RESUMEN
This study sought to develop noninvasive, in vivo imaging schemes that allow for quantitative assessment of pulmonary microvascular functional status based on the combination of pulmonary T1 mapping and dynamic contrast-enhanced (DynCE) imaging. Ultrashort-echo-time (UTE) imaging at 9.4 T of lung parenchyma was performed. Retrospective gating was based on modulation of the first point in each recorded spoke. T1 maps were obtained using a series of five consecutive images with varying RF angles and analyzed with the variable flip angle approach. The obtained mean T1 lung value of 1078 ± 38 ms correlated well with previous reports. Improved intersession variability was observed, as evident from a decreased standard deviation of motion-resolved T1 mapping (F-test = 0.051). Animals received lipopolysaccharide (LPS) and were imaged at t = 2, 6, and 12 h after administration. The nitric oxide (NO)-dependent function was assessed according to changes in lung T1 after L-NAME injection, while microvascular perfusion and oxidant stress were assessed with contrast-enhanced imaging after injection of gadolinium or 3-carbamoyl-proxyl nitroxide radical, respectively. Retrospectivel gated UTE allowed robust, motion-compensated imaging that could be used for T1 mapping of lung parenchyma. Changes in lung T1 after L-NAME injection indicated that LPS induced overproduction of NO at t = 2 and 6 h after LPS, but NO-dependent microvascular function was impaired at t = 12 h after LPS. DynCE imaging at t = 6 h after LPS injection revealed decreased microvascular perfusion, with increased vascular permeability and oxidant stress. MRI allows to visualize and quantify lung microvascular NO-dependent function and its concomitant impairment during acute respiratory distress syndrome development with high sensitivity. UTE T1 mapping appears to be sensitive and useful in probing pulmonary microvascular functional status.
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Lesión Pulmonar Aguda , Óxido Nítrico , Animales , Ratones , Estudios Retrospectivos , NG-Nitroarginina Metil Éster , Modelos Animales de Enfermedad , Lipopolisacáridos , Imagen por Resonancia Magnética/métodos , Pulmón/diagnóstico por imagen , Oxidantes , Imagenología Tridimensional/métodosRESUMEN
Nicotinamide adenine dinucleotide (NAD+ ) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide-dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD+ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5'-nucleotidase knockout mice (CD73-/-). Human non-stenotic valves (n = 10) actively converted NAD+ and NMN via both CD73 and NAD+ -glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD+ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD+ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm2 ) compared with controls. CD38 was also primarily engaged in human vascular NAD+ metabolism. Studies using specific ecto-enzyme inhibitors and CD73-/- mice confirmed that CD73 is not the only enzyme involved in NAD+ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD+ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.
RESUMEN
Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO2-) and nitrate (NO3-) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2- and NO3- co-therapy in mice with angiotensin-II (AT-II)-induced arterial hypertension. High-dose AT-II (1 mg/kg/d, 1w, s. c.) was used to induce arterial hypertension in male C57BL/6 mice. Additional inorganic nitrite (7.5 mg/kg/d, p. o.) or nitrate (150 mg/kg/d, p. o.) were administered via the drinking water. Blood pressure (tail-cuff method) and endothelial function (isometric tension) were determined. Oxidative stress and inflammation markers were quantified in aorta, heart, kidney and blood. Co-treatment with inorganic nitrite, but not with nitrate, normalized vascular function, oxidative stress markers and inflammatory pathways in AT-II treated mice. Of note, the highly beneficial effects of nitrite on all parameters and the less pronounced protection by nitrate, as seen by improvement of some parameters, were observed despite no significant increase in plasma nitrite levels by both therapies. Methemoglobin levels tended to be higher upon nitrite/nitrate treatment. Nutritional nitric oxide precursors represent a non-pharmacological treatment option for hypertension that could be applied to the general population (e.g. by eating certain vegetables). The more beneficial effects of inorganic nitrite may rely on superior NO bioactivation and stronger blood pressure lowering effects. Future large-scale clinical studies should investigate whether hypertension and cardiovascular outcome in general can be influenced by dietary inorganic nitrite therapy.
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Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Nitratos/farmacología , Nitritos/farmacología , Administración Oral , Angiotensina II/administración & dosificación , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/sangre , Presión Sanguínea/efectos de los fármacos , Hipertensión/inducido químicamente , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Nitratos/administración & dosificación , Nitratos/sangre , Nitritos/administración & dosificación , Nitritos/sangre , Estrés Oxidativo/efectos de los fármacosRESUMEN
Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran's effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2- quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.
