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Gender and racial differences in endothelial oxidative stress and inflammation in patients with symptomatic peripheral artery disease
Gender and racial differences in endothelial oxidative stress and inflammation in patients with symptomatic peripheral artery disease Andrew W. Gardner, PhD,a,d Donald E. Parker, PhD,b Polly S. Montgomery, MS,a Danuta Sosnowska, PhD,a Ana I. Casanegra, MD,c Zoltan Ungvari, MD, PhD,a Anna Csiszar, MD, PhD,a and William E. Sonntag, PhD,a Oklahoma City, Okla Background: We compared (1) cellular reactive oxygen species (ROS) production, inflammation, and apoptosis of cultured endothelial cells treated with sera and (2) circulating inflammatory measures, antioxidant capacity, vascular biomarkers, and calf muscle hemoglobin oxygen saturation (StO2) in men and women with peripheral artery disease (PAD). A secondary aim was to compare exercise performance and daily ambulatory activity between men and women. We hypothesized that women would have more impaired endothelial cellular ROS, inflammation, and apoptosis than men as well as worse systemic inflammation, antioxidant capacity, vascular biomarkers, calf muscle StO2, exercise performance, and daily ambulatory activity. Methods: The 148 symptomatic men and women with PAD were characterized on the endothelial effects of circulating factors present in the sera by a cell culture-based bioassay on primary human arterial endothelial cells. Patients were further evaluated by circulating inflammatory and vascular biomarkers, physical examination and medical history, exercise performance, and calf muscle StO2 during exercise, and ambulatory activity was monitored during 1 week. Results: Cellular ROS production was higher in African American women than in men (P [ .021), but there was no gender difference in white individuals (P [ .537). Men and women were not significantly different on endothelial cell apoptosis (P [ .833) and nuclear factor kB activity (P [ .465). For circulating factors, additional gender differences were found when comparisons were made within each race. In African Americans, women had higher intercellular adhesion molecule 1 (P [ .022) and leptin (P < .001); whereas in white individuals, women had higher matrix metallopeptidase 9 (P [ .047), higher vascular cell adhesion molecule 1 (P [ .047), and lower hepatocyte growth factor (P [ .046). Overall, women had higher apolipoprotein CIII (P [ .035), lower pain-free distance (P [ .048) and total distance (P < .001) during the 6-minute walk test, shorter time for calf muscle StO2 to reach the minimum value during exercise (P [ .027), and slower average cadence (P [ .004) during daily ambulation. Conclusions: African American women with symptomatic PAD have a heightened oxidative status, likely resulting in increased endothelial oxidative stress, compared with men. Furthermore, women exhibit a more pronounced proinflammatory profile of circulating biomarkers as well as more limited peripheral microcirculation, exercise performance, and ambulatory activity than men do. The clinical significance is that women with symptomatic PAD are in greater need than men of clinical intervention to improve oxidative stress, inflammation, and microcirculation, which may in turn have a favorable impact on their lower exercise performance and daily activity. (J Vasc Surg 2014;-:1-9.) Clinical Relevance: The clinical significance to the current study is that women with symptomatic peripheral artery disease are in greater need than men of clinical intervention to improve oxidative stress, inflammation, and microcirculation, which may in turn have a favorable impact on their lower exercise performance and daily activity. Women with symptomatic peripheral artery disease represent a vulnerable subgroup of patients who may benefit more than men from tighter medical management and more intensive behavioral therapy, such as exercise and dietary interventions, to better ameliorate vascular biomarkers and microcirculation in the calf muscle.
Peripheral artery disease (PAD) is a highly prevalent condition1 that increases in people aged 65 years and older.2 PAD is associated with increased prevalence of
coexisting diseases in other arterial beds.2,3 Concomitant cardiovascular and cerebrovascular disease in patients with PAD3 contributes to their elevated rates of cardiovascular
From the Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine,a Department of Biostatistics and Epidemiology,b and Cardiovascular Section, Department of Medicine,c University of Oklahoma Health Sciences Center; and the Veterans Affairs Medical Center.d Supported by grants from the National Institute on Aging (R01-AG-24296 to AWG; AG031085 to AC; AG038747 to WES), Oklahoma Center for the Advancement of Science and Technology (to AG, AC, WES, and ZU) and General Clinical Research Center (M01-RR-14467), the American Heart Association (to AC and ZU), the National Center for Complementary and Alternative Medicine (R01-AT006526 to ZU), and the Ellison Medical Foundation (to WES).
Author conflict of interest: none. Reprint requests: Andrew W. Gardner, PhD, Professor and Donald W. Reynolds Chair in Aging Research, University of Oklahoma Health Sciences Center, Donald W. Reynolds Department of Geriatric Medicine, 1122 NE 13th St, O’Donoghue Research Building, Ste 1200, Oklahoma City, OK 73117 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.02.045
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mortality.4,5 The cost associated with PAD averages $3.9 billion for total Medicare paid PAD-related care annually,6 which is greater than or similar to the costs associated with cardiac dysrhythmias, congestive heart failure, and cerebrovascular disease. Patients with PAD have ambulatory dysfunction and leg pain,7 impaired physical function,8,9 lower physical activity levels,10,11 and even worse healthrelated quality of life scores than in individuals with coronary artery disease and congestive heart failure.12 Furthermore, patients with PAD have increased rates of functional decline and mobility loss compared with those without PAD.8,9,13 For decades, the impact of atherosclerotic diseases in women was not fully appreciated.14 Women suffer the consequences of PAD at rates at least as high as those observed in men.15 Despite the high societal cost of PAD, clinical research to evaluate gender-based differences in the presentation and pathogenesis of PAD in women is limited. Our recent studies were the first to suggest that women with symptomatic PAD have greater ambulatory dysfunction than men, even though their ankle-brachial index (ABI) is similar.16 We found that compared with men, women have lower daily physical activity16 and slower ambulatory cadence17 in the community setting and shorter claudication onset time (COT) and shorter peak walking time (PWT) during standardized treadmill exercise in the laboratory setting.16 Greater impairment in calf muscle hemoglobin oxygen saturation (StO2) during treadmill exercise in women explained their shorter PWT,18 suggesting that women have worse microcirculation during exercise than men do. In addition, we have observed that women have a lower small artery elasticity index than men do,19 providing further support that women have impaired microvascular function. Although patients with PAD have worse endothelial function20,21 and higher levels of inflammation22 and oxidative stress23 than those without PAD, it is not clear whether there is a gender-related difference in these measurements in patients with PAD. This study was designed to test the hypotheses that gender differences exist in vascular inflammatory and oxidative status in patients with PAD. To test our hypotheses, we used a bioassay approach to assess the effects of circulating factors present in the sera on endothelial reactive oxygen species (ROS) production, inflammation, and apoptosis using cultured endothelial cells and compared circulating inflammatory and vascular biomarkers and antioxidant capacity as well as calf muscle StO2 in men and women with PAD. A secondary aim was to compare exercise performance and daily ambulatory activity between men and women. METHODS Patients Approval and informed consent. The Institutional Review Board at the University of Oklahoma Health Sciences Center and the Research and Development committee at the Oklahoma City VA Medical Center approved the
