GENDER AND RACIAL DIFFERENCES IN ENDOTHELIAL OXIDATIVE STRESS AND INFLAMMATION IN PATIENTS WITH SYMPTOMATIC PERIPHERAL ARTERY DISEASE

Abstract

Background

We compared (a) cellular reactive oxygen species (ROS) production, inflammation, and apoptosis of cultured endothelial cells treated with sera, and (b) circulating inflammatory, antioxidant capacity, vascular biomarkers, and calf muscle hemoglobin oxygen saturation (StO₂) in men and women with 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

One hundred forty-eight symptomatic men and women with PAD were characterized on the endothelial effects of circulating factors present in the sera using a cell culture-based bioassay on primary human arterial endothelial cells. Patients were further evaluated on circulating inflammatory and vascular biomarkers, physical examination and medical history, exercise performance, calf muscle StO₂ during exercise, and ambulatory activity monitored during one week.

Results

Cellular ROS production was higher in African-American women than in men (p = 0.021), but there was no gender difference in Caucasians (p = 537). Men and women were not significantly different on endothelial cell apoptois (p = 833) and NF-κB activity (p = 0.465). For circulating factors, additional gender differences were found when comparisons were made within each race. In African-Americans, women had higher intercellular cell adhesion molecule-1 (p = 0.022) and leptin (p < 0.001), whereas in Caucasians, women had higher matrix metallopeptidase 9 (p = 0.047), higher vascular cell adhesion molecule-1 (p = 0.047), and lower hepatocyte growth factor (p = 0.046). Overall, women had higher apolipoprotein CIII (p = 0.035), lower pain-free distance (p = 0.048) and total distance (p < 0.001) during the 6-minute walk test, shorter time for calf muscle StO₂ to reach the minimum value during exercise (p = 0.027), and slower average cadence (p = 0.004) during daily ambulation.

Conclusions

African-American women with symptomatic PAD have a heightened oxidative status likely resulting in increased endothelial oxidative stress than men. Furthermore, women exhibit a more pronounced pro-inflammatory profile of circulating biomarkers than men, as well as more limited peripheral microcirculation, exercise performance, and ambulatory activity than men. The clinical significance is that women with symptomatic PAD are in greater need of clinical intervention to improve oxidative stress, inflammation, and microcirculation than men, which may, in turn, favorably impact their lower exercise performance and daily activity.

INTRODUCTION

Peripheral artery disease (PAD) is a highly prevalent condition,¹ which 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/or cerebrovascular disease in patients with PAD³ contributes to their elevated rates of cardiovascular 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 health-related 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 to 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 to 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. Additionally, we have observed that women have lower small artery elasticity index than men,¹⁹ providing further support that women have impaired microvascular function. Although patients with PAD have worse endothelial function^{20, 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.

The present 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 (HSC), 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 labs and vascular clinics from the University of Oklahoma HSC and the Oklahoma City VA Medical Center referred patients for possible enrollment into an exercise rehabilitation program to treat 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 (CRC), at the University of Oklahoma HSC. 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, co-morbid conditions, claudication history, ABI, blood samples, and a list of current medications were obtained.

Inclusion and Exclusion Criteria

Patients with PAD were included in this study if they met the following criteria: (a) a history of ambulatory leg pain, (b) ambulatory leg pain confirmed by treadmill exercise,⁷ and (c) an ankle-brachial index (ABI) ≤ 0.90 at rest² or ≤ 0.73 after exercise.²⁶ Patients were excluded for the following conditions: (a) absence of PAD (ABI > 0.90 at rest and ABI > 0.73 after exercise), (b) non-compressible vessels (ABI > 1.40), (c) asymptomatic PAD, (d) use of medications indicated for the treatment of claudication (cilostazol or pentoxifylline) initiated within 3 months prior to investigation, (e) exercise limited by other diseases or conditions, (f) active cancer, (g) end stage renal disease defined as stage 5 chronic kidney disease, and, (h) 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 min after the treadmill test.^{7, 27}

Calf muscle StO₂ was measured with the treadmill test using a continuous-wave, NIRS unit (InSpectra model 325; Hutchinson Technology, Inc, Hutchinson, MN), an optical cable attached to a 25-mm probe, InSpectra software (version 2.0), and a dedicated laptop computer as previously described.²⁸

6-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 seven consecutive days using a step activity monitor (StepWatch3^™, Orthoinnovations, Inc., Oklahoma City, OK) as previously described.³⁰ 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 utilizing 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, CA, 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} Inter-individual 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 h) 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 h). Caspase activities using Caspase-Glo 3/7 assay kit (Promega, Madison, WI) were measured to assess apoptotic cell death, as previously reported.³¹

