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Microbubbles (MBs) combined with focused ultrasound (FUS) has emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery into the brain. However, the safety and biological consequences of BBB opening (BBBO) remain incompletely understood. This study aims to investigate the effects of two parameters mediating BBBO: microbubble volume dose (MVD) and mechanical index (MI). High-resolution MRI-guided FUS was employed in mouse brains to assess BBBO by manipulating these two parameters. Afterward, the sterile inflammatory response (SIR) was studied 6 h post-FUS treatment. Results demonstrated that both MVD and MI significantly influenced the extent of BBBO, with higher MVD and MI leading to increased permeability. Moreover, RNA sequencing revealed upregulation of major inflammatory pathways and immune cell infiltration after BBBO, indicating the presence and extent of SIR. Gene set enrichment analysis identified 12 gene sets associated with inflammatory responses that were significantly upregulated at higher MVD or MI. A therapeutic window was established between therapeutically relevant BBBO and the onset of SIR, providing operating regimes to avoid damage from stimulation of the NFκB pathway via TNFÉ signaling to apoptosis. These results contribute to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and further elucidate the underlying molecular mechanisms driving sterile inflammation.
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Barreira Hematoencefálica , Inflamação , Microbolhas , Barreira Hematoencefálica/metabolismo , Animais , Camundongos , Inflamação/metabolismo , Sistemas de Liberação de Medicamentos , Imageamento por Ressonância Magnética , Encéfalo/metabolismo , Encéfalo/patologia , MasculinoRESUMO
Blood-brain barrier opening (BBBO) using focused ultrasound (FUS) and microbubbles (MBs) has emerged as a promising technique for delivering therapeutics to the brain. However, the influence of various FUS and MB parameters on BBBO and subsequent sterile inflammatory response (SIR) remains unclear. In this study, we investigated the effects of MB size and composition, as well as the number of FUS sonication points, on BBBO and SIR in an immunocompetent mouse model. Using MRI-guided MB+FUS, we targeted the striatum and assessed extravasation of an MRI contrast agent to assess BBBO and RNAseq to assess SIR. Our results revealed distinct effects of these parameters on BBBO and SIR. Specifically, at a matched microbubble volume dose (MVD), MB size did not affect the extent of BBBO, but smaller (1 µm diameter) MBs exhibited a lower classification of SIR than larger (3 or 5 µm diameter) MBs. Lipid-shelled microbubbles exhibited greater BBBO and a more pronounced SIR compared to albumin-shelled microbubbles, likely owing to the latter's poor in vivo stability. As expected, increasing the number of sonication points resulted in greater BBBO and SIR. Furthermore, correlation analysis revealed strong associations between passive cavitation detection measurements of harmonic and inertial MB echoes, BBBO and the expression of SIR gene sets. Our findings highlight the critical role of MB and FUS parameters in modulating BBBO and subsequent SIR in the brain. These insights inform the development of targeted drug delivery strategies and the mitigation of adverse inflammatory reactions in neurological disorders.
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Microbubbles (MBs) combined with focused ultrasound (FUS) have emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery to the brain. However, the safety and biological consequences of BBB opening remain incompletely understood. This study investigates the effects of varying microbubble volume doses (MVD) and ultrasound mechanical indices (MI) on BBB opening and the sterile inflammatory response (SIR) using high-resolution ultra-high field MRI-guided FUS in mouse brains. The results demonstrate that both MVD and MI significantly influence the extent of BBB opening, with higher doses and mechanical indices leading to increased permeability. Moreover, RNA sequencing reveals upregulated inflammatory pathways and immune cell infiltration after BBB opening, suggesting the presence and extent of SIR. Gene set enrichment analysis identifies 12 gene sets associated with inflammatory responses that are upregulated at higher doses of MVD or MI. A therapeutic window is established between significant BBB opening and the onset of SIR, providing operating regimes for avoiding each three classes of increasing damage from stimulation of the NFκB pathway via TNFL signaling to apoptosis. This study contributes to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and sheds light on the underlying molecular mechanisms of the sterile inflammatory response. Significance Statement: The significance of this study lies in its comprehensive investigation of microbubble-facilitated focused ultrasound for blood-brain barrier (BBB) opening. By systematically exploring various combinations of microbubble volume doses and ultrasound mechanical indices, the study reveals their direct impact on the extent of BBB permeability and the induction of sterile inflammatory response (SIR). The establishment of a therapeutic window between significant BBB opening and the onset of SIR provides critical insights for safe and targeted drug delivery to the brain. These findings advance our understanding of the biological consequences of BBB opening and contribute to optimizing parameters for clinical applications, thus minimizing potential health risks, and maximizing the therapeutic potential of this technique.
