Eicosapentaenoic acid abolishes the proatherogenic effects of cholesterol: effects on migration of bovine smooth muscle and endothelial cells in vitro.

It is well known that vascular endothelial cell (EC) migration plays a major role in regeneration of the injured endothelium and also that smooth muscle cell (SMC) migration is the important step for atheromatous plaque formation. In the present study, we investigated the effects of cholesterol and eicosapentaenoic acid (EPA) on bovine carotid artery EC and SMC migration using the modified Boyden chamber technique. The migration activity of the cholesterol-enriched ECs loaded with cholesterol-rich liposomes was significantly suppressed, whereas that of the cholesterol-enriched SMCs was enhanced. Next, we examined the effects of EPA pretreatment on the migration of both cell types. When ECs and SMCs were treated with EPA (5 micrograms/ml) for 2 days, the EPA content increased from 0.55 +/- 0.04% to 11.72 +/- 0.19% and 1.22 +/- 0.09% to 9.69 +/- 0.07% in cellular phospholipids, respectively. Although pretreatment of the ECs with EPA caused a significant increase in serum-induced cell migration, pretreatment of SMCs had no effect. If both cell types were concomitantly pretreated with EPA and cholesterol-rich liposomes, EPA abolished the effects of cholesterol on the migration of both cell types, but did not affect the content of cholesterol in both cells. These data indicate the possibility that EPA counteracts the atherogenic effect of cholesterol on EC and SMC migration.

An oxidative mechanism is involved in high glucose-induced serum protein modification causing inhibition of endothelial cell proliferation.

In order to investigate the role of hyperglycemia on the development of atherosclerosis in diabetics, the effects of high glucose-induced modification of serum factors on the proliferation of bovine carotid artery endothelial cells were studied. Dialysates of high glucose-treated serum inhibit cell growth in a time- and glucose concentration-dependent manner. With 6 weeks of pretreatment, 16.7 mM glucose causes a 47.2% inhibition in cell growth compared to 5.6 mM glucose (P < 0.001). Pretreatment of serum in the presence of reduced glutathione (0.5-1.0 mM), an antioxidant, significantly prevents the high glucose-induced inhibition without inhibiting the formation of early non-enzymatic glycosylation products. Dithiothreitol (7.5 mM) treatment after preincubation with glucose fully restores the glucose-induced inhibition. When the dialysates are fractionated according to molecular mass, the high glucose-induced inhibition is maximal in the MW fraction above 100 kDa. These data suggest that high glucose conditions facilitate the susceptibility of serum proteins to sulfhydryl oxidation forming disulfide crosslinks and this oxidative process may contribute to the inhibition of endothelial cell proliferation.

Stimulatory effects of insulin and insulin-like growth factor I on migration and tube formation by vascular endothelial cells.

The effects of insulin and insulin-like growth factor I (IGF-I) on migration, proliferation and tube-forming activity of endothelial cells were investigated, by using bovine carotid artery endothelial cells. Migration was assayed by a filter membrane technique and tube formation was assayed by a quantitative angiogenesis in vitro model which we have recently developed. In this model, endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures which mimic capillaries in vivo ultrastructurally. Insulin (50-1000 microunits/ml) and IGF-I (10-200 ng/ml) significantly stimulated migration of endothelial cells in a dose-dependent manner with a maximal stimulation of 3.0-fold at 1000 microunits/ml for insulin and 3.8-fold at 200 ng/ml for IGF-I (P less than 0.01). Insulin at concentrations up to 1000 microunits/ml and IGF-I up to 100 ng/ml did not affect proliferation of endothelial cells. When insulin or IGF-I was added in culture medium on collagen gels, tube-forming activity of endothelial cells was markedly stimulated. The specific lengths of tubes significantly increased with the increase in insulin concentration from 25 to 100 microunits/ml (P less than 0.01). At 100 microunits/ml, the stimulation was 1.77-fold (P less than 0.01). IGF-I (1-100 ng/ml) also stimulated the elongation of tubes dose-dependently with a maximal stimulation of 1.96-fold at 100 ng/ml (P less than 0.01). Thus, insulin and IGF-I at pathophysiological concentrations stimulate migration and tube-forming activity of endothelial cells, suggesting that these polypeptides may stimulate repair of endothelial injury in cases such as atherosclerosis and may act as a stimulator of angiogenesis.

Eicosapentaenoic acid inhibits tube formation of vascular endothelial cells in vitro.

We previously have described a quantitative angiogenesis in vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20:5n-3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 micrograms/mL of arachidonic acid (20:4n-6), eicosapentaenoic acid or docosahexaenoic acid (22:6n-3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The context of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cell was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 micrograms/mL of eicosapentaenoic acid. At 5.0 micrograms/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition of in vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (less than 5.0 micrograms/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 micrograms/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells,at least in part, via its inhibitory effect on cellular proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of migration in bovine endothelial cells by eicosapentaenoic acid pretreatment.

It has been recognized that endothelial cell migration is an important process in the regeneration of injury in blood vessels. In this study, we examined the effects of polyunsaturated fatty acids on the migration of cultured endothelial cells using a modified Boyden chamber. When endothelial cells isolated from bovine carotid artery were pretreated for 2 days with 5 micrograms/ml of either arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid, every polyunsaturated fatty acid was incorporated substantially into cellular phospholipids. The content of arachidonic acid increased from 9.27 to 23.9% by the arachidonic acid pretreatment, and that of eicosapentaenoic acid and docosahexaenoic acid increased from 9.57 to 11.85% by the eicosapentaenoic acid pretreatment and from 5.56 to 18.40% by the docosahexaenoic acid pretreatment, respectively. Pretreatment of the cells with 0.5-5.0 micrograms/ml of eicosapentaenoic acid resulted in a dose-dependent increase in endothelial migration in response to fetal bovine serum. In contrast, pretreatment of the cells with arachidonic acid and docosahexaenoic acid had no effects on the cell migration. If eicosapentaenoic acid, however, was added directly to the migration assay system instead of the pretreatment, it did not show a profile of chemotactic factor. The eicosapentaenoic acid pretreatment also potentiated cell migration activity in response to several other chemotactic factors such as basic fibroblast growth factor, tumor necrosis factor-alpha and leukotriene C4. The effect of eicosapentaenoic acid on porcine smooth muscle cell migration was also examined. Although eicosapentaenoic acid was similarly incorporated into cellular phospholipids of smooth muscle cells by the pretreatment for 2 days, no stimulative effect was observed in the migration of smooth muscle cells at any doses (0.5-5.0 microns/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro.

In order to investigate the mechanism of angiogenesis involved in inflammatory processes, the effects of leukotrienes and prostaglandin E2 on in vitro tube formation of cultured vascular endothelial cells were examined. Endothelial cells from bovine carotid artery were cultured for 4 days between two layers of collagen gel and the lengths of organized tubes were quantitatively estimated with an image analyzer. Treatment with 10(-8)-10(-6)M of prostaglandin E2 increased the tubular lengths, and leukotriene C4 stimulated tube formation at far lower concentrations (10(-15)-10(-9)M) but leukotriene B4 and D4 were not effective on the tube formation. It was also found that endothelial cell migration was stimulated by almost the same concentrations of leukotriene C4 as those stimulating tube formation. These data suggest that leukotriene C4 is, at least, one of the important factors involved in angiogenesis during inflammatory processes.