Antonio Piccoli

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.

Neutrophils phagocytose activated platelets in vivo: A phosphatidylserine, P-selectin, and β2 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-α 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)-&agr; 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/cm2 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)F1&agr; (the hydrolysis product of prostacyclin), PGE2, and PGD2. In contrast, TNF-&agr; 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-&agr; 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-&agr; 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-&agr; generation in human endothelial cells.

Polymorphonuclear leukocytes enhance release of growth factors by cultured endothelial cells.

Porcine aortic endothelial cells (PAECs) in culture constitutively secrete polypeptide (endothelium-derived) growth factors (EDGFs) into the surrounding medium. Incubation of PAECs with human peripheral blood polymorphonuclear leukocytes (PMNs) caused a significant increase in EDGF release as assessed by [3H]thymidine incorporation into BALB/c 3T3 mouse fibroblasts and cell proliferation assay. The effect was time dependent and correlated with the number of PMNs, reaching a maximum with a 1:1 PAEC to PMN ratio. Generation of mitogenic activity was prevented by cycloheximide, indicating a requirement for de novo protein synthesis. Antibody-mediated inhibition assays suggested that mitogenic activity was due to platelet-derived growth factor and basic fibroblast growth factor. When supernatant from N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs was substituted for PMNs during incubation with PAECs, powerful mitogenic activity was generated, indicating the involvement of soluble mediators. A role for free oxygen radicals was ruled out by experiments in which superoxide dismutase and catalase did not prevent the increase in mitogenic activity. By contrast, serine protease inhibitors such as soybean trypsin inhibitor, alpha 1-antitrypsin, and eglin C reduced the PMN-stimulating activity by 70%, 80%, and 100%, respectively. The possible involvement of cathepsin G and elastase was investigated. Cathepsin G and elastase, when substituted for PMNs, increased the release of EDGFs in a dose-dependent fashion, mimicking the effect of PMNs. These findings suggest a new role for leukocyte-vessel wall interactions in the proliferative feature of atherosclerosis.

SRC-dependent outside-in signalling is a key step in the process of autoregulation of β2 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.

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.

Polymorphonuclear Leukocytes Induce PDGF Release From IL-1β–Treated Endothelial Cells Role of Adhesion Molecules and Serine Proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.

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.

Roflumilast inhibits leukocyte–platelet interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes and monocytes

Essentials Thrombosis is a major comorbidity in patients with chronic obstructive pulmonary disease (COPD). Roflumilast is a selective phosphodiesterase type‐4 (PDE4) inhibitor approved for treatment of severe COPD. PDE4 blockade by roflumilast inhibits prothrombotic functions of neutrophils and monocytes. PDE4 inhibitors may reduce thrombotic risk in COPD as well as in other vascular diseases.

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

We studied the effects of 17β-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.