RESUMO
The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.
RESUMO
Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.
Assuntos
Asma , Interleucina-33 , Animais , Humanos , Camundongos , Alarminas , Citocinas , Imunidade Inata , Inflamação , Interleucina-9 , Linfócitos , ProteômicaRESUMO
The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin.
Assuntos
DNA , Eletroporação , Humanos , Transfecção , Eletroporação/métodos , DNA/genética , Plasmídeos/genética , Colágeno/genética , FibroblastosRESUMO
Intravital microscopy allows a direct visualization of cells' behavior in their environment in a living organism with all its complexity. With appropriated models, longitudinal studies of structural and functional changes can be followed in the same animal on long period. In the field of cancer, the dorsal window chamber model is the model of choice for tumor events such as cells migration, vessels growth, and their permeability or interactions between cells and vessels. Coupled with wide-field, confocal, or multiphoton fluorescence microscopes, high spatial and temporal resolutions of the cellular events can be analyzed in vivo.
Assuntos
Microscopia Intravital , Microscopia de Fluorescência por Excitação Multifotônica , Animais , Movimento Celular , PermeabilidadeRESUMO
Electroporation, a method relying on a pulsed electric field to induce transient cell membrane permeabilization, can be used as a non-viral method to transfer genes in vitro and in vivo. Such transfer holds great promise for cancer treatment, as it can induce or replace missing or non-functioning genes. Yet, while efficient in vitro, gene-electrotherapy remains challenging in tumors. To assess the differences of gene electrotransfer in respect to applied pulses in multi-dimensional (2D, 3D) cellular organizations, we herein compared pulsed electric field protocols applicable to electrochemotherapy and gene electrotherapy and different "High Voltage-Low Voltage" pulses. Our results show that all protocols can result in efficient permeabilization of 2D- and 3D-grown cells. However, their efficiency for gene delivery varies. The gene-electrotherapy protocol is the most efficient in cell suspensions, with a transfection rate of about 50%. Conversely, despite homogenous permeabilization of the entire 3D structure, none of the tested protocols allowed gene delivery beyond the rims of multicellular spheroids. Taken together, our findings highlight the importance of electric field intensity and the occurrence of cell permeabilization, and underline the significance of pulses' duration, impacting plasmids' electrophoretic drag. The latter is sterically hindered in 3D structures and prevents the delivery of genes into spheroids' core.
RESUMO
Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.
Assuntos
Inflamassomos , Piroptose , Animais , Proteínas Reguladoras de Apoptose/genética , Caspase 1/metabolismo , Exotoxinas/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/microbiologia , Pseudomonas aeruginosa/metabolismoRESUMO
Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Células Endoteliais , Citometria de Fluxo , Imunoterapia/métodos , Camundongos , Neoplasias/terapiaRESUMO
Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79+ HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8+ T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.
Assuntos
Melanoma , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Antígeno CTLA-4 , Células Endoteliais , Humanos , Fatores Imunológicos , Imunoterapia , Linfócitos do Interstício Tumoral , Melanoma/patologia , Camundongos , Subpopulações de Linfócitos T , Vênulas/patologiaRESUMO
High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals' growth, nor it had any impact on cutaneous murine tumors' growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability.
Assuntos
Comportamento Animal , Permeabilidade Capilar , Neoplasias Experimentais/metabolismo , Ondas de Rádio , Animais , Feminino , Camundongos , Neoplasias Experimentais/patologiaRESUMO
The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.
Assuntos
Neoplasias Ovarianas , Neoplasias Peritoneais , Animais , Antígenos Glicosídicos Associados a Tumores , Feminino , Humanos , Camundongos , Recidiva Local de Neoplasia , Microambiente TumoralRESUMO
Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which have superior magnetic responsiveness and can act as efficient photothermal agents. When internalized by different cancer cell lines and normal (non-cancerous) cells, the nanochains are not toxic, as assessed on 2D and 3D cell culture models. Yet, upon irradiation with near infrared light, the nanochains become efficient cytotoxic photothermal agents. Besides, not only do they generate hyperthermia, which effectively eradicates tumor cells in vitro, but they also locally melt the collagen matrix, as we evidence in real-time, using engineered cell sheets with self-secreted extracellular matrix. By simultaneously acting as physical (magnetic and photothermal) effectors and chemical delivery systems, the nanochain-based platforms offer original multimodal possibilities for prospective cancer treatment, affecting both the cells and the extracellular matrix.
RESUMO
High power electromagnetic signals can disrupt the functioning of electronic devices. As electromagnetism plays a role in cells homeostasis, such electromagnetic signals could potentially also alter some physiological processes. Herein we report on distinct biological parameters assessment after cellular spheroids exposure to high power electromagnetic signals, such as the ones used for defense applications. Signals effects were assessed in tumor cells spheroids and in normal human dermal fibroblasts spheroids, where macroscopic aspect, growth, plasma membrane integrity, induction of apoptosis, ATP content, and mitochondrial potential were investigated after spheroids exposure to high power electromagnetic signals. No significant effects were observed, indicating that 1.5 GHz narrowband electromagnetic fields with incident amplitude level of 40 kV/m, and 150 MHz moderate-band electric fields with an amplitude of 72.5 to approximately 200 kV/m, do not cause any significant alterations of assessed parameters.
Assuntos
Campos Eletromagnéticos , Esferoides Celulares/efeitos da radiação , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos da radiação , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Processamento de Sinais Assistido por Computador , Esferoides Celulares/citologia , TemperaturaRESUMO
Dendrimers are nanosized, arborescent polymers of which size and structure are perfectly controlled. This is one reason why they are widely used for biomedical purposes. Previously, we showed that a phosphorus-based dendrimer capped with anionic azabisphosphonate groups (so-called ABP dendrimer) has immuno-modulatory and anti-inflammatory properties towards human immune cells in vitro. Thereafter, we have shown that the ABP dendrimer has a promising therapeutic efficacy to treat models of chronic inflammatory disorders. On the way to clinical translation, the biodistribution and the safety of this drug-candidate has to be thoroughly assessed. In this article, we present preliminary non-clinical data regarding biodistribution, hematological safety, genotoxicity, maximal tolerated doses, and early cardiac safety of the ABP dendrimer. One of the genotoxicity assays reveals a potential mutagen effect of the item at a concentration above 200 µM, i.e., up to 100 times the active dose in vitro on human immune cells. However, as the results obtained for all the other assays show that the ABP dendrimer has promising biodistribution and safety profiles, there is no red flag raised to hamper the regulatory pre-clinical development of the ABP dendrimer.
Assuntos
Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Dendrímeros/efeitos adversos , Dendrímeros/farmacocinética , Hidrazonas/química , Segurança , Animais , Anti-Inflamatórios/química , Dendrímeros/química , Feminino , Camundongos , Ratos , Distribuição TecidualRESUMO
BACKGROUND: Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results. METHODS: In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)). RESULTS: pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice. CONCLUSIONS: The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.
Assuntos
Eletroporação , Terapia Genética , Interleucina-12/genética , Melanoma Experimental/terapia , Animais , Apoptose , Feminino , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , PlasmídeosRESUMO
OBJECTIVE: Skin is an attractive target tissue for gene transfer due to its size, accessibility, and its immune competence. One of the promising delivery methods is gene delivery by means of electroporation (EP), i.e., gene electrotransfer (GET). To assess the importance of different effects of electroporation for successful GET we investigated: stress response and transfection efficacy upon different pulse protocols. Moreover, numerical modeling was used to explain experimental results and to test the agreement of experimental results with current knowledge about GET. METHODS: Double transgenic mice Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) were used to determine the level of stress sensed by the cell in the tissue in vivo that was exposed to EP. The effect of five different pulse protocols on stress levels sensed by the exposed cells and their efficacy for gene electrotransfer for two plasmids pEGFP-C1 (EGFP) and pCMV-tdTomato was tested. RESULTS: Quantification of the bioluminescence signal intensity shows that EP, regardless of the electric pulse parameters used, increased mean bioluminescence compared to the baseline bioluminescence signal of the non-exposed skin. The results of numerical modeling indicate that thermal stress alone is not sufficient to explain the measured bioluminescence signal. Of the tested pulse protocols, the highest expression of EGFP and tdTomato was achieved with HV-MV (high voltage - medium voltage) protocols, which agrees also with numerical model. SIGNIFICANCE: Although EP is widely used as a method for gene delivery, we show that the field could benefit from the use of mathematical modeling by introducing additional parameters such as EP induced stress and electrophoretic movement of plasmids.
Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Animais , Simulação por Computador , Camundongos , Camundongos Transgênicos , Pele/metabolismoRESUMO
The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step-by-step protocol describes the procedures to obtain an efficient GET targeted to the skin by using easy-to-use noninvasive electrodes after intradermal plasmid injection (i.d. GET). A specific pulse sequence is reported. Expression is observed by in vivo fluorescence imaging during >2 weeks as the plasmid was coding for tdTomato. The protocol is efficient for the transient expression of clinical proteins.
Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Pele/metabolismo , Animais , Feminino , Expressão Gênica/genética , Terapia Genética/métodos , Vetores Genéticos , Injeções Intradérmicas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genéticaRESUMO
Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.
Assuntos
Eletroporação/métodos , Pele/metabolismo , Animais , Eletricidade , Eletrodos , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Interleucina-12/metabolismo , Medições Luminescentes , Masculino , Camundongos Endogâmicos C57BL , Plasmídeos/metabolismo , Fatores de Tempo , Distribuição Tecidual , TransgenesRESUMO
Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.
RESUMO
Macrophage recruitment is essential for tissue homeostasis but detrimental in most cancers. Tumor-associated macrophages (TAMs) play a key role in cancer progression. Controlling their migration is, thus, potentially therapeutic. It is assumed that macrophages use amoeboid motility in vivo like other leukocytes. However, it has not yet been explored. We examined TAM migration using intravital microscopy in mouse tumors and by monitoring ex vivo tissue infiltration in human surgical samples. We demonstrated that TAMs perform protease-dependent and ROCK-independent mesenchymal migration inside mouse fibrosarcoma and breast cancer explants using their own matrix metalloproteases (MMP). In contrast, macrophages use ROCK-dependent and protease-independent amoeboid migration inside inflamed ear derma and in connective tissue at the tumor periphery. We also showed that inhibition of mesenchymal migration correlates with decreased TAM recruitment and tumor growth. In conclusion, this study elucidates how macrophages migrate in vivo, and it reveals that the MMP-dependent migration mode of TAMs provides a rationale for a new strategy in cancer immunotherapy: to target TAMs specifically through their motility. Cancer Immunol Res; 6(11); 1337-51. ©2018 AACR.
Assuntos
Neoplasias da Mama/patologia , Imunoterapia/métodos , Macrófagos/patologia , Metaloproteinases da Matriz/metabolismo , Otite/patologia , Amidas/farmacologia , Animais , Neoplasias da Mama/metabolismo , Movimento Celular , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mesoderma/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia/métodos , Técnicas de Cultura de Órgãos , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Tiofenos/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Delivery of electric field pulses, i.e. electroporation (EP), to tissues has been shown to have a blood flow modifying effect. Indeed, the diameter of blood vessels exposed to EP is immediately reduced resulting in blood flow abrogation, followed by an increase in vascular permeability. The main cause of the increased permeability remains unknown. The aim of this study was to determine whether the in vivo effects of EP on permeability of blood vessels are linked to the permeabilization of endothelial cells' membrane (EC) and/or disruption of cell-to-cell junctions. We used a dorsal window chamber model in C57Bl/6 mice coupled with multiphoton microscopy and fluorescently labelled antibodies against PECAM-1 (CD31) to visualize endothelial cell-to-cell junctions. Clinically validated EP parameters were used and behavior of cell-to-cell junctions, in combination with leakage of 70â¯kDa fluorescein isothiocyanate labelled dextran (FD), was followed in time. After EP, a constriction of blood vessels was observed and correlated with the change in the shape of ECs. This was followed by an increase in permeability of blood vessels for 70â¯kDa FD and a decrease in the volume of labelled cell-to-cell junctions. Both parameters returned to pre-treatment values in 50% of mice. For the remaining 50%, we hypothesize that disruption of cell-to-cell junctions after EP may trigger the platelet activation cascade. Our findings show for the first time in vivo that alterations in cell-to-cell junctions play an important role in the response of blood vessels to EP and explain their efficient permeabilization.