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Cardiovascular Research 2001 52(3):361-371; doi:10.1016/S0008-6363(01)00406-0
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Longchain n–3 polyunsaturated fatty acids and blood vessel function

Mahinda Y Abeywardena* and Richard J Head

CSIRO Health Sciences and Nutrition, Kintore Avenue, P.O. Box 10041, Adelaide BC, SA 5000, Australia

* Corresponding author. Tel.: +61-8-8303-8889; fax: +61-8-8303-8899 mahinda.abeywardena{at}hsn.csiro.au

Received 26 April 2001; accepted 3 July 2001


   Abstract
Top
 Abstract
1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
 References
 
The cardiovascular health benefits of longchain n–3 polyunsaturated fatty acids (PUFAs) have been reported to exert at several different cellular control mechanisms. These include, effects on lipoprotein metabolism, haemostatic function, platelet/vessel wall interactions, anti-arrhythmic actions and also inhibition of proliferation of smooth muscle cells and therefore growth of the atherosclerotic plaque. Fish oil feeding has also been found to result in moderate reductions in blood pressure and to modify vascular neuroeffector mechanisms. The majority of such cardiovascular benefits of n–3 PUFAs are likely to be mediated in the vascular wall and at the vascular endothelium level, since this monolayer of cells plays a central role in the regulation and maintenance of cardiovascular homeostasis and function. While these processes include endothelium-derived vasorelaxant and vasoconstrictor compounds, the vascular endothelium also plays host to many receptors, binding proteins, transporters and signalling mechanisms. Accordingly, endothelial dysfunction, which underlies many cardiovascular disease conditions, can trigger acute vascular events including vasospasm, thrombosis or restenosis leading to ischaemia. The longchain n–3 PUFAs have been reported to possess several properties that may positively influence vascular function. These include favourable mediator profiles (nitric oxide, eicosanoids) that influence vascular reactivity, change in vascular tone via actions on selective ion channels, and maintenance of vascular integrity. In addition to direct effects on contractility, n–3 PUFAs may affect vascular function, and the process of atherogenesis, via inhibition of vascular smooth muscle cell proliferation at the gene expression level, and by modifying expression of inflammatory cytokinesis and adhesion molecules. Collectively, these properties are consistent with pleiotropic actions of longchain n–3 PUFAs, and may explain the beneficial cardiovascular protection of this family of fatty acids that have been clearly evident through epidemiological data as well from more recent large-scale clinical trials.

KEYWORDS Endothelial function; Nitric oxide; Prostaglandins; Vasoconstriction/dilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
 References
 
The benefits of high consumption of n–3 polyunsaturated fatty acids (n–3 PUFAs) on cardiovascular disease mortality was first noted over two decades ago [1]. Now it is well recognised that longchain PUFAs of marine origin possess a multitude of actions that combat the pathogenesis of coronary heart disease (CHD; [2–4]). Although dietary intervention with fish oils failed to reduce incidence of restenosis following coronary angioplasty, and conflicting data exists for regression of atherosclerosis [3,5], significant benefits of n–3 PUFAs (eicosapentaenoic acid, 20:5 n–3, EPA; docosahexaenoic acid, 22:6 n–3, DHA) from fish oil have been observed at several different stages of CHD process. These include effects on lipoprotein metabolism, platelet/vessel wall interactions (thrombosis), cardiac arrhythmia, and ischaemic damage to heart muscle, proliferation of smooth muscle and growth of atherosclerotic plaque.

The finding that n–3 PUFAs from fish oil can produce moderate reductions in blood pressure in experimental models of hypertension and in humans has focussed attention on a potential role of n–3 PUFAs in modulating vascular contraction and vasodilatation. This review highlights some of the key observations that helped characterise the role of n–3 PUFAs on acute physiological responses in blood vessels as well as their role in modulating vascular cell to cell interactions that impinge on thrombosis formation and innervated vascular smooth muscle cell (VSMC) proliferation.


