• No results found

Cardiovascular Protective Effects of n-3 Polyunsaturated Fatty Acids With Special Emphasis on Docosahexaenoic Acid

N/A
N/A
Protected

Academic year: 2022

Share "Cardiovascular Protective Effects of n-3 Polyunsaturated Fatty Acids With Special Emphasis on Docosahexaenoic Acid"

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

308

Current Perspective

Cardiovascular Protective Effects of n-3 Polyunsaturated Fatty Acids With Special Emphasis on Docosahexaenoic Acid

Masahiko Hirafuji*, Takuji Machida, Naoya Hamaue, and Masaru Minami

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan

Received April 16, 2003

Abstract. It is widely accepted that n-3 polyunsaturated fatty acids (PUFAs) rich in fish oils protect against several types of cardiovascular diseases such as myocardial infarction, arrhythmia, atherosclerosis, or hypertension. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may be the active biological components of these effects. Although the precise cellular and molecular mechanisms underlying the beneficial effects are still uncertain, the protective effects of n-3 PUFAs are attributable to their direct effects on vascular smooth muscle cell (VSMC) functions. These n-3 PUFAs activate K+ATP channels and inhibit certain types of Ca2+ channels, probably via at least 2 distinct mechanisms. N-3 PUFAs favorably alter the eicosanoid profile and regulate cytokine-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 via mechanisms involving modulation of signaling transduction events.

N-3 PUFAs also modulate VSMC proliferation, migration, and apoptosis. These recent data suggest that modulation of these VSMC functions contribute to the beneficial effects of n-3 PUFAs on various cardiovascular disorders. Furthermore, recent studies strongly suggest that DHA has more potent and beneficial effects than EPA. However, many questions about the cellular and molecular mechanisms still remain to be answered.

Keywords: docosahexaenoic acid, eicosapentaenoic acid, vascular smooth muscle cell, cardiovascular disease

1. Introduction

A large number of epidemiological studies, clinical trials, and experimental animal studies has shown that fish oils and n-3 polyunsaturated fatty acids (PUFAs) protect against several types of cardiovascular diseases such as myocardial infarction, arrhythmia, atherosclero- sis, or hypertension (1 – 3). It is widely accepted that eicosapentaenoic acid (EPA, C20:5n-3) and docosa- hexaenoic acid (DHA, C22:6n-3) are the active bio- logical components of these effects (Fig. 1). The effects of n-3 PUFAs, particularly EPA, on functions of several types of cells such cardiac myocytes, vascular cells, platelets, leukocytes, or macrophages have been ex- plored by a number of studies in order to understand

the cellular and molecular mechanisms underlying the beneficial effects.

Although the precise mechanisms are still unclear, the protective effects of n-3 PUFAs are attributable to their direct effects on vascular endothelial and smooth muscle cell (VSMC) functions. Since endothelial cells produce and release a range of vasoactive mediators modulating vascular function and homeostasis and endothelial dysfunction is critically involved in cardio- vascular disease, many studies have focused on the influences on endothelial functions (2). However, in view of the importance of VSMC functions in vascular pathophysiology, their modulation may also be critically involved in the positive effect of n-3 PUFAs as well.

In deed, endothelium-independent vascular effects of DHA and EPA have been reported in animals and human studies (4, 5). Furthermore, recent results strongly sug- gest that DHA has more potent and beneficial effects on cardiovascular disease than EPA. DHA has been shown

*Corresponding author. FAX: +81-1332-3-1669 E-mail: hirafuji@hoku-iryo-u.ac.jp

Invited article

(2)

to be more effective than EPA in suppressing arrhythmia induced by ischemia in rats, inhibiting thromboxane-like vasoconstrictor responses in aorta from spontaneously hypertensive rats (SHR), and retarding hypertension development in SHR (6). DHA seems to be the principal n-3 PUFAs responsible for the hypotensive effect of fish oils, which was demonstrated by the first random- ized, double-blind, placebo-controlled study in humans (7). Focusing on the more recent studies, we will here discuss that the modulation of VSMC functions is involved in the cellular mechanism for the cardio- vascular protective effect of n-3 PUFAs with emphasis on the difference between EPA and DHA.