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Background: Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB opening, allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Methods: Mice were orthotopically injected with a patient-derived diffuse midline glioma (DMG) cell line, BT245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa peak negative pressure, 1 Hz pulse repetition frequency, 10-ms pulse length, 3 min treatment time)/(25 µL/kg, i.v.) targeting to the tumor location. Results: In animals receiving panobinostat (10 mg/kg, i.p.) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, without significant increase of the drug in the forebrain. In mice receiving 3 weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes (P = .01). Furthermore, we showed the first survival benefit from FUS/MB improved delivery increasing the mean survival from 21 to 31 days (P < .0001). Conclusions: Our study demonstrates that FUS-mediated BBB disruption can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.
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Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB disruption (BBBD), allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-derived DMG cell line, BT-245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa PNP, 1 Hz PRF, 10 ms PL, 3 min treatment time) / (25 µL/kg, IV) targeting to the tumor location. In animals receiving panobinostat (10 mg/kg, IP) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, with only insignificant increase of the drug in the forebrain. In mice receiving three weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes by MRI ( p = 0.01). Furthermore, FUS/MB improved the mean survival from 21 to 31 days ( p < 0.0001). Our study demonstrates that FUS-mediated BBBD can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival. One Sentence Summary: FUS and microbubbles can increase the delivery of panobinostat to a patient-derived xenograft (PDX) orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.
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Fluorescence image-guided surgery (IGS) using antibody conjugates of the fluorophore IRDye800CW have revolutionized the surgical debulking of tumors. Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, conjugated to IRDye800CW (Cet-IRDye800) is the first molecular targeted antibody probe to be used for IGS in head and neck cancer patients. In addition to surgical debulking, Cetuximab-targeted photodynamic therapy (photoimmunotherapy; PIT) is emerging in the clinic as a powerful modality for head and neck tumor photodestruction. A plethora of other photoactivable agents are also in clinical trials for photodynamic-based therapies of head and neck cancer. Considering the vascular and stromal modulating effects of sub-therapeutic photodynamic therapy, namely photodynamic priming (PDP), this study explores the potential synergy between PDP and IGS for a novel photodynamic image-guided surgery (P-IGS) strategy. To the best of our knowledge, this is the first demonstration that PDP of the tumor microenvironment can augment the tumor delivery of full-length antibodies, namely Cet-IRDye800. In this study, we demonstrate a proof-of-concept that PDP primes orthotopic FaDu human head and neck tumors in mice for P-IGS by increasing the delivery of Cet-IRDye800 by up to 138.6%, by expediting its interstitial accumulation by 10.5-fold, and by increasing its fractional tumor coverage by 49.5% at 1 h following Cet-IRDye800 administration. Importantly, PDP improves the diagnostic accuracy of tumor detection by up to 264.2% with respect to vicinal salivary glands at 1 h. As such, PDP provides a time-to-surgery benefit by reducing the time to plateau 10-fold from 25.7 h to 2.5 h. We therefore propose that a pre-operative PDP regimen can expedite and augment the accuracy of IGS-mediated surgical debulking of head and neck tumors and reduce the time-to-IGS. Furthermore, this P-IGS regimen, can also enable a forward-looking post-operative protocol for the photodestruction of unresectable microscopic disease in the surgical bed. Beyond this scope, the role of PDP in the homogenous delivery of diagnostic, theranostic and therapeutic antibodies in solid tumors is of considerable significance to the wider community.
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PURPOSE: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. EXPERIMENTAL DESIGN: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. RESULTS: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. CONCLUSIONS: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.