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Angiotensina II/efectos adversos , Antitrombinas/administración & dosificación , Dabigatrán/administración & dosificación , Ácidos Hidroxieicosatetraenoicos/sangre , Hipertensión/metabolismo , Remodelación Vascular/efectos de los fármacos , Animales , Antitrombinas/farmacología , Cromatografía Liquida , Dabigatrán/farmacología , Modelos Animales de Enfermedad , Hipertensión/sangre , Hipertensión/inducido químicamente , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Óxido Nítrico/metabolismo , Espectrometría de Masas en Tándem , Factor de von Willebrand/metabolismoRESUMEN
A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.
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Citoesqueleto/metabolismo , Cuerpos de Heinz/patología , Glutaral/toxicidad , Cuerpos de Heinz/ultraestructura , Hemo/metabolismo , Hemoglobinas/metabolismo , Humanos , Masculino , Agregado de Proteínas/fisiologíaRESUMEN
Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection. We show their potential in the detection of lung macrometastasis, in which we already observed the ECM remodelling. The CARS image revealed a dense cell fraction typical of ECM remodeling and reduction of the TPEF signal together with an increase of fluorescence lifetime predominantly due to NAD(P)H suggesting metabolic changes in the metastatic foci. FT-IR spectroscopy allowed not only for macrometastasis detection but also their stage definition based mainly on the analysis of proteins, RNA and glycogen fractions. The multimodal approach additionally suggested partial enzymatic degradation of elastin in ECM and collagen remodelling.
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Neoplasias de la Mama , Neoplasias Pulmonares , Animales , Neoplasias de la Mama/diagnóstico por imagen , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Ratones , Fotones , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría RamanRESUMEN
In fertilized fish eggs, lipids are an energy reservoir for the embryo development and substrate for organogenesis. They occur in the cytoplasmic area and form lipid droplets (LDs), but also the yolk egg is composed of lipids and proteins. Insight on the LD formation and distribution and their interactions with other cellular organelles could provide information about the role based on the egg development. For non-destructive, macro-scale visualization of biochemical components of fish eggs, such as lipids proteins and water, near-infrared (NIR) imaging is the method of choice. Mid-infrared (MIR) and Raman spectroscopy imaging were used to provide details on chemical composition of LDs and other egg organelles. NIR imaging illustrated main compartments of the egg including membrane, LDs, yolk, relative protein, and lipid content in well-localized egg structures and their interactions with water molecules. In the yolk, a co-existence of lipids and proteins with carotenoids and carbohydrates was detected by Raman spectroscopy. Results showed a prominent decrease of unsaturated fatty acids, phospholipids, and triglycerides/cholesteryl esters content in the eggs due to the embryo development. An opposite trend of changes was observed by MIR spectroscopy for the glycogen, suggesting that consumption of lipids occurred with production of this carbohydrate. The comprehensive vibrational spectroscopic analysis based on NIR, MIR, and Raman imaging is a unique tool in studying in situ dynamic biological processes.
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Gotas Lipídicas/química , Espectrofotometría Infrarroja/métodos , Espectrometría Raman/métodos , Animales , Yema de Huevo/clasificación , Huevos/análisis , Fosfolípidos/química , Cigoto/químicaRESUMEN
This work focused on a detailed assessment of lung tissue affected by metastasis of breast cancer. We used large-area chemical scanning implemented in Fourier transform infrared (FTIR) spectroscopic imaging supported with classical histological and morphological characterization. For the first time, we differentiated and defined biochemical changes due to metastasis observed in the lung parenchyma, atelectasis, fibrous, and muscle cells, as well as bronchi ciliate cells, in a qualitative and semi-quantitative manner based on spectral features. The results suggested that systematic extracellular matrix remodeling with the progress of the metastasis process evoked a decrease in the fraction of the total protein in atelectasis, fibrous, and muscle cells, as well as an increase of fibrillar proteins in the parenchyma. We also detected alterations in the secondary conformations of proteins in parenchyma and atelectasis and changes in the level of hydroxyproline residues and carbohydrate moieties in the parenchyma. The results indicate the usability of FTIR spectroscopy as a tool for the detection of extracellular matrix remodeling, thereby enabling the prediction of pre-metastatic niche formation.
