Aida Habib

Activation of human aortic smooth-muscle cells is inhibited by PPARα but not by PPARγ activators

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARγ promotes lipid storage by regulating adipocyte differentiation, PPARα stimulates the β-oxidative degradation of fatty acids. PPARα-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARα is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARα ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect,. Here we show that PPARα is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARα ligands, and not PPARγ ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARα repression of NF-κB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARα inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARα in the vascular wall may influence the process of atherosclerosis and re-stenosis.

Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid production in inflammation

To examine the role of cyclooxygenase (COX) isozymes in prostaglandin formation and oxidant stress in inflammation, we administered to volunteer subjects placebo or bolus injections of lipopolysaccharide (LPS), which caused a dose-dependent increase in temperature, heart rate, and plasma cortisol. LPS caused also dose-dependent elevations in urinary excretion of 2,3-dinor 6-keto PGF(1alpha) (PGI-M) and 11-dehydro thromboxane B(2) (Tx-M). Platelet COX-1 inhibition by chronic administration of low-dose aspirin before LPS did not alter the symptomatic and febrile responses to LPS, but the increment in urinary PGI-M and Tx-M were both partially depressed. Pretreatment with ibuprofen, a nonspecific COX inhibitor, attenuated the febrile and systemic response to LPS and inhibited prostanoid biosynthesis. Both celecoxib, a selective COX-2 inhibitor, and ibuprofen attenuated the pyrexial, but not the chronotropic, response to LPS. Experimental endotoxemia caused differential expression of the COX isozymes in monocytes and polymorphonuclear leucocytes ex vivo. LPS also increased urinary iPF(2alpha)-III, iPF(2alpha)-VI, and 8,12-iso-iPF(2alpha)-VI, isoprostane (iP) indices of lipid peroxidation, and none of the drugs blunted this response. These studies indicate that (a) although COX-2 predominates, both COX isozymes are induced and contribute to the prostaglandin response to LPS in humans; (b) COX activation contributes undetectably to lipid peroxidation induced by LPS; and (c) COX-2, but not COX-1, contributes to the constitutional response to LPS in humans.

Role of NF-κB in the Antiproliferative Effect of Endothelin-1 and Tumor Necrosis Factor-α in Human Hepatic Stellate Cells INVOLVEMENT OF CYCLOOXYGENASE-2

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype and proliferate and synthesize fibrosis components. Endothelin-1 (ET-1), which inhibited the growth of human myofibroblastic HSC, increased the formation of two NF-κB DNA binding complexes; this effect was also observed with tumor necrosis factor-α (TNF-α). The complexes were identified as the p50/p50 and p50/p65 NF-κB dimers. Activation of NF-κB was associated with the degradation of the inhibitory protein IκB-α; no IκB-β was detected. Activation of NF-κB and degradation of IκB-α were prevented by the NF-κB inhibitors sodium salicylate and MG-132. In addition to cyclooxygenase-1 (COX-1), COX-2 is also constitutively expressed in human HSC, and the use of dexamethasone and of SC-58125, a selective COX-2 inhibitor, revealed that COX-2 accounts for basal COX activity. Moreover, COX-2 mRNA and protein were up-regulated by ET-1 and TNF-α, whereas COX-1 was unaffected. Induction of COX-2 and stimulation of COX activity by ET-1 and TNF-α were prevented by sodium salicylate and MG-132, suggesting that activation of NF-κB by either factor is needed for stimulation of COX-2. Finally, SC-58125 and dexamethasone reduced the growth inhibitory effect of ET-1 and TNF-α, indicating that activation of COX-2 is required for inhibition of HSC proliferation. Taken together, our results suggest that NF-κB, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

Cyclooxygenases-1 and −2 of Endothelial Cells Utilize Exogenous or Endogenous Arachidonic Acid for Transcellular Production of Thromboxane

The presence of prostaglandin (PG) H in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1α or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1α. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF, PGE, PGF) versus transcellular (TXB) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1α, newly synthesized Cox-2 produced less TXB than 6-keto-PGF compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

Differential Signaling by the Thromboxane Receptor Isoforms via the Novel GTP-binding Protein, Gh

Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPα and TPβ) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, Gh, that can also function as a tissue transglutaminase, has been described. We investigated whether Gh functions as a signaling protein in association with thromboxane receptors. First, we sought Gh expression in cells known to express TPs. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated Gh expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells. Second, immunoprecipitation of both TPα and TPβ in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPα, but not of TPβ, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with Gh. By contrast, agonist activation of both TP isoforms resulted in Gq-mediated inositol phosphate signaling. Gh is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein.

Aspirin-insensitive thromboxane biosynthesis in essential thrombocythemia is explained by accelerated renewal of the drug target

Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A(2) (TXA(2)) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA(2) biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB(2) independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB(2) ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB(2) reduction and normalized the functional platelet response to aspirin, as assessed by urinary 11-dehydro-TXB(2) excretion and the VerifyNow Aspirin assay. Doubling the aspirin dose reduced serum TXB(2) only partially by 39% median (IQR, 29%-54%, P < .05). We conclude that the abnormal megakaryopoiesis characterizing ET accounts for a shorter-lasting antiplatelet effect of low-dose aspirin through faster renewal of platelet cyclooxygenase-1, and impaired platelet inhibition can be rescued by modulating the aspirin dosing interval rather than the dose.

