RESUMEN
Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.
Asunto(s)
Envejecimiento/genética , Angiotensina II/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Progeria/genética , Insuficiencia Renal Crónica/genética , Renina/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Endonucleasas/deficiencia , Femenino , Regulación de la Expresión Génica , Tasa de Filtración Glomerular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Riñón/metabolismo , Riñón/patología , Losartán/farmacología , Masculino , Ratones , Ratones Noqueados , Progeria/metabolismo , Progeria/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Transducción de SeñalRESUMEN
Aim: Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation. Methods and results: In Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I-V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFß reduced PGC1α levels, indicating involvement of TGFß signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator. Conclusion: Our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation.
Asunto(s)
Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Metabolismo Energético , Proteínas de la Matriz Extracelular/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Mutación , Miocitos del Músculo Liso/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aneurisma de la Aorta Torácica/patología , Respiración de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Musculares/patología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Especies Reactivas de Oxígeno/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Transducción de SeñalRESUMEN
Cellular immunotherapy targeting human tumor antigens is a promising strategy to treat solid tumors. Yet clinical results of cellular immunotherapy are disappointing. Moreover, the currently available in vitro human tumor models are not designed to study the optimization of T-cell therapies of solid tumors. Here, we describe a novel assay for multiparametric in situ analysis of therapeutic effects on individual human three-dimensional (3D) tumors. In this assay, tumors of several millimeter diameter are generated from human cancer cell lines of different tumor entities in a collagen type I microenvironment. A newly developed approach for efficient morphological analysis reveals that these in vitro tumors resemble many characteristics of the corresponding clinical cancers such as histological features, immunohistochemical staining patterns, distinct tumor growth compartments and heterogeneous protein expression. To assess the response to therapy with tumor antigen specific T-cells, standardized protocols are described to determine T-cell infiltration and tumor destruction by monitoring soluble factors and tumor growth. Human tumors engineered in 3D collagen scaffolds are excellent in vitro surrogates for avascular tumor stages allowing integrated analyses of the antitumor efficacy of cancer specific immunotherapy in situ.
Asunto(s)
Inmunoterapia , Neoplasias/terapia , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Humanos , Adhesión en Parafina , Esferoides Celulares/metabolismo , Linfocitos T Citotóxicos/metabolismo , Ingeniería de TejidosRESUMEN
AIMS: Increasing evidence supports a role for the angiotensin II-AT1-receptor axis in aneurysm development. Here, we studied whether counteracting this axis via stimulation of AT2 receptors is beneficial. Such stimulation occurs naturally during AT1-receptor blockade with losartan, but not during renin inhibition with aliskiren. METHODS AND RESULTS: Aneurysmal homozygous fibulin-4 mice, displaying a four-fold reduced fibulin-4 expression, were treated with placebo, losartan, aliskiren, or the ß-blocker propranolol from day 35 to 100. Their phenotype includes cystic media degeneration, aortic regurgitation, left ventricular dilation, reduced ejection fraction, and fractional shortening. Although losartan and aliskiren reduced hemodynamic stress and increased renin similarly, only losartan increased survival. Propranolol had no effect. No drug rescued elastic fiber fragmentation in established aneurysms, although losartan did reduce aneurysm size. Losartan also increased ejection fraction, decreased LV diameter, and reduced cardiac pSmad2 signaling. None of these effects were seen with aliskiren or propranolol. Longitudinal micro-CT measurements, a novel method in which each mouse serves as its own control, revealed that losartan reduced LV growth more than aneurysm growth, presumably because the heart profits both from the local (cardiac) effects of losartan and its effects on aortic root remodeling. CONCLUSION: Losartan, but not aliskiren or propranolol, improved survival in fibulin-4 mice. This most likely relates to its capacity to improve structure and function of both aorta and heart. The absence of this effect during aliskiren treatment, despite a similar degree of blood pressure reduction and renin-angiotensin system blockade, suggests that it might be because of AT2-receptor stimulation.
