Docosahexaenoic acid, but not eicosapentaenoic acid, improves septic shock-induced arterial dysfunction in rats.

INTRODUCTION: Long chain n-3 fatty acid supplementation may modulate septic shock-induced host response to pathogen-induced sepsis. The composition of lipid emulsions for parenteral nutrition however remains a real challenge in intensive care, depending on their fatty acid content. Because they have not been assessed yet, we aimed at determining the respective effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) during septic shock-induced vascular dysfunction. METHODS: In a peritonitis-induced septic shock model, rats were infused with EPA, DHA, an EPA/DHA mixture or 5% dextrose (D5) during 22 hours. From H18, rats were resuscitated and monitored during 4 hours. At H22, plasma, aorta and mesenteric resistance arteries were collected to perform ex vivo experiments. RESULTS: We have shown that septic rats needed an active resuscitation with fluid challenge and norepinephrine treatment, while SHAM rats did not. In septic rats, norepinephrine requirements were significantly decreased in DHA and EPA/DHA groups (10.6±12.0 and 3.7±8.0 µg/kg/min respectively versus 17.4±19.3 µg/kg/min in D5 group, p<0.05) and DHA infusion significantly improved contractile response to phenylephrine through nitric oxide pathway inhibition. DHA moreover significantly reduced vascular oxidative stress and nitric oxide production, phosphorylated IκB expression and vasodilative prostaglandin production. DHA also significantly decreased polyunsaturated fatty acid pro-inflammatory mediators and significantly increased several anti-inflammatory metabolites. CONCLUSIONS: DHA infusion in septic rats improved hemodynamic dysfunction through decreased vascular oxidative stress and inflammation, while EPA infusion did not have beneficial effects.

Effect of Xianziyizhen Recipe Capsule on PGI2-PPARδ Signaling Pathway in Embryo Implantation Dysfunction Mice.

PROBLEM: We investigated the effect of Xianziyizhen recipe capsule (XRC), a kidney-tonifying herb, on the PGI2-PPARδ signaling pathway at the maternal-fetal interface in embryo implantation dysfunction (EID) mice. METHOD OF STUDY: Intragastric administration of Progynova (estradiol) or XRC was performed in EID mouse model, following experimental induction of kidney deficiency by co-treatment with chemotherapy drug hydroxyurea and antiprogesterone mifepristone. The PPARδ and IL-11 mRNA expression in endometrium were detected by real-time relative reverse transcription-polymerase chain reaction (RT-PCR). Further, the protein expression of COX-2, PGI2, MMP-9, and TIMP-3 was detected in endometrial glandular epithelium and in stromal cells by immunohistochemical (IHC) assay. RESULTS: The results showed that hydroxyurea and mifepristone-induced EID were associated with significantly lower PPARδ and IL-11 mRNA levels in endometrium and reduced COX-2, PGI2, MMP-9, and TIMP-3 levels in endometrial glandular epithelium, compared with normal controls. However, XRC and Progynova treatment reversed these effects, leading to significant increases in PPARδ and IL-11 mRNA expression, and COX-2, PGI2, MMP-9 and TIMP-3 protein levels, when compared with the levels observed in EID mice. CONCLUSION: These results strongly suggested that XRC is beneficial in EID treatment and that XRC may mediate its effects through regulation of the PGI2-PPARδ signaling pathway.

Potassium channel openers and prostacyclin play a crucial role in mediating the vasorelaxant activity of Gynura procumbens.

BACKGROUND: Previous studies of Gynura procumbens (G. procumbens) have shown that partially purified fractions of the leaves are capable of lowering the blood pressure of rats by inhibiting angiotensin-converting enzymic activity and causing vasodilatation. The objectives of this study were therefore to further purify the active compounds that exhibited selective effects on blood vessels, determine the mechanism of actions, and to qualitatively analyse the putative compounds present. METHODS: The butanolic fraction (BU) of the crude ethanolic extract was purified using column chromatography to obtain several sub-fractions of different polarities. The in vitro effects of BU and the sub-fractions on vascular tension were subsequently determined using isolated rat thoracic aortic rings. The most potent sub-fraction (F1) alone was then investigated for its mechanisms of the vasorelaxant activity. In another experiment, thin-layer chromatography was used to qualitatively analyse the active compounds found in F1. RESULTS: The BU and the sub-fractions ranging from 10-7 to 10-2 g/ml significantly (p < 0.05) inhibited the sustained tonic contractions induced by phenylephrine and potassium chloride in a concentration-dependent manner with various degree of potency. The most potent sub-fraction (F1) antagonised the calcium-induced vasocontractions (1 x 10-4 - 1 x 10-2 M) in calcium-free with high concentration of potassium as well as in calcium- and potassium-free Krebs-Henseleit solutions. Contractions induced by noradrenaline and caffeine were not affected by F1. The vasorelaxing effect caused by F1 was significantly attenuated with preincubation of potassium channel blockers (glibenclamide and 4-aminopyridine) and prostacyclin inhibitor (indomethacin) while it was not affected by preincubation with tetraethylammonium, l-nitro-arginine methyl esther, propanolol, atropine, oxadiazolo quinoxalin one and methylene blue. The qualitative phytochemical analysis of F1 indicated the presence of flavonoids. CONCLUSION: These results confirm previous findings that G. procumbens causes vasodilatory effects by blocking calcium channels. In addition, the present study further demonstrates that the vasodilatory effect of G. procumbens may also be due to the opening of potassium channels and the stimulation of prostacyclin production. The putative compounds are probably flavonoids in nature.

