Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells

Conexina 43; Expresión génica; Translocación nuclearConnexina 43; Expressió gènica; Translocació nuclearConnexin 43; Gene expression; Nuclear translocationClassically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-β, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) under the projects HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, CENTRO-01-0145-FEDER-032179, CENTRO-01-0145-FEDER-032414, EXPL/MED-OUT/0590/2021, UIDB/04539/2020 and PPBI-Portuguese Platform of BioImaging (POCI-01-0145-FEDER-022122). This work was also supported by the European Union's Horizon 2020 research and innovation programme under grant agreement MIA-Portugal no. 857524 and the Comissão de Coordenação da Região Centro (CCDRC) through the Centro2020 Program. T.A. acknowledges funding from Instituto de Salud Carlos III, grant PI16/00772 co-financed by the ERDF, and Fundación Científica Asociación Española Contra el Cáncer (IDEAS20039AASE). This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), and the Microscopy and Bioimaging Lab (iLAB) at the Faculty of Medicine of the University of Coimbra (Coimbra, Portugal). The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. Super-resolution microscopy experiments benefited from access to CBI/IGBMC (Illkirch, France) and were supported by FRISBI (ANR-10-INBS-0005). Financial support was provided by Instruct-ERIC (PID 14677)

Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approaches

Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications.info:eu-repo/semantics/publishedVersio

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Funding Information: This work was supported by European Regional Development Fund (FEDER) through the Operational Program for Competitiveness Factors (COMPETE) [HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, POCI-01-0145-FEDER-016385, POCI-01-0145-FEDER-007440 to CNC.IBILI, POCI-01-0145-FEDER-007274 to i3S/INEB and NORTE-01-0145-FEDER-000012 to T.L.L.]; national funds through the Portuguese Foundation for Science and Technology (FCT) [PTDC/SAU-ORG/119296/2010, PTDC/ NEU-OSD/0312/2012, PESTC/ SAU/UI3282/2013-2014, MITP-TB/ECE/0013/ 2013, FCT-UID/NEU/04539/2013], PD/BD/52294/2013 to T.M.R.R., SFRH/ BD/85556/2012 (co-financed by QREN) to V.C.S]; Lisboa Portugal Regional Operational Programme (LISBOA 2020) and Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by INFARMED Autoridade Nacional do Medicamento e Produtos de Saúde, I.P. [FIS-FIS-2015-01_CCV_20150630-157]. Publisher Copyright: © 2017 The Author.Aims Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.publishersversionpublishe

Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson)

Financiado por el Departamento de Junta de Becas de la Universidad Nacional de Costa Rica y por el proyectos: MINECO (AGL2016-76143-C4-3R), MICINN (PID2020-112627RB-C32), CYTED (P117RT0522), MULTIFOREVER project, under the umbrella of ERA-NET Cofund ForestValue by ANR (FR), FNR (DE), MINCyT (AR), MINECO-AEI (ES), MMM (FI), VINNOVA (SE). ForestValue has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 773324 y al DECO (Basque government).Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15–30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 M 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 mol m2 s1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 mol m2 s1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.Pinus. ponderosa (P. Lawson y C. Lawson) es un árbol comercial y una de las especies forestales más importantes de Norteamérica. El pino ponderosa sufre dificultades cuando se somete a propagación vegetativa y, en algunos casos, se necesitan entre 15 y 30 años para alcanzar la plena reproductiva. Basándose en trabajos anteriores sobre la regeneración de P. ponderosa mediante organogénesis in vitro y tratando de mejorar los protocolos publicados, nuestro objetivo fue analizar la influencia de diferentes tipos de explantes, medios de cultivo basales, citoquininas, auxinas y tratamientos lumínicos sobre el éxito de las fases de multiplicación y enraizamiento de los brotes, multiplicación de brotes y las fases de enraizamiento. Los embriones cigóticos enteros y 44 M 6-benciladenina mostraron los mejores resultados en términos de supervivencia de los explantes. Para la organogénesis de los brotes, los embriones cigóticos enteros y medio LP (medio LP, Quoirin y Lepoivre, 1977, modificado por Aitken-Christie et al., 1988) se seleccionaron macronutrientes. Se observó una interacción positiva significativa entre los embriones cigóticos enteros y se encontró una interacción positiva significativa entre los embriones cigóticos enteros y los macronutrientes de medio LP para el porcentaje de explantes que formaban brotes. En cuanto a los tratamientos de luz aplicados, se detectó un porcentaje significativamente mayor de brotes lo suficientemente alargados como para enraizar en los brotes cultivados con LED azul, se detectó en los brotes cultivados bajo LED azul a una intensidad luminosa de 61,09 mol m2 s1. Sin embargo, el porcentaje de aclimatación fue mayor en los brotes cultivados previamente bajo luz fluorescente a una intensidad luminosa de 61,71 mol m2 s1. Los estudios anatómicos realizados con microscopía óptica y microscopía electrónica de barrido mostraron que los tratamientos de luz promovían diferencias en los aspectos anatómicos en brotes in vitro; las agujas de las plántulas expuestas a LED rojos y azules revelaron menos estomas en comparación con las agujas de las plántulas expuestas a luz fluorescente.NEIKER-BRTACentro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Centre for Functional EcologyCoimbra Institute for Clinical and Biomedical Research (iCBR)Clinical Academic Centre of Coimbra (CACC)Instituto de Investigación y Servicios Forestales (INISEFOR