procedures of this study. Written informed consent was obtained from each patient at the beginning of investigation. Recruitment. Vascular laboratories and vascular clinics from the University of Oklahoma Health Sciences Center and the Oklahoma City VA Medical Center referred patients for possible enrollment into an exercise rehabilitation program for treatment of leg pain secondary to PAD.24 The data and analyses for this study were part of the baseline assessments obtained for the exercise study. Medical screening through history and physical examination. Patients were evaluated in the morning at the Clinical Research Center at the University of Oklahoma Health Sciences Center. Patients arrived fasted but were permitted to take their usual medications. To begin the study visit, patients were evaluated with a medical history and physical examination in which demographic information, height, weight, waist circumference,25 cardiovascular risk factors, comorbid conditions, claudication history, ABI, blood samples, and list of current medications were obtained. Inclusion and exclusion criteria. Patients with PAD were included in this study if they met the following criteria: history of ambulatory leg pain; ambulatory leg pain confirmed by treadmill exercise7; and ABI # 0.90 at rest2 or #0.73 after exercise.26 Patients were excluded for the following conditions: absence of PAD (ABI > 0.90 at rest and ABI > 0.73 after exercise); noncompressible vessels (ABI > 1.40); asymptomatic PAD; use of medications indicated for the treatment of claudication (cilostazol or pentoxifylline) initiated within 3 months before investigation; exercise limited by other diseases or conditions; active cancer; end-stage renal disease, defined as stage 5 chronic kidney disease; and abnormal liver function. A series of 216 consecutive individuals were evaluated, with 148 patients eligible and 68 subjects ineligible to participate. Measurements Graded treadmill test: COT, PWT, and calf muscle StO2. Patients performed a graded treadmill test to determine study eligibility and to obtain outcome measures.7 The COT and PWT were measured and are highly reliable, as previously described.7 Ankle systolic blood pressure was obtained from the more severely diseased lower extremity before and 1 minute after the treadmill test.7,27 Calf muscle StO2 was measured with the treadmill test by a continuous-wave, near-infrared spectroscopy unit (InSpectra model 325; Hutchinson Technology, Inc, Hutchinson, Minn), an optical cable attached to a 25-mm probe, InSpectra software (version 2.0), and a dedicated laptop computer as previously described.28 Six-minute walk test. Patients performed a 6-minute walk test, and the pain-free and total walking distances were recorded.29 These measures are highly reliable, as previously described.29 Ambulatory activity monitoring. Daily ambulatory activity was assessed during 7 consecutive days with a step activity monitor (StepWatch3; Orthoinnovations, Inc, Oklahoma City, Okla), as previously described.30
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Table I. Clinical characteristics of men and women with PAD Variables
White men (n ¼ 42)
White women (n ¼ 28)
African American men (n ¼ 30)
African American women (n ¼ 48)
P value
Age, years Body mass index, kg/m2 HOMA-IR ABI Current smoking, % yes Hypertension, % yes Medication use, % No. of medications Dyslipidemia, % yes Medication use, % No. of medications Diabetes, % yes Medication use, % No. of medications Abdominal obesity, % yes Metabolic syndrome, % yes Metabolic syndrome components, n Obesity, % yes Lower extremity revascularization, % yes Coronary artery disease, % yes Myocardial infarction, % yes Cerebrovascular disease, % yes Cerebrovascular accident, % yes Chronic kidney disease, % yes Chronic obstructive pulmonary disease, % yes Dyspnea, % yes Arthritis, % yes
67 (10) 29.1 (5.6) 3.7 (4.2) 0.76 (0.25) 29 88 0.90 2.2 (0.9) 93 76 1.3 (0.5) 38 33 1.4 (0.7) 55 83 3.6 (1.3) 40 38 43 24 21 24 26 26 60 57
62 (14) 26.6 (5.4) 5.2 (8.4) 0.68 (0.27) 46 75 0.71 2.1 (0.9) 100 71 1.3 (0.4) 32 25 1.6 (0.8) 54 75 3.4 (1.3) 21 64 36 25 25 25 29 32 71 57
62 (8) 29.2 (5.1) 3.6 (4.3) 0.69 (0.22) 47 97 0.83 2.8 (1.2) 90 73 1.1 (0.3) 47 37 1.5 (0.5) 37 90 3.6 (1.1) 37 37 33 23 13 13 21 20 40 70
62 (8) 32.4 (6.0) 4.1 (4.5) 0.71 (0.25) 38 94 0.90 2.5 (1.1) 92 75 1.2 (0.5) 67 67 1.7 (0.9) 77 88 4.0 (1.2) 73 38 33 15 27 19 19 21 58 58
.045 .253 .317 .409 .563 .348 .435 .804 .460 .856 .132 .137 .052 .242 .009 .592 .278 .067 .228 .558 .466 .230 .809 .786 .845 .150 .570
ABI, Ankle-brachial index; HOMA-IR, homeostatic model assessment-insulin resistance; PAD, peripheral artery disease. Data are presented as mean (standard deviation) and percentage.
The step activity monitor is highly accurate and reliable, as previously described.30 Blood sampling. Blood was drawn by venipuncture from an antecubital vein, collected in Vacutainers, and distributed in 0.5-mL aliquots. The samples were stored at 80 C and were subsequently batched for analysis. Endothelial cell cultures. A cell culture-based bioassay approach with cultured primary human arterial endothelial cells was used to characterize the endothelial effects of circulating factors present in the sera of patients. In brief, endothelial cells (purchased from Cell Applications, Inc, San Diego, Calif, after passage 4; age of the donors is unknown) were initially cultured in MesoEndo Endothelial Cell Growth Medium (Cell Applications, Inc), followed by Endothelial Basal Medium supplemented with 10% fetal calf serum until the time of serum treatment, as described.31,32 Interindividual variance is unlikely to contribute to observed differences because detector cells used for each in vitro study were from the same donor. For treatment, fetal calf serum was replaced with serum (10%; for 24-48 hours) collected from our subjects.31 Cells cultured in Endothelial Basal Medium supplemented with 10% fetal calf serum served as an additional control. Apoptosis assay. Cultured endothelial cells were treated with sera from subjects and their respective controls (for 24 hours). Caspase activities by Caspase-Glo 3/7 assay kit (Promega, Madison, Wisc) were measured to assess apoptotic cell death, as previously reported.31
Cellular ROS production. Hydrogen peroxide production in detector endothelial cells was measured fluorometrically by the Amplex red/horseradish peroxidase assay to determine cellular oxidative stress induced by factors present in the sera.31 Transient transfection, nuclear factor k-light-chainenhancer of activated B cells (NF-kB) reporter gene assay. Transcriptional activity of NF-kB was tested in serum-treated detector endothelial cells by a reporter gene assay to determine cellular proinflammatory effects induced by factors in the sera.31 Transfections in endothelial cells were performed with the Amaxa Nucleofector technology (Amaxa, Gaithersburg, Md), as we have previously reported.31 Serum antioxidant capacity. Hydroxyl radical antioxidant capacity (HORAC) by the OxiSelect HORAC Activity Assay (Cell Biolabs Inc, San Diego, Calif) was measured from sera to determine the capacity of antioxidant enzymes and other redox molecules to counterbalance the deleterious effects of oxidative stress in the sera of subjects.31 Circulating inflammatory and vascular biomarkers. A Milliplex Human Adipokine Magnetic Bead Kit was used to determine tumor necrosis factor a, interleukin 1b, interleukin 6, interleukin 8, monocyte chemotactic protein 1, hepatocyte growth factor (HGF), and nerve growth factor. A Milliplex Human Cardiovascular Disease Panel 1 Kit was used for myeloperoxidase, matrix metallopeptidase 9 (MMP-9), E selectin, vascular cell adhesion molecule 1
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Table II. Measurements from cultured endothelial cells treated with sera in men and women with PAD Spearman partial correlation with gender Variables Apoptosis Cellular ROS production White African American NF-kB activity
Men
Women
1.11 (0.28)
1.09 (0.43)
26.39 (12.01) 26.29 (7.38) 1.40 (0.90)
26.52 (5.86) 28.57 (22.59) 1.18 (0.85)
Coefficient
P value
0.018
.833
0.077 0.308 0.066
.537 .021 .465
NF-kB, Nuclear factor kB; PAD, peripheral artery disease; ROS, reactive oxygen species. Data are presented as median (interquartile range) adjusted for age, race, and abdominal obesity.
(VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and plasminogen activity inhibitor 1. A Milliplex Human Apolipoprotein Kit was used for apolipoprotein B and apolipoprotein CIII. The Millipore kits were purchased from EMD Millipore (Billerica, Mass). Affymetrix Procarta Immunoassay was used to detect serum amyloid A, vascular endothelial growth factor A, and adiponectin. These assays were performed according to the manufacturer’s protocols. Sample protein content was determined for normalization purposes by a spectrophotometric quantification method with BCA reagent (Pierce Chemical Co, Rockford, Ill). High-sensitivity C-reactive protein. Concentration of high-sensitivity C-reactive protein was quantified from 300 mL of sera by a high-sensitivity near-infrared particle immunoassay. The Synchron LX20 (Beckman Coulter, Brea, Calif), a commercially available device, was used to perform the assay. Before each assay was performed, the Synchron system was calibrated, and a calibration curve was established.33 Oxidized low-density lipoprotein. Plasma oxidized low-density lipoprotein was measured by immunoassay (Mercodia, Uppsala, Sweden) according to the manufacturer’s protocol. Average intra-assay precision is 5%, and inter-assay precision is 8.7%. Statistical analyses The clinical and demographic characteristics were initially examined for differences in men and women with independent t-test for measurement variables and c2 test for classification variables. Analysis of covariance was used to examine gender differences for exercise performance, calf muscle StO2, 6-minute walk test, and daily ambulatory activity variables with covariates that were significantly different in men and women in the initial comparison. Within-race analyses were performed for variables for which initial analysis indicated a sex by race interaction. For all other variables, comparisons of male and female means were made with age, race, and abdominal obesity as covariates. The cultured endothelial cell and circulating inflammatory measures displayed extreme asymmetric distributions and were therefore summarized as medians and interquartile range. A technique not requiring normal distribution, the partial Spearman correlation controlling for variables that were significantly different in men and women in the initial comparison, was used to obtain a metric for the
association of each variable with gender and as a test for difference after adjustment for covariates. Within-race analyses were performed whenever initial analysis indicated different results within races for male and female comparisons. We used the NCSS statistical package for all analyses, with statistical significance set at P < .05. RESULTS The clinical characteristics of the patients with PAD are displayed in Table I. The groups were different on age (P ¼ .045) and on the prevalence of abdominal obesity (P ¼ .009). The groups were similar on the remaining variables (P > .05), but there were trends for group differences for prevalence of obesity (P ¼ .067) and for the percentage of patients taking medications for diabetes (P ¼ .052). The measurements from cultured endothelial cells treated with sera in men and women with PAD are shown in Table II. Cellular ROS production was higher in African American women than in men (P ¼ .021), but there was no gender difference in whites (P ¼ .537). Men and women were not significantly different on endothelial cell apoptosis (P ¼ .833) and NF-kB activity (P ¼ .465). Circulating inflammatory measures, antioxidant capacity, and vascular biomarkers in men and women with PAD are shown in Table III. Additional gender differences were found when comparisons were made within each race. In African Americans, women had higher ICAM-1 (P ¼ .022) and leptin (P < .001) than men did; whereas in white individuals, women had higher MMP-9 (P ¼ .047), higher VCAM-1 (P ¼ .047), and lower HGF (P ¼ .046) compared with men. Overall, women had higher apolipoprotein CIII than men did (P ¼ .035). Exercise performance and calf muscle StO2 measures during a graded treadmill test in men and women with PAD are shown in Table IV. In white individuals, COT (P ¼ .036) and PWT (P < .001) were lower in women than in men, and abdominal obesity was a significant covariate (P < .05) for PWT. In African Americans, there was no difference between men and women (P > .05) for COT and PWT, and age was a significant covariate (P < .05) for COT. For the calf muscle StO2 measures, women had a lower value at rest (P < .001), shorter time to reach the minimum StO2 value during exercise (P ¼ .027), less of an absolute drop in calf muscle StO2 during exercise
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Table III. Circulating inflammatory measures, antioxidant capacity, and vascular biomarkers in men and women with PAD Spearman partial correlation with gender Variables C-reactive protein, mg/L Tumor necrosis factor a, pg/mL Interleukin 1b, pg/mL Interleukin 6, pg/mL Interleukin 8, pg/mL Monocyte chemotactic protein 1, pg/mL Myeloperoxidase, pg/mL MMP-9, pg/mL White African American Serum amyloid A, pg/mL Oxidized low-density lipoprotein, U/L Hydroxyl radical antioxidant capacity, AU E selectin, pg/mL VCAM-1, pg/mL White African American ICAM-1, pg/mL White African American Vascular endothelial growth factor A, pg/mL Leptin, pg/mL White African American Adiponectin, pg/mL Plasminogen activator inhibitor 1, ng/mL Apolipoprotein B, ng/mL Apolipoprotein CIII, ng/mL HGF, pg/mL White African American Nerve growth factor, pg/mL
Men
Women
Coefficient
P value
3.67 53 16 24 94 1367 26
(5.00) (24) (5) (13) (52) (1797) (25)
5.30 49 15 23 98 848 28
(7.46) (30) (5) (10) (55) (1588) (42)
0.064 0.006 0.032 0.003 0.014 0.024 0.021
.447 .948 .712 .975 .873 .777 .808
703 726 9104 68 0.94 39
(235) (241) (3269) (36) (0.23) (51)
791 574 9510 68 0.97 40
(322) (417) (4319) (38) (0.21) (46)
0.248 0.154 0.040 0.037 0.023 0.097
.047 .195 .640 .661 .799 .255
2164 (784) 2262 (1035)
2367 (994) 2058 (1013)
0.247 0.051
.047 .669
1878 (1242) 2012 (1185) 29.5 (41.5)
1802 (1739) 2246 (1807) 27.0 (26.0)
0.069 0.269 0.098
.584 .022 .251
795 1054 5613 726 55 1156
1895 4268 5465 660 72 1483
0.178 0.462 0.004 0.121 0.108 0.179
.155 <.001 .967 .155 .208 .035
0.248 0.112 0.123
.046 .349 .149
(1932) (2044) (1610) (412) (56) (768)
76 (38) 72 (43) 16 (10)
(2926) (3541) (1565) (567) (69) (909)
63 (29) 75 (43) 14 (6)
HGF, Hepatocyte growth factor; ICAM-1, intercellular adhesion molecule 1; MMP-9, matrix metallopeptidase 9; PAD, peripheral artery disease; VCAM-1, vascular cell adhesion molecule 1. Data are presented as median (interquartile range) adjusted for age, race, and abdominal obesity.
(P ¼ .009), and lower StO2 at the end of exercise (P ¼ .034) than men did. After exercise, the women had longer recovery times for calf muscle StO2 to reach halfway to the resting baseline value (P ¼ .025) and to fully reach the resting baseline value (P ¼ .036), and women had a lower maximal absolute value for calf muscle StO2 (P < .001). The 6-minute walk performance and daily ambulatory activity measures in men and women with PAD are shown in Table V. For the 6-minute walk test, women had a lower pain-free distance (P ¼ .048), lower total distance (P < .001), and lower rating of perceived exertion (P ¼ .026) compared with men. For daily ambulatory activity, women had slower cadence for durations of 1 minute (P ¼ .016), 5 minutes (P ¼ .004), 20 minutes (P ¼ .041), and 60 minutes (P ¼ .038) than men did. Furthermore, women had a lower peak activity index (P ¼ .002) and a slower average cadence (P ¼ .004) than men did. DISCUSSION The primary novel findings were that circulating factors present in the sera of African American women induced
higher level of endothelial oxidative stress than those present in sera of African American men. Furthermore, African American women had higher ICAM-1 and leptin than African American men did, whereas white women had higher MMP-9, higher VCAM-1, and lower HGF than white men did. Finally, women had greater impairments in apolipoprotein CIII, calf muscle StO2, exercise performance, and daily ambulatory activity than men did. Gender differences in vascular outcomes. Several lines of evidence support the hypothesis that important gender-based distinctions exist in clinical presentation and pathogenesis of PAD, including the heightened oxidative and inflammatory status of women and greater impairment of calf muscle StO2 during exercise. ROS production by detector endothelial cells was 9% higher on treatment with sera from African American women compared with that induced by sera from African American men. The higher pro-oxidative status of women is in line with the results of our previous study, in which women had a lower small artery elasticity index than men did, and African Americans had lower small artery elasticity
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Table IV. Exercise measures during a graded treadmill test in men and women with PAD Variables
Men
Women
Exercise performance COT, seconds White 230 (27) 136 (33) African American 141 (25) 200 (20) PWT, seconds White 538 (40) 270 (49) African American 347 (42) 381 (33) Ankle SBP at rest, mm Hg 95 (4) 94 (4) Ankle SBP 1 minute after exercise, mm Hg 69 (6) 68 (6) Calf muscle StO2 measurements 57 (2) 42 (3) StO2 at rest, % saturation Minimum StO2, % saturation 17 (2) 12 (2) Time to minimum StO2, seconds 217 (33) 153 (36) 40 (3) 30 (3) Absolute drop in StO2, % saturation 70 (4) 74 (4) Percentage drop in StO2, % StO2 at PWT, % saturation 23 (2) 15 (3) Average rate of decline in StO2 from rest to minimum exercise value, % saturation/s 0.72 (0.11) 0.53 (0.12) Recovery half-time of StO2, seconds 124 (21) 185 (22) 195 (29) 279 (33) Recovery time of StO2, seconds Recovery time to maximal StO2, seconds 632 (34) 705 (37) Maximum recovery StO2, % saturation 87 (3) 66 (3)
P value
Significant covariates
.036 .097
None Agea
<.001 .569 .822 .959
AOa None None None
<.001 .113 .027 .009 .468 .034 .061 .025 .036 .154 <.001
Raceb Raceb None None Racec Raceb Racea None Agea Agea Racea
AO, Abdominal obesity; COT, claudication onset time; PAD, peripheral artery disease; PWT, peak walking time; SBP, systolic blood pressure; StO2, hemoglobin oxygen saturation. Data are presented as mean (standard error) adjusted for age, race, and abdominal obesity. a P >.05; bP < .001; cP < .01.
