Angelomaria Di Santo

Long Pentraxin PTX3 Upregulates Tissue Factor Expression in Human Endothelial Cells: A Novel Link Between Vascular Inflammation and Clotting Activation

Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1&bgr; and tumor necrosis factor-&agr;, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein I&kgr;B&agr;. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease.

Angiotensin-Converting Enzyme Inhibitors Downregulate Tissue Factor Synthesis in Monocytes

Angiotensin-converting enzyme (ACE) inhibitors reduce the risk of recurrent myocardial infarction in patients with left ventricular dysfunction. Tissue factor (TF), the initiator of blood coagulation, plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Because monocytes synthesize TF and contain several components of the renin-angiotensin system, we investigated the possibility that ACE inhibitors could modulate monocyte TF expression. Mononuclear leukocytes from healthy volunteers were incubated with endotoxin in the presence or absence of different ACE inhibitors. Captopril reduced TF expression in endotoxin-stimulated mononuclear leukocytes, as measured by a 1-stage clotting assay and ELISA analysis, by approximately 60%. The effect was dose-dependent and was attributable to ACE inhibition, given that other ACE inhibitors, such as idrapril or fosinopril, and losartan, an antagonist of the angiotensin II AT(1) receptor, caused a comparable reduction in TF activity. Reverse transcriptase-polymerase chain reaction indicated that endotoxin-mediated increased levels of TF mRNA were inhibited by ACE inhibitors. Moreover, endotoxin-induced nuclear factor-kappaB translocation to the promoter region of the gene encoding for TF was markedly inhibited by captopril. The finding that ACE inhibitors and angiotensin II AT(1) antagonists can potentially modulate TF expression by mononuclear cells has important biological and therapeutic implications for the evolution of thrombi. Our results suggest that the anti-ischemic effect of these drugs might be explained, at least in part, by their ability to reduce TF expression in monocytes.

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.

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation

Pentraxin‐3 (PTX3), an acute‐phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one‐stage clotting time. PTX3 enhanced TF activity and antigen from LPS‐stimulated monocytes in a dose‐dependent way. The effect was specific, as other PTXs, such as C‐reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF‐inducing agents such as interleukin‐1β and tumor necrosis factor α, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IκB, α phosphorylation and degradation and increased migration of the transacting factor c‐Rel/p65 into the nucleus, as determined by Western blot and electro‐mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell‐mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.

Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells

Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8‐day‐old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum‐free medium (containing 5 mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase‐mediated dUTP‐fluorescein nick end‐labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K+ (5 mM) concentrations, the potential anti‐apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10 μg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K+. Since there is evidence that low K+‐induced apoptosis in CGNs is associated with a drop in intracellular Ca2+ concentration ([Ca2+]i), activation of the cell death effector proteases caspase‐3 and caspase‐9, and of the transcription factor nuclear factor kappa B (NF‐κB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca2+]i induced by low K+, whereas it completely blocked caspase‐3, caspase‐9, and NF‐κB activation. It is concluded that CAPE could exert its anti‐apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.

Inhibition of the renin-angiotensin system downregulates tissue factor and vascular endothelial growth factor in human breast carcinoma cells

INTRODUCTIONnThe renin-angiotensin system (RAS) promotes angiogenesis and growth of neoplastic cells. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor AT1 blockers may protect against cancer. Tissue factor (TF), for its involvement in tumor growth, angiogenesis, and metastasis is considered a hallmark of cancer progression. In this study we evaluated whether RAS blockade modulates TF constitutive expression by the metastatic breast carcinoma MDA-MB-231 cell line.nnnMATERIALS AND METHODSnCell TF activity was assessed by one stage clotting time, TF and VEGF antigens and mRNA levels by ELISA and RT-PCR, respectively. AT(1) was detected by flow-cytometry and angiotensin-II levels by EIA.nnnRESULTSnCaptopril reduced in a concentration-dependent way both the strong constitutive TF activity (983.2±55.2 vs. 686.7±135.1U/5×10(5) cells with 10μg/ml captopril) and antigen (32.3±5.9 vs. 13.2±6.6ng/ml) in MDA-MB-231. Similar results were observed with enalapril. AT1 was present on cell membrane and losartan, a competitive inhibitor of AT1, reduced TF expression to a degree similar as that exerted by ACE inhibitors. Moreover, captopril and losartan downregulated the constitutive mRNA TF expression by ~35%. Similar results were observed with anti-AT1 and angiotensin II antibodies. In addition, the constitutive VEGF antigen and mRNA levels were reduced in the presence of captopril or losartan, and an anti-VEGF antibody downregulated cell TF activity by ~40%.nnnCONCLUSIONSnThese results could, at least in part, contribute to the discussion about the possible effects of ACE inhibitors and AT1 receptor antagonists in malignancy, and offer new clues to support their use for tumor control.

Paclitaxel downregulates tissue factor in cancer and host tumour-associated cells

Paclitaxel, a microtubule-stabilising compound with potent anti-tumour activity, has been clinically used in a wide variety of malignancies. Tissue factor (TF) is often expressed by tumour-associated endothelial and inflammatory cells, as well as by cancer cells themselves, and it is considered a hallmark of cancer progression. We investigated whether paclitaxel could modulate TF in human mononuclear (MN) cells, human umbilical vein endothelial cells (HUVEC) and the metastatic breast carcinoma cell line MDA-MB-231. Cells were incubated with or without paclitaxel at 37 degrees C. At the end of incubation, cells were disrupted and tested for procoagulant activity by a one-stage clotting assay, for TF antigen levels by ELISA and TF mRNA by real-time RT-PCR. IL-6 and IL-1beta were tested by ELISA in conditioned medium. Both the strong TF activity and antigen constitutively expressed by MDA-MB-231 and the TF induced by LPS, TNF-alpha and IL-1beta in MN cells and HUVEC were significantly reduced by paclitaxel. In the presence of paclitaxel, lower TF mRNA levels were also detected. Since paclitaxel has been shown to induce the expression of inflammatory genes in monocytes and tumour cells, we tested whether paclitaxel could influence IL-6 and IL-1beta release from the cells used in this paper. Neither the constitutive expression of IL-6 and IL-1beta by MDA-MB-231 nor the basal and LPS-induced release from MN cells and HUVEC was affected. Our data support the hypothesis that the anti-tumour effects of paclitaxel may, at least in part, be mediated by the capacity of this drug to modulate the procoagulant potential of cancer and host cells.

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.

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/β, 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.