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Motivated by the clinical success of gold(I) metallotherapeutic Auranofin in the effective treatment of both inflammatory and cancer diseases, we decided to prepare, characterize, and further study the [Au(kin)(PPh3)] complex (1), where Hkin = kinetin, 6-furfuryladenine, for its in vitro anti-cancer and anti-inflammatory activities. The results revealed that the complex (1) had significant in vitro cytotoxicity against human cancer cell lines (A2780, A2780R, PC-3, 22Rv1, and THP-1), with IC50 ≈ 1-5 µM, which was even significantly better than that for the conventional platinum-based drug Cisplatin while comparable with Auranofin. Although its ability to inhibit transcription factor NF-κB activity did not exceed the comparative drug Auranofin, it has been found that it is able to positively influence peroxisome-proliferator-activated receptor-gamma (PPARγ), and as a consequence of this to have the impact of moderating/reducing inflammation. The cellular effects of the complex (1) in A2780 cancer cells were also investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential, and shotgun proteomic analysis. Proteomic analysis of R2780 cells treated with complex (1) and starting compounds revealed possible different places of the effect of the studied compounds. Moreover, the time-dependent cellular accumulation of copper was studied by means of the mass spectrometry study with the aim of exploring the possible mechanisms responsible for its biological effects.
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Oro , Neoplasias Ováricas , Humanos , Femenino , Oro/farmacología , Oro/química , Cinetina/farmacología , Línea Celular Tumoral , Reguladores del Crecimiento de las Plantas/farmacología , PPAR gamma , Auranofina/farmacología , Proteómica , Neoplasias Ováricas/metabolismo , ApoptosisRESUMEN
Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a degenerative muscle disease triggered by mutations in the gene encoding for the intracellular protein dystrophin. A major source for the arrhythmias in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. The reason for ventricular conduction impairments and the associated arrhythmias in the dystrophic heart has remained unidentified. In the present study, we explored the hypothesis that dystrophin-deficient cardiac Purkinje fibers have reduced Na+ currents (INa), which would represent a potential mechanism underlying slowed ventricular conduction in the dystrophic heart. Therefore, by using a Langendorff perfusion system, we isolated Purkinje fibers from the hearts of adult wild-type control and dystrophin-deficient mdx mice. Enhanced green fluorescent protein (eGFP) expression under control of the connexin 40 gene allowed us to discriminate Purkinje fibers from eGFP-negative ventricular working cardiomyocytes after cell isolation. Finally, we recorded INa from wild-type and dystrophic mdx Purkinje fibers for comparison by means of the whole cell patch clamp technique. We found substantially reduced INa densities in mdx compared with wild-type Purkinje fibers, suggesting that dystrophin deficiency diminishes INa. Because Na+ channels in the Purkinje fiber membrane represent key determinants of ventricular conduction velocity, we propose that reduced INa in Purkinje fibers at least partly explains impaired ventricular conduction and the associated arrhythmias in the dystrophic heart.NEW & NOTEWORTHY Dystrophic cardiac Purkinje fibers have abnormally reduced Na+ current densities. This explains impaired ventricular conduction in the dystrophic heart.
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Arritmias Cardíacas/metabolismo , Trastorno del Sistema de Conducción Cardíaco/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Ramos Subendocárdicos/metabolismo , Canales de Sodio/metabolismo , Potenciales de Acción/fisiología , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/fisiopatología , Trastorno del Sistema de Conducción Cardíaco/complicaciones , Trastorno del Sistema de Conducción Cardíaco/fisiopatología , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/fisiopatología , Sodio/metabolismoRESUMEN
During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 µM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the ß2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.
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Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Interleucina-8/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Sesquiterpenos/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Antígenos CD18/antagonistas & inhibidores , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Interleucina-8/biosíntesis , Neutrófilos/fisiología , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Resveratrol, the phenolic substance isolated initially from Veratrum grandiflorum and richly present in grapes, wine, peanuts, soy, and berries, has been attracting attention of scientists and medical doctors for many decades. Herein, we review its effects on the vascular system. Studies utilizing cell cultures and pre-clinical models showed that resveratrol alleviates oxidative stress and inflammation. Furthermore, resveratrol suppresses vascular smooth muscle cell proliferation, promotes autophagy, and has been investigated in the context of vascular senescence. Pre-clinical models unambiguously demonstrated numerous vasculoprotective effects of resveratrol. In clinical trials, resveratrol moderately diminished systolic blood pressure in hypertensive patients, as well as blood glucose in patients with diabetes mellitus. Yet, open questions remain, as exemplified by a recent report which states that the intake of resveratrol might blunt certain positive effects of exercise in older persons, and further research addressing the framework for long-term use of resveratrol as a food supplement, will stay in demand.
