Omega-3 polyunsaturated fatty acids improve endothelial function in humans at risk for atherosclerosis: A review

Abstract

Epidemiology studies and clinical trials show that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can prevent atherosclerotic morbidity and evidence suggests this may be mediated by improving endothelial dysfunction. Endothelial dysfunction is characterized by reduced vasodilation and a pro-inflammatory, pro-thrombotic state, and is an early pathological event in the development of atherosclerosis. Flow-mediated dilation (FMD), a gold standard for assessing endothelial dysfunction, is a predictor of future cardiovascular events and coronary heart disease risk. Notably, risk factors for endothelial dysfunction include classic risk factors for atherosclerosis: Elevated lipids, diabetes, hypertension, elevated BMI, cigarette smoking, and metabolic syndrome. In this paper, we review the ability of n-3 PUFAs to improve endothelial dysfunction in individuals with classic risk factors for atherosclerosis, but lacking diagnosed atherosclerotic disease, with the goal of identifying those individuals that might gain the most vasoprotection from n-3 PUFA supplements. We include trials using eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or alpha-linolenic acid (ALA) alone, or EPA + DHA; and assessing endothelial function by FMD, forearm blood flow, or peripheral arterial tonometry. We found that n-3 PUFAs improved endothelial dysfunction in 16 of 17 studies in individuals with hyperlipidemia, elevated BMI, metabolic syndrome, or that smoked cigarettes, but only in 2 of 5 studies in diabetics. Further, these trials showed that use of EPA + DHA consistently improve endothelial dysfunction; ALA-enriched diets appear promising; but use of EPA or DHA alone requires further study. We conclude that individuals with hyperlipidemia, elevated BMI, metabolic syndrome, or that smoke could derive vaosprotective benefits from EPA + DHA supplementation.

1. Introduction

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) prevent atherosclerotic disease development in humans based on epidemiology studies and clinical trials [1–4]. Atherosclerotic associated-diseases include peripheral artery disease, coronary artery disease, aneurysm, and stroke. Endothelial dysfunction, which is characterized by a loss of vasodilatory and anti-inflammatory factors and a gain of vasoconstrictive and pro-inflammatory factors, is an early pathological event in the development of atherosclerotic cardiovascular (CV) diseases [5]. Risk factors for endothelial dysfunction include classic risk factors for atherosclerosis; elevated cholesterol, elevated triglycerides, diabetes, hypertension and cigarette smoking [6]. Thus, n-3 PUFAs would be considered to be a form of primary prevention since the consumption of n-3 PUFAs decreases the incidence of atherosclerotic disease as individuals age.

Endothelial dysfunction is one of the earliest events in the pathological development of atherosclerotic diseases, and flow-mediated dilation (FMD) is the non-invasive, gold standard for measuring endothelial dysfunction in the clinic. Briefly, to assess FMD the baseline brachial artery diameter is recorded via ultrasound and a blood pressure cuff is placed around the forearm and inflated for 5 min, restricting blood flow. The cuff is then deflated, causing reactive hyperemia, and the vessel diameter is continually recorded for 2 min post-cuff deflation. This shear-stress induced production of endothelial-derived vasodilatory factors, primarily nitric oxide (NO), causes the brachial artery to dilate and FMD is expressed as the percentage increase in artery diameter relative to baseline diameter [7].

Prospective studies have shown that FMD is an independent predictor of CV events, such as heart attack or stroke, in individuals without clinical CV disease or at low CV disease risk. A prospective study of 2264 women (54 ± 6 years) free of clinical CV disease demonstrated that FMD is significantly associated with CV events in a 4 year follow-up and is independent of other classic risk factors [8]. Similarly, a prospective study of 435 individuals (54 ± 12 years) without apparent coronary heart disease (CHD) show that FMD is the best independent predictor of future CV events [9]. In an earlier meta-analysis of 211 publications, FMD is significantly predictive of the 10 year risk of CHD, but only in those individuals with low Framingham risk scores [10]. Furthermore, studies have shown that improving FMD reduces the number of CV events in individuals with existing CV disease. For example, patients with hypertension and persistently impaired FMD (7.1 ± 2.5%) exhibit 3.5 non-fatal CV events/100 person-years, which is significantly higher (P < 0.0001) than 0.51 CV events/100 person-years in patients with improved FMD (13. ± 2.6%) [11]. Thus, these studies suggest that some individuals could benefit from therapies specifically targeted to improve endothelial dysfunction.

Evidence from multiple epidemiological, experimental and clinical studies suggests that n-3 PUFAs can decrease the risk of CV disease, in part, by improving vascular function. Three n-3 PUFAs have been found to be vasoprotective; eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) enriched in fish oil, and alpha-linolenic acid (ALA, 18:3n-3) enriched in certain plants. Some of these vasoprotective benefits include decreased arterial plaque buildup [12], increased anti-inflammatory properties [13], improved endothelial-dependent vasodilation as measured by FMD [14–16], decreased blood pressure [17–19], and increased antioxidant capacity [20].

2. Methods

In this article, we review the ability of n-3 PUFA supplementation to improve endothelial function in individuals with classic risk factors for atherosclerosis, including hyperlipidemia, hypertension, diabetes, smoking, elevated BMI, and metabolic syndrome with the goal of identifying those individuals that might gain the most vasoprotective benefit from n-3 PUFA supplements prior to the clinical evidence of atherosclerotic disease. In addition to FMD, we have included two other methods for assessing vascular function; peripheral arterial tonometry (PAT) and forearm blood flow (FBF). PAT measures blood flow in the index fingers using plethysmographic probes prior to, during, and after compression of blood flow in one upper arm (EndoPAT; Itamar Medical), and results are expressed as the reactive hyperemia index (RHI) [21]. FBF is assessed using strain-gauge plethysmography prior to and following upper arm occlusion or prior to and following arterial infusion of an endothelial-dependent vasodilator. While all three methods are indices of endothelial function, FMD assesses the dilation occurring in a conduit artery, while FBF and PAT assess the dilation occurring in resistance arterioles of the forearm and fingers, respectively.

