Nicole Malet

Chronic pharmacological activation of P2Y13 receptor in mice decreases HDL-cholesterol level by increasing hepatic HDL uptake and bile acid secretion.

High level of high-density lipoprotein cholesterol (HDL-cholesterol) is inversely correlated to the risk of atherosclerotic cardiovascular disease. The protective effect of HDL is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and is later delivered to the liver for further metabolism and bile excretion. We have previously demonstrated that P2Y13 receptor is critical for RCT and that intravenous bolus injection of cangrelor (AR-C69931MX), a partial agonist of P2Y13 receptor, can stimulate hepatic HDL uptake and subsequent lipid biliary secretion without any change in plasma lipid levels. In the present study, we investigated the effect of longer-term treatment with cangrelor on lipoprotein metabolism in mice. We observed that continuous delivery of cangrelor at a rate of 35μg/day/kg body weight for 3days markedly decreased plasma HDL-cholesterol level, by increasing the clearance of HDL particles by the liver. These effects were correlated to an increase in the rate of biliary bile acid secretion. An increased expression of SREBP-regulated genes of cholesterol metabolism was also observed without any change of hepatic lipid levels as compared to non-treated mice. Thus, 3-day cangrelor treatment markedly increases the flux of HDL-cholesterol from the plasma to the liver for bile acid secretion. Taken together our results suggest that P2Y13 appears a promising target for therapeutic intervention aimed at preventing or reducing cardiovascular risk.

The activation function-1 of estrogen receptor alpha prevents arterial neointima development through a direct effect on smooth muscle cells

Supplemental Digital Content is available in the text.

Mitochondrial inhibitory factor 1 (IF1) is present in human serum and is positively correlated with HDL-cholesterol.

Background Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in High Density Lipoproteins (HDL). On hepatocytes, apoA-I binds to cell surface ATP synthase (namely ecto-F1-ATPase) and stimulates its ATPase activity, generating extracellular ADP. This production of extracellular ADP activates a P2Y13-mediated HDL endocytosis pathway. Conversely, exogenous IF1, classically known as a natural mitochondrial specific inhibitor of F1-ATPase activity, inhibits ecto-F1-ATPase activity and decreases HDL endocytosis by both human hepatocytes and perfused rat liver. Methodology/Principal Findings Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 is present in the systemic circulation in humans. We first unambiguously detected IF1 in human serum by immunoprecipitation and mass spectrometry. We then set up a competitive ELISA assay in order to quantify its level in human serum. Analyses of IF1 levels in 100 normolipemic male subjects evidenced a normal distribution, with a median value of 0.49 µg/mL and a 95% confidence interval of 0.22–0.82 µg/mL. Correlations between IF1 levels and serum lipid levels demonstrated that serum IF1 levels are positively correlated with HDL-cholesterol and negatively with triglycerides (TG). Conclusions/Significance Altogether, these data support the view that, in humans, circulating IF1 might affect HDL levels by inhibiting hepatic HDL uptake and also impact TG metabolism.

PI3K signaling in arterial diseases: Non redundant functions of the PI3K isoforms.

Cardiovascular diseases are the most common cause of death around the world. This includes atherosclerosis and the adverse effects of its treatment, such as restenosis and thrombotic complications. The development of these arterial pathologies requires a series of highly-intertwined interactions between immune and arterial cells, leading to specific inflammatory and fibroproliferative cellular responses. In the last few years, the study of phosphoinositide 3-kinase (PI3K) functions has become an attractive area of investigation in the field of arterial diseases, especially since inhibitors of specific PI3K isoforms have been developed. The PI3K family includes 8 members divided into classes I, II or III depending on their substrate specificity. Although some of the different isoforms are responsible for the production of the same 3-phosphoinositides, they each have specific, non-redundant functions as a result of differences in expression levels in different cell types, activation mechanisms and specific subcellular locations. This review will focus on the functions of the different PI3K isoforms that are suspected as having protective or deleterious effects in both the various immune cells and types of cell found in the arterial wall. It will also discuss our current understanding in the context of which PI3K isoform(s) should be targeted for future therapeutic interventions to prevent or treat arterial diseases.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response

PI3Kγ plays a major role in the initiation and progression of intimal hyperplasia by specifically modulating Th1 cytokines leading to CXCL10 and RANTES production by smooth muscle cells.