2. Intracellular calcium dynamics

As recently reviewed (1, 2), many studies in experi- mental animals and human subjects with hypertension have demonstrated a moderate reduction of blood pressure after supplementation of fish oils or n-3 PUFAs. Dietary administration of fish oils to genetically hypertensive rats reduces the exaggerated contractility of the vasculature in response to sympathetic nerve stimulation or vasoconstrictors. In vitro studies also have shown that both EPA and DHA have vasorelaxant effects on isolated blood vessels, indicating that the hypotensive effects are partly related to influences on vascular reactivity, possibly by both endothelium- dependent and -independent mechanisms. There are several possibilities for the endothelium-dependent

mechanism, including the suppression of endothelium- derived contracting factors (EDCF) such as thromb- oxane A2 (TXA2) and endothelin, and enhanced release of relaxing factors such as nitric oxide (NO), prostaglan- din I2 (PGI2), and hyperpolarizing factors (EDHF). The endothelium-independent mechanisms have been also suggested by several studies (1, 2). In a recent report, DHA, but not EPA, supplementation significantly en- hanced dilatory responses and attenuated constrictor responses in the human forearm microcirculation, which were mediated by multiple mechanisms, predominantly endothelial-independent mechanisms (4). Similarly, EPA and DHA (1 – 100mM) exerted the endothelium- independent vasorelaxant effects in Wistar Kyoto rats (WKY) and SHR aortae, probably through production of prostanoids that activate K+ATP channels, and through inhibition of intracellular Ca2+ release and Ca2+ channels in VSMCs (5, 8). Since intracellular Ca2+ concentration ([Ca2+]i) in VSMCs has a pivotal role as a second messenger in the mechanism of vasoconstriction, the endothelium-independent vasorelaxant effects of n-3 PUFAs can be partly attributed to the effect on intra- cellular Ca2+ dynamics in VSMCs.

In a recent electrophysiological study using cultured cells, n-3 PUFAs (3 – 100mM), when applied acutely to the cells, activated a K+ current and effectively inhibited receptor-mediated non-selective cation currents, which are resistant to Ca2+ channel blockers, in rat A7r5 VSMCs stimulated with vasopressin and endothelin (9).

The potency of the inhibitory effect was EPA> DHA >

docosapentaenoic acid (DPA, C22:5n-3). Since n-3 PUFAs similarly inhibited the non-selective cation current induced by GTPgS, a non-hydrolyzable GTP analogue, the authors suggest that the primary site of the acute inhibitory action of n-3 PUFAs is the channel protein itself or some sites near the channels (9). These authors also reported that the long-term treatment of A7r5 VSMCs with EPA (30mM) for 7 days suppressed the resting level and rise in [Ca2+]i induced by vaso- pressin, endothelin, and platelet-derived growth factor (PDGF) (10). Under the current clamp condition, the resting membrane potential was significantly deeper in EPA-treated cells (-49.8 mV, mean) than in control cells (-44.6 mV, mean). In a study using VSMCs isolated from SHR, EPA (30mM) pretreatment attenuated the angiotensin II-induced Ca2+ transient by 95% (11). We demonstrated that in VSMCs of WKY, DHA (30mM) pretreatment for 2 days inhibited a rise in [Ca2+]i induced by 5-hydroxytryptamine (5-HT), angiotensin II, and a depolarizing concentration of KCl (12). However, DHA had no effect on [Ca2+]i in VSMCs isolated from stroke-prone SHR (SHRSP), which was higher than [Ca2+]i in VSMCs of WKY. We also found that DHA

Fig. 1. Chemical structures of arachidonic acid (C20:4n-6), EPA (C20:5n-3), and DHA (C22:6n-3). The number of carbon atoms is given before and the number of double bonds after the colon. The position of the first double bond counted from the methyl end of fatty acid discriminates PUFA as n-3 or n-6 family.

(3)

(30mM) pretreatment inhibited 5-HT-induced Ca2+ in- flux through both L-type and non L-type Ca2+ channels, while DHA had no effect on Ca2+ release from the inter- nal stores in rat VSMCs (13). The mechanism of the inhibitory effect of n-3 PUFAs incorporated into the cells by adding to the culture medium may be different from that of acutely applied n-3 PUFAs (9). As illus- trated in Fig. 2, these results strongly suggest that the specific inhibition by n-3 PUFAs of intracellular Ca2+

dynamics in VSMCs may contribute to the endothelium- independent vasorelaxant effects that had been pre- viously observed (1, 2).

3. NO Production

Besides the vasodilating effect as endothelium- derived relaxing factor (EDRF), NO has inhibitory effects on platelet aggregation and adhesion, leukocyte adhesion, and VSMC proliferation/migration and is involved in the pathophysiology of cardiovascular dis- orders. NO is produced from the metabolism of L- arginine by a family of enzymes, NO synthase (NOS).

There are three isoforms of NOS, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). eNOS is restricted to the endothelium, while iNOS is expressed in several types of cells including VSMCs following stimulation with cytokines such as interleukin-1b (IL-1b) (14).

