Sulfido-peptide leukotrienes in coronary heart disease - relationship with disease instability and myocardial ischaemia.

BACKGROUND: Urinary excretion of leukotriene (LT) E(4) is an index of LTC(4) biosynthesis and platelet-neutrophil interactions, which may occur in coronary heart disease and contribute to myocardial ischaemia. Enhanced LTC(4) biosynthesis may be a consequence of myocardial ischaemia or be linked to its pathogenetic substrate. METHODS AND RESULTS: Overnight urine collections were obtained from 17 patients with chronic stable angina, three patients with Prinzmetal's angina, 16 patients with non ST-elevation acute coronary syndromes (NSTE-ACS) and six patients with acute ST-elevation myocardial infarction (STEMI). LTE(4) excretion was measured by enzyme immunoassay after HPLC separation. Compared with healthy controls (51.1 +/- 21.3 pg mg(-1) creatinine, mean +/- SD, n = 11) and with non-coronary cardiac controls (36.6 +/- 9.8 pg mg(-1) creatinine, n = 9), LTE(4) excretion was unchanged in stable angina (40.5 +/- 25.8 pg mg(-1) creatinine), but significantly (P < 0.01) increased in NSTE-ACS (122.7 +/- 137.2 pg mg(-1) creatinine) and STEMI (213.4 +/- 172.4 pg mg(-1) creatinine). In these patients, LTE(4) excretion rapidly dropped after day 1, consistent with effective coronary reperfusion. In patients with NSTE-ACS, the increase in LTE(4) excretion was entirely restricted to patients with recent (< 48 h) spontaneous anginal episodes. Myocardial ischaemia elicited by a positive exercise stress test was not accompanied by any detectable increase in LTE(4) excretion, while a significant (P < 0.01) increase was detected after a single-vessel percutaneous coronary interventions (PCI) procedure (n = 10), as compared with diagnostic angiography (n = 9). CONCLUSIONS: In coronary heart disease, increased LTC(4) biosynthesis is restricted to ACS and not linked to myocardial ischaemia per se, but likely to the occurrence of plaque disruption.

Induction of cyclo-oxygenase-2 in human endothelial cells by SIN-1 in the absence of prostaglandin production.

Nitric oxide (NO) regulates cyclo-oxygenase (COX) activity in various cell systems and reports conflict in regard to its stimulatory versus inhibitory role. Incubation of human umbilical vein endothelial cells (HUVEC) with SIN-1 (3-morpholinosydnonimine), a donor of NO, resulted in a rapid and dose-dependent increase in the expression of COX-2 as analysed by Western and Northern blotting. Incubation of HUVEC with SIN-1 and interleukine (IL)-1alpha resulted in increased induction of COX-2 compared with IL-1alpha alone and corresponded to an additive effect. The COX-2 induction was dependent on a de novo synthesis since cycloheximide, an inhibitor of protein synthesis, blocked the enzyme expression. The increase in COX-2 expression was not accompanied by a corresponding change in prostaglandin (PG) production. However, the COX activity was partially recovered when immunoprecipitated COX-2 was incubated with arachidonic acid and haematin. Peroxynitrite, a highly reactive nitrogen molecule derived from the interaction of NO and superoxide anion, significantly increased COX-2 expression. Under these conditions and within the limit of detection of the antibody, selective antibody for nitrotyrosine failed to detect nitrated COX-2 in immunoprecipitated COX-2 when cells where incubated with SIN-1 or SIN-1+IL-1alpha. Ro 31-8220, a specific inhibitor of protein kinase (PK) C, blocked the induction of COX-2. Also, SB203580, the selective inhibitor of p38 MAP kinase, strongly blocked the induction of COX-2 by SIN-1 in the presence or absence of IL-1alpha, whereas the MEK-1 inhibitor, PD 98059, affected it to a lesser extent. These data demonstrate that SIN-1 induces COX-2 in HUVEC in the absence of PG formation and suggest a complex regulation of COX-2 expression and PG formation by NO in endothelial cells.

Heme oxygenase-1 induction by nitric oxide in RAW 264.7 macrophages is upregulated by a cyclo-oxygenase-2 inhibitor.

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.

Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment.

BACKGROUND: Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment. METHODS: Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with beta(2) agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry. RESULTS: Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups. CONCLUSIONS: These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease.

In vivo lipid peroxidation and platelet activation in cystic fibrosis.

