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1.
Am J Physiol Heart Circ Physiol ; 326(2): H370-H384, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38063811

RESUMO

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.


Assuntos
Miócitos Cardíacos , Transdução de Sinais , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Angiotensina II/farmacologia , Regulação da Expressão Gênica , Células Cultivadas , Estresse Mecânico
2.
Physiol Genomics ; 52(10): 468-477, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866086

RESUMO

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1-5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.


Assuntos
Ciclo Celular/genética , Genes Reporter , Antígeno Ki-67/genética , Plasmídeos/genética , Análise de Célula Única/métodos , Animais , Proliferação de Células/genética , Células Epiteliais/metabolismo , Corantes Fluorescentes/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Células MCF-7 , Camundongos , Microinjeções , Fenótipo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , Transdução Genética
3.
Pharmaceutics ; 12(9)2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32911688

RESUMO

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mice.

4.
Pharmacol Res Perspect ; 7(5): e00511, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31463067

RESUMO

Opioids are powerful analgesics acting via the human µ-opiate receptor (hMOR). Opioid use is associated with adverse effects such as tolerance, addiction, respiratory depression, and constipation. Two synthetic opioids, AH-7921 and U-47700 that were developed in the 1970s but never marketed, have recently appeared on the illegal drug market and in forensic toxicology reports. These agents were initially characterized for their analgesic activity in rodents; however, their pharmacology at hMOR has not been delineated. Thus, we synthesized over 50 chemical analogs based on core AH-7921 and U-47700 structures to assess for their ability to couple to Gαi signaling and induce hMOR internalization. For both the AH-7921 and U-47700 analogs, the 3,4-dichlorobenzoyl substituents were the most potent with comparable EC50 values for inhibition of cAMP accumulation; 26.49 ± 11.2 nmol L-1 and 8.8 ± 4.9 nmol L-1, respectively. Despite similar potencies for Gαi coupling, these two compounds had strikingly different hMOR internalization efficacies: U-47700 (10 µmol L-1) induced ~25% hMOR internalization similar to DAMGO while AH-7921 (10 µmol L-1) induced ~5% hMOR internalization similar to morphine. In addition, the R, R enantiomer of U-47700 is significantly more potent than the S, S enantiomer at hMOR. In conclusion, these data suggest that U-47700 and AH-7921 analogs have high analgesic potential in humans, but with divergent receptor internalization profiles, suggesting that they may exhibit differences in clinical utility or abuse potential.


Assuntos
Analgésicos Opioides/síntese química , Etilenodiaminas/síntese química , Receptores Opioides mu/metabolismo , Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Linhagem Celular , AMP Cíclico/metabolismo , Etilenodiaminas/química , Etilenodiaminas/farmacologia , Humanos , Estrutura Molecular , Receptores Opioides mu/química
5.
Nat Commun ; 10(1): 3596, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399601

RESUMO

Stem cells (SCs) receive inductive cues from the surrounding microenvironment and cells. Limited molecular evidence has connected tissue-specific mesenchymal stem cells (MSCs) with mesenchymal transit amplifying cells (MTACs). Using mouse incisor as the model, we discover a population of MSCs neibouring to the MTACs and epithelial SCs. With Notch signaling as the key regulator, we disclose molecular proof and lineage tracing evidence showing the distinct MSCs contribute to incisor MTACs and the other mesenchymal cell lineages. MTACs can feedback and regulate the homeostasis and activation of CL-MSCs through Delta-like 1 homolog (Dlk1), which balances MSCs-MTACs number and the lineage differentiation. Dlk1's function on SCs priming and self-renewal depends on its biological forms and its gene expression is under dynamic epigenetic control. Our findings can be validated in clinical samples and applied to accelerate tooth wound healing, providing an intriguing insight of how to direct SCs towards tissue regeneration.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Incisivo/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular , Linhagem da Célula , Dentina , Epigenômica , Feminino , Expressão Gênica , Homeostase , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Modelos Animais , Dente Serotino , Ratos , Ratos Wistar , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Cicatrização
6.
Nat Commun ; 10(1): 1929, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028265

RESUMO

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.