Studies in vitro have suggested that endothelium- dependent relaxation of vessel preparation by fish oils or n-3 PUFAs is due to the enhancement of NO release (1, 2). This is supported by the fact that EPA (>30 mM) stimulates NO production by endothelial cells in situ and induces endothelium-dependent relaxation of bovine coronary arteries (15). However, DHA and DPA (60mM) had no effect. The immunostaining analysis of eNOS using cultured bovine endothelial cells revealed eNOS to be co-localized with caveolin-1 in the cell membrane at a resting state, while EPA induced Ca2+- independent activation and translocation of eNOS to the cytosol and its dissociation from caveolin. This effect was comparable to that of the eNOS translocation induced by a [Ca2+]i-elevating agonist, bradykinin (10mM) (15).

An early study using cultured cells has shown that EPA also potentiates the release of NO from IL-1b- stimulated human and rat VSMCs in a concentration- dependent manner with a significant effect at concentra- tions higher than 3mM and 30 mM, respectively (16). In agreement with this study, we have recently reported that DHA (>10 mM), when added to the incubation medium, increases IL-1b-induced NO production by rat VSMCs (17). Although less potent than DHA, EPA, but not arachidonic acid, also showed the stimulatory effect, suggesting that this effect is common to n-3 PUFAs. The effect of DHA was not much remarkable, but substantial especially when IL-1b concentration was low. The effect of DHA was accompanied by poten- tiation of IL-1b-induced iNOS protein (Fig. 3) and mRNA expressions through a mechanism involving activation of the p44/42 mitogen-activated protein (MAP) kinase signaling cascade (Fig. 4). These results clearly indicate that DHA enhanced the upstream signal- ing pathways leading to iNOS translation in IL-1b- stimulated VSMCs.

NO production and iNOS expression in VSMCs are implicated in atherosclerotic vascular disease or hyper- tension (14). At the site of local vascular lesions such as atherosclerosis, IL-1b, and tumor necrosis factor-a are actively produced and released from activated macro- phages, which may induce iNOS expression in VSMCs.

Up-regulation of iNOS in VSMCs, therefore, may pri- marily function as a defensive and compensatory mecha- nism for endothelial dysfunction, by preventing the fur- ther development of pathological conditions, although excess amounts of NO radicals may cause cytotoxicity or apoptosis. It should be emphasized that recent experi- mental gene therapies by transferring an NOS gene including the iNOS gene to cardiovascular beds of various animal models of cardiovascular diseases show encouraging results and provide a novel and promising

Fig. 2. Modulation by n-3 PUFAs of intracellular Ca2+ dynamics in VSMCs as a possible cellular mechanism for n-3 PUFA-induced improved vasoreactivity. EPA and DHA can cause a decrease in intracellular calcium concentration ([Ca2+]i) via several mechanisms.

(4)

therapeutic strategy for treating cardiovascular diseases (18). Therefore, n-3 PUFAs may potentiate the primary function of iNOS-derived NO by the mild and persistent enhancing effect on iNOS induction in VSMCs.

4. Eicosanoid production

The beneficial effects of fish oils or n-3 PUFAs have been discussed largely with regard to arachidonic acid metabolism in the vessel wall as well as platelets (1, 2).

EPA is a substrate of cyclooxygenase (COX) in the arachidonate cascade, resulting in the conversion to biological active PGI3, with equipotent activities to PGI2, and inactive TXA3 (Fig. 5). DHA is not a direct substrate, rather an inhibitor in vitro, of COX, although DHA and EPA can be interconverted to each other.

Administration of fish oils containing EPA and DHA increases the n-3/n-6 PUFA ratio in the membrane phospholipids, the sum of PGI2 and PGI3, and effectively reduces TXA2 production in human studies and animal

experiments in vivo (1, 2). The origin of PGI2/PGI3 and TXA2 is considered predominantly to be endothelial cells and platelets, respectively. The in vivo increase in PGI2/PGI3 after n-3 PUFA supplementation may be explained, at least in part, by the retroconversion of DHA to EPA. DHA has been also found to antagonize prostanoid TP receptors in platelets and aorta (2).

There are two isoforms of COX, that is, COX-1 and COX-2. COX-1 is constitutively expressed in most cells, whereas COX-2 is induced by stimulation with various growth factors and cytokines such as IL-1b.

IL-1b also induces COX-2 in VSMCs, resulting in the increase in the major arachidonate metabolite, PGI2 (19).