F(2)-isoprostanes are bioactive peroxidation products of arachidonic acid whose urinary excretion provides an index of lipid peroxidation in vivo. We tested the hypothesis that formation of F(2)-isoprostanes is altered in patients with cystic fibrosis and contributes to platelet activation and pulmonary dysfunction in this setting. The urinary excretion of immunoreactive 8-iso-prostaglandin F(2alpha) (PGF(2alpha)) was significantly (p = 0.0001) higher in 36 patients with cystic fibrosis than in 36 age-matched healthy subjects: 618 +/- 406 versus 168 +/- 48 pg/mg creatinine. The urinary excretion of immunoreactive 11-dehydro-thromboxane B(2) (TXB(2)), an index of in vivo platelet activation, was also significantly (p = 0.0001) higher in patients than in control subjects: 2,440 +/- 1,453 versus 325 +/- 184 pg/mg creatinine. The excretion rate of 8-iso-PGF(2alpha) was correlated with that of 11-dehydro-TXB(2) (rho = 0.51; p = 0.0026) and inversely related to FEV(1) (rho = -0.40; p = 0.0195). Urinary 8-iso-PGF(2alpha) excretion was largely unaffected during cyclooxygenase inhibition with low-dose aspirin, nimesulide, or ibuprofen, consistent with a noncyclooxygenase mechanism of F(2)-isoprostane formation in cystic fibrosis. Increased vitamin E supplementation (from 200 to 600 mg/d) was associated with statistically significant (p = 0.005) reductions in urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion, by 42% and 29%, respectively. We conclude that enhanced lipid peroxidation is an important feature of cystic fibrosis and may contribute to persistent platelet activation and pulmonary dysfunction via generation of bioactive isoeicosanoids. Our results provide a rationale for reassessing the adequacy of vitamin E supplementation in this setting.

Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts.

1. Haem oxygenase-1 (HO-1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO-1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S-nitroso-N-acetylpenicillamine (SNAP), induced a dose- and time-dependent expression of HO-1 protein. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) accumulated in the medium and cyclo-oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX-2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO-1 and COX-2 protein expression was observed. 3. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO-1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO-1 and COX-2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.

Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness.

BACKGROUND: Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS: We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS: These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Oncostatin M induces interleukin-6 and cyclooxygenase-2 expression in human vascular smooth muscle cells : synergy with interleukin-1beta.

Oncostatin M (OSM), a cytokine first identified from activated monocytes and T lymphocytes, is one of the most potent autocrine growth factor for AIDS and Kaposi's sarcoma. Little is known about the effects of OSM on normal vascular cells. We thus exposed human aortic smooth muscle cells (hASMCs) to OSM, examined cell proliferation and morphology, and determined interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) expression. OSM had a weak antiproliferative effect. After a 4-day incubation with 100 ng/mL OSM, cell count decreased to 69+/-3% of control. However, OSM induced striking changes in hASMC morphology, characterized by a polyclonal shape, in contrast to the spindle morphological feature of control hASMCs. OSM stimulated the release of IL-6 by hASMCs in a dose-dependent way; after a 48-hour exposure, values were 8.5+/-0.7, 29.7+/-3.5, 50.9+/-4.4, and 73.8+/-7.6x10(3) U/mL (n=6) at OSM concentrations of 0, 1, 10, and 100 ng/mL, respectively. OSM induced marked expression of COX-2 protein and mRNA. Leukemia inhibitory factor had no effect on hASMCs, indicating that OSM effects on hASMCs were mediated by the OSM type II receptor and not by the leukemia inhibitory factor receptor. OSM used the JAK/STAT signaling pathway, as demonstrated by rapid phosphorylation of JAK1 and specific activation of STAT1. Interestingly, OSM acted in synergy with IL-1beta on IL-6 production and COX-2 expression. In conclusion, OSM is a novel regulator of human smooth muscle cell functions, acting in concert with IL-1beta, and OSM may play a role in major vascular diseases such as atherosclerosis.

A chronic hypercoagulable state in patients with beta-thalassaemia major is already present in childhood.

A higher than normal incidence of thromboembolic events has been observed in adult patients with beta-thalassaemia major (TM) and certain haemostatic anomalies found in these patients suggest the existence of a chronic hypercoagulable state. Thalassaemic red blood cells (RBC) were demonstrated to facilitate thrombin formation due to altered asymmetry of the membrane phospholipids with enhanced exposure of phosphatidylserine. Since RBC anomalies exist in thalassaemia from the first months of life, we studied markers of hypercoagulability and thrombophilia in 36 adult patients (range 19-38 years) and 26 children (range 2-18 years) with beta-TM. All the patients were in steady state and none had experienced clinical signs or symptoms of thrombosis. Highly elevated urinary levels of 11-dehydro-thromboxane B2 and significantly elevated plasma levels of thrombin-antithrombin III (TAT) complexes were observed to the same extent in TM children and adults. The levels of factor II were decreased while factors V, VII + X and plasminogen were within the normal range. The natural coagulation inhibitors, protein C (PC) and protein S (PS) were significantly decreased in all TM patients investigated, regardless of age, but the PS binding protein (C4bBP) and antithrombin III levels were normal. The frequency of other thrombophilic mutations was not increased. Thus, a chronic hypercoagulable state already exists in thalassaemia in childhood and may contribute to the cardiac and pulmonary anomalies and the thrombotic events which occur later.

