Mark J. Paul-Clark

Inducible cyclooxygenase may have anti-inflammatory properties

Cyclooxygenase (COX) has two isoforms. Generally, COX 1 is constitutively expressed in most tissues, where it maintains physiological processes; inducible COX 2 is considered a pro-inflammatory enzyme and a chief target for the treatment of inflammatory diseases. Here we present evidence that COX 2 may have anti-inflammatory properties. In carrageenin-induced pleurisy in rats, the predominant cells at 2 hours are polymorphonuclear leucocytes, whereas mononuclear cells dominate from 24 hours until resolution at 48 hours. In this model, COX 2 protein expression peaked initially at 2 hours, associated with maximal prostaglandin E2 synthesis. However, at 48 hours there was a second increase in COX 2 expression, 350% greater than that at 2 hours. Paradoxically, this coincided with inflammatory resolution and was associated with minimal prostaglandin E2 synthesis. In contrast, levels of prostaglandin D2, and 15deoxyΔprostaglandin J2 were high at 2 hours, decreased as inflammation increased, but were increased again at 48 hours. The selective COX 2 inhibitor NS-398 and the dual COX 1/COX 2 inhibitor indomethacin inhibited inflammation at 2 hours but significantly exacerbated inflammation at 48 hours. This exacerbation was associated with reduced exudate prostaglandin D2 and 15deoxyΔprostaglandin J2 concentrations, and was reversed by replacement of these prostaglandins. Thus, COX 2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy, dominated by polymorphonuclear leucocytes, but may aid resolution at the later, mononuclear cell-dominated phase by generating an alternative set of anti-inflammatory prostaglandins.

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Role of nuclear receptor signaling in platelets: antithrombotic effects of PPARβ

Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and then modulate at the transcriptional level the function of many target genes. Three PPARs are known: α, β (sometimes called δ), and γ. The better studied are PPARα and PPARγ, which are activated by fibrates and thiazolidinediones/glitazones, respectively. It is now believed that activation of the PPARs could be associated with the prevention of heart attack and stroke in humans. Here we report, for the first time, that human platelets contain PPARβ and that its selective activation inhibits platelet aggregation. PPARβ is a putative receptor for prostacyclin. Prostacyclin is an important antithrombotic hormone that synergizes with nitric oxide to inhibit platelet aggregation. In the current study, we show that PPARβ ligands similarly synergize with nitric oxide to inhibit platelet aggregation. These observations challenge our view of a nuclear receptor because PPARβ is present and active in nonnucleated platelets. Furthermore, these data suggest that some of the antithrombotic actions of prostacyclin may be mediated via activation of PPARs. Thus, our results identify PPARβ as a novel antiplatelet target that may mediate some of the effects of prostacyclin in blood.

Neutrophil Interaction with Inflamed Postcapillary Venule Endothelium Alters Annexin 1 Expression

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.

Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

Here, we describe the improved antiarthritic properties of a nitric oxide-releasing derivative of prednisolone that includes a sparing of the effects on bone. Glucocorticoids are widely used in the treatment of chronic inflammatory pathologies, but their use is often accompanied by side effects, including osteoporosis. Recently, a new steroid able to release low levels of nitric oxide showed potent inhibition of leukocyte trafficking and chemokine generation in models of acute inflammation. The objective of this study was to assess the anti-inflammatory activity of this nitric-oxide releasing glucocorticoid, nitro-prednisolone (NCX-1015), in parallel with the parent compound prednisolone and a control molecule lacking an NO group, (NCX-1016), in a model of rat collagen-induced arthritis. Dosing of rats with NCX-1015 (0.4–4 μmol/kg, i.p.) greatly reduced all parameters of inflammation. A significant but inferior anti-inflammatory effect also was obtained with prednisolone. Collagen-induced arthritic rats had elevated pyridinoline values (>60% over naïve rats), indicating bone and cartilage erosion; this increase was prevented by NCX-1015 but not by prednisolone or NCX-1016 treatment. In vitro, prednisolone (1 nM), but not NCX-1015, elevated bone resorbing activity of rat primary osteoclasts. In conclusion, NCX-1015 is a steroid derivative with a potential for the treatment of chronic inflammatory pathologies and that has milder side effects anticipated on the bone compartment.