Asunto(s)
Aneurisma/fisiopatología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Proteínas de la Matriz Extracelular , Insuficiencia Cardíaca/fisiopatología , Receptor de Angiotensina Tipo 1/metabolismo , Renina/metabolismo , Animales , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Ratones Transgénicos , Renina/antagonistas & inhibidoresRESUMEN
The potential risk of recurrence and progression in patients with non-muscle-invasive bladder cancer necessitates followup by cystoscopy. The risk of progression to muscle-invasive bladder cancer is estimated based on the European Organisation of Research and Treatment of Cancer score, a combination of several clinicopathological variables. However, pathological assessment is not objective and reproducibility is insufficient. The use of molecular markers could contribute to the estimation of tumor aggressiveness. We recently demonstrated that methylation of GATA2, TBX2, TBX3, and ZIC4 genes could predict progression in Ta tumors. In this study, we aimed to validate the markers in a large patient set using DNA from formalin-fixed and paraffin-embedded tissue. PALGA: the Dutch Pathology Registry was used for patient selection. We included 192 patients with pTaG1/2 bladder cancer of whom 77 experienced progression. Methylation analysis was performed and log-rank analysis was used to calculate the predictive value of each methylation marker for developing progression over time. This analysis showed better progression-free survival in patients with low methylation rates compared with the patients with high methylation rates for all markers (P<0.001) during a followup of ten-years. The combined predictive effect of the methylation markers was analyzed with the Cox-regression method. In this analysis, TBX2, TBX3, and ZIC4 were independent predictors of progression. On the basis of methylation status of TBX2 and TBX3, patients were divided into three new molecular grade groups. Survival analysis showed that only 8% of patients in the low molecular grade group progressed within 5 years. This was 29 and 63% for the intermediate- and high-molecular grade groups. In conclusion, this new molecular-grade based on the combination of TBX2 and TBX3 methylation is an excellent marker for predicting progression to muscle-invasive bladder cancer in patients with primary pTaG1/2 bladder cancer.
Asunto(s)
Carcinoma de Células Transicionales/genética , Proteínas de Dominio T Box/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/patología , Metilación de ADN , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Vejiga Urinaria/patología , Adulto JovenRESUMEN
BACKGROUND: In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an inherited deficiency of connective tissue might play a role in the combined development of pulmonary emphysema and vascular disease. METHODS: We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising pathological, functional, molecular imaging, immunological and gene expression analysis to reveal the sequence of events that culminates in pulmonary emphysema in aneurysmal Fibulin-4 deficient (Fibulin-4(R)) mice. RESULTS: Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that aneurysmal Fibulin-4(R/R) mice display severe developmental lung emphysema, whereas Fibulin-4(+/R) mice acquire alveolar breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease capacity of the lungs and ultimately results in the development of pulmonary emphysema. CONCLUSIONS: Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary inflammation as the common mechanisms in both COPD and aneurysm formation.
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Aneurisma de la Aorta/complicaciones , Aneurisma de la Aorta/patología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Enfisema Pulmonar/complicaciones , Anciano , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta/metabolismo , Estudios de Cohortes , Susceptibilidad a Enfermedades , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Neutrófilos/enzimología , Elastasa Pancreática/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , alfa 1-Antitripsina/metabolismoRESUMEN
BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. RESULTS AND LIMITATIONS: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. CONCLUSIONS: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.
Asunto(s)
Biomarcadores de Tumor/genética , Mutación , Regiones Promotoras Genéticas , Telomerasa/genética , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/genética , Anciano , Biomarcadores de Tumor/orina , Línea Celular Tumoral , Análisis Mutacional de ADN , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Humanos , Masculino , Clasificación del Tumor , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Países Bajos , Fenotipo , Valor Predictivo de las Pruebas , ARN Mensajero/orina , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , España , Telomerasa/orina , Factores de Tiempo , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/terapia , Neoplasias de la Vejiga Urinaria/orinaRESUMEN
UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.
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Andrógenos/metabolismo , Hormona de Crecimiento Humana/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Hormona de Crecimiento Humana/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Metribolona/farmacología , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/análisis , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda(-/-) ) mouse-model resembling TTD-A patients. Unexpectedly, Ttda(-/-) mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda(-/-) cells was not affected. Surprisingly, Ttda(-/-) cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda(-/-) cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda(-/-) cells as a unique class of TFIIH mutants.