Differential effects of long chain n-3 fatty acids on the expression of PGH synthase isoforms in bovine aortic endothelial cells.

Primary cultures of bovine aortic endothelial cells were used at confluency to evaluate the effect of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on the expression of both the constitutive and inducible isoforms of PGH synthase (PGHS), PGHS-1 and PGHS-2, respectively. After a 22 h period enrichment of cell lipid stores with each fatty acid, the expression of PGH synthase isoforms was measured by western blotting. EPA and DHA, but not oleate, significantly decreased the immunoreactive PGHS-1 and to a similar extent the corresponding mRNA, as measured by northern blotting. Studies on mRNA stability failed to show any difference between DHA-enriched and control cells, indicating that the decreased expression observed was likely from transcriptional origin. Under the enrichment conditions, EPA and DHA, but not oleate, moderately but significantly induced an oxidative stress as judged by malondialdehyde formation. Interestingly, hydrogen peroxide was able to mimic the effect of EPA and DHA in decreasing the expression of PGHS-1. On the other hand, the PMA-induced PGHS-2 expression could be potentiated by cell pre-enrichment with DHA, whereas hydrogen peroxide alone could induce such an expression. We conclude that the long chain n-3 fatty acids EPA and DHA may differently affect the expression of PGH synthase isoforms, possibly via an oxidative stress.

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.

Early expression of cyclo-oxygenase-2 during sporadic colorectal carcinogenesis.

Regular administration of non-steroidal anti-inflammatory drugs (NSAIDs) may reduce the incidence of colorectal cancer by targeting cyclo-oxygenase-2 (Cox-2), a key enzyme in arachidonic acid metabolism. To evaluate the role of Cox-2 in sporadic colorectal cancer development, Cox-2 expression was investigated by immunohistochemistry in 85 adenomas, 53 carcinomas, 34 hyperplastic lesions and 104 samples of histologically normal mucosa adjacent to adenoma or carcinoma. In addition, Cox-2 mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) in six adenomas and 14 carcinomas with paired grossly normal mucosa. Immunohistochemistry for the proliferation-associated antigen Ki-67 and in situ end labelling for demonstrating apoptotic bodies were also used to analyse the associations between Cox-2 expression and proliferation and apoptosis. Cox-2 protein expression was increased in 76/85 (89.4 per cent) adenomas and 44/53 (83.0 per cent) carcinomas compared with normal mucosa. Cox-2 protein expression was unrelated either to the degree of dysplasia or to the size of the adenomas (p > 0.50, p > 0.10, respectively) or to differentiation, Dukes stage or lymph node metastasis of carcinomas (all p > 0.50). Interestingly, 20/34 (58.8 per cent) hyperplastic lesions adjacent to adenomas or carcinomas displayed expression higher than in normal mucosa (18.3 per cent) (p < 0.0001) but lower than in adenomas or carcinomas (p < 10(-5), p < 0.001, respectively). There were no correlations between Cox-2 protein expression and proliferative or apoptotic index in either adenomas or carcinomas (all p > 0.25). Cox-2 mRNA expression was significantly increased in adenomas and carcinomas compared with normal mucosa (p < 0.005, p < 0.001, respectively). There were no differences between adenomas and carcinomas in either protein or mRNA levels (p > 0.25, p > 0.90, respectively). These data indicate that enhanced expression of Cox-2 occurs early during colorectal carcinogenesis and may contribute to tumour formation.

Cyclooxygenase-2 is widely expressed in atherosclerotic lesions affecting native and transplanted human coronary arteries and colocalizes with inducible nitric oxide synthase and nitrotyrosine particularly in macrophages.

