Rôle des Cellules Endothéliales Progénitrices dans la Régulation de la Fonction Plaquettaire

Les Cellules Endothéliales Progénitrices ("Endothelial Progenitor Cells", EPCs) sont des précurseurs endothéliaux qui jouent un rôle émergeant en biologie vasculaire. Les EPCs ont été localisées dans le cordon ombilical, la moelle osseuse, le sang périphérique et dans certains tissus régénérateurs. Les interactions des EPCs avec les cellules sanguines et vasculaires peuvent largement influencer leurs propriétés biologiques et dicter leur fonctionnement pendant la réparation endothéliale. Plus spécifiquement, les interactions des EPCs avec les plaquettes circulantes induisent leur migration, leur recrutement et leur différentiation en cellules endothéliales aux sites de lésions vasculaires. Cependant, l’impact d’une telle interaction sur la fonction plaquettaire n’a pas été recherché. Le but de mon projet était de :1) générer des EPCs à partir des cellules mononucléaires du sang humain périphérique ("Peripheral Blood Mononuclear Cells", PBMCs); 2) étudier les interactions adhésives entre les EPCs et les plaquettes; 3) déterminer leur impact sur la fonction plaquettaire et la formation du thrombus et 4) décrire le mécanisme d’action des EPCs sur les plaquettes et le thrombus. Mises en culture sur une surface de fibronectine dans un milieu conditionné, les PBMCs fraîchement isolées possédaient une morphologie ronde et une petite taille. Après cinq jours, les PBMCs adhérentes donnaient naissance à des colonies, puis formaient une monocouche de cellules aplaties caractéristiques des EPCs après dix jours de culture. Les EPCs différenciées étaient positives pour l’Ulex-lectine et l’Acétyle des lipoprotéines de faible densité ("Acetylated Low Density Lipoprotein", Ac-LDL), exprimaient les marqueurs progéniteurs (CD34, P-sélectine, VEGFR2, vWF et VE-Cadhérine) tandis que les marqueurs leucocytaires (CD14, PSGL-1 et L-sélectine) étaient absents. Ces EPCs interagissaient avec les plaquettes activées par un mécanisme dépendant de la P-sélectine plaquettaire, inhibaient l’activation et l’agrégation plaquettaire et réduisaient significativement l’adhésion plaquettaire, principalement par l’action de prostacycline (PGI2). En fait, ceci était associé avec une augmentation de l’expression de la cyclooxygénase-2 (COX-2) et du monoxyde d’azote (NO) synthéthase inductible (iNOS). Toutefois, les effets inhibiteurs des EPCs sur la fonction plaquettaire ont été renversés par une inhibition de la COX et non pas du NO. Bien que les EPCs fussent en mesure de lier les plaquettes via la P-sélectine, leurs effets prédominants étaient médiés essentiellement par une sécrétion paracrine, impliquant la PGI2. Néanmoins, un rapprochement étroit ou un bref contact entre les EPCs et les plaquettes était requis pour que cette fonction soit complètement réalisée. D’ailleurs, cet aspect a été investigué chez des souris déficientes en P-sélectine (P-sel-/-) et chez leurs congénères de phénotype sauvage (Wild Type, WT). Chez les souris WT, les EPCs inhibaient l’agrégation plaquettaire dans le sang complet de manière concentration-dépendante alors que dans les souris P-sel-/-, l’action des EPCs n’avait pas d’effet significatif. De plus, en utilisant un modèle murin de thrombose artérielle, nous avons démontré que l’infusion systémique des EPCs altéraient la formation du thrombus et réduisaient significativement sa masse chez les souris WT, mais non pas chez les souris P-sel-/-. En outre, le nombre des EPCs incorporées au niveau du thrombus et de la paroi vasculaire était visiblement réduit chez les P-sel-/- par rapport aux souris WT. Dans cette étude, nous sommes parvenus à différentier adéquatement des EPCs à partir des PBMCs, nous avons étudié les interactions adhésives entre les EPCs et les plaquettes, et nous avons décrit leur impact sur la fonction plaquettaire et la formation du thrombus. De plus, nous avons identifié la PGI2 comme étant le principal facteur soluble sécrété par les EPCs