New Synthesized Derivatives from N-Substituted-4-Oxo-[1] Benzopyrano [4,3-c] Pyrazole Influenced Proliferation, Viability, Spreading and Invasion of Human Liver Tumor Cells

Background/Aim: There is an unsatisfied clinical demand to develop new anticancer agents. The aim of the current study was to synthesize new coumarin derivatives using two different synthetic methodologies and to evaluate their anticancer activity. Materials and methods: Four coumarin derivatives were synthesized and evaluated for their anticancer activities. The structures of all compounds were confirmed by infrared (IR), UV-vis, Nuclear magnetic resonance (NMR) 13C NMR, 1H NMR, and high-resolution mass spectrometry (HRMS) analysis. All the synthesized compounds (4, 5, 8 and 9) were analyzed for their anti-proliferative (MTT and LDH assays and cell cycle studied with flow cytometry) and anti-invasive activity (spreading and invasion tests) on human hepatoma cell lines Huh-7 in vitro. Doxorubicin was used as control in order to compare their anti-tumoral effects. Results. All the synthesized compounds are potential inhibitors of proliferation, viability, spreading and invasion of human liver tumor cells with a 50% inhibitory Concentration range,  IC50=10.37 μM to 12.94 μM. Conclusion. This study could lead to the identification of a new target therapy for human Hepatocellular carcinoma (HCC) or other cancers

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Syndecans and enzymes involved in heparan sulfate biosynthesis and degradation are implicated in pro-angiogenic effects of low molecular weight fucoidan.

L’ischémie se définit par la réduction de la lumière d’un vaisseau, ce qui provoque dans les tissus une baisse du flux sanguin, et une hypoxie locale. Pour lutter contre l’ischémie, différentes thérapies pro-angiogéniques ont été développées afin de stimuler la formation de nouveaux vaisseaux à partir des vaisseaux préexistants. Durant ma thèse j’ai étudié l’effet pro-angiogénique du fucoïdane, un polysaccharide sulfaté provenant de l’algue brune, qui est un mimétique de glycosaminoglycanes (GAGs). Nous avons choisi le fucoïdane de bas poids moléculaire (LMWF), qui a une bonne affinité pour des facteurs pro-angiogéniques(VEGF, SDF-1/CXCL12). Puis, nous avons analysé son effet dans les cellules endothéliales humaines (HUVEC). Nos résultats montrent que LMWF est internalisé par les HUVEC en 2h par la voie d’endocytose clatherine-dépendante. De plus, LMWF stimule la viabilité, la migration des HUVEC et la formation de réseaux vasculaires. Nous avons démontré par la suite qu’en absence de GAGs sur les HUVEC, le LMWF garde toujours son potentiel pro-angiogénique. Nous avons mis en évidence que l’exostosine-2(EXT-2), l’héparanase (HPSE) et le syndécane-4 (SDC-4), sont impliqués dans l’effet pro-angiogénique du LMWF, puisque quand nous inhibons leurs expressions par ARN interférence, le pouvoir pro-angiogénique du LMWF est diminué. Nous avons démontré,dans le modèle d’hyperplasie intimale chez le rat, que le LMWF a un effet opposé sur l’expression des SDC. En effet, le LMWF induit l’expression du SDC-1 et diminue celle duSDC-4 dans la néo-intima. Nos données indiquent que l'EXT2, l'HPSE et le SDC-4 sont impliqués dans les effets pro-angiogéniques du LMWF, suggérant que les changements du métabolisme des HS liés à l'angiogenèse induite par le LMWF offrent la possibilité d'une nouvelle approche thérapeutique pour le traitement des maladies ischémiques.Induction of angiogenesis is a potential treatment for chronic ischemia. In this study we propose the analysis of pro-angiogenic treatment with fucoidan, sulfated polysaccharide from brown seaweeds, which act as glycosaminoglycans mimetics. Herein we used the low molecular weight fucoidan (LMWF), which presents a good affinity for pro-angiogenic factors(VEGF, SDF-1/CXCL12). The LMWF was mainly internalized through human vascular endothelial cell (HUVEC) clathrin-dependent endocytosis (in 2h) in which GAGs were partially involved. Our results showed that LMWF induced migration and angiogenesis in HUVEC. Interestingly, in a GAG-free HUVECs model, LMWF still kept a pro-angiogenic potential. In addition, we reported the implication of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and syndecan-4 (SDC-4) in LMWF induced angiogenesis. LMWF-treated and EXT2- or HPSE-siRNA-transfected cells shows that EXT2 or HPSE expression significantly affects the LMWF pro-angiogenic potential. In addition, LMWF increased SDC-1, but decreased SDC-4 expression. We studied the LMWF implication in SDC-1 and SDC-4 expression in rat model of intimal hyperplasia after balloon injury. Our results showed that LMWF treatment of injured artery increased SDC-1 expression, but decreased SDC-4 expression in the neointima layer. Our data indicate that EXT2, HPSE, and SDC-4 are involved in the pro-angiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic approach for ischemic diseases treatment

