RESUMO
Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI2) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI2 generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI2 generation. In conclusion, vascular LDs formation and ATGL-dependent PGI2 generation represent a universal response to vascular proinflammatory insult.
Assuntos
Epoprostenol , Ácido Oleico , Animais , Camundongos , Ácido Oleico/metabolismo , Epoprostenol/metabolismo , Gotículas Lipídicas/metabolismo , Lipopolissacarídeos/metabolismo , Lipólise , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Prostaglandinas I/metabolismoRESUMO
Endothelial inflammation is recognized as a critical condition in the development of cardiovascular diseases. TNF-induced inflammation of endothelial cells is linked to the formation of lipid droplets, augmented cortical stiffness, and nanostructural endothelial plasma membrane remodelling, but the insight into the mechanism linking these responses is missing. In the present work, we determined the formation of lipid droplets (LDs), nanomechanical, and nanostructural responses in the model of TNF-activated vascular inflammation in the isolated murine aorta using Raman spectroscopy, fluorescence imaging, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We analysed the possible role of Rac1, a major regulator of cytoskeletal organization, in TNF-induced vascular inflammation. We demonstrated that the formation of LDs, polymerization of F-actin, alterations in cortical stiffness, and nanostructural protuberances in endothelial plasma membrane were mediated by the Rac1. In particular, we revealed a significant role for Rac1 in the regulation of the formation of highly unsaturated LDs formed in response to TNF. Inhibition of Rac1 also downregulated the overexpression of ICAM-1 induced by TNF, supporting the role of Rac1 in vascular inflammation. Altogether, our results demonstrate that LDs formation, an integral component of vascular inflammation, is activated by Rac1 that also regulates nanomechanical and nanostructural alterations linked to vascular inflammation.
Assuntos
Células Endoteliais , Endotélio Vascular , Animais , Aorta , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Gotículas Lipídicas/metabolismo , CamundongosRESUMO
The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.
Assuntos
Canais Epiteliais de Sódio , Glicocálix , Receptores de Mineralocorticoides , Estresse Mecânico , Acetilcolina/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Canais Epiteliais de Sódio/metabolismo , Glicocálix/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Receptores de Mineralocorticoides/metabolismoRESUMO
Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction in vivo and in vitro. In vivo experiments were performed in 2-mo-old atherosclerosis-prone apolipoprotein E-deficient/LDL receptor-deficient (ApoE-/-/LDLR-/-) female mice and their wild-type C57BL/6J littermates. In in vitro experiments, human aortic endothelial cells (HAECs) were treated with 7K. ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and Eng and a disruption of NO metabolism. Functional analysis of the aorta demonstrated impaired vascular reactivity, and Western blot analysis revealed down-regulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased Eng expression via Krüppel-like factor 6 (KLF6), liver X nuclear receptor, and NF-κB in HAECs. 7K-induced Eng expression was prevented by the treatment with 2-hydroxypropyl-ß-cyclodextrin; 8-{[5-chloro-2-(4-methylpiperazin-1-yl) pyridine-4-carbonyl] amino}-1-(4-fluorophenyl)-4, 5-dihydrobenzo[g]indazole-3-carboxamide; or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic human leukemia promonocytic cell line cells and was prevented by Eng silencing. We concluded that hypercholesterolemia altered Eng expression and signaling, followed by endothelial or vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR-/- mice. By contrast, 7K increased Eng expression and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by Eng inhibition. Thus, we propose a relevant role for Eng in endothelial or vascular dysfunction or inflammation when exposed to cholesterol.-Vicen, M., Vitverova, B., Havelek, R., Blazickova, K., Machacek, M., Rathouska, J., Najmanová, I., Dolezelova, E., Prasnicka, A., Sternak, M., Bernabeu, C., Nachtigal, P. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.
