RESUMEN
The distribution of atherosclerotic lesions in the aorta and its branches of ApoE knockout (ApoE-/-) mice is like that of patients with atherosclerosis. By using high-resolution MALDI mass spectrometry imaging (MSI), we aimed at characterizing universally applicable physiological biomarkers by comparing the murine lipid marker profile with that of human atherosclerotic arteries. Therefore, the aorta or carotid artery of male ApoE-/- mice at different ages, human arteries with documented atherosclerotic changes originated from amputated limbs, and corresponding controls were analysed. Obtained data were subjected to multivariate statistical analysis to identify potential biomarkers. Thirty-one m/z values corresponding to individual lipid species of cholesterol esters, lysophosphatidylcholines, lysophosphatidylethanolamines, and cholesterol derivatives were found to be specific in aortic atherosclerotic plaques of old ApoE-/- mice. The lipid composition at related vessel positions of young ApoE-/- mice was more comparable with wild-type mice. Twenty-six m/z values of the murine lipid markers were found in human atherosclerotic peripheral arteries but also control vessels and showed a more patient-dependent diverse distribution. Extensive data analysis without marker preselection based on mouse data revealed lysophosphatidylcholine and glucosylated cholesterol species, the latter not being detected in the murine atherosclerotic tissue, as specific potential novel human atherosclerotic vessel markers. Despite the heterogeneous lipid profile of atherosclerotic peripheral arteries derived from human patients, we identified lipids specifically colocalized to atherosclerotic human tissue and plaques in ApoE-/- mice. These data highlight species-dependent differences in lipid profiles between peripheral artery disease and aortic atherosclerosis.
Asunto(s)
Lípidos/fisiología , Placa Aterosclerótica/metabolismo , Animales , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Colesterol/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Activation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in an increasing number of diseases, including Marfan syndrome (MFS), an inherited connective tissue disorder. mTOR-dependent reactive oxygen species (ROS) formation has also been suggested to play a role in aortic aneurysm formation in MFS patients. This study aimed to characterize the effects of mTOR inhibition by rapamycin on key redox enzymes and NADPH oxidases (NOX) in cultured vascular smooth muscle cells of a murine MFS model. Therefore, the influence of 5 and 20 nmol/L rapamycin solved in 0.1% (vol/vol) DMSO on glutathione peroxidases 1 (Gpx1) and 4 (Gpx4), superoxide dismutase 2 (Sod2), and catalase (Cat) mRNA and protein expression was investigated in isolated murine aortic smooth muscle cells. Rapamycin inhibited the mRNA expression of all redox enzymes by 30-50%, except Gpx1. In the same cells, the mRNA expression of the transcription factor NFE2-related factor-2 and peroxisome proliferator-activated receptor-γ, key factors against oxidative stress, and controlling redox gene expression were also inhibited to a comparable extent under these conditions. In addition, Nox1 but not Nox4 mRNA expression was significantly inhibited by up to 40%. DMSO alone increased nearly 2-fold the redox enzyme protein expression, which was reduced considerably to basal levels by rapamycin. Proteasomal inhibition by bortezomib could not reverse the observed decrease of GPx protein content. The rapamycin-mediated decrease in GPx protein abundance was reflected in a reduced total GPx enzymatic activity. Higher rapamycin concentrations did not further decrease but led to a renewed increase in enzymatic activity despite low GPx protein concentrations. Baseline ROS formation was slightly inhibited at 13% with 5 nmol/L rapamycin and returned to baseline levels with the higher 20 nmol/L rapamycin concentration. In conclusion, this study further characterized the mechanism of action of rapamycin. It provided an insight into how rapamycin interferes with the regulation of redox homeostasis essential for ROS-dependent signaling that does not incur cellular damage.
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Síndrome de Marfan , Animales , Ratones , Células Cultivadas , Dimetilsulfóxido/metabolismo , Dimetilsulfóxido/farmacología , Síndrome de Marfan/tratamiento farmacológico , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , ARN Mensajero/metabolismo , Sirolimus/farmacología , Sirolimus/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.
Asunto(s)
Dependovirus/genética , Terapia Genética , Insuficiencia Cardíaca/prevención & control , Hipertrofia Ventricular Izquierda/prevención & control , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/genética , Oligonucleótidos/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Endotelina-1/toxicidad , Vectores Genéticos , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/metabolismo , Oligonucleótidos/metabolismo , Ratas Wistar , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
RATIONALE: Fluid shear stress (FSS) maintains NOS-3 (endothelial NO synthase) expression. Homozygosity for the C variant of the T-786C single-nucleotide polymorphism of the NOS3 gene, which solely exists in humans, renders the gene less sensitive to FSS, resulting in a reduced endothelial cell (EC) capacity to generate NO. Decreased bioavailability of NO in the arterial vessel wall facilitates atherosclerosis. Consequently, individuals homozygous for the C variant have an increased risk for coronary heart disease (CHD). OBJECTIVE: At least 2 compensatory mechanisms seem to minimize the deleterious effects of this single-nucleotide polymorphism in affected individuals, one of which is characterized herein. METHODS AND RESULTS: Human genotyped umbilical vein ECs and THP-1 monocytes were used to investigate the role of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in vitro. Its concentration in plasma samples from genotyped patients with CHD and age-matched CHD-free controls was determined using quantitative ultraperformance LC-MS/MS. Exposure of human ECs to FSS effectively reduced monocyte transmigration particularly through monolayers of CC-genotype ECs. Primarily in CC-genotype ECs, FSS elicited a marked rise in COX (cyclooxygenase)-2 and L-PGDS (lipocalin-type prostaglandin D synthase) expression, which appeared to be NO sensitive, and provoked a significant release of 15d-PGJ2 over baseline. Exogenous 15d-PGJ2 significantly reduced monocyte transmigration and exerted a pronounced anti-inflammatory effect on the transmigrated monocytes by downregulating, for example, transcription of the IL (interleukin)-1ß gene (IL1B). Reporter gene analyses verified that this effect is due to binding of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) to 2 AREs (antioxidant response elements) in the proximal IL1B promoter. In patients with CHD, 15d-PGJ2 plasma levels were significantly upregulated compared with age-matched CHD-free controls, suggesting that this powerful anti-inflammatory prostanoid is part of an endogenous defence mechanism to counteract CHD. CONCLUSIONS: Despite a reduced capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype through an enhanced FSS-dependent release of 15d-PGJ2.
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Células Endoteliales/metabolismo , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico/sangre , Polimorfismo de Nucleótido Simple , Prostaglandina D2/análogos & derivados , Adaptación Fisiológica , Anciano , Anciano de 80 o más Años , Enfermedad Coronaria/sangre , Enfermedad Coronaria/genética , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Inducción Enzimática , Femenino , Genes Reporteros , Predisposición Genética a la Enfermedad , Hemorreología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/genética , Lipocalinas/biosíntesis , Lipocalinas/genética , Masculino , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/fisiología , Óxido Nítrico Sintasa de Tipo III/genética , Prostaglandina D2/biosíntesis , Prostaglandina D2/sangre , Prostaglandina D2/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Células THP-1RESUMEN
Monocyte extravasation into the vessel wall is a key step in atherogenesis. It is still elusive how monocytes transmigrate through the endothelial cell (EC) monolayer at atherosclerosis predilection sites. Platelets tethered to ultra-large von Willebrand factor (ULVWF) multimers deposited on the luminal EC surface following CD40 ligand (CD154) stimulation may facilitate monocyte diapedesis. Human ECs grown in a parallel plate flow chamber for live-cell imaging or Transwell permeable supports for transmigration assay were exposed to fluid or orbital shear stress and CD154. Human isolated platelets and/or monocytes were superfused over or added on top of the EC monolayer. Plasma levels and activity of the ULVWF multimer-cleaving protease ADAMTS13 were compared between coronary artery disease (CAD) patients and controls and were verified by the bioassay. Two-photon intravital microscopy was performed to monitor CD154-dependent leukocyte recruitment in the cremaster microcirculation of ADAMTS13-deficient versus wild-type mice. CD154-induced ULVWF multimer-platelet string formation on the EC surface trapped monocytes and facilitated transmigration through the EC monolayer despite high shear stress. Two-photon intravital microscopy revealed CD154-induced ULVWF multimer-platelet string formation preferentially in venules, due to strong EC expression of CD40, causing prominent downstream leukocyte extravasation. Plasma ADAMTS13 abundance and activity were significantly reduced in CAD patients and strongly facilitated both ULVWF multimer-platelet string formation and monocyte trapping in vitro. Moderate ADAMTS13 deficiency in CAD patients augments CD154-mediated deposition of platelet-decorated ULVWF multimers on the luminal EC surface, reinforcing the trapping of circulating monocytes at atherosclerosis predilection sites and promoting their diapedesis.
