Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Klin Monbl Augenheilkd ; 240(7): 878-886, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34571550

RESUMO

BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.


Assuntos
Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Etoposídeo/uso terapêutico , Neoplasias da Retina/genética
2.
Int J Mol Sci ; 22(9)2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33923095

RESUMO

Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.


Assuntos
Retinopatia Diabética/genética , Netrinas/genética , Retinite/metabolismo , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Eletrorretinografia , Regulação da Expressão Gênica , Humanos , Camundongos Transgênicos , Netrinas/metabolismo , Retina/patologia , Retina/fisiologia , Retinite/etiologia
3.
Graefes Arch Clin Exp Ophthalmol ; 258(1): 217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31729555

RESUMO

The article "Lack of netrin-4 alters vascular remodeling in the retina".

4.
Exp Eye Res ; 189: 107838, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622617

RESUMO

As many other organs, the retina has a local renin-angiotensin-system (RAS). All main elements of the RAS are active in the retina: renin, angiotensinogen, angiotensin-converting enzymes. The functional role of the intraretinal RAS is not fully understood. So far, histological and functional analysis point to a regulation of ganglion cell activity and maybe also of bipolar cell activity, but it is not clear how RAS contributes to retinal signal processing. In contrast to local RAS in other organs, the retinal RAS is clearly separated from the systemic RAS. The angiotensin-2 (AngII)/AngI ratio in the retina is different to that in the plasma. However, it appears that the retinal pigment epithelium (RPE), that forms the outer blood/retina barrier, is a major regulator of the retinal RAS by producing renin. Interestingly, comparable to the kidney, the renin production in the RPE is under control of the angiotensin-2 receptor type-1 (AT1). AT1 localizes to the basolateral membrane of the RPE and faces the blood side of the blood/retina barrier. Increases in systemic AngII reduce renin production in the RPE and therefore decrease the intraretinal RAS activity. The relevance of the local RAS for retinal function remains unclear. Nevertheless, it is of fundamental significance to understand the pathology of systemically induced retinal diseases such as hypertension or diabetes.


Assuntos
Barreira Hematorretiniana/metabolismo , Sistema Renina-Angiotensina/fisiologia , Renina/biossíntese , Epitélio Pigmentado da Retina/metabolismo , Animais , Humanos
5.
Graefes Arch Clin Exp Ophthalmol ; 257(10): 2179-2184, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31451908

RESUMO

PURPOSE: Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina. METHODS: We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG). RESULTS: Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio. CONCLUSIONS: These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.


Assuntos
Netrinas/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Remodelação Vascular/fisiologia , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Angiofluoresceinografia , Fundo de Olho , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oftalmoscopia , Neovascularização Retiniana/patologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologia
6.
Diabetologia ; 60(1): 202-211, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27704165

RESUMO

AIMS/HYPOTHESIS: Although the renin-angiotensin system plays an important role in the progression of diabetic retinopathy, its influence therein has not been systematically evaluated. Here we test the suitability of a new translational model of diabetic retinopathy, the TetO rat, for addressing the role of angiotensin-II receptor 1 (AT1) blockade in experimental diabetic retinopathy. METHODS: Diabetes was induced by tetracycline-inducible small hairpin RNA (shRNA) knockdown of the insulin receptor in rats, generating TetO rats. Systemic treatment consisted of an AT1 blocker (ARB) at the onset of diabetes, following which, 4-5 weeks later the retina was analysed in vivo and ex vivo. Retinal function was assessed by Ganzfeld electroretinography (ERG). RESULTS: Retinal vessels in TetO rats showed differences in vessel calibre, together with gliosis. The total number and the proportion of activated mononuclear phagocytes was increased. TetO rats presented with loss of retinal ganglion cells (RGC) and ERG indicated photoreceptor malfunction. Both the inner and outer blood-retina barriers were affected. The ARB treated group showed reduced gliosis and an overall amelioration of retinal function, alongside RGC recovery, whilst no statistically significant differences in vascular and inflammatory features were detected. CONCLUSIONS/INTERPRETATION: The TetO rat represents a promising translational model for the early neurovascular changes associated with type 2 diabetic retinopathy. ARB treatment had an effect on the neuronal component of the retina but not on the vasculature.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Receptor de Insulina/metabolismo , Animais , Western Blotting , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Retinopatia Diabética/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Ratos , Receptor de Insulina/genética , Receptores de Angiotensina/metabolismo , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina
7.
Clin Sci (Lond) ; 130(13): 1075-88, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27026533

