Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

Effect of hyperlipidemic serum on lipid peroxidation, synthesis of prostacyclin and thromboxane by cultured endothelial cells: protective effect of antioxidants

An increased lipid peroxides and a decreased production of prostacyclin have been shown in advanced atherosclerotic lesions and plasma. Our purpose was to determine whether the similar findings could be observed in cultured endothelial cells, and whether antioxidants could protect the cell against peroxide injury. In these experiments we have used bovine aortic endothelial cells in culture to address the issue of hyperlipidemia-induced arterial damage. Results of the present study showed that different concentration of hyperlipidemic sera from atherogenic rabbits induced a time- and dose-dependent alteration in the production of prostacyclin and levels of lipid peroxides in endothelial cells. Endothelial cells incubated with hyperlipidemic serum increased prostacyclin generation significantly during the initial stages and then continuously decreased. When endothelial cells were incubated for 36 h, TXA2 generation was also impaired and at the same time the cellular lipid peroxides content increased. There was a positive correlation between the concentration of hyperlipidemic serum and lipid peroxides and an inverse correlation with prostacyclin synthesis. The medium supplemented with antioxidant selenium or vitamin E showed a significant decrease in lipid peroxides and an increase in prostacyclin synthesis. These results suggest that both hyperlipidemic serum and lipid peroxides injury endothelial cells and inactivate prostacyclin synthetase, resulting in a decrease of prostacyclin production, while antioxidants have a protective effect. We conclude that the increase in lipid peroxides in association with hyperlipidemia results in alteration of prostacyclin synthesis that may play an important role in the pathogenesis of atherosclerosis.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

Induction of antioxidant stress proteins in vascular endothelial and smooth muscle cells: protective action of vitamin C against atherogenic lipoproteins.

Elevated levels of lipid peroxidation and increased formation of reactive oxygen species within the vascular wall in atherosclerosis can overwhelm cellular antioxidant defence mechanisms. Accumulating evidence implicates oxidatively modified low density lipoproteins (LDL) in vascular dysfunction in atherosclerosis and oxidized LDL have been localized with in atherosclerotic lesions. We here report that human oxidatively modified LDL induce expression of 'antioxidant-like' stress proteins in vascular cells, involving increases in the activity of L-cystine transport, glutathione synthesis, heme oxygenase-1 and the murine stress protein MSP23. Moreover, treatment of human arterial smooth muscle cells with the dietary antioxidant vitamin C markedly attenuates adaptive increases in endogenous antioxidant gene expression and affords protection against smooth muscle cell apoptosis induced by moderately oxidized LDL. As vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque 'necrotic' core, cap rupture and thrombosis, our findings suggest that the cytoprotective actions of vitamin C could limit plaque instability in advanced atherosclerosis.     

Acyl chain-dependent effect of lysophosphatidylcholine on endothelial prostacyclin production

Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI₂) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI₂ production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent ¹⁴C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca²⁺ concentration. In vivo, LPC increased 6-keto PGF_1α concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI₂, whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation.     

The effect on endothelial function of vitamin C during methionine induced hyperhomocysteinaemia

Background

Manipulation of total homocysteine concentration with oral methionine is associated with impairment of endothelial-dependent vasodilation. This may be caused by increased oxidative stress. Vitamin C is an aqueous phase antioxidant vitamin and free radical scavenger. We hypothesised that if the impairment of endothelial function related to experimental hyperhomocysteinaemia was free radically mediated then co-administration of vitamin C should prevent this.

Methods

Ten healthy adults took part in this crossover study. Endothelial function was determined by measuring forearm blood flow (FBF) in response to intra-arterial infusion of acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). Subjects received methionine (100 mg/Kg) plus placebo tablets, methionine plus vitamin C (2 g orally) or placebo drink plus placebo tablets. Study drugs were administered at 9 am on each study date, a minimum of two weeks passed between each study. Homocysteine (tHcy) concentration was determined at baseline and after 4 hours. Endothelial function was determined at 4 hours. Responses to the vasoactive substances are expressed as the area under the curve of change in FBF from baseline. Data are mean plus 95% Confidence Intervals.