Cyclo-oxygenase-1 and −2 contribution to endothelial dysfunction in ageing

Experiments were designed to investigate the role of cyclo‐oxygenase isoforms in endothelial dysfunction in ageing. Aortic rings with endothelium of aged and young (24 vs 4 month‐old) Wistar rats, were mounted in organ chambers for the recording of changes in isometric tension. In young rats, acetylcholine (ACh) caused a complete relaxation which was not affected by indomethacin (0.3 μM), NS‐398 (a preferential COX‐2 inhibitor; 1 μM), SQ‐29548 (a thromboxane‐receptor antagonist; 1 μM), nor valeryl‐salicylate (VAS, a preferential inhibitor of COX‐1; 3 mM). In aged rats, ACh caused a biphasic response characterized by a first phase of relaxation (0.01–1 μM ACh), followed by a contraction (3–100 μM ACh). Indomethacin, NS‐398 and SQ‐29548, but not VAS, augmented the first phase. Indomethacin, VAS, NS‐398 and SQ‐29548 decreased the contractions to high ACh concentrations. Then, the sensitivity to thromboxane receptor activation was investigated with U‐46619. The results show comparable EC50 values in young and aged rats. In aged rats, the ACh‐stimulated release of prostacyclin, prostaglandin F2α and thromboxane A2 was decreased by either indomethacin, NS‐398, VAS or endothelium removal. However, in young animals, the ACh‐stimulated release of prostacyclin and prostaglandin F2α were smaller than in older animals and remained unaffected by NS‐398. Aortic endothelial cells from aged – but not young – rats express COX‐2 isoform, while COX‐1 labelling was observed in endothelial cells from both young and aged rats. These data demonstrate the active contribution of COX‐1 and −2 in endothelial dysfunction associated with ageing.

Prognostic significance of cyclooxygenase-2 in laryngeal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) overexpression is an unfavorable prognostic marker in laryngeal squamous cell carcinoma (SCC). EGFR stimulates cyclooxygenase‐2 (COX‐2) expression in normal human keratinocytes and squamous carcinoma cells. Based on these observations a prognostic role of COX‐2 expression in laryngeal SCC can be hypothesized. Consequently, COX‐2 expression was studied in laryngeal SCC (median follow‐up = 47 months; range: 2–87 months) by quantitative immunohistochemistry (n = 61) and EGFR by binding assay (n = 51). Well‐differentiated regions of laryngeal SCC revealed strong COX‐2 immunostaining, whereas histologically normal areas neighboring tumor as well as poorly‐differentiated tumors were negative. Immunohistochemical results were confirmed by Western blot analyses. Coxs regression analysis showed that the combination of low levels of COX‐2 integrated density and high levels of EGFR covariates provided strong prediction, at 5‐year follow‐up, of both poor overall survival (χ2 = 12.905; p = 0.0016) and relapse‐free survival (χ2 = 9.209; p = 0.01). In vitro studies on CO‐K3 cell line, obtained from an EGFR positive, COX‐2 negative poorly‐differentiated laryngeal SCC, revealed that EGF stimulation failed to induce COX‐2 expression and PGE2 production suggesting a change in EGFR signaling pathway. These findings indicate that COX‐2 is overexpressed in less aggressive, low grade laryngeal SCC, whereas its expression is lost when tumors progress to a more malignant phenotype.

Rapid, Agonist-dependent Phosphorylation in Vivo of Human Thromboxane Receptor Isoforms MINIMAL INVOLVEMENT OF PROTEIN KINASE C

Thromboxane A2 (TxA2) is a potent vasoconstrictor and platelet agonist. Its biological function is tightly regulated. G protein-coupled membrane receptors transduce the effects of TxA2. However, although a single thromboxane receptor (TP) gene has been identified, two splice variants have been cloned from human placenta and megakaryocytic lines (TPα) and from human endothelial cells (TPβ). These differ in the length of their carboxyl-terminal extensions (15 versus 79 residues), which contain multiple potential sites for receptor phosphorylation. Given that TP agonists activate protein kinase C (PKC), it would seem possible that PKC-dependent phosphorylation of TPs might play a central role in homologous desensitization of these receptors. To determine if the TP isoforms were differentially phosphorylated in response to agonist in vivo, human embryonic kidney (HEK) 293 cells were stably transfected with TPα and TPβ. Isoform-specific anti-peptide antibodies were developed and used to immunoprecipitate the phosphorylated receptors. U46619, a PGH2/TxA2 mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619. Inhibition of PKC with either GF 109203X (5 μM) or RO 31-8220 (5 μM) or of protein kinase A with H-89 (50 μM) marginally influenced agonist-dependent phosphorylation of either isoform and failed to modulate homologous desensitization of agonist-induced stimulation of inositol phosphate formation. Similar results were obtained when PKC was down-regulated by long term incubation with the phorbol ester, phorbol myristate acetate. Although short term stimulation with phorbol myristate acetate caused PKC-dependent phosphorylation of TPs in vivo, thrombin stimulation of the TP-transfected HEK cells in vivo failed to phosphorylate either of the TP isoforms. Thus, despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and the likely activation of PKC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of either TP isoform.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy

We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed platelets might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with thiazole orange-positive platelets (r = 0.71, P < .001). The rate of TXA(2) biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB(2) (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB(2), were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB(2) was significantly reduced by the selective COX-2 inhibitor NS-398 added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB(2). Fourteen of the 41 patients were studied again 21 (+/- 7) months after the first visit. Serum TXB(2) was consistently reduced by approximately 30% by adding NS398 in vitro, while it was completely suppressed with 50 microM aspirin. Accelerated platelet regeneration in most aspirin-treated ET patients may explain aspirin-persistent TXA(2) biosynthesis through enhanced COX-2 activity and faster renewal of unacetylated COX-1. These findings may help in reassessing the optimal antiplatelet strategy in ET.