Alexandre Hego

Identification of a microRNA landscape targeting the PI3K/Akt signaling pathway in inflammation-induced colorectal carcinogenesis

Inflammation can contribute to tumor formation; however, markers that predict progression are still lacking. In the present study, the well-established azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced mouse model of colitis-associated cancer was used to analyze microRNA (miRNA) modulation accompanying inflammation-induced tumor development and to determine whether inflammation-triggered miRNA alterations affect the expression of genes or pathways involved in cancer. A miRNA microarray experiment was performed to establish miRNA expression profiles in mouse colon at early and late time points during inflammation and/or tumor growth. Chronic inflammation and carcinogenesis were associated with distinct changes in miRNA expression. Nevertheless, prediction algorithms of miRNA-mRNA interactions and computational analyses based on ranked miRNA lists consistently identified putative target genes that play essential roles in tumor growth or that belong to key carcinogenesis-related signaling pathways. We identified PI3K/Akt and the insulin growth factor-1 (IGF-1) as major pathways being affected in the AOM/DSS model. DSS-induced chronic inflammation downregulates miR-133a and miR-143/145, which is reportedly associated with human colorectal cancer and PI3K/Akt activation. Accordingly, conditioned medium from inflammatory cells decreases the expression of these miRNA in colorectal adenocarcinoma Caco-2 cells. Overexpression of miR-223, one of the main miRNA showing strong upregulation during AOM/DSS tumor growth, inhibited Akt phosphorylation and IGF-1R expression in these cells. Cell sorting from mouse colons delineated distinct miRNA expression patterns in epithelial and myeloid cells during the periods preceding and spanning tumor growth. Hence, cell-type-specific miRNA dysregulation and subsequent PI3K/Akt activation may be involved in the transition from intestinal inflammation to cancer.

Biological Effects of Cardiac Magnetic Resonance on Human Blood Cells

Background—Cardiac magnetic resonance (CMR) is increasingly used for the diagnosis and management of cardiac diseases. Recent studies have reported immediate post-CMR DNA double-strand breaks in T lymphocytes. We sought to evaluate CMR-induced DNA damage in lymphocytes, alterations of blood cells, and their temporal persistence. Methods and Results—In 20 prospectively enrolled healthy men (31.4±7.9 years), blood was drawn before and after (1–2 hours, 2 days, 1 month, and 1 year) unenhanced 1.5T CMR. Blood cell counts, cell death, and activation status of lymphocytes, monocytes, neutrophils, and platelets were evaluated. The first 2-hour post-CMR were characterized by a small increase of lymphocyte B and neutrophil counts and a transient drop of total lymphocytes because of a decrease in natural killer cells. Among blood cells, only neutrophils and monocytes displayed slight and transient activation. DNA double-strand breaks in lymphocytes were quantified through flow cytometric analysis of H2AX phosphorylation (&ggr;-H2AX). &ggr;-H2AX intensity in T lymphocytes did not change early after CMR but increased significantly at day 2 ⩽1 month before returning to baseline levels of 1-year post-CMR. Conclusions—Unenhanced CMR is associated with minor but significant immediate blood cell alterations or activations figuring inflammatory response, as well as DNA damage in T lymphocytes observed from day 2 until the first month but disappearing at 1-year follow-up. Although further studies are required to definitely state whether CMR can be used safely, our findings already call for caution when it comes to repeat this examination within a month.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