en culture et responsable de leurs effets inhibiteurs sur l’activation, l’adhésion et l’agrégation plaquettaire in vitro. De surcroît, nous avons élucidé le mécanisme d’action des EPCs sur l’agrégation plaquettaire et la formation du thrombus, in vivo, et nous avons souligné le rôle de la P-sélectine plaquettaire dans ce processus. Ces résultats ajoutent de nouvelles connaissances sur la biologie des EPCs et définissent leur rôle potentiel dans la régulation de la fonction plaquettaire et la thrombogenèse.Endothelial Progenitor Cells (EPCs) are believed to contribute to vascular biology and endothelial repair. EPCs have been isolated from umbilical cord, bone marrow, peripheral blood and in some regenerative tissues. Interactions of EPCs with vascular and blood cells can largely influence their functional properties and predict their destiny in the target tissues. More specifically, interactions of EPCs with circulating platelets provide the critical signal to ensure their migration and homing at the sites of vascular injury and their differentiation into endothelial cells. However, the functional consequences of such interactions on platelets remain unknown. Accordingly, this project was designed to investigate the impact of EPCs on platelet function and the specific objectives of this study were to: 1) generate EPCs from human peripheral blood mononuclear cells (PBMCs); 2) characterize the adhesive interactions between EPCs and platelets; 3) determine their impact on platelet function and thrombus formation and 4) elucidate the mechanistic action of EPCs on platelets and thrombus. Cultured on fibronectin in conditioned media, PBMCs differentiated, within ten days of culture, into EPCs, which were positive for Ulex-lectin and Ac-LDL (Acetylated Low Density Lipoprotein), and expressed progenitor markers (CD34, VEGFR2, vWF, and VE-Cadherin). These EPCs bind activated platelets through P-selectin-dependent mechanism, inhibited platelet activation, aggregation and adhesion, mainly via prostacyclin (PGI2) secretion. Indeed, this was associated with up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS). However, the effects on platelets were reversed by COX, but not by NO inhibition. Although EPCs bound platelets via platelet P-selectin, their predominant effects occurred via a paracrine secretion, implying PGI2. Nevertheless, a transitory link or brief contact between EPCs and platelets was required for this function to be fully realized. This was further depicted using a murine arterial thrombosis model in P-selectin deficient mice (P sel-/-) and their wild-type counterparts (WT). EPCs significantly impaired, in a concentration dependent-manner, collagen-induced whole blood platelet aggregation in WT mice; whereas in P-sel-/- mice, EPCs had no significant effect. Moreover, in murin model of arterial thrombosis, infusion of EPCs altered thrombus formation and significantly reduced the mass of thrombi generated in WT, but not in P-sel-/- mice. Furthermore, the number of EPCs recruited within the thrombi and along the vascular wall was visually reduced in P-sel-/- mice as compared to WT mice. In this project, we succeeded in adequately differentiating EPCs from PBMCs, we characterized the adhesive interaction between EPCs and platelets, and we addressed the impact of EPCs on platelet function and thrombus formation. Moreover, we identified PGI2 as the principal soluble factor secreted by cultured EPCs and responsible of their inhibitory effects on platelet function in vitro. In addition, using a murine model of arterial carotid injury in WT and P-sel-/- mice, we elucidate the mechanistic action of EPCs on platelet aggregation and thrombus formation, in vivo, and we highlighted the role of platelet P-selectin in this process. These findings add new insights into the biology of EPCs and reveal a potential role for EPCs in regulating platelet function, which in turn may limit thrombogenesis and maintain hemostasis at the sites of vascular injury