Assuntos
Endoglina/metabolismo , Endotélio Vascular/metabolismo , Hipercolesterolemia/metabolismo , Placa Aterosclerótica/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Células Cultivadas , Colesterol/metabolismo , Endoglina/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Indazóis/farmacologia , Ácidos Isonicotínicos/farmacologia , Fator 6 Semelhante a Kruppel/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Selectina-P/metabolismo , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Receptores de LDL/genética , Proteínas Smad/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Fiber optic Raman spectroscopy and Raman microscopy were used to investigate alterations in the aorta wall and the surrounding perivascular adipose tissue (PVAT) in the murine model of atherosclerosis (Apoe-/-/Ldlr-/- mice). Both abdominal and thoracic parts of the aorta were studied to account for the heterogenic chemical composition of aorta and its localization-dependent response in progression of atherosclerosis. The average Raman spectra obtained for both parts of aorta cross sections revealed that the chemical composition of intima-media layers along aorta remains relatively homogeneous while the lipid content in the adventitia layer markedly increases with decreasing distance to PVAT. Moreover, our results demonstrate that the increase of the lipid to protein ratio in the aorta wall correlates directly with the increased unsaturation level of lipids in PVAT and these changes occur only in the abdominal, but not in thoracic, aorta. In summary, distinct pathophysiological response in the aortic vascular wall could be uncovered by fiber optic Raman spectroscopy based on simple parameters detecting chemical contents of lipids in PVAT.
Assuntos
Aorta Abdominal/metabolismo , Aorta Torácica/metabolismo , Aterosclerose/metabolismo , Tecido Adiposo/metabolismo , Animais , Apolipoproteínas E/metabolismo , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/metabolismo , Análise Espectral Raman/métodosRESUMO
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.
Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Metástase Neoplásica/fisiopatologia , Animais , Sinalização do Cálcio/fisiologia , Feminino , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , ProteômicaRESUMO
Endothelial dysfunction is recognized as a critical condition in the development of cardiovascular disorders. This multifactorial process involves changes in the biochemical and mechanical properties of endothelial cells leading to disturbed release of vasoprotective mediators. Hypercholesterolemia and increased stiffness of the endothelial cortex are independently shown to result in reduced release of nitric oxide and thus endothelial dysfunction. However, direct evidence linking these parameters to each other is missing. Here, a novel method combining Raman spectroscopy for biochemical analysis and Atomic Force Microscopy (AFM) for analyzing the endothelial nanomechanics was established. Using this dual approach, the same areas of native ex vivo aortas were investigated, either derived from mice with endothelial dysfunction (ApoE/LDLR-/-) or wild type mice. In particular an increased intracellular lipid content and elevated cortical stiffness/elasticity were shown in ApoE/LDLR-/- aortas, demonstrating a direct link between endothelial dysfunction, the biochemical composition and the nanomechanical properties of endothelial cells.
Assuntos
Aorta/patologia , Apolipoproteínas E/genética , Endotélio Vascular/patologia , Microscopia de Força Atômica/métodos , Receptores de LDL/genética , Análise Espectral Raman/métodos , Animais , Aorta/metabolismo , Apolipoproteínas E/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Endotélio Vascular/patologia , Neoplasias Pulmonares/patologia , Pulmão/patologia , Óxido Nítrico/deficiência , Animais , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Pulmão/irrigação sanguínea , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo III/metabolismo , FosforilaçãoRESUMO
One of the new targets of untapped therapeutic potential is perivascular adipose tissue (pVAT). pVAT releases a plethora of pro- and anti-inflammatory agents and is involved in the inflammatory response of the vascular wall, playing a key role in various cardiovascular pathologies. Both fiber optic Raman spectroscopy with a high-spatial resolution probe and Raman microscopy were applied to study various types of adipose tissue with the emphasis on pVATs of the thoracic and abdominal aorta and the mesenteric artery, as well as epididymal and interscapular adipose tissue for comparison. Our results demonstrated that the lipid unsaturation degree was clearly distinct in various types of adipose tissue and was influenced by the age of animals. In particular, the basal unsaturation level of pVATs of the abdominal aorta and the mesenteric artery was considerably higher than that of the thoracic aorta and a significant increase of the unsaturation level of pVAT with age was observed showing that aging has a considerable impact on the pVAT's chemical composition. Overall, our results show that Raman spectroscopy is a sensitive tool to determine the perivascular adipose tissue chemical composition that appears to be vascular-bed specific.
Assuntos
Tecido Adiposo/química , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/metabolismo , Envelhecimento/metabolismo , Animais , Camundongos Endogâmicos C57BL , Estrutura Molecular , Análise Espectral Raman/métodos , Triglicerídeos/análise , Triglicerídeos/química , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Quitosana/química , Meios de Contraste/farmacocinética , Compostos Férricos/administração & dosagem , Fígado/patologia , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/administração & dosagem , Animais , Feminino , Compostos Férricos/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Nanopartículas de Magnetita/química , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
Endothelial dysfunction is linked to impaired endothelial-dependent vasodilatation and permeability changes. Here, we quantify both of these phenomena associated with endothelial dysfunction by MRI in vivo in mice. Endothelial function was evaluated in the brachiocephalic artery (BCA) and left carotid artery (LCA) in ApoE/LDLR(-/-) and high-fat diet (HFD)-fed mice as compared with control mice (C57BL/6J). The 3D IntraGate® FLASH sequence was used for evaluation of changes in vessels' cross-sectional area (CSA) and volume following acetylcholine (Ach) administration. Evaluation of endothelial permeability after administration of contrast agent (Galbumin, BioPAL) was based on the variable flip angle method for the assessment of parameters based on the relaxation time (T1 ) value. In order to confirm the involvement of nitric oxide (NO) in response to Ach, L-NAME-treated mice were also analyzed. To confirm that endothelial permeability changes accompany the impairment of Ach-dependent vasodilatation, permeability changes were analyzed in isolated, perfused carotid artery. In C57BL/6J mice, Ach-induced vasodilatation led to an approximately 25% increase in CSA in both vessels, which was temporarily dissociated from the effect of Ach on heart rate. In ApoE/LDLR(-/-) or HFD-fed mice Ach induced a paradoxical vasoconstriction that amounted to approximately 30% and 50% decreases in CSA of BCA and LCA respectively. In ApoE/LDLR(-/-) and HFD-fed mice endothelial permeability in BCA was also increased (fall in T1 by about 25%). In L-NAME-treated mice Ach-induced vasodilatation in BCA was lost. In isolated, perfused artery from ApoE/LDLR(-/-) mice endothelial permeability was increased. MRI-based assessment of endothelium-dependent vasodilatation induced by Ach and endothelial permeability using a retrospectively self-gated 3D gradient-echo sequence (IntraGate® FLASH) enables the reliable detection of systemic endothelial dysfunction in mice and provides an important tool for the experimental pharmacology of the endothelium in murine models of diseases in vivo. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Aterosclerose/diagnóstico por imagem , Aterosclerose/fisiopatologia , Técnicas de Imagem de Sincronização Cardíaca/métodos , Endotélio Vascular/diagnóstico por imagem , Endotélio Vascular/fisiopatologia , Imageamento Tridimensional/métodos , Angiografia por Ressonância Magnética/métodos , Animais , Permeabilidade Capilar , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador , VasodilataçãoRESUMO
Phylloquinon (PK) and menaquinones (MK) are both naturally occurring compounds belonging to vitamin K group. Present study aimed to comprehensively analyze the influence of PK in several models of vascular dysfunction to determine whether PK has vasoprotective properties, similar to those previously described for MK. Effects of PK and MK on endothelial dysfunction were studied in ApoE/LDLR-/- mice in vivo, in the isolated aorta incubated with TNF, and in vascular cells as regard inflammation and cell senescence (including replicative and stress-induced models of senescence). Moreover, the vascular conversion of exogenous vitamins to endogenous MK-4 was analyzed. PK, as well as MK, given for 8 weeks in diet (10 mg/kg) resulted in comparable improvement in endothelial function in the ApoE/LDLR-/- mice. Similarly, PK and MK prevented TNF-induced impairment of endothelium-dependent vasorelaxation in the isolated aorta. In in vitro studies in endothelial and vascular smooth muscle cells, we identified that both PK and MK displayed anti-senescence effects via decreasing DNA damage while in endothelial cells anti-inflammatory activity was ascribed to the modulation of NFκB activation. The activity of PK and MK was comparable in terms of their effect on senescence and inflammation. Presence of endogenous synthesis of MK-4 from PK in aorta and endothelial and smooth muscle cells suggests a possible involvement of MK in vascular effects of PK. In conclusion, PK and MK display comparable vasoprotective effects, which may be ascribed, at least in part, to the inhibition of cell senescence and inflammation. The vasoprotective effect of PK in the vessel wall can be related to the direct effects of PK, as well as to the action of MK formed from PK in the vascular wall.
Assuntos
Senescência Celular , Endotélio Vascular , Vitamina K 1 , Animais , Senescência Celular/efeitos dos fármacos , Camundongos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Vitamina K 1/farmacologia , Vitamina K 2/farmacologia , Vitamina K 2/análogos & derivados , Masculino , Camundongos Endogâmicos C57BL , Inflamação/metabolismo , Vasodilatação/efeitos dos fármacos , Camundongos Knockout , Aorta/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI2) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI2-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLRâ»/â» mice. We found that in the aorta of 2-month-old apoE/LDLRâ»/â» mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI2 and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLRâ»/â» mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI2 and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI2 and EDHF pathways may compensate for the loss of the biological activity of NO.
