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.

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.

Effect of pioglitazone versus metformin on cardiovascular risk markers in type 2 diabetes.

INTRODUCTION: Besides its critical role in metabolic homeostasis, peroxisome proliferator-activated receptor (PPAR)-γ modulates several cellular responses involved in atherothrombosis. This multicenter, double-blind, randomized study investigated the effects of two oral hypoglycemic agents on markers of inflammation, platelet activation, thrombogenesis, and oxidative stress in patients with type 2 diabetes. METHODS AND RESULTS: The primary objective of this study was to evaluate the effect on C-reactive protein (CRP) after a 16-week treatment period with either pioglitazone or metformin. Additionally, markers of vascular inflammatory response, platelet activation, thrombogenesis, oxidative stress, glucose, and lipid metabolism, as well as liver function, were measured. In total, 50 patients completed the study. Pioglitazone-treated patients were found to have statistically significantly larger decreases in mean CRP levels (-0.4 mg/dL) compared to those treated with metformin (-0.2 mg/dL) (P=0.04), as well as greater reductions in levels of mean fasting plasma glucose (-27 vs. -9 mg/dL; P=0.01), serum insulin (-2 vs. -1.9 mU/L; P=0.014), homeostatic model assessment (HOMA) (-1.2 vs. -0.9; P=0.015), and E-selectin (-12.4 vs. +3.4 µg/mL; P=0.01). Mean glycated hemoglobin (HbA1c) levels decreased in both treatment groups from baseline to week 16 (-0.4% in the pioglitazone group, -0.2% in the metformin group; P=0.36). Pioglitazone treatment was also found to be associated with a statistically significant increase in total cholesterol levels (+10 mg/dL in the pioglitazone arm, -3 mg/dL in the metformin arm; P=0.05) and a decrease in liver enzyme levels. CONCLUSIONS: The favorable changes in markers of systemic and vascular inflammatory response with pioglitazone suggest that it may positively influence the atherothrombotic process in type 2 diabetes.

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.

Effects of estrogen on endothelial prostanoid production and cyclooxygenase-2 and heme oxygenase-1 expression.

We studied the effects of 17ß-estradiol (E2) (10, 40 nM) on 2 vasoprotective pathways, i.e. cyclooxygenase-2 (COX-2)-dependent prostanoids and the antioxidant heme oxygenase-1 (HO-1), in human umbilical vein endothelial cells (HUVEC) exposed for 6h to steady laminar shear stress (LSS, 10 dyn/cm²), characteristic of atherosclerotic lesion-protected areas. COX-2 was induced by LSS versus static condition (SC). E2 did not significantly affect COX-2 expression in HUVEC cultured in SC or exposed to LSS. Prostacyclin (PGI2) and prostaglandin (PG)E2 were induced while PGF(2α) was reduced by LSS. E2 caused no effect or a small reduction of prostanoid biosynthesis. In HUVEC cultured in SC or exposed to LSS, E2 10 nM caused a comparable HO-1 induction (35-45%) while E2 40 nM was 5-fold more potent in LSS-exposed HUVEC than in SC (290% and 58%, respectively). PGI2 receptor antagonist RO3244794 did not affect HO-1 induction by E2. In conclusion, E2 may restrain oxidant stress in the endothelium through HO-1 induction by a mechanism independent on PGI2 signaling.

Fucans, but not fucomannoglucuronans, determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed.

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.

Platelet-leukocyte interactions in cardiovascular disease and beyond.

Platelet-leukocyte interactions define a basic cell process that is characterized by the exchange of signals between platelets and different types of leukocytes and that bridges 2 fundamental pathophysiological events: atherothrombosis and inflammatory immune reactions. When this process takes place at the site of atherosclerotic plaque development or at the site of endothelial injury, platelet-dependent leukocyte recruitment and activation contributes to the inflammatory reaction of the vessel wall, which accounts for the exacerbation of atherosclerosis and for intimal hyperplasia and plaque instability. Moreover, platelet-leukocyte interactions may have a key role in modulating a wide array of responses of both the innate and adaptive immune systems, thus contributing to the pathogenesis of inflammatory diseases and tissue damage, as well as to host defense.

Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and {beta}2 integrin-dependent cell clearance program.

Activated platelets express ligands, which are recognized by counterreceptors on neutrophils. Here, we show that the ensuing cell-to-cell interaction programs neutrophil phagocytic function, resulting in activated platelet clearance. Neutrophils that have internalized platelets circulate in the blood of patients with acute myocardial infarction, and the extent of platelet clearance correlates with expression of platelet activation, including P-selectin. Activated platelets injected intravenously in experimental animals are detectable in circulating neutrophils 60 minutes after, and within 3 hours, more than 70% circulating neutrophils have internalized platelets. Platelet clearance comprises 2 events: adhesion to neutrophils, which requires divalent cations and depends on P-selectin, on the P-selectin glycoprotein ligand-1 (PSGL-1), and on the CD11b/CD18 beta2 integrin; and internalization, which is abrogated by the phosphatidylserine-binding protein annexin A5. Adhesion to platelets causes neutrophil degranulation and is blocked by antibodies specific for P-selectin and PSGL-1, either in a synthetic medium in vitro or in the whole blood, therefore in the presence of a physiologic array of plasma cofactors and opsonins. The data suggest that the interaction between circulating platelets and neutrophils influences innate immune functions, possibly contributing to regulate vascular inflammation.

Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells.

Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2-dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-alpha generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm(2) for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (P<0.05) increase of 6-keto-prostaglandin (PG)F(1alpha) (the hydrolysis product of prostacyclin), PGE(2), and PGD(2). In contrast, TNF-alpha released in the medium in 6 hours (3633+/-882 pg) or detected in cells lysates (1091+/-270 pg) was significantly (P<0.05) reduced versus static condition (9100+/-2158 and 2208+/-300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-alpha generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2-dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF-alpha reduction by LSS. These findings suggest that inhibition of COX-2-dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF-alpha generation in human endothelial cells.

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds.

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.

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.

Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin.

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.

Prevention of cardiovascular disease in type-2 diabetes: how to improve the clinical efficacy of aspirin.

Atherosclerotic cardiovascular disease and its thrombotic complications are the principal causes of morbidity and mortality in patients with type-2 diabetes. Aspirin reduces the risk of thrombotic events in a broad range of patients with vascular disease and, in selected individuals, is beneficial for primary prevention. Although recommended by existing guidelines, in secondary and in primary prevention trials the clinical efficacy of low-dose aspirin in patients with diabetes appears to be substantially lower than in individuals without diabetes. In this review, we discuss possible mechanisms that may contribute to reduce the antithrombotic activity of aspirin in diabetes. We also discuss adjuvant therapies used in diabetic patients that may potentially improve the antithrombotic efficacy of aspirin.

SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.

In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.