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1.
Am J Physiol Heart Circ Physiol ; 314(1): H131-H137, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29212792

RESUMO

Alcohol (EtOH) consumption can variously affect cardiovascular disease. Our aim was to compare the effects of EtOH and its primary metabolite acetaldehyde (ACT) on vascular smooth muscle Notch signaling and cell growth, which are important for atherogenesis. Human coronary artery smooth muscle cells (HCASMCs) were treated with EtOH (25 mM) or ACT (10 or 25 µM). As previously reported, EtOH inhibited Notch signaling and growth of HCASMCs. In contrast, ACT treatment stimulated HCASMC proliferation (cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling, as determined by increased Notch receptor (N1 and N3) and target gene (Hairy-related transcription factor 1-3) mRNA levels. Interaction of the ligand with the Notch receptor initiates proteolytic cleavage by α- and γ-secretase, resulting in the release of the active Notch intracellular domain. Neither EtOH nor ACT had any significant effect on α-secretase activity. A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Delta-like ligand 4-stimulated γ-secretase activity in solubilized HCASMCs (similar to the effect of the control inhibitor DAPT) but no effect of ACT treatment. EtOH, but not ACT, affected the association and distribution of the γ-secretase catalytic subunit presenilin-1 with lipid rafts, as determined by dual fluorescent labeling and confocal microscopic visualization. In conclusion, ACT stimulates vascular smooth muscle cell Notch signaling and growth, effects opposite to those of EtOH. These differential actions on vascular smooth muscle cells of EtOH and its metabolite ACT may be important in mediating the ultimate effects of drinking on cardiovascular disease. NEW & NOTEWORTHY Acetaldehyde stimulates, in a Notch-dependent manner, the vascular smooth muscle cell growth that contributes to atherogenesis; effects opposite to those of ethanol. These data suggest that in addition to ethanol itself, its metabolite acetaldehyde may also mediate some of the effects of alcohol consumption on vascular cells and, thus, cardiovascular health.


Assuntos
Acetaldeído/toxicidade , Etanol/toxicidade , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Receptor Notch1/metabolismo , Receptor Notch3/metabolismo , Acetaldeído/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Etanol/metabolismo , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Presenilina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Alcohol Clin Exp Res ; 39(11): 2115-22, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26443551

RESUMO

BACKGROUND: Ethanol (EtOH) inhibits Notch-mediated vascular smooth muscle cell (SMC) proliferation, an event that is key in vessel remodeling and atherogenesis. The object of this study was to determine whether EtOH inhibits Notch signaling in SMC at the level of γ-secretase, a protease that in concert with α-secretase catalyzes the release of the intracellular domain of the Notch receptor necessary for signaling. METHODS: Human coronary artery SMCs (HCASMCs) were treated with a recombinant soluble Notch ligand, Delta-like ligand 4 (DLL4) (2 µg/ml), or transfected with a constitutively active Notch 1 intracellular domain (N1ICD), in the absence or presence of EtOH. EtOH (25 mM) treatment inhibited DLL4-stimulated CBF-1/RBP-Jk-dependent promoter activity (determined by luciferase assay) and downstream target gene HRT-3 mRNA levels. In contrast, EtOH had no effect on N1ICD-driven CBF-1/RBP-Jk-dependent promoter activity or HRT-3 expression. RESULTS: These data suggest that EtOH inhibits Notch signaling at, or prior to, Notch intracellular domain (NICD) generation. γ-Secretase activity was determined in solubilized membrane preparations from HCASMC treated with/without EtOH (25 mM) or the γ-secretase inhibitor DAPT (20 µM) using (i) a fluorometric assay and (ii) Western blot detection of cleavage products using a Flag-tagged Notch-based substrate, N100Flag. EtOH inhibited basal and DLL4-stimulated γ-secretase activity, and SMC growth to a similar extent as DAPT, whereas it had no effect on α-secretase (TACE/ADAM17) activity also determined by fluorometric assay. Moreover, EtOH treatment inhibited the expression of caveolin-1, a lipid raft protein implicated in regulating γ-secretase activity, and altered its cellular distribution in HCASMC. CONCLUSIONS: EtOH inhibits Notch signaling in vascular SMCs at the level of γ-secretase activity, possibly by affecting lipid raft function. Such a response might be expected to result in attenuation of pathologic vessel remodeling and thus may contribute to moderate alcohols' cardioprotective effects.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Etanol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Proteólise/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
3.
J Vasc Res ; 51(4): 315-24, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25322777