To identify published articles on this intervention strategy, Pubmed was searched using the following terms: (endothelial dysfunction OR flow-mediated OR FMD) AND (omega-3) AND (hyperlipidemia OR hypertriglyceridemia OR cigarette OR smoking OR hypertension OR diabetes OR metabolic syndrome) between 1951-January 2017. We excluded articles in which individuals had diagnosed peripheral artery disease, coronary artery disease, stroke, angina, atherosclerosis, acute myocardial infarction, and studies with healthy individuals who had no classic risk factors for atherosclerotic cardiovascular disease. We also excluded articles in which n-3 PUFA intake was estimated from dietary consumption of fish and treatments < 2 weeks. Finally, we identified additional articles from the reference lists and cited-searches of the publications found in PubMed. A summary of each of the 22 articles is presented in Table 1, grouped by atherosclerosis CV disease risk factor and then listed in chronological order. Additionally, we calculated a dose of n-3 PUFA consumed daily, based on the reported dose and the percentage of n-3 PUFA in the formulation.

Table 1.

Summary of study subject characteristics, study design, and outcomes on endothelial function and triglycerides (TG), following n-3 PUFA supplementation and grouped according to underlying atherosclerosis CV disease risk factor.

Study	Description of Subjects	n–3 PUFA dose [Consumed daily dose]a	Duration	Placebo control (Y/N)	Crossover design (Y/N)	Washout duration	Group size	Outcomes
Hyperlipidemia
Goodfellow et al., 2000 [15]	Hypercholesterolemic 50–56 ± 13 years old (yo) 19 Male (M), 10 Female (F)	4 g/d − 85% EPA + DHA [3.4 g/d EPA + DHA]	17 wks	Y Corn oil	N	NA	Placebo: 15 n-3 PUFA: 13	↑ FMD ↓ TG
Mori et al., 2000 [22]	Hyperlipidemic & BMI 25–30 kg/m3 Age- and BMI-matched 49–50 ± 2 yo 40 M	4 g/d − 96% EPA or 92% DHA [3.8 g/d EPA or 3.7 g/d DHA]	6 wks	Y Olive oil	N	NA	Placebo: 14 EPA: 13 DHA: 13	DHA only: ↑ FBFb DHA only: ↓ TG
Okumura et al., 2002 [23]	Hypertriglyceridemic 38–44 ± 5 yo 15 M	1.8 g/d − 98% EPA [1.8 g/d EPA]	12 wks	N	N	NA	Normolipidemic: 7 Hyperlipidemic: 8	↑ FBFb ↓ TG
Ros et al., 2004 [24]	Hypercholesterolemic 26–75 yo Sex NR	Mediterranean diet or isoenergetic diet plus 45–60 g/d walnuts [3.7–6 g/d ALA]	4 wks	N	Y	None	21	↑ FMD ↔ TG
Engler et al., 2004 [25]	Hyperlipidemic 9–19 yo Sex NR	6 g/d − 20% DHA [1.2 g/d DHA]	6 wks	Y Corn/soy oil	Y	6 wks	20	↑ FMD ↔ TG
Mindrescu et al., 2008 [26]	Hyperlipidemic 27–78 yo 23 M, 7 F	6 g/d − 43% EPA + 32% DHA and 10 mg/drosuvastatin, or 10 mg/d rosuvastatin [4.53 g/d EPA + DHA]	4 wks	N	Y	None	30	↑ FMD ↓ TG
West et al., 2010 [27]	Hypercholesterolemic 36–65 yo 20 M, 3 F (pm)c	American diet − 0.8% ALAd Linoleic acid diet − 3.6% ALA ALA diet − 6.5% ALA [15 g/d ALA]	6 wks	N	Y	≤ 3 wks	12 (subset of 23)	ALA diet: ↑ FMD ↓ TGe
Skulas-Ray et al., 2011 [28]	Hypertriglyceridemic 44 ± 10 yo (23–65) 23 M, 3 F (pm)c	1 or 4 g/d − 84% EPA + DHA [0.84 or 3.4 g/d EPA + DHA]	8 wks	Y Corn oil	Y	6 wks	26	↔ FMD ↔ RHIf ↓ TG (4 g/d)
Koh et al., 2012 [29]	Hypertriglyceridemic Age-, Sex- & BMI-matched 54–55 ± 1 yo 87 M, 58 F	2 g/d − 84% EPA + DHA or 160 mg/d fenofibrate [1.7 g/d EPA + DHA]	8 wks	Y NR	N	NA	Placebo: 49 n-3 PUFA: 50 Fenofibrate: 48	↑ FMD ↔ TG
Yamakawa et al., 2012 [30]	Hyperlipidemic Age- and sex-matched, 61.6 ± 10.6 yo 13 M, 21 F	1.8 g/d − 98% EPA [1.8 g/d EPA]	12 wks	N	N	NA	Normolipidemic: 18 Hyperlipidemic: 16	↑ FBFg ↔ TG
Oh et al., 2014 [31]	Hypertriglyceridemic 54–55 ± 9 yo 91 M, 82 F	1, 2 or 4 g/d − 84% EPA + DHA [0.84, 1.7, or 3.4 g/d EPA + DHA]	8 wks	Y NR	N	NA	Placebo: 42 1 g/d: 43 2 g/d:44 4 g/d: 44	↑ FMD (1, 2, & 4 g/d)) ↓ TG (4 g/d)
Casanova et al., 2017 [32]	Hypertriglyceridemic & hypertensive 49–59 ± 1 yo 17 M, 12 F	1.8 g/d − 60% EPA + 40% DHA or 100mg/d ciprofibrate [1.8 g/d EPA + DHA]	12 wks	N	Y	8 wks	Low CV-risk: 13	↑ FMD
							High CV-risk: 16	↔ RHI ↓ TG
Type 2 Diabetes Mellitus
McVeigh et al., 1993 [33]	T2DM 45–61 yo 20 M, 3 F	10 g/d − 30% EPA + DHA [3 g/d EPA + DHA]	6 wks	Y Olive oil	Y	6 wks	23	↑ FBFb ↔ TG
Woodman et al., 2003 [34]	T2DM & hypertensive 61.2 ± 1.2 yo (40–75) 39 M, 12 F (pm)	4 g/d − 96% EPA or 92% DHA [3.8 g/d EPA or 3.7 g/d DHA]	6 wks	Y Olive oil	N	NA	Placebo: 16 EPA: 17 DHA: 18	↔ FMD
Wong et al., 2010 [35]	T2DM 59–61 ± 9 yo 43 M, 54 F	4 g/d − 67% EPA + DHA [2.7 g/d EPA + DHA]	12 wks	Y Olive oil	N	NA	Placebo: 48 n-3 PUFA: 49	↔ FMD ↓ TG
Stirban et al., 2010 [36]	T2DM 56.8 ± 8.3 yo (37–70) Sex NR	2 g/d − 84% EPA+DHA [1.7 g/d EPA + DHA]	6 wks	Y Olive oil	Y	6 wks	32	↔ Fasting FMD Prevent Postprandial ↓ FMD ↔ Fasting TG Prevent Postprandial ↑ TG
Lobraico et al., 2015 [37]	T2DM 64.8 ± 8.8 yo 31 M, 16 F	1 g/d krill oil (% n-3 PUFA NR)h	4 wks	Y Olive oil	Y	2 wks	47	↓ RHI ↔ TG ↑ RHI
		1 g/d – krill oil	17wks	N	N	NA	34	↔ TG
Cigarette Smoking
Din et al., 2013 [38]	Cigarette smoker 28 ± 2 yo 20 M	2 g/d − 84% EPA + DHA [1.7 g/d EPA + DHA]	6 wks	Y Olive oil	Y	4 wks	20	↑ FBFb ↔ TG
Siasos et al., 2013[16]	Cigarette smoker 27.6 ± 2.6 yo 13 M, 7 F	2 g/d − 84% EPA + DHA [1.7 g/d EPA + DHA]	12 wks	Y NR	Y	4 wks	20	↑ FMD ↔ TG
Elevated BMI
Hill et al., 2007 [39]	BMI > 25 kg/m3 + ≥ 1 CV risk factor(s) 47–52 ± 2 yo 24 M, 41 F	6 g/d − 32% EPA + DHA [1.9 g/d EPA + DHA]	12 wks	Y Sunflower oil	N	NA	Placebo: 18 Placebo + Ex:14 n-3 PUFA: 17 n-3 PUFA + Ex: 16	↑ FMD ↔ TG
Dangardt et al., 2010 [40]	BMI > 30 kg/m3 16 ± 1 yo 11 M, 14 F	10 g/d − 9% EPA + 3% DHA [1.2 g/d EPA + DHA]	12 wks	Y Medium chain TG	Y	6 wks	25	↑ RHI ↔ TG
Metabolic Syndrome
Tousoulis et al., 2014 [41]	Metabolic Syndrome 44 ± 12 yo 15 M, 14 F	2 g/d − 46% EPA + 38% DHA [1.7 g/d EPA + DHA]	12 wks	Y NR	Y	8 wks	29	↑ FMD ↓ TG