   2. The vascular endothelium
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
 References
 
The endothelium plays a key role in vascular function and attempts to maintain normal homeostasis via the production of a range of biochemical mediators (Fig. 1). The endothelium is also host to many receptors, binding proteins, transporter and signalling mechanisms involved in the regulation of cellular processes including cell growth, programmed cell death (apoptosis) and cell migration. Most biochemical mediators produced by the endothelium are likely to exert modulatory actions on one or more of these processes as well as to influence actions of other mediators [6,7]. Locally generated vasoactive agents of endothelial cell origin include angiotensin II (converted from angiotensin I by the angiotensin converting enzyme (ACE) in endothelial cells), nitric oxide (NO), endothelium derived hyperpolarizing factor (EDHF), eicosanoids and polypeptide molecules such as endothelin. In certain disease states endothelium may also produce increased level of free radicals and promote abnormal contraction of blood vessels.


Figure 1
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  		Fig. 1 Vasoactive compounds released by the endothelium. A range of biochemical mediators are produced by the vascular endothelium and involved in the maintenance of normal homeostasis. NO, nitric oxide; PGI2, prostacyclin; EDHF, endothelium derived hyperpolarising factor; A-II, angiotensin II; ET, endothelin; TxA2/PGH2, thromboxaneA2/prostaglandin H2; O2·, superoxide anion.

 
Endothelial dysfunction therefore potentially reflects an imbalance between the vasoconstriction and vasodilator compounds and is associated with several cardiovascular risk factors such as hypercholesterolaemia, hypertension, diabetes and smoking. Such abnormalities in the endothelium, which underlie many cardiovascular disease conditions, may trigger acute vascular events including vasospasm, thrombosis or restenosis resulting in myocardial ischaemia and sudden death from ventricular arrhythmias (Fig. 2).


Figure 2
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  		Fig. 2 Endothelial dysfunction in cardio and cerebro-vascular disease. Abnormalities in endothelial function can trigger acute vascular events and precipitate ischaemic attacks.

 
It is conceivable that anti-thrombotic as well as anti-atherogenic actions of n–3 PUFA are at least in part mediated at the vascular endothelial cell level since this monolayer of cells not only influences platelet/vessel wall interactions but also cell proliferation, cell death and structural alterations of blood vessels known as vascular remodelling.

The multiple modes of action of n–3 PUFA may include an influence on blood vessels since at least two physiologically important biochemical mechanisms operating mainly in the vascular endothelium have been shown to be positively modulated by fish oil fatty acids. The first is the ability to modify eicosanoid biosynthesis [2], and the other is an increased endogenous nitric oxide (NO) production following supplementation with fish oil [8]. Both processes can influence vascular reactivity and likely to form the basis for the reported improvements in endothelial function and arterial elasticity observed with n–3 PUFAs [9,10]. Evidence also indicates that n–3 PUFAs may influence vascular tone by exerting action on selective ion channels [11,12] and maintain vascular integrity by influencing soluble markers of endothelial haemostatic activity [13]. Collectively, these findings provide the basis for observations in experimental animal models and human subjects which confirmed moderate reductions in blood pressure [2–4,14,15] with n–3 PUFAs suggesting altered vascular neuroeffector responses.

2.1 Neuroeffector and vascular responses
Vascular neuroeffector mechanisms refer to the physiological processes underpinning responses generated by either stimulation of the sympathetic nerves innervating the vasculature or direct stimulation of VSMC in the region of the sympathetic nerve-rich adventitia. Potential mechanisms of blood pressure lowering by n–3 PUFAs have been extensively studied in experimental animal models of hypertension particularly the spontaneously hypertensive rat (SHR) with the Wistar–Kyoto rat (WKY) serving as its control. Dietary fish oil administration reduced the enhanced vascular contractility in the hypertensive animals [14–18] and in humans [4,8,10]. In agreement with observations in animals [18], findings in humans also demonstrates potentially a greater role for DHA rather than EPA in favourably modifying vascular reactivity and lowering blood pressure [19,20].

A variety of mechanisms have been advanced and explored to seek a molecular basis for the antihypertensive and neurovascular modulatory role of dietary fish oils. Included within these approaches has been a determination of whether vasodilator/vasoconstrictor mechanisms based upon eicosanoid metabolism (prostaglandins and thromboxanes) and nitric oxide (NO) production plays a role in the modulating actions of dietary fish oils. While, there appears to be no major involvement of vasodilatory prostaglandins [15], thromboxane-A2 (TxA2) production is elevated in the SHR and modified favourably with fish oils [15–18,21].

