brachial index measurement. Using a handheld Doppler probe (Nicolet Vascular Pocket Dop II; Nicolet Biomedical Inc., Golden, Colorado), systolic blood pressures were measured in the following order: right brachial artery, right dorsalis pedis and posterior tibial arteries, left dorsalis pedis and posterior tibial arteries, and left brachial artery. Two sets of measurements were obtained. The mean of the dorsalis pedis and posterior tibial pressures in each leg was divided by the mean of all 4 brachial pressures to obtain the ankle-brachial index value. Subclavian stenosis was suspected if the right or left brachial pressure was higher in both measurement sets and there was a difference of ≥10 mm Hg between the 2 brachial pressures in either measurement set. In these cases, the average brachial pressure in the arm with highest pressure was used to calculate the ankle-brachial index.
Functional performance was measured annually with 6-minute walking distance, 4-m walking velocity at usual and fastest paces, and the Short Physical Performance Battery (SPPB). The 6-minute walk measures walking endurance and correlates with physical activity levels in patients with PAD. Participants walk up and down a 100-foot hallway for 6 minutes after instructions to cover as much distance as possible. Walking velocity was measured with a 4-m walk performed at “usual” and “fastest” paces. Each walk was performed twice. The faster walk in each pair was used in analyses.
The SPPB is a global measure of leg functioning that predicts mobility loss, nursing home placement, and mortality. To calculate the SPPB score, a 0-to-4 score is assigned for performance on 3 measures of lower-extremity functioning: usual paced 4-m walking velocity, time to rise from a seated position 5 times, and standing balance, respectively. Subjects receive a zero score for each task they are unable to complete. One to 4 scores for each task are assigned on the basis of quartiles of performance for >6,000 participants in the Established Populations for the Epidemiologic Study of the Elderly. Scores are summed to obtain the SPPB score, ranging from 0 to 12. For the 5 timed repeated chair rises, participants sit in a straight-backed chair with arms folded across their chest and stand 5 times consecutively as quickly as possible. The time to complete 5 chair rises is measured. For standing balance, participants were asked to hold 3 increasingly difficult standing positions for 10 seconds each: standing with feet together side by side and parallel (side-by-side stand), standing with feet parallel with the toes of 1 foot adjacent to and touching the heel of the opposite foot (semitandem stand), and standing with 1 foot directly in front of the other (tandem stand).
Blood specimens were collected at baseline and at each annual follow-up visit. Blood specimens were immediately processed for storage at −70°C until the time of measurement. IL-6 and sVCAM-1 were measured by enzyme-linked immunosorbent assays from R&D Systems (Minneapolis, Minnesota). Coefficient of variation percentage values were calculated from a randomly selected subset of samples in WALCS II at baseline and were 4.21% for IL-6 and 5.61% for sVCAM-1. Similar variability was observed for follow-up visits. Total cholesterol levels were measured in blood samples stored at −70°C, using enzymatic reaction with peroxidase/phenol-4-aminoiphenazone indicator reaction. High-density lipoprotein cholesterol was measured using a direct enzymatic colorimetric assay.
Algorithms developed for the Women’s Health and Aging Study and the Cardiovascular Health Study were used to document co-morbidities. These algorithms combine data from patient report, physical examination, medical record review, medications, laboratory values, and a primary care physician questionnaire. Co-morbidities assessed were angina, diabetes mellitus, myocardial infarction, stroke, heart failure, pulmonary disease, cancer, spinal stenosis, and disk disease. History of hypertension was based on patient report of physician-diagnosed hypertension or results from the primary care physician questionnaire.
The study principal investigator (M.M.M.), blinded to all other patient data, reviewed lists of medications at each visit and identified the presence or absence of use of statins, diabetes medications, cilostazol, pentoxifylline, nonsteroidal anti-inflammatory drugs, and steroids. Height was measured at baseline, and weight was measured at each study visit. Body mass index was calculated as weight divided by the square of height. Walking exercise and cigarette smoking were based on patient report. Physical activity was assessed with patient report of the number of blocks walked in the past week. Leg symptoms were measured with the San Diego Claudication Questionnaire, on the basis of a previous study.