L-Carnitine prevents the development of ventricular fibrosis and heart failure with preserved ejection fraction in hypertensive heart disease.

OBJECTIVES: Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis. METHODS AND RESULTS: Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients. CONCLUSIONS: L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF.

Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages.

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.

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.

Regulation of the expression of cyclooxygenases and production of prostaglandin I2 and E2 in human coronary artery endothelial cells by curcumin.

Curcumin regulates prostaglandin (PG) synthesis in a variety of cells. PGE2 and PGI2 are generated from arachidonic acid (AA) by cyclooxygenases 1 and 2 (COX-1 and COX-2) and the synthase (PGES and PGI2S) pathways. This study evaluates the in vitro effect of curcumin on the expression of COX-1, COX-2, PGI2S and microsomal PGES-1 (mPGES-1), and the production of PGE2 and PGI2 in human coronary artery endothelial cells (HCAEC). HCAEC monolayers were incubated with curcumin and the expression of mRNA, protein and the production of PGI2 and PGE2 were quantified. Incubation of HCAEC with curcumin led to a time and concentration-dependent increases in COX-2 mRNA with a small but significant decrease in COX-1 mRNA expression. Curcumin also stimulated the expression of PGI2S and mPGES-1 mRNA. Although curcumin stimulated COX-2, PGI2S and mPGES-1 gene expression, it failed to increase PGI2 or PGE2 production. Interestingly, supplementation of the culture medium with AA increased prostanoid production by both quiescent and curcumin-treated cells. However, in comparison to the quiescent cells, the prostanoid production by curcumin-treated cells was markedly enhanced as AA concentrations in the medium were increased, and the enhanced prostanoid production was blocked by the presence of COX-2 specific inhibitor. Taken together, these results suggest that curcumin regulates prostanoid homeostasis in HCAEC by modulating multiple steps including the expression of COX-1, COX-2, PGI2S and mPGES-1.

Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.

BACKGROUND: Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than cyclooxygenase-2-selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown. METHODS AND RESULTS: Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were significantly reduced 4 weeks after injury in mPGES-1 knockout mice compared with wild-type controls (65.6 ± 5.7 versus 37.7 ± 5.1 × 10³ pixel area and 70.5 ± 13.4% versus 47.7 ± 17.4%, respectively; P < 0.01). Induction of tenascin-C, a proproliferative and promigratory extracellular matrix protein, after injury was attenuated in the knockouts. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1-deleted vascular smooth muscle cells generated less PGE2 but more PGI2 and expressed reduced tenascin-C compared with wild-type cells. Both suppression of PGE2 and augmentation of PGI2 attenuate tenascin-C expression and vascular smooth muscle cell proliferation and migration in vitro. CONCLUSIONS: Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating tenascin-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.

Oral bioavailability, efficacy and gastric tolerability of P2026, a novel nitric oxide-releasing diclofenac in rat.

The present study was designed to evaluate, P2026 [(2-((2-(nitrooxy)ethyl)disulfanyl)ethyl 2-(2-(2,6-dichlorophenylamino)phenyl)acetate)], a novel NO (nitric oxide) donor prodrug of diclofenac for its ability to release NO and diclofenac, and whether P2026 provides advantage of improved activity/gastric tolerability over diclofenac. Oral bioavailability of P2026 was estimated from plasma concentration of diclofenac and nitrate/nitrite (NOx). Anti-inflammatory activity was evaluated in three different models of inflammation: acute (carrageenan-induced paw oedema), chronic (adjuvant-induced arthritis), and systemic (lipopolysaccharide-induced endotoxic shock). Gastric tolerability was evaluated from compound's propensity to cause gastric ulcers. P2026 exhibited dose-dependent diclofenac and NOx release. Similar to diclofenac, P2026 showed potent anti-inflammatory activity in acute and chronic model, whereas it improved activity in systemic model. Both diclofenac and P2026 inhibited gastric prostaglandin, but only diclofenac produced dose-dependent haemorrhagic ulcers. Thus, the results suggest that coupling of NO and diclofenac contribute to improved gastric tolerability while retaining the anti-inflammatory properties of diclofenac.

Curcumin prevents the oxidation and lipid modification of LDL and its inhibition of prostacyclin generation by endothelial cells in culture.

Low-density lipoprotein (LDL) was isolated from human plasma and oxidized by 5microM copper sulfate for 4h at 37 degrees C in the absence and presence of 1, 3, 5, 10, or 20microM of curcumin. LDL oxidized in the absence of curcumin (oxLDL) showed an increased levels of conjugated dienes, lipid peroxides (TBARS) and lysolecithin (lysoPC) and a significant loss of polyunsaturated fatty acids (PUFA). LDL oxidized with 5microM copper sulfate in the presence of curcumin caused a significant decrease of conjugated diene, lipid peroxides, lysoPC and significant increase of PUFA compared to oxLDL. These changes were dose dependent and reached a maximum at 5microM curcumin. Incubation of human endothelial cells (EC) with 200microg protein/ml of oxLDL caused a significant decrease of prostacyclin (PGI(2)) generation. LDL oxidized in presence of 5microM curcumin did not show any inhibition of PGI(2) generation compared to the control cells. These results indicate that curcumin is an effective chain-breaking antioxidant which prevents oxidation and lipid modification of LDL. The inhibition of oxLDL on PGI(2) is considered a contributing factor in the pathogenesis of thrombosis and atherosclerosis. Curcumin supplementation could be an effective strategy in preventing LDL oxidation and its impact on atherosclerosis and lesion formation.

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs.

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I(2), thromboxane A(2) and prostaglandin F(2alpha). The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H(2) production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.

High-dose lovastatin decreased basal prostacyclin production in cultured endothelial cells.

The effect of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on prostacyclin production was studied in cultured human umbilical vein endothelial cells. The results indicated that lovastatin induced a significant dose- and time-dependent decrease of arachidonic acid release from the cells, an inhibition of phospholipase A(2) (PLA(2)) activity, a reduction of [(3)H]choline in lysophosphatidylcholine (lysoPC), and a diminishment of (45)Ca(2+) influx into the cells. The decreased arachidonic acid release was not reversed by addition of either intermediate products during cholesterol synthesis, such as mevalonate, geranylgeraniol, farnesol, or cholesterol and lipoprotein although a reduced concentration of cholesterol in the cells, caused by lovastatin, was reversed by added cholesterol. Lovastatin, furthermore, diminished prostacyclin production and inhibited activity of cyclooxygenase-1. 27-Hydroxycholesterol (27OHC), an oxidized cholesterol, had the same effect on HMG-CoA reductase as lovastatin, but 27OHC increased arachidonic acid release and (45)Ca(2+) influx. Our results indicated that lovastatin inhibited significantly activities of PLA(2) and cyclooxygenase-1, resulting in a marked reduction in arachidonic acid release, lysoPC content and prostacyclin production in the cultured vascular endothelial cells.

Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential.

Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI(2)) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI(2) [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield ( approximately 4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells' membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (K(m)=3 microM and K(cat)=100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI(2) than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 microg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI(2), and revealed its potential for anti-thrombosis therapeutics.

The administration of folic acid reduces intravascular oxidative stress in diabetic rabbits.

There is evidence that plasma homocysteine augments angiopathy in patients with diabetes mellitus. Although lowering homocysteine with folic acid improves endothelial function, the precise mechanisms underlying this effect are unknown. To study this area further, the effect of administration of folic acid to diabetic rabbits on intraaortic oxidative stress was studied by assessing the formation of superoxide (O(2)(-)), 8-isoprostane F(2alpha) (8-IPF(2alpha)), and prostacyclin (as 6-keto-PGF(1alpha)) as well as acetylcholine-stimulated relaxation and gp47(phox) content. Nonketotic diabetes mellitus was induced in New Zealand rabbits with alloxan, and low- and high-dose folic acid was administered daily for 1 month. Rabbits were killed, aortae were excised, and rings were prepared. Rings were mounted in an organ bath, and relaxation was elicited with acetylcholine. The O(2)(-) release was measured spectrophotometrically; the gp47(phox) expression, by Western blotting; and the 8-IPF(2alpha) and 6-keto-PGF(1alpha) formation, by enzyme-linked immunosorbent assay. Blood was collected for measurement of homocysteine, red blood cell folate, and glucose. In aortae from the diabetic rabbits, acetylcholine-induced relaxation was significantly impaired compared with that in untreated controls. The O(2)(-) release, p47(phox) expression, and 8-IPF(2alpha) formation were all enhanced and 6-keto-PGF(1alpha) formation was reduced compared with controls. All these effects were reversed by both low- and high-dose folic acid. Plasma total homocysteine was reduced by high-dose, but not low-dose, folic acid. Red blood cell folate was elevated in both groups. The improvement of endothelial function in patients receiving folic acid may be due to inhibition of nicotinamide adenine nucleotide phosphate oxidase (NADPH) oxidase expression and therefore conservation of nitric oxide and prostacyclin bioavailability, 2 vasculoprotective factors.