indices than whites did.19 The fact that higher endothelial cell ROS production of women was found in African Americans but not in whites suggests that African American women represent a subgroup of patients with PAD who are particularly susceptible to progression of atherosclerosis34 and increased risk of coronary events.34,35 Circulating inflammatory markers were higher in women than in men, although the specific markers were different in African Americans and whites. In African Americans, women had higher ICAM-1 and leptin than men did, whereas white women had higher MMP-9, higher VCAM-1, and lower HGF than men did. Apolipoprotein CIII was the only marker that was higher in women regardless of race. The elevated inflammatory measures in women with PAD, coupled with their greater level of endothelial oxidative stress, suggest that they are prone to have accelerated myopathy from damaged mitochondrial electron transport chain function. Consequently, energy production is reduced and apoptosis and sarcopenia are increased.36,37 This study found that women had a shorter time to reach minimum calf muscle StO2 than men did, suggesting a greater limitation in the increase in capillary blood volume during exercise.38 The time to reach minimum calf muscle StO2 is a key measure of microcirculatory function during exercise, as this measure is positively associated with COT and PWT and negatively associated with ischemic window in patients with intermittent claudication.28 High daily ambulatory activity is associated with less impaired calf muscle StO2 during exercise in women with PAD17 and with lower inflammation in PAD,39 suggesting that
an exercise intervention may improve microcirculatory function and inflammation. Furthermore, pharmacologic therapy to improve inflammation and microcirculation of women with PAD should be assessed. Gender differences in ambulatory outcomes. Women had more impaired exercise performance than men did, supporting our previous observations.16-18 On average, women had a shorter pain-free walking distance of 36 meters and a shorter total walking distance of 63 meters during the 6-minute walk test. During the more intense treadmill test, worse exercise performance in women was observed only in white women, as both COT and PWT occurred sooner. Abdominal obesity was a significant covariate of PWT, which supports our previous finding.40 In African Americans, the lack of difference in COT and PWT was primarily due to the African American men having poor exercise performance rather than the women having good performance. In fact, the COT and PWT values of the African American men more closely resembled the values of white women than of white men. The worse exercise performance of women with symptomatic PAD is remarkable, given that their ABI values at rest and after exercise were similar to those of men, which supports previous studies.16-18 This indicates that a similar level of severity of PAD elicits greater functional limitations in women. Women had more limited daily ambulatory activity than men did, supporting our previous findings.17 The lower level of ambulatory activity in the women was observed for cadences ranging from 1 minute to 1 hour as well as for the average cadence and the peak activity
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Table V. Six-minute walk performance and daily ambulatory activity measures recorded during a 7-day monitoring period in men and women with PAD Variables Six-minute walk test Pain-free time, seconds Pain-free distance, meters Total distance, meters Rating of perceived exertion, score Walked continuously for 6 minutes, % of subjects Daily ambulatory activity Maximum 1-minute cadence, strides/min Maximum 5-minute cadence, strides/min Maximum 20-minute cadence, strides/min Maximum 30-minute cadence, strides/min Maximum 60-minute cadence, strides/min Peak activity index, strides/min Average cadence, strides/min Total strides, strides/d Total activity time, min/d
Significant covariates
Men
Women
P value
160 (11) 181 (13) 368 (10) 14.0 (0.3) 59
137 (11) 145 (13) 305 (10) 13.1 (0.3) 45
.159 .068 <.001 .026 .079
None None AOa AOb
45.8 29.8 17.5 14.6 11.0 29.9 12.0 3363 278
42.4 25.4 14.8 12.4 9.1 25.4 10.5 2820 255
.016 .004 .041 .053 .038 .002 .004 .088 .298
None None None None None None None AOb AOa
(0.9) (1.0) (0.9) (0.8) (0.6) (1.0) (0.3) (210) (14)
(1.0) (1.1) (0.9) (0.8) (0.6) (1.0) (0.4) (224) (15)
AO, Abdominal obesity; PAD, peripheral artery disease. Values are means (standard error) adjusted for age, race, and abdominal obesity. a P < .01; bP > .05.
index. In contrast, the total duration of ambulatory activity was not different between men and women. These data suggest that men and women ambulate for the same amount of time throughout the course of a day, but women do so at a slower cadence (ie, lower exercise intensity) for ambulatory activities that require short durations of 1-minute epochs as well as for ambulatory activities that occur during a prolonged period of 1 hour. The clinical implication is that the slower cadence of women, coupled with a shorter stride length, reduces their ambulatory speed in the community setting. Consequently, women may be less capable than men of performing ambulatory activities encountered throughout a typical day, such as crossing city streets at intersections, adding to their greater burden of daily ambulatory dysfunction. Limitations. One limitation is that patients volunteered to participate in this study. Thus, a self-selection bias may exist because they may represent those who had greater interest in participating, better access to transportation to the research center, and better health than PAD patients who did not volunteer. Furthermore, the results of this study are applicable only to patients with symptomatic PAD and may not be generalized to patients with less severe or more severe PAD. Our sample is well represented with African Americans, and there was a high prevalence of cardiovascular risk factors for PAD. Thus, in patients with symptomatic PAD, the findings of this study are generalizable to the large proportion of men and women with PAD who have numerous comorbid conditions. There are limitations associated with the measurement of calf muscle StO2 as previously described.28 Although calf muscle StO2 reflects a balance between oxygen delivery and use, other factors may also contribute to the StO2 measurement. There is a limitation associated with the step activity monitor, as it does not quantify nonambulatory physical
activity, and therefore it underestimates the total amount of daily physical activity accomplished to some extent. We would also like to acknowledge that detection of expression of NF-kB-driven genes, including adhesion molecules and MMP-9 (eg, by real-time quantitative reverse transcription-polymerase chain reaction), in the detector endothelial cells would be a useful additional measure of the inflammatory status of these cells. However, because of methodologic limitations (prohibitively large number of samples), these assays are not feasible presently. A final study limitation is that healthy controls within each gender and racial combination were not studied, thereby limiting the ability to determine whether the gender and racial differences found in this study are associated with PAD or whether these differences are independent of PAD. CONCLUSIONS African American women with symptomatic PAD have a heightened oxidative status, likely resulting in increased endothelial oxidative stress, compared with men. Furthermore, women exhibit a more pronounced proinflammatory profile of circulating biomarkers as well as more limited peripheral microcirculation, exercise performance, and ambulatory activity than men do. The clinical significance is that women with symptomatic PAD are in greater need than men of clinical intervention to improve oxidative stress, inflammation, and microcirculation, which may in turn have a favorable impact on their lower exercise performance and daily activity. AUTHOR CONTRIBUTIONS Conception and design: AG, ZU, AC, WS Analysis and interpretation: AG, DP, ZU Data collection: PM, DS, AIC Writing the article: AG, DP, ZU
8 Gardner et al
Critical revision of the article: AG, DP, PM, DS, AIC, ZU, AC, WS Final approval of the article: AG, DP, PM, DS, AIC, ZU, AC, WS Statistical analysis: DP Obtained funding: AG, ZU, AC, WS Overall responsibility: AG REFERENCES 1. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, et al. Executive summary: heart disease and stroke statisticsd2012 update: a report from the American Heart Association. Circulation 2012;125:188-97. 2. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:e463-654. 3. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). J Vasc Surg 2007;45(Suppl S): S5-67. 4. Brass EP, Hiatt WR. Review of mortality and cardiovascular event rates in patients enrolled in clinical trials for claudication therapies. Vasc Med 2006;11:141-5. 5. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ, et al. Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med 1992;326:381-6. 6. Hirsch AT, Hartman L, Town RJ, Virnig BA. National health care costs of peripheral arterial disease in the Medicare population. Vasc Med 2008;13:209-15. 7. Gardner AW, Skinner JS, Cantwell BW, Smith LK. Progressive vs single-stage treadmill tests for evaluation of claudication. Med Sci Sports Exerc 1991;23:402-8. 8. McDermott MM, Greenland P, Liu K, Guralnik JM, Criqui MH, Dolan NC, et al. Leg symptoms in peripheral arterial disease: associated clinical characteristics and functional impairment. JAMA 2001;286: 1599-606. 9. McDermott MM, Liu K, Greenland P, Guralnik JM, Criqui MH, Chan C, et al. Functional decline in peripheral arterial disease: associations with the ankle brachial index and leg symptoms. JAMA 2004;292:453-61. 10. McDermott MM, Liu K, O’Brien E, Guralnik JM, Criqui MH, Martin GJ, et al. Measuring physical activity in peripheral arterial disease: a comparison of two physical activity questionnaires with an accelerometer. Angiology 2000;51:91-100. 11. Sieminski DJ, Gardner AW. The relationship between free-living daily physical activity and the severity of peripheral arterial occlusive disease. Vasc Med 1997;2:286-91. 12. Ware JE Jr. The status of health assessment 1994. Annu Rev Public Health 1995;16:327-54. 13. McDermott MM, Greenland P, Liu K, Guralnik JM, Celic L, Criqui MH, et al. The ankle brachial index is associated with leg function and physical activity: the Walking and Leg Circulation Study. Ann Intern Med 2002;136:873-83. 14. Mosca L, Mochari H, Christian A, Berra K, Taubert K, Mills T, et al. National study of women’s awareness, preventive action, and barriers to cardiovascular health. Circulation 2006;113:525-34.
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15. Hirsch AT, Allison MA, Gomes AS, Corriere MA, Duval S, Ershow AG, et al. A call to action: women and peripheral artery disease: a scientific statement from the American Heart Association. Circulation 2012;125: 1449-72. 16. Gardner AW. Sex differences in claudication pain in subjects with peripheral arterial disease. Med Sci Sports Exerc 2002;34:1695-8. 17. Gardner AW, Parker DE, Montgomery PS, Khurana A, RittiDias RM, Blevins SM. Gender differences in daily ambulatory activity patterns in patients with intermittent claudication. J Vasc Surg 2010;52:1204-10. 18. Gardner AW, Parker DE, Montgomery PS, Blevins SM, Nael R, Afaq A. Sex differences in calf muscle hemoglobin oxygen saturation in patients with intermittent claudication. J Vasc Surg 2009;50:77-82. 19. Gardner AW, Montgomery PS, Blevins SM, Parker DE. Gender and ethnic differences in arterial compliance in patients with intermittent claudication. J Vasc Surg 2010;51:610-5. 20. Brendle DC, Joseph LJ, Corretti MC, Gardner AW, Katzel LI. Effects of exercise rehabilitation on endothelial reactivity in older patients with peripheral arterial disease. Am J Cardiol 2001;87:324-9. 21. Yataco AR, Corretti MC, Gardner AW, Womack CJ, Katzel LI. Endothelial reactivity and cardiac risk factors in older patients with peripheral arterial disease. Am J Cardiol 1999;83:754-8. 22. Signorelli SS, Mazzarino MC, Di Pino L, Malaponte G, Porto C, Pennisi G, et al. High circulating levels of cytokines (IL-6 and TNFa), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test. Vasc Med 2003;8:15-9. 23. Kals J, Kampus P, Kals M, Zilmer K, Kullisaar T, Teesalu R, et al. Impact of oxidative stress on arterial elasticity in patients with atherosclerosis. Am J Hypertens 2006;19:902-8. 24. Gardner AW, Parker DE, Montgomery PS, Scott KJ, Blevins SM. Efficacy of quantified home-based exercise and supervised exercise in patients with intermittent claudication: a randomized controlled trial. Circulation 2011;123:491-8. 25. Lohman TC, Roche AF, Marubini E. Anthropometric standardization reference manual. Champaign, Ill: Human Kinetics Books; 1988. p. 39-70. 26. Hiatt WR, Marshall JA, Baxter J, Sandoval R, Hildebrandt W, Kahn LR, et al. Diagnostic methods for peripheral arterial disease in the San Luis Valley Diabetes Study. J Clin Epidemiol 1990;43:597-606. 27. Gardner AW. Reliability of transcutaneous oximeter electrode heating power during exercise in patients with intermittent claudication. Angiology 1997;48:229-35. 28. Gardner AW, Parker DE, Webb N, Montgomery PS, Scott KJ, Blevins SM. Calf muscle hemoglobin oxygen saturation characteristics and exercise performance in patients with intermittent claudication. J Vasc Surg 2008;48:644-9. 29. Montgomery PS, Gardner AW. The clinical utility of a six-minute walk test in peripheral arterial occlusive disease patients. J Am Geriatr Soc 1998;46:706-11. 30. Gardner AW, Montgomery PS, Scott KJ, Afaq A, Blevins SM. Patterns of ambulatory activity in subjects with and without intermittent claudication. J Vasc Surg 2007;46:1208-14. 31. Gardner AW, Parker DE, Montgomery PS, Sosnowska D, Casanegra AI, Esponda OL, et al. Impaired vascular endothelial growth factor A and inflammation in patients with peripheral artery disease. Angiology 2013 Sep 3. [Epub ahead of print]. 32. Ungvari Z, Labinskyy N, Mukhopadhyay P, Pinto JT, Bagi Z, Ballabh P, et al. Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells. Am J Physiol Heart Circ Physiol 2009;297:H1876-81. 33. Torres JL, Ridker PM. High sensitivity C-reactive protein in clinical practice. Am Heart Hosp J 2003;1:207-11. 34. Maldonado FJ, Miralles Jde H, Aguilar EM, Gonzalez AF, Garcia JR, Garcia FA. Relationship between noninvasively measured endothelial function and peripheral arterial disease. Angiology 2009;60:725-31. 35. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation 2004;109:III27-32. 36. Pipinos II, Judge AR, Selsby JT, Zhu Z, Swanson SA, Nella AA, et al. The myopathy of peripheral arterial occlusive disease: part 1. Functional
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and histomorphological changes and evidence for mitochondrial dysfunction. Vasc Endovascular Surg 2007;41:481-9. 37. Pipinos II, Judge AR, Selsby JT, Zhu Z, Swanson SA, Nella AA, et al. The myopathy of peripheral arterial occlusive disease: part 2. Oxidative stress, neuropathy, and shift in muscle fiber type. Vasc Endovascular Surg 2008;42:101-12. 38. Mohler ER III, Lech G, Supple GE, Wang H, Chance B. Impaired exercise-induced blood volume in type 2 diabetes with or without peripheral arterial disease measured by continuous-wave near-infrared spectroscopy. Diabetes Care 2006;29:1856-9.
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39. Gardner AW, Parker DE, Montgomery PS, Blevins SM, Teague AM, Casanegra AI. Monitored daily ambulatory activity, inflammation, and oxidative stress in patients with claudication. Angiology 2013 May 21. [Epub ahead of print]. 40. Gardner AW, Montgomery PS. The effect of metabolic syndrome components on exercise performance in patients with intermittent claudication. J Vasc Surg 2008;47:1251-8.
Submitted Nov 26, 2013; accepted Feb 6, 2014.
Peripheral artery disease (PAD) is a highly prevalent condition1 that increases in people aged 65 years and older.2 PAD is associated with increased prevalence of
coexisting diseases in other arterial beds.2,3 Concomitant cardiovascular and cerebrovascular disease in patients with PAD3 contributes to their elevated rates of cardiovascular
From the Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine,a Department of Biostatistics and Epidemiology,b and Cardiovascular Section, Department of Medicine,c University of Oklahoma Health Sciences Center; and the Veterans Affairs Medical Center.d Supported by grants from the National Institute on Aging (R01-AG-24296 to AWG; AG031085 to AC; AG038747 to WES), Oklahoma Center for the Advancement of Science and Technology (to AG, AC, WES, and ZU) and General Clinical Research Center (M01-RR-14467), the American Heart Association (to AC and ZU), the National Center for Complementary and Alternative Medicine (R01-AT006526 to ZU), and the Ellison Medical Foundation (to WES).
Author conflict of interest: none. Reprint requests: Andrew W. Gardner, PhD, Professor and Donald W. Reynolds Chair in Aging Research, University of Oklahoma Health Sciences Center, Donald W. Reynolds Department of Geriatric Medicine, 1122 NE 13th St, O’Donoghue Research Building, Ste 1200, Oklahoma City, OK 73117 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.02.045
1
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2 Gardner et al
mortality.4,5 The cost associated with PAD averages $3.9 billion for total Medicare paid PAD-related care annually,6 which is greater than or similar to the costs associated with cardiac dysrhythmias, congestive heart failure, and cerebrovascular disease. Patients with PAD have ambulatory dysfunction and leg pain,7 impaired physical function,8,9 lower physical activity levels,10,11 and even worse healthrelated quality of life scores than in individuals with coronary artery disease and congestive heart failure.12 Furthermore, patients with PAD have increased rates of functional decline and mobility loss compared with those without PAD.8,9,13 For decades, the impact of atherosclerotic diseases in women was not fully appreciated.14 Women suffer the consequences of PAD at rates at least as high as those observed in men.15 Despite the high societal cost of PAD, clinical research to evaluate gender-based differences in the presentation and pathogenesis of PAD in women is limited. Our recent studies were the first to suggest that women with symptomatic PAD have greater ambulatory dysfunction than men, even though their ankle-brachial index (ABI) is similar.16 We found that compared with men, women have lower daily physical activity16 and slower ambulatory cadence17 in the community setting and shorter claudication onset time (COT) and shorter peak walking time (PWT) during standardized treadmill exercise in the laboratory setting.16 Greater impairment in calf muscle hemoglobin oxygen saturation (StO2) during treadmill exercise in women explained their shorter PWT,18 suggesting that women have worse microcirculation during exercise than men do. In addition, we have observed that women have a lower small artery elasticity index than men do,19 providing further support that women have impaired microvascular function. Although patients with PAD have worse endothelial function20,21 and higher levels of inflammation22 and oxidative stress23 than those without PAD, it is not clear whether there is a gender-related difference in these measurements in patients with PAD. This study was designed to test the hypotheses that gender differences exist in vascular inflammatory and oxidative status in patients with PAD. To test our hypotheses, we used a bioassay approach to assess the effects of circulating factors present in the sera on endothelial reactive oxygen species (ROS) production, inflammation, and apoptosis using cultured endothelial cells and compared circulating inflammatory and vascular biomarkers and antioxidant capacity as well as calf muscle StO2 in men and women with PAD. A secondary aim was to compare exercise performance and daily ambulatory activity between men and women. METHODS Patients Approval and informed consent. The Institutional Review Board at the University of Oklahoma Health Sciences Center and the Research and Development committee at the Oklahoma City VA Medical Center approved the
procedures of this study. Written informed consent was obtained from each patient at the beginning of investigation. Recruitment. Vascular laboratories and vascular clinics from the University of Oklahoma Health Sciences Center and the Oklahoma City VA Medical Center referred patients for possible enrollment into an exercise rehabilitation program for treatment of leg pain secondary to PAD.24 The data and analyses for this study were part of the baseline assessments obtained for the exercise study. Medical screening through history and physical examination. Patients were evaluated in the morning at the Clinical Research Center at the University of Oklahoma Health Sciences Center. Patients arrived fasted but were permitted to take their usual medications. To begin the study visit, patients were evaluated with a medical history and physical examination in which demographic information, height, weight, waist circumference,25 cardiovascular risk factors, comorbid conditions, claudication history, ABI, blood samples, and list of current medications were obtained. Inclusion and exclusion criteria. Patients with PAD were included in this study if they met the following criteria: history of ambulatory leg pain; ambulatory leg pain confirmed by treadmill exercise7; and ABI # 0.90 at rest2 or #0.73 after exercise.26 Patients were excluded for the following conditions: absence of PAD (ABI > 0.90 at rest and ABI > 0.73 after exercise); noncompressible vessels (ABI > 1.40); asymptomatic PAD; use of medications indicated for the treatment of claudication (cilostazol or pentoxifylline) initiated within 3 months before investigation; exercise limited by other diseases or conditions; active cancer; end-stage renal disease, defined as stage 5 chronic kidney disease; and abnormal liver function. A series of 216 consecutive individuals were evaluated, with 148 patients eligible and 68 subjects ineligible to participate. Measurements Graded treadmill test: COT, PWT, and calf muscle StO2. Patients performed a graded treadmill test to determine study eligibility and to obtain outcome measures.7 The COT and PWT were measured and are highly reliable, as previously described.7 Ankle systolic blood pressure was obtained from the more severely diseased lower extremity before and 1 minute after the treadmill test.7,27 Calf muscle StO2 was measured with the treadmill test by a continuous-wave, near-infrared spectroscopy unit (InSpectra model 325; Hutchinson Technology, Inc, Hutchinson, Minn), an optical cable attached to a 25-mm probe, InSpectra software (version 2.0), and a dedicated laptop computer as previously described.28 Six-minute walk test. Patients performed a 6-minute walk test, and the pain-free and total walking distances were recorded.29 These measures are highly reliable, as previously described.29 Ambulatory activity monitoring. Daily ambulatory activity was assessed during 7 consecutive days with a step activity monitor (StepWatch3; Orthoinnovations, Inc, Oklahoma City, Okla), as previously described.30
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Gardner et al 3
Table I. Clinical characteristics of men and women with PAD Variables
White men (n ¼ 42)
White women (n ¼ 28)
African American men (n ¼ 30)
African American women (n ¼ 48)
P value
Age, years Body mass index, kg/m2 HOMA-IR ABI Current smoking, % yes Hypertension, % yes Medication use, % No. of medications Dyslipidemia, % yes Medication use, % No. of medications Diabetes, % yes Medication use, % No. of medications Abdominal obesity, % yes Metabolic syndrome, % yes Metabolic syndrome components, n Obesity, % yes Lower extremity revascularization, % yes Coronary artery disease, % yes Myocardial infarction, % yes Cerebrovascular disease, % yes Cerebrovascular accident, % yes Chronic kidney disease, % yes Chronic obstructive pulmonary disease, % yes Dyspnea, % yes Arthritis, % yes
67 (10) 29.1 (5.6) 3.7 (4.2) 0.76 (0.25) 29 88 0.90 2.2 (0.9) 93 76 1.3 (0.5) 38 33 1.4 (0.7) 55 83 3.6 (1.3) 40 38 43 24 21 24 26 26 60 57
62 (14) 26.6 (5.4) 5.2 (8.4) 0.68 (0.27) 46 75 0.71 2.1 (0.9) 100 71 1.3 (0.4) 32 25 1.6 (0.8) 54 75 3.4 (1.3) 21 64 36 25 25 25 29 32 71 57
62 (8) 29.2 (5.1) 3.6 (4.3) 0.69 (0.22) 47 97 0.83 2.8 (1.2) 90 73 1.1 (0.3) 47 37 1.5 (0.5) 37 90 3.6 (1.1) 37 37 33 23 13 13 21 20 40 70
62 (8) 32.4 (6.0) 4.1 (4.5) 0.71 (0.25) 38 94 0.90 2.5 (1.1) 92 75 1.2 (0.5) 67 67 1.7 (0.9) 77 88 4.0 (1.2) 73 38 33 15 27 19 19 21 58 58
.045 .253 .317 .409 .563 .348 .435 .804 .460 .856 .132 .137 .052 .242 .009 .592 .278 .067 .228 .558 .466 .230 .809 .786 .845 .150 .570
ABI, Ankle-brachial index; HOMA-IR, homeostatic model assessment-insulin resistance; PAD, peripheral artery disease. Data are presented as mean (standard deviation) and percentage.
The step activity monitor is highly accurate and reliable, as previously described.30 Blood sampling. Blood was drawn by venipuncture from an antecubital vein, collected in Vacutainers, and distributed in 0.5-mL aliquots. The samples were stored at 80 C and were subsequently batched for analysis. Endothelial cell cultures. A cell culture-based bioassay approach with cultured primary human arterial endothelial cells was used to characterize the endothelial effects of circulating factors present in the sera of patients. In brief, endothelial cells (purchased from Cell Applications, Inc, San Diego, Calif, after passage 4; age of the donors is unknown) were initially cultured in MesoEndo Endothelial Cell Growth Medium (Cell Applications, Inc), followed by Endothelial Basal Medium supplemented with 10% fetal calf serum until the time of serum treatment, as described.31,32 Interindividual variance is unlikely to contribute to observed differences because detector cells used for each in vitro study were from the same donor. For treatment, fetal calf serum was replaced with serum (10%; for 24-48 hours) collected from our subjects.31 Cells cultured in Endothelial Basal Medium supplemented with 10% fetal calf serum served as an additional control. Apoptosis assay. Cultured endothelial cells were treated with sera from subjects and their respective controls (for 24 hours). Caspase activities by Caspase-Glo 3/7 assay kit (Promega, Madison, Wisc) were measured to assess apoptotic cell death, as previously reported.31
Cellular ROS production. Hydrogen peroxide production in detector endothelial cells was measured fluorometrically by the Amplex red/horseradish peroxidase assay to determine cellular oxidative stress induced by factors present in the sera.31 Transient transfection, nuclear factor k-light-chainenhancer of activated B cells (NF-kB) reporter gene assay. Transcriptional activity of NF-kB was tested in serum-treated detector endothelial cells by a reporter gene assay to determine cellular proinflammatory effects induced by factors in the sera.31 Transfections in endothelial cells were performed with the Amaxa Nucleofector technology (Amaxa, Gaithersburg, Md), as we have previously reported.31 Serum antioxidant capacity. Hydroxyl radical antioxidant capacity (HORAC) by the OxiSelect HORAC Activity Assay (Cell Biolabs Inc, San Diego, Calif) was measured from sera to determine the capacity of antioxidant enzymes and other redox molecules to counterbalance the deleterious effects of oxidative stress in the sera of subjects.31 Circulating inflammatory and vascular biomarkers. A Milliplex Human Adipokine Magnetic Bead Kit was used to determine tumor necrosis factor a, interleukin 1b, interleukin 6, interleukin 8, monocyte chemotactic protein 1, hepatocyte growth factor (HGF), and nerve growth factor. A Milliplex Human Cardiovascular Disease Panel 1 Kit was used for myeloperoxidase, matrix metallopeptidase 9 (MMP-9), E selectin, vascular cell adhesion molecule 1
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4 Gardner et al
Table II. Measurements from cultured endothelial cells treated with sera in men and women with PAD Spearman partial correlation with gender Variables Apoptosis Cellular ROS production White African American NF-kB activity
Men
Women
1.11 (0.28)
1.09 (0.43)
26.39 (12.01) 26.29 (7.38) 1.40 (0.90)
26.52 (5.86) 28.57 (22.59) 1.18 (0.85)
Coefficient
P value
0.018
.833
0.077 0.308 0.066
.537 .021 .465
NF-kB, Nuclear factor kB; PAD, peripheral artery disease; ROS, reactive oxygen species. Data are presented as median (interquartile range) adjusted for age, race, and abdominal obesity.
(VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and plasminogen activity inhibitor 1. A Milliplex Human Apolipoprotein Kit was used for apolipoprotein B and apolipoprotein CIII. The Millipore kits were purchased from EMD Millipore (Billerica, Mass). Affymetrix Procarta Immunoassay was used to detect serum amyloid A, vascular endothelial growth factor A, and adiponectin. These assays were performed according to the manufacturer’s protocols. Sample protein content was determined for normalization purposes by a spectrophotometric quantification method with BCA reagent (Pierce Chemical Co, Rockford, Ill). High-sensitivity C-reactive protein. Concentration of high-sensitivity C-reactive protein was quantified from 300 mL of sera by a high-sensitivity near-infrared particle immunoassay. The Synchron LX20 (Beckman Coulter, Brea, Calif), a commercially available device, was used to perform the assay. Before each assay was performed, the Synchron system was calibrated, and a calibration curve was established.33 Oxidized low-density lipoprotein. Plasma oxidized low-density lipoprotein was measured by immunoassay (Mercodia, Uppsala, Sweden) according to the manufacturer’s protocol. Average intra-assay precision is 5%, and inter-assay precision is 8.7%. Statistical analyses The clinical and demographic characteristics were initially examined for differences in men and women with independent t-test for measurement variables and c2 test for classification variables. Analysis of covariance was used to examine gender differences for exercise performance, calf muscle StO2, 6-minute walk test, and daily ambulatory activity variables with covariates that were significantly different in men and women in the initial comparison. Within-race analyses were performed for variables for which initial analysis indicated a sex by race interaction. For all other variables, comparisons of male and female means were made with age, race, and abdominal obesity as covariates. The cultured endothelial cell and circulating inflammatory measures displayed extreme asymmetric distributions and were therefore summarized as medians and interquartile range. A technique not requiring normal distribution, the partial Spearman correlation controlling for variables that were significantly different in men and women in the initial comparison, was used to obtain a metric for the
association of each variable with gender and as a test for difference after adjustment for covariates. Within-race analyses were performed whenever initial analysis indicated different results within races for male and female comparisons. We used the NCSS statistical package for all analyses, with statistical significance set at P < .05. RESULTS The clinical characteristics of the patients with PAD are displayed in Table I. The groups were different on age (P ¼ .045) and on the prevalence of abdominal obesity (P ¼ .009). The groups were similar on the remaining variables (P > .05), but there were trends for group differences for prevalence of obesity (P ¼ .067) and for the percentage of patients taking medications for diabetes (P ¼ .052). The measurements from cultured endothelial cells treated with sera in men and women with PAD are shown in Table II. Cellular ROS production was higher in African American women than in men (P ¼ .021), but there was no gender difference in whites (P ¼ .537). Men and women were not significantly different on endothelial cell apoptosis (P ¼ .833) and NF-kB activity (P ¼ .465). Circulating inflammatory measures, antioxidant capacity, and vascular biomarkers in men and women with PAD are shown in Table III. Additional gender differences were found when comparisons were made within each race. In African Americans, women had higher ICAM-1 (P ¼ .022) and leptin (P < .001) than men did; whereas in white individuals, women had higher MMP-9 (P ¼ .047), higher VCAM-1 (P ¼ .047), and lower HGF (P ¼ .046) compared with men. Overall, women had higher apolipoprotein CIII than men did (P ¼ .035). Exercise performance and calf muscle StO2 measures during a graded treadmill test in men and women with PAD are shown in Table IV. In white individuals, COT (P ¼ .036) and PWT (P < .001) were lower in women than in men, and abdominal obesity was a significant covariate (P < .05) for PWT. In African Americans, there was no difference between men and women (P > .05) for COT and PWT, and age was a significant covariate (P < .05) for COT. For the calf muscle StO2 measures, women had a lower value at rest (P < .001), shorter time to reach the minimum StO2 value during exercise (P ¼ .027), less of an absolute drop in calf muscle StO2 during exercise
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Gardner et al 5
Table III. Circulating inflammatory measures, antioxidant capacity, and vascular biomarkers in men and women with PAD Spearman partial correlation with gender Variables C-reactive protein, mg/L Tumor necrosis factor a, pg/mL Interleukin 1b, pg/mL Interleukin 6, pg/mL Interleukin 8, pg/mL Monocyte chemotactic protein 1, pg/mL Myeloperoxidase, pg/mL MMP-9, pg/mL White African American Serum amyloid A, pg/mL Oxidized low-density lipoprotein, U/L Hydroxyl radical antioxidant capacity, AU E selectin, pg/mL VCAM-1, pg/mL White African American ICAM-1, pg/mL White African American Vascular endothelial growth factor A, pg/mL Leptin, pg/mL White African American Adiponectin, pg/mL Plasminogen activator inhibitor 1, ng/mL Apolipoprotein B, ng/mL Apolipoprotein CIII, ng/mL HGF, pg/mL White African American Nerve growth factor, pg/mL
Men
Women
Coefficient
P value
3.67 53 16 24 94 1367 26
(5.00) (24) (5) (13) (52) (1797) (25)
5.30 49 15 23 98 848 28
(7.46) (30) (5) (10) (55) (1588) (42)
0.064 0.006 0.032 0.003 0.014 0.024 0.021
.447 .948 .712 .975 .873 .777 .808
703 726 9104 68 0.94 39
(235) (241) (3269) (36) (0.23) (51)
791 574 9510 68 0.97 40
(322) (417) (4319) (38) (0.21) (46)
0.248 0.154 0.040 0.037 0.023 0.097
.047 .195 .640 .661 .799 .255
2164 (784) 2262 (1035)
2367 (994) 2058 (1013)
0.247 0.051
.047 .669
1878 (1242) 2012 (1185) 29.5 (41.5)
1802 (1739) 2246 (1807) 27.0 (26.0)
0.069 0.269 0.098
.584 .022 .251
795 1054 5613 726 55 1156
1895 4268 5465 660 72 1483
0.178 0.462 0.004 0.121 0.108 0.179
.155 <.001 .967 .155 .208 .035
0.248 0.112 0.123
.046 .349 .149
(1932) (2044) (1610) (412) (56) (768)
76 (38) 72 (43) 16 (10)
(2926) (3541) (1565) (567) (69) (909)
63 (29) 75 (43) 14 (6)
HGF, Hepatocyte growth factor; ICAM-1, intercellular adhesion molecule 1; MMP-9, matrix metallopeptidase 9; PAD, peripheral artery disease; VCAM-1, vascular cell adhesion molecule 1. Data are presented as median (interquartile range) adjusted for age, race, and abdominal obesity.
(P ¼ .009), and lower StO2 at the end of exercise (P ¼ .034) than men did. After exercise, the women had longer recovery times for calf muscle StO2 to reach halfway to the resting baseline value (P ¼ .025) and to fully reach the resting baseline value (P ¼ .036), and women had a lower maximal absolute value for calf muscle StO2 (P < .001). The 6-minute walk performance and daily ambulatory activity measures in men and women with PAD are shown in Table V. For the 6-minute walk test, women had a lower pain-free distance (P ¼ .048), lower total distance (P < .001), and lower rating of perceived exertion (P ¼ .026) compared with men. For daily ambulatory activity, women had slower cadence for durations of 1 minute (P ¼ .016), 5 minutes (P ¼ .004), 20 minutes (P ¼ .041), and 60 minutes (P ¼ .038) than men did. Furthermore, women had a lower peak activity index (P ¼ .002) and a slower average cadence (P ¼ .004) than men did. DISCUSSION The primary novel findings were that circulating factors present in the sera of African American women induced
higher level of endothelial oxidative stress than those present in sera of African American men. Furthermore, African American women had higher ICAM-1 and leptin than African American men did, whereas white women had higher MMP-9, higher VCAM-1, and lower HGF than white men did. Finally, women had greater impairments in apolipoprotein CIII, calf muscle StO2, exercise performance, and daily ambulatory activity than men did. Gender differences in vascular outcomes. Several lines of evidence support the hypothesis that important gender-based distinctions exist in clinical presentation and pathogenesis of PAD, including the heightened oxidative and inflammatory status of women and greater impairment of calf muscle StO2 during exercise. ROS production by detector endothelial cells was 9% higher on treatment with sera from African American women compared with that induced by sera from African American men. The higher pro-oxidative status of women is in line with the results of our previous study, in which women had a lower small artery elasticity index than men did, and African Americans had lower small artery elasticity
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6 Gardner et al
Table IV. Exercise measures during a graded treadmill test in men and women with PAD Variables
Men
Women
Exercise performance COT, seconds White 230 (27) 136 (33) African American 141 (25) 200 (20) PWT, seconds White 538 (40) 270 (49) African American 347 (42) 381 (33) Ankle SBP at rest, mm Hg 95 (4) 94 (4) Ankle SBP 1 minute after exercise, mm Hg 69 (6) 68 (6) Calf muscle StO2 measurements 57 (2) 42 (3) StO2 at rest, % saturation Minimum StO2, % saturation 17 (2) 12 (2) Time to minimum StO2, seconds 217 (33) 153 (36) 40 (3) 30 (3) Absolute drop in StO2, % saturation 70 (4) 74 (4) Percentage drop in StO2, % StO2 at PWT, % saturation 23 (2) 15 (3) Average rate of decline in StO2 from rest to minimum exercise value, % saturation/s 0.72 (0.11) 0.53 (0.12) Recovery half-time of StO2, seconds 124 (21) 185 (22) 195 (29) 279 (33) Recovery time of StO2, seconds Recovery time to maximal StO2, seconds 632 (34) 705 (37) Maximum recovery StO2, % saturation 87 (3) 66 (3)
P value
Significant covariates
.036 .097
None Agea
<.001 .569 .822 .959
AOa None None None
<.001 .113 .027 .009 .468 .034 .061 .025 .036 .154 <.001
Raceb Raceb None None Racec Raceb Racea None Agea Agea Racea
AO, Abdominal obesity; COT, claudication onset time; PAD, peripheral artery disease; PWT, peak walking time; SBP, systolic blood pressure; StO2, hemoglobin oxygen saturation. Data are presented as mean (standard error) adjusted for age, race, and abdominal obesity. a P >.05; bP < .001; cP < .01.