Antiplatelet Actions of Statins and Fibrates Are Mediated by PPARs

Objectives—Statins and fibrates are hypolipidemic drugs which decrease cardiac events in individuals without raised levels of cholesterol. These drugs inhibit platelet function, but the mechanisms by which this pleiotropic effect is exerted are not known. Methods and Results—We used a range of approaches to show statins inhibit human platelet activation in vitro while engaging PPAR&agr; and PPAR&ggr;. The effects of simvastatin were prevented by the PPAR&ggr; antagonist GW9662 or the PPAR&agr; antagonist GW6471. In a small-scale human study fluvastatin activated PPAR&agr; and PPAR&ggr; in platelets and reduced aggregation in response to arachidonic acid ex vivo. The effects of fenofibrate were prevented by PPAR&agr; antagonism with GW6471. Fenofibrate increased bleeding time in wild-type, but not in PPAR&agr;−/− mice. The inhibitory effect of fenofibrate, but not simvastatin, on aggregation was prevented by deletion of PPAR&agr; in murine platelets. PKC&agr;, which influences platelet activation, associated and immune-precipitated with PPAR&ggr; in platelets stimulated with statins and with PPAR&agr; in platelets stimulated with fenofibrate. Conclusions—This study is the first to provide a unifying explanation of how fibrates and statins reduce thrombotic and cardiovascular risk. Our findings that PPARs associate with PKC&agr; in platelets also provide a mechanism by which these effects are mediated.

Glucocorticoid Receptor Nitration Leads to Enhanced Anti-Inflammatory Effects of Novel Steroid Ligands

It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4′-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1–10 μM NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [3H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.

21-NO-prednisolone is a novel nitric oxide-releasing derivative of prednisolone with enhanced anti-inflammatory properties

Anti‐inflammatory effects of a novel derivative of the glucocorticoid prednisolone were investigated. NCX‐1015 (prednisolone 21‐[(4′‐nitrooxymethyl)benzoate]) incubation in human platelet‐rich plasma produced a time (0–60u2003min) and concentration (3–300u2003μM) dependent release of nitrite, that was mirrored by accumulation of cyclic guanosine monophosphate in the human platelets. Intraperitoneal injection of NCX‐1015 to mice (up to 27.7u2003μmolu2003kg−1) produced nitrite accumulation in the peritoneal cavity maximal at 60u2003min. NCX‐1015 dose‐dependently induced the steroid sensitive cell surface marker CD163 in human peripheral blood mononuclear cells (PBMCs). NCX‐1015 was more potent than prednisolone in inducing CD163. Similarly, lipopolysaccharide induced interleukin‐1β release from these cells was inhibited by NCX‐1015 with higher potency than prednisolone. In the zymosan peritonitis model, NCX‐1015 was more active than prednisolone in suppressing neutrophil extravasation (ED50 of 5.5 and 25.8u2003μmolu2003kg−1, respectively), nitrite accumulation (ED50 of 1.38 and 22.2u2003μmolu2003kg−1, respectively) and release of the chemokine KC (ED50 of 5.5 and 27.7u2003μmolu2003kg−1, respectively) as determined at the 4u2003h time‐point. No differences were measured for the levels of interleukin‐1β or prostaglandin E2. NCX‐1015 administered orally was also found to be equally active. Co‐administration of the nitric oxide donors NOC‐18 ((z)‐1‐[(2‐aminoethyl)‐N‐(2‐aminoethyl)amino] diazen‐1‐ium‐1, 2‐diolate; 7.9u2003μmolu2003kg−1) or sodium nitroprusside (13.8u2003μmolu2003kg−1) with prednisolone resulted in an additive anti‐migratory action. In a chronic model of granulomatous tissue inflammation, administration of NCX‐1015 (13.9u2003μmolu2003kg−1) from day 1 (i.e. after induction of inflammation) was more effective than prednisolone in reducing the granuloma dry weight, and this was associated to a lower anti‐angiogenic effect. In conclusion we show that NCX‐1015 is more potent than prednisolone in controlling several, though not all, parameters of acute and chronic inflammation, and propose that this effect may be due to a co‐operation between the steroid moiety and nitric oxide or related species released in biological fluids. Whereas this aspect needs to be further clarified, we propose NCX‐1015 as the first member of a novel class of anti‐inflammatory compounds, the nitro‐steroids.

Nitric Oxide Synthase Inhibitors Have Opposite Effects on Acute Inflammation Depending on Their Route of Administration

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor l-N5(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1–6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B4 levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2

Summary.u2002 Background:u2002Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A2 formation is non‐linear and TXA2 generation must be inhibited by at least 95% to inhibit TXA2‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives:u2002To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA2 generation and platelet activity. Method:u2002Platelet aggregation, thrombi adhesion and TXA2 production in response to arachidonic acid (0.03–1u2003mmolu2003L−1), collagen (0.1–30u2003μgu2003mL−1), epinephrine (0.001–100 μmol L−1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30u2003μmolu2003L−1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75u2003mg, o.d.) for 7u2003days. Results:u2002Platelet aggregation, adhesion and TXA2 production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC50 values that did not differ. A linear relationship existed between aggregation and TXA2 production for all combinations of arachidonic acid or collagen and aspirin (Pu2003<u20030.01; R2 0.92; nu2003=u2003224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions:u2002These studies demonstrate a linear relationship between inhibition of platelet TXA2 generation and TXA2‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.