Key role of PI3Kγ in monocyte chemotactic protein-1-mediated amplification of PDGF-induced aortic smooth muscle cell migration

Vascular smooth muscle cell (SMC) migration within the arterial wall is a crucial event in atherogenesis and restenosis. Monocyte chemotactic protein‐1/CC‐chemokine receptor 2 (MCP‐1/CCR2) signalling is involved in SMC migration processes but the molecular mechanisms have not been well characterized. We investigated the role of PI3Kγ in SMC migration induced by MCP‐1.

Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2.

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.

PI3Kβ Plays a Key Role in Apolipoprotein A-I-Induced Endothelial Cell Proliferation Through Activation of the Ecto-F 1 -ATPase/P2Y 1 Receptors

Background/Aims: High-density lipoproteins (HDL) exert multiple cardioprotective functions on the arterial wall, including the promotion of endothelial cell survival and proliferation. Among mechanism contributing to endothelial protection, it has been reported that apolipoprotein A-I (apoA-I), the major protein in HDL, binds and activates the endothelial ecto-F1-ATPase receptor. This generates extracellular ADP, which in turn promotes endothelial cell survival. In this study we aimed to further investigate the signaling pathway involved downstream of apoA-I-induced ecto-F1-ATPase activation. Methods: In human umbilical vein endothelial cells (HUVECs), pharmacological and gene silencing approaches were used to study pathways involved downstream ecto-F1-ATPase activation by apoA-I. Results: ApoA-I and HDL both induced Akt phosphorylation. F1-ATPase inhibitors such as inhibitory factor 1 and oligomycin completely blocked apoA-I-induced Akt phosphorylaton and significantly blocked HDL-induced phosphorylation, indicating that this signaling pathway is dependent on ecto-F1-ATPase activation by apoA-I. Further, we were able to specify roles for the P2Y1-ADPreceptor and the PI3Kβ isoform in this pathway since pharmacological inhibition and silencing of these proteins dramatically inhibited apoA-I-induced Akt phosphorylation and cell proliferation. Conclusion: Altogether, these data highlight a key role of the P2Y1/PI3Kβ axis in endothelial cell proliferation downstream of ecto-F1-ATPase activation by apoA-I. Pharmacological targeting of this pathway could represent a promising approach to enhance vascular endothelial protection.

0377 : Phosphoinositide 3-kinase gamma: a potential clinical target in the prevention of vascular damages inuced by arterial injury

Context Angioplasty with stent placement, used to treat symptomatic atherosclerosis plaques, is frequently complicated by restenosis. This pathology is characteried by fibroproliferative and immuno-inflammatory mechanisms of the arterial wall, called intimal hyperplasia (IH). Aims Our previous results demonstrated that phosphoinositide 3-kinase (??(PI3K(??) was a key mediator of inflammatory processes of the arterial wall leading to atherosclerosis. We now proposed this enzyme as a potential clinical target to prevent vascular damages occuring after arterial lesion leading to IH. As cellular and molecular aspects of IH are still incompletely described, we intended to offer a better understanding ofimmune responses involved in this pathology. Results Using a mouse model of arterial mechanical injury, we showed that PI3K(?-deficient mice and mice expressing a catalytically-inactive PI3K(?(PI3K(?KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around site of vascular lesion. The transfer of PI3K(?KD CD4 + T cells into Rag2-deficient mice greatly reduced vascular occlusion compared to WT cells, clearly demonstrating the involvement of PI3K(?in CD4 + T cells during IH formation. In addition, we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3K(?activity, leading to decreased chemokine production by smooth muscle cells. Finally, we show that a short period of treatment with a PI3K(?inhibitor decreased IH development in arterial mechanical injury in mice but also in a rat carotid artery balloon injury model demonstrating the therapeutic potential of this inhibitor. Conclusion Our work pintpoint PI3K(?as a good target for IH prevention and brings new insights in the immune biology of the disease.