Implication des interactions plaquettes-neutrophiles dans la sécrétion des métalloprotéinases

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Novel CD44-downstream signaling pathways mediating breast tumor invasion

CD44, also known as homing cell adhesion molecule is a multi-structural cell molecule involved in cell-cell and cell-extracellular matrix communications. CD44 regulates a number of central signaling pathways, including PI3K/AKT, Rho GTPases and the Ras-MAPK pathways, but also acts as a growth/arrest sensor, and inhibitor of angiogenesis and invasion, in response to signals from the microenvironment. The function of CD44 has been very controversial since it acts as both, a suppressor and a promoter of tumor growth and progression. To address this discrepancy, we have previously established CD44-inducible system both in vitro and in vivo. Next, using microarray analysis, we have identified and validated Survivin, Cortactin and TGF-β2 as novel CD44-downstream target genes, and characterized their signaling pathways underpinning CD44-promoted breast cancer (BC) cell invasion. This report aims to update the literature by adding and discussing the impact of these novel three signaling pathways to better understand the CD44-signaling pathways involved in BC tumor cell invasion

Recent Developments in Nanomaterials-Based Drug Delivery and Upgrading Treatment of Cardiovascular Diseases

Cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. However, despite the recent developments in the management of CVDs, the early and long outcomes vary considerably in patients, especially with the current challenges facing the detection and treatment of CVDs. This disparity is due to a lack of advanced diagnostic tools and targeted therapies, requiring innovative and alternative methods. Nanotechnology offers the opportunity to use nanomaterials in improving health and controlling diseases. Notably, nanotechnologies have recognized potential applicability in managing chronic diseases in the past few years, especially cancer and CVDs. Of particular interest is the use of nanoparticles as drug carriers to increase the pharmaco-efficacy and safety of conventional therapies. Different strategies have been proposed to use nanoparticles as drug carriers in CVDs; however, controversies regarding the selection of nanomaterials and nanoformulation are slowing their clinical translation. Therefore, this review focuses on nanotechnology for drug delivery and the application of nanomedicine in CVDs

Impacts on Student Learning and Skills and Implementation Challenges of Two Student-Centered Learning Methods Applied in Online Education

Online education became more prevalent during the COVID-19 pandemic in many countries around the world, including the Gulf Cooperation Council (GCC) countries. This study aims at assessing the impacts on learning and skills of two student-centered instructional strategies (problem-based learning (PBL) and just-in-time teaching (JiTT)) used online and their implementation challenges. The PBL and JiTT were implemented in modules taught in various courses delivered at different bachelor’s study levels and disciplines. The research used a mixed design research method. Quantitative data were collected from exam scores and two self-administered surveys. Qualitative data were collected using individual structured interviews. The lecture-based learning method was used for comparisons. A total of 134 students participated in the quizzes and exams, 85 students completed the self-perceived impacts on learning and skills survey, and 82 students completed the implementation challenges survey. Ten students participated in the structured interviews. Tests and survey scores showed that both online PBL and JiTT had significant impacts on students learning and skills and that these effects are consistent across various disciplines. A non-conducive online learning climate, internet connectivity problems, heavy workloads, and time management issues were reported as the implementation challenges. The PBL and JiTT can be considered as effective teaching/learning strategies in online education.This research was conducted under a grant from Qatar National Research Fund (a member of Qatar foundation) (RRC02-0824-210043)

Flavin Oxidase-Induced ROS Generation Modulates PKC Biphasic Effect of Resveratrol on Endothelial Cell Survival.