Assuntos
Células Endoteliais/patologia , Proteínas Hedgehog/genética , Prolapso da Valva Mitral/patologia , Valva Mitral/patologia , Células-Tronco Pluripotentes/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Proteínas Relacionadas a Caderinas , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Endocárdio/metabolismo , Endocárdio/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Valva Mitral/metabolismo , Prolapso da Valva Mitral/genética , Prolapso da Valva Mitral/metabolismo , Prolapso da Valva Mitral/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína Wnt3A/farmacologia
7.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30523147

RESUMO

Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly-disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases.


Assuntos
Antígeno AC133/metabolismo , Cílios/metabolismo , Incisivo/citologia , Antígeno AC133/genética , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Incisivo/metabolismo , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Transporte Proteico , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo
8.
Front Cell Neurosci ; 12: 279, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30237761

RESUMO

Peripheral endothelial cells are capable of erythrophagocytosis, but data on brain endothelial erythrophagocytosis are limited. We studied the relationship between brain endothelial erythrophagocytosis and cerebral microhemorrhage, the pathological substrate of MRI-demonstrable cerebral microbleeds. To demonstrate the erythrophagocytic capability of the brain endothelium, we studied the interactions between brain endothelial cells and red blood cells exposed to oxidative stress in vitro, and developed a new in vitro cerebral microbleeds model to study the subsequent passage of hemoglobin across the brain endothelial monolayer. Using multiple approaches, our results show marked brain endothelial erythrophagocytosis of red blood cells exposed to oxidative stress compared with control red blood cells in vitro. This brain endothelial erythrophagocytosis was accompanied by passage of hemoglobin across the brain endothelial monolayer with unaltered monolayer integrity. In vivo and confocal fluorescence microscopy studies confirmed the extravasation of RBC exposed to oxidative stress across brain endothelium. These findings, demonstrating erythrophagocytosis mediated by the brain endothelial monolayer and the subsequent passage of iron-rich hemoglobin in vitro and RBC in vivo, may have implications for elucidating mechanisms involved in the development of cerebral microbleeds that are not dependent on disruption of the microvasculature.

9.
J Physiol ; 595(17): 5797-5813, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28688178

RESUMO

KEY POINTS: Changes in gene expression that occur within hours of exposure to hypoxia in in vivo skeletal muscles remain unexplored. Two hours of hypoxia caused significant down-regulation of extracellular matrix genes followed by a shift at 6 h to altered expression of genes associated with the nuclear lumen while respiratory and blood gases were stabilized. Enrichment analysis of mRNAs classified by stability rates suggests an attenuation of post-transcriptional regulation within hours of hypoxic exposure, where PI3K-Akt signalling was suggested to have a nodal role by pathway analysis. Experimental measurements and bioinformatic analyses suggested that the dephosphorylation of Akt after 2 h of hypoxic exposure might deactivate RNA-binding protein BRF1, hence resulting in the selective degradation of mRNAs. ABSTRACT: The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia-tolerant tissues like skeletal muscles. We used RNA-seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down-regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down-regulated genes had mRNA half-lives longer than 2 h, suggesting a role for post-transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K-Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K-Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K-Akt targets, 75% could be explained by a deactivation of adenylate-uridylate-rich element (ARE)-binding protein BRF1, a target of PI3K-Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K-Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the dephosphorylation of ARE-binding protein BRF1.


Assuntos
Hipóxia/genética , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais
10.
Aging Cell ; 16(1): 82-92, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28090760

RESUMO

Aging causes cardiac dysfunction, often leading to heart failure and death. The molecular basis of age-associated changes in cardiac structure and function is largely unknown. The fruit fly, Drosophila melanogaster, is well-suited to investigate the genetics of cardiac aging. Flies age rapidly over the course of weeks, benefit from many tools to easily manipulate their genome, and their heart has significant genetic and phenotypic similarities to the human heart. Here, we performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. We also examined gene expression in single fly hearts and found significant variability as has been reported in rodents. We propose that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, our data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging.