It is established that activation of NO production in the vascular endothelium induces vasodilatation. In the SHR, NO mediated relaxation in the aorta is impaired and restored with fish oil administration [17]. This could be due to a suppression of vasoconstriction by NO in the normotensive state and an absence of a similar influence in the SHR. However, it would seem unlikely that the modulation of contractile responses by fish oil treatment in the SHR is solely attributable to a primary role of n–3 PUFAs in restoring endothelium dependent relaxation. For instance, endothelium independent relaxant effects of EPA and DHA have been reported in animal and human studies [20,22,23]. While the basic mechanism is unclear in the human setting, vasorelaxation in the WKY and SHR induced by EPA and DHA are thought to involve prostanoid mediated activation of K+-ATP channels, and mobilisation of intracellular Ca2+ in VSMC via L-type (DHA) and non L-type (EPA) Ca2+ channels [22,23].

Therefore, it appears that fish oils may influence receptor function, transduction processes and membrane ion channels in the vasculature. There is growing evidence (discussed later) supporting a role of fish oils in altering ion transport to promote a hyperpolarising action. Further focus should also be directed toward understanding the potential influence of specific n–3 PUFAs on vascular remodelling and favourably influencing vascular neuroeffector mechanisms (Fig. 3). In this context it should noted that EPA inhibits VSMC proliferation [24,25], and DHA has been reported to trigger VSMC apoptosis implicating a role in vascular remodelling [25,26].


Figure 3
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  		Fig. 3 Beneficial modulation of vascular abnormalities in hypertension by longchain n–3 PUFAs. Studies with fish oils in experimental animal models and human subjects demonstrate n–3 PUFAs to exert a wide range of protective actions on the vascular structure and function.

 

   3. Biochemical mediators
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
 References
 
3.1 Eicosanoids
EPA and DHA can act as alternative substrates, for both cyclooxygenase (COX) and lipoxygenase (LOX) enzyme complexes giving rise to 3-series prostaglandins and thromboxanes, and 5-series leukotrienes respectively (Fig. 4). Although these metabolites bear considerable structural resemblances to those produced by the preferred substrate for eicosanoid biosynthesis — n–6 polyunsaturated arachidonic acid (AA, 20:4 n–6) — the overall cardiovascular benefits exerted by products derived from n–3 PUFAs are more favourable due to differences in biological activities (Fig. 4; [2,27–29]).


Figure 4
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  		Fig. 4 Major eicosanoid metabolites derived from n–3 and n–6 polyunsaturated fatty acid substrates. Different families of eicosanoids with varied biological potencies are produced by n–3 PUFAs. COX, cyclooxygenase; LOX, lipoxygenase; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; AA, arachidonic acid; LT, leukotrienes; PG, prostaglandin; Tx, thromboxane.

 
The observed anti-thrombotic actions of fish oil PUFAs are generally explained by the inhibition of platelet TxA2 and parallel changes in the clotting mechanisms. Significant reduction in TxA2 production following longchain n–3 PUFAs has been observed both in vitro and in vivo studies both in experimental animal models and in human subjects [2,28]. In vitro estimation of prostacyclins — PGI2+PGI3 — also shows a reduction after n–3 PUFA feeding, albeit smaller in magnitude than TxA2. In contrast, in vivo studies exhibited either no change or even an increased generation of PGI2 following n–3 PUFAs [27,29,30] providing evidence for a possible differential modulation of thromboxane and prostacyclin production by the n–3 PUFAs [31]. Differences between in vitro and in vivo production, as measured by the excretion of urinary metabolites, have also been reported for E and F series prostaglandins [32]. The vascular endothelium is the main source of anti-aggregatory and vasodilatory prostacyclins, therefore preservation of beneficial prostacyclins at the expense of thromboxanes could form a key mechanism for the reported benefits of n–3 PUFA on vascular function.

This specific inhibitory action n-PUFA on TxA2 synthesis is not explained by the changes in the availability of precursor fatty acids alone and may perhaps be exerted at the biosynthetic enzyme level [31]. In addition, DHA and EPA have been shown to act as antagonists at the TxA2/PGH2 receptor in human platelets [33]. In the latter study, DHA was found to be more potent than EPA in blocking the activation of platelets induced by the stable TxA2 mimetic U46619. [GenBank] However, DHA was the only PUFA to show competitive antagonism at the TxA2/PGH2 receptor in rat aorta [34].