Although the arachidonate metabolite PGE2 is normally considered to be a pro-inflammatory mediator, PGI2 /PGI3 regulates vasoconstriction, platelet aggregation /adhesion, leukocyte adhesion, and VSMC prolifera- tion/migration, sharing these cardioprotective effects with NO. In certain pathophysiological situations, there- fore, COX-2 and iNOS co-expressed in VSMCs may function in concert to potentiate the defensive and compensatory mechanism at the site of vascular injury, particularly in atherosclerosis or thrombosis.

Supporting the increased PGI2/PGI3 production in

Fig. 3. Effects of PUFAs on IL-1b-induced iNOS protein expres- sion in VSMCs. Cells were stimulated with 3 ng / ml IL-1b for 24 h in the absence or the presence of each PUFA as indicated. A: DHA dose-dependently enhanced IL-1b-induced iNOS expression. B: EPA had a less potent effect, whereas arachidonic acid had no effect.

Each PUFA was at 30mM. C: Summary of densitometric analysis expressed as a percentage, taking the control as 100%. Each column represents the mean± S.E.M. of replicate experiments. **P<0.01 vs control. Adopted from Ref. 17 with permission.

Fig. 4. Effect of DHA on p44/42 MAP kinase activation induced by IL-1b in VSMCs. Expression of phosphorylated p44/42 MAP kinase protein (p-p44 / 42) was determined by Western blot analysis.

A: Representative Western blot image. Cells were stimulated with 3 ng / ml IL-1b in the absence or the presence of DHA (30 mM) for the indicated time periods. B: Summary of densitometric analysis for p-p44 / 42 MAP kinase, expressed as a percentage, taking the control (IL-1b alone for 10 min) as 100%. Mean ± S.E.M. of 4 separate experiments. *P<0.05 vs control. Adopted from Ref. 17 with permission.

(5)

vivo, it is reported that PGI2 production is enhanced in rat VSMCs cultured with a triglycerol (TG) emulsified form of EPA (EPA-TG, >80 mM) to simulate them under the in vivo condition, whereas DHA-TG has no effect (20). Since EPA-TG did not alter COX-1 and 10% fetal calf serum-induced COX-2 protein expres- sions, they concluded that low levels of lipid peroxides activate COX activities. A more recent study demon- strated that IL-1b-induced PGE2 production and expres- sions of the type IIA secreted phospholipase A2 and COX-2 genes in rat VSMCs were inhibited by preload- ing cells with EPA and DHA (50mM) for 24 h, while these were enhanced with arachidonic acid (21). COX-2 protein expression was not evaluated in this study.

Interestingly, the authors also showed that IL-1b- induced p44/42 MAP kinase phosphorylation is en- hanced by arachidonic acid and slightly enhanced by DHA, but not by EPA. However, in contrast to their study, we have recently observed that when simulta- neously added to the culture medium, DHA (30mM) and, to a lesser extent, EPA potentiated IL-1b-induced COX-2 protein and mRNA expressions in rat VSMCs, while arachidonic acid had no effect (22). DHA had no effect on the COX-1 expression, while DHA also potentiated phorbol ester-induced COX-2 induction.

PD98059, an inhibitor of p44/42 MAP kinase kinase, inhibited IL-1b-induced COX-2 protein expression, and the potentiating effect of DHA on p44/42 MAP kinase activation correlated well with the result on COX-2

expression. We have no clear explanation for these inconsistent results, but these may be due to the experi- mental conditions such as the applied form and concen- tration of n-3 PUFAs, experimental protocol, or stimuli used. Free forms of n-3 PUFAs are susceptible to oxi- dation producing lipid peroxides, which also have a significant effect on the activity of the arachidonate cascade (20).

5. Proliferation, migration, and apoptosis

Human and experimental animal studies have shown that supplementation of n-3 PUFAs or fish oils signifi- cantly inhibit the development of atherosclerotic lesions, via mechanisms independent of their lipid lowering effect (23). Since VSMC proliferation and migration play an important role in the pathogenesis of athero- sclerosis, or postangioplasty restenosis as well as hyper- tension, these mechanisms can be proposed to include the direct effect on VSMC proliferation and migration.

In fact, EPA-TG (>40 mM) and, to a lesser degree, DHA-TG inhibited VSMC proliferation through modu- lating various steps of the signal transduction pathway by PDGF (24). These included the inhibitions of PDGF binding on its receptors and activation of protein kinase C. EPA-TG also suppressed c-fos mRNA expres- sion, one of the immediate early genes, through partly inhibiting c-fos transcription. EPA (20mM), but not DHA, also significantly inhibited basal and angiotensin

Fig. 5. Modulation by n-3 PUFAs of eicosanoid profile in VSMCs as a possible mechanism for their cardiovascular protec- tive effects. Supplementation of fish oils containing n-3 PUFAs may increase the n-3 / n-6 PUFA ratio in the membrane phospholipids, the sum of PGI2 and PGI3, and effectively reduces TXA2 production in vivo. Furthermore, DHA may antagonize prostanoid TP receptors in platelets and blood vessels. PGI synthase, prostaglandin I synthase; TX synthase, thromboxane syn- thase; LPO, lipid peroxides.