Etude des effets pro-angiogéniques du fucoïdane de bas poids moléculaire : implication des syndécannes et des enzymes impliquées dans la biosynthése et la dégradation des héparanes sulfates

Induction of angiogenesis is a potential treatment for chronic ischemia. In this study we propose the analysis of pro-angiogenic treatment with fucoidan, sulfated polysaccharide from brown seaweeds, which act as glycosaminoglycans mimetics. Herein we used the low molecular weight fucoidan (LMWF), which presents a good affinity for pro-angiogenic factors(VEGF, SDF-1/CXCL12). The LMWF was mainly internalized through human vascular endothelial cell (HUVEC) clathrin-dependent endocytosis (in 2h) in which GAGs were partially involved. Our results showed that LMWF induced migration and angiogenesis in HUVEC. Interestingly, in a GAG-free HUVECs model, LMWF still kept a pro-angiogenic potential. In addition, we reported the implication of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and syndecan-4 (SDC-4) in LMWF induced angiogenesis. LMWF-treated and EXT2- or HPSE-siRNA-transfected cells shows that EXT2 or HPSE expression significantly affects the LMWF pro-angiogenic potential. In addition, LMWF increased SDC-1, but decreased SDC-4 expression. We studied the LMWF implication in SDC-1 and SDC-4 expression in rat model of intimal hyperplasia after balloon injury. Our results showed that LMWF treatment of injured artery increased SDC-1 expression, but decreased SDC-4 expression in the neointima layer. Our data indicate that EXT2, HPSE, and SDC-4 are involved in the pro-angiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic approach for ischemic diseases treatment.L’ischémie se définit par la réduction de la lumière d’un vaisseau, ce qui provoque dans les tissus une baisse du flux sanguin, et une hypoxie locale. Pour lutter contre l’ischémie, différentes thérapies pro-angiogéniques ont été développées afin de stimuler la formation de nouveaux vaisseaux à partir des vaisseaux préexistants. Durant ma thèse j’ai étudié l’effet pro-angiogénique du fucoïdane, un polysaccharide sulfaté provenant de l’algue brune, qui est un mimétique de glycosaminoglycanes (GAGs). Nous avons choisi le fucoïdane de bas poids moléculaire (LMWF), qui a une bonne affinité pour des facteurs pro-angiogéniques(VEGF, SDF-1/CXCL12). Puis, nous avons analysé son effet dans les cellules endothéliales humaines (HUVEC). Nos résultats montrent que LMWF est internalisé par les HUVEC en 2h par la voie d’endocytose clatherine-dépendante. De plus, LMWF stimule la viabilité, la migration des HUVEC et la formation de réseaux vasculaires. Nous avons démontré par la suite qu’en absence de GAGs sur les HUVEC, le LMWF garde toujours son potentiel pro-angiogénique. Nous avons mis en évidence que l’exostosine-2(EXT-2), l’héparanase (HPSE) et le syndécane-4 (SDC-4), sont impliqués dans l’effet pro-angiogénique du LMWF, puisque quand nous inhibons leurs expressions par ARN interférence, le pouvoir pro-angiogénique du LMWF est diminué. Nous avons démontré,dans le modèle d’hyperplasie intimale chez le rat, que le LMWF a un effet opposé sur l’expression des SDC. En effet, le LMWF induit l’expression du SDC-1 et diminue celle duSDC-4 dans la néo-intima. Nos données indiquent que l'EXT2, l'HPSE et le SDC-4 sont impliqués dans les effets pro-angiogéniques du LMWF, suggérant que les changements du métabolisme des HS liés à l'angiogenèse induite par le LMWF offrent la possibilité d'une nouvelle approche thérapeutique pour le traitement des maladies ischémiques

Etude des intéractions entre les cellules tumorales et l'endothélium vasculaire ( rôles des contraintes de cisaillement )