Further evidence in support of increased PGI2 production was reported by Saito et al. [35] who observed enhanced synthesis in cultured rat VSMC enriched with a triglycerol emulsified form of EPA. Interestingly, DHA-TG was without effect. Activation of COX by EPA via the generation of low lipid peroxide level has been put forward as a potential mechanism although the failure of DHA to similarly activate the COX enzyme complex remains unclear. In contrast, Achard et al. [36] found in bovine aortic endothelial cells that both EPA and DHA reduce PGH synthase-1 expression, perhaps at the transcriptional level, thereby inhibiting the synthesis of PGI2. n–3 PUFAs however, did not inhibit the endothelial cell PGI2 synthase. Both EPA and DHA caused approximately a 50% inhibition in PGI2 production in response to a range of exogenous stimulants such as bradykinin, calcium ionophore and AA.

Docosapentaenoic acid (DPA; 22:5 n–3) has also been shown to be inhibitory in the system but the effect may be attributed to retroconversion of DPA to EPA [37]. Whether this is a reflection of what occurs in vivo is an open question since as indicated earlier, several studies have reported differences between in vitro and in vivo generation of beneficial prostacyclins (PGI2+PGI3) by n–3 PUFAs. Further studies exploring the effects of n–3 PUFAs on platelet thromboxane biosynthetic enzymes, both at the biochemical and at the gene expression level, would be needed to comprehensively define the modulation, including any preferential production of prostacyclins by n–3 PUFAs [31,38] in maintaining an anti-thrombotic state.

3.2 Nitric oxide
It could be argued that recent observations of increased urinary excretion of NO metabolites in humans after fish oil supplementation [8] may be the primary mechanism responsible for the anti-thrombotic effect of n–3 PUFAs in that enhanced endogenous NO may offset vasoconstrictor influences in general as well as any reductions in vasodilatory influence of PGI2 by these polyenoic fatty acids. Nevertheless, it is worth noting that no increase in endogenous NO production was seen with EPA and that DHA appears to be the active component in mediating this effect [8]. This latter observation however, is possibly at odds with an anti-thrombotic action based on a differential modulation of eicosanoids since both EPA and DHA have been shown to be effective in achieving a favourable prostacyclin/thromboxane ratio. Furthermore, preparations enriched in EPA as well as DHA have been shown to either maintain or even increase the generation of prostacyclins in experimental animals and in humans [29,30,38].

The observation that EPA was ineffective in increasing NO is an intriguing one since several studies reported that DHA may play a more prominent role than EPA in conferring protection against several cardiovascular disease indices. These include lipids and lipoproteins, hypertension, cardiac arrhythmia, heart rate, vascular reactivity and hypertension induced renal damage [4,18]. It is possible that a prominent role of DHA when contrasted to EPA in providing this vasoprotection may perhaps be related to a differential influence on endothelial cell NO [8]. However, in vitro treatment of rat aortic rings with either EPA or DHA augmented the endothelium dependent vasorelaxation to a similar extent due to an enhanced release of EDRF and vasodilator prostaglandins [39]. Furthermore, Omura et al. [40] observed EPA, but not DPA or DHA stimulated NO production and induced endothelium-dependant relaxation in bovine coronary arteries precontracted with thromboxane mimetic U46619. [GenBank] In contrast, both EPA and DHA caused significant inhibition of vasoconstriction in the isolated perfused rabbit ear induced by E2 and F2 isoprostanes and U46619 [GenBank] [41]. More studies are needed in relation to the mechanisms of vasorelaxation induced by n–3 PUFAs since both EPA and DHA have also been reported to antagonise TxA2/PGH2 receptors [33,34] as well as to induce relaxation via endothelium independent mechanisms [20,22,23].

In addition to enhancing vasodilatory influence, n–3 PUFAs may inhibit the release of vasoconstrictor agents. Similarly to the effects on vasoconstrictor eicosanoids [2,28], EPA has been found to cause a dose dependent inhibition of endothelin (ET-1) production in bovine mesangial cells [42].