(6)

II- and phorbol ester-induced DNA synthesis in rat VSMCs, but did not affect PDGF-stimulated DNA synthesis (25). The authors also showed that EPA exerted specific inhibition of exaggerated growth of VSMCs of SHR through the suppression of transforming growth factor-b mRNA expression and cyclin-depen- dent kinase 2 activity. Thus, EPA seems to have more potent anti-proliferative effect than DHA. However, it has been reported that a low concentration (0.33 – 3.3mM) of EPA and DHA equipotently block the proliferation induced by TXA2, 5-HT, and PDGF (26, 27) in canine VSMCs. When EPA and DHA were combined in the ratio they were present in fish oils, there was a synergistic interaction in the inhibitory effect. Furthermore, these n-3 PUFAs suppressed 5-HT- induced upregulation of 5-HT2 receptor mRNA expres- sion (27). N-3 PUFA pretreatment also inhibited PDGF- induced migration of human (28) and rat A7r5 (10) VSMCs. Thus, n-3 PUFAs have been reported to regu- late VSMC proliferation/migration induced by several mitogens via multiple mechanisms, as summerized in Fig. 6.

VSMC apoptosis plays an important role in cardio- vascular diseases by influencing vascular remodeling, restenosis or plaque rupture, although the precise role still remains unclear (29). DHA (40mM) has been demonstrated to induce apoptosis through translocation of plasma membrane phosphatidylserine and disruption of mitochondrial transmembrane potential, followed by

increased bax expression and caspase 3 activation in rat VSMCs (30). DHA also induced apoptosis through more than 2 distinct mechanisms: p38 MAP kinase-dependent pathways that regulate peroxisome proliferator-activated receptor-a and p38 MAP kinase-independent pathways via dissipation of mitochondrial transmembrane poten- tial and cytochrome c release (31). These studies suggest that by triggering VSMC apoptosis, DHA may play a pathophysiological role in cardiovascular diseases. N-3 PUFAs may also modulate VSMC proliferation, migra- tion, or apoptosis indirectly by modulating eicosanoid and NO production at the site of vascular injury (Fig. 6).

6. Mechanisms of cellular actions

PUFAs are major components of membrane phospho- lipids and play a key role in membrane functions.

When added to the culture medium, n-3 PUFAs can be easily incorporated into the membrane phospholipids or triglycerides and increase membrane cholesterol efflux in VSMCs (32). It is reported that DHA has a greater effect than EPA in increasing membrane fluidity of endothelial cells cultured from rat thoracic aorta (33).

The greater effect of DHA was considered to be due to its greater ability to decrease membrane cholesterol content and/or the cholesterol/phospholipid molar ratio and also to its greater ability to elevate the unsaturated index in the plasma membrane. These physicochemical alterations in the membrane properties may directly or

Fig. 6. Anti-proliferative and pro-apoptotic effects of n-3 PUFAs on VSMCs as a possible mechanism for their anti-atherogenic effect. EPA and DHA have been reported to regulate VSMC proliferation / migration induced by several mitogens. These n-3 PUFAs, DHA in particular, can also cause apoptosis via multiple mechanisms. Indirect autocrine effects via the modu- lation of PGI2/PGI3 and NO production may also contribute to the anti-atherogenic mechanism. PKC, protein kinase C; TGF-b, transforming growth factor-b;

Cdk2, cyclin-dependent kinase 2; PS, phosphatidyl- serine; DYm, mitochondrial transmembrane potential;

PPAR-a, peroxisome proliferator-activated receptor-a.

(7)

indirectly influence functions of membrane-bound pro- teins such as receptors, GTP binding proteins, ion channels, and various enzymes. Since signaling events such as the MAP kinase cascade is initiated also at the plasma membrane and modified by the membrane lipid microenvironment, the alteration in the membrane properties may affect downstream signaling pathways after receptor stimulation or expression of ion channel proteins.

In addition, ion channels seem to be modulated by n-3 PUFAs by another mechanism that does not require their incorporation into the membrane lipid pools.

The inhibitory effects of acutely applied n-3 PUFAs on receptor-mediated non-selective cation current in agonist-stimulated rat A7r5 VSMC are acutely irrevers- ible after washing out by a buffer containing albumin, suggesting that incorporation of n-3 PUFA is not required for the ion channel inhibition (9). The authors suggested that the primary site of the acute inhibitory action of n-3 PUFAs is the channel protein itself or some site near the channels. Interestingly, a single point mutation in the a subunit of human myocardial Na+ channel transiently expressed in human embryonic kidney 293 cells significantly decreased the inhibitory effect of n-3 PUFAs on the voltage-gated Na+ currents, which may play a significant role in the antiarrythmic actions of n-3 PUFAs (34). Although this interactive or binding site is sensitive not only to n-3 PUFAs, but also to monounsaturated and saturated fatty acids, such an approach may provide a new insight into the mecha- nism through which n-3 PUFAs modulate ion channel activity.

It is worth noting that an effective concentration (30mM) of DHA or EPA in VSMCs isolated from WKY had no significant effects on Ca2+ influx and iNOS/COX-2 expression in VSMCs isolated from SHRSP (Ref. 12 and M. Hirafuji et al., unpublished data). The reason why VSMCs of SHRSP are resistant to these n-3 PUFAs is unclear. It may be related to the alterations in uptake, intracellular transport, metabolism, or composition of PUFA, or in function of target molecules in the plasma membrane of VSMCs derived from SHRSP. Regardless of the mechanisms responsible for the cellular effects of n-3 PUFAs seen in VSMCs of normotensive WKY, it remains to be clarified why VSMCs of SHRSP are refractory to n-3 PUFAs.

There are some differences in the cellular effects of EPA and DHA in VSMCs, partly due to the differences in chain length and number of double bonds. Differential effects of EPA and DHA have been also shown not only in blood pressure and vascular reactivity, but also in heart rate and lipid/lipoprotein metabolism in humans (35). However, it is unclear whether these differences

have relevance to the different potency between DHA and EPA in modulating VSMC functions. Conflicting results concerning the potency of EPA and DHA may be partly due to the difference in the form applied to the cells, that is, free acid form, esterified form, albumin- bound form, or TG-emulsified form.

7. Conclusion

There are accumulating evidences that support the modulation of VSMC functions as one of the possible mechanisms underlying the cardiovascular protective effects of DHA and EPA. These n-3 PUFAs activate K+ATP channels and inhibit certain types of Ca2+ chan- nels, probably via at least 2 distinct mechanisms. The suppression of VSMC excitability may partly explain the reduction of vascular response and endothelium- independent vasorelaxant effects of n-3 PUFAs. The effects on ion channel activities by n-3 PUFAs may further affect the subsequent signaling cascade, resulting in the modulation of other cellular functions such as proliferation or migration. N-3 PUFAs alter the

Fig. 7. Modulation by EPA and DHA (n-3 PUFAs) of VSMC functions as a possible cellular mechanism for their cardiovascular protective effects. These multiple and diverse cellular effects may lead to improved vasoreactivity, anti-thrombogenic and anti-athero- genic effects, and maintained vascular integrity. All these properties may contribute to the beneficial effects of n-3 PUFAs rich in fish oils on several cardiovascular diseases.

(8)

eicosanoid profile by decreasing TXA2 but also by enhancing PGI2/PGI3 production. N-3 PUFAs also modulate IL-1b-induced expression of iNOS and COX- 2 via a mechanism involving the p44/42 MAP kinase signaling cascade. Since these enzymes synthesize important mediators, NO and eicosanoids, respectively, these effects may have particular relevance to the pro- tective effect against local vascular injury such as atherosclerosis or thrombosis. N-3 PUFAs also modu- late VSMC proliferation/migration or apoptosis directly or indirectly through the autocrine effect of these media- tors at the site of vascular injury. As illustrated in Fig. 7, all these cellular effects seem to be favorable to the beneficial effects of n-3 PUFAs on cardiovascular diseases. Nevertheless, many questions still remain to be answered as to whether the effects may be exerted merely through the physicochemical changes in the membrane properties, through interacting with specific target proteins in the plasma membrane, or through interacting with other functional cytoplasmic or nuclear molecules.

Recent results strongly suggest that DHA has more potent and beneficial effect on cardiovascular diseases than EPA. DHA has been also reported to prevent and be effective for treating senile dementia, depression, certain visual dysfunction, arthritis, diabetes mellitus, and some cancers, some of which EPA is not effective for. It should be noted that DHA, a natural occurring product, has so many diverse beneficial effects without clinically significant adverse effects, when given at the recommended dose. The progress in studies for the precise cellular and molecular mechanism of DHA may provide useful knowledge for the development of promising new and safe drugs.

Acknowledgments

This work was supported in part by Grants-in-Aid for Science Research from Japan Society for the Promotion of Science; the Academic Science Frontier Project of the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and the Akiyama Foundation.

References

1 Horrocks LA, Yeo YK. Health benefits of docosahexaenoic acid (DHA). Pharmacol Res. 1999;40:211–225.

2 Abeywardena MY, Head RJ. Longchain n-3 polyunsaturated fatty acids and blood vessel function. Cardiovasc Res. 2001;

52:361–371.

3 Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation.

2002;106:2747–2757.

4 Mori TA, Watts GF, Burke V, Hilme E, Puddey IB, Beilin LJ.

Differential effects of eicosapentaenoic acid and docosa- hexaenoic acid on vascular reactivity of the forearm micro- circulation in hyperlipidemic, overweight men. Circulation.

2000;102:1264–1269.

5 Engler MB, Engler MM, Browne A, Sun Y-P, Sievers R.

Mechanisms of vasorelaxation induced by eicosapentaenoic acid (20:5n-3) in WKY rat aorta. Br J Pharmacol. 2000;131:1793–

1799.

6 McLennan P, Howe P, Abeywardena M, et al. The cardio- vascular protective role of docosahexaenoic acid. Eur J Pharma- col. 1996;300:83–89.

7 Mori TA, Bao, DQ, Burke V, Puddey IB, Beilin LJ. Docosa- hexaenoic acid but not eicosapentaenoic acid lowers ambulatory blood pressure and heart rate in humans. Hypertension. 1999;

34:253–260.

8 Engler MB, Engler MM. Docosahexaenoic acid-induced vaso- relaxation in hypertensive rats: mechanisms of action. Biol Res Nurs. 2000;2:85–95.

9 Asano M, Nakajima T, Iwasawa K, et al. Inhibitory effects of w-3 polyunsaturated fatty acids on receptor-mediated non- selective cation currents in rat A7r5 vascular smooth muscle cells. Br J Pharmacol. 1997;120:1367–1375.

10 Asano M, Nakajima T, Hazama H, et al. Influence of cellular incorporation of n-3 eicosapentaenoic acid on intracellular Ca2+ concentration and membrane potential in vascular smooth muscle cells. Atherosclerosis. 1998;138:117–127.

11 Engler MB, MA YH, Engler MM. Calcium-mediated mecha- nisms of eicosapentaenoic acid-induced relaxation in hyper- tensive rat aorta. Am J Hypertens. 1999;12:1225–1235.

12 Hirafuji M, Ebihara T, Kawahara F, et al. Effect of docosa- hexaenoic acid on intracellular calcium dynamics in vascular smooth muscle cells from normotensive and genetically hyper- tensive rats. Res Commun Mol Pathol Pharmacol. 1998;102:29–

42.

13 Hirafuji M, Ebihara T, Kawahara F, Hamaue N, Endo T, Minami M. Inhibition by docosahexaenoic acid of receptor-mediated Ca2+ influx in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine. Eur J Pharmacol. 2001;427:195–201.

14 Dusting GJ. Nitric oxide in cardiovascular disorders. J Vasc Res. 1995;32:143–161.

15 Omura M, Kobayashi S, Mizutani Y, et al. Eicosapentaenoic acid (EPA) induces Ca2+-independent activation and trans- location of endothelial nitric oxide synthase and endothelium- dependent vasorelaxation. FEBS Lett. 2001;487:361–366.

16 Schini VB, Durante W, Catovsky S, Vanhoutte PM. Eicosapen- taenoic acid potentiates the production of nitric oxide evoked by interleukin-1b in cultured vascular smooth muscle cells.

J Vasc Res. 1993;30:209–217.

17 Hirafuji M, Machida T, Tsunoda M, Miyamoto A, Minami M.

Docosahexaenoic acid potentiates interleukin-1b induction of nitric oxide synthase through mechanism involving p44 / 42 MAPK activation in rat vascular smooth muscle cells. Br J Pharmacol. 2002;136:613–619.

18 Chen AF, Ren J, Miao C-Y. Nitric oxide synthase gene therapy for cardiovascular disease. Jpn J Pharmacol. 2002;89:327–336.

19 Pritchard KA Jr, O’Banion MK, Miano JM, et al. Induction of cyclooxygenase-2 in rat vascular smooth muscle cells in vitro and in vivo. J Biol Chem. 1994;269:8504–8509.

20 Saito J, Terano T, Hirai A, Shiina T, Tamura Y, Saito Y. Mecha- nisms of enhanced production of PGI2 in cultured rat vascular

(9)

smooth muscle cells enriched with eicosapentaenoic acid.

Atherosclerosis. 1997;131:219–228.

21 Bousserouel S, Brouillet A, Béréziat G, Raymondjean M, Andréani M. Different effects of n-6 and n-3 polyunsaturated fatty acids on the activation of rat smooth muscle cells by inter- leukin-1b. J Lipid Res. 2003;44:601–611.

22 Machida T, Hirafuji M, Sakai M, et al. Docosahexaenoic acid potentiates IL-1b-induced COX-2 expression via p44/42 MAPK activation in rat vascular smooth muscle cells (Abstract).

J Pharmacol Sci. 2003;91 Suppl I:114P.

23 De Caterina R, Zampolli A. N-3 fatty acids: antiatherosclerotic effects. Lipids. 2001;36:S69–S78.

24 Terano T, Shiina T, Tamura Y. Eicosapentaenoic acid sup- pressed the proliferation of vascular smooth muscle cells through modulation of various steps of growth signals. Lipids.

1996;31:S-301–S-304.

25 Nakayama M, Fukuda N, Watanabe Y, et al. Low dose of eicos- apentaenoic acid inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats through suppression of transforming growth factor-b. J Hyper- tens. 1999;17:1421–1430.

26 Pakala R, Pakala R, Benedict CR. Thromboxane A2 fails to induce proliferation of smooth muscle cells enriched with eicosapentaenoic acid and docosahexaenoic acid. Prostaglandins Leukotr Essent Fatty Acids. 1999;60:275–281.

27 Pakala R, Pakala R, Sheng WL, Benedict CR. Vascular smooth muscle cells preloaded with eicosapentaenoic acid and docosa- hexaenoic acid fail to respond to serotonin stimulation. Athero-

sclerosis. 2000;153:47–57.

28 Mizutani M, Asano M, Roy S, et al. w-3 Polyunsaturated fatty acids inhibit migration of human vascular smooth muscle cells in vitro. Life Sci. 1997;61:PL269–PL274.

29 Walsh K, Smith RC, Kim H-S. Vascular cell apoptosis in remodeling, restenosis, and plaque ruptures. Circ Res. 2000;87:

184–188.

30 Diep QN, Intengan HD, Schiffrin EL. Endothelin-1 attenuates w3 fatty acid-induced apoptosis by inhibition of caspase 3.

Hypertension. 2000;35:287–291.

31 Diep QN, Touyz RM, Schiffrin EL. Docosahexaenoic acid, a peroxisome proliferator-activated receptor-a ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase. Hypertension. 2000;36:

851–855.

32 Dusserre E, Pulcini T, Bourdillon MC, Ciavatti M, Berthezene F. w-3 Fatty acids in smooth muscle cell phospholipids increase membrane cholesterol efflux. Lipids. 1995;30:35–41.

33 Hashimoto M, Hossain S, Yamasaki H, Yazawa K, Masumura S.

Effects of eicosapentaenoic acid and docosahexaenoic acid on plasma membrane fluidity of aortic endothelial cells. Lipids.

1999;34:1297–1304.

34 Xiao Y-F, Ke Q, Wang S-Y, et al. Single point mutations affect fatty acid block of human myocardial sodium channel a subunit Na+ channels. Proc Natl Acad Sci USA. 2001;98:3606–3611.

35 Mori TA, Beilin LJ. Long-chain omega 3 fatty acids, blood lipids and cardiovascular risk reduction. Curr Opin Lipidol.

2001;12:11–17.

References

Related documents

According to the New York State Education Department, the five boroughs of New York City have over 2,100 schools that serve 1.1 million students from pre-kindergarten to high school,

We invite unpublished novel, original, empirical and high quality research work pertaining to recent developments &amp; practices in the areas of Computer Science &amp;

Yeats, of course, wrote political poems which are not poems of witness: celebrations of heroism like &#34;Easter, 1916,&#34; polemical poems like &#34;September, 1913,&#34;

framework for analyzing disclosure requirements in Milavetz , 559 U.S. There attorneys brought a First Amendment challenge to a requirement that professionals assisting

Perhaps some of the most important achieve- ments of TCR can be seen in consumer and marketing jour- nals where consumer welfare issues have become more prom- inent and the results

It addresses a knowledge gap that constrains service provision for a growing market of older people and which underestimates older people’s potential contribution in the early phases

An overall feasibility study may be conducted to establish the business reasons for proceeding, followed by a comparison of alternative technical solutions from

By providing more light to the driver of all vehicle types and improving the sign legibility for disadvantaged signs, 3M’s DG 3 sheeting is seen as a breakthrough in improving the