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Sistema Cardiovascular/efectos de los fármacos , Resveratrol/farmacología , Animales , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/patología , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Humanos , Resveratrol/uso terapéuticoRESUMEN
OBJECTIVE: Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. APPROACH AND RESULTS: We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1-/- bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. CONCLUSIONS: We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1.
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Macrófagos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteolisis , Fibrosis , Humanos , Pulmón/enzimología , Pulmón/patología , Metaloproteinasa 14 de la Matriz/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.
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BACKGROUND: Angiogenesis, the formation of new blood vessels, is an essential process under physiological and pathological conditions. METHOD: Here, we improved the directed in vivo angiogenesis assay (DIVAA®) test, which is based on the usage of small Matrigel-filled tubes that are implanted into mice subcutaneously for a period of up to 15 days. The subsequent ex vivo assessment of neoangiogenesis within the silicon tubes is then achieved by fluorometry. RESULTS: We showed that the immunohistochemical quantification of the ingrowth of endothelial cells, based on CD31, was superior to the fluorometric quantification advised in the manufacturer's instructions. We optimised the explantation procedure, ensuring the complete recovery of the ingrown vessels. Using this modified protocol, we investigated the effect of the length of stay of the implanted tubes as well as of the concentration of the growth factors VEGF and FGF on the assay. CONCLUSION: Our improved protocol offered an effective and reliable alternative to the original assay, which is expected to facilitate in vivo research on angiogenesis and, thus, might drive the development of novel therapeutic agents.
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Inductores de la Angiogénesis/administración & dosificación , Bioensayo/métodos , Colágeno/administración & dosificación , Células Endoteliales/efectos de los fármacos , Inmunohistoquímica , Laminina/administración & dosificación , Neovascularización Fisiológica/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteoglicanos/administración & dosificación , Tejido Subcutáneo/irrigación sanguínea , Animales , Biomarcadores/metabolismo , Combinación de Medicamentos , Células Endoteliales/metabolismo , Fluorometría , Procesamiento de Imagen Asistido por Computador , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT-PCR, we found CCL7, a chemokine ligand known to interact with multiple C-C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non-lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod-induced psoriasis-like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro-psoriatic cytokines such as CCL20, IL-12p40 and IL-17C, while its blockade led to an increase in the antipsoriatic cytokine IL-4. In humans receiving the TNF-α-blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF-α-dependent Th1/Th17-mediated inflammation in lesional psoriatic skin.
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Quimiocina CCL7/metabolismo , Psoriasis/etiología , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Animales , Estudios de Casos y Controles , Quimiocina CCL7/genética , Dermatitis Atópica/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Endoteliales/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Infliximab/farmacología , Interleucina-1beta/metabolismo , Queratinocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Psoriasis/inmunología , Psoriasis/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Enfermedades de la Piel/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto JovenRESUMEN
RATIONALE: Innate and adaptive immune responses alter numerous homeostatic processes that are controlled by nuclear hormone receptors. NR4A1 is a nuclear receptor that is induced in vascular pathologies, where it mediates protection. OBJECTIVE: The underlying mechanisms that regulate the activity of NR4A1 during vascular injury are not clear. We therefore searched for modulators of NR4A1 function that are present during vascular inflammation. METHODS AND RESULTS: We report that the protein encoded by interferon stimulated gene 12 (ISG12), is a novel interaction partner of NR4A1 that inhibits the transcriptional activities of NR4A1 by mediating its Crm1-dependent nuclear export. Using 2 models of vascular injury, we show that ISG12-deficient mice are protected from neointima formation. This effect is dependent on the presence of NR4A1, as mice deficient for both ISG12 and NR4A1 exhibit neointima formation similar to wild-type mice. CONCLUSIONS: These findings identify a previously unrecognized feedback loop activated by interferons that inhibits the vasculoprotective functions of NR4A nuclear receptors, providing a potential new therapeutic target for interferon-driven pathologies.