3.3 Nitric oxide, eicosanoids and isoprostanes
There is a potential interplay between NO and eicosanoids [43] and the role of n–3 PUFAs in modulating vascular function should be measured against this interaction. For example, it was demonstrated in hypertensive rats that inhibition of endothelial cell NO production unmasked a vasoconstrictor response that was sensitive to COX inhibitors as well as following the blockade of TxA2/PGH2 receptors [44,45]. However, this abnormal TxA2-like constrictor response is mainly mediated via prostaglandin endoperoxides (PGG2/PGH2) intermediates as inhibitors of TxA2 synthetase was without effect [45]. Furthermore, it was observed that pre-feeding of hypertensive rats with purified DHA attenuated this response whilst dietary EPA failed to alter this abnormality [18]. It appears that this differential action of DHA is mediated via two distinct mechanisms (Fig. 5) — via an inhibitory effect at the TxA2 synthetase level [31] and by antagonism at the vascular TxA2/PGH2 receptor [34]. This receptor antagonism was only observed for DHA and not shared by other n–3 PUFAs and is in agreement with observations following dietary administration of fish oil [18,34].


Figure 5
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  		Fig. 5 Differential modulation of PGI2 and TxA2 by longchain n–3 PUFAs. Fish oil fatty acids may preferentially reduce the effects of vasoconstrictor mediators (e.g. thromoboxane-A2, isoprostanes) via distinct mechanisms (see text).

 
It is likely vascular relaxation promoted by fish oils [9,10,14,16] is exerted mainly at the biochemical mediator level including the inhibition of TxA2-like constrictor response [18] whilst preferentially preserving or increasing the prostacyclins [27–31], or by directly modulating NO [8,40,46,47]. Current knowledge also suggests that isoprostanes — prostaglandin-like compounds formed non-enzymically from free radical mediated oxidation of membrane lipids [48,49] — as potent modulators of vascular contractility [48–50]. Studies also indicate the production of isoprostanes via COX and NO pathways [51] and thus assign a link between NO, prostanoids, free radicals and isoprostanes for the regulation of vascular tone [43]. The production and/or physiological effects of isoprostanes that may result from longchain n–3 PUFAs are yet to be established due mainly to analytical limitations in characterising the different yet structurally closely resembling families of different isoprostanes and prostaglandins.

Another possibility is that although longchain n–3 polyenoic fatty acids have generally been regarded as susceptible to oxidation due to the presence of number of double bonds, it is equally likely that in situ these PUFAs may act as a sink and/or scavengers to remove specific free radicals. Acute additions of n–3 PUFAs to human neutrophils have been reported to decrease superoxide anion generation primarily via a prostaglandin dependant pathway [52], and fish oil supplementation has been shown to attenuate free radical generation following coronary occlusion and reperfusion in rabbits [53]. In vivo assessment of oxidant stress in humans — as quantified by urinary F2 isoprostanes — have confirmed these earlier reports and also demonstrated both EPA and DHA to equally reduce the endogenous free radical status [54]. Therefore, n–3 PUFAs in addition to exerting direct actions at the substrate, enzyme and receptor levels may also be to modulate key mediator pathways in the vasculature including COX, NO and isoprostane production, all of which are dependant on the endogenous free radical status [48,49,55,56]. Collectively these mechanisms argue for the role of n–3 PUFAs in mediating a shift from vasoconstriction to vasodilatation (Figs. 4 and 5Go).


   4. Vascular membrane ion channels
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
5. Growth and proliferation...
 6. Summary
 References
 
A role of n–3 PUFAs is modulating ion channels in cell membranes has arisen from studies on excitable cardiac muscle cells. The n–3 PUFAs prevent abnormal beating in rat cardiomyocytes exposed to several arrhythmogenic stimulants [57]. It has been proposed that the n–3 PUFAs exert this influence by influencing Ca2+ availability by inhibition of voltage dependent L-type Ca2+ channels and voltage dependent Na+ currents [58–60]. A major focus of the role of n–3 PUFAs and cardiac muscle cell excitability has been directed toward ischaemia induced arrhythmias as these fatty acids prevent the development of ventricular fibrillation (VF) associated with coronary artery ligation in several animal models [61–64]. Work using isolated mammalian cell lines [65], myocytes [57–60] and studies on heart rate variability (HRV) in humans [66,67] have allowed a better understanding of anti-arrhythmic actions of n–3 PUFAs. HRV which is a measure of cardiac autonomic tone, indicates that modulation of electrophysiological properties of the myocardium by n–3 PUFAs as the most likely mechanism for their anti-arrhythmic actions. n–3 PUFAs have been shown to increase HRV, which is reflected as a higher VF threshold thus reducing the vulnerability to arrhythmia [66,67]. In view of the importance of ion channels in vascular smooth muscle function it is not surprising that attention has been drawn to an interaction of n–3 PUFAs and ion channels and vascular function.