Assuntos
Envelhecimento/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Animais , Biologia Computacional , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ontologia Genética , Genes de Insetos , Mamíferos/genética , Microfluídica , Nanotecnologia , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo , Transcrição Gênica
11.
Aging Cell ; 16(1): 93-103, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28090761

RESUMO

Heart performance declines with age. Impaired protein quality control (PQC), due to reduced ubiquitin-proteasome system (UPS) activity, autophagic function, and/or chaperone-mediated protein refolding, contributes to cardiac deterioration. The transcription factor FOXO participates in regulating genes involved in PQC, senescence, and numerous other processes. Here, a comprehensive approach, involving molecular genetics, novel assays to probe insect cardiac physiology, and bioinformatics, was utilized to investigate the influence of heart-restricted manipulation of dFOXO expression in the rapidly aging Drosophila melanogaster model. Modest dFOXO overexpression was cardioprotective, ameliorating nonpathological functional decline with age. This was accompanied by increased expression of genes associated predominantly with the UPS, relative to other PQC components, which was validated by a significant decrease in ubiquitinated proteins. RNAi knockdown of UPS candidates accordingly compromised myocardial physiology in young flies. Conversely, excessive dFOXO overexpression or suppression proved detrimental to heart function and/or organismal development. This study highlights D. melanogaster as a model of cardiac aging and FOXO as a tightly regulated mediator of proteostasis and heart performance over time.


Assuntos
Envelhecimento/genética , Envelhecimento/patologia , Proteínas de Drosophila/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Miocárdio/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Genes de Insetos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Transcrição Gênica , Ubiquitina/metabolismo
12.
Mol Ther Methods Clin Dev ; 6: 16069, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27990449

RESUMO

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

13.
J Clin Invest ; 125(8): 3256-68, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26193633

RESUMO

The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.


Assuntos
Proliferação de Células/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas com Homeodomínio LIM/metabolismo , Contração Miocárdica/fisiologia , Nó Sinoatrial/embriologia , Fatores de Transcrição/metabolismo , Animais , Anquirinas/genética , Anquirinas/metabolismo , Sobrevivência Celular , Cromatina/genética , Cromatina/metabolismo , Deleção de Genes , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Transgênicos , Ligação Proteica , Síndrome do Nó Sinusal/embriologia , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/patologia , Nó Sinoatrial/citologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética
14.
Dev Cell ; 33(5): 507-21, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-26028220

RESUMO

Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proliferação de Células , Embrião de Mamíferos/citologia , Feto/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/fisiopatologia , Miócitos Cardíacos/citologia , Proteína Supressora de Tumor p53/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Feto/metabolismo , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
15.
Mol Pharmacol ; 88(1): 181-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25737495

RESUMO

G protein-coupled receptors (GPCRs), the largest family of signaling receptors in the human genome, are also the largest class of targets of approved drugs. Are the optimal GPCRs (in terms of efficacy and safety) currently targeted therapeutically? Especially given the large number (∼ 120) of orphan GPCRs (which lack known physiologic agonists), it is likely that previously unrecognized GPCRs, especially orphan receptors, regulate cell function and can be therapeutic targets. Knowledge is limited regarding the diversity and identity of GPCRs that are activated by endogenous ligands and that native cells express. Here, we review approaches to define GPCR expression in tissues and cells and results from studies using these approaches. We identify problems with the available data and suggest future ways to identify and validate the physiologic and therapeutic roles of previously unrecognized GPCRs. We propose that a particularly useful approach to identify functionally important GPCRs with therapeutic potential will be to focus on receptors that show selective increases in expression in diseased cells from patients and experimental animals.