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Neoplasias de la Mama/patología , Matriz Extracelular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Espectroscopía Infrarroja por Transformada de Fourier , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Femenino , Humanos , Inmunohistoquímica , RatonesRESUMEN
Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation. We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-ß-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways. This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.
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Adenosina Desaminasa/genética , Aorta/metabolismo , Aterosclerosis/genética , Inflamación/genética , Adenosina/genética , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/patología , Membrana Celular/efectos de los fármacos , Colesterol/genética , Colesterol/metabolismo , Células Endoteliales/enzimología , Exocitosis/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Inflamación/enzimología , Inflamación/patología , Molécula 1 de Adhesión Intercelular/genética , Interleucina-6/genética , Quinasas Janus/genética , Lipopolisacáridos/farmacología , Metabolismo/genética , Ratones , Pentostatina/farmacología , Ratas , Factores de Transcripción STAT/genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
INTRODUCTION: The tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma. MATERIALS AND METHODS: The studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery). RESULTS: Palpable primary tumors were noted in the 2nd week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1st week and progressed during the 2nd week, accompanied by increased concentration of 2-OH-E+, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1st week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2nd week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz). CONCLUSION: The analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.
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Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Metástasis de la Neoplasia/fisiopatología , Animales , Señalización del Calcio/fisiología , Femenino , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/fisiopatología , Ratones , Ratones Endogámicos BALB C , ProteómicaRESUMEN
BACKGROUND: The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis. METHODS: Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics. RESULTS: Based on primary tumour growth and pulmonary metastases, 1-2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3-4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate ("Warburg effect"). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins. CONCLUSIONS: The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Animales , Arginasa/metabolismo , Arginina/análogos & derivados , Arginina/sangre , Arginina/metabolismo , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Línea Celular Tumoral/trasplante , Cromatografía Líquida de Alta Presión/métodos , Modelos Animales de Enfermedad , Metabolismo Energético , Femenino , Humanos , Metabolismo de los Lípidos , Lípidos/sangre , Metabolómica/métodos , Ratones , Ratones Endogámicos BALB C , Estadificación de Neoplasias , Poliaminas/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Mesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial-mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice. METHODS: NO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1-5 weeks after 4T1 cancer cell inoculation in Balb/c mice. RESULTS: Phosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation. CONCLUSIONS: Decreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis.
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Neoplasias de la Mama/patología , Endotelio Vascular/patología , Neoplasias Pulmonares/patología , Pulmón/patología , Óxido Nítrico/deficiencia , Animales , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Pulmón/irrigación sanguínea , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo III/metabolismo , FosforilaciónRESUMEN
The combination of FT-IR and Raman spectroscopies allowed the biochemical profiling of lungs in the early stage of pulmonary metastasis in the murine model of breast cancer. Histological staining was used as a reference. Raman spectroscopy was especially useful in the detection and semi-quantitative analysis of the vitamin A content in lung lipofibroblasts, whereas the IR technique provided semi-quantitative information on the contents of nucleic acids, carbohydrates including glycogen, and lipids as well as changes in the secondary structures of tissue proteins. Our spectroscopic results suggest that the early phase of metastasis in the lung is characterized by a decrease in the endogenous retinoid content in combination with a decrease in the content of glycogen and lipids.
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Neoplasias de la Mama/patología , Neoplasias Pulmonares/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Animales , Línea Celular Tumoral , Glucógeno/análisis , Lípidos/análisis , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Retinoides/análisisRESUMEN
Bioreactivity of superparamagnetic iron oxide nanoparticles (SPION) coated with thin layers of either cationic or anionic chitosan derivatives and serving as contrast agents in magnetic resonance imaging (MRI) was studied in vivo using BALB/c mouse model. Synthesized dual-modal fluorescing SPION were tracked in time using both fluorescent imaging and MRI. Although SPION started to be excreted by kidneys relatively shortly after administration they were uptaken by liver enhancing MRI contrast even up to 7 days. Importantly, chitosan-coated SPION caused only mild activation of acute phase response not affecting biochemical parameters of blood. Liver histology indicated the presence of SPION and modest increase in the number of Kupffer cells. The overall results indicated that SPION coated with ultrathin layers of chitosan ionic derivatives can serve as T2 contrast agents for diagnosis of liver diseases or imaging of other organs assuming the dose is optimized according to the need.
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Quitosano/química , Medios de Contraste/farmacocinética , Compuestos Férricos/administración & dosificación , Hígado/patología , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/administración & dosificación , Animales , Femenino , Compuestos Férricos/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Nanopartículas de Magnetita/química , Ratones , Ratones Endogámicos BALB C , Distribución TisularRESUMEN
Numerous cellular and extracellular components should be analyzed in sections of atherosclerotic plaques to assess atherosclerosis progression and vulnerability. Here, we combined orcein (O) staining for elastic fibers and martius scarlet blue (MSB) polychrome to visualize various morphological contents of plaque in brachiocephalic arteries (BCA) of apoE/LDLR-/- mice. Elastic fibers (including broken elastic laminae and 'buried' fibrous caps) were stained purple and they could be easily distinguished from collagen fibers (blue). Orcein allowed clear identification of even the finest elastic fibers. Erythrocytes were stained yellow and they could easily be discerned from mature fibrin (red). Old fibrin tends to acquire blue color. The method of OMSB staining is simple, takes less than 1 h to perform and can be adapted to automatic stainers. Most importantly, the color separation is good enough to allow digital automatic segmentation of specific components in tissue section and quantitative analysis of the plaque constituents. OMSB was used to compare atherosclerotic plaques in proximal and distal regions of BCA in apoE/LDLR-/- mice. In conclusion, OMSB staining represents a novel staining that could be routinely used for qualitative and quantitative microscopic assessments of formaldehyde-fixed and paraffin-embedded sections of arteries with atherosclerotic lesions.
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Apolipoproteínas E/deficiencia , Compuestos Azo/análisis , Tronco Braquiocefálico/patología , Oxazinas/análisis , Placa Aterosclerótica/patología , Receptores de LDL/deficiencia , Coloración y Etiquetado , Animales , Compuestos Azo/química , Ratones , Ratones Noqueados , Oxazinas/química , Investigación CualitativaRESUMEN
1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)-deficient mice. ApoE/LDLR(-/-) mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1 α and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR(-/-) mice, an effect associated with an improvement in prostacyclin- and nitric oxide-dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.
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Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Niacina/uso terapéutico , Niacinamida/análogos & derivados , Receptores de LDL/deficiencia , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Niacina/farmacología , Niacinamida/farmacología , Niacinamida/uso terapéutico , Técnicas de Cultivo de Órganos , Resultado del TratamientoRESUMEN
ApoE/LDLR(-/-) mice represent a reliable model of atherosclerosis. However, it is not clear whether cardiac performance is impaired in this murine model of atherosclerosis. Here, we used MRI to characterize cardiac performance in vivo in apoE/LDLR(-/-) mice with advanced atherosclerosis. Six-month-old apoE/LDLR(-/-) mice and age-matched C57BL/6J mice (control) were examined using highly time-resolved cine-MRI [whole-chamber left ventricle (LV) imaging] and MR tagging (three slices: basal, mid-cavity and apical). Global and regional measures of cardiac function included LV volumes, kinetics, time-dependent parameters, strains and rotations. Histological analysis was performed using OMSB (orceine with Martius, Scarlet and Blue) and ORO (oil red-O) staining to demonstrate the presence of advanced coronary atherosclerosis. MR-tagging-based strain analysis in apoE/LDLR(-/-) mice revealed an increased frequency of radial and circumferential systolic stretch (25% and 50% of segments, respectively, p ≤ 0.012), increased radial post-systolic strain index (45% of segments, p = 0.009) and decreased LV untwisting rate (-30.3° (11.6°)/cycle, p = 0.004) when compared with control mice. Maximal strains and LV twist were unchanged. Most of the cine-MRI-based LV functional and anatomical parameters also remained unchanged in apoE/LDLR(-/-) mice, with only a lower filling rate, longer filling time, shorter isovolumetric contraction time and slower heart rate observed in comparison with control mice. The coronary arteries displayed severe atherosclerosis, as evidenced by histological analysis. Using comprehensive MRI methods, we have demonstrated that, despite severe coronary atherosclerosis in six-month-old apoE/LDLR(-/-) mice, cardiac performance including global parameters, twist and strains, was well preserved. Only subtle diastolic alterations, possibly of ischemic background, were uncovered. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Técnicas de Imagen Cardíaca , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/fisiopatología , Imagen por Resonancia Cinemagnética/métodos , Volumen Sistólico , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/fisiopatología , Animales , Enfermedad de la Arteria Coronaria/complicaciones , Progresión de la Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Disfunción Ventricular Izquierda/etiologíaRESUMEN
OBJECTIVE: To assess alterations in perfusion and liver function in the concanavalin A (ConA)-induced mouse model of acute liver failure (ALF) using two magnetic resonance imaging (MRI)-based methods: dynamic contrast-enhanced MRI (DCE-MRI) with Gd-EOB-DTPA contrast agent and arterial spin labelling (ASL). MATERIALS AND METHODS: BALB/c mice were studied using a 9.4 T MRI system. The IntraGateFLASHTM and FAIR-EPI pulse sequences were used for optimum mouse abdomen imaging. RESULTS: The average perfusion values for the liver of the control and ConA group were equal to 245 ± 20 and 200 ± 32 ml/min/100 g (p = 0.008, respectively). DCE-MRI showed that the time to the peak of the image enhancement was 6.14 ± 1.07 min and 9.72 ± 1.69 min in the control and ConA group (p < 0.001, respectively), while the rate of the contrast wash-out in the control and ConA group was 0.037 ± 0.008 and 0.021 ± 0.008 min-1 (p = 0.004, respectively). These results were consistent with hepatocyte injury in the ConA-treated mice as confirmed by histopathological staining. CONCLUSIONS: Both the ASL and DCE-MRI techniques represent a reliable methodology to assess alterations in liver perfusion and hepatocyte integrity in murine hepatitis.
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Hepatitis/diagnóstico por imagen , Hepatocitos/patología , Hígado/fisiopatología , Imagen por Resonancia Magnética , Enfermedad Aguda , Animales , Concanavalina A/química , Medios de Contraste/química , Gadolinio DTPA/química , Hepatitis/fisiopatología , Humanos , Hígado/diagnóstico por imagen , Fallo Hepático Agudo/diagnóstico por imagen , Fallo Hepático Agudo/fisiopatología , Ratones , Ratones Endogámicos BALB C , Perfusión , Estudios Retrospectivos , Marcadores de SpinRESUMEN
AIM: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. METHODS: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. RESULTS: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. CONCLUSION: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.
Asunto(s)
Angiotensina II , Enfermedades Vasculares , Ratones , Masculino , Animales , Angiotensina II/farmacología , Angiotensina II/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/farmacología , Análisis de la Onda del Pulso , Trombina/metabolismo , Trombina/farmacología , Ratones Endogámicos C57BL , Enfermedades Vasculares/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Endotelio Vascular , Óxido Nítrico/metabolismoRESUMEN
Vascular ageing is associated with increased arterial stiffness and cardiovascular mortality that might be linked to altered vascular energy metabolism. The aim of this study was to establish a Seahorse XFe96 Analyzer-based methodology for the reliable, functional assessment of mitochondrial respiration and glycolysis in single murine aortic rings and to validate this functional assay by characterising alterations in vascular energy metabolism in aged mice. Healthy young and old C57BL/6 mice were used for the analyses. An optimised setup consisting of the Seahorse XFe96 Analyzer and Seahorse Spheroid Microplates was applied for the mitochondrial stress test and the glycolysis stress test on the isolated murine aortic rings, supplemented with analysis of NAD content in the aorta. To confirm the age-dependent stiffness of the vasculature, pulse wave velocity was measured in vivo. In addition, the activity of vascular nitric oxide synthase and vascular wall morphology were analysed ex vivo. The vascular ageing phenotype in old mice was confirmed by increased aortic stiffness, vascular wall remodelling, and nitric oxide synthase activity impairment. The rings of the aorta taken from old mice showed changes in vascular energy metabolism, including impaired spare respiratory capacity, maximal respiration, glycolysis, and glycolytic capacity, as well as a fall in the NAD pool. In conclusion, optimised Seahorse XFe96-based analysis to study energy metabolism in single aortic rings of murine aorta revealed a robust impairment of functional vascular respiratory and glycolytic capacity in old mice linked to NAD deficiency that coincided with age-related aortic wall remodelling and stiffness.