Dietary intake of natural antioxidants is thought to impart protection against oxidative-associated cardiovascular diseases. Despite many in vivo studies and clinical trials, this issue has not been conclusively resolved. Resveratrol (RES) is one of the most extensively studied dietary polyphenolic antioxidants. Paradoxically, we have previously demonstrated that high RES concentrations exert a pro-oxidant effect eventually elevating ROS levels leading to cell death. Here, we further elucidate the molecular determinants underpinning RES-induced oxidative cell death. Using human umbilical vein endothelial cells (HUVECs), the effect of increasing concentrations of RES on DNA synthesis and apoptosis was studied. In addition, mRNA and protein levels of cell survival or apoptosis genes, as well as protein kinase C (PKC) activity were determined. While high concentrations of RES reduce PKC activity, inhibit DNA synthesis and induce apoptosis, low RES concentrations elicit an opposite effect. This biphasic concentration-dependent effect (BCDE) of RES on PKC activity is mirrored at the molecular level. Indeed, high RES concentrations upregulate the proapoptotic , while downregulating the antiapoptotic , at both mRNA and protein levels. Similarly, high RES concentrations downregulate the cell cycle progression genes, , ornithine decarboxylase and cyclin D1 protein levels, while low RES concentrations display an increasing trend. The BCDE of RES on PKC activity is abrogated by the ROS scavenger Tempol, indicating that this enzyme acts downstream of the RES-elicited ROS signaling. The RES-induced BCDE on HUVEC cell cycle machinery was also blunted by the flavin inhibitor diphenyleneiodonium (DPI), implicating flavin oxidase-generated ROS as the mechanistic link in the cellular response to different RES concentrations. Finally, PKC inhibition abrogates the BCDE elicited by RES on both cell cycle progression and pro-apoptotic gene expression in HUVECs, mechanistically implicating PKC in the cellular response to different RES concentrations. Our results provide new molecular insight into the impact of RES on endothelial function/dysfunction, further confirming that obtaining an optimal benefit of RES is concentration-dependent. Importantly, the BCDE of RES could explain why other studies failed to establish the cardio-protective effects mediated by natural antioxidants, thus providing a guide for future investigation looking at cardio-protection by natural antioxidants

Impaired Liver Size and Compromised Neurobehavioral Activity are Elicited by Chitosan Nanoparticles in the Zebrafish Embryo Model

The use of chitosan nanoparticles (ChNPs) in various biological and environmental applications is attracting great interest. However, potential side effects related to ChNP toxicity remain the major limitation hampering their wide application. For the first time, we investigate the potential organ-specific (cardiac, hepatic, and neuromuscular) toxicity of ChNPs (size 100–150 nm) using the zebrafish embryo model. Our data highlight the absence of both acute and teratogenic toxic effects of ChNPs (~100% survival rate) even at the higher concentration employed (200 mg/L). Although no single sign of cardiotoxicity was observed upon exposure to 200 mg/L of ChNPs, as judged by heartbeat rate, the corrected QT interval (QTc, which measures the time between the start of the Q wave and the end of the T wave in the heart's electrical cycle), maximum cardiac arrest, and ejection fraction assays, the same dosage elicited the impairment of both liver size (decreased liver size, but without steatosis and lipid yolk retention) and neurobehavioral activity (increased movement under different light conditions). Although the observed toxic effect failed to affect embryo survival, whether a prolonged ChNP treatment may induce other potentially harmful effects remains to be elucidated. By reporting new insights on their organ-specific toxicity, our results add novel and useful information into the available data concerning the in vivo effect of ChNPs.This work was supported by the NPRP grant [#9-254-2-120] from the Qatar National Research Fund, a Member of Qatar Foundation. The study was also partially supported by the grants [GCC-2017-001] given to G.K.N. and [QUCG-CHS-2018n2019-1] given to G.P