Asunto(s)
Reparación del ADN , Síndromes de Tricotiodistrofia , Animales , Síndrome de Cockayne , Humanos , Mutación , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , Transcripción Genética , Síndromes de Tricotiodistrofia/genéticaRESUMEN
PURPOSE: Bladder tumors in patients younger than 20 years show a low incidence of the genetic and epigenetic aberrations typically found in older patients. One of the most common epigenetic aberrations in human malignancies is DNA hypermethylation. Polycomb group complexes have an important role during lineage choices in embryogenesis and their target genes are 12 times more likely to be methylated than nonpolycomb group target genes. We hypothesized that methylation of polycomb group target genes is an early event in urothelial carcinogenesis and thus might be observed in young patients. MATERIALS AND METHODS: We stratified 167 patients by age into 4 groups, including age less than 20 years in 14, 20 to 40 in 48, 40 to 60 in 47 and greater than 60 in 58. Five previously identified polycomb group target genes (MEIS1, ONECUT2, OTX1, PCDH7 and SOX21) were selected for methylation analysis. Methylation ratios were calculated by using the unmethylated and methylated signal. The outcome represented the fraction of methylated cells within one tumor. Genes with similar methylation ratios in all age groups were considered as potential bladder cancer initiating candidates. RESULTS: Three genes showed higher methylation ratios in tumors from older patients, including ONECUT2, SOX21 and OTX1 (each p <0.001). MEIS1 showed a similar methylation ratio in all groups but the median methylation ratio was low. PCDH7 showed a similar median methylation percent in all age categories, ie 54% at less than 20, 59% at 20 to 40, 59% at 40 to 60 and 67% at greater than 60 years (p = 0.1). CONCLUSIONS: Tumors from young patients showed less methylation for most markers. PCDH7 showed high methylation ratios in all age categories. Therefore, it could have an important role in early urothelial carcinogenesis.
Asunto(s)
Cadherinas/genética , Carcinoma de Células Transicionales/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Vejiga Urinaria/genética , Adolescente , Adulto , Factores de Edad , Anciano , Carcinoma de Células Transicionales/diagnóstico , Estudios de Cohortes , Metilación de ADN/fisiología , Femenino , Marcadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Proteínas del Grupo Polycomb/genética , Pronóstico , Regiones Promotoras Genéticas/genética , Protocadherinas , Medición de Riesgo , Sensibilidad y Especificidad , Neoplasias de la Vejiga Urinaria/diagnóstico , Adulto JovenRESUMEN
Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Transducción de Señal , Trombopoyetina/genética , Trombopoyetina/metabolismoRESUMEN
Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR-deficient Csa(-/-) and Csb(-/-) CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER-deficient Xpa(-/-) and Xpc(-/-) XP mice, but also occurred in Xpd(XPCS) mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR-deficient mice are compatible with focal dysmyelination in CS patients. Both TCR-deficient and NER-deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa(-/-), Csb(-/-)) or highly sporadic (Xpa(-/-), Xpc(-/-)) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR-deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa(-/-) and Csb(-/-) TCR-deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR-deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival.
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Reparación del ADN/genética , Degeneración Nerviosa/genética , Neuronas/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Síndrome de Cockayne/genética , Trastornos por Deficiencias en la Reparación del ADN , Modelos Animales de Enfermedad , Humanos , Leucoencefalopatías/genética , Ratones , Vaina de Mielina/genética , Vaina de Mielina/patología , Degeneración Nerviosa/metabolismo , Neuronas/patología , Mutación Puntual , Xerodermia Pigmentosa/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT(1)) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4(+/R)) and 4-fold (homozygous Fibulin-4(R/R)) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-ß signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-ß. Tissue levels of Ang II, a regulator of TGF-ß signaling, were increased. Prenatal treatment with the AT(1) receptor antagonist losartan, which blunts TGF-ß signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4(R/R) mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT(1) receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease.
Asunto(s)
Aorta Torácica/fisiopatología , Aneurisma de la Aorta/fisiopatología , Proteínas de la Matriz Extracelular/deficiencia , Vasoconstricción/fisiología , Angiotensina II/genética , Angiotensina II/metabolismo , Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Animales Recién Nacidos , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/prevención & control , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Losartán/farmacología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenilefrina/farmacología , Embarazo , Receptor de Angiotensina Tipo 1/fisiología , Proteína Smad2/genética , Proteína Smad2/metabolismo , Transcriptoma , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Vasoconstrictores/farmacologíaRESUMEN
BACKGROUND: We imaged the protease activity of matrix metalloproteinases (MMPs) upregulated during aneurysm formation, using protease-activatable near-infrared fluorescence probes. We tested whether these protease-activatable sensors can directly report the in vivo activity of the key biomarkers in aneurysm, using our genetically modified fibulin-4 mouse models for aneurysm formation. Mice homozygous for the fibulin-4 reduced-expression allele (fibulin-4(R/R)) show dilatation of the ascending aorta and a tortuous, stiffened aorta resulting from disorganized elastic fiber networks. Strikingly, even a moderate reduction in expression of fibulin-4 in the heterozygous fibulin-4(+/R) mice occasionally results in modest aneurysm formation. METHODS AND RESULTS: Aorta transcriptome and protein expression analysis of fibulin-4(+/R) and fibulin-4(R/R) animals identified excessive transforming growth factor-ß signaling as the critical event in the pathogenesis of aneurysm formation. To determine whether a perturbed elastin lamellar structure arose from induction of transforming growth factor-ß-regulated MMPs, we performed gelatin zymography and used a protease-activatable near-infrared fluorescence probe to monitor and quantify MMP upregulation in animals, using various in vivo optical imaging modules and coregistration of the fluorescence signal with CT images of the same animals. Gelatin zymography demonstrated a significant increase in the presence of the active form of MMP-9 in the aortic arch of fibulin-4(R/R) mice. In vivo analysis of MMP upregulation using the near-infrared fluorescence probe and subsequent isosurface concentration mapping from reconstructed tomographic images from fibulin-4(+/R) and fibulin-4(R/R) mice revealed a graded increase in activation of MMPs within the aneurysmal lesions. CONCLUSIONS: We aimed to develop molecular imaging procedures for faster, earlier, and easier recognition of aortic aneurysms. We show that in vivo coregistration of MMP activity by noninvasive tomographic imaging methods allows the detection of increased MMP activity, even before the aneurysm has actually formed.
Asunto(s)
Aorta Torácica/patología , Aneurisma de la Aorta Torácica/diagnóstico , Proteínas de la Matriz Extracelular/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Imagen Molecular , Animales , Aorta Torácica/diagnóstico por imagen , Aorta Torácica/enzimología , Aneurisma de la Aorta Torácica/enzimología , Aneurisma de la Aorta Torácica/genética , Aortografía/métodos , Biomarcadores/metabolismo , Técnicas Biosensibles , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Diagnóstico Precoz , Elastina , Activación Enzimática , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Fluorescencia , Angiografía por Resonancia Magnética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Molecular/métodos , Valor Predictivo de las Pruebas , Transducción de Señal , Espectroscopía Infrarroja Corta , Tomografía Computarizada por Rayos X , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
UNLABELLED: We determined the renal radiation dose of a series of (111)In-labeled peptides using animal SPECT. Because the animals' health deteriorated, renal toxicity was assessed. METHODS: Wild-type and megalin-deficient mice were imaged repeatedly at 3- to 6-wk intervals to quantify renal retention after injection of 40-50 MBq of (111)In-diethylenetriaminepentaacetic acid-labeled peptides (octreotide, exendin, octreotate, neurotensin, and minigastrin analogs), and the absorbed kidney radiation doses were estimated. Body weight, renal function parameters, and renal histology were determined at 16-20 wk after the first scan and compared with those in naive animals. RESULTS: Because of high renal retention, (111)In-diethylenetriaminepentaacetic acid-exendin-4 scans resulted in a 70-Gy kidney radiation dose in wild-type mice. Megalin-deficient kidneys received 20-40 Gy. The other peptides resulted in much lower renal doses. Kidney function monitoring indicated renal damage in imaged animals. CONCLUSION: Micro-SPECT enables longitudinal studies in 1 animal. However, long-term nephrotoxic effects may be induced after high renal radiation doses, even with (111)In-labeled radiotracers.