Assuntos
Perfilação da Expressão Gênica/métodos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Terapia de Alvo Molecular , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Distribuição Tecidual
16.
Circ Res ; 116(5): e28-39, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25605649

RESUMO

RATIONALE: Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE: To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS: Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS: Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cardiomegalia/enzimologia , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias Cardíacas/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cardiomegalia/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Dilatada/prevenção & controle , Células Cultivadas , Progressão da Doença , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Insuficiência Cardíaca/fisiopatologia , Canais Iônicos/biossíntese , Canais Iônicos/genética , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , PPAR alfa/biossíntese , PPAR alfa/genética , Mutação Puntual , Pressão , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Espécies Reativas de Oxigênio , Análise de Sequência de RNA , Sulfonamidas/farmacologia , Transfecção , Proteína Desacopladora 3
17.
Sci Rep ; 4: 6425, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25236849

RESUMO

Cells secrete and assemble extracellular matrix throughout development, giving rise to time-dependent, tissue-specific stiffness. Mimicking myocardial matrix stiffening, i.e. ~10-fold increase over 1 week, with a hydrogel system enhances myofibrillar organization of embryonic cardiomyocytes compared to static hydrogels, and thus we sought to identify specific mechanosensitive proteins involved. Expression and/or phosphorylation state of 309 unique protein kinases were examined in embryonic cardiomyocytes plated on either dynamically stiffening or static mature myocardial stiffness hydrogels. Gene ontology analysis of these kinases identified cardiogenic pathways that exhibited time-dependent up-regulation on dynamic versus static matrices, including PI3K/AKT and p38 MAPK, while GSK3ß, a known antagonist of cardiomyocyte maturation, was down-regulated. Additionally, inhibiting GSK3ß on static matrices improved spontaneous contraction and myofibril organization, while inhibiting agonist AKT on dynamic matrices reduced myofibril organization and spontaneous contraction, confirming its role in mechanically-driven maturation. Together, these data indicate that mechanically-driven maturation is at least partially achieved via active mechanosensing at focal adhesions, affecting expression and phosphorylation of a variety of protein kinases important to cardiomyogenesis.


Assuntos
Sistema de Sinalização das MAP Quinases/genética , Miócitos Cardíacos/metabolismo , Proteínas Quinases/biossíntese , Rigidez Vascular/genética , Animais , Comunicação Celular/genética , Embrião de Galinha , Regulação da Expressão Gênica , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Análise Serial de Tecidos
18.
J Clin Invest ; 124(7): 2921-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24937432

RESUMO

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.


Assuntos
Insuficiência Cardíaca/patologia , Miocárdio/patologia , Animais , Biomarcadores/metabolismo , Pressão Sanguínea , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem da Célula , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Endocárdio/metabolismo , Endocárdio/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Perfilação da Expressão Gênica , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Pericárdio/metabolismo , Pericárdio/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
BMC Bioinformatics ; 15: 69, 2014 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-24621103

RESUMO

BACKGROUND: Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. RESULTS: We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila.MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. CONCLUSIONS: MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Metanálise como Assunto , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Animais , Drosophila , Camundongos , Controle de Qualidade
20.
Procedia Comput Sci ; 29: 2162-2167, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26605000

RESUMO

Increasing numbers of genomic technologies are leading to massive amounts of genomic data, all of which requires complex analysis. More and more bioinformatics analysis tools are being developed by scientist to simplify these analyses. However, different pipelines have been developed using different software environments. This makes integrations of these diverse bioinformatics tools difficult. Kepler provides an open source environment to integrate these disparate packages. Using Kepler, we integrated several external tools including Bioconductor packages, AltAnalyze, a python-based open source tool, and R-based comparison tool to build an automated workflow to meta-analyze both online and local microarray data. The automated workflow connects the integrated tools seamlessly, delivers data flow between the tools smoothly, and hence improves efficiency and accuracy of complex data analyses. Our workflow exemplifies the usage of Kepler as a scientific workflow platform for bioinformatics pipelines.

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