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Sarcoma de Ewing , Linhagem Celular Tumoral , Plasticidade Celular , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVES: Three-dimensional (3D) H-scan is a new ultrasound (US) technique that images the relative size of acoustic scatterers. The goal of this research was to evaluate use of 3D H-scan US imaging for monitoring early breast cancer response to neoadjuvant therapy using a preclinical murine model of breast cancer. MATERIALS AND METHODS: Preclinical studies were conducted using luciferase-positive breast cancer-bearing mice (n = 40). Anesthetized animals underwent US imaging at baseline before administration with an apoptosis-inducing drug or a saline control. Image data were acquired using a US scanner equipped with a volumetric transducer following either a shorter- or longer-term protocol. The later included bioluminescent imaging to quantify tumor cell viability. At termination, tumors were excised for ex vivo analysis. RESULTS: In vivo results showed that 3D H-scan US imaging is considerably more sensitive to tumor changes after apoptosis-inducing drug therapy as compared with traditional B-scan US. Although there was no difference at baseline (P > 0.99), H-scan US results from treated tumors exhibited progressive decreases in image intensity (up to 62.2% by day 3) that had a significant linear correlation with cancer cell nuclear size (R2 > 0.51, P < 0.001). Results were validated by histological data and a secondary longitudinal study with survival as the primary end point. DISCUSSION: Experimental results demonstrate that noninvasive 3D H-scan US imaging can detect an early breast tumor response to apoptosis-inducing drug therapy. Local in vivo H-scan US image intensity correlated with cancer cell nuclear size, which is one of the first observable changes of a cancer cell undergoing apoptosis and confirmed using histological techniques. Early imaging results seem to provide prognostic insight on longer-term tumor response. Overall, 3D H-scan US imaging is a promising technique that visualizes the entire tumor and detects breast cancer response at an early stage of therapy.
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Neoplasias da Mama , Terapia Neoadjuvante , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Feminino , Humanos , Imageamento Tridimensional/métodos , Estudos Longitudinais , Camundongos , Ultrassonografia/métodosRESUMO
Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.
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Proteínas do Citoesqueleto/metabolismo , Gânglios Espinais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Vasodilatação/fisiologia , Animais , Proteínas do Citoesqueleto/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Nociceptividade/fisiologia , Nociceptores/fisiologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologiaRESUMO
Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.
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Osso e Ossos/metabolismo , Diferenciação Celular , Linhagem da Célula , Células-Tronco Mesenquimais/metabolismo , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismo , Osteogênese , Adipócitos/metabolismo , Animais , Condrócitos/metabolismo , Modelos Animais de Doenças , Expressão Ectópica do Gene , Epigenômica , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Análise de Célula Única , Tendões/metabolismoRESUMO
Liver disease is increasing in prevalence across the globe. We present here a multiparametric ultrasound (mpUS) imaging approach for assessing nonalcoholic fatty liver disease (NALFD). This study was performed using rats (N = 21) that were fed either a control or methionine and choline deficient (MCD) diet. A mpUS imaging approach that includes H-scan ultrasound (US), shear wave elastography, and contrast-enhanced US measurements were then performed at 0 (baseline), 2, and 6 weeks. Thereafter, animals were euthanized and livers excised for histological processing. A support vector machine (SVM) was used to find a decision plane that classifies normal and fatty liver conditions. In vivo mpUS results from control and MCD diet fed animals reveal that all mpUS measures were different at week 6 (P < 0.05). Principal component analysis (PCA) showed that the H-scan US data contributed the highest percentage to the classification among the mpUS measurements. The SVM resulted in 100% accuracy for classification of normal and high fat livers and 92% accuracy for classification of normal, low fat, and high fat livers. Histology findings found considerable steatosis in the MCD diet fed animals. This study suggests that mpUS examinations have the potential to provide a comprehensive estimation of the main components of early stage NAFLD.
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Técnicas de Imagem por Elasticidade , Alimentos Formulados/efeitos adversos , Fígado/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Animais , Deficiência de Colina , Metionina/deficiência , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this research project was to evaluate the use of 3-dimensional (3-D) super-resolution ultrasound (SR-US) imaging to assess any early changes in breast cancer after treatment with a vascular-disrupting agent (VDA). A Vevo 3100 ultrasound system (FUJIFILM VisualSonics Inc) equipped with an MX 201 transducer was used for image acquisition. A total of 2.5 × 107 microbubbles (MBs) were injected into the tail vein of anesthetized breast cancer-bearing mice using repeat bolus injections every 5 min. A total of 10 stacks of ultrasound images were collected as the transducer was mechanically moved across the tumor at 0.6 mm intervals yielding a 6-mm thick volume. At each tumor location, a stack contained 1 × 104 frames of ultrasound data that were acquired at 463 frames/sec and stored as in-phase/quadrature (IQ) format. After motion correction, each temporal stack of ultrasound images was processed separately for clutter signal removal, which was followed by MB localization and enumeration before generation of the final SR-US image. After reconstruction of the 3-D SR-US volume dataset, the tumor microvasculature was enhanced using a multiscale vessel enhancement filter. Vessels from the resultant microvascular network were then segmented using an adaptive thresholding method. Finally, mean microvascular density (MVD) measurements from each tumor volume were computed as a summarizing statistic. While no differences were found between baseline SR-US image-derived measures of MVD (p = 0.76), these same measurements were significantly lower at 24 h after VDA treatment (p < 0.001). Overall, 3-D SR-US imaging detected early tumor changes following treatment with a vascular-targeted drug.