Assuntos
Aorta/fisiopatologia , Apolipoproteínas E/deficiência , Fatores Biológicos/metabolismo , Endotélio Vascular/fisiopatologia , Epoprostenol/metabolismo , Óxido Nítrico/metabolismo , Receptores de LDL/deficiência , Animais , Aorta/patologia , Apolipoproteínas E/metabolismo , Ciclo-Oxigenase 2/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Peróxido de Hidrogênio/metabolismo , Hipercolesterolemia/sangue , Hipercolesterolemia/fisiopatologia , Técnicas In Vitro , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/metabolismo , Regulação para Cima , VasodilataçãoRESUMO
Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2'-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imaging of cell proliferation with the use of "click chemistry" reaction with a fluorescent azide. Here, we utilized EdU as a click-free Raman probe for tracking endothelial cell proliferation. Raman imaging of EdU was performed in live endothelial cells, showing an advantage over fluorescence imaging of EdU, as this technique did not require sample fixation and permeabilization. To validate Raman-based imaging of EdU to study endothelial cell proliferation, we showed that when endothelial cells were treated with cycloheximide or doxorubicin to impair the proliferation of endothelial cells, the Raman-based signal of EdU was diminished. Furthermore, endothelial cells proliferation detected using EdU-labelled Raman imaging was compared with fluorescence imaging. Finally, the method of Raman-based EdU imaging was used in the isolated murine aorta ex vivo. Altogether, our results show that Raman-based imaging of EdU provides a novel alternative for fluorescence-based assay to assess endothelial proliferation and regeneration.
Assuntos
Azidas , Técnicas Biossensoriais , Alcinos , Animais , Proliferação de Células , Cicloeximida , DNA , Doxorrubicina , Células Endoteliais , CamundongosRESUMO
Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.
RESUMO
Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg-1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.
RESUMO
AIMS: Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs). METHODS AND RESULTS: Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid]. CONCLUSION: In the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.
Assuntos
Insuficiência Cardíaca , Doenças Vasculares , Acetilcolina/metabolismo , Animais , Arginase/metabolismo , Doença Crônica , Modelos Animais de Doenças , Eicosanoides/metabolismo , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Dissulfeto de Glutationa/metabolismo , Camundongos , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , VasodilataçãoRESUMO
Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6-91.3%) with an important content of arachidonic acid (8.7-19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.
Assuntos
Aorta Torácica/metabolismo , Células Endoteliais/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Ácido Oleico/farmacologia , Imagem Óptica , Análise Espectral RamanRESUMO
Lipid droplets (LDs) play regulatory role in various cells but their significance in endothelial pathophysiology is still not well understood. Here, we studied LDs in in situ endothelial cells (ECs) in isolated blood vessels stimulated with pro-inflammatory or pro-apoptotic stimuli using Raman and fluorescence imaging. Endothelial inflammation induced by murine TNF-α (mTNF-α) was featured by overexpression of ICAM-1, vWF, increased production of PGI2, and was associated with the formation of low number of LDs. However in the presence of atglistatin, the inhibitor of triacyclglycerols hydrolysis, the number of LDs significantly increased. In contrast, in endothelium stimulated by human TNF-α (hTNF-α) or FasL, apart from endothelial inflammation, displayed also apoptosis as evidenced by high annexin expression and significant LDs formation. Raman imaging confirmed that LDs were localized in endothelium and revealed significant heterogeneity in biochemical composition of endothelial LDs that dependent on endothelial stimuli. Repertoire of LDs included LDs rich in highly unsaturated lipids, assigned to the inflammation, as well as LDs featured by more saturated lipids linked to apoptosis, where Raman signals indicating content of cholesterol and phospholipids were higher for endothelial apoptosis in comparison to endothelial inflammation. The heterogeneity in chemical composition of LDs suggested more complex pathophysiological role of endothelial LDs then previously appreciated.
Assuntos
Proteína Ligante Fas/farmacologia , Inflamação/metabolismo , Gotículas Lipídicas/química , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Células Endoteliais/química , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular , Camundongos , Compostos de Fenilureia/farmacologia , Prostaglandinas I/metabolismo , Análise Espectral Raman , Fator de von Willebrand/metabolismoRESUMO
Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.