RESUMO

Severe hypertension destroys eyesight. The RAS (renin-angiotensin system) may contribute to this. This study relied on an established angiotensin, AngII (angiotensin II)-elevated dTGR (double-transgenic rat) model and same-background SD (Sprague-Dawley) rat controls. In dTGRs, plasma levels of AngII were increased. We determined the general retinal phenotype and observed degeneration of ganglion cells that we defined as vascular degeneration. We also inspected relevant gene expression and lastly observed alterations in the outer blood-retinal barrier. We found that both scotopic a-wave and b-wave as well as oscillatory potential amplitude were significantly decreased in dTGRs, compared with SD rat controls. However, the b/a-wave ratio remained unchanged. Fluorescence angiography of the peripheral retina indicated that exudates, or fluorescein leakage, from peripheral vessels were increased in dTGRs compared with controls. Immunohistological analysis of blood vessels in retina whole-mount preparations showed structural alterations in the retina of dTGRs. We then determined the general retinal phenotype. We observed the degeneration of ganglion cells, defined vascular degenerations and finally found differential expression of RAS-related genes and angiogenic genes. We found the expression of both human angiotensinogen and human renin in the hypertensive retina. Although the renin gene expression was not altered, the AngII levels in the retina were increased 4-fold in the dTGR retina compared with that in SD rats, a finding with mechanistic implications. We suggest that alterations in the outer blood-retinal barrier could foster an area of visual-related research based on our findings. Finally, we introduce the dTGR model of retinal disease.


Assuntos
Retinopatia Hipertensiva/fisiopatologia , Sistema Renina-Angiotensina/genética , Angiotensina II/metabolismo , Angiotensinogênio/genética , Animais , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Retinopatia Hipertensiva/genética , Masculino , Ratos Transgênicos , Renina/metabolismo
9.
Invest Ophthalmol Vis Sci ; 65(4): 5, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38558091

RESUMO

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.


Assuntos
Neovascularização de Coroide , Degeneração Macular , Humanos , Camundongos , Animais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Sacarina/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Edulcorantes , Estudos Transversais , Cromatografia Líquida , Espectrometria de Massas em Tandem , Neovascularização de Coroide/metabolismo , Degeneração Macular/metabolismo , RNA Mensageiro/genética , Injeções Intravítreas , Inibidores da Angiogênese/uso terapêutico
10.
Exp Eye Res ; 94(1): 157-73, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22182671

RESUMO

Differences in transient receptor potential (TRP) and cannabinoid receptor type 1 (CB1) expression levels can serve as prognostic factors for retinoblastoma (RB) tumor progression. We hypothesized in RB tissue that such differences are also indicators of whether or not they are sensitive to etoposide. Accordingly, we compared in malignant etoposide-sensitive and etoposide-resistant WERI-Rb1 cells TRPV1, TRPM8 and TRPA1 subtype and CB1 gene expression pattern levels and accompanying functional activity using quantitative real-time RT-PCR, immunohistochemistry, immunofluorescence microscopy, calcium imaging as well as patch-clamp technology. Gene expression patterns were evaluated in enucleated human RB tissues (n = 4). Both etoposide-resistant and etoposide-sensitive WERI-Rb1 cells expressed all of the aforementioned channels based on responses to known activators and thermal challenges. However, TRPA1 was absent in the etoposide-resistant counterpart. Even though both types of RB cells express TRPV1 as well as TRPM8 and CB1, the capsaicin (50 µM) (CAP)-induced Ca(2+) rise caused by TRPV1 activation was prompt and transient only in etoposide-resistant RB cells (n = 8). In this cell type, the inability of CB1 activation (10 µM WIN) to suppress Ca(2+) responses to CAP (50 µM; n = 4) may be attributable to the absence of TRPA1 gene expression. Therefore, using genetic approaches to upregulate TRPA1 expression could provide a means to induce etoposide sensitivity and suppress RB cell tumorigenesis.


Assuntos
Cálcio/metabolismo , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias da Retina/metabolismo , Retinoblastoma/metabolismo , Canais de Potencial de Receptor Transitório/genética , Capsaicina/farmacologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/genética , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico
11.
Mol Cell Biol ; 27(4): 1370-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17130236

RESUMO

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.


Assuntos
Marcação de Genes , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Visão Ocular/fisiologia , Oxirredutases do Álcool , Animais , Eletrorretinografia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Estresse Oxidativo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/enzimologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retinal Desidrogenase/deficiência , Retinal Desidrogenase/imunologia , Retinoides/análise , Visão Ocular/genética
12.
PLoS One ; 13(3): e0193961, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29547662

