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3.
PLoS One ; 14(8): e0221277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31433825

RESUMO

BACKGROUND: Impaired cardiac vagal function, quantified preoperatively as slower heart rate recovery (HRR) after exercise, is independently associated with perioperative myocardial injury. Parasympathetic (vagal) dysfunction may also promote (extra-cardiac) multi-organ dysfunction, although perioperative data are lacking. Assuming that cardiac vagal activity, and therefore heart rate recovery response, is a marker of brainstem parasympathetic dysfunction, we hypothesized that impaired HRR would be associated with a higher incidence of morbidity after noncardiac surgery. METHODS: In two prospective, blinded, observational cohort studies, we established the definition of impaired vagal function in terms of the HRR threshold that is associated with perioperative myocardial injury (HRR ≤ 12 beats min-1 (bpm), 60 seconds after cessation of cardiopulmonary exercise testing. The primary outcome of this secondary analysis was all-cause morbidity three and five days after surgery, defined using the Post-Operative Morbidity Survey. Secondary outcomes of this analysis were type of morbidity and time to become morbidity-free. Logistic regression and Cox regression tested for the association between HRR and morbidity. Results are presented as odds/hazard ratios [OR or HR; (95% confidence intervals). RESULTS: 882/1941 (45.4%) patients had HRR≤12bpm. All-cause morbidity within 5 days of surgery was more common in 585/822 (71.2%) patients with HRR≤12bpm, compared to 718/1119 (64.2%) patients with HRR>12bpm (OR:1.38 (1.14-1.67); p = 0.001). HRR≤12bpm was associated with more frequent episodes of pulmonary (OR:1.31 (1.05-1.62);p = 0.02)), infective (OR:1.38 (1.10-1.72); p = 0.006), renal (OR:1.91 (1.30-2.79); p = 0.02)), cardiovascular (OR:1.39 (1.15-1.69); p<0.001)), neurological (OR:1.73 (1.11-2.70); p = 0.02)) and pain morbidity (OR:1.38 (1.14-1.68); p = 0.001) within 5 days of surgery. CONCLUSIONS: Multi-organ dysfunction is more common in surgical patients with cardiac vagal dysfunction, defined as HRR ≤ 12 bpm after preoperative cardiopulmonary exercise testing. CLINICAL TRIAL REGISTRY: ISRCTN88456378.

4.
J Muscle Res Cell Motil ; 40(2): 151-157, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31222588

RESUMO

Tissue engineering is a complex field where the elements of biology and engineering are combined in an attempt to recapitulate the native environment of the body. Tissue engineering has shown one thing categorically; that the human body is extremely complex and it is truly a difficult task to generate this in the lab. There have been varied attempts at trying to generate a model for the heart with numerous cell types and different scaffolds or materials. The common underlying theme in these approaches is to combine together matrix material and different cell types to make something similar to heart tissue. Multi-cellularity is an essential aspect of the heart and therefore critical to any approach which would try to mimic such a complex tissue. The heart is made up of many cell types that combine to form complex structures like: deformable chambers, a tri-layered heart muscle, and vessels. Thus, in this review we will summarise how tissue engineering has progressed in modelling the heart and what gaps still exist in this dynamic field.

5.
J Med Chem ; 62(9): 4731-4741, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-30964292

RESUMO

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

6.
Stem Cell Reports ; 11(5): 1226-1243, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30392975

RESUMO

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.


Assuntos
Actinas/genética , Cardiomiopatia Hipertrófica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Miócitos Cardíacos/patologia , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Sistemas CRISPR-Cas/genética , Cálcio/metabolismo , Sinalização do Cálcio , Cardiomiopatia Hipertrófica/fisiopatologia , Edição de Genes , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Engenharia Tecidual
7.
Genome Biol Evol ; 10(9): 2151-2167, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29986009