MicroRNAs in Valvular Heart Diseases: Potential Role as Markers and Actors of Valvular and Cardiac Remodeling

miRNAs are a class of over 5000 noncoding RNAs that regulate more than half of the protein-encoding genes by provoking their degradation or preventing their translation. miRNAs are key regulators of complex biological processes underlying several cardiovascular disorders, including left ventricular hypertrophy, ischemic heart disease, heart failure, hypertension and arrhythmias. Moreover, circulating miRNAs herald promise as biomarkers in acute myocardial infarction and heart failure. In this context, this review gives an overview of studies that suggest that miRNAs could also play a role in valvular heart diseases. This area of research is still at its infancy, and further investigations in large patient cohorts and cellular or animal models are needed to provide strong data. Most studies focused on aortic stenosis, one of the most common valvular diseases in developed countries. Profiling and functional analyses indicate that miRNAs could contribute to activation of aortic valve interstitial cells to a myofibroblast phenotype, leading to valvular fibrosis and calcification, and to pressure overload-induced myocardial remodeling and hypertrophy. Data also indicate that specific miRNA signatures, in combination with clinical and functional imaging parameters, could represent useful biomarkers of disease progression or recovery after aortic valve replacement.

Purinergic control of inflammation and thrombosis: Role of P2X1 receptors

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/ − mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/ − mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.

Dual-Specificity Phosphatase 3 Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis

Background— A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. Methods and Results— This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride–induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase C&ggr;2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2–induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions— DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2–mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.

Sepsis prediction in critically ill patients by platelet activation markers on ICU admission: a prospective pilot study

BackgroundPlatelets have been involved in both immune surveillance and host defense against severe infection. To date, whether platelet phenotype or other hemostasis components could be associated with predisposition to sepsis in critical illness remains unknown. The aim of this work was to identify platelet markers that could predict sepsis occurrence in critically ill injured patients.MethodsThis single-center, prospective, observational, 7-month study was based on a cohort of 99 non-infected adult patients admitted to ICUs for elective cardiac surgery, trauma, acute brain injury, and post-operative prolonged ventilation and followed up during ICU stay. Clinical characteristics and severity score (SOFA) were recorded on admission. Platelet activation markers, including fibrinogen binding to platelets, platelet membrane P-selectin expression, plasma soluble CD40L, and platelet-leukocytes aggregates were assayed by flow cytometry at admission and 48xa0h later, and then at the time of sepsis diagnosis (Sepsis-3 criteria) and 7xa0days later for sepsis patients. Hospitalization data and outcomes were also recorded.MethodsOf the 99 patients, 19 developed sepsis after a median time of 5xa0days. These patients had a higher SOFA score at admission; levels of fibrinogen binding to platelets (platelet-Fg) and of D-dimers were also significantly increased compared to the other patients. Levels 48xa0h after ICU admission no longer differed between the two patient groups. Platelet-Fg % was an independent predictor of sepsis (Pu2009=u20090.0031). By ROC curve analysis, cutoff point for Platelet-Fg (AUCu2009=u20090.75) was 50%. In patients with a SOFA cutoff of 8, the risk of sepsis reached 87% when Platelet-Fg levels were above 50%. Patients with sepsis had longer ICU and hospital stays and higher death rate.ConclusionsPlatelet-bound fibrinogen levels assayed by flow cytometry within 24xa0h of ICU admission help identifying critically ill patients at risk of developing sepsis.

Advances in Pathophysiology of Calcific Aortic Valve Disease Propose Novel Molecular Therapeutic Targets.

Calcific Aortic Valve Disease (CAVD) is the most common heart valve disease and its incidence is expected to rise with aging population. No medical treatment so far has shown slowing progression of CAVD progression. Surgery remains to this day the only way to treat it. Effective drug therapy can only be achieved through a better insight into the pathogenic mechanisms underlying CAVD. The cellular and molecular events leading to leaflets calcification are complex. Upon endothelium cell damage, oxidized LDLs trigger a proinflammatory response disrupting healthy cross-talk between valve endothelial and interstitial cells. Therefore, valve interstitial cells transform into osteoblasts and mineralize the leaflets. Studies have investigated signaling pathways driving and connecting lipid metabolism, inflammation and osteogenesis. This review draws a summary of the recent advances and discusses their exploitation as promising therapeutic targets to treat CAVD and reduce valve replacement.

Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis

Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets.

AMPK-ACC signaling modulates platelet phospholipids content and potentiates platelet function and thrombus formation

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.