RESUMO

Animal models of disease are an indispensable element in our quest to understand pathophysiology and develop novel therapies. Ex vivo studies have severe limitations, in particular their inability to study individual disease progression over time. In this respect, non-invasive in vivo technologies offer multiple advantages. We here used bilateral common carotid artery occlusion (BCCAO) in mice, an established model for ischemic retinopathy, and performed a multimodal in vivo and ex vivo follow-up. We used scanning laser ophthalmoscopy (SLO), ocular coherence tomography (OCT) and electroretinography (ERG) over 6 weeks followed by ex vivo analyses. BCCAO leads to vascular remodeling with thickening of veins starting at 4 weeks, loss of photoreceptor synapses with concomitant reduced b-waves in the ERG and thinning of the retina. Mononuclear phagocytes showed fluctuation of activity over time. There was large inter-individual variation in the severity of neuronal degeneration and cellular inflammatory responses. Ex vivo analysis confirmed these variable features of vascular remodeling, neurodegeneration and inflammation. In summary, we conclude that multimodal follow-up and subgroup analysis of retinal changes in BCCAO further calls into question the use of ex vivo studies with distinct single end-points. We propose that our approach can foster the understanding of retinal disease as well as the clinical translation of emerging therapeutic strategies.


Assuntos
Arteriopatias Oclusivas/patologia , Doenças das Artérias Carótidas/patologia , Retina/patologia , Vasos Retinianos/patologia , Animais , Artéria Carótida Primitiva/patologia , Modelos Animais de Doenças , Progressão da Doença , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oftalmoscopia/métodos , Degeneração Retiniana/patologia , Tomografia de Coerência Óptica/métodos
14.
Invest Ophthalmol Vis Sci ; 47(8): 3523-30, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16877424

RESUMO

PURPOSE: In a spontaneous mutant substrain of C57BL/10 mice, severely affected retinal ribbon-type synapses have been described. The retinopathy was accompanied by a substantial loss in the activities of the second-order neurons. Rod photoreceptor responses were maintained with reduced amplitude, whereas cone activities were absent. This study was conducted to identify the genetic defect underlying this hitherto unknown autosomal recessive cone-rod dysfunction. METHODS: Genome-wide linkage analysis and screening of positional candidate genes were used to identify the causative mutation. Tissue-specific transcriptional activity of the defective gene was determined by Northern blot analysis and RT-PCR approaches. The number of cone photoreceptors was estimated by immunohistochemistry. RESULTS: The mutation was localized to a 275-kb region of chromosome 6. Within this candidate interval, a homozygous frameshift mutation (c.2367insC) was identified in the Cacna2d4 gene of affected animals. This gene codes for an L-type calcium channel auxiliary subunit of the alpha2delta type. The mutation introduces a premature stop codon that truncates one third of the predicted Cacna2d4 protein. A severe reduction in Cacna2d4 transcript levels observed in mutant retinas probably results in the lack of Cacna2d4 protein. The mutation leads to significant loss of rods, whereas the number of cone cells remains unaffected until 6 weeks of age. CONCLUSIONS: The Cacna2d4 mutation underlies a novel channelopathy leading to cone-rod dysfunction in the visual system of mice and provides a new candidate gene for human retinal disorders including night blindness, retinitis pigmentosa, and cone-rod dystrophies.


Assuntos
Canais de Cálcio Tipo L/genética , Mutação da Fase de Leitura , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Análise Mutacional de DNA , Eletrorretinografia , Ligação Genética , Genótipo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica
15.
Sci Rep ; 6: 18828, 2016 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-26732856

RESUMO

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4(-/-) mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4(-/-) mice. In OIR, Ntn-4(-/-) mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4(-/-) mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4(-/-) mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization.


Assuntos
Oftalmopatias/etiologia , Oftalmopatias/patologia , Neovascularização Patológica/genética , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/genética , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Oftalmopatias/diagnóstico , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Netrinas , Neuroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Retina/metabolismo , Retina/patologia , Doenças Retinianas/diagnóstico , Doenças Retinianas/etiologia , Doenças Retinianas/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Cell Signal ; 26(1): 56-69, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24084605

RESUMO

Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca(2+) channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca(2+) permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca(2+) transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca(2+) transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La(3+), capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca(2+) transients, which were suppressed by La(3+) and CPZ whereas CAP-induced Ca(2+) transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.


Assuntos
Cálcio/metabolismo , Melanoma/genética , Melanoma/patologia , Temperatura , Canais de Potencial de Receptor Transitório/genética , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Benzoxazinas/farmacologia , Capsaicina/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Morfolinas/farmacologia , Naftalenos/farmacologia , Pirimidinonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo
17.
Invest Ophthalmol Vis Sci ; 54(4): 2582-9, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23518774

RESUMO

PURPOSE: Therapeutic approaches to retinal disease require a continuous monitoring of functional improvement over lesion areas that sometimes cannot be shown in full-field ERG. The aim of this study was to assess the usefulness of multifocal electroretinograms (mfERGs) under visual control using scanning laser ophthalmoscopy (SLO) for evaluation of local retinopathy in mice. METHODS: mfERGs were optimized for recordings in C57BL/6 mice by varying dark steps between each stimuli, background intensity, and the numbers of hexagons. Local retinopathy was induced by argon laser photocoagulation with different spot sizes and retinal irradiances. mfERG recordings were performed before, and 10 days and 4 weeks after laser treatment. In each recording, the central hexagon was positioned on the optic nerve head visualized by SLO images. The amplitudes of the P1 response components were analyzed as a function of retinal location. RESULTS: The mfERG amplitudes depended on stimulus condition. The P1 amplitudes increased with increasing number of dark frames in the m-sequence and with decreasing number of hexagons. A stimulus with 19 hexagons and four dark frames was chosen because substantial response amplitudes could be achieved while preserving sufficient spatial resolution. In the untreated eyes, the response to the central hexagon, stimulating the optic nerve head, was smaller than those to the surrounding hexagons. The responses to hexagons stimulating photocoagulated areas were reduced compared with the responses of surrounding areas. The amplitude reduction was more pronounced when the coagulated areas were larger and when higher energies were used. CONCLUSIONS: Areas with decreased sensitivities to light stimulation (either the optic nerve head or damaged retinal areas) can be detected and correlated with the retinal images and in the mfERG responses. We demonstrate that the mfERG technique is able to reproducibly detect the functional consequences of a local treatment.


Assuntos
Eletrorretinografia/métodos , Disco Óptico/fisiopatologia , Retina/fisiopatologia , Doenças Retinianas/diagnóstico , Animais , Modelos Animais de Doenças , Fundo de Olho , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa/métodos , Doenças Retinianas/fisiopatologia
18.
EMBO Mol Med ; 4(9): 980-91, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22903875

RESUMO

ß-Secretase (BACE1) is a major drug target for combating Alzheimer's disease (AD). Here we show that BACE1(-/-) mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial-derived factor, a known regulator of γ-secretase, inhibits vascular endothelial growth factor (VEGF)-induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ-secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age-related macular degeneration.


Assuntos
Secretases da Proteína Precursora do Amiloide/deficiência , Ácido Aspártico Endopeptidases/deficiência , Lipofuscina/metabolismo , Retina/patologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Neovascularização de Coroide/patologia , Inibidores Enzimáticos/administração & dosagem , Feminino , Inativação Gênica , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
19.
PLoS One ; 7(6): e38310, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22701626

RESUMO

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.


Assuntos
Modelos Animais de Doenças , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Fenótipo , Degeneração Retiniana/patologia , Análise de Variância , Animais , Temperatura Corporal , Encéfalo/patologia , Eletrorretinografia , Comportamento Exploratório/fisiologia , Feminino , Ferritinas/sangue , Genótipo , Coração/crescimento & desenvolvimento , Imuno-Histoquímica , Linfócitos/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/complicações , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Tamanho do Órgão , Consumo de Oxigênio/fisiologia , Degeneração Retiniana/etiologia
20.
Invest Ophthalmol Vis Sci ; 51(2): 1106-15, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20007830

RESUMO

PURPOSE: To establish mouse models for RPGR-associated diseases by generating and characterizing an Rpgr mutation (in-frame deletion of exon 4) in two different genetic backgrounds (BL/6 and BALB/c). METHODS: Gene targeting in embryonic stem (ES) cells was performed to introduce a in-frame deletion of exon 4 in the Rpgr gene (Rpgr(DeltaEx4)). Subsequently, the mutation was introduced in two different inbred mouse strains by successive breeding. Mutant and wild-type mice of both strains were characterized by electroretinography (ERG) and histology at five time points (1, 3, 6, 9, and 12 months). RPGR transcript amounts were assessed by quantitative RT-PCR. A variety of photoreceptor proteins, including RPGR-ORF15, RPGRIP, PDE6delta/PrBPdelta, rhodopsin, and cone opsin, were localized on retinal sections by immunohistochemistry. RESULTS: Mislocalization of rhodopsin and cone opsin was an early pathologic event in mutant mice of both lines. In contrast, RPGR-ORF15 as well as RPGRIP1 and PDE6delta/PrBPdelta showed similar localizations in mutant and wild-type animals. Functional and histologic studies revealed a mild rod-dominated phenotype in mutant male mice on the BL/6 background, whereas a cone-dominated phenotype was observed for the same mutation in the BALB/c background. CONCLUSIONS: Both Rpgr mutant mouse lines developed retinal disease with a striking effect of the genetic background. Cone-specific modifiers might influence the retinal phenotype in the BALB/c strain. The two lines provide models to study RPGR function in rods and cones, respectively.


Assuntos
Proteínas de Transporte/genética , Modelos Animais de Doenças , Proteínas do Olho/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Proteínas de Transporte/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas do Citoesqueleto , Eletrorretinografia , Células-Tronco Embrionárias/metabolismo , Éxons/genética , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Opsinas/metabolismo , Proteínas/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Doenças Retinianas/metabolismo , Doenças Retinianas/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA