n-3 Polyunsaturated fatty acids supplementation in peripheral artery disease: the OMEGA-PAD trial

Abstract

Despite current consensus guidelines recommending intensive cardiovascular risk factor management for peripheral artery disease (PAD), patients suffering from PAD continue to experience significant morbidity and mortality. This excess morbid burden is at least partially related to impaired vascular function and systemic inflammation. Interventions bridging this gap are critical. Dietary supplementation of n-3 polyunsaturated fatty acids (n-3 PUFA) has been shown to improve endothelial function and reduce inflammation in different cohorts, as well as to decrease cardiovascular events in secondary prevention trials in patients with coronary artery disease. Their effects in the PAD population are, however, less well understood. The OMEGA-PAD trial is a double-blinded, randomized, placebo-controlled trial that examines the impact of a high-dose, short-duration dietary oral supplementation of n-3 PUFA on vascular function and inflammation in patients with established PAD. The purpose of this article is to provide a detailed description of the design and methods of the OMEGA-PAD trial, and a summary of baseline characteristics of the cohort. ClinicalTrials.gov Identifier: NCT01310270

Keywords

fatty acids peripheral artery disease randomized controlled trials

Introduction and rationale

Peripheral artery disease (PAD) is an epidemic with significant impact on the health of our society. Classically, PAD affects more than 12% of people aged over 65 years, and more than 20% of those aged above 75.¹ However, a more recent study suggests that in a primary care population, one-third of patients aged 70 years and above suffer from PAD.² Once PAD is diagnosed, the annual mortality rate is 4–6%,^3,4 with a 20–60% increased risk of myocardial infarction^5,6 and a 40% increased risk of stroke. With further progression of the disease to its most advanced stage (critical limb ischemia), the 1-year mortality is increased to 25% and may be as high as 45% in those who have undergone an amputation.^3,7,8 Overall, the public remains poorly informed about PAD, and there are major knowledge gaps with regards to the definition of PAD, the risk factors and symptoms of the disease, as well as the association of the disease with the risk of amputation or mortality.⁹ In 2001, a total of $4.37 billion was spent by Medicare on PAD-related treatment and 88% of these expenditures were for inpatient care.¹⁰ Interestingly, the international Reduction of Atherothrombosis for Continued Health (REACH) Registry reported PAD incurring higher annual mean medication and hospitalization costs annually than coronary artery disease (CAD) and cerebrovascular disease.¹¹ Interventions to improve the morbidity and mortality of patients with PAD are therefore critical.

n-3 Polyunsaturated fatty acids (PUFA) have long been recognized to reduce cardiovascular disease,^12,13 with a high tissue ratio of n-3/n-6 PUFA leading to a reduced risk of coronary events.¹⁴ Greenland Eskimos who consume a diet high in n-3 PUFA¹⁵ have a low incidence of CAD. Randomized trials evaluating the impact of oily fish or fish oil supplementation on cardiovascular health have demonstrated a decrease in total mortality, a decrease in cardiovascular death, a decrease in sudden cardiac death and a reduction in non-fatal cardiovascular events.^16
–19 There is presently more controversy about the benefits of n-3 PUFA in patients with cardiovascular disease. For example, in a recent meta-analysis published last year by Rizos et al., there were no significant benefits of n-3 PUFA on all-cause mortality, cardiac death, sudden death, myocardial infarction, or stroke.²⁰ However, in another meta-analysis published the same year, n-3 PUFA were found to be protective against vascular death.²¹ One of the largest trials, the JELIS study, randomized 18,000 hypercholesterolemic patients to statin and n-3 PUFA supplementation versus statins alone. The group with n-3 PUFA supplementation experienced significantly fewer non-fatal coronary events.¹⁸ Furthermore, n-3 PUFA supplementation was associated with a 55% reduction in major coronary events in the PAD subgroup compared to an 18% relative reduction in the patients without PAD.²² Since patients with PAD continue to experience a higher risk of cardiovascular events compared to those with CAD,^23,24 they could potentially benefit from improved medical therapy, warranting further investigations on n-3 PUFA in this patient population.

Part of the clinically relevant benefit of fish-oil or n-3 PUFA supplementation is thought to be due to improvement in endothelial function and reduction in inflammation. Supporting evidence of this proposition includes an improvement in endothelial function in healthy volunteers,^25
–27 in obese adolescents²⁸ and in patients with chronic heart failure,²⁹ as well as a dramatic reduction in inflammation as measured by C-reactive protein (CRP) in healthy individuals,^30
–36 male smokers,³⁷ obese adolescents,²⁸ and in patients with rheumatoid arthritis³⁸ or CAD.³⁹ Still, evidence for the beneficial effects of n-3 PUFA in patients with PAD is controversial.⁴⁰ Although there is some evidence suggesting that consumption of n-3 FAs in the diet may be associated with a decreased prevalence of PAD,⁴¹ the effects of n-3 PUFA on PAD have not been as thoroughly investigated. Several relatively small prospective studies have aimed to assess the effects of fish oil and n-3 PUFA on different parameters related to PAD,^42
–49 with different conclusions. One meta-analysis studied the effects of n-3 PUFA in intermittent claudication⁵⁰ and concluded that blood viscosity decreases with n-3 PUFA supplementation, but that there is no evidence it significantly affects walking distance, the ankle–brachial index or blood pressure. At the present time, there is also insufficient evidence to know if n-3 PUFA can improve endothelial function and inflammation in patients with PAD.

The OMEGA-PAD trial aims to assess the impact of high-dose, short-term, n-3 PUFA supplementation on endothelial function and inflammation in patients with established symptomatic PAD (Figure 1). The results obtained will be correlated with the omega-3 index, a validated marker of tissue n-3 PUFA content and an independent graded risk factor for death from CAD.^51,52 The scientific knowledge obtained from this study could have a significant clinical impact resulting in improved treatment and outcomes for patients suffering from PAD. The results of this study will also provide valuable mechanistic insight into PUFA as well as inform on this class of agents for cardiovascular outcome trials. At the present time, despite guidelines from the American Heart Association (AHA) of n-3 PUFA and diet in general,^53,54 the evidence for PAD remains elusive. If successful, the present study will provide direct evidence of a beneficial impact of dietary n-3 PUFA supplementation on the vascular health of the PAD population, and help define a treatment goal (omega-3 index threshold) to guide such recommendations.

Figure 1.

OMEGA-PAD trial.

Methods

Study design

The OMEGA-PAD trial is a randomized, double-blind, placebo-controlled trial. A total of 80 patients aged 50 years or above with lower extremity PAD in the form of intermittent claudication and an ankle–brachial index (ABI) of <0.9 presenting to the vascular surgery clinic at the Veterans Affairs Medical Center in San Francisco (SFVAMC) will be randomized to 2.2 g oral n-3 PUFA twice daily (total of 4.4 g/day) or a matched placebo for 1 month. Measurements will be done at baseline and after 1 month (Figure 2 and Table 1). The primary endpoint is a change in endothelial function measured by brachial artery flow-mediated, endothelium-dependent vasodilation (FMD). Secondary endpoints include a change in inflammatory and anti-inflammatory markers such as high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), soluble intracellular adhesion molecule-1 (sICAM-1), tumor necrosis factor-α (TNF-α) and 15-epimeric lipoxin A4 (15-epi-LXA4), and improvement in lipid profile (low-density lipoprotein (LDL), triglycerides, high-density lipoprotein (HDL)), blood pressure and patient-perceived walking impairment as determined by the Walking Impairment Questionnaire.⁵⁵ The omega-3 index will be measured to correlate observed effects with changes in the red blood cell (RBC) content of n-3 PUFA.

Figure 2.

Study protocol.

Table 1.

Testing measurements.

Measures	Baseline	1-month follow-up
1. Demographics, anthropometrics, medical history	X
2. Hemodynamic measurements
3. Brachial flow-mediated vasodilation (FMD) study	X	X
4. Arterial tonometry	X	X
5. Ankle–brachial index
6. Body fat composition
7. Inflammatory markers	X	X
8. Omega-3 index	X	X
9. Lipid, renal, and metabolic markers	X	X
10. Walking Impairment Questionnaire	X	X
11. Nutritional Questionnaire (Block 2005 Food Frequency Questionnaire)	X	X
12. Three post-traumatic stress disorder/depression questionnaires (PHQ-9, CIV-C, PTSD Scale)	XXX	X

Ethics

Institutional review board approval was granted for this study by the Committee on Human Research at the University of California, San Francisco (UCSF). The study was registered with ClinicalTrials.org (NCT01310270).

Study population, site and recruitment

The subjects recruited for this study will include males and females with PAD, age 50 years and above. The patients targeted are those with intermittent claudication (Rutherford grade 1–3). Inclusion criteria include: (1) intermittent claudication (mild-severe); (2) resting or exercise ABI<0.9; and (3) age ≥ 50. Exclusion criteria include: (1) critical limb ischemia (rest pain or tissue loss); (2) hypersensitivity/allergies to fish or seafood; (3) significant renal, hepatic or inflammatory disorder; (4) concurrent severe infections; (5) acute illness or major surgery within 30 days; and (6) receiving immunosuppressive medications. Eighty subjects will be recruited from the vascular surgery clinic at the SFVAMC. All patients will undergo a complete history and physical examination. Demographic information, cardiovascular history (e.g. coronary artery disease, cerebrovascular disease), risk factors (hypertension, diabetes, hypercholesterolemia, cigarette smoking, renal insufficiency), medications, and pertinent cardiovascular examination findings will be documented.

Randomization procedures and nutritional intervention

After an individual is recruited and determined to be eligible, the participant will be randomized to one of two groups: n-3 PUFA or placebo. n-3 PUFA supplementation is achieved with four capsules of ProOmega^® twice daily (Nordic Naturals, Watsonville, CA, USA), corresponding to a total of 4.4 g/day. Each ProOmega capsule contains 325 mg of eicosapentaenoic acid (EPA) and 225 mg of docosahexaenoic acid (DHA). We chose the dose of 4 g/day, which corresponds with the AHA’s recommendations for the treatment of hypertriglyceridemia.^40,41 In order to achieve this target dose, a total of eight tabs of ProOmega (325 mg EPA/225 mg DHA) are used, corresponding to a total dose of 4.4 g/day of n-3 PUFA. The placebo group will take the same number of capsules containing an inactive substance (soybean; Nordic Naturals). The placebo capsules are designed to be the same color and shape as the treatment capsules. Prior to the randomization visit, project staff will contact the assigned research pharmacist who will generate the block randomization process. Four subjects will be randomized per block (total of 20 blocks) with a ratio of 1:1 for each block. A 1-month period was chosen in order to assess the impact of acute oral supplementation of fish oil, as other investigators have demonstrated that 1 month of treatment was sufficient to see changes in the lipid profile.⁴⁴

Study protocol

Individuals who are interested in the study will undergo an initial screening questionnaire with the study staff in clinic or over the phone. Those meeting the inclusion criteria will be invited to enter the research study, at which time informed consent will be obtained by study staff in accordance with requirements of the Committee for Human Research.

Subjects will come into the hospital for an initial visit for baseline measurements and then commence the study drug or placebo for 1 month (Figure 2). At the end of the month, they will return for a second visit when study measurements will be repeated (Table 1). In addition to the primary and secondary endpoints described above, other measurements will include bilateral ABIs, bioelectric impedance analysis (BIA), and arterial tonometry to correlate with FMD results. Subjects will also be instructed to complete a nutrition questionnaire (Block 2005 Food Frequency Questionnaire; NutritionQuest, Berkeley, CA, USA) based on their diet,⁵⁶ two surveys for post-traumatic stress disorders,^57,58 and one 8-item patient health questionnaire for depression.⁵⁹

Measurements

Demographics, anthropometrics, medical history, and hemodynamic measurements

Demographic data (including age, race, sex, hip and waist circumference, body mass index (BMI), prior supplement use, exercise frequency), cardiovascular history (e.g. CAD, cerebrovascular disease, previous procedures), risk factors (hypertension, diabetes, hypercholesterolemia, cigarette smoking, renal insufficiency), medications, and pertinent cardiovascular examination findings will be recorded. Blood pressure will be recorded by an indirect sphygmomanometer.

Vascular reactivity of brachial arteries

Flow-mediated vasodilation will be performed according to current guidelines and standards^60,61 and as already described by our group⁶² and other investigators.^63
–66 Subjects will be asked to be fasting (≥ 8 hours) and free of nicotine (≥ 4 hours). A history of recent medications will be recorded. Subjects will be allowed to rest for 10 minutes in a supine position in a darkened room at 23°C. The subject’s arm will be extended onto a movement-constraining pillow with the palmar aspect oriented anteriorly. A 5 cm tourniquet blood pressure cuff will be placed on the upper arm distal to the insertion of the deltoid. The length of the brachial artery will be surveyed by B-mode ultrasound (Philips HD11) using a broadband linear array transducer with a 3–12 MHz range (Philips L12-3) until a straight segment with a visible registration structure can be located. The probe will be oriented so that the artery is at least 3 cm deep to the surface of the skin, the focus aligned with the deep boundary of the vessel, and clearly demarcated intima/lumen boundaries are visible.