RESUMO

The diversity of mechanisms and capacity for regeneration across the Metazoa present an intriguing challenge in evolutionary biology, impacting on the burgeoning field of regenerative medicine. Broad taxonomic sampling is essential to improve our understanding of regeneration, and studies outside of the traditional model organisms have proved extremely informative. Within the historically understudied Spiralia, the Annelida have an impressive variety of tractable regenerative systems. The biomeralizing, blastema-less regeneration of the head appendage (operculum) of the serpulid polychaete keelworm Spirobranchus (formerly Pomatoceros) lamarcki is one such system. To profile potential regulatory mechanisms, we classified the homeobox gene content of opercular regeneration transcriptomes. As a result of retrieving several difficult-to-classify homeobox sequences, we performed an extensive search and phylogenetic analysis of the TALE and PRD-class homeobox gene content of a broad selection of lophotrochozoan genomes. These analyses contribute to our increasing understanding of the diversity, taxonomic extent, rapid evolution, and radical flexibility of these recently discovered homeobox gene radiations. Our expansion and integration of previous nomenclature systems helps to clarify their cryptic orthology. We also describe an unusual divergent S. lamarcki Antp gene, a previously unclassified lophotrochozoan orphan gene family (Lopx), and a number of novel Nk class orphan genes. The expression and potential involvement of many of these lineage- and clade-restricted homeobox genes in S. lamarcki operculum regeneration provides an example of diversity in regenerative mechanisms, as well as significantly improving our understanding of homeobox gene evolution.


Assuntos
Genes Homeobox , Poliquetos/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Anelídeos/genética , Evolução Molecular , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Filogenia , Alinhamento de Sequência
8.
Acta Diabetol ; 55(10): 1029-1036, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29931424

RESUMO

AIMS: Prior exposure to insulin-induced hypoglycemia was shown to increase glial acetate metabolism (GAM) during subsequent exposure to hypoglycemia in diabetic individuals. However, it remained unclear whether this effect was dependent on the disease state or the antecedent cause of hypoglycemia. We aimed to establish whether exposure to fasting-induced hypoglycemia was sufficient to produce alterations in GAM in non-diabetic individuals. METHODS: GAM was measured via carbon-13 magnetic resonance spectroscopy during infusion of [1-13C] acetate before and after a 72-h fast in six metabolically healthy men. All participants were male, aged 18-40 years, with a Body Mass Index of 20.0-27.9 kg/m2, who consented to reside at Pennington Biomedical Research Center for 4 days. The main outcome measure was the percent enhancement of cerebral [1-13C] bicarbonate (the primary metabolic byproduct of glial oxidation of [1-13C] acetate). Continuous glucose monitoring was used to measure hypoglycemic episodes during the 72-h fast. RESULTS: As expected, 72 h of fasting significantly reduced blood glucose levels and resulted in a high frequency of hypoglycemic episodes. Steady-state GAM increased from 53.5 ± 3.7 to 61.9 ± 1.7% following the 72-h fast (p = 0.005). This increase correlated with greater duration of hypoglycemia experienced during the fast (r = 0.967). In addition, subjects with greater GAM at baseline experienced a greater increase in the duration of hypoglycemia experienced during the 72-h fast (r = 0.979). CONCLUSIONS: GAM has potential as a biomarker for susceptibility to hypoglycemic episodes. TRAIL REGISTRATION: Clinicaltrials.gov ID: NCT02690168.


Assuntos
Acetatos/metabolismo , Hipoglicemia/metabolismo , Neuroglia/metabolismo , Adolescente , Adulto , Glicemia/metabolismo , Automonitorização da Glicemia , Suscetibilidade a Doenças , Jejum/metabolismo , Feminino , Humanos , Hipoglicemia/fisiopatologia , Insulina/metabolismo , Masculino , Adulto Jovem
9.
Sci Rep ; 8(1): 9414, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925900

RESUMO

The Pax3/7 transcription factor family is integral to developmental gene networks contributing to important innovations in vertebrate evolution, including the neural crest. The basal chordate lineage of amphioxus is ideally placed to understand the dynamics of the gene regulatory network evolution that produced these novelties. We report here the discovery that the cephalochordate lineage possesses two Pax3/7 genes, Pax3/7a and Pax3/7b. The tandem duplication is ancestral to all extant amphioxus, occurring in both Asymmetron and Branchiostoma, but originated after the split from the lineage leading to vertebrates. The two paralogues are differentially expressed during embryonic development, particularly in neural and somitic tissues, suggesting distinct regulation. Our results have implications for the study of amphioxus regeneration, neural plate and crest evolution, and differential tandem paralogue evolution.


