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1.
J Neurosci ; 32(19): 6600-10, 2012 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-22573682

RESUMO

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Coristoma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Núcleo Vestibular Lateral/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Núcleo Vestibular Lateral/citologia , Núcleo Vestibular Lateral/crescimento & desenvolvimento
2.
Sci Rep ; 4: 6885, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25363458

RESUMO

Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doxiciclina/farmacologia , Células Ciliadas Auditivas Internas/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Engenharia Genética , Humanos , Camundongos Transgênicos , Especificidade de Órgãos
3.
Am J Physiol Regul Integr Comp Physiol ; 296(3): R715-21, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19118096

RESUMO

In obesity, skeletal muscle blood flow during exercise (functional hyperemia) is impaired. We have indirectly demonstrated that an altered arachidonic acid metabolism is responsible for the impaired functional vasodilation in the obese Zucker rat (OZR), a model of obesity. In this study, we tested the hypothesis that there is an impaired release of PGI(2) due to a nitration of PGI(2) synthase (PGIS), which is associated with a decreased prostanoid receptor expression. PGI(2), PGE(2), and thromboxane A(2) (TXA(2)) release were determined in vitro using ELISA under basal conditions and in response to arachidonic acid (AA) administration (50 microM). Immunofluorescence of PGI(2) and TXA(2) receptors (IP and TP, respectively) was determined in dispersed vascular smooth muscle cells (VSMCs). Nitration of tyrosine residues of the PGIS enzyme was determined using immunoprecipitation and Western blot analysis. Following AA administration, PGI(2) and PGE(2) release were attenuated in OZR compared with lean Zucker rats (LZR; controls). Basal and AA-induced TXA(2) release were not significantly different between groups. IP and TP immunofluorescence were not significantly different between OZR and LZR groups. OZR exhibited elevated nitration of tyrosine residues of PGIS compared with LZR. These results suggest that alterations in the PGI(2) pathway (attenuated PGI(2) synthesis), and not the TXA(2) pathway (normal TXA(2) synthesis/no change in TP receptor expression), underlie the attenuated functional hyperemia in the OZR.


Assuntos
Epoprostenol/biossíntese , Obesidade/metabolismo , Animais , Ácido Araquidônico/metabolismo , Western Blotting , Separação Celular , Imunofluorescência , Técnicas In Vitro , Masculino , Microcirculação/fisiologia , Miócitos de Músculo Liso/metabolismo , Nitratos/metabolismo , Condicionamento Físico Animal/fisiologia , Ratos , Ratos Zucker , Receptores de Epoprostenol/biossíntese , Receptores de Tromboxano A2 e Prostaglandina H2/biossíntese , Tirosina/metabolismo
4.
Microcirculation ; 15(6): 485-94, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19086258

RESUMO

OBJECTIVE: Skeletal muscle blood flow during exercise is impaired in obesity. We tested the hypothesis that the attenuated vasodilation in skeletal muscle arterioles of obese Zucker rats (OZR) is due to altered K(ATP) channel-mediated vasodilation. MATERIALS AND METHODS: K(ATP) channel function was determined in isolated skeletal muscle arterioles in response to the K(ATP) opener cromakalim (0.1-10 microM) during normal myogenic tone and alpha-adrenergic-mediated tone (0.1 microM phenylephrine). The spinotrapezius muscle was prepared and the vasodilatory responses to muscle stimulation or iloprost (0.028-2.8 microM) were observed before and after the application of the K(ATP) inhibitor, glibenclamide (10 microM). Channel subunit expression was determined by using western blot analyses. RESULTS: Cromakalim concentration-response curves were shifted in OZR as compared to lean controls. OZR exhibited impaired functional and iloprost-induced vasodilation as compared to the lean controls. Glibenclamide inhibited the functional and iloprost-induced dilation in the lean rats with no effects in the obese a nimals. Channel subunit expression was similar in femoral arteries. CONCLUSION: The impaired functional vasodilation in the OZR is associated with altered K(ATP) channel sensitivity.


Assuntos
Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Canais de Potássio/biossíntese , Vasodilatação , Animais , Arteríolas/metabolismo , Arteríolas/fisiopatologia , Cromakalim/farmacologia , Relação Dose-Resposta a Droga , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Iloprosta/farmacologia , Masculino , Obesidade/fisiopatologia , Fenilefrina/farmacologia , Ratos , Ratos Zucker , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
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