indices than whites did.19 The fact that higher endothelial cell ROS production of women was found in African Americans but not in whites suggests that African American women represent a subgroup of patients with PAD who are particularly susceptible to progression of atherosclerosis34 and increased risk of coronary events.34,35 Circulating inflammatory markers were higher in women than in men, although the specific markers were different in African Americans and whites. In African Americans, women had higher ICAM-1 and leptin than men did, whereas white women had higher MMP-9, higher VCAM-1, and lower HGF than men did. Apolipoprotein CIII was the only marker that was higher in women regardless of race. The elevated inflammatory measures in women with PAD, coupled with their greater level of endothelial oxidative stress, suggest that they are prone to have accelerated myopathy from damaged mitochondrial electron transport chain function. Consequently, energy production is reduced and apoptosis and sarcopenia are increased.36,37 This study found that women had a shorter time to reach minimum calf muscle StO2 than men did, suggesting a greater limitation in the increase in capillary blood volume during exercise.38 The time to reach minimum calf muscle StO2 is a key measure of microcirculatory function during exercise, as this measure is positively associated with COT and PWT and negatively associated with ischemic window in patients with intermittent claudication.28 High daily ambulatory activity is associated with less impaired calf muscle StO2 during exercise in women with PAD17 and with lower inflammation in PAD,39 suggesting that
an exercise intervention may improve microcirculatory function and inflammation. Furthermore, pharmacologic therapy to improve inflammation and microcirculation of women with PAD should be assessed. Gender differences in ambulatory outcomes. Women had more impaired exercise performance than men did, supporting our previous observations.16-18 On average, women had a shorter pain-free walking distance of 36 meters and a shorter total walking distance of 63 meters during the 6-minute walk test. During the more intense treadmill test, worse exercise performance in women was observed only in white women, as both COT and PWT occurred sooner. Abdominal obesity was a significant covariate of PWT, which supports our previous finding.40 In African Americans, the lack of difference in COT and PWT was primarily due to the African American men having poor exercise performance rather than the women having good performance. In fact, the COT and PWT values of the African American men more closely resembled the values of white women than of white men. The worse exercise performance of women with symptomatic PAD is remarkable, given that their ABI values at rest and after exercise were similar to those of men, which supports previous studies.16-18 This indicates that a similar level of severity of PAD elicits greater functional limitations in women. Women had more limited daily ambulatory activity than men did, supporting our previous findings.17 The lower level of ambulatory activity in the women was observed for cadences ranging from 1 minute to 1 hour as well as for the average cadence and the peak activity
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Gardner et al 7
Table V. Six-minute walk performance and daily ambulatory activity measures recorded during a 7-day monitoring period in men and women with PAD Variables Six-minute walk test Pain-free time, seconds Pain-free distance, meters Total distance, meters Rating of perceived exertion, score Walked continuously for 6 minutes, % of subjects Daily ambulatory activity Maximum 1-minute cadence, strides/min Maximum 5-minute cadence, strides/min Maximum 20-minute cadence, strides/min Maximum 30-minute cadence, strides/min Maximum 60-minute cadence, strides/min Peak activity index, strides/min Average cadence, strides/min Total strides, strides/d Total activity time, min/d
Significant covariates
Men
Women
P value
160 (11) 181 (13) 368 (10) 14.0 (0.3) 59
137 (11) 145 (13) 305 (10) 13.1 (0.3) 45
.159 .068 <.001 .026 .079
None None AOa AOb
45.8 29.8 17.5 14.6 11.0 29.9 12.0 3363 278
42.4 25.4 14.8 12.4 9.1 25.4 10.5 2820 255
.016 .004 .041 .053 .038 .002 .004 .088 .298
None None None None None None None AOb AOa
(0.9) (1.0) (0.9) (0.8) (0.6) (1.0) (0.3) (210) (14)
(1.0) (1.1) (0.9) (0.8) (0.6) (1.0) (0.4) (224) (15)
AO, Abdominal obesity; PAD, peripheral artery disease. Values are means (standard error) adjusted for age, race, and abdominal obesity. a P < .01; bP > .05.
index. In contrast, the total duration of ambulatory activity was not different between men and women. These data suggest that men and women ambulate for the same amount of time throughout the course of a day, but women do so at a slower cadence (ie, lower exercise intensity) for ambulatory activities that require short durations of 1-minute epochs as well as for ambulatory activities that occur during a prolonged period of 1 hour. The clinical implication is that the slower cadence of women, coupled with a shorter stride length, reduces their ambulatory speed in the community setting. Consequently, women may be less capable than men of performing ambulatory activities encountered throughout a typical day, such as crossing city streets at intersections, adding to their greater burden of daily ambulatory dysfunction. Limitations. One limitation is that patients volunteered to participate in this study. Thus, a self-selection bias may exist because they may represent those who had greater interest in participating, better access to transportation to the research center, and better health than PAD patients who did not volunteer. Furthermore, the results of this study are applicable only to patients with symptomatic PAD and may not be generalized to patients with less severe or more severe PAD. Our sample is well represented with African Americans, and there was a high prevalence of cardiovascular risk factors for PAD. Thus, in patients with symptomatic PAD, the findings of this study are generalizable to the large proportion of men and women with PAD who have numerous comorbid conditions. There are limitations associated with the measurement of calf muscle StO2 as previously described.28 Although calf muscle StO2 reflects a balance between oxygen delivery and use, other factors may also contribute to the StO2 measurement. There is a limitation associated with the step activity monitor, as it does not quantify nonambulatory physical
activity, and therefore it underestimates the total amount of daily physical activity accomplished to some extent. We would also like to acknowledge that detection of expression of NF-kB-driven genes, including adhesion molecules and MMP-9 (eg, by real-time quantitative reverse transcription-polymerase chain reaction), in the detector endothelial cells would be a useful additional measure of the inflammatory status of these cells. However, because of methodologic limitations (prohibitively large number of samples), these assays are not feasible presently. A final study limitation is that healthy controls within each gender and racial combination were not studied, thereby limiting the ability to determine whether the gender and racial differences found in this study are associated with PAD or whether these differences are independent of PAD. CONCLUSIONS African American women with symptomatic PAD have a heightened oxidative status, likely resulting in increased endothelial oxidative stress, compared with men. Furthermore, women exhibit a more pronounced proinflammatory profile of circulating biomarkers as well as more limited peripheral microcirculation, exercise performance, and ambulatory activity than men do. The clinical significance is that women with symptomatic PAD are in greater need than men of clinical intervention to improve oxidative stress, inflammation, and microcirculation, which may in turn have a favorable impact on their lower exercise performance and daily activity. AUTHOR CONTRIBUTIONS Conception and design: AG, ZU, AC, WS Analysis and interpretation: AG, DP, ZU Data collection: PM, DS, AIC Writing the article: AG, DP, ZU
8 Gardner et al
Critical revision of the article: AG, DP, PM, DS, AIC, ZU, AC, WS Final approval of the article: AG, DP, PM, DS, AIC, ZU, AC, WS Statistical analysis: DP Obtained funding: AG, ZU, AC, WS Overall responsibility: AG REFERENCES 1. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, et al. Executive summary: heart disease and stroke statisticsd2012 update: a report from the American Heart Association. Circulation 2012;125:188-97. 2. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:e463-654. 3. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). J Vasc Surg 2007;45(Suppl S): S5-67. 4. Brass EP, Hiatt WR. Review of mortality and cardiovascular event rates in patients enrolled in clinical trials for claudication therapies. Vasc Med 2006;11:141-5. 5. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ, et al. Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med 1992;326:381-6. 6. Hirsch AT, Hartman L, Town RJ, Virnig BA. National health care costs of peripheral arterial disease in the Medicare population. Vasc Med 2008;13:209-15. 7. Gardner AW, Skinner JS, Cantwell BW, Smith LK. Progressive vs single-stage treadmill tests for evaluation of claudication. Med Sci Sports Exerc 1991;23:402-8. 8. McDermott MM, Greenland P, Liu K, Guralnik JM, Criqui MH, Dolan NC, et al. Leg symptoms in peripheral arterial disease: associated clinical characteristics and functional impairment. JAMA 2001;286: 1599-606. 9. McDermott MM, Liu K, Greenland P, Guralnik JM, Criqui MH, Chan C, et al. Functional decline in peripheral arterial disease: associations with the ankle brachial index and leg symptoms. JAMA 2004;292:453-61. 10. McDermott MM, Liu K, O’Brien E, Guralnik JM, Criqui MH, Martin GJ, et al. Measuring physical activity in peripheral arterial disease: a comparison of two physical activity questionnaires with an accelerometer. Angiology 2000;51:91-100. 11. Sieminski DJ, Gardner AW. The relationship between free-living daily physical activity and the severity of peripheral arterial occlusive disease. Vasc Med 1997;2:286-91. 12. Ware JE Jr. The status of health assessment 1994. Annu Rev Public Health 1995;16:327-54. 13. McDermott MM, Greenland P, Liu K, Guralnik JM, Celic L, Criqui MH, et al. The ankle brachial index is associated with leg function and physical activity: the Walking and Leg Circulation Study. Ann Intern Med 2002;136:873-83. 14. Mosca L, Mochari H, Christian A, Berra K, Taubert K, Mills T, et al. National study of women’s awareness, preventive action, and barriers to cardiovascular health. Circulation 2006;113:525-34.
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Submitted Nov 26, 2013; accepted Feb 6, 2014.