Cellular ROS Production

Hydrogen peroxide (H₂O₂) production in detector endothelial cells was measured fluorometrically using the Amplex Red/horseradish peroxidase assay to determine cellular oxidative stress induced by factors in present in the sera.³¹

Transient Transfection, Nuclear Factor K-Light-Chain-Enhancer of Activated B Cells (NF-κB) Reporter Gene Assay

Transcriptional activity of NF-κB was tested in serum-treated detector endothelial cells by a reporter gene assay, to determine cellular pro-inflammatory effects induced by factors in the sera.³¹ Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported.³¹

Serum Antioxidant Capacity

Hydroxyl Radical Antioxidant Capacity (HORAC) using the OxiSelect HORAC Activity Assay (Cell Biolabs Inc., San Diego, CA) 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 Millipex Human Adipokine Magnetic Bead Kit was used for determining tumor necrosis factor alpha (TNF α), interleukin-1b (IL-1b), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), hepatocyte growth factor (HGF), and nerve growth factor (NGF). A Millipex Human Cardiovascular Disease (CVD) Panel 1 Kit was used for myeloperoxidase (MPO), matrix metallopeptidase 9 (MMP-9), E selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and plasminogen activity inhibitor-1 (PAI-1). A Millipex Human Apolipoprotein Kit was used for apolipoprotein B and apolipoprotein CIII. The Millipore kits were purchased from EMD Millipore, Billerica, MA. Affymetrix Procarta Immunoassay was used to detect serum amyloid A (SAA), vascular endothelial growth factor-A (VEGF-A) and adiponectin. These assays were performed according to manufacturer’s protocols. Sample protein content was determined for normalization purposes by a spectrophotometric quantification method using BCA reagent (pierce Chemical Co., Rockford, IL).

High-Sensitivity C-Reactive Protein (hsCRP)

Concentration of hsCRP was quantified from 300 μl of sera using a high-sensitivity Near Infrared Particles Immunoassay. The SYNCHRON LX-20 (Beckman-Coulter; California, USA), a commercially available device, was used to perform the assay. Prior to performing each assay, the SYNCHRON system was calibrated, and a calibration curve was established.³³

Oxidized LDL

Plasma oxidized LDL 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

Initially the clinical and demographic characteristics were examined for differences in men and women with using independent t-test for measurement variables and chi square test for classification variables. ANCOVA was used to examine gender differences for exercise performance, calf muscle StO_2, 6-minute walk test, and daily ambulatory activity variables using covariates that were significantly different in men and women in the initial comparison. Within race analyses were performed for variables where 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 inter quartile 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 analysis 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 < 0.05.

RESULTS

The clinical characteristics of the patients with PAD are displayed in Table I. The groups were different on age (p = 0.045), and on the prevalence of abdominal obesity (p = 0.009). The groups were similar on the remaining variables (p > 0.05), but there were trends for group differences for prevalence of obesity (p = 0.067) and for the percentage of patients taking medications for diabetes (p = 0.052).

Table I.

Clinical characteristics of men and women with peripheral artery disease. Values are means (SD) and percentages.

Variables	Caucasian Men (n = 42)	Caucasian Women (n = 28)	African- American Men (n = 30)	African- American Women (n = 48)	P Value
Age (years)	67 (10)	62 (14)	62 (8)	62 (8)	0.045
Body Mass Index (kg/m2)	29.1 (5.6)	26.6 (5.4)	29.2 (5.1)	32.4 (6.0)	0.253
HOMA-IR	3.7 (4.2)	5.2 (8.4)	3.6 (4.3)	4.1 (4.5)	0.317
Ankle/Brachial Index	0.76 (0.25)	0.68 (0.27)	0.69 (0.22)	0.71 (0.25)	0.409
Current Smoking (% yes)	29	46	47	38	0.563
Hypertension (% yes)	88	75	97	94	0.348
Medication Use (% yes)	0.90	0.71	0.83	0.90	0.435
Number of Medications (n)	2.2 (0.9)	2.1 (0.9)	2.8 (1.2)	2.5 (1.1)	0.804
Dyslipidemia (% yes)	93	100	90	92	0.460
Medication Use (%)	76	71	73	75	0.856
Number of Medications (n)	1.3 (0.5)	1.3 (0.4)	1.1 (0.3)	1.2 (0.5)	0.132
Diabetes (% yes)	38	32	47	67	0.137
Medication Use (%)	33	25	37	67	0.052
Number of Medications (n)	1.4 (0.7)	1.6 (0.8)	1.5 (0.5)	1.7 (0.9)	0.242
Abdominal Obesity (% yes)	55	54	37	77	0.009
Metabolic Syndrome (% yes)	83	75	90	88	0.592
Metabolic Syndrome Components (n)	3.6 (1.3)	3.4 (1.3)	3.6 (1.1)	4.0 (1.2)	0.278
Obesity (% yes)	40	21	37	73	0.067
Lower Extremity Revascularization (% yes)	38	64	37	38	0.228
Coronary Artery Disease (% yes)	43	36	33	33	0.558
Myocardial Infarction (% yes)	24	25	23	15	0.466
Cerebrovascular Disease (% yes)	21	25	13	27	0.230
Cerebrovascular Accident (% yes)	24	25	13	19	0.809
Chronic Kidney Disease (% yes)	26	29	21	19	0.786
Chronic Obstructive Pulmonary Disease (% yes)	26	32	20	21	0.845
Dyspnea (% yes)	60	71	40	58	0.150
Arthritis (% yes)	57	57	70	58	0.570