NR – Not reported; NA – Not applicable

^an-3 PUFA dose consumed daily is based on the weight of capsules consumed daily (g/d) multiplied by the percentage of n-3 PUFA in the formulation. For example, from [15]: 4 g/d x 84% EPA+DHA = n-3 PUFA dose consumed daily of 3.4 g/d EPA+DHA.

^bFBF – Forearm blood flow was assessed following intra-arterial infusion of the endothelial-dependent vasodilator, acetylcholine.

^cPM – All females in the cohort were postmenopausal.

^dPercent energy in the diet derived from ALA.

^eReported in [42].

^fRHI – Reactive hyperemia index was assessed in the digital microcirculation using PAT.

^gFBF – Forearm blood flow was assessed following reactive hyperemia.

^hA consumed daily dose could not be calculated without information on the percentage of EPA and DHA in the capsules.

3. Hyperlipidemia

Since elevated cholesterol and triglycerides are risk factors for endothelial dysfunction [43] and atherosclerosis [44], we reviewed those papers that assessed the ability of n-3 PUFA supplementation to improve endothelial dysfunction in individuals with hyperlipidemia. Additionally, since n-3 PUFAs are one prescription therapy approved for patients with very high triglycerides (≥500 mg/dL) [45] and the American Heart Association recommends that consuming 2–4 g/d EPA+DHA can achieve triglyceride-lowering effects [46], we also report the outcome of n-3 PUFA supplementation on triglyceride levels when available.

In a small study, triglycerides were lowered and FMD was improved in subjects with hypercholesterolemia (total cholesterol > 575 mg/dL), who were given placebo or EPA + DHA for 17 weeks [16]. The n-3 PUFA treatment significantly decreased triglycerides, compared to placebo (placebo: 202.67 ± 184.97 mg/dL; n-3 PUFA: 153.11 ± 84.08 mg/dL, P < 0.05) and significantly improved FMD (placebo: 0.04 ± 0.10 mm, n-3 PUFA: 0.12 ± 0.07 mm, P < 0.05).

In another study, DHA, but not EPA, improved FBF and significantly lowered triglycerides in overweight and mildly hyperlipidemic men [22]. In this double-blind, placebo-controlled trial, men who were overweight (BMI, 25–30 kg/m³) and hyperlipidemic (total cholesterol ≥232 mg/dL, triglycerides ≥159 mg/dL, or both) were randomly assigned to placebo, EPA or DHA. After 6 weeks, FBF, measured in response to infusion of acetylcholine, was significantly increased in the DHA treatment group, compare to baseline (P < 0.047) and compared placebo at 6 weeks (P < 0.04). In contrast, the EPA group showed no improvement in FBF. In addition, DHA reduced triglycerides by 20% post-intervention (P < 0.006), while EPA showed a non-significant reduction of 18.4% (P < 0.068).

In another small study of men with hypertriglyceridemia, EPA supplementation improved FBF and significantly reduced triglycerides [23]. Men with elevated triglycerides (150–500 mg/dL) were given EPA for 12 weeks. Acetylcholine-induced FBF was significantly lower at baseline, compared to men with normal triglycerides (P < 0.034), and was significantly increased, compared to baseline (P < 0.011). Additionally, EPA significantly reduced serum triglycerides, compared to baseline (baseline: 274 ± 27 mg/dL; 12 weeks: 188.9 ± 34.5 mg/dL; P < 0.025).

In another study, subjects with hypercholesterolemia treated with walnuts exhibited significant increases in FMD [24]. Walnuts are highly enriched in the n-3 PUFA, ALA, the metabolic precursor to EPA and DHA. In this study, subjects with moderate hypercholesterolemia (LDL cholesterol > 130 mg/dL and triglycerides < 250 mg/dL) were randomized into a control Mediterranean-type diet or an isoenergetic diet plus walnuts for 4 weeks followed by crossover without washout. The walnut-enriched diet significantly improved FMD, compared to the control diet (control diet: 3.6 ± 3.3%, walnut diet: 5.9 ± 3.3%; P = 0.043). There was no significant difference in triglycerides between groups.

While the above studies were conducted in adults, the following study highlights that n-3 PUFA supplementation can improve FMD in children with possible hypercholesterolemia or hyperlipidemia hereditary factors [25]. In a double-blind, randomized, placebo-controlled crossover study, offspring of family members with hypercholesterolemia or the phenotype of the familial combined hyperlipidemia were placed on a National Cholesterol Education Program Step II (NCEP-II) diet for 6 weeks. Subjects were then randomized into placebo or DHA groups for 6 weeks, followed by washout, and subsequent cross over to the other treatment arm, while remaining on the NCEP-II diet throughout. There were no significant differences in serum triglycerides among any of the groups. While placebo + diet significantly improved FMD over baseline, only the DHA supplement + diet significantly improved FMD (7.9 ± 2.9%), compared to all other groups (baseline: 5.9 ± 2.3%, P < 0.001; diet only: 6.3 ± 2.6%, P < 0.002; placebo + diet: 6.8 ± 2.4%, P < 0.012; washout + diet: 6.5 ± 2.7, P < 0.001).

In another study, the combination of an n-3 PUFA supplement with a statin significantly improved endothelial function and decreased triglycerides, compared to the statin alone [26]. In this study, individuals with hyperlipidemia (LDL cholesterol > 100 mg/dL and/or triglycerides > 150 mg/dL) were treated with rosuvastatin plus EPA + DHA (Group 1) or rosuvastatin alone (Group 2). After 4 weeks, lipids and FMD were measured and the groups crossed over to the other treatment arm without washout. In Group 1, triglycerides were significantly decreased after 4 weeks, compared to baseline (baseline: 139 ± 57 mg/dL; rosuvastatin + EPA + DHA: 91 ± 40 mg/dL; P < 0.05). After n-3 PUFA supplementation was stopped, triglycerides significantly increased (99 ± 27 mg/dL; P < 0.05). In Group 2, triglycerides did not significantly change after 4 weeks, compared to baseline (baseline: 137 ± 71 mg/dL; rosuvastatin alone: 141 ± 71 mg/dL). However, after the n-3 PUFA supplementation was added, triglycerides significantly decreased (103 ± 36 mg/dL, P < 0.05). FMD of Group 1 was significantly increased after 4 weeks of rosuvastatin + n-3 PUFA, compared to baseline (baseline: −1.42 ± 3.27%, 4 weeks: 11.36 ± 4.33%); however, after the n-3 PUFA supplement was discontinued, FMD decreased (0.59 ± 8.32%). FMD of Group 2 did not differ after 4 weeks of rosuvastatin alone; however, after n-3 PUFA supplement was added to the statin therapy, the FMD significantly increased, compared to baseline (14.73 ± 10.77%, P < 0.05).

In another study using a diet enriched in walnuts, walnut oil and flaxseed oil, subjects with elevated cholesterol exhibited a significant improvement in FMD [27]. Participants with elevated cholesterol (200–240 mg/dL) were randomly assigned to a sequence of three diets (American, Linoleic acid, ALA) that were each consumed for 6 weeks, followed by washout and crossover to the next diet. FMD was assessed in a subset (n = 12) of individuals at the end of each diet period. Individuals consuming the ALA diet exhibited the highest FMD when all diets were compared (American diet: 6.1 ± 1.1%; Linoleic acid diet: 6.7 ± 1.0%; ALA diet: 8.2 ± 1.0%; P < 0.02 diet effect by ANOVA). Notably, in an earlier study using the same diets and experimental design, both the linoleic acid diet and ALA diet significantly reduced triglycerides [42].

In one placebo-controlled study, n-3 PUFAs significantly reduced high triglycerides, but failed to improve FMD or RHI assessed by PAT [28]. In this study, subjects with high triglycerides (140–339 mg/dL) were enrolled in a 3-period crossover study of 8-week treatment periods with 6 week washout periods. The treatments included placebo or a low or high dose of EPA+DHA. Triglyceride levels in those treated with the high dose decreased significantly by 27% (173.7 ± 17.5 mg/dL, P = 0.002), compared to placebo (237.3 ± 17.5 mg/dL), while there was no change in triglyceride levels in the low dose group (215.3 ± 17.5 mg/dL). However, neither post-treatment FMD nor RHI differed among the three treatment groups.

In a randomized, single-blind, placebo-controlled study of hyperlipidemic subjects, comparison of n-3 PUFA supplementation versus fenofibrate showed that both treatments effectively improved FMD and reduced triglycerides [29]. Subjects with high triglycerides (≥150 mg/dL) were treated for 8 weeks with placebo, EPA+DHA or fenofibrate alone. Both n-3 PUFAs and fenofibrate significantly improved FMD, compared to baseline (n-3 PUFA: baseline 4.72 ± 0.27% vs 8 weeks 7.28 ± 0.33%, P < 0.001; fenofibrate: baseline 4.63 ± 0.26% vs 8 weeks 6.97 ± 0.28%, P < 0.001). Additionally, both n-3 PUFAs and fenofibrate significantly reduced triglycerides, compared to baseline (n-3 PUFA: baseline 290 ± 12 mg/dL vs 8 weeks 226 ± 16 mg/dL, P < 0.001; fenofibrate: baseline 274 ± 19 mg/dL vs 8 weeks 174 ± 11 mg/dL, P < 0.001), but only fenofibrate reduced triglycerides, compared to placebo. In a subsequent trial of similar design with hyperlipidemic subjects, the combination of EPA+DHA plus fenofibrate did not result in significant improvements in FMD or triglycerides over EPA+DHA or fenofibrate alone [47].