Assuntos
Cefalocordados/embriologia , Cefalocordados/metabolismo , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX7/metabolismo , Animais , Teorema de Bayes , Evolução Molecular , Éxons/genética , Duplicação Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Crista Neural/embriologia , Crista Neural/metabolismo , Placa Neural/embriologia , Placa Neural/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX7/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vertebrados/embriologia , Vertebrados/metabolismo
10.
Brain Behav Immun ; 67: 47-53, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28807718

RESUMO

OBJECTIVE: Experimental animal models demonstrate that autonomic activity regulates systemic inflammation. By contrast, human studies are limited in number and exclusively use heart rate variability (HRV) as an index of cardiac autonomic regulation. HRV measures are primarily dependent on, and need to be corrected for, heart rate. Thus, independent autonomic measures are required to confirm HRV-based findings. Here, the authors sought to replicate the findings of preceding HRV-based studies by using HRV-independent, exercise-evoked sympathetic and parasympathetic measures of cardiac autonomic regulation to examine the relationship between autonomic function and systemic inflammation. METHODS: Sympathetic function was assessed by measuring heart rate changes during unloaded pedaling prior to onset of exercise, divided into quartiles; an anticipatory heart rate (AHRR) rise during this period is evoked by mental stress in many individuals. Parasympathetic function was assessed by heart rate recovery (HRR) 60s after finishing cardiopulmonary exercise testing, divided into quartiles. Parasympathetic dysfunction was defined by delayed heart rate recovery (HRR) ≤12.beats.min-1, a threshold value associated with higher cardiovascular morbidity/mortality in the general population. Systemic inflammation was primarily assessed by neutrophil-lymphocyte ratio (NLR), where a ratio >4 is prognostic across several inflammatory diseases and correlates strongly with elevated plasma levels of pro-inflammatory cytokines. High-sensitivity C-reactive protein (hsCRP) was also measured. RESULTS: In 1624 subjects (65±14y; 67.9% male), lower HRR (impaired vagal activity) was associated with progressively higher NLR (p=0.004 for trend across quartiles). Delayed HRR, recorded in 646/1624 (39.6%) subjects, was associated with neutrophil-lymphocyte ratio >4 (relative risk: 1.43 (95%CI: 1.18-1.74); P=0.0003). Similar results were found for hsCRP (p=0.045). By contrast, AHRR was not associated with NLR (relative risk: 1.24 (95%CI: 0.94-1.65); P=0.14). CONCLUSIONS: Delayed HRR, a robust measure of parasympathetic dysfunction, is independently associated with leukocyte ratios indicative of systemic inflammation. These results further support a role for parasympathetic modulation of systemic inflammation in humans.


Assuntos
Inflamação/fisiopatologia , Sistema Nervoso Parassimpático/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Idoso , Estudos de Coortes , Teste de Esforço , Feminino , Frequência Cardíaca , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
11.
Am J Physiol Heart Circ Physiol ; 313(6): H1213-H1226, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28887330

RESUMO

Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin ( ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca2+ sensitivity and sudden death in a proportion (up to 40%) of young (28-40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28-40 days old) and when they were adult (8-12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca2+ transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca2+ waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca2+ content, a proportion of these cells had larger Ca2+ spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca2+ background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca2+ homeostasis. NEW & NOTEWORTHY Mice with the actin Glu99Lys hypertrophic cardiomyopathy mutation ( ACTC E99K) are prone to sudden cardiac death around 40 days, associated with increased Ca2+ transients, spark mass, and fibrosis. However, adult survivors have normal Ca2+ transients and spark density accompanied by hypertrophy. Penetrance of the sudden cardiac death phenotype depends on the genetic background of the mouse. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/calcium-regulation-in-e99k-mouse-heart/ .


Assuntos
Sinalização do Cálcio , Cardiomiopatia Hipertrófica/metabolismo , Morte Súbita Cardíaca , Patrimônio Genético , Actinas/genética , Fatores Etários , Animais , Cardiomiopatia Hipertrófica/genética , Células Cultivadas , Colágeno/metabolismo , Coração/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mutação de Sentido Incorreto , Contração Miocárdica , Miócitos Cardíacos/metabolismo
12.
J Cell Physiol ; 231(1): 72-83, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25899717

RESUMO

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-ß1 and TGF-ß receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.


Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Animais , Densidade Óssea/fisiologia , Remodelação Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular/genética , Proteínas do Olho/genética , Glicoproteínas de Membrana/genética , Camundongos Transgênicos , Osteogênese/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
13.
Sci Rep ; 5: 18050, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26657400

RESUMO

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.