Le processus métastatique se déroule en une séquence d'évènements inter-reliée conduisant à la dissémination de la tumeur dans l'organisme, via la circulation lymphatique ou sanguine. Ce phénomène fait intervenir des interactions entre les cellules tumorales (CT) et l'endothélium vasculaire, essentielles à sa réalisation. En conditions physiologiques, ces interactions ont lieu en présence de l'écoulement sanguin. Cependant, l'influence des forces hémodynamiques sur la dissémination tumorale reste peu caractérisée. L'objectif de cette thèse est d'analyser, d'un point de vue morphologique, biochimique, et moléculaire, l'effet des contraintes de cisaillement sur les interactions entre les CT et les cellules endothéliales (CE), dans un modèle d'étude in vitro. Nos résultats indiquent que l'écoulement module la morphologie et l'étalement des CT, et régule l'expression protéique ainsi que l'activité intracellulaire des CE. De plus, la coculture des CT avec des CE, sous un écoulement modéré ou nul, stimule les CE via l'activation de la voie de signalisation NFKB. Cela induit la surexpression des molécules d'adhérence cellulaire endothéliales, facilitant ainsi l'adhésion et la migration des CT. En revanche, l'application de contraintes élevées, inhibent l'activation des CE induite par la coculture ou par le TNFa, ce qui met en exergue les propriétés anti-inflammatoires des fortes contraintes de cisaillement. Cette thèse démontre pour la première fois, l'existence d'un seuil de contrainte de cisaillement. De part et d'autre de ce seuil, l'écoulement exercerait un effet différentiel sur la morphologie cellulaire, l'expression des protéines membranaires et l'activité intracellulaire. Nos résultats suggèrent également, que la formation des métastases se déroulerait préférentiellement dans les régions de l'organisme où les contraintes de cisaillement sont faibles ou modérées (veines, capillaires, canaux lymphatiques, bifurcations artérielles).Cancer metastasis is a multistep process which involves interactions between Tumor Cells (TC) and Endothelial Cells (EC), and leads to tumoral dissemination through Iymphatic or blood circulation. However, the mechanism by which fluid shear forces modulate tumoral dissemination remains still uncIear. The goal of this thesis was to analyze morphological, biochemical and molecular effects of shear stress on TC-EC interactions, using an original in vitro assay. Our results indicated that shear stress level modulated TC morphology and spreading, and regulated protein expression and intracellular activity in ECs. Furthermore, the coculture of TCs with ECs under moderate flow or static conditions, up-regulated endothelial cell adhesion molecules via NFKB activation. This, in tum, enhanced TC adhesion and transmigration through ECs. ln contrast, high shear stress totally abolished the stimulation of ECs induced by coculture or by pro-inflammatory agents, demonstrating the anti-inflammatory properties ofhigh shear stress. This work suggests for the first time, the existence of a shear stress threshold with differential effects on cell morphology, protein expression and the activation of signaling pathways. These findings partially explain why low shear regions (veins, capillaries, bifurcations ofarteries, lymphatic vessels) are prone to metastasis, unlike the relatively anti-inflammatory high shear regions.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

The impact of methanol mass transport on its conversion for the production of hydrogen and oxygenated reactive species in sono-irradiated aqueous solution

This study aims principally to assess numerically the impact of methanol mass transport (i.e., evaporation/condensation across the acoustic bubble wall) on the thermodynamics and chemical effects (methanol conversion, hydrogen and oxygenated reactive species production) of acoustic cavitation in sono-irradiated aqueous solution. This effect was revealed at various ultrasound frequencies (from 213 to 1000 kHz) and acoustic intensities (1 and 2 W/cm2) over a range of methanol concentrations (from 0 to 100%, v/v). It was found that the impact of methanol concentration on the expansion and compression ratios, bubble temperature, CH3OH conversion and the molar productions inside the bubble is frequency dependent (either with or without consideration of methanol mass transport), where this effect is more pronounced when the ultrasound frequency is decreased. Alternatively, the decrease in acoustic intensity decreases clearly the effect of methanol mass transport on the bubble sono-activity. When methanol mass transfer is eliminated, the decrease of the bubble temperature, CH3OH conversion and the molar yield of the bubble with the rise of methanol concentration was found to be more amortized as the wave frequency is reduced from 1 MHz to 213 kHz, compared to the case when the mass transport of methanol is taken into account. Our findings indicate clearly the importance of incorporating the evaporation and condensation mechanisms of methanol throughout the numerical simulations of a single bubble dynamics and chemical activity

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFkappaB: differential role of the shear stress.

International audienceCancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 hours. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFkappaB inhibitor prior to coculture, fully prevented coculture-induced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished coculture-induced as well as TNFalpha-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFkappaB activation, and that this action can be modulated by shear stress

Low molecular weight fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through the modulation of matrix metalloproteinase-2 expression.

International audienceThe therapeutic potential of low molecular-weight fucoidan (LMWF), a sulfated polysaccharide extracted from brown seaweed was investigated on vascular smooth muscle cell (VSMC) and human vascular endothelial cell (HUV-EC-C) proliferation and migration in vitro and in a rat model of intimal hyperplasia. Sprague-Dawley rats were subjected to balloon injury in the thoracic aorta followed by two weeks' treatment with either LMWF (5mg/kg/day) or vehicle. Morphological analysis and proliferating cell nuclear antigen immunostaining at day 14 indicated that LMWF prevented intimal hyperplasia in rat thoracic aorta as compared with vehicle (neo-intima area, 3±0.50 versus 5±0.30mm, <0.01). In situ zymography showed that LMWF significantly decreased the activity of matrix metalloproteinase (MMP)-2 in the neo-intima compared to vehicle. The in vitro study demonstrated that 10μg/ml LMWF increased HUV-EC-C migration by 45±5% but reduced VSMC migration by 40±3%. LMWF also increased MMP-2 mRNA expression in HUV-EC-Cs and reduced it in VSMCs. MMP-2 level in the conditioned medium from cells incubated with 10μg/ml LMWF was 5.4-fold higher in HUV-EC-C, but 6-fold lower in VSMCs than in untreated control cells. Furthermore, decreasing MMP-2 expression in HUV-EC-Cs or VSMCs by RNA interference resulted in reduced LMWF-induced effects on cell migration