Prior to cuff inflation, the baseline diameter of the vessel and blood-flow velocity will be recorded for 60 seconds using electrocardiogram (EKG)-gated image capture software (Brachial Imager; Medical Imaging Applications LLC, Coralville, IA, USA). Baseline blood-flow velocity will be recorded for 60 seconds using an insonation angle of 60°. The Doppler sample gate will be positioned to cover the center, but not the edges, of the lumen. The probe will not be moved between measurements.

The blood pressure cuff will then be inflated to the greater of 250 mmHg or 50 mmHg above the subject’s systolic blood pressure for a period of 5 minutes. Recording of the B-mode images will begin 10 seconds prior to cuff release. Blood-flow velocity will be assessed for a period of 30 seconds post-cuff release using the methods described above. B-mode images will be recorded until 3 minutes post-cuff release.

Analysis of the images will be performed using continuous edge-detection software (Brachial Analyzer; Medical Imaging Applications LLC). Baseline diameter will be recorded as the mean of 60 seconds of data. From hyperemia recordings, the exact moment of cuff release will be noted. Hyperemia diameter will be calculated using a pre-determined time window (55–65 seconds post-cuff release). FMD% will be calculated as [(60 s hyperemia diameter – avg baseline diameter/avg baseline diameter)*100]. The vasoreactivity index was calculated by normalizing FMD to brachial stimulus ratio, with brachial stimulus ratio corresponding to hyperemia flow divided by baseline flow. FMD in healthy subjects is expected to be above 7%⁶⁰ and has been reported to range between 0.20 and 19.2%.⁶⁷ Our preliminary data are comparable to other investigators in the PAD population (5–9%).^62,63,65

Time-averaged velocity measurements will be obtained using the peak-velocity method. Average velocity at baseline will be obtained from 60 seconds of data. Velocity of the hyperemia stimulus will be calculated as the mean velocity of the first four heart beats following cuff-release. Both mean velocity and the velocity time integral will be recorded.

Quality control will be assessed at each point of the measurements. Image quality will be evaluated by a second observer and graded on a 6-point scale that includes: registration structure (landmark), horizontally directed artery, correct longitudinal alignment, clearly visualized near wall intimal medial thickness (IMT) and far wall IMT, and at least 5 mm of clearly visualized artery.

Tonometry

Measurements of arterial stiffness will be performed with an applanation tonometer (SphygmoCor CP (Tonometry System; Itasca, IL, USA) and the provided analysis software. After measuring the subject’s blood pressure and applying EKG leads, the applanation tonometer probe will be placed over the subject’s radial, carotid, and femoral arteries for about 3 minutes to capture pulse wave tracings of the artery of interest. The tracings will be used in the Fourier transform algorithm to calculate several parameters including ascending aortic blood pressure, aortic pulse pressure and augmentation index, ejection duration, and pulse wave velocity between any two sites in the vasculature.

Ankle–brachial index

Ankle–brachial indices will be measured using current guidelines and standards.⁶⁸ Systolic blood pressures of the brachial, posterior tibial and dorsalis pedis arteries will be measured bilaterally. For each lower extremity, the highest systolic pressure of the two pedal pulses will be divided by the highest systolic pressure of the two brachial arteries.

Body fat composition

Body fat composition will be measured using bioelectric impedance analysis (RJL Quantum 101Q; RJL Sciences, Clinton Township, MI, USA). Resistance and reactance will be used to calculate impedance [(resistance²+reactance²)^0.5], which is used to calculate fat free mass (FFM) [(0.8787*ht²)/(impedance^0.4851*25.22)+(0.0813*wt)+0.071]. FFM is used to calculate body fat mass [wt–FFM] and fat percentage [(body fat mass/wt)*100].

Inflammatory markers

Inflammation will be measured through inflammatory biomarkers including hsCRP, IL-6, sICAM-1, TNF-α and 15-epi-LXA4.^69,70 Ten milliliters of whole venous blood will be collected in a fasting state in a tiger-top tube, clotted for a minimum of 30 minutes at room temperature, and centrifuged at 2800 rpm for 10 minutes at 4°C. Serum will be stored at −80°C until assayed for IL-6, sICAM-1, and TNF-α per standard kit protocol (R&D Systems Inc., Minneapolis, MN, USA). The typical coefficients of variation for IL-6, sICAM-1, and TNF-α are 7.4%, 4.6%, and 5.4%, respectively. The lower limits of detection are 0.04 pg/ml, 0.1 ng/ml, and 0.11 pg/ml, respectively. Plasma will be assayed for hsCRP the same day as collection by the SFVAMC lab per standard methodology (Analyzer; Beckman Coulter, Miami, FL, USA). The coefficient of variation for hsCRP using this procedure is 5.1%. In healthy individuals, CRP is <1.0 mg/dl.

n-Fatty acid measurements

The omega-3 index represents the red blood cell (RBC) content of the two major long-chain n-3 FAs, EPA and DHA.⁷¹ It equates to EPA+DHA as a percent of total RBC FAs and has been identified as a biomarker for increased risk for CAD, especially sudden cardiac death. It is read as high, intermediate, and low-risk levels of the omega-3 index (<4%, 4–8%, >8%, respectively). Ten milliliters of whole venous blood will be collected in a fasting state in an EDTA tube, and centrifuged at 2800 rpm for 10 minutes at 4°C within 30 minutes of collection. Packed RBCs will be stored at −80°C until assayed for n-3 FAs, n-6 FAs, arachidonic acid (AA), EPA, DHA, omega-3 index, saturated fat, trans fat, and monounsaturated fat. The RBCs will be analyzed according to the HS-Omega-3 Index^® methodology.⁷² Briefly, fatty acid methyl esters are generated from RBCs by acid transesterification with boron trifluoride and analyzed by capillary gas chromatography using a GC-2010 Gas Chromatograph (Shimadzu Corporation, Columbia, MD, USA) equipped with a SP2560, 100-m column (Supelco, Inc., Bellefonte, PA, USA). Fatty acids are identified by comparison with a standard mixture of fatty acids characteristic of erythrocytes and reported as a percentage of total identified fatty acids after response factor correction. Trans fats are defined as the sum of the trans isomers of oleic acid (C18:1) and linoleic acid (C18:2n-6; i.e. the C18 trans). The typical coefficient of variation for the HS-Omega-3 Index (EPA+DHA) using this procedure is 3% and 7% for trans fats. The average omega-3 index in the US population is 4.5%, with values ranging from 2.7% in the lowest 5^th percentile to 8.8% in the highest 95^th percentile.⁷³

