Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis.

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

Mechanisms of endothelial cell dysfunction in cystic fibrosis.

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans­endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a ß2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.

The natural phosphoinositide derivative glycerophosphoinositol inhibits the lipopolysaccharide-induced inflammatory and thrombotic responses.

Inflammatory responses are elicited through lipid products of phospholipase A2 activity that acts on the membrane phospholipids, including the phosphoinositides, to form the proinflammatory arachidonic acid and, in parallel, the glycerophosphoinositols. Here, we investigate the role of the glycerophosphoinositol in the inflammatory response. We show that it is part of a negative feedback loop that limits proinflammatory and prothrombotic responses in human monocytes stimulated with lipopolysaccharide. This inhibition is exerted both on the signaling cascade initiated by the lipopolysaccharide with the glycerophosphoinositol-dependent decrease in IκB kinase α/ß, p38, JNK, and Erk1/2 kinase phosphorylation and at the nuclear level with decreased NF-κB translocation and binding to inflammatory gene promoters. In a model of endotoxemia in the mouse, treatment with glycerophosphoinositol reduced TNF-α synthesis, which supports the concept that glycerophosphoinositol inhibits the de novo synthesis of proinflammatory and prothrombotic compounds and might thus have a role as an endogenous mediator in the resolution of inflammation. As indicated, this effect of glycerophosphoinositol can also be exploited in the treatment of manifestations of severe inflammation by exogenous administration of the compound.

Phosphodiesterase type 4 blockade prevents platelet-mediated neutrophil recruitment at the site of vascular injury.

OBJECTIVE: Platelet-neutrophil interactions play a key role in cardiovascular disease and inflammatory processes. Src family kinases mediate P-selectin glycoprotein ligand-1-Mac-1 cross talk necessary for firm platelet-neutrophil adhesion. Because Src family kinase activity can be regulated by cAMP-dependent pathways, in this work, we evaluated the role of phosphodiesterases in the signaling events that are required to sustain platelet-neutrophil interactions and neutrophil recruitment at the site of vascular injury. APPROACH AND RESULTS: In neutrophils exposed to P-selectin, selective phosphodiesterase 4 (PDE4) inhibition prevented Src family kinase-mediated phosphorylation of the proline-rich tyrosine kinase 2 on Tyr579/Tyr580. The effects of PDE4 inhibition required protein kinase A, likely through protein kinase A-mediated activation of COOH-terminal Src kinase, a major negative regulator of Src family kinases. PDE4, but not other phosphodiesterase inhibitors, reduced platelet-neutrophil conjugates as well as neutrophil firm adhesion on spread platelets under flow conditions. The effect of PDE4 inhibition on neutrophil adhesion was primarily mediated by downregulation of P-selectin-induced activation of Mac-1. In a murine model of endovascular injury, selective inhibition of PDE4 significantly reduced neutrophil recruitment at the site of vascular damage. CONCLUSIONS: This study identifies PDE4 as a central node in the signaling network that mediates platelet-neutrophil adhesion and suggests that pharmacological inhibition of PDE4 may represent a novel therapeutic avenue for the treatment of cardiovascular disease.

Prasugrel inhibits platelet-leukocyte interaction and reduces inflammatory markers in a model of endotoxic shock in the mouse.

Prasugrel, through its active metabolite, reduces atherothrombosis and its clinical manifestations by inhibiting platelet activation and aggregation. Platelets also contribute to inflammation through interaction with different classes of leukocytes. We investigated whether the inhibitory effect of prasugrel on platelets also counteract inflammatory responses. The effect of prasugrel active metabolite, R-138727, was investigated on platelet P-selectin expression, platelet adhesion to polymorphonuclear leukocytes (PMN) and monocytes (MN) and Mac-1 expression in PMN and MN, in vitro, in human cells. The ex vivo effect of prasugrel administration on P-selectin, thromboxane (TXB)2 formation, platelet-PMN conjugates and Mac-1 expression in PMN triggered by PAR-4 agonist peptide was examined in whole blood from healthy mice as well as from mice in which an acute inflammatory reaction was induced by treatment with endotoxin. The effect of prasugrel on inflammatory markers in endotoxin-treated animals was also tested in vivo. R-138727 inhibited agonist-stimulated expression of platelet P-selectin, platelet-PMN and platelet-MN adhesion and platelet-dependent Mac-1 expression in leukocytes. Addition of aspirin did not modify the inhibitory effect elicited by R-138727. Treatment of mice with prasugrel resulted in a profound inhibition of platelet P-selectin expression, TXB2 production, platelet-PMN adhesion and Mac-1 expression in PMN induced by ex vivo stimulation with PAR-4 agonist peptide of whole blood from healthy or endotoxin-treated mice. Measurement of markers revealed that prasugrel reduced TXB2 and tumour necrosis factor-α synthesis and increased nitric oxide metabolites in endotoxin-treated mice in vivo. In conclusion, prasugrel reduces platelet interactions with PMN and MN. Through these effects prasugrel may curb platelet-mediated inflammatory responses.

Src family kinases mediate neutrophil adhesion to adherent platelets.

Polymorphonuclear leukocyte (PMN)-platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of "outside-in" signaling by Src-family tyrosine kinases (SFKs) in the regulation of alphaMbeta2-integrin-dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in beta2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from alphaMbeta2(-/-), hck(-/-)fgr(-/-), and hck(-/-)fgr(-/-)lyn(-/-) mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of alphaMbeta2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for alphaMbeta2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin-PSGL-1-mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

The lipoxygenase-cyclooxygenase inhibitor licofelone prevents thromboxane A2-mediated cardiovascular derangement triggered by the inflammatory peptide fMLP in the rabbit.

Licofelone is an analogue of arachidonic acid that inhibits 5-lipoxygenase (LOX), cyclooxygenase (COX)-1 and COX-2. We investigated the effects of licofelone on cardiovascular derangements and production of thromboxane (Tx)A(2) induced by the inflammatory agonist n-formyl-methionyl-leucyl-phenylalanine (fMLP) in the rabbit, in comparison with those of aspirin or rofecoxib, inhibitors of COX-1 and COX-2, respectively. In control rabbits, injection of fMLP (30 nmol/kg) in the jugular vein evokes ischemic electrocardiographic (ECG) changes in the first 1-5 min, i.e. a profound depression of the ST segment and inversion of the T wave. Simultaneously, fMLP induces bradycardia and hypotension and increases TxB(2) blood levels. All changes are transient. Licofelone (60 mg/kg/5 days, p.os) prevented fMLP-induced ECG ischemic changes in all treated animals, reverted bradycardia and hypotension, and significantly reduced TxB(2). Aspirin (10 mg/kg/5 days, p.os) prevented ischemic ECG alterations in 2 out of 5 treated animals and did not modify either bradycardia or hypotension. One rabbit died two min after fMLP. In 2 rabbits, aspirin reduced TxB(2) levels by more than 80% respect to mean control values; the remaining two rabbits produced an amount of TxB(2) similar to controls. These two rabbits also showed ischemic ECG changes. Rofecoxib (10 mg/kg/5 days, p.os) did not prevent fMLP-induced ischemic ECG alteration, bradycardia and hypotension, and did not significantly modify the increase of TxB(2). These results indicate that the capacity of licofelone to efficiently suppress TxA(2) production, is responsible for the protection from the cardiovascular derangement triggered by an inflammatory stimulus.

Clopidogrel inhibits platelet-leukocyte adhesion and platelet-dependent leukocyte activation.

Clopidogrel is considered to be an important therapeutic advance in anti-platelet therapy. We investigated whether inhibition by clopidogrel results in a reduced capacity of platelets to adhere and stimulate pro-atherothrombotic and inflammatory functions in polymorphonuclear leukocytes (PMN) and in monocytes (MN). An eventual effect on these processes could further substantiate anti-atherothrombotic properties of this drug. The effects of clopidogrel or of its active metabolite were investigated on ADP or thrombin receptor-induced platelet activation and on platelet-leukocyte interactions ex vivo in the mouse or in vitro in isolated human cells or whole blood, respectively. Clopidogrel inhibited platelet aggregation, expression of P-selectin, platelet-PMN adhesion and platelet-dependent ROS production in mouse PMN. Similarly pretreatment of human platelets with the active metabolite of clopidogrel in vitro resulted in a profound inhibition of platelet P-selectin expression, platelet-PMN adhesion and production of ROS by PMN. Pretreatment with the active metabolite of clopidogrel significantly impaired the ability of platelets to up-regulate the expression of TF procoagulant activity in MN, in a washed cell system. Moreover, the active metabolite of clopidogrel inhibited rapidTF exposure on platelet as well as on leukocyte surfaces in whole blood. By reducing platelet-dependent up-regulation of inflammatory and pro-atherothrombotic functions in leukocytes, clopidogrel may reduce inflammation that underlies the chronic process of atherosclerosis and its acute complications.

Pharmacokinetic and pharmacodynamic differences between two low dosages of aspirin may affect therapeutic outcomes.