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Traumatismos de las Arterias Carótidas/prevención & control , Arteria Femoral/metabolismo , Inflamación/prevención & control , Proteínas de la Membrana/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteínas/metabolismo , Lesiones del Sistema Vascular/prevención & control , Transporte Activo de Núcleo Celular , Animales , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Retroalimentación Fisiológica , Arteria Femoral/lesiones , Arteria Femoral/patología , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interferones/metabolismo , Carioferinas/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Tiempo , Transcripción Genética , Transfección , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/inmunología , Lesiones del Sistema Vascular/metabolismo , Lesiones del Sistema Vascular/patología , Proteína Exportina 1RESUMEN
Alterations in cardiac impulse conduction may exert both beneficial and detrimental effects. The assessment of ventricular conduction properties is of paramount importance both in clinical and in experimental settings. Currently the duration of the QRS complex is regarded as hallmark of in-vivo assessment of global ventricular conduction time. In addition, the amplitude of the QRS complex has been suggested to reflect ventricular conduction time in man and in rats. Here, for the first time, we systematically investigated the relationship between QRS duration ("QRS") and QRS amplitude ("RS-height"; RSh) in the murine ECG obtained during anesthesia. In mice harbouring a homozygous knockout of the transmembrane protein podoplanin (PDPN-/-; n = 10) we found both a shorter QRS and a greater RSh than in wild-type animals (n = 13). In both genotypes cumulative i.p. administration of 5 mg/kg and 10 mg/kg of the Na channel blocker flecainide resulted in dose-dependent QRS increase and RSh decrease, whereby the drug-induced changes in RSh were greater than in QRS. In both genotypes the flecainide-induced changes in QRS and in RSh were significantly correlated with each other (R = -0.56, P = 0.004). Whereas dispersion of QRS and RSh was similar between genotypes, dispersion of the ratio QRS/RSh was significantly smaller in PDPN-/- than in wild-types. We conclude that in the murine ECG QRS is inversely related to RSh. We suggest that both parameters should be considered in the analysis of ventricular conduction time in the murine ECG.
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Flecainida , Sistema de Conducción Cardíaco , Ratas , Animales , Ratones , Flecainida/farmacología , Electrocardiografía , Ventrículos Cardíacos , Frecuencia CardíacaRESUMEN
Atherosclerosis is a chronic, inflammatory disease of the vessel wall where triggered immune cells bind to inflamed endothelium, extravasate and sustain local inflammation. Leukocyte adhesion and extravasation are mediated by adhesion molecules expressed by activated endothelial cells, like intercellular adhesion molecule 1 (ICAM-1). Extracellular adherence protein (Eap) from Staphylococcus aureus binds to a plethora of extracellular matrix proteins, including ICAM-1 and its ligands macrophage-1 antigen (Mac-1, αMß2) and lymphocyte function-associated antigen 1 (LFA-1, αLß2), thereby disrupting the interaction between leukocytes and endothelial cells. We aimed to use Eap to inhibit the interaction of leukocytes with activated endothelial cells in settings of developing and established atherosclerosis in apolipoprotein E (ApoE) deficient mice on high-fat diet. In developing atherosclerosis, Eap treatment reduced circulating platelet-neutrophil aggregates as well as infiltration of T cells and neutrophils into the growing plaque, accompanied by reduced formation of neutrophil extracellular traps (NETs). However, plaque size did not change. Intervention treatment with Eap of already established plaques did not result in cellular or morphological plaque changes, whereas T cell infiltration was increased and thereby again modulated by Eap. We conclude that although Eap leads to cellular changes in developing plaques, clinical implications might be limited as patients are usually treated at a more advanced stage of disease progression. Hence, usage of Eap might be an interesting mechanistic tool for cellular infiltration during plaque development in basic research but not a clinical target.
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Aterosclerosis , Placa Aterosclerótica , Ratones , Animales , Molécula 1 de Adhesión Intercelular/genética , Staphylococcus aureus/metabolismo , Células Endoteliales/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , FenotipoRESUMEN
Pre-clinical studies from the recent past have indicated that senescent cells can negatively affect health and contribute to premature aging. Targeted eradication of these cells has been shown to improve the health of aged experimental animals, leading to a clinical interest in finding compounds that selectively eliminate senescent cells while sparing non-senescent ones. In our study, we identified a senolytic capacity of statins, which are lipid-lowering drugs prescribed to patients at high risk of cardiovascular events. Using two different models of senescence in human vascular endothelial cells (HUVECs), we found that statins preferentially eliminated senescent cells, while leaving non-senescent cells unharmed. We observed that the senolytic effect of statins could be negated with the co-administration of mevalonic acid and that statins induced cell detachment leading to anoikis-like apoptosis, as evidenced by real-time visualization of caspase-3/7 activation. Our findings suggest that statins possess a senolytic property, possibly also contributing to their described beneficial cardiovascular effects. Further studies are needed to explore the potential of short-term, high-dose statin treatment as a candidate senolytic therapy.