RESUMO

UNLABELLED: We previously reported that ethanol (EtOH) stimulates endothelial angiogenic activity mediated via a notch- and angiopoietin-1 (Ang-1) pathway. As crosstalk exists between notch and vascular endothelial growth factor (VEGF) signaling, we examined whether the VEGF receptor (VEGFR) Flk-1 (fetal liver kinase 1) mediates EtOH-stimulated notch signaling and angiogenic activity. METHODS AND RESULTS: Treatment of human coronary artery endothelial cells (HCAECs) with EtOH (1-50 mM, 24 h) dose-dependently increased Flk-1 expression with a maximum increase observed at 25 mM EtOH. Ethanol treatment activated both Flk-1 and Flt-1 (FMS-like tyrosine kinase 1) as indicated by their phosphorylation, and subsequent stimulation of Akt. EtOH activation of Flk-1 was inhibited by the VEGFR inhibitor SU5416. Gene silencing of Flk-1 using small interfering RNA inhibited the EtOH-induced increase in notch receptors 1 and 4 and notch target gene (hairy enhancer of split-related transcription factor 1) mRNA. Knockdown of Flk-1 inhibited EtOH-induced Ang-1/Tie-2 mRNA expression and blocked EtOH-induced HCAEC network formation on Matrigel, a response that was restored by notch ligand, notch ligand delta-like ligand 4, treatment. In vivo, moderate alcohol feeding increased vascular remodeling in mouse ischemic hindlimbs. CONCLUSIONS: These data demonstrate that EtOH activates Flk-1 and Flt-1 receptors in HCAECs and promotes angiogenic activity via an Flk-1/notch pathway. These effects of EtOH may be relevant to the influence of moderate alcohol consumption on cardiovascular health.


Assuntos
Células Endoteliais/efeitos dos fármacos , Etanol/farmacologia , Isquemia/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Depressores do Sistema Nervoso Central/farmacologia , Vasos Coronários/citologia , Células Endoteliais/citologia , Artéria Femoral , Membro Posterior/irrigação sanguínea , Humanos , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , RNA Interferente Pequeno/genética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
4.
Arterioscler Thromb Vasc Biol ; 33(8): 1960-4, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23766265

RESUMO

OBJECTIVE: To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. APPROACH AND RESULTS: In vitro, human coronary arterial smooth muscle cells were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression while concomitantly decreasing human coronary arterial smooth muscle cell growth to that similar under normal flow conditions. In vivo, ligation injury-induced low flow increased vascular smooth muscle cell growth and vascular remodeling, while increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow-induced increases in Ptc-1/Notch expression and markedly reduced the subsequent vascular remodeling. CONCLUSIONS: These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.


Assuntos
Vasos Coronários/lesões , Vasos Coronários/fisiologia , Neointima/fisiopatologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Adulto , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Células Cultivadas , Circulação Coronária/fisiologia , Vasos Coronários/citologia , Humanos , Hiperplasia , Técnicas In Vitro , Músculo Liso Vascular/citologia , Músculo Liso Vascular/lesões , Músculo Liso Vascular/fisiologia , Neointima/metabolismo , Neointima/patologia , Receptores Patched , Receptor Patched-1 , Fluxo Pulsátil/fisiologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores Notch/genética , Receptores Notch/metabolismo
5.
PLoS One ; 9(1): e84122, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24416200

RESUMO

OBJECTIVES: To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling. METHODS AND RESULTS: Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown. CONCLUSION: These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.


Assuntos
Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Receptor Notch1/genética , Receptor Notch1/metabolismo , Animais , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Proliferação de Células , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
6.
Dev Dyn ; 238(2): 358-66, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18773497

RESUMO

Fibroblast growth factors play important roles in inner ear development. Previous studies showed that mouse Fgf16 is expressed asymmetrically during the otic cup and vesicle stages of development, suggesting roles in regulating or responding to anteroposterior axial cues. Here, we studied otic Fgf16 expression throughout embryonic development and found transcripts in the developing cristae and in a few cells in the lateral wall of the cochlear duct. To determine the otic function of Fgf16 and to follow the fate of Fgf16-expressing cells, we generated an Fgf16(IRESCre) allele. We show that Fgf16 does not have a unique role in inner ear development and that the Fgf16 lineage is found throughout the three cristae, in portions of the semicircular canal ducts, and in the cochlear spiral prominence epithelial cells. This strain will be useful for gene ablations in these tissues.


