Prenylcysteine oxidase 1, an emerging player in atherosclerosis.

The research into the pathophysiology of atherosclerosis has considerably increased our understanding of the disease complexity, but still many questions remain unanswered, both mechanistically and pharmacologically. Here, we provided evidence that the pro-oxidant enzyme Prenylcysteine Oxidase 1 (PCYOX1), in the human atherosclerotic lesions, is both synthesized locally and transported within the subintimal space by proatherogenic lipoproteins accumulating in the arterial wall during atherogenesis. Further, Pcyox1 deficiency in Apoe-/- mice retards atheroprogression, is associated with decreased features of lesion vulnerability and lower levels of lipid peroxidation, reduces plasma lipid levels and inflammation. PCYOX1 silencing in vitro affects the cellular proteome by influencing multiple functions related to inflammation, oxidative stress, and platelet adhesion. Collectively, these findings identify the pro-oxidant enzyme PCYOX1 as an emerging player in atherogenesis and, therefore, understanding the biology and mechanisms of all functions of this unique enzyme is likely to provide additional therapeutic opportunities in addressing atherosclerosis.

Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation.

AIM: The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. METHODS AND RESULTS: PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (-80.36±11.5% and -95.53±4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (-45.55±1.37% and -53.39±9.8%), thus pointing to a protective effect independent of a hematopoietic cell's derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. CONCLUSION: PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis.

In vivo prostacyclin biosynthesis and effects of different aspirin regimens in patients with essential thrombocythaemia.

Essential thrombocythaemia (ET) is characterised by enhanced platelet generation and thrombosis. Once daily (od) aspirin incompletely inhibits platelet thromboxane (TX)A2 production in ET. A twice daily (bid) dosing is necessary to fully inhibit TXA2. Whether this dosing regimen affects in vivo prostacyclin (PGI2) biosynthesis is unknown. PGI2 biosynthesis was characterised in 50 ET patients on enteric-coated (EC) aspirin 100 mg od, by measuring its urinary metabolite, 2,3-dinor-6-keto-PGF1α (PGI-M). Moreover, in a crossover study 22 patients poorly responsive to standard aspirin based on serum TXB2 levels (≥4 ng/ml) were randomised to different seven-day aspirin regimens: EC aspirin 100 mg od, 100 mg bid, 200 mg od, or plain aspirin 100 mg od. PGI-M measured 24 hours after the last aspirin intake (EC, 100 mg od) was similar in patients and healthy subjects both on (n=10) and off (n=30) aspirin. PGI-M was unrelated to in vivo TXA2 biosynthesis, and not affected by EC aspirin 100 mg bid or 200 mg od as compared to EC 100 mg od. PGI2 biosynthesis in aspirin-treated ET patients appears unrelated to TXA2 biosynthesis, and not affected by an improved aspirin regimen, demonstrating its vascular safety for future trials.

Mitochondrial reactive oxygen species: a common pathway for PAR1- and PAR2-mediated tissue factor induction in human endothelial cells.

BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. OBJECTIVES: To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real-time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2'-7'-dichlorofluorescein diacetate. RESULTS: ROS generated by the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF-kappaB activation has been identified. CONCLUSIONS: Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.

15-deoxy-delta12,14-Prostaglandin J2 inhibits tissue factor expression in human macrophages and endothelial cells: evidence for ERK1/2 signaling pathway blockade.

Basic and clinical evidence has provided insight into the molecular events that link inflammation and coagulation. Increased expression of tissue factor (TF) by circulating and vascular cells has been indicated as responsible for the thrombotic complications associated with acute and chronic inflammation. TF is indeed inducible in circulating and vascular cells by cytokines and bacterial lipopolysaccharide (LPS) and its expression triggers the coagulation. The cyclopentenone prostaglandins are naturally occurring prostaglandin D2 (PGD2) derivatives that comprises prostaglandin J2 (PGJ2) and its metabolites delta12-PGJ2 and 15-deoxy- delta12,14-prostaglandin J2 (15d-PGJ2). These compounds, detected in vivo in a later phase of the inflammatory response, are characterized by anti-inflammatory activity and participate to the resolution of inflammation. We have here investigated the effect of 15d-PGJ2 on TF expression in human macrophages and endothelial cells (HUVEC). Our results indicate that 15d-PGJ2 down-regulates LPS- and TNFalpha-induced TF activity, protein and mRNA through inhibition of TF gene transcription. The effect of 15d-PGJ2 is targeted to the NF-kappaB/I-kappaB pathway and to the mitogen activated protein kinase ERK1/2. A role of PPAR-gamma activation in TF inhibition by 15d-PGJ2 was excluded. We conclude that 15d-PGJ2 negatively affects TF expression in macrophages and endothelial cells through a PPARgamma-independent mechanism. This down-regulation may be crucial to limit excessive blood clotting activation in immuno-inflammatory diseases.