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Senescencia Celular , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Animales , Humanos , Anciano , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Endoteliales , Anoicis , SenoterapéuticosRESUMEN
Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.
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Regulación hacia Abajo , Fosfatasa 1 de Especificidad Dual/metabolismo , Fosfatasa 6 de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de LDL/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Lactoferrina/farmacología , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Factores de Tiempo , Vitronectina/metabolismoRESUMEN
During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin(+) lymph sacs and cardinal veins, but not in podoplanin(-/-) embryos. Thus, podoplanin(-/-) mice develop a "nonseparation" phenotype, characterized by a blood-filled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin-blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.
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Plaquetas/fisiología , Vasos Sanguíneos/embriología , Vasos Linfáticos/embriología , Glicoproteínas de Membrana/fisiología , Animales , Antiinfecciosos/farmacología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Proteínas del Citoesqueleto/fisiología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endotelio Linfático/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/metabolismo , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Agregación Plaquetaria , Embarazo , Ácido Salicílico/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Paulownia tomentosa Steud., a traditional Chinese medicinal plant, was used for many centuries in Chinese herbal medicine as a component of remedies for many illnesses, including inflammatory diseases. It is a rich source of phenolic compounds, mainly geranylated flavonoids, which are currently studied for their promising biological activities. AIM OF THE STUDY: The study aimed to isolate minor geranylated flavanones and flavones from P. tomentosa fruit and evaluate their cytotoxicity and possible anti-inflammatory effects in a cell-based model of inflammation. MATERIALS AND METHODS: Chromatographic separation of chloroform portion of the ethanolic extract of P. tomentosa fruit led to the isolation of twenty-seven flavonoids (1-27), twenty-six of them geranylated with different modifications and one non-geranylated flavanone, and two phenolic compounds. Compounds were identified using UV, IR, HRMS, NMR, and CD spectroscopy. Ten of these compounds (7-10, 12, 21, 22, 24, 25, and 27) were determined to be new flavonoid derivatives obtained from a natural source for the first time. Selected compounds were analyzed for cytotoxicity and anti-inflammatory potential to affect the activation of nuclear factor κB/activator protein 1 (NF-κB/AP-1) after lipopolysaccharide (LPS) stimulation. RESULTS: All the test compounds (1-21 and 23-26) reduced the activation of NF-κB/AP-1 24 h after the addition of LPS. Eight compounds (5, 14-18, 21, and 26) were more active than prednisone, a widely used anti-inflammatory drug. However, this effect was not seen significantly on the level of TNF-α and IL-1ß, which can be explained by the plurality of possible outcomes of activation of the NF-κB pathway in cells. CONCLUSIONS: Results of the presented study confirmed that constituents from traditional Chinese medicinal plant P. tomentosa Steud. have promising anti-inflammatory activities and can serve as a potential source of inspiration for new anti-inflammatory medications.
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Lamiales , Plantas Medicinales , Antiinflamatorios/química , Flavonoides/análisis , Frutas/química , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Extractos Vegetales/análisis , Extractos Vegetales/farmacología , Plantas Medicinales/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Background: Extravasation during chemotherapy administration can lead to dangerous adverse effects ranging from pain to tissue necrosis. Evidence-based data about prevention and treatment of extravasation injuries of some clinically used compounds still remains elusive. This work aimed to investigate, in a preclinical mouse model, the effects of extravasation of two chemotherapeutic agents, nanoliposomal irinotecan (nal-Iri) and trabectedin. In addition, we aimed to study treatment options for injuries induced by extravasation of these substances. Methods: Mice were subcutaneously injected with nal-Iri or trabectedin applied in clinically used concentration. Doxorubicin was used as a positive control. In subsequently performed experiments, hyaluronidase, DMSO and tacrolimus were tested as potential treatments against extravasation-induced injuries by trabectedin. Systemic effects were analyzed by observation and documentation of the health status of mice and local reactions were measured and graded. In addition, hematoxylin-eosin stained histological sections of the treated skin areas were analyzed. Results: Of the two tested substances, only trabectedin showed vesicant effects. Subcutaneous injection of trabectedin caused erythema formation in mice by day two that was progressing to skin ulcerations by day five. Furthermore, we found that topical treatment of mice with tacrolimus or DMSO reduced the vesicant effects of trabectedin. The results observed in vivo were supported microscopically by the analysis of histological sections. Conclusions: We recommend classifying trabectedin as a vesicant agent and nal-Iri as a non-vesicant agent. Furthermore, our results obtained in a preclinical model suggest that tacrolimus and DMSO might be suitable treatment options of trabectedin extravasations, a finding that might be further utilized in clinical studies.