Tertiles of IL-6 and sVCAM-1 were defined for the baseline visit and at each annual follow-up visit, respectively, for up to 4 visits. Participants were classified according to the number of visits at which their IL-6 levels decreased within the highest and lowest tertiles. Participants whose IL-6 values fell within the lowest tertile of IL-6 in ≥3 of the 4 study visits (i.e., persistently low inflammation) were classified in category 1 for IL-6. Participants whose IL-6 values fell within the highest tertile of IL-6 in ≥3 study visits (persistently high inflammation) were classified in category 3 for IL-6. Remaining participants, who had intermittently high or low levels of each biomarker and did not meet criteria for category 1 or category 3, were classified in category 2. Similar categories were defined for sVCAM-1 values. We used tertiles to define persistently high and low inflammation because tertiles allowed us to distinguish between participants with high versus low levels of the biomarkers while also maximizing sample size in the 3 categories of inflammation defined.
Baseline characteristics of participants were compared across the 3 categories of IL-6 and sVCAM-1 using analysis of covariance, adjusting for age. In comparing change in functional performance across the groups, a longitudinal or repeated-measures analysis of covariance was carried out using a mixed-model approach, in which the within-subject correlations are modeled with a subject-specific random effect. The primary dependent variables for each analysis were the successive annual differences in the particular functional performance measure of interest (i.e., the difference in functioning between baseline and the first follow-up visit, between the first and second follow-up visits, and between the second and third follow-up visits). Associations of each IL-6 category with change in each functional performance measure were compared using analysis of covariance, adjusting for baseline covariates (gender, age, race, co-morbidities, body mass index, pack-years of smoking, high-density lipoprotein cholesterol, total cholesterol, statin use, walking exercise, and physical activity) and previous-year performance (model 1). Covariates in model 1 were selected because they were potential confounders of the associations of the inflammation categories with average annual decrease in functional performance. Model 1 analyses were repeated with additional adjustment for time-dependent ankle-brachial index, smoking status, statin use, physical activity, diabetes medication use, and walking exercise (model 2). These additional time-dependent covariates in model 2 were selected to determine whether changes in lower-extremity atherosclerosis severity and participant behaviors overtime influenced the significant associations identified in model 1 analyses. For models 1 and 2, pairwise comparisons between category 1 and category 3 were performed using analysis of covariance, adjusting for covariates as defined for models 1 and 2. Model 2 analyses were repeated with additional adjustment for cilostazol, pentoxifylline, nonsteroidal anti-inflammatory medications, and steroid medications. Model 1 and model 2 analyses were repeated for the sVCAM-1 categories. Statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, North Carolina).
Results
Among 478 WALCS II participants with PAD, 255 had blood drawn for ≥3 annual study visits and were included in analyses. WALCS II participants excluded because they did not have ≥3 annual blood draws were older (75.9 ± 8.99 vs 74.4 ± 7.54 years, p = 0.049) and walked fewer blocks during the past week (17.7 ± 31.1 vs 37.5 ± 74.0, p <0.001) compared to WALCS II participants included in analyses.
Fifty-seven participants met criteria for persistently low IL-6 levels (category 1), 150 met criteria for category 2, and 48 met criteria for persistently high IL-6 levels (category 3). Sixty-five participants met criteria for persistently low sVCAM-1 levels (category 1), 121 met criteria for category 2, and 69 met criteria for persistently high sVCAM-1 levels (category 3). The average ages of the participants in categories 1, 2, and 3 for IL-6 were 72.1 ± 7.3, 74.9 ± 7.7, and 75.7 ± 6.9 years, respectively (p for trend = 0.011). The average ages of participants in categories 1, 2, and 3 for sVCAM-1 were 70.4 ± 6.9, 74.0 ± 7.3, and 78.8 ± 6.1 years, respectively (p for trend <0.001).