Inflammation appears to have a major role in the development of atherosclerotic lesions affecting native and transplanted coronary arteries. The subsequent risk of plaque rupture and acute ischemic events correlates with the degree of inflammation and may be modified by aspirin, an anti-inflammatory cyclooxygenase inhibitor. Cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) are involved in the inflammatory response via the rapid and exaggerated production of prostanoids and nitric oxide, both of which may have proatherosclerotic effects. These effects may be mediated by the formation of peroxynitrite in the case of nitric oxide and involve "cross talk" between the two enzyme systems. This study aimed to investigate native and transplant atherosclerosis for the presence and distribution of Cox-2 and iNOS. Immunocytochemical studies were performed on atherosclerotic lesions from patients with native (n=12) and transplant (n=5) coronary disease by using antibodies to Cox-2, iNOS, and nitrotyrosine (an indicator of peroxynitrite production). Control tissue was obtained from unused donor hearts and at the time of autopsy. Cox-2 and iNOS colocalized predominantly in macrophages/foam cells in both types of atherosclerosis. Cox-2 expression was also detected in medial smooth muscle cells and endothelial cells, including those of the vasa vasorum. Nitrotyrosine was found in the same distribution as that of iNOS and was colocalized with Cox-2 in macrophages. Cox-2 and iNOS are coexpressed in native and transplant atherosclerosis, possibly allowing for interaction between the enzymes and suggesting an alternative mechanism for the benefits of aspirin via inhibition of Cox-2 activity.

Effects of antiaggregant and antiinflammatory doses of aspirin on coronary hemodynamics and myocardial reactive hyperemia in conscious dogs.

Clinical studies have shown that low doses of aspirin (<300 mg/day) inhibit thromboxane A2 production and platelet aggregation but preserve prostacyclin synthesis. In contrast, high doses of aspirin (>1,000 mg/day) suppress the synthesis of both eicosanoids. Because the consequences of aspirin administration have never been investigated on coronary vasomotor tone in vivo, we investigated the effects of low and high doses of aspirin on systemic and coronary hemodynamics under basal conditions and after myocardial reactive hyperemia in conscious dogs. Dogs were instrumented with a Doppler flow probe and a hydraulic occluder. Coronary blood flow was measured in the conscious state at baseline and during myocardial reactive hyperemia after 10, 20, and 30 s of coronary occlusion. Thromboxane B2 serum concentrations, an index of platelet aggregation, decreased by >90% after long-term i.v. administration of aspirin, 100 mg/day for 7 days (low dose). Neither systemic and coronary hemodynamics nor reactive hyperemia were affected by the drug. After combined administration of this low dose of aspirin and of the nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA, 30 mg/kg/day/7 days), reactive hyperemia decreased to the same extent as when L-NNA was administered alone. After administration of a unique high-dose aspirin (1,000 mg, i.v.), myocardial reactive hyperemia was markedly reduced, and this effect was still observed after previous blockade of NOS and cyclooxygenase by L-NNA and diclofenac, respectively. Thus long-term treatment with a low antiaggregant dose of aspirin does not alter the ability of coronary vessels to dilate during myocardial reactive hyperemia in conscious dogs. In contrast, short-term administration of a high antiinflammatory dose of aspirin severely blunts myocardial reactive hyperemia through a mechanism that is independent of both cyclooxygenase and nitric oxide metabolic pathways.

The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis.

Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1, 738 +/- 274 vs. 1,002 +/- 107 pmol/ micromol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.

Cytokine-induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide-dependent and -independent mechanism in rat osteoblasts in vitro.

Osteoblasts respond to stimulation with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) by production of nitric oxide and prostaglandins (PGs). In this study the relationship between nitric oxide and PG synthesis was investigated after cytokine stimulation of cultured rat osteoblasts. IL-1, TNF-alpha, IFN-gamma, and exogenous sodium nitroprusside, a nitric oxide donor, all stimulated PGE2 production in a dose-dependent manner. PGE2 production was blocked by L-nitro-arginine methyl ester, an inhibitor of nitric oxide production, after IFN-gamma stimulation and was partially blocked after TNF-alpha stimulation. However, IL-1-induced PGE2 was unaffected. Similarly, expression of the cyclooxygenase-2 protein was stimulated by cytokines, and IFN-gamma-induced expression was again blocked by L-nitro-arginine methyl ester. In contrast, all cytokines induced the cyclooxygenase-2 mRNA expression independently of nitric oxide production, although exogenous sodium nitroprusside was able to induce the cyclooxygenase-2 mRNA in the absence of cytokines. The results show that nitric oxide can induce PG synthesis and cyclooxygenase-2 expression and may regulate cyclooxygenase-2 expression at both transcriptional and post-transcriptional levels. In addition, the data show the existence of both nitric oxide-dependent and -independent pathways of PG synthesis after cytokine stimulation of osteoblasts. The results suggest that nitric oxide may be an important mediator of PG production in inflammatory bone diseases.

Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets.

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.