Results

Following oral methionine tHcy concentration increased significantly versus placebo. At this time endothelial-dependent responses were significantly reduced compared to placebo (31.2 units [22.1-40.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo). Endothelial-independent responses were unchanged. Co-administration of vitamin C did not alter the increase in homocysteine or prevent the impairment of endothelial-dependent responses (31.4 [19.5-43.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo)

Conclusions

This study demonstrates that methionine increased tHcy with impairment of the endothelial-dependent vasomotor responses. Administration of vitamin C did not prevent this impairment and our results do not support the hypothesis that the endothelial impairment is mediated by adverse oxidative stress.     

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2‐min) half‐life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX‐1‐10aa‐PGIS, which links human cyclooxygenase‐1 (COX‐1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2‐secreting strains (PGI2‐EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT‐PCR, and Western blot analyses confirmed the overexpression of COX‐1‐10aa‐PGIS in PGI2‐EPCs. PGI2‐EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2‐EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell‐ and paracrine‐mediated in vitro angiogenesis was attenuated when COX‐1‐10aa‐PGIS protein expression was knocked down. Whole‐cell patch‐clamp studies showed that 4‐aminopyridine‐sensitive K⁺ current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2‐EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2‐EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine‐mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia. J. Cell. Physiol. 227: 2907–2916, 2012. © 2011 Wiley Periodicals, Inc.     

Recycling of vitamin C from its oxidized forms by human endothelial cells

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.     

Studies on the effect of diadenlyated nucleotides on calcium mobilization and prostacyclin synthesis in bovine aortic endothelial cells

Extracellular adenine dinucleotides are modulators of blood vessel tone. We have previously demonstrated that Ap(2)A and Ap(4)A induce the synthesis of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) while Ap(3)A and Ap(5)A do not [FEBS Lett. 427 (1998) 320; Arch. Biochem. Biophys. 364 (1999) 280.]. In this communication we determine the effect of Ap(x)As (x=2-5) on prostacyclin (PGI(2)) synthesis and Ca(2+) mobilization in BAEC. Ap(2)A and Ap(4)A significantly enhanced the synthesis of PGI(2) while Ap(3)A and Ap(5)A do not. These data support the notion that Ap(2)A and Ap(4)A are vasodilators. All four dinucleotides significantly enhanced Ca(2+) mobilization over basal levels. Ap(5)A and Ap(3)A enhanced 2.0 and 1.6 times more Ca(2+) release than Ap(4)A, respectively. Since neither Ap(5)A nor Ap(3)A enhanced the synthesis of either PGI(2) or NO but did mobilize Ca(2+), these data support the hypothesis that in BAEC Ca(2+) release is localized or compartmentalized.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.     

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.     

Shear stress enhances prostacyclin release from endocardial endothelial cells

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.     

Stimulatory effect of vitamin C on autophagy in glial cells

Intracellular accumulation of damaged or abnormal proteins is a common event associated with numerous neurodegenerative diseases and other age-related pathologies. Increasing the activity of the intracellular proteolytic systems normally responsible for the removal of these abnormal proteins might be beneficial in lessening the severity or development of those pathologies. In this study we have used human astrocyte glial cells to investigate the effect of vitamin C (ascorbate) on the intracellular turnover of proteins. Supplementation of the culture medium with physiological concentrations of vitamin C did not affect protein synthesis, but did increase the rate of protein degradation by lysosomes. Vitamin C accelerated the degradation of intra- and extracellular proteins targeted to the lysosomal lumen by autophagic and heterophagic pathways. At the doses analyzed, vitamin C lowered and stabilized the acidic intralysosomal pH at values that result in maximum activation of the lysosomal hydrolases.