Think like a Virus: Toward Improving Nanovaccine Development against SARS-CoV-2

There is no doubt that infectious diseases present global impact on the economy, society, health, mental state, and even political aspects, causing a long-lasting dent, and the situation will surely worsen if and when the viral spread becomes out of control, as seen during the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Despite the considerable achievements made in viral prevention and treatment, there are still significant challenges that can be overcome through careful understanding of the viral mechanism of action to establish common ground for innovating new preventative and treatment strategies. Viruses can be regarded as devil nanomachines, and one innovative approach to face and stop the spread of viral infections is the development of nanoparticles that can act similar to them as drug/vaccine carriers. Moreover, we can use the properties that different viruses have in designing nanoparticles that reassemble the virus conformational structures but that do not present the detrimental threats to human health that native viruses possess. This review discusses the current preventative strategies (i.e., vaccination) used in facing viral infections and the associated limitations, highlighting the importance of innovating new approaches to face viral infectious diseases and discussing the current nanoapplications in vaccine development and the challenges that still face the nanovaccine field.This research was funded by RC 47/2020 from IRCCS Burlo Garofolo/Italian Ministry of Health and by the QUCG-CAS-22/23-499 Collaborative Grant from Qatar University

Multiomics integration in skin diseases with alterations in notch signaling pathway: PlatOMICs phase 1 deployment

The high volume of information produced in the age of omics was and still is an important step to understanding several pathological processes, providing the enlightenment of complex molecular networks and the identification of molecular targets associated with many diseases. Despite these remarkable scientific advances, the majority of the results are disconnected and divergent, making their use limited. Skin diseases with alterations in the Notch signaling pathway were extensively studied during the omics era. In the GWAS Catalog, considering only studies on genomics association (GWAS), several works were deposited, some of which with divergent results. In addition, there are thousands of scientific articles available about these skin diseases. In our study, we focused our attention on skin diseases characterized by the impairment of Notch signaling, this pathway being of pivotal importance in the context of epithelial disorders. We considered the pathologies of five human skin diseases, Hidradenitis Suppurativa, Dowling Degos Disease, Adams-Oliver Syndrome, Psoriasis, and Atopic Dermatitis, in which the molecular alterations in the Notch signaling pathway have been reported. To this end, we started developing a new multiomics platform, PlatOMICs, to integrate and re-analyze omics information, searching for the molecular interactions involved in the pathogenesis of skin diseases with alterations in the Notch signaling pathway.This work was supported by a grant from the Institute for Maternal and Child Health IRCCS “Burlo Garofolo/Italian Ministry of Health” (BioHub 03/20), by the grant Interreg Italia-Slovenia, ISE-EMH 07/2019 and by CNPq (311415/2020-2)

Inositol 1,4,5-Trisphosphate Receptors in Hypertension.

Chronic hypertension remains a major cause of global mortality and morbidity. It is a complex disease that is the clinical manifestation of multiple genetic, environmental, nutritional, hormonal, and aging-related disorders. Evidence supports a role for vascular aging in the development of hypertension involving an impairment in endothelial function together with an alteration in vascular smooth muscle cells (VSMCs) calcium homeostasis leading to increased myogenic tone. Changes in free intracellular calcium levels ([Ca] ) are mediated either by the influx of Ca from the extracellular space or release of Ca from intracellular stores, mainly the sarcoplasmic reticulum (SR). The influx of extracellular Ca occurs primarily through voltage-gated Ca channels (VGCCs), store-operated Ca channels (SOC), and Ca release-activated channels (CRAC), whereas SR-Ca release occurs through inositol trisphosphate receptor (IPR) and ryanodine receptors (RyRs). IPR-mediated SR-Ca release, in the form of Ca waves, not only contributes to VSMC contraction and regulates VGCC function but is also intimately involved in structural remodeling of resistance arteries in hypertension. This involves a phenotypic switch of VSMCs as well as an alteration of cytoplasmic Ca signaling machinery, a phenomena tightly related to the aging process. Several lines of evidence implicate changes in expression/function levels of IPR isoforms in the development of hypertension, VSMC phenotypic switch, and vascular aging. The present review discusses the current knowledge of these mechanisms in an integrative approach and further suggests potential new targets for hypertension management and treatment.This publication was made possible by an MPP fund (#320133) from the American University of Beirut to AE