In a small hyperlipidemic cohort with hypercholesterolemia (243 ± 29 mg/dL) and/or hypertriglyceridemia (209 ± 92 mg/dL), n-3 PUFA supplementation improved FBF, but failed to reduce triglyceride levels [30]. In this study, subjects were given EPA and serum lipids and FBF were measured at 0, 4 and 12 weeks of treatment. In the hyperlipidemic group there was no significant change in triglycerides after treatment, compared to baseline. Notably, FBF was significantly lower in the hyperlipidemic group at baseline, compared to the normolipidemic group (hyperlipidemic baseline: 15.4 ± 6.1 mL/min/100 g; normolipidemic: 22.8 ± 1.2 mL/min/100 g, P < 0.01). However, after 12 weeks of EPA treatment FBF was significantly improved in the hyperlipidemic group, compared to baseline, (21.7 ± 4.4 mL/min/100 g; P = 0.046).

In one placebo-controlled study, n-3 PUFAs significantly reduced borderline high triglycerides and simultaneously improved FMD [31]. In this study, subjects with hypertriglyceridemia were treated for 8 weeks with placebo, or 1, 2, or 4 g/d EPA + DHA. Triglycerides in the groups treated with 1 or 2 g/d were not different from placebo, but levels in individuals treated with 4 g/d were significantly decreased (191 ± 117 mg/dL, P < 0.05), compared to placebo (247 ± 102 mg/dL). However, all three groups treated with EPA+DHA had significantly improved FMD, compared to baseline (P < 0.001), and to the placebo group (placebo: 6.31 ± 1.56%, 1 g/d: 7.61 ± 1.68%, 2 g/d: 7.64 ± 1.74%, and 4 g/d: 8.37 ± 1.51%; P < 0.05).

In a recent randomized, crossover study, comparison of n-3 PUFA supplementation versus ciprofibrate in individuals with both hypertriglyceridemia and hypertension showed that both treatments effectively improved FMD and reduced triglycerides, but not RHI [32]. In this study, individuals taking hypertension medication and with high triglycerides (150–499 mg/dL) were classified as having low (< 7.5%, n = 13) or high (≥7.5%, n = 16) CV risk, based on their estimated 10 year risk for atherosclerotic CV disease (ASCVD) [48], and randomized to receive EPA + DHA or ciprofibrate for 12 weeks. This was followed by washout and subsequent crossover to the other treatment. In the low-risk subjects, FMD was significantly improved with both n-3 PUFA supplementation and ciprofibrate treatment, compared to baseline (n-3 PUFA: baseline: 10.1 ± 1.5%; 12 weeks: 13.5 ± 1.2, P = 0.012; ciprofibrate: baseline: 8.7 ± 1.5%; 12 weeks: 14.0 ± 1.9, P = 0.036). There were no significant changes in RHI in either treatment group as measured by PAT. Triglycerides were significantly decreased after n-3 PUFA supplementation and ciprofibrate treatment (n-3 PUFA: baseline: 259 ± 29 mg/dL; 12 weeks: 200 ± 26 mg/dL, P = 0.040; ciprofibrate: baseline: 250 ± 26 mg/dL; 12 weeks: 152 ± 28 mg/dL, P ≤ 0.001). In the high-risk subjects, FMD was significantly increased with n-3 PUFA supplementation, compared to baseline (n-3 PUFA: baseline: 11.1 ± 1.6%; 12 weeks: 13.5 ± 1.2, P = 0.010), but there was no improvement after ciprofibrate treatment alone. There were no significant changes in RHI in either treatment group. Triglycerides were significantly decreased after n-3 PUFA supplementation and ciprofibrate treatment (n-3 PUFA: baseline: 255 ± 20 mg/dL; 12 weeks: 199 ± 36 mg/dL, P = 0.020; ciprofibrate: baseline: 244 ± 16 mg/dL; 12 weeks: 149 ± 19 mg/dL, P ≤ 0.001).

Among the twelve studies enrolling hyperlipidemic subjects, eleven studies showed that n-3 PUFA supplementation, including formulations (EPA+DHA, EPA alone, DHA alone, and ALA alone), improved endothelial dysfunction as assessed by FMD (8 studies) or FBF (3 studies) and seven studies showed n-3 PUFA supplementation also reduced triglycerides. The reasons why one study failed to observe an improvement in FMD is not clear [28]. The study was a randomized, double-blind, placebo-controlled, crossover design with six weeks washout between treatment arms. The investigators used a high dose EPA + DHA for an eight-week duration and observed a significant decrease in triglycerides. However, while the enrolled group size was similar to other studies (n = 26), the age range was large and disproportionately male. It has been reported that the age-related decline in endothelial function occurs significantly earlier in men (41 yo) than in women (58 yo) [49]. Thus, it is possible that the age and sex characteristics of the enrolled cohort resulted in the inability of n-3 PUFA supplementation to improve FMD. Additionally, two studies using an EPA + DHA formulation also assessed endothelial dysfunction by PAT; however, neither study observed an improvement in RHI [28,32]. Taken together these results showed that n-3 PUFA supplementation in hyperlipidemic individuals can improve endothelial dysfunction, particularly as assessed by FMD, and this improvement can occur even in the absence of a reduction in triglycerides.

4. Type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a pathological contributor to both endothelial dysfunction and atherosclerosis [50,51]. Thus, we also reviewed those studies that assessed the ability of n-3 PUFA supplementation to improve endothelial dysfunction in individuals with T2DM. One study has shown that n-3 PUFA supplements cannot improve fasting FMD, but can improve postprandial FMD in subjects with T2DM [36]. In this study, subjects with controlled T2DM were randomly divided into placebo or EPA + DHA for 6 weeks followed by washout and crossover. FMD was measured following a 12 h fast and again at 2, 4 and 6 h after eating a high-fat meal. Fasting FMD did not differ between placebo and n-3 PUFA treatment groups. While postprandial FMD decreased in both groups with a maximal reduction at 4 h, the decrease was only significant in the placebo group. In the placebo group, postprandial FMD was significantly lower at 4 h, compared to fasting FMD, (FMD – fasting: 5.54 ± 0.55% vs 4 h postprandial: 3.45 ± 0.54%; −38%, p < 0.05). In contrast, in the n-3 PUFA group postprandial FMD at 4 h did differ from fasting FMD (FMD – fasting: 4.85 ± 0.6% vs 4 h postprandial: 4.23 ± 0.48%; −13%). Further, the overall postprandial decrease in FMD (expressed as area under the curve, AUC% x hr) was significantly greater in the placebo group, compared to the n-3 PUFA group (placebo: −8.24 ± 2.20% x hr; n-3 PUFA: −2.31 ± 1.83% x hr, P < 0.05). While fasting triglycerides did not differ between placebo and n-3 PUFA treatment groups, the postprandial increase in triglycerides was significantly attenuated in the n-3 PUFA group at 4 h (placebo: 233 ± 26 mg/dl; n-3 PUFA: 198 ± 28 mg/dL, P < 0.05) and when integrated across the 6 h post prandial period (placebo: 587 ± 62 mg/dL x hr; n-3 PUFA: 518 ± 58 mg/dL x hr; P < 0).