RESUMEN
Nicotine addiction develops predominantly during human adolescence through smoking. Self-administration experiments in rodents verify this biological preponderance to adolescence, suggesting evolutionary-conserved and age-defined mechanisms which influence the susceptibility to nicotine addiction. The hippocampus, a brain region linked to drug-related memory storage, undergoes major morpho-functional restructuring during adolescence and is strongly affected by nicotine stimulation. However, the signaling mechanisms shaping the effects of nicotine in young vs. adult brains remain unclear. MicroRNAs (miRNAs) emerged recently as modulators of brain neuroplasticity, learning and memory, and addiction. Nevertheless, the age-dependent interplay between miRNAs regulation and hippocampal nicotinergic signaling remains poorly explored. We here combined biophysical and pharmacological methods to examine the impact of miRNA-132/212 gene-deletion (miRNA-132/212-/-) and nicotine stimulation on synaptic functions in adolescent and mature adult mice at two hippocampal synaptic circuits: the medial perforant pathway (MPP) to dentate yrus (DG) synapses (MPP-DG) and CA3 Schaffer collaterals to CA1 synapses (CA3-CA1). Basal synaptic transmission and short-term (paired-pulse-induced) synaptic plasticity was unaltered in adolescent and adult miRNA-132/212-/- mice hippocampi, compared with wild-type controls. However, nicotine stimulation promoted CA3-CA1 synaptic potentiation in mature adult (not adolescent) wild-type and suppressed MPP-DG synaptic potentiation in miRNA-132/212-/- mice. Altered levels of CREB, Phospho-CREB, and acetylcholinesterase (AChE) expression were further detected in adult miRNA-132/212-/- mice hippocampi. These observations propose miRNAs as age-sensitive bimodal regulators of hippocampal nicotinergic signaling and, given the relevance of the hippocampus for drug-related memory storage, encourage further research on the influence of miRNAs 132 and 212 in nicotine addiction in the young and the adult brain.
Asunto(s)
Envejecimiento/genética , Hipocampo/fisiología , MicroARNs/metabolismo , Plasticidad Neuronal/genética , Nicotina/farmacología , Acetilcolinesterasa/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Giro Dentado/efectos de los fármacos , Giro Dentado/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Plasticidad Neuronal/efectos de los fármacos , Fosforilación/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
OBJECTIVE: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. METHODS AND RESULTS: I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. CONCLUSIONS: I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.
Asunto(s)
Arteriopatías Oclusivas/prevención & control , Proliferación Celular , Indoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Oximas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Arteriopatías Oclusivas/metabolismo , Arteriopatías Oclusivas/patología , Becaplermina , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Constricción Patológica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Arteria Femoral/efectos de los fármacos , Arteria Femoral/metabolismo , Arteria Femoral/patología , Citometría de Flujo , Humanos , Hiperplasia , Interferón gamma/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Trombina/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Based on the traditional use and scientific reports on the anti-inflammatory potential of red sandalwood, i.e., the heartwood of Pterocarpus santalinus L., we investigated its activity in a model of IL-1 stimulated endothelial cells. Endothelial cells were stimulated with IL-1 with or without prior incubation with a defined sandalwoodextract (PS), and analyzed for the expression of selected pro-inflammatory genes. The activity of NF-κB, a transcription factor of central importance for inflammatory gene expression was assessed by reporter gene analysis, Western blotting of IκBα, and nuclear translocation studies. In addition, microarray studies were performed followed by verification of selected genes by qPCR and supplemented by bioinformatics analysis. Our results show that PS is able to suppress the induction of E-selectin and VCAM-1, molecules that mediate key steps in the adhesion of leukocytes to the endothelium. It also suppressed the activity of an NF-κB reporter, IκBα phosphorylation and degradation, and the nuclear translocation of NF-κB RelA. In contrast, it stimulated JNK phosphorylation indicating the activation of the JNK signaling pathway. Gene expression profiling revealed that PS inhibits only a specific subset of IL-1 induced genes, while others remain unaffected. Most strongly suppressed genes were the signal transducer TRAF1 and the chemokine CX3CL1, whereas IL-8 was an example of a non-affected gene. Notably, PS also stimulated the expression of certain genes, including ones with negative regulatory function, e.g., members of the NR4A family, the mRNA destabilizing protein TTP as well as the transcription factors ATF3 and BHLHB40. These results provide mechanistic insight into the anti-inflammatory activity of PS, and suggest that it acts through the interplay of negative and positive regulators to achieve a differential inhibition of inflammatory gene expression.