Assuntos
Orelha Interna/embriologia , Fatores de Crescimento de Fibroblastos/biossíntese , Animais , Padronização Corporal , Linhagem da Célula/fisiologia , Ducto Coclear/embriologia , Ducto Coclear/metabolismo , Orelha Interna/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Camundongos , Camundongos Mutantes , Canais Semicirculares/embriologia , Canais Semicirculares/metabolismo
7.
Development ; 134(20): 3615-25, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17855431

RESUMO

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. We show in mouse that Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. We provide new morphologic data on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, we show that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems.


Assuntos
Padronização Corporal , Orelha Interna , Epitélio , Fator 3 de Crescimento de Fibroblastos/metabolismo , Morfogênese , Animais , Biomarcadores/metabolismo , Orelha Interna/anormalidades , Orelha Interna/citologia , Orelha Interna/embriologia , Orelha Interna/metabolismo , Epitélio/anatomia & histologia , Epitélio/embriologia , Epitélio/metabolismo , Fator 3 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A
8.
Development ; 134(1): 167-76, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17164422

RESUMO

Mitogen-activated protein kinase (MAPK) pathways are major mediators of extracellular signals that are transduced to the nucleus. MAPK signaling is attenuated at several levels, and one class of dual-specificity phosphatases, the MAPK phosphatases (MKPs), inhibit MAPK signaling by dephosphorylating activated MAPKs. Several of the MKPs are themselves induced by the signaling pathways they regulate, forming negative feedback loops that attenuate the signals. We show here that in mouse embryos, Fibroblast growth factor receptors (FGFRs) are required for transcription of Dusp6, which encodes MKP3, an extracellular signal-regulated kinase (ERK)-specific MKP. Targeted inactivation of Dusp6 increases levels of phosphorylated ERK, as well as the pERK target, Erm, and transcripts initiated from the Dusp6 promoter itself. Finally, the Dusp6 mutant allele causes variably penetrant, dominant postnatal lethality, skeletal dwarfism, coronal craniosynostosis and hearing loss; phenotypes that are also characteristic of mutations that activate FGFRs inappropriately. Taken together, these results show that DUSP6 serves in vivo as a negative feedback regulator of FGFR signaling and suggest that mutations in DUSP6 or related genes are candidates for causing or modifying unexplained cases of FGFR-like syndromes.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Alelos , Animais , Linhagem Celular , Fosfatase 6 de Especificidade Dupla , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Feminino , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Gravidez , Proteínas Tirosina Fosfatases/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transcrição Gênica
9.
Dev Dyn ; 228(2): 267-72, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14517998

RESUMO

The inner ear, which mediates hearing and equilibrium, develops from an ectodermal placode located adjacent to the developing hindbrain. Induction of the placode and its subsequent morphogenesis and differentiation into the inner ear epithelium and its sensory neurons, involves signalling interactions within and between otic and non-otic tissues. Several members of the fibroblast growth factor (FGF) family play important roles at various stages of otic development; however, there are additional family members that have not been evaluated. In this study, we surveyed the expression patterns of 18 mouse Fgf and 3 Fgf receptor (Fgfr) genes during early otic development. Two members of the Fgf family, Fgf4 and Fgf16, and all three tested members of the Fgfr family, Fgfr2c, Fgfr3c, and Fgfr4, were expressed in tissues relevant to inner ear development. Fgf4 transcripts were expressed in the preplacodal and placodal ectoderm, suggesting potential roles in placode induction and/or maintenance. Fgf16 was expressed in the posterior otic cup and vesicle, suggesting roles in otic cell fate decisions and/or axis formation.


Assuntos
Orelha Interna/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Orelha/embriologia , Ectoderma/metabolismo , Fatores de Crescimento de Fibroblastos/classificação , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/classificação , Receptores de Fatores de Crescimento de Fibroblastos/genética
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