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Radioisótopos de Indio , Riñón/efectos de la radiación , Péptidos , Tomografía Computarizada de Emisión de Fotón Único/efectos adversos , Animales , Exenatida , Femenino , Inyecciones , Riñón/patología , Riñón/fisiopatología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Especificidad de Órganos , Ácido Pentético/química , Péptidos/administración & dosificación , Péptidos/química , Dosis de Radiación , Riesgo , Factores de Tiempo , Tomografía Computarizada por Rayos X , Ponzoñas/químicaRESUMEN
BACKGROUND: The molecular characteristics and the clinical disease course of bladder cancer (BC) in young patients remain largely unresolved. All patients are monitored according to an intensive surveillance protocol and we aim to gain more insight into the molecular pathways of bladder tumors in young patients that could ultimately contribute to patient stratification, improve patient quality of life and reduce associated costs. We also determined whether a biomarker-based surveillance could be feasible. CASE PRESENTATION: We report a unique case of a 26-year-old Caucasian male with recurrent non-muscle invasive bladder tumors occurring at a high frequency and analyzed multiple tumors (maximal pTaG2) and urine samples of this patient. Analysis included FGFR3 mutation detection, FGFR3 and TP53 immunohistochemistry, mircosatellite analysis of markers on chromosomes 8, 9, 10, 11 and 17 and a genome wide single nucleotide polymorphism-array (SNP). All analyzed tumors contained a mutation in FGFR3 and were associated with FGFR3 overexpression. None of the tumors showed overexpression of TP53. We found a deletion on chromosome 9 in the primary tumor and this was confirmed by the SNP-array that showed regions of loss on chromosome 9. Detection of all recurrences was possible by urinary FGFR3 mutation analysis. CONCLUSIONS: Our findings would suggest that the BC disease course is determined by not only a patient's age, but also by the molecular characteristics of a tumor. This young patient contained typical genetic changes found in tumors of older patients and implies a clinical disease course comparable to older patients. We demonstrate that FGFR3 mutation analysis on voided urine is a simple non-invasive method and could serve as a feasible follow-up approach for this young patient presenting with an FGFR3 mutant tumor.
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Predisposición Genética a la Enfermedad/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Humanos , MasculinoRESUMEN
Heparan sulfate glycosaminoglycans (HS-GAGs) are not only the structural elements of tissue architecture but also regulate the bioavailability and transduction pathways of heparan sulfate-bound polypeptides released by cells or the extracellular matrix. Heparan sulfate-bound polypeptides include inflammatory mediators, chemokines, angiogenic factors, morphogens, and growth-promoting factors that induce cell migration, proliferation, and differentiation in wound healing. OTR4120, a polymer engineered to mimic the properties of HS-GAGs, is used to replace the natural HS-GAGs that are degraded during wound repair, and enhance the tissue regeneration by preserving the cellular microenvironment and the endogenous signals needed for tissue regeneration. We previously demonstrated that OTR4120 treatment had a long-term effect on increasing breaking strength and vasodilation in healing rat full-thickness excisional wounds. The present study investigates the underlying mechanisms of the effects of OTR4120 treatment in improving the quality of cutaneous wound repair. We found that OTR4120 treatment stimulated inflammation resolution and increased neovascularization. OTR4120 treatment also promoted epidermal migration and proliferation during reepithelialization. Moreover, the granulation tissue formation and collagen maturation were improved in OTR4120-treated wounds. Three months after wounding, the effects of OTR4120 treatment on vascularization and inflammation resolution were normalized, except for an improved neodermis. We conclude that OTR4120 is a potential matrix therapeutic agent that ensures the quality of normal cutaneous wound repair and may restore impaired wound healing characterized by deficient angiogenesis and prolonged inflammation.
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Antiinflamatorios/farmacología , Colágeno/efectos de los fármacos , Glicosaminoglicanos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Masculino , Neovascularización Fisiológica/fisiología , Ratas , Úlcera Cutánea/fisiopatología , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
INTRODUCTION: External beam radiotherapy for prostate cancer leads to erectile dysfunction in 36%-43% of patients. The underlying mechanism is largely unknown, although some clinical studies suggest that the arterial supply to the corpora cavernosa is responsible. Two animal experimental studies reported on the effects of a single fraction of prostate irradiation on the penile structures. However, irradiation in multiple fractions is more representative of the actual clinical treatment. AIM: The present prospective, controlled study was initiated to investigate the effect of fractionated prostate irradiation on the arteries of the corpora cavernosa. MAIN OUTCOME MEASURES: Histological evaluation of the penile tissue in comparison with control rats at 2, 4, and 9 weeks after irradiation. METHODS: The prostate of twelve rats was treated with external beam radiation in 5 daily fractions of 7.4 gray. Three control rats were treated with sham irradiation. Prostatic and penile tissue was evaluated for general histology (hematoxylin-eosin). The penile tissue was further evaluated after combined staining for collagen (resorcin fuchsin) and alpha-smooth muscle actin (SMA) (Biogenex). RESULTS: The prostate showed adequate irradiation with fibrosis occurring at 9 weeks after irradiation. The corpora cavernosa showed arteries that had developed loss of smooth muscle cells expressing SMA, thickening of the intima, and occlusions. All the control rats maintained normal anatomy. CONCLUSION: This is the first animal experimental study that demonstrates changes in the arteries of the corpora cavernosa after fractionated irradiation to the prostatic area. The preliminary data suggests that erectile dysfunction after radiotherapy might be caused by radiation damage to the arterial supply of the corpora cavernosa.