[Effect of Bushen Antai Recipe on prostaglandin I2 expression and its nuclear receptor at implantation site in mice with blastocyst implantation dysfunction].

OBJECTIVE: To observe the effect of Bushen Antai Recipe (BAR) on expression of prostaglandin I2 (PGI2) and its nuclear receptor peroxisome proliferators-activated receptor delta (PPARdelta) at implantation site in mice with blastocyst implantation dysfunction. METHODS: Pregnant mice were divided into three groups randomly, the normal group, the model group and the BAR group. The pregnant uterus of all mice was cut off on the 4th (D4), 5th (D5), 6th (D6) and 8th (D8) day of pregnancy for determining the PGI2 expression with radio immunoassay; and the mRNA and protein expression of PPARdelta with RT-PCR and immunohistochemistry at implantation site. RESULTS: PGI2 expression in the model group was obviously lower than that in the normal group (P < 0.01), and also lower than that in the BAR group (P < 0.01), while the index was insignificantly different between the normal and the BAR group. Compared with the normal group, the expression of PPARdelta in the model group was delayed temporally and spatially (P < 0.05), while that in the BAR group was not significantly different. CONCLUSION: BAR can improve the implantation in mice with blastocyst implantation dysfunction through promoting the PGI2 expression and its nuclear receptor PPARdelta at implantation site.

Honokiol up-regulates prostacyclin synthease protein expression and inhibits endothelial cell apoptosis.

Honokiol is a bioactive compound extracted from the Chinese medicinal herb Magnolia officinalis. We recently demonstrated that honokiol inhibited arterial thrombosis through stimulation of prostacyclin (PGI2) generation and endothelial cell protection. The current study is designed to investigate its mechanism of stimulation of PGI2 generation and cell protection. 6-keto-PGF1alpha, the stable metabolite of PGI2, in the media of rat aortic endothelial cells was measured with radioimmunoassay kits. Indomethacin, an inhibitor of cyclooxygenase (COX) and tranylcypromine, a prostacyclin synthease inhibitor were used to ascertain the target enzyme affected by honokiol. Prostacyclin synthease protein levels in endothelial cells were determined by Western blot analysis using an anti-PGI2 synthease rabbit polyclonal antibody. Flow cytometry was used to quantify the apoptotic cells and spectrophotometry was used to test the caspase-3 activity. Honokiol (0.376-37.6 microM) increased the level of 6-keto-PGF1alpha in the media of normal endothelial cells. It counteracted the inhibitory effect of tranylcypromine on the PGI2 generation, but did not influence the effect of indomethacin; evidently, honokiol up-regulated the expression of prostacyclin synthease in the endothelial cells. These effects showed perfect concentration-dependent behavior. In addition, at lower concentration (0.376-3.76 microM), honokiol significantly decreased the percentage of apoptotic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) and significantly lowered the activity of caspase-3 stimulated by ox-LDL. A high dose of honokiol (37.6 microM), however, failed to influence either of them. In conclusion, honokiol augments PGI2 generation in normal endothelial cells; its effect on PGI2 generation attributes to up-regulation of prostacyclin synthease expression; its cell protection may be correlated with its inhibition on apoptosis of endothelial cells. These findings have partly revealed the molecular mechanism of honokiol on inhibiting arterial thrombosis.

BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells.

A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.

Effects of phytoestrogens genistein and daidzein on prostacyclin production by human endothelial cells.