Two additional studies in diabetic subjects also failed to show an improvement in fasting FMD following n-3 PUFA treatment. In one study individuals with controlled T2DM and diagnosed hypertension were randomly assigned to placebo, EPA or DHA for 6 weeks [34]. FMD, assessed at baseline and during the final 2 weeks of treatment, did not differ among the three groups. In a second study individuals with controlled T2DM were divided between placebo or EPA+DHA for 12 weeks [35]. FMD was not significantly different between the placebo and n-3 PUFA groups (P = 0.95); however, triglycerides were significantly decreased in the n-3 PUFA group, compared to the placebo (placebo group: 141.6 ± 88.5 mg/dL; n-3 PUFA: 106.2 ± 44.25 mg/dL; P = 0.01).

Despite these negative outcomes, two studies showed an improvement in endothelial dysfunction. The first study assessed endothelial function by FBF following acetylcholine infusion [33]. In a double-blind, placebo controlled, crossover study, individuals with T2DM were randomized into placebo or EPA + DHA for 6 weeks, followed by washout and then crossover. FBF was measured following intra-arterial infusion of increasing doses of the endothelial-dependent vasodilator, acetylcholine. FBF in response to acetylcholine was significantly increased at all doses when taking n-3 PUFA supplements, compared to baseline and to placebo (P < 0.01).

In another double-blind, placebo controlled, crossover study, endothelial function was significantly improved in individuals with T2DM consuming krill oil (percentage of EPA and DHA not specified) [37]. Participants were randomized into placebo or krill oil for 4 weeks followed by washout and crossover, and then a subset of the same individuals (n = 34) participated in an additional 17 week krill oil supplementation period. RHI was significantly increased after 4 weeks of krill oil supplementation, compared to placebo (placebo: 1.83 ± 0.56; krill oil: 2.04 ± 0.52; P = 0.025), and after 17 weeks of krill oil supplementation, compared to baseline (baseline: 1.90 ± 0.54; krill oil: 2.16 ± 0.73; P = 0.041).

Of the five studies reviewed in individuals with T2DM, all were blinded, randomized and placebo-controlled; four used an EPA + DHA combination and one did a direct comparison of EPA versus DHA. Two studies using an EPA + DHA combination observed an improvement of endothelial dysfunction. In contrast, two studies using an EPA + DHA combination and one study comparing EPA to DHA failed to observe an improvement. The one notable difference between the studies with positive and negative outcomes on endothelial dysfunction was the method used to assess endothelial function. The two studies with positive outcomes used FBF following acetylcholine infusion and PAT, respectively, whereas the three studies with negative outcomes used FMD. FMD of the brachial artery is primarily mediated by NO, while FBF and PAT of the forearm and digital microvasculature, respectively, likely involve a combination of NO, prostaglandins and endothelial-derived hyperpolarizing factors. Despite the fact that each method is differentially associated with CV risk factors, typically the three methods do not correlate with each other [52,53]. These limited data suggest that n-3 PUFA supplementation in individuals with T2DM may be able to improve endothelial dysfunction in the microvasculature, but not in conduit vessels, such as the brachial artery.

5. Cigarette smoking

Cigarette smoking is a risk factor for endothelial dysfunction and is one of the single biggest independent risk factors for atherosclerosis [54–56]. Thus, we reviewed those studies that assessed the ability of n-3 PUFA supplementation to improve endothelial dysfunction in current cigarette smokers. In a prospective, double-blind, crossover, placebo-controlled, randomized study, n-3 PUFAs were shown to improve FBF in response to endothelium dependent vasodilators [38]. Healthy male smokers (≥5 cigarettes/day), were placed on placebo or EPA + DHA for 6 weeks followed by washout and crossover. Dose-dependent responses in FBF were measured following infusion of two endothelium-dependent vasodilators; acetylcholine and substance P. Individuals taking n-3 PUFA supplements had increased FBF, compared to placebo, but only acetylcholine-induced vasodilation was significantly increased (acetylcholine: P = 0.0032; substance P: P = 0.056).

In another study n-3 PUFAs were shown to improve FMD and reduce triglycerides in a double-blind, crossover, placebo-controlled study in current cigarette smokers (> 20 cigarettes per day for > 5 years), lacking any other classic risk factors of CV disease [16]. A baseline FMD was measured before randomly assigning each subject to receive placebo or EPA+DHA for 12 weeks followed by washout and crossover. FMD was measured again after 4 and 12 weeks. The n-3 PUFA supplement non-significantly increased FMD after 4 weeks, compared to baseline, (baseline: 7.27 ± 2.56%, 4 weeks n-3 PUFA: 8.53 ± 3.55%), but significantly improved it after 12 weeks (9.98 ± 5.30%, P < 0.05), compared to baseline. Additionally, compared to placebo, individuals taking the n-3 PUFA supplement exhibited significantly higher FMD at both 4 weeks (P < 0.05) and 12 weeks (P < 0.001).

6. Elevated body mass index (BMI)

Elevated BMI has been shown to be a risk factor for endothelial dysfunction [57,58] and for atherosclerosis [59]. We reviewed two studies that investigated the relationship between n-3 PUFA supplementation, elevated BMI and endothelial dysfunction. In the first study, individuals were recruited that were overweight or obese (BMI > 25 kg/m²) and had one or more CV risk factors, including hypertension (140/90–160/100 mm Hg), increased plasma triglycerides (> 142 mg/dL) or elevated total cholesterol (> 212 mg/dL) [39]. Subjects were randomly divided into four groups. Two groups consumed EPA + DHA and two groups consumed placebo for 12 weeks. One n-3 PUFA group and one placebo group were required to run or walk 3 times/week for 45 minutes. Individuals taking the n-3 PUFA supplement exhibited a significant time-dependent improvement in FMD, compared to placebo, (treatment x time interaction, P < 0.05), and a significantly higher FMD, compared to the placebo group, after 12 weeks (P < 0.01). There was no effect of exercise status on this outcome. The n-3 n-3 PUFA supplement also significantly reduced triglycerides at both 6 and 12 weeks, compared to placebo (P < 0.05).

While the previous study assessed n-3 PUFA supplementation in overweight or obese adults, this trial assessed effects in adolescents. In a double-blind, placebo-controlled, crossover study obese adolescents (BMI > 30 kg/m³) were randomly assigned to placebo or EPA + DHA for 12 weeks, followed by washout and crossover [40]. Endothelial function, assessed by PAT, and serum triglycerides were measured at the end of each treatment period. n-3 PUFA supplementation had no effect on triglycerides, compared to placebo. However, pair-wise comparison of RH response curves showed that n-3 PUFAs significantly improved endothelial dysfunction (P = 0.01).

7. Metabolic syndrome

Metabolic syndrome is defined as a cluster of conditions that increase risk for atherosclerotic cardiovascular disease and diabetes. These conditions include: dyslipidemia, hypertension, impaired fasting glucose, and abdominal fat [60,61]. We reviewed one study where participants with diagnosed metabolic syndrome were enrolled in a double-blind, placebo-controlled, crossover trial [41]. Individuals were randomized into placebo or EPA + DHA for 12 weeks. This was followed by washout and then crossover. FMD was significantly improved after 12 weeks of n-3 PUFA supplementation, compared to baseline, and exhibited significant improved overtime (baseline: 3.67 ± 3.57%, 4 weeks: 5.13 ± 4.51%, 12 weeks: 7.72 ± 4.17%, P < 0.05 baseline vs 12 wks; P < 0.001 time-dependent trend). There were no significant differences observed in the placebo group. n-3 PUFA supplementation also significantly decreased triglycerides over time (baseline: 180 ± 22 mg/dL; 4 weeks: 175 ± 21 mg/dL; 12 weeks: 166 ± 17 mg/dL; P < 0.001 trend), in contrast to no changes in the placebo group over time.

8. Discussion

The review of these published studies reveals that n-3 PUFA supplementation can successfully improve endothelial dysfunction in individuals with traditional risk factors for atherosclerotic CV disease, including hyperlipidemia, cigarette smoking, elevated BMI, and metabolic syndrome. Of the 22 studies, n-3 PUFA supplementation improved endothelial dysfunction in 18 of them, including 11 of 12 studies of hyperlipidemic individuals, 2 of 2 studies of young cigarette smokers, 2 of 5 studies of individuals with T2DM, and 3 of 3 studies of individuals with elevated BMI or metabolic syndrome.

Since the n-3 PUFA dose, composition, and treatment duration used in these studies varied, we devised an approach to more easily compare the outcomes across all trials. First, we calculated an n-3 PUFA dose consumed daily by multiplying the capsule weight consumed per day (g/d) by the percentage of n-3 PUFA in the capsule (Table 1). We then calculated a cumulative dose consumed by multiplying the dose consumed daily (g/d) by 7 days/wk by the number of weeks of treatment, and then grouped the results by the composition of the formulation (Table 2). One study was excluded from this analysis due to incomplete information to complete the calculations [37]. This approach illustrated that endothelial dysfunction was consistently improved by cumulative doses of ≥ 95 g EPA + DHA and that triglycerides were consistently reduced by cumulative doses of ≥ 151 g EPA + DHA. This suggests that higher doses of EPA + DHA may be required to reduce triglycerides than are needed to needed to improve endothelial dysfunction, which would be consistent with the approved prescription dose of 4 g/d (84% EPA+DHA, e.g. Lovaza^®, Vacepa^®) for reducing triglycerides in patients with very high triglycerides (≥ 500 mg/dL). However, these data also suggest that endothelial dysfunction can be improved in the absence of a reduction in triglycerides.

Table 2.

Summary of n-3 PUFA cumulative dose, outcomes on endothelial function and triglycerides (TG), and atherosclerosis CV disease risk factor, grouped according to the n-3 PUFA formulation.

n-3 PUFA Formulation	Cumulative Dose (g)a	Outcomesb		Atherosclerosis CV Disease Risk Factor	Reference
		Improved Endothelial Dysfunction	Reduced TG
EPA + DHA	47	− (FMD)	−	Hypertriglyceridemia	[28]
	47	+ (FMD)	−	Hypertriglyceridemia	[31]
	71	+ (FBF)	−	Smoking	[38]
	71	− (FMD)	−	T2DM	[36]
	95	+ (FMD)	−	Hypertriglyceridemia	[29]
	95	+ (FMD)	−	Hypertriglyceridemia	[31]
	101	+ (RHI)	−	Elevated BMI	[40]
	126	+ (FBF)	−	T2DM	[33]
	126	+ (FMD)	+	Hyperlipidemia	[26]
	143	+ (FMD)	−	Smoking	[16]
	143	+ (FMD)	+	Metabolic syndrome	[41]
	151	+ (FMD)	+	Hypertriglyceridemia & hypertension	[32]
	160	+ (FMD)	+	Elevated BMI	[39]
	190	− (FMD)	+	Hypertriglyceridemia	[28]
	190	+ (FMD)	+	Hypertriglyceridemia	[31]
	227	− (FMD)	+	T2DM	[35]
	405	+ (FMD)	+	Hypercholesterolemia	[15]
EPA	151	+ (FBF)	−	Hyperlipidemia	[30]
	151	+ (FBF)	+	Hypertriglyceridemia	[23]
	160	− (FMD)	−	Hyperlipidemia & elevated BMI	[22]
	160	− (FMD)	NR	T2DM	[34]
DHA	50	+ (FMD)	−	Hyperlipidemia	[25]
	155	− (FMD)	NR	T2DM	[34]
	155	+ (FMD)	+	Hyperlipidemia & elevated BMI	[22]
ALA	104–168	+ (FMD)	−	Hypercholesterolemia	[24]
	630c	+ (FMD)	+	Hypercholesterolemia	[27]

^aCumulative dose was calculated by multiplying the dose of n-3 PUFA consumed daily in g/d (Table 1) by 7 d/wk by the number of weeks of treatment. For example, from [15]: 3.4 g/d EPA + DHA × 7 d/wk × 17 wk = cumulative dose of 405 g.