Assuntos
Microscopia de Fluorescência/métodos , Desenho de Equipamento/métodos , Imagem Tridimensional/métodos , Luz , Iluminação/métodos , Agulhas , Dispositivos Ópticos , Fibras Ópticas
14.
PLoS One ; 8(5): e64903, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23741415

RESUMO

Mechanosensory hair cells (HCs) and surrounding supporting cells (SCs) in the mouse cochlea are important for hearing and are derived from the same prosensory progenitors. Notch1 signaling plays dual but contrasting and age-dependent roles in mouse cochlear development: early lateral induction and subsequent lateral inhibition. However, it has been difficult to directly visualize mouse cochlear cells experiencing various levels of Notch1 activity at single cell resolution. Here, we characterized two knock-in mouse lines, Notch1(Cre (Low)/+) and Notch1(Cre (High)/+) , with different Cre recombinase activities, that can detect Notch1 receptor proteolysis or Notch1 activity at high and low thresholds, respectively. Using both lines together with a highly sensitive Cre reporter line, we showed that Notch1 activity is nearly undetectable during lateral induction but increases to medium and high levels during lateral inhibition. Furthermore, we found that within the neonatal organ of Corti, the vast majority of cells that experience Notch1 activity were SCs not HCs, suggesting that HCs kept undetectable Notch1 activity during the entire lineage development. Furthermore, among SC subtypes, ∼85-99% of Deiters' and outer pillar cells but only ∼19-38% of inner pillar cells experience medium and high levels of Notch1 activity. Our results demonstrate that Notch1 activity is highly heterogeneous: 1) between lateral induction and inhibition; 2) between HC and SC lineages; 3) among different SC subtypes; 4) among different cells within each SC subtype. Such heterogeneity should elucidate how the development of the cochclear sensory epithelium is precisely controlled and how HC regeneration can be best achieved in postnatal cochleae.


Assuntos
Cóclea/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Alelos , Animais , Cóclea/embriologia , Expressão Gênica , Genes Reporter , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Camundongos , Camundongos Transgênicos , Proteólise , Receptor Notch1/genética
15.
PLoS One ; 8(1): e52345, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23326329

RESUMO

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.


Assuntos
Espinhas Dendríticas/efeitos dos fármacos , Glicina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de AMPA/fisiologia , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/fisiologia , Proteína 4 Homóloga a Disks-Large , Endopeptidases/genética , Endopeptidases/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Immunoblotting , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
16.
J Neurosci ; 32(31): 10530-40, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22855803

RESUMO

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27(Kip1), a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27(Kip1), p27(Kip1)-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27(Kip1) gradually declined with age. In addition, deletion of Sox2 or p27(Kip1) did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27(Kip1) promoter support that Sox2 directly activates p27(Kip1) transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27(Kip1) to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27(Kip1) expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.


Assuntos
Cóclea/citologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p27/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOXB1/genética , Tamoxifeno/farmacologia , Transfecção , Proteínas Supressoras de Tumor/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-22514534

RESUMO

We examined in vivo evidence of axonal degeneration in association with neuronal pathology in Alzheimer's disease (AD) through analysis of fornix microstructural integrity and measures of hippocampal subfield atrophy. Based on known anatomical topography, we hypothesized that the local thickness of subiculum and CA1 hippocampus fields would be associated with fornix integrity, reflecting an association between AD-related injury to hippocampal neurons and degeneration of associated axon fibers. To test this hypothesis, multi-modal imaging, combining measures of local hippocampal radii with diffusion tensor imaging (DTI), was applied to 44 individuals clinically diagnosed with AD, 44 individuals clinically diagnosed with mild cognitive impairment (MCI), and 96 cognitively normal individuals. Fornix microstructural degradation, as measured by reduced DTI-based fractional anisotropy (FA), was prominent in both MCI and AD, and was associated with reduced hippocampal volumes. Further, reduced fornix FA was associated with reduced anterior CA1 and antero-medial subiculum thickness. Finally, while both lesser fornix FA and lesser hippocampal volume were associated with lesser episodic memory, only the hippocampal measures were significant predictors of episodic memory in models including both hippocampal and fornix predictors. The region-specific association between fornix integrity and hippocampal neuronal death may provide in vivo evidence for degenerative white matter injury in AD: axonal pathology that is closely linked to neuronal injury.