BACKGROUND: Meta-analyses of the prevention of major vascular events by aspirin suggest therapeutic equivalence of all dosages. However, the optimal dosage still remains problematic, and a recent trial found aspirin 160 mg/day to be more effective than 80 mg/day for secondary prevention of ischaemic stroke. OBJECTIVE: To evaluate two low dosages of aspirin in terms of pharmacokinetics and pharmacodynamics (inhibition of platelet thromboxane generation and urinary excretion of thromboxane and prostacyclin metabolites). DESIGN AND PARTICIPANTS: A randomised cross-over study was performed in 16 healthy volunteers (9 women and 7 men, 33.8 +/- 5.1 years old) given enteric-coated aspirin 80 or 160 mg/day for 7 days. METHODS: Plasma concentrations of salicylate and aspirin were measured by high-performance liquid chromatography (HPLC) after both the first and the last dose (days 1 and 7). The usual pharmacokinetic parameters were then derived. Serum thromboxane B2 (TxB2) was measured by radioimmunoassay. The urinary excretion of 11-dehydro-TxB2 and 2,3-dinor-6-keto-prostaglandin F1alpha were measured on 8-hour urine samples by immunoassay after extraction and HPLC separation, both before and after 7 days of drug administration. RESULTS: With the 160 mg dosage, but not with the 80 mg dosage, higher concentrations of aspirin were found at day 7 compared with day 1. For aspirin 80 mg/day, 24-hour area under the concentration-time curve (AUC24) was similar on days 1 and 7 (569 +/- 339 vs 605 +/- 377 microg. h/L), but increased from 904 +/- 356 microg. h/L on day 1 to 1355 +/- 883 microg. h/L on day 7 with the higher dosage. Similarly, the AUC24 for salicylate was similar on days 1 and 7 with the lower dosage, but significantly increased from day 1 to day 7 after the higher dosage. This paralleled inhibition of serum TxB2 levels (99% vs 95% average inhibition by 160 and 80 mg/day) and of urinary excretion of thromboxane metabolite (77% vs 61% average inhibition by 160 and 80 mg/day), without altering the excretion of prostacyclin metabolite. CONCLUSIONS: Inhibition of serum TxB2 generation and of thromboxane metabolite urinary excretion by the lower dosage of aspirin, although substantial, still appeared incomplete. The small but significant further increase of serum TxB2 inhibition by the higher dosage was accompanied by an even greater inhibition of urinary excretion. We suggest that in some instances this difference would translate into a greater clinical benefit with the higher aspirin dosage. Our findings may also contribute to better definition of the recent concept of 'aspirin resistance'.

Licofelone, a dual lipoxygenase-cyclooxygenase inhibitor, downregulates polymorphonuclear leukocyte and platelet function.

Polymorphonuclear leukocytes are strongly implicated in the pathogenesis of inflammatory disease. Polymorphonuclear leukocyte recruitment at sites of inflammation, mainly sustained by the beta2-integrins, is followed by the synthesis and release of inflammatory mediators, such as leukotrienes, proteolytic enzymes and reactive oxygen species. Functional and metabolic interactions between polymorphonuclear leukocytes and platelets can contribute to and exacerbate the process. The effects of the dual 5-lipoxygenase and cyclooxygenase inhibitor licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) were studied on arachidonic acid transcellular metabolism occurring between polymorphonuclear leukocytes and platelets. The formation of leukotriene C(4), a leukotriene A(4)-derived metabolite, by mixed polymorphonuclear leukocyte/platelet suspensions stimulated with 10 microM A23187 was inhibited by licofelone with an IC(50) of 3.8 +/- 0.07 microM. The formation of 5,12-di-hydroxy-eicosatetraenoic acid (HETE) was abolished at concentrations > or = 10 microM. Licofelone also inhibited the generation of reactive oxygen species by polymorphonuclear leukocytes stimulated with 1 microM n-formyl-methionyl-leucyl-phenylalanine (fMLP), 10 nM complement fraction 5a (C5a) and 1 microM platelet activating factor (PAF) with IC(50)s of 24.4 +/- 0.6, 11.0 +/- 1.5 and 11.7 +/-1.2 microM; elastase release induced by the three agonists was inhibited with IC(50)s of 12.2 +/- 2.2, 23.5 +/- 8 and 2.6 +/- 1 microM, respectively. Homotypic polymorphonuclear leukocyte aggregation induced by fMLP, C5A and PAF was inhibited by licofelone with IC(50)s of 23.7 +/- 4.8, 15.6 +/- 3.4 and 15.4 +/- 4 microM, respectively. The present study extends the anti-lipoxygenase and anti-cyclooxygenase activities of licofelone to the production of arachidonic acid metabolites generated as a consequence of polymorphonuclear leukocyte-platelet transcellular metabolism and to polymorphonuclear leukocyte responses relevant to the pathogenesis of inflammation. The coexistence within the same molecule of a wide spectrum of anti-inflammatory properties is of interest.