^b“+” indicates improved endothelial dysfunction and reduced TG, while “−” indicates no improvement in endothelial dysfunction and no reduction in TG.

^cBased on the report of 3.3 g/d ALA consumed from walnuts, 1.5 g/d ALA consumed from walnut oil, and the assumption of 10.4 g/d of ALA consumed from flaxseed oil.

The data for formulations containing only EPA or DHA alone were limited with highly variable outcomes. EPA successfully improved endothelial dysfunction measured by FBF in two studies at a cumulative dose of 151 g [23,30], but failed to improve endothelial dysfunction measured by FMD in two studies at a cumulative dose of 160 g [22,34]. In contrast, DHA improved endothelial dysfunction as measured by FMD at cumulative doses of 50 and 155 g [22,25], but not at 160 g in diabetics [34]. Lastly, two studies of diets enriched in ALA suggested a low cumulative dose (100–200 g) improved endothelial dysfunction, but did not reduce triglycerides, while a much higher cumulative dose (630 g) improved both outcomes.

The one notable atherosclerosis CV disease risk factor for which n-3 PUFA supplementation did not consistently improve endothelial dysfunction was in individuals with T2DM. n-3 PUFA supplementation failed to improve endothelial dysfunction in three of the five studies conducted in diabetic subjects [34–36]. All three of the studies with negative outcomes were placebo-controlled and assessed endothelial function using the gold standard, FMD. One study used a cumulative dose as high as 227 g EPA + DHA [35], which is higher than doses that improved endothelial dysfunction in ten other trials in non-diabetic subjects. Further, the lack of improvement in FMD occurred despite significant increases in platelet n-3 PUFAs in one study [34] and significant decreases in triglycerides in another [35]. It is noteworthy that the two studies in which EPA+DHA improved endothelial dysfunction in diabetic subjects assessed endothelial dysfunction in microvascular beds of the fingers and forearm [33,37]. Thus, it is possible that n-3 PUFA supplementation only improves endothelial function in microvascular arterioles, but not conduit arteries in diabetic subjects. Potentially, this difference could reflect the mechanisms by which (1) T2DM induces endothelial dysfunction, (2) n-3 PUFAs improve endothelial dysfunction, and (3) endothelial-dependent vasodilation is regulated in micro- versus macro-vasculature. Future studies of all these mechanisms are needed to understand the potential vasoprotective benefit of n-3 PUFA supplementation in individuals with T2DM.

The mechanisms underlying the improvement of endothelial dysfunction by n-3 PUFA supplementation in human subjects have not been fully elucidated. However, numerous studies suggest that n-3 PUFAs may improve endothelial function by increasing NO levels [62]. For example, EPA increases NO in endothelial cells in situ and stimulates endothelial and NO-dependent dilation in bovine coronary arteries ex vivo [63]. Further, both EPA and DHA activate endothelial nitric oxide synthase (eNOS) in cultured human endothelial cells [63,64] and dietary n-3 PUFAs significantly increase eNOS activation in the mouse aorta [65]. DHA also increases NO by increasing interleukin-1B-induced inducible nitric oxide synthase (iNOS) mRNA by activation of p44/42 mitogen-activated protein kinase signaling [66]. Furthermore, this allows for increased extracellular calcium release from vascular smooth muscle cells, which improves vasoreactivity [67]. In vivo, dietary n-3 PUFA supplementation normalizes endothelial dysfunction in mouse mesenteric arterioles that is induced by cigarette smoke exposure [14]. Notably, however, the improvement in mesenteric arteriolar FMD is mediated by an increase in NO-independent dilation, suggesting that n-3 PUFAs also can increase the expression and/or activity of other endothelial-derived vasodilators, in addition to NO. This mechanism may account for differences in the vasodilatory benefits of n-3 PUFA supplementation between microvascular arterioles versus conduit arteries.

Another possible mechanism underlying the improvement of endothelial function by n-3 PUFAs includes decreasing reactive oxygen species (ROS). Vascular ROS can reduce NO bioavailability and increase endothelial-derived vasoconstrictors, thus impairing endothelial-dependent vasodilation. It has been shown that n-3 PUFAs decrease ROS in doxorubicin-treated cardiomyocytes and in endothelial cells exposed to environmental particulates in vitro [68,69]. In a mouse model, dietary n-3 PUFA supplementation significantly reduces cigarette smoke-induced increases in two markers of oxidative stress, 8-isoprostane and heme oxygenase-1 mRNA [14]. In a menopausal rat model, n-3 PUFA supplementation is associated with decreased ROS production through modulation of NADPH oxidase and iNOS [70]. In one clinical study, n-3 PUFA intake is positively correlated with in total antioxidant capacity, but unfortunately endothelial function was not assessed in these participants [20].

Lastly, endothelial function also is impaired by endothelial activation, inflammation, and hypertension, which are all risk factors for the development of atherosclerosis. Studies show that n-3 PUFAs can inhibit endothelial activation, and are anti-inflammatory and anti-hypertensive. Endothelial activation is associated with increases in surface expression of adhesion molecules which promotes leukocyte adhesion and inflammation. Both EPA and DHA reduce adhesion molecule expression and leukocyte adhesion to endothelial cells in vitro [71–73]. Further, clinical studies also show that n-3 PUFA supplementation reduces monocyte activation, markers of inflammation and hypertension, all of which can contribute to endothelial dysfunction [19,74,75].

9. Conclusions

Based on our review of the literature, we conclude that individuals with traditional risk factors for atherosclerotic CV disease, including hyperlipidemia, cigarette smoking, elevated BMI, and metabolic syndrome, could derive vasoprotective benefits from n-3 PUFA supplementation, particularly from an EPA+DHA formulation resulting in cumulative dose ≥95 g over a minimum of 4 weeks. While results from ALA-enriched diets appear promising, currently the evidence is inadequate to conclude whether formulations containing only ALA, EPA, or DHA have similar benefit as formulations of EPA+DHA. Given that endothelial dysfunction, as measured by FMD, is an independent predictor of future CV events and an early predictor of CHD risk, evidence from these studies suggest that n-3 PUFA supplementation could serve as a primary prevention strategy for atherosclerotic disease for many individuals at risk. This conclusion is consistent with the recommendations of the American Heart Association, the World Health Organization, and other health agencies that individuals without cardiovascular disease can derive cardiovascular disease risk reduction by intake of n-3 PUFAs [76].