Lauroylated histidine-enriched S4(13)-PV peptide as an efficient gene silencing mediator in cancer cells

PurposeThis study aimed to endow the cell-penetrating peptide (CPP) S4(13)-PV with adequate features towards a safe and effective application in cancer gene therapy.MethodsPeptide/siRNA complexes were prepared with two new derivatives of the CPP S4(13)-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S4(13)-PV peptide, being named C12-H-5-S4(13)-PV and H-5-S4(13)-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum.ResultsPeptide/siRNA complexes prepared with the C12-H-5-S4(13)-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H-5-S4(13)-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes.ConclusionsThese encouraging results pave the way for a potential application of the C12-H-5-S4(13)-PV peptide as a promising tool in cancer gene therapy.European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme [CENTRO-010145-FEDER-000008]European Regional Development Fund (ERDF), through the COMPETE 2020 -Operational Programme for Competitiveness and InternationalisationPortuguese national funds via FCT - Fundacao para a Ciencia e a Tecnologia [POCI-01-0145-FEDER-016390: CANCEL STEM, POCI-01-0145-FEDER-007440, UIDB/04539/2020]Fundacao para a Ciencia e Tecnologia (FCT, Portugal)Portuguese Foundation for Science and Technology [UID/QUI/50006/2013, UID/MULTI/04378/2013, IF/00092/2014, SFRH/BD/79077/2011, PD/BD/106035/2015]info:eu-repo/semantics/publishedVersio

Corrigendum : Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication

Ischaemia alters the effects of cardiomyocyte-derived extracellular vesicles on macrophage activation

We thank Dr Nuno Alves (Cardiology Department, CHUC‐HG) who performed the collection of human blood samples and Doctor Francisco Caramelo (iCBR/FMUC) for helping with the statistical analysis. This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) [under the projects PAC “NETDIAMOND” POCI‐01‐0145‐FEDER‐016385; HealthyAging2020 CENTRO‐01‐0145‐ FEDER‐000012‐N2323; POCI‐01‐0145‐FEDER‐007440, CENTRO‐01‐ 0145‐FEDER‐032179, CENTRO‐01‐0145‐FEDER‐032414 and FCT‐ UID/NEU/04539/2013 to CNC.IBILI]. TMM was supported by PD/ BD/106043/2015 and TRR by PD/BD/52294/2013 from Fundação para a Ciência e a Tecnologia (FCT). JS was supported by Horizon2020 ERC‐2016‐COG EVICARE (725229).Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell-cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte-derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro-inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL-1β and IL-6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL-1β, IL-6, IL-10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum-circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte-derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post-infarction remodelling.publishersversionpublishe

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs)