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1.
Sci Rep ; 11(1): 10962, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040115

RESUMO

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.


Assuntos
Proteínas do Olho/fisiologia , Neurogênese/fisiologia , Neuropeptídeos/fisiologia , Receptores de Somatostatina/fisiologia , Retina/citologia , Animais , Relação Dose-Resposta a Droga , Proteínas do Olho/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Idade Gestacional , Humanos , Ligantes , Camundongos , Camundongos Knockout , Neuropeptídeos/agonistas , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/farmacologia , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de Somatostatina/deficiência , Receptores de Somatostatina/efeitos dos fármacos , Retina/embriologia , Transdução de Sinais/fisiologia , Análise de Célula Única
2.
Aging (Albany NY) ; 13(1): 460-476, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33291074

RESUMO

Drugs or compounds have been shown to promote longevity in various approaches. We used Drosophila to explore novel natural compounds can be applied to anti-aging. Here we reported that a flavonoid named Dihydromyricetin can increase stress that tolerance and lipid levels, slow down gut dysfunction and extend Drosophila lifespan. Dihydromyricetin can also lessen pERK and pAKT signaling, consequently activating FOXO and AOP to modulate longevity. Our results suggested that DHM could be used as an effective compound for anti-aging intervention, which could likely be applied to both mammals and humans.


Assuntos
Envelhecimento/efeitos dos fármacos , Proteínas de Drosophila/efeitos dos fármacos , Proteínas do Olho/efeitos dos fármacos , Flavonóis/farmacologia , Fatores de Transcrição Forkhead/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Proteínas Repressoras/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas do Olho/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Repressoras/metabolismo
3.
Neurotox Res ; 38(2): 487-497, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32495312

RESUMO

An increased number of patients with residual hearing are undergoing cochlear implantation. A subset of these experience delayed hearing loss post-implantation, and the aetiology of this loss is not well understood. Our previous studies suggest that electrical stimulation can induce damage to hair cells in organ of Corti (OC) organotypic cultures. Dexamethasone has the potential to protect residual hearing due to its multiple effects on cells and tissue (e.g., anti-inflammatory, free radical scavenger). We therefore hypothesized that dexamethasone treatment could prevent electrical stimulation induced changes in the OC. Organ of Corti explants from neonatal rats (P2-4) were cultured for 24 h with two different concentrations of dexamethasone. Thereafter, OC were subjected to a charge-balanced biphasic pulsed electrical stimulation (0.44-2 mA) for a further 24 h. Unstimulated dexamethasone-treated OC served as controls. Outcome analysis included immunohistochemical labelling of ribbon synapses, histochemical analysis of free reactive oxygen species and morphological analysis of stereocilia bundles. Overall, the protective effects of dexamethasone on electrically induced damage in cochlear explants were moderate. High-dose dexamethasone protected bundle integrity at higher current levels. Low-dose dexamethasone tended to increase ribbon density in the apical region.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Órgão Espiral/efeitos dos fármacos , Estereocílios/efeitos dos fármacos , Animais , Estimulação Elétrica , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Fármacos Neuroprotetores , Técnicas de Cultura de Órgãos , Órgão Espiral/metabolismo , Órgão Espiral/ultraestrutura , Ratos , Espécies Reativas de Oxigênio/metabolismo , Estereocílios/ultraestrutura , Sinapses/efeitos dos fármacos , Sinapses/metabolismo
4.
Exp Mol Med ; 49(12): e411, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29244789

RESUMO

Clinical expansion of mesenchymal stem cells (MSCs) is hampered by the lack of knowledge regarding how to prevent MSC apoptosis and promote their proliferation in serum-free medium. Our in vitro studies demonstrated that human umbilical cord MSCs (HUCMSCs) underwent apoptosis in the serum-free medium. When HUCMSCs were co-cultured with retinal pigment epithelial cells (ARPE19), however, HUCMSCs exhibited normal growth and morphology in serum-free medium. Their colony formation was promoted by the conditioned medium (CM) of ARPE19 cells on Matrigel. Proteomics analysis showed that pigment epithelium-derived factor (PEDF) was one of the most abundant extracellular proteins in the ARPE19 CM, whereas enzyme-linked immunosorbent assay confirmed that large amounts of PEDF was secreted from ARPE19 cells. Adding anti-PEDF-blocking antibodies to the co-culture of HUCMSCs with ARPE19 cells increased apoptosis of HUCMSCs. Conversely, treatment with PEDF significantly reduced apoptosis and increased proliferation of HUCMSCs in serum-free medium. PEDF was further demonstrated to exert this anti-apoptotic effect by inhibiting P53 expression to suppress caspase activation. In vivo studies demonstrated that co-injection of HUCMSCs with ARPE19 cells in immunocompromised NOD-SCID mice also increased survival and decreased apoptosis of HUCMSCs. PEDF also showed no negative effect on the mesoderm differentiation capability of HUCMSCs. In conclusion, this study is the first to demonstrate that PEDF promotes HUCMSC proliferation and protects them from apoptosis by reducing p53 expression in the serum-free medium. This study provides crucial information for clinical-scale expansion of HUCMSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas do Olho/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Fatores de Crescimento Neural/efeitos dos fármacos , Serpinas/efeitos dos fármacos , Cordão Umbilical/citologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Combinação de Medicamentos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Laminina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Proteoglicanas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Proteína Supressora de Tumor p53/genética
5.
Exp Eye Res ; 155: 91-98, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28153738

RESUMO

Glaucoma is the leading cause of irreversible blindness and is usually classified as angle closure and open angle glaucoma (OAG). Primary open angle glaucoma represents the most frequent clinical presentation leading to ganglion cell death and optic nerve degeneration as a main consequence of an intraocular pressure' (IOP) increase. The mechanisms of this IOP increase in such pathology remain unclear but one protein called Myocilin could be a part of the puzzle in the trabecular meshwork (TM). Previously described to be transcriptionally regulated by glucocorticoids, the comprehension of the trabecular regulation of Myocilin' expression has only weakly progressed since 15 years. Due to the essential molecular and cellular implications of retinoids' pathway in eye development and physiology, we investigate the potential role of the retinoic acid in such regulation and expression. This study demonstrates that the global retinoids signaling machinery is present in immortalized TM cells and that Myocilin (MYOC) expression is upregulated by retinoic acid alone or combined with a glucocorticoid co-treatment. This regulation by retinoic acid acts through the MYOC promoter which contains a critical cluster of four retinoic acid responsive elements (RAREs), with the RARE-DR2 presenting the strongest effect and binding the RARα/RXRα heterodimer. All together, these results open up new perspectives for the molecular understanding glaucoma pathophysiology and provide further actionable clues on Myocilin gene regulation.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , RNA/genética , Malha Trabecular/metabolismo , Tretinoína/farmacologia , Western Blotting , Células Cultivadas , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/efeitos dos fármacos , Proteínas do Olho/biossíntese , Proteínas do Olho/efeitos dos fármacos , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Pressão Intraocular/fisiologia , Ceratolíticos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologia
6.
J Ocul Pharmacol Ther ; 29(3): 363-5, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23046435

RESUMO

PURPOSE: To investigate the effect of intravitreal triamcinolone acetonide (TA) on aqueous humor levels of vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule 1 (sICAM-1), and pigment epithelium-derived factor (PEDF) in patients with central retinal vein occlusion (CRVO) and macular edema. METHODS: We measured VEGF, sICAM-1, and PEDF levels in aqueous humor samples from 2 eyes of 2 CRVO patients during injection of TA. RESULTS: In both patients, the VEGF and sICAM-1 levels in aqueous humor samples obtained during initial injection of TA were higher than at the time of reinjection. Conversely, the initial PEDF levels were lower than those at reinjection. CONCLUSIONS: Aqueous humor levels of VEGF and sICAM-1 were decreased by TA treatment in 2 CRVO patients, while PEDF was increased. Intravitreal TA could be an option for CRVO patients with a low PEDF level and/or moderate VEGF and sICAM-1 levels.


Assuntos
Glucocorticoides/uso terapêutico , Edema Macular/tratamento farmacológico , Oclusão da Veia Retiniana/tratamento farmacológico , Triancinolona Acetonida/uso terapêutico , Idoso , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/metabolismo , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Feminino , Glucocorticoides/administração & dosagem , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Injeções Intravítreas , Edema Macular/patologia , Masculino , Fatores de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Oclusão da Veia Retiniana/patologia , Serpinas/efeitos dos fármacos , Serpinas/metabolismo , Triancinolona Acetonida/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Curr Eye Res ; 37(11): 1025-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22906018

RESUMO

PURPOSE: To investigate the influence of lactate on expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in rat retina. METHODS: Retinal explants from neonatal Sprague Dawley rats were incubated with media containing 10, 20, or 30 mM of lactic acid. The 10 mM group was used as a control. At 24 h after incubation, retinas were sectioned for light microscopy, and expressions of VEGF and PEDF measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: The architecture of cultured retinas appeared to be intact. Compared with control, both RT-PCR and Western blot analysis showed that 30 mM of lactic acid significantly increased the levels of VEGF, but not PEDF. CONCLUSIONS: Stimulation of production of retinal VEGF by lactate is dependent on the concentration of lactate. Lactate has no effect on the expression of PEDF in rat retinal explants.


Assuntos
DNA/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácido Láctico/farmacologia , Fatores de Crescimento Neural/genética , Retina/metabolismo , Serpinas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Animais Recém-Nascidos , Western Blotting , Proteínas do Olho/biossíntese , Proteínas do Olho/efeitos dos fármacos , Masculino , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/biossíntese , Serpinas/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
8.
Invest Ophthalmol Vis Sci ; 53(7): 3572-83, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22577078

RESUMO

PURPOSE: Oxidative stress induces retinal damage and contributes to vision loss in progressive retinopathies. Carcinine (ß-alanyl-histamine) is a natural imidazole-containing peptide derivative with antioxidant activity. It is predicted to scavenge 4-hydroxynonenal (4-HNE), a toxic product of lipid oxidation. The aim of this study was to confirm the 4-HNE scavenging effect and evaluate the neuroprotective effect of carcinine in mouse retina subjected to oxidative stress. METHODS: HPLC coupled with mass spectrometry was used to analyze carcinine and 4-HNE-carcinine adduct. Protection of retinal proteins from modification by 4-HNE was tested by incubating carcinine with retinal protein extract and 4-HNE. Modified retinal proteins were quantified by dot-blot analysis. Mice were treated with carcinine (intravitreal injection and gavage) and exposed to bright light to induce oxidative damage in the retina. Photoreceptor degeneration was measured by histology and electroretinography. Retinal levels of retinol dehydrogenase 12 (RDH12) were measured by immunoblot analysis, after exposure to bright light and in retinal explants after exposure to 4-HNE. RESULTS: The ability of carcinine to form an adduct with 4-HNE, as well as to prevent and even reverse the adduction of retinal proteins by the toxic aldehyde was demonstrated in vitro. Carcinine, administered by intravitreal injection or gavage, strongly protected mouse retina against light-induced photoreceptor degeneration and had a protective effect on RHD12, a protein found specifically in photoreceptor cells. CONCLUSIONS: This study suggests that carcinine can be administered noninvasively to efficiently protect photoreceptor cells from oxidative damage. Carcinine could be administered daily to prevent vision loss in progressive retinopathies.


Assuntos
Aldeídos/metabolismo , Carnosina/análogos & derivados , Proteínas do Olho/efeitos dos fármacos , Retina/efeitos dos fármacos , Degeneração Retiniana/patologia , Retinal Desidrogenase/efeitos dos fármacos , Animais , Células 3T3 BALB , Carnosina/farmacologia , Cromatografia Líquida de Alta Pressão , Eletrorretinografia , Proteínas do Olho/metabolismo , Espectrometria de Massas , Camundongos , Fármacos Neuroprotetores , Estresse Oxidativo/efeitos dos fármacos , Retinal Desidrogenase/metabolismo
9.
Eye Contact Lens ; 38(1): 36-42, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22146703

RESUMO

OBJECTIVES: Tear film proteins have antimicrobial and other functions that may be lost after denaturation during contact lens wear. A new multipurpose solution has recently become available (Biotrue, Bausch + Lomb Inc., Rochester, NY), which contains protein-stabilizing agents including hyaluronic acid, poloxamine, and sulfobetaine 10, the latter used previously as a laboratory tool to renature proteins. We examine whether this new multipurpose solution formulation can prevent the denaturation of human lactoferrin and lysozyme at physiologic levels in response to a powerful denaturing challenge. METHODS: Human lactoferrin and lysozyme were treated with sodium dodecyl sulfate (SDS) either with or without an investigational version of the new multipurpose solution (without its two disinfectant agents) (investigational multipurpose solution [iMPS]). The structure was assessed by native-polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorometry; additionally, antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus was measured. RESULTS: The iMPS prevented an SDS-induced shift in the native-PAGE banding position of lactoferrin. The SDS treatment substantially altered the lactoferrin DSC and fluorescence spectra, indicating that the protein had denatured. This change did not occur in the presence of iMPS. Lactoferrin and lysozyme showed antibacterial and bacteriolytic activity, which was abolished after SDS treatment; this loss of activity did not occur for proteins treated with iMPS. CONCLUSIONS: These data clearly show that the iMPS prevents the denaturation of physiologic levels of human lactoferrin and lysozyme by the strongly denaturing surfactant SDS and that stabilized proteins retain their function. We conclude that this solution has the capacity to stabilize the structure and function of tear proteins.


