The putative cardioprotective effects of omega-3 fatty acids have been documented in epidemiologic and case-control studies, but the mechanisms for these effects have not been conclusively demonstrated. A new study suggested that treatment with omega-3 fatty acids results in stabilization of advanced plaques by reducing the number of foam cells, macrophages, and T cells in the plaques, thus reducing plaque inflammation. Prof. Calder, lead investigator of the Omacor Carotid EndArterectomy iNtervention (OCEAN) study, believes that these effects may contribute to reduced mortality in patients who consume omega-3 fatty acids.
OCEAN: Rationale
The premise of the OCEAN study, a double-blind comparison of treatment with omega-3 fatty acids vs placebo in patients awaiting carotid endarterectomy, was based on evidence from epidemiologic and case-control studies that consumption of fish, oily fish, and long-chain omega-3 fatty acids reduces cardiovascular events and mortality, most likely slowing or limiting atherosclerosis due to risk-factor reduction. For example, in the GISSI Prevenzione trial, treatment with omega-3 fatty acids resulted in significant reductions in mortality and cardiovascular mortality, with these effects becoming apparent within only a few months after starting treatment.
A previous randomized controlled trial carried out by Prof. Calder's group in patients awaiting carotid endarterectomy showed a positive relationship between plaque stability and the omega-3 fatty acid content of the plaque, with incorporation of omega-3 fatty acids into carotid plaque fractions that increased after intake of fish oil. Morphologic properties of the plaque were altered: fewer plaques from patients being treated with fish oil having thin fibrous caps and signs of inflammation and more plaques having thick fibrous caps and no signs of inflammation compared with plaques in control patients. In addition, the number of macrophages in plaques from patients receiving fish oil was lower than in control plaques.
The OCEAN trial was set up to provide evidence that omega-3 fatty acids enter carotid plaques and induce changes indicative of increased plaque stability and that they reduce the expression of inflammatory mediators in the plaque.
Patients and Treatment
Patients aged > 18 years who were awaiting carotid endarterectomy were identified at 3 UK centers. Patients taking fish oil or evening primrose oil or consuming more than 2 oily fish meals per week or were due for surgery within 7 days were excluded from enrollment. A total of 121 patients (average age, 73 years; 65% men) were randomized to treatment with omega-3 fatty acids 2 g/day (n = 60) or placebo (n = 61) before surgery. The omega-3 fatty acids were administered as Omacor capsules (Solvay Healthcare Ltd, Southampton, United Kingdom). Each 1-g capsule contained 90% omega-3 acid ethyl esters, including eicosapentaenoic acid (EPA) (46%) and docosahexaenoic acid (DHA) (38%).
Baseline characteristics, including lipid levels (Table 1), were similar in each treatment group. Most patients were overweight, hypertensive, and current or former smokers. The majority of patients were being treated with aspirin (81%) and statins (84%), and 56% were on angiotensin-converting enzyme (ACE) inhibitors, 35% on calcium channel blockers, 35% on beta blockers, and 20% on anticoagulants.
Table 1. OCEAN: Baseline Lipids
| Lipid | Placebo | Omega-3 |
|---|---|---|
| Serum triglycerides -- mmol/L (mg/dL) | 1.6 (142) | 1.4 (124) |
| Serum cholesterol -- mmol/L (mg/dL) | 4.8 (186) | 4.8 (186) |
| Serum LDL cholesterol -- mmol/L (mg/dL) | 2.8 (108) | 2.7 (105) |
| Serum C-reactive protein | 5.5 | 6.5 |
Patients remained in the study until they went to surgery. When the study was designed, the average time until surgery was 40-50 days, but by the time the study was carried out it had been shortened so that the median duration of treatment was 21 days in both treatment groups.
Plaques were obtained at surgery and the morphology, histology, and cellular infiltration studied with immunohistochemistry. mRNA expression of inflammatory matrix metalloproteinases (MMPs), adhesion molecules, and cytokines in the plaques was measured.
Primary Endpoint
EPA and DHA were both rapidly taken up into the plaques in the treated patients. Compared with the placebo group, treated patients showed a significant 100% increase in EPA (P < .001) and a nonsignificant 10% increase in DHA (P = .20).
Fifty patients on placebo and 45 in the treatment group were evaluable for the primary endpoint, a composite of histologic and immunohistochemical parameters, including:
Size of the lipid core;
Number of foam cells;
Presence of hemorrhage;
Number of macrophages (CD68+) in the plaque;
Number of macrophages (CD68+) in the fibrous caps;
Overall density of inflammation in the plaque; and
Overall density of inflammation in the fibrous caps.
The number of foam cells was significantly lower in patients treated with omega-3 fatty acids than those in the placebo group (P = .039). Most of the other elements of the endpoint were lower in the treatment group compared with placebo, although the differences did not reach statistical significance. The composite mean score was also lower in the treatment group vs the placebo group (P = .20).
There were highly significant inverse correlations between the amount of EPA in the plaque and plaque inflammation, instability, and T-cell infiltration. The combination of EPA plus DHA also demonstrated a highly significant inverse correlation with inflammation, instability, and T-cell infiltration, and a tendency toward a negative correlation with macrophage infiltration (Table 2).
Table 2. Correlation Between Omega-3 Fatty Acids and Plaque Features (P Values)
| Plaque Feature | EPA | DHA | EPA-DHA |
|---|---|---|---|
| Inflammation | .011 | .096 | .033 |
| Calcification | -- | .022 | .057 |
| Instability | .021 | .086 | .037 |
| T cells (CD3+) | .010 | .091 | .031 |
| Macrophages (CD68+) | .109 | .090 | .069 |
| Macrophages in fibrous cap | .105 | -- | -- |
The mean score that was based on the histologic and immunohistochemical analyses of the component of the primary endpoint was also highly significantly correlated with the EPA content of the plaque (Table 3). "The more EPA in the plaque, the less inflamed and more stable it is," Prof. Calder explained.
Table 3. Correlation Between Primary Endpoint and Plaque EPA
| Histology Feature | Plaque EPA* | P Value |
|---|---|---|
| Mean score | -.021 | .043 |
EPA = eicosapentaenoic acid MMPs
Because MMPs released from macrophages, foam cells, and smooth muscle cells degrade the plaque cap, making it more vulnerable to rupture, mRNA expression for MMPs 1, -3, -7, -8, -9, -12, and -13 was measured by real-time polymerase chain reaction (RT-PCR). Expression of 3 MMPs associated with plaque instability was significantly lower after active treatment compared with placebo. MMP-9, which is highly expressed in carotid plaques and involved in collagen breakdown and cap weakening, was 40% lower in the treatment group vs placebo (P = .005). Expression of MMP-7 and MMP-12 mRNA was also significantly lower in the treatment group. Expression of the other 4 MMPs did not differ significantly between the 2 treatment groups.
Expression of plasma intercellular adhesion molecule (ICAM)-1 was significantly lower in the omega-3 fatty acids group (P = .014). ICAM-1 is an adhesion molecule that is involved in recruitment of monocytes into the vessel wall, and increased expression is associated with progression of atherosclerosis, Prof. Calder explained. The decrease seen in macrophage numbers could be related to lower ICAM-1 expression, he speculated.
Expression of the inflammatory cytokine interleukin (IL)-6 was significantly lower in the treatment group compared with placebo (P = .039).
Implications
OCEAN has shown that increased availability of omega-3 fatty acids results in their incorporation, particularly EPA, resulting in fewer macrophages, foam cells, and T cells, and lower expression of inflammatory markers, ICAM-1, IL-6, and selected MMPs, Prof. Calder concluded. Histologically this results in a plaque that appears to be less inflamed and more stable.
Prof. Calder has been involved in the development of therapy with omega-3 fatty acids for many years. He believes that because of their anti-inflammatory properties, these agents may have other applications in chronic and acute inflammation; in diseases, such as cystic fibrosis; and for prevention of inflammatory response, such as sepsis in postsurgical patients.
References
Cawood AL, Ding R, Napper FL, et al. Long chain omega-3 fatty acids enter advanced atherosclerotic plaques and are associated with decreased inflammation and decreased inflammatory gene expression. Atherosclerosis. 2006;7(suppl):160. Abstract Tu-W20:3.
Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet. 1999;354:447-455. Abstract
Thies F, Garry JM, Yaqoob P, et al. Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial. Lancet. 2003;361:477-485. Abstract