The molecular mechanisms of the vascular effects of phytoestrogens are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. This study examine the effects of two phytoestrogens, the isoflavones genistein and daidzein, on prostacyclin production by cultured human umbilical vein endothelial cells (HUVECs) and the possible role of not only estrogen receptors but also both COX isoforms. The two phytoestrogens significantly increased prostacyclin release in a time- and dose-dependent (0.01-1 microM) manner, being higher than control after 24 h. Selective inhibitors of COX-1, SC-560 [5-(4-chlorophenyl)-1-(4-methoxypjenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2, NS-398 (N-[2-(cyclohexyloxy)-4 nitrophenyl]-methanesulfonamide), were used to investigate the relative contribution of each enzyme. Both inhibitors decreased basal production of prostacyclin, but only COX-2 inhibition completely abolished the isoflavone-stimulated prostacyclin production. Phytoestrogens also increased COX-2 mRNA expression and protein content without affecting COX-1 levels. All these effects were mediated through estrogen receptor activation since treatment of cells with the estrogen receptor antagonist ICI 182780 [7alpha-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta diol] completely abolished the isoflavone-induced increase in prostacyclin production, COX-2 mRNA expression, and COX-2 protein content. The results clearly support the hypothesis that genistein and daidzein increased HUVEC prostacyclin production through estrogen receptor-dependent mechanism, which involved the enhancement of COX-2 protein and activity.

Peroxynitrite provides the peroxide tone for PGHS-2-dependent prostacyclin synthesis in vascular smooth muscle cells.

Endotoxin-treated vascular smooth muscle cells (VSMCs) were recently shown to release high amounts of prostacyclin (PGI2) dependent on the induction of prostaglandin endoperoxide synthase-2 (PGHS-2). In contrast to endothelial PGI2-synthase, for which nitration and inhibition by peroxynitrite was reported, addition of SIN-1 as a peroxynitrite-generating system did not cause inhibition but rather doubled PGI2 release by VSMC. The hypothesis of peroxynitrite supplementing an unsaturated peroxide tone for PGHS-2 was supported by H2O2 exerting the same effect. Studies performed with purified PGHS-2 revealed maximal elevation of enzyme activity in the presence of equimolar concentrations of *NO and *O2-, which together form peroxynitrite, while excessive production of either one radical was inhibitory. Most importantly, 6-keto-PGF1alpha formation by intact VSMC depended on a nearly equimolar generation of *NO and *O2- for providing the endogenous peroxide tone. These findings, together with the observation that an excess of exogenously added *NO, as well as uric acid as a scavenger of peroxynitrite potently reduced PGI2 release, underlined the role of peroxynitrite as the dominating and rate-limiting intracellular mediator of peroxide tone in VSMC. The results allow us to postulate a new cross-talk between the *NO and the prostanoid pathways with a crucial role for peroxynitrite in providing the peroxide tone for a continuous activation of PGHS-2.

Nitroaspirins and morpholinosydnonimine but not aspirin inhibit the formation of superoxide and the expression of gp91phox induced by endotoxin and cytokines in pig pulmonary artery vascular smooth muscle cells and endothelial cells.

BACKGROUND: Although nonsteroidal antiinflammatory drugs (NSAIDs) are ineffective in treating acute respiratory distress syndrome (ARDS), inhalational NO has proved to be useful. NO-donating NSAIDs may therefore be more effective in treating ARDS than NSAIDs alone. Because oxidant stress is central to the pathophysiology of ARDS, the effect of nitroaspirins (NCX 4016, NCX 4040, and NCX 4050) compared with morpholinosydnonimine (SIN-1; an NO donor) and aspirin (ASA) on superoxide (O2*-) formation and gp91phox (an active catalytic subunit of NADPH oxidase) expression in pig pulmonary artery vascular smooth muscle cells (PAVSMCs) and endothelial cells (PAECs) was investigated. METHODS AND RESULTS: Cultured PAVSMCs and PAECs were incubated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1alpha (with or without NO-ASA, SIN-1, or ASA) for 16 hours, and O2*- release was measured by use of the reduction of ferricytochrome c. The expression of gp91(phox) was assessed by use of Western blotting. LPS, TNF-alpha, and IL-1alpha all stimulated the formation of O2*- and expression of gp91(phox) in both PAVSMCs and PAECs, an effect inhibited by NADPH oxidase inhibitors, diphenyleneiodonium, and apocynin. SIN-1, NCX 4016, and NCX 4050 but not ASA alone inhibited the formation of O2*- and expression of gp91(phox). CONCLUSIONS: LPS and cytokines promote the formation of O2*- in PAVSMCs and PAECs through an augmentation of NADPH oxidase activity, which in turn is prevented by NO. Thus, NO may play a protective role in preventing excess O2*- formation, but its negation by O2*- may augment the progress of ARDS. The inhibitory effect of nitroaspirins suggests that they may be therapeutically useful in treating ARDS through the suppression of NADPH oxidase upregulation and O2*- formation.