The key finding of this study is that a brief 28-day supplementation with EPA+DHA led to a significant reduction in plasma levels of cytokines and angiogenesis factors implicated in atherosclerosis. To the best of our knowledge, no prior studies have compared the effects of aspirin and EPA+DHA, alone, and in combination, on all of these analytes simultaneously. EPA and DHA have been shown to exert potent anti-angiogenic effects by inhibiting the production of important inflammatory and angiogenic mediators, namely VEGF, PDGF, platelet-derived endothelial cell growth factor (PD-ECGF), COX-2, prostaglandin-E2 (PGE2), nitric oxide, NF-κB, matrix metalloproteinases and beta-catenin[
8]. PDGF concentrations in plasma in our study were reduced by consumption of 3.4g/d of EPA+DHA. This is in contrast to the results of an earlier study of 63 healthy participants in which the consumption of 0.3, 0.6 or 0.9 g/day of EPA+DHA for 8 weeks had no effect on serum PDGF levels[
9]. A dose of 0.6 mg/day is consistent with slightly more than 2 portions of fatty fish per week, and 1 g/day is consistent with about 4 fatty fish meals per week. These differences in the effects of EPA+DHA on PDGF suggest that doses of EPA+DHA higher than those typically achieved through diet may be necessary to achieve downregulation of PDGF. In support of this, a dose of 7 g/day for 4 weeks has been shown to suppress adherence-activated and non-activated mononuclear cell-mediated production of PDGF and MCP in humans[
10]. To date, the modulation of blood concentrations of angiogenesis activator b-FGF in humans by EPA and DHA has not been reported.
It is known from in vitro studies that EPA and DHA downregulate the production of TNF-α, IL-6, IL-8, and IL-1ß via modulation of nuclear factor (NF)-B[
11], while also downregulating monocyte[
12] and MCP-1[
13] activity. However, the doses used and duration of administration vary considerably from study-to-study in humans. While some of the studies provided <2 g EPA+DHA per day[
11], others have examined the effects of higher doses[
14]. Perhaps because of these differences, the amount of EPA+DHA required to exert beneficial effects is not clear. Most of these previous studies used
ex vivo techniques to examine immune cell function, unlike the current study in which we directly measured plasma levels in human subjects taking approved doses of EPA+DHA.
The formation of vasa vasorum through angiogenesis has been associated with plaque instability and rupture, as micro-vessel formation has a predilection for the shoulder regions of atherosclerotic plaques[
15]. Stimulators of angiogenesis that would induce the growth of new blood vessels and thus potentially reduce ischemic burden in the heart and limbs have been considered promising[
16], however, despite promising results in preclinical models, data from clinical trials have been inconclusive, and evidence suggests that angiogenic factors actually promote atherosclerosis and potentially destabilize coronary plaques[
2]. EPA and DHA have been shown to inhibit pathologic angiogenesis[
17].
In contrast to our expectations, IL-10 concentrations decreased with EPA+DHA treatment. In an observational study involving 1123 subjects[
18], lower DHA plasma levels were strongly associated with lower IL-10 concentrations. To our knowledge, there are no published data on the effects of pharmaceutical-grade EPA+DHA given at FDA-approved doses on IL-10. Interestingly, the inhibitory effect of EPA+DHA on plasma IL-10 levels was more modest in our study than for most of the other cytokines. Although we cannot know if oral doses of DHA alone would alter IL-10 levels, nor whether, in our study, it was the high dose of omega-3 fatty acids or the provision of EPA that lowered this marker, it seems possible that EPA and/or DHA on IL-10 may affect this cytokine differently depending on the dose and preparation used.
Our study found no effect of a 650mg dose of aspirin alone on plasma cytokines and pro-angiogenesis factors. This dose of aspirin was chosen because we originally wanted to study the potential effects of aspirin and EPA+DHA on platelet function[
19]. It remains possible that other doses of aspirin, or ingestion of aspirin for longer time points, might affect some of the inflammatory cytokines or proangiogenesis factors measured. Alternatively, while the clearance of many cytokines and angiogenesis factors occurs quickly with half-lives of < 3 hours, it is possible that the effects of aspirin on molecules with longer half-lives could be detected in studies with longer periods of aspirin use. In addition, the data in this study should be cautiously interpreted as the number of participants was relatively small. Their age was also quite low and they were quite healthy, limiting the ability to predict the process of atherosclerosis and clinical events. Thus, different results could be found in a much larger cohort with older, or diseased, participants. In addition, we were not able to determine if the angiogenesis stimulating factors of cigarette smoking[
20] and endothelial progenitor cells[
21] influence the effects that EPA+DHA or aspirin had on the pro-angiogenesis molecules that we measured. Future studies will be needed to investigate these possibilities.
In conclusion, our findings support the idea that the omega-3 fatty acids EPA+DHA have anti-inflammatory and anti-angiogenesis effects in vivo, which may contribute to the beneficial effects of fish oil supplementation in susceptible human subjects who take or do not take aspirin.
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