Renal, lipid and metabolic measurements

Blood samples will be collected in a fasting state for measurement of creatinine (Cr), estimated glomerular filtration rate (eGFR), albumin, hemoglobin A1c (HbA1c) as well as total cholesterol, triglycerides, LDL and HDL. Plasma will be assayed for these analytes the same day as collection by the SFVAMC lab per standard methodology (Beckman Coulter Analyzer). Serum will be isolated at the same time points for homocysteine and assayed the same day as collection by the SFVAMC lab per standard methodology (Architect i1000 Analyzer; Abbott Laboratories, Lake Forest, IL, USA).

Walking Impairment Questionnaire (WIQ)

The Walking Impairment Questionnaire (WIQ) is a validated instrument that assesses the patient-perceived walking impairment.⁵⁵ This questionnaire will be administered by research staff and measures patient-perceived walking capacity and limitation across three domains: distance, speed, and stair-climbing. The WIQ Distance score is calculated after the patient ranks, on an ordinal Likert scale, his or her degree of difficulty walking specific distances (indoors at home to five blocks). The WIQ Speed score uses a similar method: patients are asked to rank their degree of difficulty walking a fixed distance at difference speeds (walking slowly to jogging). The WIQ Stair score asks patients to rank their difficulty climbing one to three flights of stairs. To score the WIQ, the individual items are multiplied by a weighted factor, the products are then summed and divided by the maximum possible score yielding a percent. For example, the WIQ Distance multiplies different distances (20 to 1500 feet; 6.1 to 457.2 meters). Each distance is multiplied by the respondent’s difficulty ranking (0 to 4). The sum of these is divided by the total possible score. The WIQ Speed score assigns weights to different speeds (1 to 5 mph; 1.6 to 8 km/h). The WIQ Stair score assigns a proportional weighting to the number of flights of stairs (one to three).

Adverse events

Adverse events will be regularly assessed by telephone every week as well as at the end of the study. All events will be recorded, whether elicited by the study staff or reported by the subject at clinic visits or via telephone contact. Additionally, all participants will be asked to contact study staff in the event of hospitalization, emergency room visits or any other unexpected medical event.

Statistical analysis

The sample size for this study was calculated using a paired t-test. Our study evaluates the hypothesis that n-3 PUFA supplementation is associated with an improvement in brachial artery FMD. Based on the available data from the literature, the closest estimate of an effect size that can be expected is a 40% increase in FMD. For example, Schiano et al. demonstrated a 3.3% (6.7–10.0%) absolute increase in FMD with n-3 PUFA supplementation,⁴⁸ which corresponds to a 49% relative increase in FMD. In the hypercholesterolemic patients, Goodfellow et al. demonstrated a doubling of FMD with n-3 PUFA supplementation.⁷⁴ Hence, a 40% increase in FMD would be considered conservative. Based on data from a similar patient population at Brigham and Women’s Hospital,⁷⁵ a baseline value of 9.21±4.6% is expected in the PAD population. With a power of 0.9 and a 40% increase in brachial artery diameter, a sample size of 33 patients per group (66 total) should lead to significance. Considering a possible 10% loss of patients during the study due to loss of follow-up, discontinuing participation in the study, we plan to randomize 40 patients per group for a total of 80 patients participating in the study.

For subsequent analysis, all variables will be summarized by appropriate descriptive statistics at each time point. The intervention group will be compared to the placebo group for homogeneity on demographic and baseline clinical variables via the chi-squared test or Student’s t-test (depending on the type of variable). Through stratified randomization, these variables are anticipated to be comparable in both groups. All analyses will be based on intention to treat (all participants randomized will be analyzed based upon their assigned group). The significance level will be preset at 0.05 and two-sided tests will be performed. Results will be correlated with the change in the omega-3 index after intervention. Statistical analyses will be performed using Stata/SE 12 (StataCorp, College Station, TX, USA).

Results

Baseline characteristics of the first 50 randomized patients in the OMEGA-PAD trial

Recruitment for the OMEGA-PAD trial started in April 2011. Fifty male veterans have been enrolled and randomized (Table 2). The mean age is 67±9 years. The mean index limb ABI is 0.80±0.19. CAD is present in 36%, hypertension in 92%, and diabetes mellitus in 32%. A total of 47% are current smokers and 45% are former smokers. The mean HbA1c is 8±1, LDL 93±37 mg/dl, triglycerides 160±98 mg/dl and HDL 43±12 mg/dl. The baseline brachial FMD is 7±4%, indicating an overall impairment of endothelial function in this cohort. The inflammatory burden of this cohort is substantial – as evidenced by hsCRP 4.5±4.8 mg/l. The baseline omega-3 index is 4.9±0.2%.

Table 2.

Baseline characteristics of the first 50 individuals recruited for the OMEGA-PAD trial.

General characteristics	Frequency count (%)	Mean	SD
Age, years		67	9
Male	50 (100%)
Caucasian	36 (72%)
Veteran	47 (100%)
Left ABI		0.80	0.19
Right ABI		0.82	0.20
Rutherford
Mild claudication	12 (24%)
Moderate claudication	16 (32%)
Severe claudication	22 (44%)
BMI		28	4
Waist:hip ratio		1.01	0.06
Omega index (%)		4.9	0.2
Comorbidities
Coronary artery disease	18 (36%)
Hypertension	46 (92%)
Hyperlipidemia	45 (90%)
Diabetes mellitus	16 (32%)
Surgical history
Index bypass	7 (14%)
Index percutaneous	13 (27%)
Systolic blood pressure (mmHg)		136	18
Diastolic blood pressure (mmHg)		76	9
Medications
Aspirin	28 (58%)
Ace inhibitor	18 (38%)
B-blocker	26 (54%)
Statin	43 (90%)
PAD risk factors
Smoking, current	23 (47%)
Smoking, former	22 (45%)
Pack years, years*packs/day		51.36	37.58
Cholesterol, mg/dl		166.24	41.36
Triglycerides, mg/dl		160	98
HDL, mg/dl		43	12
LDL, mg/dl		93	37
Serum creatinine (CR), mg/dl		1.05	0.34
eGFR, ml/min		79	23
Albumin, g/dl		4.02	0.35
Homocysteine, µmol/l		13.62	4.41
HbA1c, %		8	1
Inflammation
hsCRP, mg/l		4.5	4.8
IL-6, pg/ml		1.6	0.9
ICAM-1, ng/ml		12.6	5.0
TNF-α, pg/ml		2.3	1.6
Brachial FMD and tonometry
Brachial FMD, %		7.19	4.19
Brachial artery baseline diameter, cm		0.38	0.06
Brachial artery mean velocity, m/s		0.15	0.07
Brachial artery baseline flow, ml/min		103.46	56.06
Brachial artery baseline shear stress, dynes/cm²		11.49	5.58
Brachial artery reactive hyperemia diameter, cm		0.40	0.06
Brachial artery reactive hyperemia velocity, m/s		0.67	0.28
Brachial reactive hyperemia flow, ml/min		513.48	248.29
Brachial reactive hyperemia shear stress, dynes/cm²		47.37	20.34
Stimulus ratio		4.88	2.31
Patient-perceived walking performance
Walking distance (score: 0–100)		26	23
Walking speed (score: 0–100)		26	24
Stairs (score: 0–100)		32	25

ABI, ankle–brachial index; BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; eGFR, estimated glomerular filtration rate; hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin-6; ICAM-1, intracellular adhesion molecule-1; TNF-α, tumor necrosis factor-α; FMD, flow-mediated, endothelium-dependent vasodilation.

Discussion

PAD is an epidemic with significant impact on the health of the aging population. In addition to better understanding the effects of n-3 PUFA supplementation on vascular health in patients with symptomatic PAD, we expect that this study will serve as a pilot study for a larger randomized trial assessing the effects of n-3 PUFA supplementation on disease progression and clinical events in patients with PAD. Successful prevention of critical limb ischemia and treatment of the disease at an early stage could have a tremendous impact in terms of the economic benefits to the individual as well as the healthcare system, and more importantly a significant improvement to the quality of life of thousands of patients.

Significance of the study and contribution to knowledge development

This study is innovative in several areas of PAD research. First, it is presently unknown whether a short-term dietary intervention (high-dose n-3 PUFA oral supplementation) has the potential to reverse key features (endothelial dysfunction and inflammation) of vasculopathy associated with chronic PAD. Second, this study captures clinically relevant physiological endpoints of dietary supplement (fish oil) which could potentially become an alternative to drug therapies for the treatment of PAD. Current medical therapies for PAD are limited to antiplatelet and lipid-lowering medications, and there is a great need for additional pharmacotherapy to reduce the burden of this disease. It is known that elderly patients have a lower compliance to statins compared to younger patients.^76,77 n-3 PUFA supplementation may therefore represent a cheaper and better tolerated alternative for that population, with the overall goal of slowing and potentially reversing disease progression.

The definition of a relationship between dietary omega-3 supplementation and inflammatory markers in a PAD cohort is another novel aspect of this trial. Epidemiologic evidence linking inflammation to the future development and severity of PAD has been established.^78
–81 CRP is a classical acute-phase marker and a member of the pentraxin family of innate immune response proteins.⁸² It is synthesized by the liver and plasma concentrations can increase 1000-fold in response to an inflammatory stimulus.⁸³ Inflammation in general, and CRP in particular, has been correlated with impaired endothelial function in vivo and in vitro,^84,85 and hsCRP levels have been inversely correlated with acetylcholine-induced, endothelial-dependent forearm blood flow.⁸⁶ hsCRP as well as IL-6 and ICAM have been found to be increased in patients with PAD.^75,87,88 Emerging evidence suggests that omega-3 FAs are biologically linked to the endogenous resolution pathway of PAD^89
–91 and 15-epi-LXA4 levels have been found to be reduced in PAD patients.⁸⁸ This study will allow us to correlate levels of pro- and anti-inflammatory markers in response to omega-3 supplementation in a cohort with PAD. Furthermore, the inflammatory markers chosen for this trial have already been found to be correlated with clinical events in PAD patients in our group and in others.^75,88,92

Another unique aspect of our study is the correlation of endothelial function and inflammatory changes with the actual incorporation of n-3 PUFA in the RBC membrane using the omega-3 index, a validated marker of tissue n-3 content. Impaired FMD of the brachial artery independently predicts cardiovascular events in patients undergoing vascular surgery,^93,94 emphasizing the relationship between markers of inflammation, thrombosis, and endothelial function.⁹⁵ Our study will provide direct evidence linking n-3 PUFA supplementation to vascular phenotype via changes in cellular lipid metabolism.

Lastly, the preliminary data accumulated during the proposed study may lead to the development of a laboratory treatment target for the use of n-3 PUFA supplements in PAD. The omega-3 index has been correlated with CAD. A subsequent trial based on the data acquired in this study could validate a suggested threshold level of the omega-3 index in a larger population with PAD. Overall, we feel that this trial will potentially bring another paradigm to clinical practice with the potential addition of n-3 PUFA supplementation to the medical management of patients, particularly elderly patients with PAD. Future trials could evaluate the effects of n-3 PUFA on the functional status of patients with PAD.

Brachial artery FMD in this cohort is significantly depressed compared with other cohorts and therefore we feel this represents a vulnerable cohort for incident and recurrent cardiovascular events. It is not currently known whether or not improvement of endothelial function in this cohort will translate into improvement in clinical outcomes and this represents one of the first attempts to mitigate risk in this population. A recent meta-analysis suggested that the association between FMD and the estimated 10-year risk of CAD assessed by the Framingham risk score was strongest in low-risk populations compared to medium or high-risk populations.⁹⁶ In the first 50 patients randomized to this study, the Framingham risk score (10-year cardiovascular risk) is 19%. Therefore, we feel studies in high-risk populations such as ours will provide clarity on this issue and guide future investigations.

Additional limitations of study/design/methods

It is possible that patients may experience an aftertaste after taking the fish oil capsules, possibly affecting their compliance with treatment. However, we will perform a pill count when patients return after the treatment period. Furthermore, the omega-3 index should help us confirm that the n-3 PUFA supplementation was taken by quantifying the incorporation into the RBC membrane. Additionally, hsCRP and other biomarkers may be affected by concurrent infectious or inflammatory illness unrelated to vascular disease per se. However, with proper selection and exclusion criteria as outlined, the baseline levels should be minimally affected by such factors. The magnitude of an important association may be less than hypothesized, in which case an inadequate sample size may fail to detect potentially significant relationships between markers and endothelial function.