Asunto(s)
Proteína ADAMTS13/metabolismo , Plaquetas/metabolismo , Antígenos CD40/metabolismo , Comunicación Celular/fisiología , Células Endoteliales/metabolismo , Factor de von Willebrand/metabolismo , Adolescente , Adulto , Anciano , Animales , Aterosclerosis/metabolismo , Células Cultivadas , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Monocitos/metabolismo , Agregación Plaquetaria/fisiología , Estrés Mecánico , Adulto JovenRESUMEN
Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of CâC double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 µm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 µm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.
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Benzofenonas/química , Compuestos de Bencilo/química , Diagnóstico por Imagen/métodos , Lípidos/análisis , Piridinas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Técnicas Biosensibles , Cerebelo/metabolismo , Femenino , Técnicas Histológicas , Isomerismo , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/metabolismoRESUMEN
The anatomic arrangement of microvascular endothelial cells and cardiomyocytes in vivo enables close interactions among these cells. In our in vitro co-culture system, ANP and BNP expression in the mouse atrial cardiomyocyte cell line HL-1 and subsequent ANP release were significantly upregulated when co-cultured with mouse cardiac microvascular endothelial cells or exposed to endothelial cell-conditioned medium. Endothelin-1 (ET-1) activation of endothelial cells remarkably enhanced their paracrine effect on cardiomyocyte gene expression, suggesting that ET-1 stimulation of endothelial cells affects expression of fetal genes such as ANP and BNP in adult cardiomyocytes through paracrine signalling. Exposure of HL-1â¯cells and murine induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to authentic angiopoietin-2 (Ang2) caused a concentration-dependent decrease in ANP expression while ET-1-induced ANP expression was augmented by low but inhibited by high concentrations of Ang2. FK506-mediated inhibition of the calcineurin-NFAT pathway in the HL-1â¯cells selectively inhibited the stimulatory effect of the conditioned medium derived from ET-1-pre-stimulated endothelial cells on cardiomyocyte fetal gene expression. Combined with previous results indicating a crucial role for ANP and BNP in cardiac homeostasis, our findings provide further evidence that paracrine signalling by cardiac microvascular endothelial cells modulates cardiomyocyte function.
Asunto(s)
Comunicación Celular/genética , Células Endoteliales/fisiología , Expresión Génica , Miocitos Cardíacos/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Expresión Génica/efectos de los fármacos , Ratones , Miocardio/metabolismo , Comunicación Paracrina/genéticaRESUMEN
BACKGROUND: Transplant vasculopathy (TV) is the main limiting factor for long-term graft survival characterized by fibrosis, myofibroblast, and smooth muscle cell (SMC) proliferation. Decoy oligodeoxynucleotide (dODN) against the transcription factor activator protein-1 (AP-1) might interfere with the expression of AV-related genes that govern neointima formation. METHODS: Aortic allografts from DBA/2 mice were incubated with control buffer, consensus, or mutated control AP-1 dODN and were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight [BW]) was administered daily. Explantation and histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography. RESULTS: Intima-to-media (I/M) ratio and neointima formation were significantly reduced in the consensus AP-1 dODN treatment group by 37% (p < 0.05) and 67% (p < 0.01), respectively. SMC α-actin-2 staining and macrophage marker expression revealed a marked reduction in the neointima. I/M ratio was found to correlate with the number of tissue macrophages (p < 0.05). MMP and fibrosis marker expression were not significantly altered. CONCLUSION: Intraoperative AP-1dODN utilization might be a strategy to preserve graft function after transplantation.