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Arterias/efectos de la radiación , Endotelio Vascular/efectos de la radiación , Fibrosis/etiología , Músculo Liso/efectos de la radiación , Pene/irrigación sanguínea , Próstata/irrigación sanguínea , Animales , Fraccionamiento de la Dosis de Radiación , Masculino , Pene/efectos de la radiación , Proyectos Piloto , Estudios Prospectivos , Próstata/efectos de la radiación , Ratas , Ratas Sprague-DawleyRESUMEN
Non-muscle invasive bladder cancers (NMI-BCs) represent 75% of bladder cancers upon presentation. After removal of the primary tumour by transurethral resection, multiple recurrences continue to develop in 70% of patients. Consequently, prolonged and costly surveillance by cystoscopy is required. Mutations in the FGFR3 oncogene are common in NMI-BCs and are associated with a lower chance of progression to muscle-invasive disease. Here we analysed the consistency of FGFR3 mutations in primary and recurrent tumours. This knowledge is of crucial importance if FGFR3 mutation analysis on urinary cells is to be used as an alternative for cystoscopical surveillance. To this end, we monitored the disease process and FGFR3 mutation status of primary and recurrent tumours in 118 patients with NMI-BC. During median follow-up of 8.8 years, these patients underwent 2133 cystoscopies and 80 patients developed 414 recurrences. FGFR3 mutations were equally prevalent in primary and recurrent tumours (63%). Patients can have different types of FGFR3 mutations in different tumours. Recurrence risk was not significantly different for patients with a mutant or wild-type primary tumour. Recurrence rates varied widely between patients but were constant for a patient and were unrelated to FGFR3 status. In the mutant patient group, in contrast to the wild-type group, recurrences continued to develop after 10 years. In 81% of the recurrences of patients with a mutant primary tumour, a mutation was found. Moreover, recurrences in this patient group were of lower stage and grade than those of patients with a wild-type primary tumour (p < 0.001). These results suggest that surveillance by FGFR3 mutation analysis on voided urine in combination with a reduced cystoscopy frequency of patients presenting with an FGFR3 mutant tumour is worth investigating.
Asunto(s)
Análisis Mutacional de ADN , Recurrencia Local de Neoplasia/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Distribución de Chi-Cuadrado , Células Clonales , Cistoscopía , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Regressions of metastatic solid tumors after allogeneic human leukocyte antigen (HLA)-matched stem cell transplantation (SCT) are often associated with detrimental graft-versus-host disease (GVHD). The graft-versus-host reaction of the HLA-matched donor is directed mainly against the multiple mismatched minor histocompatibility antigens (mHags) of the patient. mHags are strong HLA-restricted alloantigens with differential tissue distribution. Ubiquitously expressed mHags are the prime in situ targets of GVHD. The mHag HA-1 is hematopoiesis restricted, but displays additionally an aberrant expression on solid tumors. Thus, HA-1 might be an excellent target to boost the anti-solid tumor effect of allogeneic SCT without inducing severe GVHD. Here, we show that cytotoxic T lymphocytes (CTLs) solely targeting the human mHag HA-1 are capable of eradicating 3-dimensional human solid tumors in a highly mHag-specific manner in vitro, accompanied by interferon-gamma release. In vivo, HA-1-specific CTLs distribute systemically and prevent human breast cancer metastases in immunodeficient mice. Moreover, HA-1-specific CTLs infiltrate and inhibit the progression of fully established metastases. Our study provides the first proof for the efficacy of a clinically applicable concept to exploit single mismatched mHags with hematopoiesis- and solid tumor-restricted expression for boosting the anti-solid tumor effect of allogeneic SCT.