Another major limitation is related to unknown aspects of the dosing scheme. A high-dose and short-duration intervention was chosen but a longer treatment and/or higher dose may be required to have a beneficial effect. Furthermore, the very short-term follow-up period will limit the ability to discern effects that may require more time to be observed. These unknown aspects could, however, be studied in further trials.

The study population, composed of mostly male veterans who are predominantly Caucasian, may not be fully representative of the wider PAD population. Furthermore, the inflammatory phenotype of the population studied at the SFVAMC may be skewed compared to the typical PAD patient with regards to levels of psychological and psychosocial factors affecting overall stress and inflammation.⁹⁷

Conclusion

The OMEGA-PAD trial will test the novel hypothesis that short-term, intensive n-3 PUFA supplementation improves functional and inflammatory parameters in a cohort at high vascular risk. The results of this study will provide valuable mechanistic insight into PUFA as well as inform on this class of agents for cardiovascular outcome trials to ultimately decrease morbidity burden in this patient population.

Footnotes

Declaration of conflicting interest

The authors have no conflicts of interest to declare.

Funding

This work was supported by start-up funds from the University of California, San Francisco and the Northern California Institute for Research and Education. The project described was supported by a Society for Vascular Surgery Seed Grant and by Award Number KL2RR024130 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The funding organizations were not involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript. This publication was also supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through UCSF-CTSI Grant Number KL2 TR000143. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

References

Criqui

Fronek

Barrett-Connor

Klauber

Gabriel

Goodman

. The prevalence of peripheral arterial disease in a defined population. Circulation 1985; 71: 510–515.

Hirsch

Criqui

Treat-Jacobson

. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001; 286: 1317–1324.

Criqui

Langer

Fronek

. Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med 1992; 326: 381–386.

McKenna

Wolfson

Kuller

. The ratio of ankle and arm arterial pressure as an independent predictor of mortality. Atherosclerosis 1991; 87: 119–128.

Smith

Shipley

Rose

. Intermittent claudication, heart disease risk factors, and mortality. The Whitehall Study. Circulation 1990; 82: 1925–1931.

Leng

Lee

Fowkes

. Incidence, natural history and cardiovascular events in symptomatic and asymptomatic peripheral arterial disease in the general population. Int J Epidemiol 1996; 25: 1172–1181.

Kazmers

Perkins

Jacobs

. Major lower extremity amputation in Veterans Affairs medical centers. Ann Vasc Surg 2000; 14: 216–222.

Dormandy

Heeck

Vig

. The fate of patients with critical leg ischemia. Semin Vasc Surg 1999; 12: 142–147.

Hirsch

Murphy

Lovell

. Gaps in public knowledge of peripheral arterial disease: the first national PAD public awareness survey. Circulation 2007; 116: 2086–2094.

10.

Hirsch

Hartman

Town

Virnig

. National health care costs of peripheral arterial disease in the Medicare population. Vasc Med 2008; 13: 209–215.

11.

Mahoney

Wang

Cohen

. One-year costs in patients with a history of or at risk for atherothrombosis in the United States. Circ Cardiovasc Qual Outcomes 2008; 1: 38–45.

12.

Kromhout

Bosschieter

de Lezenne Coulander

. The inverse relation between fish consumption and 20-year mortality from coronary heart disease. N Engl J Med 1985; 312: 1205–1209.

13.

Daviglus

Stamler

Orencia

. Fish consumption and the 30-year risk of fatal myocardial infarction. N Engl J Med 1997; 336: 1046–1053.

14.

Harris

Poston

Haddock

. Tissue n-3 and n-6 fatty acids and risk for coronary heart disease events. Atherosclerosis 2007; 193: 1–10.

15.

Bang

Dyerberg

Nielsen

. Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos. Lancet 1971; i: 1143–1145.

16.

Burr

Fehily

Gilbert

. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 1989; ii: 757–761.

17.

Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico. Lancet 1999; 354: 447–455.

18.

Yokoyama

Origasa

Matsuzaki

. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet 2007; 369: 1090–1098.

19.

Marik

Varon

. Omega-3 dietary supplements and the risk of cardiovascular events: a systematic review. Clin Cardiol 2009; 32: 365–372.

20.

Rizos

Ntzani

Bika

Kostapanos

Elisaf

. Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic review and meta-analysis. JAMA 2012; 308: 1024–1033.

21.

Kotwal

Jun

Sullivan

Perkovic

Neal

. Omega 3 Fatty acids and cardiovascular outcomes: systematic review and meta-analysis. Circ Cardiovasc Qual Outcomes 2012; 5: 808–818.

22.

Ishikawa

Yokoyama

Saito

. Preventive effects of eicosapentaenoic acid on coronary artery disease in patients with peripheral artery disease. Circ J 2010; 74: 1451–1457.

23.

Cotter

Cannon

McCabe

. Prior peripheral arterial disease and cerebrovascular disease are independent predictors of adverse outcome in patients with acute coronary syndromes: are we doing enough? Results from the Orbofiban in Patients with Unstable Coronary Syndromes–Thrombolysis In Myocardial Infarction (OPUS-TIMI) 16 study. Am Heart J 2003; 145: 622–627.

24.

Golomb

Dang

Criqui

. Peripheral arterial disease: morbidity and mortality implications. Circulation 2006; 114: 688–699.

25.

Tousoulis

Papageorgiou

Antoniades

. Acute effects of different types of oil consumption on endothelial function, oxidative stress status and vascular inflammation in healthy volunteers. Br J Nutr 2010; 103: 43–49.

26.

Khan

Elherik

Bolton-Smith

. The effects of dietary fatty acid supplementation on endothelial function and vascular tone in healthy subjects. Cardiovasc Res 2003; 59: 955–962.

27.

Fahs

Yan

Ranadive

. The effect of acute fish-oil supplementation on endothelial function and arterial stiffness following a high-fat meal. Appl Physiol Nutr Metab 2010; 35: 294–302.

28.

Dangardt

Osika

Chen

. Omega-3 fatty acid supplementation improves vascular function and reduces inflammation in obese adolescents. Atherosclerosis 2010; 212: 580–585.

29.

Morgan

Dixon

Hanratty

. Effects of dietary omega-3 fatty acid supplementation on endothelium-dependent vasodilation in patients with chronic heart failure. Am J Cardiol 2006; 97: 547–551.

30.

Bloomer

Larson

Fisher-Wellman

Galpin

Schilling

. Effect of eicosapentaenoic and docosahexaenoic acid on resting and exercise-induced inflammatory and oxidative stress biomarkers: a randomized, placebo controlled, cross-over study. Lipids Health Dis 2009; 8: 36.

31.

Liu

Daviglus

. Associations of dietary long-chain n-3 polyunsaturated fatty acids and fish with biomarkers of inflammation and endothelial activation (from the Multi-Ethnic Study of Atherosclerosis [MESA]). Am J Cardiol 2009; 103: 1238–1243.

32.

Lopez-Garcia

Schulze

Manson

. Consumption of (n-3) fatty acids is related to plasma biomarkers of inflammation and endothelial activation in women. J Nutr 2004; 134: 1806–1811.

33.

Micallef

Munro

Garg

. An inverse relationship between plasma n-3 fatty acids and C-reactive protein in healthy individuals. Eur J Clin Nutr 2009; 63: 1154–1156.

34.

Poudel-Tandukar

Nanri

Matsushita

. Dietary intakes of alpha-linolenic and linoleic acids are inversely associated with serum C-reactive protein levels among Japanese men. Nutr Res 2009; 29: 363–370.

35.

Niu

Hozawa

Kuriyama

. Dietary long-chain n-3 fatty acids of marine origin and serum C-reactive protein concentrations are associated in a population with a diet rich in marine products. Am J Clin Nutr 2006; 84: 223–229.

36.

Murakami

Sasaki

Takahashi

. Total n-3 polyunsaturated fatty acid intake is inversely associated with serum C-reactive protein in young Japanese women. Nutr Res 2008; 28: 309–314.

37.

Ohsawa

Itai

Onoda

. Dietary intake of n-3 polyunsaturated fatty acids is inversely associated with CRP levels, especially among male smokers. Atherosclerosis 2008; 201: 184–191.

38.

Sundrarjun

Komindr

Archararit

. Effects of n-3 fatty acids on serum interleukin-6, tumour necrosis factor-alpha and soluble tumour necrosis factor receptor p55 in active rheumatoid arthritis. J Int Med Res 2004; 32: 443–454.

39.

Farzaneh-Far

Harris

Garg

Whooley

. Inverse association of erythrocyte n-3 fatty acid levels with inflammatory biomarkers in patients with stable coronary artery disease: The Heart and Soul Study. Atherosclerosis 2009; 205: 538–543.

40.

Grenon

Hughes-Fulford

Rapp

Conte

. Polyunsaturated fatty acids and peripheral artery disease. Vasc Med 2012; 17: 51–63.

41.

Lane

Magno

Lane

Chan

Hoyt

Greenfield

. Nutrition impacts the prevalence of peripheral arterial disease in the United States. J Vasc Surg 2008; 48: 897–904.

42.

Woodcock

Smith

Lambert

. Beneficial effect of fish oil on blood viscosity in peripheral vascular disease. Br Med J 1984; 288: 592–594.

43.

Gans

Bilo

Weersink

. Fish oil supplementation in patients with stable claudication. Am J Surg 1990; 160: 490–495.

44.

Mori

Vandongen

Mahanian

Douglas

. Plasma lipid levels and platelet and neutrophil function in patients with vascular disease following fish oil and olive oil supplementation. Metabolism 1992; 41: 1059–1067.

45.

Leng

Lee

Fowkes

. Randomized controlled trial of gamma-linolenic acid and eicosapentaenoic acid in peripheral arterial disease. Clin Nutr 1998; 17: 265–271.

46.

Carrero

Lopez-Huertas

Salmeron

Baro

Ros

. Daily supplementation with (n-3) PUFAs, oleic acid, folic acid, and vitamins B-6 and E increases pain-free walking distance and improves risk factors in men with peripheral vascular disease. J Nutr 2005; 135: 1393–1399.

47.

Conway

Dillon

Evans

Howells-Jones

Price

Harding

Hill

. A doubled-blinded, randomized study to determine the effect of omega-3 marine triglycerides on intermittent claudication. Yearbook 2005. London: Vascular Society of Great Britain and Ireland, 86 A.

48.

Schiano

Laurenzano

Brevetti

. Omega-3 polyunsaturated fatty acid in peripheral arterial disease: effect on lipid pattern, disease severity, inflammation profile, and endothelial function. Clin Nutr 2008; 27: 241–247.

49.

Madden

Brunner

Dastur

. Fish oil induced increase in walking distance, but not ankle brachial pressure index, in peripheral arterial disease is dependent on both body mass index and inflammatory genotype. Prostaglandins Leukot Essent Fatty Acids 2007; 76: 331–340.

50.

Sommerfield

Price

Hiatt

. Omega-3 fatty acids for intermittent claudication. Cochrane Database Syst Rev 2007; (4): CD003833.

51.

Harris

. The omega-3 index as a risk factor for coronary heart disease. Am J Clin Nutr 2008; 87: 1997S–2002S.

52.

Harris

Von Schacky

. The Omega-3 Index: a new risk factor for death from coronary heart disease? Prev Med 2004; 39: 212–220.

53.

Lichtenstein

Appel

Brands

. Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation 2006; 114: 82–96.

54.

Kris-Etherton

Harris

Appel

. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 2002; 106: 2747–2757.

55.

Nicolai

Kruidenier

Rouwet

Graffius

Prins

Teijink

. The walking impairment questionnaire: an effective tool to assess the effect of treatment in patients with intermittent claudication. J Vasc Surg 2009; 50: 89–94.

56.

Block

Woods

Potosky

Clifford

. Validation of a self-administered diet history questionnaire using multiple diet records. J Clin Epidemiol 1990; 43: 1327–1335.

57.

Blanchard

Jones-Alexander

Buckley

Forneris

. Psychometric properties of the PTSD Checklist (PCL). Behav Res Ther 1996; 34: 669–673.

58.

Foa

Cashman

Jaycox

Perry

. The validation of a self-report measure of PTSD: The Posttraumatic Diagnostic Scale. Psychological Assessment 1997; 9: 445–451.

59.

Kroenke

Spitzer

Williams

. The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med 2001; 16: 606–613.

60.

Corretti

Anderson

Benjamin

. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002; 39: 257–265.

61.

Thijssen

Black

Pyke

. Assessment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol 2011; 300: H2–H12.

62.

Owens

Wake

Conte

Gerhard-Herman

Beckman

. In vivo human lower extremity saphenous vein bypass grafts manifest flow mediated vasodilation. J Vasc Surg 2009; 50: 1063–1070.

63.

McDermott

Ades

Guralnik

. Treadmill exercise and resistance training in patients with peripheral arterial disease with and without intermittent claudication: a randomized controlled trial. JAMA 2009; 301: 165–174.

64.

Golledge

Leicht

Crowther

. Determinants of endothelial function in a cohort of patients with peripheral artery disease. Cardiology 2008; 111: 51–56.

65.

Husmann

Dorffler-Melly

Kalka

Diehm

Baumgartner

Silvestro

. Successful lower extremity angioplasty improves brachial artery flow-mediated dilation in patients with peripheral arterial disease. J Vasc Surg 2008; 48: 1211–1216.

66.

Payvandi

Dyer

McPherson

. Physical activity during daily life and brachial artery flow-mediated dilation in peripheral arterial disease. Vasc Med 2009; 14: 193–201.

67.

Bots

Westerink

Rabelink

de Koning

. Assessment of flow-mediated vasodilatation (FMD) of the brachial artery: effects of technical aspects of the FMD measurement on the FMD response. Eur Heart J 2005; 26: 363–368.

68.

Aboyans

Criqui

Abraham

. Measurement and interpretation of the ankle-brachial index: a scientific statement from the American Heart Association. Circulation 2012; 126: 2890–2909.

69.

Serhan

Hamberg

Samuelsson

. Lipoxins: novel series of biologically active compounds formed from arachidonic acid in human leukocytes. Proc Natl Acad Sci U S A 1984; 81: 5335–5339.

70.

Spite

Owens

. Aspirin-triggered lipoxin and resolvin E1 modulate vascular smooth muscle phenotype and correlate with peripheral atherosclerosis. Am J Pathol 2010; 177: 2116–2123.

71.

Harris

. The omega-3 index as a risk factor for coronary heart disease. Am J Clin Nutr 2008; 87: 1997S–2002S.

72.

Farzaneh-Far

Lin

Epel

Harris

Blackburn

Whooley

. Association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease. JAMA 2010; 303: 250–257.

73.

Harris

Pottala

Varvel

Borowski

Ward

McConnell

. Erythrocyte omega-3 fatty acids increase and linoleic acid decreases with age: observations from 160,000 patients. Prostaglandins Leukot Essent Fatty Acids 2013; 88: 257–263.

74.

Goodfellow

Bellamy

Ramsey

Jones

Lewis

. Dietary supplementation with marine omega-3 fatty acids improve systemic large artery endothelial function in subjects with hypercholesterolemia. J Am Coll Cardiol 2000; 35: 265–270.

75.

Owens

Wake

Conte

Gerhard-Herman

Beckman

. In vivo human lower extremity saphenous vein bypass grafts manifest flow mediated vasodilation. J Vasc Surg 2009; 50: 1063–1070.

76.

Benner

Glynn

Mogun

Neumann

Weinstein

Avorn

. Long-term persistence in use of statin therapy in elderly patients. JAMA 2002; 288: 455–461.

77.

Jackevicius

Mamdani

. Adherence with statin therapy in elderly patients with and without acute coronary syndromes. JAMA 2002; 288: 462–467.

78.

Ridker

Cushman

Stampfer

Tracy

Hennekens

. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998; 97: 425–428.

79.

Ridker

Stampfer

Rifai

. Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease. JAMA 2001; 285: 2481–2485.

80.

Beckman

Preis

Ridker

Gerhard-Herman

. Comparison of usefulness of inflammatory markers in patients with versus without peripheral arterial disease in predicting adverse cardiovascular outcomes (myocardial infarction, stroke, and death). Am J Cardiol 2005; 96: 1374–1378.

81.

Tzoulaki

Murray

Lee

Rumley

Lowe

Fowkes

. C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study. Circulation 2005; 112: 976–983.

82.

Westhuyzen

Healy

. Review: Biology and relevance of C-reactive protein in cardiovascular and renal disease. Ann Clin Lab Sci 2000; 30: 133–143.

83.

Gauldie

Richards

Northemann

Fey

Baumann

. IFN beta 2/BSF2/IL-6 is the monocyte-derived HSF that regulates receptor-specific acute phase gene regulation in hepatocytes. Ann N Y Acad Sci 1989; 557: 46–58; discussion 59.

84.

Verma

Wang

. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 2002; 106: 913–919.

85.

Owens

Ridker

Belkin

. Elevated C-reactive protein levels are associated with postoperative events in patients undergoing lower extremity vein bypass surgery. J Vasc Surg 2007; 45: 2–9; discussion 9.

86.

Fichtlscherer

Rosenberger

Walter

Breuer

Dimmeler

Zeiher

. Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease. Circulation 2000; 102: 1000–1006.

87.

Conen

Rexrode

Creager

Ridker

Pradhan

. Metabolic syndrome, inflammation, and risk of symptomatic peripheral artery disease in women: a prospective study. Circulation 2009; 120: 1041–1047.

88.

Spite

Owens

. Aspirin-triggered lipoxin and resolvin E1 modulate vascular smooth muscle phenotype and correlate with peripheral atherosclerosis. Am J Pathol 2010; 177: 2116–2123.

89.

Serhan

Brain

Buckley

. Resolution of inflammation: state of the art, definitions and terms. FASEB J 2007; 21: 325–332.

90.

Serhan

Chiang

Van Dyke

. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 2008; 8: 349–361.

91.

Serhan

Hong

Gronert

. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 2002; 196: 1025–1037.

92.

Pande

Perlstein

Beckman

Creager

. Association of insulin resistance and inflammation with peripheral arterial disease: the National Health and Nutrition Examination Survey, 1999 to 2004. Circulation 2008; 118: 33–41.

93.

Gokce

Keaney

Jr Hunter

. Predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease. J Am Coll Cardiol 2003; 41: 1769–1775.

94.

Gokce

Keaney

Jr Hunter

Watkins

Menzoian

Vita

. Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study. Circulation 2002; 105: 1567–1572.

95.

Brevetti

Silvestro

Di Giacomo

. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg 2003; 38: 374–379.

96.

Witte

Westerink

de Koning

van der Graaf

Grobbee

Bots

. Is the association between flow-mediated dilation and cardiovascular risk limited to low-risk populations? J Am Coll Cardiol 2005; 45: 1987–1993.

97.

Grenon

Hiramoto

Smolderen

Vittinghoff

Whooley

Cohen

. Association between depression and peripheral artery disease: insights from the heart and soul study. J Am Heart Assoc 2012; 1: e002667.