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Hox genes are indispensable for the proper patterning of the skeletal morphology of the axial and appendicular skeleton during embryonic development. Recently, it has been demonstrated that Hox expression continues from embryonic stages through postnatal and adult stages exclusively in a skeletal stem cell population. However, whether Hox genes continue to function after development has not been rigorously investigated. We generated a Hoxd11 conditional allele and induced genetic deletion at adult stages to show that Hox11 genes play critical roles in skeletal homeostasis of the forelimb zeugopod (radius and ulna). Conditional loss of Hox11 function at adult stages leads to replacement of normal lamellar bone with an abnormal woven bone-like matrix of highly disorganized collagen fibers. Examining the lineage from the Hox-expressing mutant cells demonstrates no loss of stem cell population. Differentiation in the osteoblast lineage initiates with Runx2 expression, which is observed similarly in mutants and controls. With loss of Hox11 function, however, osteoblasts fail to mature, with no progression to osteopontin or osteocalcin expression. Osteocyte-like cells become embedded within the abnormal bony matrix, but they completely lack dendrites, as well as the characteristic lacuno-canalicular network, and do not express SOST. Together, our studies show that Hox11 genes continuously function in the adult skeleton in a region-specific manner by regulating differentiation of Hox-expressing skeletal stem cells into the osteolineage.
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Osso e Ossos/embriologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Animais , Osso e Ossos/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Feminino , Membro Anterior/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Genes Homeobox/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Esqueleto/embriologia , Fatores de Transcrição/metabolismoRESUMO
mRNA function is controlled by RNA-binding proteins. The specificity of RNA-binding factors for their targets is critical in that it enables all subsequent regulation. Despite widespread recognition of the pervasive role RNA-binding proteins play in development and disease, they remain challenging to target with small molecules. A renaissance in RNA therapeutics has led to the identification of modifications that substantially increase RNA stability. When combined with information regarding specificity, a new class of oligonucleotide mimics has emerged as a means to competitively disrupt the regulation of endogenous substrates. These decoys have been used to inhibit RNA-binding proteins in living animals. Decoys will likely provide new insights into the expansive roles of RNA-binding proteins in biology and disease. Here, we describe examples where they have been used and discuss how they could be applied to new targets.
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Oligonucleotídeos/farmacologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Mimetismo Molecular , Ligação Proteica/efeitos dos fármacos , Estabilidade de RNA , RNA Mensageiro/químicaRESUMO
BACKGROUND & AIMS: Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the 'stemness' of ISCs are still not well defined. Here, we investigated the role of Krüppel-like factor 5 (KLF5) in regulating ISC functions. METHODS: We performed studies in adult Lgr5EGFP-IRES-creERT2;Rosa26LSLtdTomato (Lgr5Ctrl) and Lgr5EGFP-IRES-creERT2;Klf5fl/fl;Rosa26LSLtdTomato (Lgr5ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5ΔKlf5 but not Lgr5Crtl mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5ΔKlf5 or Lgr5Crtl mice and examined by immunofluorescence stain. RESULTS: Lgr5+ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5+ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5+ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls. CONCLUSION: We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.
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Células-Tronco Adultas/fisiologia , Mucosa Intestinal/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Lesões por Radiação/patologia , Regeneração/genética , Células-Tronco Adultas/efeitos da radiação , Animais , Estudos de Casos e Controles , Linhagem da Célula/genética , Autorrenovação Celular/genética , Células Cultivadas , Colite/etiologia , Colite/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Modelos Animais de Doenças , Enterite/etiologia , Enterite/patologia , Epigênese Genética , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos da radiação , Fatores de Transcrição Kruppel-Like/análise , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Transgênicos , Organoides , Cultura Primária de Células , RNA-Seq , Receptores Acoplados a Proteínas G/genética , Ativação Transcricional , Irradiação Corporal Total , Via de Sinalização Wnt/genéticaRESUMO
Multipotent mesenchymal stromal cells (MSCs) are required for skeletal formation, maintenance, and repair throughout life; however, current models posit that postnatally arising long-lived adult MSCs replace transient embryonic progenitor populations. We previously reported exclusive expression and function of the embryonic patterning transcription factor, Hoxa11, in adult skeletal progenitor-enriched MSCs. Here, using a newly generated Hoxa11-CreERT2 lineage-tracing system, we show Hoxa11-lineage marked cells give rise to all skeletal lineages throughout the life of the animal and persist as MSCs. Hoxa11 lineage-positive cells give rise to previously described progenitor-enriched MSC populations marked by LepR-Cre and Osx-CreER, placing them upstream of these populations. Our studies establish that Hox-expressing cells are skeletal stem cells that arise from the earliest stages of skeletal development and self-renew throughout the life of the animal.