Asunto(s)
Aorta/trasplante , Enfermedades de la Aorta/prevención & control , Supervivencia de Injerto , Oligodesoxirribonucleótidos/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Modelos Animales de Enfermedad , Femenino , Fibrosis , Hiperplasia , Macrófagos/metabolismo , Macrófagos/patología , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neointima , Oligodesoxirribonucleótidos/genética , Factores de Tiempo , Factor de Transcripción AP-1/genética , Remodelación VascularRESUMEN
BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.
Asunto(s)
Carnosina/química , Carnosina/metabolismo , Polímeros/química , Piruvaldehído/química , Animales , Anserina/análisis , Anserina/química , Anserina/metabolismo , Carnosina/análisis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Glutatión/análisis , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Estrés Oxidativo/efectos de los fármacos , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Polímeros/metabolismo , Piruvaldehído/toxicidad , Albúmina Sérica/química , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Diabetes mellitus is a metabolic disease characterized by, among others, elevated blood glucose levels. Hyperglycaemia as well as enhanced levels of glucose-derived reactive metabolites contribute to the development of diabetic complications partly via increased generation of reactive oxygen species (ROS). ROS are not only part of signaling pathways themselves but also lead to carbonylation of particular amino acid side chains by direct metal-catalyzed oxidation. In addition, carbonyl groups can be introduced into proteins indirectly by non-oxidative covalent adduction of reactive carbonyl species generated by the oxidation of lipids or carbohydrates. Both direct and indirect carbonylation mechanisms may affect protein conformation, activity, and function. Herein we introduce the different mechanisms of the carbonylation reaction, discuss degradation mechanisms, and the fate of proteins modified this way and how the overall degree of carbonylation affects protein homeostasis and function differently. The role of protein carbonylation in metabolic control systems and cell signaling are also summarized. Finally, current diagnostic and antioxidant therapeutic options in diabetes are discussed.
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Glucemia/metabolismo , Diabetes Mellitus/metabolismo , Metabolismo Energético , Estrés Oxidativo , Carbonilación Proteica , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/uso terapéutico , Glucemia/efectos de los fármacos , Diabetes Mellitus/sangre , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/tratamiento farmacológico , Metabolismo Energético/efectos de los fármacos , Humanos , Hipoglucemiantes/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , ProteolisisRESUMEN
Macrophages are large phagocytes playing a crucial role in the development and progression of atherosclerosis. The phenotypic polarization and activation of macrophages in atherosclerotic plaques depends on their complex micro-environment and at the same time has a major impact on the vulnerability or stability of advanced atherosclerotic lesions. Many in vitro and in vivo studies have been designed to define markers for macrophage subtypes to better understand the mechanism of plaque progression but they have rather added to the confusion. Nonetheless, some of the in vitro defined macrophage subtypes, like the pro-inflammatory M1 or the anti-inflammatory M2a/b/c macrophage, have been shown to be present in atherosclerotic plaques. Herein, we developed a comprehensive workflow to distinguish between human in vitro differentiated pro-inflammatory M1 and anti-inflammatory M2a and M2c macrophages. The cells were analyzed using qPCR and FACS analyses for defining suitable markers on the transcript (mRNA) and protein level as well as MALDI MSI for the assignment of metabolic markers, which can be used for the identification of the corresponding macrophage subtypes in atherosclerotic plaques. Data obtained using both qPCR and FACS analyses were in agreement with the literature. For the analysis of the macrophages with MALDI MSI, a comprehensive workflow was developed and the obtained data were subjected to different statistical analysis methods like principal component analysis (PCA) to define markers for each macrophage type. Our MALDI MSI results revealed that the method produces reliable and reproducible results but that the heterogeneity of the monocytes derived from different donors is too high to define universal markers on the metabolic level. Moreover, the results show that a sample set of three biological replicates is not sufficient to obtain representative data and therefore we recommend performing ring experiments in which the samples are measured by different laboratories.
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Diferenciación Celular , Macrófagos/citología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Antiinflamatorios , Biomarcadores , Células Cultivadas , Humanos , Monocitos/citología , Placa Aterosclerótica/inmunologíaRESUMEN
Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation.
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Melanoma/metabolismo , Agregación Plaquetaria , Factor de von Willebrand/metabolismo , Proteínas ADAM/sangre , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Animales , Coagulación Sanguínea , Plaquetas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Activación Enzimática , Fibrinolíticos/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Melanoma/sangre , Melanoma/patología , Ratones , Ratones Transgénicos , Microvasos/metabolismo , Neovascularización Patológica/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-ret/metabolismo , Tinzaparina , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.
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Dipeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos de Sulfhidrilo/farmacología , Regulación Alostérica/efectos de los fármacos , Dipeptidasas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
Carnosinase 1 (CN1) contributes to diabetic nephropathy by cleaving histidine-dipeptides which scavenge reactive oxygen and carbonyl species and increase nitric oxide (NO) production. In diabetic mice renal CN1 activity is increased, the regulatory mechanisms are unknown. We therefore analysed the in vitro and in vivo regulation of CN1 activity using recombinant and human CN1, and the db/db mouse model of diabetes. Glucose, leptin and insulin did not modify recombinant and human CN1 activity in vitro, glucose did not alter renal CN1 activity of WT or db/db mice ex vivo. Reactive metabolite methylglyoxal and Fenton reagent carbonylated recombinant CN1 and doubled CN1 efficiency. NO S-nitrosylated CN1 and decreased CN1 efficiency for carnosine by 70 % (p < 0.01), but not for anserine. Both CN1 cysteine residues were nitrosylated, the cysteine at position 102 but not at position 229 regulated CN1 activities. In db/db mice, renal CN1 mRNA and protein levels were similar as in non-diabetic controls, CN1 efficiency 1.9 and 1.6 fold higher for carnosine and anserine. Renal carbonyl stress was strongly increased and NO production halved, CN1 highly carbonylated and less S-nitrosylated compared to WT mice. GSH and NO2/3 concentrations were reduced and inversely related with carnosine degradation rate (r = -0.82/-0.85). Thus, reactive metabolites of diabetes upregulate CN1 activity by post-translational modifications, and thus decrease the availability of reactive metabolite-scavenging histidine dipeptides in the kidney in a positive feedback loop. Interference with this vicious circle may represent a new therapeutic target for mitigation of DN.
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Carnosina/metabolismo , Diabetes Mellitus/metabolismo , Óxido Nítrico/metabolismo , Piruvaldehído/metabolismo , Animales , Carnosina/genética , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Dipeptidasas/genética , Dipeptidasas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Ratones , Ratones Mutantes , MutaciónRESUMEN
Steroid-refractory graft-versus-host disease is a life-threatening complication after allogeneic stem cell transplantation. Evidence is accumulating that steroid-refractory graft-versus-host disease is associated with endothelial distress. Endothelial cell homeostasis is regulated by nitric oxide, and serum nitrates are derived from nitric oxide synthase activity or dietary sources. In this retrospective study based on 417 patients allografted at our institution we investigated whether quantification of serum nitrates could predict steroid-refractory graft-versus-host disease. Elevated pre-transplant levels of serum nitrates (>26.5 µM) predicted steroid-refractory graft-versus-host disease (P=0.026) and non-relapse mortality (P=0.028), particularly in combination with high pre-transplant angiopoietin-2 levels (P=0.0007 and P=0.021, respectively). Multivariate analyses confirmed serum nitrates as independent predictors of steroid-refractory graft-versus-host disease and non-relapse mortality. Differences in serum nitrate levels did not correlate with serum levels of tumor necrosis factor or C-reactive protein or expression of inducible nitric oxide synthase in blood cells. Patients with high pre-transplant nitrate levels had significantly reduced rates of refractory graft-versus-host disease (P=0.031) when pravastatin was taken. In summary, patients at high risk of developing steroid-refractory graft-versus-host disease could be identified prior to transplantation by serum markers linked to endothelial cell function. Retrospectively, statin medication was associated with a reduced incidence of refractory graft-versus-host disease in this endothelial high-risk cohort.
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Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Nitratos/sangre , Adolescente , Adulto , Anciano , Angiopoyetina 2/sangre , Activación Enzimática , Femenino , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa/metabolismo , Evaluación del Resultado de la Atención al Paciente , Pronóstico , Recurrencia , Estudios Retrospectivos , Riesgo , Esteroides/uso terapéutico , Adulto JovenRESUMEN
Foam cells in atheroma are engorged with lipid droplets (LDs) that contain esters of regulatory lipids whose metabolism remains poorly understood. LD-associated hydrolase (LDAH) has a lipase structure and high affinity for LDs of foam cells. Using knockout and transgenic mice of both sexes, here we show that LDAH inhibits atherosclerosis development and promotes stable lesion architectures. Broad and targeted lipidomic analyzes of primary macrophages and comparative lipid profiling of atheroma identified a broad impact of LDAH on esterified sterols, including natural liver X receptor (LXR) sterol ligands. Transcriptomic analyzes coupled with rescue experiments show that LDAH modulates the expression of prototypical LXR targets and leads macrophages to a less inflammatory phenotype with a profibrotic gene signature. These studies underscore the role of LDs as reservoirs and metabolic hubs of bioactive lipids, and suggest that LDAH favorably modulates macrophage activation and protects against atherosclerosis via lipolytic mobilization of regulatory sterols.
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Aterosclerosis , Gotas Lipídicas , Receptores X del Hígado , Macrófagos , Ratones Noqueados , Animales , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/prevención & control , Aterosclerosis/patología , Receptores X del Hígado/metabolismo , Receptores X del Hígado/genética , Ratones , Masculino , Ligandos , Femenino , Gotas Lipídicas/metabolismo , Macrófagos/metabolismo , Esteroles/metabolismo , Células Espumosas/metabolismo , Ratones Transgénicos , Ratones Endogámicos C57BL , Humanos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Activación de Macrófagos , Esterol EsterasaRESUMEN
Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells in vitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.
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Hemodynamic forces are important effectors of endothelial cell phenotype and function. Because CD40-CD154 interactions between endothelial cells and mononuclear leukocytes or activated platelets play an important role in vascular dysfunction, we investigated the effects of cyclic stretch on CD40 expression in human cultured endothelial cells. Short-term stretch transiently up-regulated CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein which was prevented by inhibition of the 20S proteasome or scavenging of peroxynitrite. Tyrosine nitration of CD40 also occurred under static conditions on addition of authentic peroxynitrite, and according to mass spectrometry analysis Tyr-82 but not Tyr-31 was its target in the native protein. Immunofluorescence analysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent redistribution of the protein from the cell membrane to the cytoplasm, which was prevented by methyl-ß-cyclodextrin. Moreover, CD154-stimulated IL-12p40 and E-selectin expression markedly decreased after exposure to authentic peroxynitrite or cyclic stretch, respectively. Coimmunoprecipitation demonstrated a decreased binding of TRAF2 and TRAF6 to the CD40 protein after tyrosine nitration. Through this posttranslational oxidative modification of an important costimulatory molecule, endothelial cells are able to quickly adapt to unfavorable hemodynamics and maintain their anti-inflammatory phenotype.
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Antígenos CD40/genética , Células Endoteliales/metabolismo , Nitratos/metabolismo , Estrés Mecánico , Tirosina/metabolismo , Antígenos CD40/química , Antígenos CD40/metabolismo , Células Cultivadas , Células Endoteliales/fisiología , Regulación de la Expresión Génica , Hemodinámica/genética , Hemodinámica/fisiología , Humanos , Inflamación/metabolismo , Inflamación/patología , Nitrocompuestos/metabolismo , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Resistencia a la Tracción/fisiologíaRESUMEN
BACKGROUND: Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA. RESULTS: ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD. CONCLUSIONS: We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.
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Proteína ADAM17 , Antígenos CD40 , Humanos , Proteína ADAM17/genética , Proteína C-Reactiva , Antígenos CD40/metabolismo , Ligando de CD40/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Interleucina-6 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND AND AIMS: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors. METHODS: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells. RESULTS: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L. CONCLUSIONS: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment.