RESUMEN
RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.
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Leucemia , Empalmosomas , Humanos , Empalmosomas/genética , Estructuras R-Loop , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Reparación del ADN , Leucemia/tratamiento farmacológico , Leucemia/genética , Factores de Empalme de ARN/genética , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
Aims: The aim of this study was to evaluate the role of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography-computed tomography (PET-CT) scan in the detection of axillary lymph node (ALN) involvement and comparison with sentinel lymph node biopsy (SLNB) in operable early-stage breast cancer (EBC). Settings and Design: It is a retrospective analysis of staging PET-CT scan of EBC. Methods: A total of 128 patients with histopathologically proven breast cancer (BC) were included in the study. Preoperative mammography supplemented with ultrasonography and staging 18F-FDG PET-CT scan was done for all patients. Surgery was done within 30 (mean ± standard deviation = 13.8 ± 10.5) days of staging. SLNB was performed in patients without PET-positive ALNs. All patients with positive sentinel nodes and PET-positive ALNs underwent axillary lymph node dissection (ALND). Statistical Analysis Used: The comparison between categorical variables was made by Chi-square/Fisher's exact test as applicable. For continuous variables comparisons, Student's t-test and one-way analysis of variance tests were used. Results: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PET-CT scan for detection of ALN involvement were 41.7%, 93.2%, 92.1%, and 45.6%, respectively. Sensitivity, specificity, PPV, and NPV of mammography were 84.5%, 54.5%, 78.0%, and 68.6%, respectively. Sixteen out of 46 (34.7%) patients with negative ALNs in PET-CT scan finally showed involvement in histopathology report after SLNB resulting in upstage of the disease. The size of tumor deposits in sentinel nodes was significantly smaller than PET-positive ALNs (P = 0.01). Our observations correlate with the results of earlier studies published in the literature. Conclusions: 18F-FDG PET-CT scan cannot substitute SLNB for ALN screening in EBC. The limitations are most marked in smaller and micrometastatic tumor deposits in ALNs and may be attributed to limitations of PET resolution. However, PET-positive nodes showed good specificity for disease involvement in our study. Therefore, ALND can safely be performed by omitting SLNB in such cases.
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Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.
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Leucemia Mieloide Aguda , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Polycythemia Vera (PV) is a chronic myeloproliferative neoplasm resulting from an acquired driver mutation in the JAK2 gene of hematopoietic stem and progenitor cells resulting in the overproduction of mature erythrocytes and abnormally high hematocrit, in turn leading to thromboembolic complications. Therapeutic phlebotomy is the most common treatment to reduce the hematocrit levels and consequently decrease thromboembolic risk. Here we demonstrate that, by using the iron restrictive properties of the antisense oligonucleotides against Tmprss6 mRNA, we can increase hepcidin to achieve effects equivalent to therapeutic phlebotomy. We provide evidence that this less invasive approach could represent an additional therapeutic tool for the treatment of PV patients.
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Proteínas de la Membrana/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Policitemia Vera/tratamiento farmacológico , Animales , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Oligonucleótidos Antisentido/genética , Policitemia Vera/genética , Policitemia Vera/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
We present a rare case of an aberrant right vertebral artery originating from the arch of aorta distal to the origin of the left subclavian artery. The incidence of this particular variant of aberrant origin of the right vertebral artery is extremely uncommon with only seventeen cases reported in literature to date. This case was incidentally detected on a staging positron emission tomography-computerized tomography (PET-CT) scan for lung cancer. We review the incidence, embryological mechanism, and clinical importance of this aberrant course of the right vertebral artery.
RESUMEN
Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-ß family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.
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Miositis Osificante , Osificación Heterotópica , Activinas/genética , Animales , Condrogénesis , Ratones , Osificación Heterotópica/genética , OsteogénesisRESUMEN
Emerging evidences highlight importance of epigenetic regulation and their integration with transcriptional and cell signaling machinery in determining tissue resident adult pluripotent mesenchymal stem/stromal cell (MSC) activity, lineage commitment, and multicellular development. Histone modifying enzymes and large multi-subunit chromatin remodeling complexes and their cell type-specific plasticity remain the central defining features of gene regulation and establishment of tissue identity. Modulation of transcription factor expression gradient ex vivo and concomitant flexibility of higher order chromatin architecture in response to signaling cues are exciting approaches to regulate MSC activity and tissue rejuvenation. Being an important constituent of the adult bone marrow microenvironment/niche, pathophysiological perturbation in MSC homeostasis also causes impaired hematopoietic stem/progenitor cell function in a non-cell autonomous mechanism. In addition, pluripotent MSCs can function as immune regulatory cells, and they reside at the crossroad of innate and adaptive immune response pathways. Research in the past few years suggest that MSCs/stromal fibroblasts significantly contribute to the establishment of immunosuppressive microenvironment in shaping antitumor immunity. Therefore, it is important to understand mesenchymal stromal epigenome and transcriptional regulation to leverage its applications in regenerative medicine, epigenetic memory-guided trained immunity, immune-metabolic rewiring, and precision immune reprogramming. In this review, we highlight the latest developments and prospects in chromatin biology in determining MSC function in the context of lineage commitment and immunomodulation.
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Ensamble y Desensamble de Cromatina/inmunología , Células Madre Hematopoyéticas/inmunología , Histonas/inmunología , Células Madre Mesenquimatosas/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Nicho de Células Madre/inmunología , Animales , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
Significance: Iron is an essential element required for sustaining a normal healthy life. However, an excess amount of iron in the bloodstream and tissue generates toxic hydroxyl radicals through Fenton reactions. Henceforth, a balance in iron concentration is extremely important to maintain cellular homeostasis in both normal hematopoiesis and erythropoiesis. Iron deficiency or iron overload can impact hematopoiesis and is associated with many hematological diseases. Recent Advances: The mechanisms of action of key iron regulators such as erythroferrone and the discovery of new drugs, such as ACE-536/luspatercept, are of potential interest to treat hematological disorders, such as ß-thalassemia. New therapies targeting inflammation-induced ineffective erythropoiesis are also in progress. Furthermore, emerging evidences support differential interactions between iron and its cellular antioxidant responses of hematopoietic and neighboring stromal cells. Both iron and its systemic regulator, such as hepcidin, play a significant role in regulating erythropoiesis. Critical Issues: Significant pre-clinical studies are on the way and new drugs targeting iron metabolism have been recently approved or are undergoing clinical trials to treat pathological conditions with impaired erythropoiesis such as myelodysplastic syndromes or ß-thalassemia. Future Directions: Future studies should explore how iron regulates hematopoiesis in both benign and malignant conditions. Antioxid. Redox Signal. 35, 415-432.
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Receptores de Activinas Tipo II/farmacología , Eritropoyesis/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/farmacología , Hierro/metabolismo , Síndromes Mielodisplásicos/tratamiento farmacológico , Hormonas Peptídicas/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Talasemia beta/tratamiento farmacológico , Humanos , Síndromes Mielodisplásicos/metabolismo , Talasemia beta/metabolismoRESUMEN
We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation. For complete details on the use and execution of this protocol, please refer to Sinha et al. (2020).
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Bioensayo/métodos , Técnicas de Cocultivo/métodos , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Antígenos CD34/metabolismo , Separación Celular , Centrifugación por Gradiente de Densidad , Células Clonales , Humanos , Separación Inmunomagnética , Modelos BiológicosRESUMEN
The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2'-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.
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Bone morphogenic protein (BMP)/transforming growth factor ß (TGF-ß) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfßrII affecting BMP-early-responsive gene expression. Gain of function of BmprIß, TgfßrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-ß signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis.
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Proteínas Morfogenéticas Óseas/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular , Humanos , Transducción de SeñalAsunto(s)
Proteínas Morfogenéticas Óseas/deficiencia , Factores de Diferenciación de Crecimiento/deficiencia , Proteínas Recombinantes de Fusión/farmacología , Talasemia beta/metabolismo , Anemia/sangre , Anemia/tratamiento farmacológico , Anemia/etiología , Animales , Biomarcadores , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Modelos Animales de Enfermedad , Factores de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/metabolismo , Ratones , Ratones Transgénicos , Resultado del Tratamiento , Talasemia beta/sangre , Talasemia beta/tratamiento farmacológico , Talasemia beta/genéticaRESUMEN
Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.
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Anemia Aplásica/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/deficiencia , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Complejos Multiproteicos/genética , Síndromes Mielodisplásicos/genética , Factores de Transcripción/deficiencia , Adolescente , Adulto , Anciano , Anemia Aplásica/metabolismo , Anemia Aplásica/patología , Médula Ósea/patología , Células Clonales/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Estudios de Seguimiento , Regulación de la Expresión Génica , Hematopoyesis , Humanos , Masculino , Persona de Mediana Edad , Complejos Multiproteicos/biosíntesis , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Subunidades de Proteína , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Adulto JovenRESUMEN
Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1â¯month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3â¯months, articular cartilage consisted of a 2-3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72â¯h, respectively. Notably, EdU injection in 3â¯month-old mice labeled both progenitors and maturing chondrocytes by 96â¯h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling.
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Cartílago Articular/citología , Condrocitos/citología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Animales , Animales Recién Nacidos , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis , Ratones , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Articulación Temporomandibular/citología , Articulación Temporomandibular/metabolismo , Tenascina/metabolismoRESUMEN
BACKGROUND: Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. METHODS: Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. RESULTS: Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. CONCLUSIONS: TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.
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Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inhibidores , Vasos Coronarios/efectos de los fármacos , Propofol/farmacología , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Vasodilatadores/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Células Cultivadas , Vasos Coronarios/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/genética , Canales de Potencial de Receptor Transitorio/genética , Vasoconstrictores/antagonistas & inhibidores , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.
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Exostosis Múltiple Hereditaria/genética , N-Acetilglucosaminiltransferasas/genética , Osteocondroma/genética , Proteína Smad1/genética , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Médula Cervical/metabolismo , Médula Cervical/patología , Condrogénesis/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Exostosis Múltiple Hereditaria/diagnóstico por imagen , Exostosis Múltiple Hereditaria/tratamiento farmacológico , Exostosis Múltiple Hereditaria/patología , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , Heparitina Sulfato/biosíntesis , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Mutación , Osteocondroma/diagnóstico por imagen , Osteocondroma/patología , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Tomografía Computarizada de EmisiónRESUMEN
Heterotopic ossification (HO) consists of ectopic cartilage and bone formation following severe trauma or invasive surgeries, and a genetic form of it characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Recent mouse studies showed that HO was significantly inhibited by systemic treatment with a corticosteroid or the retinoic acid receptor γ agonist Palovarotene. Because these drugs act differently, the data raised intriguing questions including whether the drugs affected HO via similar means, whether a combination therapy would be more effective or whether the drugs may hamper each other's action. To tackle these questions, we used an effective HO mouse model involving subcutaneous implantation of Matrigel plus rhBMP2, and compared the effectiveness of prednisone, dexamathaosone, Palovarotene or combination of. Each corticosteroid and Palovarotene reduced bone formation at max doses, and a combination therapy elicited similar outcomes without obvious interference. While Palovarotene had effectively prevented the initial cartilaginous phase of HO, the steroids appeared to act more on the bony phase. In reporter assays, dexamethasone and Palovarotene induced transcriptional activity of their respective GRE or RARE constructs and did not interfere with each other's pathway. Interestingly, both drugs inhibited the activity of a reporter construct for the inflammatory mediator NF-κB, particularly in combination. In good agreement, immunohistochemical analyses showed that both drugs markedly reduced the number of mast cells and macrophages near and within the ectopic Matrigel mass and reduced also the number of progenitor cells. In sum, corticosteroids and Palovarotene appear to block HO via common and distinct mechanisms. Most importantly, they directly or indirectly inhibit the recruitment of immune and inflammatory cells present at the affected site, thus alleviating the effects of key HO instigators.
Asunto(s)
Antiinflamatorios/uso terapéutico , Osificación Heterotópica/tratamiento farmacológico , Pirazoles/uso terapéutico , Retinoides/agonistas , Estilbenos/uso terapéutico , Animales , Antiinflamatorios/farmacología , Peso Corporal/efectos de los fármacos , Cartílago/efectos de los fármacos , Cartílago/patología , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Genes Reporteros , Macrófagos/efectos de los fármacos , Macrófagos/patología , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Osificación Heterotópica/patología , Prednisona/farmacología , Prednisona/uso terapéutico , Pirazoles/farmacología , Estilbenos/farmacología , Transfección , Resultado del TratamientoRESUMEN
We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.
RESUMEN
BACKGROUND: Transient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol is an intravenous anesthetic known to cause vasorelaxation. Our objectives were to examine the extent to which TRPA1 and/or TRPV1 ion channels mediate propofol-induced depressor responses in vivo and to delineate the signaling pathway(s) involved. METHODS: Mice were subjected to surgery under 1.5-2.5% sevoflurane gas with supplemental oxygen. After a stable baseline in mean arterial pressure (MAP) was achieved propofol (2.5, 5.0, 10.0 mg/kg/min) was administered to assess the hemodynamic actions of the intravenous anesthetic. The effect of nitric oxide synthase (NOS) inhibition with L-NAME and/or calcium-gated K+ channel (BKCa) inhibition with Penetrim A (Pen A), alone and in combination, on propofol-induced decreases in mean arterial pressure were assessed in control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). RESULTS: Propofol decreased MAP in control mice and this effect was markedly attenuated in TRPA1-/- and TRPAV-/- mice but unaffected in TRPV1-/-mice. Moreover, pretreatment with L-NAME or Pen A attenuated the decrease in MAP in control and TRPV1-/- mice, and combined inhibition abolished the depressor response. In contrast, the markedly attenuated propofol-induced depressor response observed in TRPA1-/- and TRPAV-/- mice was unaffected by pre-treatment with Pen A or L-NAME when used either alone or in combination. CONCLUSION: These data demonstrate for the first time that propofol-induced depressor responses in vivo are predominantly mediated by TRPA1 ion channels with no involvement of TRPV1 ion channels and includes activation of both NOS and BKCa channels.