Another potential mechanism by which n–3 PUFAs may regulate vascular tone was reported by Asano et al. [11]. Using cultured rat VSMC and voltage clamp technique, these investigators found that n–3 PUFAs — EPA, DPA and DHA, inhibit the receptor mediated non-selective cation current and in addition, activate a K+ current in a concentration dependent manner. Although the ability of n–3 PUFAs to block the Na+ channel in rat ventricular myocytes has been reported previously [59,60], these findings on the effectiveness of n–3 PUFAs to activate K+ channels indicate that this hyperpolarizing action may also contribute to the vasorelaxant actions of fish oil fatty acids.

The effects of long-term treatment to permit incorporation of n–3 PUFAs within cellular pools, as compared to acute challenge, on electrophysiological properties of VSMC was also recently reported by [12]. Treatment of rat A7r5 VSMC cells with EPA for 7 days was associated with a partial inhibition of resting intracellular calcium concentration [Ca2+]i and agonist induced rise in [Ca2+]i. In addition, EPA treatment tended to hyperpolarize resting membrane potential through an increase in outward currents generated via the activation of K+ channel and Na+/K+ pump. These findings are of considerable interest since the hyperpolarizing effects of n–3 PUFAs following long-term treatment may suppress or inactivate voltage dependent Ca2+ channels resulting in a lower [Ca2+]i and inhibit the agonist induced increases in [Ca2+]i in excitable cells providing an electrophysiological basis for the reported anti-arrhythmic, vasorelaxant as well as anti-atherogenic actions of n–3 PUFAs. In support of the this latter speculation for anti-atherogenic actions, the authors observed that parallel to the reduction in membrane potential and [Ca2+]i, EPA pre-treatment also inhibited the PDGF induced migration of VSMC.

There is accumulating evidence to suggest that n–3 PUFAs have an ability to act at the cellular ion channel levels to alter the electrophysiology of excitable cells and therefore to directly influence physiological parameters including cardiac rhythm and vascular tone.


   5. Growth and proliferation of vascular cells
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
6. Summary
 References
 
5.1 Cell proliferation
In addition to their direct influence on vascular contractility and on more acute cellular events, n–3 PUFAs may also influence vascular function and the process of atherogenesis by influencing the growth and proliferation of VSMC [24–26,68]. Interestingly, the potency for this anti-proliferative action has been reported to be greater for EPA than DHA [69], which differs from the observations for cardiovascular protection where DHA appears to play a more prominent role [8,18–20].

The inhibition of VSMC proliferation by EPA is achieved at various steps of the signal transduction pathway for growth factors. For example, EPA has been shown to prevent the binding of PDGF to its surface receptor [24], to suppress Protein Kinase C activation and mRNA expression of the early growth gene in the nucleus, c-fos, by inhibiting c-fos transcription. In addition, cyclins and their catalytic subunits; cyclin-dependent kinases, which control the progression of the cell cycle via DNA synthesis, have also been reported to be inhibited by the longchain n–3 PUFAs, EPA and DHA [69]. EPA also been reported to suppress the transforming growth factor-β and inhibit the exaggerated growth of VSMC of SHR [25]. Taken together, it appears that these longchain polyenoics may inhibit the proliferation of VSMC by more than one mechanism - modulation of various steps of growth signals as well by inhibiting DNA synthesis.