Assuntos
Soluções para Lentes de Contato/química , Proteínas do Olho/química , Lactoferrina/química , Muramidase/química , Análise de Variância , Contagem de Colônia Microbiana , Soluções para Lentes de Contato/farmacologia , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Proteínas do Olho/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Dodecilsulfato de Sódio/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
10.
Br J Ophthalmol ; 95(9): 1309-15, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21719569

RESUMO

AIM: To explore the antiangiogenic property of isoliquiritigenin (ISL) on in vivo and in vitro models. DESIGN: Laboratory investigation. METHODS: The effect of ISL on angiogenesis development was investigated using ex ovo chick chorioallantoic membrane model. Its effect on pathological angiogenesis was examined by (1) silver nitrate cauterisation-induced corneal neovascularisation in BALB/c mice, followed by topical ISL (0.2-50 µM) and CD31 immunofluorescence of corneal blood vessels; (2) argon laser photocoagulation-induced choroidal neovascularisation in C57BL/6 mice, followed by intravitreal ISL (10-200 µM) and fundus fluorescein angiography and immunofluorescence with Griffonia simplicifolia isolectin-B4 (GSA I-B4); and (3) oxygen-induced retinopathy in C57BL/6J mice pups, followed by intravitreal ISL (1-100 µM) and GSA I-B4 immunofluorescence. The vascular area was quantified and analysed by one-way analysis of variance and Student t test. Expression of vascular endothelial growth factor (VEGF) and pigment-epithelium-derived factor in human umbilical vein endothelial cells was analysed by western blotting. RESULTS: Ex ovo chick chorioallantoic membrane assay showed that ISL dose-dependently suppressed VEGF-induced vessel growth. In vivo experiments illustrated that topical ISL alleviated corneal neovascularisation (IC(50)=7.14 µM, day 7) and intravitreal ISL reduced vessel leakage and GSA I-B4-positive vascular area in choroidal and retinal neovascularisation. ISL was found to dose-dependently suppress VEGF and induce pigment epithelium derived factor expression in cultured endothelial cells. CONCLUSION: Using various experimental models of ocular neovascularisation, the authors have demonstrated that ISL from licorice extract has an antiangiogenic effect. The authors' findings suggest that ISL may be a potential antiangiogenic molecule in the development of therapy for neovascularisation diseases.


Assuntos
Chalconas/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Neovascularização da Córnea/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Glycyrrhiza , Neovascularização Retiniana/tratamento farmacológico , Animais , Animais Recém-Nascidos , Western Blotting , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/embriologia , Membrana Corioalantoide/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/biossíntese , Proteínas do Olho/efeitos dos fármacos , Feminino , Angiofluoresceinografia , Imunofluorescência , Fundo de Olho , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/efeitos dos fármacos , Soluções Oftálmicas , Plantas , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Serpinas/biossíntese , Serpinas/efeitos dos fármacos , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese
11.
PLoS One ; 6(1): e16271, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-21249158

RESUMO

BACKGROUND: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. METHODOLOGY/PRINCIPAL FINDINGS: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. CONCLUSIONS/SIGNIFICANCE: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.


Assuntos
Retinopatia Diabética/metabolismo , Proteínas do Olho/efeitos dos fármacos , Insulina/farmacologia , Proteômica/métodos , Retina/química , Animais , Glicemia , Diabetes Mellitus Experimental , Retinopatia Diabética/tratamento farmacológico , Proteínas do Olho/biossíntese , Perfilação da Expressão Gênica , Insulina/administração & dosagem , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , Ratos , Retina/metabolismo
12.
Osteoporos Int ; 22(2): 703-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20414641

RESUMO

UNLABELLED: The purpose of this study is to investigate the dose-dependent effects of SWH on bone properties and the mechanism involved in mediating the osteoprotective actions of SWH. The results indicated that SWH could improve bone properties by inhibiting the process of bone resorption and stimulating the process of bone formation. INTRODUCTION: Our previous study showed that Sambucus williamsii HANCE (SWH) improved trabecular bone mass and cortical bone strength in ovariectomized (OVX) rats. The purpose of this study is to investigate the dose-dependent effects of SWH on bone properties and the mechanism involved in mediating the osteoprotective actions of SWH. METHODS: Three-month-old C57BL/6J mice were fed a phytoestrogen-free diet and subjected to either ovariectomy or sham operation. OVX mice were treated with genistein (50 mg/kg), or a low (200 mg/kg), medium (500 mg/kg), or high (1,000 mg/kg) dose of SWH extract. RESULTS: SWH could dose-dependently decrease urinary Ca excretion and increase serum Ca level in OVX mice. It could increase tibial bone mineral density and exert beneficial effects on the microarchitecture of trabecular bone in the OVX mice. SWH suppressed the ovariectomy-induced expression of Cbfa1 mRNA and cathepsin K mRNA and enhanced the ratio of OPG/RANKL mRNA expression in the tibia. In vitro study showed that SWH dramatically reduced the number of TRAP-positive cells in RANKL-induced RAW 264.7 cells. CONCLUSIONS: The present study indicated that SWH could improve bone properties by inhibiting the process of bone resorption and stimulating the process of bone formation.


Assuntos
Reabsorção Óssea/metabolismo , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Sambucus , Fosfatase Ácida/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Cálcio/sangue , Cálcio/metabolismo , Estudos de Casos e Controles , Catepsina K/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Feminino , Membro Posterior , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoprotegerina/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ovariectomia , Ligante RANK/metabolismo , Fosfatase Ácida Resistente a Tartarato , Tíbia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Resultado do Tratamento
13.
Trends Pharmacol Sci ; 31(6): 284-95, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20435355

RESUMO

Knowledge about retinal photoreceptor signal transduction and the visual cycle required for normal eyesight has increased exponentially over the past decade. Substantial progress in human genetics has facilitated the identification of candidate genes and complex networks underlying inherited retinal diseases. Natural mutations in animal models that mimic human diseases have been characterized and advanced genetic manipulation can now be used to generate small mammalian models of human retinal diseases. Pharmacological repair of defective visual processes in animal models not only validates their involvement in vision, but also provides great promise for the development of improved therapies for millions who are progressing towards blindness or are almost completely robbed of their eyesight.


Assuntos
Cegueira/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Doenças Retinianas/tratamento farmacológico , Retinaldeído/administração & dosagem , Retinoides/administração & dosagem , Animais , Cegueira/genética , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Modelos Biológicos , Células Fotorreceptoras de Vertebrados/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismo , Retinoides/farmacocinética , Retinoides/farmacologia , Visão Ocular/fisiologia , Percepção Visual/fisiologia
14.
J Cardiovasc Pharmacol ; 55(1): 110-5, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19920768

RESUMO

Bestrophin 3 (Best3), a member of bestrophin Cl channel family, is a CaCl(cGMP) channel candidate in vascular smooth muscle cells. The mechanism for its activation remains unclear. In previous studies, we reported that an autoinhibitory domain ((356)IPSFLGS(362)) existed in Best3 C-terminus and when the autoinhibitory domain was mutated, the Best3 channel was dramatically activated. In this study, we further dissected the roles of the C-terminal sequence in Best3 activation. We found that there were eight basic amino acids downstream of the AI domain within the region (384-397), which were also involved in Best3 activation. Mutations of these basic amino acids significantly activated Best3 as a Cl channel. Led by the assumption that the basic amino acids may be involved in the Best3 C-terminal membrane association through binding to membranous phospholipids, we discovered that PI3Kalpha inhibitor IV could strongly activate Best3.


Assuntos
Canais de Cloreto/metabolismo , Proteínas do Olho/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Sequência de Aminoácidos , Animais , Linhagem Celular , GMP Cíclico/metabolismo , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutação
15.
Neuroreport ; 20(8): 755-8, 2009 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-19398933

RESUMO

Light is one of the most important time cues for entrainment of the circadian clock. Drosophila circadian photoreception is mediated by cryptochrome in clock neurons and by rhodopsins in photic organs. We generated Rh5 mutants to elucidate circadian photoreception by rhodopsins. The Rh1, Rh5 and Rh6 mutants were combined with cry, and entrained to a 6-h delayed photoperiod. The cry, Rh1, Rh5 and Rh6 quadruple mutant became entrained by white light. In contrast, reentrainment to green and yellow light was abolished in the cry, Rh1, Rh5 and Rh6 quadruple mutant, and remarkably slowed in the cry, Rh1 and Rh6 triple mutant. These results suggest that cry, Rh1, Rh5 and Rh6 are essential for circadian photoentrainment to green and yellow light.


Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas do Olho/metabolismo , Fotoperíodo , Receptores Acoplados a Proteínas G/metabolismo , Rodopsinas Sensoriais/metabolismo , Animais , Relógios Biológicos/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Criptocromos , Drosophila/genética , Proteínas de Drosophila/efeitos dos fármacos , Proteínas de Drosophila/genética , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/genética , Luz , Mutação/genética , Estimulação Luminosa , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Rodopsinas Sensoriais/genética , Rodopsinas Sensoriais/efeitos da radiação
16.
Aging Cell ; 8(2): 162-77, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19239420

RESUMO

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-beta (Abeta) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Abeta(1-42) - OAbeta(1-42) - on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAbeta(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAbeta(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAbeta(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Abeta in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Abeta may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Degeneração Macular/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Humanos , Hipertrofia/induzido quimicamente , Hipertrofia/metabolismo , Hipertrofia/fisiopatologia , Degeneração Macular/induzido quimicamente , Degeneração Macular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , cis-trans-Isomerases
17.
Mol Vis ; 15: 45-59, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19145251

RESUMO

PURPOSE: The primate retina contains a specialized, cone-rich macula, which mediates high acuity and color vision. The spatial resolution provided by the neural retina at the macula is optimized by stereotyped retinal blood vessel and ganglion cell axon patterning, which radiate away from the macula and reduce shadowing of macular photoreceptors. However, the genes that mediate these specializations, and the reasons for the vulnerability of the macula to degenerative disease, remain obscure. The aim of this study was to identify novel genes that may influence retinal vascular patterning and definition of the foveal avascular area. METHODS: We used RNA from human fetal retinas at 19-20 weeks of gestation (WG; n=4) to measure differential gene expression in the macula, a region nasal to disc (nasal) and in the surrounding retina (surround) by hybridization to 12 GeneChip microarrays (HG-U133 Plus 2.0). The raw data was subjected to quality control assessment and preprocessing, using GC-RMA. We then used ANOVA analysis (Partek) Genomic Suite 6.3) and clustering (DAVID website) to identify the most highly represented genes clustered according to "biological process." The neural retina is fully differentiated at the macula at 19-20 WG, while neuronal progenitor cells are present throughout the rest of the retina. We therefore excluded genes associated with the cell cycle, and markers of differentiated neurons, from further analyses. Significantly regulated genes (p<0.01) were then identified in a second round of clustering according to molecular/reaction (KEGG) pathway. Genes of interest were verified by quantitative PCR (QRT-PCR), and 2 genes were localized by in situ hybridization. RESULTS: We generated two lists of differentially regulated genes: "macula versus surround" and "macula versus nasal." KEGG pathway clustering of the filtered gene lists identified 25 axon guidance-related genes that are differentially regulated in the macula. Furthermore, we found significant upregulation of three anti-angiogenic factors in the macula: pigment epithelium derived factor (PEDF), natriuretic peptide precurusor B (NPPB), and collagen type IValpha2. Differential expression of several members of the ephrin and semaphorin axon guidance gene families, PEDF, and NPPB was verified by QRT-PCR. Localization of PEDF and Eph-A6 mRNAs in sections of macaque retina shows expression of both genes concentrates in the ganglion cell layer (GCL) at the developing fovea, consistent with an involvement in definition of the foveal avascular area. CONCLUSIONS: Because the axons of macular ganglion cells exit the retina from around 8 WG, we suggest that the axon guidance genes highly expressed at the macula at 19-20 WG are also involved in vascular patterning, along with PEDF and NPPB. Localization of both PEDF and Eph-A6 mRNAs to the GCL of the developing fovea supports this idea. It is possible that specialization of the macular vessels, including definition of the foveal avascular area, is mediated by processes that piggyback on axon guidance mechanisms in effect earlier in development. These findings may be useful to understand the vulnerability of the macula to degeneration and to develop new therapeutic strategies to inhibit neovascularization.