18.
PLoS One ; 7(3): e34123, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22448289

RESUMO

BACKGROUND: During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling. METHODOLOGY/PRINCIPAL FINDINGS: We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG(CreER+); Rosa26-NICD(loxp/+) mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICD-negative). CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additional pathways that could synergistically interact with Notch1 pathway to produce HCs at postnatal ages.


Assuntos
Cóclea/patologia , Orelha Interna/citologia , Células Ciliadas Auditivas/citologia , Receptores Notch/fisiologia , Regeneração/fisiologia , Transdução de Sinais , Fatores Etários , Animais , Animais Recém-Nascidos , Comunicação Celular , Cóclea/metabolismo , Orelha Interna/embriologia , Orelha Interna/metabolismo , Feminino , Imunofluorescência , Células Ciliadas Auditivas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas/fisiologia , RNA não Traduzido
19.
Dev Dyn ; 241(4): 684-96, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22354878

RESUMO

BACKGROUND: Notch signaling is active in mouse cochlear prosensory progenitors but declines in differentiating sensory hair cells (HCs). Overactivation of the Notch1 intracellular domain (NICD) in progenitors blocks HC fate commitment and/or differentiation. However, it is not known whether reactivation of NICD in differentiating HCs also interrupts their developmental program and reactivates its downstream targets. RESULTS: By analyzing Atoh1(CreER+) ; Rosa26-NICD(loxp/+) or Atoh1(CreER+) ; Rosa26-NICD(loxp/+) ; RBP-J(loxp/loxp) mice, we demonstrated that ectopic NICD in differentiating HCs caused reactivation of Sox2 and Prox1 in an RBP-J-dependent manner. Interestingly, Prox1 reactivation was exclusive to outer HCs (OHCs). In addition, lineage tracing analysis of Prox1(CreER/+) ; Rosa26-EYFP(loxp/+) and Prox1(CreEGFP/+) ; Rosa26-EYFP(loxp/+) mice showed that nearly all HCs experiencing Prox1 expression were OHCs. Surprisingly, these HCs still matured normally with expression of prestin, wild-type-like morphology, and uptake of FM4-64FX dye at adult ages. CONCLUSIONS: Our results suggest that the developmental program of cochlear differentiating HCs is refractory to Notch reactivation and that Notch is an upstream regulator of Sox2 and Prox1 in cochlear development. In addition, our results support that Sox2 and Prox1 should not be the main blockers for terminal differentiation of HCs newly regenerated from postnatal cochlear SCs that still maintain Sox2 and Prox1 expression.


Assuntos
Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/fisiologia , Receptores Notch/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula/fisiologia , Células Ciliadas Auditivas/fisiologia , Camundongos , Transdução de Sinais
20.
Crit Rev Eukaryot Gene Expr ; 21(1): 43-69, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21967332

RESUMO

Connective tissue growth factor (CTGF) is a 38 kDa, cysteine rich, extracellular matrix protein composed of 4 domains or modules. CTGF has been shown to regulate a diverse array of cellular functions and has been implicated in more complex biological processes such as angiogenesis, chondrogenesis, and osteogenesis. A role for CTGF in the development and maintenance of skeletal tissues first came to light in studies demonstrating its expression in cartilage and bone cells, which was dramatically increased during skeletal repair or regeneration. The physiological significance of CTGF in skeletogenesis was confirmed in CTGF-null mice, which exhibited multiple skeletal dysmorphisms as a result of impaired growth plate chondrogenesis, angiogenesis, and bone formation/mineralization. Given the emerging importance of CTGF in osteogenesis and chondrogenesis, this review will focus on its expression in skeletal tissues, its effects on osteoblast and chondrocyte differentiation and function, and the skeletal implications of ablation or over-expression of CTGF in knockout or transgenic mouse models, respectively. In addition, this review will examine the role of integrin-mediated signaling and the regulation of CTGF expression as it relates to skeletogenesis. We will emphasize CTGF studies in bone or bone cells, and will identify opportunities for future investigations concerning CTGF and chondrogenesis/osteogenesis.


Assuntos
Desenvolvimento Ósseo/fisiologia , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Peptídeos/metabolismo , Animais , Desenvolvimento Ósseo/genética , Condrogênese/genética , Condrogênese/fisiologia , Fator de Crescimento do Tecido Conjuntivo/genética , Eptifibatida , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Osteogênese/genética , Osteogênese/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
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