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Adipócitos/citologia , Condrócitos/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Animais , Regeneração Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/embriologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Receptores para Leptina/genética , Fator de Transcrição Sp7/genéticaRESUMO
mRNA function is meticulously controlled. We provide an overview of the integral role that posttranscriptional controls play in the perception of painful stimuli by sensory neurons. These specialized cells, termed nociceptors, precisely regulate mRNA polarity, translation, and stability. A growing body of evidence has revealed that targeted disruption of mRNAs and RNA-binding proteins robustly diminishes pain-associated behaviors. We propose that the use of multiple independent regulatory paradigms facilitates robust temporal and spatial precision of protein expression in response to a range of pain-promoting stimuli. This article is categorized under: RNA in Disease and Development > RNA in Disease Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability.
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Dor/genética , Dor/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , HumanosRESUMO
Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.
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The limb musculoskeletal system provides a primary means for locomotion, manipulation of objects and protection for most vertebrate organisms. Intricate integration of the bone, tendon and muscle tissues are required for function. These three tissues arise largely independent of one another, but the connections formed during later development are maintained throughout life and are re-established following injury. Each of these tissues also have mesenchymal stem/progenitor cells that function in maintenance and repair. Here in, we will review the major events in the development of limb skeleton, tendon, and muscle tissues, their response to injury, and discuss current knowledge regarding resident progenitor/stem cells within each tissue that participate in development, repair, and regeneration in vivo.
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Extremidades/embriologia , Extremidades/fisiologia , Desenvolvimento Musculoesquelético/fisiologia , Regeneração/fisiologia , Animais , Osso e Ossos/citologia , Osso e Ossos/embriologia , Osso e Ossos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Músculos/citologia , Músculos/embriologia , Músculos/fisiologia , Desenvolvimento Musculoesquelético/genética , Regeneração/genética , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Tendões/citologia , Tendões/embriologia , Tendões/fisiologiaRESUMO
PURPOSE: To evaluate refractive outcomes of cataract surgery in previously vitrectomized eyes and compare the accuracy of intraocular lens (IOL) calculation formulas in this population. SETTING: Stony Brook University Hospital, Stony Brook, New York, USA. DESIGN: Retrospective case series. METHODS: All electronic medical records of patients who had phacoemulsification from 2013 to 2017 were reviewed. Patients who had previously had pars plana vitrectomy in the same eye were selected. Patients with a history of refractive surgery, silicone oil in the eye, or other factors preventing accurate IOL power calculation were excluded. RESULTS: The mean postoperative spherical equivalent was -0.16 diopter (D) ± 0.81 (SD) (n = 61). The mean prediction errors were 0.30 ± 0.82 D, -0.09 ± 0.76 D, 0.23 ± 0.76 D, 0.25 ± 0.81 D, 0.04 ± 0.85 D, 0.30 ± 0.82 D, 0.33 ± 0.79 D, and 0.45 ± 0.80 D with the Holladay 1, Wang/Koch (WKA) adjusted Holladay 1 (n = 14), Holladay 2 (n = 48), SRK/T, WKA SRK/T (n = 14), Hill-Radial Basis Function (n = 50), and Ladas formulas, respectively. The formula with the highest percentage of predictions within ±0.50 D of the postoperative outcome was the Holladay 2 (60.42%). Statistically significant differences between the predicted and actual refractive outcomes were found with all formulas (P < .05) except the WKA Holladay and WKA SRK/T. Intraclass correlation showed low repeatability (<0.50) for all formulas. CONCLUSIONS: Refractive outcomes after cataract extraction in vitrectomized patients can be variable and more hyperopic than the predicted outcomes using traditional and newer IOL calculation methods. Thus, predicting refractive outcomes in this population is challenging, and patients should be counseled accordingly.