HOMA-IR = homeostatic model assessment – insulin resistance.

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 women African- Americans than in men (p = 0.021), but there was no gender difference in Caucasians (p = 0.537). Men and women were not significantly different on endothelial cell apoptosis (p = 0.833) and NF-κB activity (p = 0.465).

Table II.

Measurements from cultured endothelial cells treated with sera in men and women with peripheral artery disease. Values are medians (interquartile ranges) adjusted for age, race, and abdominal obesity.

Variables	Men	Women	Spearman Partial Correlation with Gender
			Coefficient	p-value
Apoptosis	1.11 (0.28)	1.09 (0.43)	0.018	0.833
Cellular ROS production
Caucasian	26.39 (12.01)	26.52 (5.86)	0.077	0.537
African-American	26.29 (7.38)	28.57 (22.59)	−0.308	0.021
NF-κB activity	1.40 (0.90)	1.18 (0.85)	0.066	0.465

ROS = reactive oxygen species.

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 = 0.022) and leptin (p < 0.001) than men, whereas in Caucasians, women had higher MMP-9 (p = 0.047), higher VCAM-1 (p = 0.047), and lower HGF (p = 0.046) than compared to men. Overall, women had higher apolipoprotein CIII than men (p = 0.035).

Table III.

Circulating inflammatory measures, antioxidant capacity, and vascular biomarkers in men and women with peripheral artery disease (PAD). Values are medians (interquartile ranges) adjusted for age, race, and abdominal obesity.

Variables	Men	Women	Spearman Partial Correlation with Gender
			Coefficient	p-value
C-Reactive Protein (mg/L)	3.67 (5.00)	5.30 (7.46)	−0.064	0.447
Tumor necrosis factor alpha (pg/ml)	53 (24)	49 (30)	0.006	0.948
Interleukin-1b (pg/ml)	16 (5)	15 (5)	0.032	0.712
Interleukin-6 (pg/ml)	24 (13)	23 (10)	0.003	0.975
Interleukin-8 (pg/ml)	94 (52)	98 (55)	0.014	0.873
Monocyte chemotactic protein-1 (pg/ml)	1367 (1797)	848 (1588)	0.024	0.777
Myeloperoxidase (pg/ml)	26 (25)	28 (42)	0.021	0.808
Matrix metallopeptidase 9 (pg/ml)
Caucasian	703 (235)	791 (322)	−0.248	0.047
African-American	726 (241)	574 (417)	0.154	0.195
Serum Amyloid A (pg/ml)	9104 (3269)	9510 (4319)	−0.040	0.640
Oxidized Low Density Lipoprotein (U/L)	68 (36)	68 (38)	0.037	0.661
Hydroxyl Radical Antioxidant Capacity (AU)	0.94 (0.23)	0.97 (0.21)	0.023	0.799
E selectin (pg/ml)	39 (51)	40 (46)	0.097	0.255
Vascular Cell Adhesion Molecule-1 (pg/ml)
Caucasian	2164 784	2367 (994)	−0.247	0.047
African-American	2262 (1035)	2058 (1013)	0.051	0.669
Intercellular Cell Adhesion Molecule-1 (pg/ml)
Caucasian	1878 (1242)	1802 (1739)	−0.069	0.584
African-American	2012 (1185)	2246 (1807)	−0.269	0.022
Vascular Endothelial Growth Factor-A (pg/ml)	29.5 (41.5)	27.0 (26.0)	0.098	0.251
Leptin (pg/ml)
Caucasian	795 (1932)	1895 (2926)	−0.178	0.155
African-American	1054 (2044)	4268 (3541)	−0.462	< 0.001
Adiponectin (pg/ml)	5613 (1610)	5465 (1565)	0.004	0.967
Plasminogen Activator Inhibitor-1 (ng/ml)	726 (412)	660 (567)	0.121	0.155
Apolipoprotein B (ng/ml)	55 (56)	72 (69)	−0.108	0.208
Apolipoprotein CIII (ng/ml)	1156 768	1483 (909)	−0.179	0.035
Hepatocyte Growth Factor (pg/ml)
Caucasian	76 (38)	63 (29)	0.248	0.046
African-American	72 (43)	75 (43)	−0.112	0.349
Nerve Growth Factor (pg/ml)	16 (10)	14 (6)	0.123	0.149

Exercise performance and calf muscle StO₂ measures during a graded treadmill test in men and women with PAD are shown in Table IV. In Caucasians, COT (p = 0.036) and PWT (p <0.001) were lower in women than in men, and abdominal obesity was a significant covariate (p < 0.05) for PWT. In African-Americans there was no difference between men and women (p > 0.05) for COT and PWT, and age was a significant covariate (p < 0.05) for COT. For the calf muscle StO₂ measures, women had a lower value at rest (p < 0.001), shorter time to reach the minimum StO₂ value during exercise (p = 0.027), less of an absolute drop in calf muscle StO₂ during exercise (p = 0.009), and lower StO₂ at the end of exercise (p = 0.034) than men. Following exercise, the women had longer recovery times for calf muscle StO₂ to reach half way to the resting baseline value (p = 0.025) and to fully reach the resting baseline value (p = 0.036), and women had a lower maximal absolute value for calf muscle StO₂ (p < 0.001).

Table IV.

Exercise measures during a graded treadmill test in men and women with peripheral artery disease. Values are means (SE) adjusted for age, race, and abdominal obesity.

Variables	Men	Women	P Value	Significant Covariates
Exercise Performance
Claudication Onset Time (sec)
Caucasian	230 (27)	136 (33)	0.036	None
African-American	141 (25)	200 (20)	0.097	Age*
Peak Walking Time (sec)
Caucasian	538 (40)	270 (49)	< 0.001	AO*
African-American	347 (42)	381 (33)	0.569	None
Ankle SBP at rest (mm Hg)	95 (4)	94 (4)	0.822	None
Ankle SBP 1 min after exercise (mm Hg)	69 (6)	68 (6)	0.959	None
Calf Muscle StO2 Measurements
StO2 at rest (% saturation)	57 (2)	42 (3)	< 0.001	Race***
Minimum StO2 (% saturation)	17 (2)	12 (2)	0.113	Race***
Time to minimum StO2 (sec)	217 (33)	153 (36)	0.027	None
Absolute Drop in StO2 (% saturation)	40 (3)	30 (3)	0.009	None
Percentage Drop in StO2 (%)	70 (4)	74 (4)	0.468	Race**
StO2 at Peak Walking Time (%saturation)	23 (2)	15 (3)	0.034	Race***
Average Rate of Decline in StO2 from rest to minimum exercise value (% saturation/sec)	0.72 (0.11)	0.53 (0.12)	0.061	Race*
Recovery Half-Time of StO2 (sec)	124 (21)	185 (22)	0.025	None
Recovery Time of StO2 (sec)	195 (29)	279 (33)	0.036	Age*
Recovery Time to Maximal StO2 (sec)	632 (34)	705 (37)	0.154	Age*
Maximum recovery StO2 (% saturation)	87 (3)	66 (3)	< 0.001	Race*

AO = abdominal obesity.

^*p > 0.05,

^**p < 0.01,

^***p < 0.001.

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 = 0.048), lower total distance (p < 0.001), and lower rating of perceived exertion (p = 0.026) than compared to men. For daily ambulatory activity, women had slower cadence for durations of 1 minute (p = 0.016), 5 minutes (p = 0.004), 20 minutes (p = 0.041), and 60 minutes (p = 0.038) than men. Furthermore, women had a lower peak activity index (p = 0.002) and a slower average cadence (p = 0.004) than men.

Table V.

6-minute walk performance and daily ambulatory activity measures recorded during a 7-day monitoring period in men and women with peripheral artery disease. Values are means (SE) adjusted for age, race, and abdominal obesity.