5.2 Adhesion molecules
The adhesion of circulating leukocytes to vascular endothelium and subsequent recruitment and infiltration of monocytes into the vascular wall is a major event in the biological events underpinning atherogenesis and inflammation. This complex series of events are primarily regulated by the expression of a range of adhesion molecules on vascular endothelial cells aided by the release of various chemo-attractant factors. Whilst a normal healthy endothelium tends to repel the adhesion of leukocytes, an activated endothelium may promote adhesion processes. A range of compounds of different origin including, oxidised low-density lipoproteins, lipopolysaccharides and inflammatory cytokines (e.g. interleukins, tumour necrosis factor alpha) have been found to cause ‘endothelial activation’. Significant modulation of endothelial adhesion molecules by n–3 PUFAs has also been reported [70] and reviewed [71]. The expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) has been reported to be reduced by DHA. In addition, DHA exerted a time and dose dependent reduction in the expression of endothelial cell adhesion molecule-1 (ELAM-1/E-selectin), Intracellular Adhesion Molecule-1 (ICAM-1), interleukins (IL-6 and IL-8) after challenging with various stimuli and the extent of reduction paralleled the incorporation of DHA into cellular phospholipids. Further to the reduced expression of adhesion molecules and leukocyte recruitment agents, DHA was found to reduce the adhesion of human monocytes and monocratic U937 cells to activated endothelial cells [71–73]. In human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1-beta (IL-1b), the expression of ICAM-1, VCAM-1 and E-selectin mRNA levels was reduced by EPA and DHA.

It is noteworthy that these modulatory effects appear to be totally independent of DHA metabolism to cyclooxygenase products [71,73]. Similarly, only DHA not EPA reduced the cytokine stimulated VCAM-1 expression and resulted in a greater reduction in pro-inflammatory cytokine production. Adhesion of human lymphocytes to endothelial cells was reported to be inhibited by either the addition or pre-treatment with n–3 PUFAs [72]. Several studies have found DHA to possess greater potency in comparison to EPA although the two PUFAs in association may act synergistically [73,74].

5.3 Fatty acid structure
Structural requirements for unsaturated fatty acids in relation to endothelial activation by pro-inflammatory cytokines have recently been identified [71,75]. In summary, the expression of VCAM-1 in endothelial cells activated with pro-inflammatory cytokines such as IL-1, TNF or bacterial lipopolysaccharide, was found to be directly related to the presence (or absence) of double bonds in the fatty acid molecule rather than the type of unsaturation (i.e. n–3 vs. n–6). These investigators also concluded that a double bond is the minimum necessary and sufficient requirement for fatty acid inhibition of endothelial activation. Therefore, the highest potency was seen with DHA which accommodates the highest number (six) of double bonds whilst both DPA and EPA, although differing in chain length, have the same number of double bonds and yielded identical results. Similarly, inhibition by AA was lower than EPA and the fatty acids with the same chain length but with variable number as well as the type of unsaturation of double bonds — oleic (18:1 n–9), elaidic (18:1 n–9 trans isomer of oleic acid), linoleic (18:2 n–6), {alpha}-linolenic ({alpha}18:3 n–3) and {gamma}-linolenic ({gamma}18:3 n–6) — all yielded inhibitions that were reflective of the number of double bonds rather than the type of unsaturation. Whilst monounsaturated palmitoleic acid (16:1) was similar to oleic and elaidic acids, saturated fatty acids palmitic (16:0) or stearic (18:0), failed to provide any protection. Furthermore, the protective effects of unsaturated fatty acids required the incorporation of the fatty acid in question to specific fatty acid pools within the endothelial cell. This is in contrast to an apparent lack of requirement for incorporation in studies reported for the inhibition of ion channels following the acute addition n–3 PUFAs as in the case for anti-arrhythmic activity in cultured myocytes [58–60] or opening of K+ channels in rat VSMC cells [11].

These findings may imply that altered fatty acid composition is a pre-requisite for the modulation of gene expression for adhesion molecules. However, it is also clear that this effect is not mediated via a general alteration in the physico-chemical properties or ‘fluidity’ of the membrane, since fatty acids with either cis or trans double bonds resulted in same inhibitory potencies despite the wide differences in physical properties that are known to exist between these two configurations [76]. It was concluded that n–3 PUFA will have the largest beneficial effect among unsaturates for a given chain length since they accommodate a greater number of double bonds and in addition serves as poor substrates for eicosanoids biosynthesis [75].

In vascular endothelial cells, DHA has been found to increase membrane fluidity more than EPA [77]. In cardiomyocytes, however, any alterations in packing of membrane phospholipids by n–3 PUFAs are found not to be responsible for their anti-arrhythmic properties [78]. Such observations may add a further dimension to the currently held view regarding the potential for increased lipid peroxidation in highly polyunsaturated fatty acids. The findings could suggest that the higher the number of double bonds in the fatty acid molecules, the greater the protection against endothelial activation, which can trigger inflammation and the process of atherogenesis. It appears that increased number of double bonds in the fatty acid molecule may be effective in conferring a higher protection. Conceivably this could be achieved by the PUFAs from by acting as a sink for the damaging effects of reactive free radicals.

Collectively, these data suggests that n–3 PUFAs have the ability to modulate certain key biologically active proteins involved in the pathogenesis of atherosclerosis through gene expression as well as at DNA and protein synthesis levels in a manner independent of their modulatory effects on eicosanoid metabolism. Similarly, further studies on structure–activity relationships of the n–3 PUFAs induced changes in electrophysiology with resultant effects on intracellular distribution of ions may in a similar fashion further our understanding with regard to the specific anti-arrhythmic and vasorelaxant actions of n–3 PUFAs.


   6. Summary
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
References
 
When reviewing the influence of n–3 PUFAs on cardiovascular function it is apparent that these fatty acids influence a wide range of biochemical and physiological functions. This is not surprising in view of the pivotal role fatty acids play in membrane function and integrity as well as their related role in the synthesis of biologically active lipid mediators. However what does emerge are two distinct actions which may provide insights into the precise nature of the interaction of PUFAs in the cardiovascular system. Firstly the n–3 PUFAs exert beneficial effects on several different cardiovascular risk factors including favourable influences on plasma triglycerides, blood pressure, platelet and leukocyte function and coagulation and fibrinolysis processes [2–5,13]. In addition, the n–3 PUFAs provide protection during the acute events associated with, for example, myocardial ischaemia [61–64]. In blood vessels it emerges that n–3 PUFAs decrease either the expression or activity of the processes that favour platelet aggregation and induction of abnormal vascular growth (Fig. 1). Concurrently n–3 PUFAs favour vasodilatory mechanisms over vasoconstrictive processes by a variety of possibly interlinked processes (Fig. 6). The question remains as to why these longchain n–3 PUFAs consistently produce an influence on widely differing processes consistent with cardiovascular disease prevention as clearly demonstrated in several large clinical trials [3,79]. What is evident is that in addition to being protective against acute cardiovascular events and consequent deleterious effects of myocardial ischaemia including cardiac arrhythmia and tissue damage [18,64,80], the n–3 PUFAs act to prevent pathological and physiological processes that precipitate ischaemic episodes.


Figure 6
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  		Fig. 6 Pleiotropic actions of longchain n–3 PUFAs on the vasculature. Summary of reported beneficial effects exerted by fish oil fatty acids.

 
In addition to the established cardiovascular benefits of n–3 PUFAs [2–5], more recent studies have identified additional protective actions of longchain n–3 polyenoic fatty acids on a myriad of different cellular mechanisms of pathophysiological consequence. Although, most of these discoveries have been made in vitro, using cultured cell lines in acute experiments under defined conditions, it is likely that such protective actions may in deed be extended to chronic disease processes such as atherogenesis and the development of coronary artery disease.

It can also be speculated that the ability of longchain n–3 PUFAs to exert such multiple modes of action (Fig. 7) may provide the biological basis for their wide ranging cardiovascular protection when compared to specific pharmacological agents directed towards individual cardiovascular pathophysiological mechanisms. Such spectrum of potential benefits (Figs. 6 and 7Go) also suggests longchain n–3 PUFAs to possess substantial pleiotropic actions, akin to those observed with certain pharmacological interventions [81]. It remains to be determined what additional vascular benefits may be derived from these longchain n–3 PUFAs.


Figure 7
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  		Fig. 7 Cardiovascular benefits of longchain n–3 PUFAs. Several key pathophysiological events in the cardiovascular disease cascade are beneficially modulated by longchain n–3 PUFAs of marine origin.

 
Time for primary review 19 days.


   References
Top
 Abstract
 1. Introduction
 2. The vascular endothelium
 3. Biochemical mediators
 4. Vascular membrane ion...
 5. Growth and proliferation...
 6. Summary
 References
 

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