Persistently high levels of IL-6 were associated with higher body mass index, greater smoking pack-years, and a greater number of cardiovascular diseases compared to persistently lower levels of IL-6, adjusting for age ( Table 1 ). Persistently high levels of sVCAM-1 were associated with a higher prevalence of men and a lower prevalence of African Americans compared to persistently low levels of sVCAM-1, adjusting for age ( Table 1 ).
| Variable | Persistently Low | Fluctuating | Persistently High | p Value for Trend |
|---|---|---|---|---|
| IL-6 levels | (n = 57) | (n = 150) | (n = 48) | |
| Men | 61% | 50% | 67% | 0.562 |
| African Americans | 16% | 18% | 9% | 0.372 |
| Ankle-brachial index | 0.64 ± 0.02 | 0.63 ± 0.01 | 0.64 ± 0.02 | 0.979 |
| Body mass index (kg/m 2 ) | 27.11 ± 0.63 | 28.05 ± 0.39 | 29.00 ± 0.68 | 0.044 |
| Diabetes mellitus | 28% | 31% | 41% | 0.197 |
| Current smokers | 9% | 11% | 17% | 0.190 |
| Number of cardiovascular diseases (range 0–4) † | 0.56 ± 0.15 | 1.13 ± 0.09 | 1.31 ± 0.17 | <0.001 |
| Pulmonary disease | 35% | 42% | 45% | 0.336 |
| Cancer | 26% | 19% | 18% | 0.344 |
| Statin use | 56% | 55% | 54% | 0.878 |
| Blocks walked during the past week | 46.7 ± 9.8 | 34.9 ± 6.0 | 34.6 ± 10.6 | 0.384 |
| Classic intermittent claudication | 24.6% | 24.7% | 27.1% | |
| Asymptomatic PAD § | 17.5% | 21.3% | 18.8% | 0.912 ‡ |
| Exertional leg symptoms other than intermittent claudication | 58.0% | 54.0% | 54.2% | |
| sVCAM-1 levels | (n = 65) | (n = 121) | (n = 69) | |
| Men | 47% | 54% | 66% | 0.048 |
| African Americans ⁎ | 29% | 13% | 8% | 0.005 |
| Ankle-brachial index | 0.60 ± 0.02 | 0.64 ± 0.01 | 0.64 ± 0.02 | 0.145 |
| Body mass index (kg/m 2 ) | 27.08 ± 0.61 | 28.38 ± 0.43 | 28.26 ± 0.60 | 0.189 |
| Diabetes mellitus | 25% | 32% | 39% | 0.113 |
| Current smokers | 14% | 11% | 12% | 0.764 |
| Number of cardiovascular diseases (range 0–4) | 0.89 ± 0.15 | 1.03 ± 0.11 | 1.20 ± 0.15 | 0.171 |
| Pulmonary disease | 34% | 43% | 42% | 0.384 |
| Cancer | 20% | 25% | 13% | 0.265 |
| Statin use | 49% | 62% | 48% | 0.943 |
| Blocks walked during the past week | 36.3 ± 9.5 | 39.4 ± 6.7 | 35.2 ± 9.3 | 0.935 |
| Classic intermittent claudication | 30.8% | 24.0% | 21.7% | |
| Asymptomatic PAD § | 9.2% | 18.2% | 33.3% | 0.061 ‡ |
| Exertional leg symptoms other than intermittent claudication | 60.0% | 57.8% | 45.0% |
⁎ p = 0.002 for pairwise comparison between category 1 and category 3.
† p = 0.001 for pairwise comparison between category 1 and category 3.
‡ Overall p value for the association of the inflammation category with leg symptoms.
Figure 1 shows associations of IL-6 categories with average annual change in 6-minute walking distance. Adjusting for age, gender, race, previous-year performance, co-morbidities, high-density lipoprotein, total cholesterol, smoking, body mass index, ankle-brachial index, statin use, and physical activity (model 1, Figure 1 ), participants with persistently high levels of IL-6 (category 3) had greater average annual decline in 6-minute walking distance compared to those with fluctuating IL-6 levels (category 2) and persistently low levels of IL-6 (category 1). After additional adjustment of model 1 for annual changes in ankle-brachial index, statin use, walking exercise, smoking status, physical activity, and diabetes medication use, persistently high levels of IL-6 remained associated with faster decline in 6-minute walking distance ( Figure 1 , model 2). In model 2, those in category 3 for IL-6 had significantly greater average annual decreases in 6-minute walking distance compared to those in category 1.