Variables	Men	Women	P Value	Significant Covariates
6-Minute Walk Test
Pain-Free Time (sec)	160 (11)	137 (11)	0.159	None
Pain-Free Distance (meters)	181 (13)	145 (13)	0.068	None
Total Distance (meters)	368 (10)	305 (10)	< 0.001	AO**
Rating of Perceived Exertion (score)	14.0 (0.3)	13.1 (0.3)	0.026	AO*
Walked Continuously for 6 Minutes (%of subjects)	59	45	0.079
Daily Ambulatory Activity
Maximum 1-minute cadence (strides/min)	45.8 (0.9)	42.4 (1.0)	0.016	None
Maximum 5-minute cadence (strides/min)	29.8 (1.0)	25.4 (1.1)	0.004	None
Maximum 20-minute cadence (strides/min)	17.5 (0.9)	14.8 (0.9)	0.041	None
Maximum 30-minute cadence (strides/min)	14.6 (0.8)	12.4 (0.8)	0.053	None
Maximum 60-minute cadence (strides/min)	11.0 (0.6)	9.1 (0.6)	0.038	None
Peak Activity Index (strides/min)	29.9 (1.0)	25.4 (1.0)	0.002	None
Average Cadence (strides/min)	12.0 (0.3)	10.5 (0.4)	0.004	None
Total Strides (strides/day)	3363 (210)	2820 (224)	0.088	AO*
Total Activity Time (min/day)	278 (14)	255 (15)	0.298	AO**

AO = abdominal obesity.

^*p > 0.05,

^**p < 0.01,

^***p < 0.001.

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 from African-American men. Furthermore, African-American women had higher ICAM-1 and leptin than African-American men, whereas Caucasian women had higher MMP-9, higher VCAM-1, and lower HGF than Caucasian men. Finally, women had greater impairments in apolipoprotein CIII, calf muscle StO₂, exercise performance, and daily ambulatory activity than men.

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 upon treatment with sera from African-American women as compared to 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 lower small artery elasticity index than men, and African-Americans had lower small artery elasticity indices than Caucasians.¹⁹ The fact that higher endothelial cell ROS production of women was found in African-Americans but not in Caucasians suggests that African-American women represent a subgroup of patients with PAD who are particularly susceptible to progression of atherosclerosis,³⁴ 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 Caucasians. In African-Americans, women had higher ICAM-1 and leptin than men, whereas Caucasian women had higher MMP-9, higher VCAM-1, and lower HGF than men. 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}

The current study found that women had a shorter time to reach minimum calf muscle StO₂ than men, 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 PAD,¹⁷ 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, 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 only observed in Caucasians, 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 Caucasian women than of Caucasian men. The worse exercise performance of women with symptomatic PAD is remarkable given that their ABI values at rest and following exercise were similar to 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, supporting our previous findings.¹⁷ The lower level of ambulatory activity in the women was observed for cadences ranging from one minute to one hour, as well as the average cadence and the peak activity 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 (i.e., lower exercise intensity) for ambulatory activities that require short durations of 1 minute epochs as well as ambulatory activities that occur over a prolonged period of time of one 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 of performing ambulatory activities encountered throughout a typical day than men, 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 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 only applicable 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 the present study are generalizable to the large proportion of men and women with PAD who have numerous co-morbid 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 utilization, other factors may also contribute to the StO₂ measurement. There is a limitation associated with the step activity monitor, as it does not quantify non-ambulatory 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-κB -driven genes, including adhesion molecules and MMP-9 (e.g. by real-time quantitative RT-PCR) in the detector endothelial cells would be a useful additional measure of the inflammatory status of these cells. However, due to methodological 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 the present study were associated with PAD or whether these differences are independent of PAD.

Conclusion and clinical significance

In conclusion, African-American women with symptomatic PAD have a heightened oxidative status likely resulting in increased endothelial oxidative stress than men. Furthermore, women exhibit a more pronounced pro-inflammatory profile of circulating biomarkers than men, as well as more limited peripheral microcirculation, exercise performance, and ambulatory activity than men. The clinical significance is that women with symptomatic PAD are in greater need of clinical intervention to improve oxidative stress, inflammation, and microcirculation than men, which may, in turn, favorably impact their lower exercise performance and daily activity.

Clinical Relevance.

The clinical significance to the current study is that women with symptomatic PAD are in greater need of clinical intervention to improve oxidative stress, inflammation, and microcirculation than men, which may, in turn, favorably impact their lower exercise performance and daily activity. Women with symptomatic PAD represent a vulnerable subgroup of patients who may benefit more 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 than men.