Assuntos
Inibidores da Angiogênese/genética , Axônios/metabolismo , Perfilação da Expressão Gênica , Macula Lutea/embriologia , Macula Lutea/metabolismo , Adulto , Inibidores da Angiogênese/metabolismo , Animais , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Macaca , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Família Multigênica , Fatores de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Controle de Qualidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serpinas/efeitos dos fármacos , Serpinas/metabolismo
18.
J Endocrinol ; 200(2): 207-21, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19036760

RESUMO

Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5'-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0.1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.


Assuntos
Colágeno/efeitos dos fármacos , Fibrose/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Vitamina D/farmacologia , Animais , Proteína Morfogenética Óssea 7/efeitos dos fármacos , Células Cultivadas , Etanol/farmacologia , Proteínas do Olho/efeitos dos fármacos , Folistatina/efeitos dos fármacos , Metaloproteinase 8 da Matriz/efeitos dos fármacos , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C3H , Fatores de Crescimento Neural/efeitos dos fármacos , Receptores de Calcitriol/efeitos dos fármacos , Serpinas/efeitos dos fármacos , Fatores de Tempo , Fator de Crescimento Transformador beta1/efeitos dos fármacos
19.
Basic Clin Pharmacol Toxicol ; 101(6): 459-64, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17927688

RESUMO

Rottlerin has been widely accepted as a specific inhibitor of protein kinase C delta (PKC delta); however, recent data suggest that the specificity of this compound become a question. Herein, we address this issue using a lens organ culture system, as PKC delta might regulate the gap junction permeability in lens. Interestingly, we found that rottlerin induced the degradation of connexin50 more rapidly than that of PKC delta. Furthermore, comparison of rottlerin with a protonophore, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) that shares many characteristics with rottlerin, showed that both rottlerin and FCCP dramatically increased lens weight over time. This increase in lens weight was partially reversed by depletion of extracellular calcium with ethyleneglycoltetraacetic acid (EGTA) or by blocking L-type calcium channels with verapamil, suggesting rottlerin may induce calcium influx. Indeed, the rapid degradation of connexin50 (but not PKC delta) induced by rottlerin and FCCP was blocked by EGTA. In addition, rottlerin and FCCP also induced degradation of connexin46, filensin, vimentin and CP49. In order to determine whether this protein degradation is associated with the decrease of ATP due to uncoupling mitochondria by rottlerin, ATP content in lenses with different treatments were examined. The result indicated that EGTA had no effect on lens ATP content. Taken together, these data suggest that rottlerin, like FCCP, induces calcium influx, leading to protein degradation and cleavage in the lens, and that this effect is unrelated to the inhibition of PKC delta. Thus, extreme caution must be taken when considering use of rottlerin as a PKC delta inhibitor.


Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Cristalino/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Conexinas/efeitos dos fármacos , Conexinas/metabolismo , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/efeitos dos fármacos , Proteínas de Filamentos Intermediários/metabolismo , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Técnicas de Cultura de Órgãos , Tamanho do Órgão , Proteína Quinase C-delta/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Vimentina/efeitos dos fármacos , Vimentina/metabolismo
20.
J Int Med Res ; 35(3): 427-32, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17593873

RESUMO

Pigment epithelium-derived factor (PEDF) may have a protective role in atherosclerosis and is associated with the presence of components of the metabolic syndrome. Since oxidative stress has been postulated to play an important role in the pathogenesis of vascular injury in the metabolic syndrome, this study investigated the effects of hydrogen peroxide (H2O2) on PEDF in the immortalized human hepatocyte cell line OUMS-29. PEDF gene expression was measured using quantitative real-time reverse transcription-polymerase chain reaction and PEDF protein expression was analysed by Western blot. H2O2 upregulated PEDF mRNA levels and increased PEDF protein production in OUMS-29 cells in time- and dose-dependent manners. The anti-oxidant N-acetylcysteine significantly blocked H2O2-induced PEDF overexpression in OUMS-29 cells. These results suggest that hepatic PEDF levels may be elevated to counteract the effects of oxidative stress. H2O2-induced PEDF overproduction in the liver may act as a negative feedback system against vascular damage in the metabolic syndrome.


Assuntos
Proteínas do Olho/genética , Hepatócitos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Fatores de Crescimento Neural/genética , Serpinas/genética , Linhagem Celular Transformada , Proteínas do Olho/efeitos dos fármacos , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fatores de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Serpinas/efeitos dos fármacos , Serpinas/metabolismo
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